Comparison of four different immunosuppression protocols without long-term steroid therapy in kidney recipients monitored by surveillance biopsy: Five-year outcomes

Transplant Immunology 20 (2008) 32–42

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Transplant Immunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t r i m

Comparison of four different immunosuppression protocols without long-term steroid therapy in kidney recipients monitored by surveillance biopsy: Five-year outcomes Mysore S. Anil Kumar a,⁎, M. Irfan Saeed a, Karthik Ranganna b, Gregory Malat a, Nedjema Sustento-Reodica c, Arjun M.S. Kumar a, William C. Meyers a a b c

Department of Surgery and Division of Transplantation, Drexel University College of Medicine and Hahnemann University Hospital Philadelphia PA 19102, United States Department of Medicine and Division of Nephrology, Drexel University College of Medicine and Hahnemann University Hospital Philadelphia PA 19102, United States Department of Pathology, Drexel University College of Medicine and Hahnemann University Hospital Philadelphia PA 19102, United States

a r t i c l e

i n f o

Article history: Received 27 May 2008 Received in revised form 1 August 2008 Accepted 8 August 2008 Keywords: Immunosuppression protocol Kidney transplant Steroid withdrawal

a b s t r a c t Induction and maintenance immunosuppression protocols with or without long-term steroid therapy in kidney transplant recipients are variable and are transplant center-specific. The aim of this prospective randomized pilot study was to compare 5-year outcomes in kidney recipients maintained on 4 different calcineurin inhibitor (CNI)-based immunosuppression protocols without long-term steroid therapy. Two hundred consenting patients who received kidney transplants between June 2000 and October 2004 were enrolled in 4 immunosuppression protocol groups, with 50 patients in each group: cyclosporine (CSA)/mycophenolate mofetil (MMF), CSA/ sirolimus (SRL), tacrolimus (TAC)/MMF, and TAC/SRL. Induction therapy was done with basiliximab and methylprednisolone. Steroids were withdrawn on post-transplant day 2, and long-term steroid therapy was not used. Demographic characteristics among the four groups were comparable; approximately 50% of the recipients were African American and ≥80% of the kidneys transplanted were from deceased donors. Clinical acute rejection (CAR) was confirmed by biopsy and treated with intravenous pulse steroid therapy. Steroid-unresponsive CAR was treated with Thymoglobulin. Surveillance biopsies were performed at 1, 6, 12, 24, 36, 48, and 60 months to evaluate subclinical acute rejection (SCAR), chronic allograft injury (CAI), and other pathological changes per the Banff 2005 schema. The primary end point was CAR, and secondary end points were 5-year patient and graft survival rates, renal function, SCAR, CAI, and adverse events. In the first year post-transplant, the incidence of CAR was 18% in the CSA/MMF group, 8% in the CSA/SRL group,14% in the TAC/MMF group, and 4% in the TAC/SRL group (CSA/MMF vs. TAC/SRL; p = 0.05). The incidence of SCAR was 22% in the CSA/MMF group, 8% in the CSA/SRL group, 16% in the TAC/MMF group, and 6% in the TAC/SRL group (CSA/MMF vs. CSA/SRL and TAC/SRL; p = 0.05). After the first year, the incidences of CAR and SCAR decreased and were comparable in all 4 groups. At 5 years posttransplant, cumulative CAI due to interstitial fibrosis/tubular atrophy (IF/TA), hypertension (HTN), and chronic calcineurin inhibitor (CNI) toxicity was observed in 54%, 48%, and 8% of the CSA/MMF group vs. 16%, 36%, and 12% of the CSA/SRL group vs. 38%, 24% and 6% of the TAC/MMF group vs. 14%, 25% and 12% of the TAC/SLR group (IF/TA: CSA/MMF vs. CSA/SRL and TAC/SRL; p = 0.04, HTN: CSA/MMF vs. TAC/MMF and TAC/SRL; p = 0.05, CNI toxicity: TAC/SRL and CSA/SRL vs. TAC/MMF; p = 0.05). Five-year patient and graft survival rates were 82% and 60% in the CSA/MMF group, 82% and 60% in the CSA/SRL group, 84% and 62% in the TAC/MMF group, and 82% and 64% in the TAC/SRL group (p = 0.9). Serum creatinine levels and creatinine clearances at 5 years were comparable among the groups. Our data show that the rates of CAR and SCAR in the first year post-transplant were significantly lower in the CSA/SRL and TAC/SRL groups and that cumulative CAI rates due to IF/TA and HTN at 5 years were significantly lower in the TAC/MMF, TAC/SRL, and CSA/SRL groups than in the CSA/MMF group. Despite significant differences in the incidences of CAR and SCAR and prevalence of different types of CAI at 5 years, renal function and patient and graft survival rates at 5 years were comparable among kidney recipients maintained on 4 different immunosuppression protocols without long-term steroid therapy. © 2008 Elsevier B.V. All rights reserved.

1. Introduction ⁎ Corresponding author. Drexel University College of Medicine, MS 417, 5th Floor, Feinstein Bldg, Broad and Vine, Philadelphia PA 19102, United States. Tel.: +1 215 762 1856; fax: +1 215 762 1621. E-mail address: [email protected] (M.S. Anil Kumar). 0966-3274/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2008.08.005

The present standard-of-care immunosuppression protocols in most transplant programs in the United States consist of induction with IL-2R or a lymphocyte-depleting antibody and maintenance with

