Y. Ueda; H. Marusawa; T. Kaido; Y. Ogura; F. Oike; A. Mori; K. Ogawa; A. Yoshizawa; E. Hatano; A. Miyagawa-Hayashino; H. Haga; H. Egawa; Y. Takada; S. Uemoto; T. Chiba
Posted: 02/07/2012; J Viral Hepat. 2012;19(1):32-38. © 2012 Blackwell Publishing
Abstract and Introduction
Approximately 30% of patients who have recurrent hepatitis C after liver transplantation achieve sustained virological response (SVR) by taking a combination therapy of pegylated interferon and ribavirin. For the remaining non-SVR patients, an effective management treatment has not yet been established. In this study, efficacy of long-term peginterferon maintenance therapy for non-SVR patients was evaluated. Forty patients who had previously received the combination therapy for hepatitis C after living donor liver transplantation were classified into one of the following three groups: the SVR group (n = 11); the non-SVR-IFN group (n = 17), which received low-dose peginterferon maintenance therapy for non-SVR patients; and the non-SVR-Withdrawal group (n = 12), which discontinued the interferon treatment. We then compared histological changes among these three groups after 2 or more years follow-up. Activity grade of liver histology improved or remained stable in patients in the SVR and non-SVR-IFN groups, but deteriorated in half of the patients in the non-SVR-Withdrawal group. Fibrosis improved or remained stable in 10 of 11 SVR patients and in 13 of 17 non-SVR-IFN patients, but deteriorated in all non-SVR-Withdrawal patients. Mean changes in fibrosis stage between pretreatment and final liver biopsy were −0.18, +0.06 and +2.2 in the SVR, non-SVR-IFN and non-SVR-Withdrawal groups, respectively. Fibrosis stage deteriorated to F3 or F4 significantly more rapidly in the non-SVR-Withdrawal group than in the other two groups. In conclusion, continuing long-term maintenance therapy with peginterferon prevented histological progression of hepatitis C in patients who had undergone living donor liver transplantation.
Cirrhosis and hepatocellular carcinoma caused by hepatitis C virus (HCV) infection is the leading indication for liver transplantation in Japan, the United States and western Europe. However, liver allograft infection with HCV following liver transplantation is universal, and almost all patients develop recurrent liver injury.[1–6] The progression of recurrent hepatitis C is often accelerated and, without appropriate antiviral therapy, 10–25% of patients develop cirrhosis within 5 years after transplantation, resulting in poorer prognosis for HCV-positive recipients than HCV-negative recipients.
To prevent the progression of hepatitis C after liver transplantation, a combined therapy of pegylated interferon plus ribavirin is commonly administered.[8,9] However, the efficacy of this combination therapy is limited: The mean sustained virological response (SVR) rate among patients with recurrent hepatitis C after liver transplantation was only 30% (range, 8–50%). Effective management of the remaining 70% of the patients who are unable to achieve SVR has not been established.
We recently reported the change in liver histology after combination therapy with interferon plus ribavirin in patients who have recurrent hepatitis C after living donor liver transplantations (LDLT). Among patients who did not achieve SVR, activity grade was not improved and fibrosis stage deteriorated. On the other hand, SVR was associated with reduced hepatic inflammation and suppression of liver fibrosis progression. Because the histological progression of non-SVR patients occurred mainly after interferon therapy was discontinued, we hypothesized that long-term, continuous interferon administration might be effective in slowing the progression of liver damage in these patients. Therefore, after our previous study, we prescribed a low-dose peginterferon maintenance therapy for non-SVR patients. Here, we evaluated the efficacy of this treatment by investigating long-term histological changes in these patients, as well as comparing them to the changes observed in SVR patients and non-SVR patients who did not receive maintenance treatment.
Eighty patients who had previously received the combination therapy with interferon and ribavirin (n = 40) or peginterferon and ribavirin (n = 40) for recurrent hepatitis C after LDLT at Kyoto University between January 2001 and April 2007 were retrospectively analysed.
Between March 1999 and December 2006, 141 patients with HCV-related liver diseases underwent LDLT at Kyoto University. Of these, 100 patients had been followed up for more than 6 months after LDLT in our hospital. Antiviral therapy was given to 80 patients with recurrent hepatitis C between January 2001 and April 2007. The remaining 20 patients did not receive the antiviral therapy because of no histological recurrence of hepatitis C in the follow-up period. To evaluate the histological progression caused by hepatitis C, patients who were diagnosed as having other causes of liver injury, such as biliary complications, chronic rejection, and de novo autoimmune hepatitis (AIH), were excluded. Patients who discontinued the treatment within 3 months because of worsening of liver function caused by hepatitis C were also excluded, because the rapid progression of these patients is not comparable to the long-term progression and inclusion of these patients would have led to overestimation of the progression in the patients who discontinued treatment. Patients were also excluded if they did not have a liver biopsy more than 2 years after the initiation of treatment, because this prevented an analysis of long-term histological changes.
