1Department of Medicine, College of Medicine, Hawler Medical University, Erbil, Iraq; 2Department of Clinical Haematology, Nanakaly Hospital for Blood Diseases & Oncology, Erbil, Iraq E-mail: mah_kawa@yahoo.com اجلوانب السريرية )اإلكلينيكية(، حتليل النمط املناعي الظاهري ومعدل البقاء على قيد احلياة ملرضى سرطان الدم الليمفاوي املزمن يف مدينة أربيل، العراق كاوه حممدامني ح�شن abstract: Objectives: Chronic lymphocytic leukaemia (CLL) is characterised by an accumulation of clonal B cells in the blood, bone marrow and lymphatic tissue. This study aimed to evaluate the clinical and immunophenotypic characteristics and survival rate of CLL patients. Methods: This retrospective study was conducted at the Nanakaly Hospital for Blood Diseases & Oncology in Erbil, Iraq, between January 2011 and December 2017. A total of 105 CLL patients were assessed to determine clinical presentation and staging, immunophenotype and survival rate. Results: The median age of the patients was 65 years and 63.8% were male. The main clinical presentations were splenomegaly (64.8%), pallor (61.9%) and lymphadenopathy (60%). More than half of the patients presented at an advanced clinical stage according to the Rai and Binet staging systems (59.1% and 55.2%, respectively). All CLL cases expressed both cluster of differentiation (CD)19 and CD5, 67.6% had monoclonal kappa light chains and 21% expressed CD38. The five-year overall survival (OS) rate was 61.3%. The mean duration of five-year survival was 41.3 months (95% confidence interval: 36.4–46.3 months). There were no correlations between survival and sociodemographic, clinical or laboratory characteristics. Conclusion: In comparison to the existing Western literature, Iraqi CLL patients more frequently presented with hepatosplenomegaly and at a more advanced clinical stage. In addition, the five-year OS rate was much lower. Keywords: Lymphoproliferative Disorders; Chronic Lymphocytic Leukemia; Immunophenotyping; Survival Rates; Iraq. هدفت الليمفاوي. والن�شيج العظام ونخاع الدم يف الن�شيلة ب خاليا برتاكم املزمن الليمفاوي الدم �رصطان يتميز الهدف: امللخ�ص: هذه الدرا�شة ايل تقييم اخل�شائ�س ال�رصيرية و النمط املناعي الظاهري ومعدل البقاء على قيد احلياة للمر�شى امل�شابني ب�رصطان الدم الليمفاوي املزمن. الطريقة: اأجريت هذه الدرا�شة الأ�شرتجاعية يف م�شت�شفى ناناكايل لأمرا�س الدم والأورام يف اأربيل، العراق، بني يناير 2011 ودي�شمرب 2017. مت تقييم ما جمموعه 105 م�شابا مبر�س �رصطان الدم الليمفاوي املزمن لتحديد العر�س ال�رصيري و مرحلة املر�س وكانت الذكور. من بينهم 63.8% �شنة املر�شى 65 عمر متو�شط كان احلياة. النتائج: قيد على البقاء ومعدل الظاهري املناعي النمط و العالمات ال�رصيرية الرئي�شية عبارة عن ت�شخم الطحال )%64.8(، �شحوب )%61.9( واعتالل العقد اللمفاوية )%60(. كان اأكرث من ن�شف املر�شى يف مرحلة �رصيرية متقدمة وفقا جلداول راي وبينيت )%59.1 و %55.2 على التوايل(. اأظهر حتليل النمط املناعي الظاهري جلميع املر�شى كال من جمموعة التمايز 19 و 5، بينما اأظهر %67.6 من املر�شى �شال�شل كابا اخلفيفة وحيدة الن�شيلة و %21 منهم كان لديهم جمموعة التمايز 38. وكان املعدل العام للبقاء على قيد احلياة ملدة خم�س �شنوات هو %61.3. وكان متو�شط مدة البقاء على قيد احلياة 36.4–46.3 اأ�شهر(. مل تكن هناك ارتباط بني معدل البقاء على قيد احلياة وال�شفات ملدة خم�س �شنوات 41.3 �شهرا )فا�شل الثقة 95%: الجتماعية والدميوغرافية وال�رصيرية واملخربية للمر�شى. اخلال�صة: باملقارنة مع املطبوعات الغربية احلالية، فاإن مر�شى �رصطان الدم الليمفاوي املزمن العراقيني لديهم بن�شبة اكرث من ت�شخم الطحال والكبد ويتم ت�شخي�شهم يف مرحلة �رصيرية اأكرث تقدًما. بالإ�شافة اإىل ذلك، كان معدل البقاء على قيد احلياة ملدة خم�س �شنوات فيهم اأقل بكثري. الكلمات املفتاحية: ال�شطرابات التكاثرية الليمفاوية؛ �رصطان الدم الليمفاوي املزمن؛ النمط املناعي الظاهري؛ معدلت البقاء على قيد احلياة؛ العراق. Clinical Aspects, Immunophenotypic Analysis and Survival Rate of Chronic Lymphocytic Leukaemia Patients in Erbil City, Iraq Kawa M. Hasan1,2 clinical & basic research Sultan Qaboos University Med J, November 2018, Vol. 18, Iss. 4, pp. e461–467, Epub. 28 