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a calcineurin inhibitor (CNI) in combination with mycophenolate mofetil (MMF) or sirolimus (SRL) and long-term prednisone therapy [1–6]. Recent studies show that early withdrawal of steroids, that is, between 2 and 7 days after kidney transplant, is safe and equally efficacious compared with long-term steroid therapy but has the advantage of avoiding the wide-ranging side effects associated with long-term steroid administration [7–9]. Few prospective studies in kidney recipients compare different immunosuppression protocols with or without long-term steroid therapy [10–12]. The current CNIbased combination immunosuppression protocols are potent in decreasing acute rejections but are ineffective in preventing the development and progression of chronic allograft injury (CAI), which presently is one of the major causes of graft loss after a kidney is transplanted. Surveillance biopsy is a useful tool to monitor CAI in kidney transplant recipients. Additionally, CNI-based therapy has significant morbidity due to nephrotoxicity and other side effects that include malignancy, infection, and metabolic diseases [13,14]. Both MMF and SRL have antiproliferative properties and may be beneficial in delaying or minimizing CAI [15,16]. Prospective studies comparing the long-term outcomes of combination CNI-based therapy without long-term steroid therapy are lacking. 2. Objective The aim of this pilot study was to prospectively compare and evaluate long-term outcomes in 200 kidney transplant recipients randomized into four different CNI-based immunosuppression protocols with early steroid withdrawal monitored by surveillance biopsy. 3. Patients and methods This study was approved by the institutional review board of Drexel University College of Medicine and Hahnemann University Hospital (Drexel university project # 1000182). This study was registered as a randomized trial with the Cochrane Renal Group (# CRG 1106000095). The protocol was designed to randomly assign 200 de novo kidney transplant recipients to one of 4 groups, with 50 patients per group and with each group receiving one of four immunosuppressive regimens: cyclosporine (CSA) with MMF, CSA with SRL, tacrolimus (TAC) with MMF, and TAC with SRL. Randomization was completed online using the first generator plan at Randomization.com (accessed at http://www.randomization.com). The study was open for randomization until 50 de novo kidney recipients were enrolled in each of the four groups for a total of 200 patients. All recipients in this study received transplants between June 2000 and October 2004. Selection criteria were as follows: recipients were older than 20 years of age, were able to sign an informed consent form, were HIV and HBV negative, had deceased and living donor kidneys, and were of any ethnicity. This report is an analysis of the subgroup of patients randomized into one of four groups of immunosuppression protocols treated without long-term steroid therapy. A cohort of patients in this study was reported previously to compare ethnicity [African American (AA) vs. non-African American (non-AA) kidney recipients] and to compare early steroid withdrawal and long-term steroid therapy in kidney transplant recipients but without comparing the outcomes of the four different immunosuppression protocols [9,17]. A cohort of the CSA/MMF group from the present study was described in a previous publication comparing early steroid withdrawal with long-term steroid therapy in kidney transplant recipients [18]. The present study is the first report of results comparing kidney transplant recipients randomized to four different CNI-based immunosuppression protocols with early steroid withdrawal. 3.1. Immunosuppression protocols and infection prophylaxis All recipients were given induction therapy with 2 doses of 20 mg basiliximab on days 0 and 4 and 2 doses of methylprednisolone, 250 mg

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on day 0 and 125 mg on day 1. Steroid therapy was discontinued completely after the second dose of methylprednisolone [9]. TAC or CSA was initiated on day 1. TAC was initiated at 0.02 mg/kg body weight per day and the dose was increased rapidly to achieve trough blood levels of 15–18 ng/ml by day 4; these levels were maintained for 1 month. Beginning with the second month, the TAC blood levels were gradually reduced to 10 ng/ml by the end of 1 year. CSA was initiated at 3 mg/kg body weight per day, and the dose was rapidly increased to maintain C2 blood levels (cyclosporine blood levels 2 h after the dose) of 1000–1200 ng/ml at 1 month and then gradually tapered to maintain 700 ng/ml at 1 year. MMF was initiated on day 1 at 2 g/day in divided doses, and trough blood levels of mycophenolic acid were maintained between 1 and 3 µ/ml. SRL was initiated on day 4 at 2 mg/day; the dose was then adjusted to maintain trough blood levels of 5 to 10 ng/ml. During the initial study period, in the first 10 patients, clinical observations and surveillance biopsy results showed that with the above-described CNI blood levels, recipients in the CSA/SRL and TAC/SRL groups showed lower renal function and acute CNI toxicity confirmed by biopsy. After these clinical and biopsy findings were noted in the first 10 patients in each of the CSA/SRL and TAC/SRL groups, beginning 1 month after transplantation, the protocol was modified in subsequent patients to maintain lower blood levels of CSA and TAC. In the CSA/SRL and TAC/SRL groups, the daily doses of CSA and TAC were adjusted to maintain C2 blood levels of CSA of between 500 and 800 ng/ml and trough levels of TAC of between 5 and 9 ng/ml of blood. We did not use a loading dose of SRL, and initiation of SRL was delayed to day 4 to minimize wound-healing complications. Steroid therapy was discontinued after the second dose of methylprednisolone, and long-term steroid therapy was not used in these patients. All participants except donor/recipient seronegative pairs received valganciclovir for prophylaxis for cytomegalovirus (CMV) infection. Trimethoprim-sulfamethoxazole was given for prophylaxis for Pneumocystis carinii. Both of these prophylactic agents were given for 100 days post-transplant. 3.2. Diagnosis and treatment of clinical acute rejection Clinical acute rejection (CAR) was diagnosed as a persistently elevated level of serum creatinine that was 15% or more above baseline. All CARs were confirmed by percutaneous biopsy of the transplanted kidney. Patients with CAR confirmed by biopsy were treated with pulse doses of methylprednisolone: 1 g for 2 days or 500 mg for 4 days [9]. Recipients with steroid unresponsive rejections were re-biopsied and treated with Thymoglobulin (Genzyme Corp., Cambridge MA). Table 1 Demographic characteristics of donors and recipients Patient characteristics Recipients Age in years (mean ± SD) Male gender (%) African American (%) Diabetes (%) BMI (mean ± SD) HLA antigen mismatch (mean ± SD)

CSA/MMF CSA/SRL group group

TAC/MMF group

TAC/SRL group

p value

51 ± 14 35 (70%) 25 (50%) 12 (24%)⁎ 28.0 ± 4.8 4.0 ± 1.9

56 ± 13 37 (74%) 25 (50%) 26 (52%)⁎ 28.2 ± 5.8 4.1 ± 2.0

48 ± 14⁎ 34 (68%) 27 (54%) 13 (26%) 29.8 ± 8.4 4.0 ± 2.1

59 ± 12⁎ 34 (68%) 26 (52%) 23 (46%)⁎ 27.1 ± 5.3 4.1 ± 1.8

⁎0.05 0.08 0.9 ⁎0.03 0.88 0.9

43 (86%) 11 (22%) 15 ± 6

44(88%) 11 (22%) 16 ± 8

43 (86%) 13 (26%) 17 ± 8⁎

0.90 0.89 ⁎0.04

Donor demography Deceased donors (%) 41(82%) Expanded criteria donors (%) 12 (24%) Cold ischemia time in 14 ± 8⁎ hours (mean ± SD) Age in years (mean ± SD) 42 ± 18 BMI (mean ± SD) 26.1 ± 6.3 Male gender (%) 29 (58%) * Significant p value.

