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Abstract Background and aim In patients with chronic hepatitis C, 8 weeks of glecaprevir and pibrentasvir (GLE/PIB) treatment for chronic hepatitis (non‐cirrhosis) and 12 weeks for cirrhosis have been approved in Japan. However, whether 8 weeks of treatment for cirrhosis may reduce treatment efficacy has not been adequately investigated. Methods This prospective, nationwide, multicenter cohort study enrolled 1275 patients with chronic hepatitis C who received GLE/PIB therapy. The effect of liver fibrosis and treatment periods on the efficiency of GLE/PIB therapy was investigated. The primary endpoint was the sustained virological response (SVR) rate in patients with chronic hepatitis (non‐cirrhosis) and cirrhosis. The association between treatment periods and liver fibrosis on the SVR after 12 weeks of treatment rate was investigated. Results The SVR rates in patients with chronic hepatitis with 8 weeks of treatment, chronic hepatitis with 12 weeks of treatment, cirrhosis with 8 weeks of treatment, and cirrhosis with 12 weeks of treatment were 98.9% (800/809), 100% (87/87), 100% (166/166), and 99.1% (211/213), respectively, and were was not different among these groups (P = 0.4). Conclusion GLE/PIB therapy for chronic hepatitis C had high efficacy regardless of liver fibrosis status and treatment periods. Periods of GLE/PIB therapy could be chosen with available modalities, and high SVR rates could be achieved regardless of the decision.

Abstract Background and Aim Hepatocellular carcinoma (HCC) surveillance in low‐risk patients (annual incidence

Here the authors demonstrate that de novo DNA methylation mediated by DNMT3B is regulated by nitric oxide (NO). They also isolate a unique modulator (DBIC) that inhibits S-nitrosylation of DNMT3B, which mitigates cell proliferation and tumorigenic conversion in vivo.

Abstract Background and Aim To validate a composite predictive model for hepatocellular carcinoma (HCC) development in patients with advanced liver fibrosis associated with chronic hepatitis C virus (HCV) who have received direct‐acting antiviral (DAA) therapy and achieved sustained virologic response (SVR). Methods This study included 1258 patients with advanced liver fibrosis associated with HCV genotype 1, 2, or both. General evaluation score (GES), which is based on sex, age, fibrosis stage, albumin, and α‐fetoprotein, was used as a composite predictive model. Results There were 645 (51.3%) patients in the low‐risk group, 228 (18.1%) in the intermediate‐risk group, and 385 (30.6%) in the high‐risk group based on GES categories. The 12‐, 36‐, and 60‐month cumulative incidence of HCC was 0.7%, 5.3%, and 13.0%, respectively. Multivariable analysis with Cox proportional hazards models showed that male sex (hazard ratio [HR], 1.863; 95% confidence interval [CI], 1.204–2.883), F4 fibrosis stage (HR, 3.199; 95% CI, 1.696–6.036), and albumin (HR, 0.489; 95% CI, 0.288–0.828) are independently associated with HCC development. The incidence of HCC differed significantly by GES‐based risk category (P

Background. Although HBV infection is a serious health issue worldwide, the landscape of HBV genome dynamics in the host has not yet been clarified. This study aimed to determine the continuous genome sequence of each HBV clone using a single-molecule real-time sequencing platform, and clarify the dynamics of structural abnormalities during persistent HBV infection without antiviral therapy. Patients and Methods. Twenty-five serum specimens were collected from 10 untreated HBV-infected patients. Continuous whole-genome sequencing of each clone was performed using a PacBio Sequel sequencer; the relationship between genomic variations and clinical information was analyzed. The diversity and phylogeny of the viral clones with structural variations were also analyzed. Results. The whole-genome sequences of 797,352 HBV clones were determined. The deletion was the most common structural abnormality and concentrated in the preS/S and C regions. Hepatitis B e antibody (anti-HBe)-negative samples or samples with high alanine aminotransferase levels have significantly diverse deletions than anti-HBe-positive samples or samples with low alanine aminotransferase levels. Phylogenetic analysis demonstrated that various defective and full-length clones evolve independently and form diverse viral populations. Conclusions. Single-molecule real-time long-read sequencing revealed the dynamics of genomic quasispecies during the natural course of chronic HBV infections. Defective viral clones are prone to emerge under the condition of active hepatitis, and several types of defective variants can evolve independently of the viral clones with the full-length genome.