Mar 19 Submitted 18 Jul 18 Revision Req. 3 Sep 18; Revision Recd. 27 Sep 18 Accepted 18 Oct 18 doi: 10.18295/squmj.2018.18.04.006 Advances in Knowledge - This study provides the clinical and immunophenotypic findings and the survival rate of chronic lymphocytic leukaemia (CLL) patients in Erbil City, Iraq. - The current sample of CLL patients more frequently presented with hepatosplenomegaly, although less frequently with lymphadenopathy, compared to populations reported from Western countries. In addition, the Iraqi patients tended to present at a more advanced clinical stage and had lower survival rates than Western patients. Application to Patient Care - The current results highlight the importance of implementing more advanced work-up tools for Iraqi CLL patients, such as a thorough cytogenetic evaluation. Clinical Aspects, Immunophenotypic Analysis and Survival Rate of Chronic Lymphocytic Leukaemia Patients in Erbil City, Iraq e462 | SQU Medical Journal, November 2018, Volume 18, Issue 4 Chronic lymphocytic leukaemia (cll) is the most common type of leukaemia among adults in Western countries; however, it is relat- ively rare in Asia.1,2 It is characterised by the build-up of lymphocytes in the blood, bone marrow and lymphatic tissue. The median age at diagnosis ranges from 67–72 years and the condition is more common in males.1,2 The clinical course of CLL is highly variable, ranging from no symptoms to the rapid development of features of high-risk disease.3 Lymph node swelling is the most common presenting feature of CLL, although fever, night sweats and weight loss are sometimes seen. The most common physical findings are lymphadenopathy, splen- omegaly and, less frequently, hepatomegaly.3 A diagnosis of CLL can be established via a complete blood count (CBC) showing the progressive accumulation of clonal B cells (>5,000 B lymphocytes/mL) over a period of at least three months and an immunophenotypic study demonstrating clonal lymphocytes expressing B cell mar- kers and cluster of differentiation (CD)5.4 Both the mod- ified Rai and Binet clinical staging systems are widely used to classify CLL patients into different prognostic groups according to the extent of lymph node involve- ment, enlargement of the liver and/or spleen and blood findings (i.e. anaemia and thrombocytopaenia).5,6 The median survival rate for patients with CLL ranges from 18 months to more than 10 years, depending on prognostic factors like age, gender, disease stage, performance status, lymphocyte doubling time, absolute lymphocyte count (ALC) and β-2 microglobulin serum levels.7 Furthermore, the expression of certain biological markers such as CD38 or CD49d, deletion of chromo- some 17p13 and the mutation status of the immuno- globulin heavy-chain variable region, tumour protein- 53 (TP53), Notch homolog 1, splicing factor-3B subunit-1 (SF3B1), baculoviral inhibitor of apoptosis protein-1 repeat-containing protein-3 (BIRC3), myeloid different- iation primary response 88 and zeta chain T cell receptor- associated protein kinase-70 (ZAP70) genes also have a prognostic impact.8 The current study aimed to assess and compare the clinical characteristics, immunopheno- typic findings and survival rate of CLL patients in Erbil, Iraq, with previously reported international and regional data. Methods This retrospective study was carried out between January 2011 and December 2017 at the Nanakaly Hospital for Blood Diseases & Oncology, a regional referral hospital for adult and paediatric patients with benign and malig- nant haematology and solid oncology disorders in Erbil, Iraq. All patients attending the hospital during the study period and diagnosed with CLL as confirmed by CBC and immunophenotyping were invited to participate in the study. The main clinical presentation and characteristics of each patient was documented, including the presence of lymphadenopathy, pallor, hepatomegaly and spleno- megaly. In