KIDNEY TRANSPLANTATION Effect of Age on Conversion to Everolimus with Calcineurin Inhibitor Minimization at A Late Post-Transplant Stage Junji Uchida1*, Shunji Nishide1, Kazuya Kabei1, Hisao Shimada1, Akihiro Kosoku1, Tomoaki Iwai1, Nobuyuki Kuwabara1, Toshihide Naganuma1, Norihiko Kumada2, Yoshiaki Takemoto1, Tatsuya Nakatani1 Purpose: The purpose of this study was to identify the risk factors for everolimus discontinuation in kidney trans- plant recipients converted to everolimus with calcineurin inhibitor (CNI) minimization at a late post-transplant stage. Materials and Methods: An observational retrospective cohort study was conducted on a total of 38 recipients of kidney transplantation at our institution from June 2012 to March 2015 who were converted from antimetabolites to everolimus at a late post-transplant stage and followed for 1 year. We divided the patients into two groups to evaluate the factors affecting everolimus discontinuation after conversion: everolimus continuation group (n = 23), patients in whom everolimus maintained, and everolimus discontinuation group (n = 15), patients in whom everolimus were stopped within 1 year after conversion. Results: Age at conversion was significantly older in the everolimus discontinuation group compared to the everolimus continuation group (57.9 ± 12.0 years in the everolimus discontinuation group vs 45.7 ± 11.2 years in the everolimus continuous group; P = .0062). Multivariate cox proportional hazard regression analysis revealed that age at conversion significantly correlated with everolimus discontinuation (P = .012). Receiver operating char- acteristic curve of age at conversion showed that the cut-off value was 55 years old for the everolimus discontinu- ation group [area under curve 0.804, 95% confidence interval (0.654-0.954), sensitivity 86.7%, specificity 65.2%]. Conclusion: Our results indicated that late conversion to everolimus with CNI minimization in elderly recipients older than 55 years of age may be associated with more frequent adverse events and discontinuations. Keywords: age; calcineurin inhibitor minimization; everolimus; immunosuppressive agent; kidney transplantation INTRODUCTION Kidney transplantation is the most preferable renal replacement therapy in improving life expectan- cy and quality of life for patients with end-stage renal disease. The central issue in kidney transplantation re- mains to be the suppression of allograft rejection. The aim of immunosuppression therapy is to reduce the risk of rejection and to prolong patient and graft survival. Current immunosuppressive protocols, consisting of calcineurin inhibitors (CNIs), mycophenolate mofetil (MMF), and steroid, have appreciably improved short- and medium-term graft survival(1). However, improve- ments in long-term graft survival are restricted by nephrotoxicity associated with CNI administration(2). Immunosuppressive regimens that minimize exposure to CNIs following kidney transplantation have been widely investigated in order to reduce the burden of CNI-related complications(3). Among them, there have been several published clinical studies on conversion to everolimus in maintenance transplants. The AC- ERTAIN study revealed that conversion to everoli- mus with CNI minimization or elimination at a late post-transplant stage was associated with more frequent adverse effects and discontinuation(4). Our previous pi- lot study showed that recipients with good graft func- tion may benefit from conversion to everolimus with 1Department of Urology, Osaka City University Graduate School of Medicine, Osaka, Japan. 2Department of Urology, Suita Municipal Hospital, Suita, Japan. *Correspondence: Department of Urology, Osaka City University Graduate School of Medicine 1-4-3, Asahi-machi, Abeno-ku, Osaka, 545-8585, Japan. Phone: +81-6-6645-3857, Fax: +81-6-6647-4426. E-mail: m9492120@msic.med.osaka-cu.ac.jp. Received December 2017 & Accepted July 2018 CNI minimization at a late post-transplant stage, as an improvement in graft function compared to baseline was observed in everolimus maintenance patients(5). Everolimus plus CNI minimization may provide some advantage to the renal function of recipients in whom everolimus could be maintained. The aim of this study was to identify the risk factors for everolimus discontin- uation after conversion to everolimus with CNI minimi- zation at a late post-transplant stage. MATERIALS AND METHODS Study Population We began to convert patients on MMF to everolimus with CNI minimization at our institution in June 2012. We have also applied everolimus to ABO-incompatible kidney transplant recipients(6) and patients with rela- tively good graft