Ryoko Yamada

Frequency of, and factors associated with, hepatitis B virus reactivation in hepatitis C patients treated with all-oral direct-acting antivirals: Analysis of a Japanese prospective cohort

Several case reports have shown that hepatitis B virus (HBV) reactivation developed in hepatitis C patients with a current or previous HBV infection during direct‐acting antiviral (DAA) treatment, which led to severe hepatitis or death in some cases. However, its precise frequency and risk factors are not entirely clear. We analyzed a prospective cohort.

Significance of liver stiffness measurement by acoustic radiation force impulse (ARFI) among hepatitis C patients

The degree of liver fibrosis is strongly associated with the antiviral effect of interferon on chronic hepatitis C patients. In this study, the accuracy of acoustic radiation force impulse (ARFI) in assessing liver fibrosis and the association between liver stiffness using ARFI and antiviral effects were investigated. The 124 patients with chronic hepatitis C enrolled in this study included 94 with HCV genotype 1 and 40 (30%) with moderate fibrosis (METAVIR fibrosis score ≥ F2). Sixty‐one patients received pegylated interferon (peg‐IFN) plus ribavirin combination therapy and the treatment responses were assessed. The shear wave velocity (Vs value) by ARFI had a strong correlation with the histological fibrosis stage (P < 0.001). The AUROC of the Vs value, aspartate aminotransferase platelet ratio index and FIB4 for the diagnoses of moderate fibrosis (≥F2) were 0.890, 0.779, and 0.737, respectively. HCV genotype 1 patients with the TT allele of IL28B and with a low Vs value (<1.40 m/sec) who were treated with peg‐IFN plus ribavirin therapy achieved a sustained virologic response at a rate of 79% (15/19), while all patients with the TG/GG allele of IL28B and a high Vs value (≥1.40 m/sec) experienced a non‐virologic response (6/6). The Vs value measured by ARFI could not predict the treatment response for patients with HCV genotype 2. It is concluded that the combination of ARFI at cut off of 1.4 m/sec and IL28B may be useful for patients with chronic hepatitis C with genotype 1 treated with peg‐IFN/ribavirin combination therapy. J. Med. Virol. 86:241–247, 2014.

Baseline quasispecies selection and novel mutations contribute to emerging resistance-associated substitutions in hepatitis C virus after direct-acting antiviral treatment

Resistance-associated substitutions (RASs) in hepatitis C virus (HCV) appear upon failure of treatment with direct-acting antivirals (DAAs). However, their origin has not been clarified in detail. Among 11 HCV genotype 1b patients who experienced virologic failure with asunaprevir (ASV)/daclatasvir (DCV), 10 had major NS5A L31M/V-Y93H variants after treatment. L31M/V-Y93H variants were detected as a minor clone before therapy in 6 patients and were the most closely related to the post-treatment variants by phylogenetic tree analysis in 4 patients. Next, to consider the involvement of a trace amount of pre-existing variants below the detection limit, we analysed human hepatocyte chimeric mice infected with DAA-naïve patient serum. L31V-Y93H variants emerged after treatment with ledipasvir (LDV)/GS-558093 (nucleotide NS5B inhibitor) and decreased under the detection limit, but these variants were dissimilar to the L31V-Y93H variants reappearing after ASV/DCV re-treatment. Finally, to develop an infection derived from a single HCV clone, we intrahepatically injected full-genome HCV RNA (engineered based on the wild-type genotype 1b sequence) into chimeric mice. A new Y93H mutation actually occurred in this model after LDV monotherapy failure. In conclusion, post-treatment RASs appear by 2 mechanisms: the selection of pre-existing substitutions among quasispecies and the generation of novel mutations during therapy.

Risk factors for hepatocellular carcinoma in hepatitis C patients with normal alanine aminotransferase treated with pegylated interferon and ribavirin

Pegylated interferon (Peg‐IFN) plus ribavirin combination therapy is effective in patients with hepatitis C virus (HCV) infection and normal alanine aminotransferase levels (NALT). However, it remains unclear whether the risk of hepatocellular carcinoma (HCC) incidence is actually reduced in virological responders. In this study, HCC incidence was examined for 809 patients with NALT (ALT ≤ 40 IU/mL) treated with Peg‐IFN alpha‐2b and ribavirin for a mean observation period of 36.2 ± 16.5 months. The risk factors for HCC incidence were analysed by Kaplan–Meier method and Cox proportional hazards model. On multivariate analysis among NALT patients, the risk of HCC incidence was significantly reduced in patients with sustained virological response (SVR) or relapse compared with those showing nonresponse (NR) (SVR vs NR, hazard ratio (HR): 0.16, P = 0.009, relapse vs NR, HR: 0.11, P = 0.037). Other risk factors were older age (≥65 years vs <60 years, HR: 6.0, P = 0.032, 60–64 vs <60 years, HR: 3.2, P = 0.212) and male gender (HR: 3.9, P = 0.031). Among 176 patients with PNALT (ALT ≤ 30 IU/mL), only one patient developed HCC and no significant risk factors associated with HCC development were found. In conclusion, antiviral therapy for NALT patients with HCV infection can lower the HCC incidence in responders, particularly for aged and male patients. The indication of antiviral therapy for PNALT (ALT ≤ 30 IU/mL) patients should be carefully determined.

The suppressive effect of nucleos(t)ide analogue treatment on the incidence of hepatocellular carcinoma in chronic hepatitis B patients

The development of nucleos(t)ide analogues (NA) has influenced hepatitis B virus management. However, the annual incidence rate during NA treatment has been reported to be 0.3–1.2% in non‐cirrhosis cases and 1.8–6.0% in cirrhosis cases, indicating that the suppressive effect of NA treatment on hepatocellular carcinoma (HCC) would be insufficient. Past studies, including one randomized control trial that compared lamivudine treatment with placebo, have revealed that NA treatment could suppress the incidence of HCC in patients with advanced fibrosis. However, it remains unknown whether NA treatment can suppress the incidence of HCC in chronic hepatitis patients without advanced fibrosis. The HCC incidence in patients treated with entecavir was similar to that of those treated with lamivudine, although entecavir exhibits a stronger viral suppression than lamivudine. The following risk factors related to the incidence of HCC during NA treatment have been identified: older age, male gender, pre‐existing cirrhosis, a family clustering of hepatitis B virus, lower platelet counts, and higher hepatitis B core‐related antigens as baseline factors and higher alpha fetoprotein levels as an on‐treatment factor. Conversely, the loss of the hepatitis B surface antigen (HBsAg) by interferon or NA was correlated with a lower HCC incidence rate. Because interferon treatment has much more effects on reducing HBsAg levels compared with NA treatment, a combination treatment with NA and pegylated interferon can bring additional reduction of HBsAg levels compared with NA monotherapy. Further study is needed to clarify this.

The real impact of telaprevir dosage on the antiviral and side effects of telaprevir, pegylated interferon and ribavirin therapy for chronic hepatitis C patients with HCV genotype 1

Triple therapy with telaprevir, pegylated interferon and ribavirin has been reported to improve antiviral efficacy but have potentially severe adverse effects in patients with chronic hepatitis C. To avoid the severe effects of telaprevir, lowering the dose has been suggested. However, impact of dosage changes on antiviral and adverse effects remains unclear. One hundred and sixty‐six Japanese patients with HCV genotype 1 were treated with triple therapy. The drug exposure of each medication was calculated by averaging the dose actually taken. The overall SVR rate was 82%. The telaprevir discontinuation rate was 26%. The factors associated with discontinuation were an older age (≥65 y.o.) and a higher average dose during treatment. The telaprevir discontinuation rates were 42%, 25% and 14% in patients at ≥35, 25–35 and <25 mg/kg/day of telaprevir and 58% in older patients at ≥35 mg/kg/day of TVR. The factors associated with SVR were treatment‐naïve, relapse to previous treatment, higher average telaprevir dose during treatment and completion of treatment. The SVR rate was higher, at 91%, in patients at 25–35 mg/kg/day of telaprevir than the 71% and 78% observed in those at <25 and ≥35 mg/kg/day of drug. In Japanese patients, a mean telaprevir dose of 25–35 mg/kg/day during treatment can augment its efficacy in triple therapy for patients with HCV genotype 1.

Nonstructural protein 5A/P32 deletion after failure of ledipasvir/sofosbuvir in hepatitis C virus genotype 1b infection

Nonstructural protein 5A (NS5A) inhibitors play an essential role in the combination treatment of direct-acting antivirals (DAAs) against chronic hepatitis C (CHC). Daclatasvir, a prototype of this class, combined with asunaprevir, has been widely used since 2014 in Asia, where genotype 1b infection is prevalent. Well-known resistance-associated substitutions (RASs) for this treatment include NS5A-L31M/V and Y93H. In addition, recent studies have reported the emergence of a deletion mutant at NS5A-P32 in a subset of patients at the time of virological failure (VF) with this treatment. This mutant is attracting much attention because replicon studies indicate that, compared with NS5A-Y93H, it confers extremely higher resistance to NS5A inhibitors. Moreover, retreatment with DAAs in patients carrying this mutant after daclatasvir/asunaprevir treatment has been reported to be ineffective. Here, we report on a case of NS5A-P32 deletion (P32del) after ledipasvir/sofosbuvir treatment. We treated 1,031 Japanese patients with genotype 1 CHC with ledipasvir/sofosbuvir, and 12 cases exhibited VF. Among these cases, sera from 10 patients available were analyzed for NS5A RAS by deep sequencing, as described (Table 1). All patients had genotype 1b infection and, except for case no. 1, exhibited the NS5A Y93H substitution at baseline or after VF. Case no. 1 showed P32del at the time of VF; the P32del was not detected at baseline (0.1% deep sequencing cutoff), but appeared 4 weeks after completion of 12 weeks of treatment and was continuously detected at a high frequency for 52 weeks (Fig. 1A). After the appearance of the P32del, nucleotide sequences of NS5A domain-I in a major clone were completely consistent at any point. In addition, known RASs to sofosbuvir, including S282T, were not detected by deep sequencing during the entire course. The patient was a 75-year-old treatment-na€ıve man with compensated cirrhosis with a treatment history for hepatocellular carcinoma. We performed phylogenetic tree analysis to clarify the relationship between pretreatment virus and posttreatment virus (Fig. 1B). This analysis indicated that the most frequent wild-type virus at baseline (A1 in Fig. 1B) acquired a deletion of three bases at position P32, becoming virus B18 posttreatment; one more sense mutation resulted in the most frequent virus, B1, posttreatment, which served as the starting point for the subsequent expansion of diversity. Both clinical trials and real-world data have shown that the viral clearance rate of ledipasvir/sofosbuvir is

Ultra-deep sequencing analysis of resistance-associated variants during retreatment with simeprevir-based triple therapy after failure of telaprevir-based triple therapy in patients with genotype 1 hepatitis C virus infection

Simeprevir (SMV)‐based triple therapy is an effective retreatment option following failure of telaprevir (TVR)‐based triple therapy. However, it is unclear whether the persistence of resistance‐associated variants (RAVs) induced by TVR‐based therapy may reduce the treatment effect of SMV‐based therapy.

CTGF Mediates Tumor–Stroma Interactions between Hepatoma Cells and Hepatic Stellate Cells to Accelerate HCC Progression

Connective tissue growth factor (CTGF) is a matricellular protein related to hepatic fibrosis. This study aims to clarify the roles of CTGF in hepatocellular carcinoma (HCC), which usually develops from fibrotic liver. CTGF was overexpressed in 93 human HCC compared with nontumorous tissues, primarily in tumor cells. Increased CTGF expression was associated with clinicopathologic malignancy of HCC. CTGF was upregulated in hepatoma cells in hepatocyte-specific Kras-mutated mice (Alb-Cre KrasLSL-G12D/+). Hepatocyte-specific knockout of CTGF in these mice (Alb-Cre KrasLSL-G12D/+ CTGFfl/fl) decreased liver tumor number and size. Hepatic stellate cells (HSC) were present in both human and murine liver tumors, and α-SMA expression, a marker of HSC activation, positively correlated with CTGF expression. Forced expression of CTGF did not affect growth of PLC/PRF/5 cells, a hepatoma cell line with little CTGF expression, but facilitated their growth in the presence of LX-2 cells, an HSC line. The growth of HepG2 cells, which express high levels of CTGF, was promoted by coculture with LX-2 cells compared with monoculture. Growth promotion by LX-2 cells was negated by an anti-CTGF antibody in both culture and xenografts. Coculturing LX-2 cells with HepG2 cells drove LX-2-derived production of IL6, which led to STAT-3 activation and proliferation of HepG2 cells. An anti-CTGF antibody reduced IL6 production in LX-2 cells and suppressed STAT-3 activation in HepG2 cells. In conclusion, our data identify tumor cell-derived CTGF as a keystone in the HCC microenvironment, activating nearby HSC that transmit progrowth signals to HCC cells, and this interaction is susceptible to inhibition by an anti-CTGF antibody.Significance: Protumor cross-talk between cancer cells and hepatic stellate cells presents an opportunity for therapeutic intervention against HCC.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/17/4902/F1.large.jpg Cancer Res; 78(17); 4902-14. ©2018 AACR.

Impact of ribavirin dosage in chronic hepatitis C patients treated with simeprevir, pegylated interferon plus ribavirin combination therapy

The factors associated with sustained virologic response (SVR) in chronic hepatitis C (CH‐C) genotype 1 patients treated with simeprevir (SMV), pegylated interferon (Peg‐IFN) plus ribavirin (RBV) triple therapy have not been fully investigated. Two hundred and twenty‐nine treatment‐naïve CH‐C patients treated with SMV triple therapy were enrolled in this study. The overall SVR rate was 87% in per‐protocol analysis. In multivariate analysis, the interleukin (IL) 28B genotype (rs8099917, TT vs. non‐TT, odds ratio [OR]: 0.044, P = 0.001) and RBV dose (< 10/10–12/ ≥ 12 mg/kg/day, OR: 4.513, P = 0.041) were significant factors associated with SVR. In patients with the IL28B non‐TT genotype, RBV dose affected SVR dose‐dependently in stratified analysis of RBV dose (P = 0.015); it was 44% (8/18) for patients administered <10 mg/kg/day of RBV, 78% (14/18) for those administered 10–12 mg/kg/day of RBV, and 100% (3/3) for those administered ≥12 mg/kg/day of RBV, whereas in patients with the IL28B TT genotype, a significant correlation between SVR and RBV dose was not observed (P = 0.229). Regarding RBV dose reduction of less than 10 mg/kg/day, the inosine triphosphate pyrophosphatase (ITPA) genotype (rs1127354, CC vs. non‐CC, OR: 0.239, P = 0.003) and age (by 1 y.o., OR: 1.084, P = 0.002) were significant independent factors. RBV dosage affected SVR dose‐dependently in patients with the IL28B non‐TT genotype treated with SMV triple therapy. Special attention to anemia progression and RBV dosage should be paid to aged patients with the ITPA CC genotype. J. Med. Virol. 88:1776–1784, 2016.