Selection and use of immunosuppressive therapies after liver transplantation: current German practice

Transcription

1 Clin Transplant 2016: 30: DOI: /ctr John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Clinical Transplantation Review Article Selection and use of immunosuppressive therapies after liver transplantation: current German practice Herzer K, Strassburg CP, Braun F, Engelmann C, Guba M, Lehner F, Nadalin S, Pascher A, Scherer MN, Schnitzbauer AA, Zimmermann T, Nashan B, Sterneck M. Selection and use of immunosuppressive therapies after liver transplantation: current German practice. Abstract: In recent years, immunosuppression (IS) after liver transplantation (LT) has become increasingly diversified as the choice of agents has expanded and clinicians seek to optimize the balance of immunosuppressive potency with the risk of adverse events in individual patients. Calcineurin inhibitors (CNIs) are the primary agents used for patients undergoing liver transplantation. Other therapeutic agents like interleukin-2 receptor antagonists are not universally administered, but can be considered for the delay or reduction in CNI exposure. An early addition of mycophenolate mofetil (MMF) or the mtor inhibitor everolimus also allows for the reduction in the CNI dose. To reduce the risk of malignancy, in particular of skin tumors, as well as to prevent the deterioration of renal function, everolimus-based therapy may be advantageous. Apart from patients with autoimmune hepatitis, steroids are withdrawn within 3 6 months after transplantation. Overall, immunosuppression can only be standardized in a limited proportion of patients due to specific clinical requirements and risk factors. Future studies should attempt to refine accurate individualization of the immunosuppressive regimen in specific difficult-to-treat patient subpopulations. Kerstin Herzer a,b, Christian P. Strassburg c, Felix Braun d, Cornelius Engelmann e, Markus Guba f, Frank Lehner g, Silvio Nadalin h, Andreas Pascher i, Marcus N. Scherer j, Andreas A. Schnitzbauer k, Tim Zimmermann l,bj orn Nashan m and Martina Sterneck n a Department of General, Visceral and Transplantation Surgery, b Department of Gastroenterology and Hepatology, University Hospital Essen, University Duisburg-Essen, Essen, c Department for Gastroenterology and Hepatology, University Hospital Bonn, Bonn, d Department for Transplantation Surgery, University Hospital Kiel, Kiel, e Department for Gastroenterology and Rheumatology, Section Hepatology, University of Leipzig, Leipzig, f Department for Transplant Surgery, University Hospital Munich, Munich, g Department for Transplant Surgery, University Hospital Hannover, Hannover, h Department for General, Visceral and Transplant Surgery, University Hospital Tuebingen, Tuebingen, i Department of Visceral and Transplant Surgery, Charite-Universit atsmedizin Berlin, Berlin, j Department for General-, Visceral- and Transplant Surgery, University Hospital Regensburg, Regensburg, k Clinic for General and Visceral Surgery, Frankfurt University Hospitals, Goethe University Frankfurt/Main, Frankfurt/Main, l Department for Gastroenterology and Hepatology, University Hospital Mainz, Mainz, m Department for Hepatobiliary Surgery and Transplantation, University Hospital Hamburg, Hamburg, and n University Transplant CenterUniversity Hospital Hamburg, Hamburg, Germany Key words: antiproliferative drugs calcineurin inhibitors everolimus immunosuppression induction liver transplantation mycophenolate mofetil steroids 487

2 Herzer et al. Corresponding author: Kerstin Herzer MD, Professor of Medicine, Department of General, Visceral and Transplantation Surgery and Department of Gastroenterology and Hepatology, University Hospital Essen, University Duisburg-Essen, Hufelandstrasse 55, Essen, Germany. Tel.: ; fax: ; Accepted for publication 1 February 2016 The calcineurin inhibitors (CNI), cyclosporine (CsA), and tacrolimus remain the standard of care for immunosuppressive therapy in liver transplantation (1), but their long-term use can lead to severe adverse effects, which include nephrotoxicity, neurotoxicity, and an increased cardiovascular risk. Combination therapy with inosine monophosphate dehydrogenase (IMPDH) inhibitors or the mammalian target of rapamycin (mtor) inhibitor everolimus has emerged as a strategy to reduce CNI-related adverse events. However, regimens vary widely and accepted general recommendations for immunosuppressive protocols in this setting are lacking. Furthermore, induction therapy, which is advised in kidney transplant recipients, is a matter of debate in liver transplantation. In March 2015, a group of 13 German transplant surgeons and hepatologists met to discuss the current aspects of practice for immunosuppressive strategies after liver transplantation. Information on immunosuppressive strategies at the authors centers was collected to provide an insight into current practice at German transplant units. Data were provided from eleven centers. The key points from the panel board discussion are reflected below and discussed based on the available literature, clinical trial data, and personal experience. Induction therapy The use of induction therapy is currently diverse. Fewer than half the authors centers use induction therapy regularly, typically antithymocyte globulin (ATG) or basiliximab, while the other centers never use induction. Published evidence in kidney transplantation is based on the KDIGO guidelines that recommend IL-2 receptor antibody induction as standard of care and the use of lymphocyte depleting agents (e.g., ATG) in highly immunized patients (2). The role of induction therapy in liver transplantation is less clear-cut (3). In the United States, only 10% of de novo liver transplant patients receive induction therapy (4), although this is becoming more frequent in order to reduce the risk of rejection or, more often, to support CNI minimization or steroid avoidance (5). Of the available induction agents, such as rabbit ATG (Thymoglobulin Ò and ATG-Fresenius), the IL-2 receptor antibody basiliximab, and alemtuzumab (3, 4), only ATG-Fresenius is licensed for use as induction therapy in liver transplant patients. Induction therapy to reduce risk of rejection A meta-analysis of 12 randomized controlled trials, involving 3251 liver transplant recipients, has investigated the effect of induction therapy on biopsy-proven acute rejection (BPAR) episodes (6). Induction with IL-2 receptor