EASL Clinical Practice Guidelines

Immunosuppression

Standard regimens

The liver is considered a privileged organ in terms of immunological interactions. Spontaneous resolution of severe acute rejection episodes has been described in patients after LT, and these findings have switched the clinician’s aim in using immunosuppression from a complete suppression of acute rejection to a reduction of immunosuppression-related side effects particularly renal toxicity. Therefore long-term outcome for patients is becoming the main concern for clinicians, as long-term direct and indirect side effects of immunosuppressive therapy are a major cause of morbidity and mortality. New immunosuppressive protocols have been adopted using combination of drugs with different modes of action, but this has not necessarily resulted in lower immunopotency despite lower doses of each drug. Moreover, new agents with promising results are entering clinical practice.

CNIs are the principal choice for immunosuppression after LT both in Europe and in the US, with nearly 97% of liver transplanted patients discharged from the hospital on CNIs [263]. Both cyclosporine (CsA) and tacrolimus (Tac) bind to cytoplasmic receptors (cyclophilin and FK-binding protein 12, respectively), and the resulting complexes inactivate calcineurin, a pivotal enzyme in T cell receptor signalling. Calcineurin inhibition prevents IL2 gene transcription, thereby inhibiting T cell IL production.

Among CNIs, Tac is the drug of choice in almost 90% of liver transplanted patients, resulting in a significant increase in its use since 1998 to date.

The best evidence for comparison of the two CNIs is derived from a meta-analysis [[264], [265]] including 3813 patients, which shows immunosuppression with Tac reduces mortality at 1- and 3-years post-transplant, reduced graft loss, reduced rejection and steroid-resistant rejection.

A prolonged-release formulation of Tac has been developed to provide once-daily dosing, with similar efficacy and safety to the twice-daily formulation [[266], [267]]. This formulation seems to have also a positive impact on adherence to immunosuppressive therapy [268].

Azathioprine (AZA) and mycophenolate mofetil (MMF) are the two antimetabolites used in LT. AZA is a prodrug of 6-mercaptopurine that inhibits inosine monophosphate dehydrogenase (IMPDH) and reduces purine synthesis, affecting T and B lymphocyte proliferation [269]. Mycophenolic acid is the active metabolite of MMF and is a selective, non-competitive inhibitor of IMPDH. It is used for both treatment and prevention of rejection in combination with CNI [270].

Their use has constantly increased in the last two decades, due to the clinical need to reduce CNI doses in order to minimize side effects such as nephrotoxicity. Since its introduction MMF has progressively become the most used antimetabolite agent, replacing AZA. However, the evidence for a significant benefit in terms of preventing acute cellular rejection using MMF rather than AZA is very poor.

Only two randomized controlled trials (RCTs) directly compared MMF with AZA [[270], [271]], with one update [272], and no difference was found between MMF and AZA in terms of patient and graft survival [270].

An enteric-coated formulation of mycophenolate sodium (EC-MPS) has been developed to reduce the gastrointestinal side effects by delaying mycophenolic acid (MPA, the active metabolite of MMF) release until the small intestine. Bioequivalence has been shown in renal transplantation for both pharmacokinetics [[273], [274], [275]] and a RCT [276]. In LT EC-MPS use is limited [[277], [278]].

Sirolimus (SRL) and everolimus (EVR) are inhibitors of the mammalian target of rapamycin (mTOR). Their immunosuppressive activity is related to the blockade of IL-2 and IL-15 induction of proliferation of T and B lymphocytes.

SRL was first approved for renal transplantation; however, a black box warning was placed on its use in LT after two multicentre trials (Wyeth 211 and 220) found that SRL was associated with increased incidence of early hepatic artery thrombosis, and with excess mortality and graft loss after LT. However, since 2000, several studies have been performed on de novo mTOR inhibitor use after LT showing either a reduced or a similar incidence of hepatic artery thrombosis in patients receiving SRL compared to controls [[279], [280], [281]]. SRL is a promising alternative that may be equivalent to CNI in preventing graft rejection. The adverse effects of SRL include dose-dependent hyperlipidaemia, thrombocytopenia, anaemia, leukopenia, with the absence of neurotoxicity, nephrotoxicity and diabetogenesis, but it has adverse effects on wound healing [282]. Further studies are needed to assess the value of SRL as the primary immunosuppressor after LT, either as a single agent or in combination with other agents.

