A20 in hepatic stellate cells suppresses chronic hepatitis by inhibiting DCLK1-JNK pathway-dependent chemokines.

Hepatic stellate cells (HSCs) are responsible for liver fibrosis accompanied by its activation into myofibroblasts and the abundant production of extracellular matrix. However, the HSC contribution to progression of liver inflammation has been less known. We aimed to elucidate the mechanism in HSCs underlying the inflammatory response and the function of tumor necrosis factor α-related protein A20 (TNFAIP3). We established A20 conditional knockout (KO) mice crossing Twist2-Cre and A20 floxed mice. Using these mice, the effect of A20 was analyzed in mouse liver and HSCs. The human HSC line LX-2 was also used to examine the role and underlying molecular mechanism of A20. In this KO model, A20 was deficient in >80% of HSCs. Spontaneous inflammation with mild fibrosis was found in the liver of the mouse model without any exogenous agents, suggesting that A20 in HSCs suppresses chronic hepatitis. Comprehensive RNA sequence analysis revealed that A20-deficient HSCs exhibited an inflammatory phenotype and abnormally expressed chemokines. A20 suppressed JNK pathway activation in HSCs. Loss of A20 function in LX-2 cells also induced excessive chemokine expression, mimicking A20-deficient HSCs. A20 overexpression suppressed chemokine expression in LX-2. In addition, we identified DCLK1 in the genes regulated by A20. DCLK1 activated the JNK pathway and upregulates chemokine expression. DCLK1 inhibition significantly decreased chemokine induction by A20-silencing, suggesting that A20 controlled chemokine expression in HSCs via the DCLK1-JNK pathway. In conclusion, A20 suppresses chemokine induction dependent on the DCLK1-JNK signaling pathway. These findings demonstrate the therapeutic potential of A20 and the DCLK1-JNK pathway for the regulation of inflammation in chronic hepatitis.

Prognostic significance of C-reactive protein in unresectable hepatocellular carcinoma treated with atezolizumab and bevacizumab.

AIM: C-reactive protein (CRP) is both an inflammatory and prognostic marker in various cancers. This study aimed to elucidate the characteristics of CRP and the prognostic factors in patients who were administered with atezolizumab plus bevacizumab (ATZ + BEV) for unresectable hepatocellular carcinoma (HCC). METHODS: A total of 213 patients who received ATZ + BEV for HCC from November 2020 to March 2023 at 15 hospitals were enrolled in this retrospective study. The prognosis was analyzed by subdividing the patients based on baseline characteristics, radiologic response, and treatment lines. Accuracy of survival prediction was assessed using CRP, alpha fetoprotein (AFP), C-reactive protein and alpha fetoprotein in immunotherapy (CRAFITY), and Glasgow Prognostic Score. RESULTS: Compared with patients with baseline CRP <1 mg/dL, those with baseline CRP ≥1 mg/dL (n = 45) had a significantly higher baseline albumin-bilirubin score and AFP levels, significantly lower disease control rate (62.2%), and significantly shorter median overall survival (hazards ratios 2.292; 95% confidence interval 1.313-5.107; log-rank test, p < 0.001). Multivariate analysis identified CRP ≥1 mg/dL, AFP ≥100 ng/mL, and modified albumin-bilirubin grade as the significant prognostic factors. The baseline CRP, AFP, CRAFITY, and Glasgow Prognostic Score demonstrated higher discrimination for 1-year survival prediction after first-line ATZ + BEV administration, compared with beyond second line, with area under the receiver operating characteristic curves of 0.759, 0.761, 0.805, and 0.717, respectively. CONCLUSIONS: CRP was a significant biomarker in patients treated with ATZ + BEV for HCC. Elevated CRP levels may indicate aggressive cancer progression and potential resistance to ATZ + BEV therapy.

High serum gamma-glutamyltransferase level after hepatitis C virus elimination is a risk factor for the development of hepatocellular carcinoma.