addition, the patients underwent a CBC and immunophenotyping study by flow cytometry using a FACSCanto II flow cytometer device (BD Biosciences, San Jose, California, USA) and/or immunohistochemistry. The laboratory analysis included a reticulocyte count, erythrocyte sedimentation rate, direct antiglobulin test (DAT), bone marrow examination, serology tests (i.e. to determine lactate dehydrogenase [LDH] levels) and liver and renal function tests. In indicated cases, patients also underwent various imaging studies, including ultra- sonography of the abdomen, a chest X-ray and computed tomography. In each case, clinical stage was determined Table 1: Baseline characteristics of chronic lymphocytic leukaemia patients in Erbil, Iraq (N = 105) Characteristic n (%) Age in years <50 10 (9.5) 50–59 21 (20) 60–69 40 (38.1) ≥70 34 (32.4) Gender Male 67 (63.8) Female 38 (36.2) Clinical presentation* Splenomegaly† 68 (64.8) Pallor 65 (61.9) Peripheral lymphadenopathy 63 (60) Hepatomegaly 23 (21.9) Rai clinical stage 0 14 (13.3) I 9 (8.6) II 20 (19) III 19 (18.1) IV 43 (41) Binet clinical stage A 29 (27.6) B 18 (17.1) C 58 (55.2) *Percentages do not add up to 100% as some patients may have had more than one clinical presentation. †For patients with splenomegaly, the mean longitudinal axis of the spleen was 14.7 ± 3.4 cm. Kawa M. Hasan Clinical and Basic Research | e463 according to both the Rai and Binet staging systems.5,6 Survival rates were calculated, including the overall survival (OS) rate over the study period and the five- year OS rate. Data were analysed using the Statistical Package for the Social Sciences (SPSS), Version 22.0 (IBM Corp., Armonk, New York, USA). A Chi-squared test was used to compare associations between proportions. Kaplan- Meier survival curves were plotted and the log-rank Mantel-Cox test was used to compare mean survival times. A P value of ≤0.050 was considered statistically significant. This study was approved by the Scientific and Ethical Committees of the College of Medicine, Hawler Medical University, Erbil. Results A total of 143 CLL patients attended the hospital during the study period. However, 38 patients were excluded due to a lack of adequate data or follow-up. As such, Figure 1: Immunophenotypic findings of chronic lymphocytic leukaemia patients in Erbil, Iraq (N = 105). CD = cluster of differentiation; Bcl2 = B cell lymphoma 2; FMC7 = Flinder Medical Centre-7. Table 2: Laboratory results of chronic lymphocytic leukaemia patients in Erbil, Iraq (N = 105) Investigation Mean ± SD Median Range Normal range Haemoglobin in g/dL 10.6 ± 2.3 11 5–15.4 13.5–17.5/11.5–15.5* Whole blood count × 109/L 94.8 ± 106.6 65 8.9–491 4–11 Absolute lymphocyte count × 109/L 87 ± 104.5 58.5 6–462.5 1–4.8 Absolute neutrophil count × 109/L 6.7 ± 5.7 6 0.2–54 1.8–7.7 Platelet count × 109/L 140 ± 74.2 115 30–382 150–400 Reticulocytes, % 2.1 ± 2.0 1.7 0.1–10 0.5–1.5 Erythrocyte sedimentation rate in mm/hour 44.3 ± 33.3 37 2–145 0–10/3–15* Lactate dehydrogenase in U/L 400.1 ± 159.9 378 108–905 100–250 Aspartate aminotransferase in U/L 24 ± 10.9 22 8.9–99 8–48 Alanine transaminase in U/L 18.1 ± 9.9 17 4–50.3 7–55 Blood urea in mg/dL 44.2 ± 21.3 41 16.3–160 15–40 Serum creatinine in mg/dL 1 ± 0.4 1 0.3–3 0.7–1.3 SD = standard deviation. *Males/females. Table 3: Treatment provided to chronic lymphocytic leukaemia patients in Erbil, Iraq (N = 105) Treatment* n (%) FCR 24 (22.9) BR 19 (18.1) FC 2 (1.9) Chlorambucil 9 (8.6) Chlorambucil plus prednisolone 10 (9.5) Rituximab and chlorambucil 10 (9.5) COP 4 (3.8) Rituximab and COP 5 (4.8) Rituximab and CHOP 7 (6.7) Rituximab 2 (1.9) No treatment 17 (16.2) FCR = fludarabine, cyclophosphamide and rituximab; BR = bendam- ustine plus rituximab; FC = fludarabine plus cyclophosphamide; COP = cyclo- phosphamide, oncovin and prednisolone; CHOP =cyclophosphamide, hydro- xydaunomycin, oncovin and prednisolone. *Percentages do not add up to 100% as