Targeted DNA and RNA sequencing of fine-needle biopsy FFPE specimens in patients with unresectable hepatocellular carcinoma treated with sorafenib.

The multi-kinase inhibitor sorafenib is now used as standard therapy for advanced hepatocellular carcinoma (HCC). Predictive biomarkers of response to sorafenib are thus necessary. The purpose of this study was to assess the feasibility of using targeted DNA and RNA sequencing to elucidate candidate biomarkers of sorafenib response using fine-needle biopsy, formalin-fixed paraffin-embedded (FFPE) specimens in patients with HCC. Targeted DNA and RNA deep sequencing were feasible for the evaluation of fine-needle biopsy FFPE specimens obtained from 46 patients with HCC treated with sorafenib. Frequent mutations of suppressor genes, such as CTNNB1 (34.8%) and TP53 (26.1%), were detected in the HCC tumors. After excluding these suppressor genes, the average numbers of detected oncogene mutations differed significantly between the non-PD and PD groups (P = 0.0446). This result suggests that the oncogene mutational burden in the tumor might be associated with the clinical response to sorafenib. We have identified candidate gene expression (TGFa, PECAM1, and NRG1) in tumor for the prediction of sorafenib response and PFS by RNA sequencing. Our findings provide new insights into biomarkers for sorafenib therapy and allow us to discuss future therapeutic strategies.

Proposal of Japan Red Cross score for sorafenib therapy in hepatocellular carcinoma.

AIM: There have been no established predictors of the outcome on sorafenib therapy for hepatocellular carcinoma (HCC) patients. We aimed to establish a new prognostic model suitable for sorafenib in HCC. METHODS: Among 465 HCC patients treated with sorafenib in 14 hospitals, we formed a training cohort with 270 patients at seven hospitals located in West Japan and a validation cohort with 167 patients at seven hospitals located in East Japan. In the training cohort, we examined the relationship between overall survival (OS) and pretreatment clinical factors, and structured a new prognostic model. We verified this model in the validation cohort and compared with four existing staging models. RESULTS: Multivariate analysis demonstrated distant metastases, portal invasion, intrahepatic tumor burden of more than 50%, serum α-fetoprotein of 150 ng/dL or more, des-γ-carboxyprothrombin of 1200 mAU/mL or more, albumin of 3.5 g/dL or less and total bilirubin of more than 1.0 mg/dL were significant independent adverse prognostic factors. We calculated a Japan Red Cross (JRC) score with these factors and classified three groups: low-, intermediate- or high-risk. Their median OS were well stratified (18.0, 8.8 and 3.7 months, respectively, P < 0.001) in the training cohort. In the validation cohort, OS were also statistically stratified (23.9, 10.3 and 2.9 months, P < 0.001). C-statistics of the JRC score was 0.755, the highest in the five models, indicating its novel predictability. CONCLUSION: Our proposed JRC score well predicts the prognosis of sorafenib therapy, and would be useful to plan individualized strategies for unresectable HCC.

Risk of hepatocellular carcinoma in cirrhotic hepatitis B virus patients during nucleoside/nucleotide analog therapy.

AIM: Some patients develop hepatocellular carcinoma (HCC) during nucleoside/nucleotide analog (NA) therapy even if alanine aminotransferase (ALT) or hepatitis B virus (HBV) DNA levels are sufficiently reduced. The aim of this study is to identify the risk factors of development of HCC during NA therapy. METHODS: Six hundred and two patients were analyzed who were continuously receiving NA for chronic HBV infection. The patients who developed HCC previously or within 1 year of therapy were excluded. In the patients studied, the median duration of therapy was 90 months. A total of 492 patients had chronic hepatitis (CH) and 110 had liver cirrhosis (LC). RESULTS: In 602 patients, the rate of normalization of ALT, loss of serum HBV DNA and development of HCC were 90.4%, 55.4%, and 6.1%, respectively. The significant risk factors of development of HCC were LC status and duration of therapy. The annual incidence of HCC in LC patients was 2.53%/year, compared with 0.34%/year in CH patients. When the relation between the incidence of HCC and the response to therapy was evaluated, in patients with normalization of ALT level, loss of HBV DNA by real-time polymerase chain reaction or hepatitis B e-antigen seroconversion, the incidences of HCC was reduced to some extent. However, none of the patients who achieved hepatitis B surface antigen (HBsAg) seroclearance during NA therapy developed HCC. CONCLUSION: LC status was the significant risk factor of development of HCC during NA therapy. However, none of the patients who showed HBsAg seroclearance developed HCC. The ultimate goal of therapy for reduced risk of HCC may be HBsAg seroclearance.

