Yasuhiro Asahina

α-fetoprotein levels after interferon therapy and risk of hepatocarcinogenesis in chronic hepatitis C.

The effects of interferon (IFN) treatment and the post‐IFN treatment α‐fetoprotein (AFP) levels on risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C (CHC) are unknown. To determine the relationship between AFP and alanine transaminase (ALT) levels and HCC risk, a cohort consisting of 1,818 patients histologically proven to have CHC treated with IFN were studied. Cumulative incidence and HCC risk were analyzed over a mean follow‐up period of 6.1 years using the Kaplan‐Meier method and Cox proportional hazard analysis. HCC developed in 179 study subjects. According to multivariate analysis, older age, male gender, advanced fibrosis, severe steatosis, lower serum albumin levels, non sustained virological response (non‐SVR), and higher post‐IFN treatment ALT or AFP levels were identified as independent factors significantly associated with HCC development. Cutoff values for ALT and AFP for prediction of future HCC were determined as 40 IU/L and 6.0 ng/mL, respectively, and negative predictive values of these cutoffs were high at 0.960 in each value. The cumulative incidence of HCC was significantly lower in patients whose post‐IFN treatment ALT and AFP levels were suppressed to less than the cutoff values even in non‐SVR patients. This suppressive effect was also found in patients whose post‐IFN treatment ALT and AFP levels were reduced to less than the cutoff values despite abnormal pretreatment levels. Conclusion: Post‐IFN treatment ALT and AFP levels are significantly associated with hepatocarcinogenesis. Measurement of these values is useful for predicting future HCC risk after IFN treatment. Suppression of these values after IFN therapy reduces HCC risk even in patients without HCV eradication. (Hepatology 2013;58:1253–1262)

Genome-Wide Association Study Confirming Association of HLA-DP with Protection against Chronic Hepatitis B and Viral Clearance in Japanese and Korean

Hepatitis B virus (HBV) infection can lead to serious liver diseases, including liver cirrhosis (LC) and hepatocellular carcinoma (HCC); however, about 85–90% of infected individuals become inactive carriers with sustained biochemical remission and very low risk of LC or HCC. To identify host genetic factors contributing to HBV clearance, we conducted genome-wide association studies (GWAS) and replication analysis using samples from HBV carriers and spontaneously HBV-resolved Japanese and Korean individuals. Association analysis in the Japanese and Korean data identified the HLA-DPA1 and HLA-DPB1 genes with Pmetau200a=u200a1.89×10−12 for rs3077 and Pmetau200a=u200a9.69×10−10 for rs9277542. We also found that the HLA-DPA1 and HLA-DPB1 genes were significantly associated with protective effects against chronic hepatitis B (CHB) in Japanese, Korean and other Asian populations, including Chinese and Thai individuals (Pmetau200a=u200a4.40×10−19 for rs3077 and Pmetau200a=u200a1.28×10−15 for rs9277542). These results suggest that the associations between the HLA-DP locus and the protective effects against persistent HBV infection and with clearance of HBV were replicated widely in East Asian populations; however, there are no reports of GWAS in Caucasian or African populations. Based on the GWAS in this study, there were no significant SNPs associated with HCC development. To clarify the pathogenesis of CHB and the mechanisms of HBV clearance, further studies are necessary, including functional analyses of the HLA-DP molecule.

Mutations in the core promoter region of hepatitis B virus in patients with chronic hepatitis B

The core promoter region of hepatitis B virus genomes regulates transcription of the precore and pregenomic mRNAs encoding hepatitis B e antigen (HBeAg) and core antigen that contain target epitopes for cytotoxic T lymphocytes. The prevalence and clinical significance of mutations in this region were investigated. DNA was extracted from six asymptomatic carriers positive for HBeAg, eight asymptomatic carriers positive for an anti‐HBe antibody, and 24 patients with chronic liver disease. The core promoter and precore regions of hepatitis B virus genomes were amplified by polymerase chain reaction, and predominant sequences were determined by direct sequencing. Mutations were found in none of the HBeAg‐positive asymptomatic carriers but in all of the anti‐HBe‐positive asymptomatic carriers and the patients with chronic liver disease. Especially, A to T mutations at nucleotide 1762 and G to A mutations at nucleotide 1764 were found in five anti‐HBe‐positive asymptomatic carriers, and 22 patients with chronic liver disease. These two mutation hot spots were located within binding sites of the nuclear factors, and nucleotide 1762 was also involved in the A, T rich sequence that is located 28 base pairs upstream of the precore mRNA initiation site. Serum HBeAg and DNA polymerase levels were significantly lower in patients with these mutations than those without these mutations, and five individuals with these mutations were positive for anti‐HBe despite the absence of the precore stop codon mutation. These mutants may be selected by host immune response to HBeAg and/or core antigen.

