Long-term Combination of Interferon Alfa-2b and Ribavirin for Hepatitis C Recurrence in Liver Transplant Patients

Authors


Abstract

The purpose of this study was to evaluate the feasibility, tolerability and efficacy of long-term combination therapy with interferon-alfa 2b (IFNα-2b) and ribavirin (Rb) for recurrent hepatitis C after liver transplantation. Fifteen patients with histologically confirmed hepatitis C after liver transplantation were treated. After a basic course of 12 months (IFNα-2b 3 MU/3 times a week; Rb 3 × 200 mg/day), patients achieving clearance of viremia underwent maintenance therapy with ribavirin (3 × 200 mg/day). Patients without virological response continuously received combination therapy. Levels of HCV RNA, aminotransferases and bilirubin were followed. Therapy led to a significant decline of transaminases and bilirubin in all patients (p < 0.05). Sixty-four per cent of patients had clearance of viremia after 12 months. Sustained virological response was 88%. In patients without virological response, continuation of combination therapy prevented another biochemical relapse of hepatitis. Treatment was accompanied by severe hematological side-effects, requiring medical support in a majority of patients. In two patients (13.5%), therapy finally had to be withdrawn because of major hematological disorders. These results indicate that long-term combination therapy with IFNα-2b and Rb is effective in the treatment of recurrent hepatitis C and in preventing further relapse of disease after liver transplantation, but side-effects may require cessation of therapy.

Introduction

Hepatitis C virus (HCV)-related liver failure is the leading indication for liver transplantation (LT) (1). HCV reinfection after transplantation is a universal process that may result in a more rapid and aggressive course of disease than in the nontransplantation setting (2,3). More than 50% of liver graft recipients develop chronic active hepatitis, which can process to cirrhosis and subsequent graft failure (4). Interferon (IFN)α-2b with its spectrum of antiviral, antiproliferative and immunomodulatory effects, is capable of temporarily decreasing serum HCV RNA titers (5). But its application as therapy or prophylaxis for recurrent hepatitis C after LT was not effective in achieving sustained virological response (6–8). The combination with ribavirin (Rb), a synthetic nucleoside analogue, seems to have more potential in the treatment of recurrent hepatitis C post-transplantation (9). As experience with this treatment is limited, the most effective duration of therapy is not known. In previous studies, therapy lasted between 6 and 12 months and was associated with severe side-effects (10–12). In addition, the clinical course of patients after completion of therapy and the efficacy of reinitiation because of disease progression have not yet been reported. The aim of this study was to evaluate the feasibility, tolerability and efficacy of a long-term combination of IFNα-2b and Rb in the treatment of histologically confirmed recurrent hepatitis C after LT, with the longest follow-up being 48 months.

Patients and Methods

After institutional approval by the local Ethics Committee and subsequent informed consent, 15 patients with biopsy-confirmed recurrent hepatitis C after LT were enrolled in the study between December 1995 and December 2000. Elevation of aminotransferases to at least twice the upper normal limit led to further histological evaluation to exclude other causes of hepatic dysfunction. All patients revealed antibodies to HCV by enzyme-linked immunosorbent assay and had positive polymerase chain reaction (PCR) for HCV RNA in serum. HCV RNA levels were determined in duplicate by a commercial bcDNA signal amplification assay (COBAS AMPLICOR HCV test, version 2.0, Roche Diagnostics, Branchburg, NJ, USA), as already described (13,14). Quantitation of HCV RNA in the serum was expressed in thousand genomequivalents (Keq) per milliliter. HCV genotype was determined in all patients (INNO-LIPA HCV II, Innogenetics, Zwijnaarde, Belgium). The immunosuppressive regimen consisted of a quadruple regimen (induction with anti-T-lymphocyte globulin, cyclosporin A or tacrolimus, prednisone, azathioprine or mycophenolat mofetil). Patients underwent a basic treatment course of 12 months with a combination of subcutaneous IFNα-2b (Intron A®, Essex Pharma, Munich, Germany) 3 MU three times a week and oral Rb (Rebetol®, Essex Pharma GmbH, Munich, Germany) 600 mg daily. After finishing the basic treatment course, patients achieving biochemical and virological responses (normalization of serum aminotransferases and HCV RNA level below detection limit, < 100 eq/mL) received monotherapy with Rb (600 mg/day) without temporal limitation. In patients with positive HCV RNA PCR, combination therapy was continued, even in the case of biochemical response. Persistent cytopenia (leukocyte count < 1500 Gpt/L, platelet count < 40 000 Gpt/L) despite application of granulocyte-colony stimulating factor (g-CSF, Neupogen®, Amgen, Thousand Oaks, CA, USA) 15 MU 1 − 2 times weekly led to a dose reduction of IFNα-2b. The dose of Rb was reduced for anemia resistant to treatment with erythropoietin (Erypo®, Janssen-Cilag, Neuss, Germany) 12 000 U/week and transfusion. Biochemical response was evaluated by determination of aminotransferases [aspartate aminotransferase (AST) alanine aminotransferase (ALT)], bilirubin and prothrombin time. Virological response was assessed by determination of HCV RNA levels every month. Protocol liver biopsy was scheduled to be performed after 6 months of therapy, or any time when liver function was deteriorating. Histological specimens were reviewed and scored according to the histological activity index (HAI), described by Knodell et al. (15). Continuous variables were expressed as mean ± SD, and compared by the Student t-test or the Wilcoxon test, if necessary. A p-value of < 0.05 was considered significant.