There has been a gradual, but constant, increase in the use of induction agents, particularly in the last ten years. This has been done to reduce immunosuppression toxicity by minimizing CNIs and steroid use. This has paralleled the introduction of the MELD allocation system, which has resulted in more patients with renal impairment undergoing LT and a greater risk of renal toxicity.

Among induction agents, IL-2 receptor (CD25) monoclonal antibodies (daclizumab and basiliximab) have been the ones mostly used. They are chimeric and humanized antibodies that act on a receptorial subunit, expressed only on activated T lymphocytes, and selectively inhibit their proliferation. Daclizumab has been recently removed from the market, because of diminishing demand.

In a sub-analysis of the basiliximab registration trial no difference was found in death/acute rejection/graft loss between patients receiving basiliximab (52.8%) compared to placebo (44.1%) (both in association with CsA and steroids). When HCV negative patients were evaluated separately, patients treated with basiliximab had a significantly lower incidence of acute rejection at 6 months compared to placebo [283].

These data were confirmed in a recent literature review including 18 studies showing that liver transplanted patients, receiving IL-2R antagonists, experienced lower albumin creatinine ratio at 12 months or later, less steroid-resistant acute rejection, less renal dysfunction, when associated with reduced or delayed, and less incidence of post-transplant diabetes mellitus. No difference was found in patient and graft survival [284]. However, these agents should always be used in combination with CNIs to avoid high incidence of acute rejection, as shown in some studies [[285], [286]].

The other group of induction agents is represented by anti-thymocyte (ATG) and anti-lymphocyte (ALG) polyclonal antibodies. These are heterologous preparations consisting of an infusion of rabbit- or equine-derived antibodies against human T cells. In two retrospective studies [[287], [288]], a three-day induction with ATG in combination with standard CNI dosage was associated with better renal function, but no difference in terms of post-transplant survival. In one study [288] the rate of albumin creatinine ratio was lower in the ATG group.

Between 2000 and 2010, the Food and Drug Administration approved several generic formulations of CNIs (both CsA and Tac) and antimetabolites (both MMF and AZA). Despite the indisputable economic benefits provided by generic drugs, concerns still persist on their use in clinical practice [[289], [290], [291]].

The general consensus in the transplant community is that immunosuppressive drugs should be classified as critical-dose drugs, and such generic drugs should be subjected to different standards for approval [292].

Current opinion among the transplant community is that the use of generic immunosuppressive therapy is safe compared with branded drugs; however, precautions have to be taken [293]. It is mandatory to be aware of the lack of proven bioequivalence between different generic compounds, and that stringent therapeutic drug monitoring is in place during the initial switch phase [294]. Additional studies are needed to assess the true impact of generic immunosuppression.

Recommendations

Regimens for specific categories of recipients (with renal failure, HCV positive, at risk of infections, at risk of metabolic syndrome, with de novo tumours, etc.)

Immunosuppression in patients with renal impairment

Chronic renal dysfunction, defined as a GFR of ⩽29 ml/min/1.73 m2 of body-surface area or the development of ESRD, occurs approximately in 18% of liver transplant recipients by five years post-transplant [295]. The most important risk factor for the development of nephrotoxicity is the use of CNIs. CNI-induced nephrotoxicity has a component of reversible renal vasoconstriction. Eventually, tubulointerstitial chronic fibrosis and irreversible change can develop [296].

In patients with renal dysfunction the administration of induction agents and in particular IL-2R antibody can be used together with delayed introduction of CNIs [[297], [298], [299]].

Three multicentre, RCTs [[297], [298], [299]] evaluated the use of IL-2R antibodies as part of a CNI-sparing strategy in patients with kidney dysfunction after LT. In these studies IL-2R antibodies were given in association with MMF followed by delayed introduction of Tac at standard dose [299] or at reduced dose [298]. Patients receiving IL-2R antibodies with delayed and low dose Tac plus MMF and steroids had significant GFR preservation in one study [298], and a significant improvement in the GFR at 1 and at 6 months after LT compared with the control group in another [299]. Conversely an open, randomized, multicentre trial did not find any benefit in terms of renal function using immunosuppressive protocols based on daclizumab induction with delayed Tac [297].

The association of MMF with CNI reduction (at least 50%) or CNI withdrawal is associated with a significant improvement in renal function and a low risk of biopsy-proven acute rejection [[300], [301], [302], [303], [304], [305]]. The combination of MMF with CNI withdrawal [[306], [307], [308], [309], [310]], despite the improvement of renal function in nearly 60%-80% of patients, is associated with a significantly increased risk of acute rejection (between 3% and 30%) [311], too high for current standards.

