HBV with precore and basal core promoter mutations exhibits a high replication phenotype and causes ER stress-mediated cell death in humanized liver chimeric mice.

BACKGROUND AND AIMS: Mutations within the precore (PC) and basal core promoter (BCP) regions of the HBV genome are associated with fulminant hepatitis and HBV reactivation. These mutations may enhance viral replication, but little is known about whether they directly induce damage to the liver. We investigated mechanisms of direct cytopathic effects induced by the infection with PC/BCP mutants in the absence of immune response in vitro and in vivo . APPROACH AND RESULTS: Mice with humanized livers and hepatocytes derived from humanized mice were infected with either wild-type or mutant-type PC/BCP HBV, and the HBV replication and human hepatocyte damage were evaluated. HBV proliferated vigorously in mice with PC/BCP-mutant infection, and the severe loss of human hepatocytes with a slight human ALT elevation subsequently occurred only in PC/BCP mutant mice. In PC/BCP mutant infection, the accumulation of HBsAg in humanized livers colocalized with the endoplasmic reticulum, leading to apoptosis through unfolded protein response in HBV-infected hepatocytes. RNA-sequencing revealed the molecular characteristics of the phenotype of PC/BCP mutant infection in a humanized mouse model. Reduced ALT elevation and higher HBV DNA levels in this model are consistent with characteristics of HBV reactivation, indicating that the hepatocyte damage in this model might mimic HBV reactivation followed by hepatocyte damage under immunosuppressive conditions. CONCLUSION: PC and BCP mutations were associated with enhanced viral replication and cell death induced by ER stress using HBV infection models. These mutations might be associated with liver damage in patients with fulminant hepatitis or HBV reactivation.

Time trend of outcomes according to systemic therapy for patients with unresectable hepatocellular carcinoma: A single-institution study.

BACKGROUND: We have been able to use molecular targeted agents for unresectable hepatocellular carcinoma since 2009, and immune checkpoint inhibitors have been approved in recent years. We assessed the efficacy of systemic therapy in Hiroshima University Hospital by each era. METHODS: A total of 357 patients who were treated with sorafenib, lenvatinib, atezolizumab plus bevacizumab combination therapy, or durvalumab plus tremeliumab combination therapy as first-line systemic therapy in our hospital from November 2009 to December 2023 were enrolled in this retrospective cohort study. We divided the years from 2009 to 2023 into the following three periods: cohort I, 2009-2016, the single-molecular targeted agent era; cohort II, 2017-2020, the multi-molecular targeted agent era; and cohort III, 2020-2023, the immuno-oncology era. RESULTS: The median survival time was 9.5 months in cohort I, 15.8 months in cohort II, and 20.2 months in cohort III. The median survival time in cohort III was significantly (p < 0.01) longer than in the other cohorts. The overall response rate by mRECIST was 4.1% in cohort I, 28.7% in cohort II, and 47.2% in cohort III. The disease control rate was 41.6% in cohort I, 61.2% in cohort II, and 73.6% in cohort III. Both overall response rate and disease control rate significantly increased by era. CONCLUSIONS: We consider that advancements in systemic therapy, along with changes in treatment strategies, such as sequential therapy after progression, contribute to the prolonged prognosis across different eras.

Significance of changes in tumor markers in patients treated with durvalumab plus tremelimumab combination therapy as a surrogate marker for tumor response to unresectable hepatocellular carcinoma.

AIM: When evaluating response to immune checkpoint inhibitor therapy, the tumor sometimes initially swells before shrinking and ultimately responding, also called pseudo-progression. In this study, we analyzed whether tumor markers were useful for reflecting the treatment response. METHODS: Thirty-three patients who were treated with durvalumab plus tremelimumab combination therapy (Dur + Tre) were enrolled. Their functional reserve was Child-Pugh grade A. Their tumor markers α-fetoprotein (AFP), des-γ-carboxy prothrombin (DCP), or AFP-Lectin 3 fraction (AFP-L3) were positive. Tumor markers were evaluated before treatment and at 1, 4, and 8 weeks after the start of treatment. The first radiological evaluation was carried out at 4 weeks and the second evaluation at 8-12 weeks. The responders included those with complete response and partial response and the nonresponders included those with stable disease (SD) and progression disease at best response evaluated by Response Evaluation Criteria in Solid Tumors. RESULTS: In the responder group, the change ratio of AFP, DCP, and AFP-L3 specifically decreased at 8 weeks. In the nonresponder group, the change ratio of DCP specifically increased at 4 weeks. The optimal cut-off value to divide responders and nonresponders at 4 weeks was approximately -40%. The ratio of responders was 72.7% in the patients whose AFP or DCP decreased over 40% at 4 weeks. CONCLUSIONS: The change in tumor markers is a more useful predicter of tumor response to Dur + Tre than imaging evaluation alone.