47 ± 11⁎ 38 ± 19⁎ 45 ± 25 ⁎0.04 28.0 ± 6.1 28.6 ± 15.3 27.2 ± 13.6 0.88 26 (52%)⁎ 33 (66%) 34 (68%)⁎ ⁎0.05

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3.3. Surveillance biopsy Surveillance biopsies were completed at 1, 6, 12, 24, 36, 48, and 60 months. The renal tissue was evaluated for subclinical acute rejection (SCAR) and CAI per the Banff 2005 schema [19]. The biopsies were performed percutaneously in the transplant outpatient office using an 18-gauge needle (Easy Core Biopsy Systems™). Usually one biopsy core was obtained, but in many cases 2 cores were obtained to permit a detailed examination of renal tissue with hematoxylin and eosin, Masson's trichrome, periodic Schiff,

Jones metanamine silver, and C4D stains. After the biopsy, patients were kept under observation and on absolute bed rest for 4 h and were discharged home the same day. Acute CNI toxicity was primarily a pathological diagnosis based on the results of renal biopsies showing isometric vacuolization of tubular cells. Biopsies were performed on demand in recipients with graft dysfunction. Acute CNI toxicity was also diagnosed from the results of surveillance biopsies from patients without renal dysfunction in the presence of isometric vacuolization of tubular cells and in the absence of other pathological features.

Fig. 1. Number of kidney recipients treated for 1 to 5 years with CSA/MMF vs. CSA/SRL vs. TAC/MMF vs. TAC/SRL who had indicated biopsies, incidence of clinical acute rejection, acute CNI toxicity, acute pyelonephritis, and acute tubular necrosis.

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3.4. Diagnosis and treatment of SCAR

3.5. CAI

SCAR was defined as acute rejection of Banff grade 1 or more in the absence of concurrent functional deterioration [20] and was observable at biopsy. SCAR was treated with methylprednisolone in a manner similar to that used for acute rejection. Thymoglobulin was not used to treat SCAR.

Surveillance biopsies were monitored for CAI and other incidental pathological changes. CAI was diagnosed and reported as per the Banff schema 2005 [19]. In recipients of extended criteria donor (ECD) kidneys, the pre-existing structural changes observed in pre-transplant baseline biopsy material were taken into consideration while scoring

Fig. 2. Patient and graft survival rates in kidney recipients treated with CSA/MMF, CSA/SRL, TAC/MMF, and TAC/SRL immunosuppression therapy.

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CAI in surveillance biopsy. The final CAI score in ECD kidneys was derived by comparing and subtracting the pre-existing structural changes seen prior to transplant from the overall changes seen in surveillance biopsies [12].

by counting all glomeruli and assessing all arterioles and interstitium for fibrosis in at least 2 sections of the wedge biopsy. Only those kidneys with less than 15% structural damage were accepted for transplant.

3.6. Steroid protocol and maintenance immunosuppression in recipients with CAR and SCAR

3.9. Monitoring of kidney function

After pulse doses of steroids were given to recipients with CAR and SCAR, steroid therapy was discontinued completely. These patients did not receive long-term steroid therapy. In addition to treating CAR and SCAR with a short course of pulse steroid therapy, the daily dose of TAC or CSA was increased to maintain elevated blood levels. TAC blood levels were increased by 3 to 5 ng/ml, and CSA blood levels were increased by 50 to 100 ng/ml from the pre-rejection baseline levels. These higher levels were maintained for 3 months after CAR and SCAR resolved as described previously [9]. 3.7. Delayed graft function Delayed graft function (DGF) was diagnosed if the patient's urine volume was less than 1000 ml, if serum creatinine levels decreased slowly by less than 25% from pre-transplant levels in the first 24 h after transplant, or if the patient required dialysis in the first week after the transplant [21]. A biopsy was performed at 7 to10 days in all recipients with DGF to rule out associated acute rejection. 3.8. ECD kidneys ECD kidneys were transplanted according to our previously described protocol [22]. All ECD kidneys were biopsied prior to transplant into recipients matched by donor age and body mass index. The pre-transplant biopsy specimens from ECD kidneys were evaluated for the presence of glomerulosclerosis, interstitial fibrosis, arterial injury, and interstitial inflammation. The wedge biopsy performed at the time of donor nephrectomy was large enough to contain at least 50 glomeruli or more. In cases with insufficient specimens, wedge biopsies were repeated to obtain specimens adequate for pre-transplant evaluation of ECD kidneys. Biopsy results usually showed 60 to 100 glomeruli; the injury was evaluated

Kidney function was assessed by regular monitoring of serum creatinine levels and estimated creatinine clearances using the Cockcroft–Gault formula. 3.10. Surveillance of proteinuria Recipients were monitored for proteinuria at surveillance and at indicated biopsy points of time. Random urine samples were collected prior to biopsy, and the protein:creatinine ratio was measured; creatinine was measured using the Jaffe reaction and protein, by the pyrogallol red method [23,24]. The definition of proteinuria for this study is protein loss of N300 mg/day [25]. 3.11. Subgroup analysis In our center, approximately 50% of kidney recipients are of African American (AA) descent. The long-term outcomes in AA kidney recipients are generally inferior compared with those from non-AA recipients [26]. We compared AA with non-AA recipients in each of the four groups to assess the effect of race on long-term outcomes with different immunosuppression protocols. 3.12. Conversion of maintenance immunosuppression This study intended to continue the baseline randomized immunosuppression protocol and to treat patients to obtain the best possible outcomes. However, recipients who were unable to tolerate their randomized immunosuppression protocols due to severe and distressing side effects, to persistent acute rejections, or to severe CAI injury were converted to immunosuppression protocols that best matched their tolerance and response. All recipients receiving conversion therapy were included in their baseline randomized immunosuppression group for final analysis of outcomes.

Fig. 3. Kidney recipients completing surveillance biopsies and incidence of subclinical acute rejection in groups treated with CSA/MMF vs. CSA/SRL.