Only three studies have explored the role of AZA in association with CNI reduction or withdrawal [[312], [313], [314]] showing an improvement in renal function, but again this increased the risk of rejection in some cases [314]. To date no RCTs have been performed directly comparing MMF and AZA with respect to renal function [315].

SRL has been used in liver recipients with renal dysfunction, in order to reduce or stop CNI use. However, the role of mTOR inhibitors in patients with CNI-induced renal impairment is controversial.

In a recent meta-analysis, based on 11 studies (including three RCTs), SRL was not associated with an improvement in renal function at 1 year with a statistically significant increase in infection, rash, mouth ulcers, and discontinuation of therapy [316].

A large prospective, open-label, randomized trial evaluated conversion from CNI to SRL-based immunosuppression for preservation of renal function in LT patients. Overall, 607 patients were randomized early after transplant (within 24 h) and converted from CNI to SRL (n = 393) or CNI continuation for up to 6 years (n = 214). Changes in baseline-adjusted mean Cockcroft–Gault GFR at 12 months were not significant between the two groups [317]. In a more recent prospective, open-label, multicentre study, patients were randomized 4 to 12 weeks after transplantation to receive SRL plus MMF (n = 148) or CNI plus MMF (n = 145). Immunosuppression based on SRL plus MMF was associated with a significantly greater renal function improvement from baseline with a mean percentage change in GFR compared with CNI plus MMF [318].

Data on EVR in combination with CNI withdrawal or reduction are encouraging but not completely conclusive.

The application of an immunosuppressive protocol with EVR and the withdrawal of CNIs has been associated with an initial improvement of renal function tests without an increase in the risk of rejection [319]. However, in a prospective, randomized, multicentre study the mean change in creatinine clearance from baseline to 6 months was similar between patients treated with EVR in association with CNI reduction or discontinuation groups and patients using CNI at standard dose [320].

Further RCTs confirmed that early EVR-based CNI-free immunosuppression is feasible following LT, and patients benefit from sustained preservation of renal function vs. patients on CNI for at least 3 years [[321], [322]]. In a 24-month prospective, randomized, multicentre, open-label study the adjusted change in estimated GFR from randomization to month 24 was superior with EVR plus reduced Tac vs. Tac control (p <0.001). However, the randomization to Tac elimination was stopped prematurely due to a significantly higher rate of treated biopsy-proven acute rejection [[323], [324]].

Recommendations

Immunosuppression in HCV liver transplanted patients

Immunosuppression for HCV patients represents a fine balance between suppressing immunity and maintaining optimal host viral responses. However, the use of highly efficacious IFN-free regimens to cure HCV infection will most likely be unnecessary to individualize immunosuppressive therapy in this setting.

CsA has been shown to have a suppressive effect on the HCV replicon RNA level and HCV protein expression in a HCV sub-genomic replicon cell culture system [325]. However, there is still controversy about the effect of CsA on HCV replication in vivo, in the setting of clinical organ transplantation.

A meta-analysis including five RCTs did not find any significant differences in terms of mortality, graft survival, biopsy-proven acute rejection, corticoresistant acute rejection or fibrosis cholestatic hepatitis between Tac-based vs. CsA-based immunosuppression in HCV liver transplant recipients [326].

Considering the potential influence of CsA on the efficacy of antiviral therapy in transplant recipients, several studies explored this field with controversial results. In the only randomized controlled study available to date the antiviral effect of CsA during therapy with PegIFNα-2a and RBV in liver transplant recipients with HCV recurrence (Ishak Fibrosis Stage = 2) was assessed. In patients who switched from Tac to CsA, SVR was higher than in patients on Tac receiving PegIFN/RBV therapy, but the difference was not statistically significant [327].

Although the data on the increase of HCV viral loads due to steroid boluses are convincing [[328], [329]], the effects of steroid maintenance are still controversial. The link between steroid therapy and viral replication after LT in HCV recipients prompted many centres to advocate steroid therapy withdrawal. However, robust data are limited as to the efficacy of this approach. A rapid reduction in the dose of steroid dosage may be harmful for HCV recurrence [330].

Short-term maintenance with steroids (<6 months) with slow tapering has been shown to be associated with less fibrosis progression [[331], [332], [333]].

