Nathalie Boyer

Long-Term Histologic Improvement and Loss of Detectable Intrahepatic HCV RNA in Patients with Chronic Hepatitis C and Sustained Response to Interferon-α Therapy

Chronic hepatitis C is a major cause of cirrhosis and hepatocellular carcinoma. Spontaneous remission of the disease seems to be rare, but interferon- therapy induces a response characterized by normalization of aminotransferase levels and improvement of liver histologic findings in 38% to 48% of patients [1-6]. However, more than half of responders have relapse and a reincrease of serum aminotransferase levels within 6 months after withdrawal of interferon- therapy. Less than 20% of treated patients have a sustained response with persistently normal aminotransferase levels during the 6-month period after treatment. Long-term outcome in patients with sustained response is not well known. A few studies of small numbers of patients with follow-up periods of 1 to 4 years have suggested a long-term benefit in some patients; however, late relapse was seen in other studies [7-13]. Little information is available on histologic outcome, and the question of the long-term benefit of interferon- therapy with regard to hepatic viral clearance and histologic improvement remains to be answered. To address this question, we assessed the long-term biochemical, virologic, and histologic outcomes of 80 patients with chronic hepatitis C who had a sustained response during the 6 months after interferon- therapy. Methods Patients Among 450 patients with chronic hepatitis C who received interferon- therapy in our center between 1987 and 1993, 80 consecutive patients who had a sustained response after therapy were included in our study. Sustained response was defined as 1) strictly normal serum alanine aminotransferase [ALT] levels each month for the first 6 months after the end of therapy and 2) negative results on testing for serum hepatitis C virus (HCV) RNA 6 months after treatment. The 80 patients were followed for at least 1 year after the end of treatment; further follow-up was considered long-term follow-up. Of these 80 patients, 50 were treated in six randomized, controlled trials of interferon-. Twenty-two received recombinant interferon-2b(Intron A, Schering-Plough, Levallois, France) at a dose of 3 or 5 million U [4, 6, 14]; 19 received lymphoblastoid interferon- (Wellferon, Wellcome, Issy-les-Moulineaux, France) at a dose of 3 or 5 million U [15, 16]; and 9 received recombinant interferon-alpha2a (Roferon-A, Produits Roche, Neuilly, France) at a dose of 3 million U [17]. The six trials were approved by the ethical committee, and all patients gave informed consent. The 30 patients who did not participate in the trials received the licensed standard schedule for interferon- therapy: recombinant interferon-alpha2a or -alpha2b, 3 million U three times a week for 6 months. Treatment schedules are shown in Table 1. Table 1. Demographic, Biochemical, Virologic, and Histologic Characteristics before Treatment and Treatment Schedule in 80 Patients with Chronic Hepatitis C and Sustained Response to Interferon- Therapy* All patients had chronic hepatitis C shown on biopsy within 6 months before treatment; the mean histology score (SD) was 7.9 2.2 (range, 3 to 13). All patients had serum ALT levels greater than 1.5 times the upper limit of normal for at least 6 months, and all were negative for hepatitis B surface antigen and antibodies to HIV. Follow-up Follow-up included clinical assessment every 6 months, measurement of serum ALT levels at least every 3 months, and detection of serum HCV RNA by polymerase chain reaction (PCR) every 6 months. Antibodies to Hepatitis C Virus Before treatment, all patients were positive for antibodies to HCV. Antibody testing (done retrospectively for patients treated before 1990) was done by using second-generation enzyme-linked immunoassay and Recombinant Immunoblast Assay (Ortho Diagnostic Systems, Levallois, France). Serum Hepatitis C Virus RNA We tested for the presence of serum HCV RNA in all 80 patients on serum specimens that were kept frozen and collected 6 months after withdrawal of interferon- therapy and then every 6 months during the entire follow-up period. Serum HCV RNA was detected by using PCR (Amplicor HCV, Produits Roche) [18]. Serum HCV RNA quantitation was performed with the quantitative branched-DNA signal amplification assay (Quantiplex HCV RNA, Chiron Diagnostics, Eragny, France) before treatment in 62 patients for whom pretreatment serum HCV RNA measurements were available and during follow-up in patients with persistent or fluctuating detectable HCV RNA levels [19]. Genotype and Serotype of Hepatitis C Virus We did HCV genotyping in 62 patients for whom pretreatment serum measurements were available. Genotyping was done by using the reverse hybridization assay (LiPA, InGeN, Rungis, France) after amplification with the PCR assay [20]. The classification system of Simmonds and colleagues was used [21]. In 18 patients who had no pretreatment serum aliquots available and whose post-treatment