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3.13. Definition of adverse events Adverse events monitored in this study were new onset diabetes mellitus (NODM), gastrointestinal side effects, hyperlipidemia, complications of wound healing, complications of biopsy, severe infections requiring hospitalization, de novo malignancies, and anemia requiring prolonged erythropoietin therapy. NODM was defined according to the guidelines of the American Diabetes Association, with de novo elevation of fasting blood sugar of N125 mg/dl and/or requirement for oral hypoglycemic agents or insulin after transplant [27]. 3.14. Patient and graft survival and statistical analysis Patient and graft survival rates were calculated by the Kaplan–Meier estimate, Student's t-test was used to compare continuous variables, and chi-square analysis was used to compare categorical demographic variables and outcome differences among patient groups. Statistical analyses were performed using SPSS® (Statistical Package for the Social Services, SPSS, Inc., Chicago, IL, version 6.14) software. 4. Results All surviving patients have completed 4 years post-transplant, and 170 patients (85%) have completed 5 to 8 years post-transplant. Table 1 shows the demographic characteristics of patients and donors in the four groups. All recipients including those with CAR and SCAR have remained free of long-term steroid therapy. 4.1. CAR and other incidental pathological findings in indicated biopsies Clinically acute increase in the serum creatinine level was an indication for biopsy. We performed 164 indicated biopsies: 71 were performed in the first year; 32, in the second year; 23 each, in the third and fourth years; and 15, in the fifth year post-transplant. In the first year, CAR was diagnosed in 18% of recipients in the CSA/MMF, 8% in the CSA/SRL, 14% in the TAC/MMF, and 4% in the

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TAC/SRL groups (CSA/MMF vs. TAC/SRL, p = 0.05). In the subsequent 2 to 5 years, 6% or fewer patients had CAR in each of the 4 groups annually. In most recipients, other pathological conditions caused elevation of serum creatinine levels. Fig. 1 shows the incidence of CAR and other pathological conditions diagnosed from the results of indicated biopsies in each of the four groups from 1 year through 5 years. During the 5-year transplant period, the total number of indicated biopsies performed was 54 in the CSA/MMF group, 38 in the CSA/SRL group, 45 in the TAC/MMF group, and 27 in the TAC/SRL group (CSA/MMF vs. TAC/SRL, p = 0.04). 4.2. Patient and graft survival rates The patient and graft survival rates in the 4 groups were actual at 4 years and actuarial at 5 years. At 5 years, the patient survival rates were 82%, 82%, 84%, and 82%; graft survival rates were 60%, 60%, 62%, and 64% in the CSA/MMF, CSA/SRL, TAC/MMF, and TAC/SRL groups, respectively (p = 0.9). Fig. 2 shows the patient and graft survival rates for all four groups for 1 through 5 years. 4.3. Surveillance biopsy and SCAR Surveillance biopsy was completed in most of the patients at the scheduled intervals. A few patients could not be biopsied at the scheduled intervals because they were receiving anticoagulation therapy or because they refused to have the procedure. Surveillance biopsy at 1 month was completed in 100% of recipients. The incidence of SCAR was high in the first year and decreased over the 5-year period. At each biopsy point, from 1 through 5 years, 97% or more of the surviving patients with functioning grafts had surveillance biopsies. In the first year, the incidence of SCAR was significantly lower in the TAC/SRL and CSA/SRL groups than in the CSA/MMF group (p = 0.05). In years 2 through 5, the incidence of SCAR decreased in all 4 groups. At 5 years, SCAR was diagnosed in 10% or fewer of the surviving recipients. Fig. 3 shows the number of recipients with functioning grafts, the number and percent of recipients completing surveillance biopsies at 1, 2, 3, 4, and 5 years, and the incidence of SCAR in the four groups of recipients. 4.4. CAI Surveillance biopsies were performed in all patients at 1, 2, 3, 4, and 5 years to monitor for CAI. Banff 2005 criteria were used for pathological classification and

Fig. 4. CAI identified by surveillance biopsies in kidney recipients treated for 1 to 5 years with CSA/MMF, CSA/SRL, TAC/MMF, or TAC/SRL.

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Fig. 4 (continued).

grading of CAI (19). Chronic hypertensive changes were based on pathological findings associated with long-standing mean arterial pressures ≥85; chronic CNI toxicity was based on pathological findings associated with mean arterial pressures ≤80. Interstitial fibrosis and tubular atrophy (IF/TA) levels were scored and reported separately regardless of etiology. Fig. 4 shows CAI due to hypertension (chronic hypertensive changes included arterial and/or fibrointimal thickening with reduplication of elastica usually affecting small artery and arteriolar hyaline changes); chronic CNI toxicity and IF/TA; viral infections; pyelonephritis; chronic obstruction; chronic T-cell-mediated rejection and chronic active antibody-mediated rejection; recurrent disease observed in material obtained from surveillance biopsies at 1 to 5 years; and severity of IF/TA.

4.6. Change in maintenance immunosuppression In each of the 4 groups, some of the recipients did not tolerate their randomized combination of immunosuppressive agents. These patients were converted to one of the other combinations, depending on the side effects of one or both of their randomized immunosuppressive combinations. Table 2 shows the number of recipients in each group with a change in immunosuppressive agents and the number of recipients able to continue the original randomized immunosuppression therapy. 4.7. Daily dose and blood levels of CSA, TAC, MMF, and SRL

4.5. DGF DGF was diagnosed in 46%, 84%, 42%, and 74% of patients from the CSA/MMF, CSA/SRL, TAC/MMF, and TAC/SRL groups respectively (CSA/MMF and TAC/MMF vs. CSA/SRL and TAC/SRL, p = 0.05). The mean length of DGF was 6.9 ± 3.3, 10.8 ± 2.6, 6.7 ± 2.9, and 9.8 ± 2.1 days in the CSA/MMF, CSA/SRL, TAC/MMF, and TAC/SRL groups, respectively (CSA/MMF and TAC/MMF vs. CSA/SRL and TAC/SRL groups p = 0.05).