Clinical features associated with radiological response to sorafenib in unresectable hepatocellular carcinoma: a large multicenter study in Japan.

BACKGROUND & AIMS: There have been no established predictive factors of responders to sorafenib in patients with unresectable hepatocellular carcinoma (HCC). This study aimed to investigate the factors predicting a good response to sorafenib in Japanese patients with HCC. METHODS: A total of 465 patients with unresectable HCC in the Japanese Red Cross Liver Study Group were treated with sorafenib between January 2008 and August 2013, and 316 patients with sufficient clinical data were analysed. To determine the factors predicting a good response, the relationships between radiological response and the following clinicopathological factors were analysed: age, gender, performance status, liver function, tumour status and decrease in serum alpha-foetoprotein (AFP) level after 1 month. RESULTS: This study included 259 males and 57 females with a median age of 70 years (range, 37-90 years), of which 191 (60.4%) were classified as Barcelona Clinic Liver Cancer stage C, and 271 (85.8%) had Child-Pugh class A liver function. The median overall survival time was 307 days and progression-free survival time was 109 days. According to the modified Response Evaluation Criteria In Solid Tumours, four patients achieved a complete response, 51 achieved a partial response, 136 had stable disease and 125 had progressive disease. Multivariate analysis identified female gender (P = 0.003) and decreased serum AFP level after 1 month (P = 0.042) as independent predictors of a complete or partial response. CONCLUSION: Our results suggest female gender and a decrease in serum AFP level are independent predictors of good response to sorafenib.

Comparison of standard-dose and half­dose sorafenib therapy on clinical outcome in patients with unresectable hepatocellular carcinoma in field practice: A propensity score matching analysis.

The aims of the present study were to examine whether unresectable hepatocellular carcinoma (HCC) patients treated with initial dose of sorafenib of 400 mg/day (half-dose group) had comparable treatment efficacy, safety and survival merit as compared with those treated with initial dose of sorafenib of 800 mg/day (standard-dose group) in a multicenter large study. For reducing the bias in patient selection, we compared clinical outcomes of these two groups using propensity score matching analysis. A total of 465 patients were treated with sorafenib at fourteen hospitals in Japanese Red Cross Liver Study Group from 2008 to 2013. After propensity score matching, 139 matched HCC patients were selected for analysis in both groups. We retrospectively compared overall survival (OS), progression-free survival (PFS), best treatment response and sorafenib related serious adverse events (SAEs) in the two groups. There were no relevant differences in terms of OS (median OS intervals: 9.2 months in the standard-dose group and 9.7 months in the half­dose group, P=0.350), PFS (median PFS intervals: 3.4 months in the standard-dose group and 3.2 months in the half-dose group, P=0.729) and best treatment efficacy (objective response rate: P=0.416; disease control rate: P=0.719). Grade 3 or more SAEs were observed in 37 patients (26.6%) in the standard-dose group and 33 patients (23.7%) in the half-dose group (P=0.580). Furthermore, in all subgroup analyses according to Child-Pugh classification and Barcelona Clinic Liver Cancer stage, there were no significant differences in the two groups. In conclusion, unresectable HCC patients treated with initial half­dose sorafenib had comparable prognosis compared with those treated with initial standard-dose sorafenib.

Multicenter study of pegylated interferon α-2a monotherapy for hepatitis C virus-infected patients on hemodialysis: REACH study.