Treatment Protocol and Definition of Responses to Treatment
After liver transplantation, patients with recurrent HCV liver disease underwent treatment with interferon–α–2b (3 or 6 mega units thrice weekly) plus ribavirin (400–800 mg/day orally) for the first 6 months. This was followed by interferon monotherapy for 6 months. This treatment protocol was employed between January 2001 through April 2004 inclusive. From May 2004 to April 2007, patients underwent combination antiviral therapy comprising peginterferon–α–2b (1.5 μg/kg body weight, weekly) and ribavirin (400–800 mg/day orally). Patients who became negative for serum HCV RNA within 12 months after initiating the treatment continued to receive the full (initial) dose for 8–22 months to achieve SVR; then, the treatment ended. Patients who were negative for serum HCV RNA for more than 6 months after completion of interferon therapy were defined as achieving SVR.
Patients who did not become negative for serum HCV RNA within 12 months of initiating the combination therapy, as well as patients who experienced a relapse after transient discontinuation of the treatment, continued to receive a low-dose peginterferon maintenance therapy (0.5–0.75 μg/kg of peginterferon-α-2b with or without ribavirin at 200 mg/day). Treatments occurred during the study period, May 2005–December 2009. During this time, the therapy was discontinued in patients with severe adverse events. Additionally, peginterferon treatments were discontinued when neutrophil and platelet counts fell below 500 and 30 000/μL, respectively, and ribavirin was discontinued when haemoglobin levels fell below 8 g/dL.
Liver biopsies were performed when patients' alanine aminotransferase (ALT) levels were more than twice the upper limit of normal, or at yearly intervals, with informed consent. Biopsy specimens were evaluated by two pathologists (H.H. and A.M.) with extensive experience in the pathology of liver transplantation. Necroinflammatory activity (A0–A3) and fibrosis stage (F0–F4) were assessed using METAVIR scores.[14,15] Grading was defined as A0 (no activity), A1 (mild activity), A2 (moderate activity) or A3 (severe activity); staging was defined as F0 (no fibrosis), F1 (mild fibrosis), F2 (moderate fibrosis), F3 (severe fibrosis) or F4 (cirrhosis).[14,15]
The following equations were used to analyse the histological changes:
- Changes in activity grade = grade at final biopsy − grade at pretreatment biopsy, and
- Changes in fibrosis stage = stage at final biopsy − stage at pretreatment biopsy.
Tacrolimus and low-dose steroid therapies were administered to induce immunosuppression.[12,13,16] The lower limit of the target for whole blood tacrolimus level was 10–15 ng/mL during the first 2 weeks, 10 ng/mL during weeks 2–8 and 5–8 ng/mL thereafter. Four patients received cyclosporine microemulsions, rather than tacrolimus, to induce immunosuppression (Table 1). Steroid therapy was initiated at a dose of 10 mg/kg before graft reperfusion and then tapered from 1 mg/kg per day on the first day to 0.3 mg/kg per day until the end of the first month, followed by 0.1 mg/kg per day until the end of the third month. After that, steroid administration was terminated. Mycophenolate mofetil (MMF) was administered to patients who experienced refractory rejection or required reduction in tacrolimus or cyclosporine doses because of adverse events.
Hepatitis C virus genotype was determined using a genotyping system based on polymerase chain reaction (PCR) of the core region using genotype-specific PCR primers. Serum HCV RNA load was evaluated once a month during treatment and 24 weeks after treatment, using PCR and an Amplicor HCV assay (Cobas Amplicor HCV Monitor; Roche Molecular Systems, Pleasanton, CA, USA).
Wilcoxon and Kruskal–Wallis tests, chi-square tests and t-tests were used to analyse the continuous variables, categorical variables and histological changes, respectively. The Kaplan–Meier method was used to estimate the rates of patients who showed a progression of fibrosis to stage F3 or F4 after the initiation of the interferon therapy; log-rank tests were used to compare these rates among groups. Significance was defined as P < 0.05.
Characteristics of Patients
Hepatitis C virus RNA concentrations and histological evidence were used to diagnose 80 patients with recurrent hepatitis C after LDLT. These patients were given one of two combination therapies: interferon and ribavirin (n = 40) or peginterferon and ribavirin (n = 40) at Kyoto University between January 2001 and April 2007. Thirty-one of the 80 patients who received the combination therapy achieved SVR (Fig. 1). Among the remaining 49 non-SVR patients, 23 (47%) received the low-dose peginterferon maintenance therapy, while 26 (53%) discontinued treatment within 12 months and did not receive low-dose peginterferon maintenance therapy as this was the patients' wish (n = 4), because of general fatigue (n = 4), recurrent hepatocellular carcinoma (n = 4), worsening of liver function (n = 3), biliary complications (n = 3), heart failure (n = 2), brain haemorrhage (n = 1), dementia (n = 1), sinusitis (n = 1), anaemia (n = 1), neutropenia (n = 1), and haemosputum (n = 1).