Comprehensive genomic profiling testing in Japanese castration-resistant prostate cancer patients: results of a single-center retrospective cohort study.

OBJECTIVE: Comprehensive genomic profiling testing using a hybrid-capture next-generation sequencing is commonly used in clinical practice to employ precision medicine in cancer treatment worldwide. In this study, we aimed to analyze the profiles obtained using comprehensive genomic profiling testing that was performed in Japanese castration-resistant prostate cancer patients and to discuss the genetic findings in a real-world setting. METHODS: A total of 60 cases and 57 castration-resistant prostate cancer patients underwent comprehensive genomic profiling testing between 1 January 2021 and 31 December 2022. Four types of comprehensive genomic profiling testing were selected, and clinically significant cancer-specific gene alterations were identified. RESULTS: The median age of patients was 74 years, and the median prostate-specific antigen value at the time of submission was 18.6 ng/ml. Fifty-seven (95%) of 60 cases were metastatic castration-resistant prostate cancers, and 3 cases (5%) were non-metastatic. Among all genetic alterations, androgen-receptor alteration was the most frequently detected in 17 cases (28.3%), followed by 15 cases of TP53 (25.0%), 14 cases of CDK12 (23.3%), 10 cases of phosphatase and tensin homolog (16.7%) and 9 cases of ATM (15.0%) mutations. A total of 13 patients (21.7%) received systemic therapy according to the comprehensive genomic profiling testing results. Overall, the survival rate was significantly greater in the group treated through systemic therapy based on comprehensive genomic profiling testing compared with the group without new therapeutic treatment (P = 0.041). CONCLUSIONS: Comprehensive genomic profiling testing is recommended in castration-resistant prostate cancer patients identified as resistant to standard therapy as this can provide a new therapeutic option.

Impact and usefulness of the transition to the new MAFLD classification for non-B, non-C HCC: a retrospective cohort study.

BACKGROUND: Metabolic dysfunction-associated fatty liver disease (MAFLD) represents a new classification system for fatty liver disease. In this study, we investigated the clinical characteristics of patients with MAFLD-hepatocellular carcinoma (HCC) in comparison with those with nonalcoholic fatty liver disease (NAFLD) and considered the validity and challenges of the new criteria. METHODS: This study included 237 untreated non-B, non-C HCC patients with hepatic steatosis. We examined the profile and laboratory findings of patients with MAFLD-HCC and NAFLD-HCC. We also classified MAFLD-HCC patients according to the factors on which the diagnosis was based and compared their clinical characteristics. RESULTS: A total of 222 (94%) and 101 (43%) patients were diagnosed with MAFLD and NAFLD, respectively. MAFLD-HCC patients were more likely to be male than NAFLD-HCC, but there were no significant differences in metabolic indices, noninvasive liver fibrosis score or HCC status. In a study of MAFLD-HCC patients by diagnostic factor, those with overweight only were younger and had advanced liver fibrosis histologically, and when limited to patients younger than 70 years, the majority were overweight. Redefinition of overweight as BMI ≥ 25 reduced the number of MAFLD-HCC patients by only 5, from 222 to 217. CONCLUSIONS: MAFLD accounted for the majority of non-B, non-C HCC cases with hepatic steatosis. Examination of additional cases and revision of the detailed criteria is needed so that it can be used to efficiently select patients with fatty liver who are at high risk of developing HCC.

Correlation between serum pro-inflammatory cytokine levels and the prognosis of the patients with acute liver failure.

