Assessment Of the Safety and Benefit of Prospective Immunosuppressive Drug Withdrawal in Liver Transplantation and Prediction of Operational Tolerance
Assessment Of the Safety and Benefit of Prospective Immunosuppressive Drug Withdrawal in Liver Transplantation and Prediction of Operational Tolerance
In liver transplantation up to 20% of recipients can completely discontinue immunosuppressive
 therapy maintaining normal graft function, and are conventionally considered as operationally
 tolerant. Discontinuation of immunosuppressive drugs in operationally tolerant recipients
 could lessen the side effects of chronic immunosuppressive therapy. However, this strategy
 results in the development of rejection in a high proportion of recipients who require
 lifelong immunosuppression. Thus, there is a need to identify predictive factors of
 successful drug withdrawal and to define the clinical and histological outcomes of
 operationally tolerant liver recipients.

 The main objective of this study is to establish the safety of attempting immunosuppressive
 (IS) drug withdrawal in stable liver transplant recipients, using standard clinical,
 biological and histopathological methods, to screen and follow-up patients, and to confirm
 the benefit of maintaining immunosuppressive drug interruption in patients who are tolerant
 to their liver transplant. The secondary objective of this study is to identify predictive
 factors of operational tolerance and to attempt to develop a multi-parameter "decision rule"
 to predict patient tolerance or non-tolerance in order to improve patient screening and
 follow-up. In a diagnostic observational sub-study, peripheral blood and liver tissue samples
 collected before immunosuppressive drug withdrawal will be employed to validate the
 diagnostic accuracy of a previously identified set of tolerance biomarkers and to identify
 potential new biomarkers capable of predicting the outcome of the immunosuppressive
 withdrawal protocol.
1. This is a prospective study in which liver transplant recipients on maintenance
 immunosuppression (IS) will undergo carefully supervised IS weaning over a period of
 approximately 6-9 months. Patients will be gathered from a consortium of European liver
 transplant units including: Hospital Clínic Barcelona (Dr A. Sánchez-Fueyo), University
 Tor Vergata Rome (Dr G.Tisone), University Hospitals Leuven (Dr J.Pirenne). Patients
 will be followed-up for a total of 48 months after inclusion.

 2. Patients will be visited every 2-3 weeks, and immunosuppressive drugs will be gradually
 discontinued with the aim of achieving 50% decrease in drug dosages by month 3, and
 complete withdrawal by month 6 after initiation of the study. Following drug
 discontinuation, patients will continue to be followed every 2-3 weeks for 3 months, and
 monthly thereafter until month 12 after initiation of the study. Liver function tests
 will be obtained at every clinical follow-up visit. Patients will be considered
 operationally tolerant if no rejection takes place during the weaning procedure and the
 12-month period after complete drug discontinuation. All patients who fail to completely
 discontinue IS, or in whom drugs have to be reintroduced after a period of complete
 withdrawal, are considered as IS-dependent or non-tolerant. All biopsies will be blindly
 reviewed at the end of the study by a single pathologist to ensure diagnostic
 consistency between the two institutions.

 3. Management of liver function test alterations:

 -  Increases in liver function tests below 2-fold normal levels for AST/ALT/GGT,
 1.5-fold normal levels for ALP, or 2 mgIdL for bilirubin will result in no further
 decreases in drug dosages, and performance of new liver function tests in 14 days.
 Worsening or persistence of liver function test alterations will constitute
 indication for liver biopsy.

 -  Increases in liver function tests beyond 2-fold normal levels for AST/ALT/GGT,
 1.5-fold normal levels for ALP, or 2 mg/dL for bilirubin will result in liver
 biopsy.

 4. Diagnosis of liver graft rejection: will be based on the finding of 2 out of 3 of the
 following histological criteria: portal inflammation, injury to bile duct epithelium,
 and endothelitis. The finding of a mixed portal/lobular lymphocytic infiltrate not
 attributable to any other cause and responding to an increase in immunosuppressive drug
 doses will also be considered as a rejection.

 5. Management of rejection episodes: Specific therapy will be decided by the corresponding
 clinician/institution. Patients undergoing graft rejection will finalize their
 participation in the study. Worsening of HCV hepatitis or appearance of portal/lobular
 inflammation not attributable to rejection or viral etiology may also justify withdrawal
 from the study according to each institution's experience.

 6. Peripheral blood samples will be collected before weaning starts and every time routine
 liver function tests are measured. These samples will be employed to conduct microarray
 gene expression experiments. A portion of all liver biopsies collected will also be
 cryopreserved to conduct gene expression analyses. This experiments will constitute the
 basis of an observational sub-study (described in a different protocol) entitled:
 "Search for the immunological signature of operational tolerance in liver
 transplantation".

 7. Sample size has been estimated in order to be able to replicate the microarray gene
 expression results obtained by our group when comparing tolerant and non-tolerant
 recipients. In the current project we have followed a novel method recently proposed by
 Tibshirani et al. and have performed a permutation-based analysis of our pilot
 microarray data obtained from tolerant and non-tolerant samples. According to this
 analysis, at least 45 samples (20% tolerant, 80% non-tolerant) are required in order to
 be able to discriminate between tolerant and non-tolerant HCV-positive samples with a
 false discovery rate <0.05. Based on this analysis, we expect to enrol a total of 80
 patients in order to have at least 60 patients evaluable, who will be randomly allocated
 into training (45 patients) and testing (15 patients) sets for data analysis purposes.
Inclusion Criteria:

 -  Liver transplantation performed for non-autoimmune liver disease performed at least 3
 years before IS weaning.

 -  Absence of acute and/or chronic rejection episodes during the 12 months before
 weaning.

 -  Basal liver biopsy without signs of acute and/or chronic rejection.

 -  No evidences of autoimmune liver disease.

 -  Absence of acute and/or chronic rejection episodes during the 12 months before
 weaning.

 -  Basal liver biopsy without signs of acute and/or chronic rejection.

 -  Low dose immunosuppression (monotherapy with calcineurin inhibitors, mTOR inhibitors
 or mycophenolate mofetil, or combined therapy with 2 drugs at very low doses).

 -  Absence of medical or psychological disturbances that preclude the safe performance of
 the trial.

 -  Stability of liver graft function, defined as: normal liver function tests (AST, ALT,
 ALP, GGT) during at least 6 months, or, alternatively, minor alterations in liver
 function tests that have not changed over the previous 6 months (AST/ALT < 2 fold
 normal levels; ALP < 1.5 fold normal levels; GGT < 2 fold normal levels; bilirubin < 2
 mg/dL).

 -  Patients exhibiting at least one of the following characteristics: a) severe side
 effects of immunosuppressive drugs (diabetes, renal failure, hyperlipidemia,
 hypertension); b) risk of neoplasm development defined by history of previous
 non-hepatocarcinoma neoplasms or history of any of the following risk factors: tobacco
 or alcohol consumption, age greater than 60 years; c) chronic liver disease due to
 hepatitis C virus infection in patients not receiving anti-viral treatment.

 -  Signature of informed consent.