Calcineurin Inhibitor-Sparing Strategies in Renal Transplantation ...

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Calcineurin Inhibitor-Sparing Strategies in Renal Transplantation: Where Are We? A Comprehensive Review of the Current Evidence Brian Camilleri,1,2 Julie M. Bridson,2 Ahmed Halawa2,3 Abstract The introduction of the calcineurin inhibitors cyclosporine and tacrolimus in the immunosuppressive regimens for kidney transplant has been associated with substantial reductions in the incidence of acute rejection, with a subsequent improvement in 1-year graft survival. However, this has not directly correlated with improvements in long-term allograft survival. Immunosuppressive medications are associated with toxicities related directly to immunosuppressive effects, and these are similar among different agents. In addition, there are other toxicities that are unique for each drug. Immunosuppressive minimization strategies have attempted to address both of these toxicities. Calcineurin inhibitors have been associated with chronic nephrotoxicity, and various calcineurin inhibitor-sparing strategies have been used to address this issue with the aim of improving long-term outcomes. However, there has been a paradigm shift over the past 10 to 15 years, with the appreciation that calcineurin inhibitor nephrotoxicity is not the major cause of late graft failure. Studies have now shown that chronic immune injury mediated by donorspecific antibodies may account for most late graft losses. Although some patients do benefit from calcineurin inhibitor-sparing approaches, others may have late allograft loss from chronic and subacute immune-mediated injury. Unfortunately, the vast majority of calcineurin inhibitor-sparing studies have short-term follow-up and have not explored the change in the donor-specific antibody profile. One of the biggest challenges that we face is being able to distinguish among patients who will benefit from this strategy and those who will not. In this study, we From the 1Renal Unit, Ipswich Hospital NHS Trust, Ipswich, United Kingdom; the 2Faculty of Health and Science, Institute of Learning and Teaching, University of Liverpool, United Kingdom; and the 3Sheffield Teaching Hospitals, Sheffield, United Kingdom Acknowledgements: The authors have no conflicts of interest to declare, and no funding was received for this study. Corresponding author: Brian Camilleri, Ipswich Hospital NHS Trust, Heath Road, Ipswich UK IP4 5PD Phone: +44 147 370 2593 E-mail: [email protected]

Experimental and Clinical Transplantation (2016) 5: 471-483

Copyright © Başkent University 2016 Printed in Turkey. All Rights Reserved.

review the various strategies used to limit or avoid the use of calcineurin inhibitors and address the benefits and pitfalls associated in pursuing such strategies. Key words: Calcineurin avoidance, Calcineurin conversion, Calcineurin minimization, Calcineurin withdrawal Introduction The introduction of cyclosporine in the early 1980s in the field of renal transplantation led to a substantial reduction in the incidence of acute rejection with a subsequent improvement in 1-year graft survival.1 Tacrolimus was approved for kidney transplant in 1997 with improvement in clinical outcomes.2 Unfortunately, the reduction in acute rejection rates has not directly translated into an improvement in allograft survival.3 At the beginning of the 2000s, nephrotoxicity caused by the calcineurin inhibitors (CNIs), cyclosporine, and tacrolimus was thought to be an important cause of this lack of improvement in long-term graft survival.4 Introduction of new immunosuppression drugs without nephrotoxicity such as mycophenolate mofetil (MMF) in 1995, sirolimus in 1999, everolimus in 2010, and belatacept in 2011 has led to efforts directed toward CNI sparing. Calcineurin inhibitor-sparing regimens can be applied in various ways.5 In this study, we review the triggers for these strategies and the various ways in which they have been pursued and we highlight the potential pitfalls that may accompany these regimens. Calcineurin inhibitor mode of action and adverse effects Cyclosporine and tacrolimus pass freely across the cell membrane into the cell and bind to immunophilins: cyclosporine binds to cyclophilin and tacrolimus binds to FK506-binding protein 12. DOI: 10.6002/ect.2015.0283

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Brian Camilleri et al/Experimental and Clinical Transplantation (2016) 5: 471-483

The CNI-immunophilin complex binds the protein phosphatase calcineurin, competitively inhibiting interaction with its substrates, including nuclear factor of activated T cells. Inhibition of nuclear factor of activated T-cell dephosphorylation prevents its translocation to the cell nucleus, leading to reduced expression of genes required for T-cell activation such as interleukin 2.6 Acute toxicities associated with CNIs include hypertension, tremors, seizures, thrombotic microangiopathy, and renal dysfunction. Cyclosporine leads to afferent arteriolar constriction within the kidney, which may not necessarily lead to irreversible nephrotoxicity.7 Calcineurin inhibitors have also been implicated in the pathogenesis of posttransplant type 2 diabetes mellitus and hyperlipidemia; this is relevant because cardiovascular disease is the leading cause of death in renal transplant patients. Calcineurin inhibitors are also associated with cosmetic stigmata, which may promote noncompliance and which has been associated with late allograft loss.8 There are differences between the CNI toxicity profiles. Tacrolimus is more commonly associated with glucose intolerance, tremor, and hair loss, whereas hypertension, lipid abnormalities, and hirsutism are more common with cyclosporine.9 Tacrolimus may be less nephrotoxic; however, this remains controversial. Chronic nephrotoxicity has been the main complication with long-term use of CNIs. This complication was initially identified in cardiac transplant recipients and later reported in other nonrenal organ transplants. Characteristic histologic features have been observed in both renal transplant recipients and in patients treated with CNIs for autoimmune disease.4 Histologic features include striped interstitial fibrosis, tubular atrophy, medial arteriolar hyalinosis, and tubular microcalcification. Nankivell and associates demonstrated similar lesions in serial protocol biopsies of kidney allografts treated with CNIs.4 These findings, together with the introduction of nonnephrotoxic immunosuppression agents, led to various attempts at reducing or eliminating the use of CNIs in renal transplant. Calcineurin inhibitor-sparing strategies Calcineurin inhibitor avoidance Complete omission of CNI is usually planned de novo and involves using another immunosuppressant (Table 1).

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Antibody induction, mycophenolate mofetil, and steroids Initial attempts at CNI avoidance did not use an alternative immunosuppressant agent, and this was clearly ineffective. Using daclizumab as an induction agent, Vincenti and associates reported a 6-month acute rejection rate of 48% using MMF and steroids.11 Grinyo and associates used antithymocyte globulin with high-dose MMF and steroids without CNI in suboptimal donors.12 Their results showed that 24% of patients had acute rejection, necessitating reintroduction of CNI in several patients. The high dose of MMF (3 g/d postoperatively) was not well tolerated and was reduced in 93% of the patients due to adverse events. Use of de novo sirolimus In phase II studies by Groth and associates13 and Kreis and associates,14 attention was shifted toward the mammalian target of rapamycin (mTOR) inhibitor sirolimus as a CNI avoidance strategy. Sirolimus irreversibly inhibits mTOR kinase activity, interfering with signal 3 of T-cell activation,6 blocking lymphocyte proliferation in response to many cytokines and growth factors. Groth and associates randomized patients to receive either sirolimus or cyclosporine (Sandimmune) in combination with azathioprine and steroids; at 2 years, patients showed similar acute rejection rates, with higher calculated glomerular filtration rate (GFR) in the sirolimus group.13 Kreis and associates showed similar results using MMF.14 Flechner and associates reported on de novo renal transplant recipients who received basiliximab, MMF 2 g/day, steroids, and either sirolimus or cyclosporine.15 At 2 years, the sirolimus group had better renal function and reduced prevalence of chronic allograft nephropathy. Larson and associates compared sirolimus with tacrolimus.16 Graft function and acute rejection rates were comparable, but protocol biopsies revealed a higher incidence of chronic vascular changes in the tacrolimus group. The ELITE-SYMPHONY study was designed to evaluate the effect and safety of 4 immunosuppressive regimens, including a CNI-free arm.17 The authors randomized 1645 renal transplant recipients to either standard-dose cyclosporine, MMF, and steroids or daclizumab induction, MMF, and steroids in combination with low-dose cyclosporine, low-dose tacrolimus, and low-dose