Considering steroid-free immunosuppressive regimens, three prospective, randomized studies did not find a significant difference with regard to liver fibrosis and viral loads when steroid maintenance was compared with steroid-free regimens in HCV liver transplanted patients [[334], [335], [336]]. These data were confirmed in a meta-analysis. However, HCV recurrence was assessed heterogeneously and data on fibrosis progression and on steroids dose and withdrawal were not reported. Moreover, no individual trial reached statistical significance [337].

When MMF and AZA are compared with respect to their potential impact on HCV recurrence after LT, there is little evidence supporting the use of MMF over AZA, and indeed AZA appears better. In a recent review of the literature 70% of the studies found that severity of HCV recurrence was decreased using AZA, whereas only three studies showed similar severity in HCV recurrence whether AZA was used or not. No study showed that AZA was associated with increased severity of recurrent HCV. Conversely six out of 17 studies, which used MMF, showed an increased severity of HCV recurrence, whereas nine out of 17 showed no effect [315].

Wiesner et al. [270] directly compared MMF and AZA in HCV positive liver transplanted patients. A significant reduction in the incidence of acute hepatic allograft rejection or graft loss in the MMF group compared with the AZA group was seen at 6 months after LT. The incidence of HCV recurrence, defined histologically and in the presence of HCV RNA, was 18.5% in the MMF group and 29.1% in the AZA group at 6 months after LT, but no long-term data is available.

Recently Kornberg et al. [338] performed a prospective study revealing that in patients treated with MMF, recurrent disease was diagnosed earlier than in the AZA group, but they experienced less severe allograft fibrosis at diagnosis. However, the stage of fibrosis significantly increased in the MMF group during 6-months of antiviral treatment compared to the AZA group.

The anti-fibrogenic properties of mTOR inhibitors have been shown in animal models of liver disease where fibrosis progression was attenuated with a low dose of SRL, with SRL and EVR being associated with significantly less fibrosis progression and portal hypertension than treatment with CNIs [339]. Moreover, mTOR inhibitors may affect HCV progression by reducing HCV replication [340]. In vivo data are scarse and mainly based on retrospective studies showing that SRL reduces the incidence of advanced fibrosis (stage ⩾2) both at 1- and 2-years after LT in HCV transplanted patients receiving de novo SRL compared to a control group [341]. Very few data are available on EVR and HCV recurrence after LT [[320], [342]].

Considering ATG, in a randomized study comparing thymoglobulin induction plus Tac monotherapy vs. Tac plus steroids without induction HCV recurrence was similar in the two groups, but the mean time to histologic recurrence was shorter in the thymoglobulin group [343]. ATG during the induction phase was associated with a lower frequency of recurrence of HCV in patients undergoing LT. This, however, did not affect the 1- and 2-year survival and the frequency of acute rejection, infections, or neoplasms [344].

No significant difference with regard to liver fibrosis and viral loads were found in HCV liver transplanted patients treated with induction therapy based on daclizumab/basiliximab [[283], [334], [336]].

A cross-sectional study evaluated the use of alemtuzumab (anti-CD52) in liver transplanted recipients. HCV positive patients did significantly worse than those who were HCV negative, both in the induction and the control group. Moreover, increased HCV viral replication was worse with alemtuzumab, but there was no data on histological recurrence [345].

Recommendations

Immunosuppression in patients with HCC

The immunosuppression plays a central role in the increased risk of cancer after LT, including the recurrence of HCC.

In vitro studies and animal models have shown that CNIs increase the production of TGF-β in a dose-dependent fashion, promoting tumour cell invasiveness and resistance to apoptosis. In vitro data also showed that CsA can induce an invasive phenotype in adenocarcinoma cells through a TGF-β-mediated mechanism [346]. Moreover, in rats with HCC, treatment with CsA was associated with reduced survival and increased metastasis [347].

In retrospective studies a dose-dependent relationship between CNIs and recurrence of HCC after LT was found [[348], [349]].

When CsA is compared to Tac in terms of HCC, recurrence data are not conclusive, and is based on a retrospective study. There are some evidence that CsA is associated with increased 5-year disease-free survival [350] and reduced recurrence rate [351], but these data were not confirmed in subsequent studies [348].

The studies evaluating the role of immunosuppression on HCC recurrence showed no influence of MMF [[348], [351]]. No data are available on the influence of AZA on HCC recurrence after LT.

mTOR inhibitors in LT have a potential anticancer effect. This is due to their inhibitory effect on cancer stem cell self-renewal, on cancer cell growth/proliferation and on tumour angiogenesis. These properties could make mTOR inhibitors the potential immunosuppression of choice in patients transplanted for HCC. To date several studies have been performed to test the impact of SRL on HCC recurrence and on patient survival after LT, however no RCTs have been published. Although most of these studies showed beneficial effect in using SRL, the available evidence is based on clinical reports and retrospective studies.

