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Legalon SIL ( SIL) is a chemically hydrophilized version of silibinin, an extract of milk thistle ( Silybum marianum) seeds that has exhibited hepatoprotective and antiviral effectiveness against hepatitis C virus ( HCV) in patients leading to viral clearance in combination with ribavirin. To elucidate the incompletely understood mode of action of SIL against HCV, mathematical modelling of HCV kinetics and human hepatocyte gene expression studies were performed in uPA- SCID-chimeric mice with humanized livers. Chronically HCV-infected mice ( n = 15) were treated for 14 days with daily intravenous SIL at 469, 265 or 61.5 mg/kg. Serum HCV and human albumin ( hAlb) were measured frequently, and liver HCV RNA was analysed at days 3 and 14. Microarray analysis of human hepatocyte gene expression was performed at days 0, 3 and 14 of treatment. While hAlb remained constant, a biphasic viral decline in serum was observed consisting of a rapid 1st phase followed by a second slower phase (or plateau with the two lower SIL dosings). SIL effectiveness in blocking viral production was similar among dosing groups (median ε = 77%). However, the rate of HCV-infected hepatocyte decline, δ, was dose-dependent. Intracellular HCV RNA levels correlated ( r = 0.66, P = 0.01) with serum HCV RNA. Pathway analysis revealed increased anti-inflammatory and antiproliferative gene expression in human hepatocytes in SIL-treated mice. The results suggest that SIL could lead to a continuous second-phase viral decline, that is potentially viral clearance, in the absence of adaptive immune response along with increased anti-inflammatory and antiproliferative gene expression in human hepatocytes. [ABSTRACT FROM AUTHOR]

Pathogen-specific mi RNA profiles might reveal potential new avenues for therapy. To identify mi RNAs directly associated with hepatitis B virus ( HBV) in hepatocytes, we performed a mi RNA array analysis using urokinase-type plasminogen activator (u PA)-severe combined immunodeficiency ( SCID) mice where the livers were highly repopulated with human hepatocytes and human immune cells are absent. Mice were inoculated with HBV-infected patient serum samples. Eight weeks after HBV infection, human hepatocytes were collected from liver tissues, and mi RNAs were analysed using the Toray 3 D array system. The effect of mi RNAs on HBV replication was analysed using HBV-transfected Hep G2 cells. Four mi RNAs, hsa-mi R-486-3p, hsa-mi R-1908, hsa-mi R-675 and hsa-mi R-1231 were upregulated in mouse and human livers with HBV infection. These mi RNAs were associated with immune response pathways such as inflammation mediated by chemokine and cytokine signalling. Of these mi RNAs, hsa-mi R-1231, which showed high homology with HBV core and HBx sequences, was most highly upregulated. In HBV-transfected Hep G2 cells, overexpression of hsa-mi R-1231 resulted in suppression of HBV replication with HBV core reduction. In conclusion, a novel interaction between hsa-mi R-1231 and HBV replication was identified. This interaction might be useful in developing new therapeutic strategies against HBV. [ABSTRACT FROM AUTHOR]

Complementary DNA microarray analysis of human livers cannot exclude the influence of the immunological response. In this study, complementary DNA microarray analysis was performed under immunodeficient conditions with human hepatocyte chimeric mice, and gene expression profiles were analyzed by hepatitis B virus (HBV) infection and/or interferon treatment. The expression levels of 183 of 525 genes upregulated by interferon treatment were significantly suppressed in response to HBV infection. Suppressed genes were statistically significantly associated with the interferon signaling pathway and pattern recognition receptors in the bacteria/virus recognition pathway (P = 1.0 x 10(-8) and P = 1.2 x 10(-8), respectively). HBV infection attenuated virus recognition and interferon response in hepatocytes, which facilitated HBV escape from innate immunity. [ABSTRACT FROM AUTHOR]

The cover image, by Kazuaki Chayama et al., is based on the Original Article Hepatitis C virus dynamics and cellular gene expression in uPA‐SCID chimeric mice with humanized livers during intravenous silibinin monotherapy, DOI: 10.1111/jvh.12551. [ABSTRACT FROM AUTHOR]

Recent studies indicate that treatment of chronic hepatitis D virus (HDV) with either pegylated interferon (IFN)λ or pegylated IFNα monotherapy leads to a dramatic decline in HDV RNA. Herein, we investigated the innate antiviral efficacy of IFNλ and IFNα in humanized mice that lack an adaptive immune response. Humanized mice were either co-infected with hepatitis B virus (HBV) and HDV simultaneously, or HDV infection was performed subsequent to HBV infection (i.e., superinfected). After steady viral replication was achieved, mice received either IFNλ (n = 6) or IFNα (n = 7) for 12 (or 13) weeks. Pretreatment median levels of serum HBV DNA (8.8 [IQR:0.2] log IU/ml), HDV RNA (9.8 [0.5] log IU/ml), HBsAg (4.0 [0.4] log IU/ml) and human albumin, hAlb (6.9 [0.1] log ng/mL) were similar between mice treated with IFNα or IFNλ and between those coinfected versus superinfected. Compared to mice treated with IFNλ, mice treated with IFNα had a significantly greater decline in HBV, HDV, and HBsAg levels. In conclusion, IFNα induces stronger inhibition of HBV and HDV than IFNλ in humanized mice that lack an adaptive immune response. Further studies are needed to assess the respective role of the combined innate-and adaptive-immune systems in the treatment of HBV and HDV with IFNα and IFNλ., Competing Interests: Declaration of competing interest Kazuaki Chayama has received honoraria from Bristol-Myers Squibb and MSD K.K., AbbVie, Gilead Science, Dainippon Sumitomo Pharma and Mitsubishi Tanabe Pharma and research funding from Gilead Science, Dainippon Sumitomo Pharma, MSD K.K., AbbVie, Eisai, TORAY, Otsuka Pharma, Chugai Pharma, Takeda Pharma and Roche. Jeffrey Glenn, director, and equity holder of Eiger Biopharmaceuticals. Ohad Etzion has received consulting fees from Eiger Biopharmaceuticals. The other authors declare no conflicts of interest that pertain to this work., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background/aim: Renal dysfunction necessitates S-1 dose reduction. However, decreased dihydropyrimidine dehydrogenase (DPD) activity may lead to adverse events due to 5-FU. The guidelines provided by pharmaceutical companies state that total bilirubin (T-Bil) should be ≤upper limit of normal (ULN)×1.5 as a reference value for safely taking S-1. Nevertheless, the relationship between the degree of liver dysfunction and S-1 dose reduction has not been clearly established., Patients and Methods: This study focused on patients who received S-1 monotherapy for various types of cancer. The primary outcome was defined as the variation between blood sampling results on the test day and the subsequent test. The variation data were categorized based on the difference in T-Bil: Low T-Bil group (≤2.25) and High T-Bil group (>2.25)., Results: The number of patients that underwent S-1 monotherapy was 883 and the running number was 7,511; Low T-Bil group included 7,245 and High T-Bil group included 266. Examination of the effect of the T-Bil Group on clinical outcomes revealed a correlation with red blood cell (RBC) count, platelet (PLT) count, and T-Bil level. When the impact of the interaction between the T-Bil Group and any of the clinical outcomes, such as the RBC count, PLT count, and T-Bil level, was determined, each outcome showed a significant decrease in the High T-Bil group compared with the Low T-Bil group., Conclusion: S-1 administration in patients with liver dysfunction accompanied by elevated T-Bil levels may cause thrombocytopenia., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Surgery avoidance is an important goal in Crohn's disease (CD) treatment and predicting the risk of subsequent surgery is important to determine adequate therapeutic strength for patients with newly diagnosed CD. Herein, we aimed to construct a prediction model for the risk of subsequent surgery based on disease characteristics at the patients' initial visit. We retrospectively collected disease characteristic data from 93 patients with newly diagnosed CD. A logistic regression model with a brute force method was used to maximize the area under the receiver operating characteristic curve (auROC) by employing a combination of potential predictors from 14 covariates (16,383). The auROC remained almost constant when one to 12 covariates were considered, reaching a peak of 0.89 at four covariates (small-bowel patency, extensive small-bowel lesions, main lesions, and the number of poor prognostic factors), and it decreased with increasing covariate size. The most significant predictors were small-bowel patency, extensive small-bowel lesions, and age or major lesions. Therefore, this prediction model using covariates may be helpful in determining the likelihood that a patient with newly diagnosed CD will require surgery, which can aid in appropriate treatment selection for high-risk patients.