Per the protocol, the targeted blood levels of CSA, TAC, MMF, and SRL were similar in all 4 groups. However, both indicated and surveillance biopsies showed that, with comparable blood levels of CSA and TAC, both short- and long-term CNI nephrotoxicity significantly increased in recipients maintained on combinations containing SRL (CSA/SRL and TAC/SRL) compared to those containing MMF (CSA/MMF and TAC/MMF). To overcome the CNI nephrotoxicity, the daily doses of CSA and TAC were reduced to maintain

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Fig. 4 (continued). significantly lower blood levels of CSA and TAC combined with SRL compared with those recipients receiving combinations containing MMF. Six months after the transplant, the mean daily dose of CSA was 375 ± 35 mg in the CSA/MMF group and 189 ± 23 mg in the CSA/SRL group (p = 0.05); the mean daily dose of TAC was 11.8 ± 2.9 mg in the TAC/MMF group and 6.5 ± 1.3 mg in the TAC/SRL group (p = 0.04). The mean daily dose of MMF was 1.82 ± 0.4 g in the CSA/MMF group and 1.15 ± 0.2 g in the TAC/MMF group (p = 0.05). The mean daily dose of SRL was 3.8 ± 1.0 mg in the CSA/SRL group and 3.7 ± 0.9 mg in the TAC/SRL group (p = 0.9). At 5 years, the mean CSA dose was 225 ±22 mg/day in the CSA/MMF group and 142 ± 15 mg/day in the CSA/SRL group (p = 0.02); the mean TAC dose was 8.9 ± 1.6 mg/day in the TAC/MMF group and 4.9±0.9 mg/day in the TAC/SRL group (space) (p=0.01); the mean MMF dose was 1.74±0.3 g/day in the CSA/MMF group and 1.39 ± 05 g/day in the TAC/MMF group (p = 0.05); and the mean SRL dose was 2.1 ±0.5 mg/day in the CSA/SRL group and 1.8±0.3 mg/day in the TAC/SRL group (p = 0.08), respectively. At 6 months post-transplant in the CSA/MMF group, the mean C2 blood levels of CSA were 1062 ± 121 ng/ml and trough MPA levels were 2.8 ± 09 µg/ml; in the CSA/SRL group, C2 blood levels were 756 ± 65 ng/ml and SRL was 6.9 ± 2.2 ng/ml of blood. In the TAC/MMF group, trough TAC levels were 11.3 ± 2.8 ng/ml and MPA levels were 1.9 ± 0.8 µg/ml. In the TAC/SRL group, TAC blood levels were 6.1 ± 2.7 ng/ml and SRL levels were 6.8 ± 1.7 ng/ml (C2 levels: CSA/MMF vs. CSA/SRL; p = 0.03, TAC levels: TAC/MMF vs. TAC/SRL; p = 0.04, MPA levels: CSA/MMF vs. TAC/MMF; p = 0.05 and SRL levels: CSA/SRL vs. TAC/SRL p = 0.6). At 5 years, the mean CSA C2 levels were 524 ± 87 ng/ml in the CSA/MMF group and 389 ± 26 ng/ml in the CSA/SRL group (p = 0.03); the mean trough TAC levels were 7.9± 1.6 ng/ml in the TAC/MMF group and 4.8 ± 1.3 ng/ml in the TAC/SRL group (p = 0.04); the mean trough levels of MPA were 1.9± 0.9 µg/ml in the CSA/MMF group and 1.6 µg/ml in the TAC/MMF group (p = 0.05); and the mean trough SRL levels were 4.8 ± 0.8 ng/ml in the CSA/SRL group and 4.5 ± 0.6 ng/ml in the TAC/SRL group (p = 0.6). Both the MMF dose and MPA blood levels were lower in the TAC/MMF group than in the CSA/MMF group because patients in the TAC/MMF group could not tolerate equivalent doses of MMF compared to recipients in the CSA/MMF group because of severe upper and lower gastrointestinal side effects. 4.8. Kidney function Kidney function was monitored by serial measurements of serum creatinine levels and estimated creatinine clearances at monthly intervals in the first year and then quarterly for 5 years. Fig. 5 shows the mean serum creatinine levels and mean creatinine clearances at 1, 2, 3, 4, and 5 years in the 4 groups of kidney recipients. At 5 years, a significant difference existed in the serum creatinine levels (p = 0.04) and creatinine clearances (p = 0.05) of the CSA/MMF and TAC/SRL groups. 4.9. Subgroup analysis of AA and non-AA recipients In each of the four groups, approximately 50% were AA recipients. The outcomes of the AA and non-AA recipients in each of the four groups were analyzed. Table 3 shows the incidence of CAR, SCAR, the prevalence of interstitial fibrosis/tubular atrophy, hypertensive changes, and patient and graft survival rates at 5 years. In each of the 4 groups, subgroup analysis of AA and non-AA recipients showed the following: the incidence of CAR was comparable; the incidence of SCAR was higher in AA recipients throughout the 5-year period; the prevalence of CAI due to hypertension and interstitial

fibrosis/tubular atrophy was significantly higher at 5 years in AA recipients than in nonAA recipients in all 4 groups. Five-year patient and graft survival rates were comparable between AA and non-AA recipients in all 4 groups. 4.10. Surveillance proteinuria During the 5 years post-transplant, proteinuria was present in 34 (17%) patients: 6 in the CSA/MMF group, 11 in the CSA/SRL group, 6 in the TAC/MMF group, and 11 in the TAC/SRL group. The proteinuria was in the nephrotic range of N3500 mg/day in 2 patients in the CSA/MMF group (both had pre-transplant diabetes); in 9 patients in the CSA/SRL group (4 had pre-transplant diabetes, 2 had recurrent renal disease, 3 had no predisposing factors); in 3 patients in the TAC/MMF group (2 patients had recurrent disease and 1 had diabetes); and in 8 patients (4 had diabetes, 1 had recurrent disease, and 3 had no predisposing factors) in the TAC/SRL group (CSA/SRL and TAC/SRL vs. CSA/MMF and TAC/MMF; p = 0.05). A nephrotic range of proteinuria developed at various time points after transplantation; in the CSA/MMF group, both recipients developed proteinuria between 2 and 4 years; in the CSA/SRL group, 3 recipients developed proteinuria at 1 year, 1 at 2 years, and 5 at 3 years; in the TAC/MMF group, 1 recipient developed proteinuria at 2 years and 2, at 3 years; and in the TAC/SRL group, 3 patients developed proteinuria at 2 years, 2 recipients at 3 years, and 3 at 4 years. In recipients of all 4 groups with a nephrotic range of proteinuria, biopsy findings included podocyte injury, chronic active antibody-mediated rejection, and peritubular capillary basement multilayering. Most of these patients had pre-transplant diabetes mellitus or recurrent renal disease. All recipients were treated with angiotensin receptor blocking drugs to reduce proteinuria. Eight recipients who received SRL treatment lost the grafts between 1 and 4 years. The remaining 9 recipients with reduced proteinuria continue to have graft function at 5 years. In the MMF-treated group, 3 patients lost the grafts between 1 and 4 years and 2 continue to have graft function at 5 years. 4.11. Adverse events The mean body mass index from pre-transplant baseline to 3 years post-transplant increased by approximately 2% to 3% in each of the four groups of patients. NODM was seen in 6% of patients in the TAC/MMF group; in 4% of those in the TAC/SRL group, in 0% of those in the CSA/MMF group, and in 4% of those in the CSA/SRL group at 5 years posttransplant (p = ns). NODM was observed within the first 12 months after transplant in all groups of recipients. There was no correlation between NODM and pulse doses of methylprednisolone used to treat CAR or SCAR. Severe infections diagnosed included bacterial sepsis, CMV infection, and Candida infection. Severe bacterial infections were seen in 18% of patients in the TAC/MMF group, in 24% in the TAC/SRL group, in 10% in the CSA/MMF group, and in 16% in the CSA/SRL group (CSA/MMF vs. TAC/SRL groups p = 0.05). CMV infections were seen in one patient each in the TAC/MMF and CSA/MMF groups, and Candida pyelonephritis was seen in one patient in the TAC/SRL group. Mouth ulcers were seen in 3 (6%) patients in the CSA/SRL group and in 2 (4%) in the TAC/SRL group. Endoscope-proven reflux gastroesophagitis was seen in 1 (2%) patient in the CSA/MMF group and in 5 (10%) patients in the TAC/MMF group. Severe diarrhea causing dehydration and acute renal failure was observed in 3 (6%) patients in the CSA/MMF group and in 4 (8%) in the TAC/MMF group.

