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Keywords:

  • Hepatitis C;
  • liver transplantation;
  • MMF

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients, Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Hepatitis C virus (HCV) recurrence after orthotopic liver transplantation (OLT) is almost universal. The optimal immunosuppression for these patients is still under discussion. We designed a retrospective case-control study to evaluate the effect of mycophenolate mofetil (MMF) treatment in patients with recurrent hepatitis C.

Forty patients with histologically proven hepatitis C recurrence after OLT were treated with MMF and calcineurin inhibitor (CNI) taper for 24 months and matched with 40 non-MMF-treated positive liver transplant recipients. Liver biopsies were obtained prior to MMF treatment and after a mean follow-up of 24 months. Histological changes were evaluated utilizing the Metavir score.

Comparison of fibrosis/inflammation showed no impairment of histological findings during MMF treatment. In contrast, histological findings of the 40 non-MMF patients showed a significant increase of severity for inflammation/fibrosis. Viral load was similar in both groups. The course of alanin amino transferase (ALT) levels measured during MMF treatment showed a significant decrease.

MMF in combination with CNI taper showed a positive effect on fibrosis progression, graft inflammation and ALT levels and may improve the clinical course of HCV after OLT, however, the antiviral properties of MMF are still unconfirmed.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients, Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Hepatitis C virus (HCV) infection and cirrhosis are the most common indications for liver transplantation in the United States and Europe (1,2). Posttransplant recurrence of hepatitis C, defined by the detection of serum HCV-RNA, is almost 100% (3). In approximately 50% of HCV-infected transplant recipients, graft hepatitis is apparently histo logical (4). Allograft failure leading to graft loss due to graft reinfection occurs in up to 40%, with increasing severity of recurrence during the last years (5).

The inositol-monophosphate-dehydrogenase inhibitor mycophenolate mofetil (MMF) is increasingly being used as a standard immunosuppressant after orthotopic liver transplantation (OLT).

The results of MMF combined with cyclosporine A (CsA) or tacrolimus (Tac) after OLT are promising (6). Several studies showed that MMF as prophylactic treatment for prevention of acute rejection delayed the onset or reduced the incidence of acute rejection (7). MMF is reported to be potent and safe in patients with failed primary immunosuppression for acute cellular rejection (ACR), chronic rejection (CR) and calcineurin inhibitor (CNI)—induced toxicities after OLT (8).

It had also been suggested that MMF has antiviral and antifibrotic effects (9–11). Herrine et al. achieved a significant virological response after 24 weeks by combining pegylated interferon with MMF in patients with chronic hepatitis C who had relapsed on interferon plus ribavirin (12).

Mitogenic responses in fibroblasts and endothelial cells are inhibited, and may lead to a reduction of inflammation and fibrosis (13,14). The aim of the study was to elucidate whether MMF has a positive effect on the histological and clinical course of recurrent HCV after OLT when combined with simultaneous tapering of CNIs.

Patients, Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients, Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Study design

We have retrospectively reviewed the medical records of 80 patients with hepatitis C recurrence who were transplanted in the era between 1995 and 2000. For that we evaluated 40 transplant recipients with histological proven hepatitis C recurrence who received MMF with special emphasis on the histological course using a retrospective case-control analysis. Liver biopsies were therefore obtained and scored routinely prior to the start of MMF treatment and after a mean follow-up of 24 months. These findings were then compared with the results of liver biopsies of 40 patients with hepatitis C recurrence without MMF treatment. In the MMF and control group liver biopsies were performed yearly after OLT. All liver biopsies were from the same posttransplant period and all patients showed histologically proven graft hepatitis with corresponding rising serum HCV-RNA as shown in Figure 1. The two groups were matched for age, sex and mean time after OLT and immunosuppression. The study was approved by the departmental ethics committee. Written informed consent was obtained from each patient before start of MMF therapy.

image

Figure 1. Rising HCV-RNA serum levels in mEq/mL 90 days after OLT and 800 days after OLT for MMF patients and for non-MMF patients.

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Standard immunosuppression

Standard immunosuppression refers either to CsA- or Tac-based regimens. Tac-based immunosuppressive regimens consisted of dual (Tac, steroids), triple (Tac, azathioprine, steroids) or quadruple drug induction regimens including antithymocyte globulin (ATG). CsA-based protocols consisted of triple (CsA, azathioprine, steroids) or quadruple drug induction regimens, including an ATG preparation (Fresenius, Bad Homburg, Germany), or mono clonal antiinterleukin 2-receptor antibodies.

