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Human hepatitis B virus (HBV) infection remains a serious health problem worldwide. However, the mechanism for the maintenance of HBV in a latent state within host cells remains unclear. Here, using single-cell RNA sequencing analysis, we identified four genes linked to the maintenance of HBV in a liver cell line expressing HBV RNA at a low frequency. These genes included DOCK11 and DENND2A, which encode small GTPase regulators. In primary human hepatocytes infected with HBV, knockdown of these two genes decreased the amount of both HBV DNA and covalently closed circular DNA to below the limit of detection. Our findings reveal a role for DOCK11 and DENND2A in the maintenance of HBV.

Background & aims: Genome-wide association studies in primary biliary cholangitis (PBC) have failed to find X chromosome (chrX) variants associated with the disease. Here, we specifically explore the chrX contribution to PBC, a sexually dimorphic complex autoimmune disease. Methods: We performed a chrX-wide association study, including genotype data from 5 genome-wide association studies (from Italy, United Kingdom, Canada, China, and Japan; 5244 case patients and 11,875 control individuals). Results: Single-marker association analyses found approximately 100 loci displaying P < 5 × 10-4, with the most significant being a signal within the OTUD5 gene (rs3027490; P = 4.80 × 10-6; odds ratio [OR], 1.39; 95% confidence interval [CI], 1.028-1.88; Japanese cohort). Although the transethnic meta-analysis evidenced only a suggestive signal (rs2239452, mapping within the PIM2 gene; OR, 1.17; 95% CI, 1.09-1.26; P = 9.93 × 10-8), the population-specific meta-analysis showed a genome-wide significant locus in East Asian individuals pointing to the same region (rs7059064, mapping within the GRIPAP1 gene; P = 6.2 × 10-9; OR, 1.33; 95% CI, 1.21-1.46). Indeed, rs7059064 tags a unique linkage disequilibrium block including 7 genes: TIMM17B, PQBP1, PIM2, SLC35A2, OTUD5, KCND1, and GRIPAP1, as well as a superenhancer (GH0XJ048933 within OTUD5) targeting all these genes. GH0XJ048933 is also predicted to target FOXP3, the main T-regulatory cell lineage specification factor. Consistently, OTUD5 and FOXP3 RNA levels were up-regulated in PBC case patients (1.75- and 1.64-fold, respectively). Conclusions: This work represents the first comprehensive study, to our knowledge, of the chrX contribution to the genetics of an autoimmune liver disease and shows a novel PBC-related genome-wide significant locus.

[BACKGROUND & AIMS] Primary biliary cholangitis (PBC) is a chronic liver disease in which autoimmune destruction of the small intra-hepatic bile ducts eventually leads to cirrhosis. Many patients have inadequate response to licensed medications, motivating the search for novel therapies. Previous genome-wide association studies (GWAS) and meta-analyses (GWMA) of PBC have identified numerous risk loci for this condition, providing insight into its aetiology. We undertook the largest GWMA of PBC to date, aiming to identify additional risk loci and prioritise candidate genes for in silico drug efficacy screening. [METHODS] We combined new and existing genotype data for 10, 516 cases and 20, 772 controls from five European and two East Asian cohorts. [RESULTS] We identified 56 genome-wide significant loci (20 novel) including 46 in European, 13 in Asian, and 41 in combined cohorts; and a 57th genome-wide significant locus (also novel) in conditional analysis of the European cohorts. Candidate genes at newly identified loci include FCRL3, INAVA, PRDM1, IRF7, CCR6, CD226, and IL12RB1, each having key roles in immunity. Pathway analysis reiterated the likely importance of pattern recognition receptor and TNF signalling, Jak-STAT signalling, and differentiation of TH1 and TH17 cells in the pathogenesis of this disease. Drug efficacy screening identified several medications predicted to be therapeutic in PBC, some well-established in the treatment of other autoimmune disorders. [CONCLUSIONS] This study has identified additional risk loci for PBC, provided a hierarchy of agents that could be trialled in this condition, and emphasised the value of genetic and genomic approaches to drug discovery in complex disorders. [Lay summary] Primary biliary cholangitis (PBC) is a chronic liver disease that eventually leads to cirrhosis. In this study, we analysed genetic information from 10, 516 people with PBC and 20, 772 healthy individuals recruited in Canada, China, Italy, Japan, UK, or USA. We identified several genetic regions associated with PBC. Each of these regions contains several genes. For each region, we used diverse sources of evidence to help us choose the gene most likely to be involved in causing PBC. We used these ‘candidate genes’ to help us identify medications that are currently used for treatment of other conditions, which might also be useful for treatment of PBC., 原発性胆汁性胆管炎のゲノムワイド関連解析 --国際メタ解析による新規疾患感受性遺伝子と治療薬候補の同定--. 京都大学プレスリリース. 2021-06-28.

Cancer stemness evinces interest owing to the resulting malignancy and poor prognosis. We previously demonstrated that hepatic stem cell-like hepatocellular carcinoma (HpSC-HCC) is associated with high vascular invasion and poor prognosis. Dickkopf-1 (DKK-1), a Wnt signaling regulator, is highly expressed in HpSC-HCC. Here, we assessed the diagnostic and prognostic potential of serum DKK-1. Its levels were significantly higher in 391 patients with HCC compared with 205 patients with chronic liver disease. Receiver operating characteristic curve analysis revealed the optimal cutoff value of DKK-1 to diagnose HCC and predict the 3-year survival as 262.2 and 365.9 pg/mL, respectively. HCC patients with high-serum DKK-1 levels showed poor prognosis. We evaluated the effects of anti-DKK-1 antibody treatment on tumor growth in vivo and of recombinant DKK-1 on cell proliferation, invasion, and angiogenesis in vitro. DKK-1 knockdown decreased cancer cell proliferation, migration, and invasion. DKK-1 supplementation promoted angiogenesis in vitro; this effect was abolished by an anti-DKK-1 antibody. Co-injection of the anti-DKK-1 antibody with Huh7 cells inhibited their growth in NOD/SCID mice. Thus, DKK-1 promotes proliferation, migration, and invasion of HCC cells and activates angiogenesis in vascular endothelial cells. DKK-1 is a prognostic biomarker for HCC and a functional molecule for targeted therapy.

Nonalcoholic fatty liver disease (NAFLD) is associated with metabolic syndrome, which includes diabetes mellitus and hyperlipidemia. A fraction of NAFLD patients develop nonalcoholic steatohepatitis, leading to cirrhosis associated with various serious complications, including hepatocellular carcinoma, gastroesophageal varices, cardiovascular events, and other organ malignancy. Although the incidence of chronic viral hepatitis with associated complications has gradually decreased as highly effective antiviral therapies have been established, the number of patients with steatohepatitis has been increasing. This retrospective study examined data of 229 patients from 22 hospitals in our region. We examined 155 cases of chronological data and assessed the development of liver fibrosis and evaluated hepatic reserve-related markers such as platelet count, FIB-4 index, prothrombin time, and serum albumin concentration. We analyzed the relationship of these chronological changes and the incidence of NAFLD related serious complications. Data related to liver fibrosis progression, albumin, and prothrombin time were significantly associated with the occurrence of serious complications associated with cirrhosis. We compared 22 event and 133 nonevent cases of chronological changes in the data per year and found that serum albumin concentration was significantly lower in the group that developed serious complications (event cases: -0.21 g/dL/year, nonevent cases: –0.04 g/dL/year (P

Aim Sequential administration of sorafenib followed by regorafenib or lenvatinib is effective against advanced hepatocellular carcinoma (HCC). In this study, we compared the safety profiles and anti-tumor effects of sequential sorafenib and regorafenib or lenvatinib therapy in patients with HCC. Methods We investigated adverse events, treatment responses and dose intensities in patients with HCC who were consecutively treated with sorafenib followed by regorafenib or lenvatinib at the individual level. Results Each group included 20 patients. The safety profiles of regorafenib and sorafenib were similar. The severity of hypophosphatemia, palmar-plantar erythrodysesthesia syndrome, and decreased neutrophil counts associated with regorafenib or sorafenib was similar in 12 patients. Conversely, the incidences and grades of adverse events differed between sorafenib and lenvatinib treatment. The anti-tumor effects of regorafenib and lenvatinib compared with sorafenib were significantly different for each patient. The response to treatment and progression-free survival were comparable for regorafenib and lenvatinib. The median relative dose intensities during the first 56 days of regorafenib and lenvatinib treatment were 83.6 and 80.0%, respectively. Conclusions Similar adverse events were experienced by patients during consecutive treatment with sorafenib and regorafenib, which was not observed during treatment with sorafenib and lenvatinib. The obtained safety profile of sorafenib provided meaningful insights for selecting sequential therapy for patients with advanced HCC.