Abstract Background and Aim This study aimed to evaluate the long‐term clinical course of patients achieving a sustained virologic response (SVR) with daclatasvir plus asunaprevir (DCV/ASV) therapy. Methods A total of 911 patients who achieved SVR with DCV/ASV were assessed. To evaluate pretreatment factors contributing to hepatocellular carcinoma (HCC) after SVR, univariate and multivariate analyses were performed in all patients, in those with preexisting HCC, and in those without preexisting HCC. We selected a low‐risk group of HCC cases after SVR. Finally, we evaluated liver function after achieving SVR. Results In multivariable analyses, male sex, older age, patients with a history of HCC treatment, excess alcohol use, lower albumin, and low platelet count remained significant in the overall group; male sex and low albumin remained significant in patients with a history of HCC treatment; and male sex, older age, excess alcohol use, low platelet count, high alpha‐fetoprotein (AFP), and high des‐γ‐carboxy prothrombin (DCP) remained significant in those without a history of HCC treatment. Patients who had not received treatment for HCC, females, those under 70 years of age, and those with platelet count ≥13 (×104/μL), AFP

Abstract The emergence of hepatitis C virus (HCV) with resistance-associated substitution (RAS), produced by mutations in the HCV genome, is a major problem in direct acting antivirals (DAA) treatment. This study aimed to clarify the mutational spectrum in HCV-RNA and the substitution pattern for the emergence of RASs in patients with chronic HCV infection. HCV-RNA from two HCV replicon cell lines and the serum HCV-RNA of four non-liver transplant and four post-liver transplant patients with unsuccessful DAA treatment were analyzed using high-accuracy single-molecule real-time long-read sequencing. Transition substitutions, especially A>G and U>C, occurred prominently under DAAs in both non-transplant and post-transplant patients, with a mutational bias identical to that occurring in HCV replicon cell lines during 10-year culturing. These mutational biases were reproduced in natural courses after DAA treatment. RASs emerged via both transition and transversion substitutions. NS3-D168 and NS5A-L31 RASs resulted from transversion mutations, while NS5A-Y93 RASs was caused by transition substitutions. The fidelity of the RNA-dependent RNA polymerase, HCV-NS5B, produces mutational bias in the HCV genome, characterized by dominant transition mutations, notably A>G and U>C substitutions. However, RASs are acquired by both transition and transversion substitutions, and the RASs-positive HCV clones are selected and proliferated under DAA treatment pressure.

The identification of patients with advanced fibrosis who do not need any further hepatocellular carcinoma (HCC) surveillance after the eradication of hepatitis C is pivotal. In this study, we developed a simple serum‐based risk model that could identify patients with low‐risk HCC. This was a nationwide multicenter study involving 16 Hospitals in Japan. Patients with advanced fibrosis (1,325 in a derivation cohort and 508 in a validation cohort) who achieved sustained virological responses at 24 weeks after treatment (SVR24) were enrolled. The HCC risk model at any point after SVR24 and its change were evaluated, and subsequent HCC development was analyzed. Based on the multivariable analysis, patients fulfilling all of the factors (GAF4 criteria: gamma‐glutamyl transferase

Abstract Background and Aim The pathogenic process underlying the development of hepatocellular carcinoma (HCC) is not yet clear in patients with hepatitis C virus (HCV) who have received direct‐acting antiviral (DAA) therapy and achieved sustained virological response (SVR). This study validated a composite predictive model for HCC in these patients. Methods This study included 3058 patients in whom HCV was eradicated with DAA therapy. After DAAs recommendation for surveillance (ADRES) score, which is based on sex, FIB‐4 index, and α‐fetoprotein, was used as a composite predictive model for HCC development. Results The 1‐, 3‐, and 5‐year cumulative incidence rates of HCC were 0.9, 4.5, and 15.2%, respectively. Multivariate analysis with Cox proportional hazards models showed that male sex (hazard ratio [HR], 2.646; 95% confidence interval [CI], 1.790–3.911), FIB‐4 index >3.25 (HR, 2.891; 95% CI, 1.947–4.293), and α‐fetoprotein >5 ng/mL (HR, 2.835; 95% CI, 1.914–4.200) are independently associated with HCC development. The incidence of HCC differed significantly by ADRES score (P

