Significance of serum IP-10/CXCL10 measurement in predicting post-direct acting antiviral treatment liver function in patients with HCV-decompensated liver cirrhosis.

BACKGROUND: Recently, with the advent of sofosbuvir/velpatasvir therapy, sustained virological response (SVR) can now be achieved even in patients with decompensated cirrhosis (dLC). However, the prognosis after SVR does not always improve in dLC, and appropriate indicators enabling prediction of prognosis is desired. PATIENTS AND METHODS: Serum IP-10/CXCL10 levels were measured in 47 patients (15 chronic hepatitis [CH], 17 compensated cirrhosis [cLC], and 15 dLC) receiving direct acting antiviral (DAA) therapy, and their changes during the therapy were examined. RESULTS: All the patients achieved SVR. In patients with CH, the average IP-10 level was 367, 102, and 68 pg/ml respectively at baseline, at the end of therapy and at 12 weeks after SVR (SVR12), and was decreased upon DAA therapy (P < 0.001). In patients with cLC, IP-10 was respectively 215, 91, and 77 pg/ml, and was decreased upon DAA therapy (P < 0.001) while it was 283, 131, and 182 pg/ml in patients with dLC and there was no evident decrease (P = 0.55). When patients with dLC were further classified depending on the difference in Child-Pugh (CP) score improvement at SVR12, a significant decrease in IP-10 was observed after treatment in those with improvement (P = 0.023) while a significant increase was observed in those without improvement (P = 0.016). CONCLUSION: While serum IP-10 level was decreased in patients with CH/cLC and dLC with post-SVR-CP improvement following SVR, it was increased in patients with dLC without post-SVR CP improvement. The result indicates that IP-10 dynamics may be useful for predicting liver function after DAA therapy.

Deep sequencing analysis of serum hepatitis B virus-RNA during nucleot(s)ide analogue therapy.

AIM: Recently, serum hepatitis B virus (HBV)-RNA has been reported to be detectable even when HBV particle production is inhibited by nucleot(s)ide analogues (NAs). However, the dynamics of the HBV-RNA sequence compared with those of HBV-DNA during the emergence of antiviral resistance are yet to be elucidated. METHODS: First, we quantified serum HBV-RNA in 181 infected patients, and its relationships with clinical characteristics as well as HBV markers were investigated. Next, we undertook simultaneous deep sequencing of HBV-RNA/HBV-DNA and their dynamics among four patients receiving NA therapy who were experiencing viral breakthrough. RESULTS: Serum HBV-RNA was detected in 25% (31/123) of cases among patients with HBV without NAs, and the detection rate was significantly high in hepatitis B e antigen-positive cases with high viral activity. In patients with chronic hepatitis, hepatitis B core-related antigen was significantly correlated with serum HBV-RNA irrespective of NA use. In the analysis of the four patients experiencing viral breakthrough, no NA resistance mutation was detected in the serum HBV-RNA immediately before the breakthrough. However, NA-resistant sequences appeared at the rates of 0%, 3%, 14%, and 100%, and the NA-resistant HBV-RNA sequence rate was correlated with the peak HBV-DNA titer multiplied by the HBV-DNA detection duration during the breakthrough (R2 = 0.978) observed before redisappearance of HBV-DNA following the addition of new NA. CONCLUSION: Serum HBV-RNA could reflect the transcriptional activity of covalently closed circular DNA and hepatitis B core-related antigen. The dynamics of HBV-RNA could help understanding of the turnover process of HBV covalently closed circular DNA in the liver.

MiR-10a in Pancreatic Juice as a Biomarker for Invasive Intraductal Papillary Mucinous Neoplasm by miRNA Sequencing.