BACKGROUND AND AIM: The prognosis of acute liver failure (ALF) remains poor, and liver transplantation is an alternative treatment option. Assessing the prognosis of ALF is important in determining treatment strategies. Here, we investigated clinical factors including serum pro-inflammatory cytokine levels that are associated with the prognosis of ALF. METHODS: Sixty-six patients who developed ALF were enrolled in this study. Serum concentrations of 12 pro-inflammatory cytokines were measured on admission. The prognosis and factors associated with survival and development of hepatic coma were analyzed. RESULTS: Of 66 patients, 4 patients underwent liver transplantation, and 49 patients were rescued without liver transplantation, while the remaining 13 patients died. Serum concentrations of interleukin (IL)-1ß, IL-4, IL-6, IL-8, IL-13, TNF, IFN -γ, IP-10, and G-CSF were significantly elevated in ALF patients. IL-4 and IL-8 levels were higher in patients who underwent liver transplantation or died than in rescued patients. Multivariable analysis identified age ≥ 55 years and IL-4 ≥ 1.2 pg/mL on admission as independent factors for mortality. Serum IL-8 levels were higher in patients with hepatic coma, and prothrombin-international normalized ratio ≥ 3.5 and IL-8 ≥ 77.2 pg/mL on admission were associated with development of hepatic coma after admission. CONCLUSION: Serum levels of several pro-inflammatory cytokines were elevated in ALF patients. IL-4 and IL-8 were correlated with survival and development of hepatic coma after admission, respectively. Measurement of serum pro-inflammatory cytokines seems to be useful for the management of ALF.

Untying relaxed circular DNA of hepatitis B virus by polymerase reaction provides a new option for accurate quantification and visualization of covalently closed circular DNA.

Hepatitis B virus (HBV) is a small hepatotropic DNA virus that replicates via an RNA intermediate. After entry, the virus capsid carries relaxed circular DNA (rcDNA) into the nucleus where the viral genome is converted into covalently closed circular DNA (cccDNA), which serves as the template for all viral transcripts. To monitor cccDNA levels, preprocessing methods to eliminate rcDNA have emerged for quantitative PCR, although Southern blotting is still the only method to discriminate cccDNA from other DNA intermediates. In this study, we have established a robust method for untying mature rcDNA into double stranded linear DNA using specific polymerases. Untying rcDNA provides not only an alternative method for cccDNA quantification but also a sensitive method for visualizing cccDNA. We combined this method with plasmid-safe DNase and T5 exonuclease preprocessing and revealed that accurate quantification requires cccDNA digestion by a restriction enzyme because heat stability of cccDNA increases after T5 exonuclease treatment. In digital PCR using duplex TaqMan probes, fewer than 1000 copies of cccDNA were successfully visualized as double positive spots that were distinct from single positives derived from untied rcDNA. This method was further applied to the infection model of primary hepatocytes treated with nucleoside analogues and a core protein allosteric modulator to monitor cccDNA levels. Relative quantification of cccDNA by human genome copy demonstrated the possibility of precise evaluation of cccDNA level per nucleus. These results clearly indicate that the sequential reaction from untying rcDNA is useful to investigate cccDNA fates in a small fraction of nuclei.

Prevalence of immune-related adverse events and anti-tumor efficacy following immune checkpoint inhibitor therapy in Japanese patients with various solid tumors.

BACKGROUND: While immune checkpoint inhibitors (ICIs) occasionally cause immune-related adverse events (irAEs) in various organs, the prevalence of irAEs and potential risk factors have not been clarified. We identified irAE predictive factors and examined the relationship between the effect of ICIs and irAEs for patients with malignancies. METHODS: A total of 533 cases treated with ICIs, including programmed death 1 (PD-1), PD-ligand 1 (PD-L1), and cytotoxic T-lymphocyte antigen 4 (CTLA-4), for various malignancies were included retrospectively. We recorded irAEs from medical records and graded them using the Common Terminology Criteria for Adverse Events version 5. Prevalence and predictive factors associated with immune-related liver injury and the relationship between irAE and treatment response were analyzed. RESULTS: During a median of 10 (1-103) cycles with a median follow-up after several ICI initiations of 384 (21-1715) days, irAEs with all grades and with grade ≥ 3 developed in 144 (27.0%) and 57 (10.7%) cases. Cumulative irAE development rates were 21.9, 33.5, and 43.0% in all grades and 8.8, 14.9, and 20.7% in grade ≥ 3 at 5, 10, and 20 cycles, respectively. Patients who received anti-CTLA4 therapy were more likely to develop irAEs compared to those who received anti-PD-1 or anti-PD-L1 monotherapy. Liver injury was the most common irAE. Multivariate analysis identified the combination of PD-1 and anti-CTL-4 antibodies (hazard ratio [HR], 17.04; P < 0.0001) and baseline eosinophil count ≥130/µL (HR, 3.01 for < 130; P = 0.012) as independent risk factors for the incidence of immune-related liver injury with grade ≥ 2. Patients who developed irAEs had a higher disease control rate (P < 0.0001) and an increased overall survival rate compared to those without irAEs (P < 0.0001). CONCLUSION: Combination therapy with anti-PD-1 and anti-CTL-4 antibodies resulted in higher a frequency of irAEs. Baseline absolute eosinophil count was found to be a predictive factor for immune-related liver injury. Occurrence of irAEs may be associated with higher efficacy of ICI treatment and longer survival among patients who receive ICI therapy.

