Clinicopathological and genomic features of superficial esophageal squamous cell carcinomas in nondrinker, nonsmoker females.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is sometimes detected in non-drinker and non-smoker females who are considered to have very low risk of ESCC development in daily practice. This study examined the clinicopathological and genomic characteristics of ESCCs in females with no history of drinking and smoking. METHODS: The sample comprised 118 ESCC lesions occurring in 95 female patients who underwent endoscopic submucosal dissection at our department between January 2008 and December 2019. The patients were categorized into two groups: 51 lesions in 49 patients with no history of drinking and smoking (nondrinker/nonsmoker [NDNS] group) and 69 lesions in 45 patients with a history of drinking or smoking (drinker/smoker [DS] group). We analyzed the differences in clinicopathological and cancerous genomic characteristics between the groups. Significant genomic alterations were validated using immunohistochemistry. RESULTS: Multiple logistic regression revealed that older age, fewer multiple Lugol-voiding lesions (LVLs), and reflux esophagitis (RE) were independently associated with the occurrence of ESCCs in the NDNS group. ESCC lesions in the NDNS group were predominantly located in the mid-thoracic esophagus, posterior wall side, with 0-IIa, the aspect ratio of the lesion >2 (vertical/horizontal), and endoscopic keratinization. Genetic analysis showed that CDKN2A driver alterations were significantly more frequent and KMT2D alterations were significantly less frequent in the NDNS group than in the DS group. KMT2D alterations were strongly correlated with immunostaining. CONCLUSION: Older nondrinker, nonsmoker females with RE and fewer multiple LVLs may develop longitudinal 0-IIa ESCC with keratinization of the posterior wall of the mid-thoracic esophagus. ESCCs in nondrinker, nonsmoker females had fewer KMT2D alterations and more CDKN2A alterations, which may be a biomarker for treatment.

Multi-omics analysis of a fatty liver model using human hepatocyte chimeric mice.

We developed a fatty liver mouse model using human hepatocyte chimeric mice. As transplanted human hepatocytes do not respond to mouse growth hormone (GH) and tend to accumulate fat, we hypothesized that addition of human GH would alter lipid metabolism and reduce accumulation of fat in the liver even when fed a high-fat diet. Six uPA/SCID chimeric mice were fed a high-fat GAN diet to induce fatty liver while six were fed a normal CRF1 diet, and GH was administered to three mice in each group. The mice were euthanized at 8 weeks, and human hepatocytes were extracted for RNA-Seq, DIA proteomics, and metabolomics analysis. Abdominal echocardiography revealed that the degree of fatty liver increased significantly in mice fed GAN diet (p < 0.001) and decreased significantly in mice treated with GH (p = 0.026). Weighted gene correlation network analysis identified IGF1 and SEMA7A as eigengenes. Administration of GH significantly reduced triglyceride levels and was strongly associated with metabolism of amino acids. MiBiOmics analysis identified perilipin-2 as a co-inertia driver. Results from multi-omics analysis revealed distinct gene expression and protein/metabolite profiles in each treatment group when mice were fed a high-fat or normal diet with or without administration of GH.

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.

Lenvatinib activates anti-tumor immunity by suppressing immunoinhibitory infiltrates in the tumor microenvironment of advanced hepatocellular carcinoma.