serum specimens were negative on PCR, HCV genotyping was not possible and HCV serotyping was done. Serotyping was performed with an immunoenzymatic assay that shows antibodies directed to peptides encoded by the NS4 region of the HCV genome, which are specific for serotypes 1, 2, 3, 4, 5, and 6 (Murex Diagnostics, Chatillon, France) [22]. These serotypes are classified according to the respective genotypes in the classification system of Simmonds and colleagues [21]. Liver Histologic Studies Liver biopsy was done at the end of interferon- therapy or 6 or 12 months after therapy, according to the relevant protocol, in patients who participated in the randomized trials. Another biopsy was done 1 to 6 years after therapy in all patients. Biopsy specimens obtained before and after treatment were assessed for fibrosis (score, 0 to 4) and activity (score, 0 to 18) according to the scoring system of Knodell and coworkers [23]. Chronic hepatitis C was defined as mild if the total histology score was less than 6, moderate if the score was between 6 and 9, and severe if the score was greater than 9. Biopsy specimens obtained by using a percutaneous Menghini needle were at least 10 mm long. Specimens were coded and were assessed by one pathologist. After interferon- therapy, 109 liver biopsies were done in 69 of the 80 patients (32 patients had 2 biopsies, and 4 patients had 3 biopsies); 22 were done at the end of treatment and 87 were done during follow-up. In 48 of the 69 patients, the last post-treatment biopsy was done at least 1 year after the end of interferon- treatment (2.2 1.3 years [range, 1 to 6.2 years]). In these 48 patients, histologic outcome was assessed by comparing the pretreatment biopsy specimen with the last biopsy specimen obtained after treatment. Histologic outcome was defined as improved if the total histology score was at least 2 points lower in the post-treatment specimen, as no change if the score was 1 point lower in the post-treatment specimen or was the same in the two specimens, and as deterioration if the score was higher in the post-treatment specimen. Liver Hepatitis C Virus RNA A liver tissue specimen was collected 1 to 5 years after treatment in 27 patients (2 years after treatment in 10 patients); 13 of the 27 had a liver tissue specimen collected before treatment. All tissue samples were immediately frozen in liquid nitrogen and kept at 80C. All tubes and tissue grinders used for the liver HCV RNA extraction procedure were disposable and sterile and were autoclaved. Liver RNA was extracted with the guanidium isothiocyanate procedure (Trizol, Appligen, Illkirch, France), and PCR was performed in the 5NC region of the HCV genome according to the methods of Martinot-Peignoux and associates [24]. The quality of the extracted liver RNA was ascertained with the amplification of the cellular 28S ribosomal messenger RNA (rRNA) [25]. The 28S rRNA represents 80% of the total cellular RNA; the presence of 28S rRNA attested to the good preservation of the liver specimens. Specimens without amplifiable 28S rRNA were excluded from our study. All PCR assays were done in the presence of negative and positive controls, which were liver tissue specimens collected during the period in which the study specimens were collected. Negative controls were obtained from patients without antibodies to HCV and patients without detectable serum HCV RNA who had chronic liver disease. Positive controls were obtained from patients with untreated chronic hepatitis C. At each step of the procedure, a negative specimen without nucleic acid was added. All PCR procedures on liver and serum were done with strict application of the measures described by Kwok and Higuchi [26]. Role of the Funding Source The funding source did not influence the analysis or interpretation of the data or the decision to submit the paper for publication. Results In our 80 patients with chronic hepatitis C, a sustained response was defined by 1) a persistently normal serum ALT level throughout the 6-month post-treatment follow-up period and 2) negative results on serum HCV RNA testing 6 months after treatment. Patients were followed for 1 to 7.6 years after the end of interferon- therapy (mean SD, 4.0 2.0 years). The characteristics of the 80 persons with sustained response are shown in Table 1. The numbers of patients contributing data at each time point during follow-up are shown in Table 2. Table 2. Patients Contributing Data at Each Time Point during Follow-up* Clinical Outcomes At the end of follow-up, 79 of the 80 persons with sustained response were fully active and alive; 1 had died of peritoneal carcinomatosis related to colon carcinoma. Of the 5 patients with cirrhosis, none developed decompensated liver disease or hepatocellular carcinoma as assessed by abdominal ultrasonography done every 6 months. Of the 75 patients who did not have cirrhosis before receiving interferon- therapy, none developed cirrhosis. All patients who had fatigue before treatment (60%) said that the fatigue completely disappeared after tre

