1 Service d'Hépatologie, Hôpital Beaujon AP-HP, Clichy, 2 Service d'Hépatologie, 3 Service de Virologie, Hôpital Necker AP-HP, Paris, 4 Département de Biostatistiques et Information Médicale, Hôpital St Louis AP-HP, Paris, 5 Service d'Hépatologie, Hopital de Bicêtre AP-HP, Le Kremlin Bicêtre, 6 Service d'Hépatologie, Hôpital Pellegrin Bordeaux, 7 Service d'Anatomie et de Cytologie Pathologiques, Hôpital Beaujon AP-HP, Clichy, 8 Service d'Anatomo-pathologie, Hôpital Necker AP-HP, Paris and 9 Direction Régionale de la Recherche Clinique, AP-HP, Hôpital Saint Louis, Paris, France
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Abstract |
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Methods. We had planned to include 120 patients with HCV RNA detectable by polymerase chain reaction (PCR) (Amplicor Roche) and histologically documented chronic hepatitis. The dose of -interferon was 3 million units (MU) three times weekly (TTW), to be reduced to 1.5 MU TTW in case of side-effects. Tolerance was evaluated monthly; virological efficacy was evaluated by PCR. A liver biopsy was performed at month 18 (M18).
Results. (a) Tolerance. After 37 patients had been included, the study was discontinued by the promoting institution because of severe side-effects requiring that treatment be stopped in 19 patients. The side-effects were: cardiac (4) neuropsychiatric (2), digestive (3), acute necrosis of the graft (1), severe asthenia (9), minor side-effects were observed in 22 patients. A complete 12-month course was completed in 12 patients for the 3 MU TTW dose and in six patients for the 1.5 MU TTW reduced dose. Normal ALT level (OR, 0.16; CI 95%, 0.030.89) at inclusion was associated with interruption of treatment (univariate analysis). (b) Efficacy. Sustained virological response was observed in only seven (18.9%), of the 18 patients who completed the treatment (38%). Increased ALT at inclusion (OR, 1.04; CI 95%, 1.011.09) and cumulated doses of interferon (OR, 1.01; CI 95%, 1.0041.026) were jointly associated with a sustained response, while positive PCR at M2 was strongly predictive of treatment failure.
Conclusion: Tolerance of interferon is poor in haemodialysis patients. Sustained response is fairly high in patients who have 12 months of treatment and seems to be based on the immune status of the patients (ALT) and the cumulative doses of interferon.
Keywords: hepatitis C virus; interferon-; polymerase chain reaction; prospective multicentre study; side-effects; virological efficacy
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Introduction |
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Therefore we decided to evaluate the efficacy and tolerance of -interferon in a prospective multicentre cohort study in HCV-infected dialysis patients.
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Subjects and methods |
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Patients were excluded if they met at least one of the following criteria: (i) age <18 or >60 years, (ii) receiving immunosuppressive therapy or other treatments, namely anti-histaminics, omeprazole, non-steroidal anti-inflammatory drugs, aciclovir, or amiodarone, (iii) presence of co-infection with hepatitis B virus or human immunodeficiency virus, (iv) active drug addiction, (v) alcohol consumption >40 g/day, (vi) previous treatment for HCV infection, (vii) evidence of hepatocellular carcinoma ( fetoprotein >100 ng/ml), (viii) haemophiliacs, and (ix) patients with contra-indication to interferon. All patients provided an informed consent form. The study was approved by the Ethics Committee of Pitié Salpetrière, Paris, France.
Study design and protocol
Patients were scheduled to receive 3 MU s.c. of interferon -2b (Schering Plough, Kenilworth, USA) at the end of the haemodialysis procedure session thrice weekly for 48 weeks.