Results

Patient characteristics

None of the patients died or had to be retransplanted. There was no case of acute or chronic rejection. Eleven patients were male. The mean age of all patients was 50.3 years at the beginning of therapy (range: 29–70 years). Follow-up after initiation of therapy ranged from 9 to 48 months. Mean HCV RNA levels were 659 × Keq/mL at the beginning of therapy. Fourteen of 15 patients were genotype 1b. Mean AST and ALT levels were 1.1 ± 1.0 µmol/L and 1.4 ± 1.1 µmol/L, respectively. The mean HAI at the time of diagnosis was 6.8 ± 0.9 (Table 1).

Table 1. : Characteristics of patients with recurrent hepatitis C after liver transplantation (LT)
 MeanMinimumMaximum
  1. AST, aspartate aminotransferase; ALT, alanine aminotransferase; HAI, histological activity index.

Age (years)502970
Beginning of therapy
 post-treatment (weeks)
383128
Follow-up
 post-treatment (months)
20948
AST (µmol/L)1.20.43.9
ALT (µmol/L)1.50.54.0
Bilirubin (µmol/L)691538.8
HCV RNA (Keq/mL)6591.21120
HAI6.96.08.0

Effects on graft function

Fourteen patients reached a minimum follow-up of 12 months after beginning treatment. In all patients improvement of graft function was recorded (biochemical response: 100%). Mean levels of aminotransferases declined significantly during the first month of therapy (AST: 1.1 µmol/mL/0.22 µmol/L, p = 0.001, normal range: 0.08–0.37 µmol/L; ALT: 1.51 µmol/L/0.32 µmol/mL, p < 0.001, normal range: 0.10–0.47 µmol/L) (Figure 1). Bilirubin levels dropped significantly during the first 3 months of therapy (upper limit: 17 µmol/L) (Figure 2). Prothrombin time, a marker of metabolic graft viability, increased continuously, but differences of changes were not significant.

Figure 1.

Course of aspartate aminotransferase (AST) level after initiation of combination therapy, ★ p < 0.05 (AST normal range: 0.08–0.30 µmol/L).

Figure 2.

Course of bilirubin level after initiation of therapy, ★ p < 0.05 (bilirubin upper limit: 17 mmol/L).

Virological response

HCV RNA was detected and quantified in serum of all 15 patients before treatment. HCV RNA level declined significantly during the basic course of combination therapy (Figure 3). Forty per cent (6/15) and 64% (9/14) of patients achieved clearance of viremia after 6 and 12 months. Nevertheless, in patients with positive PCR, aminotransferases and HCV RNA levels declined significantly too (Table 2). Patients responding after 12 months of the basic course revealed lower pretransplant (577 ± 509 Keq/mL/1052 ± 162 Keq/mL, p < 0.05) and pretreatment (609 ± 384 Keq/mL/658 ± 298 Keq/mL, p = 0.5) viremia than patients not responding. In 5 of 14 patients (35.7%), the combination therapy was continued after 12 months of the basic course, because of positive PCR. In two of them, therapy had to be withdrawn because of severe side-effects. Both patients relapsed, as biochemically and histologically confirmed, 6 months after withdrawal of therapy. The HCV RNA levels of the other patients under continued combination therapy did not show a further significant decline, but they are still free from another biochemical and histological relapse of disease more than 24 months after initiation of treatment. Eight of nine patients remained HCV PCR positive during Rb maintenance therapy with a follow-up of 12–48 months after initiation of therapy (sustained virological response 88%). One patient relapsed to positive PCR6 months after dose reduction of Rb monotherapy, and a second basic course of combination therapy was started. However, due to continued anemia and leukocytopenia despite supportive therapy, full-dose combination therapy was no longer feasible. At present, 48 months after beginning the primary basic treatment course and 24 months after re-initiation of therapy, the patient is still free of another biochemical relapse, despite positive HCV RNA PCR.