antagonists resulted in a significant reduction in BPAR (23.1% vs. 28.1% without induction; relative risk [RR] 0.82, 95% CI [ ]; p = 0.04), although there was no significant effect on mortality or graft loss (Table 1). Furthermore, a single-center, retrospective analysis in basiliximab-treated patients compared to historical controls without induction reported acute rejection in only 7% of patients given basiliximab vs. 34% in the control group (p = 0.001) (9). One double-blind, multicenter, randomized, controlled trial showed a significant reduction in early rejection among 381 liver transplant recipients when IL-2 receptor antagonist induction was added to a standard immunosuppressive regimen comprising CsA and steroids (10). There was a significant difference in the rate of early BPAR across the overall population of 381 patients during the first six wk post-transplant, which was maintained in the HCV-negative subpopulation at month 6 (n = 248; 33.1% vs. 47.6%; p = 0.034). Although no longer commercially available, an abbreviated two-dose regimen of daclizumab also appeared effective when added to a standard regimen of CNI and steroids in a single-arm study (11). 488

3 Immunosuppression after liver transplant Table 1. Summary of meta-analyses on immunosuppressive regimens after liver transplantation Meta-analysis Study Design N Strategy Intervention Groups Patient survival (%) Graft survival Acute rejection (%) Wang et al., 2010 (6) McAlister et al., 2006 (7) Segev et al., 2008 (8) RCT 12 Induction Daclizumab or basiliximab Ind + CNI MMF+/S vs. CNI MMF+/S RCT 16 Standard TAC or CsA TAC Aza/MMF+S vs. CsA Aza/MMF+S RCT 19 Steroidsparing TAC or CsA with or without steroids Steroid-free vs. steroid-based Mortality: RR % CI [ ] p = 0.33 Mortality: 13.4 vs RR % CI [ ] p = 0.03 Mortality: RR % CI [ ] Graft loss: RR % CI [ ] p = 0.64 Graft loss: 17.0 vs RR % CI [ ] p = Graft loss: RR % CI [ ] 23.1 vs RR % CI [ ] p = vs RR % CI [ ] p < RR % CI [ ] without steroid replacement RR % CI [ ] With steroid replacement Aza, azathioprine; CI, confidence interval; CNI, calcineurin inhibitor; CsA, cyclosporine; Ind, induction; MMF, mycophenolate mofetil; RCT, randomized, controlled trial; RR, relative risk; S, steroids; TAC, tacrolimus. Induction therapy to support CNI delay or reduction Three randomized trials have demonstrated that the IL-2 receptor antagonist daclizumab permits CNI delay or reduction in liver transplant patients without increasing the risk of rejection (Table 2) (12 14). Equivalent studies have not been performed for basiliximab, the only commercially available IL-2 receptor antagonist, but there is no reason to expect substantially different results. Four retrospective analyses have evaluated the effect of ATG (Thymoglobulin Ò ) induction on rejection prophylaxis in delayed-cni regimens (24 27). Two studies showed significantly lower rejection rates in patients with CNI delay after ATG induction compared to immediate CNI without induction therapy (24, 25). Soliman et al. (24) compared introduction of either CsA or tacrolimus at day 3 post-transplant after ATG induction vs. standard CNI without induction in 391 patients, all of whom received steroids. After one yr, the rejection rate was 14.5% vs. 31.8% (p < 0.001) and estimated GFR was significantly higher in the delayed-cni group (mean 81 ml/min vs. 75 ml/ min, p = 0.020). Horton et al. (25) analyzed data from 443 patients over a 10-yr observation period and found rejection rates of 34.2% for ATG induction with CNI delay vs. 66.6% for standard CNI therapy (p 0.01), using either CsA or tacrolimus. Background immunosuppression consisted of mycophenolate mofetil (MMF) or azathioprine plus steroids. A third retrospective study, of 52 patients, analyzed tacrolimus minimization with ATG induction and observed no difference in rejection rates vs. a standard tacrolimus regimen (26). Finally, a retrospective analysis of 198 patients found no significant difference in the incidence of rejection episodes between ATG induction and delayed CsA or tacrolimus vs. a conventional regimen (16% vs. 26%, ), but mean estimated GFR was higher in the ATG/CNI delayed group (57.4 ml/min/1.73 m 2 vs ml/ min/1.73 m 2,p< 0.001) (27). Although controlled trials are lacking, the consistency of these results suggests that ATG (Thymoglobulin Ò ) induction can support delayed-cni initiation without loss of immunosuppressive efficacy and may preserve renal function. The multicenter, randomized, controlled DIA- MOND trial assessed the effect of different prolonged-release tacrolimus regimens with or without basiliximab induction on renal function as well as on transplant rejection 24 wk post-transplant. The results of the study showed that prolonged-release tacrolimus at a reduced dose ( mg/kg/ d) plus basiliximab given at days 0 and 4 was associated with a significantly reduced number of patients affected by BPAR compared to a regimen with a higher dose of prolonged-release tacrolimus (0.2 mg/kg/d) without basiliximab (12.1% vs. 17.9%, respectively; p = 0.016) immediately posttransplant and compared to a regimen of prolonged-release tacrolimus (0.2 mg/kg/d) delayed until day 5 (16.8%; p = 0.039). All patients 489

4 Herzer et al. Table 2. Summary of randomized controlled studies of induction for CNI delay or minimization and steroid avoidance in liver transplantation Study Design N Duration Strategy Induction Groups Patient survival (%) Graft survival (%) Acute rejection (%) Calmus et al., 2010 (12) Neuberger et al., 2009 (13) Yoshida et al., 2005 (14) Benitez et al., 2010 (15) Trunecka et al., 2015 (16) Otero et al., 2009 (17) Klintmalm et al., 2007 (18) Boillot et al., 2005 (19) Eason et al., 2003 (20) Llado et al., 2008 (21) Filipponi et al., 2004 (22) Pageaux et al., 2004 (23) randomized single-center open-label Single-center Double-blind Double-blind m CNI delay (pod 5) Dac Ind+del TAC+MMF+S TAC+MMF+S m CNI delay (pod 5) and minimization m CNI delay (pod 5) and minimization m CNI minimization/ withdrawal from month 3 Dac Dac ATG Ind+red/del TAC+MMF+S red TAC+MMF+S TAC+S Ind+red/del TAC+MMF+S TAC+MMF+S Ind+red/free TAC TAC+S wk CNI minimization Bas TAC+MMF red TAC+MMF del TAC+MMF m Steroid-sparing Dac Ind+TAC+MMF TAC+S m Steroid-sparing Dac Ind+TAC+MMF TAC+MMF+S TAC+S m Steroid-sparing Dac Ind+TAC TAC+S m (mean) Steroid-sparing ATG Ind+TAC+MMF TAC+MMF+S m Steroid-sparing Bas Ind+CsA Ind+CsA+S m Steroid-sparing Bas Ind+ CsA+Aza Ind+CsA+Aza+S m Steroid-sparing