The follow-up of the patients was in the charge of both nephrologists and hepatologists. The patients were carefully followed up three times a week by the nephrologists, during haemodialysis procedures with weekly haematological and biochemical tests, and key points of the protocol were decided together with the referent hepatologist; a visit was planned with the hepatologist at week 4 to evaluate the treatment tolerance. If important asthenia (auto-evaluated on visual analogue scale, VAS) or other side-effects were noted, the dose was reduced to 1.5 MU TTW. Interruption of treatment was considered in case of severe side-effects or continued side-effects with the reduced dose (Figure 1). Scheduled follow-up was 72 weeks (18 months) for each patient. Epidemiological data were recorded before inclusion, and patients were followed both by nephrologists during haemodialysis procedures, and by a hepatologist at weeks 4, 8, and every 2 months until the 18th month. Blood pressure, pulse, body weight and asthenia (by VAS) were evaluated at each visit, and side-effects were recorded. The study started in October 1995, the last patient was included in May 1997, and inclusion of further patients was interrupted by the promoting institution (DRRC, Assistance Publique-Hopitaux de Paris) in June 1997, due to the frequency (30%) and the severity of side-effects. We therefore report our experience on the treatment of the 37 included patients, followed-up prospectively for 18 months.
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Methods |
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Virological analysis
Serum samples for virological studies were collected in each centre, and centrally stored at -80°C.
Serological assays
Serum antibodies to HCV (anti-HCV) were detected with third-generation HCV enzyme-linked immunosorbent assay (ELISA associated to analytic RIBA3 test (Ortho Diagnosis Systems, Raritan, NJ, USA) according to the manufacturer's instructions.
Quantitative detection of serum HCV RNA
HCV RNA in the serum was quantified with the Amplicor-HCV Monitor assay version 1.0 (Roche SA, Neuilly/ Seine, France). To do this longitudinal study, all sequential samples from each patient were tested with the same run of the technique. The Roche test 1.0 was constantly used along this study so that the results could be compared to the initial values previously obtained with the same methodology. Before RNA extraction, 100 µl of serum was added to 900 µl of RPMI and ultracentrifuged at 23500 g for 1 h at 4°C, to avoid PCR inhibitors that are frequently present in the sera of haemodialysis patients. The supernatant was discarded and the pellet was lysed with lysis buffer according to the manufacturer's instructions. The Roche Monitor assay was based on reverse transcription and amplification of the HCV RNA with primers from the 5'NCR in the presence of an internal control that shared primers with HCV. Both competitor and sample DNAs were detected by hybridization to a biotin-labelled probe, followed by incubation with enzymatically labelled streptavidin and detection in a colorimetric assay.
Genotyping of HCV strains
Genotyping was performed using a commercially available line probe assay (Innolipa, Innogenetics, Gent, Belgium).
Histological analysis
All patients underwent at least one liver biopsy. Liver biopsy samples were embedded in paraffin. After staining by H&E and Perls' stain, biopsies were blindly reviewed by the same pathologist. Chronic liver disease was classified as chronic hepatitis or cirrhosis. Liver disease activity was evaluated according to the Knodell score [16]. The Knodell score provides semi-quantification of four indices: periportal necrosis (range 010), intralobular degeneration and focal necrosis, portal inflammation and fibrosis (range 04). Liver biopsies were performed before treatment and 18 months after entering the study. Specimens were analysed in each centre, and graded using the Knodell scoring system by histology activity index according to the degree of periportal necrosis, portal and lobular inflammation (total activity), and fibrosis.
Endpoints
The main endpoint was classically defined as negative HCV RNA by PCR at 18 months, defining long-term response [6]. Responders and non-responders were defined as patients with negative or positive PCR by the end of treatment respectively. Relapse was defined as negative PCR at the end of treatment but positive PCR at the end of follow-up. Sustained response was defined as negative PCR at the end of treatment and at the end of follow-up (18 months). Histological response was based on a liver biopsy at month 18.
Statistical analysis
Actuarial occurrences of dose reduction, dose discontinuation and severe side-effects over time were separately analysed using the KaplanMeier method.
Predictive factors for treatment discontinuation and for sustained response were analysed separately, using univariate then multivariate modelling, both based on the logistic regression model. The association between covariates and outcome were represented by estimated odds ratio (OR) with 95% confidence intervals (CI 95%). Two-sided tests were computed with P value <0.05 defining statistical significance. SAS software package (SAS Inc, Cary, NC, USA) was used for the statistical analysis.
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Results |
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Efficacy
End of treatment response was evaluated in 12/18 of those patients who completed treatment (66%). The sustained response was evaluated 6 months after treatment discontinuation and occurred in 7/37 patients (18.9%), among whom five of the 29 patients had genotype 1 (17%). However, in patients who underwent 12-month treatment, the sustained virological response was observed in 7/18 (38%). All patients with a sustained response had negative PCR at month 2, while no end of treatment or sustained response was observed in patients with positive PCR at month 2.