AIM: Gamma-glutamyltransferase (GGT) is known as an oxidative stress marker, induced by alcohol consumption and metabolic disorders, and is reported as a predictor of hepatocellular carcinoma (HCC) development after hepatitis C virus (HCV) elimination. However, it is not clear whether GGT serves simply as a surrogate marker for overlapping metabolic diseases or reflects HCV-specific carcinogenicity. We investigated the association between GGT and hepatocarcinogenesis after achieving a sustained viral response (SVR), accounting for drinking habits or diabetes, and examined predisposing factors associated with GGT levels after SVR. METHODS: This is a prospective, multicenter, and observational study using the database of 1001 patients after HCV eradication with direct-acting antiviral agents. The association of GGT at SVR with cumulative HCC development was examined in a multivariate analysis using Cox proportional hazard models after adjustment for covariates including alcohol and diabetes. The association between oxidative stress markers or genetic factors and GGT levels was analyzed. RESULTS: High GGT levels at SVR were associated with HCC development (HR] 2.38, 95% CI 1.10-5.17). This association was also significant when restricted to patients without alcohol consumption or diabetes (HR 8.38, 95% CI 2.87-24.47). GGT levels were correlated with serum growth differentiation factor 15 levels, a marker of mitochondrial dysfunction. Single-nucleotide polymorphisms of ZNF827 and GDF15 were associated with high GGT levels. CONCLUSIONS: High GGT levels at SVR were associated with HCC development after accounting for alcohol consumption and diabetes. GGT levels are influenced by genetic predisposition and may reflect mitochondrial dysfunction after HCV eradication.

Factors associated with liver injury and prognosis in advanced cancer patients treated with immune checkpoint inhibitors.

AIM: The use of immune checkpoint inhibitors (ICIs) has increased remarkably, and immune-related adverse events (irAEs) have also increased. This study aimed to identify factors associated with immune-related liver injury (irLI), and the relationship between the grades of irLI and overall survival (OS) in patients treated with ICIs. METHODS: A total of 571 patients who had been treated for advanced malignancies with ICIs between January 2015 and March 2022 were retrospectively recruited. The presence of liver injury was determined by the aspartate aminotransferase and alanine aminotransferase elevation. The irLI grading was based on Common Terminology Criteria for Adverse Events version 5.0. RESULTS: A total of 50 (8.8%) patients had grade ≥2 irLI and 24 (4.2%) had grade ≥3 irLI. Treatment with anti-cytotoxic T-lymphocyte-associated protein-4 agents and baseline grade 1 aspartate aminotransferase/alanine aminotransferase elevation were independent predictive factors of grade ≥2 irLI. Treatment with anti-cytotoxic T-lymphocyte-associated protein-4 was the only independent predictive factor of grade ≥3 irLI. The median OS for patients who experienced any irAEs was significantly longer than of those without irAEs (hazard ratio 0.503, 95% CI 0.398-0.636, p < 0.001). The median OS in patients with grade ≥2 irLI was significantly longer (HR 0.570, 95% CI 0.387-0.838, p = 0.022). There was no significant difference between the median OS in patients with grade ≥3 irLI and the others (p = 0.11). CONCLUSION: The incidence of irLI was significantly higher in patients treated with anti-cytotoxic T-lymphocyte-associated protein-4 agents. Even in patients with pre-existing grade 1 aspartate aminotransferase/alanine aminotransferase elevation, appropriate follow-up and control of the irLI can improve the prognosis.

Time course alterations of virus sequences and immunoglobulin titers in a chronic hepatitis E patient.

AIM: Hepatitis E virus (HEV) can cause chronic infection in immunocompromised hosts. However, the dynamics of HEV during persistent infection is not well understood. To elucidate time course alterations in virus sequences and anti-HEV antibodies during persistent infection, we analyzed the HEV sequences and titers of anti-HEV antibodies from a chronic hepatitis E patient. METHODS: Serum samples were obtained from a chronic hepatitis E patient under corticosteroid therapy for neurological disease. The titers of anti-HEV antibodies (immunoglobulin A, immunoglobulin M, and immunoglobulin G) in serum samples were detected by enzyme immunoassay. The full or near-full nucleotide sequences of HEV isolated from consecutive serum samples were identified and compared. Phylogenetic analysis was also performed. RESULTS: Alterations of anti-HEV antibodies from a chronic hepatitis E patient were different from those previously reported in acute hepatitis E patients. The virus sequence was unchanged in the period without treatment, but nucleotide mutations were observed after ribavirin treatment was started. In addition, the sequence of this strain had extremely high identity to that isolated from swine liver in Japan. CONCLUSIONS: Virus mutations in HEV emerged after ribavirin treatment was started. Sequence analysis may useful for deciding the treatment strategy for chronic hepatitis E patients who did not eliminate the virus with 3 months of RBV treatment and inferring the origin of the infection. This report provides insights into the chronicity of hepatitis E, and the impact of persistent infection and ribavirin treatment on the emergence of virus mutations.