Figure 3.

HCV RNA titer declined significantly during a 12-month basic course of combined therapy, ★ p = 0.03; * p = 0.028.

Table 2. : In both, virological responder and nonresponder, levels of aminotransferases declined comparably; therapy led to a decrease of HCV RNA levels in patients with still positive PCR after 12 months of combination therapy
Months
post-initiation
AST (µmol/mL)
PCR+/PCR–
HCV-RNA-titer (Keq/mL)
PCR pre-/post-treatment
  1. AST, aspartate aminotransferase.

60.17/0.211000/640
120.18/0.22658/225

Effects on liver pathology

Liver biopsies of all 15 patients before treatment revealed histological signs of active hepatitis. In 10 patients, the control biopsy was performed 6 months after beginning combination therapy. The mean HAI declined significantly from 6.8 ± 0.9 to 4.1 ± 0.7 (p < 0.001). There was no difference in HAI between virological responders (n = 4, mean HAI declined from 6.3 to 4.1) and patients with positive HCV RNA PCR (n = 6, mean HAI decreased from 6.8 to 3.9) after 6 months of basic treatment course.

Side-effects

Combination therapy was temporarily accompanied by severe side-effects. The application of IFNα-2b led to flu-like symptoms, such as fatigue, headache and fever, in all patients. Many of them suffered from hematological disorders, especially during the first weeks of treatment (Table 3). Sixty-six per cent (10/14) of patients had to be treated with g-CSF at a dose of 15 MU 1–2 times weekly because of consistent leukocytopenia (leukocytes < 2 gigaparticles [Gpt]/L). Numbers of leukocytes before and after 12 months of combination therapy were 6.4 ± 1.6 Gpt/L and 3.6 ± 0.6 GpT/L (p < 0001). During the following 6 months of Rb monotherapy (n = 8), the leukocyte count increased significantly from 4.1 ± 0.6 Gpt/L to 5.8 ± 0.2 Gpt/L (p = 0.005). After initiation of Rb, 46% of patients (7/15) developed signs of hemolysis and anemia. All of them were temporarily treated with subcutaneous erythropoietin at a dose of 12 000 IU per week. In five patients (33%), transfusion became necessary; in three of them, the dose of Rb finally had to be reduced to 2 × 200 mg/d (Table 3). Hemoglobin levels declined from 7.2 ± 1.0 Gpt/L to 4.9 ± 0.4 Gpt/L after 12 months of combination therapy (p < 0.001). As mentioned above, therapy had to be completely withdrawn in two patients after 12 and 14 months of treatment because of progressive hemolysis and leukocytopenia. One patient additionally suffered from severe mental depressive syndrome.

Table 3. : Therapy was complicated by severe side-effects that had to be treated in a majority of patients
Side-effectsnTherapyn
Anemia (< 4 Ggpt/L)7 (46%)Erythropoietin/transfusion7/5 (46%/33%)
Leukocytopenia (< 2 Gpt/L)14 (93%)g-CSF10 (66.7%)
Thrombocytopenia5 (33%)Transfusion0
Flu-like symptoms15 (100%)Paracetamol, acetylsalicylate15 (100%)

Discussion

As survival after LT is improving, allograft failure due to HCV recurrence may become an increasing problem (16). Retransplantation is then the only useful option, but results for retransplantation in general are inferior to those of primary grafting. In addition, the dramatic shortage of donor organs forces us to perform a critical selection of appropriate patients. These facts underline the need for sufficient therapy of recurrent hepatitis C after LT. Recently, the combination of IFNα-2b and Rb proved to be more efficient in reaching HCV RNA clearance than monotherapy in the pretransplant setting (17–19). But there is little experience with this concept in treating recurrent disease after LT. Early in the 1990s, Wright et al. and Feray et al. already reported some success with interferon monotherapy after LT (6,7). Biochemical and virological response could be achieved in only 10–20% of patients. Even early prophylaxis with interferon after LT did not decrease the incidence or severity of HCV hepatitis (8). In a pilot study by Cattral and coworkers, Rb monotherapy for 3 months led to biochemical improvement but not to clearance of viremia in nine patients (20). Early after cessation of treatment all nine patients relapsed biochemically. Bizollon et al. were the first to report a favorable 45% HCV-RNA clearance rate and significantly improved graft morphology after 6 months of combined therapy, followed by Rb maintenance for another 6 months (9). Rb monotherapy had to be stopped because of anemia in only 3 of 21 patients. Mazzaferro and his group reported beneficial effects of early prophylaxis with IFNα-2b and Rb in 21 patients after LT (21). Nine patients (41%) achieved clearance of viremia, and only one patient revealed morphologic signs of chronic active hepatitis after 12 months of therapy. Other studies failed to confirm these favorable effects of combination therapy. In a study of Götz and colleagues, clearance of viremia was achieved in only one of ten patients after combination treatment lasting 12 weeks (10). Ben-Ari et al. reported severe side-effects, leading to complete withdrawal of therapy in four of five patients (11). In a study by Fisher and coworkers, only five of eight patients could finish a 6-month treatment course (12). In four patients therapy had to be reinitiated because of recurrent disease. Two patients died from liver failure, one during the first course of treatment, the other after reinitiation. But in none of these studies are there exact statements about the achievement of long-term sustained biochemical and virological response, the incidence of re-recurrent hepatitis C after completion of treatment and the possible long-term effect of reinitiation.

