Effect of Calcineurin Inhibitors on Survival and Histologic Disease Severity in HCV-Infected Liver Transplant Recipients

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1 LIVER TRANSPLANTATION 12: , 2006 ORIGINAL ARTICLE Effect of Calcineurin Inhibitors on Survival and Histologic Disease Severity in HCV-Infected Liver Transplant Recipients Marina Berenguer, 1 Victoria Aguilera, 1 Martín Prieto, 1 Fernando San Juan, 2 José M. Rayón, 3 Salvador Benlloch, 1 and Joaquín Berenguer 1 1 HepatoGastroenterology Service, 2 Liver Transplantation and Surgery Unit, and 3 Pathology Service, Hospital Universitario La Fe, Valencia, Spain The severity of recurrent hepatitis C virus (HCV) is likely related to several factors. Controversial results have been reported regarding the effect of specific calcineurin-inhibitors. The aim of this research was to determine whether there are differences on posttransplantation outcome in HCV-infected patients based on initial immunosuppression. Prospective randomized trial comparing tacrolimus vs. cyclosporine-based immunosuppression in a cohort of patients undergoing primary orthotopic liver transplantation between 2001 and 2003 was used. Yearly biopsies were performed. Patients with at least 1 protocol biopsy and those with very severe recurrence despite a follow-up of less than 1 yr (cholestatic hepatitis, progression to bridging fibrosis/cirrhosis) were included. Baseline characteristics (demographics, liver function at transplantation, genotype distribution, donor, surgery, immunosuppression except for the type of calcineurin inhibitor) did not differ between the 2 groups. Severe disease (defined as bridging fibrosis, cirrhosis, cholestatic hepatitis, and/or death due to recurrent disease in the first year) was present in 27 in 90 (30%), and was equally distributed in the cyclosporine and tacrolimus groups (15/46 vs. 12/44, respectively). A total of 33 in 90 (37%) patients had no fibrosis in the first year biopsy with no difference between the cyclosporine and tacrolimus groups (36.5 vs. 37%). The percentage of patients developing recurrent acute hepatitis was also similar (32% vs 35%); time to acute hepatitis though was shorter in the tacrolimus group (59 days [35-185] vs. 92 days [39-343] in the cyclosporin group; P 0.02). Cholestatic hepatitis was observed in 4 of 44 and 5 of 46 patients under cyclosporine and tacrolimus, respectively (P not significant). In conclusions, the short-term posttransplantation course of hepatitis C is not related to the calcineurin inhibitor used. Liver Transpl 12: , AASLD. Received August 8, 2005; accepted September 22, See Editorial on Page 710 Hepatitis C virus (HCV)-cirrhosis is the most frequent diagnosis in patients undergoing liver transplantation. 1 Unfortunately, recurrence of HCV has shown to negatively impact graft and patient survival. 2-4 Few simple variables, including old donor age and potent immunosuppression have been shown to be associated with the outcome The exact definition of potent immunosuppression, however, is difficult to translate in terms of type, doses, and duration of immunosuppressive agents. Indeed, results from published studies show controversial results regarding the specific effect of the different immunosuppressive agents on recurrent hepatitis C This may be explained by the study design (mainly retrospective studies), the presence of confounding factors (genotype distribution, type of donor), the multiplicity of immunosuppressive combinations and the lack of protocol biopsies. In fact, there are not yet studies evaluating prospectively the effect of calcineurin inhibitors on the histologic severity of recurrent hepatitis C using a homogeneous population and yearly protocol biopsies. We therefore decided to prospectively randomize liver transplant patients in 2 groups, a first group receiving Abbreviations: HCV, hepatitis C virus. Supported in part by a grant from the Instituto de Salud Carlos III (C03/02). S.B. has a contract from the Instituto de Salud Carlos III, Ministerio de Sanidad (CM04/00217). Address reprint requests to Dr. Marina Berenguer, Servicio de HepatoGastroenterología, Hospital Universitario La Fe, Avenida Campanar, 21, Valencia, Spain. Telephone: ; FAX: ; mbhaym@teleline.es DOI /lt Published online in Wiley InterScience ( American Association for the Study of Liver Diseases.