Bas Ind+CsA Ind+CsA+S p = % 12.1% p = % p = p = n/a p = p = 0.03 ATG, antithymocyte globulin; Aza, azathioprine; Bas, basiliximab; CsA, cyclosporine A; Dac, daclizumab; Del, delayed; Ind, induction; m, months; MMF, mycophenolate mofetil; n/a, not available; ns, not significant; pod, postoperative day; red, reduced; S, steroids; TAC, tacrolimus. received MMF plus a corticosteroid bolus. Additionally, the reduced and the delayed dose of tacrolimus were associated with a higher estimated GFR compared to the immediate post-transplant tacrolimus regimen (p = and p = 0.047, respectively) (16). CNI withdrawal after ATG induction, however, does not appear to be beneficial. In a small pilot study, 37 patients were randomized to standarddose tacrolimus plus steroids or ATG induction (ATG-Fresenius) plus reduced-dose tacrolimus monotherapy with attempted tacrolimus withdrawal in rejection-free patients with stable liver function at three months post-transplant (15). Acute rejection was more frequent in the ATG induction/tacrolimus-withdrawal group at one yr compared to the standard arm (66.7% vs. 31.2%, p = 0.033). Induction therapy to support steroid avoidance Several single-center and multicenter studies have evaluated whether induction therapy permits steroid avoidance after liver transplantation without an increased risk of rejection (Table 2) (17 23). Three open-label, multicenter studies investigated induction with the IL-2 receptor antagonist daclizumab. In a trial of 157 patients by Otero et al. 490

5 Immunosuppression after liver transplant (17), the rejection rate was significantly lower in steroid-free patients compared to the cohort without induction who received standard steroids (11.5% vs. 26.6%, p = 0.017). All patients received tacrolimus, but MMF was given only in the daclizumab arm, which renders the interpretation of these results more complicated. Similarly, in a trial of 295 patients, Klintmalm et al. (18) included MMF in the steroid-free regimen for patients given daclizumab with tacrolimus, but not in the induction-free control arm (tacrolimus with steroids); at two yr post-transplant, rates of rejection were similar in both groups. The largest study, involving 698 patients, directly compared daclizumab with tacrolimus vs. tacrolimus with steroids and found that steroid-free immunosuppression in the presence of an IL-2 receptor antagonist was not associated with an increase in rejection (26.5% vs. 25.4% for tacrolimus/steroids, p = 0.727) (19). Similar results were obtained in a single-center study comparing ATG induction (Thymoglobulin Ò ), tacrolimus, and MMF without steroids vs. standard immunosuppression with tacrolimus, MMF, and steroids (20). However, a double-blind trial of basiliximab induction with CsA, in which 140 patients were randomized to steroid withdrawal at day 14 or to standard steroids, found an increased rate of rejection in the steroid-withdrawal group (39.4% vs. 24.3%, p = 0.04) (22). In contrast, another randomized trial, in 89 hepatitis C-positive patients, found similar rates of rejection at two yr posttransplant with or without steroids in combination with basiliximab induction and CsA (21). Conclusions There is robust evidence to support the use of induction therapy in specific settings after liver transplantation. Induction therapy is useful for the reduction of CNI exposure and prevention of acute rejection in liver transplant patients. Induction-facilitated CNI reduction needs to be evaluated on an individual patient basis, for example, in patients with renal impairment. Induction therapy with an IL-2 receptor antagonist in combination with tacrolimus can facilitate steroid avoidance in liver transplant patients. Calcineurin inhibitors All the authors centers use CNI-based immunosuppressive regimens in all patients in the de novo situation, with all but one center preferring to use tacrolimus instead of CsA as initial therapy in the majority of patients. There was agreement from all centers regarding the use of CsA as the second-line CNI in specific indications. In the maintenance setting, CsA is used in about 10 40% of patients at the German centers. In general, tacrolimus has become the preferred CNI in de novo liver transplant patients based on the evidence of lower rates of renal deterioration, mortality, graft loss, and incidence of acute rejection (7, 28). One reason for a higher efficacy under tacrolimus may possibly be related to relatively poor early absorption of CsA (29). Additionally, once-daily extended-release tacrolimus is a promising new development. Several studies have suggested that conversion from twiceto once-daily tacrolimus is safe in stable liver transplant recipients and improves compliance (30, 31). In addition, a retrospective analysis of the European Liver Transplant Registry (ELTR) that included 4367 patients on twice-daily dosing of conventional tacrolimus and 528 on the once-daily extended-release formulation (32), indicated that the twice-daily regimen was associated with a higher risk for graft loss (risk ratio: 1.81; p = 0.001) and death (risk ratio: 1.72; p = 0.004) over long-term follow-up (up to five yr). It was proposed that this may be due to reduced variability in tacrolimus trough levels and/or improved treatment compliance. However, these registry data have to interpreted with caution. Data on treatment of de novo liver transplant patients with the once-daily extended-release formulation are still scarce, although the available results have indicated high efficacy and safety (33). Tacrolimus is associated with an increased risk for developing post-transplantation diabetes mellitus vs. CsA. One meta-analysis of randomized controlled trials found the relative risk for de novo diabetes to be 1.38 with tacrolimus vs. CsA (95% confidence interval [CI] ) (7), and a 12- month pilot study observed resolution of newonset diabetes in 36% of liver transplant patients switched from tacrolimus to CsA (34). Neurotoxicity is also more frequent under tacrolimus (35), but other CNI-related adverse effects, however, may be milder under tacrolimus. In a prospective study of 80 patients experiencing renal toxicity and nonrenal side effects such as hypertension under CsA, conversion to tacrolimus led to a decrease in mean arterial blood pressure (from to 95.9 mmhg, p < 0.001) and in total cholesterol (from to mg/dl, p < 0.001) (35). Both agents, however, are associated with acute and chronic nephrotoxicity caused by afferent arteriolar vasoconstriction and subsequent renal hypoxia 491