Hepatitis B virus (HBV) infection kinetics in immunodeficient mice reconstituted with humanized livers from inoculation to steady state is highly dynamic despite the absence of an adaptive immune response. To recapitulate the multiphasic viral kinetic patterns, we developed an agent-based model that includes intracellular virion production cycles reflecting the cyclic nature of each individual virus lifecycle. The model fits the data well predicting an increase in production cycles initially starting with a long production cycle of 1 virion per 20 hours that gradually reaches 1 virion per hour after approximately 3-4 days before virion production increases dramatically to reach to a steady state rate of 4 virions per hour per cell. Together, modeling suggests that it is the cyclic nature of the virus lifecycle combined with an initial slow but increasing rate of HBV production from each cell that plays a role in generating the observed multiphasic HBV kinetic patterns in humanized mice., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: YI, HI and CT are Phoenix Bio employees., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Purpose: To investigate parental readiness for their child's transition to adulthood among pediatric patients with chronic disease in Japan., Design and Methods: In this cross-sectional study, parents of children with chronic diseases attending a pediatric outpatient hospital completed an anonymous self-administered questionnaire to investigate demographics and parental readiness for children's transition to adulthood. Logistic regression analysis was conducted to determine whether parents' readiness differed according to their children's disease types., Results: A total of 179 parents responded to the survey. Of these, 60% confirmed awareness and knowledge of their children's disease and treatment with their children. They also thought and accepted that there would be a time when their children would move from pediatrics to an adult department. More than half of parents had discussions with their children about the children's interests and would discuss with each other if their children's choice differed from the parents' opinion. <20% of parents had gathered information about the transition and made sure their children saw the outpatient clinic alone or encouraged them to write a record of their visits. The readiness of parents of children with diabetes and of children with cardiovascular disease was higher than those of children with cancer and blood diseases on several items., Conclusions: Parental readiness to support their children's transitions did not progress well, suggesting the need for relevant interventions., Practice Implications: Children and parents should be supported in acquiring information about the transition to adulthood and in implementing child-oriented outpatient visits., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Recently, the importance of achieving clinical and deep remissions with mucosal healing (MH) has been demonstrated as a therapeutic goal to avoid Crohn's disease (CD) surgical operations. Although ileocolonoscopy (CS) is considered the gold standard, there are increasing reports on the benefits of capsule endoscopy (CE) and serum leucine-rich α2-glycoprotein (LRG) for evaluating small-bowel lesions in CD. We evaluated the data of 20 patients with CD who underwent CE in our department between July 2020 and June 2021 and whose serum LRG level was measured within 2 months. Concerning the mean LRG value, there was no significant difference between the CS-MH and CS-non-MH groups. Conversely, the mean LRG level was 10.0 μg/mL in seven patients in the CE-MH group and 15.2 μg/mL in 11 patients in the CE-non-MH group with a significant difference between the two groups ( p = 0.0025). This study's findings show that CE can sufficiently determine total MH in most cases, and LRG is useful for evaluating CD small-bowel MH because of its correlation with CE-MH. Furthermore, satisfying CS-MH criteria and a cut-off value of 13.4 μg/mL for LRG suggests its usefulness as a CD small-bowel MH marker, which could be incorporated into the treat-to-target strategy.

Capsule endoscopy has been widely used as a non-invasive diagnostic tool for small or large intestinal lesions. In recent years, automated lesion detection systems using machine learning have been devised. This study aimed to develop an automated system for capsule endoscopic severity in patients with ulcerative colitis along the entire length of the colon using ResNet50. Capsule endoscopy videos from patients with ulcerative colitis were collected prospectively. Each single examination video file was partitioned into four segments: the cecum and ascending colon, transverse colon, descending and sigmoid colon, and rectum. Fifty still pictures (576 × 576 pixels) were extracted from each partitioned video. A patch (128 × 128 pixels) was trimmed from the still picture at every 32-pixel-strides. A total of 739,021 patch images were manually classified into six categories: 0) Mayo endoscopic subscore (MES) 0, 1) MES1, 2) MES2, 3) MES3, 4) inadequate quality for evaluation, and 5) ileal mucosa. ResNet50, a deep learning framework, was trained using 483,644 datasets and validated using 255,377 independent datasets. In total, 31 capsule endoscopy videos from 22 patients were collected. The accuracy rates of the training and validation datasets were 0.992 and 0.973, respectively. An automated evaluation system for the capsule endoscopic severity of ulcerative colitis was developed. This could be a useful tool for assessing topographic disease activity, thus decreasing the burden of image interpretation on endoscopists., Competing Interests: The authors have declared that no competing interests exist.