2 CALCINEURIN INHIBITORS AND HCV POSTTRANSPLANTATION 763 neoral cyclosporine and a second group immunosuppressed with tacrolimus. We hypothesized that the outcome would not depend on the type of immunosuppressive drug used. The aim of this study was therefore to determine whether there are differences in histologic outcome with regard to the calcineurin inhibitor used. In order to test this, we compared the rate of severe disease within the first year between 2 groups of patients prospectively randomized to receive neoral cyclosporine vs. tacrolimus. We present here the preliminary results since this is an ongoing study that we plan to continue for another 2 yr. PATIENTS AND METHODS Patients Between October 2001 and June 2004, 122 adult patients underwent primary liver transplantation at our institution for HCV-related cirrhosis without hepatitis B virus infection. This is an open label randomized study in which 62 patients were assigned to cyclosporine-based immunosuppression and 60 to tacrolimus-based regime. Only HCV-RNA positive patients with at least 1-yr protocol biopsy performed in the absence of prior antiviral therapy and/or patients with an earlier clinical indicated biopsy showing severe recurrent disease (defined as cholestatic hepatitis and/or progression to bridging fibrosis) were included in this study. With the exception of one patient, HCV-infected patients did not receive pre and/or early posttransplantation antiviral therapy. Standard or pegylated interferon in combination with ribavirin were used as treatment of histologically-proven recurrent hepatitis C, generally evaluated in the first-year liver biopsy. Antiviral therapy was initiated at earlier time points in cases of aggressive recurrent disease, defined as fibrosing cholestatic hepatitis and/or progression to fibrosis 3 or 4 before the first-year protocol biopsy. The criteria used for selecting patients with cirrhosis and a localized hepatocellular carcinoma are those proposed previously. 2 The follow-up of this preliminary analysis was terminated at the time of either the patient s death, retransplantation or at the end of the observation period (May 2005). Histological Assessment Protocol liver biopsies were performed yearly ( 4 months). Additional biopsies were performed when clinically indicated. All biopsy specimens were reviewed by a single pathologist (J.M.R.) in a blinded fashion, and only those obtained before any antiviral therapy was instituted were evaluated in this study. Sections were stained routinely with hematoxylin-eosin, reticulin, Perls, and Orcein stains. Liver biopsies classified as hepatitis were scored evaluating both the stage of fibrosis and the degree of necroinflammatory activity, as previously reported. 2 The grade was determined by combining the histologic activity index scores for periportal necrosis, lobular degeneration, and necrosis and portal inflammation, and was defined as follows: 1 to 2, minimal; 3 to 6, mild; 7 to 10, moderate; 11 to 14, severe. The stage corresponded to the original histologic activity index fibrosis score: 0, none; 1, fibrous portal expansion; 3, bridging fibrosis; and 4, cirrhosis. Cholestatic hepatitis was defined following recent recommendations. 12 Immunosuppression During the study period, all patients undergoing liver transplantation at our institution were prospectively randomized to receive cyclosporine neoral steroids vs. tacrolimus steroids. Additional therapies were used in cases of early posttransplantation complications that required a substantial reduction in calcineurin inhibitor doses. Initial doses were as follows: methylprednisolone given intravenously with tapering of the dose from 200 to 20 mg at day 6, at which time 20 mg/day of prednisone were administered orally; cyclosporine (trough levels of ng/ml the first month, ng/ml the second and the third months, ng/ml until the end of the first year, and around 100 ng/ml thereafter); tacrolimus (trough levels of 5-15 ng/ml the first 3 months, 5-10 ng/ml thereafter). Prednisone dose was started at 20 mg 1 week after transplantation and tapered down at a slow rate with final withdrawal after 9 to 12 months from transplantation. Only in cases where cholestatic hepatitis was diagnosed or in patients with severe side-effects related to the use of corticosteroids, prednisone was tapered down more rapidly. Cytomegalovirus (CMV) Prophylaxis Gancyclovir or valgancylocir were administered under the following circumstances: 1) positive donor and negative recipient; 2) retransplantation; 3) use of monoclonal or polyclonal antibodies; or 4) surgery complicated with high blood-product requirements. Outcome Variables Progression to severe disease within the first year (bridging fibrosis, cirrhosis, cholestatic hepatitis, death due to recurrent hepatitis) was used as the primary end-point. Secondary end-points included: 1) progression to fibrosis 1 in the first-year liver biopsy; 2) percentage of patients developing acute hepatitis and time to hepatitis; 3) percentage of patients developing cholestatic hepatitis; and 4) graft/patient survival. Factors Analyzed and Compared Between the 2 Groups These included: 1) Demographics: age at transplantation and sex distribution. 