Road to elimination of HCV: Clinical challenges in HCV management.

Since its discovery in 1989, the road to a cure for hepatitis C virus (HCV) has been slow, but most patients can now expect to achieve a sustained virological response (SVR). With direct-acting antiviral (DAA) combination therapies such as glecaprevir/pibrentasvir and velpatasvir/sofosbuvir, 98% of patients successfully eradicate the virus, even if previous treatments failed or if resistance-associated substitutions (RASs) are present. Adverse events are rare or mild, and patients with compensated cirrhosis and other co-morbidities are often eligible for treatment. However, a small number of patients fail to eradicate the virus even after retreatment. The cause of failure is mainly due to emergence of NS5A P32 deletion mutants after initial DAA therapy in genotype 1b patients, although the reason is unknown for some patients. Alternative therapies that do not rely on NS5A inhibitors, such as sofosbuvir plus ribavirin, can be attempted in these patients. While scaled-up treatment efforts present a challenge, another problem is that many carriers are unaware of their infection. Long-term damage to the liver becomes irreversible, and patients who are not diagnosed in time can develop liver cancer or liver failure even after eliminating the virus. The long-term costs of treatment of advanced liver disease in undiagnosed patients relative to the immediate costs of DAA therapy should be considered. As no vaccine is yet available, eventual elimination of the virus requires identifying and treating undiagnosed cases and screening of high-risk populations such as injection drug users and men who have sex with men and female sex workers.

Underestimation of impaired glucose tolerance and usefulness of a continuous glucose monitoring system in chronic liver disease.

BACKGROUND AND AIM: The prevalence of glucose intolerance in chronic liver disease patients is high, but glucose intolerance may be overlooked in a single blood test. The purpose of this study is to evaluate blood glucose variability in patients with chronic liver disease by a continuous glucose monitoring system (CGMS) and to examine the discrepancy between hemoglobin A1c (HbA1c) levels estimated from average blood glucose levels and HbA1c. METHODS: This study included 335 patients with chronic liver disease associated with glucose intolerance. A fasting blood test and 72-h CGMS were performed. The estimated HbA1c was calculated from the average blood glucose level, and the correlation between hepatic functional reserve and blood glucose-related parameters was analyzed. From the obtained data, we created a new formula to calculate HbA1c without using CGMS. RESULTS: As hepatic functional reserve decreased, average blood glucose and insulin resistance increased while HbA1c decreased (P < 0.0001). The discrepancy between the estimated HbA1c calculated from the mean blood glucose level and the serum HbA1c (ΔHbA1c) increased as the liver reserve decreased. Using multiple regression analysis, a formula based on fasting blood glucose, HbA1c, body mass index, albumin, and liver function was constructed, and its validity was demonstrated in a study using a different control group. CONCLUSIONS: Hemoglobin A1c may be underestimated because of decreased hepatic functional reserve. CGMS was useful in assessing accurate glycemic control of blood glucose and in detecting postprandial hyperglycemia and nocturnal hypoglycemia. Patients with chronic hepatic impairment should be corrected for hepatic functional reserve before glycemic control.

Novel drug resistance-associated substitutions against pibrentasvir emerged in genotype 1b hepatitis C virus-infected human hepatocyte transplanted mice.