Early serum HBsAg drop: A strong predictor of sustained virological response to pegylated interferon alfa‐2a in HBeAg‐negative patients

Pegylated interferon alfa‐2a (PEG‐IFN) may induce sustained virological response (SVR) in 20% of hepatitis B e antigen (HBeAg)‐negative chronic hepatitis B (CHB) patients. In addition, loss of hepatitis B surface antigen (HBsAg) is achieved with a 10% yearly rate after treatment cessation in sustained responders. The aim of this study was to assess on‐treatment serum HBsAg kinetics to predict SVR in HBeAg‐negative patients treated with PEG‐IFN. Forty‐eight consecutive patients were treated with PEG‐IFN (180 μg/week) for 48 weeks. Serum hepatitis B virus (HBV) DNA (COBAS TaqMan) and HBsAg (Abbott Architect HBsAg QT assay) were assessed at baseline, during treatment (weeks 12, 24, and 48), and during follow‐up (weeks 72 and 96). SVR was defined as undetectable serum HBV DNA (<70 copies/mL) 24 weeks after treatment cessation. Twenty‐five percent of patients achieved SVR. They were not different from those who failed treatment regarding age, sex, ethnicity, HBV genotype, baseline serum HBV DNA and HBsAg levels, or liver histology. During treatment, serum HBsAg levels decreased only in patients who developed SVR, with mean decreases of 0.8 ± 0.5, 1.5 ± 0.6, and 2.1 ± 1.2 log10 IU/mL at weeks 12, 24, and 48, respectively. A decrease of 0.5 and 1 log10 IU/mL in serum HBsAg levels at weeks 12 and 24 of therapy, respectively, had high predictive values of SVR (negative predictive value [NPV] 90%, positive predictive value [PPV] 89% for week 12; NPV 97%, PPV 92% for week 24). HBsAg loss was observed in three patients, all with SVR. Conclusion: Early serum HBsAg drop has high predictive values of SVR to PEG‐IFN in HBeAg‐negative CHB patients. Serum quantitative HBsAg may be a useful tool to optimize the management of PEG‐IFN therapy in these patients. (HEPATOLOGY 2009.)

Liver gene expression signature to predict response to pegylated interferon plus ribavirin combination therapy in patients with chronic hepatitis C

Background and Aims: The gold standard treatment of chronic hepatitis C (CHC) is combined pegylated interferon and ribavirin. Considering side effects and treatment cost, prediction of treatment response before therapy is important. The aim of this study was to identify a liver gene signature to predict sustained virological response in patients with CHC. Methods: Group A (training set) comprised 40 patients with CHC including 14 non-responders (NRs) and 26 sustained virological responders (SVRs). Group B (validation set) comprised 29 patients including 9 NRs and 20 SVRs. Eleven responder–relapsers were also included. A total of 58 genes associated with liver gene expression dysregulation during CHC were selected from the literature. Real-time quantitative RT-PCR assays were used to analyse the mRNA expression of these 58 selected genes in liver biopsy specimens taken from the patients before treatment. Results: From the Group A data, three genes whose expression was significantly increased in NRs compared with SVRs were identified: IFI-6-16/G1P3, IFI27 and ISG15/G1P2. These three genes also showed significant differences in their expression profiles between NRs and SVRs in the independent sample (Group B). Supervised class prediction analysis identified a two-gene (IFI27 and CXCL9) signature, which accurately predicted treatment response in 79.3% (23/29) of patients from the validation set (Group B), with a predictive accuracy of 100% (9/9) and of 70% (14/20) in NRs and SVRs, respectively. The expression profiles of responder–relapsers did not differ significantly from those of NRs and SVRs, and 73% (8/11) of them were predicted as SVRs with the two-gene classifier. Conclusion: NRs and SVRs have different liver gene expression profiles before treatment. The most notable changes occurred mainly in interferon-stimulated genes. Treatment response could be predicted with a two-gene signature (IFI27 and CXCL9).