2) Pretransplantation variables: presence of hepatocellular carcinoma, Child- Turcotte-Pugh classification, history of significant alcohol consumption, and history of failed interferon

3 764 BERENGUER ET AL. TABLE 1. Baseline Characteristics of the Study Population Neoral-based IS (n 44) Tac-based IS (n 46) P value Gender (% male) 31 (70%) 33 (72%) ns Age (median, range) 54 (28 66) 59 (37 67) ns HCC (%) 21 (48%) 16 (35%) ns Alcohol (%) 9 (20.5%) 9 (20%) ns Child classification (%) 9 (5 13) 10 (5 14) ns A 12 (27%) 7 (15%) B 13 (30%) 16 (35%) C 19 (43%) 23 (50%) Prior failed IFN therapy (%) 14 (32%) 13 (28%) ns Genotype (% 1a/1b) 30 (83%) 35 (90%) ns Donor gender (% male) 24 (55%) 31 (67%) ns Donor age (median, range) 55 (12 83) 57 (16 81) ns Warm ischemia time (minute) 37.5 (20 65) 40 (15 80) ns Cold ischemia time (minute) 300 ( ) 340 ( ) Initial graft function (% moderate-severe dysfunction) 8 (18%) 13 (28%) ns Additional IS agents as induction (%)* 3 (7%) 6 (13%) ns Induction with MMF (%) 2 (4.5%) 5 (11%) ns Use of MMF 8 (18%) 7 (15%) ns MP boluses (% yes) 3 (7%) 1 (2%) ns Prednisone doses (mg) at: 1 month 15 (10 20) 17.5 (0 20) ns 3 months 12.5 (10 20) 12.5 (0.15) ns 6 months 7.5 (0 15) 7.5 (0 15) ns Abbreviations: HCC, hepatocellular carcinoma; IFN, interferon; MP, methyprednisolone; IS, immunosuppression; MMF, mycophenolate mofetil. *Additional immunosuppressive drugs: those added to calcineurin inhibitors and steroids. Use of mycophenolate mofetil either as induction regime or as maintenance during the first year post-transplantation. therapy in the past. 3) Donor-related variables: age and gender. 4) Surgically-related variables: duration of cold preservation and rewarming time, duration of intervention, and initial graft function. 5) History of acute recurrent hepatitis evidenced histologically, and time to acute hepatitis. An initial liver biopsy was typically performed when liver enzymes rose to twice the upper limit of normality. If, in these cases, changes compatible with HCV-related acute hepatitis were present, the patient was included in the group of patients with a history of acute hepatitis C. 6) Immunosuppression-related variables: histologically-diagnosed rejection episodes requiring methylprednisolone boluses, use of additional immunosuppressive drugs either for induction immunosuppression and/or rejection treatment, and prednisone doses at 1, 3, and 6 months posttransplantation. 7) Biochemically-related variables: alanine aminotransferase levels at 1, 6, and 12 months. Statistical Analysis Categorical data were compared using a 2 test or Fisher s exact test when indicated. When categorical variables were ordered, comparisons were done using a 2 test for trend. Continuous variables were expressed as median and range and compared by the Mann-Whitney test. HCV-related severe disease was defined by the presence of bridging fibrosis, cirrhosis, fibrosing cholestatic hepatitis, and death due HCV occurring within the first year posttransplantation. All statistical analyses were performed with SPSS 9.0 (SPSS, Chicago, IL). RESULTS Patient Demographics Ninety patients out of the 122 patients transplanted between October 2001 and June 2004 fulfilled the inclusion criteria (Table 1). The remaining 32 liver transplant recipients were excluded due to: negativity of HCV ribonucleic acid following transplantation (n 1); associated medical conditions (Budd-Chiari posttransplantation (n 2); biliary and/or arterial complications (n 4); recurrence of hepatocellular carcinoma (n 1); lack of adequate first-year liver biopsy (n 3); or death within the first year (n 21, none related to HCV). The causes of death included: sepsis (n 13), cardiovascular complications (n 3), de novo tumor (n 2), neurological complications (n 1) and surgically-related (n 2). Excluded patients were similar to those included in the study in terms of demographics, donor and surgical-related variables, genotype distribution, and initial immunosuppression (data not shown).