Hepatitis C virus NS4B protein targets STING and abrogates RIG‐I–mediated type I interferon‐dependent innate immunity

Hepatitis C virus (HCV) infection blocks cellular interferon (IFN)‐mediated antiviral signaling through cleavage of Cardif by HCV‐NS3/4A serine protease. Like NS3/4A, NS4B protein strongly blocks IFN‐β production signaling mediated by retinoic acid–inducible gene I (RIG‐I); however, the underlying molecular mechanisms are not well understood. Recently, the stimulator of interferon genes (STING) was identified as an activator of RIG‐I signaling. STING possesses a structural homology domain with flaviviral NS4B, which suggests a direct protein‐protein interaction. In the present study, we investigated the molecular mechanisms by which NS4B targets RIG‐I–induced and STING‐mediated IFN‐β production signaling. IFN‐β promoter reporter assay showed that IFN‐β promoter activation induced by RIG‐I or Cardif was significantly suppressed by both NS4B and NS3/4A, whereas STING‐induced IFN‐β activation was suppressed by NS4B but not by NS3/4A, suggesting that NS4B had a distinct point of interaction. Immunostaining showed that STING colocalized with NS4B in the endoplasmic reticulum. Immunoprecipitation and bimolecular fluorescence complementation (BiFC) assays demonstrated that NS4B specifically bound STING. Intriguingly, NS4B expression blocked the protein interaction between STING and Cardif, which is required for robust IFN‐β activation. NS4B truncation assays showed that its N terminus, containing the STING homology domain, was necessary for the suppression of IFN‐β promoter activation. NS4B suppressed residual IFN‐β activation by an NS3/4A‐cleaved Cardif (Cardif1‐508), suggesting that NS3/4A and NS4B may cooperate in the blockade of IFN‐β production. Conclusion: NS4B suppresses RIG‐I–mediated IFN‐β production signaling through a direct protein interaction with STING. Disruption of that interaction may restore cellular antiviral responses and may constitute a novel therapeutic strategy for the eradication of HCV. (HEPATOLOGY 2013)

Prevalence and Significance of Naturally Occurring Mutations in the Surface and Polymerase Genes of Hepatitis B Virus

The prevalence and clinical significance of naturally occurring mutations in the full-length surface and overlapping polymerase genes of hepatitis B virus (HBV) were analyzed in 42 patients with chronic hepatitis. Mutations were observed in 10 patients (24%) in the a determinant region, which is the neutralizing epitope within the major hydrophilic region of the surface gene. A high proportion of these mutations (17/18; 94%) occurred in the first loop, unlike mutations induced by immunization. The presence of serum antibody to hepatitis B surface antigen was significantly associated with these mutations. No other region of the surface gene contained any cluster of mutations. These results suggest that escape mutations commonly contribute to persistency in the natural course of HBV infection. In contrast, mutations affecting the major catalytic domains of the polymerase gene, which could alter susceptibility to antiviral nucleoside analogues, were not detected at all.

Association of gene expression involving innate immunity and genetic variation in interleukin 28B with antiviral response

Innate immunity plays an important role in host antiviral response to hepatitis C viral (HCV) infection. Recently, single nucleotide polymorphisms (SNPs) of IL28B and host response to peginterferon α (PEG‐IFNα) and ribavirin (RBV) were shown to be strongly associated. We aimed to determine the gene expression involving innate immunity in IL28B genotypes and elucidate its relation to response to antiviral treatment. We genotyped IL28B SNPs (rs8099917 and rs12979860) in 88 chronic hepatitis C patients treated with PEG‐IFNα‐2b/RBV and quantified expressions of viral sensors (RIG‐I, MDA5, and LGP2), adaptor molecule (IPS‐1), related ubiquitin E3‐ligase (RNF125), modulators (ISG15 and USP18), and IL28 (IFNλ). Both IL28B SNPs were 100% identical; 54 patients possessed rs8099917 TT/rs12979860 CC (IL28B major patients) and 34 possessed rs8099917 TG/rs12979860 CT (IL28B minor patients). Hepatic expressions of viral sensors and modulators in IL28B minor patients were significantly up‐regulated compared with that in IL28B major patients (≈3.3‐fold, P < 0.001). However, expression of IPS‐1 was significantly lower in IL28B minor patients (1.2‐fold, P = 0.028). Expressions of viral sensors and modulators were significantly higher in nonvirological responders (NVR) than that in others despite stratification by IL28B genotype (≈2.6‐fold, P < 0.001). Multivariate and ROC analyses indicated that higher RIG‐I and ISG15 expressions and RIG‐I/IPS‐1 expression ratio were independent factors for NVR. IPS‐1 down‐regulation in IL28B minor patients was confirmed by western blotting, and the extent of IPS‐1 protein cleavage was associated with the variable treatment response. Conclusion: Gene expression involving innate immunity is strongly associated with IL28B genotype and response to PEG‐IFNα/RBV. Both IL28B minor allele and higher RIG‐I and ISG15 expressions and RIG‐I/IPS‐1 ratio are independent factors for NVR. (Hepatology 2012)