New biomarkers are needed to further stratify the risk of malignancy in intraductal papillary mucinous neoplasm (IPMN). Although microRNAs (miRNAs) are expected to be stable biomarkers, they can vary owing to a lack of definite internal controls. To identify universal biomarkers for invasive IPMN, we performed miRNA sequencing using tumor-normal paired samples. A total of 19 resected tissues and 13 pancreatic juice samples from 32 IPMN patients were analyzed for miRNA expression by next-generation sequencing with a two-step normalization of miRNA sequence data. The miRNAs involved in IPMN associated with invasive carcinoma were identified from this tissue analysis and further verified with the pancreatic juice samples. From the tumor-normal paired tissue analysis of the expression levels of 2792 miRNAs, 20 upregulated and 17 downregulated miRNAs were identified. In IPMN associated with invasive carcinoma (INV), miR-10a-5p and miR-221-3p were upregulated and miR-148a-3p was downregulated when compared with noninvasive IPMN. When these findings were further validated with pancreatic juice samples, miR-10a-5p was found to be elevated in INV (p = 0.002). Therefore, three differentially expressed miRNAs were identified in tissues with INV, and the expression of miR-10a-5p was also elevated in pancreatic juice samples with INV. MiR-10a-5p is a promising additional biomarker for invasive IPMN.

Cancer-related genetic changes in multistep hepatocarcinogenesis and their correlation with imaging and histological findings.

AIM: The landscape of cancer-related genetic aberrations in hepatocellular carcinoma (HCC) has gradually become clear through recent next-generation sequencing studies. However, it remains unclear how genetic aberrations correlate with imaging and histological findings. METHODS: Using 117 formalin-fixed paraffin-embedded specimens of primary liver tumors, we undertook targeted next-generation sequencing of 50 cancer-related genes and digital polymerase chain reaction of hTERT. After classifying tumors into several imaging groups by hierarchal clustering with the information from gadoxetic acid enhanced magnetic resonance imaging, contrast-enhanced computed tomography, contrast-enhanced ultrasound, and diffusion-weighted imaging magnetic resonance imaging, the correlation between genetic aberrations and imaging and histology were investigated. RESULTS: Most frequent mutations were hTERT (61.5%), followed by TP53 (42.7%), RB1 (24.8%), and CTNNB1 (18.8%). Liver tumors were classified into six imaging groups/grades, and the prevalence of hTERT mutations tended to increase with the advancement of imaging/histological grades (P = 0.026 and 0.13, respectively), whereas no such tendency was evident for TP53 mutation (P = 0.78 and 1.00, respectively). Focusing on the mutations in each tumor, although the variant frequency (VF) of hTERT did not change (P = 0.36 and 0.14, respectively) in association with imaging/histological grades, TP53 VF increased significantly (P = 0.004 and <0.001, respectively). In multivariate analysis, stage III or IV (hazard ratio, 3.64; P = 0.003), TP53 VF ≥ 50% (hazard ratio, 3.79; P = 0.020) was extracted as an independent risk for recurrence in primary HCC patients. CONCLUSIONS: Increased prevalence of hTERT mutation and increased TP53 mutation VF are characteristic features of HCC progression, diagnosed with imaging/histological studies.

Form-Vessel Classification of Cholangioscopy Findings to Diagnose Biliary Tract Carcinoma's Superficial Spread.

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.

Identification of early genetic changes in well-differentiated intramucosal gastric carcinoma by target deep sequencing.

BACKGROUND AND AIMS: The recent advancement of next-generation sequencing (NGS) has enabled the identification of cancer-related somatic aberrations in advanced gastric cancer. However, these remain unclear in early gastric cancers, especially in intramucosal gastric cancers. PATIENTS AND METHODS: Thirty-one well-differentiated (tub1) intramucosal gastric cancers obtained by endoscopic submucosal dissection (ESD) from 29 patients were analyzed. After precise collection of tumors and non-tumors from formalin-fixed paraffin-embedded tissues using laser-captured microdissection (LCM), target sequencing analysis of 50 cancer-related genes was performed using an Ion-Proton sequencer. RESULTS: The most frequent hotspot mutation was found in TP53 (17 lesions, 54.8%) followed by the Wnt-signaling pathway genes, APC and CTNNB1 (6 lesions, 19.4%). The mutations in TP53 and the Wnt-signaling genes were mutually exclusive (p = 0.004). There was a tendency that H. pylori infection (p = 0.082) and macroscopic protrusion (p = 0.095) was associated with the presence of these mutations. Only 10 lesions (59%) among 17 lesions with proven TP53 mutations were positive for p53 immunostaining demonstrating the superiority of the mutational analysis. In addition, focal gene amplification of ERBB2 (16%) was found frequently in these early stage lesions. CONCLUSIONS: Using LCM and NGS, mutations in TP53 and the Wnt-signaling pathway were frequently found and were mutually exclusive in the earliest stage of gastric carcinogenesis.