As induction therapy five patients of the MMF group received ATG, and seven received monoclonal antiinterleukin 2-receptor antibodies. In the non-MMF group, 11 patients received ATG as induction therapy and 3 patients received OKT3.

HCV-RNA quantitation

Absolute levels of serum HCV-RNA were quantitated using a quantitative competitive RT-PCR analysis (Amplicor, Roche Molecular Systems, Inc., Branchgurg, NJ). The Department of Molecular Hepatology of the Charité performed all quantitation assays for each patient with this assay. Recurrent hepatitis C was defined by following criteria: HCV-RNA detectable by PCR in serum, elevated transaminase levels and histological signs of reinfection. Other viral infections such as hepatitis B and cytomegalovirus were excluded by serum analyses.

Patients with MMF treatment

The median age of 30 male and 10 female patients receiving MMF was 54 years (range: 27–73). Thirty-three patients received MMF in combination with Tac (FK506). Five patients received MMF in combination with CsA, one patient received MMF in combination with rapamune and one patient was treated with MMF monotherapy. MMF dosage ranged from 500 to 2000 mg/day. The median time of treatment after liver transplantation was 24 ± 2.5 months. The serum levels of CNIs during treatment are shown in Figure 2. The mean time after OLT until start of MMF treatment was 848 ± 996 days. None of the patients died during the follow-up. The therapy was started with a mean daily Tac dose of 5.14 ± 2.56 mg, and after a mean time of 24 months the daily Tac dose was tapered down to 2.06 ± 1.26 mg (p < 0.002). Demographics and immunosuppression of the MMF patients are listed in Table 1.

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Figure 2. The course of Tac during MMF treatment showed a significant decrease in Tac through levels expressed in nanograms per ml (p < 0.001). In contrast, no significant decrease could be seen in the non-MMF group (p < 0.24).

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Table 1.  Characteristics of 40 patients with MMF treatment and 40 patients without MMF treatment after liver transplantation
VariablesMMF groupControl groupp
  1. *p < 0.01;**p < 0.24.

n4040n.s.
Age (year)54 (27–73)57 (32–80)n.s.
Male (%)7567.5n.s.
Mean time after OLT (days)848 ± 996887 ± 452n.s.
 Prior treatment (start of MMF)Post treatment (after 24 months)First controlAfter 24 months 
Viral load3.99 ± 8.452.44 ± 4.86.34 ± 12.767.03 ± 10.67n.s.
 (mEq/mL) mEq/mL mEq/mL mEq/mL mEq/mL 
Inflammation score1.5 ± 0.11.5 ± 0.130.8 ± 0.611.5 ± 0.96MMF versus control p < 0.001
Fibrosis score1 ± 0.21 ± 0.40.5 ± 0.742.05 ± 1.02MMF versus control p < 0.013
Mean daily Tac dose (mg)5.14 ± 2.562.06 ± 1.26*4.81 ± 2.094.18 ± 1.96** 
ImmunosuppressionImmunosuppression
Tac/MMFn = 33Tacn = 29
CsA/MMFn = 5Tac/AZAn = 1
Rap/MMFn = 1CsAn = 8
MMFn = 1CsA/Predn = 1
 CsA/AZAn = 1

Thirty patients received MMF due to reinfection, and six due to acute rejection episodes. Diagnosis of acute rejection was based on elevated liver enzymes. Features consistent with acute rejection were scored based on the Banff rejection activity index (15). For management of CNI-associated nephrotoxic side effects, three patients received MMF. In one patient MMF was started for management of CNI-associated neurotoxic side effects.

Tapering protocol for CNIs (MMF Group)

The mean daily dose of Tac was reduced to 2 mg within 24 months according to the following tapering protocol: We started reduction of Tac 4 weeks after beginning of MMF treatment. The step-by-step reduction was triggered by serum transaminases and therefore tapering was discontinued if transaminases increased. Tacrolimus was reduced by 1 mg within the first 4 weeks, followed by a weekly tapering of 0.5 mg. The target levels were 50% of the initial daily Tac dose.