four patients were treated with more than one protocol. Clinical Aspects, Immunophenotypic Analysis and Survival Rate of Chronic Lymphocytic Leukaemia Patients in Erbil City, Iraq e464 | SQU Medical Journal, November 2018, Volume 18, Issue 4 105 patients were included in the final analysis. Of these, 67 (63.8%) were male and 38 (36.2%) were female. The mean age at presentation was 63.3 ± 10.7 years. Most patients (70.5%) were ≥60 years, with only 9.5% <50 years. The age at diagnosis ranged from 28–81 years old, with a median of 65 years. The main clinical presentations were splenomegaly (64.8%) pallor (61.9%) and lymphadenopathy (60%). Hepatomegaly was observed in 21.9% of patients. Modified Rai staging showed a high risk (stage III or IV) of CLL in 59.1%, an intermediate risk (stage I or II) in 27.6% and a low risk (stage 0) in 13.3% of patients. In terms of Binet staging, 55.2% of patients were stage C, 17.1% were stage B and 27.6% were stage A [Table 1]. Most patients (72.4%) were from Erbil and other cities in the Kurdistan region of Iraq, while the rest were from other northern and western Iraqi cities. The mean haemoglobin level was 10.6 ± 2.3 g/dL (range: 5–15.4 g/dL). Mean platelet and white blood cell counts were 140 ± 74.2 × 109/L (range: 30–382 × 109/L) and 94.8 ± 106.6 × 109/L (range: 8.9–491 × 109/L), respectively. The mean ALC was 87 ± 104.5 × 109/L (range: 6–462.5 × 109/L) [Table 2]. Overall, LDH levels were elevated in 82.8% of patients and 11.4% had autoimmune haemolytic anaemia (AIHA) with positive DAT results. Anaemia was observed in 83 (79.1%) patients, comprising 83.6% and 71.1% of male and female patients, respectively. A total of 63 (60%) and 28 (26.7%) patients presented with thrombocytopaenia and incidental lymph- ocytosis, respectively. None of the patients had Richter’s syndrome. All patients underwent immunophenotyping an- alysis via flow cytometry and/or immunohistochemistry from either peripheral blood (79.1%) or bone marrow (21%) samples. All cases expressed both CD19 and CD5, while the vast majority expressed CD23 (94.3%), CD45 (84.8%) and B cell lymphoma 2 (82.9%). About two- thirds of the patients (67.6%) expressed monoclonal kappa light chains, while just under one-third (32.4%) expressed lambda chains. Only 21% of cases expressed CD38 [Figure 1]. In terms of treatment, most patients received either fludarabine, cyclophosphamide and rituximab (22.9%) or bendamustine plus rituximab (18.1%). The rest underwent various other chemotherapy prot- ocols (46.7%) or did not receive any treatment at all (16.2%) [Table 3]. The mean follow-up period was 27.7 months. A total of 34 (32.4%) patients died during the study period, of which 24 (70.6%) were male and 10 (29.4%) were female, resulting in an OS rate of 67.6%. Although survival was greater among those aged 50–59 years (90.5% versus 60–62.5% in other age groups) and among females (73.7% versus 64.2% in males), these differences were not statistically significant (P = 0.098 and 0.163, Table 4: Correlation between survival rate and selected characteristics among chronic lymphocytic leukaemia patients in Erbil, Iraq (N = 105) Characteristic n (%) P value Survived (n = 71) Died (n = 34) Age in years <50 6 (60) 4 (40) 0.098 50–59 19 (90.5) 2 (9.5) 60–69 25 (62.5) 15 (37.5) ≥70 21 (61.8) 13 (38.2) Gender Male 43 (64.2) 24 (35.8) 0.163 Female 28 (73.7) 10 (26.3) Lymphadenopathy Yes 41 (65.1) 22 (34.9) 0.496 No 30 (71.4) 12 (28.6) Splenomegaly Yes 46 (67.6) 22 (32.4) 0.993 No 25 (67.6) 12 (32.4) Hepatomegaly Yes 14 (60.9) 9 (39.1) 0.434 No 57 (69.5) 25 (30.5) Rai clinical stage 0 12 (85.7) 2 (14.3) 0.190 I 4 (44.4) 5 (55.6) II 15 (75) 5 (25) III 14 (73.7) 5 (26.3) IV 26 (60.5) 17 (39.5) Binet clinical stage A 21 (72.4) 8 (27.6) 0.648B 13 (72.2) 5 (27.8) C 37 (63.8) 21 (36.2) Hb level in g/dL ≤11 54 (65.9) 28 (34.1) 0.465 >11 17 (73.9) 6 (26.1) WBC × 109/L <65 37 (69.8) 16 (30.2) 0.628 ≥65 34 (65.4) 18 (34.6) Platelet count × 109/L <150 44 (69.8) 19 (30.2) 0.551 ≥150 27 (64.3) 15 (35.7) LDH in U/L <250 17 (81) 4 (19) 0.144 ≥250 54 (64.3) 30 (35.7) Hb = haemoglobin; WBC = white blood cells; LDH = lactate dehydrogenase. Kawa M. Hasan Clinical and Basic Research | e465 respectively). Other clinical and laboratory characteristics were also not significantly correlated with survival (P >0.050 each) [Table 4]. The five-year OS rate was 61.3%. The mean duration of five-year survival was 60 months (95% confidence interval: 36.4–46.3 months); however, the median survival could not be estimated and could have occurred beyond the study period. The mean duration of survival was longer among females and those aged 50–59 years old; however, these differences were not statistically significant (P = 0.185 and 0.163, respectively) [Figure 2]. Discussion In the current study, the median age at diagnosis of CLL patients in Erbil, Iraq, was similar to that reported in neighbouring countries such as Iran and Turkey (65 years versus 60.73 and 64 years, respectively).2,9 In contrast, the median age at diagnosis in Western countries is much higher, ranging from 67–72 years old.1,10,11 However, the frequency of male CLL patients in the current study was identical to that reported in Italy (64%).12 In terms of clinical presentation, spleno- megaly (64.8% versus 54%) and hepatomegaly (21.9% versus 14%) were reported more frequently in the current study compared to Western patients, while lymphadenopathy was less frequent (60% versus 87%).3 In Turkey, rates of splenomegaly, hepatomegaly and lymphadenopathy differed at 55.4%, 54.6% and 79.2%, respectively.9 In Iran, Payandeh et al. also reported varying rates of organomegaly (34%) and lymphaden- opathy (38.7%).2 The diversity of clinical presentations in CLL and correlations with ethnicity have been well documented in previous research.13 The incidence of anaemia and AIHA in the current study (78.1% and 11%, respectively) was comp- arable to that reported in previous research in which AIHA was documented in up to 11% of late-stage CLL patients, with positive DAT results seen in up to 15% of patients.14 In the present study, 60% of patients had thrombocytopaenia; this finding is understandable given that most patients presented at an advanced clinical stage, such as Rai stage III or IV (59.1%) or Binet stage C (55.2%). According to data from other developing countries such as Iran, Turkey and India, patients in this region are often first seen at more advanced stages (38.5%, 33.3% and 41%, respectively).2,15,16 However, in developed countries, only 10–20% of patients present at an advanced stage.17 These findings may indicate that routine CBC analysis is less frequently performed in developing countries or that hospitals in this region lack proper evaluation and management protocols or experience delays in the referral of patients with incidental lymphocytosis. It is also possible that there is a difference in the biology of the disease (e.g. in terms of such patients having a less mutated subtype of CLL).4 In the present study, most cases expressed CD19, CD5 and CD23, with CD79b less frequently expressed; these findings are consistent with the characteristic profile of CLL disease.18 For those patients not expressing CD23—considered atypical CLL cases—other markers could help to differentiate CLL from mantle cell lymph- oma, such as the negative expression of Flinder Medical Figure 2: Kaplan-Meier survival curves showing (A) five- year overall survival (OS), (B) five-year OS by age group (P = 0.185) and (C) five-year OS by gender (P = 0.163) among chronic lymphocytic leukaemia patients in Erbil, Iraq (N = 105). Clinical Aspects, Immunophenotypic Analysis and Survival Rate of Chronic Lymphocytic Leukaemia Patients in Erbil City, Iraq e466 | SQU Medical Journal, November 2018, Volume 18, Issue 4 Centre-7.19 Overall, CD38 expression at any level is regarded as a