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Table 1. Calcineurin Inhibitor Avoidance Studies Author (Year) and Study

Intervention Arm

Control Arm

Study Length, Outcome Renal Function mo Studies With Antibody Induction, Mycophenolate Mofetil, and Steroids

Vincenti et al (2001)11 Grinyo et al (2003)12

Daclizumab, MMF, steroids Thymoglobulin, MMF, steroids

None

6

AR 48%

None

6

AR 24%

Groth et al (1999)13

Sirolimus, azathioprine, steroids Sirolimus, MMF, steroids

At 1 year, creatinine was 113 µmol/L in nonrejectors and 154 µmol/L in rejectors At 1 year, mean serum creatinine was 178 µmol/L

Studies With Sirolimus De Novo

Kreis et al (2000)14

Cyclosporine, azathioprine, steroids Cyclosporine, MMF, steroids

12

Similar patient and graft survival; similar AR rates (38% vs 41%)

Serum creatinine was significantly lower with sirolimus at 3 and 4 mo

12

Similar patient and graft survival; similar AR rates (27.5% vs 18.4%)

From month 2 onward, calculated GFR was consistently higher in sirolimus group Significantly higher GFR (80.4 vs 63.4 mL/min)

Flechner et al (2004)15

Basiliximab, Basiliximab, 24 Similar patient and graft survival sirolimus, MMF, cyclosporine, and AR rates steroids MMF, steroids Larson et al Sirolimus, MMF, Tacrolimus, 12 Similar patient and graft survival; No difference in GFR (63 vs 61 mL/min) (2006)16 steroids MMF, steroids similar AR rates (13% vs 10%) Ekberg et al Daclizumab, MMF, Standard-dose 12 Low-dose tacrolimus had the GFR of 59.4 (low-dose cyclosporine) (2007)17 steroids, and lowcyclosporine, lowest AR (12.3%) and highest vs 65.4 (low-dose tacrolimus) vs ELITE-SYMPHONY dose cyclosporine MMF, steroids allograft survival (94.2%) 56.7 (low-dose sirolimus) vs 57.1 Study low-dose tacrolimus (standard-dose cyclosporine) mL/min or low-dose sirolimus Buchler et al rATG, sirolimus, rATG, 12 Similar patient and graft survival Similar estimated GFR (60 vs (2007)18 MMF, steroids cyclosporine and AR rates 57 mL/min) SPEISSER Trial MMF, steroids Flechner et al All groups: 12 Group 2 had higher than expected Similar GFR at 1 and 2 years (59.1 vs (2011)20 daclizumab, AR rates (25.7% vs 6.5% in the 59.3 vs 62.0 mL/min) ORION Study steroids tacrolimus plus MMF arm) leading Group 1: sirolimus + to termination of this arm; similar tacrolimus (wk 13, graft and patient survival and AR elimination) rates for groups 1 and 3 Group 2: sirolimus + MMF Group 3: tacrolimus + MMF Sharif et al Meta-analysis of 52 studies including 11 337 renal transplant patients. Use of mTOR inhibitor in combination with MMF increases the odds 21 (2011) of graft failure (odds ratio of 1.43) Studies With Belatacept

Vincenti et al (2005)25 Vincenti et al (2010)26 BENEFIT Study

Durrbach et al (2010)29 BENEFIT-EXT Rostaing et al (2011)32

Basiliximab, belatacept MI or LI, MMF, steroids Basiliximab, belatacept MI or LI, MMF, steroids

Basiliximab, cyclosporine, MMF, steroids Basiliximab, cyclosporine, MMF, steroids

Basiliximab, belatacept MI or LI, MMF, steroids Switch to belatacept

Basiliximab, cyclosporine, MMF, steroids Continue with CNI (44% cyclosporine, 54% tacrolimus)

6

Similar AR rates (MI: 7% vs LI: 6% vs cyclosporine: 8%)

12

Similar patient and graft survival rates; belatacept groups had higher incidence of AR (MI: 22 vs LI: 17 vs cyclosporine: 7%) and higher grade of AR; PTLD more common in belatacept groups Similar patient and graft survival; similar AR rates (MI: 18% vs LI: 18% vs cyclosporine: 14%) 7% within belatacept group had AR, all within 6 mo

12

12

At 12 mo, significantly higher GFR in belatacept groups (MI: 66.3, LI: 62.1, and cyclosporine: 53.5 mL/min) Superior renal function in belatacept groups (GFR for MI: 65, LI: 63, cyclosporine: 50 mL/min)

Mean measured GFR was significantly better in MI belatacept group vs cyclosporine group (52 vs 45 mL/min) Mean change from baseline in calculated GFR was higher in the belatacept group (7 vs 2 mL/min); improvement was greater with baseline GFR 40-60 mL/min

Abbreviations: AR, acute rejection; GFR, glomerular filtration rate; LI, less intensive; MI, more intensive; MMF, mycophenolate mofetil; mTOR, mammalian target of rapamycin inhibitor; PTLD, posttransplant lymphoproliferative disease; rATG, rabbit antithymocyte globulin All results showing intervention arm vs control arm, unless otherwise stated. Significant results P ≤ .05.

sirolimus. Glomerular filtration rate at 1 year was significantly higher in the tacrolimus arm than in the other treatment groups. The tacrolimus arm had the lowest rate of biopsy-proven acute rejection (BPAR) and the best allograft survival. The sirolimus arm had the highest BPAR and the lowest allograft

survival, which was similar to standard dose cyclosporine. Patients in the sirolimus arm had more adverse events and a greater rate of treatment failure (35.8% vs 12% in the low-dose tacrolimus group; P < .001), despite a relatively low sirolimus trough level (trough levels of 4-8 ng/mL).