Hepatitis B virus (HBV)-infected patients with low hepatitis B surface antigen and high hepatitis B core-related antigen titers have a high risk of HBV-related hepatocellular carcinoma.

AIM: Although the viral markers hepatitis B surface antigen (HBsAg) and hepatitis B core-related antigen (HbcrAg) could reflect intrahepatic hepatitis B virus (HBV) replication activity and constitute important biomarkers for hepatocellular carcinoma (HCC), the value of using these two markers in combination for assessing HCC risk has not been clarified in detail. METHODS: Four hundred and forty-nine consecutive patients with chronic HBV infection were included in the study and the association of HBsAg and HBcrAg with HCC risk was investigated cross-sectionally, as well as longitudinally. RESULTS: When the high value cut-offs of HBsAg and HBcrAg were defined as 3.0 log IU/mL and 3.0 log U/mL, respectively, patients with a history of HCC were found frequently in the low HBsAg group (P = 0.002) and high HBcrAg group (P < 0.001). When HBsAg and HBcrAg were combined, an HCC history was most frequent in the subset with low HBsAg and high HBcrAg, among the HBeAg-negative patients (odds ratio [OR], 7.83; P < 0.001), irrespective of nucleos(t) ide analogue (NA) therapy (NA: OR, 4.76; P < 0.001; non-NA: OR, 9.60; P < 0.001). In a longitudinal analysis of the subsequent development of HCC, carried out on the 338 patients without an HCC history at enrollment, HCC developed significantly more frequently in the low HBsAg/high HBcrAg group (P = 0.005). CONCLUSIONS: Patients with low HBsAg/high HBcrAg values are at high risk of developing HBV-related HCC, according to this cross-sectional and longitudinal analysis, indicating that the combination of HBsAg and HBcrAg values is an excellent biomarker for assessing HCC risk.

Deep sequencing and phylogenetic analysis of variants resistant to interferon-based protease inhibitor therapy in chronic hepatitis induced by genotype 1b hepatitis C virus.

UNLABELLED: Because of recent advances in deep sequencing technology, detailed analysis of hepatitis C virus (HCV) quasispecies and their dynamic changes in response to direct antiviral agents (DAAs) became possible, although the role of quasispecies is not fully understood. In this study, to clarify the evolution of viral quasispecies and the origin of drug-resistant mutations induced by interferon (IFN)-based protease inhibitor therapy, the nonstructural-3 (NS3) region of genotype 1b HCV in 34 chronic hepatitis patients treated with telaprevir (TVR)/pegylated interferon (PEG-IFN)/ribavirin (RBV) was subjected to a deep sequencing study coupled with phylogenetic analysis. Twenty-six patients (76.5%) achieved a sustained viral response (SVR), while 8 patients did not (non-SVR; 23.5%). When the complexity of the quasispecies was expressed as the mutation frequency or Shannon entropy value, a significant decrease in the IFNL3 (rs8099917) TT group and a marginal decrease in the SVR group were found soon (12 h) after the introduction of treatment, whereas there was no decrease in the non-SVR group and no significant decrease in mutation frequency in the IFNL3 TG/GG group. In the analysis of viral quasispecies composition in non-SVR patients, major populations greatly changed, accompanied by the appearance of resistance, and the compositions were unlikely to return to the pretreatment composition even after the end of therapy. Clinically TVR-resistant variants were observed in 5 non-SVR patients (5/8, 62.5%), all of which were suspected to have acquired resistance by mutations through phylogenetic analysis. In conclusion, results of the study have important implications for treatment response and outcome in interferon-based protease inhibitor therapy. IMPORTANCE: In the host, hepatitis C virus (HCV) consists of a variety of populations (quasispecies), and it is supposed that dynamic changes in quasispecies are closely related to pathogenesis, although this is poorly understood. In this study, recently developed deep sequencing technology was introduced, and changes in quasispecies associated with telaprevir (TVR)/pegylated interferon (PEG-IFN)/ribavirin (RBV) triple therapy and their clinical significance were investigated extensively by phylogenetic tree analysis. Through this study, the associations among treatment response, changes in viral quasispecies complexity in the early stage of treatment, changes in the quasispecies composition, and origin of TVR-resistant variant HCV were elucidated.

Clinical characteristics and prognosis of non-B non-C hepatocellular carcinoma patients with modest alcohol consumption.

AIM: Alcoholic hepatocellular carcinoma (ALD-HCC) accounts for the majority of non-B non-C HCC (NBNC-HCC) cases. Although alcohol is a potent carcinogen, there have been few reports on the influence of modest alcohol consumption in NBNC-HCC. This study aimed to investigate the clinical characteristics and prognosis of NBNC-HCC patients with modest alcohol consumption. METHODS: From 2007 to 2010, 2283 HCC patients were evaluated at 10 hospitals. We collected detailed etiology data of 588 NBNC-HCC patients and compared the clinical characteristics and prognosis between ALD-HCC and modest alcohol-HCC patients. RESULTS: There were 69 HCC patients with modest alcohol consumption, accounting for 3% of all HCC patients evaluated. This patient group had significantly more women and higher prevalence of Child-Pugh class A, hypertension and advanced disease stage, and were diagnosed with HCC at an older age than the ALD-HCC group (266 patients). Additionally, among the modest alcohol-HCC patients, diabetes was significantly more common in the anti-hepatitis B core (HBc) negative subgroup than in the anti-HBc positive subgroup. However, no significant difference in survival was observed between the two patient groups regardless of significant differences in tumor staging. Alcohol consumption and metabolic factors were not significant independent predictors of survival. CONCLUSION: The clinical characteristics of modest alcohol-HCC included advanced staging, favorable liver reserve capacity and older age at diagnosis. HCC development in patients with modest alcohol consumption may relate to metabolic factors. Although approximately 30% of the evaluated HCC cases were in advanced stages, the prognosis of NBNC-HCC patients with modest alcohol consumption was relatively favorable.

Liver stiffness measurement for risk assessment of hepatocellular carcinoma.

AIM: Liver fibrosis is a risk factor for hepatocellular carcinoma (HCC), but at what fibrotic stage the risk for HCC is increased has been poorly investigated quantitatively. This study aimed to determine the appropriate cut-off value of liver stiffness for HCC concurrence by FibroScan, and its clinical significance in hepatitis B virus (HBV), hepatitis C virus (HCV) and non-B, non-C (NBNC) liver disease. METHODS: Subjects comprised 1002 cases (246 with HCC and 756 without HCC) with chronic liver disease (HBV, 104; HCV, 722; and NBNC, 176). RESULTS: Liver stiffness was significantly greater in all groups with HCC, and the determined cut-off value for HCC concurrence was more than 12.0 kPa in those with HCV, more than 8.5 kPa in those with HBV and more than 12.0 kPa in those with NBNC. Liver stiffness of more than 12.0 kPa was an independent risk factor for new HCC development in HCV. For HCV, risk factors for HCC concurrence were old age, male sex, low albumin, low platelets and liver stiffness, while for HBV they were old age, low platelets and liver stiffness, and for NBNC they were old age, elevated α-fetoprotein and liver stiffness. CONCLUSION: Liver stiffness cut-off values and their association with HCC concurrence were different depending on the etiology. In HCV, liver stiffness of more than 12.0 kPa was an independent risk factor for new HCC development. Collectively, determining the fibrotic cut-off values for HCC concurrence would be important in evaluating HCC risks.

Semiannual Imaging Surveillance Is Associated with Better Survival in Patients with Non-B, Non-C Hepatocellular Carcinoma.

Since it remains elusive whether and how the imaging surveillance affects the survival in patients with non-B, non-C hepatocellular carcinoma (NBNC-HCC), we conducted this retrospective study which investigated the association between the semiannual surveillance prior to HCC diagnosis and the survival in patients with the initial diagnosis of HCC induced by hepatitis B virus (HBV) and/or hepatitis C virus (HCV) infections (N = 141) and non-B, non-C etiology (N = 30). It was demonstrated that surveillance was less frequently performed in the NBNC-HCC patients compared to that in HCC patients with HBV and/or HCV infections (B/C-HCC patients), and the survival was unfavorable in NBNC-HCC patients. On the other hand, the survival of NBNC-HCC patients with semiannual surveillance was significantly favorable than those patients without semiannual surveillance, and the survival was similar between B/C-HCCs and NBNC-HCCs with semiannual surveillance. In conclusion, though NBNC-HCC patients compared to B/C-HCC patients had poorer prognosis overall, these NBNC-HCC patients with semiannual surveillance had a better survival almost equivalent to the survival of B/C-HCC patients with semiannual surveillance, demonstrating the clinical utility of the semiannual imaging surveillance program for NBNC-HCCs.

Deep-sequencing analysis of the association between the quasispecies nature of the hepatitis C virus core region and disease progression.

Variation of core amino acid (aa) 70 of hepatitis C virus (HCV) has been shown recently to be closely correlated with liver disease progression, suggesting that the core region might be present as a quasispecies during persistent infection and that this quasispecies nature might have an influence on the progression of disease. In our investigation, the subjects were 79 patients infected with HCV genotype 1b (25 with chronic hepatitis [CH], 29 with liver cirrhosis [LC], and 25 with hepatocellular carcinoma [HCC]). Deep sequencing of the HCV core region was carried out on their sera by using a Roche 454 GS Junior pyrosequencer. Based on a plasmid containing a cloned HCV sequence (pCV-J4L6S), the background error rate associated with pyrosequencing, including the PCR procedure, was calculated as 0.092 ± 0.005/base. Deep sequencing of the core region in the clinical samples showed a mixture of "mutant-type" Q/H and "wild-type" R at the core aa 70 position in most cases (71/79 [89.9%]), and the ratio of mutant residues to R in the mixture increased as liver disease advanced to LC and HCC. Meanwhile, phylogenetic analysis of the almost-complete core region revealed that the HCV isolates differed genetically depending on the mutation status at core aa 70. We conclude that the core aa 70 mixture ratio, determined by deep sequencing, reflected the status of liver disease, demonstrating a significant association between core aa 70 and disease progression in CH patients infected with HCV genotype 1b.

Deep sequencing analysis of variants resistant to the non-structural 5A inhibitor daclatasvir in patients with genotype 1b hepatitis C virus infection.

AIM: Daclatasvir, a non-structural (NS)5A replication complex inhibitor, is a potent and promising direct antiviral agent (DAA) for hepatitis C virus (HCV), being most effective in genotype 1b infection. Although it is known that genotype 1b viruses with Y93H and/or L31M/V/F mutations have strong resistance to daclatasvir, it is not known whether there are some clinical background conditions that favor the occurrence of HCV carrying those NS5A mutations. METHODS: In this study, we carried out deep sequencing analysis of stored sera to determine the presence and significance of daclatasvir-resistant mutants in 110 genotype 1b HCV-infected patients with no previous daclatasvir treatment. RESULTS: Deep sequencing analysis revealed that the NS5A L31M/V/F and Y93H mutations were present in 13 (11.8%) and 34 (30.9%) of the 110 patients, respectively, and significantly more frequently than in the control plasmid. Simultaneous L31M/V/F and Y93H mutations were detected in four of the 110 patients (3.6%). When the clinical relevance of NS5A resistance was investigated, Y93H was significantly correlated with the IL28B major (TT) genotype of the host (P = 0.042). CONCLUSION: Y93H was detected frequently by deep sequencing in daclatasvir treatment-naïve patients. Importantly, it seems that the IL28B status of the patients may influence the presence of Y93H mutations, resulting in different treatment responses to daclatasvir.

Hepatocellular carcinoma risk assessment using gadoxetic acid-enhanced hepatocyte phase magnetic resonance imaging.

AIM: To investigate whether the patients with hypovascular liver nodules determined on the arterial phase and hypointensity on the hepatocyte phase gadoxetic acid-enhanced magnetic resonance imaging (hypovascular hypointense nodules) are at increased risk of hepatocarcinogenesis, we assessed subsequent typical hepatocellular carcinoma (HCC) development at any sites of the liver with and without such nodules. METHODS: One hundred and twenty-seven patients with chronic hepatitis B or C and without a history of HCC, including 68 with liver cirrhosis, were divided into those with (non-clean liver group, n = 18) and without (clean liver group, n = 109) hypovascular hypointense nodules. All the patients were followed up for 3 years, and HCC development rates and risk factors were analyzed with the Kaplan-Meier method and the Cox proportional hazard model, respectively. RESULTS: A total of 17 patients (10 in the non-clean liver group and seven in the clean liver group) developed typical HCC. Cumulative 3-year rates of HCC development were 55.5% in the non-clean liver group and 6.4% in the clean liver group (P < 0.001), and those at the different sites from the initial nodules was also higher in the non-clean liver group (22.2%) than the clean liver group (6.4%) (P = 0.003). Multivariate analysis identified older age (P = 0.024), low platelet counts (P = 0.017) and a non-clean liver (P < 0.001) as independent risk factors for subsequent HCC development. CONCLUSION: Patients with hypovascular hypointense liver nodules are at a higher risk for HCC development at any sites of the liver than those without such nodules.

Comprehensive analysis for viral elements and interleukin-28B polymorphisms in response to pegylated interferon plus ribavirin therapy in hepatitis C virus 1B infection.

UNLABELLED: To comprehensively characterize the contribution of virological factors as well as interleukin-28B (IL28B) single-nucleotide polymorphisms (SNPs) in determining treatment responses in pegylated-interferon plus ribavirin (Peg-IFN/RBV) therapy for chronic hepatitis C virus (HCV)-1b infection, we undertook a retrospective cohort analysis for the pretreatment dominant complete HCV open reading frame (ORF) amino-acid (aa) sequence study in 103 consecutive HCV-1b Japanese patients. The dominant HCV sequences classified by the response were subjected to systematic sliding-window comparison analysis to characterize response-specific viral sequences, along with IL28B SNP analyses (rs8099917). In each comparison of the patients between with and without rapid viral response (RVR), nonearly viral response (nEVR), sustained virological response (SVR), or relapse, the following regions were extracted as most significantly associated with the different responses respectively: nonstructural protein 5A (NS5A) aa.2224-2248 (P = 1.2E-07); core aa.70 (P = 4E-04); NS5A aa.2340-2382 (P = 7.0E-08); and NS5A aa.2360-2377 (P = 1.1E-05). Those NS5A regions nearly coincided with the interferon (IFN) sensitivity-determining region (NS5A aa.2209-2248) and the IFN/RBV resistance-determining region (NS5A aa.2339-2379). In a multivariate analysis, the IL28B SNP (odds ratio [OR] = 16.8; P = 0.009) and NS5A aa.2340-2382 (OR = 13.8; P = 0.0003) were extracted as the two most-significant independent variables contributing to the final outcome. CONCLUSION: In Peg-IFN/RBV therapy, polymorphisms in IL28B, NS5A aa.2224-2248, core aa.70, and, most important, NS5A aa.2340-2382 have a tremendous influence on treatment response in association with viral kinetics, resulting in significantly different outcomes in chronic HCV-1b infection.

Serum RANTES level influences the response to pegylated interferon and ribavirin therapy in chronic hepatitis C.