A 45-year-old Japanese male patient who was diagnosed with celiac disease (CeD) developed type I enteropathy-associated T-cell lymphoma (EATL). In 2013, the patient was admitted to our hospital with worsening of diarrhea and weight loss. Pathological examination of biopsy specimens from the duodenum and ileum led to a diagnosis of suspected EATL. A previous total colonoscopy (TCS) indicated villous atrophy in the terminal ileum. The patient was changed to a gluten-free diet, and the nutritional status gradually improved. In September 2014, he experienced acute right lower abdominal pain. He underwent urgent surgery, and a perforation was identified in the ileum. A diagnosis of type I EATL was made following histopathological examination. After eight courses of CHOP therapy, the patient entered complete remission. TCS and esophagogastroduodenoscopy with magnifying narrow-band imaging performed in 2015 identified villous regrowth in the distal ileum and duodenum. Capsule endoscopy also found villous regrowth in the entire small intestine. To our knowledge, this is the first case of type I EATL following CeD with villous atrophy before EATL occurrence in a Japanese HLA-DQ2 carrier. The possibility of type I EATL occurring after CeD should be recognized, although CeD is quite rare in Japan.

Background and Aims: The clinical course and responsiveness to antiviral treatments differs among hepatitis B virus (HBV) genotypes. However, the cause of these differences is unclear. In the present study, we compared mRNA expression profiles in human hepatocyte chimeric mice infected with HBV genotypes A and C., Methods: Fifteen chimeric mice were prepared and divided into the following three groups: uninfected control mice, HBV genotype A-infected mice, and HBV genotype C-infected mice. Human hepatocytes were collected from these mouse livers and gene expression analyses were performed using next-generation RNA sequencing., Results: Although similar pathways were influenced by HBV infection, including inflammation mediated by chemokine and cytokine signaling, p53, and integrin signaling pathways, expression levels of up-regulated genes by HBV genotype A or C infection were quite different. In HBV genotype A-infected hepatocytes, 172 genes, including KRT23 and C10orf54, were significantly more highly expressed than in HBV genotype C-infected cells, whereas 10 genes, including SPX and IER3, were expressed at significantly lower levels. Genes associated with the p53 pathway and the inflammation mediated by chemokine and cytokine signaling pathway were more highly expressed in cells with HBV genotype A infection, whereas genes associated with CCKR signaling map and oxidative stress response were more highly expressed in cells with HBV genotype C infection., Conclusion: Several differences in gene expression with respect to HBV genotype A and C infection were detected in human hepatocytes. These differences might be associated with genotypic difference in the clinical course or responsiveness to treatment.

The activation of hepatitis B virus (HBV)-related hepatitis is associated with both natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). We analyzed the association between the immune response and changes in the proportion of Pre-S deletion variants. We quantified Pre-S deleted HBV (HBV-del) and wild-type HBV (HBV-wt) DNA levels in sera obtained from HBV-infected mice and chronic hepatitis B patients. In chronic hepatitis B patients, the HBV-del proportion usually increased during or after ALT elevation but did not occur during all ALT elevations. To clarify this difference in the immunological responses, we performed in vivo analyses using HBV-infected human hepatocyte chimeric mice. Although HBV-del proportions did not change in mice with NK cell-associated hepatitis or in mice treated with entecavir, the proportions sharply increased in mice with CTL-associated hepatitis. Furthermore, the number of patients in which HBV-del proportions were greater than 5% was significantly higher in chronic hepatitis B patients than in asymptomatic carriers (P = 0.023). We identified associations between virological response in chronic hepatitis B patients and two different immune responses. The proportion of HBV-del variants could be a useful biomarker for distinguishing between chronic hepatitis and asymptomatic carriers., (© 2018 John Wiley & Sons Ltd.)

The events in the immune response to hepatitis B virus (HBV) remain unclear. We analyzed the direct influence of HBV on gene expression in human hepatocytes under immunodeficient conditions using a human hepatocyte chimeric mouse model. HBV-infected or non-infected chimeric mouse livers were collected, and gene expression profiles were compared. Since IL-8 was the most significantly up-regulated gene at 8 weeks after HBV infection, we focused on IL-8 and found that HBx and the large HBs (L-HBs) protein induce transcription of IL-8 via endoplasmic reticulum stress. This stress induces IL-8 transcription via NFAT activation and contributes to suppression of interferon responsiveness in HBV-infected human hepatocytes. In the present study, we identified a novel regulatory mechanism in which the L-HBs protein activates IL-8 via endoplasmic reticulum stress, suggesting a key role for IL-8 in the immune response to HBV and a potential new target for antiviral treatments of HBV infection., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Combined daclatasvir (DCV)/asunaprevir (ASV) plus beclabuvir (BCV) treatment shows a high virological response for genotype 1b chronic hepatitis C patients. However, its efficacy for patients for whom previous direct-acting antiviral (DAA) therapy failed is not known. We analysed the efficacy of DCV/ASV/BCV treatment for HCV-infected mice and chronic hepatitis patients. Human hepatocyte chimaeric mice were injected with serum samples obtained from either a DAA-naïve patient or a DCV/ASV treatment failure and were then treated with DCV/ASV alone or in combination with BCV for 4 weeks. DCV/ASV treatment successfully eliminated the virus in DAA-naïve-patient HCV-infected mice. DCV/ASV treatment failure HCV-infected mice developed viral breakthrough during DCV/ASV treatment, with the emergence of NS5A-L31V/Y93H HCV resistance-associated variants (RAVs) being observed by direct sequencing. DCV/ASV/BCV treatment inhibited viral breakthrough in NS5A-L31V/Y93H-mutated HCV-infected mice, but HCV relapsed with the emergence of NS5B-P495S variants after the cessation of the treatment. The efficacy of the triple therapy was also analysed in HCV-infected patients; one DAA-naïve patient and four prior DAA treatment failures were treated with 12 weeks of DCV/ASV/BCV therapy. Sustained virological response was achieved in a DAA-naïve patient and one of the DCV/ASV treatment failures through DCV/ASV/BCV therapy; however, HCV relapse occurred in the other patients with prior DCV/ASV and/or sofosbuvir/ledipasvir treatment failures. DCV/ASV/BCV therapy seems to have limited efficacy for patients with NS5A RAVs for whom prior DAA treatment has failed.