function(5). For this study, a total of 38 recipients of kidney transplantation at our institution from June 2012 to March 2015 who were converted from antimetabolites to everolimus for 1 year ending in March 2016 were investigated. The inclusion criteria for conversion were as follows:(1) at least 3 months af- ter transplantation,(2) renal function defined as a serum creatinine (S-Cr) value < 2.5 mg/dl,(3) no acute rejec- tion episodes for more than 3 months, and(4) normal or slightly increased albuminuria defined as a urinary al- Kidney Transplantation 266 Vol 15 No 05 September-October 2018 267 bumin excretion rate (the ratio of spot urine albumin to Cr)< 300 mg/g Cr. Treatment with everolimus was stopped due to adverse events in 15 patients (39.5%). Seven patients with general fatigue, 2 with interstitial pneumonia, 2 with peripheral edema, 1 with menoxen- ia, 1 with redness and itching of face, 1 with colon di- verticulitis, and 1 with cholecystitis were led to discon- tinuation of everolimus. Median time from conversion to discontinuation was 119 days, with a range between 17 and 271 days. There were no graft failures or ap- parent clinical rejection during the observation period. This study analyzed retrospectively the risk factors for everolimus discontinuation after late conversion of sta- ble kidney transplant recipients from antimetabolites with standard exposure CNIs to everolimus with very low exposure CNIs as a 1-year pilot study. We retro- spectively compared the clinical parameters such as age at conversion, gender, estimated glomerular filtration rate at conversion, urinary albumin excretion at conver- sion, type of calcineurin inhibitors, dialysis duration, and period from transplantation to conversion between the two groups to analyze the risk factors for everoli- mus discontinuation. We divided the patients into two groups to evaluate the factors leading to everolimus discontinuation: the everolimus continuation group (n = 23), patients in whom everolimus was maintained for 1 year after conversion, and the everolimus discontinu- ation group (n = 15), patients in whom everolimus was stopped within 1 year after conversion. The number of patients at month 1, 3, 6, and 12 in the everolimus continuation group and the everolimus discontinuation group is shown in Table 1. Protocols of conversion to CNI minimization On the day of conversion, MMF or mizoribine was discontinued and everolimus was started at a dose of 1.5 mg/day (0.75 mg, twice a day) in the patients who received cyclosporine (CsA group) or 3.0 mg/day (1.5 mg, twice a day) in the patients who received tacroli- mus (Tac group). The CNI dose was simultaneously reduced to 40-60% below baseline values. Dose adjust- ments started from 1 week onward to target an everoli- mus trough level of 3 to 8 ng/ml and a CsA trough level of 25-50 ng/ml or a Tac trough level of 2-4 ng/ ml. Everolimus trough levels were assessed at 1 week and every month until 1 year after conversion. Baseline doses of methylprednisolone were continued unaltered in all patients. Clinical and biochemical measurement and concentra- tion of CNIs and everolimus At baseline, clinical parameters including age, gender, cause of end-stage renal disease, duration of dialysis, time to transplantation, donor type, and ABO-compat- ibility were collected. At baseline and at 1, 3, 6, and 12 months after conversion, fasting blood samples were obtained in the early morning for biochemical studies, including total cholesterol, triglycerides, high density lipoprotein cholesterol, low density lipoprotein cholesterol, and trough levels of CNI and everolimus. Estimated glomerular filtration rate (eGFR) was calcu- lated using the modified Modification of Diet in Renal Disease equation using the new Japanese coefficient(7). Urinary albumin excretion rate (the ratio of spot urine concentrations of albumin to creatinine) was measured at baseline and at 1, 3, 6, and 12 months after conver- sion. We evaluated these clinical parameters at baseline compared to 12 months after conversion between the two groups. All subjects provided informed consent pri- or to enrollment in this study, which was approved by the Human Ethics Committee of Osaka City University Hospital. All procedures were in accordance with the Helsinki Declaration of 1975. Statistical analysis Statistical analysis was conducted using EZR (Saita- ma Medical Center, Jichi Medical University, Saita- ma, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Australia). More precisely, it is a modified version of R commander designed to add statistical functions fre- quently used in biostatistics(8). The results are expressed as mean ± standard deviation or median with ranges and as proportions for categorical variables. Changes were evaluated with the paired t test or Wilcoxon test. Differences between the two groups were analyzed by Student’s t-test or Mann-Whitney U-test. Categorical variables were compared using chi-squared analysis or Fisher's exact test. Univariate association between var- iables was assessed by cox proportional hazard regres- sion analysis, and multivariate cox proportional hazard regression analysis was performed to determine the fac- tors related to everolimus discontinuation. Cut-off val- ues of the factors related to everolimus discontinuation were calculated by the receiver operating characteris- tics curve (ROC curve). Statistical significance was set as p < 0.05. Table1. Number of patients Postconversion Everolimus Everolimus continuation group discontinuation group month 1 36 2 month3 34 4 month 6 27 11 month 12 23 15 Figure 1. Changes in estimated glomerular filtration rate and uri- nary albumin excretion before and after conversion. Recipients in whom everolimus was maintained for 1 year after conversion (everolimus continuation group) or recipients in whom everolimus was stopped (everolimus discontinuation group). NS=not signif- icant Age at conversion to everolimus with CNI minimization-Uchida et al. RESULTS Baseline patient characteristics The demographic and clinical characteristics at base- line of the everolimus continuation and discontinuation groups are presented in Table 2. Age at transplant and conversion was significantly older in the everolimus discontinuation group compared to the everolimus con- tinuation group. No significant differences were ob- served between the two groups with regard to the other clinical parameters. Renal function and urinary albumin excretion (Figure 1) There were no significant differences in eGFR and urinary albumin excretion at baseline between the two groups (Table 2). In the everolimus continuation group, the mean eGFR value was significantly elevated from 46.7±12.6 ml/min/1.73m2 at baseline to 49.8 ± 14.3 ml/min/1.73m2 at 12 months after conversion. In the everolimus discontinuation group, there was no signifi- cant difference in the mean eGFR between baseline and 12 months after conversion. Furthermore, there was a significant difference in the change of eGFR between the two groups. In the everolimus continuation group, the log [urinary albumin excretion] was significantly increased from 1.06 ± 0.29 at baseline to 1.29 ± 0.44 at 12 months after conversion. In the everolimus discon- tinuation group, there was no change in the log [urinary albumin excretion] between baseline and 12 months af- ter conversion. Lipid profile There were no significant differences in total choles- terol and low-density lipoprotein at baseline between the two groups. There were no significant differences in total cholesterol and low-density lipoprotein between baseline and 12 months after conversion in both groups. Relationship between everolimus discontinuation and clinical parameters Univariate cox proportional hazard regression analysis revealed that discontinuation of everolimus correlated with urinary albumin excretion and age at conversion. We selected age at conversion, urinary albumin excre- tion, and eGFR as variables associated with everoli- mus discontinuation for the multivariate analysis. Age Table 2. Comparison of clinical parameters between Everolimus continuous and discontinuous group Everolimus continuation group Everolimus discontinuation group P-value n 23 15 Age at transplant (year) 45.7 ± 11.2 57.9 ± 12.0 .00622 Gender (male: female) 13:10 10:5 .736 HD duration (months) 7 7.2 ± 100 67.6 ± 73.8 .786 Calcineurin inhibitor 14:9 9:6 1.0 (Cyclosporin: Tacrolimus) Cause of end stage renal disease CGN; 5, IgA N; 6, CGN; 3, IgA N; 1, DM N; 3, .415 DM N; 2, Renal sclerosis; 2, ADPKD; 0, Renal sclerosis; 1, Unknown; 5, Others; 1 ADPKD; 2, Unknown; 5, Others; 2 Donor age (year) 55.0 ± 11.8 56.9 ± 9.7 .634 Donor type (living: deceased) 19:4 14:1 .63 Donor relation Spouses; 10, Parent/Child; 8, Sister;1 Spouses; 11, Parent/Child; 3, Sister;0 .344 HLA mismatch (antigen) 3.5 ± 1.1 4.1±1.6 .0675 ABO-compatibility compatible; 13, incompatible; 10 compatible; 4, incompatible; 11 1.0 Period from transplant to conversion (months) 41.7 ± 54.5 44.8 ± 32.1 .848 Age at conversion (year) 48.9 ± 12.0 61.9 ± 11.5 .00563 eGFR (ml/min/1.73m2) 46.7 ± 12.6 47.9 ± 12.6 .786 Urinary albumin excretion (mg/g Cr) 13.8 ± 7.4 20.9 ± 13.4 .0776 Total cholesterol (mg/dL) 201.8 ± 26.9 198.8 ± 34.5 .779 Triglyceride (mg/dL) 121.5 ± 52.2 108.8 ± 42.9 .464 Low density lipoprotein (mg/dL) 103.3 ± 26.0 106.4 ± 17.0 .692 