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Other groups attempted CNI avoidance with de novo sirolimus in combination with MMF and steroids using antithymocyte globulin as the induction agent.18,19 In the SPIESSER Trial, there was no statistically significant difference in patient and graft survival and incidence of BPAR.18 Glomerular filtration rate was significantly higher in the sirolimus group. Similar results were obtained by Glotz and associates.19 In both studies, premature withdrawal and adverse events were higher in the sirolimus group. Patients were divided into 3 arms for the ORION study: sirolimus plus tacrolimus, sirolimus plus MMF, and tacrolimus plus MMF.20 The study sponsor removed patients receiving CNI-free regimen due to the high rate of BPAR within the first 6 months. A meta-analysis of 2688 patients from 16 randomized controlled trials comparing mTOR inhibitor with CNI-based regimens for initial immunosuppression suggested that the use of an mTOR inhibitor as de novo therapy was associated with an increased rate of graft failure (odds ratio of 1.43; P = .01).21 The primary outcome measure in this meta-analysis was overall graft failure (composite of death-censored graft loss and death with graft function) at the main study endpoint (most commonly 1 year). Observational studies have also raised concerns with de novo use of mTOR inhibitor. The Analysis of the Scientific Registry of Renal Transplant Recipients showed that the combination of sirolimus and MMF was associated with the highest 6-month acute rejection rates, a significantly lower graft survival at 5 years, and a 75% increased risk of patient death relative to the tacrolimus and MMF discharge regimen.22 In an analysis of 139 370 kidney transplant patients between 1999 and 2010, de novo use of an mTOR inhibitor was associated with increased allograft loss and mortality throughout the 8 years of longitudinal follow-up.23 A further concern with use of mTOR inhibitor therapy as de novo therapy is its effect on delayed wound healing.24 Calcineurin inhibitor avoidance with newer drugs (belatacept) Interest in CNI avoidance has reemerged with the introduction of belatacept, a second-generation cytotoxic T lymphocyte-associated antigen 4 immunoglobulin, which blocks the costimulatory

Exp Clin Transplant

pathway of T-cell activation. It is a fusion protein consisting of the extracellular domain of cytotoxic T lymphocyte-associated antigen 4 and the Fc domain of human IgG1. Belatacept binds avidly to CD80 and CD86 present on the surface of the antigenpresenting cells, blocking signal 2 in the T-cell activation pathway.6 In a phase II study, belatacept was given as more intensive and less intensive regimens and compared to cyclosporine in kidney transplant recipients.25 Belatacept was not inferior to prevention of acute rejection at 6 months, and both belatacept groups showed a higher GFR than the cyclosporine group. The cyclosporine-treated patients demonstrated an increased incidence of chronic allograft nephropathy. Belatacept continued to preserve GFR for up to 5 years, although it resulted in more episodes of BPAR. Similarly, the BENEFIT Study assessed 666 standard criteria kidney transplant recipients treated with a more intensive or less intensive belatacept-based therapy versus cyclosporine.26 There were fewer patients in the belatacept groups having renal impairment at 1 year. The incidence of chronic allograft nephropathy was lower in both belatacept arms compared with the cyclosporine arm (more intensive: 18% and less intensive: 24% vs cyclosporine: 32%). Belatacept-treated patients had a higher incidence of BPAR and more Banff IIB rejections, especially in the more intensive group. Three-year follow-up data by the same author group showed similar outcomes.27 More recently, 7-year follow-up results showed that patients who had received belatacept had a statistically significant 43% relative risk reduction of death or graft loss and significantly better renal function (calculated GFR of 78 mL/min/1.73 m2 for belatacept vs 51 mL/min/1.73 m2 for cyclosporine).28 The BENEFIT-EXT study (compared similar regimens; of note, the study included 595 expanded criteria donors and high-immunologic risk patients.29 The belatacept groups were not inferior compared with those who received cyclosporine. The cyclosporine group developed renal impairment more frequently. Biopsy-proven chronic allograft nephropathy was similar between groups, and acute rejection was comparable in all arms. There were more patients with BANFF IIB acute rejection in the more intensive treatment group. At 3 years, graft survival was similar between groups, but the estimated GFR was lower in the cyclosporine arm.30

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Belatacept was associated with sustained improvement in renal function at 7 years.31 The main safety risk for belatacept-based therapy highlighted in these studies is posttransplant lymphoproliferative disease, and it should not be used in patients who are Epstein-Barr virus negative. Most studies with belatacept have been in de novo renal transplant patients; however, switching from CNI-based regimen to a belatacept-based regimen has also been studied in stable renal transplant recipients. In a study by Rostaing and associates, patients who were more than 6 months but less than 36 months after transplant were randomized to either switch to belatacept or continue

CNI treatment.32 At 1 year, switching to belatacept was associated with a significant improvement in renal function using calculated GFR versus the CNI group. Calcineurin inhibitor minimization This refers to using a lower dose of CNI. This is usually planned de novo or as a result of an adverse event (Table 2). Calcineurin inhibitor minimization with mycophenolic mofetil Patients in the CAESAR Study were treated with low-dose cyclosporine, MMF, and steroids; standard-

Table 2. Calcineurin Inhibitor Minimization Studies Author (Year) and Study

Intervention Arm

Control Arm

Study Length, mo

Outcome

Renal Function

Ekberg et al (2007)33 CAESAR Study

Daclizumab, MMF, steroids, and cyclosporine withdrawal by month 6 or lowdose cyclosporine

Standard-dose cyclosporine, MMF, steroids

12

AR significantly higher in cyclosporine withdrawal group (38%) vs low-dose cyclosporine (25.4%) and standard-dose cyclosporine (27.5%)

Similar GFR results between cyclosporine withdrawal and low-dose cyclosporine vs standard-dose cyclosporine (50.9 vs 48.6mL/min)

Ekberg et al (2007)17 ELITESYMPHONY Study

Daclizumab, MMF, steroids and low-dose cyclosporine or low-dose tacrolimus or low-dose sirolimus

Standard-dose cyclosporine, MMF, steroids

12

Low-dose tacrolimus had highest GFR (65.4 mL/min), lowest AR (12.3%), and highest allograft survival (94.2%)

GFR of 59.4 (low-dose cyclosporine) vs 65.4 (low-dose tacrolimus) vs 56.7 (lowdose sirolimus) vs 57.1 (standard-dose cyclosporine) mL/min

Gaston et al (2009)34 Opticept Trial

Group A: concentrationcontrolled MMF lowdose cyclosporine, steroids Group B: MMF, standard-dose cyclosporine, steroids

Group C: Fixed-dose MMF, standarddose cyclosporine, steroids

24

Group A had fewer treatment failures (including AR, graft loss, and death)

Group A had 12.3% increase in estimated GFR from baseline compared with 5.4% increase in group B and 8.2% in group C

Tedesco Silva et al (2010)37 A2309 Study

Basiliximab, lowdose cyclosporine, steroids, and EVL 1.5 mg/d or EVL 3 mg/d

Basiliximab, MPA, steroids standard-dose cyclosporine

Watson et al (2005)39

Alemtuzumab, lowdose cyclosporine

Cyclosporine, azathioprine, steroids (retrospective comparison)

Ciancio et al (2005)40

Group A: rATG, tacrolimus, MMF, steroids Group B: alemtuzumab, low-dose tacrolimus, MMF Group C: daclizumab, tacrolimus, MMF, steroids

3C Study (2014)42

Alemtuzumab, low-dose tacrolimus, MMF

Studies With Mycophenolic Mofetil

Studies With Mammalian Target of Rapamycin Inhibitor Immunosuppression

24

Efficacy failure rate (AR, graft loss, and death) was noninferior in the groups (25.3% vs 21.9% vs 24.2%)

Mean estimated GFR was noninferior in the EVL groups vs MPA group (54.6 vs 51.3 vs 52.2 mL/min)

Studies With Alemtuzumab

Basiliximab, standard-dose tacrolimus, MMF, steroids

60

Similar patient and graft survival; AR similar at 5 y (31.5% vs 33.6%)

No significant difference in serum creatinine concentration between groups at any time point during 5 y

15

Similar patient and graft survival; AR at 1 y was equivalent (16.6% in all 3 groups)

Similar renal function at 1 y

6

AR was significantly lower in the alemtuzumab arm (7% vs 16%)

Similar graft function at 6 mo (50.1 vs 49.8 mL/min)

Abbreviations: AR, acute rejection; EVL, everolimus; GFR, glomerular filtration rate; MMF, mycophenolate mofetil; MPA, mycophenolic acid; rATG, rabbit antithymocyte globulin All results showing intervention arm vs control arm, unless otherwise stated. Significant results P ≤ 0.05.