Figure 1. Flow diagram showing the outcome of interferon therapy for patients with recurrent hepatitis C after living donor liver transplantation and indicating the classification of patients in this study.
Of the 31 SVR patients, five were excluded because of chronic rejection (n = 3), biliary complications (n = 1) and de novo AIH (n = 1). Fifteen patients did not have liver biopsies more than 2 years after the initiation of the interferon therapy, mainly because liver function tests were normal. The remaining 11 patients were classified as the SVR group for analysis in this study. Among the 23 patients who received maintenance therapy, one patient with biliary complications and five patients who did not have liver biopsy more than 2 years after the initiation of therapy were excluded from the study. The remaining 17 patients were classified into the non-SVR-IFN group. Among the 26 patients who discontinued treatment within 12 months, three patients who initially experienced worsening of liver function were excluded because of the rapid progression of HCV; an additional three patients were excluded because of biliary complications. Eight patients were excluded because they had no liver biopsies taken more than 2 years after the initiation of the treatment. The remaining 12 patients were classified into the non-SVR-Withdrawal group. Cumulatively, we analysed the long-term histological changes of 40 patients: 11 in the SVR group (27.5% of the total), 17 in the non-SVR-IFN group (42.5% of the total) and 12 in the non-SVR-Withdrawal group (30% of the total).
There were no significant differences in the baseline characteristics among patients in the SVR, non-SVR-IFN, and non-SVR-Withdrawal groups (Table 1). The median age of patients at the beginning of therapy was 56.5 years (range, 15–70 years). The treatment started at a median of 9.5 months (range, 1.1–85.3 months) after LDLT. Thirty-five patients (88%) were infected with HCV genotype 1b. HCV genotypes of the remaining patients were 2a (n = 3), 2b (n = 1) and undetermined (n = 1). Median serum HCV RNA load was 2290 kIU/mL (range, 73.7–5000 kIU/mL); i.e. most patients had an extremely high viral load. Before the treatment, the necroinflammatory activity of all patients was A1 or greater, and 33 patients (83%) had a fibrosis score of F1 or greater. Among patients receiving tacrolimus for immunosuppression, the median serum trough level was 5.95 ng/mL (range, 3.3–10.9).
Effect of Maintenance Interferon Therapy on Liver Histology
To evaluate the efficacy of long-term peginterferon therapy on histological changes, we compared scores between final biopsy samples (median, 44.0 months; range, 24.0–81.3 months) and those taken prior to treatment. Five patients in the non-SVR-IFN group discontinued maintenance therapy between 26.5 and 53.1 months after the initiation of the treatment because of the adverse events. For these patients, the biopsies taken just before or within 3 months after discontinuation of the treatment were analysed as final biopsies. Despite the variation in time between pretreatment and final biopsy sample collection, there were no significant differences in the duration among the three groups (P = 0.547). Median duration from initiation of interferon therapy to final liver biopsy was 41.9 months (range, 24.0–81.3 months) in the SVR group, 41.7 months (range, 26.5–68.4 months) in the non-SVR-IFN group and 46.5 months (range, 30.4–79.6 months) in the non-SVR-Withdrawal group.
There were no significant differences in baseline activity grades or fibrosis stages of patients in the three treatment groups when they were first diagnosed with recurrent hepatitis C (Table 1). However, there were noticeable differences among the three groups by the end of treatment (Fig. 2a). The activity grade of all patients in the SVR and non-SVR-IFN groups improved or remained stable, whereas it deteriorated in 6 (50%) of 12 patients in the non-SVR-Withdrawal group. The fibrosis stage deteriorated in all patients in the non-SVR-Withdrawal group; nine of these patients (75%) deteriorated by more than one stage. In contrast, only four patients (24%) in the non-SVR-IFN group deteriorated, all by only a single stage. Furthermore, three patients actually improved. In the SVR group, fibrosis stage decreased or remained stable in 10 of 11 patients (91%).