Loss of fibrocystin promotes interleukin-8-dependent proliferation and CTGF production of biliary epithelium.

BACKGROUND & AIMS: Congenital hepatic fibrosis (CHF) is a genetic liver disease resulting in abnormal proliferation of cholangiocytes and progressive hepatic fibrosis. CHF is caused by mutations in the PKHD1 gene and the subsequent dysfunction of the protein it encodes, fibrocystin. However, the underlying molecular mechanism of CHF, which is quite different from liver cirrhosis, remains unclear. This study investigated the molecular mechanism of CHF pathophysiology using a genetically engineered human induced pluripotent stem (iPS) cell model to aid the discovery of novel therapeutic agents for CHF. METHODS: PKHD1-knockout (PKHD1-KO) and heterozygously mutated PKHD1 iPS clones were established by RNA-guided genome editing using the CRISPR/Cas9 system. The iPS clones were differentiated into cholangiocyte-like cells in cysts (cholangiocytic cysts [CCs]) in a 3D-culture system. RESULTS: The CCs were composed of a monolayer of cholangiocyte-like cells. The proliferation of PKHD1-KO CCs was significantly increased by interleukin-8 (IL-8) secreted in an autocrine manner. IL-8 production was significantly elevated in PKHD1-KO CCs due to mitogen-activated protein kinase pathway activation caused by fibrocystin deficiency. The production of connective tissue growth factor (CTGF) was also increased in PKHD1-KO CCs in an IL-8-dependent manner. Furthermore, validation analysis demonstrated that both the serum IL-8 level and the expression of IL-8 and CTGF in the liver samples were significantly increased in patients with CHF, consistent with our in vitro human iPS-disease model of CHF. CONCLUSIONS: Loss of fibrocystin function promotes IL-8-dependent proliferation of, and CTGF production by, human cholangiocytes, suggesting that IL-8 and CTGF are essential for the pathogenesis of CHF. IL-8 and CTGF are candidate molecular targets for the treatment of CHF. LAY SUMMARY: Congenital hepatic fibrosis (CHF) is a genetic liver disease caused by mutations of the PKHD1 gene. Dysfunction of the protein it encodes, fibrocystin, is closely associated with CHF pathogenesis. Using an in vitro human induced pluripotent stem cell model and patient samples, we showed that the loss of fibrocystin function promotes proliferation of cholangiocytes and the production of connective tissue growth factor (CTGF) in an interleukin 8 (IL-8)-dependent manner. These results suggest that IL-8 and CTGF are essential for the pathogenesis of CHF.

Diabetic Retinopathy as a Risk Factor Associated with the Development of Hepatocellular Carcinoma in Nonalcoholic Fatty Liver Disease.