4 CALCINEURIN INHIBITORS AND HCV POSTTRANSPLANTATION 765 TABLE 2. Outcome of Cyclosporine vs Tacrolimus-Based Patients Neoral-based IS (n 44) Tac-based IS (n 46) P value Acute hepatitis (%) 14 (32%) 16 (35%) ns Time to acute hepatitis (days)* 92 (39 343) 59 (35 185) 0.02 Cholestatic hepatitis (%) 4 (9%) 5 (11%) ns Severe hepatitis (F3-F4, cholestatic hepatitis, 12 (27%) 15 (32%) ns death due to recurrent HCV) (%) Fibrosis stage in the 1 st year liver biopsy F0 16 (36%) 17 (37%) ns F1 16 (36%) 15 (33%) F3 8 (20%) 9 (18%) F4 4 (9%) 5 (11%) ALT levels (IU)* 1 month 48 (9 609) 64 (17 573) ns 6 months 86 (21 635) 84 (21 488) 12 months 89 (14 555) 91 (21 683) Death (%) 6 (13.5%) 6 (13%) ns Recurrent HCV-disease 4 (67%) 6 (100%) Sepsis 1 (10.5%) 0 Other 1 (16.5%) 0 Abbreviations: IS, immunosuppression; ALT, alanine aminotransferase; Tac, tacrolimus. *median, range. Comparison Between the Cyclosporine and Tacrolimus Groups Randomization of the 90 patients was as follows: Neoral cyclosporine arm (n 44), tacrolimus-based arm (n 46). There were no differences in baseline characteristics, including patient and donor demographics, genotype distribution, surgical-related factors, and immunosuppression besides the calcineurin inhibitor used (Table 1). Clinical Outcome in Those Under Tacrolimus vs. Cyclosporine-Based Immunosuppression Biochemical evolution: Serum alanine aminotransferase levels were similar in both groups at 1, 6, and 12 months (Table 2). Histological outcome: The rate of severe hepatitis was similar in the cyclosporine vs. the tacrolimus group (12/44 vs. 15/46, respectively). In addition, the percentage of patients with a stage of fibrosis 1 in their first-year liver biopsy was the same in both groups (63%). Finally, a diagnosis of acute hepatitis in their first clinically-indicated liver biopsy was similarly found in both groups (32% vs. 35%, respectively). In addition, the same number of patients developed cholestatic hepatitis in both groups (4/44 in the cyclosporine-based group vs. 5/46 in the tacrolimus-based arm). Time to acute hepatitis was shorter in the tacrolimus group compared to the cyclosporine group (59 days [35-185] vs. 92 days [39-343] in the cyclosporine group; P 0.02). Clinical outcome: Graft and patient survival did not differ between the 2 groups (Fig. 1). The causes of death were also similar with recurrent disease accounting for Figure 1. Patient survival in tacrolimus vs. cyclosporinebased immunosuppression. Note: Only patients with at least 1-yr protocol liver biopsy and those with very severe recurrence despite a follow-up of less than 1 yr (cholestatic hepatitis, progression to bridging fibrosis/cirrhosis) were included. Log-rank comparison of treatment groups, P the majority of deaths in these patients (67% in the cyclosporine group vs. 100% in the tacrolimus arm). DISCUSSION Recurrent hepatitis C, presenting as chronic hepatitis in the graft and/or cholestatic hepatitis, is a major problem in liver transplant units, due to both its high frequency and its aggressivity. 1 In the recent consensus conference on this topic held in Phoenix, AZ in March

5 766 BERENGUER ET AL. 2003, the experts raised the need to further explore the effect of immunosuppression on the severity of recurrent hepatitis C. 12 Indeed, there are several indirect findings that suggest that immunosuppression is likely the major factor in determining variations in clinical outcome: 1) the higher rate of HCV-related fibrosis progression in immunosuppressed populations, such as liver transplant recipients or HIV-coinfected patients, compared to immune competent patients; 1,5 2) the higher aggressivity of compensated cirrhosis in transplant recipients compared to nonimmunosuppressed patients with HCV-cirrhosis; 13 3) the known detrimental effect of high methyl-prednisolone boluses on the severity of recurrent hepatitis C; 1,9-11 and 4) the potential implication of more potent immunosuppressive drugs in the recent worsening of recurrent hepatitis C. 2,7,10 Unfortunately, the data on the effect of specific immunosuppressive agents on recurrent hepatitis C are generally controversial. 