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dose cyclosporine, MMF, steroids; or cyclosporine withdrawal (cyclosporine tapered starting at month 4).33 Patients in the low-dose cyclosporine and cyclosporine withdrawal arms received daclizumab induction. There was no difference in GFR at 12 months between the groups. The BPAR rates were significantly higher in the cyclosporine withdrawal group. There did not seem to be an advantage in using a low-dose cyclosporine regimen, and withdrawal of cyclosporine was associated with an increased risk of rejection. Other studies that looked at CNI minimization with mycophenolic mofetil include the EliteSymphony Study described above.17 In this study, patients treated with low-dose tacrolimus had the lowest acute rejection rate at 1 year and had significantly better renal function. The Opticept Trial compared a concentration-controlled dose of MMF with a fixed-dose regimen.34 Patients received either the concentration-controlled MMF and reduced-level CNI, concentration-controlled MMF and standardlevel CNI, or fixed-dose MMF and standard-level CNI. There were no major differences at 2 years between the fixed-dose and the concentrationcontrolled MMF groups, indicating the potential utility of concentration-controlled MMF in CNIsparing regimens. Overall, CNI minimization seems to be effective, with BPAR and graft survival rates similar to that shown in patients who received standard CNI doses. However, GFR improvements were only small and not significant. In addition, these studies had short-term follow-up and lacked data on antibody-mediated rejections, circulating donor-specific antibodies (DSAs), and staining for C4d in renal biopsy specimens. Calcineurin inhibitor minimization with mammalian target of rapamycin inhibitor immunosuppression Mammalian target of rapamycin inhibitors have been used in combination with CNI, but they can potentiate CNI nephrotoxicity when coadministered with CNI.35 The combination of mTOR inhibitors and CNIs may lead to enhanced renal tissue exposure despite adequate whole blood levels of either drug, which seems to be mediated by P-glycoprotein inhibition in renal tubular cells.36 Everolimus is a metabolite of sirolimus, with similar immunosuppressant properties but a shorter half-life, and it has been used in CNI minimization regimens. The A2309 Study is the largest prospective

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renal transplant trial (n = 833 patients) of an mTOR inhibitor plus reduced-exposure cyclosporine to date.37,38 Renal transplant recipients were randomized to everolimus, with target trough concentration of 3 to 8 or 6 to 12 μg/L plus reducedexposure cyclosporine or to mycophenolate sodium plus standard exposure cyclosporine. All patients received basiliximab and steroids. An everolimus trough concentration of 3 to 8 μg/L was associated with comparable efficacy and renal function versus mycophenolate sodium plus standard exposure cyclosporine over 2 years. There was a significantly higher incidence of adverse events, leading to discontinuation in the everolimus groups compared with mycophenolate sodium. Other studies have included everolimus with CNI minimization; however, no overall differences in renal function, rejection rates, or survival were observed. Calcineurin inhibitor minimization with alemtuzumab Calcineurin inhibitor minimization has also been attempted in combination with alemtuzumab, a humanized monoclonal antibody specific for CD52 (a cell surface antigen expressed on T and B lymphocytes, monocytes, and macrophages). Watson and associates reported no difference in a 5-year follow-up study of patients who received alemtuzumab as induction therapy versus patients who did not receive alemtuzumab.39 The alemtuzumab group received half-level cyclosporine, no antimetabolite agents, and minimal steroids. Treated patients were compared in a retrospective contemporaneous-controlled manner with recipients who received conventional immunosuppression with cyclosporine, azathioprine, and prednisolone. Acute rejection at 5 years was similar with no differences in graft loss. Ciancio and associates compared antithymocyte globulin, alemtuzumab, and daclizumab.40 Maintenance therapy was with tacrolimus, MMF, and steroids; however, patients in the alemtuzumab group also received half-dose tacrolimus and no steroids 1 week after transplant. Acute rejection at 1 year was similar, occurring in 5 patients of 30 in all 3 groups. Recipients receiving alemtuzumab had higher chronic allograft nephropathy at 24 months.41 It is difficult to extrapolate concrete conclusions from the studies mentioned above due to the limitations of the study designs and the small sample sizes. More recently, in 2014, the results of the 3C

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Study were reported.42 In this study, 426 patients were randomly assigned to alemtuzumab-based treatment and 426 patients to basiliximab-based treatment. Alemtuzumab-based induction treatment was followed by low-dose tacrolimus and MMF without steroids, whereas the basiliximab-based induction treatment regimen was followed by standard-dose tacrolimus, MMF, and steroids. At 6 months after transplant, patients in the alemtuzumab-based treatment group had a 58% reduction in BPAR. Calcineurin inhibitor withdrawal This refers to tapering of CNI dose until eliminated. This is usually planned de novo or as a result of an adverse event (Table 3).

Calcineurin inhibitor withdrawal from azathioprine and steroid regimen Studies of cyclosporine withdrawal leading to dual immunosuppression with azathioprine and steroids have been associated with high risk of acute rejection.43,44 In a meta-analysis by Kasiske and associates, cyclosporine withdrawal was associated with an increased risk of acute rejection but no significant risk of graft failure.43 Cyclosporine withdrawal in select patients seems to impart little risk of long-term graft failure. One of the speculations by the authors was that the deleterious effects of acute rejection after cyclosporine withdrawal are balanced by the beneficial effects of reduced nephrotoxicity. However, these results may

Table 3. Calcineurin Inhibitor Withdrawal Studies Author (Year) and Study

Intervention Arm

Control Arm

Study Length, mo

Outcome

Renal Function

Kasiske et al (2000)43

Meta-analysis of 10 cyclosporine withdrawal trials; there was a statistically significant increase in AR [0.11 (0.07-0.15; P < .001)]; in 12 trials, the relative risk of graft failure was not increased

Gallagher et al (2004)44

Group 2: cyclosporine alone or Group 3: cyclosporine for 3 mo, then azathioprine, steroids

Abramowicz et al (2002)45

Cyclosporine withdrawal, MMF, steroids

Cyclosporine, MMF, steroids

6

AR occurred in 10.6% in cyclosporine withdrawal vs 2.4%, with no graft loss

There was a statistically significant increase in creatinine clearance in the per-protocol population (7.5 mL/min)

Dudley et al (2005)46 Creeping Creatinine Study

Cyclosporine withdrawal and introduction of MMF

Continue with cyclosporine

6

No AR in the cyclosporine withdrawal group

Statistically significant improvement or stabilization of renal function in the cyclosporine withdrawal group (58% vs 32%)