6 Herzer et al. (36, 37). Results from large-scale retrospective analyses in up to 1100 patients who received CNI therapy have shown the incidence of end-stage renal disease after yr to be between 4% and 9% (38 40), with 43% of patients experiencing renal dysfunction after 10 yr (41). Further CNIrelated complications can include de novo malignancies (42) and cardiovascular and metabolic disorders (43). Calcineurin inhibitor-sparing regimens CNI minimization strategies include delayed-cni introduction, early dose minimization, and, rarely, CNI withdrawal. As discussed above, induction therapy supports delayed-cni therapy with no loss of immunosuppressive potency and can improve post-transplant renal function (13, 24, 27). Early minimization of CNI by additional application of antiproliferative agents (see Antiproliferative agents ) offers an effective treatment option in de novo liver transplant patients and is advantageous for preserving renal function (44, 45). In several studies, CNI-free regimens have been associated with a higher frequency of rejection rates vs. CNI-containing protocols and are thus not generally recommended (45 48). However, a recent pilot study indicated that CNI-free immunosuppressive therapy with basiliximab, steroids, MMF, and sirolimus can be safely performed in selected patients (49). Also, the PROTECT (50) and the RESCUE (51) studies indicated that CNIfree immunosuppression is feasible in de novo and long-term liver transplant patients without an increased risk of rejection, if the everolimus dose is adjusted to achieve a target level between 6 and 8 ng/ml, and if CNI is weaned very gradually. Usually, CNI therapy is only discontinued when there is a clinical imperative, most commonly in patients with post-transplant lymphoproliferative disease (PTLD). Conclusions CNI therapy is still the cornerstone of immunosuppression after liver transplantation, with tacrolimus achieving better patient and graft survival than CsA. The side effect profile of CNIs necessitates protocols, which balance the prevention of rejection and the avoidance of serious complications. Regimens should be tailored to the individual needs of the patient, taking into account comorbidities, side effects, and efficacy. Delayed-CNI therapy with IL-2 receptor antagonist induction and reduced CNI protocols that include everolimus or MMF appear to reduce side effects, in particular the loss of renal function, without increasing the risk for rejection. Despite a potential for renoprotection, CNIfree regimens cannot be generally recommended. Antiproliferative agents Antiproliferative immunosuppressive therapies (MMF and azathioprine) block the cell cycle via inhibition of purine synthesis. This restricts the proliferation of various cell types, particularly T- lymphocytes. MMF and azathioprine are approved for use in liver transplant recipients. MMF is only licensed for use in combination with CsA, although it is commonly used in combination with tacrolimus. In current German practice, between 50% and 100% of de novo patients receive an antiproliferative medication within the first four wk post-transplant. MMF is most commonly used by all centers, while azathioprine is reserved for the few patients with additional autoimmune diseases. Mycophenolate mofetil Generally, MMF is employed in liver transplantation to increase immunosuppressive potency or to enable CNI reduction. De novo MMF is effective when CNI dose reductions are planned. In the RESPECT study (n = 525), rejection rates were comparable using low-dose tacrolimus plus MMF or standard tacrolimus without MMF (29.2% and 27.6%, ), and renal function showed no benefit in the low-dose tacrolimus group in this trial (13). In another multicenter study of 195 de novo liver transplant patients, the group randomized to standard tacrolimus without MMF had a higher rate of acute rejection at week 48 than that given low-dose tacrolimus with MMF (46% vs. 30%; hazard ratio [HR] 0.59; 95% CI: [ ]; p = 0.024), and importantly, estimated GFR was higher (mean 90 ml/min vs. 78 ml/min, p = 0.018) in the MMF/tacrolimus minimization cohort (44). However, a renal advantage has not been confirmed elsewhere. An earlier, smaller randomized trial in 60 patients found no difference in rejection rates using standard-dose tacrolimus vs. reduced-dose tacrolimus, both with concomitant MMF, but also no significant improvement in renal function under the CNI minimization regimen (52). Introduction of MMF also allows for CNI reduction in the maintenance setting, for example, in response to renal impairment or the develop- 492

7 Immunosuppression after liver transplant ment of cardiovascular risk factors. However, there are only small pilot studies indicating a benefit in long-term liver transplant patients. In a small randomized study, liver transplant recipients on maintenance immunosuppression with CNIrelated renal dysfunction received either MMF with reduced CNI (n = 50), or continued their maintenance CNI dose (n = 25) (53). At 12 months postrandomization, mean estimated GFR increased significantly from 38.8 to 47.0 ml/ min/1.73 m 2 in the MMF group (p < 0.001) but remained largely unchanged in the control arm (40.4 vs ml/min/1.73 m 2 in the control group: the between-group difference was statistically significant [p = 0.01]). Furthermore, blood pressure decreased only in the CNI reduction group (p = 0.001) (53). Another small multicenter study of 56 maintenance therapy patients with CNI-related renal dysfunction randomized patients to start MMF with up to 50% reduction in their CNI dose or to receive no MMF, but with the CNI dose reduced no more than 25% (54). Creatinine clearance increased significantly in the MMF/CNI group during the 12-month study (from 42.6 to 51.7 ml/min) but not in the standard CNI group (42.8 ml/min at baseline, 44.8 ml/min at month 12); again, the intergroup difference was significant (p = 0.04). Acute rejection did not occur in any MMF-treated patients, but developed in one control patient (54). The appropriate MMF dose when given in combination with CNI remains a matter for discussion. Dosing decisions should take into account the different effects of concomitant CsA or tacrolimus described in kidney transplant recipients. CsA inhibits enterohepatic recirculation of mycophenolic acid glucuronide, the key MMF metabolite (55), an effect which can reduce MMF exposure by 30 40% compared to