Chimeric urokinase type plasminogen activator (uPA)/severely severe combined immunodeficiency (SCID) mice reconstituted with humanized livers are useful for studying hepatitis B virus (HBV) infection in the absence of an adaptive immune response. However, the detailed characterization of HBV infection kinetics necessary to enable in-depth mechanistic studies in this in vivo HBV infection model is lacking. To characterize HBV kinetics post-inoculation (p.i.) to steady state, 42 mice were inoculated with HBV. Serum HBV DNA was frequently measured from 1 minute to 63 days p.i. Total intrahepatic HBV DNA, HBV covalently closed circular DNA (cccDNA), and HBV RNA was measured in a subset of mice at 2, 4, 6, 10, and 13 weeks p.i. HBV half-life (t 1/2 ) was estimated using a linear mixed-effects model. During the first 6 hours p.i., serum HBV declined in repopulated uPA/SCID mice with a t 1/2 = 62 minutes (95% confidence interval [CI] = 59-67). Thereafter, viral decline slowed followed by a 2-day lower plateau. Subsequent viral amplification was multiphasic with an initial mean doubling time of t 2 = 8 ± 3 hours followed by an interim plateau before prolonged amplification (t 2 = 2 ± 0.5 days) to a final HBV steady state of 9.3 ± 0.3 log copies (cps)/mL. Serum HBV and intrahepatic HBV DNA were positively correlated (R 2 = 0.98)., Conclusion: HBV infection in uPA/SCID chimeric mice is highly dynamic despite the absence of an adaptive immune response. Serum HBV t 1/2 in humanized uPA/SCID mice was estimated to be ∼1 hour regardless of inoculum size. The HBV acute infection kinetics presented here is an important step in characterizing this experimental model system so that it can be effectively used to elucidate the dynamics of the HBV life cycle and thus possibly reveal effective antiviral drug targets. (Hepatology 2018)., (© 2018 by the American Association for the Study of Liver Diseases.)

Background and Aims: The risk factors in the progression of nonalcoholic fatty liver disease (NAFLD) have not been fully clarified. Porphyromonas gingivalis (P.g) has been considered to be a confounding risk factor for systemic diseases. We aimed to evaluate the effect of P.g infection on risk of progression to NASH., Methods: (1) Serum IgG antibody titers against P.g fimbriae (fimA) in 200 biopsy-proven NAFLD patients were measured by ELISA and compared with histological findings. (2) C57BL/6J mice were fed a control diet (CD) or high-fat diet (HFD) with or without P.g-odontogenic infection and analyzed histologically. Mouse livers were analyzed using CE-TOFMS and LC-TOFMS., Results: (1) A significant correlation between fibrosis progression and antibody titers against P.g possessing fimA type 4 was identified (P = 0.0081). Multivariate analysis identified older age and type 4 P.g-positivity as risk factors for advanced fibrosis. (2) Fibrosis and steatosis were more severe in HFD P.g(+) mice compared with HFD P.g(-) mice. In metabolome analysis, fatty acid metabolism was significantly disrupted with HFD in P.g-infected mouse livers. Monounsaturated/saturated fatty acid ratios were significantly higher in the HFD P.g(+) group than in the HFD P.g(-) group (P < 0.05). Moreover, expression levels of SCD1 and ELOVL6 were significantly reduced., Conclusions: These results suggest that P.g infection is an important risk factor for pathological progression in NAFLD. Increase in the monounsaturated/saturated fatty acid ratio may be an important change that facilitates progression of NAFLD.

We report the preparation of new C 3 - and C S -symmetrical molecules constructed on a triazine (TAZ) template. Anti-herpes simplex virus type 1 (anti-HSV-1) and cytotoxic activities against Vero cells of synthesized TAZ derivatives were evaluated. The results suggested that the presence of an electron-donating group(s) on the benzene ring in benzylamine groups on the TAZ template is an important structural factor for expressing a high level of anti-HSV-1 activity and low cytotoxicity for these C 3 types of TAZ derivatives. Among the tested TAZ derivatives, compounds 4f and 7h showed the highest anti HSV-1 activities (EC 50 =0.98 and 1.23 µM, respectively) and low cytotoxic activities to Vero cells (50% cytotoxic concentration (CC 50 )=292.2 and >200 µM, respectively).

Nucleot(s)ide analogues and peginterferon (PEG-IFN) treatment are the only approved therapies for chronic hepatitis B virus (HBV) infection. However, complete eradication of the virus, as indicated by persistent loss of hepatitis B surface antigen (HBsAg), is rare among treated patients. This is due to long-term persistence of the HBV genome in infected hepatocytes in the form of covalently closed circular DNA (cccDNA). In this study, we investigated whether administration of a large dose of a nucleoside analogue in combination with PEG-IFN can achieve long-term loss of HBsAg in human hepatocyte chimeric mice. Mice were treated with a high dose of entecavir and/or PEG-IFN for 6 weeks. High-dose combination therapy with both drugs resulted in persistently negative HBV DNA in serum. Although small amounts of HBV DNA and cccDNA (0.1 and 0.01 copy/cell, respectively) remained in the mouse livers, some of the mice remained persistently negative for serum HBV DNA at 13 weeks after cessation of the therapy. Serum HBsAg and hepatitis B core-related antigen (HBcrAg) continued to decrease and eventually became negative at 12 weeks after cessation of the therapy. Analysis of the HBV genome in treated mice showed accumulation of G-to-A hypermutation and CpG III island methylation. Persistent loss of serum HBV DNA and loss of HBV markers by high-dose entecavir and PEG-IFN combination treatment in chimeric mice suggests that control of HBV can be achieved even in the absence of a cellular immune response., (Copyright © 2017 American Society for Microbiology.)

Although nucleot(s)ide analogues and pegylated interferon alpha 2a (PEG-IFN-α2a) can suppress hepatitis B virus (HBV) replication, it is difficult to achieve complete HBV elimination from hepatocytes. A novel site-specific pegylated recombinant human IFN-β (TRK-560) was recently developed. In the present study, we evaluated the antiviral effects of TRK-560 on HBV replication in vitro and in vivo. In vitro and in vivo HBV replication models were treated with antivirals including TRK-560, and changes in HBV markers were evaluated. To analyze antiviral mechanisms, cDNA microarray analysis and an enzyme-linked immunoassay (ELISA) were performed. TRK-560 significantly suppressed the production of intracellular HBV replication intermediates and extracellular HBV surface antigen (HBsAg) ( P < 0.001 and P < 0.001, respectively), and the antiviral effects of TRK-560 were enhanced in combination with nucleot(s)ide analogues, such as entecavir and tenofovir disoproxil fumarate. The reduction in HBV DNA levels by TRK-560 treatment was significantly higher than that by PEG-IFN-α2a treatment both in vitro and in vivo ( P = 0.004 and P = 0.046, respectively), and intracellular HBV covalently closed circular DNA (cccDNA) reduction by TRK-560 treatment was also significantly higher than that by PEG-IFN-α2a treatment in vivo ( P = 0.0495). cDNA microarrays and ELISA for CXCL10 production revealed significant differences between TRK-560 and PEG-IFN-α2a in the induction potency of interferon-stimulated genes. TRK-560 shows a stronger antiviral potency via higher induction of interferon-stimulated genes and stronger stimulation of immune cell chemotaxis than PEG-IFN-α2a. As HBsAg loss and HBV cccDNA eradication are important clinical goals, these results suggest a potential role for TRK-560 in the development of more effective treatment for chronic hepatitis B infection., (Copyright © 2017 American Society for Microbiology.)

Urokinase-type plasminogen activator/severe combined immunodeficiency (uPA/SCID) mice transplanted with human hepatocytes are permissive for hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. However, one of the problems affecting uPA transgenic mice is the expansion of mouse hepatocyte colonies due to homologous recombination of the uPA gene. In this study, we attempted to infect HBV and HCV in humanized cDNA-uPA/SCID mice, a novel uPA transgenic mouse model designed to overcome this disadvantage. Three hundred and eighty-six uPA/SCID and 493 cDNA-uPA/SCID mice were transplanted with human hepatocytes and then injected with either HBV- or HCV-positive human serum samples or HBV-transfected cell culture medium. Twelve weeks after human hepatocyte transplantation, the mouse serum concentration of human albumin, which is correlated with the degree of repopulation by human hepatocytes, was significantly higher in cDNA-uPA/SCID mice compared with uPA/SCID mice. HBV-infected cDNA-uPA/SCID mice showed significantly greater and more persistent viraemia, and similar virological effects by entecavir treatment were achieved in both systems. HCV-infected cDNA-uPA/SCID mice developed more frequent and significantly higher viraemia compared with uPA/SCID mice. The present study using a large number of mice showed that cDNA-uPA/SCID mice transplanted with human hepatocytes developed high and long-term persistent viraemia following HBV and HCV infection, and a higher survival rate was observed in cDNA-uPA/SCID compared with uPA/SCID mice. These mice may be a useful animal model for the study of HBV and HCV virology and the analysis of the effect of antiviral drugs.

Interferon (IFN) inhibits hepatitis C virus (HCV) replication through up-regulation of intrahepatic IFN-stimulated gene expression but also through activation of host immune cells. In the present study, we analyzed the immune cell-mediated antiviral effects of IFN-α using HCV-infected mice. Urokinase-type plasminogen activator (uPA)-severe combined immunodeficiency (SCID) mice with transplanted human hepatocytes were infected with genotype 1b HCV and injected with human peripheral blood mononuclear cells (PBMCs). IFN-α treatment following human PBMC transplantation resulted in a significant reduction in serum HCV RNA titers and a higher human CD45-positive mononuclear cell chimerism compared to mice without human PBMC transplantation. In mice with human PBMCs treated with IFN-α, serum concentrations of IFN-γ increased, and natural killer T (NKT) cells, especially type I NKT cells, produced IFN-γ. Mice in which IFN-γ signaling was blocked using antibody or in which transplanted PBMCs were depleted for type I NKT cells showed similar levels of anti-HCV effect compared with mice treated only with IFN-α. These results show that IFN-α stimulates IFN-γ expression in type 1 NKT cells and enhances the inhibition of HCV replication. We propose that type 1 NKT cells might represent a new therapeutic target for chronic hepatitis C patients.

Background:  Although treatment-emergent NS3/4A protease inhibitor (PI)-resistant variants typically decrease in frequency after cessation of PI therapy in patients with chronic hepatitis C virus (HCV) infection, HCV susceptibility to PIs in patients who have not responded to previous PI therapy has not been addressed., Methods:  Patients with chronic HCV genotype 1 infection were treated either with simeprevir plus interferon or with daclatasvir plus asunaprevir. Frequencies of drug-resistant mutations among patients with treatment failure were analyzed by deep sequencing. Human hepatocyte chimeric mice were injected with serum samples obtained from either treatment-naive patients or nonresponders to treatment with daclatasvir plus asunaprevir and then were treated with simeprevir and sofosbuvir., Results:  Virological response to daclatasvir plus asunaprevir treatment was significantly lower in patients with simeprevir treatment failure as compared to those without previous treatment. Deep-sequencing analysis showed that the frequency of PI treatment-emergent NS3-D168 mutations gradually decreased and were completely replaced by wild-type genes after cessation of therapy. However, mice injected with serum obtained from a patient with PI treatment failure rapidly developed NS3-D168 mutations at significantly higher frequencies following either simeprevir or sofosbuvir treatment., Conclusions:  The virological response to daclatasvir plus asunaprevir treatment was low in patients with simeprevir treatment failure. PI resistance remains even after disappearance of mutant strains by deep sequencing., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Aim: Polymorphisms in the ITPA gene influence anemia during peg-interferon (PEG-IFN) and ribavirin (RBV) therapy, but their effects during triple therapy with simeprevir, PEG-IFN, and RBV are not sufficiently known., Methods: We analyzed 212 patients with genotype 1 chronic hepatitis C, who were treated with simeprevir plus PEG-IFN/RBV triple therapy, and assessed the effect of the ITPA polymorphism on hemoglobin levels and RBV dose reduction. ITPA (rs1127354) and IFNL4 (ss469415590) polymorphisms were genotyped using the Invader assay. A stepwise multivariate regression analysis was carried out to identify factors associated with outcome of the therapy., Results: Reduction of hemoglobin levels was similar between patients treated with simeprevir plus PEG-IFN/RBV and those treated with PEG-IFN/RBV therapy. In simeprevir plus PEG-IFN/RBV-treated patients, decreases in hemoglobin levels were faster and greater, and the cumulative proportion of patients with ribavirin dose reduction was significantly greater in ITPA genotype CC patients than in CA/AA patients. The total dose of simeprevir and PEG-IFN was similar between ITPA genotype CC and CA/AA patients. In contrast, the total dose of RBV was lower in patients with the CC genotype. Multivariate analysis showed that the IFNL4 TT/TT genotype, but not the ITPA SNP genotype, treatment history (treatment-naive or relapse during prior treatment), and treatment completion were significantly associated with outcome of therapy., Conclusion: ITPA polymorphism influences hemoglobin levels and incidence of RVB dose reduction during simeprevir triple therapy, indicating the importance of monitoring anemia during treatment, particularly for ITPA genotype CC patients., (© 2016 The Japan Society of Hepatology.)