Many studies have reported poor vital prognosis in hepatitis C virus (HCV)-infected dialysis patients. The rate of HCV-infected dialysis patients in Japan is as high as 9.8%, and antiviral therapy is believed to be important for improving vital prognosis. We conducted a multicenter study to examine the administration method for pegylated interferon α-2a (PEG-IFNα-2a) monotherapy in HCV-infected dialysis. We studied 56 patients: 14 with low viral loads (HCV RNA < 5.0 log IU/mL) were treated with 90 µg PEG-IFNα-2a weekly, 42 with high viral loads (HCV RNA ≥ 5.0 log IU/mL) were treated with 135 µg PEG-IFNα-2a weekly. We examined the sustained virological response (SVR), factors affecting the SVR, and treatment safety. The overall SVR rate was 39% (22/56); that for genotype 1, genotype 2, low viral loads, and high viral loads was 29%, 67%, 93%, and 21%, respectively. From receiver operating characteristic (ROC) analysis, the HCV RNA cutoff values likely to achieve SVR for genotypes 1 and 2 were <5.7 log IU/mL (SVR rate: 64% 9/14) and <6.5 log IU/mL (SVR rate: 88% 7/8), respectively. If there was HCV RNA negativation at 4 weeks (rapid virological response), the SVR rate was 94% (16/17), whereas it was 6% (1/16) if there was HCV RNA positivity at 24 weeks. The rate of treatment discontinuation from adverse events or aggravated complications was 25% (14/56). High SVR rates can potentially be achieved with PEG-IFN monotherapy by identifying the target patients, based on virus type and viral load before initiating treatment and by modifying therapy during treatment according to responsiveness.

Sorafenib Therapy for BCLC Stage B/C Hepatocellular Carcinoma; Clinical Outcome and Safety in Aged Patients: A Multicenter Study in Japan.

BACKGROUND AND AIMS: We aimed to compare clinical outcomes and safety after sorafenib therapy between patients with Barcelona Clinic Liver Cancer (BCLC) stage B or C hepatocellular carcinoma (HCC) aged ≥75 years (aged group, n=179) and those with BCLC stage B or C HCC aged <75 years (control group, n=279). PATIENTS AND METHODS: We compared overall survival (OS), progression free survival (PFS), best treatment response and sorafenib related serious adverse events (SAEs) of grade 3 or more in the two groups. Furthermore, for reducing the selection bias, we compared clinical outcome of these two groups using propensity score matching analysis. RESULTS: The median OS and PFS intervals were 9.7 and 3.8 months in the aged group and 8.2 and 3.3 months in the control group (P=0.641 for OS and P=0.068 for PFS). Disease control rates were 49.2% (88/179) in the aged group and 49.1% (137/279) in the control group (P>0.999). Objective response rates were 15.1% (27/179) in the aged group and 14.3% (40/279) in the control group (P=0.892). Treatment related SAEs of grade 3 or more were observed in 51 patients (28.5%) in the aged group and in 69 patients (24.7%) in the control group (P=0.385). In the propensity score matched cohort (132 pairs), no significant difference in the two groups was observed in terms of OS (P=0.898) and PFS (P=0.407). CONCLUSION: In BCLC stage B or C HCC patients treated with sorafenib, life expectancy, disease progression, treatment efficacy and SAEs are unaffected by age over 75 years.

New susceptibility and resistance HLA-DP alleles to HBV-related diseases identified by a trans-ethnic association study in Asia.

Previous studies have revealed the association between SNPs located on human leukocyte antigen (HLA) class II genes, including HLA-DP and HLA-DQ, and chronic hepatitis B virus (HBV) infection, mainly in Asian populations. HLA-DP alleles or haplotypes associated with chronic HBV infection or disease progression have not been fully identified in Asian populations. We performed trans-ethnic association analyses of HLA-DPA1, HLA-DPB1 alleles and haplotypes with hepatitis B virus infection and disease progression among Asian populations comprising Japanese, Korean, Hong Kong, and Thai subjects. To assess the association between HLA-DP and chronic HBV infection and disease progression, we conducted high-resolution (4-digit) HLA-DPA1 and HLA-DPB1 genotyping in a total of 3,167 samples, including HBV patients, HBV-resolved individuals and healthy controls. Trans-ethnic association analyses among Asian populations identified a new risk allele HLA-DPB1*09 ∶ 01 (P = 1.36 × 10(-6); OR= 1.97; 95% CI, 1.50-2.59) and a new protective allele DPB1*02 ∶ 01 (P = 5.22 × 10(-6); OR = 0.68; 95% CI, 0.58-0.81) to chronic HBV infection, in addition to the previously reported alleles. Moreover, DPB1*02 ∶ 01 was also associated with a decreased risk of disease progression in chronic HBV patients among Asian populations (P = 1.55 × 10(-7); OR = 0.50; 95% CI, 0.39-0.65). Trans-ethnic association analyses identified Asian-specific associations of HLA-DP alleles and haplotypes with HBV infection or disease progression. The present findings will serve as a base for future functional studies of HLA-DP molecules in order to understand the pathogenesis of HBV infection and the development of hepatocellular carcinoma.

Imatinib plasma levels during successful long-term treatment of metastatic-gastrointestinal stromal tumors.

BACKGROUND/AIMS: To investigate whether imatinib dosage correlated with effective plasma levels and clinical characteristics for Japanese patients undergoing long-term (≥2 years) imatinib therapy for GISTs. METHODOLOGY: Twenty-five patients who received imatinib for a metastatic pathologically diagnosed GISTs at our hospital were enrolled. Imatinib response was assessed according to Choi's criteria. Blood samples were collected 22­26 h after the previous imatinib dose before the next scheduled dose. Results: Fourteen patients were male and the median age was 65 years. The median duration of imatinib therapy was 3.8 years (range, 2.0­11.5 years). The median plasma level of imatinib was 1098 ng/ml and the minimal plasma level after ≥5 years of therapy was 789 ng/ml. Imatinib dosage was significantly correlated with history of gastrectomy. The minimum body surface area of patients who received 400-mg/day imatinib dosage was 1.560 m2. CONCLUSIONS: The minimum level in all patients showing response for ≥5 years of treatment was 789 ng/ml, suggesting an effective plasma imatinib level of ≥800 ng/ml. Our results suggest that imatinib dosage of 400 mg/day is recommended for a patient with a large BSA (≥1.56 m2) and that of 300 mg/day might be sufficient for patients who have undergone a gastrectomy.

Serum interferon-gamma-inducible protein-10 concentrations and IL28B genotype associated with responses to pegylated interferon plus ribavirin with and without telaprevir for chronic hepatitis C.

AIM: Several studies have shown that high pretreatment concentrations of serum interferon-γ-inducible protein-10 (IP-10) are correlated with non-response to pegylated interferon (PEG-IFN) plus ribavirin (RBV) for chronic hepatitis C (CHC). However, there are few reports on their effect on the Asian population. METHODS: We enrolled 104 Japanese genotype 1 CHC individuals treated with PEG-IFN/RBV and 45 with PEG-IFN/RBV/telaprevir, and evaluated the impact of pretreatment serum IP-10 concentrations on their virological responses. RESULTS: The pretreatment serum IP-10 concentrations were not correlated with IL28B genotype. The receiver-operator curve analysis determined the cut-off value of IP-10 for predicting a sustained virological response (SVR) as 300 pg/mL. In multivariate analysis, the IL28B favorable genotype and IP-10 concentration of less than 300 pg/mL were independent factors for predicting SVR. In a subgroup of patients with the IL28B favorable genotype, the SVR rate was higher in the patients with IP-10 of less than 300 than in those with 300 pg/mL or more, whereas no patient with the IL28B unfavorable genotype and IP-10 of 300 pg/mL or more achieved SVR. Among the patients treated with PEG-IFN/RBV/telaprevir, low pretreatment concentrations of serum IP-10 were associated with a very rapid virological response, defined as undetectable HCV RNA at week 2 after the start of therapy. CONCLUSION: Pretreatment serum IP-10 concentrations are associated with treatment efficacy in PEG-IFN/RBV and with early viral kinetics of hepatitis C virus in PEG-IFN/RBV/telaprevir therapy.

Novel nasogastric tube-related criteria for urgent endoscopy in nonvariceal upper gastrointestinal bleeding.

BACKGROUND: Patients with active upper gastrointestinal bleeding (UGIB) require urgent endoscopy, but appropriate criteria for urgent endoscopy in these patients have not yet been established. AIMS: The goal of this study is to establish a simple system for the selection of UGIB patients who may benefit from urgent endoscopy. METHODS: Of the 335 patients who required emergency hospitalization for UGIB from May 2010 to March 2012 at Nagoya Daini Red Cross Hospital, 166 patients who underwent placement of a nasogastric tube (NGT) were retrospectively identified. Active bleeding on the endoscopic image was used as an endpoint that reflected the need for urgent endoscopy. RESULTS: The ratio of the heart rate to the systolic blood pressure (HR/SBP ratio) and aspiration of fresh or dark red fluid from the NGT [NGT(+)] were significant predictors of active bleeding in the univariate analysis [HR/SBP ratio, P=0.016; NGT(+), P<0.001]. The HR/SBP ratio [odds ratio (OR) 8.118; 95% confidence intervals (CI) 1.696-38.850; P=0.009] and NGT(+) (OR 4.630; 95% CI 2.092-10.204; P<0.001) were also significantly associated with active bleeding in the multivariate analysis. Moreover, receiver operating characteristic analysis revealed a setting with HR/SBP ratio>1.4 or NGT(+) to be optimal criteria to predict active bleeding. These criteria were associated with a sensitivity of 64.9% (24/37) and a specificity of 76.7% (99/129) for the prediction of active bleeding; consequently, they are superior to the sensitivity and specificity of previously proposed criteria. CONCLUSIONS: A novel and simple criteria system using NGT(+) and HR/SBP is a good predictor of the need for urgent endoscopy in patients with nonvariceal UGIB.

Quantitation of HBsAg predicts response to entecavir therapy in HBV genotype C patients.

AIM: To analysis the factors that predict the response to entecavir therapy in chronic hepatitis patients with hepatitis B virus (HBV) genotype C. METHODS: Fifty patients [hepatitis B e antigen (HBeAg)-negative:HBeAg-positive = 26:24] with HBV genotype C, who received naïve entecavir therapy for > 2 years, were analyzed. Patients who showed HBV DNA levels ≥ 3.0 log viral copies/mL after 2 years of entecavir therapy were designated as slow-responders, while those that showed < 3.0 log copies/mL were termed rapid-responders. Quantitative hepatitis B surface antigen (HBsAg) levels (qHBsAg) were determined by the Architect HBsAg QT immunoassay. Hepatitis B core-related antigen was detected by enzyme immunoassay. Pre-C and Core promoter mutations were determined using by polymerase chain reaction (PCR). Drug-resistance mutations were detected by the PCR-Invader method. RESULTS: At year 2, HBV DNA levels in all patients in the HBeAg-negative group were < 3.0 log copies/mL. In contrast, in the HBeAg-positive group, 41.7% were slow-responders, while 58.3% were rapid-responders. No entecavir-resistant mutants were detected in the slow-responders. When the pretreatment factors were compared between the slow- and rapid-responders; the median qHBsAg in the slow-responders was 4.57 log IU/mL, compared with 3.63 log IU/mL in the rapid-responders (P < 0.01). When the pretreatment factors predictive of HBV DNA-negative status at year 2 in all 50 patients were analyzed, HBeAg-negative status, low HBV DNA levels, and low qHBsAg levels were significant (P < 0.01). Multivariate analysis revealed that the low qHBsAg level was the most significant predictive factor (P = 0.03). CONCLUSION: Quantitation of HBsAg could be a useful indicator to predict response to entecavir therapy.

Genome-wide association study confirming association of HLA-DP with protection against chronic hepatitis B and viral clearance in Japanese and Korean.

Hepatitis B virus (HBV) infection can lead to serious liver diseases, including liver cirrhosis (LC) and hepatocellular carcinoma (HCC); however, about 85-90% of infected individuals become inactive carriers with sustained biochemical remission and very low risk of LC or HCC. To identify host genetic factors contributing to HBV clearance, we conducted genome-wide association studies (GWAS) and replication analysis using samples from HBV carriers and spontaneously HBV-resolved Japanese and Korean individuals. Association analysis in the Japanese and Korean data identified the HLA-DPA1 and HLA-DPB1 genes with P(meta) = 1.89×10⁻¹² for rs3077 and P(meta) = 9.69×10⁻¹° for rs9277542. We also found that the HLA-DPA1 and HLA-DPB1 genes were significantly associated with protective effects against chronic hepatitis B (CHB) in Japanese, Korean and other Asian populations, including Chinese and Thai individuals (P(meta) = 4.40×10⁻¹9 for rs3077 and P(meta) = 1.28×10⁻¹5 for rs9277542). These results suggest that the associations between the HLA-DP locus and the protective effects against persistent HBV infection and with clearance of HBV were replicated widely in East Asian populations; however, there are no reports of GWAS in Caucasian or African populations. Based on the GWAS in this study, there were no significant SNPs associated with HCC development. To clarify the pathogenesis of CHB and the mechanisms of HBV clearance, further studies are necessary, including functional analyses of the HLA-DP molecule.

Development of new IL28B genotyping method using Invader Plus assay.

IL28B polymorphism is associated with the response to pegylated interferon-α with ribavirin (PEG-IFN-α/RBV) treatment in chronic hepatitis C patients. As a genotyping assay for IL28B single nucleotide polymorphisms (SNPs) in clinical practice, the Invader Plus assay was developed. The accuracy, intra-assay, inter-assay precision, and the limit of detection of the Invader Plus assay were evaluated. Two SNPs (rs8099917 and rs12979860) associated with IL28B were genotyped by the Invader Plus and TaqMan assay in 512 Japanese patients. In comparison with direct sequencing, the Invader Plus assay showed 99% accuracy in rs8099917 and 100% accuracy in rs12979860. Intra-assay and inter-assay precision were sufficient to use in clinical practice and the detection limit was 1ngDNA/assay. Genotyping by rs8099917 showed that 361 (71%), 144 (28%) and seven (1%) of the patients were major homozygous, heterozygous and minor homozygous types, respectively. Five of the 512 cases (1%) had haplotype differences, but none showed differences between the two genotyping methods. For patients with HCV genotype 1, the prevalence of responders in the major homozygous type was 83.3%, and that of non-responders in the minor heterozygous/homozygous type was 72.5%. A convenient IL28B genotyping method using the Invader Plus assay could be useful to predict the treatment outcome in clinical practice.

Clinical factors related to long-term administration of sorafenib in patients with hepatocellular carcinoma.

BACKGROUND: Sorafenib has been approved in the indication of unresectable hepatocellular carcinoma, but there are many cases in which administration of the drug is discontinued due to severe side effects. In this study, we compared the characteristics of patients who continued and discontinued sorafenib. METHODS: Ninety-six patients (75 men and 21 women) were initiated on sorafenib from July 2009 through September 2011. The patient characteristics of interest included gender, age, etiology, Child-Pugh classification, treatment history and frequency, and levels of α-fetoprotein, des- gamma-carboxy prothrombin, aspartate amino acid transferase, and alanine aminotransferase. Duration of administration of sorafenib and reasons for its discontinuation were compared. RESULTS: Median overall survival was 11.8 months. Discontinuation of sorafenib within 90 days was identified as an independent prognostic factor for overall survival on multivariate analysis (P < 0.0001). Transarterial chemoembolization performed six times or more (P = 0.013) was also identified as an independent factor contributing to discontinuation of sorafenib within 90 days in multivariate analysis. Patients who received sorafenib for ≥90 days had significantly longer overall survival than those who discontinued it (P < 0.0001). CONCLUSION: Prolonged treatment with sorafenib is an important factor in achieving extended overall survival. We recommend starting sorafenib before latent liver damage has occurred as a result of too many transarterial chemoembolization procedures.

The rs8099917 polymorphism, when determined by a suitable genotyping method, is a better predictor for response to pegylated alpha interferon/ribavirin therapy in Japanese patients than other single nucleotide polymorphisms associated with interleukin-28B.

We focused on determining the most accurate and convenient genotyping methods and most appropriate single nucleotide polymorphism (SNP) among four such polymorphisms associated with interleukin-28B (IL-28B) in order to design tailor-made therapy for patients with chronic hepatitis C virus (HCV) patients. First, five different methods (direct sequencing, high-resolution melting analysis [HRM], hybridization probe [HP], the InvaderPlus assay [Invader], and the TaqMan SNP genotyping assay [TaqMan]) were developed for genotyping four SNPs (rs11881222, rs8103142, rs8099917, and rs12979860) associated with IL-28B, and their accuracies were compared for 292 Japanese patients. Next, the four SNPs associated with IL-28B were genotyped by Invader for 416 additional Japanese patients, and the response to pegylated interferon/ribavirin (PEG-IFN/RBV) treatment was evaluated when the four SNPs were not in linkage disequilibrium (LD). HRM failed to genotype one of the four SNPs in five patients. In 2 of 287 patients, the results of genotyping rs8099917 by direct sequencing differed from the results of the other three methods. The HP, TaqMan, and Invader methods were accurate for determination of the SNPs associated with IL-28B. In 10 of the 708 (1.4%) patients, the four SNPs were not in LD. Eight of nine (88.9%) patients whose rs8099917 was homozygous for the major allele were virological responders, even though one or more of the other SNPs were heterozygous. The HP, TaqMan, and Invader methods were suitable to determine the SNPs associated with IL-28B. The rs8099917 polymorphism should be the best predictor for the response to the PEG-IFN/RBV treatment among Japanese chronic hepatitis C patients.

Accuracy of the Tokyo Guidelines for the diagnosis of acute cholangitis and cholecystitis taking into consideration the clinical practice pattern in Japan.

PURPOSE: Three years have passed since the publication of the Tokyo Guidelines for the management of acute cholangitis and cholecystitis, and we believe that the time has come to assess their validity. METHODS: In this study, we validated the diagnostic accuracy of these criteria in 74 patients with an initial diagnosis of acute cholangitis and 81 patients with an initial diagnosis of acute cholecystitis. We also statistically compared the accuracy of the diagnosis made based on the Tokyo Guidelines with that based on the presence of Charcot's triad for acute cholangitis and Murphy's sign for acute cholecystitis with use of the sign test to assess differences. RESULTS: The results revealed that the diagnostic sensitivity and specificity of the Tokyo Guidelines for suspected or definitive acute cholangitis were 72.1 and 38.5%, respectively, and the corresponding values for definitive cholangitis alone were 63.9 and 69.2%, respectively. For definitive acute cholecystitis, the diagnostic sensitivity and specificity of the Tokyo Guidelines were 84.9 and 50.0%, respectively. The accuracy of diagnosis based on the Tokyo Guidelines was significantly higher than that based on the presence of Charcot's triad (acute cholangitis, p < 0.001 by the sign test) or Murphy's sign (acute cholecystitis, p < 0.001 by the sign test). CONCLUSIONS: It was therefore concluded that the Tokyo Guidelines should be used more widely for the diagnosis of acute cholangitis and cholecystitis in the twenty-first century. Hereafter, various efforts should be made to improve the sensitivity and specificity of the diagnostic criterion of the Tokyo Guidelines.

Mechanism of entecavir resistance of hepatitis B virus with viral breakthrough as determined by long-term clinical assessment and molecular docking simulation.

The mechanism by which entecavir resistance (ETVr) substitutions of hepatitis B virus (HBV) can induce breakthrough (BT) during ETV therapy is largely unknown. We conducted a cross-sectional study of 49 lamivudine (LVD)-refractory patients and 59 naïve patients with chronic hepatitis B. BT was observed in 26.8% of the LVD-refractory group during weeks 60 to 144 of ETV therapy. A line probe assay revealed ETVr substitutions only in the LVD-refractory group, i.e., in 4.9% of patients at baseline, increasing to 14.6%, 24.4%, and 44.8% at weeks 48, 96, and 144, respectively. Multivariate logistic regression analysis adjusted for age, gender, HBV DNA levels, and LVD resistance (LVDr) (L180M and M204V, but not M204I) indicated that T184 substitutions and S202G (not S202C) were a significant factor for BT (adjusted odds ratio [OR], 141.12, and 95% confidence interval [CI], 6.94 to 2,870.20; OR, 201.25, and 95% CI, 11.22 to 3608.65, respectively). Modeling of HBV reverse transcriptase (RT) by docking simulation indicated that a combination of LVDr and ETVr (T184L or S202G) was characterized by a change in the direction of the D205 residue and steric conflict in the binding pocket of ETV triphosphate (ETV-TP), by significantly longer minimal distances (2.2 A and 2.1 A), and by higher potential energy (-117 and -99.8 Kcal/mol) for ETV-TP compared with the wild type (1.3 A; -178 Kcal/mol) and LVDr substitutions (1.5 A; -141 Kcal/mol). Our data suggest that the low binding affinity of ETV-TP for the HBV RT, involving conformational change of the binding pocket of HBV RT by L180M, M204V plus T184L, and S202G, could induce BT.

Effect of ursodeoxycholic acid on serum liver enzymes and bile acid metabolism in chronic active hepatitis C virus infection.

AIM: Many reports have revealed ursodeoxycholic acid (UDCA) to be effective against chronic hepatitis C virus (HCV). However, some cases resist this therapy and the mechanism of action remains unclear. In this study, UDCA was administered to patients with chronic HCV and the correlation between the bile acids of the biliary bile and serum and the drug efficacy was investigated. METHODS: Fifteen patients were given 600 mg/day of UDCA for more than 24 weeks. The serum bile acid concentrations and biliary and serum bile acid were collected before and after 24 weeks of UDCA treatment, and composition determined by high-performance liquid chromatography. RESULTS: The treatment was effective in nine cases (ALT decreased to less than twice the normal values 80 IU/L) and ineffective in six cases. There was no significant difference in the serum bile acid concentrations before and after UDCA treatment between the values of both cases. After UDCA treatment, the serum percentage of UDCA (effective, 62.5 +/- 2.0; ineffective, 53.5 +/- 2.5, (P = 0.02)) and the percentage of chenodeoxycholic acid (CDCA) showed no remarkable changes. In the biliary bile the percentage of CDCA (effective, 30.9 +/- 2.0; ineffective, 20.0 +/- 3.0, (P = 0.007)) and the percentage of UDCA showed no remarkable changes. CONCLUSION: In the effective cases, the percentage of UDCA in the serum and the percentage of CDCA in biliary bile were significantly higher than in the ineffective cases. This indicates that, when effective, CDCA decreases in hepatocytes and this reduction contributes to hepatoprotection.

Combination therapy with direct hemoperfusion using polymyxin B-immobilized fiber and oral vancomycin improves fulminant pseudomembranous colitis by reducing the elevated endogenous cannabinoids and inflammatory cytokines: report of a case.

This paper reports a case of fulminant pseudo-membranous colitis which did not lead to septic shock. The case was improved by combination therapy with direct hemoperfusion using polymyxin B-immobilized fiber and oral vancomycin. Direct hemoperfusion using polymyxin B-immobilized fiber has been demonstrated to have excellent therapeutic effects for the treatment of septic shock by removing circulating lipopolysaccharide. In the present case, the combination therapy dramatically improved clinical status of the patient. The clinical improvement occurred in parallel with a decrease in APACHE II score (from 20 to 14 points), serum levels of endogenous cannabinoids (anandamide and 2-arachidonylglycerol), and inflammatory cytokine (interleukin-6). Thus, direct hemoperfusion is strongly recommended in cases of fulminant pseudomembranous colitis, because direct hemoperfusion using polymyxin B-immobilized fiber reduces inflammatory cytokines by absorbing endogenous cannabinoids and, thereby, improves the patient's condition.