JSH Guidelines for the Management of Hepatitis C Virus Infection: A 2016 update for genotype 1 and 2

THE JAPAN SOCIETY of Hepatology and Drafting Committee for Hepatitis Management Guidelines produced the first clinical practice guidelines for the management of hepatitis C virus (HCV) infection in 2012, followed by frequent updates. As English versions, we published JSH guidelines in 2013, and a 2014 update for genotype 1 in 2014. Thereafter several interferon-free regimens with direct acting antivirals (DAAs) have been launched in the clinical setting both for genotype 1 and 2 and treatment recommendations have been greatly changed with these progresses. In this year 2016, the Drafting Committee for Hepatitis Management Guidelines lauched a 2016 update for genotype 1 and 2. These JSH guidelines are intended to assist physicians and other healthcare providers to assist their decision making in the clinical process. The Committee defenitely hope these guideline help patients infected with HCV, their families and other interested individuals to overcome HCV infection and improve the outcome and quality of life with assistance of physicians and other healthcare providers. In these updated version, we focused on newly-available IFN-free DAAs and the current treatment recommendations. Please refer to the previous versions when IFN, ribavirin, and other IFN-based DAAs (telaprevir, simeprevir) are of interst. Correspondence: Atsushi Tanaka, Department of Medicine, Teikyo University School of Medicine, 2-11-1, Kaga, Itabashi-ku, Tokyo 173-8605. Email: a-tanaka@med.teikyo-u.ac.jp Conflicts of Interest: Conflicts of interest of the “Hepatitis C Treatment Guidelines (Third Version)”Hepatitis Treatment Guideline Preparation Committee Members 1 Financial compensation (1,000,000 yen or more per year from a single company/organization) None 2 Profits from shares (1,000,000 yen or more, or 5% or more of said shares, per year from a single company)No stock owned 3 Patent use fees (1,000,000 yen or more per year for a single patent) SRL Inc. 4 Speaking fees (1,000,000 yen or more per year from a single company or organization) MSD, Dainippon Sumitomo Pharma Company, Ltd., Bristol-Myers K.K., Mitsubishi Tanabe Pharma Corporation, Toray Industries, Janssen Pharmaceutica, Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Pharmaceutical Co., Ltd., Bayer Yakuhin, Ltd. 5 Manuscript fees (1,000,000 yen or more per year from a single company or organization) None 6 Total amount of research fees, grants, etc. (2,000,000 yen or more per year total paid from affiliated departments in a single company or organization sharing research expenses (e.g., courses, fields, or laboratories)) None *Drafting Committee for Hepatitis Management Guidelines, the Japan Society of Hepatology (in alphabetical order):Yasuhiro Asahina, Department of Gastroenterology and Hepatology, School of Medicine, Tokyo Medical and Dental University; Namiki Izumi, Department of Gastroenterology and Hepatology, Musashino Red Cross Hospital; Kumada Hiromitsu, Department of Hepatology, Toranomon Hospital; Masayuki Kurosaki, Department of Gastroenterology and Hepatology, Musashino Red Cross Hospital; **Kazuhiko Koike, Department ofGastroenterology, Graduate School of Medicine, The University of Tokyo; Fumitaka Suzuki, Department of Hepatology, Toranomon Hospital; * Hajime Takikawa, Department of Medicine, Teikyo University School of Medicine; Atsushi Tanaka, Department of Medicine, Teikyo University School of Medicine; Eiji Tanaka, The Second Department of Internal Medicine, ShinshuUniversity School ofMedicine; Yasuhito Tanaka, Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Science;Hirohito Tsubouchi, KagoshimaCityHospital, Norio Hayashi, Kansai-Rosai Hospital; Naoki Hiramatsu, Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine; Hiroshi Yotsuyanagi, Department of Infectious Diseases, Graduate School of Medicine, The University of Tokyo (* Committee chair; ** Special committee member) 7 Total amount of scholarship (support) payments received (2,000,000 yen or more per year paid by affiliated departments (e.g., courses, fields, laboratories) in a single company or organization sharing a scholarship budget) MSD, Mitsubishi Tanabe Pharma Corporation, Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Pharmaceutical Co., Ltd. 8 Sponsored courses provided by companies, etc. (noted when there is an affiliation with a course sponsored by a company, etc.) MSD, Dainippon Sumitomo Pharma Company, Ltd., Bristol-Myers, K.K., Toray Industries, Chugai Pharmaceutical Co., Ltd. 9 Receipt of travel expenses, gifts, etc. (50,000 yen or more per year from a single company or organization) None