Background/Aims. Underwater endoscopic mucosal resection (UEMR) has recently attracted attention as a safe and effective method for superficial nonampullary duodenal epithelial tumors (SNADETs). However, water is a fluid and it does not remain in all areas, and because it mixes easily with other substances, it sometimes becomes cloudy and reduces visibility. A new endoscopic gel product that does not mix with fluids and tends to remain in the injected area has recently been applied to EMR and is known as gel immersion EMR (GIEMR) or under-gel EMR. Here, we evaluated the endoscopic outcomes of UEMR and GIEMR. Materials and Methods. This was a retrospective cohort study conducted in two municipal hospitals. We identified 24 patients with SNADETs of 3–18 mm in diameter who underwent UEMR or GIEMR. One lesion was excluded from the analysis because it was found to be in the stomach after surgery. The primary outcome was procedure time. Results. GIEMR significantly reduced the procedure time compared with UEMR (5 min vs. 10 min, P=0.016). There was no significant difference between the UEMR and GIEMR groups for en bloc resection rate (93% vs. 100%, P=1.0) and R0 resection rate (57% vs. 80%, P=0.39). No serious complications were observed in either group. Conclusions. GIEMR of SNADET has the potential to reduce procedure time compared with UEMR and may be particularly effective in areas where immersion in water is difficult.

Hepatocellular carcinoma (HCC) arises in the background of chronic liver diseases, including hepatitis and liver cirrhosis caused by hepatitis C virus (HCV) infection. It is well known that HCV eradication using antiviral drugs can efficiently inhibit hepatocarcinogenesis. Recent advances in and development of direct-acting antiviral (DAA) drugs has revolutionized the treatment of HCV infection, and the vast majority of HCV patients can achieve HCV eradication using DAAs. However, mounting evidence clearly indicates that HCC inevitably occurs in a subset of patients after successful viral eradication using DAA therapy. Cancer is a genetic disease, and the accumulation of genetic and epigenetic aberrations may cause hepatocarcinogenesis in chronically damaged liver, even after virus elimination. In this review, we highlight HCC development after HCV eradication and discuss the current understanding of the molecular mechanisms of tumorigenesis after virus elimination, focusing on the genetic and epigenetic background of chronically damaged liver tissues.

Background & Aims: We aimed to clarify the features of resistance-associated substitutions (RASs) after failure of multiple interferon (IFN)-free regimens in HCV genotype 1b infections. Methods: A total of 1,193 patients with HCV for whom direct-acting antiviral (DAA) treatment had failed were enrolled from 67 institutions in Japan. The RASs in non-structural protein (NS)3, NS5A, and NS5B were determined by population sequencing. Results: Failure of 1, 2, and 3 regimens was observed in 1,101; 80; and 12 patients, respectively. Among patients with failure of 1 regimen, Y56H and D168V in NS3 were more frequently detected after failure of paritaprevir, whereas D168E was more frequently detected after failure of regimens including asunaprevir. R30H and L31-RAS in NS5A were frequently detected after failure of regimens including daclatasvir. The prevalence of Y93-RAS was high irrespective of the regimen. S282T RAS in NS5B was detected in 3.9% of ledipasvir/sofosbuvir failures. The prevalence of D168-RAS increased significantly according to the number of failed regimens (p

Epidemiological, clinical, and biological studies convincingly demonstrate that chronic inflammation predisposes to the development of human cancers. In digestive organs, inflammation-associated cancers include colitis-associated colorectal cancers, Helicobacter pylori-associated gastric cancer, as well as Barrett’s esophagus and esophageal adenocarcinoma associated with chronic duodenogastric-esophageal reflux. Cancer is a genomic disease, and stepwise accumulation of genetic and epigenetic alterations of tumor-related genes leads to the development of tumor cells. Recent genome analyses show that genetic alterations, which are evoked by inflammation, are latently accumulated in inflamed epithelial cells of digestive organs. Production of reactive oxygen and aberrant expression of activation-induced cytidine deaminase, a nucleotide-editing enzyme, could be induced in inflamed gastrointestinal epithelial cells and play a role as a genomic modulator of inflammation-associated carcinogenesis. Understanding the molecular linkage between inflammation and genetic alterations will open up a new field of tumor biology and provide a novel strategy for the prevention of inflammation-associated tumorigenesis.