poor prognostic indicator; it was expressed in 21% of cases in the present study, which is comparable to the findings of Hojjat Farsangi et al. (27.6%).20 In the current study, 67.6% of patients had monoclonal kappa light chains, with 32.4% expressing lambda chains. Hojjat Fersangi et al. reported rates of expression of kappa and lambda chains to be 71.3% and 28.7%, respectively, among Iranian CLL patients.20 Stamatopoulos et al. also reported similar findings in Greece.21 The OS (67.6%) in the current study was comp- arable to data reported from Iran (64%), although the five-year OS rate (61.3%) was higher than results from Turkey (36.5%) and India (51%).2,22,23 However, data from Germany and the USA indicate much higher five-year relative survival rates (80.2% and 82.4%, respectively).24 The mean duration of five-year survival in the current study was 41.3 months, which is similar to data reported from Iran (38.5 months).2 This may be related to variations in a number of patient-related characteristics, such as clinical stage at diagnosis. The reason why the five-year OS rate in the present study was lower than the overall OS is likely because patients whom were more recently diagnosed (i.e. in 2015–2017) had not yet received their five-year follow-up at the time of the analysis. Early diagnosis plays a major role in improving survival in CLL patients.25 Other possible causes for low five-year OS rates are the lack of availability of equipment to conduct thorough cytogenetic evaluations for factors known to have a prognostic impact—such as deletions of chromosomes 11q, 13q, 17p and trisomy 12 as well as TP53, NOTCH1, SF3B1 and BIRC3 gene mutations.8 In addition, insufficient access to adequate or novel chemotherapy agents like obinutuzumab, ibru- tinib, idelalisib and venetoclax as a frontline treatment or for relapsed/refractory cases may negatively affect survival.26 No significant correlations were noted between survival rates and selected sociodemographic, clinical or laboratory characteristics in the present study. These findings partially agree with those previously reported by Shvidel et al.; however, they contradict de Faria et al.’s study.27,28 The difference between these findings is probably due to variations in sample size, the lack of genetic assessment conducted in the current study and the well-known impact of ethnicity.13 In addition, data regarding CD49d and ZAP70 expression, which have a major prognostic impact on CLL patients, were not available.8 Further research is therefore recommended to evaluate these factors. Conclusion The median age, male-to-female ratio and clinical and immunophenotypic characteristics of Iraqi CLL patients in the current study did not differ greatly from previous regional and international data; however, patients more frequently presented at an advanced stage and had a lower five-year OS rate compared to Western populations. In addition, Iraqi patients more frequently presented with organomegaly, including hepatomegaly, although less frequently with lymphadenopathy. c o n f l i c t o f i n t e r e s t The authors declare no conflicts of interest. f u n d i n g No funding was received for this study. References 1. Watson L, Wyld P, Catovsky D. Disease burden of chronic lymph- ocytic leukaemia within the European Union. Eur J Haematol 2008; 81:253–8. https://doi.org/10.1111/j.1600-0609.2008.01114.x. 2. Payandeh M, Sadeghi E, Sadeghi M. Survival and clinical aspects for patients with chronic lymphocytic leukemia in Ker- manshah, Iran. Asian Pac J Cancer Prev 2015; 16:7987–90. https://doi.org/10.7314/APJCP.2015.16.17.7987. 3. Abbott BL. Chronic lymphocytic leukemia: Recent advances in diagnosis and treatment. Oncologist 2006; 11:21–30. https://doi. org/10.1634/theoncologist.11-1-21. 4. Hallek M. Chronic lymphocytic leukemia: 2017 update on diagnosis, risk stratification, and treatment. 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