Hepatitis B Virus-KMT2B Integration Drives Hepatic Oncogenic Processes in a Human Gene-edited Induced Pluripotent Stem Cells-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.

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.

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.

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.

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.

Endoscopic Closure of an Acute Duodenal Perforation Occurring during Endoscopic Ultrasound Using Endoclips and Polyglycolic Acid Sheets with Fibrin Glue.

Acute duodenal perforation during endoscopic ultrasound (EUS) is a serious complication. The conventional endoscopic treatment for duodenal perforations such as endoscopic clipping is unsatisfactory; recently, the effectiveness of over-the-scope clipping (OTSC) has been reported. A 91-year-old woman was referred to our hospital with the chief complaint of jaundice. Contrast-enhanced computed tomography showed a 2-cm mass in the pancreatic head; we planned EUS-guided fine-needle aspiration. During exploration for a puncture route from the duodenal bulb using a linear echoendoscope under carbon dioxide insufflation, the duodenal lumen was suddenly filled with blood. A perforation <15 mm was identified in the superior duodenal horn. We attempted an endoscopic closure with multiple endoclips but could not completely close the perforation site. Strips of bioabsorbable polyglycolic acid (PGA) sheets were placed over the gaps between the endoclips with biopsy forceps and fixed in place with fibrin glue, completely covering the perforation site. Two days after the procedure, the perforation site had closed. Nine days later, endoscopic biliary stenting was performed. The patient was diagnosed with pancreatic cancer through bile cytology, and the optimal supportive care for her age was selected. Endoscopic tissue shielding with PGA sheets and fibrin glue is increasingly being reported for use during gastrointestinal endoscopic procedures. In this case, surgery was avoided due to successful endoscopic treatment using endoclips and PGA sheets with fibrin glue without OTSC. This method may be useful for repairing acute duodenal perforations during EUS and should therefore be known to pancreatobiliary endoscopists.