Background. Primary sclerosing cholangitis (PSC) is a progressive cholestatic liver disease, with liver transplantation being the sole life-saving treatment for end-stage PSC-related liver disease. However, recurrence of PSC after liver transplantation is a common complication, with the risk factors for recurrence being controversial. Methods. We conducted a retrospective chart review of 45 patients who had undergone liver transplantation for PSC at our institute. The risk factors for PSC recurrence and graft failure after liver transplantation were analyzed. Results. The graft survival rates were 55.4% at 5 years and 32.8% at 10 years after liver transplantation for PSC. PSC recurrence was diagnosed in 16 (40%) of 40 patients, at a median 30 months (range, 9-70 months) after liver transplantation. The cumulative incidence rate of PSC recurrence was 24.5% at 3 years, 39.3% at 5 years, and 45.8% at 6 years. Among the 16 patients diagnosed with PSC recurrence, the graft survival rate was 56.3% at 5 years, and 21.9% at 10 years after the recurrence. Active inflammatory bowel disease after liver transplantation was identified as an independent risk factor for PSC recurrence. Age younger than 30 years at the time of PSC diagnosis and bacteremia were factors significantly associated with graft failure after liver transplantation on multivariate analysis. Conclusions. PSC frequently recurred and progressed to graft failure after liver transplantation for PSC. Maintaining an inactive status of inflammatory bowel disease might offer protection against PSC recurrence.

Hepatocellular carcinoma (HCC) is a common complication of chronic viral hepatitis. In support of this notion, we have reported that hepatitis B surface antigen (HBsAg)-specific CD8+ T lymphocytes critically contribute to inducing chronic liver cell injury that exerts high carcinogenic potential in a hepatitis B virus (HBV) transgenic mouse model. The dynamics of the molecular signatures responsible for hepatocellular carcinogenesis are not fully understood. The current study was designed to determine the serial changes in gene expression profiles in a model of chronic immune-mediated hepatitis.Three-month-old HBV transgenic mice were immunologically reconstituted with bone marrow cells and splenocytes from syngeneic nontransgenic donors. Liver tissues were obtained every three months until 18 months at which time all mice developed multiple liver tumors. Nitrative DNA lesions and hepatocyte turnover were assessed immunohistochemically. Gene expression profiles were generated by extracting total RNA from the tissues and analyzing by microarray.The nitrative DNA lesions and the regenerative proliferation of hepatocytes were increased during the progression of chronic liver disease. In a gene expression profile analysis of liver samples, the chemokine- and T cell receptor (TCR)-mediated pathways were enhanced during chronic hepatitis, and the EGF- and VEGF-mediated pathways were induced in HCC. Among these molecules, the protein levels of STAT3 were greatly enhanced in all hepatocyte nuclei and further elevated in the cytoplasm in HCC tissue samples at 18 months, and the levels of phosphorylated TP53 (p-p53-Ser 6 and -Ser 15) were increased in liver tissues.HBV-specific immune responses caused unique molecular signatures in the liver tissues of chronic hepatitis and triggered subsequent carcinogenic gene expression profiles in a mouse model. The results suggest a plausible molecular basis responsible for HBV-induced immune pathogenesis of HCC.

Hepatitis B virus (HBV) infection is one of the most prevalent infectious diseases associated with various human liver diseases, including acute, fulminant and chronic hepatitis; liver cirrhosis; and hepatocellular carcinoma. Despite the availability of an HBV vaccine and the development of antiviral therapies, there are still more than 350 million chronically infected people worldwide, approximately 5% of the world population. To understand the virus biology and pathogenesis in HBV-infected patients, several animal models have been developed to mimic hepatic HBV infection and the immune response against HBV, but the narrow host range of HBV infection and lack of a full immune response spectrum in animal models remain significant limitations. Accumulating evidence obtained from studies using a variety of mouse models that recapitulate hepatic HBV infection provides several clues for understanding host-virus immunologic interactions during HBV infection, whereas the determinants of the immune response required for HBV clearance are poorly defined. Therefore, adequate mouse models are urgently needed to elucidate the mechanism of HBV elimination and identify novel targets for antiviral therapies.