Suwelack et al (2004)47

MMF, steroids

CNI, MMF, steroids

32 (stopped due to ethical reasons)

No AR occurred

Renal function improved on dual therapy

McGrath et al (2001)48

MMF, steroids

Tacrolimus, azathioprine, steroids

8.1

No AR in both groups

Improvement in renal function in the MMF group

Johnson et al (2001)49

Sirolimus, steroids, cyclosporine withdrawn at 3 mo

Sirolimus, cyclosporine, steroids

12

No difference in patient and graft survival; AR rates were higher in the sirolimus-steroid group (9.8 vs 4.2%)

Renal function was better in the sirolimus-steroid group (calculated GFR 63 vs 57 mL/min)

Oberbauer et al (2005)50 Rapamune Maintenance Regimen Study

Sirolimus, steroids, cyclosporine withdrawn at 3 mo

Sirolimus, cyclosporine, steroids

48

Sirolimus-steroid group had better graft survival; AR rates were similar (10.2% vs 6.5%)

Calculated GFR was better in the sirolimus-steroid group (58.3 vs 43.8 mL/min)

Russ et al (2005)51 Rapamune Maintenance Regimen Study

Sirolimus, steroid, cyclosporine withdrawn at 3 mo

Sirolimus, cyclosporine, steroid

48

Cyclosporine withdrawal led to beneficial outcomes regardless of renal function

Enhanced benefit of cyclosporine withdrawal if baseline GFR was < 45 mL/min

Mulay et al (2005)52

A systematic review of randomized trials of CNI withdrawal from sirolimus-based regimen. CNI withdrawal was associated with an increased risk of AR (risk difference 6%; P = .002) in the short term but a higher creatinine clearance at 1 y (mean difference 7.49 mL/min; P < .001) and lower blood pressure (relative risk 0.56; P = .001).

Studies From Azathioprine-Steroid Regimen

Group 1: azathioprine, steroids

180

Significant difference in graft survival (group 1: 58% vs group 2: 51% vs group 3: 70%)

Cyclosporine-only group had lower mean creatinine levels at all time points beyond 3 mo

Studies From MMF-Steroid Regimen in Patients With or Without Renal Dysfunction

Studies From Sirolimus-Based Regimen

Abbreviations: AR, acute rejection; CNI, calcineurin inhibitor; GFR, glomerular filtration rate; MMF, mycophenolate mofetil All results showing intervention arm vs control arm, unless otherwise stated. Significant results P ≤ .05.

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be different with longer follow-up, and in these studies there is no information about the development of DSAs. Calcineurin inhibitor withdrawal from mycophenolate mofetil and steroid regimen in patients with or without renal dysfunction In an randomized controlled trial by Abramowicz and associates involving patients with stable renal function on cyclosporine and steroid maintenance with or without azathioprine, MMF was added on for patients on dual therapy or MMF replaced azathioprine over a 3-month cycle.45 Patients were then randomized to continue with cyclosporine plus MMF and steroids or have cyclosporine withdrawn. There was a statistically significant improvement in renal function in the cyclosporine withdrawal group, but there was an increase in acute rejection at 6 months in the cyclosporine withdrawal group. Calcineurin inhibitor elimination in the context of MMF immunosuppression has also been attempted in patients with deteriorating renal function.46–48 In the 2005 Creeping Creatinine Study by Dudley and associates, 143 patients with significant deterioration in renal function more than 6 months after transplant were maintained on cyclosporine-based immunosuppression or withdrawn from cyclosporine and maintained only on MMF and steroids.46 After cyclosporine withdrawal, there was no increased acute rejection rate (patients had a renal biopsy before enrolment to ensure there was no subclinical rejection), and there was a significant increase in GFR (6.7 ± 3.2 mL/min). Calcineurin inhibitor withdrawal from sirolimusbased regimen In 2001, Johnson and associates randomized renal transplant patients at 3 months to discontinue cyclosporine from their sirolimus-cyclosporinesteroid regimen, which resulted in a significantly better renal function at 12 months in the CNI withdrawal group.49 In the 2005 Rapamune Maintenance Regimen Trial, patients were randomized at 3 months to either continue with sirolimus-cyclosporine-steroids or to have cyclosporine withdrawn with a higher targeted sirolimus level.50,51 Overall graft survival, death with a functioning graft, and death-censored graft survival were significantly better in the sirolimussteroid arm at 48 months. Calculated GFR was

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significantly higher with the withdrawal of cyclosporine, and acute rejection was similar in both groups. Mulay and associates published a systematic review of randomized trials on CNI withdrawal using sirolimus-based therapy.52 Calcineurin inhibitor withdrawal was associated with an increased risk of acute rejection but higher creatinine clearance and reduced blood pressure. Although promising, these results are not unexpected. The combination of cyclosporine and sirolimus has synergistic nephrotoxic effects, and it is not really surprising that removal of cyclosporine from regimens using both cyclosporine and sirolimus leads to improved renal function or allograft histology. Calcineurin inhibitor conversion This refers to replacing CNI with another immunosuppressant. This is usually a result of an adverse event but can be planned de novo (Table 4). Conversion late after transplant Another strategy to improve allograft outcome is to start an mTOR inhibitor during CNI withdrawal. In the 2009 CONVERT trial, 830 renal allograft recipients, 6 to 120 months after transplant, commenced a CNI-based regimen (along with steroids and either MMF or azathioprine); patients were randomized to substitute the CNI for sirolimus or continue on their current regimen.53 The subgroup of patients with an estimated GFR of 20 to 40 mL/min had to be stopped because 8 of 48 patients (17%) in the sirolimus arm reached the primary safety outcome of BPAR, graft loss, or death. In the group with an estimated GFR > 40 mL/min, there was no difference in estimated GFR, acute rejection, or mortality. Proteinuria increased significantly in the sirolimus arm. However, patients in the subgroup with baseline GFR > 40 mL/min and urine proteinto-creatinine ratio ≤ 0.11 showed substantially less of an increment in this ratio after sirolimus conversion, had significantly better renal function, and had lower graft loss compared with the overall sirolimus conversion group. Diekmann and associates noted that, in patients with proteinuria > 800 mg/day, conversion to sirolimus was associated with a marked increase in urinary proteinuria.54 These observations suggest that conversion to sirolimus

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Table 4. Calcineurin Inhibitor Conversion Studies Author (Year) and Study

Intervention Arm

Control Arm

Study Length, mo

Outcome

Renal Function

Late after Transplant Studies

Schena et al (2009)53 CONVERT Trial

Convert CNI to sirolimus at 6120 mo

Continue with CNI

24

No difference in AR

Superior renal function with sirolimus if baseline GFR above 40 mL/min and no proteinuria

Lebranchu et al (2009)55 CONCEPT Study

Convert cyclosporine to sirolimus at 3 mo, MMF, and steroids (discontinued at month 8)

Cyclosporine, MMF, and steroids (discontinued at month 8)

52

Similar patient and graft survival; AR rate higher in sirolimus group (17% vs 8%; P = .071), mainly after discontinuing steroids

Estimated creatinine clearance better with sirolimus (68.9 vs 64.4 mL/min)

Guba et al (2010)56 SMART Study

Convert cyclosporine to sirolimus at 10-24 d

rATG, cyclosporine, MMF, steroids

12

Similar patient and graft survival and AR

Estimated GFR better in the sirolimus group (64.5 vs 53.4 mL/min)

Weir et al (2011)57 Spare The Nephron Study

Convert CNI to sirolimus at 30-180 d

CNI, MMF

24

Similar AR rates; similar rates of treatment failure (patient death, graft loss, adverse events)

Calculated GFR was significantly greater in the sirolimus group

Budde et al (2012)58 ZEUS Study

Convert cyclosporine to EVL at 4.5 mo

Basiliximab, cyclosporine, EC-MPS, steroids

36

AR rate was higher in the EVL group (13% vs 4.8%); similar patient and graft survival rates

GFR was significantly higher in the EVL group (69.2 vs 61.3 mL/min)

3C Study (2014)42

Results of the second randomization are still awaited. This entails a switch to sirolimus or continuing on tacrolimus in both alemtuzumab and basiliximab arms. Conversion to occur at 5-7 mo after transplant.

