Although recurrence of hepatitis C after liver transplantation (LT) is almost universal, the clinical outcome of posttransplantation hepatitis C is highly variable, with as many as 30% of patients progressing to severe disease within 5 yr after transplantation, while the majority have minimal or nonprogressive liver injury.1–3 Once the diagnosis of recurrent cirrhosis has been made, the risk of decompensation has been estimated to be approximately 50% in 1 yr.3 This course of the disease is clearly accelerated when compared with that observed in the nontransplant population and decreases survival. In this respect, data from the United Network for Organ Sharing registry indicate that LT in hepatitis C virus (HCV)-positive recipients is associated with an increased rate of death and graft failure as compared with patients who are HCV-negative.4 In addition, although individual host and viral characteristics have been associated with poor patient and graft survival,4–9 no model exists that can reliably identify those recipients at greatest risk for either patient mortality or development of severe recurrent disease after transplantation. Moreover, a further complicating factor is that the rate of recurrent cirrhosis appears to have increased in recent years and the reasons for these worse results have been associated with the increasing age of donors and the use of more potent immunosuppressive protocols.10, 11 Given this background, the present study had 2 main aims:
1to assess patient survival and fibrosis progression in different transplant years; and
2to model the effects of pre- and posttransplantation variables on both patient survival and severity of the recurrent disease defined as the development of a Ishak fibrosis score >3.
To do this, we evaluated a large number of HCV-positive recipients transplanted in 3 European centers, each center having similar transplant activity over the years and adopting protocol liver biopsies during the follow-up.
Between January 1990 and December 2002, 502 consecutive patients with HCV-related cirrhosis and without B or Delta coinfections were transplanted in 3 different European Centers (Niguarda Hospital in Milan (center 1), Royal Free Hospital in London (center 2), and University Hospital in Padua (center 3). All 502 patients were included in the overall survival analysis, while only the 354 patients with a follow-up longer than 1 yr were considered for the analysis of predictors of both disease progression and patient survival from 1 yr onward. HCV infection was defined as positivity of serum HCV-ribonucleic acid by reverse transcription polymerase chain reaction.
Protocol liver biopsies were performed at least at 1, 3, and 5 yr posttransplantation, independently of liver biochemistry. Beyond this interval, a late liver biopsy was usually performed between 7 and 10 yr after transplantation. Considering only the scheduled liver biopsies performed after the first posttransplantation year, a total of 847 biopsy specimens were available for histological analysis with a mean of 2.43 per patient (median 2, range 1-5). Liver biopsies were reviewed by 3 local pathologists (E.M. in Milan, A.Q. in London, and M.G. in Padua) who agreed to utilize the Ishak score for assessing the stage of fibrosis and the degree of necroinflammatory activity.12 Protocol liver biopsies were available in 342 of 354 (97%), 224 of 251 (89%), 156 of 175 (89%), 83 of 116 (72%), and 42 of 47 (89%) recipients who were followed up for 1, 3, 5, 7, and 10 yr after orthotopic LT. Notably, overall expected liver biopsies at 3 centers were 90%, 84%, and 92%. All liver biopsies were blindly reevaluated at each site for the purpose of this study and the pathologists agreed on these 2 main points: 1) to recognize the parameter fibrosis S4-6 (Ishak fibrosis score 4-6); and 2) to consider as inadequate liver biopsies with less than 6 complete portal spaces.
Induction immunosuppression did not change over the years and consisted of quadruple therapy with rabbit antithymocyte globulins (ATG; Fresenius, Bad Homburgh, Germany) for the first 5 days, azathioprine for the first 30 days, steroids that were generally withdrawn within 3 months from transplant, and cyclosporine maintained long-term. Protocol biopsies were not used to evaluate rejection.
Induction protocols were changed over the years: for the first 5 yr of transplant activity, most patients received standard triple therapy (cyclosporine, azathioprine, and steroids), whereas in the following years, tacrolimus was substituted for cyclosporine or tacrolimus was used as monotherapy. Steroids were usually withdrawn within 6 months, whereas azathioprine was continued long-term unless side effects or complications developed. Secondary drugs, including azathioprine, mycophenolate mofetil, and steroids, were discontinued within the first year after transplantation. Protocol biopsies were used to evaluate rejection.
Double-drug induction (cyclosporine or tacrolimus and corticosteroids) was the standard protocol. Steroids were withdrawn within 6 months from transplant and progressively earlier in recent years. Protocol biopsies were not used to evaluate rejection.
In case of renal impairment (defined as a plasma creatinine >1.5 mg/dL) all 3 centers adopted the policy of reducing target cyclosporine or tacrolimus levels, which were in general halved, while either azathioprine or mycophenolate acid were continued or added.