concomitant tacrolimus (56, 57). Therefore, the full MMF dose of 2 g/d may not be necessary in tacrolimustreated patients. Conversion to CNI-free therapy with MMF, and particularly the use of MMF monotherapy, can increase rejection rates. In a randomized study, maintenance patients receiving CNI monotherapy or CNI with steroids were switched to MMF monotherapy or continued their CNI-based regimen (58). After a follow-up of five yr, rejection occurred in 11.1% of the MMF group (n = 72) compared to 2.9% in the standard CNI group (n = 70) (p = 0.055) and CNI was reintroduced in over 30% of MMF-treated patients, mostly in response to rejection. Similar results were obtained from a small randomized study of 28 patients, with 21% of patients experiencing BPAR within six months of switching to MMF (46). One randomized trial of liver transplant recipients switched to MMF monotherapy due to CNI-related toxicity had to be prematurely terminated because of increased rejection rates in the experimental arm (59). Recently, the Spare-the-Nephron trial reported a higher rate of rejection in maintenance liver transplant patients given a CNI-free regimen composed of MMF and the mtor inhibitor sirolimus compared with a standard CNI regimen (12.2% vs. 4.1% at one yr, p = 0.02) (46). The CNI-free group showed a greater improvement in renal function from baseline to year 1 vs. controls (mean increase in estimated GFR 19.7% vs. 1.2%, p = 0.001) (48). However, there are also reports of successful outcomes following conversion to MMF in which the increase in risk of rejection after CNI withdrawal was less marked, and renal insufficiency improved (58). Interestingly, one retrospective analysis of 47 liver transplant patients with steroid-resistant acute rejection found that within two wk after the addition of MMF to the immunosuppressive regimen, liver function normalized in 38 cases (60), indicating a role for MMF as rescue therapy in this setting. One caveat to the introduction of MMF is the potential for an increased risk of cytomegalovirus (CMV) infection and CMV-related disease (61). If organ manifestations of CMV-related disease develop, MMF should be discontinued. Other MMF-related side effects, notably hematological adverse events such as leukopenia and anemia, also need to be taken into account and closely monitored. Conclusions MMF is not formally approved but, however, recommended in de novo and maintenance patients to support reduced-cni protocols. Proof of a renal benefit in liver transplantation awaits confirmation. MMF can be used to increase the level of immunosuppression in cases of steroid-resistant rejection. Combination therapy with MMF and an mtor inhibitor is not standard practice, but may represent an option in individual cases. CNI-free therapy with MMF monotherapy, or MMF plus steroids, carries a higher risk of rejection episodes and should only be used in special clinical situations. MMF maybe discontinued in the event of CMV infections, particularly if organ manifestations are present. 493

8 Herzer et al. Azathioprine Once a key component of post-transplant regimens (62), azathioprine is now only rarely used in transplant recipients, because MMF and mtor inhibitors are characterized by more potent antiproliferative effects and have been shown to be useful in CNI-sparing regimens. Azathioprine is now largely restricted to specific indications such as recurrent or de novo autoimmune hepatitis (AIH) (63), or chronic inflammatory bowel disease (IBD) (64). Of note, a US registry analysis of solid organ transplant recipients, including liver transplants, showed that azathioprine may be associated with an increased risk for development of hepatocellular carcinoma (HCC) (RR 1.3; 95% CI [ ]) and cholangiocarcinoma (RR 2.0; 95% CI [ ]) (65). However, US registry data also indicated a reduced risk for Burkitt-lymphoma (RR 0.56, 95%-CI [ ]) under azathioprine (66), although the methodology was suboptimal and this observation has not been confirmed elsewhere. Conclusions Azathioprine may be beneficial in specific situations such as the management of IBD or AIH, but valid evidence is lacking. Patients already receiving azathioprine for these indications should be continued on this regimen. mtor Inhibitors Everolimus is currently the only mtor inhibitor approved for use in liver transplant patients. It is licensed for use in combination with tacrolimus in this setting but in common practice is also used in combination with CsA. All the authors centers use everolimus in 10 60% of their patients as maintenance therapy, in combination with tacrolimus. Immediate use of everolimus after liver transplantation is not yet established and mostly applied in ongoing clinical trials such as HEPHAISTOS or by named-patient approval only. De novo patients An early study of everolimus administered at a fixed dose ( mg d) with standard CsA confirmed it to have an acceptable safety and tolerability profile in de novo liver transplant recipients (67). Subsequent trials have focused on a potential renal benefit in this setting. Clear evidence regarding a nephroprotective effect for everolimus with or without low-dose CNI therapy in de novo liver transplantation is available from multi- and singlecenter prospective randomized studies (Table 3) and in addition is supported by several retrospective single-center analyses (71). The H2304 study compared everolimus with tacrolimus minimization or withdrawal vs. a standard tacrolimus regimen in 719 de novo liver transplant patients (45, 68). Everolimus (targeting a trough concentration of 3 8 ng/ml) was introduced at day 30 posttransplant. The rate of BPAR was significantly lower with everolimus and reduced tacrolimus compared to the standard tacrolimus regimen (6.1% vs. 13.3%, p = 0.01) but tacrolimus withdrawal resulted in a higher incidence of acute rejection (26.4%). Preservation of renal function was significantly better in the everolimus/reduced tacrolimus group, with mean estimated GFR of 81.1, 80.9, and 74.7 ml/min/1.73 m 2 at randomization, year 1 and 2, respectively, compared to 78.0, 70.3, and 67.8 ml/min/1.73 m 2 in the control arm. Another European randomized multicenter study (PROTECT) in 203 de novo patients analyzed renal end points following CNI withdrawal after conversion to everolimus between days 30 and 60 (50). In contrast to the H2304 study, the incidence of acute rejections was comparable between the CNI-free/everolimus and the standard CNI group (17.7% vs. 