Figure 2. Effect of maintenance interferon therapy on liver histology: (a) Changes in activity grade (upper) and fibrosis score (lower) of individual patients before interferon therapy (Pre) and at final biopsy (final). (b) Mean changes of liver activity grade (left) and fibrosis stage (right) between pretreatment liver biopsy and the final liver biopsy in each of the three treatment groups. The error bars represent 2 SEs. (c) Kaplan–Meier estimates of the progression rates among patients whose fibrosis advanced to F3 or F4. The dashed line indicates the sustained virological response (SVR) group, the solid line indicates the non-SVR-IFN group and the dotted line indicates the non-SVR-Withdrawal group
In patients in the SVR and non-SVR-IFN groups, the mean activity grade was markedly reduced in the final biopsy, compared to the pretreatment biopsy (Fig. 2b). In contrast, patients in the non-SVR-Withdrawal group experienced an increase in activity grade. The differences between the non-SVR-Withdrawal group and both the SVR and the non-SVR-IFN groups were statistically significant (P <0.001). The mean changes in fibrosis stage in the SVR and non-SVR-IFN groups were −0.18 and +0.06, respectively, suggesting that fibrosis did not change during the follow-up period. However, there was an obvious increase (+2.2) among patients in the non-SVR-Withdrawal group, indicating marked progression of fibrosis.
The Kaplan–Meier analysis allowed us to investigate whether patients in the three treatment groups experienced different progression rates to late-stage fibrosis (Fig. 2c). No patient in the SVR group and only 1 patient (6%) in the non-SVR-IFN group developed fibrosis stage F3 or F4, whereas nine patients (75%) in the non-SVR-Withdrawal group progressed to these stages. The rates of fibrosis progression were significantly higher in the non-SVR-Withdrawal group than in the non-SVR-IFN and SVR groups (P = 0.0049 and P = 0.0086, respectively). There was no significant difference between the SVR group and the non-SVR-IFN group (P = 0.3980). Five-year progression rates to F3 or F4 were 0% in the SVR group, 14% in the non-SVR-IFN group and 54% in the non-SVR-Withdrawal group.
Safety and Tolerability of Maintenance Interferon Therapy
Five of 17 patients (29%) who received low-dose maintenance peginterferon treatment discontinued interferon therapy because of biliary complications (n = 2), neutropenia (n = 1), anaemia (n = 1) and de novo AIH (n = 1), between 26.5 and 53.1 months after its initiation. The biliary complications were not related to interferon therapy. Patients with neutropenia and anaemia recovered after discontinuing interferon therapy and were able to resume therapy within months (3 and 10, respectively). Steroid therapy alleviated the de novo AIH, but the patients did not resume interferon therapy.
Studies have repeatedly shown the benefits of achieving SVR via interferon therapy after liver transplantation. For instance, the durability of the SVR is associated with improvements in hepatic inflammation and histological regression of fibrosis over the long-term.[18–23] In contrast, efficacy of interferon therapy for non-SVR patients after liver transplantation had not previously been investigated. Here, we have demonstrated that long-term peginterferon maintenance therapy suppresses histological progression of recurrent hepatitis C after LDLT.
Maintenance interferon therapy was recently shown to have no influence on either histological or clinical outcomes in patients with nontransplant hepatitis C. This conclusion was drawn after observing that the rate of fibrosis progression was similar between treatment and control groups following a 3.5-year randomized controlled trial of low-dose peginterferon. As a large number of patients with advanced fibrosis were enrolled in the randomized controlled trial, it is difficult to compare with our study in which the number of patients studied is much smaller and patients with advanced fibrosis were not enrolled. In the current study after liver transplantation, however, we demonstrated that low-dose maintenance interferon therapy reduced necroinflammatory activity and fibrosis scores in non-SVR patients to levels similar to those in SVR patients. Furthermore, we found that non-SVR patients who discontinued treatment had significantly worse scores once no longer receiving therapy.
Although these results clearly suggest that low-dose peginterferon maintenance therapy is beneficial for non-SVR patients with recurrent hepatitis C after liver transplantation, the mechanism behind this positive response is unknown. Progression of hepatitis C and development of fibrosis after discontinuation of interferon treatment has been shown to proceed more rapidly in patients who have undergone liver transplantation.[20,21] Our results, indicating that activity grade and fibrosis stage markedly deteriorated in non-SVR patients who discontinued maintenance treatment, support these previous findings. Thus, such a rapid progression of recurrent hepatitis C in patients who discontinued interferon therapy may have highlighted the beneficial effect of the low-dose peginterferon maintenance therapy.
Another issue is the tolerability and safety of long-term peginterferon maintenance treatment. In this study, five patients (29%) discontinued the treatment during the peginterferon maintenance treatment, but only three did so for reasons directly related to the treatment. While two of these patients recovered simply by discontinuing the treatment, the third did require steroid pulse therapy to treat de novo AIH. Overall, however, the maintenance therapy did not result in the incidence of major adverse events, suggesting that it is both a tolerable and a safe treatment method.
Our work shows that long-term, low-dose peginterferon administration is an effective method for inhibiting the progression of liver damage for recurrent hepatitis C after liver transplantation. Unfortunately, this was not a randomized control study, and only a small number of patients were eligible for research. Therefore, we recommend further work to more fully explore the effects of this treatment and to improve the outcomes for patients who do not achieve SVR.
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