Euvrard et al (2008)60 TUMORAPA Study

Sirolimus

Early After Transplant Studies

Calcineurin Inhibitor Conversion to Sirolimus Due to Adverse Effects Other Than Nephrotoxicity

CNI

24

Survival free of cutaneous squamous cell cancer was significantly longer in sirolimus group

Graft function remained stable in both study groups

Abbreviations: AR, acute rejection; CNI, calcineurin inhibitor; EC-MPS, enteric-coated mycophenolate sodium; GFR, glomerular filtration rate; MMF, mycophenolate mofetil; rATG, rabbit antithymocyte globulin All results showing intervention arm vs control arm, unless otherwise stated. Significant results P ≤ .05.

should occur before significant renal parenchymal injury, as manifested by reduced GFR and proteinuria, has occurred. Conversion early after transplant Several studies investigated earlier conversion using an mTOR inhibitor. There was a scientific opinion after the CONVERT Study that substitution of mTOR inhibitor for CNI should occur after extensive allograft dysfunction for any benefit to be appreciated, hence the move toward earlier conversion. In 2009, Lebranchu and associates reported on the CONCEPT Study, in which 237 patients were enrolled to remain in the triple therapy with cyclosporine, MMF, and steroids or to switch cyclosporine to sirolimus by month three.55 All patients stopped steroids by month 8. The sirolimus group had a higher incidence of acute rejection episodes but did not reach statistical significance. Renal function was significantly better in the sirolimus group. Significantly more patients in the sirolimus group developed aphthous stomatitis (46% vs 5%; P ≤ .001), diarrhea (30% vs 9%; P < .001), and/or acne (19% vs 5%; P = .004).

In the 2010 SMART Study, antithymocyte globulin induction therapy was used, with maintenance therapy with cyclosporine, MMF, and steroids.56 Conversion from cyclosporine to sirolimus occurred early at 10 to 24 days. After 1 year, patients who had been switched to sirolimus had higher GFR with no change in BPAR rates. At 36 months, renal function remained higher in the sirolimus group; however, more patients discontinued therapy in the sirolimus group. In the 2011 Spare the Nephron Study, kidney transplant patients who had started on a CNI-based regimen (primarily tacrolimus) and MMF were converted to sirolimus at 30 to 180 days after transplant.57 The sirolimus trough levels were defined as 5 to 10 μg/L in combination with 1 to 1.5 g MMF and steroids. After 2 years, renal function in the CNI withdrawal group was significantly better, with similar BPAR and graft loss rates. Serious adverse events were reported in 53.2% of patients in the lowdose sirolimus group compared with 43.4% to 44.3% in the other study groups (P < .05 for all comparisons and for between-group comparisons with low-dose sirolimus).

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In the 2012 ZEUS Study, cyclosporine was withdrawn at 4.5 months and switched to everolimus.58 Glomerular filtration rate was higher in the everolimus group and the acute rejection rate was higher in the everolimus group, but it did not exert a deleterious effect on renal function by 3 years. A significant improvement in renal function with a mean estimated GFR difference of 5.3 mL/min/1.73 m2 in favor of everolimus was reported at 5 years.59 The increase in early mild acute rejection did not affect long-term graft function or survival.59 Results of the second randomization in the 2014 3C Study are still awaited; patients were randomized at 6 months to continue with tacrolimus or switch to sirolimus.42 Calcineurin inhibitor conversion to sirolimus due to adverse effects other than nephrotoxicity In a randomized controlled trial reported by Euvrard and associates, renal transplant recipients with a prior history of squamous cell carcinoma who had been receiving a CNI were randomized to continue with the CNI or to substitute sirolimus for the CNI.60 Patients on sirolimus had a significant delay in the median time to recurrent squamous cell carcinoma over a 2-year follow-up (7 vs 15 mo; P = .002). Potential Complications and Unknowns with Calcineurin Inhibitor-Sparing Strategies Although the best outcomes in renal transplant to date have been realized in the CNI era, there has been a drive for CNI-sparing strategies to reduce their intrinsic nephrotoxic effects. Mammalian target of rapamycin inhibitors certainly have a role to play in reducing nephrotoxicity, especially when used within the first year after transplant, but a significant number of patients do not tolerate them. Belatacept is a promising new agent, but longer follow-up is required before there is adequate evidence to demonstrate that it truly improves long-term patient outcomes. Removal of a CNI and especially cyclosporine from immunosuppressive regimens usually results in lower creatinine levels and probably merely reflects the removal of the CNI constrictor effect on the afferent arteriole. However, this does not necessarily affect preexisting histologic lesions that can continue to progress over time. The study by Nankivell and associates in 2003 is observational and does not establish CNI-related lesions as the

Exp Clin Transplant

immediate and predominant cause of progressive renal dysfunction.4 Snanoudj and associates later showed histologic lesions associated with CNI toxicity present in the kidneys of patients who were never exposed to CNIs.61 In another study by Nankivell and associates, lesions similar to CNI toxicity were less common in MMF-treated patients receiving cyclosporine and steroids versus those on azathioprine.62 In the study by Naesens and associates,63 in which biopsies were obtained from recipients taking tacrolimus, MMF, and steroids, the primary determinants of chronic pathologic lesions in the renal allograft were low tacrolimus exposure and subclinical acute rejections. These studies suggested an underlying immunologic process leading to both the histologic changes and graft failure. Several recent studies have investigated the causes of late graft failure, and a common theme seems to be that chronic antibody-mediated rejection is the predominant cause.70-73 In the 2010 DeKAF Study,66 patients were divided into 4 groups based on the presence of C4d and DSA as shown by allograft biopsies with new-onset dysfunction: no C4d and no DSA, only DSA, only C4d, and both C4d and DSA. Diagnosis of CNI nephrotoxicity was spread across all 4 of the groups, with no effect on the risk of late graft failure. Patients with a histologic diagnosis of CNI nephrotoxicity were significantly less likely to lose their allografts than those without such a diagnosis. Evidence of antibody-mediated injury (C4d and DSA) was common and present in 57% of patients with new-onset late kidney allograft dysfunction, and the risk of graft failure was significantly worse in the presence of C4d staining. Sellares and associates studied recipients who had an indication biopsy from 6 to 32 years after transplant,67 with results showing that the major causes of graft failure in 60 kidneys were antibodymediated rejection, probable antibody-mediated rejection, or mixed rejection. Nonadherence to immunosuppressive therapy was present in approximately 50% of patients; there was evidence of antibody-mediated rejection in 18 of 19 nonadherent patients who lost their graft. In the study by Nankivell and associates, DSA and C4d staining techniques were not available, making it difficult to interpret the link between pathologic lesions and graft failure.4 In 2012, Loupy and associates68 reviewed the effect of DSA on long-term graft survival, with