Sequential changes in full-length genomes of hepatitis B virus accompanying acute exacerbation of chronic hepatitis B

BACKGROUND/AIMnDuring the course of persistent hepatitis B virus infection, viral replication markedly decreases after acute exacerbation of liver inflammation accompanied by emergence of antihepatitis B e antibody (anti-HBe) and/or anti-hepatitis B surface antibody (anti-HBs). In some cases, however, persistent viral replication continues even after such exacerbation with or without HBeAg/anti-HBe seroconversion. The aim of the present study was to investigate the extent of genetic variations of HBV in this phenomenon.nnnMETHODSnFull-length HBV genomes were amplified by polymerase chain reaction from sera of three patients before and after acute exacerbation and were directly sequenced.nnnRESULTSnIn the whole genomes of 3215 nucleotides, only six nucleotide mutations for six amino acid substitutions (2 in the surface gene, 2 in the X gene, 1 in the core gene and 1 in the polymerase gene) were observed in patient 1, 15 mutations for 14 amino acid substitutions (1 in the pre-core codon 28, 4 in the surface gene, 4 in the core gene and 5 in the polymerase gene) were observed in patient 2, and 5 mutations for 6 amino acid substitutions (2 in the surface gene, 2 in the X gene, pre-core stop codon mutation and 1 in the polymerase gene) were observed in patient 3. Substitution in the a determinant of the surface gene, which encodes target epitopes for neutralizing antibodies, as well as those in the pre-core/core gene, which encodes epitopes for cytotoxic T cells, were mainly found.nnnCONCLUSIONnHBV that remained after the emergence of anti-HBe and anti-HBs are considered to possess mutations in epitopes for both humoral and cellular immunity. These mutant HBV may be involved in the pathogenesis of persistent hepatic injury after acute exacerbation.

Changes in plasma vascular endothelial growth factor at 8 weeks after sorafenib administration as predictors of survival for advanced hepatocellular carcinoma

A new predictive biomarker for determining prognosis in patients with hepatocellular carcinoma (HCC) who receive sorafenib is required, because achieving a reduction in tumor size with sorafenib is rare, even in patients who have a favorable prognosis. Vascular endothelial growth factor (VEGF) receptor is a sorafenib target. In the current study, the authors examined changes in plasma VEGF concentrations during sorafenib treatment and determined the clinical significance of VEGF as a prognostic indicator in patients with HCC.

Effect of interferon-based and -free therapy on early occurrence and recurrence of hepatocellular carcinoma in chronic hepatitis C

BACKGROUND AND AIMSnAlthough treatment for hepatitis C virus has been dramatically improved by the development of direct-acting antiviral agents (DAAs), whether interferon (IFN)-free therapy reduces hepatocarcinogenesis in an equivalent manner to IFN-based therapy remains controversial. The aims of this study were to evaluate the occurrence and recurrence of hepatocellular carcinoma (HCC) in chronic hepatitis C (CHC) patients treated with DAAs and to identify biomarkers of HCC development after antiviral treatment.nnnMETHODSnA restrospective review of a prospective database of 1,897 CHC patients who were treated with IFN-based (1,145) or IFN-free therapies (752) was carried out. Cumulative HCC occurrence and recurrence rates were compared using propensity score-matched analysis. Predictors of HCC development after viral eradication were identified by multivariate analysis.nnnRESULTSnPropensity score-matched analysis showed no significant difference in HCC occurrence (p=0.49) and recurrence rates (p=0.54) between groups treated with IFN-based or IFN-free therapies. In multivariate analysis, higher levels of post-treatment α-fetoprotein (AFP) or Wisteria floribunda agglutinin positive Mac-2 binding protein (WFA+M2BP) were independently associated with HCC occurrence and recurrence after viral eradication. Only post-treatment WFA+M2BP level was significantly associated with HCC occurrence and recurrence among patients without severe fibrosis. The area under the receiver operating characteristic (ROC) curve for WFA+M2BP levels was greater than that for AFP levels in ROC analysis.nnnCONCLUSIONnThe risks of early HCC occurrence and recurrence after viral eradication were similar between IFN-based and IFN-free therapies. Post-treatment levels of WFA+M2BP may be helpful screening biomarkers for assessing the risk of HCC after IFN-free therapy. Patients with high WFA+M2BP levels after antiviral treatment, even without severe fibrosis, must be followed up carefully for HCC development. Lay summary: The risks of early HCC occurrence and recurrence after viral eradication were similar between IFN-based and IFN-free therapies. Post-treatment levels of WFA+M2BP may be helpful screening biomarkers for assessing the risk of HCC after IFN-free therapy.