BACKGROUND: The risk factors associated with the development of hepatocellular carcinoma (HCC) in nonalcoholic fatty liver disease (NAFLD) are still unclear. The aim of the present study was to identify such risk factors in NAFLD patients who developed HCC. METHODS: Between April 2000 and -December 2016, a total of 182 patients with NAFLD were enrolled in this study; of these, only 22 patients had HCC. To identify risk factors, univariate and multivariate analyses were performed. To identify risk factors other than the degree of fibrosis, propensity matched analysis adjusted by the NAFLD fibrosis score (NFS) was carried out on 44 patients. Multivariate and survival analyses were also performed in HCC patients. RESULTS: In 182 patients, multivariate analysis highlighted the NFS (OR 2.275; p < 0.001) and hypertension (OR 5.868; p = 0.037) as independent factors that were significantly associated with the development of HCC. After adjustment for the NFS, multivariate analysis identified diabetic retinopathy (OR 8.654; p = 0.017) as an independent factor that was significantly associated with the development of HCC. For predicting the development of HCC, the area under the receiver operating characteristic curve of diabetic retinopathy was significantly higher than that of diabetes (0.731 vs. 0.615; p < 0.001). In patients with HCC, multivariate analysis indicated that the NFS were significantly associated with diabetic retinopathy. CONCLUSIONS: Diabetic retinopathy as well as liver fibrosis is a risk factor that associates with the development of HCC in NAFLD patients. Therefore, NAFLD patients with diabetic retinopathy should undergo careful screening for HCC.

Association of serum interferon-λ3 levels with hepatocarcinogenesis in chronic hepatitis C patients treated with direct-acting antiviral agents.

AIM: Although the efficacy of hepatitis C virus (HCV) treatment is improved dramatically by direct-acting antiviral agents (DAAs), the assessment of hepatocellular carcinoma (HCC) remains important. Interferon lambda 3 (IFN-λ3) is associated with liver fibrosis and inflammation in chronic hepatitis C (CHC) patients, but its impact on carcinogenesis remains controversial and little is known about its effects after viral clearance. To determine the contribution of IFN-λ3 to hepatocarcinogenesis after HCV clearance, we analyzed IFNL3 genotypes and serial serum IFN-λ3 levels in CHC patients who achieved sustained virologic responses (SVR). METHODS: This study comprised 201 CHC patients treated with DAAs. Serum samples were collected sequentially and IFN-λ3 levels were quantified by chemiluminescence enzyme immunoassay. The IFNL3 polymorphism (rs8099917) was genotyped in 195 patients. RESULTS: One hundred and twenty-five patients were rs8099917 T/T and 70 were non-T/T. Serum IFN-λ3 levels did not differ significantly with IFNL3 genotype, dropped markedly by 1 week and remained low up to 24 weeks after the end of treatment. Interferon-λ3 levels were significantly higher after viral clearance in patients who developed HCC and were associated with a higher potential for hepatocarcinogenesis, such as a higher frequency of non-hypervascular hypointensive nodules (P = 0.046), higher stages of liver fibrosis (P < 0.001), and higher post-treatment levels of Wisteria floribunda agglutinin positive Mac-2 binding protein (P < 0.001) and alanine aminotransferase (P < 0.001). CONCLUSIONS: Serum IFN-λ3 levels after HCV clearance are associated with the potential for HCC development. Interferon-λ3 could be helpful for elucidating the relationships among immunologic status, liver fibrosis, liver inflammation, and hepatocarcinogenesis, after achieving SVR.

Comprehensive genetic analysis of cholangiolocellular carcinoma with a coexistent hepatocellular carcinoma-like area and metachronous hepatocellular carcinoma.

AIM: The genetic profile of cholangiolocellular carcinoma (CLC) and its origin in relation to hepatocellular carcinoma (HCC) remain unclear. To elucidate the genetic profile of CLC, a comprehensive analysis of genetic mutations was carried out in a case of CLC with an HCC-like focal area and metachronous HCC. METHOD: Liver tissue was obtained from CLC, a co-existent HCC-like area, and metachronously developed HCC by laser capture microdissection of formalin-fixed paraffin-embedded specimens obtained by hepatectomy. Gene mutational profiles were analyzed comprehensively by next-generation sequencing and digital PCR. Relationships among gene profiles, immunohistochemistry, and clinicopathological findings were investigated. RESULTS: Mutations in EGFR, PTEN, RB1, TP53, and ERBB2 were found in CLC, whereas mutations in KIT, BRAF, PTEN, TP53, and SMAD4 were found in the coexistent HCC-like area. Only the mutation in PTEN has a common Catalogue of Somatic Mutations in Cancer ID in the CLC and coexistent HCC-like area, and is related to the kinase-RAS module. In contrast, no cancer-related mutations were found in the metachronous HCC. No TERT mutations were found in any of the regions by digital PCR. Immunohistochemical staining for p53 was negative in CLC, although ≤10% positive in the coexistent HCC-like area. Immunostaining of C-kit, HER2, PTEN, and SMAD4 were negative. CONCLUSION: The genomic features of CLC and the focal area of an HCC-like region differ, but are related to the kinase-RAS module. The development of carcinogenesis in the CLC and HCC-like areas in this case might differ, following a common PTEN mutation, although alteration of the kinase-RAS module is the most common molecular event in CLC.