Combination therapy with glecaprevir and pibrentasvir (PIB) has high efficacy for patients with hepatitis C virus (HCV) infection except among those who experienced NS5A-P32 deletion (del) mutation during prior DAA treatment failure. However, some patients fail to achieve SVR through combination treatment even in the absence of NS5A-P32del. We analyzed emergence of NS5A resistance-associated substitutions (RASs) against PIB using HCV-infected mice. Male human hepatocyte transplanted mice were infected with genotype 1b wild-type HCV. Mice were treated with PIB, resulting in a transient decrease in serum HCV RNA levels but followed by relapse during the treatment. Direct sequence analysis showed emergences of various mutations in the NS5A region, including L31V/P32del, L31F/P32del/Y93H, NS5A-P29del/Y85C, and NS5A-F37Y. PIB was less effective in mice with NS5A-F37Y mutations compared to mice with wild-type HCV. NS5A-F37Y showed 5.4-fold resistance to PIB relative to wild-type based on analysis using HCV subgenomic replicon systems. The present in vivo and in vitro studies identified NS5A-F37Y as a novel RAS against PIB and showed the possibility of emergence of various NS5A RASs including P29del, P32del and F37Y following PIB treatment. These mutations might emerge and lead to failure to respond to DAA therapies including PIB-based regimens in chronic hepatitis C patients.

Serum PreS1 and HBsAg ratio reflects liver fibrosis and predicts the development of hepatocellular carcinoma in chronic hepatitis B patients.

While the preS1 region of the large hepatitis B surface protein plays an essential role in hepatitis B virus (HBV) infection, the effect of preS1 on liver fibrosis and hepatocarcinogenesis in chronic hepatitis B (CHB) patients is not well known. In this study, we measured serum preS1 levels by chemiluminescent immunoassay technology in 690 CHB patients and evaluated the correlation between serum preS1 levels and HBV, liver function markers and liver inflammation, fibrosis assessed by histological findings. Predictive factors for hepatocellular carcinoma (HCC) development in patients who had no previous history of HCC at the time of preS1 level measurement were also analysed. Median hepatitis B surface antigen (HBsAg) and preS1 levels were 3.08 log IU/mL and 98 ng/mL, respectively. PreS1 values were significantly correlated with serum HBsAg (p <0.001), hepatitis B core-related antigen (HBcrAg) (p <0.001) and HBV DNA levels (p <0.01). PreS1 values were also significantly correlated with serum alanine aminotransferase levels (p <0.001) and were significantly higher in patients who had higher grading of liver inflammatory activity (p <0.05). HBsAg level was correlated, but preS1/HBsAg ratio reflected liver fibrosis staging more directly than HBsAg alone. Multivariate analysis identified age ≥53 years (hazard ratio [HR], 18.360 for <53 years; p = 0.021) and preS1/HBsAg ratio ≥0.12 (HR, 6.205 for <0.12; p = 0.040) as significant and independent factors for HCC development in CHB patients. The preS1/HBsAg ratio directly reflects liver fibrosis, and the ratio might be a predictive marker for HCC development in CHB patients.

Clinical Usefulness of Serum Autotaxin for Early Prediction of Relapse in Male Patients with Type 1 Autoimmune Pancreatitis.

BACKGROUND: Serum IgG4 level is a useful diagnostic marker for autoimmune pancreatitis (AIP), but it is difficult to use to predict relapse. AIMS: We investigated whether serum autotaxin (ATX) level is predictive of AIP relapse after steroid therapy. METHODS: Fifty-six patients with type 1 AIP were investigated. We measured serum ATX at the time of diagnosis. We selected 24 males for whom serum samples during steroid therapy had been obtained and measured serum ATX at steroid therapy for induction of remission and at maintenance therapy. In the relapse group, we also measured ATX at the time of relapse. RESULTS: ATX was significantly higher in female patients than in male patients. In order to clarify changes in ATX during steroid therapy, we focused on 24 male patients. We found that ATX decreased significantly during steroid therapy for induction of remission and at the time of maintenance therapy. In half of all patients who relapsed during maintenance therapy, ATX was significantly elevated at the time of relapse compared with that of induction therapy (P = 0.039). When we compared ATX at the time of maintenance therapy between patients with relapse and without, we observed significantly higher ATX in the former (P = 0.024). We found that the combination of ATX and elastase-1 could predict relapse with high accuracy (95%). CONCLUSIONS: Preliminary evidence suggests that serum ATX might serve as a candidate biomarker to predict relapse of AIP as well as to monitor the effect of steroid therapy.

Signal Activation of Hepatitis B Virus-Related Hepatocarcinogenesis by Up-regulation of SUV39h1.