481

Table 5. Recommended Patient Course

a) b) c) d)

e)

Calcineurin inhibitor-based immunosuppression remains the cornerstone of our current immunosuppression protocols9 The current mainstay immunosuppression protocol is the one used in the ELITE-SYMPHONY study (ie, antibody induction, low-dose tacrolimus, MMF, and steroids)17 Avoid use of sirolimus de novo17,21,22,24 Some patients will benefit from conversion of tacrolimus to sirolimus at 6 months after transplant; these patients should: i. be low immunologic risk (exclude if loss of previous kidney transplant within 6 months not due to technical reasons and/or biopsy-proven acute rejection in the previous 1 month)55–57 ii. have estimated GFR > 40 mL/min and no significant proteinuria (ie, below 800 mg/day)53,54 Some patients will benefit from belatacept25,26,29; this should be considered in patients who have CNI nephrotoxicity, persistent acute tubular necrosis, or thrombotic microangiopathy and who are unable to: i. switch to sirolimus (early after transplant, acute tubular necrosis, thrombotic microangiopathy, proteinuria, severe renal dysfunction) ii. undertake CNI elimination (high risk of acute rejection [eg, < 6 months after transplant], previous acute rejection, high immunologic risk [eg, more than 1B and DR mismatch, donor-specific antibodies, highly sensitized; unable to tolerate high-dose MMF, eg, at least 1500 mg/d]

Abbreviations: CNI, calcineurin inhibitor; GFR, glomerular filtration rate; MMF, mycophenolate mofetil

antibody-mediated rejection now recognized as the major cause of graft failure, superseding the historic dogma of CNI toxicity as the main cause.66,67 Other findings have shown that 15% to 20% of patients on standard immunosuppressive agents including CNI, MMF, and prednisolone develop de novo DSA over a period of 5 years after transplant.69,70 Few CNIsparing studies have investigated de novo formation of DSA levels. Patients treated with belatacept had surprisingly low rates of DSA at 3 years (5%-6%)27 and 7 years (3.1% vs 11.6% for cyclosporine).71 However, no statistical comparison was performed and no distinction was made between pretransplant DSA and de novo DSA. In a ZEUS substudy, Liefeldt and associates found an increase of circulating DSAs and antibody-mediated rejection in the cohort of patients with conversion from cyclosporine to everolimus.72 Their results showed that 10.8% of patients on cyclosporine developed DSA after a median of 991 days, whereas 23% of patients who had been randomized to everolimus developed DSA after 551 days (log-rank test, P = .048). In 2015, De Sandes-Freitas and associates reported similar findings in patients converted to sirolimus.73 In contrast, there was no increase in de novo DSA in kidney and other organ transplant recipients when everolimus was used late after transplant in combination with low-dose CNI.74 It is possible that CNI-sparing strategies carry the risk of increasing acute or chronic antibody-mediated rejection; unfortunately, the vast majority of CNI-sparing studies have short-term follow-up and do not include the search for DSA. Conclusions Immunosuppressive minimization strategies try to address both the overall immunosuppressive effect common to all of these agents and toxicities unique

for the compound. Unfortunately, we do not have reliable tests to quantify the level of immunosuppression achieved cumulatively or even for single drugs. By reducing immunosuppression, we can reduce or eliminate these toxicities; however, any such strategy has the potential for underimmunosuppression and consequently acute or chronic rejection and potentially premature graft loss and death. There seems to have been a paradigm shift over the past 10 to 15 years with the appreciation that CNI nephrotoxicity is not the major cause of late graft failure. Studies have now shown that chronic immune injury mediated by DSAs may account for the majority of late graft losses. This raises yet other questions and the search for immunosuppressants able to control chronic humoral antidonor injury. In view of this, although some patients do benefit from CNI-sparing approaches, others may have late allograft loss from chronic and subacute immunemediated injury. One of the biggest challenges that we face is to be able to distinguish who benefits from this strategy and who will not. Until such time, we recommend the outlined course shown in Table 5. References 1. Zand MS. Immunosuppression and immune monitoring after

renal transplantation. Semin Dial. 18(6):511-519. 2. Fung JJ. Tacrolimus and transplantation: a decade in review. Transplantation. 2004;77(9 Suppl):S41-S43. 3. Meier-Kriesche HU, Schold JD, Srinivas TR, Kaplan B. Lack of Improvement in Renal Allograft Survival Despite a Marked Decrease in Acute Rejection Rates over the Most Recent Era. Am J Transplant. 2004;4(3):378-383. 4. Nankivell BJ, Borrows RJ, Fung CL-S, O’Connell PJ, Allen RDM, Chapman JR. The natural history of chronic allograft nephropathy. N Engl J Med. 2003;349(24):2326-2333. 5. Cortazar F, Diaz-Wong R, Roth D, Isakova T. Corticosteroid and calcineurin inhibitor sparing regimens in kidney transplantation. Nephrol Dial Transplant. 2013;28(11):2708-2716. 6. Halloran PF. Immunosuppressive Drugs for Kidney Transplantation. N Engl J Med. 2004;351(26):2715-2730.