1,8-12 Several reasons may explain discrepancies between studies, including the fact that most studies are retrospective and hence potentially biased by confounding factors. In addition, few studies are based on prospective protocol liver biopsies performed in the totality of patients analyzed. In some of our earlier studies, the use of cyclosporine appeared to have a beneficial effect on recurrent HCV. 2,7 In addition, a potential antiviral effect of cyclosporine has been recently suggested. 14 Our earlier studies though were retrospective and were aimed at determining which factors were associated with progressive disease. 2,7 In addition, the antiviral effect attributed to cyclosporine derives only from in vitro studies. 14 We therefore decided to randomize new liver transplant patients into 2 arms of immunosuppression: cyclosporine-neoral steroids vs. tacrolimus steroids. The major conclusions from this prospective study may be summarized as follows: 1) cyclosporineneoral based immunosuppression is similar to tacrolimus with regards to the severity of recurrent standard chronic hepatitis C, at least in the short term; 2) cholestatic hepatitis occurs at the same rate, regardless of the calcineurin inhibitor used. We chose to evaluate the outcome at 1 yr since it has become increasingly common to start antiviral therapy based on the results of the first-year protocol liver biopsy. 1,6,15 Given the progressive nature of recurrent hepatitis C, we cannot exclude that the outcome would have been different if the length of histologic follow-up had been extended to 2-3 yr. However, we found unethical to follow our patients longer than 1 yr without any therapeutic intervention on our part for 2 reasons. Firstly a clear relationship between the stage of fibrosis at the first-year biopsy and the risk of subsequent progression to cirrhosis. 6,15 Secondly, the new pegylated interferons are associated with an increased success rate in the liver transplant population. 16,17 In terms of immunosuppression, and since there are data suggesting that a rapid steroid tapering may also be, in part, responsible for the recent worsening in disease progression due to partial immune reconstitution, 2,7,18 we decided to slowly taper steroids with reduction to 5 mg over the first 6 months and subsequent withdrawal during the following 3 to 6 months. Only in cases with severe corticosteroid-related side effects or in those developing fibrosing cholestatic hepatitis, a form of recurrence thought to be caused by a direct effect of the virus, steroids were more rapidly withdrawn. Our results are in accordance to many studies which although not specifically aimed at evaluating the effect of these compounds on histologically-assessed HCVrelated disease progression, found no differences in the overall outcome The results are also similar to those recently described in a prospective study in which however, no data were presented regarding the effect of calcineurin inhibitors on the severity of histologic recurrence. 19 The only statistical difference found between the 2 groups relates to the time to acute hepatitis, shorter in the tacrolimus group compared to the cyclosporine group, an observation recently reported in a multicenter study. 