BACKGROUND: Hepatitis B virus (HBV) X (HBx) protein is associated with hepatocellular carcinogenesis via the induction of malignant transformation and mitochondrial dysfunction. However, the association between HBx and histone methyltransferase in carcinogenesis has not been fully clarified. In the current study, we analyzed the association between HBx and the histone methyltransferase suppressor of variegation 3-9 homolog 1 (SUV39h1) using HBV replication models. METHODS: We constructed several HBx and SUV39h1 expression plasmids and analyzed the association between HBx and SUV39h1 with respect to HBV replication and hepatocarcinogenesis. RESULTS: SUV39h1 up-regulation was observed in HBV-infected humanized mouse livers and clinical HBV-related hepatocellular carcinoma tissues, indicating that SUV39h1 expression might be regulated by HBV infection. Through in vitro analysis, we determined that the coactivator domain of HBx interacts with the PSET (PostSET) and SET (Su(var)3-9, Enhancer-of-zeste, Trithorax) domains of SUV39h1. The expression levels of 4 genes, activating transcription factor 6, α-fetoprotein, growth arrest and DNA damage-inducible 45a, and dual-specificity phosphatase 1, known to induce carcinogenesis via HBx expression, were up-regulated by HBx and further up-regulated in the presence of both HBx and SUV39h1. Furthermore, histone methyltransferase activity, the main function of SUV39h1, was enhanced in the presence of HBx. CONCLUSIONS: We demonstrated that SUV39h1 and HBx enhance each other's activity, leading to HBx-mediated hepatocarcinogenesis. We propose that regulation of this interaction could help suppress development of hepatocellular carcinoma.

Ribavirin induces hepatitis C virus genome mutations in chronic hepatitis patients who failed to respond to prior daclatasvir plus asunaprevir therapy.

Ribavirin (RBV) induces nucleotide (nt) substitutions in hepatitis C virus (HCV) genome nonstructural (NS) regions. Although emergence of drug resistance-associated variants is associated with direct-acting antiviral treatment failure, the effect of RBV on genome substitutions in such patients is unknown. Genotype 1b HCV subgenomic replicon cells were treated with RBV for 120 hours. Six patients with chronic genotype 1b with HCV-infected patients who failed to respond to prior daclatasvir plus asunaprevir (DCV/ASV) therapy were treated with 12 weeks of sofosbuvir and ledipasvir plus RBV after 4 weeks of RBV monotherapy. RBV-induced genome mutations in the HCV NS region (nt3493-9301) in replicon cells and in patients during 4 weeks of RBV monotherapy were analyzed by deep sequencing. RBV-associated G-to-A and C-to-U transitions increased in a dose-dependent manner in HCV replicon cells after the RBV treatment. In patients with prior DCV/ASV treatment failures, the median serum HCV RNA level was 6.25 ± 0.31 log IU/mL at the start of RBV therapy and decreased significantly to 5.95 ± 0.4 log IU/mL (P = .03) after 4 weeks of RBV monotherapy. Although predominant HCV genome substitutions rates were similar between nontreatment and RBV-treatment periods (0.042 and 0.031 per base pair, respectively; P = .248), the frequencies of G-to-A and C-to-U transitions significantly increased after RBV monotherapy. These transitions were enriched, particularly within the HCV NS3 region in all patients. RBV treatment induces G-to-A and C-to-U transitions in the HCV genome even in chronic patients with hepatitis C with prior DCV/ASV treatment failures.

Liver fibrosis assessments using FibroScan, virtual-touch tissue quantification, the FIB-4 index, and mac-2 binding protein glycosylation isomer levels compared with pathological findings of liver resection specimens in patients with hepatitis C infection.

BACKGROUND: Evaluation of fibrosis stage is important to monitor progression of liver disease and risk of hepatocellular carcinoma (HCC). While liver biopsy is the gold standard, the method is invasive and faces several limitations. The aim of this study was to determine correlations among METAVIR scores and FibroScan, Virtual-Touch tissue quantification (VTQ), fibrosis index based on four factors (FIB-4 index), and Mac-2 binding protein glycosylation isomer (M2BPGi) level, and for examine differences in the reliability of non-invasive methods to evaluate fibrosis. METHODS: We used liver resection specimens from patients with hepatitis C virus (HCV), correlations were assessed between METAVIR scores and non-invasive method. Receiver operating characteristic (ROC) curves were generated to determine the sensitivity, specificity, and cut off values of the methods. RESULTS: All Patients group: In F0-2 vs F3-4, the areas under the ROC curve (AUC) (0.85) of FibroScan was significantly higher than that (0.67) of FIB-4 index (p = 0.002) and that (0.67) of M2BPGi (p = 0.001). The AUC (0.83) of VTQ was significantly higher than that (0.67) of FIB-4 index (p = 0.01) and that (0.67) of M2BPGi (p = 0.002). In F0-3 vs F4, the AUC (0.86) of VTQ was significantly higher than that (0.65) of FIB-4 index (p = 0.04). The AUC (0.89) of FibroScan was significantly higher than that (0.65) of FIB-4 index (p = 0.002) and that (0.76) of M2BPGi (p = 0.02). Non-SVR group: In F0-2 vs F3-4, the AUC (0.85) of FibroScan was significantly higher than that (0.84) of FIB-4 index (p = 0.02) and that (0.73) of M2BPGi (p = 0.003). The AUC (0.84) of VTQ was significantly higher than that (0.74) of FIB-4 index (p = 0.04). In F0-3 vs F4, the AUC (0.91) of FibroScan was significantly higher than that (0.67) of FIB-4 index (p = 0.003) and that (0.78) of M2BPGi (p = 0.02). The AUC (0.88) of VTQ was significantly higher than that of FIB-4 index (0.67) and that of M2BPGi (0.78) (p = 0.04). CONCLUSIONS: FibroScan and VTQ best reflected the results of hepatic fibrosis diagnosis using liver resection specimens among the four examination methods evaluated.

Impact of viral eradication by direct-acting antivirals on the risk of hepatocellular carcinoma development, prognosis, and portal hypertension in hepatitis C virus-related compensated cirrhosis patients.

AIM: We analyzed the impact of hepatitis C virus eradication by direct-acting antiviral (DAA) therapy on the risk of development of hepatocellular carcinoma (HCC), prognosis, and portal hypertension in patients with liver cirrhosis. METHODS: The rate of HCC development and overall survival after achievement of sustained virological response (SVR) in 173 DAA-treated compensated cirrhosis patients without HCC history were retrospectively compared with that of 125 cirrhosis patients who achieved SVR by interferon (IFN)-based therapy or that of 85 cirrhosis patients who failed to respond to anti-HCV therapy. Changes in esophagogastric varices (EGV) and incidence of portosystemic encephalopathy were analyzed in 87 consecutive cirrhosis patients. RESULTS: The cumulative HCC development rates at 1, 3, and 5 years were 2%, 7%, and 7% for the DAA-SVR group, significantly lower than the 3%, 7%, and 18% for the non-SVR group (log-rank, P < 0.001). The cumulative overall survival rates were also significantly improved in the DAA-SVR group compared to the non-SVR group (log-rank, P < 0.001). These rates were similar between DAA-SVR and IFN-SVR groups (P = 0.121 and 0.261, respectively). Esophagogastric varices were aggravated, and portosystemic encephalopathy occurred in a subset of cirrhosis patients who achieved SVR by DAA therapy. These events were more frequent in patients with large feeding vessels for EGV and portosystemic shunts at the time of SVR. CONCLUSION: Achievement of SVR by DAA therapy reduces the risk of HCC development and prolongs survival, similar to theresults achieved with IFN-based therapy, but portal hypertension is not immediately improved in compensated liver cirrhosis patients.

Real-world efficacy of sofosbuvir plus velpatasvir therapy for patients with hepatitis C virus-related decompensated cirrhosis.

AIM: Combination therapy with sofosbuvir (SOF) plus velpatasvir (VEL) is approved for patients with hepatitis C virus (HCV)-related decompensated cirrhosis. We analyzed the real-world efficacy of SOF/VEL therapy. METHODS: Thirty-three patients with HCV-related decompensated cirrhosis (25 and eight patients with Child B and C, respectively) were treated with SOF/VEL for 12 weeks. The HCV non-structural protein (NS)5A and NS5B drug resistance-associated substitutions (RASs) were determined by direct sequencing. RESULT: Thirty-two of 33 patients completed the treatment, but the remaining patient discontinued the therapy during third week of the treatment due to aggravation of hepatic encephalopathy. Serum HCV-RNA became negative during the treatment in all patients but relapsed after the end of therapy in five patients. In total, 28 out of 33 patients (85%) achieved sustained virological response 12 weeks following completion of treatment (SVR12). The SVR12 rate was 96% in patients with Child B, but significantly lower, at 50%, in patients with Child C (P < 0.05). In genotype 1b HCV-infected patients, all eight patients without baseline NS5A RASs, but only three of seven patients with RASs, achieved SVR12. Multivariate analysis identified Child B (odds ratio, 35.8 for Child C; P = 0.045) as an independent predictor of SVR12. Median serum albumin levels significantly increased only in patients who achieved SVR12. Child-Pugh scores improved in 16 of 28 patients (57%) following achievement of SVR12. CONCLUSION: The effect of SOF/VEL therapy is lower for patients with Child C. Improvement of hepatic function is expected after viral eradication with SOF/VEL therapy in patients with decompensated cirrhosis.