BACKGROUND: Lenvatinib, a multiple receptor tyrosine kinase inhibitor, might exert antitumor effects via tumor immune modulation. However, changes in the tumor immune microenvironment induced by lenvatinib are poorly understood. We investigated the effect of lenvatinib on immune features in clinical samples from patients with hepatocellular carcinoma. METHODS: Fifty-one patients with advanced hepatocellular carcinoma who received lenvatinib monotherapy as first-line treatment were enrolled. We collected blood sample (n = 51) and tumor tissue (n, baseline/four weeks after treatment initiation/post-progression = 50/8/12). DNA, RNA, and proteins extracted from the tissues were subjected to multi-omics analysis, and patients were classified into two groups according to baseline immune status. Each group was investigated in terms of the dynamics of tumor signaling. We also longitudinally analyzed circulating immune proteins and chemokines in peripheral blood. RESULTS: Here we show that lenvatinib has similar anti-tumor efficacy with objective response rate and progression-free survival in both Immune-Hot and Immune-Cold subtypes. Immune signatures associated with T-cell functions and interferon responses are enriched in the early phase of treatment, while signatures associated with immunoinhibitory cells are downregulated along with efficient vascular endothelial growth factor receptor and fibroblast growth factor receptor blockades. These findings are supported by imaging mass cytometry, T-cell receptor repertoire analysis and kinetics of circulating proteins. We also identify interleukin-8 and angiopoietin-2 as possible targets of intervention to overcome resistance to existing immunotherapies. CONCLUSIONS: Our findings show the ability of lenvatinib to modulate tumor immunity in clinical samples of hepatocellular carcinoma.

Two types of therapy for liver cancer are immunotherapy and anti-angiogenic therapy. Immunotherapy helps the patient's immune system to attack the tumor. Anti-angiogenic therapy blocks the formation of new blood vessels (angiogenesis) in the tumor, and this type of therapy might also impact the immune system. We analyzed changes in the immune characteristics of human liver cancer samples induced by lenvatinib, an anti-angiogenic therapy. Patient outcomes on lenvatinib did not depend on the immune features of the tumor before treatment. However, immune characteristics of the tumors did change after treatment, and this may mean these tumors become easier to treat with immunotherapies. These findings help us to understand the effects of lenvatinib in liver cancer and whether, for example, it might be useful to combine this drug with immunotherapy.

Deficiency of SCAP inhibits HBV pathogenesis via activation of the interferon signaling pathway.

Hepatitis B virus (HBV) infects the liver and is a major risk factor for liver cirrhosis and hepatocellular carcinoma. Approaches for an effective cure are thwarted by limited knowledge of virus-host interactions. Herein, we identified SCAP as a novel host factor that regulates HBV gene expression. SCAP, sterol regulatory element-binding protein (SREBP) cleavage-activating protein, is an integral membrane protein located in the endoplasmic reticulum. The protein plays a central role in controlling lipid synthesis and uptake by cells. We found that gene silencing of SCAP significantly inhibited HBV replication; furthermore, knockdown of SREBP2 but not SREBP1, the downstream effectors of SCAP, reduced HBs antigen production from HBV infected primary hepatocytes. We also demonstrated that knockdown of SCAP resulted in activation of interferons (IFNs) and IFN stimulated genes (ISGs). Conversely, ectopic expression of SREBP2 in SCAP-deficient cells restored expression of IFNs and ISGs. Importantly, expression of SREBP2 restored HBV production in SCAP knockdown cells, suggesting that SCAP participates in HBV replication through an effect on IFN production via its downstream effector SREBP2. This observation was further confirmed by blocking IFN signaling by an anti-IFN antibody, which restored HBV infection in SCAP-deficient cells. This led to the conclusion that SCAP regulates the IFN pathway through SREBP, thereby affecting the HBV lifecycle. This is the first study to reveal the involvement of SCAP in regulation of HBV infection. These results may facilitate development of new antiviral strategies against HBV.

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.

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.

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.

The burden of Hepatitis B virus infection in Kenya: A systematic review and meta-analysis.