Side effects of MMF treatment

MMF-associated side effects were observed in 8 out of 40 patients. Gastrointestinal disorders such as nausea and diarrhea were seen in three patients and infections in another three patients. Two patients showed leukopenia. A dose reduction was sufficient in all cases. Withdrawal of MMF was not required.

Patients without MMF treatment (control group)

Forty patients with histological proven hepatitis C reinfection without MMF treatment were used as a control group. Data were collected from matched pairs. The characteristics of the non-MMF patients were: 27 males and 13 females with a median age of 57 (range: 32–80). The median time after OLT was 887 ± 452 days. At the beginning of the study none of these patients had cirrhosis or experienced episodes of acute rejection. None of the patients received interferon. The immunosuppressive therapy consisted of Tac (n = 29), cyclosporin (n = 8), Tac combined with azathioprine (n = 1) and cyclosporine combined with prednisolone (n = 1). Tapering of CNIs was not carried out in non-MMF patients and the mean daily Tac dose in these patients did not change during the observation period. The baseline characteristics of non-MMF patients are shown in Table 1.

Histological examination

Liver biopsies were performed routinely prior to and after 2 years of treatment. All paraffin-embedded liver biopsies were stained with hematoxylin-eosin, Masson-goldner, iron and periodic Schiff reagent. Biopsies were interpreted by an experienced pathologist who was blinded to the clinical status, except for the knowledge that the patient had undergone transplantation for end-stage liver disease as a result of HCV. ACR was scored based on the Banff rejection activity index (RAI) (16).

Histological recurrence of HCV was based on histopathological findings such as necroinflammatory activity and fibrosis, inflammatory infiltrates (portal/periportal/lobular) and inflammatory cell types (lymphocytes/eosinophils/plasma cells). All biopsy specimens were scored for inflammation and fibrosis by a semiquantitative histological score modified according to the simplified method of histological classification and quantitation of chronic hepatitis (17). Before scoring all specimens were re-read in a blind fashion. The findings were scored according to the amount of portal/periportal inflammation, lobular inflammation activity, degenerative liver cell changes and portal fibrosis using a scale of 0–3 for the criterion ‘inflammatory activity’ (0: absent, 0.5: minimal, 1: mild, 2: moderate, 3: severe) and a scale from 0 to 4 for the criterion ‘portal fibrosis’ (0: absent, 1: mild, 2: moderate without septa, 3: moderate with septa, 4: cirrhosis). The same procedure was performed in the control group.

Statistical analysis

Data were collected from a prospective database (Microsoft Access 2.0, Microsoft, Corporation, USA). Mann-Whitney U test and Wilcoxon signed rank test were applied using SPSS statistical software (SPSS, Inc., Chicago, IL). Categorical data were analyzed using the Fisher´s exact test for simple cross tables and likelihood ratio chi-square test for higher dimensional (2 × 3) tables. Statistical results were expressed as mean ± standard error. Changes over the time in each group were evaluated by one-way repeated measurements ANOVA. A p-value of <0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients, Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Immunosuppresssive treatment and acute rejection

In the MMF group, the mean daily dose of Tac was reduced from 5.14 ± 2.56 mg to 2.06 ± 1.26 mg after 24 months (p < 0.002), whereas in the control group the dose of Tac was not tapered. This led to a significant decrease of Tac through levels in the MMF group. The average Tac serum level was 8.78 ± 2.67 at the start of MMF therapy and decreased to 5.42 ± 1.57 ng/mL during MMF therapy (p < 0.001). In contrast, the course of Tac in the non-MMF group showed no significant decrease (p < 0.24). The average daily Tac serum level was 8.65 ± 3.16 ng/mL to 8.20 ± 3.13 ng /mL (Figure 2).

Despite the reduction of CNI, none of the patients in the MMF group developed acute rejection episodes. Also, none of the patients in the control group required treatment for acute rejection episodes.

Course of HCV-RNA during MMF therapy and without MMF therapy

No significant changes were seen in viral load measured by HCV-RNA during MMF therapy. The mean viral load at start of therapy was 3.99 ± 8.45 mEq/mL. After 24 months of treatment, the viral load decreased to 2.44 ± 4.8 mE/mL (p < 0.39).

There was no significant change in viral load in the non-MMF group. The viral load at the beginning of the observation was 6.34 ± 12.76 mEq/mL, respectively, 7.03 ± 10.67 mEq/mL about 24 months later (p < 0.89) (Table 1).