Background and Aims: Interferon and nucleotide/nucleoside analogues are the main treatments for chronic hepatitis B. These drugs effectively reduce serum hepatitis B virus (HBV) DNA titers but fail to sufficiently reduce hepatitis B surface antigen (HBsAg) levels. Following the recent identification of sodium taurocholate cotransporting polypeptide as a receptor for HBV entry, inhibition of HBV entry has become an attractive therapeutic target for chronic hepatitis B treatment. We therefore evaluated the antiviral effects of antibody to HBsAg (anti-HBs) immunoglobulin (HBIG), which can inhibit HBV entry, by in an vivo study and a clinical trial., Methods: In the in vivo study, HBV-infected mice were generated from human hepatocyte chimeric mice and treated with HBIG. A clinical trial evaluating HBIG therapy in patients was also performed., Results: In the mouse study, HBV DNA titers were reduced and serum HBsAg titers decreased to undetectable levels following high-dose HBIG injection. On the basis of this result, eight chronic hepatitis B patients, who had received long-term nucleotide analogue treatment, were treated with monthly HBIG injections as an additional treatment. After 1 year of treatment, an HBsAg level reduction of more than 1 log IU/mL was observed in four patients, and three patients became anti-HBs positive. No adverse events occurred during HBIG therapy., Conclusion: These results suggest that monthly HBIG injection might benefit patients with chronic hepatitis B whose HBsAg titer becomes lower following long-term nucleotide/nucleoside analogue treatment.

Aim: Although interferon (IFN)-free antiviral therapy is expected to improve the treatment response for chronic hepatitis C, the effect on liver function and liver fibrosis is unknown. In this study, we analyzed the long-term follow up of liver function parameters and liver fibrosis markers in genotype 1b hepatitis C virus (HCV)-infected patients treated with daclatasvir and asunaprevir., Methods: Thirty patients were treated with daclatasvir and asunaprevir for 24 weeks, and 26 patients achieved sustained virological response (SVR). We measured liver function parameters, serum alanine aminotransferase (ALT) and albumin levels and liver fibrosis markers, hyaluronic acid, type IV collagen and Mac-2-binding protein (M2BPGi) before and after (median, 27 months; range, 17-47) completion of the treatment in SVR and non-SVR patients. We also measured serum α-fetoprotein (AFP) levels during the therapy and follow-up period., Results: Pretreatment serum ALT and albumin levels and liver fibrosis markers were similar between SVR and non-SVR patients. Twenty-seven months after treatment, serum ALT and albumin levels significantly improved only in SVR patients. Although there was no change in non-SVR patients, platelet count and serum liver fibrosis markers significantly improved in SVR patients. Serum AFP levels rapidly decreased during the treatment in both SVR and non-SVR patients, but the change was significant only in SVR patients., Conclusion: Successful viral eradication by IFN-free daclatasvir and asunaprevir therapy could lead to improved liver function parameters and reduced liver fibrosis markers and AFP levels. This treatment has the potential to improve liver fibrosis and decrease the incidence of hepatocarcinogenesis., (© 2015 The Japan Society of Hepatology.)

Scavenger receptor class B type 1 (SR-B1) and low-density lipoprotein receptor (LDLR) are known to be involved in entry of hepatitis C virus (HCV), but their precise roles and their interplay are not fully understood. In this study, deficiency of both SR-B1 and LDLR in Huh7 cells was shown to impair the entry of HCV more strongly than deficiency of either SR-B1 or LDLR alone. In addition, exogenous expression of not only SR-B1 and LDLR but also very low-density lipoprotein receptor (VLDLR) rescued HCV entry in the SR-B1 and LDLR double-knockout cells, suggesting that VLDLR has similar roles in HCV entry. VLDLR is a lipoprotein receptor, but the level of its hepatic expression was lower than those of SR-B1 and LDLR. Moreover, expression of mutant lipoprotein receptors incapable of binding to or uptake of lipid resulted in no or slight enhancement of HCV entry in the double-knockout cells, suggesting that binding and/or uptake activities of lipid by lipoprotein receptors are essential for HCV entry. In addition, rescue of infectivity in the double-knockout cells by the expression of the lipoprotein receptors was not observed following infection with pseudotype particles bearing HCV envelope proteins produced in non-hepatic cells, suggesting that lipoproteins associated with HCV particles participate in the entry through their interaction with lipoprotein receptors. Buoyant density gradient analysis revealed that HCV utilizes these lipoprotein receptors in a manner dependent on the lipoproteins associated with HCV particles. Collectively, these results suggest that lipoprotein receptors redundantly participate in the entry of HCV.

Signal-peptide peptidase (SPP) is an intramembrane protease that participates in the production of the mature core protein of hepatitis C virus (HCV). Here we show that SPP inhibition reduces the production of infectious HCV particles and pathogenesis. The immature core protein produced in SPP-knockout cells or by treatment with an SPP inhibitor is quickly degraded by the ubiquitin-proteasome pathway. Oral administration of the SPP inhibitor to transgenic mice expressing HCV core protein (CoreTg) reduces the expression of core protein and ameliorates insulin resistance and liver steatosis. Moreover, the haploinsufficiency of SPP in CoreTg has similar effects. TRC8, an E3 ubiquitin ligase, is required for the degradation of the immature core protein. The expression of the HCV core protein alters endoplasmic reticulum (ER) distribution and induces ER stress in SPP/TRC8 double-knockout cells. These data suggest that HCV utilizes SPP cleavage to circumvent the induction of ER stress in host cells.