The diagnosis of hepatitis C was based on the detection of anti-HCV and HCV-ribonucleic acid in the serum (HCV amplicor kit; Roche Diagnostic Systems, Branchburgh, NJ) prior to transplantation. HCV genotypes were typed according to different standardized methods. In 20% of patients, genotyping results were not available mainly due to lack of serum samples. The viral load was not evaluated as a predictive factor, because the tests used to quantify viremia have changed substantially over the years, and correctly stored serum samples were not usually available.
Follow-up time was defined as the number of months from LT to clinical events, death, or the last contact with the patient up to December 31, 2003. Follow-up data were obtained through periodic controls at outpatient clinics. Clinical examination and routine laboratory investigations were performed at least every 6 months in all cases. The primary outcome was survival. The secondary outcome was severe fibrosis progression, defined as the development of an Ishak fibrosis score >3.
Continuous variables were expressed as mean ± standard deviation. The χ2 and Student's t-test were performed as appropriate, all P values were 2-tailed. The Kaplan–Meier method was used to estimate the length of survival and of survival without severe fibrosis progression. Different variables regarding the recipient, the donor, and the virus were considered for univariate analysis. Variables with a P value <0.10 at univariate analysis were included in the final multivariate model. The following variables were evaluated by univariate analysis for both patient survival and development of stage 4–6 fibrosis. 1) Host risk factors: age at transplantation and gender, presence of hepatocellular carcinoma either known or incidental. 2) Viral factors: genotype (genotype 1 or 4 vs. non-1 non-4). 3) Donor-related variables: age, gender, and donor/recipient gender mismatch. In accord with data coming from the literature,10 donor age was also categorized in the following cohorts: <50; 50–59, and >60 yr. 4) External risk factors: including the type of immunosuppression (cyclosporine vs. tacrolimus; induction with antilymphocytic preparations; use of steroid pulses during the first 6 months; exposure yes/no and length of administration of azathioprine, steroids, and mycophenolate mofetil) and antiviral therapy with at least 6 months of interferon and ribavirin. 5) Year of transplantation both as a continuous variable and categorized as follows: 1990–1992; 1993–1994; 1995–1996; 1997–98; 1999–2000; and 2001–2002.
The Cox proportional hazard model was used to assess survival and survival without severe fibrosis progression in a multiple regression analysis. All analyses were conducted with SAS version 6.08 (SAS Institute, Cary, NC). For the Cox regression, the SAS PHREG procedure was used. All P values were 2-tailed, and all the confidence intervals (CIs) were 95%. We verified that the assumption of proportional hazard in the Cox model, was not violated; this was also checked with analytical and graphical methods. We also performed a test of trend in the hazard ratio by adding into the model a new variable representing the interaction effect between the prognostic variable and the follow-up time. If this variable is not statistically significant, this represents a strong evidence that there is no trend (increase or decrease) over time in the hazard ratio. To estimate expected survival time for a hypothetical patient with a combination of prognostic factors, the estimate survival function was computed.
Patient Characteristics at Baseline
All 502 patients were included in the overall survival analysis while only the 354 patients with a follow-up longer than 1 yr were considered for the analysis of predictors of both disease progression and patient survival from 1 yr onward (Fig. 1). Of the 148 patients excluded, 72 died within the first 3 postoperative months (early post-LT mortality), usually from a combination of poor graft function and sepsis and none from HCV recurrence. A total of 33 patients died between 3 months and 1 yr from transplant and were excluded because they did not survive long enough to reach the time of the 1-yr protocol liver biopsy. The causes of death of these 33 patients were: recurrent hepatocellular carcinoma (6), vascular accidents (6), hematological neoplasms (3), chronic rejection (3), a combination of sepsis and poor graft function (12), HCV cholestatic recurrence (2), and cryptogenic cirrhosis (1). Of the remaining 43 excluded patients who survived beyond 1 yr, 33 were “dropouts,” either due to lack of adequate liver histology during follow-up (25 patients) or were lost to follow-up (8 patients). Finally, the remaining 10 patients were excluded for the presence of ongoing confounding factors (4 patients with ongoing biliary problems, 3 with de novo hepatitis B virus, 2 with arterial occlusion, and 1 with portal thrombosis)
The baseline characteristics of the subgroup of 354 patients eligible for the analysis of predictors are shown in Table 1. Notably, no differences were found between the baseline clinical features of the whole cohort of 502 patients and those of the 354 patients considered in the analysis of predictors.