Table 2 Conversion of baseline immunosuppression due to intolerance, persistent side effects, and unresponsive acute rejection Recipient group

Persistent side effects and intolerance

Unresponsive acute rejections

Total conversions

Conversion therapy

Number of recipients maintained on original therapy

CSA/MMF CSA/SRL

CSA in 2, MMF in 4 CSA in 3, SRL in 3

3 2

9 8

41 (82%) 42 (84%)

TAC/MMF

TAC in 3, MMF in 4

2

9

TAC/SRL

TAC in 2, SRL in 2

1

5

TAC/MMF in 5, CSA/SRL in 4 TAC/MMF in 6 CSA/MMF in 2 CSA/MMF in 3 TAC/SRL in 6 CSA/SRL in 3 TAC/MMF in 2

41 (82%) 45 (90%)

40

M.S. Anil Kumar et al. / Transplant Immunology 20 (2008) 32–42

Fig. 5. Renal function: Serum creatinine levels and estimated creatinine clearances at 1, 2, 3, 4, and 5 years in kidney recipients receiving CSA/MMF, CSA/SRL, TAC/MMF, TAC, or SRL. Erythropoietin treatment was given to patients with a hemoglobin level of less than 11 g/dl: 6% of patients in the TAC/MMF group, 22% in the TAC/SRL group, 4% in the CSA/MMF group, and 8% in the CSA/SRL group (p =0.02; TAC/SRL vs. TAC/MMF, p =0.02; TAC/SRL vs. CSA/MMF and p= 0.05; TAC/SRL vs. CSA/SRL groups). Table 3 Incidence of CAR and SCAR, prevalence of interstitial fibrosis/tubular atrophy, hypertensive changes, and 5-year patient and graft survival rates in African American vs. non-African American recipients in each of 4 groups of immunosuppression protocols Recipient group

CSA/MMF group

CSA/SRL group

TAC/MMF group

TAC/SRL group

Ethnicity

AA

Non-AA

AA

Non-AA

AA

Non-AA

AA

Non-AA

Number of recipients

25

25

25

25

27

23

26

24

Recipients with CAR 1 year 2 years 3 years 4 years 5 years

11 3 1 4 2

7 3 3 2 2

5 2 2 2 0

3 2 2 0 0

7 1 2 3 2

7 3 2 1 0

3 1 1 1 2

1 1 1 3 0

Recipients with SCAR 1 year 2 years 3 years 4 years 5 years

16 10 8 7 6

6 4 2 3 2

6 7 8 6 9

2 3 2 2 1

12 9 9 6 5

4 3 1 2 1

5 9 6 7 5

1 1 2 1 1

Hypertensive changes 1 year 2 years 3 years 4 years 5 years

14 18 26 29 38

8 10 10 12 14

6 12 14 14 22

4 6 6 8 14

6 10 10 14 16

4 6 6 8 8

4 8 10 12 18

4 6 6 6 8

Interstitial fibrosis/tubular atrophy 1 year 12 4 2 years 16 6 3 years 20 8 4 years 28 14 5 years 36 18

4 4 4 8 14

4 4 4 4 6

6 12 18 22 28

2 6 6 10 14

4 4 4 8 14

4 4 4 4 6

Patient survival 1 year 2 years 3 years 4 years 5 years

92 89 86 80 78

94 94 90 84 82

94 90 88 83 80

94 90 90 86 82

94 90 88 85 82

96 92 88 88 84

93 90 88 82 79

95 93 89 83 80

Delayed wound healing or infection of the initial transplant surgery incision was seen in 2 (4%) patients in the CSA/MMF group, in 3 (6%) in the CSA/SRL group, in 3 (6%) in the TAC/MMF group, and in 3 (6%) in the TAC/SRL group. The lipid levels were measured prior to transplant in all recipients and included serum cholesterol, triglycerides, and LDL and HDL fractions. Approximately 20% of patients in each group were maintained on lipid-lowering agents prior to transplant. In the first year after transplant, lipid-lowering agents were used in 80%, 100%, 74%, and 82% in the CSA/MMF, CSA/SRL, TAC/MMF, and TAC/SRL groups, respectively (TAC/MMF vs. CSA/SRL, p = 0.05). In the post-transplant period ranging from 1 to 8 years, de novo malignancies were seen in 23 recipients: 9 in the CSA/MMF group (2 each of post-transplant lymphoproliferative disease and colon and thyroid cancers, one each of leukemia, renal cell carcinoma, and malignant parotid tumor); 2 in the CSA/SRL group (1 each of thyroid and lung cancers); 10 in the TAC/MMF group (2 each of thyroid and breast cancers, 1 each of bladder, colon, prostate, lung, and renal cell cancer and 1 post-transplant lymphoproliferative disease); and 2 in the TAC/SRL group (1 each of breast cancer and post-transplant lymphoproliferative disease) ( CSA/SRL and TAC/SRL vs. CSA/MMF and TAC/MMF; p = 0.04).