Regarding the natural history of this disease and the special situation of immunosuppression, reinfection of hepatitis C after LT must assumed to be a lifetime problem (22). An appropriate therapy should protect the patient from further relapse of progressive, histologically proven hepatitis C, which may accelerate the development of liver cirrhosis and subsequent graft failure. The current study has evaluated the efficacy and tolerability of long-term combination therapy with IFNα-2b and Rb. We hypothesized that combination treatment should be continued in patients without clearance of viremia, since HCV RNA titer correlates well with severity of histological recurrence of disease (23,24). In fact, none of our patients with uninterrupted long-term combination treatment experienced another biochemical relapse, despite continuously positive viral PCR. However, withdrawal of treatment immediately led to increasing viremia and to biopsy-confirmed progression of disease in two of our patients. The discrepancy between the biochemical response and the unchanged viremia is a well-known phenomenon and has already been described in the pre- and post-transplant period (25–27). It suggests that therapy does not work solely by inhibition of viral replication in the graft. Interferon is known to modulate antiviral T-cell responses and cytokine production, and increases the expression of HLA class I antigens within the allograft (28,29). Rb depresses interleukin (IL)-4, an inhibitor of cytotoxic T lymphocyte activity, while supporting IL-2 and IFN-γ activity (30,31). The combination of both drugs may augment the immunomodulatory effects of each. Thus, in patients without clearance of viremia, the beneficial impact of therapy may be best reflected by sustained biochemical response. Nevertheless, clearance of HCV RNA remains the principal aim of therapy, because a sustained virological response results in reduction of hepatic inflammation and an improved outcome in patients after LT (18,32). Determination of HCV RNA levels in the graft could provide further useful information about the long-term impact of therapy, but we did not perform additional elective liver biopsies, which is a major limitation of this study.

There are several possible explanations for our favorable results. First, in 11 of 15 patients, initiation of combination therapy was started within 6 months after LT, before the development of severe liver damage. Second, immunosuppression was reduced progressively towards monotherapy with calcineurin inhibitor during the first 6 months post-transplantation. Nevertheless, long-term application of IFNα-2b did not lead to allograft rejection. In addition, Rb monotherapy was maintained continuously (without temporal limitation), which differs from recent studies. To reduce severe hematological disorders, we introduced supportive cytoproliferative therapy early. Thus, we were able to implement a more aggressive policy of antiviral treatment. Nevertheless, dose reduction or withdrawal of therapy was necessary in a significant number of patients. Nonetheless, the prevention of further disease progression must be a principal aim of antiviral treatment, since re-initiation may require higher doses, which is not feasible in view of reported side-effects (32,33).

There are still several questions that need to be answered. The most appropriate management of patients with progression of disease after dose reduction or withdrawal of therapy is unknown. Most of these patients will refuse the re-initiation of another course of treatment or even an increase in the dose because of recently experienced symptoms. The introduction of pegylated IFNα-2b with prolonged half-life and enhanced efficacy could provide new therapeutic options (34,35). In addition, the most beneficial duration of both Rb maintenance therapy and continuation of combination therapy in virological nonresponders, is not clear and finally, the long-term impact of therapy on morphological disease progression and graft/patient survival has not been established. Although we noticed morphological improvement in our early biopsies, the impact on progression of graft fibrosis must be further evaluated. The lack of morphological differences between virological responders and nonresponders in our series may be due to the short follow-up of 6 months. Thus, the performance of long-term control biopsies could be useful.

In conclusion, this study, though uncontrolled, suggests that long-term combination therapy with IFNα-2b and Rb is effective in the treatment of recurrent hepatitis C disease and in preventing threatening relapse of disease after LT. Consequent monotherapy with Rb after successful combination therapy seems to maintain sustained virological response. However, because of associated hematological side-effects, a significant number of patients need continuous supportive treatment to reach target doses and to keep them on antiviral treatment.

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