15.3%, p = 0.7). At month 12 post-transplant, mean estimated GFR based on the Modification of Diet in Renal Disease (MDRD) formula was significantly higher in the CNI-free everolimus group, a difference that was preserved at three yr of follow-up (69). Other randomized trials have also demonstrated a renal benefit after early switch to everolimus (70) or sirolimus (48), although BPAR was more frequent after switch to sirolimus in the Spare-the-Nephron study (48). Notably, none of these studies included patients with advanced renal dysfunction and a nephroprotective effect of mtor inhibitor-facilitated CNI reduction/withdrawal has yet to be demonstrated in this situation. Whether mtor inhibition prolongs recurrencefree survival in patients transplanted for HCC remains unconfirmed, but several single-center retrospective analyses indicate a lower rate of recurrence. In one study, HCC recurrence-free survival at five yr was 78.8% in patients treated with sirolimus (n = 45) compared to 54% with standard CNI therapy (n = 52) (RR 0.622; 95% CI [0.385; 0.954; p = 0.03) (72). Other small studies have reported similar results (73, 74). The results of a prospective multicenter study of sirolimus in patients with HCC are anticipated (75). 494

9 Immunosuppression after liver transplant Table 3. Summary of randomized controlled de novo and maintenance mtor inhibitor studies Study Design and duration N Groups EVR introduction and dose CNI dose Patient survival/ Death (%) Graft survival/ Graft loss Acute rejection (%) GFR ml/min/1.73 m 2 mean SD De novo De Simone et al., 2012 (68) Saliba et al., 2013 (45) H2304 Fischer et al., (50) Sterneck et al., (69) PROTECT Masetti et al., 2010 (70) 24 month 12 months 12 months Single-center 719 Red TAC C ng/ml (introduction day 30) C ng/ml Death 5.2 TAC control C ng/ml until month 4 and C ng/ml thereafter TAC withdrawal C ng/ml (introduction day 30) 203 EVR C ng/ml with TAC C ng/ml with CsA (introduction day 30 60) Graft loss 3.9 BPAR 6.1 RND/year 1/year Elimination at month Tapering to 70%, complete withdrawal after 2 months p = (red TAC vs. control) Death 4.2 p = (red TAC vs. control) Graft loss 2.1 p = 0.01 (red TAC vs. control) BPAR 17.7 p < year 1 p = year 2 (red TAC vs. control; MDRD) RND/year 1/year Control Continue initial CNI dose p = 1.0 p = 1.0 p = 0.7 p = year 1 78 CNI withdrawal C ng/ml until day 30, 8 12 ng/ml until the end of month 6 and 6 10 ng/ml thereafter (introduction day 10) CsA C ng/ml until day 30, then ng/ml until the end of month 6 and ng/ml thereafter Patient survival 90.4 Not reported BPAR 5.7 p = year 3 (MDRD) Extension study population N = 81 RND/12 months p = 0.87 p < (MDRD) 495

10 Herzer et al. Table 3. Continued Study Design and duration N Groups EVR introduction and dose CNI dose Patient survival/ Death (%) Graft survival/ Graft loss Acute rejection (%) GFR ml/min/1.73 m 2 mean SD Maintenance De Simone et al., 2009 (51) RESCUE 6 months Watson et al., 2007 (47) 12 months Single-center Teperman et al., 2013 (48) Spare-the-Nephron 12 months 145 EVR C ng/ml (red CNI) C ng/ml (CNI-free) 50% CNI reduction CNI withdrawal Control Continue previous CNI regimen Death SRL C ng/ml CNI withdrawal Death 0/13 CNI Continue previous CNI regimen 294 SRL C ng/ml CNI withdrawal Death 2.7 CNI Continue previous CNI regimen Graft loss 0 BPAR 1.4% RND/6 months (CrCl) % p = Not reported 2/13 (15.4%) Median baseline 49.8 Mean change month /17 0/17 Median baseline Graft loss (PP) 3.4 BPAR Mean change month p = 0.07 RND/12 months (CrCl) p = 0.1 p = 0.04 p = 0.02 p < (change from baseline) BPAR, biopsy-proven acute rejection; CNI, calcineurin inhibitor; CrCl, creatinine clearance; CsA, cyclosporine A; EVR, everolimus; GFR, glomerular filtration rate; MDRD, Modification of Diet in Renal Disease; ns, not significant; PP, per protocol; red, reduced; RND, randomization; SD, standard deviation; SRL, sirolimus; TAC, tacrolimus. 496

11 Immunosuppression after liver transplant Conclusions Everolimus with low-dose CNI is a safe, nephroprotective regimen for de novo liver transplant patients. Early switch to a CNI-free everolimus-based regimen carries a higher risk for rejection Further long-term studies are needed to evaluate the benefit of an mtor inhibitor-based immunosuppressive regimen with respect to reducing the risk of HCC recurrence. mtor inhibition in maintenance patients The main indications for introduction of everolimus in the maintenance setting are renal insufficiency, CNI-induced neurotoxicity, de novo or recurring malignancy, and chronic or refractory rejection (76). However, no prospective clinical study has to date demonstrated an improvement in kidney function following a switch from CNI to everolimus in long-term liver transplant patients with established renal dysfunction. The six-month multicenter, randomized RESCUE study found no difference in creatinine clearance in maintenance patients switched from a CNI-based immunosuppression to everolimus in a CNI-free or reduced- CNI regimen, although reduction in CNI exposure in the control arm, and the wide variations in time since transplantation complicated the data analysis and interpretation (51). Encouragingly, CNI elimination was feasible without any increase in rejection rates (1.4% in both groups at month 6). Patients with malignancies may also benefit from switch to an mtor inhibitor-based immunosuppressive regimen (77, 78). Several case reports of renal transplant patients have reported regression of Kaposi s sarcoma after conversion to sirolimus (78 81), but high-quality studies have not been performed. A retrospective study of 83 liver transplant patients with de novo solid tumors reported improved survival after patients were converted to everolimus: five-yr survival was 35.2% in the everolimus group (n = 39) compared to 19.4% in controls (n = 44; p = 0.003) (82). Single-center retrospective studies have also suggested that mtor inhibitors may improve outcomes in recurrent HCC, with or without concomitant sorafenib therapy (83, 84), but confirmatory evidence is yet to be reported. Conclusions There is no published evidence yet that the conversion to everolimus improves renal function in long-term patients. However, this may be considered as rescue therapy. CNI should be slowly weaned to prevent rejection episodes. Switch to an mtor inhibitor should be considered in patients with recurring malignant skin tumors, especially in Kaposi s sarcoma. At present, there is insufficient evidence for