Low sense of coherence is associated with anxiety among adults: Results based on data from all 47 prefectures of Japan.

BACKGROUND: Anxiety disorder is a major societal, economic, and healthcare burden, so it is essential to identify underlying risk factors. Sense of coherence (SOC), defined as an individual's perceived ability to control events and cope with challenges, is strongly associated with healthcare outcomes. METHODS: This study investigated the association between SOC and anxiety among adults in all 47 prefectures of Japan using a cross-sectional online panel survey. SOC was assessed using the University of Tokyo Health Sociology version of the SOC Scale and anxiety was assessed using the Generalized Anxiety Disorder-7 (GAD-7) scale. RESULTS: Among 2100 participants (1051 males and 1049 females), 475 (22.6%) were identified with anxiety (scoring 8 points or higher on the GAD-7 scale). Before adjustment for potential confounders, there was a significant negative association between SOC and presence of anxiety. Even after adjustment, the presence of anxiety was significantly higher among the lower SOC subgroup than the higher SOC subgroup. CONCLUSIONS: These results indicate that approximately one-fifth of adults in Japan suffer from anxiety and that low SOC is a significant risk factor.

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.

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.

Unique clinical courses of transfusion-transmitted hepatitis E in patients with immunosuppression.

BACKGROUND: The high prevalence of specific immunoglobulin G for hepatitis E virus (HEV) in Japanese people raises the possibility of a high incidence of HEV-viremic blood donors and therefore frequent transfusion-transmitted HEV (TT-HEV). STUDY DESIGN AND METHODS: TT-HEV cases established in Japan through hemovigilance and those published in the literature were collected. Infectivity of HEV-contaminated blood components and disease severity in relation to immunosuppression were investigated. RESULTS: Twenty established TT-HEV cases were recorded over the past 17 years. A lookback study verified that five of 10 patients transfused with known HEV-contaminated blood components acquired HEV infection. The minimal infectious dose of HEV through transfusion was 3.6 × 104 IU. Nine of the 19 TT-HEV cases analyzed had hematologic diseases. Only two cases showed the maximal alanine aminotransferase level of more than 1000 U/L. Two patients with hematologic malignancy and two liver transplant recipients had chronic liver injury of moderate severity. CONCLUSION: The infectivity of HEV-contaminated components was 50%. Immunosuppression likely causes the moderate illness of TT-HEV, but it may lead to the establishment of chronic sequelae. Transfusion recipients, a population that is variably immunosuppressed, are more vulnerable to chronic liver injury as a result of TT-HEV than the general population is as a result of food-borne 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. METHODS: A functional role for BMP-4 in proliferation and terminal differentiation of murine hepatoblasts was validated by assay of colony formation, biliary luminal formation, and hepatic maturation using primary hepatoblasts in vitro. Molecular mechanisms regulating such effects of BMP-4 on primary hepatoblasts were also analyzed. RESULTS: Stimulation of BMP-4 upregulated phosphorylation of Smad1/5 in hepatoblasts. Bone morphogenetic protein-4 significantly suppressed colony formation of primary hepatoblasts in a dose-dependent manner, significantly suppressed cholangiocytic luminal formation of hepatoblasts, and promoted hepatic maturation of primary hepatoblasts. Stimulation of BMP-4 regulated the activation of several mitogen-activated protein kinases, such as extracellular signal-regulated kinase, Akt, p38 mitogen-activated protein kinase, and calcium/calmodulin-dependent protein kinase IIα in primary hepatoblasts. Moreover, Wnt5a, a molecule regulating cholangiocytic luminal formation, and BMP-4 coordinately suppressed proliferation and cholangiocytic luminal formation of hepatoblasts. CONCLUSION: This study shows that BMP-4-mediated signaling controls proliferation and terminal differentiation of fetal hepatic stem/progenitor cells.