5. Discussion The calcineurin inhibitors have remained the mainstay of immunosuppression therapy in patients undergoing kidney transplants despite their nephrotoxicity and the chronic attrition of transplanted kidneys due to CAI [20]. In non-sensitized patients, most transplant centers in the United States use a single immunosuppression protocol consisting of induction with an IL-2R antibody or a lymphocyte-depleting antibody and maintenance with a calcineurin inhibitor in combination with either MMF or SRL and long-term steroid therapy. Long-term steroid therapy has wide-ranging side effects and may potentiate the metabolic and hypertensive side effects of calcineurin inhibitors [13,28]. Previous reports show that early steroid withdrawal after kidney transplant is feasible in kidney transplant recipients [7–9]. Earlier reports show that transplanted kidneys undergo structural changes despite stable graft function [20]. CARs associated with graft dysfunction are widely reported by all transplant centers. On the other hand, SCAR is diagnosed only from the results of surveillance biopsies and is reported by few centers practicing surveillance biopsy as standard-of-care management. Previous studies have shown that SCAR may affect short- and long-term graft function and survival [29,30]. Continuous monitoring of kidney transplant recipients by periodic surveillance biopsy at pre-determined intervals is a useful tool for the diagnosis of SCAR and to study the prevalence of CAI [17,29]. Randomized studies show that treatment of SCAR is beneficial in prolonging survival

M.S. Anil Kumar et al. / Transplant Immunology 20 (2008) 32–42

[29,30]. Previous studies compared 1- to 3-year outcomes in kidney recipients maintained on two to three different calcineurin-based immunosuppression protocols with or without long-term steroid therapy but without continuously monitoring structural changes by serial surveillance biopsies soon after transplantation [10,11]. At present, no prospective studies compare long-term outcomes of early steroid withdrawal in 4 different calcineurin-based immunosuppression protocols in kidney recipients monitored for renal structural changes by surveillance biopsy. This prospective randomized study with predominantly deceased donor kidney recipients and approximately 50% AA recipients evaluated 5-year outcomes in kidney transplant recipients with early steroid withdrawal and maintained on four calcineurin-based immunosuppression protocols: CSA/MMF, CSA/SRL, TAC/MMF, and TAC/SRL. All recipients were continuously monitored for incidence of CAR, long-term patient and graft survival, incidence of SCAR, incidence and prevalence of CAI, and renal function. The primary end point of this study was CAR confirmed by biopsy. More than 80% of recipients continued with their assigned immunosuppression regimen. All recipients have remained free of long-term steroid therapy. In this study, the incidence of CAR, SCAR, type of CAI, adverse events, and daily dose and blood levels of CNIs varied in the 4 immunosuppression regimens. The rate of DGF was significantly higher and the duration longer in the CSA/SRL and TAC/SRL groups than in the CSA/MMF and TAC/MMF groups. This finding may be explained by the increased nephrotoxicity of CNI with the SRL combination. We delayed initiation of SRL to 4 days post-transplant and started with a low initial dose of 2 mg/day without a loading dose. Despite this precaution, DGF was significantly higher in the CSA/SRL and TAC/SRL groups. The incidence of CAR and SCAR was highest in the CSA/MMF group of recipients in the first year after transplant. The incidence of CAR in the first year after transplant in kidney recipients treated with CSA/MMF in this study is comparable and consistent with that in previous reports [18,31]. CAR was significantly higher in the CSA/MMF group than in the TAC/MMF group because the daily dose of MMF was 2 g. It is possible that many AA recipients in the CSA/MMF group may have experienced a higher incidence of CAR because we did not use 3 g of MMF per day. Our initial experience showed that most of our patients, including AA recipients, could not tolerate 3 g/day of MMF due to the severe gastrointestinal side effects. Five-year patient and graft survival rates were comparable among the four groups. Evaluation of CAI showed a significantly increased prevalence of changes due to hypertension and IF/TA in the CSA/MMF group. Chronic CNI toxicity was high in the CSA/SRL and TAC/SRL groups. Because of higher incidence of CNI toxicity, both daily doses and blood levels of CSA and TAC were significantly lower in the CSA/SRL and TAC/SRL groups than in the CSA/MMF and TAC/MMF groups. A previous report shows that chronic allograft nephropathy eventually develops in all transplanted kidneys at 10 years in recipients maintained on CSA-based immunosuppression [20]. In our study, it is possible that the lower prevalence of CAI due to hypertension and IF/TA in the CSA/SRL and TAC/SRL groups may be due to lower doses and lower blood levels of CSA and TAC. Calcineurin inhibitors have been implicated for some of the chronic structural changes observed in transplanted kidneys, and SRL has been reported to limit chronic vascular changes in native kidneys and ameliorate arteriolar injury [32,33]. A recent report of a randomized study shows that the vascular changes of CAI were significantly less in kidney recipients maintained on a non calcineurin inhibitor-based immunosuppression regimen consisting of SRL, MMF, and prednisone compared with a TAC, MMF, and prednisone regimen [34]. Progressive chronic allograft injury results in decreased graft function and predicts early graft loss [20,35]. This study monitored the chronic structural changes due to toxicity, immunological, and non-immunological injuries with different combinations of immunosuppressive drugs from 1 through 5 years.