AIM: Prediction of treatment responses to pegylated interferon (PEG IFN) plus ribavirin (RBV) therapy is uncertain for genotype 1b chronic hepatitis C. METHODS: In this study, 96 patients were investigated for the correlation between 36 pretreatment serum chemokine/cytokine levels and PEG IFN/RBV treatment efficacy by a sandwich enzyme-linked immunoassay (ELISA) and a bead array. RESULTS: First, chemokines/cytokines were measured semiquantitatively by sandwich ELISA in 31 randomly-selected patients and the serum regulated on activation normal T-cell expressed and secreted (RANTES) level was found to be significantly higher in the sustained virological response (SVR) group than the non-SVR group (P = 0.048). Precise RANTES measurement in all 96 patients using a bead array confirmed this correlation (P = 0.002). However, the genetic RANTES haplotype was not significantly related to the serum level. The serum RANTES level was extracted by multivariate analysis (odds ratio = 4.09, 95% confidence interval = 1.02-16.5, P = 0.048) as an independent variable contributing to SVR. CONCLUSION: The serum RANTES level is an important determinant influencing the virological response to PEG IFN/RBV therapy in chronic hepatitis C.

Correlation between pretreatment viral sequences and the emergence of lamivudine resistance in hepatitis B virus infection.

The emergence of amino acid or nucleotide substitutions leads to lamivudine resistance in hepatitis B virus (HBV) infected patients. The aim of this study was to investigate whether viral sequences help predict the emergence of lamivudine resistance. The study subjects comprised 59 consecutive patients infected with HBV treated with daily therapy of 100 mg lamivudine. Among those, 32 patients with adequate pretreatment serum preservation were investigated for the correlation between viral amino acid substitutions and the appearance of lamivudine resistance with consideration of clinical background by determining dominant HBV full open reading frames. Viral resistance to lamivudine emerged in 28 of 59 patients (47%) in a median period of 2.45 years. Sequence comparisons of HBV genomes between patients who later developed lamivudine resistance and patients who did not revealed the existence of significant differences between the two groups in the pre-S1 84 (P = 0.042), pre-S2 1 (P = 0.017) and 22 (P = 0.015), and polymerase tp 95 (P = 0.046), judged by a log-rank test. Viral sequence analyses revealed the presence of amino acid substitutions in HBV pre-S1 and pre-S2 that may be associated with the emergence of lamivudine resistance during chronic HBV infection.

The Impact of Antiviral Therapy for Hepatitis C Virus on the Survival of Patients after Hepatocellular Carcinoma Treatment.

Objective Owing to advances in direct-acting antiviral (DAA) therapy, a considerable number of patients with hepatitis C virus (HCV)-positive hepatocellular carcinoma (HCC) are now able to achieve a sustained viral response (SVR) after curative treatment of HCC. However, the beneficial effect of a DAA-SVR on the survival remains unclear. Methods A total of 205 patients with HCC who were HCV-positive with Child-Pugh A at the onset from 2008 to 2018 were categorized into 2 groups: 140 patients untreated for HCV throughout the entire course after HCC development (untreated group) and 65 patients treated for HCV with DAAs following HCC treatment who achieved an SVR (SVR group). After propensity score matching, 63 patients from each group were selected. Using these patients, the survival and maintenance of Child-Pugh A after HCC treatment were compared between the untreated group and SVR group. Results There was a significant difference in the overall survival (p<0.001) and the rate of maintaining Child-Pugh A (p<0.001) between the groups. The 5-year survival rates were 96% (SVR group) and 60% (untreated group), and the proportions of patients with Child-Pugh A at 5 years after HCC treatment were 96% (SVR group) and 38% (untreated group). Conclusion In patients with HCV-positive HCC, achieving a DAA-SVR after HCC treatment significantly improved the overall survival rate compared with HCV-untreated patients. The contribution of DAA-SVR during the course of HCC treatment to a longer survival is mainly due to the prevention of the progression of Child-Pugh A to B/C. Further research is needed to determine whether aggressive antiviral therapy is also effective for HCC patients with Child-Pugh B/C.

Analysis of direct-acting antiviral-resistant hepatitis C virus haplotype diversity by single-molecule and long-read sequencing.

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.