improved survival in other solid tumors to justify conversion to everolimus. Relative contraindications, such as proteinuria, unresponsive hyperlipidemia, leukopenia, or pancytopenia, must be taken into account and the resulting risk:benefit ratio critically assessed. Steroid therapy In all but one center, almost all de novo liver transplant patients initially receive steroids, but steroids are usually withdrawn within 3 6 months. Longterm triple therapy including steroids is reserved for a small proportion of patients and is not common German practice. Use of modern immunosuppressive agents can generally obviate the long-term need for steroids after liver transplantation. According to a metaanalysis of 19 randomized trials published in 2008, complete steroid avoidance without a compensatory addition of other immunosuppressive agents leads to a higher risk of BPAR (RR 1.31; 95% CI [1.04; 1.64]; p = 0.02) (8), but some more recent studies have contradicted this. A randomized, double-blind study in 156 patients found that steroidfree immunosuppression did not affect rejection rates compared to a steroid-containing regimen in patients receiving tacrolimus monotherapy (13.2% vs. 8.2%; p = 0.38) (85). Another double-blind study, in 110 patients treated with tacrolimus, has also reported that rejection was similar with or without steroids (34.2% vs. 48.2%; p = 0.116) after one yr (86). Of note, a large randomized trial of 602 patients showed similar rates of BPAR using a steroid-free regimen and tacrolimus maintenance therapy with or without an adjunct administration of MMF (19.7% vs. 16.2%; ) (87). Long-term steroid therapy is continued in some patients due to a perceived benefit in terms of the risk of late rejection, recurrence of AIH, renal impairment, and concomitant medical conditions that require steroids, although the evidence base for this decision is weak (88). The strongest data relate to the avoidance of AIH recurrence, which occurs in approximately 20 30% of patients and can develop at any time after liver transplantation (89). Steroid continuation or reintroduction is required in the majority of patients transplanted for AIH (90), and withdrawal should only be considered in the presence of high CNI dosing (89). 497

12 Herzer et al. Successful steroid avoidance or withdrawal depends on the risk profile of the patient, time point after transplantation, and the employed basic immunosuppressive regimen. Timing as well as the speed of steroid taper rate should be individually adjusted based upon the clinical indication for withdrawal, with a rapid withdrawal in cases of severe infection (e.g., septicemia) and a more gradual withdrawal in the presence of metabolic or cardiovascular risk factors. This should be influenced and adapted based upon the previous dose and the duration of treatment. Conclusions Other than in patients with AIH, in whom long-term steroids should be considered, steroids can be withdrawn within 3 6 months after transplantation to reduce chronic side effects. Summary There is a widespread disparity in the application of most immunosuppressive substances following liver transplantation between transplant centers. However, there is also broad consensus on some aspects of immunosuppressive protocols for liver transplant patients. Inclusion of CNI therapy is utilized almost universally, and steroids can be withdrawn successfully. The use of azathioprine, in contrast to MMF, is appropriate only in patients with IBD or other autoimmune diseases. Approaches differ more widely, however, regarding the use and type of induction therapy, and the introduction of MMF or everolimus at the time of transplant. Only the combination of CNI with everolimus has so far demonstrated a longterm nephroprotective effect for de novo liver transplant patients. However, randomized studies comparing everolimus with MMF are lacking. Thus, a standardized immunosuppressive protocol can only be applied to a limited number of patients. Future prospective trials, addressing specific subgroups of difficult-to-treat patients, complemented by retrospective registry analyses addressing individual open questions, are necessary and awaited. These data would help to further refine an adaption of the immunosuppressive regimen according to the individual profile of each patient. Acknowledgements Editorial assistance was provided by Dr. Karin Eichele and Caroline Dunstall. Source of support The authors travel costs to the meeting venue, and medical writing support, were covered by a grant from Novartis Pharma GmbH. Authors contributions All authors participated in the meeting at which the content of this article was discussed, contributing data, experience, and opinions for inclusion in a first draft. All authors critically reviewed, amended, and approved the current version prior to publication. Conflict of interest Kerstin Herzer received speaker s honoraria, travel grants, research funding, and/or membership of advisory boards from Novartis, Astellas, Chiesi, Roche, Biotest, and BMS; Christian P. Strassburg received speaker s honoraria and travel grants from BMS, AbbVie, Novartis, and Biotest. Felix Braun received speaker s honoraria, travel grants, research funding, and/or membership of advisory boards from Novartis, Astellas, Roche, and Biotest; Cornelius Engelmann received speaker s honoraria, travel grants, research funding, and/or membership of advisory boards from Novartis, and received travel grants from Astellas and Biotest; Markus Guba received speaker s honoraria, travel grants, research funding, and/or membership of advisory boards from Novartis, Pfizer, and Astellas; Frank Lehner received speaker s honoraria, travel grants, research funding, and/or membership of advisory boards from Novartis, Astellas, Chiesi, Roche, Pfizer, and Hexal; Silvio Nadalin received speaker s honoraria, travel grants, research funding, and/or membership of advisory boards from Novartis and Astellas; Andreas Pascher received speaker s honoraria, travel grants, research funding, and/or membership of advisory boards from Novartis, Astellas, and NPS; Marcus N. Scherer received speaker s honoraria, travel grants, research funding, and/or membership of advisory boards from Novartis, Astellas, Biotest, MSD, Pfizer, Hexal, Roche, Aesculap, Sanofi-Aventis, Merck, Ethicon, and Chiesi; Andreas A. Schnitzbauer received speaker s honorary, honoraria for memberships in advisory boards, and travel grants from Novartis and Astellas; Tim Zimmermann received speaker s honoraria, travel grants, research funding, and/or membership of advisory boards from Novartis, Astellas, Biotest, Roche, Pfizer, Panacea, and TEVA; Bj orn Nashan received speaker s honoraria, travel grants, research funding, and/or mem- 498