High density lipoprotein (mg/dL) 65.8 ± 16.0 68.1 ± 20.6 .734 Abbrebiations: HD, hemodialysis; CGN, chronic glomeronephritis; IgA N, IgA nephropathy; DM N, Diabetic nephropathy; ADPKD, autosomal dominant polycystic kidney disease; HLA, human leukocyte antigen; eGFR, estimated glomerular filtration rate. aDifferences between the two groups were analyzed by Student’s t-test or Mann-Whitney U-test. bCategorical variables were compared using chi-squared analysis or Fisher's exact test. Figure 2. A threshold of age at conversion associated with discon- tinuation of everolimus by use of receiver operating characteristic curve analysis. Age at conversion to everolimus with CNI minimization-Uchida et al. Kidney Transplantation 268 Vol 15 No 05 September-October 2018 269 at conversion was selected as a variable by the back- ward model. Previous reports showed that conversion to everolimus was advised in patients with proteinuria or not good graft function(4,9). Multivariate cox propor- tional hazard regression analysis indicated that age at conversion was independently associated with discon- tinuation of everolimus (Table 3). Cut-off value of everolimus discontinuation The ROC curve of age at conversion for the everolimus discontinuation group showed that the cut-off value was 55 years old [area under curve 0.804, 95 % confidence interval (0.654-0.954), sensitivity 86.7 %, specificity 65.2%] (Figure 2). DISCUSSION In this study, the risk factors for everolimus discon- tinuation after conversion to everolimus with CNI minimization in the kidney transplant recipients with good renal function were analyzed. Age at conversion was significantly older in the everolimus discontinua- tion group (average age: 61.9 years) compared to the everolimus continuation group and significantly corre- lated with discontinuation of everolimus by multivari- ate analysis. Moreover, we revealed that late conversion to everolimus with CNI minimization in elderly recipi- ents older than 55 years of age may be associated with more frequent adverse events and discontinuations by the ROC curve, although the specificity was relatively low because of the small sample size. To our knowl- edge, there have been no reports on the safety and ef- ficacy of everolimus in elderly recipients, although the cut-off point for elderly patients differs among various countries. Our results may be useful to explore patients who could be converted to everolimus with CNI mini- mization at a late post-transplant stage. Increasing age is associated with structural and func- tional changes in body compartments and tissue that alter absorptive capacity, volume of distribution, he- patic metabolic function, and ultimately drug disposi- tion. Age-related changes may appear in most organs and can alter pharmacodynamics responses to med- ications(10). Although no data have been published on elderly changes, elderly recipients may be more suscep- tible to developing adverse effects related to immuno- suppressive drugs, especially everolimus. That is, elder- ly recipients may not be eligible for conversion from MMF to everolimus at a late post-transplant stage. The present study showed that eGFR in the everolimus continuation group was significantly improved com- pared with that at baseline. Moreover, there was a sig- nificant difference in the change in eGFR between the two groups. In the post hoc analysis of the Zeus study, the renal benefit increased slightly from year 1 for liv- ing donor kidney transplant recipients who remained on everolimus-maintained immunosuppression, and for living donor kidney transplant recipients who dis- continued everolimus, the renal benefit was lost(11). Our study showed that selected recipients with good renal function may acquire renal benefit, if they remained on everolimus-maintained immunosupression. The nephrotoxic effect of CNIs can limit long-term sur- vival(2). Recent strategies to avoid or reduce exposure to CNIs have focused on immunosuppressive drugs that are generally considered non-nephrotoxic, such as mTOR inhibitors. Everolimus has shown potent anti- proliferative effects and has prevented allograft rejec- tion in preclinical models(12). In experimental models, everolimus has been shown to ameliorate progression of chronic allograft nephropathy, not only when admin- istered prophylactically from the time of transplanta- tion but also in advanced disease(13,14). Even conversion to everolimus in maintenance transplants may lead to renal benefit. However, late conversion everolimus in recipients with high baseline proteinuria has been re- ported to induce a decline in graft function and poor graft prognosis in previous clinical trials(15). The introduction of everolimus with CNI minimization at a late post-transplant stage may have some benefits due to its pleiotropic effects. Everolimus exhibits an- ti-neoplastic, anti-viral, anti-atherosclerotic, and an- ti-proliferative properties. It is well known that kidney transplant recipients receiving mTOR inhibitors have a lower risk of developing cytomegalovirus infec- tion(16). The CONVERT trial revealed that mTOR in- hibitor-based immunosuppression was associated with a lower rate of malignancy at 2 years postconversion compared with CNI-based immunosuppression(17,18). Chronic antibody-mediated rejection is considered to play a major role in late allograft loss(2,19). Although everolimus-based immunosuppression in early con- version from CNI was reported to be associated with an increased risk of developing denovo donor-specific antibodies and antibody-mediated rejection(20), a recent review demonstrated that late conversion to CNI-free immunosuppressive regimen with mTOR inhibitors did not appear to affect the risk of denovo donor-specific Table 3. Cox hazard regression analysis of risk factors associated with everolimus discontinuation Univariate Multivariate Variable Hazard ratio (95% CI) P-value Hazard ratio (95% CI) P-value Gender 1.478 (0.505-4.327) .476 Age at conversion 1.080 (1.025-1.136) .00416 1.075 (1.016-1.137) .012 Period from transplant to conversion (months) 1.001 (0.992-1.011) .815 HD duration 0.999 (0.993-1.004) .626 CsA/Tac 1.023 (0.364-2.877) .965 Donor age 0.978 (0.921-1.04) .468 HLA mismatch 1.508 (0.961-2.361) .074 ABO-incompatiblity 2.808 (0.893-8.84) .077 eGFR at conversion 1.003 (0.965-1.042) .252 1.017 (0.934-1.062) .454 Urinary albumin excretion 1.041 (1.003-1.079) .0318 1.028 (0.988-1.068) .173 Abbreviations: HD, hemodialysis; CsA, cyclosporin; Tac, tacrolimus; eGFR, estimated glomerular filtration rate. Age at conversion to everolimus with CNI minimization-Uchida et al. antibodies(21). Moreover, in human cell cultures, it was reported that everolimus was equally effective as tac- rolimus in suppressing humoral alloimmunity(22). Late conversion to everolimus may be a favorable strategy in the expectation of avoiding MMF toxicity or reducing CNI-associated long-term toxicities, because it may not elicit the development of denovo donor-specific anti- bodies, if the patient remained on everolimus treatment. The recipients who remained on everolimus in this study showed a significant increase in urinary albumin excretion compared to the recipients in whom everoli- mus was stopped. mTOR inhibitor use has been associ- ated with proteinuria/albuminuria in kidney transplant recipients(23). Potential mechanisms for mTOR-associ- ated proteinuria/albuminuria include decreased vascu- lar endothelial growth factor synthesis and inhibition of key podocyte proteins that comprise the glomerular slit diaphragm, including nephrin(23). In kidney transplant recipients, microalbuminuria predicts graft loss and all-cause mortality(24). However, the impact of mTOR inhibitor-induced proteinuria/albuminuria on graft out- come has remained unclear. In this study, albuminuria was slightly elevated after late conversion to everoli- mus in patients in whom everolimus was maintained (median value of urinary albumin excretion: 16 mg/g Cr (6-126 mg/g Cr)). Slightly increased albuminuria may well induce an undesirable effect for long-term graft and patient survival. The present study might have limitations because of the small sample size and because it is a retrospective study. However, there have been few reports on everolimus in elderly patients receiving kidney transplantation. It is not yet established whether everolimus is safe and ef- fective for elderly recipients. To our knowledge, this is the first demonstration to identify the possible risk fac- tors for discontinuation of everolimus at late conversion by multivariate analysis, although the present study is a pilot. Further prospective well-controlled and long- term follow-up trials with a larger number of patients are needed to confirm our results. CONCLUSIONS In conclusion, the present study identified the possibili- ty that late conversion to everolimus with CNI minimi- zation in elderly recipients older than 55 years of age may be associated with more frequent adverse events and discontinuations. The recipients enrolled in this study had relative good graft function with less albu- minuria. Therefore, recipients whose ages are less than 55 years and who have relatively good graft function with little chronic allograft damage may be available for late conversion to everolimus. 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