Pathogenesis, diagnosis and management of hepatitis C

The hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide. It is estimated that about 170 million people are chronically infected with HCV. Chronic hepatitis C is a major cause of cirrhosis and hepatocellular carcinoma and HCV-related end-stage liver disease is, in many countries, the first cause of liver transplantation. HCV infection is characterized by its propensity to chronicity. Because of its high genetic variability, HCV has the capability to escape the immune response of the host. HCV is not directly cytopathic and liver lesions are mainly related to immune-mediated mechanisms, which are characterized by a predominant type 1 helper cell response. Co-factors influencing the outcome of the disease including age, gender and alcohol consumption are poorly understood and other factors such as immunologic and genetic factors may play an important role. Recent studies have shown that the combination therapy with alpha interferon and ribavirin induces a sustained virological response in about 40% of patients with chronic hepatitis C. The sustained response rates are mainly dependent on the viral genotype (roughly 60% in genotype non-1 and 30% in genotype 1). Reliable diagnostic tools are now available and useful for detecting HCV infection, to quantify viral load and to determine the viral type. The assessment of the viral quasispecies and the characterization of viral sequences might be clinically relevant but standardized and simple techniques are needed. The lack of animal models and of in vitro culture systems hampers the understanding of the pathogenesis of chronic hepatitis C and the development of new antivirals. New therapeutic schedules with higher and/or daily doses of alpha interferon do not seem to improve the efficacy greatly. The conjugation with polyethylene glycol (PEG) improved the pharmacodynamics and the efficacy of alpha interferon. Emerging new therapies include inhibitors of viral enzymes (protease, helicase and polymerase), cytokines (IL-12 and IL-10), antisense oligonucleotides and ribozymes. The first candidate compounds should be available in the next few years. The development of an effective vaccine remains the most difficult and pressing challenge. Because of the high protein variability of HCV, protective vaccines could be extremely difficult to produce and therapeutic vaccines seem more realistic. Considerable progress has been made in the field of HCV since its discovery 10 years ago but a major effort needs to be made in the next decade to control HCV-related liver disease.

Serum hepatitis B virus DNA levels and liver histology in inactive HBsAg carriers

BACKGROUND/AIMS A recent NIH research workshop on hepatitis B virus (HBV) revisited the definition of healthy HBsAg carriers. The new definition inactive surface antigen (HBsAg) carriers includes an estimated serum HBV DNA level below 105 copies/ml. However, this cut-off value needs to be confirmed. METHODS Eighty-five consecutive patients, HBsAg-positive/HBeAg-negative with persistently normal alanine aminotransferase (ALT) and undetectable serum HBV DNA with standard assay (Versant HBV DNA Assay (bDNA), Bayer) were prospectively followed for 3.2+/-2.6 (range 0.5-11) years; 58 underwent a liver biopsy. Serum HBV DNA was quantified with a sensitive polymerase chain reaction assay (Cobas Amplicor HBV Monitor, Roche) (sensitivity 200 copies/ml), and liver histology was assessed using the Ishak scoring system. RESULTS The median serum HBV DNA level was 1300 copies/ml (<200-179 x 10(3) copies/ml), 16% of the subjects had no detectable serum HBV DNA and 98% had levels below 10(5) copies/ml. Histologic lesions were mild (total score <7) in all cases. Loss of HBsAg was observed in three patients, three patients experienced a transient increase in ALT (<2 x upper limit of normal), and serum HBV DNA levels remained stable (1-6 years) in 97% of the 38 patients retested. CONCLUSIONS In our study of inactive HBsAg carriers, the median serum HBV DNA level was 1300 copies/ml, the serum HBV DNA level was below 10(5) copies/ml in 98% of the patients, and remained stable; histological lesions were mild in all cases.

Efficacy of peginterferon alpha-2b in chronic hepatitis delta: relevance of quantitative RT-PCR for follow-up.

Hepatitis delta virus (HDV) can cause severe acute and chronic liver disease in patients infected by hepatitis B virus. Interferon alpha at high doses, although poorly efficient, is the only treatment reported to provide some benefit in chronic hepatitis delta. Pegylated interferon alpha (PEG‐IFN) has not yet been evaluated. Treatment is usually monitored by the qualitative detection of HDV‐RNA in serum. In this study, safety and efficacy of PEG‐IFN were assessed in chronic hepatitis delta, and serum HDV‐RNA kinetics were determined using quantitative RT‐PCR. Fourteen patients with chronic hepatitis delta received subcutaneous PEG‐IFN alpha‐2b during 12 months (1.5 μg/kg per week). Serum HDV‐RNA was quantified at initiation and during the course of therapy, and during the posttreatment follow‐up period, which ranged from 6 to 42 months (median 16 months). PEG‐IFN alpha‐2b was well tolerated, inducing no serious adverse effect. Sustained biochemical response was obtained in 8 patients (57%). At the end of treatment, 8 patients (57%) had achieved virological response (undetectable HDV‐RNA). Sustained virological response throughout the posttreatment follow‐up period was observed in 6 patients (43%). HDV‐RNA kinetics were predictive of the response: after 3 months of PEG‐IFN, HDV‐RNA levels were significantly lower in the responders than in the nonresponders group (P = .018). After 6 months of therapy, a negative HDV‐RNA was predictive of sustained response (P = .021). In conclusion, this preliminary study indicates that PEG‐IFN alpha‐2b is safe and efficient for treatment of chronic hepatitis delta. The follow‐up of HDV‐RNA levels during therapy, which allows the differentiation of various profiles of virological responses, improves treatment monitoring. (HEPATOLOGY 2006;44:728–735.)

Cryoglobulinemia with vasculitis associated with hepatitis C virus infection

Essential mixed cryoglobulinemia is frequently associated with chronic hepatitis. This report presents four cases of cryoglobulinemia with vasculitis associated with chronic hepatitis related to hepatitis C virus infection. Hepatitis C virus infection was ascertained in the four patients by both the presence in the serum of anti-HCV antibodies detected by the four-antigen recombinant immunoblot assay and of HCV RNA detected by polymerase chain reaction. In two patients tested, anti-HCV antibodies were not detected after centrifugation in the purified cryoglobulin but were detected in the supernatant. HCV RNA was detected in the purified cryoglobulin in all four patients and was detected in the supernatant in three patients. In one patient receiving recombinant interferon alfa, serum aminotransferases normalized and cryoglobulin disappeared; in another patient receiving recombinant interferon alfa, serum aminotransferases remained high and the cryoglobulin persisted. The presence of HCV RNA in the cryoglobulin and the parallelism of the changes of the cryoglobulinemia and of the serum aminotransferases during recombinant interferon alfa administration suggest that HCV infection is responsible for the production of cryoglobulinemia and vasculitis. It is proposed that HCV infection is a cause of cryoglobulinemia associated with chronic hepatitis.

High rates of HBsAg seroconversion in HBeAg-positive chronic hepatitis B patients responding to interferon: A long-term follow-up study

