Next generation sequencing (NGS) has revealed a great deal about cancer-related somatic changes in esophageal squamous cell neoplasia; however, the changes in the very early stages remain unclear.

Combination therapy with ribavirin, interferon, and viral protease inhibitors could be expected to elicit a high level of sustained virologic response in patients infected with hepatitis C virus (HCV). However, several severe side effects of this combination therapy have been encountered in clinical trials. In order to develop more effective and safer anti-HCV compounds, we employed the replicon systems derived from several strains of HCV to screen 84 extracts from 54 organisms that were gathered from the sea surrounding Okinawa Prefecture, Japan. The ethyl acetate-soluble extract that was prepared from marine sponge Amphimedon sp. showed the highest inhibitory effect on viral replication, with EC₅₀ values of 1.5 and 24.9 µg/ml in sub-genomic replicon cell lines derived from genotypes 1b and 2a, respectively. But the extract had no effect on interferon-inducing signaling or cytotoxicity. Treatment with the extract inhibited virus production by 30% relative to the control in the JFH1-Huh7 cell culture system. The in vitro enzymological assays revealed that treatment with the extract suppressed both helicase and protease activities of NS3 with IC₅₀ values of 18.9 and 10.9 µg/ml, respectively. Treatment with the extract of Amphimedon sp. inhibited RNA-binding ability but not ATPase activity. These results suggest that the novel compound(s) included in Amphimedon sp. can target the protease and helicase activities of HCV NS3.

Genetic indicators of endoscopic resection for colorectal carcinoma remain inconclusive. This study analyzed genetic changes in early colorectal tumors that could inform decisions for endoscopic procedures.

Hepatitis C virus (HCV) is a causative agent of acute and chronic hepatitis, leading to the development of hepatic cirrhosis and hepatocellular carcinoma. We prepared extracts from 61 marine organisms and screened them by an in vitro fluorescence assay targeting the viral helicase (NS3), which plays an important role in HCV replication, to identify effective candidates for anti-HCV agents. An ethyl acetate-soluble fraction of the feather star Alloeocomatella polycladia exhibited the strongest inhibition of NS3 helicase activity, with an IC(50) of 11.7 µg/mL. The extract of A. polycladia inhibited interaction between NS3 and RNA but not ATPase of NS3. Furthermore, the replication of the replicons derived from three HCV strains of genotype 1b in cultured cells was suppressed by the extract with an EC(50) value of 23 to 44 µg/mL, which is similar to the IC(50) value of the NS3 helicase assay. The extract did not induce interferon or inhibit cell growth. These results suggest that the unknown compound(s) included in A. polycladia can inhibit HCV replication by suppressing the helicase activity of HCV NS3. This study may present a new approach toward the development of a novel therapy for chronic hepatitis C.

We aimed to evaluate a newly developed peroral cholangioscopy (POCS) classification system by comparing classified lesions with histological and genetic findings. We analyzed 30 biopsied specimens from 11 patients with biliary tract cancer (BTC) who underwent POCS. An original classification of POCS findings was made based on the biliary surface's form (F factor, 4 grades) and vessel structure (V-factor, 3 grades). Findings were then compared with those of corresponding biopsy specimens analyzed histologically and by next-generation sequencing to identify somatic mutations. In addition, the histology of postoperative surgical stumps and preoperative POCS findings were compared. Histological malignancy rate in biopsied specimens increased with increasing F- and V-factor scores (F1, 0%; F1, 25%; F3, 50%; F4, 62.5%; p = 0.0015; V1, 0%; V2, 20%; V3, 70%; p < 0.001). Furthermore, we observed a statistically significant increase of the mutant allele frequency of mutated genes with increasing F- and V-factor scores (F factor, p = 0.0050; V-factor, p < 0.001). All surgical stumps were accurately diagnosed using POCS findings. The F-V classification of POCS findings is both histologically and genetically valid and will contribute to the methods of diagnosing the superficial spread of BTC tumors.

Although comprehensive gene analyses of pancreatic cancer provide new knowledge on molecular mechanisms, the usefulness and possibility of the analyses in routinely available clinical samples remain unclear. We assessed the possibility and utility of target sequencing of endoscopically obtained pancreatic cancer samples. Fifty-eight pancreatic cancer patients who underwent EUS-FNA or endoscopic biopsy were enrolled. The extracted DNA quantity was assessed and used for next-generation sequencing (NGS) of 50 cancer-related genes from which gene mutations, copy number alterations, and microsatellite instability (MSI) were extracted via secondary analysis. A median of 19.2 ng (3.8-228) of DNA was extracted from formalin-fixed paraffin-embedded samples. Gene alterations were detected in 55 of 58 samples (94.8%), including all samples with a DNA concentration below the detection limit (n = 11). Four frequently altered genes were KRAS (83%), TP53 (66%), SMAD4 (26%), and PTEN (17%), and molecular targetable genes were detected in 13 cases (22.4%). Five samples (8.6%) had many mutations and suspected MSI with impaired mismatch repair genes. A Cox regression analysis revealed that metastasis (p < 0.005, hazard ratio [HR] 10.1), serum CEA >5 ng/ml (p = 0.01, HR 2.86), ≤10 detected hotspot mutations (p = 0.03, HR 9.86), and intact Ras signaling (p < 0.005, HR 5.57) were associated with a poor pancreatic cancer prognosis. We performed small, targeted sequencing of pancreatic cancer using available samples from real clinical practice and determined the relationship between gene alterations and prognosis to help determine treatment choices.

Routinely available clinical samples of all stages of pancreatic cancer are used in the present study to elucidate its molecular mechanisms and identify novel therapeutic targets. We evaluated the use of next-generation sequencing (NGS) of endoscopically obtained pancreatic cancer tissues. We enrolled 147 patients who underwent endoscopic ultrasound-guided fine-needle aspiration or endoscopic biopsy. The quantity and quality of the extracted DNA was assessed. Tissue samples were used for NGS of 78 cancer-related genes, from which gene alterations and microsatellite instability (MSI) were extracted. NGS was successful in 141 out of 147 (96%) cases. Gene alterations were detected in 134 out of 141 (91%) samples, among which eight out of 10 samples with a DNA concentration below the detection limit had some type of gene alteration. Targetable genes were detected in 28 (19.9%) cases. MSI and germline mutations in homologous recombination repair associated genes were detected in 5% and 3% of cases, respectively. Cox regression analysis revealed that metastasis (P < .005; hazard ratio [HR], 3.30) was associated with poor prognosis in all pancreatic cancer patients. In addition, fewer than three mutations (P = .03; HR, 2.48) and serum carcinoembryonic antigen levels >5 ng/mL (P < .005; HR, 3.94) were associated with worse prognosis in cases without and with metastasis, respectively. Targeted sequencing of all stages of pancreatic cancer using available samples from real clinical practice could be used to determine the relationship between gene alterations and prognosis to help determine treatment choices.

Although the usefulness of liquid biopsy as a biomarker in the treatment of hepatocellular carcinoma (HCC) has been suggested, its usefulness in transcatheter arterial chemoembolization (TACE) or tyrosine kinase inhibitor (TKI) therapies has not been reported in detail. In this study, we investigated the clinical value of a cell-free (cf)DNA quantification system targeting the human telomerase reverse transcriptase (hTERT) promoter mutation in advanced HCC treatment. Plasma from 67 patients with advanced HCC, treated with TACE and TKI, was used for extraction of cfDNA. We defined cfDNA with the hTERT promoter C228T mutation as circulating mutant DNA (mutant DNA) and without the mutation as circulating wild-type DNA (wild-type DNA). We analyzed the changes in mutant and wild-type DNA levels during HCC treatment and examined the relationship between changes in the cfDNA level and the clinical course. Mutant DNA was detected in 73.1% (49/67) of the patients during HCC treatment. In univariate analysis, factors associated with detection of mutant DNA before treatment were the intrahepatic maximum tumor diameter (P = 0.015) and protein induced by vitamin K absence (PIVKAII) (P = 0.006). The degree of mutant DNA change after TACE was significantly correlated with tumor volume (P < 0.001), reflecting the treated tumor volume. Responders with peak cfDNA levels within 1 week of TKI initiation had significantly better progression-free survival than nonresponders (P = 0.004). Conclusion: Changes in blood hTERT promoter mutant DNA levels during TACE or TKI treatment indirectly reflect the amount of HCCs and are useful for predicting long-term treatment responses.

Sodium taurocholate cotransporting polypeptide (NTCP) has been reported as a functional receptor for hepatitis B virus (HBV) infection. However, HBV could not efficiently infect HepG2 cells expressing NTCP (NTCP-HepG2 cells) under adherent monolayer-cell conditions. In this study, NTCP was mainly detected in the basolateral membrane region, but not the apical site, of monolayer NTCP-HepG2 cells. We hypothesized that non-adherent cell conditions of infection would enhance HBV infectivity. Non-adherent NTCP-HepG2 cells were prepared by treatment with trypsin and EDTA, which did not degrade NTCP in the membrane fraction. HBV successfully infected NTCP-HepG2 cells at a viral dose 10 times lower in non-adherent phase than in adherent phase. Efficient infection of non-adherent NTCP-HepG2 cells with blood-borne or cell-culture-derived HBV was observed and was remarkably impaired in the presence of the myristoylated preS1 peptide. HBV could also efficiently infect HepaRG cells under non-adherent cell conditions. We screened several compounds using our culture system and identified proscillaridin A as a potent anti-HBV agent with an IC50 value of 7.2 nM. In conclusion, non-adherent host cell conditions of infection augmented HBV infectivity in an NTCP-dependent manner, thus providing a novel strategy to identify anti-HBV drugs and investigate the mechanism of HBV infection.

The current treatments of chronic hepatitis B (CHB) face a limited choice of vaccine, antibody and antiviral agents. The development of additional antiviral agents is still needed for improvement of CHB therapy. In this study, we established a screening system in order to identify compounds inhibiting the core promoter activity of hepatitis B virus (HBV). We prepared 80 extracts of marine organisms from the coral reefs of Indonesia and screened them by using this system. Eventually, two extracts showed high inhibitory activity (>95%) and low cytotoxicity (66% to 77%). Solvent fractionation, column chromatography and NMR analysis revealed that 3,5-dibromo-2-(2,4-dibromophenoxy)-phenol (compound 1) and 3,4,5-tribromo-2-(2,4-dibromophenoxy)-phenol (compound 2), which are classified as polybrominated diphenyl ethers (PBDEs), were identified as anti-HBV agents in the extracts. Compounds 1 and 2 inhibited HBV core promoter activity as well as HBV production from HepG2.2.15.7 cells in a dose-dependent manner. The EC50 values of compounds 1 and 2 were 0.23 and 0.80 µM, respectively, while selectivity indexes of compound 1 and 2 were 18.2 and 12.8, respectively. These results suggest that our cell-based HBV core promoter assay system is useful to determine anti-HBV compounds, and that two PBDE compounds are expected to be candidates of lead compounds for the development of anti-HBV drugs.

Caffeic acid phenethyl ester (CAPE) has been reported as a multifunctional compound. In this report, we tested the effect of CAPE and its derivatives on hepatitis C virus (HCV) replication in order to develop an effective anti-HCV compound. CAPE and CAPE derivatives exhibited anti-HCV activity against an HCV replicon cell line of genotype 1b with EC50 values in a range from 1.0 to 109.6 µM. Analyses of chemical structure and antiviral activity suggested that the length of the n-alkyl side chain and catechol moiety are responsible for the anti-HCV activity of these compounds. Caffeic acid n-octyl ester exhibited the highest anti-HCV activity among the tested derivatives with an EC50 value of 1.0 µM and an SI value of 63.1 by using the replicon cell line derived from genotype 1b strain Con1. Treatment with caffeic acid n-octyl ester inhibited HCV replication of genotype 2a at a similar level to that of genotype 1b irrespectively of interferon signaling. Caffeic acid n-octyl ester could synergistically enhance the anti-HCV activities of interferon-alpha 2b, daclatasvir, and VX-222, but neither telaprevir nor danoprevir. These results suggest that caffeic acid n-octyl ester is a potential candidate for novel anti-HCV chemotherapy drugs.

The 78-kDa glucose-regulated protein (GRP78) is an important molecular chaperone in the endoplasmic reticulum (ER) induced by various stresses. This study showed that stimulation with anti-CD3 mAb, PMA plus ionomycin, or an antigen increased the levels of GRP78 mRNA in primary T cells, which was inhibited by Ca(2+) chelators EGTA and BAPTA-AM and by an inhibitor of calcineurin FK506. In addition, the specific knockdown of GRP78 protein expression induced apoptosis in mouse EL-4 T cell line associated with CHOP induction and caspase-3 activation. Furthermore, overexpression of GRP78 inhibited PMA/ionomycin-induced cell death in EL-4 cells. Collectively, GRP78 expression is induced by TCR activation via a Ca(2+)-dependent pathway and may play a critical role in maintaining T cell viability in the steady and TCR-activated states. These results suggest a novel regulatory mechanism and an essential function of GRP78 in T cells.

Presarcopenia is a prognostic factor in patients with hepatocellular carcinoma (HCC). The Japan integrated staging (JIS) score is a prognostic method that combines the Child-Turcotte-Pugh classification and the tumor-node-metastasis (TNM) staging for HCC. We investigated the relationship between presarcopenia, the JIS score, and prognosis in patients with primary HCC. This retrospective study included 153 patients with primary HCC who were hospitalized from October 2011 to March 2018 at Municipal Hospital of Kofu. The skeletal muscle mass was measured using simplified psoas muscle mass index (PMI) based on CT imaging, and PMI using the volume analyzer SYNAPSE VINCENT ver3.0. We diagnosed presarcopenia based on the cut off value according to the assessment criteria for sarcopenia in liver disease defined by the Japan Society of Hepatology. Forty-three patients (28%) were diagnosed with presarcopenia. The median event-free survival was significantly worse in patients with presarcopenia than those without presarcopenia (P = 0.016). In multivariate analysis, presence of presarcopenia, JIS score ≥3, alpha-fetoprotein ≥200 ng/ml, and prothrombin induced by vitamin K absence-II ≥ 200 mAU/ml were significant prognostic factors. Among the patients with JIS scores ≥3, there was no difference in the event occurrence rate with presence of presarcopenia (P = 0.96). Among the patients with JIS scores ≤2, the median event-free-survival was significantly shorter in those with presarcopenia than those without presarcopenia (P = 0.045). Presarcopenia was an independent prognostic factor in patients with primary HCC. In patients with JIS scores ≤2, the median event-free survival was significantly shorter in those with presarcopenia compared to those without presarcopenia. In the patients with JIS scores ≥3, there was no difference in the event occurrence rates in those with and without presarcopenia.

Liquid biopsy is a method that can efficiently detect tumor genetic abnormalities from body fluids such as blood and urine. Detection sensitivity and the available number of mutations in cell-free DNA (cfDNA) are limited. In this study, we develop a highly sensitive and comprehensive method to detect mutations from cfDNA by concentrating tumor fractions of small cfDNA in advanced colorectal cancers.

The method of analyzing individual resistant hepatitis C virus (HCV) by a combination of haplotyping and resistance-associated substitution (RAS) has not been fully elucidated because conventional sequencing has only yielded short and fragmented viral genomes. We performed haplotype analysis of HCV mutations in 12 asunaprevir/daclatasvir treatment-failure cases using the Oxford Nanopore sequencer. This enabled single-molecule long-read sequencing using rolling circle amplification (RCA) for correction of the sequencing error. RCA of the circularized reverse-transcription polymerase chain reaction products successfully produced DNA longer than 30 kilobase pairs (kb) containing multiple tandem repeats of a target 3 kb HCV genome. The long-read sequencing of these RCA products could determine the original sequence of the target single molecule as the consensus nucleotide sequence of the tandem repeats and revealed the presence of multiple viral haplotypes with the combination of various mutations in each host. In addition to already known signature RASs, such as NS3-D168 and NS5A-L31/Y93, there were various RASs specific to a different haplotype after treatment failure. The distribution of viral haplotype changed over time; some haplotypes disappeared without acquiring resistant mutations, and other haplotypes, which were not observed before treatment, appeared after treatment. Conclusion: The combination of various mutations other than the known signature RAS was suggested to influence the kinetics of individual HCV quasispecies in the direct-acting antiviral treatment. HCV haplotype dynamic analysis will provide novel information on the role of HCV diversity within the host, which will be useful for elucidating the pathological mechanism of HCV-related diseases.

The clinical applicability of digital next-generation sequencing (dNGS), which eliminates polymerase chain reaction (PCR) and sequencing error-derived noise by using molecular barcodes (MBs), has not been fully evaluated. We evaluated the utility of dNGS of cell-free DNA (cfDNA) in liquid biopsies obtained from patients with pancreatic cancer.

The chaperone system is known to be exploited by viruses for their replication. In the present study, we identified the cochaperone FKBP6 as a host factor required for hepatitis C virus (HCV) replication. FKBP6 is a peptidyl prolyl cis-trans isomerase with three domains of the tetratricopeptide repeat (TPR), but lacks FK-506 binding ability. FKBP6 interacted with HCV nonstructural protein 5A (NS5A) and also formed a complex with FKBP6 itself or FKBP8, which is known to be critical for HCV replication. The Val(121) of NS5A and TPR domains of FKBP6 were responsible for the interaction between NS5A and FKBP6. FKBP6 was colocalized with NS5A, FKBP8, and double-stranded RNA in HCV-infected cells. HCV replication was completely suppressed in FKBP6-knockout hepatoma cell lines, while the expression of FKBP6 restored HCV replication in FKBP6-knockout cells. A treatment with the FKBP8 inhibitor N-(N', N'-dimethylcarboxamidomethyl)cycloheximide impaired the formation of a homo- or hetero-complex consisting of FKBP6 and/or FKBP8, and suppressed HCV replication. HCV infection promoted the expression of FKBP6, but not that of FKBP8, in cultured cells and human liver tissue. These results indicate that FKBP6 is an HCV-induced host factor that supports viral replication in cooperation with NS5A.

Deletions are observed frequently in the preS1/S2 region of hepatitis B virus (HBV) genome, in association with liver disease advancement. However, the most significant preS1/S2 region and its influences on viral markers are unclear.

We have reported previously that synthetic small interfering RNA (siRNA) and DNA-based siRNA expression vectors efficiently and specifically suppress hepatitis C virus (HCV) replication in vitro. In this study, we investigated the effects of the siRNA targeting HCV-RNA in vivo.

The genetic changes underlying carcinogenesis in patients with risk factors of gallbladder carcinoma (GBC) remains controversial, especially in patients with pancreaticobiliary maljunction (PBM). This study aimed to clarify the association between risk factors of GBC and genetic changes using next-generation sequencing (NGS).