Histological analysis of liver biopsies for the MMF group compared to the control group

At the beginning of the study, patients in the MMF group showed a significantly higher Metavir score of inflammation compared to patients without MMF therapy (p < 0.001). In both groups, no differences were seen with regard to fibrosis. The analysis of histological findings with regard to inflammation showed an average of 1.5 ± 0.1 points in MMF patients prior to MMF treatment. After 24 months of treatment, the histological scores showed no significant changes (1.5 ± 0.13; p < 0.1). Fibrosis stage was 1.0 ± 0.2 points prior to MMF treatment and showed no impairment after 24 months of treatment (1 ± 0.4; p < 0.06).

In comparison, the histological findings of inflammation showed an average of 0.8 ± 0.61 in the first biopsy of non-MMF patients and a significant impairment of 1.5 ± 0.96 after 24 months (p < 0.001). Fibrosis stages increased from 0.5 ± 0.74 to 2.05 ± 1.02 in the control group (p < 0.001). Compared to the non-MMF group, fibrosis stages were significant lower in MMF patients (p < 0.001). The same findings were seen for inflammation. Compared to the control group, MMF patients showed a significantly lower Metavir score after MMF treatment (p < 0.013) (Table 1).

Biochemical course of transaminases prior to and after MMF treatment

Comparisons of the alanin amino transferase (ALT) levels prior and after MMF treatment showed a significant decrease during therapy compared to the non-MMF group, as demonstrated in Figure 3 (p < 0.002). At the start of MMF therapy, the average ALT level was 80 ± 90 U/L and decreased to 27 ± 15 U/L after 24 month of treatment. The non-MMF group however showed no decrease, as also demonstrated in Figure 3.

image

Figure 3. ALT levels (U/L) during MMF treatment showed a significant decrease (p < 0.002). In contrast, the ALT levels (U/L) for the control group showed no changes within 24 months.

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Treatment of acute rejection

Six patients were included in the study for treatment of an acute rejection episode to avoid steroid pulse therapy with methylprednisolone. Patients received MMF 2000 mg/day for 7 days. After initiation of MMF treatment, the therapy was continued with MMF 1000 mg/day for 24 months. Five of the six patients showed a good response to MMF treatment, with normalization of liver serum enzymes and no longer requiring steroid therapy. One patient required pulse therapy of 500 mg methylprednisolone for 3 days. None of the patients developed signs of CR.

Treatment of CsA and FK506 side effects

Three patients with recurrent HCV infection showed nephrotoxic side effects due to CsA/FK506. Normalization of serum creatinine levels after MMF treatment was seen in all three cases. Clinical symptoms of neurotoxicity significantly improved in one patient when Tac was stopped and MMF therapy was initiated (Figure 4).

image

Figure 4. Course of serum creatinine levels in patients after MMF treatment for CNI nephrototxicity (mg/dL).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients, Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

The severity of recurrent hepatitis C in transplant recipients has increased in recent years. Recurrence of hepatitis C after OLT is universal. An accelerated course leading to cirrhosis within 5 years has been described in up to 40% (18). Especially, HCV genotype 1b infected liver recipients are at a high risk of developing graft cirrhosis within the first 4–5 years following transplantation (19). It has been indicated that stronger immunosuppressive treatment plays a leading role in this issue (20). In addition to its well-documented immunosuppressive potency, MMF is supposed to have antiviral properties and exerts antifibrotic affects (21,22). Data regarding MMF and prognosis of inflammation/fibrosis after OLT for HCV are still to date controversially discussed (23). The impact of MMF on hepa titis C recurrence still remains unclear (24). We therefore evaluated the impact of MMF combined with simultaneous tapering of CNIs, with special emphasize to the histological course after liver transplantation in patients with histologically proven hepatitis C recurrence and graft hepatitis.