Table 1. Baseline Clinical Features of the 354 Patients Eligible for the Analysis of Predictors of Severe Fibrosis Development (Ishak Fibrosis Score >3) in the 3 Centers
Overall Survival Analysis on the Whole Population of 502 Patients: Effect of Year of Transplantation
The overall survival rates were 78.7%, 66.3%, and 58.6%, at 12, 60, and 120 months, respectively, without a significant difference among the 3 participating centers (P = 0.2 by log-rank test). In more detail, overall patient survival at 1, 5, and 10 yr after transplantation was: 74%, 64%, and 58% at Center 1; 78%, 68%, and 61% at Center 2; and 83%, 75%, and 65% at Center 3.
A favorable trend over the years was observed when the survival analysis was performed according to Kaplan-Meier (Fig. 2). We also arbitrarily split our patient cohort into 2 subgroups (patients transplanted before and after December 31, 1998) to make our results comparable with those of the Valencia group that reported a very unfavorable outcome after December 31, 1998. When comparing patients receiving transplants before and after December 31, 1998, we observed a reduced mortality both at 3 and 12 months in patients receiving transplants after 1998 (P = 0.09 and P = 0.001) (Table 2).
Table 2. Mortalities at 3 and 12 Months Post-LT in the Overall Cohort of 502 Patients: Comparison Between LT Performed Before and After December 31, 1998
Mortality at 3 months (%)
Mortality at 12 months (%)
Variables Associated With Patient Survival (From 1 Yr) and With Severe Fibrosis Progression
A total of 59 patients died during the follow-up period. Severe HCV recurrence was responsible for more than one-half of the deaths (33/59, 56%). The causes of death in the remaining 26 patients were as follows: de novo tumor (8), recurrent hepatocellular carcinoma (4), sepsis (3), chronic rejection (3), vascular accidents (3), and other various causes (5). Notably, the Ishak fibrosis scores in the 26 patients who died of causes unrelated to HCV ranged between 1 and 3.
A total of 89 of the 354 (25.1%) transplant recipients developed an Ishak fibrosis score 4-6 after a median time of 35 months posttransplantation. The overall 1-, 3-, 5-, and 10-yr cumulative incidence of Ishak fibrosis score 4-6 was 10%, 18%, 27%, and 32%, respectively. Notably, this cumulative incidence of Ishak fibrosis score 4-6 did not change significantly over the years (P = 0.6).
By univariate analysis, 5 variables (presence of hepatocellular carcinoma, advanced donor age, therapy of acute rejection, fibrosis progression beyond stage 3, and earlier year of transplantation [both as a continuous and categorical variable]) were significantly associated with reduced patient survival and were included in a Cox proportional hazards model. Of these 5 variables, only 3 (advanced donor age, fibrosis progression beyond stage 3, and earlier year of transplantation) remained significant after Cox multivariate analysis (Table 3).
Table 3. Predictors of Death and of Development Severe Fibrosis (Ishak Score >3)
By univariate analysis, 5 variables (recipient female gender, advanced age of the donor, female gender of the donor, donor/recipient gender mismatch, and avoidance of induction therapy with antilymphocytic preparations) were significantly associated with the development of Ishak fibrosis score 4-6 and were included in the Cox proportional hazards model. Of these 5 variables, only 3 (recipient female gender, advanced age of the donor, and avoidance of antilymphocytic preparations for induction) remained significant after Cox multivariate analysis (Table 3).
The prognostic effect of donor age on fibrosis progression was confirmed in all 3 subcohorts of each participating center. As for female gender of the recipient, all 3 subgroups analyzed showed a similar “effect size” but no statistical significance (Center 1: hazard ratio [HR] 0.54, 95% CI 0.25-1.16, P = 0.11; Center 2: HR 0.52, 95% CI 0.27-1.005, P = 0.051; Center 3: HR 0.59, 95% CI 0.22-1.158, P = 0.29). The third variable “induction with antibody” could not be retested in the 3 separated subcohorts as it was mainly utilized in Center 1. Upon replacing Ishak fibrosis score 5-6 as the dependent variable in the multivariate analysis, we obtained a model in which donor age (HR 1.034; 95% CI 1.018-1.050) emerged as the only significant predictor while recipient gender (HR 0.62; 95% CI 0.38-1.022) and induction with rabbit antithymocyte globulins (HR 0.65; 95% CI 0.40-1.080) became of marginal statistical significance.
We finally combined the 2 significant prognostic factors that can easily be obtained before transplantation (recipient gender and donor age) looking for possible interactions. The estimated probabilities of severe fibrosis progression for hypothetical recipients according to their gender and age of their donors are shown in Figure 3. Notably, a female recipient receiving a graft from a 60-yr-old donor had an almost doubled risk of worsening severity of hepatic fibrosis than a male recipient receiving a graft from the same 60-yr-old donor.