Background: Chronic Hepatitis B virus (HBV) infection causes liver cirrhosis and cancer and is a major public health concern in Kenya. However, so far no systematic review and meta-analysis has been conducted to estimate the burden of disease in the country. A better understanding of HBV infection prevalence will help the government implement efficient strategies at eliminating the disease. This systematic review and meta-analysis was therefore conducted to summarize and update the available information on the burden of HBV in Kenya. Method: We systematically searched PubMed, Science Direct, Web of Science, Scopus, African Journals OnLine, and Google Scholar databases to retrieve primary studies conducted between January 1990 and June 2021 that assessed the prevalence of HBV infection in Kenya based on measurement of the Hepatitis B Surface Antigen (HBsAg). Meta-analysis was performed using the random effects model where HBsAg prevalence was estimated at a 95% confidence interval (CI) after simple pooling analysis. Potential sources of heterogeneity were also investigated. Results: Fifty studies were included in the meta-analysis with a sample size of 108448. The overall pooled prevalence estimate of HBV in Kenya was 7.8% (95% CI: 5.8-10.1). Subgroup analysis revealed the highest prevalence among patients presenting with jaundice at 41.7% (95% CI: 13.5-73.3) whereas blood donors had the lowest prevalence at 4.1% (95% CI: 2.4-6.3). Prevalence in Human Immunodeficiency Virus (HIV)-infected individuals was 8.2% (95% CI: 5.8-11.0). An estimate of the total variation between studies revealed substantial heterogeneity (I2 = 99%) which could be explained by the study type, the risk status of individuals, and the region of study. Conclusion: We present the first systematic review and meta-analysis of the prevalence of HBV in Kenya. Our results show that the burden of HBV in Kenya is still enormous. This calls for an urgent need to implement public health intervention measures and strategic policies that will bring the disease under control and lead to final elimination. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=264859, identifier: CRD42021264859.

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.

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.

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.

Construction of an anti-hepatitis B virus preS1 antibody and usefulness of preS1 measurement for chronic hepatitis B patients: Anti-HBV PreS1 antibody.

OBJECTIVES: The preS1 region plays an essential role in hepatitis B virus (HBV) infection. We construct an antibody that binds to preS1 and a measurement system for serum preS1 in chronic HBV-infected patients. METHODS: Hybridoma clones that produce anti-preS1 antibodies were obtained by the iliac lymph node method. Epitope mapping was conducted, and an enzyme-linked immunosorbent assay (ELISA)-based method was developed. Using this ELISA system, serum preS1 levels were measured in 200 chronic HBV-infected patients. RESULTS: Eight types of hybridomas were obtained, of which antibody 3-55 using amino acids 38-47 as the epitope showed high binding affinity to preS1. Serum preS1 levels measured by ELISA using 3-55 antibody were correlated with HBsAg, HBcrAg and HBV DNA levels. Among HBeAg-negative patients without antiviral therapeutic objective (HBV DNA <3.3 log IU/mL or alanine aminotransferase ≤30 U/L), preS1 was significantly higher in subjects who had progressed to the point of requiring antiviral therapy compared to subjects who had maintained their status for the preceding three years (p<0.01). CONCLUSIONS: We constructed an antibody against preS1 and an ELISA system capable of measuring serum preS1 levels. PreS1 may serve as a novel tool to predict the need for antiviral therapy in HBeAg-negative HBV-infected patients.

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.

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.

Identification and monitoring of mutations in circulating cell-free tumor DNA in hepatocellular carcinoma treated with lenvatinib.

BACKGROUND: There has been a recent surge in interest in predicting biological effects associated with genomic alterations in order to implement personalized cancer treatment strategies. However, no reports have yet evaluated the utility of profiling blood-based circulating tumor DNA (ctDNA) in hepatocellular carcinoma (HCC) patients treated with lenvatinib (LEN). METHOD: We retrospectively performed ctDNA next-generation sequencing (NGS) analysis in 24 patients with advanced HCC at baseline and 4 weeks after initiation of LEN. Association of the changes in variant allele frequencies (VAFs) during treatment and clinical outcome were evaluated. RESULTS: In total, 131 single nucleotide variants, 17 indels, and 23 copy number variations were detected as somatic alterations in 28, 6, and 12 genes, respectively in 23 of 24 patients. The most frequently altered genes were TP53 (54%), CTNNB1 (42%), TERT (42%), ATM (25%), and ARID1A (13%). The reduction in the mean frequency of variants (VAFmean) following 4 weeks of LEN treatment was associated with longer progression-free survival. The specificity and sensitivity of the reduction of VAFmean for predicting partial response were 0.67 and 1.0, respectively, which were higher than those of serum α-fetoprotein level (0.10 and 0.93, respectively). No association between the mutation status at baseline and the effectiveness of LEN was observed. CONCLUSION: Our study demonstrated that somatic alterations could be detected in the majority of advanced HCC patients by ctDNA profiling and that ctDNA-kinetics during LEN treatment was a useful marker of disease progression. These results suggest that ctDNA profiling is a promising method that provides valuable information in clinical practice.