Risk factors for histological progression of non-alcoholic steatohepatitis analyzed from repeated biopsy cases.

BACKGROUND AND AIM: The most important prognostic factor for non-alcoholic steatohepatitis (NASH) is liver fibrosis. The aim of this study is to examine clinical parameters involved in pathological progression in NASH patients who underwent repeated liver biopsy and to analyze the response to treatment with respect to NASH-related single nucleotide polymorphisms (SNPs). We performed longitudinal analysis of genetic and clinical factors associated with progression of NASH. METHODS: Eighty NASH patients who had undergone serial liver biopsies were enrolled in this retrospective cohort study. Histological exacerbation was determined based on non-alcoholic fatty liver disease activity score (NAS) and liver fibrosis. RESULTS: About 22.5% had progression of fibrosis, 22.5% had improvement of fibrosis, and 55.0% had no change. NAS increased in 12.5%, decreased in 61.3%, and remained stable in the remaining 26.3%. We examined factors associated with histological progression versus non-progression. Poor response of alanine aminotransferase (ALT) levels, increase in HbA1c levels, and presence of the tumor necrosis factor risk allele in the rs1799964 SNP were identified as independent risk factors contributing to histological progression in NASH patients. In addition, we found that the histological progression rate varies with ALT response, HbA1c levels, and rs1799964 genotype. CONCLUSIONS: In this study, we clarified the serum ALT level and the clinical significance of HbA1c to evaluate the progression of fibrosis in Japanese NASH patients. Furthermore, the tumor necrosis factor SNP was more likely to be involved in the response than PNPLA3 SNP. By simultaneously evaluating three factors, it is possible to estimate the risk of histological progression more accurately.

Efficacy of glecaprevir and pibrentasvir treatment for genotype 1b hepatitis C virus drug resistance-associated variants in humanized mice.

Combination therapy with glecaprevir (GLE) and pibrentasvir (PIB) has high efficacy for pan-genotypic hepatitis C virus (HCV)-infected patients. However, the efficacy for patients who acquired potent NS5A inhibitor resistance-associated variants (RAVs) as a result of failure to respond to previous direct-acting antiviral (DAA) therapies is unclear. We investigated the efficacy of GLE/PIB treatment for genotype 1b HCV strains containing RAVs using subgenomic replicon systems and human hepatocyte transplanted mice. Mice were injected with serum samples obtained from a DAA-naïve patient or daclatasvir plus asunaprevir (DCV/ASV) treatment failures including NS5A-L31M/Y93H, -P58S/A92K or -P32 deletion (P32del) RAVs, then treated with GLE/PIB. HCV was eliminated by GLE/PIB treatment in mice with wild-type and NS5A-L31M/Y93H but relapsed in mice with NS5A-P58S/A92K, followed by emergence of additional NS5A mutations after cessation of the treatment. In NS5A-P32del-infected mice, serum HCV RNA remained positive during the GLE/PIB treatment. NS5A-P58S/A92K showed 1.5-fold resistance to PIB relative to wild-type based on analysis using HCV subgenomic replicon systems. When mice were administered various proportions of HCV wild-type and P32del strains and treated with GLE/PIB, serum HCV RNA remained positive in mice with high frequencies of P32del. In these mice, the P32del was undetectable by deep sequencing before GLE/PIB treatment, but P32del strains relapsed after cessation of the GLE/PIB treatment. GLE/PIB is effective for wild-type and NS5A-L31M/Y93H HCV strains, but the effect seems to be low for P58S/A92K and NS5A-P32del RAVs. Although NS5A-P32del was not detected, the mutation may be present at low frequency in DCV/ASV treatment failures.