We previously reported that interferon (IFN)-free direct-acting antiviral combination treatment succeeded in eradicating genotype 1b hepatitis C virus (HCV) in human hepatocyte chimeric mice. In this study, we examined the effect of vaniprevir (MK7009, NS3/4A protease inhibitor) and BMS-788329 (NS5A inhibitor) combination treatment on HCV genotype 1b and the expression of IFN-stimulated genes (ISGs) using a subgenomic replicon system and the same animal model. Combination treatment with vaniprevir and BMS-788329 significantly reduced HCV replication compared to vaniprevir monotherapy in HCV replicon cells (Huh7/Rep-Feo cells). HCV genotype 1b-infected human hepatocyte chimeric mice were treated with vaniprevir alone or in combination with BMS-788329 for four weeks. Vaniprevir monotherapy reduced serum HCV RNA titers in mice, but viral breakthrough was observed in mice with high HCV titers. Ultra-deep sequence analysis revealed a predominant replacement by drug-resistant substitutions at 168 in HCV NS3 region in these mice. Conversely, in mice with low HCV titers, HCV was eradicated by vaniprevir monotherapy without viral breakthrough. In contrast to monotherapy, combination treatment with vaniprevir and BMS-788329 succeeded in completely eradicating HCV regardless of serum viral titer. IFN-alpha treatment significantly increased ISG expression; however, vaniprevir and BMS-788329 combination treatment caused no increase in ISG expression both in cultured cells and in mouse livers. Therefore, combination treatment with vaniprevir and BMS-788329 eliminated HCV via a non-ISG-mediated mechanism. This oral treatment might offer an alternative DAA combination therapy for patients with chronic hepatitis C., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background & Aims: Tenofovir disoproxil fumarate (TDF) has been approved for chronic hepatitis B treatment, and favorable susceptibility of hepatitis B virus (HBV) has been indicated. However, differences in TDF susceptibility among HBV genotypes and drug-resistant strains are unclear. In this study, TDF susceptibilities between genotypes A and C were evaluated in vitro and in vivo using several drug-resistant HBV clones., Methods: HBV expression plasmids were constructed from sera of HBV carriers, and drug-resistant substitutions were introduced by site-directed mutagenesis. TDF susceptibility was evaluated by changes of core-associated HBV replication intermediates in vitro or by change of serum HBV DNA in human hepatocyte chimeric mice carrying each HBV clone in vivo., Results: TDF susceptibilities of lamivudine-resistant clones (rtL180M/M204V) and lamivudine plus entecavir-resistant clones (rtL180M/S202G/M204V) were similar to wild type clones in vitro. However, lamivudine plus adefovir-resistant clones (rtA181T/N236T) acquired tolerance to TDF, and the rtN236T mutation was considered to be a causal substitution for TDF resistance. Furthermore, genotypic differences in TDF susceptibility were also observed between genotypes A and C in vitro, and the differences could be confirmed in vivo (p = 0.023)., Conclusions: The present study indicates that TDF susceptibility varies among HBV genotypes and drug-resistant HBV clones., (Copyright © 2015 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Background: Although the frequency of emergent drug-resistant strains of HCV in patients who failed to respond to simeprevir plus pegylated interferon (PEG-IFN) and ribavirin (RBV) decreased after cessation of the treatment, it is not clear whether or not the NS3-D168 variants affect the outcome of NS5A and NS3 inhibitor combination therapy. In this study, we investigated the relationship between the effect of daclatasvir plus asunaprevir treatment and the frequencies of NS3-D168 variants., Methods: HCV genotype-1b-infected human hepatocyte chimeric mice with various frequencies of NS3-D168 amino acid substitutions were treated with asunaprevir alone or in combination with daclatasvir for 4 weeks. Frequencies of NS3-D168 substitutions at baseline were analysed by ultra-deep sequencing. Some mice with NS3-D168 substitutions were treated with PEG-IFN or telaprevir for 4 weeks., Results: Mice with high frequencies of NS3-D168 showed low susceptibility to asunaprevir treatment and failed to respond to daclatasvir plus asunaprevir therapy. In contrast, mice with a low frequency (less than approximately 14%) of NS3-D168 showed a similar susceptibility to wild-type HCV-infected mice and achieved viral eradication with daclatasvir plus asunaprevir therapy. Although treatment with either telaprevir or PEG-IFN resulted in reduction of serum HCV RNA levels, no significant decrease in the frequency of NS3-D168 substitutions was achieved., Conclusions: Daclatasvir and asunaprevir treatment could eliminate NS3-D168 variant HCV if the frequency was low. It is necessary to confirm that the frequency of NS3-D168 variants has decreased sufficiently before adopting daclatasvir plus asunaprevir therapy in patients with simeprevir plus PEG-IFN/RBV treatment failure.

Although interferon-free antiviral treatment is expected to improve treatment of hepatitis C, it is unclear to what extent pre-existing drug-resistant amino acid substitutions influence response to therapy. The impact of pre-existing drug-resistant substitutions on virological response to daclatasvir and asunaprevir combination therapy was studied in genotype 1b hepatitis C virus (HCV)-infected patients. Thirty-one patients were treated with daclatasvir and asunaprevir for 24 weeks. Twenty-six patients achieved sustained virological response (SVR), three patients experienced viral breakthrough, and two patients relapsed. Direct sequencing analysis of HCV showed the existence of daclatasvir-resistant NS5A-L31M or -Y93H/F variants in nine out of 30 patients (30%) prior to treatment, while asunaprevir-resistant NS3-D168 mutations were not detected in any patient. All 21 patients with wild-type NS5A-L31 and -Y93 achieved SVR, whereas only four out of nine patients (44%) with L31M or Y93F/H substitutions achieved SVR (P = 0.001). Ultra-deep sequencing analysis showed that treatment failure was associated with the emergence of both NS5A-L31/Y93 and NS3-D168 variants. NS5A-L31/Y93 variants remained at high frequency through post-treatment weeks 103 through 170, while NS3-D168 variants were replaced by wild-type in all patients. In conclusion, pre-existence of NS5A inhibitor-resistant substitutions compromised the response to daclatasvir and asunaprevir combination therapy, and treatment failure was associated with the emergence of both NS5A-L31/Y93 and NS3-D168 variants. While asunaprevir-resistant variants that emerged during therapy returned to wild-type, daclatasvir-resistant variants tended to persist in the absence of the drug., (© 2015 Wiley Periodicals, Inc.)

Unlabelled: Hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) are critical in eliminating infection. We developed an animal model in which HBV-infected human hepatocytes are targeted by HBV-specific CTLs. After HBV inoculation in human hepatocyte-transplanted herpes simplex virus type-1 thymidine kinase-NOG mice, human peripheral blood mononuclear cells (PBMCs) were administered, and albumin, HBV DNA, alanine aminotransferase (ALT), and cytokine levels were analyzed. Histopathological and flow-cytometric analysis of infiltrating human immune cells were performed, and the efficacy of CTL-associated antigen-4 immunoglobulin (CTLA4Ig) against liver damage was evaluated. PBMC treatment resulted in massive hepatocyte damage with elevation of ALT, granzyme A, and gamma interferon and decrease in albumin and HBV DNA. The number of liver-infiltrating human lymphocytes and CD8-positive cells was significantly higher in HBV-infected mice. HBV-specific CTLs were detected by core and polymerase peptide-major histocompatibility complex-tetramer, and the population of regulatory T cells was significantly decreased in HBV-infected mice. Serum hepatitis B surface (HBs) antigen became negative, and HBs antibody appeared. CTLA4Ig treatment strongly inhibited infiltration of mononuclear cells. CTLA4Ig treatment will be used to treat patients who develop severe acute hepatitis B to prevent liver transplantation or lethality. This animal model is useful for virological and immunological analysis of HBV infection and to develop new therapies for severe acute hepatitis B., Importance: Without liver transplantation, some HBV-infected patients will die from severe liver damage due to acute overreaction of the immune system. No effective treatment exists, due in part to the lack of a suitable animal model. An animal model is necessary to investigate the mechanism of hepatitis and to develop therapeutic strategies to prevent acute liver failure in HBV infection. We developed an animal model in which HBV-infected human hepatocytes are targeted by human HBV-specific CTLs. In this model, HBV-infected human hepatocytes were transplanted into severely immunodeficient NOG mice in order to reconstruct elements of the human immune system. Using this model, we found that CTL-associated antigen-4 immunoglobulin was able to suppress damage to HBV-infected hepatocytes, suggesting an approach to treatment. This animal model is useful for virological and immunological analysis of HBV infection and to develop new therapies for severe acute hepatitis B., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background & Aims: Single nucleotide polymorphisms within the interferon lambda 4 (IFNL4) locus are strongly associated with spontaneous clearance of hepatitis C virus (HCV) infection and early viral response to interferon therapy. Interaction between host genotype and amino acid substitutions might also influence the risk of antiviral resistance in interferon-free direct acting antiviral (DAA) therapies., Methods: The relationship between IFNL4 genotype and HCV substitutions was analyzed in 929 patients with chronic HCV genotype 1b infection. Ultra-deep sequencing and quasispecies reconstruction was performed on the N-terminal region of NS5A in 57 patients., Results: IFNL4 genotype was strongly associated with HCV NS5A Y93 and core protein substitutions, and the number and diversity of predicted quasispecies was marginally greater in IFNL4 TT/TT patients compared to TT/ΔG, ΔG/ΔG patients. RNA secondary structure prediction of the NS5A region suggests that variable sites are more likely to occupy unpaired, high entropy positions., Conclusions: HCV infection is proposed to induce a more efficient antiviral response in individuals with the IFNL4 TT/TT genotype that results either in viral clearance or selection for viral adaptations. The association between IFNL4 TT/TT genotype and Y93 substitutions may impact the risk of antiviral resistance in NS5A inhibitors in DAA therapy., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Daclatasvir and asunaprevir dual oral therapy is expected to achieve high sustained virological response (SVR) rates in patients with HCV genotype 1b infection. However, presence of the NS5A-Y93H substitution at baseline has been shown to be an independent predictor of treatment failure for this regimen. By using the Invader assay, we developed a system to rapidly and accurately detect the presence of mutant strains and evaluate the proportion of patients harboring a pre-treatment Y93H mutation. This assay system, consisting of nested PCR followed by Invader reaction with well-designed primers and probes, attained a high overall assay success rate of 98.9% among a total of 702 Japanese HCV genotype 1b patients. Even in serum samples with low HCV titers, more than half of the samples could be successfully assayed. Our assay system showed a better lower detection limit of Y93H proportion than using direct sequencing, and Y93H frequencies obtained by this method correlated well with those of deep-sequencing analysis (r = 0.85, P <0.001). The proportion of the patients with the mutant strain estimated by this assay was 23.6% (164/694). Interestingly, patients with the Y93H mutant strain showed significantly lower ALT levels (p=8.8 x 10-4), higher serum HCV RNA levels (p=4.3 x 10-7), and lower HCC risk (p=6.9 x 10-3) than those with the wild type strain. Because the method is both sensitive and rapid, the NS5A-Y93H mutant strain detection system established in this study may provide important pre-treatment information valuable not only for treatment decisions but also for prediction of disease progression in HCV genotype 1b patients.

Background & Aims: Circulating tumor DNA (ctDNA) carrying tumor-specific sequence alterations has been found in the cell-free fraction of blood. Liver cancer tumor specimens are difficult to obtain, and noninvasive methods are required to assess cancer progression and characterize underlying genomic features., Methods: We analyzed 46 patients with hepatocellular carcinoma who underwent hepatectomy or liver transplantation and for whom whole-genome sequencing data was available. We designed personalized assays targeting somatic rearrangements of each tumor to quantify serum ctDNA. Exome sequencing was performed using cell-free DNA paired primary tumor tissue DNA from a patient with recurrent liver cancer after transcatheter arterial chemoembolization (TACE)., Results: We successfully detected ctDNA from 100 μL of serum samples in 7 of the 46 patients before surgery, increasing with disease progression. The cumulative incidence of recurrence and extrahepatic metastasis in the ctDNA-positive group were statistically significantly worse than in the ctDNA-negative group ( P  = .0102 and .0386, respectively). Multivariate analysis identified ctDNA (OR 6.10; 95% CI, 1.11-33.33, P  = .038) as an independent predictor of microscopic vascular invasion of the portal vein (VP). We identified 45 nonsynonymous somatic mutations in cell-free DNA after TACE and 71 nonsynonymous somatic mutations in primary tumor tissue by exome sequencing. We identified 25 common mutations in both samples, and 83% of mutations identified in the primary tumor could be detected in the cell-free DNA., Conclusions: The presence of ctDNA reflects tumor progression, and detection of ctDNA can predict VP and recurrence, especially extrahepatic metastasis within 2 years. Our study demonstrated the usefulness of ctDNA detection and sequencing analysis of cell-free DNA for personalized treatment of liver cancer.