Fukiko Kawai-Kitahata

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

BACKGROUND AND AIMS Although 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. METHODS A 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. RESULTS Propensity 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. CONCLUSION 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. 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.

ITPA gene variation and ribavirin-induced anemia in patients with genotype 2 chronic hepatitis C treated with sofosbuvir plus ribavirin

Sofosbuvir (SOF) and ribavirin (RBV) combination therapy produces a sustained response in many patients with genotype 2 chronic hepatitis C. However, RBV‐induced anemia is a troublesome side‐effect that may limit this treatment. Genetic variation leading to inosine triphosphatase (ITPA) deficiency is known to protect against RBV‐induced hemolytic anemia. This study aimed to evaluate the relationships between the efficacy and safety of SOF/RBV treatment and ITPA gene variants.

Efficacy of additional radiofrequency ablation after transcatheter arterial chemoembolization for intermediate hepatocellular carcinoma

For intermediate hepatocellular carcinoma (HCC), transcatheter arterial chemoembolization (TACE) therapy is recommended in the guidelines as a monotherapy, although TACE is a non‐curative therapy. The aims of the present study were to evaluate the efficacy of adding radiofrequency ablation (RFA) to TACE in patients with intermediate HCC, and to identify the factors that were associated with favorable survival in these patients.

Impaired induction of interleukin 28B and expression of interferon λ 4 associated with nonresponse to interferon‐based therapy in chronic hepatitis C

Interferon (IFN) λ plays an important role in innate immunity to protect against hepatitis C viral (HCV) infection. Single nucleotide polymorphisms (SNPs) near IL28B (IFNλ3) are strongly associated with treatment response to IFNα therapy in chronic hepatitis C (CHC) patients. Recently, IFNλ4 related to IL28B‐unfavorable allele was discovered. However, the impact of IFNλs on CHC is unknown. We aimed to investigate the mechanism underlying responsiveness to IFN‐based therapy in CHC associated with SNPs near IL28B.

Human induced pluripotent stem cell-derived hepatic cell lines as a new model for host interaction with hepatitis B virus

Hepatitis B virus (HBV) is not eradicated by current antiviral therapies due to persistence of HBV covalently closed circular DNA (cccDNA) in host cells, and thus development of novel culture models for productive HBV infection is urgently needed, which will allow the study of HBV cccDNA eradication. To meet this need, we developed culture models of HBV infection using human induced pluripotent stem cell-derived hepatocyte lineages, including immature proliferating hepatic progenitor-like cell lines (iPS-HPCs) and differentiated hepatocyte-like cells (iPS-Heps). These cells were susceptible to HBV infection, produced HBV particles, and maintained innate immune responses. The infection efficiency of HBV in iPS-HPCs predominantly depended on the expression levels of sodium taurocholate cotransporting polypeptide (NTCP), and was low relative to iPS-Heps: however, long-term culture of iPS-Heps was difficult. To provide a model for HBV persistence, iPS-HPCs overexpressing NTCP were established. The long-term persistence of HBV cccDNA was detected in iPS-HPCs overexpressing NTCP, and depended on the inhibition of the Janus-kinase signaling pathway. In conclusion, this study provides evidence that iPS-derived hepatic cell lines can be utilized for novel HBV culture models with genetic variation to investigate the interactions between HBV and host cells and the development of anti-HBV strategies.

Matrix metalloproteinase-14 mediates formation of bile ducts and hepatic maturation of fetal hepatic progenitor cells

Fetal hepatic stem/progenitor cells, called hepatoblasts, play central roles in liver development; however, the molecular mechanisms regulating the phenotype of these cells have not been completely elucidated. Matrix metalloproteinase (MMP)-14 is a type I transmembrane proteinase regulating pericellular proteolysis of the extracellular matrix and is essential for the activation of several MMPs and cytokines. However, the physiological functions of MMP-14 in liver development are unknown. Here we describe a functional role for MMP-14 in hepatic and biliary differentiation of mouse hepatoblasts. MMP-14 was upregulated in cells around the portal vein in perinatal stage liver. Formation of bile duct-like structures in MMP-14-deficient livers was significantly delayed compared with wild-type livers in vivo. In vitro biliary differentiation assays showed that formation of cholangiocytic cysts derived from MMP-14-deficient hepatoblasts was completely impaired, and that overexpression of MMP-14 in hepatoblasts promoted the formation of bile duct-like cysts. In contrast, the expression of molecules associated with metabolic functions in hepatocytes, including hepatic nuclear factor 4α and tryptophan 2,3-dioxygenase, were significantly increased in MMP-14-deficient livers. Expression of the epidermal growth factor receptor and phosphorylation of mitogen-activated protein kinases were significantly upregulated in MMP-14-deficient livers. We demonstrate that MMP-14-mediated signaling in fetal hepatic progenitor cells promotes biliary luminal formation around the portal vein and negatively controls the maturation of hepatocytes.

Hepatic IFNL4 expression is associated with non‐response to interferon‐based therapy through the regulation of basal interferon‐stimulated gene expression in chronic hepatitis C patients

Single nucleotide polymorphisms (SNPs) within or near interferon lambda 4 (IFNL4) gene located upstream of IFNL3 are associated with response to anti‐HCV therapy both in interferon (IFN)‐based and IFN‐free regimens. IFNL4 encodes IFNλ4, a newly discovered type III IFN, and its expression is controlled by rs368234815‐TT/ΔG, which is in strong linkage disequilibrium (LD) with other tag SNPs within or near IFNL4 such as rs12979860 and rs8099917. Intrahepatic expression levels of IFN‐stimulated genes (ISGs) affect the responsiveness to IFNα and are also associated with IFNL4 genotype. However, IFNL4 expressions and its role in intrinsic antiviral innate immunity remain unclear. This study evaluated the effect of IFNL4 on intrahepatic ISG expression and investigated its relationship with treatment outcomes in liver samples obtained from 49 chronic hepatitis C patients treated with pegylated (PEG)‐IFN/ribavirin therapy. IFNL4 mRNA was detected in 11 of 22 patients with IFNL4‐unfavorable SNPs but not in patients with favorable genotypes. IFNL4 expression was associated with non‐response to PEG‐IFN/ribavirin therapy. Intrahepatic expression of antiviral ISGs (ISG15 and MX1) was significantly higher in IFNL4‐unfavorable patients with detectable IFNL4 mRNA than in patients with undetectable IFNL4 mRNA, whereas the expression of suppressive ISGs (RNF125, SOCS1, SOCS3, and RNF11) was lower in patients with detectable IFNL4 mRNA. In summary, intrahepatic expression of IFNL4 was associated with increased antiviral ISG expression and decreased suppressive ISG expression at baseline, resulting in poor responsiveness to IFNα‐based therapy in HCV infection.

Bone morphogenetic protein-4 modulates proliferation and terminal differentiation of fetal hepatic stem/progenitor cells.

Fetal hepatic stem/progenitor cells, called hepatoblasts, play central roles in liver organogenesis; however, molecular mechanisms regulating proliferation and terminal differentiation of such cells have not been completely elucidated. Bone morphogenetic protein‐4 (BMP‐4) is essential for the development of stem cells in various tissues, but its function in regulating the phenotype of hepatoblasts after the mid‐gestational fetal stage remains unclear. The aim of this study is to clarify a functional role for BMP‐4 in proliferation and terminal differentiation of murine hepatoblasts in mid‐gestational fetal livers.

Evaluation of Interferon Resistance in Newly Established Genotype 1b Hepatitis C Virus Cell Culture System.

Background and Aims: The hepatitis C virus (HCV) genotype 1b is known to exhibit treatment resistance with respect to interferon (IFN) therapy. Substitution of amino acids 70 and 91 in the core region of the 1b genotype is a significant predictor of liver carcinogenesis and poor response to pegylated-IFN-α and ribavirin therapy. However, the molecular mechanism has not yet been clearly elucidated because of limitations of the HCV genotype 1b infectious model. Recently, the TPF1-M170T HCV genotype 1b cell culture system was established, in which the clone successfully replicates and infects Huh-7-derived Huh7-ALS32.50 cells. Therefore, the purpose of this study was to compare IFN resistance in various HCV clones using this system. Methods: HCV core amino acid substitutions R70Q and L91M were introduced to the TPF1-M170T clone and then transfected into Huh7-ALS32.50 cells. To evaluate the production of each virus, intracellular HCV core antigens were measured. Results were confirmed with Western blot analysis using anti-NS5A antibodies, and IFN sensitivity was subsequently measured. Results: Each clone was transfected successfully compared with JFH-1, with a significant difference in intracellular HCV core antigen (p < 0.05), an indicator of continuous HCV replication. Among all clones, L91M showed the highest increase in the HCV core antigen and HCV protein. There was no significant resistance against IFN treatment in core substitutions; however, IFN sensitivity was significantly different between the wildtype core and JFH-1 (p < 0.05). Conclusions: A novel genotype 1b HCV cell culture was constructed with core amino acid substitutions, which demonstrated IFN resistance of genotype 1b. This system will be useful for future analyses into the mechanisms of HCV genotype 1b treatment.