Background aims Immunotherapy is effective for many types of cancer, but its benefits in advanced pancreatic cancer, which has a poor prognosis, are not well established. In this study, the authors examined the effects of adoptive T-cell immunotherapy (ATI) on immune cell profiles and prognosis in patients with unresectable advanced pancreatic cancer. Methods Seventy-seven patients with unresectable advanced pancreatic cancer were treated with six cycles of αβ T cells alone or in combination with chemotherapy or chemoradiation. Immune cell profiles in peripheral blood samples obtained before and after treatment were comprehensively evaluated by flow cytometry. Furthermore, associations between changes in immune cell frequencies and prognosis were determined. Results ATI prolonged survival to 18.7 months compared with previous estimates of 6.2–11.1 months for patients treated with chemotherapy alone. ATI decreased CD3+CD4+CD8− T cell frequency in peripheral blood and increased CD3+CD4−CD8+ T cell frequency. An increase in CD3+ T cells and CD3+TCRγδ− T cells in peripheral blood after treatment was associated with a good prognosis. Conclusions ATI altered the immune profile in peripheral blood, including CD3+CD4−CD8+ T cells, and improved prognosis in pancreatic cancer.

A 79-year-old man with metastatic melanoma of the right maxillary sinus and multiple liver metastases received a single dose of nivolumab. Eight days later, he experienced impaired consciousness, accompanied by abnormal laboratory and electrocardiographic findings. He was therefore diagnosed with tumor lysis syndrome (TLS). Laboratory and electrocardiographic findings improved immediately after continuous hemodiafiltration; however, he died 22 days after receiving nivolumab. Autopsy revealed massive tumor necrosis in the liver. There are few case reports of TLS associated with immune checkpoint inhibitors, indicating that we should be prepared to manage especially in a patient with liver involvement of high tumor burden.

Background We aimed to determine the optimal approach with endoscopic biliary drainage (EBD) and corticosteroid (CS) for the treatment of IgG4-related sclerosing cholangitis (ISC). Methods To evaluate the safety of EBD for treatment of biliary stricture caused by ISC, we assessed the risk of stent dislodgement and sought to determine the most appropriate time for stent removal. We also assessed the safety of treatment with CS alone for patients with obstructive jaundice, and the rate of and risk factors for biliary tract complications. Results Sixty-nine patients with ISC treated with CS were enrolled. Twenty-eight patients (40.6%) were treated with EBD for biliary stricture before CS initiation. Intentional stent removal was performed in thirteen (46.4%) after confirming CS-induced improvement. Eleven of thirteen patients (84.6%) underwent stent removal within 1 month after CS initiation and all their stent removals were safely carried out without early (within two weeks) recurrence of obstructive jaundice. Ten of twenty-eight patients (35.7%) experienced spontaneous stent dislodgement after CS initiation, and seven (70%) of them developed stent dislodgement two weeks to two months after CS initiation. Among forty-one patients treated with CS alone without EBD, 10 patients had obstructive jaundice at the time of CS initiation and all of them achieved clinical improvement without biliary tract infection. During the follow-up, three patients (4.3%), all of whom had undergone EBD, developed bile-duct stones, while none of those treated with CS alone developed bile-duct stones (p = 0.032). Long-term biliary stenting was a risk factor for bile-duct stones. Conclusions Biliary stent removal should be carried out within 2 weeks after CS initiation if biliary stricture improves to prevent stent dislodgement. Obstructive jaundice can be treated safely with CS alone in patients without infection. Clinicians should be aware of the possibility of bile-duct stones in patients treated with EBD.

Background & Aims: To eliminate hepatitis B virus (HBV) infection, scale-up of testing and treatment in resource-limited countries is crucial. However, access to nucleic acid testing to quantify HBV DNA, an essential test to examine treatment eligibility, remains severely limited. We assessed the performance of a novel immunoassay, HBV core-related antigen (HBcrAg), as a low-cost (less than US $15/assay) alternative to nucleic acid testing to indicate clinically important high viremia in chronic HBV patients infected with different genotypes. Methods: We searched Medline, Embase, Scopus, and Web of Science databases through June 27, 2018. Three reviewers independently selected studies measuring HBV DNA and HBcrAg in the same blood samples. We contacted authors to provide individual participant data (IPD). We randomly allocated each IPD to a derivation or validation cohort. We applied optimal HBcrAg cut-off values derived from the derivation set to the validation set to estimate sensitivity/specificity. Results: Of 74 eligible studies, IPD were obtained successfully for 60 studies (81%). Meta-analysis included 5591 IPD without antiviral therapy and 4806 treated with antivirals. In untreated patients, the pooled area under the receiver operating characteristic curve and optimal cut-off values were as follows: 0.88 (95% CI, 0.83–0.94) and 3.6 log U/mL to diagnose HBV DNA level of 2000 IU/mL or greater; and 0.96 (95% CI, 0.94–0.98) and 5.3 log U/mL for 200,000 IU/mL or greater, respectively. In the validation set, the sensitivity and specificity were 85.2% and 84.7% to diagnose HBV DNA level of 2000 IU/mL or greater, and 91.8% and 90.5% for 200,000 IU/mL or greater, respectively. The performance did not vary by HBV genotypes. In patients treated with anti-HBV therapy the correlation between HBcrAg and HBV DNA was poor. Conclusions: HBcrAg might be a useful serologic marker to indicate clinically important high viremia in treatment-naïve, HBV-infected patients.

Background Liver cirrhosis results from chronic hepatitis, and is characterized by advanced fibrosis due to long-term hepatic inflammation. Cirrhosis ultimately leads to manifestations of jaundice, ascites, and encephalopathy, and increases the risk of hepatocellular carcinoma. Once cirrhosis is established, resulting in hepatic failure, no effective treatment is available. Therefore, novel therapies to inhibit disease progression of cirrhosis are needed. Objective The objective of this investigator-initiated clinical trial is to assess the safety and efficacy of autologous adipose tissue-derived regenerative (stem) cell therapy delivered to the liver via the hepatic artery in patients with liver cirrhosis. Methods Through consultation with the Japan Pharmaceuticals and Medical Devices Agency, we designed a clinical trial to assess a therapy for liver cirrhosis based on autologous adipose tissue-derived regenerative (stem) cells, which are extracted using an adipose tissue dissociation device. The primary endpoints of the trial are the serum albumin concentration, prothrombin activity, harmful events, and device malfunction. Results Enrollment and registration were initiated in November 2017, and the follow-up period ended in November 2019. Data analysis and the clinical study report will be completed by the end of March 2020. Conclusions Completion of this clinical trial, including data analysis, will provide data on the safety and efficacy of this novel liver repair therapy based on autologous adipose tissue-derived regenerative (stem) cells using an adipose tissue dissociation device. Trial Registration UMIN Clinical Trials Registry UMIN000022601; https://tinyurl.com/w9uqw3q International Registered Report Identifier (IRRID) DERR1-10.2196/17904

α-Fetoprotein (AFP) is considered a good target for immunotherapy strategies against hepatocellular carcinoma (HCC); however, no immunodominant AFP-derived MHC class II-restricted helper T-lymphocyte (HTL) epitope has been reported. Therefore, we identified novel AFP-derived HTL epitopes possessing high immunogenicity. HTL epitopes were predicted using the online service, and peptides were subsequently synthesized. Four newly synthesized peptides showed positive reactivity in >20% patients on ELISPOT using peripheral blood mononuclear cells (PBMCs). Among these, the highest rate was shown by AFP1 (MKWVESIFLIFLLNFTESRT), which also showed the highest positive rate in cell proliferation assays. Binding assays demonstrated that AFP1 had strong binding properties toward MHC molecules. Further, blocking assays performed using an anti-HLA-DR antibody showed that immune response decreased, confirming the binding of AFP1 to HLA-DR molecules. Furthermore, the survival rates of patients with stages II–IV HCC indicated that T cell response against AFP1 led to significantly greater survival that of patients without T cell response. When evaluating immune response against AFP1 before and after HCC treatment, an increase in the frequency of peptide-specific T cells was observed after treatment in patients with HLA-DRB1*1502, *0405, and *0901 alleles. In conclusion, the identified epitopes may be useful for immunotherapy strategies against HCC.

Background: Hyperuricemia drives the development of nonalcoholic fatty liver disease (NAFLD). Pharmacological inhibition of xanthine oxidase (XO), a rate-limiting enzyme for uric acid (UA) production, has been demonstrated to improve hepatic steatosis in diet-induced obese mice. However, it remains unclear whether inhibition of XO could improve nonalcoholic steatohepatitis (NASH), a more advanced form of NAFLD, in terms of both liver inflammation and fibrosis. Methods: To investigate the effects of febuxostat and allopurinol, XO inhibitors clinically used for gout, on diet-induced NASH, C57BL/6J mice were fed a high-fat, high-cholesterol, and cholate diet (CL diet) with or without either 0.001% febuxostat or 0.003% allopurinol for 18 weeks. Systemic glucose tolerance and insulin sensitivity, liver histology, inflammation, and oxidative stress were tested. Furthermore, we conducted a single-arm, open-label intervention study with febuxostat for NAFLD patients with hyperuricemia. Findings: Despite a similar hypouricemic effect of the XO inhibitors on blood UA level, febuxostat, but not allopurinol, significantly decreased hepatic UA levels in CL diet-fed mice. This reduction in hepatic UA levels was accompanied by alleviated insulin resistance, lipid peroxidation, and classically activated M1-like macrophage accumulation in the liver. Furthermore, in NAFLD patients with hyperuricemia, treatment with febuxostat for 24 weeks decreased the serum UA level, accompanied by a reduction in serum levels of liver enzymes, alanine aminotransferase and aspartate aminotransferase. Interpretation: XO may represent a promising therapeutic target in NAFLD/NASH, especially in patients with hyperuricemia. Trial Registration: The study has been registered with UMIN-CTR (#UMIN000008686). Funding Statement: This work was supported by Grants-in-aid for Scientific Research (B) (25282017) and Challenging Exploratory Research (15K12698) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and the Japan Diabetes Foundation (to T.O.). This study was also supported by Teijin Pharma Limited. Declaration of Interests: The authors declare no conflict of interest associated with this manuscript. Ethics Approval Statement: The study was approved by the Ethics Committee of Kanazawa University Hospital. All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Kanazawa University, Japan.

BACKGROUND Liver cirrhosis results from chronic hepatitis, and is characterized by advanced fibrosis due to long-term hepatic inflammation. Cirrhosis ultimately leads to manifestations of jaundice, ascites, and encephalopathy, and increases the risk of hepatocellular carcinoma. Once cirrhosis is established, resulting in hepatic failure, no effective treatment is available. Therefore, novel therapies to inhibit disease progression of cirrhosis are needed. OBJECTIVE The objective of this investigator-initiated clinical trial is to assess the safety and efficacy of autologous adipose tissue-derived regenerative (stem) cell therapy delivered to the liver via the hepatic artery in patients with liver cirrhosis. METHODS Through consultation with the Japan Pharmaceuticals and Medical Devices Agency, we designed a clinical trial to assess a therapy for liver cirrhosis based on autologous adipose tissue-derived regenerative (stem) cells, which are extracted using an adipose tissue dissociation device. The primary endpoints of the trial are the serum albumin concentration, prothrombin activity, harmful events, and device malfunction. RESULTS Enrollment and registration were initiated in November 2017, and the follow-up period ended in November 2019. Data analysis and the clinical study report will be completed by the end of March 2020. CONCLUSIONS Completion of this clinical trial, including data analysis, will provide data on the safety and efficacy of this novel liver repair therapy based on autologous adipose tissue-derived regenerative (stem) cells using an adipose tissue dissociation device. CLINICALTRIAL UMIN Clinical Trials Registry UMIN000022601; https://tinyurl.com/w9uqw3q INTERNATIONAL REGISTERED REPORT DERR1-10.2196/17904

Osteopontin (OPN) is involved in cell proliferation, migration, inflammation, and tumor progression in various tissues. OPN induces stemness by interacting with CD44, but the functional relevance of OPN-mediated interferon (IFN) signaling and hepatitis C virus (HCV) replication in stem cell populations remains unclear. In this study, we investigated the effect of OPN on HCV replication and IFN signaling in cancer stem cells (CSCs) positive for epithelial cell adhesion molecule (EpCAM) and CD44. We show that the EpCAM+/CD44+ CSCs show marked HCV replication when compared to EpCAM−/CD44− cells. In addition, OPN significantly enhances this HCV replication in EpCAM+/CD44+ CSCs and markedly suppresses IFN-stimulated gene expression. The GSK-3β inhibitor BIO increases the EpCAM+/CD44+ CSC population and OPN expression and impairs IFN signaling via STAT1 degradation. Taken together, our data suggest that OPN enhances HCV replication in the EpCAM+/CD44+ CSCs, while it also negatively regulates the IFN signaling pathway via inhibition of STAT1 phosphorylation and degradation. Therefore, OPN may represent a novel therapeutic target for treating HCV-related hepatocellular carcinoma.

BackgroundPortal vein thrombosis (PVT) is a common complication of cirrhosis. However, in patients with PVT and cirrhosis, there is no clear evidence supporting effective treatment modalities. In this study, we examined the effectiveness and safety of anticoagulation therapy using danaparoid sodium for PVT in patients with cirrhosis.MethodsThis retrospective study assessed 52 cirrhotic patients with PVT treated with danaparoid sodium for 2 weeks between November 2008 and September 2018. The primary outcome measure was the post-treatment status of PVT assessed by reduction in thrombus volume and safety of the therapeutic intervention. PVT status was evaluated with contrast-enhanced computed tomography (CECT). All patients received 1250 units of danaparoid sodium twice daily by intravenous injection for 14 days. Patients on antithrombin III (AT-III) combination therapy were additionally administered 1500 units of AT-III on days 1–5 and days 8–12. Effectiveness was evaluated by CECT from between days 13 and 18. The secondary outcome measure was the prognosis of PVT.ResultsAll patients showed reduction in PVT volume without complications. Return of plasma AT-III level to > 70% during the treatment period contributes to ≥75% reduction of PVT volume. The prognosis in PVT patients depends on hepatic reserve capacity. When limited to Child-Pugh B and C liver cirrhosis patients, a ≥ 75% reduction of PVT volume improved the prognosis.ConclusionsDanaparoid sodium-based anticoagulation therapy was effective and safe for PVT in patients with cirrhosis. Return of plasma AT-III level to the normal range during the treatment period contributes to reduction of PVT volume. A reduction of ≥75% in PVT volume may improve the prognosis of Child-Pugh B and C decompensated cirrhosis patients with PVT.

We report three cases of genotype 1b hepatitis C virus (HCV) reinfection after liver transplantation. When antiviral treatment was considered, all three patients had renal dysfunction and had been treated with immunosuppressive agents for a long time; one with tacrolimus (TAC) and the others with cyclosporine A (CyA). Therefore, the possible antiviral regimens among direct-acting antivirals (DAA) were limited and so we treated all three patients with ombitasvir/paritaprevir/ritonavir (OBV/PTV/r). Because ritonavir is known to markedly increase the blood concentration of TAC and CyA through drug-drug interactions, close monitoring of blood concentrations of TAC or CyA and dose adjustments of immunosuppressive agents were needed. Sustained virus response was achieved in all the patients treated, and there were no adverse effects or transplant rejection. OBV/PTV/r might be a useful DAA regimen for patients with genotype 1 HCV reinfection in the setting of renal dysfunction.

AIM This study aimed to investigate liver functional reserves during sorafenib treatment for advanced hepatocellular carcinoma (HCC), to identify predictive factors for maintaining them, and to analyze the proportion of candidates for regorafenib, which has been proven to improve patients' outcomes in the RESORCE trial. METHODS We retrospectively investigated Child-Pugh scores during and after sorafenib treatment and evaluated their effects on second-line treatment and outcomes of 125 patients with advanced HCC. RESULTS Pretreatment Child-Pugh A was maintained in 59/90 (65.6%) patients and pretreatment Child-Pugh B was improved to Child-Pugh A in 10/35 (28.6%) patients when sorafenib ceased. A Child-Pugh score = 5 and aspartate amino transferase

403 Background: Time to progression (TTP) is widely used as the endpoint in early-phase trials for advanced hepatocellular carcinoma (HCC). However, the relevance of using TTP as a surrogate marker for overall survival (OS) in pivotal trials remains uncertain. Methods: The PubMed database and ASCO meeting library were searched for reports of randomized controlled trials that investigated patients with advanced HCC, included data for both OS and TTP, and were launched between 2009 and 2016. Correlation between hazard ratios (HRs) for TTP and OS was determined using weighted linear regression. Correlations between median OS and TTP, and between median OS and post-progression survival (PPS), defined as the period obtained by subtracting median TTP from median OS, were also evaluated. Results: The database search yielded 24 trials with 50 arms. Overall, TTP HR correlated with OS HR (R = 0.73); however, the coefficient in the regression equation was 0.48. When trials were stratified by treatment line, TTP HR was more strongly correlated with OS HR in second-line (R = 0.91) than in first-line (R = 0.77) settings. Correlation between median OS and median TTP was weak (R = 0.50), whereas the correlation between median OS and median PPS was strong (R = 0.78). Conclusions: In advanced HCC, the OS HR can be predicted from the TTP HR, especially in second-line trials, which is useful when considering whether to proceed to a pivotal trial based on the results of early-phase trials. However, TTP may not be appropriate as a primary endpoint in a pivotal trial if the trial aims to evaluate the survival benefit of novel agents because improvement of TTP cannot fully reflect improvement of OS because of the impact of PPS on OS.

Nucleos(t)ide analogues therapies are currently approved for the treatment of chronic hepatitis B virus (HBV) infection, which effectively suppress HBV replication and correlate with the anti-HBV-specific immune response. Notch signaling serves pleiotropic roles in the immune system that also contribute to virus-specific immunity. In this study, we assessed Notch signal-related gene expression after administrating nucleoside or nucleotide analogues to HBV-replicating cells and clinical liver tissues. We found distinct Notch signaling expression patterns under nucleos(t)ide analogues therapies, with high expression for nucleotide analogues (adefovir pivoxil or tenofovir disoproxil fumarate) and low expression for nucleoside analogues (lamivudine or entecavir) in the presence of HBV infection. Furthermore, activation of mammalian target of rapamycin (mTOR)-Akt (Ser473) phosphorylation was also observed after nucleotide analogue treatment. In conclusion, nucleoside and nucleotide analogues displayed different patterns of Notch signaling activity under HBV infection, and the induction of mTORC2-Akt (Ser473) phosphorylation may contribute to nucleotide analogues-mediated Notch signaling activation.

We have performed a genome-wide association study (GWAS) including 473 Japanese HBV (hepatitis B virus)-positive HCC (hepatocellular carcinoma) patients and 516 HBV carriers including chronic hepatitis and asymptomatic carrier individuals to identify new host genetic factors associated with HBV-derived HCC in Japanese and other East Asian populations. We identified 65 SNPs with P values −4 located within the HLA class I region and three SNPs were genotyped in three independent population-based replication sets. Meta-analysis confirmed the association of the three SNPs (rs2523961: OR = 1.73, P = 7.50 × 10−12; rs1110446: OR = 1.79, P = 1.66 × 10−13; and rs3094137: OR = 1.73, P = 7.09 × 10−9). We then performed two-field HLA genotype imputation for six HLA loci using genotyping data to investigate the association between HLA alleles and HCC. HLA allele association testing revealed that HLA-A * 33:03 (OR = 1.97, P = 4.58 × 10−4) was significantly associated with disease progression to HCC. Conditioning analysis of each of the three SNPs on the HLA class I region abolished the association of HLA-A*33:03 with disease progression to HCC. However, conditioning the HLA allele could not eliminate the association of the three SNPs, suggesting that additional genetic factors may exist in the HLA class I region.

Background The relationship between specific genome alterations and hepatocellular carcinoma (HCC) cancer stem cells (CSCs) remains unclear. In this study, we evaluated the relationship between somatic mutations and epithelial cell adhesion molecule positive (EpCAM+) CSCs. Methods Two patient-derived HCC samples (HCC1 and HCC2) were sorted by EpCAM expression and analyzed by whole exome sequence. We measured PCDH18 expression level in eight HCC cell lines as well as HCC1 and HCC2 by real-time quantitative RT-PCR. We validated the identified gene mutations in 57 paired of HCC and matched non-cancerous liver tissues by Sanger sequence. Results Whole exome sequencing on the sorted EpCAM+ and EpCAM− HCC1 and HCC2 cells revealed 19,263 nonsynonymous mutations in the cording region. We selected mutations that potentially impair the function of the encoded protein. Ultimately, 60 mutations including 13 novel nonsense and frameshift mutations were identified. Among them, PCDH18 mutation was more frequently detected in sorted EpCAM+ cells than in EpCAM− cells in HCC1 by whole exome sequences. However, we could not confirm the difference of PCDH18 mutation frequency between sorted EpCAM+ and EpCAM− cells by Sanger sequencing, indicating that PCDH18 mutation could not explain intracellular heterogeneity. In contrast, we found novel PCDH18 mutations, including c.2556_2557delTG, c.1474C>G, c.2337A>G, and c.2976G>T, were detected in HCC1 and 3/57 (5.3%) additional HCC surgical specimens. All four HCCs with PCDH18 mutations were EpCAM-positive, suggesting that PCDH18 somatic mutations might explain the intertumor heterogeneity of HCCs in terms of the expression status of EpCAM. Furthermore, EpCAM-positive cell lines (Huh1, Huh7, HepG2, and Hep3B) had lower PCDH18 expression than EpCAM-negative cell lines (PLC/PRL/5, HLE, HLF, and SK-Hep-1), and PCDH18 knockdown in HCC2 cells slightly enhanced cell proliferation. Conclusions Our data suggest that PCDH18 is functionally suppressed in a subset of EpCAM-positive HCCs through somatic mutations, and may play a role in the development of EpCAM-positive HCCs. Electronic supplementary material The online version of this article (doi:10.1186/s12935-017-0467-x) contains supplementary material, which is available to authorized users.

Stromal cells in adipose tissue are useful for repair/regenerative therapy as they harbor a substantial number of mesenchymal stem cells; therefore, freshly isolated autologous uncultured adipose tissue derived stromal cells (u-ADSCs) are useful for regenerative therapy, and obviate the need for mesenchymal stem cells. We evaluated the therapeutic effect of murine u-ADSCs and sorted subsets of u-ADSCs in a concanavalin A (ConA) induced murine model of hepatitis, as well as their characteristics. We found that 10-20% of u-ADSCs expressed the CD45 leukocyte-related antigen. CD68, which is a marker of macrophages (MΦs), was expressed by 50% of CD45+ u-ADSCs. About 90% of CD68+ CD45+ cells expressed CD206 antigen, which is a marker of inhibitory M2-type MΦs. Genes related to M2-type MUs were especially more highly expressed by CD45+ CD206+ u-ADSCs than by CD45- u-ADSCs. CD45+ u-ADSCs inhibited the expression of cytokines/chemokines and suppressed the proliferation of splenocytes stimulated with ConA. We observed that not only whole u-ADSCs, but also the CD45+ subset of u-ADSCs ameliorated the ConA-induced hepatitis in mice. In conclusion, we show that freshly isolated murine u-ADSCs were effective against acute hepatitis, and CD45+ u-ADSCs acting phenotypically and functionally like M2-type MΦs, contributed to the repair of liver tissue undergoing inflammation.

The pathogenesis of non-alcoholic steatohepatitis (NASH) is still unclear and the prevention of the development of hepatocellular carcinoma (HCC) has not been established. We established an atherogenic and high-fat diet mouse model that develops hepatic steatosis, inflammation, fibrosis, and liver tumors at a high frequency. Using two NASH-HCC mouse models, we showed that peretinoin, an acyclic retinoid, significantly improved liver histology and reduced the incidence of liver tumors. Interestingly, we found that peretinoin induced autophagy in the liver of mice, which was characterized by the increased co-localized expression of microtubule-associated protein light chain 3B-II and lysosome-associated membrane protein 2, and increased autophagosome formation and autophagy flux in the liver. These findings were confirmed using primary mouse hepatocytes. Among representative autophagy pathways, the autophagy related (Atg) 5-Atg12-Atg16L1 pathway was impaired; especially, Atg16L1 was repressed at both the mRNA and protein level. Decreased Atg16L1 mRNA expression was also found in the liver of patients with NASH according to disease progression. Promoter analysis revealed that peretinoin activated the promoter of Atg16L1 by increasing the expression of CCAAT/enhancer-binding-protein-alpha. Interestingly, Atg16L1 overexpression in HepG2 cells inhibited palmitate-induced NF-kB activation and interleukin-6-induced STAT3 activation. We showed that Atg16L1 induced the de-phosphorylation of Gp130, a receptor subunit of interleukin-6 family cytokines, which subsequently repressed phosphorylated-STAT3 (Tyr705) levels, and this process might be independent of autophagy function. Thus, peretinoin prevents the progression of NASH and the development of HCC through activating the autophagy pathway by increased Atg16L1 expression, which is an essential regulator of autophagy and anti-inflammatory proteins.

BACKGROUND: Cancer stem cells (CSCs) are considered a pivotal target for the eradication of hepatocellular carcinoma (HCC). Recently, we reported that the CSC markers epithelial cell adhesion molecule (EpCAM) and CD90 are expressed independently in primary HCCs and cell lines, and CD90+ cells share features of metastatic vascular endothelial cells and express the vascular endothelial marker CD105, a co-receptor of transforming growth factor-beta. METHODS: The EpCAM+ cell lines HuH1 and HuH7 were treated with 5-fluorouracil (5-FU) or epirubicin in vitro. Gene and protein expression levels were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and fluorescence-activated cell sorting, respectively. The expression of CD105 in primary HCC was evaluated by immunohistochemistry. The relationship of CD105 expression status and HCC prognosis was evaluated using 85 surgically resected HCC tissues by Kaplan–Meier survival analysis. RESULTS: 5-FU or epirubicin treatment resulted in the generation of CD90+ and CD105+ cells in vitro in HuH1 and HuH7 cells, which originally contain no CD90+ or CD105+ cells. This phenomenon was validated by qRT-PCR analysis with activation of the epithelial-mesenchymal transition (EMT) program regulators Snail family zinc finger 1 (SNAI1) and SNAI2. Immunohistochemical analysis indicated that CD105+ cells were morphologically identical to vascular endothelial cells in untreated primary HCCs. However, surgically resected specimens after transcatheter arterial chemoembolization clearly indicated that CD105+ cancer cells survived at the peripheral edge of the tumor. Kaplan–Meier survival analysis indicated that HCCs expressing CD105 showed poor prognosis after surgery with statistical significance. CONCLUSIONS: Taken together, our data highlight the role of CD105+ HCC cells with activation of the EMT program generated de novo after cytotoxic therapy on the prognosis of HCC patients.

// Kai Takegoshi 1 , Masao Honda 1, 2 , Hikari Okada 1 , Riuta Takabatake 1 , Naoto Matsuzawa-Nagata 3 , Jean S. Campbell 4 , Masashi Nishikawa 1 , Tetsuro Shimakami 1 , Takayoshi Shirasaki 1 , Yoshio Sakai 1 , Taro Yamashita 1 , Toshinari Takamura 3 , Takuji Tanaka 5 , Shuichi Kaneko 1, 3 1 Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan 2 Department of Advanced Medical Technology, Kanazawa University Graduate School of Health Medicine, Kanazawa, Japan 3 Department of Disease Control and Homeostasis, Kanazawa University Graduate School of Medicine, Kanazawa, Japan 4 Department of Pathology, University of Washington School of Medicine, Seattle, WA, USA 5 The Tohkai Cytopathology Institute: Cancer Research and Prevention, Gifu, Japan Correspondence to: Masao Honda, email: mhonda@m-kanazawa.jp Keywords: branched-chain amino acids, nonalcoholic steatohepatitis, mammalian target of rapamycin complex 1, hepatocellular carcinoma, transforming growth factor β1 Received: May 12, 2016 Accepted: December 27, 2016 Published: February 13, 2017 ABSTRACT Oral supplementation with branched-chain amino acids (BCAA; leucine, isoleucine, and valine) in patients with liver cirrhosis potentially suppresses the incidence of hepatocellular carcinoma (HCC) and improves event-free survival. However, the detailed mechanisms of BCAA action have not been fully elucidated. BCAA were administered to atherogenic and high-fat (Ath+HF) diet-induced nonalcoholic steatohepatitis (NASH) model mice. Liver histology, tumor incidence, and gene expression profiles were evaluated. Ath+HF diet mice developed hepatic tumors at a high frequency at 68 weeks. BCAA supplementation significantly improved hepatic steatosis, inflammation, fibrosis, and tumors in Ath+HF mice at 68 weeks. GeneChip analysis demonstrated the significant resolution of pro-fibrotic gene expression by BCAA supplementation. The anti-fibrotic effect of BCAA was confirmed further using platelet-derived growth factor C transgenic mice, which develop hepatic fibrosis and tumors. In vitro, BCAA restored the transforming growth factor (TGF)-β1-stimulated expression of pro-fibrotic genes in hepatic stellate cells (HSC). In hepatocytes, BCAA restored TGF-β1-induced apoptosis, lipogenesis, and Wnt/β-Catenin signaling, and inhibited the transformation of WB-F344 rat liver epithelial stem-like cells. BCAA repressed the promoter activity of TGFβ1R1 by inhibiting the expression of the transcription factor NFY and histone acetyltransferase p300. Interestingly, the inhibitory effect of BCAA on TGF-β1 signaling was mTORC1 activity-dependent, suggesting the presence of negative feedback regulation from mTORC1 to TGF-β1 signaling. Thus, BCAA induce an anti-fibrotic effect in HSC, prevent apoptosis in hepatocytes, and decrease the incidence of HCC; therefore, BCAA supplementation would be beneficial for patients with advanced liver fibrosis with a high risk of HCC.

Hepatocellular carcinoma (HCC) usually develops in chronically damaged liver. We investigated hepatic reserves during chemotherapy of patients with advanced HCC and compensated liver function to evaluate the effect on patients' outcomes of maintaining hepatic reserve after chemotherapy. We retrospectively reviewed the medical records of 190 patients with Child-Pugh A with advanced HCC who were treated with sorafenib or hepatic arterial infusion chemotherapy (HAIC). We investigated the Child-Pugh score and albumin-bilirubin grade for hepatic reserve, and evaluated the effect of the change in Child-Pugh scores on patients' outcomes. Subjects were treated with sorafenib (n = 59) or HAIC (n = 131). Of patients with Child-Pugh data, 66.7% maintained or improved their Child-Pugh score after 4 weeks. Treatment with HAIC was the only factor that significantly contributed to maintaining Child-Pugh scores after 4 weeks. The overall survival of patients with a higher Child-Pugh score after 4 weeks was shorter than that of patients whose Child-Pugh classification was unchanged. Multivariate analysis demonstrated that an increased Child-Pugh score after 4 weeks was one of the independent unfavorable prognostic factors. The change of hepatic reserve as a function of albumin-bilirubin grade did not significantly correlate with patients' outcomes. Maintaining the Child-Pugh score during chemotherapy benefits the outcomes of patients with advanced HCC, even those with sufficient hepatic reserve.

Aim Most patients with advanced hepatocellular carcinoma (HCC) have underlying chronic liver disease, which potentially deteriorated the liver functional reserve that often affects the patients' clinical course. We investigated and compared the changes in liver functional reserve during lenvatinib or sorafenib therapy in patients with advanced HCC. Methods We prospectively collected medical information about patients with advanced HCC with a Child-Pugh score of 5-7 to compare the liver functional reserve during treatment in those who were treated with lenvatinib or sorafenib. We also evaluated the effect of the change in the liver functional reserve on patients' outcome. Moreover, we analyzed the contributing factors for maintaining the liver functional reserve during treatment. Results Patients were treated with lenvatinib (n = 45) or sorafenib (n = 157). Forty-five patients in the lenvatinib group and 135 patients in the sorafenib group were selected through a propensity score matching analysis. More patients treated with lenvatinib had a Child-Pugh score that was maintained or improved after 4 and 12 weeks compared with those treated with sorafenib (P = 0.048, P = 0.036, respectively). Lenvatinib was identified as one of the variables that was associated with maintaining Child-Pugh scores. Multivariate analysis revealed that a worsened Child-Pugh score after 4 weeks was an independent unfavorable predictive factor for overall survival. Conclusions More patients treated with lenvatinib for advanced HCC maintained their liver functional reserves compared with those treated with sorafenib. Maintaining the liver functional reserve contributed to better outcomes for patients with advanced HCC.

Background & Aims Sorafenib is a multireceptor tyrosine kinase inhibitor that can prolong overall survival in patients with advanced hepatocellular carcinoma (HCC). Although most HCC patients who receive sorafenib ultimately show disease progression, it still is unclear whether and how HCC cells acquire chemoresistance during sorafenib treatment in human beings. Methods We analyzed surgically resected HCC tissues from a patient who received sorafenib for prevention of HCC recurrence after surgery (Adjuvant Sorafenib for Hepatocellular Carcinoma after Resection or Ablation trial) and established patient-derived HCC cells. Whole-exome sequence analysis was performed to detect mutations in sorafenib-resistant clones. We examined 30 advanced HCC cases immunohistochemically and 140 HCC cases enrolled in the Adjuvant Sorafenib for Hepatocellular Carcinoma after Resection or Ablation trial using microarray analysis to evaluate the association of Capicua Transcriptional Repressor (CIC) status with sorafenib treatment response. Results We found a CIC mutation in recurrent HCC specimens after sorafenib. CIC encodes Capicua, a general sensor of receptor tyrosine kinase signaling. HCC cells established from the recurrent tumor specimen showed chemoresistance to sorafenib in vitro and in vivo. Established sorafenib-resistant Huh1 and Huh7 cell lines showed reduced expression of Capicua without mutations. Immunohistochemical analysis showed that HCC patients with low Capicua expression showed poor overall survival. Microarray analysis showed that the CIC gene signature could predict the preventive effect of adjuvant sorafenib treatment on HCC recurrence. Intriguingly, although CIC knockdown induced sorafenib resistance in HCC cell lines, regorafenib suppressed growth of sorafenib-resistant, Capicua-inactivated HCC cells and inhibited extracellular signal-regulated kinase phosphorylation. Conclusions Evaluation of Capicua status may be pivotal to predict response to sorafenib, and regorafenib treatment could be effective to treat HCC with functional Capicua impairment.

Hepatitis C virus (HCV) cell culture systems have facilitated the development of efficient direct-acting antivirals against HCV. Huh-7.5, a subline of the human hepatoma cell line Huh-7, has been used widely to amplify HCV because HCV can efficiently replicate in these cells due to a defect in innate antiviral signalling. Recently, we established a novel cell line, KH, derived from human hepatocellular carcinoma, which showed atypical uptake of gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) in a Gd-EOB-DTPA-enhanced magnetic resonance imaging study. KH cells expressed hepatocyte markers including microRNA-122 (miR-122) at a lower level than Huh-7.5 cells. We demonstrated that KH cells could support the entire life cycle of HCV; however, HCV replicated at a lower rate in KH cells compared to Huh-7.5 cells, and virus particles produced from KH cells seemed to have some disadvantages in viral assembly compared with those produced from Huh-7.5 cells. KH cells had more robust interferon-stimulated gene expression and induction upon HCV RNA transfection, interferon-α2b addition, and HCV infection than Huh-7.5 cells. Interestingly, both miR-122 supplementation and IRF3 knockout in KH cells boosted HCV replication to a similar level as in Huh-7.5 cells, suggesting that intact innate antiviral signalling and lower miR-122 expression limit HCV replication in KH cells. KH cells will enable a deeper understanding of the role of the innate immune response in persistent HCV infection.

Notch1 is regulated by E3 ubiquitin ligases, with proteasomal degradation of the Notch intracellular domain affecting the transcription of target genes. cAMP response element-binding protein (CREB) mediates the transcription of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA). We assessed the relationship between HBV cccDNA and Notch signaling activities. HBV cccDNA levels and relative gene expression were evaluated in HBV-replicating cells treated with Jagged1 shRNA and a γ-secretase inhibitor. The effects of these factors in surgically resected clinical samples were also assessed. Notch inhibition suppressed HBV cccDNA and CREB-related expression but increased ITCH and NUMB levels. Proteasome inhibitor augmented HBV cccDNA, restored Notch and CREB expression, and inhibited ITCH and NUMB function. Increased HBV cccDNA was observed after ITCH and NUMB blockage, even after treatment with the adenylate cyclase activator forskolin; protein kinase A (PKA) inhibitor had the opposite effect. Notch activation and E3 ligase inactivation were observed in HBV-positive cells in clinical liver tissue. Collectively, these findings reveal that Notch signaling activity facilitates HBV cccDNA transcription via CREB to trigger the downstream PKA-phospho-CREB cascade and is regulated by E3 ubiquitin ligase-modulation of the Notch intracellular domain.

Article, Scientific reports.9 :102(2019)

ObjectiveThe clinical significance of polymorphisms in the interleukin-28B gene encoding interferon (IFN)-λ3, which has antiviral effects, is known in chronic HCV but not in HBV infection. Thus, we measured IFN-λ3 levels in patients with HBV and investigated its clinical significance and association with nucleos(t)ide (NUC) analogue administration.DesignSerum IFN-λ3 level was measured in 254 patients with HBV with varying clinical conditions using our own high sensitivity method. The resulting values were compared with various clinical variables. In addition, cell lines originating from various organs were cultured with NUCs, and the production of IFN-λ3 was evaluated.ResultsHigher serum IFN-λ3 levels were detected in the patients treated with nucleotide analogues (adefovir or tenofovir) compared with those treated with nucleoside analogues (lamivudine or entecavir). There were no other differences in the clinical background between the two groups. A rise in the serum IFN-λ3 levels was observed during additional administration of the nucleotide analogues. In vitro experiments showed that the nucleotide analogues directly and dose-dependently induced IFN-λ3 production only in colon cancer cells. Furthermore, the supernatant from cultured adefovir-treated colon cancer cells significantly induced IFN-stimulated genes (ISGs) and inhibited hepatitis B surface antigen (HBsAg) production in hepatoma cells, as compared with the supernatant from entecavir-treated cells.ConclusionsWe discovered that the nucleotide analogues show an additional pharmacological effect by inducing IFN-λ3 production, which further induces ISGs and results in a reduction of HBsAg production. These findings provide novel insights for HBV treatment and suggest IFN-λ3 induction as a possible target.

金沢大学先進予防医学研究センター / 金沢大学附属病院, There is no established treatment for patients with advanced hepatocellular carcinoma (HCC) with Child–Pugh class B cirrhosis. The aim of the present study was to assess the efficacy of hepatic arterial infusion chemotherapy (HAIC) according to Child–Pugh score (CPS) and to evaluate the correlation of a patient's response to HAIC with hepatic reserve and outcome. We retrospectively reviewed the medical records of 377 patients treated with HAIC between March 2003 and February 2015. Subjects included 179 with Child–Pugh class B. Median overall survival was 12.1 months for patients with CPS = 7 (n = 75) and 11.9 months for patients with CPS = 8 (n = 58), which were significantly longer compared with those of patients with CPS = 9 (n = 46, 6.3 months). The objective response rates of patients with CPS = 7, 8 and 9 were 26.7%, 27.6% and 6.5%, respectively. The CPS of responders improved significantly after HAIC, whereas those of nonresponders did not. A multivariate analysis demonstrated that improved CPS, responses to HAIC and absence of extrahepatic lesions were independent favorable prognostic factors. Patients with CPS = 7 or 8 tolerated HAIC, but nine (19.6%) of patients with CPS = 9 were unable to complete one course. HAIC is effective and safe for patients with a CPS = 7 or 8 and improved hepatic reserve of responders significantly. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

金沢大学先進予防医学研究センター / 金沢大学医薬保健研究域医学系, Notch signaling abnormalities are reported to be involved in the acceleration of malignancy in solid tumors and stem cell formation or regeneration in various organs. We analyzed specific genes for DNA copy number variations in liver cancer cells and investigated whether these factors relate to clinical outcome. Chromosome 20p, which includes the ligand for Notch pathways, Jagged1, was found to be amplified in several types of hepatoma cells, and its mRNA was up-regulated according to α-fetoprotein gene expression levels. Notch inhibition using Jagged1 shRNA and γ-secretase inhibitors produced significant suppression of cell growth in α-fetoprotein–producing cells with suppression of downstream genes. Using invivo hepatoma models, the administration of γ-secretase inhibitors resulted in reduced tumor sizes and effective Notch inhibition with widespread apoptosis and necrosis of viable tumor cells. The γ-secretase inhibitors suppressed cell growth of the epithelial cell adhesion molecule–positive fraction in hepatoma cells, indicating that Notch inhibitors could suppress the stem cell features of liver cancer cells. Even in clinical liver cancer samples, the expression of α-fetoprotein and Jagged1 showed significant correlation, and amplification of the copy number of Jagged1 was associated with Jagged1 mRNA expression and poor survival after liver cancer surgical resection. In conclusion, amplification of Jagged1 contributed to mRNA expression that activates the Jagged1-Notch signaling pathway in liver cancer and led to poor outcome. © 2016 American Society for Investigative Pathology, Embargo Period 12 months

Dendritic cell (DC)–based immunotherapies are believed to help eradicate residual tumor cells, including hepatocellular carcinoma (HCC). Here, we assessed the safety and clinical response to OK432-stimulated monocyte-derived DCs (MoDCs) in treating HCC after radiofrequency ablation (RFA). MoDCs were derived from 30 HCC patients in the presence of interleukin-4 and granulocyte-macrophage colony stimulating factor for 5 days and then cultured for 2 more days in the medium (basic protocol) or stimulated with OK432. On day 7, DCs were harvested and percutaneously injected into HCC tumors after RFA. We observed no grade 3 or 4 National Cancer Institute Common Toxicity Criteria adverse events. Kaplan-Meier analysis indicated that patients treated with RFA + OK432-stimulated DCs transfer had longer recurrence-free survival than those treated with RFA + basic-protocol DCs (median: 24.8 vs 13.0 months; P = .003). RFA with DC infusion can enhance various tumor-associated antigen (TAA)–specific T-cell responses. Additionally, the 5-year RFS rate for patients with significantly increased TAA-specific T-cell responses was much higher than for other patients (50.0% vs. 7.7%; P = .030). Our study provides useful information for development of HCC immunotherapies (trial registration: UMIN000001701).

金沢大学疾患モデル総合研究センター, Background Pancreatic ductular adenocarcinoma (PDAC) is among the most dreadful of malignancies, in part due to the lack of efficacious chemotherapy. Immune checkpoint inhibitors, including anti-programmed cell death 1 (anti-PD-1) antibodies, are novel promising forms of systemic immunotherapy. In the current study, we assessed whether gemcitabine (GEM) combined with anti-PD-1 antibody treatment was efficacious as immunochemotherapy for advanced PDAC using a murine model of liver metastasis. Methods The murine model of PDAC liver metastasis was established by intrasplenically injecting the murine pancreatic cancer cell line PAN02 into immunocompetent C57BL/6J mice. The mice were treated with an anti-PD-1 antibody, GEM, or a combination of GEM plus anti-PD-1 antibody, and compared with no treatment (control); liver metastases, immune cell infiltration, gene expression, immune cell response phenotypes, and overall survival were investigated. Results In the metastatic tumor tissues of mice treated with GEM plus anti-PD-1 antibody, we observed the increased infiltration of Th1 lymphocytes and M1 macrophages. Gene expression profile analysis of peripheral blood cells obtained from mice treated with GEM plus anti-PD-1 antibody clearly highlighted T cell and innate immune signaling pathways. Survival of PDAC liver metastasis mice was significantly prolonged by the combination therapy (median survival, 66 days) when compared with that of GEM alone treatment (median survival, 56 days). Expanded lymphocytes, which were isolated from the splenocytes of PDAC liver metastasis mice treated with GEM plus anti-PD-1 antibody, had an increased number of M1 macrophages. Conclusion The combination of anti-PD-1 antibody immunotherapy with GEM was beneficial to treat a murine model of PDAC liver metastasis by enhancing the immune response mediated by Th1 lymphocytes and M1 macrophages and was associated with CD8+ T cells. ©

Background and aim The pathological features of non-alcoholic steatohepatitis (NASH) have not been determined, so fundamental treatment has not been established. Adipose-tissue-derived stromal/stem cells (ADSCs) are beneficial for repair/regenerative therapy of impaired organs because of their immuno-modulatory capability. In this study, we assessed how liver damage progresses during the early development phase of the murine NASH model and investigated whether ADSCs are preventatively efficacious against the fibrosis progression of NASH. Methods C57BL/6J mice were fed with atherogenic high fat or high-fat diet 60 developing into NASH or simple steatosis. Their hepatic inflammatory cells (HICs) were analyzed by cDNA microarray. NASH mice were treated with ADSCs injected into spleen when hepatic inflammation was initially observed, and liver samples were analyzed. The effect of ADSCs on the mice hepatic stellate cell (HSC) line stimulated by recombinant IL-17 and HICs from NASH mice was analyzed. Results The gene expression features of HICs implicated as humoral cytokine mediators of lymphoid cells during NASH development, compared with a simple steatosis model. One of the featured cytokines was IL-17. The development of hepatic fibrosis was alleviated when NASH mice were treated with ADSCs as well as treated with anti-IL-17 antibody, and the frequency of IL-17-secreting HICs decreased. NASH-HICs enhanced proliferation of HSCs, in which proliferation was sensitive to IL-17 stimulation. The stimulatory effect of NASH-HICs on the activation of HSCs was attenuated by co-culture with ADSCs. Conclusion ADSCs treatment prevented progression of NASH fibrosis by suppressing IL-17-mediated inflammation, which was associated with HSCs activation.

Nonalcoholic steatohepatitis (NASH) is associated with liver inflammation in patients with nonalcoholic fatty liver disease, and it can progress to liver fibrosis at an advanced stage, as well as hepatocellular carcinoma (HCC) and portal hypertension. Although liver fibrosis is accurately diagnosed via biopsy, noninvasive methods are preferable. Aldo-keto reductase family 1 member B10 (AKR1B10) is associated with HCC and is secreted into the blood by liver cells via a lysosome-mediated nonclassical pathway. Accordingly, we analyzed whether secretion of AKR1B10 protein is associated with advanced NASH.We performed histological staging in 85 Matteoni classification type III and IV NASH patients and evaluated the incidence of HCC, formation of gastroesophageal varices, and prognosis according to serum AKR1B10 and Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA(+)-M2BP)(M2BPGi) and by comparison with conventional markers of fibrosis. A positive correlation was found between the Brunt classification and serum AKR1B10 level. In Brunt stage 4 patients, AKR1B10 levels were higher than those of other liver fibrosis markers, with higher specificity. The cutoff values for AKR1B10 and WFA(+)-M2BP for stage 4 fibrosis were 1.03 and 3.11, respectively. The rates of stage 4 fibrosis, HCC incidence, and gastroesophageal varix formation were significantly different between the two groups subdivided according to these cutoff levels. Moreover, the patients in the higher value group had significantly worse prognosis after NASH diagnosis CONCLUSION: AKR1B10 is a useful serum biomarker for advanced liver fibrosis in NASH and, combined with serum WFA(+)-M2BP, can predict HCC development, gastroesophageal varix formation, and poor prognosis.

Simeprevir is a novel NS3/4A protease inhibitor (PI) of hepatitis C virus (HCV). The baseline polymorphism NS3-Q80K is frequently observed in genotype (GT) 1a HCV and often associated with treatment failure in simeprevir-containing regimens. We aimed to elucidate mechanisms of treatment failure due to NS3-Q80K. We included a Q80R mutation in our study and generated a series of Huh-7.5 cell lines, each of which harbored either wild-type GT 1a strain H77S.3 or the Q80K or Q80R variant. The cells were cultured with increasing concentrations of simeprevir, and NS3 domain sequences were determined. The mutations identified by sequence analyses were subsequently introduced into H77S.3. The sensitivity of each mutant to the NS3/4A PIs simeprevir, asunaprevir, grazoprevir, and paritaprevir was analyzed. We introduced the mutations into GT 1b strain N.2 and compared the sensitivity to simeprevir with that of GT 1a strain H77S.3. While simeprevir treatment selected mutations at residue D168, such as D168A/V in the wild-type virus, an additional mutation at residue R155, R155K, was selected in Q80K/R variants at simeprevir concentrations of

Significance Hepatocellular carcinoma (HCC) is a highly lethal cancer, partly because of its high rate of recurrence, which is caused by the presence of liver cancer stem cells (CSCs). Here, using a selective chemopreventive agent, acyclic retinoid (ACR), as a bioprobe, we identified MYCN, which is mostly recognized as an oncogene in neuroblastoma, as a therapeutic target of ACR for HCC through a selective deletion of MYCN+ liver CSCs. We also demonstrated that the expression of MYCN in HCC served as a prognostic biomarker and positively correlated with recurrence of de novo HCC after curative treatment. Our study highlighted MYCN as a biomarker and therapeutic target in drug discovery for screening chemopreventive agents against the recurrence of HCC., Hepatocellular carcinoma (HCC) is a highly lethal cancer that has a high rate of recurrence, in part because of cancer stem cell (CSC)-dependent field cancerization. Acyclic retinoid (ACR) is a synthetic vitamin A-like compound capable of preventing the recurrence of HCC. Here, we performed a genome-wide transcriptome screen and showed that ACR selectively suppressed the expression of MYCN, a member of the MYC family of basic helix–loop–helix–zipper transcription factors, in HCC cell cultures, animal models, and liver biopsies obtained from HCC patients. MYCN expression in human HCC was correlated positively with both CSC and Wnt/β-catenin signaling markers but negatively with mature hepatocyte markers. Functional analysis showed repressed cell-cycle progression, proliferation, and colony formation, activated caspase-8, and induced cell death in HCC cells following silencing of MYCN expression. High-content single-cell imaging analysis and flow cytometric analysis identified a MYCN+ CSC subpopulation in the heterogeneous HCC cell cultures and showed that these cells were selectively killed by ACR. Particularly, EpCAM+ cells isolated using a cell-sorting system showed increased MYCN expression and sensitivity to ACR compared with EpCAM− cells. In a long-term (>10 y) follow-up study of 102 patients with HCC, MYCN was expressed at higher levels in the HCC tumor region than in nontumor regions, and there was a positive correlation between MYCN expression and recurrence of de novo HCC but not metastatic HCC after curative treatment. In summary, these results suggest that MYCN serves as a prognostic biomarker and therapeutic target of ACR for liver CSCs in de novo HCC.

Background. Colorectal cancer (CRC), the most common malignancy worldwide, causes inflammation. We explored the inflammatory pathophysiology of CRC by assessing the peripheral blood parameters. Methods. The differences in gene expression profiles of whole blood cells and cell subpopulations between CRC patients and healthy controls were analyzed using DNA microarray. Serum cytokine/chemokine concentrations in CRC patients and healthy controls were measured via multiplex detection immunoassays. In addition, we explored correlations between the expression levels of certain genes of peripheral CD4+ cells and serum chemokine concentrations. Results. The gene expression profiles of peripheral CD4+ cells of CRC patients differed from those of healthy controls, but this was not true of CD8+ cells, CD14+ cells, CD15+ cells, or CD19+ cells. Serum IL-8 and eotaxin-1 levels were significantly elevated in CRC patients, and the levels substantially correlated with the expression levels of certain genes of CD4+ cells. Interestingly, the relationships between gene expression levels in peripheral CD4+ cells and serum IL-8 and eotaxin-1 levels resembled those of monocytes/macrophages, not T cells. Conclusions. Serum IL-8 and eotaxin-1 concentrations increased and were associated with changes in the gene expression of peripheral CD4+ cells in CRC patients.

The liver is a multifunctional organ with roles in metabolism, detoxification, and the immune response. Liver secretory selenoprotein P (SeP), a hepatokine, causes insulin resistance in the liver and peripheral muscle. We showed hepatitis C virus (HCV) infection increased serum SeP levels, which may explain the increased risk of type 2 diabetes in patients with HCV infection. Increased SeP mRNA (SEPP1 mRNA) in hepatocytes inhibited type 1 interferon response by means of undefined unique mechanisms. We revealed SEPP1 mRNA directly bound to retinoic acidinducible gene I (RIG-I), a virus RNA sensor, and inhibited RIG-I activity. The robust induction of interferon-stimulated genes (>1000-fold) and repression of HCV replication (>10-fold) were observed in hepatocytes in which SEPP1 mRNA was knocked down. Clinically, high SeP serum levels were significantly associated with treatment failure of direct actingantivirals for HCV. Thus, SeP regulates insulin resistance and innate immunity, possibly inducing immune tolerance in the liver.

金沢大学先進予防医学研究科, Background: Sorafenib is a multiple receptor tyrosine kinase inhibitor known to prolong overall survival in patients with advanced hepatocellular carcinoma (HCC). Predicting this drug's survival benefits is challenging because clinical responses are rarely measurable during treatment. In this study, we hypothesized that serum cytokines levels could predict the survival of advanced HCC patients, as sorafenib targets signaling pathways activated in the tumor stromal microenvironment and potentially affects serum cytokine profiles. Methods: Of 143 patients with advanced-stage HCC, 104 who were recruited between 2003 and 2007 received hepatic arterial infusion chemotherapy (HAIC) that mainly targets tumor epithelial cells at S-phase (cohort 1); additionally, 39 recruited between 2010 and 2012 received sorafenib, which primarily targets the stromal vascular endothelial cells. Serum samples were collected and aliquoted prior to the treatment. Serum EGF, bFGF, HGF, IFN-γ, IL-10, IL-12, IL-2, IL-4, IL-5, IL-6, IL-8, IP-10, MIG, PDGF-BB, SCF, SDF1, TGF-β, TGF-α, TNF-α, and VEGF-A were measured via enzyme-linked immunosorbent assays. The Modified Response Evaluation Criteria in Solid Tumors were used to assess tumor responses. Results: The median survival time of HCC patients in cohorts 1 (HAIC-treated) and 2 (sorafenib-treated) were 12.0 and 12.4 months, respectively. Kaplan-Meier analysis revealed no significant survival differences between the 2 groups. Patients who survived more than 2 years after sorafenib treatment exhibited higher serum levels of IL-10, IL-12, TNF-a, IL-8, SDF-1, EGF, PDGF-BB, SCF, and TGF-α. Furthermore, cohort 2 patients with higher serum IL-5 (>12 pg/mL), IL-8 (>10 pg/mL), PDGF-BB (>300 pg/mL), and VEGF-A (>50 pg/mL) levels achieved longer survival; cohort 1 patients did not. Hierarchical cluster analysis of 6 cytokines robustly enriched for comparison analysis between cohorts 1 and 2 (IL-5, IL-8, TGF-α, PDGF-BB, CXCL9, and VEGF-A) revealed that elevation of these cytokines correlated with better survival when treated with sorafenib but not with HAIC. Conclusions: Patients who exhibited survival benefits owing to sorafenib treatment tended to present higher serum cytokines levels, potentially reflecting the activation of stromal signaling in the tumor microenvironment. Our study thus introduces novel biomarkers that may identify advanced HCC patients who may experience survival benefits with sorafenib treatment. © 2017 The Author(s).

BACKGROUND Although nucleos(t)ide analog (NA) therapy effectively reduces the hepatitis B virus (HBV) DNA load in the serum of patients with chronic hepatitis B, it does not completely reduce the incidence of hepatocellular carcinoma (HCC). METHODS AND RESULTS A total of 109 patients who had chronic hepatitis B and were receiving NA therapy were analyzed. Multivariate Cox regression analysis showed that age (>60 years had a hazard ratio [HR] of 2.66), FIB-4 index (an index of >2.1 had a HR of 2.57), and the presence of HBV core-related antigen (HBcrAg; HR, 3.53) during treatment were significantly associated with the development of HCC. The amount of HBV DNA and pregenomic RNA in liver were significantly higher in 16 HBcrAg-positive patients, compared with 12 HBcrAg-negative patients, suggesting active HBV replication in HBcrAg-positive livers. Hepatic gene expression profiling showed that HBV-promoting transcriptional factors, including HNF4α, PPARα, and LRH1, were upregulated in HBcrAg-positive livers. HepAD38 cells overexpressing LRH1 increased HBV replication, characterized by higher HBV DNA and pregenomic RNA levels, during long-term exposure to entecavir. Conversely, overexpression of precore/core in HepG2 cells increased levels of these transcriptional factors. Metformin efficiently repressed HBV replication in primary human hepatocytes. CONCLUSIONS Modulating HBV transcriptional factors by metformin in combination with NA therapy would potentiate anti-HBV activity and reduce the incidence of HCC in HBcrAg-positive patients.

Aim Although sorafenib is a standard drug for advanced hepatocellular carcinoma (HCC), little is known about a patient's clinical course after treatment. We investigated the effect of post-progression survival (PPS) and progression-free survival (PFS) on overall survival (OS) in patients whose advanced HCC was treated by sorafenib. Methods We searched in the PubMed database for reports with survival data of patients with HCC treated with sorafenib monotherapy, and selected reports with 20 or more patients each that provided data for both OS and PFS or time to progression (TTP). Median PPS (mPPS) was defined as the period obtained by subtracting median PFS or TTP (mPFS/TTP) from median OS (mOS). We identified 56 reports with 5803 patients. We investigated the correlation of mOS and either mPPS or mPFS/TTP using weighted linear regression. Results Median PPS correlated with mOS (r = 0.834) very strongly, whereas mPFS/TTP did not correlate with mOS as highly as PPS did (r = 0.546). When we stratified survival data by Child-Pugh classification, a significantly greater average percentage of mPPS to mOS was seen in Child-Pugh class A (54.4 ± 17.6%) than in Child-Pugh class B (32.0 ± 11.6%) (P = 0.015). Conclusion PPS highly correlated with OS, and its importance should be more emphasized for advanced HCC patients treated after sorafenib therapy, whereas we need to take more care in interpreting the results of PFS to evaluate treatment efficacy in clinical trials of advanced HCC.

Background & Aims Hepatocellular carcinoma is composed of a subset of cells with enhanced tumorigenicity and chemoresistance that are called cancer stem (or stem-like) cells. We explored the role of chromodomain-helicase-DNA-binding protein 4, which is encoded by the CHD4 gene and is known to epigenetically control gene regulation and DNA damage responses in EpCAM + liver cancer stem cells. Methods Gene and protein expression profiles were determined by microarray and immunohistochemistry in 245 and 144 hepatocellular carcinoma patients, respectively. The relationship between gene/protein expression and prognosis was examined. The functional role of CHD4 was evaluated in primary hepatocellular carcinoma cells and in cell lines in vitro and in vivo . Results CHD4 was abundantly expressed in EpCAM + hepatocellular carcinoma with expression of hepatic stem cell markers and poor prognosis in two independent cohorts. In cell lines, CHD4 knockdown increased chemosensitivity and CHD4 overexpression induced epirubicin chemoresistance. To inhibit the functions of CHD4 that are mediated through histone deacetylase and poly (ADP-ribose) polymerase, we evaluated the effect of the histone deacetylase inhibitor suberohydroxamic acid and the poly (ADP-ribose) polymerase inhibitor AG-014699. Treatment with either suberohydroxamic acid or AG-014699 reduced the number of EpCAM + liver cancer stem cells in vitro , and suberohydroxamic acid and AG-014699 in combination successfully inhibited tumor growth in a mouse xenograft model. Conclusions CHD4 plays a pivotal role in chemoresistance and the maintenance of stemness in liver cancer stem cells and is therefore a good target for the eradication of hepatocellular carcinoma.