13 Immunosuppression after liver transplant bership of advisory boards from Astellas, Bristol Meyer-Squibb, Chiesi, Hexal, and Novartis; Martina Sterneck received speaker s honoraria, travel grants, research funding, and/or membership of advisory boards from Novartis, received travel grants from Biotest, Astellas, and Roche, and received speaker s honoraria and travel grants from Biotest. References 1. OJO AO, HELD PJ, PORT FK et al. Chronic renal failure after transplantation of a nonrenal organ. N Engl J Med 2003: 349: T URK TR, WITZKE O, ZEIER M. KDIGO-Leitlinien zur Betreuung von Nierentransplantatempf angern. Nephrologe 2010: 5: ROSTAING L, SALIBA F, CALMUS Y, DHARANCY S, BOILLOT O. Review article: use of induction therapy in liver transplantation. Transplant Rev 2012: 26: TURNER AP, KNECHTLE SJ. Induction immunosuppression in liver transplantation: a review. Transpl Int 2013: 26: KIM WR, STOCK PG, SMITH JM et al. OPTN/SRTR 2011 annual data report: liver. Am J Transplant 2013: 13(Suppl 1): WANG XF, LI JD, PENG Y, DAI Y, SHI G, XU W. Interleukin-2 receptor antagonists in liver transplantation: a meta-analysis of randomized trials. Transplant Proc 2010: 42: MCALISTER VC, HADDAD E, RENOUF E, MALTHANER RA, KJAER MS, GLUUD LL. Cyclosporin versus tacrolimus as primary immunosuppressant after liver transplantation: a meta-analysis. Am J Transplant 2006: 6: SEGEV DL, SOZIO SM, SHIN EJ et al. Steroid avoidance in liver transplantation: meta-analysis and meta-regression of randomized trials. Liver Transpl 2008: 14: RAMIREZ CB, DORIA C, DI FRANCESCO F, IARIA M, KANG Y, MARINO IR. Basiliximab induction in adult liver transplant recipients with 93% rejection-free patient and graft survival at 24 months. Transplant Proc 2006: 38: NEUHAUS P, CLAVIEN PA, KITTUR D et al. Improved treatment response with basiliximab immunoprophylaxis after liver transplantation: results from a double-blind randomized placebo-controlled trial. Liver Transpl 2002: 8: NIEMEYER G, KOCH M, LIGHT S, KUSE ER, NASHAN B. Long-term safety, tolerability and efficacy of daclizumab (Zenapax) in a two-dose regimen in liver transplant recipients. Am J Transplant 2002: 2: CALMUS Y, KAMAR N, GUGENHEIM J et al. Assessing renal function with daclizumab induction and delayed tacrolimus introduction in liver transplant recipients. Transplantation 2010: 89: NEUBERGER JM, MAMELOK RD, NEUHAUS P et al. Delayed introduction of reduced-dose tacrolimus, and renal function in liver transplantation: the ReSpECT study. Am J Transplant 2009: 9: YOSHIDA EM, MAROTTA PJ, GREIG PD et al. Evaluation of renal function in liver transplant recipients receiving daclizumab (Zenapax), mycophenolate mofetil, and a delayed, low-dose tacrolimus regimen vs. a standard-dose tacrolimus and mycophenolate mofetil regimen: a multicenter randomized clinical trial. Liver Transpl 2005: 1064: BENITEZ CE, PUIG-PEY I, LOPEZ M et al. ATG-Fresenius treatment and low-dose tacrolimus: results of a randomized controlled trial in liver transplantation. Am J Transplant 2010: 10: TRUNECKA P, KLEMPNAUER J, BECHSTEIN WO et al. Renal function in de novo liver transplant recipients receiving different prolonged-release tacrolimus regimens-the DIA- MOND Study. Am J Transplant 2015: 1843: OTERO A, VARO E, DE URBINA JO et al. A prospective randomized open study in liver transplant recipients: daclizumab, mycophenolate mofetil, and tacrolimus versus tacrolimus and steroids. Liver Transpl 2009: 15: KLINTMALM GB, WASHBURN WK, RUDICH SM et al. Corticosteroid-free immunosuppression with daclizumab in HCV(+) liver transplant recipients: 1-year interim results of the HCV-3 study. Liver Transpl 2007: 13: BOILLOT O, MAYER DA, BOUDJEMA K et al. Corticosteroid-free immunosuppression with tacrolimus following induction with daclizumab: a large randomized clinical study. Liver Transpl 2005: 11: EASON JD, NAIR S, COHEN AJ, BLAZEK JL, LOSS GE Jr. Steroid-free liver transplantation using rabbit antithymocyte globulin and early tacrolimus monotherapy. Transplantation 2003: 75: LLADO L, FABREGAT J, CASTELLOTE J et al. Impact of immunosuppression without steroids on rejection and hepatitis C virus evolution after liver transplantation: results of a prospective randomized study. Liver Transpl 2008: 14: FILIPPONI F, CALLEA F, SALIZZONI M et al. Double-blind comparison of hepatitis C histological recurrence rate in HCV+ liver transplant recipients given basiliximab+ steroids or basiliximab+ placebo, in addition to cyclosporine and azathioprine. Transplantation 2004: 78: PAGEAUX GP, CALMUS Y, BOILLOT O et al. Steroid withdrawal at day 14 after liver transplantation: a doubleblind, placebo-controlled study. Liver Transpl 2004: 10: SOLIMAN T, HETZ H, BURGHUBER C et al. Short-term induction therapy with anti-thymocyte globulin and delayed use of calcineurin inhibitors in orthotopic liver transplantation. Liver Transpl 2007: 1039: HORTON PJ, TCHERVENKOV J, BARKUN JS et al. Antithymocyte globulin induction therapy in hepatitis C-positive liver transplant recipients. J Gastrointest Surg 2005: 9: DE RUVO N, CUCCHETTI A, LAURO A et al. Preliminary results of a prope tolerogenic regimen with thymoglobulin pretreatment and hepatitis C virus recurrence in liver transplantation. Transplantation 2005: 80: BAJJOKA I, HSAIKY L, BROWN K, ABOULJOUD M. Preserving renal function in liver transplant recipients with rabbit anti-thymocyte globulin and delayed initiation of calcineurin inhibitors. Liver Transpl 2008: 14: OJO AO. Renal disease in recipients of nonrenal solid organ transplantation. Semin Nephrol 2007: 27: L UCK R, B OGER J, KUSE E, KLEMPNAUER J, NASHAN B. Achieving adequate cyclosporine exposure in liver transplant recipients: a novel strategy for monitoring and dosing using intravenous therapy. Liver Transpl 2004: 10: BECKEBAUM S, IACOB S, SWEID D et al. Efficacy, safety, and immunosuppressant adherence in stable liver transplant patients converted from a twice-daily tacrolimus-based regimen to once-daily tacrolimus extended-release formulation. Transpl Int 2011: 24: KIM JM, KWON CH, JOH JW et al. The conversion of once-daily extended-release tacrolimus is safe in stable liver transplant recipients: a randomized prospective study. Liver Transpl 2016: 22: