M. Martinot-Peignoux

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.)

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.)

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.)

In vivo hepatic endoplasmic reticulum stress in patients with chronic hepatitis C.

In hepatocytes, the accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress and the unfolded protein response (UPR), mediated by the ER‐resident stress sensors ATF‐6, IRE1, and PERK. UPR‐responsive genes are involved in the fate of ER‐stressed cells. Cells carrying hepatitis C virus (HCV) subgenomic replicons exhibit in vitro ER stress and suggest that HCV inhibits the UPR. Since in vivo ER homeostasis is unknown in livers with chronic HCV infection, we investigated ER stress and the UPR in liver samples from untreated patients with chronic hepatitis C (CHC), in comparison with normal livers. Electron microscopy, western blotting, and real‐time RT‐PCR were used in liver biopsy specimens. Electron microscopy identified features showing ER stress in hepatocyte samples from patients with CHC; however, ‘ER‐stressed’ hepatocytes were found in clusters (3‐5 cells) that were scattered in the liver parenchyma. Western blot analysis confirmed the existence of hepatic ER stress by showing activation of the three ER stress sensors ATF‐6, IRE1, and PERK in CHC. Real‐time RT‐PCR showed no significant induction of UPR‐responsive genes in CHC. In contrast, genes involved in the control of diffuse processes such as liver proliferation, inflammation, and apoptosis were significantly induced in CHC. In conclusion, livers from patients with untreated CHC exhibit in vivo hepatocyte ER stress and activation of the three UPR sensors without apparent induction of UPR‐responsive genes. This lack of gene induction may be explained by the inhibiting action of HCV per se (as suggested by in vitro studies) and/or by our finding of the localized nature of hepatocyte ER stress. Copyright

Relapse of hepatitis C virus–associated mixed cryoglobulinemia vasculitis in patients with sustained viral response

OBJECTIVE To investigate the clinical characteristics, outcomes, and results of hepatitis C virus (HCV) RNA analyses in a group of patients with HCV-associated mixed cryoglobulinemia (MC) vasculitis who experienced a relapse of vasculitis despite achieving a sustained viral response to treatment with antiviral agents. METHODS HCV RNA testing was performed by the transcription-mediated amplification (TMA) method in sera and cryoprecipitates (detection limit 2.5 IU/ml). HCV replication was assessed in peripheral blood mononuclear cells (PBMCs) by a modified real-time polymerase chain reaction assay (detection limit 15 IU/10(6) cells). RESULTS We identified 8 patients with relapse of HCV-MC vasculitis despite their having achieved a sustained viral response to treatment. Relapse appeared early after the end of treatment (mean +/- SD 2.5 +/- 3.5 months) and included mainly purpura (n = 7) and arthralgia (n = 5). Relapse was associated with an increase in serum cryoglobulin levels as compared with end-of-treatment levels (mean +/- SD 0.3 +/- 0.09 gm/liter and 0.08 +/- 0.04 gm/liter, respectively; P < 0.01) and a decrease in C4 levels. In most patients, the relapse was brief, and the MC vasculitis manifestations subsided. A search for HCV RNA by TMA was negative in all patients tested (7 of 8 patients), both in sera and in cryoprecipitates. HCV replication was not found in PBMCs from any of the patients tested (6 of 8 patients). In 3 patients, the MC vasculitis symptoms persisted and were associated with elevated cryoglobulin levels. B cell lymphoma was diagnosed in 2 of these 3 patients. CONCLUSION Relapse of MC vasculitis does occur in a few patients with HCV infection, despite achieving a sustained viral response, and this relapse is not related to persistence of virus. Relapse is short-lived and may be induced by the withdrawal of interferon alfa therapy. However, in patients with persistent MC vasculitis symptoms, a different underlying condition should be considered, especially B cell lymphoma.

Cytokines as predictors for sustained response and as markers for immunomodulation in patients with chronic hepatitis C.

OBJECTIVES (i) To characterize serum cytokine levels of tumor necrosis factor alpha (TNF alpha), interleukin 6 (IL 6), IL 8 and IL 12 in non-cirrhotic patients with chronic hepatitis C, (ii) to correlate the levels of these cytokines with the degree of the disease at the basal level, (iii) to correlate these levels with the response to therapy, (iv) to compare profiles of cytokines in monotherapy (MT) versus combination therapy (CT), and (v) to compare the immunomodulatory effects of MT versus CT. DESIGN AND METHODS 47 patients were enrolled in the study. The controls were 120 volunteers (recruited from students and staff) that did not present HCV RNA positive and were not known to suffer any other metabolic disease. Thirty patients formed the other group of controls, with alcoholic liver disease (ALD). Serum cytokine levels were assessed using enzyme-linked immunosorbent assay (ELISA). RESULTS The sustained responders (SRs) have basal values much lower than relapsed responders (RRs) and non-responders (NRs) regardless of the therapy. CONCLUSIONS Cytokines can be used as non-invasive markers for sustained response and as monitors for the outcome of therapy.

Kinetics of serum cytokines reflect changes in the severity of chronic hepatitis C presenting minimal fibrosis

Our aims were to measure the kinetics of serum tumour necrosis alpha (TNF‐α) and transforming growth factor beta (TGF‐β) levels as markers of progression of disease in nontreated chronic hepatitis C virus (HCV)‐infected patients with minimal or no fibrosis and minimal histology activity index (HAI) scores. Our study group consisted of 56 patients diagnosed with minimal (1) or no fibrosis (0) and minimal HAI (0–1) on their first biopsy as defined by Knodell and METAVIR scores. We compared their initial (entry of study) cytokine levels with a group of 103 HCV controls with minimal (0–1) to mild fibrosis (0–3) and mild HAI (5.5). Serum TNF‐α and TGF‐β levels were measured by enzyme‐linked‐immunosorbent‐assay. A significant difference was seen in TNF‐α levels at baseline in the study group vs. controls. Regardless of their HAI, there was a correlation between TGF‐β and degree of fibrosis. As shown by their biopsies, during the 3 years (from entry to follow up), many of the patients that initially had minimal fibrosis progressed to higher degree of fibrosis. This progression is paralleled by an increase in TGF‐β levels when comparing initial and follow‐up levels. In conclusion, serum TNF‐α reflects the progression of inflammation as seen in liver biopsies and TGF‐β reflects the degree of fibrosis in HCV patients.

Significant gene expression differences in histologically “Normal” liver biopsies: Implications for control tissue

Gene expression technologies allow the analysis of gene networks whose expression is associated with specific pathological conditions compared with normal tissue. We hypothesized that histologically normal tissue obtained in different ways (percutaneous or surgical liver biopsies), usually used as normal controls in gene expression studies, could have different gene expression patterns. Group A comprised percutaneous liver biopsies in 14 patients with mildly elevated alanine aminotransferase in whom all causes of liver disease had been ruled out. Group B comprised 14 surgical liver biopsies of nontumoral livers. All 28 specimens were histologically normal. Real‐time quantitative reverse‐transcription polymerase chain reaction were used to compare the messenger RNA expression of 240 selected genes in these two groups. Expression of 26 of the 240 genes was significantly different between groups A and B; 23 genes were up‐regulated in group A, while three were down‐regulated in group B. The most notable changes occurred in the inflammatory response family genes. Eight genes discriminated perfectly between groups A and B: seven up‐regulated genes (PAI1, THBS1, IL8, PTGS2, CXCR4, JUN, and FOS), and one down‐regulated gene (IHH). In chronic hepatitis C liver samples, a lower or higher expression of a IL8 was found depending on whether the controls were obtained percutaneously or surgically. Conclusion: Our study demonstrates that histologically normal liver tissue obtained in two different ways (percutaneous or surgical) has different gene expression patterns emphasizing the importance of an adequate selection of histologically normal controls to prevent discordant results in gene expression studies. (HEPATOLOGY 2008.)