20 Whether this statistical difference though makes a difference from a clinical point of view is debatable. In conclusion, the results from this prospective study confirm previous assumptions that the choice of calcineurin inhibitors, when they are used in dual therapy along with steroids, does not impact the histologic severity of recurrent hepatitis C, at least in the shortterm. It is likely that the pattern of recurrence and the progression of the disease is more a reflection of overall excess immunosuppression than the direct effect of a specific immunosuppressive agent. The development of better in vivo and in vitro systems to evaluate the antiviral effect of different drugs, including immunosuppressive agents will likely help in solving this dilemma. REFERENCES 1. Berenguer M, Lopez-Labrador FX, Wright TL. Hepatitis C and liver transplantation. J Hepatol 2001;35: Berenguer M, Prieto M, San Juan F, Rayón M, Martinez F, Carrasco D, et al. Contribution of donor age to the recent decrease in patient survival among HCV-infected liver transplant recipients. 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6 CALCINEURIN INHIBITORS AND HCV POSTTRANSPLANTATION 767 current hepatitis C after liver transplantation. J Hepatol 2005;42: McCaughan GW, Zekry A. Impact of immunosuppression on immunopathogenesis of liver damage in hepatitis C virus-infected recipients following liver transplantation. Liver Transpl 2003;9(Suppl 3.:S21 S Samonakis DN, Triantos CK, Thalheimer U, Quaglia A, Leandro G, Teixeira R, et al. Immunosuppression and donor age with respect to severity of HCv recurrence after liver transplantation. Liver Transpl 2005;11: Lake JR. The role of immunosuppression in recurrence of hepatitis C. Liver Transpl 2003;9:S63 S International Liver Transplantation Society HCV Consensus Group. Report of the First International Liver Transplant Society Consensus Conference on Liver Transplantation and Hepatitis C. Liver Transpl 2003;9(Suppl 3.:S1 S Berenguer M, Prieto M, Rayón JM, Mora J, Pastor M, Ortiz V, et al. Natural history of clinically compensated HCVrelated graft cirrhosis following liver transplantation. Hepatology 2000;32: Watashi K, Hijikata M, Hosaka M, Yamaji M, Shimotohno K. Cyclosporin A suppresses replication of hepatitis C virus genome in cultured hepatocytes. Hepatology 2003; 38: Firpi RJ, Abdelmalek MF, Soldevila-Pico C, Cabrera R, Shuster JJ, Theriaque D, et al. One-year protocol liver biopsy can stratify fibrosis progression in liver transplant recipients with recurrent hepatitis C infection. Liver Transpl 2004;10: Dumortier J, Scoaxec JY, Chevallier P, Boillot O. Treatment of recurrent hepatitis C after liver transplantation: a pilot study of peginterferon alfa 2b and ribavirin combination. J Hepatol 2004;40: Rodríguez-Luna H, Khatib A, Sharma P, et al. Treatment of recurrent hepatitis C infection after liver transplantation with combination of pegylated interferon alfa 2b and ribavirin: an open label series. Transplantation 2004;77: Brillanti S, Vivarelli M, De Ruvo N, Aden AA, Camaggi V, D Errico A et al. Slowly tapering off steroids protects the graft against hepatitis C recurrence after liver transplantation. Liver Transpl 2002;8: Martin P, Busuttil RW, Goldstein RM, Crippin JS, Klintmalm GB, Fitzsimmons WE, et al. Impact of tacrolimus versus cyclosporine in hepatitis C virus-infected liver transplant recipients on recurrent hepatitis C: a prospective randomized trial. Liver Transpl 2004;10: Levy G, Villamil F, Samuel D, Sanjuan F, Grazi GL, Wu Y, et al. Results of list2t, a multicenter, randomized study comparing cyclosporine microemulsion with C2 monitoring and tacrolimus with C0 monitoring in de novo liver transplantation. Transplantation 2004;77: