Ultra-deep sequencing analysis of resistance-associated variants during retreatment with simeprevir-based triple therapy after failure of telaprevir-based triple therapy in patients with genotype 1 hepatitis C virus infection.

AIM: Simeprevir (SMV)-based triple therapy is an effective retreatment option following failure of telaprevir (TVR)-based triple therapy. However, it is unclear whether the persistence of resistance-associated variants (RAVs) induced by TVR-based therapy may reduce the treatment effect of SMV-based therapy. METHODS: The factors associated with the treatment effect, including RAVs in the NS3 region, were examined in 21 patients with genotype 1b HCV infection who were treated with SMV-based therapy after failure of TVR-based therapy. Ultra-deep sequencing was carried out to detect RAVs. RESULTS: With the exception of one patient who discontinued treatment owing to adverse events, the sustained virologic response (SVR) rate was 50% (10/20). Ultra-deep sequencing at the start of SMV-based therapy revealed that TVR-resistant variants were detected in six patients (29%), and no variants were observed at position 168. Cross-resistance between TVR and SMV with low frequency was detected in only one patient, and this patient achieved SVR. Higher SVRs for SMV-based therapy were attained in patients who discontinued treatment owing to the adverse effects of prior TVR-based therapy (discontinuation 100% vs. non-discontinuation 29%, P = 0.005), and patients who relapsed following prior pegylated interferon plus ribavirin therapy (relapse 100% vs. non-response 20%, P = 0.007). CONCLUSIONS: In this study, ultra-deep sequencing analysis revealed that TVR and/or SMV-resistant variants may have no influence on the effect of SMV-based therapy after failure of TVR-based therapy. Patients who discontinued treatment owing to adverse effects of TVR-based therapy and relapsers to previous pegylated interferon/ribavirin therapy would be good candidates for retreatment with SMV-based therapy.

Declining incidence of hepatocellular carcinoma in Osaka, Japan, from 1990 to 2003.

BACKGROUND: Japan has the highest incidence rate of primary liver cancer attributed to chronic hepatitis C virus (HCV) infection among developed countries. Molecular clock analysis of HCV sequences revealed that the spread of HCV took place earlier in Japan than in other countries. This might influence recent temporal trends in hepatocellular carcinoma (HCC) incidence. OBJECTIVE: To characterize the contribution of HCV-related hepatocellular carcinoma (HCC) to recent changes in HCC incidence in Osaka, Japan. DESIGN: Population-based survey. SETTING: Osaka Cancer Registry and 10 hospitals in Osaka. PARTICIPANTS: 63,862 patients with HCC that was diagnosed between 1981 and 2003 in Osaka Prefecture, including 5253 HCV-seropositive patients with HCC that was diagnosed between 1990 and 2003 at 10 hospitals. MEASUREMENTS: Incidence of HCC and estimated incidence rate of HCV-related HCC, measured by multiplying the prevalence of anti-HCV by the corresponding HCC incidence rate. RESULTS: Between 1981 and 2003, peak incidence of HCC among men age 50 to 59 years, 60 to 69 years, and 70 to 79 years occurred in 1986, 1995, and 2000, respectively, with marked downward trends thereafter (average annual change, -7.9, -22.3, and -12.4 per 100,000 persons, respectively). Similar trends were observed in women. Estimated sex- and age-specific incidence of HCV-related HCC (per 100,000 persons) decreased from 255 to 92 cases at the maximum in men age 60 to 69 years and from 61 to 34 cases in women age 60 to 69 years, whereas estimated incidence of non-HCV-related HCC did not change between 1990 and 2003. LIMITATION: Infection was determined only by HCV seropositivity. CONCLUSION: The incidence of HCC in Osaka started to decrease by 2000, mainly because of decreased HCV-related HCC.

POGLUT1, the putative effector gene driven by rs2293370 in primary biliary cholangitis susceptibility locus chromosome 3q13.33.

Primary biliary cholangitis (PBC) is a chronic and cholestatic autoimmune liver disease caused by the destruction of intrahepatic small bile ducts. Our previous genome-wide association study (GWAS) identified six susceptibility loci for PBC. Here, in order to further elucidate the genetic architecture of PBC, a GWAS was performed on an additional independent sample set, then a genome-wide meta-analysis with our previous GWAS was performed based on a whole-genome single nucleotide polymorphism (SNP) imputation analysis of a total of 4,045 Japanese individuals (2,060 cases and 1,985 healthy controls). A susceptibility locus on chromosome 3q13.33 (including ARHGAP31, TMEM39A, POGLUT1, TIMMDC1, and CD80) was previously identified both in the European and Chinese populations and was replicated in the Japanese population (OR = 0.7241, P = 3.5 × 10-9). Subsequent in silico and in vitro functional analyses identified rs2293370, previously reported as the top-hit SNP in this locus in the European population, as the primary functional SNP. Moreover, e-QTL analysis indicated that the effector gene of rs2293370 was Protein O-Glucosyltransferase 1 (POGLUT1) (P = 3.4 × 10-8). This is the first study to demonstrate that POGLUT1 and not CD80 is the effector gene regulated by the primary functional SNP rs2293370, and that increased expression of POGLUT1 might be involved in the pathogenesis of PBC.

HLA-DRB1 and DQB1 alleles in Japanese type 1 autoimmune hepatitis: The predisposing role of the DR4/DR8 heterozygous genotype.

OBJECTIVE: Autoimmune hepatitis (AIH) is a chronic progressive liver disease. AIH is composed predominantly of type 1 in Japanese populations. The genetic and environmental factors are associated with the pathogenesis of AIH. HLA-DRB1*03:01 and *04:01 are associated with type 1 AIH in European and *04:05 in Japanese populations. Here, we conducted an HLA association study in order to find HLA alleles or haplotypes predisposing or protective for Japanese AIH. METHODS: HLA-DRB1 and DQB1 genotyping of 360 type 1 AIH patients and 1026 healthy controls was performed. RESULTS: The predisposing association of DRB1*04:01 (P = 0.0006, corrected P [Pc] = 0.0193, odds ratio [OR] 2.97, 95% confidence interval [CI] 1.62-5.43), DRB1*04:05 (P = 1.89×10-21, Pc = 5.86×10-20, OR 3.41, 95% CI 2.65-4.38), and DQB1*04:01 (P = 4.66×10-18, Pc = 6.99×10-17, OR 3.89, 95% CI 2.84-5.33) and the protective association of DRB1*13:02 (P = 0.0003, Pc = 0.0080, OR 0.48, 95% CI 0.32-0.72) with Japanese type 1 AIH were observed. An association of the DR4/DR8 heterozygous genotype with Japanese AIH was identified for the first time (P = 3.12×10-9, OR 3.52, 95% CI 2.34-5.29). Susceptible diplotypes were DRB1*04:05-DQB1*04:01/DRB1*08:02-DQB1*03:02 (P = 0.0004, OR 24.77, 95% CI 1.45-424.31) and DRB1*04:05-DQB1*04:01/DRB1*08:03-DQB1*06:01 (P = 1.18×10-6, OR 10.64, 95% CI 3.19-35.46). Serum levels of Immunoglobulin G and Immunoglobulin M, International Autoimmune Hepatitis Group score, positive rate of anti-smooth muscle antibodies, and the rate of definite AIH were higher in AIH patients with DRB1*04:05 than without. CONCLUSIONS: The important roles of specific combinations of DRB1 and DQB1 alleles or haplotypes in the pathogenesis of type 1 AIH were suggested. The association of DR4/DR8 heterozygous genotype suggested the pathologic importance of trans-complementing DQα-ß heterodimer molecules encoded by DQA1 allele of one haplotype and the DQB1 allele of the other haplotype, as it was proposed in the HLA association studies of Type 1 diabetes.

Classification of ultrasonographic images of small hepatocellular carcinoma using galactose-based contrast agent: relation between image patterns and histologic features.

PURPOSE: The aim of this study was to establish the relation between observed ultrasonographic (US) images produced with a galactose-based contrast agent and histologic characteristics of small hepatocellular carcinomas (HCCs). MATERIALS AND METHODS: A total of 64 nodules in 64 patients, 22 well differentiated and 42 moderately differentiated with a histologically proven HCC, smaller than 3.0 cm in diameter and who had undergone hepatectomy were consecutively examined by contrast-enhanced US using a galactose-based contrast agent. Perfusion images were acquired by intermittent high-intensity, harmonic power Doppler sonography using a high pulse-repetition frequency and high-pass filter setting. Perfusion images of the arterial and late phases were classified into several patterns and compared with the histologic findings obtained from resected specimens. RESULTS: Most of the well- and moderately differentiated resected HCCs showed hyperechoic change during the arterial phase. However, 13 (59%) of the well-differentiated HCCs showed isoechoic change and 27 (64%) of the moderately differentiated HCCs showed hypoechoic change during the late phase. The difference is statistically significant (P < 0.0001). In a comparison of microscopic portal invasion (vp) of HCCs using enhanced US patterns, both vp(-) and vp(+) groups showed a high incidence of the hypervascular pattern during the arterial phase; in contrast, during the late phase 11 (73%) of 15 vp(+) nodules showed hypoechoic change with spotty signals. This difference is statistically significant (P < 0.0001) when compared with a high incidence (52%) of signal defect in the vp(-) group. The existence of well-differentiated components associated with the periphery of moderately differentiated HCCs also correlated closely with patterns during the late phase (P < 0.01). CONCLUSIONS: Late-phase contrast-enhanced US images of small HCCs with a galactose-based contrast agent are useful for predicting specific histologic characteristics.

Frequent impairment of the spindle assembly checkpoint in hepatocellular carcinoma.

BACKGROUND: Chromosomal instability (CI) leading to aneuploidy is one form of genetic instability, a characteristic feature of various types of cancers. Recent work has suggested that CI can be induced by a spindle assembly checkpoint defect. The aim of the current study was to determine the frequency of a defect of the checkpoint in hepatocellular carcinoma (HCC) and to establish whether alterations of genes encoding the checkpoint were associated with CI in HCC. METHODS: Aneuploidy and the function of the spindle assembly checkpoint were examined using DNA flow cytometry and morphologic analysis with microtubule disrupting drugs. To explore the molecular basis, the authors examined the expression and alterations of the mitotic checkpoint gene, BUB1, using Northern hybridization and direct sequencing in 8 HCC cell lines and 50 HCC specimens. Furthermore, the authors examined the alterations of other mitotic checkpoint genes, BUBR1, BUB3, MAD2B, and CDC20, using direct sequencing in HCC cell lines with aneuploidy. RESULTS: An impaired spindle assembly checkpoint was found in five (62.5%) of the eight aneuploid cell lines. Transcriptional expressions of the BUB1 gene appeared in all cell lines. While some polymorphic base changes were noted in BUB1, BUBR1, and CDC20, no mutations responsible for impairment of the mitotic checkpoint were found in either the HCC cell lines or HCC specimens, which suggests that these genes did not seem to be involved in tumor development in HCC. CONCLUSIONS: The loss of spindle assembly checkpoint occurred with a high frequency in HCC with CI. However, other mechanisms might also contribute to CI in HCC.