Two recent meta-analysis [[352], [353]] demonstrated lower HCC recurrence and lower overall mortality in patients treated with SRL.

The results from the only prospective, multicentre, randomized, open-label trial (SILVER trial) showed that SRL improves recurrence-free survival and overall survival in the first 3 to 5 years in low risk patients with HCC within Milan criteria [[354], [355]].

Considering there are no randomized controlled studies on EVR this suggests a protective effect against HCC recurrence. Data from phase I and phase I/II clinical studies suggest that EVR monotherapy may stabilize advanced HCC progression [[356], [357]].

Recommendations

Immunosuppression in patients with de novo tumours

The risk of de novo malignancy should be considered similarly in clinical practice with Tac or CsA-based immunosuppressive regimens. In only one single centre study patients treated with CsA had an increased risk of malignancy compared with Tac treated patients [358]. However, the lower rejection rates detected in CsA group suggests higher immunosuppressive potency with CsA in this series. The risk of malignancy related to CNI in clinical practice may come from the dosage rather than the type of CNI used, as shown in a RCT performed in kidney transplant recipients [359].

To date there is no evidence suggesting a link between the use of MMF and de novo malignancy after LT. Data on MMF and de novo malignancies are available only in renal transplanted [309] and heart transplanted patients [360]. In heart transplanted patients the use of MMF had a protective effect against de novo malignancy.

There are no published RCTs evaluating the effect of mTOR inhibitors in preventing de novo malignancy after LT. The available evidence is based on clinical reports and retrospective studies, thus making it difficult to extract solid conclusions. There are reports of improved outcome of lymphoproliferative disorders and Kaposi sarcoma after switching to an mTOR inhibitor [361]. Despite this, many transplant centres frequently add or convert to an mTOR inhibitor when there are risk factors for malignancy after LT, or even when a tumour has been diagnosed.

Recommendations

Total withdrawal of immunosuppression

The main aspiration of transplant clinicians is the acceptance of the graft by the recipient without any long-term pharmacological help [[362], [363], [364]]. Long-term survivors following LT are often systematically and excessively immunosuppressed. Consequently, drug weaning is a strategy which should be considered providing it is done gradually under careful physician surveillance. Several studies have explored the possibility to completely withdraw immunosuppression in liver transplant recipients [[365], [366], [367], [368], [369], [370], [371], [372], [373], [374], [375]]. In these studies, the complete withdrawal of immunosuppression was achieved in nearly 20% of patients, on average. However, the incidence of acute rejection was significantly high with percentages ranging between 12% and 76.4%. Moreover, in two cases, chronic rejection led to graft loss among patients undergoing immunosuppression weaning protocols [[369], [373]].

Patients achieving immunosuppression withdrawal experienced a reduced infection rate, less medication requirement to treat comorbidities [376] and an improvement in creatinine, glucose and uric acid serum levels [377] compared with patients who failed immunosuppressive drug withdrawal.

Despite these promising results, most of the studies exploring immunosuppression withdrawal are based on retrospective analysis, small sample size and on single centre experience. Moreover, the lacking of a specific and well-defined protocol of immunosuppression withdrawal and patient monitoring, make these data not applicable to general clinical practice [378].

More recently the first two prospective multicentre trials of immunosuppression withdrawal in paediatric and adult patients have been performed [[368], [379]]. In the paediatric multicentre study, 20 stable paediatric recipients of parental living donor liver transplants underwent immunosuppression withdrawal at a median age of 8 years and 6 months. Immunosuppression withdrawal was achieved gradually over a minimum of 36 weeks, and patients were followed-up for a median of 32.9 months. Of 20 paediatric patients, 12 maintained normal allograft function for a median of 35.7 months after discontinuing immunosuppression therapy. Of interest, patients with operational tolerance initiated immunosuppression withdrawal later after transplantation compared with patients without operational tolerance [368]. In the adult trial, stable liver recipients at least 3 years after transplantation were included. Among the 98 recipients evaluated, 41 successfully discontinued all immunosuppressive drugs, whereas 57 experienced acute rejection. Tolerance was associated with time since transplantation, recipient age and male gender. No benefits in terms of renal function, diabetes and hypertension were seen in patients who underwent immunosuppression withdrawal [379].

Recommendation