BACKGROUND/AIMS To assess the HBsAg seroconversion rate and its impact on the long-term outcome in chronic hepatitis B patients treated with conventional interferon, and to analyze the serum HBsAg concentration prior to seroconversion. METHODS Ninety-seven HBeAg-positive patients were retrospectively evaluated. Sustained virological response (SVR) was defined as HBeAg seroconversion and undetectable serum HBV-DNA 48 weeks after treatment discontinuation. HBsAg level was assessed at yearly intervals until seroconversion in SVRs. RESULTS Twenty-five patients (26%) achieved SVR. By multivariate analysis, SVR was associated with low serum HBV DNA level and severe liver fibrosis. During a median follow-up of 14 years (range, 5-20 years), 28 patients (29%) developed HBsAg seroconversion including 16 SVRs (64%) and 12 non-SVRs (16%), p < 0.001. HBsAg quantification showed a major decrease (median = 46%, range = 19-100%) in the first year after interferon starting in SVR patients. Six patients developed hepatocellular carcinoma, none of them had undergone HBsAg seroconversion. Liver fibrosis improved in 70% of patients with HBsAg seroconversion compared to 30% of those without HBsAg seroconversion (p < 0.01). CONCLUSIONS HBsAg seroconversion is achieved with a high steady rate in patients responding to interferon, and associated with excellent outcome. Prospective studies are needed to clarify the utility of on-treatment quantitative serum HBsAg in interferon-based therapy.

Twelve weeks posttreatment follow-up is as relevant as 24 weeks to determine the sustained virologic response in patients with hepatitis C virus receiving pegylated interferon and ribavirin.

A sustained virologic response (SVR) in patients with chronic hepatitis C receiving pegylated interferon (PEG‐IFN) plus ribavirin is defined as undetectable serum HCV‐RNA at 24 weeks (W+24) posttreatment follow‐up. Viral load outcome in patients with virological relapse (VR) has not been explored. This study evaluated whether the assessment of serum HCV‐RNA 12 weeks (W+12) after the end of treatment was as relevant as W+24 to evaluate SVR in 573 patients who received combination PEG‐IFN and ribavirin and had a virological response at the end of treatment. Serum HCV‐RNA was measured, using a new assay based on transcription‐mediated amplification (TMA) with a lowest detection limit of 5‐10 IU/mL, at W+12 and W+24 after the end of treatment. VR was defined as reappearance of detectable HCV‐RNA at W+24 posttreatment follow‐up. The positive predictive value (PPV) of undetectable serum HCV‐RNA at W+12 was evaluated to identify patients with SVR, and the viral load outcome was measured in relapse patients. At the W+24 posttreatment follow‐up, 408 (71%) patients had an SVR, 181 (71.2%) were treated with PEG‐IFNα‐2a and ribavirin, and 227 (71.1%) were treated with PEG‐IFNα‐2b and ribavirin. At W+12, serum HCV‐RNA was undetectable in 409 patients, and 408 patients were SVR (PPV 99.7%, 95% confidence interval 99.1‐100). In relapse patients, serum HCV‐RNA levels were 5.623 ± 0.748, 4.979 ± 0.870, and 5.216 ± 0.758 log10 IU/mL at baseline, W+12, and W+24, respectively. Conclusion: Our results show that the assessment of serum HCV‐RNA 12 weeks after the end of treatment, using the highly sensitive TMA assay (PPV 99.7%), is as relevant as after 24 weeks to predict SVR and make decisions on the management of treated patients, suggesting a new definition for SVR. (HEPATOLOGY 2010.)

Predictors of sustained response to alpha interferon therapy in chronic hepatitis C

OBJECTIVES To utilize cytokine levels to predict sustained response (SR) to alpha interferon (IFN alpha) therapy in chronic hepatitis C patients, and to determine the relationship between serum tumor necrosis factor alpha (TNF alpha), interleukin (IL) IL 6, IL 8, IL 12, transforming growth factor beta (TGF beta 1) and the degree of liver damage as reflected by traditional markers. DESIGN AND METHODS Serum cytokine levels were assessed using ELISA in 18 patients included in a controlled clinical trial of IFN alpha. RESULTS Of the 18 patients, 27% were sustained responders (SR), 27% were response and relapse responders (RR), and 46% were non-responders (NR). Multivariate analysis showed that a low serum TNF alpha level and high serum IL 8 levels were independent factors associated with SR to IFN alpha therapy. Serum TNF alpha level highly correlated with viral load and genotype predictive values (p < 0.001). Therapy lowered the IL 6 and IL 12 profile. TGF beta 1 levels in serum are positively correlated with fibrinogenesis. CONCLUSIONS IFN alpha therapy modulates immune response to hepatitis C virus, contributing to sustained response.