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

AIM: 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. METHODS: Ninety patients with genotype 2 chronic hepatitis C treated with SOF/RBV were studied. The relationships among genetic polymorphisms of ITPA and the decline in hemoglobin levels from baseline, RBV dose reduction, and sustained virological response (SVR) rates were analyzed. RESULTS: Overall SVR at 12 weeks was 94.4% (85/90). Patients with the ITPA CA/AA genotypes had a lower degree of anemia throughout the therapy than those with the ITPA CC genotype. The percentage of patients requiring RBV dose reduction was significantly lower for those with the ITPA CA/AA variation, a difference even more apparent when the pretreatment hemoglobin level was <12 g/dL. The dose reduction of RBV and serum albumin level were significantly associated with SVR. CONCLUSIONS: Patients with the ITPA CA/AA genotype were less likely to develop anemia than those with the ITPA CC genotype and were more likely to complete SOF/RBV therapy. These results may provide a valuable pharmacogenetic diagnostic tool to predict drug-induced adverse events, particularly in patients with pre-existing anemia.

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.

HBV-KMT2B integration drives hepatic oncogenic processes in a human gene-edited iPSC-derived model.

BACKGROUND & AIMS: Hepatitis B virus (HBV)-DNA integration into the host genome contributes to hepatocellular carcinoma (HCC) development. KMT2B is the second most frequent locus of HBV-DNA integration in HCC, however its role and function remain unclear. We aimed to clarify the impact of HBV-KMT2B integration in HCC development using a human genome-edited induced pluripotent stem cells (iPSCs) model. METHODS: Based on the genetic information on HBV-KMT2B integration in HCC, we determined its complete DNA sequence and transcript variants. To exclude the effect of other oncogenic mutations, we reproduced HBV integration in healthy donor iPSCs with an intact genome and analyzed its effects using iPSC-derived hepatic progenitor cells (HPCs) and hepatocytes (iPS-Heps). RESULTS: The reproduced HBV-KMT2B integration significantly upregulated the proliferation of hepatic cells. Comprehensive transcriptional and epigenetic analyses revealed enhanced expression of cell cycle-related genes in hepatic cells with HBV-KMT2B integration based on perturbation of histone 3 lysine 4 tri-methylation(H3K4me3), mimicking that in the original HCC sample. Long-read RNA-sequence detected the common KMT2B transcript variants in the HCC sample and HPCs. Overexpression of the truncated variant significantly enhanced proliferation of hepatic cells, whereas HBV-KMT2B fusion transcripts did not enhance proliferation. HBV-KMT2B-integrated HPCs exhibited replication stress and DNA damage, indicating that our model initiated the process of hepatocarcinogenesis due to abnormally promoted KMT2B function. CONCLUSIONS: Our disease model using genetically engineered iPSCs provides the first insight into both the KMT2B function in HCC development and the oncogenic processes by HBV-KMT2B integration. We clarified the novel oncogenic mechanism in HBV-related HCC due to aberrant KMT2B function.

Analysis of prognosis and background liver disease in non-advanced hepatocellular carcinoma in two decades.

BACKGROUND/AIM: Antiviral hepatitis and systemic therapies for hepatocellular carcinoma (HCC) remarkably progressed in the recent 10 years. This study aimed to reveal the actual transition and changes in the prognosis and background liver disease in non-advanced HCC in the past 20 years. METHODS: This retrospectively recruited 566 patients who were diagnosed with non-advanced HCC from February 2002 to February 2022. The prognosis was analyzed by subdividing according to the diagnosis date (period I: February 2002-April 2009 and period Ⅱ: May 2009-February 2022). RESULTS: Patients in period II (n = 351) were significantly older, with lower albumin-bilirubin (ALBI) scores and alpha-fetoprotein (AFP) and more anti-viral therapy, systemic therapy, and hepatic arterial infusion chemotherapy as compared with those in period I (n = 215). The etiology ratio of the background liver disease revealed decreased hepatitis C virus from 70.6% to 49.0% and increased non-B, non-C from 17.7% to 39.9% from periods I to Ⅱ. The multivariate analysis revealed older age and higher ALBI score in Barcelona Clinic Liver Cancer (BCLC) 0/A stage, AFP of >20 ng/mL, and higher ALBI score in BCLC B stage as independent prognosis factors. Fine-Gray competing risk model analysis revealed that liver-related deaths significantly decreased in period II as compared to period I, especially for BCLC stage 0/A (HR: 0.656; 95%CI: 0.442-0.972, P = 0.036). CONCLUSION: The characteristics of patients with non-advanced HCC have changed over time. Appropriate background liver management led to better liver-related prognoses in BCLC 0/A.

Cholestatic liver fibrosis and toxin-induced fibrosis are exacerbated in matrix metalloproteinase-2 deficient mice.

Matrix metalloproteinase (MMP) plays an important role in homeostatic regulation of the extracellular environment and degradation of matrix. During liver fibrosis, several MMPs, including MMP-2, are up-regulated in activated hepatic stellate cells, which are responsible for exacerbation of liver cirrhosis. However, it remains unclear how loss of MMP-2 influences molecular dynamics associated with fibrogenesis in the liver. To explore the role of MMP-2 in hepatic fibrogenesis, we employed two fibrosis models in mice; toxin (carbon tetrachloride, CCl4)-induced and cholestasis-induced fibrosis. In the chronic CCl4 administration model, MMP-2 deficient mice exhibited extensive liver fibrosis as compared with wild-type mice. Several molecules related to activation of hepatic stellate cells were up-regulated in MMP-2 deficient liver, suggesting that myofibroblastic change of hepatic stellate cells was promoted in MMP-2 deficient liver. In the cholestasis model, fibrosis in MMP-2 deficient liver was also accelerated as compared with wild type liver. Production of tissue inhibitor of metalloproteinase 1 increased in MMP-2 deficient liver in both models, while transforming growth factor ß, platelet-derived growth factor receptor and MMP-14 were up-regulated only in the CCl4 model. Our study demonstrated, using 2 experimental murine models, that loss of MMP-2 exacerbates liver fibrosis, and suggested that MMP-2 suppresses tissue inhibitor of metalloproteinase 1 up-regulation during liver fibrosis.

Mac-2 binding protein glycosylation isomer as a novel predictive biomarker for patient survival after hepatitis C virus eradication by DAAs.

BACKGROUND: It is crucial to identify risk factors for life prognosis after hepatitis C virus (HCV) eradication among patients with or without a high risk of liver cancer or complications. METHODS: This is a prospective, multicenter and observational study using the database of 1031 patients after HCV eradication by direct-acting antiviral agents (DAAs) to evaluate the development of hepatocellular carcinoma (HCC) and patients' survival after a sustained virological response (SVR). The Cox proportional hazards regression model was used to estimate hazard ratios associated with HCC development and survival. RESULTS: AFP at SVR was significantly associated with HCC recurrence in the adjusted model. Liver fibrosis, Mac-2 binding protein glycosylation isomer (M2BPGi) at SVR and smoking status before treatment were positively associated with the development of HCC and M2BPGi was positively associated with HCC recurrence, although not reaching statistical significance. Among patients without a history of HCC, M2BPGi and estimated glomerular filtration rate (eGFR) at SVR were significantly associated with death after viral eradication [M2BPGi (HR 4.07, 95% CI 1.22, 13.57), eGFR (HR 0.97, 95% CI 0.94, 0.99)]. Strikingly, of 16 patients who died, among participants without a history of HCC, only two died of liver cancer associated with HCV, whereas 11 died of non-HCV- related cancer or cardiovascular diseases. CONCLUSION: M2BPGi at SVR is a potential predictor for patients' survival and a candidate biomarker for detecting individuals who are at greater risk of death due to cancer-related and unrelated to HCV, as well as cardiovascular diseases, after viral eradication.