Evaluation of the associations of interlukin-7 genetic variants with toxicity and efficacy of immune checkpoint inhibitors: A replication study of a Japanese population, based on the findings of a European genome-wide association study.

AIM: Recent genome-wide association studies of European populations have identified rs16906115, a single-nucleotide polymorphism in the interleukin-7 gene, as a predictor of immune-related adverse events (irAEs) and the therapeutic efficacy of immune checkpoint inhibitors. We evaluated this single-nucleotide polymorphism in a Japanese population. METHODS: From January 2021, we stored host DNA from individuals who received various types of immune checkpoint inhibitors. From this population, we categorized 510 participants into cases (grade ≥2 irAEs) and controls (received ≥3 immune checkpoint inhibitor doses, follow-up ≥12 weeks, no irAEs), and divided 339 hepatocellular carcinoma patients treated with atezolizumab/bevacizumab into responders and non-responders, evaluated using the modified response evaluation criteria in solid tumors. We compared the minor allele frequencies of rs16906115 between cases and controls, and responders and non-responders. RESULTS: In the irAE prediction analysis of 234 cases and 276 controls, the minor allele frequency was 0.244 in the case group and 0.265 in the control group. This difference is not significant. In the analysis predicting the therapeutic efficacy for hepatocellular carcinoma patients, the responders had a significantly lower minor allele frequency of 0.220, compared with 0.300 for the non-responders (p = 0.022). Univariate and multivariate analyses identified the minor allele homozygosity as a significant predictor of treatment response, with odds ratios of 0.292 (p = 0.015) in the univariate analysis and 0.315 (p = 0.023) in the multivariate analysis. CONCLUSIONS: In our Japanese cohort, no association was found between the rs16906115 minor allele and irAEs or treatment efficacy. The minor allele homozygosity may be associated with a negative therapeutic outcome. CLINICAL TRIAL REGISTRATION: UMIN Clinical Trials Registry with the number UMIN000043798.

A Phase 2, Prospective, Multicenter, Single-Arm Trial of Transarterial Chemoembolization Therapy in Combination Strategy with Lenvatinib in Patients with Unresectable Intermediate-Stage Hepatocellular Carcinoma: TACTICS-L Trial.

Introduction: Transarterial chemoembolization (TACE) is the standard treatment for unresectable intermediate-stage hepatocellular carcinoma (HCC), but recurrence after TACE is common. The present phase 2, prospective, multicenter, single-arm trial, the TACTICS-L trial, investigated the efficacy and safety of TACE plus lenvatinib (LEN), a drug that more strongly promotes vascular normalization and has a better objective response rate (ORR) than sorafenib (jRCTs031180074). Methods: Participants were patients with HCC who had not previously received systemic therapy, hepatic arterial infusion chemotherapy, or immunotherapy and who were ineligible for resection or percutaneous ablation therapy. LEN was to be administered 14-21 days before the first TACE, stopped 2 days before TACE, and resumed 3 days after TACE. Key inclusion criteria were unresectable HCC, Child-Pugh A liver function, 0-2 prior TACE sessions, tumor size ≤10 cm, number of tumors ≤10, and ECOG performance status 0-1. Key exclusion criteria were vascular invasion and extrahepatic spread. The primary endpoint was progression-free survival (PFS) by RECICL, and secondary endpoints were time to untreatable progression, ORR, overall survival (OS), and safety. Results: A total of 62 HCC patients were enrolled in this trial. The median age was 72 years, 77.4% of patients were men, and 95.2% had PS 0. The primary endpoint of median PFS was 28.0 months (90% confidence interval [CI] 25.1-31.0) after a minimum 24 months of follow-up. The secondary endpoint of median OS was not reached (90% CI 35.5 months-NR). LEN-TACE achieved a high response rate and high complete response (CR) rate (4 weeks after the first TACE: ORR 79.0%, CR rate 53.2%; best response: ORR 88.7%, CR rate 67.7%) by RECICL. Exploratory subgroup analyses showed that the characteristics of responders/nonresponders (ORR and CR rate) were similar and that LEN-TACE would be effective in all subgroups, including the population in whom TACE alone would be less likely to be curative (e.g., patients with the non-simple nodular type or a high tumor burden). The relative dose intensity of LEN before the first TACE was important for achieving higher CR rate/ORR by LEN-TACE. No new safety concerns were observed. Conclusion: The results of this trial provide encouraging evidence, supporting the efficacy and favorable safety profile of LEN-TACE in patients who are ineligible for locoregional therapy.

Therapeutic Modifications without Discontinuation of Atezolizumab Plus Bevacizumab Therapy Are Associated with Favorable Overall Survival and Time to Progression in Patients with Unresectable Hepatocellular Carcinoma.

We retrospectively evaluated the impact of therapeutic modifications of atezolizumab (Atezo) plus bevacizumab (Bev) therapy (Atezo/Bev), including the interruption or discontinuation of both Atezo and Bev, and the reduction or discontinuation of Bev, on the outcome of patients with unresectable hepatocellular carcinoma (uHCC) (median observation period: 9.40 months). One hundred uHCC from five hospitals were included. Therapeutic modifications without discontinuation of both Atezo and Bev (n = 46) were associated with favorable overall survival (median not reached; hazard ratio (HR): 0.23) and time to progression (median: 10.00 months; HR: 0.23) with no therapeutic modification defined as the reference. In contrast, the discontinuation of both Atezo and Bev without other therapeutic modifications (n = 20) was associated with unfavorable overall survival (median: 9.63 months; HR: 2.72) and time to progression (median: 2.53 months; HR: 2.78). Patients with modified albumin-bilirubin grade 2b liver function (n = 43) or immune-related adverse events (irAEs) (n = 31) discontinued both Atezo and Bev without other therapeutic modifications more frequently (30.2% and 35.5%, respectively) than those with modified albumin-bilirubin grade 1 (10.2%) and without irAEs (13.0%). Patients with objective response (n = 48) experienced irAEs more frequently (n = 21) than those without (n = 10) (p = 0.027). Avoiding the discontinuation of both Atezo and Bev without other therapeutic modifications may be the optimal management of uHCC.

Safety and Effectiveness of Lenvatinib in Patients with Unresectable Hepatocellular Carcinoma in Real-World Clinical Practice: An Observational Post-Marketing Study in Japan.

BACKGROUND: Lenvatinib was approved for use in unresectable hepatocellular carcinoma (uHCC) in Japan in 2018. Patients with diverse clinical characteristics receive lenvatinib treatment in clinical practice. Thus, it is crucial to evaluate the safety and effectiveness of lenvatinib in real-world clinical settings. OBJECTIVE: This study aimed to evaluate the real-world safety and effectiveness of lenvatinib for uHCC in clinical practice in Japan. PATIENTS AND METHODS: Between July 2018 and January 2019, patients with uHCC who were administered lenvatinib for the first time were enrolled in this prospective, multicenter, observational post-marketing study (NCT03663114). Patients were orally administered lenvatinib and followed up for 12 months. For safety, adverse drug reactions (ADRs) were evaluated. For effectiveness, the objective response rate (ORR) was calculated to evaluate tumor response. Overall survival (OS) was estimated using the Kaplan-Meier method. RESULTS: Data of 703 patients (median age, 73 years; 80.2% males) were analyzed. The median (range) treatment duration was 25.3 (0.3-68.9) weeks. The mean ± standard deviation initial dose was 7.37 ± 1.65 mg in patients with body weight < 60 kg and 10.43 ± 2.49 mg in those with body weight ≥ 60 kg. ADRs (any grade) were reported in 84.9% of the patients, with Grade ≥ 3 ADRs reported in 42.5% of the patients. The most common ADRs (> 10%) were decreased appetite, fatigue, hypertension, proteinuria, palmar-plantar erythrodysesthesia, hypothyroidism, and diarrhea. The median OS of the 703 patients was 498.0 days. In 494 patients assessed using the modified Response Evaluation Criteria in Solid Tumors (mRECIST), the ORR was 39.5% (95% confidence interval: 35.1-43.9%). Patients with better liver or renal function at baseline achieved significantly higher ORR than those with worse liver or renal function. CONCLUSIONS: In patients with uHCC in real-world clinical practice in Japan, treatment with lenvatinib was generally well tolerated, and no new safety concerns were identified. The ORR and median OS were similar to or better than the results of the Japanese subset of the global Phase III REFLECT trial. Our results demonstrated that clinically meaningful treatment responses were achieved with lenvatinib in real-world clinical practice.

DEB-TACE combined with hepatic artery infusion chemotherapy might be an affordable treatment option for advanced stage of HCC.

Alternative treatment modalities are necessary because of the low response rates and unsuitability of molecular-targeted agents (MTA) and/or immune checkpoint inhibitors (iCIs) in HCC patients. Therefore, we analyzed whether drug-eluting beads (DEB)-transcatheter arterial chemoembolization (TACE) with low-dose-FP (Ultra-FP) therapy could improve the efficacy and safety of treatment in difficult-to-treat HCC patients, especially those with advanced stage HCC. From November 2017 to April 2021, 118 consecutive patients with non-resectable difficult-to-treat HCC were included in this study. All patients were treated with Ultra-FP therapy. After the weak DEB-TACE procedure, we administered low-dose FP for 2 weeks followed by resting for 4 weeks. The numbers of HCC patients CR/PR/SD/PD induced by Ultra-FP therapy were 36/52/17/13 (Modified RECIST) patients, respectively. The objective response rate of Ultra-FP therapy was 74.6% (88/118 patients). Tumor marker reduction was observed in 81.4% (96/118 patients). The objective response rate (ORR) in the HCC patients with portal vein tumor thrombosis (PVTT) was 75% (18/24 patients). Median overall survival (mOS) of all included HCC patients was 738 days. The mOS of HCC patients with PVTT (-)/PVTT (+) was 816 days/718 days. The proportion of patients based on ALBI grade system was not significantly different between pre- and after 3 course Ultra-FP therapy. Ultra-FP therapy might be an affordable treatment option for difficult-to-treat advanced HCC. ORR and overall survival after receiving Ultra-FP therapy were remarkable in comparison to various kinds of systemic therapy including MTA and iCIs.

Anti-nuclear antibody and a granuloma could be biomarkers for iCIs-related hepatitis by anti-PD-1 treatment.

It has been reported that various kinds of immune checkpoint inhibitors (iCIs) could induce immune-related liver damage. We should focus on the programmed cell death-receptor-1 (PD-1) antibody and non-small cell lung cancer (NSCLC) to analyze the characteristics of hepatitis related to iCIs and find factors that could be useful biomarkers for the diagnosis. A single-center retrospective study of 252 NSCLC patients who received PD-1 antibody (nivolumab or pembrolizumab). Some of the biochemical markers and immunological markers were analyzed during PD-1-antibody treatment with or without ALT elevation. Histopathological features were reviewed by a single expert of hepatic pathology focusing on the following features: fibrosis, portal inflammation, lobular inflammation, lobular necrosis. The formation of macro- and micro-granulomas was also evaluated. The frequency of liver damage induced by nivolumab including grade 1 to 4 (ALT) was 41.9% (78/186 patients). The positive rate of anti-nuclear antibody in the nivolumab group with iCIs-related hepatitis was significantly higher than that in the nivolumab group without iCIs-related hepatitis (p = 0.00112). Granulomatous changes were significantly increased in patients with iCIs-related hepatitis compared with DILI and AIH patients (p < 0.05). The ratios of inflammatory cells CD4/CD8, and CD138/CD3 in ICIs-related hepatitis were significantly lower than those in AIH or DILI patients (p < 0.05). We demonstrated that the pre-existing ANA and characteristic liver histology including CD8+ cells dominancy and granulomatous hepatitis could be biomarkers for the diagnosis of iCIs-related hepatitis in the NSCLC with anti-PD-1 therapy.

Identification of microRNA-96-5p as a postoperative, prognostic microRNA predictor in nonviral hepatocellular carcinoma.

AIM: The microRNA (miR) clusters miR-183/96/182 and miR-217/216a/216b are significantly upregulated in nonviral hepatocellular carcinoma (NBNC-HCC). Here, we investigate the impact of each member of these clusters on the clinical outcome of NBNC-HCC and analyze the antitumor effects of miR-96-5p. METHODS: The association between recurrence-free survival of 111 NBNC-HCC patients and the levels of miR-183-5p, miR-96-5p, miR-182-5p, miR-217-5p, miR-216a-5p, and miR-216b-5p in tumor and adjacent tissues was investigated. The impact of miR-96-5p on apoptosis and invasion of a hepatoma cell line, HepG2, was investigated by cell counting, Transwell assay, and flow cytometry, respectively. RESULTS: MicroRNA-183-5p, miR-96-5p, miR-182-5p, miR-217-5p, and miR-216b-5p were significantly upregulated in tumor tissues compared to the adjacent tissues (p = 0.0005, p = 0.0030, p = 0.0002, p = 0.0011, and p = 0.0288, respectively). By multivariate Cox regression analysis, high tumor/adjacent ratios of miR-182-5p (p = 0.007) and miR-217-5p (p = 0.008) were associated with poor recurrence-free survival. In contrast, a low tumor/adjacent ratio of miR-96-5p (p < 0.001) was associated with poor recurrence-free survival. It suggested that further upregulation of miR-96-5p in tumors might have an inhibitory effect on recurrence. Transfection of miR-96-5p mimic significantly induced apoptosis of HepG2 cells, in association with downregulation of Nucleophosmin 1 (NPM1) and a decrease of phosphorylated AKT protein. Interestingly, simultaneous knockdown of the NPM1 and AKT genes induced apoptosis. MicroRNA-96-5p also suppressed proliferation and invasion, which inhibited epithelial-to-mesenchymal transition of HCC cells. CONCLUSION: MicroRNA-96-5p as a tumor suppressor would be valuable to stratify NBNC-HCC patients at high risk of recurrence.

Transcriptomics Identify Thrombospondin-2 as a Biomarker for NASH and Advanced Liver Fibrosis.

BACKGROUND AND AIMS: NAFLD is the most common liver disease worldwide. NASH, the progressive form of NAFLD, and advanced fibrosis are associated with poor outcomes. We searched for their noninvasive biomarkers. APPROACH AND RESULTS: Global RNA sequencing of liver tissue from 98 patients with biopsy-proven NAFLD was performed. Unsupervised hierarchical clustering well distinguished NASH from nonalcoholic fatty liver (NAFL), and patients with NASH exhibited molecular abnormalities reflecting their pathological features. Transcriptomic analysis identified proteins up-regulated in NASH and/or advanced fibrosis (stage F3-F4), including matricellular glycoprotein thrombospondin-2 (TSP-2), encoded by the thrombospondin 2 (THBS2) gene. The intrahepatic THBS2 expression level showed the highest areas under the receiver operating characteristic curves (AUROCs) of 0.915 and 0.957 for diagnosing NASH and advanced fibrosis, respectively. THBS2 positively correlated with inflammation and ballooning according to NAFLD activity score, serum aspartate aminotransferase and hyaluronic acid (HA) levels, and NAFLD Fibrosis Score (NFS). THBS2 was associated with extracellular matrix and collagen biosynthesis, platelet activation, caspase-mediated cleavage of cytoskeletal proteins, and immune cell infiltration. Serum TSP-2 expression was measured in 213 patients with biopsy-proven NAFLD, was significantly higher in NASH than in NAFL, and increased parallel to fibrosis stage. The AUROCs for predicting NASH and advanced fibrosis were 0.776 and 0.856, respectively, which were comparable to Fibrosis-4 index, serum HA level, and NFS in advanced fibrosis diagnosis. Serum TSP-2 level and platelet count were independent predictors of NASH and advanced fibrosis. Serum TSP-2 levels could stratify patients with NAFLD according to the risk of hepatic complications, including liver cancer and decompensated cirrhotic events. CONCLUSIONS: TSP-2 may be a useful biomarker for NASH and advanced fibrosis diagnosis in patients with NAFLD.

Establishing Efficient Systems through Electronic Medical Records to Promote Intra-hospital Referrals of Hepatitis Virus Carriers to Hepatology Specialists: A Multicenter Questionnaire-based Survey of 1,281 Healthcare Professionals.

Objective Persistent hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are major causative factors of hepatic cirrhosis and hepatocellular carcinoma. However, the development of antiviral treatment has enabled their suppression. Therefore, the early detection and treatment of these infections are important. The objective of this study was to assess the level of awareness among healthcare professionals about hepatitis virus infection and electronic medical records alert system. Methods We surveyed healthcare professionals from 10 institutions with electronic medical records alert systems. All participants attended a lecture about the reactivation risk due to HBV infections, the most recent antiviral treatment for HCV infections, and the electronic medical records alert system. They participated in a questionnaire-based survey about their awareness of these infections, current status of intra-hospital referral, need for intra-hospital referrals before and after the lecture, and reasons for non-referral of patients to specialists. Results Responses were received from 1,281 healthcare professionals. Physicians and pharmacists had a high level of awareness about HBV and HCV. Among physicians, the level of awareness of those in the surgical field and other fields was significantly lower than that of the professionals in the internal medicine field. The awareness of the need to refer patients to hepatologists increased from 84.7-85.4% before to 93.0% after the lecture. The most frequent reasons for not referring patients previously were "I had no knowledge and/or interest" (28.1% of responses) and "All I did was explain the results orally" (24.2%). Conclusion More widespread education of healthcare personnel is important to increase the number of individuals receiving appropriate treatment from specialist physicians.

Immune Checkpoint Inhibitor Can Reduce HCV-RNA without Liver Damage.

Recently, immune checkpoint inhibitors (iCIs) have been used to treat cancers. Once some of the iCIs for the treatment of hepatocellular carcinoma (HCC) are certified in clinical trials, they are likely be administered to HCC patients with hepatitis C virus (HCV). However, the immunopathogenesis of HCV after the administration of iCIs has not been clarified. We experienced a lung cancer patient with HCV infection treated by nivolumab, programmed cell death 1 (PD-1) antibody. HCV-RNA gradually decreased after the start of nivolumab treatment. However, no increase in transaminase was observed during the decline of HCV-RNA. It was thought that HCV-specific cytotoxic T lymphocytes (CTLs) were activated by iCIs.

[Cutting Edge of Hepatic Artery Infusion Chemotherapy for Hepatocellular Carcinoma].

Recently, the choices of treatments for hepatocellular carcinoma(HCC)were increasing since the several molecular targeting agents(MTA)were approved for treatment of advanced HCC patients. On the other hand, the transarterial chemoembolization( TACE)and hepatic artery infusion chemotherapy(HAIC)have been improved by various kinds of methods. The liver resection, radiofrequency ablation(RFA)and microwave coagulation(MWA)might achieve complete cure. However, the treatment indication of liver resection, RFA and MWA should be limited. Therefore, multidisciplinary treatment including HAIC, TACE, MTA, RFA, MWA, and liver resection should be considered to control HCC.

Hepatitis C virus infection suppresses hepatitis B virus replication via the RIG-I-like helicase pathway.

Mechanisms of hepatitis B virus (HBV) reactivation after hepatitis C virus (HCV) elimination by direct-acting antiviral (DAA) treatment in HBV/HCV-co-infected patients remain unclear. We examined RIG-I-like helicase (RLH) pathway activation by HBV mono-infection, HCV mono-infection or HBV/HCV co-infection and interference between HBV and HCV in primary human hepatocytes. Interference between HBV and HCV and HBV reactivation after DAA treatment in humanized-liver mice were assessed. HCV infection activated RLH pathway, as evidenced by RIG-I, ISG15 and ISG56 expression induction; HBV caused only RIG-I induction in vitro. RLH activation was also found in HBV/HCV-co-infected cells, and HBV replication were suppressed in HBV/HCV-co-infected than in HBV-mono-infected cells. siRNA-mediated double knockdown of ISG15 and ISG56 increased HBV replication in HBV/HCV-co-infected cells. HCV infection activated RLH pathway and suppressed HBV replication in humanized-liver mice. Subsequent elimination of HCV by DAA administration downregulated RLH pathway and upregulated HBV replication in mice. RLH pathway was activated in livers of chronic hepatitis C patients compared to those of chronic hepatitis B or non-B, non-C patients. The RLH pathway activation was downregulated by HCV elimination. In conclusion, HCV infection activated RLH pathway and suppressed HBV replication in human hepatocytes. HCV elimination upregulated HBV replication, probably through RLH pathway downregulation.

Complete Atrioventricular Block Associated with Pembrolizumab-induced Acute Myocarditis: The Need for Close Cardiac Monitoring.

Pembrolizumab, a humanized monoclonal IgG4 antibody directed against programmed death-1, is an immune checkpoint inhibitor that has been introduced for the treatment of non-small-cell lung cancer. However, immune checkpoint inhibitors may cause severe immune-related adverse events. We herein present a case of lung cancer with complete atrioventricular block associated with acute myocarditis, which developed 16 days after the administration of pembrolizumab. The clinical course of this case suggested a strong need for close cardiac monitoring when pembrolizumab is administered on an outpatient basis.

Selective insulin resistance with differential expressions of IRS-1 and IRS-2 in human NAFLD livers.

BACKGROUND/OBJECTIVE: Insulin signals, via the regulation of key enzyme expression, both suppress gluconeogenesis and enhance lipid synthesis in the liver. Animal studies have revealed insulin signaling favoring gluconeogenesis suppression to be selectively impaired in steatotic livers. However, whether, and if so how, such selective insulin resistance occurs in human steatotic livers remains unknown. Our aim was to investigate selective insulin resistance in human livers with non-alcoholic fatty liver disease (NAFLD). SUBJECTS/METHODS: We examined mRNA expressions of key molecules for insulin signaling, gluconeogenesis and lipogenesis in human liver biopsy samples obtained from 51 non-diabetic subjects: 9 healthy controls and 42 NAFLD patients, and analyzed associations of these molecules with each other and with detailed pathological and clinical biochemistry data. RESULTS: In NAFLD patients, insulin receptor substrate (IRS)-2 expression was decreased, while those of key enzymes for gluconeogenesis were increased. These alterations of IRS-2 and gluconeogenesis enzymes were induced both in simple steatosis (SS) and non-alcoholic steatohepatitis (NASH), while these expression levels did not differ between SS and NASH. Furthermore, alterations in the expressions of IRS-2 and gluconeogenesis enzymes showed strong negative correlations and were concurrently induced in the early histological stage of NAFLD. In contrast, fatty acid synthase (FAS) expression was not decreased in NAFLD, despite IRS-2 downregulation, but correlated strongly with IRS-1 expression. Furthermore, no histological scores were associated with these molecules. Thus, IRS-1 signaling, which is not impaired in NAFLD, appears to modulate FAS expression. CONCLUSION: These analyses revealed that selective insulin resistance is present in human NAFLD livers and occurs in its early phases. The effect of insulin, during the IRS step, on gene expressions for lipogenesis and gluconeogenesis are apparently distinct and preferential downregulation of IRS-2 may contribute to selective resistance to the suppressive effects of insulin on gluconeogenesis.

Hepatitis C infected patients need vitamin D3 supplementation in the era of direct acting antivirals treatment.

It has been reported that the serum level of vitamin D3 (VitD3) could affect the natural course of chronic hepatitis C (CH-C) and the response to treatment with pegylated interferon (Peg-IFN) and ribavirin. Although several mechanisms for the favorable effects of VitD3 supplementation were reported, the total effect of VitD3 supplementation remains unclear. Previously, we reported that supplementation with 1(OH)VitD3 could enhance the Th1 response inducing not only a favorable immune response for viral eradication but also HCC control. Recently, the main treatment of CH-C should be direct acting antivirals (DAAs) without Peg-IFN. Peg-IFN is a strong immune-modulator. Therefore, an immunological analysis should be carried out to understand the effect of VitD3 after treatment of DAAs without Peg-IFN. The induction of a favorable immune response by adding VitD3 might be able to suppress the hepatocarcinogenesis after achieving SVR, especially in children and elderly patients with severe fibrosis lacking sufficient amounts of VitD3.

Genome-Wide Association Study Identifies TLL1 Variant Associated With Development of Hepatocellular Carcinoma After Eradication of Hepatitis C Virus Infection.

BACKGROUND & AIMS: There is still a risk for hepatocellular carcinoma (HCC) development after eradication of hepatitis C virus (HCV) infection with antiviral agents. We investigated genetic factors associated with the development of HCC in patients with a sustained virologic response (SVR) to treatment for chronic HCV infection. METHODS: We obtained genomic DNA from 457 patients in Japan with a SVR to interferon-based treatment for chronic HCV infection from 2007 through 2015. We conducted a genome-wide association study (GWAS), followed by a replication analysis of 79 candidate single nucleotide polymorphisms (SNPs) in an independent set of 486 patients in Japan. The study end point was HCC diagnosis or confirmation of lack of HCC (at follow-up examinations until December 2014 in the GWAS cohort, and until January 2016 in the replication cohort). We collected clinical and laboratory data from all patients. We analyzed expression levels of candidate gene variants in human hepatic stellate cells, rats with steatohepatitis caused by a choline-deficient L-amino acid-defined diet, and a mouse model of liver injury caused by administration of carbon tetrachloride. We also analyzed expression levels in liver tissues of patients with chronic HCV infection with different stages of fibrosis or tumors vs patients without HCV infection (controls). RESULTS: We found a strong association between the SNP rs17047200, located within the intron of the tolloid like 1 gene (TLL1) on chromosome 4, and development of HCC; there was a genome-wide level of significance when the results of the GWAS and replication study were combined (odds ratio, 2.37; P = 2.66 × 10-8). Multivariate analysis showed rs17047200 AT/TT to be an independent risk factor for HCC (hazard ratio, 1.78; P = .008), along with male sex, older age, lower level of albumin, advanced stage of hepatic fibrosis, presence of diabetes, and higher post-treatment level of α-fetoprotein. Combining the rs17047200 genotype with other factors, we developed prediction models for HCC development in patients with mild or advanced hepatic fibrosis. Levels of TLL1 messenger RNA (mRNA) in human hepatic stellate cells increased with activation. Levels of Tll1 mRNA increased in liver tissues of rodents with hepatic fibrogenesis compared with controls. Levels of TLL1 mRNA increased in liver tissues of patients with progression of fibrosis. Gene expression levels of TLL1 short variants, including isoform 2, were higher in patients with rs17047200 AT/TT. CONCLUSIONS: In a GWAS, we identified the association between the SNP rs17047200, within the intron of TLL1, and development of HCC in patients who achieved an SVR to treatment for chronic HCV infection. We found levels of Tll1/TLL1 mRNA to be increased in rodent models of liver injury and liver tissues of patients with fibrosis, compared with controls. We propose that this SNP might affect splicing of TLL1 mRNA, yielding short variants with high catalytic activity that accelerates hepatic fibrogenesis and carcinogenesis. Further studies are needed to determine how rs17047200 affects TLL1 mRNA levels, splicing, and translation, as well as the prevalence of this variant among other patients with HCC. Tests for the TLL1 SNP might be used to identify patients at risk for HCC after an SVR to treatment of HCV infection.

Activation of the Hypoxia Inducible Factor 1α Subunit Pathway in Steatotic Liver Contributes to Formation of Cholesterol Gallstones.

BACKGROUND & AIMS: Hypoxia-inducible factor 1α subunit (HIF1A) is a transcription factor that controls the cellular response to hypoxia and is activated in hepatocytes of patients with nonalcoholic fatty liver disease (NAFLD). NAFLD increases the risk for cholesterol gallstone disease by unclear mechanisms. We studied the relationship between HIF1A and gallstone formation associated with liver steatosis. METHODS: We performed studies with mice with inducible disruption of Hif1a in hepatocytes via a Cre adenoviral vector (inducible hepatocyte-selective HIF1A knockout [iH-HIFKO] mice), and mice without disruption of Hif1a (control mice). Mice were fed a diet rich in cholesterol and cholate for 1 or 2 weeks; gallbladders were collected and the number of gallstones was determined. Livers and biliary tissues were analyzed by histology, quantitative reverse-transcription polymerase chain reaction, immunohistochemistry, and immunoblots. We measured concentrations of bile acid, cholesterol, and phospholipid in bile and rates of bile flow. Primary hepatocytes and cholangiocytes were isolated and analyzed. HIF1A was knocked down in Hepa1-6 cells with small interfering RNAs. Liver biopsy samples from patients with NAFLD, with or without gallstones, were analyzed by quantitative reverse-transcription polymerase chain reaction. RESULTS: Control mice fed a diet rich in cholesterol and cholate developed liver steatosis with hypoxia; levels of HIF1A protein were increased in hepatocytes around central veins and 90% of mice developed cholesterol gallstones. Only 20% of the iH-HIFKO mice developed cholesterol gallstones. In iH-HIFKO mice, the biliary lipid concentration was reduced by 36%, compared with control mice, and bile flow was increased by 35%. We observed increased water secretion from hepatocytes into bile canaliculi to mediate these effects, resulting in suppression of cholelithogenesis. Hepatic expression of aquaporin 8 (AQP8) protein was 1.5-fold higher in iH-HIFKO mice than in control mice. Under hypoxic conditions, cultured hepatocytes increased expression of Hif1a, Hmox1, and Vegfa messenger RNAs (mRNAs), and down-regulated expression of AQP8 mRNA and protein; AQP8 down-regulation was not observed in cells with knockdown of HIF1A. iH-HIFKO mice had reduced inflammation and mucin deposition in the gallbladder compared with control mice. Liver tissues from patients with NAFLD with gallstones had increased levels of HIF1A, HMOX1, and VEGFA mRNAs, compared with livers from patients with NAFLD without gallstones. CONCLUSIONS: In steatotic livers of mice, hypoxia up-regulates expression of HIF1A, which reduces expression of AQP8 and concentrates biliary lipids via suppression of water secretion from hepatocytes. This promotes cholesterol gallstone formation. Livers from patients with NAFLD and gallstones express higher levels of HIF1A than livers from patients with NAFLD without gallstones.

Hepatitis C virus has a genetically determined lymphotropism through co-receptor B7.2.

B-cell infection by hepatitis C virus (HCV) has been a controversial topic. To examine whether HCV has a genetically determined lymphotropism through a co-receptor specific for the infection by lymphotropic HCV, we established an infectious clone and chimeric virus of hepatotropic and lymphotropic HCV strains derived from an HCV-positive B-cell lymphoma. The viral envelope and 5'-UTR sequences of the lymphotropic HCV strain were responsible for the lymphotropism. Silencing of the virus sensor, RIGI, or overexpression of microRNA-122 promoted persistent viral replication in B cells. By cDNA library screening, we identified an immune cell-specific, co-stimulatory receptor B7.2 (CD86) as a co-receptor of lymphotropic HCV. Infection of B cells by HCV inhibited the recall reaction to antigen stimulation. Together, a co-receptor B7.2 enabled lymphotropic HCV to infect memory B cells, leading to inhibition of memory B-cell function and persistent HCV infection in HCV-infected hosts.

PD-L1+MDSCs are increased in HCC patients and induced by soluble factor in the tumor microenvironment.

Myeloid-derived suppressor cells (MDSCs) could have important roles in immune regulation, and MDSCs can be induced in patients with various malignant tumors. The immune-suppressive functions of MDSCs in hepatocellular carcinoma (HCC) patients have not been clarified. Therefore, we tried to analyze the biological significance of MDSCs in HCC patients. We quantified PD-L1+MDSCs of HCC patients in various conditions by using multi-color flow cytometry analysis. PBMCs from HCC patients contained significantly higher percentages of PD-L1+MDSCs in comparison to those from healthy subjects (p < 0.001). The percentages of PD-L1+MDSCs were reduced by curative treatment for HCC (p < 0.05), and the percentages of PD-L1+MDSCs before treatment were inversely correlated with disease-free survival time. After we cocultivated PBMCs and several liver cancer cell lines in a transwell coculture system, the percentages of PD-L1+MDSCs were significantly increased compared with control (p < 0.05). The expression of M-CSF and VEGFA was higher in the cell lines that strongly induced PD-L1+MDSCs. Peripheral blood from HCC patients had significantly higher percentages of PD-L1+MDSCs in comparison to those of healthy subjects, and the percentages of PD-L1+MDSCs were reduced by HCC treatment, suggesting that we might use PD-L1+MDSCs as a new biomarker of HCC.

Global, cancer-specific microRNA cluster hypomethylation was functionally associated with the development of non-B non-C hepatocellular carcinoma.

BACKGROUND: While hepatitis B and C viral infection have been suppressed, non-B non-C hepatocellular carcinoma (NBNC-HCC) is considered to be rising in incidence terms in some developed countries where prevalence of those viral infections among HCC patients had been very high (such as Japan, Korea, and Italy). To elucidate critical molecular changes in NBNC-HCC, we integrated three large datasets relating to comprehensive array-based analysis of genome-wide DNA methylation (N = 43 pairs) and mRNA/miRNA expression (N = 15, and 24 pairs, respectively) via statistical modeling. RESULTS: Hierarchical clustering of DNA methylation in miRNA coding regions clearly distinguished NBNC-HCC tissue samples from relevant background tissues, revealing a remarkable tumor-specific hypomethylation cluster. In addition, miRNA clusters were extremely hypomethylated in tumor samples (median methylation change for non-clustered miRNAs: -2.3%, clustered miRNAs: -24.6%). The proportion of CpGs hypomethylated in more than 90% of the samples was 55.9% of all CpGs within miRNA clusters, and the peak methylation level was drastically shifted from 84% to 39%. Following statistical adjustment, the difference in methylation levels within miRNA coding regions was positively associated with their expression change. Receiver operating characteristic (ROC) analysis revealed a great discriminatory ability in respect to cluster-miRNA methylation. Moreover, miRNA methylation change was negatively correlated with corresponding target gene expression amongst conserved and highly matched miRNA sites. CONCLUSIONS: We observed a drastic negative shift of methylation levels in miRNA cluster regions. Changes in methylation status of miRNAs were more indicative of target gene expression and pathological diagnosis than respective miRNA expression changes, suggesting the importance of genome-wide miRNA methylation for tumor development. Our study dynamically summarized global miRNA hypomethylation and its genome-wide scale consequence in NBNC-HCC.