41

Subgroup analysis of the AA and non-AA recipients in each of the four groups showed that CAR rates were comparable, SCAR rates were significantly higher, and CAI changes due to hypertension and interstitial fibrosis/tubular atrophy were significantly greater in AA recipients than in non-AA recipients in their respective immunosuppression protocols. The mean arterial pressures were ≥85 in most of the AA recipients compared with those in the non-AA recipients; this difference may explain the higher incidence of chronic hypertensive changes in AA recipients. The higher incidences of SCAR and CAI did not affect long-term patient and graft survival rates in AA recipients compared with non-AA recipients in their respective regimens. Subgroup analysis shows that, despite the small numbers, AA recipients in each of the four immunosuppression groups had comparable rates of CAR but that the rates of side effects from SCAR and CAI, hypertension and interstitial fibrosis/tubular atrophy, were significantly higher in the AA than in the non-AA recipients. Although the number of recipients analyzed in each of the four groups in this study is too small to provide any meaningful statistical significance, these results are consistent with those from our previous report on a larger group of recipients in which we compared AA with non-AA recipients (17). The incidence of severe infections was significantly higher in the TAC/SRL group compared to that in the CSA/MMF group, suggesting that dose reduction of one or both drugs in the TAC/SRL combination may be beneficial in lowering the number of serious infections. Delayed wound healing was comparable in all groups. It has been reported that SRL causes delayed wound healing [36]. We initiated SRL on the 4th transplant day, and we did not use a loading dose of SRL. It is possible that we did not observe a higher incidence of wound complications in SRL-treated patients because of early steroid withdrawal, lack of loading dose of SRL, and delayed initiation of SRL after transplant. A significantly higher proportion of patients in the TAC/SRL group required erythropoietin treatment in the post-transplant period than in the other 3 groups of patients. SRL has been shown to affect hematopoietic cell growth and survival [37]. It is possible that antiproliferative and bone marrow suppression properties of SRL were more severely expressed when used in combination with TAC to cause anemia. The incidence of NODM in kidney recipients treated with TAC in combination with MMF and prednisone is reported to be as high as 21% [38]. Calcineurin inhibitors and long-term steroid therapy are independent risk factors for NODM in kidney transplant recipients, and a combination of these 2 has the potential to increase the risk for NODM. Early steroid withdrawal significantly reduces NODM [9]. In this series NODM was 6% or less at 5 years in all 4 groups. The incidence of de novo malignancies was significantly higher in the CSA/MMF and TAC/MMF groups compared with the CSA/SRL and TAC/SRL groups. CNIs are reported to increase malignancy [14]. On the other hand, sirolimus has known antitumor effects [39,40]. Two factors may have decreased de novo malignancies in the CSA/SRL and TAC/SRL groups: (1) the direct antitumor effect of sirolimus in these patients and (2) the fact that recipients were maintained on significantly lower daily doses and lower blood levels of CSA and TAC compared with recipients in the CSA/MMF and TAC/MMF groups. This study has certain limitations: It was a single-center trial with a small number of patients in each of the four groups. Despite a longer follow-up period after transplant, a significant difference among the groups may not become apparent when one has a small cohort of patients in each group. In each of the four groups, approximately 20% to 25% of recipients were transplanted with ECD kidneys with age-associated structural changes. These kidneys may be more susceptible to preservation injury, CNI toxicity, and immunological injuries and may have affected the outcomes of CAI, renal function, and survival compared to standard criteria donor kidneys. The results of this pilot prospective randomized trial in kidney recipients treated with four different calcineurin inhibitor-based therapies, CSA/MMF, CSA/SRL, TAC/MMF, and TAC/SRL and early

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steroid withdrawal 2 days post-transplant, show that 5-year patient and graft survival rates are comparable among the four groups. The overall incidence of NODM remains low in all groups. The incidence of acute rejection, the prevalence of CAI, and the adverse events vary in each of the immunosuppression protocols analyzed in this study. Despite significant differences in the incidence of CAR, SCAR, severity of CAI, and adverse events, 5-year renal function and patient and graft survival rates were comparable in all four groups of patients. References [1] Calne RY, White DJG, Evans DB, et al. Cyclosporin A in cadaveric organ transplantation. Br Med J 1981;282:934–6. [2] Laskow DA, Vincenti F, Neylon J, et al. An open-label, concentration-ranging trial of FK506 in primary kidney transplantation: a report of the United States Multicenter FK506 Kidney Transplant Group. Transplantation 1996;62:900–5. [3] Sollinger HW. Mycophenolate mofetil for the prevention of acute rejection in primary cadaveric renal allograft recipients. Transplantation 1995;60:225–32. [4] Kahan BD, Podbielski J, Napoli KL, et al. Immunosuppressive effects and safety of a sirolimus/cyclosporine combination regimen for renal transplantation. Transplantation 1998;66:1040–6. [5] Brennan DC, Flavin K, Lowell JA, et al. A randomized, double-blinded comparison of Thymoglobulin versus Atgam for induction immunosuppressive therapy in adult renal transplant recipients. Transplantation 1999;67(7):1011–8. [6] Nashan B, Moore R, Amlot P, et al. Randomized trial of basiliximab versus placebo for control of acute cellular rejection in renal allograft recipients. Lancet 1997;350:1193–8. [7] Matas AJ, Kandaswamy R, Humar A, et al. Long-term immunosuppression without maintenance prednisone after kidney transplantation. Ann Surg 2004;240:510–7. [8] Hricik DE, Knauss TC, Bodziak KA, et al. Withdrawal of steroid therapy in African American kidney transplant recipients receiving tacrolimus and sirolimus. Transplantation 2003;76:938–42. [9] Kumar MSA, Heifets M, Moritz MJ, et al. Safety and efficacy of steroid withdrawal 2 days after kidney transplantation: analysis of results at 3 years. Transplantation 2006;81:832–9. [10] Ciancio G, Burke GW, Gaynor JJ, et al. A randomized long-term trial of tacrolimus/ sirolimus versus tacrolimus/mycophenolate mofetil versus cyclosporine/sirolimus in renal transplantation: three year analysis. Transplantation 2006;81(6):845–52. [11] Kandaswamy R, Melancon JK, Dunn TY, et al. A prospective randomized trial of steroid-free maintenance regimens in kidney transplant recipients: an interim analysis. Am J Transplant 2005;5:1529–36. [12] Anil Kumar MS, Heifets, Fyfe B, et al. Comparison of steroid avoidance in tacrolimus/ mycophenolate mofetil and tacrolimus/sirolimus combination in kidney transplantation monitored by surveillance biopsy. Transplantation 2005;80:807–14. [13] Kahan BD, Flechner SM, Lorber MI, et al. Complications of cyclosporine-prednisone immunosuppression in 402 renal allograft recipients exclusively followed at a single center for from one to five years. Transplantation 1987;43:197–204. [14] Hojo M, Morimoto T, Maluccio M, et al. Cyclosporine induces cancer progression by a cell-autonomous mechanism. Nature 1999;397:530–4. [15] Sollinger HW. Mycophenolate mofetil for the prevention of acute rejection in primary cadaveric renal allograft recipients. Transplantation 1995;60:225–32. [16] Kahan BD, Podbielski J, Napoli KL, et al. Immunosuppressive effects and safety of a sirolimus/cyclosporine combination regimen for renal transplantation. Transplantation 1998;66:1040–6. [17] Kumar MSA, Khan S, Ranganna K, et al. Long-term outcome of early steroid withdrawal after kidney transplantation in African American recipients monitored by surveillance biopsy. Am J Transplant 2008;8:574–5.

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