Although the main limits of the present study, as in many other retrospective studies, are related to the heterogeneity of the posttransplantation immunosuppressive regimens and the presence of many other confounding variables, nevertheless, it has 2 major strengths as it considers a large cohort of HCV liver transplant recipients followed up for a long period of time and it evaluates fibrosis progression after LT using protocol liver biopsies scheduled up to 10 yr after transplantation. Our main findings can be summarized as follows: 1) patients receiving transplants in more recent years have a significant improved outcome in spite of the increased utilization of older donors; 2) older donors and the female gender of the recipients are the 2 major risk factor for the development of severe recurrent disease, while the adoption of an induction therapy with antibody preparations is associated with a less aggressive course.
These observations stand in sharp contrast to what has been previously reported by some groups who showed a dramatic worsening in the outcome of HCV patients transplanted after 1998,3, 5, 11 with only a 60% survival at 1 yr in 1 center.10, 11 One explanation given was possibly the combined detrimental effect of the increasing age of the donors and the use of more potent induction immunosuppression, followed by a rapid reconstitution of the immune system with discontinuation of secondary drugs at earlier time points than was previously done. The present study confirms that the age of the donor is an important determinant of both patient survival and fibrosis progression.10, 13, 14 However, the survival improved over the years despite the fact that the median age of donors in our cohort was even higher than that reported by the Valencia group and there did not appear to be a worsening of the fibrosis progression over the years. The improved survival in the present study is in keeping with the recent analysis of the United Network for Organ Sharing database on 6,441 HCV patients transplanted after 1994,15 showing that graft and patient survival was not lower in recent years compared with earlier time periods. Moreover, another study based on strict histologic surveillance of 183 HCV recipients undergoing LT in Berlin over the last 15 yr did not see any increase in fibrosis progression in patients transplanted after 1996 when compared with those transplanted before 1996.16 Notably, a very recent work from the Valencia group,17 which considered patients transplanted between 2001 and 2004, reported a significant improvement in the outcome of HCV infected patients in line with this and many other experiences.
Our finding that the use of T-cell-depleting drugs in the induction phase of immunosuppression is associated with a protective effect on fibrosis progression is rather intriguing. These regimens, in fact, result in very low rates of acute cellular rejection. One can speculate that avoidance of cellular rejection may be crucial in these patients as it will spare the utilization of steroid pulses, which have been frequently associated with severe recurrent disease.18–20
A key result of our analysis is that female recipients have worse HCV-induced fibrosis progression than male recipients and, importantly, this finding was observed in all 3 subcohorts of patients from the 3 participating centers. Although to our knowledge this association has never been reported in the past, data from the United Network for Organ Sharing registry have clearly indicated that HCV-positive female recipients have worse survival than males.4 It is difficult to explain such a negative association between female gender and fibrosis progression, particularly if we consider that these results are at odds with several studies of HCV progression in nontransplant patients, in which male gender and not female is associated with a worse outcome. However, postmenopausal females lose this advantage in the pretransplantation setting, which may represent a better comparison with the posttransplantation population. Preliminary data in immunocompetent individuals with chronic hepatitis C suggest that steatosis is frequently encountered in postmenopausal women and is an important cofactor for disease progression.21 Since the majority of our female recipients are in the postmenopausal state, this association needs to be reinvestigated in appropriately-designed studies. To evaluate the clinical relevance of our findings, we evaluated the estimated risk of severe recurrent disease due to the combination of 2 prognostic factors, both of which can be easily obtained before transplantation: recipient gender and donor age. The results demonstrate a very important synergy such that the allocation of an old donor (>60 yr) to a female recipient with hepatitis C leads to severe fibrosis progression within 5 yr from transplantation in about 90% of cases. On the contrary, the allocation of the same old donor to a male recipient allows much better results, with less than 50% of the male patients progressing beyond Ishak fibrosis score 3 at 5 yr from transplantation. Currently, prioritization of recipients for LT in the United States is based solely on recipient characteristics (Model for End-Stage Liver Disease score, which combines serum bilirubin, creatinine, and international normalized ratio of prothrombin time), which does not take into consideration the etiology of the disease in the recipient nor their gender. Neither does it considers any donor factor. Although it is too early to suggest changes of the allocation procedures based on our findings, which need to be confirmed by others, nevertheless this study strongly suggests that the combination of a female recipient receiving an old graft should be considered a strong risk factor for the development of severe recurrence.