Iterative 6-month post-treatment liver biopsy (performed at the end of follow-up, 72 weeks) could be obtained in 20/37 patients, (including seven sustained responders). The Knodell fibrosis scores were stable in 14/20 (70%) and improved in 2/20 (10%). By contrast, increase of the fibrosis score (one point) was noted in 4/20 cases, none of the four patients being a sustained responder.
Knodell activity scores were stable or improved in most cases (12/20 (60%) and 2/20 (10%) respectively). Iron overload was minor in all patients.
Body mass index, age, transaminase activity >normal, and cumulated doses of interferon were related to sustained response in the univariate analysis (Table 3). Due to the small number of events (seven patients with sustained response), we did not perform multivariate analysis.
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Discussion |
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The poor tolerance of interferon in haemodialysis patients may be related to the pharmacokinetics of the drug. Prior pharmacokinetic studies performed with natural interferon showed that the clearance of interferon was not modified by haemodialysis [17]. However, we and others have performed pharmacokinetic studies of recombinant interferon and shown that the serum concentrations of interferon were significantly higher in dialysis than in patients with normal renal function [1315]; then providing a possible explanation of the poor tolerance of the drug. Side-effects were noted at all periods of the drug administration of the drug and occurred in many cases. However, this frequency should be considered in relation to the spontaneous occurrence of cardiac and neoplastic events in these patients, who have an annual mortality rate of approximately 15% [18]. Special attention must be paid to the possible acute necrosis of the non-functional kidney graft, which is immunologically reactivated by interferon, and nephrectomy of the graft must be discussed prior to treatment when necessary. Moreover, our results did not provide predictive factors of the interruption of treatment, except ALT activity above the upper limit of normal and normal body mass index, both factors that probably reflect a good condition in the patients.
The sustained effect of therapy is higher, even if it is still low, in haemodialysis patients than that in patients with normal renal function and genotype 1 [6], not only if the results are analysed on an intent-to-treat basis (18.9%) but more strikingly if the patients reaching the end of treatment period are taken into account (7/18, 38%). Moreover these patients were mostly infected by genotype 1, which is related to a lower response (from 3 to 18%) in immunocompetent patients [6].
Results of univariate analysis
Increased ALT activity was found to be predictive of sustained response. This probably reflects the immune response of the host to HCV infection, and patients with chronic hepatitis and normal ALT probably have important immune response impairment. All patients with a sustained response had negative PCR at month 2, while positive PCR at month 2 was never associated with a sustained response. However, we did not include a negative PCR at month 2 in the logistic model since negative PCR was already included in the definition of the end-point (sustained response). If, due to marked side-effects, a dose reduction, or interruption of treatment is discussed in patients treated with interferon, the efficacy of treatment should be evaluated in relation to negative PCR after 2 months, so that therapy will only be continued in responders, since this subgroup has better chance of long-term sustained response. Treatment would then be interrupted in patients with positive PCR and side-effects.
The consequences of HCV infection are a matter of concern during haemodialysis and the transplantation periods. The prevalence of HCV infection in haemodialysis patients is nearly 20%, due to either blood transfusion, with a markedly reduced risk due to administration of erythropoietin, nosocomial transmission, or the transplanted kidney [19]. Nosocomial transmission of HCV is still observed, even if rarely, in haemodialysis units [2]. HCV infection is an important issue in the long-term survival of patients treated by renal transplantation [35]. Antiviral therapy with interferon has been shown to be potentially dangerous, inducing severe rejection episodes [7], and is no longer used in transplanted patients. Therefore, interferon administration must be undertaken before transplantation.
In conclusion, this prospective study showed the poor tolerance of interferon in haemodialysis patients. Therefore this treatment should only be offered to patients who are waiting for kidney transplantation, to avoid the severe evolution of liver disease under immunosuppressive therapy, or in those with severe liver disease to limit development of cirrhosis. When the treatment has been decided, the physician and the patient must be aware that there is a risk of severe side-effects, leading to the interruption of treatment, and that occurs at any time during the course of treatment. However, these limitations are counterbalanced by a fairly high chance of sustained response in patients reaching 12 month of treatment.
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Acknowledgments |
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Notes |
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References |
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