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.

A case of acute liver failure with echovirus infection diagnosed by a multi-virus real-time PCR system.

BACKGROUND: Multi-virus real-time polymerase chain reaction (PCR) system is able to simultaneously detect 163 viruses using a multiplex Taqman real-time PCR system. We present a case of acute liver failure (ALF) of unknown etiology diagnosed with　echovirus 30 infection via multi-virus real-time PCR. CASE PRESENTATION: A previously healthy 66-year-old man had a persistent fever and developed ALF of unclear etiology. Although viral infection was suspected, serological screening showed no evidence of acute viral infections such as hepatitis A, B, C and E, Epstein-Barr virus, herpes simplex virus, and varicella zoster virus. Multi-virus real-time PCR revealed the presence of enterovirus and echovirus 30 genomes, and reverse transcription-PCR using enterovirus-specific primers confirmed the presence of enterovirus genome in serum samples at the time of admission. Anti-echovirus antibody titers showed an increase in paired sera. In spite of multimodality treatment, the patient died due to multiple organ failure. Histological analysis in autopsy revealed extensive coagulative necrosis of the hepatocytes and immunohistochemical analysis showed the expression of enterovirus antigens in necrotic hepatocytes. CONCLUSIONS: We present here a case of echovirus 30 associated with ALF. Multi-virus real-time PCR is useful for detection of virus for patients with ALF of unknown etiology suspected of harboring a viral infection.

Efficacy of Lusutrombopag for Thrombocytopenia in Patients with Chronic Liver Disease Scheduled to Undergo Invasive Procedures.

Objective Lusutrombopag is a thrombopoietin receptor agonist that improves thrombocytopenia in patients with chronic liver disease scheduled to undergo invasive procedures. However, information on the efficacy of repeated lusutrombopag treatment and factors associated with the treatment is scarce. We analyzed the efficacy of repeated lusutrombopag treatment and the factors associated with a response to lusutrombopag. Methods Thirty-nine patients with chronic liver disease who received lusutrombopag treatment before undergoing invasive procedures were enrolled in this retrospective study. Of the 39 patients, 10 received lusutrombopag treatment multiple times for a total of 53 regimens of lusutrombopag treatment. Changes in platelet counts, the effects of repeated lusutrombopag treatment, and factors associated with response to lusutrombopag were analyzed. Results The median platelet count increased significantly from 4.5×104/µL before lusutrombopag treatment to 7.2×104/µL before the invasive procedure (p<0.01), and patients undergoing 49 of the 53 (92%) treatment regimens succeeded in undergoing invasive procedures without needing platelet transfusions. In patients who received lusutrombopag treatment repeatedly, the median platelet count significantly increased following the second administration of lusutrombopag, and the effects of lusutrombopag were similar between the first and second administration. A multivariate analysis identified the absence of diabetes mellitus (odds ratio, 5.56 for presence; p=0.04) as a significant and independent predictor of a response to lusutrombopag. Conclusion Lusutrombopag treatment significantly increased platelet counts in patients with chronic liver disease, making it possible to receive invasive procedures. The treatment produced identical effects when it was repeated. The efficacy of lusutrombopag might be decreased in patients with diabetes mellitus.

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.