It is well known that high levels of immunosuppression cause cholestatic HCV disease by suppressing immune and inflammatory response (25). Steroids are also known to play an important role in the development of fibrosis. The incidence and severity of recurrent hepatitis C are related to steroids when used as maintenance immunosuppression or in the context of treatment of acute rejection as reported by Gane et al. (26). Sreekumar et al. showed that pulse steroid therapy for treatment of acute rejection markedly increased HCV-RNA levels in the early posttransplantation period (27). This correlates with more aggressive recurrence of hepatitis and an increase in hepatic fibrosis (28). To avoid steroid pulse therapy with methylprednisolone, MMF was administered to patients with an acute rejection episode. Five out of six patients responded well to the MMF treatment, with normalization of liver serum enzymes, and did not require further steroid therapy. To date none of these patients showed signs of CR. In our study the combined therapy of MMF treatment and CNI taper led to a significantly decelerated impairment of liver graft histology compared to the matched control group. During the follow-up, the MMF group showed no inflammation and fibrosis progression. In contrast, non-MMF patients without CNI tapering showed a significant increase in severity of inflammation and fibrosis. This data stand in strong contrast to previously reported data from Jain et al., who found a significant impairment of liver histology after MMF treatment without tapering of CNIs (29). A possible explanation for these different results could be the 50% reduction of CNIs in MMF patients carried out in the present study. Interestingly, this was possible without an increased risk for the development of acute rejection.

MMF blocks the de novo-biosynthesis of guanine nucleotides, which are required for DNA synthesis. This results in an inhibition of lymphocyte proliferation, which may lead to a decrease of inflammatory activity. The use of specific inhibitors of the enzyme IMPDH may provide an alternative treatment for patients with chronic HCV infection (30). The proposed independent antifibrotic effects of IMPDH inhibitors and described mitogenic responses in fibroblasts and endothelial cells may lead to a reduction of inflammation and fibrosis as reported by Lau et al. (31).

The antiviral properties of MMF post liver transplantation still remain unclear. Faola et al. reports on decreased 3-month HCV-RNA levels after induction with MMF after liver transplantation (32). Our study could not confirm any antiviral effect of MMF. Although MMF has been shown to inhibit the replication of yellow fever, parainfluenza, coxsackievirus B4 and human immunodeficiency virus in vitro, in our study MMF had no influence on HCV-RNA (33,34).

Especially, MMF showed an histological benefit, although it had no impact on viral load. Therefore, it should be emphasized that liver biopsies in HCV patients are indispensable for determining the clinical course after OLT.

There are confusing and controversial data regarding the impact of primary immunosuppression on the outcome of patients who undergo transplantation for hepatitis C. The cell-mediated immunity seems to play a major role in controlling viral activity and determine the outcome of hepatitis C infection. The direct T-cell response to foreign MHC antigens has been thought to be the distinguishing feature of cell-mediated allogenic immunity (35). Cell-mediated immunity is important in controlling viral activity and determine the outcome of HCV infection. It is well known that CNIs impair T-helper cells function (36). An imbalance between viral factors and the host immune system induced by immunosuppression represents the most crucial factor to explain that patients treated with stronger immunosuppression have a more aggressive course of HCV infection. The competence of the immune system during the acute phase of HCV infection seems to be particularly important for the progression of the disease in nontransplant patients (37). The findings of significantly reduced inflammation and fibrosis scores in MMF patients are possibly due to the lesser CNI levels maintained in these patients and not due to a direct MMF effect. However, MMF plays an important role in permitting CNI reduction without an increased risk of acute rejection episodes.

Our results of MMF treatment and simultaneous lowered through levels of CNI offer the therapeutic option of a steriod-free immunosuppressive protocol. This could however also be a tool for avoiding steroid pulse therapies in patients with unclear elevation of transaminases. It is known that the diagnosis between acute rejection and hepatitis C recurrence can be challenging. Due to this problem, additional MMF treatment could be a steroid-sparing alternative. On the other hand, MMF could have the potential to realize a steroid-free immunosuppressive regimen after OLT for HCV-positive transplant recipients. Our results show that MMF could play a role in the immunosuppressive induction regimen. MMF induction therapy without steroids could be safe and possibly be beneficial for the course of recurrent hepatitis C after OLT. We therefore have started an immunosuppressive trial comparing Tac/MMF with Tac/steriods in patients with HCV graft hepatitis after OLT.

In conclusion, the introduction of MMF and simultaneous taper of CNIs in patients with recurrent hepatitis C is well tolerated and not associated with an increased risk of acute rejection. CNI toxicity instead was ameliorated. Since inflammation and fibrosis progression were significantly reduced, it can be assumed that long-term outcome of HCV-positive recipients will be improved.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients, Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

We are grateful to Sylvia Albrecht for excellent secretarial assistance.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients, Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References