BackgroundGiven the limited efficacy and high adverse event rate associated with treatment of recurrent hepatitis C after liver transplantation, an individualized treatment strategy should be considered. The aim of this study was to identify predictors of response to antiviral therapy for hepatitis C after living donor liver transplantation (LDLT) and to study the associated adverse events.MethodsA retrospective chart review was performed on 125 hepatitis C virus (HCV)-positive LDLT recipients who received interferon plus ribavirin and/or peginterferon plus ribavirin therapy at Kyoto University between January 2001 and June 2011.ResultsSerum HCV RNA reached undetectable levels within 48 weeks in 77 (62%) of 125 patients, and these patients were defined as showing virological response (VR). Of 117 patients, 50 (43%) achieved sustained VR (SVR). Predictive factors associated with both VR and SVR by univariate analysis included low pretransplant serum HCV RNA levels, a non-1 HCV genotype, and low pretreatment serum HCV RNA levels. In addition, LDLT from ABO-mismatched donors was significantly associated with VR, and white cell and neutrophil counts before interferon therapy were associated with SVR. Multivariate analysis showed that 2 variables-pretransplant serum HCV RNA level less than 500 kIU/mL and a non-1 HCV genotype-remained in models of both VR and SVR and that an ABO mismatch was associated with VR. No variables with a significant effect on treatment withdrawal were found.ConclusionsVirological response to antiviral therapy in patients with hepatitis C recurring after LDLT can be predicted prior to transplant, based on pretransplant serum HCV-RNA levels and HCV genotype. LDLT from ABO-mismatched donors may contribute to more efficacious interferon therapy.Trial registrationUMIN-CTR UMIN000003286.

Background and aimsAlthough the advent of ultra-deep sequencing technology allows for the analysis of heretofore-undetectable minor viral mutants, a limited amount of information is currently available regarding the clinical implications of hepatitis B virus (HBV) genomic heterogeneity.MethodsTo characterize the HBV genetic heterogeneity in association with anti-viral therapy, we performed ultra-deep sequencing of full-genome HBV in the liver and serum of 19 patients with chronic viral infection, including 14 therapy-naïve and 5 nucleos(t)ide analogue(NA)-treated cases.ResultsMost genomic changes observed in viral variants were single base substitutions and were widely distributed throughout the HBV genome. Four of eight (50%) chronic therapy-naïve HBeAg-negative patients showed a relatively low prevalence of the G1896A pre-core (pre-C) mutant in the liver tissues, suggesting that other mutations were involved in their HBeAg seroconversion. Interestingly, liver tissues in 4 of 5 (80%) of the chronic NA-treated anti-HBe-positive cases had extremely low levels of the G1896A pre-C mutant (0.0%, 0.0%, 0.1%, and 1.1%), suggesting the high sensitivity of the G1896A pre-C mutant to NA. Moreover, various abundances of clones resistant to NA were common in both the liver and serum of treatment-naïve patients, and the proportion of M204VI mutants resistant to lamivudine and entecavir expanded in response to entecavir treatment in the serum of 35.7% (5/14) of patients, suggesting the putative risk of developing drug resistance to NA.ConclusionOur findings illustrate the strong advantage of deep sequencing on viral genome as a tool for dissecting the pathophysiology of HBV infection.

The hepatitis C virus (HCV) invariably shows wide heterogeneity in infected patients, referred to as a quasispecies population. Massive amounts of genetic information due to the abundance of HCV variants could be an obstacle to evaluate the viral genetic heterogeneity in detail.Using a newly developed massive-parallel ultra-deep sequencing technique, we investigated the viral genetic heterogeneity in 27 chronic hepatitis C patients receiving peg-interferon (IFN) α2b plus ribavirin therapy.Ultra-deep sequencing determined a total of more than 10 million nucleotides of the HCV genome, corresponding to a mean of more than 1000 clones in each specimen, and unveiled extremely high genetic heterogeneity in the genotype 1b HCV population. There was no significant difference in the level of viral complexity between immediate virologic responders and non-responders at baseline (p = 0.39). Immediate virologic responders (n = 8) showed a significant reduction in the genetic complexity spanning all the viral genetic regions at the early phase of IFN administration (p = 0.037). In contrast, non-virologic responders (n = 8) showed no significant changes in the level of viral quasispecies (p = 0.12), indicating that very few viral clones are sensitive to IFN treatment. We also demonstrated that clones resistant to direct-acting antivirals for HCV, such as viral protease and polymerase inhibitors, preexist with various abundances in all 27 treatment-naïve patients, suggesting the risk of the development of drug resistance against these agents.Use of the ultra-deep sequencing technology revealed massive genetic heterogeneity of HCV, which has important implications regarding the treatment response and outcome of antiviral therapy.