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7. Bennett W, DeMattos A, Meyer M, Andoh T, Barry J. Chronic cyclosporine nephropathy in renal transplantation. Transpl Proc. 1996;28:2100-2103. 8. Dew MA, DiMartini AF, De Vito Dabbs A, et al. Rates and risk factors for nonadherence to the medical regimen after adult solid organ transplantation. Transplantation. 2007;83(7):858-873. 9. Webster AC, Woodroffe RC, Taylor RS, Chapman JR, Craig JC. Tacrolimus versus ciclosporin as primary immunosuppression for kidney transplant recipients: meta-analysis and meta-regression of randomised trial data. BMJ. 2005;331(7520):810. 10. Feutren G, Mihatsch M. Risk factors for cyclosporine-induced nephropathy in patients with autoimmune diseases. N Engl J Med. 1992;326:1654-1660. 11. Vincenti F, Ramos E, Brattstrom C, et al. Multicenter trial exploring calcineurin inhibitors avoidance in renal transplantation. Transplantation. 2001;71(7):1282-1287. 12. Grinyó JM, Gil-Vernet S, Cruzado JM, et al. Calcineurin inhibitor-free immunosuppression based on antithymocyte globulin and mycophenolate mofetil in cadaveric kidney transplantation: Results after 5 years. Transpl Int. 2003;16(11):820-827. 13. Groth CG, Bäckman L, Morales JM, et al. Sirolimus (rapamycin)based therapy in human renal transplantation: similar efficacy and different toxicity compared with cyclosporine. Sirolimus European Renal Transplant Study Group. Transplantation. 1999;67(7):10361042. 14. Kreis H, Cisterne JM, Land W, et al. Sirolimus in association with mycophenolate mofetil induction for the prevention of acute graft rejection in renal allograft recipients. Transplantation. 2000;69(7): 1252-1260. 15. Flechner SM, Kurian SM, Solez K, et al. De Novo kidney transplantation without use of calcineurin inhibitors preserves renal structure and function at two years. Am J Transplant. 2004;4 (11): 1776-1785. 16. Larson TS, Dean PG, Stegall MD, et al. Complete avoidance of calcineurin inhibitors in renal transplantation: A randomized trial comparing sirolimus and tacrolimus. Am J Transplant. 2006;6(3): 514522. 17. Ekberg H, Tedesco-Silva H, Demirbas A, et al. Reduced exposure to calcineurin inhibitors in renal transplantation. N Engl J Med. 2007; 357:2562-2575. 18. Büchler M, Caillard S, Barbier S, et al. Sirolimus versus cyclosporine in kidney recipients receiving thymoglobulin®, mycophenolate mofetil and a 6-month course of steroids. Am J Transplant. 2007;7(11):2522-2531. 19. Glotz D, Charpentier B, Abramovicz D, et al. Thymoglobulin induction and sirolimus versus tacrolimus in kidney transplant recipients receiving mycophenolate mofetil and steroids. Transplantation. 2010;89(12):1511-1517. 20. Flechner SM, Glyda M, Cockfield S, et al. The ORION study: Comparison of two sirolimus-based regimens versus tacrolimus and mycophenolate mofetil in renal allograft recipients. Am J Transplant. 2011;11(8):1633-1644. 21. Sharif a., Shabir S, Chand S, Cockwell P, Ball S, Borrows R. MetaAnalysis of Calcineurin-Inhibitor-Sparing Regimens in Kidney Transplantation. J Am Soc Nephrol. 2011;22(11):2107-2118. 22. Dickinson DM, Bryant PC, Williams MC, et al. Transplant data: sources, collection, and caveats. Am J Transplant. 2004;4 Suppl 9:13-26. 23. Isakova T, Xie H, Messinger S, et al. Inhibitors of mTOR and risks of allograft failure and mortality in kidney transplantation. Am J Transplant. 2013;13(1):100-110. 24. Pengel LHM, Liu LQ, Morris PJ. Do wound complications or lymphoceles occur more often in solid organ transplant recipients on mTOR inhibitors? A systematic review of randomized controlled trials. Transpl Int. 2011;24(12):1216-1230. 25. Vincenti F, Larsen C, Durrbach A, et al. Costimulation blockade with belatacept in renal transplantation. N Engl J Med. 2005; 353(8):770781. 26. Vincenti F, Charpentier B, Vanrenterghem Y, et al. A phase III study of belatacept-based immunosuppression regimens versus cyclosporine in renal transplant recipients (BENEFIT Study). Am J Transplant. 2010;10(3):535-546.

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27. Vincenti F, Larsen CP, Alberu J, et al. Three-year outcomes from BENEFIT, a randomized, active-controlled, parallel-group study in adult kidney transplant recipients. Am J Transplant. 2012;12(1):210217. 28. Vincenti F, Grinyo J, Rostaing L, et al. Belatacept-treated patients had better graft survival at 7-years post-transplant compared with cyclosporine-treated patients: final results from BENEFIT. Am J Transplant. 2015;15(S3). 29. Durrbach a., Pestana JM, Pearson T, et al. A phase III study of belatacept versus cyclosporine in kidney transplants from extended criteria donors (BENEFIT-EXT Study). Am J Transplant. 2010;10(3):547-557. 30. Pestana JOM, Grinyo JM, Vanrenterghem Y, et al. Three-year outcomes from BENEFIT-EXT: A phase III study of belatacept versus cyclosporine in recipients of extended criteria donor kidneys. Am J Transplant. 2012;12(3):630-639. 31. Florman S, Medina Pestana J, Rial M, et al. Final results from the BENEFIT-EXT Trial: A 7 year follow-up of belatacept treated patients. Am J Transplant. 2015;15(S3). 32. Rostaing L, Massari P, Garcia VD, et al. Switching from calcineurin inhibitor-based regimens to a belatacept-based regimen in renal transplant recipients: A randomized phase II study. Clin J Am Soc Nephrol. 2011;6(2):430-439. 33. Ekberg H, Grinyó J, Nashan B, et al. Cyclosporine sparing with mycophenolate mofetil, daclizumab and corticosteroids in renal allograft recipients: The CAESAR study. Am J Transplant. 2007;7(3): 560-570. 34. Gaston RS, Kaplan B, Shah T, et al. Fixed- or controlled-dose mycophenolate mofetil with standard- or reduced-dose calcineurin inhibitors: The opticept trial. Am J Transplant. 2009;9(7): 1607-1619. 35. Kaplan B, Qazi Y, Wellen JR. Strategies for the management of adverse events associated with mTOR inhibitors. Transplant Rev. 2014;28(3):126-133. 36. Barbari A, Maawad M, Kassouf HK, Kamel G. Mammalian Target of Rapamycin Inhibitors and Nephrotoxicity: Fact or Fiction. Exp Clin Transplant. 2015;(5):377-386. 37. Tedesco Silva H, Cibrik D, Johnston T, et al. Everolimus plus reduced-exposure CsA versus mycophenolic acid plus standardexposure CsA in renal-transplant recipients. Am J Transplant. 2010;10(6):1401-1413. 38. Cibrik D, Silva HT, Vathsala A, et al. Randomized trial of everolimusfacilitated calcineurin inhibitor minimization over 24 months in renal transplantation. Transplantation. 2013;95(7):933-942. 39. Watson CJE, Bradley JA, Friend PJ, et al. Alemtuzumab (CAMPATH 1H) induction therapy in cadaveric kidney transplantation Efficacy and safety at five years. Am J Transplant. 2005;5(6):13471353. 40. Ciancio G, Burke GW, Gaynor JJ, et al. A randomized trial of three renal transplant induction antibodies: early comparison of tacrolimus, mycophenolate mofetil, and steroid dosing, and newer immune-monitoring. Transplantation. 2005;80(4): 457-465. 41. Ciancio G, Burke GW, Gaynor JJ, et al. A randomized trial of thymoglobulin vs. alemtuzumab (with lower dose maintenance immunosuppression) vs. daclizumab in renal transplantation at 24 months of follow-up. Clin Transplant. 2008;22(2):200-210. 42. 3C Study Collaborative Group, Haynes R, Harden P, et al. Alemtuzumab-based induction treatment versus basiliximabbased induction treatment in kidney transplantation (the 3C Study): a randomised trial. Lancet. 2014;384(9955):1684-1690. 43. Kasiske BL, Chakkera HA, Louis TA, Ma JZ. A Meta-Analysis of Immunosuppression Withdrawal Trials in Renal Transplantation. J Am Soc Neph. 2000;11:1910-1917. 44. Gallagher MP, Hall B, Craig J, Berry G, Tiller DJ, Eris J. A randomized controlled trial of cyclosporine withdrawal in renal-transplant recipients: 15-year results. Transplantation. 2004;78(11):1653-1660. 45. Abramowicz D, Manas D, Lao M, et al. Cyclosporine withdrawal from a mycophenolate mofetil-containing immunosuppressive regimen in stable kidney transplant recipients: a randomized, controlled study. Transplantation. 2002;74(12):1725-1734.


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