Yoichi Hiasa

Genome-wide association of IL28B with response to pegylated interferon-α and ribavirin therapy for chronic hepatitis C

The recommended treatment for patients with chronic hepatitis C, pegylated interferon-α (PEG-IFN-α) plus ribavirin (RBV), does not provide sustained virologic response (SVR) in all patients. We report a genome-wide association study (GWAS) to null virological response (NVR) in the treatment of patients with hepatitis C virus (HCV) genotype 1 within a Japanese population. We found two SNPs near the gene IL28B on chromosome 19 to be strongly associated with NVR (rs12980275, P = 1.93 × 10−13, and rs8099917, 3.11 × 10−15). We replicated these associations in an independent cohort (combined P values, 2.84 × 10−27 (OR = 17.7; 95% CI = 10.0–31.3) and 2.68 × 10−32 (OR = 27.1; 95% CI = 14.6–50.3), respectively). Compared to NVR, these SNPs were also associated with SVR (rs12980275, P = 3.99 × 10−24, and rs8099917, P = 1.11 × 10−27). In further fine mapping of the region, seven SNPs (rs8105790, rs11881222, rs8103142, rs28416813, rs4803219, rs8099917 and rs7248668) located in the IL28B region showed the most significant associations (P = 5.52 × 10−28–2.68 × 10−32; OR = 22.3–27.1). Real-time quantitative PCR assays in peripheral blood mononuclear cells showed lower IL28B expression levels in individuals carrying the minor alleles (P = 0.015).

Liver Fibrosis in Patients with Chronic Hepatitis C: Noninvasive Diagnosis by Means of Real-time Tissue Elastography—Establishment of the Method for Measurement

PURPOSE To prospectively measure liver stiffness with real-time tissue elastography in patients with chronic hepatitis C and to compare the results with those of clinical assessment of fibrosis by using histologic stage as the reference standard. MATERIALS AND METHODS All subjects gave informed consent, and the study was approved by the institutional ethics committee. Seventy hospitalized patients (46 men, 24 women; mean age, 65.5 years ± 11.7 [standard deviation]; age range, 33-87 years) with chronic hepatitis C underwent real-time elastography between January 2009 and September 2009. Elastography was performed at four liver locations by two independent observers. The elastic ratio (ratio of the value in the intrahepatic venous small vessels divided by the value in the hepatic parenchyma) was calculated and was compared with histologic fibrosis stage at liver biopsy. The elastic ratio and clinical fibrosis markers were assessed by using receiver operating characteristic (ROC) analysis. The differences between body site and observers were assessed with κ statistics and intraclass correlation coefficients (ICCs). RESULTS Real-time tissue elastography cutoff values were 2.73 for F of 2 or greater, 3.25 for F of 3 or greater, and 3.93 for F of 4. No site differences were observed (κ = 0.835, ICC = 0.966), and the elastic ratio measurement was correlated between the two examiners (r(2) = 0.869, P < .0001). The areas under the ROC curves for elastic ratio, hyaluronic acid, type IV collagen, aspartate aminotransferase-to-platelet ratio index, FibroIndex, Forns score, and Hepascore were 0.95, 0.32, 0.73, 0.76, 0.76, 0.87, and 0.70, respectively; the elastic ratio performed better than the serum fibrosis markers and other scores. CONCLUSION Real-time tissue elastography is not invasive and could be used to evaluate liver fibrosis in patients with chronic hepatitis C. SUPPLEMENTAL MATERIAL http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.10100319/-/DC1.

FGF3/FGF4 amplification and multiple lung metastases in responders to sorafenib in hepatocellular carcinoma

The response rate to sorafenib in hepatocellular carcinoma (HCC) is relatively low (0.7%‐3%), however, rapid and drastic tumor regression is occasionally observed. The molecular backgrounds and clinico‐pathological features of these responders remain largely unclear. We analyzed the clinical and molecular backgrounds of 13 responders to sorafenib with significant tumor shrinkage in a retrospective study. A comparative genomic hybridization analysis using one frozen HCC sample from a responder demonstrated that the 11q13 region, a rare amplicon in HCC including the loci for FGF3 and FGF4, was highly amplified. A real‐time polymerase chain reaction–based copy number assay revealed that FGF3/FGF4 amplification was observed in three of the 10 HCC samples from responders in which DNA was evaluable, whereas amplification was not observed in 38 patients with stable or progressive disease (P = 0.006). Fluorescence in situ hybridization analysis confirmed FGF3 amplification. In addition, the clinico‐pathological features showed that multiple lung metastases (5/13, P = 0.006) and a poorly differentiated histological type (5/13, P = 0.13) were frequently observed in responders. A growth inhibitory assay showed that only one FGF3/FGF4‐amplified and three FGFR2‐amplified cancer cell lines exhibited hypersensitivity to sorafenib in vitro. Finally, an in vivo study revealed that treatment with a low dose of sorafenib was partially effective for stably and exogenously expressed FGF4 tumors, while being less effective in tumors expressing EGFP or FGF3. Conclusion: FGF3/FGF4 amplification was observed in around 2% of HCCs. Although the sample size was relatively small, FGF3/FGF4 amplification, a poorly differentiated histological type, and multiple lung metastases were frequently observed in responders to sorafenib. Our findings may provide a novel insight into the molecular background of HCC and sorafenib responders, warranting further prospective biomarker studies. (HEPATOLOGY 2013)

Genome-wide association study identified ITPA/DDRGK1 variants reflecting thrombocytopenia in pegylated interferon and ribavirin therapy for chronic hepatitis C

Hematologic abnormalities during current therapy with pegylated interferon and ribavirin (PEG-IFN/RBV) for chronic hepatitis C (CHC) often necessitate dose reduction and premature withdrawal from therapy. The aim of this study was to identify host factors associated with IFN-induced thrombocytopenia by genome-wide association study (GWAS). In the GWAS stage using 900K single-nucleotide polymorphism (SNP) microarrays, 303 Japanese CHC patients treated with PEG-IFN/RBV therapy were genotyped. One SNP (rs11697186) located on DDRGK1 gene on chromosome 20 showed strong associations in the minor-allele-dominant model with the decrease of platelet counts in response to PEG-IFN/RBV therapy [P = 8.17 × 10(-9); odds ratio (OR) = 4.6]. These associations were replicated in another sample set (n = 391) and the combined P-values reached 5.29 × 10(-17) (OR = 4.5). Fine mapping with 22 SNPs around DDRGK1 and ITPA genes showed that rs11697186 at the GWAS stage had a strong linkage disequilibrium with rs1127354, known as a functional variant in the ITPA gene. The ITPA-AA/CA genotype was independently associated with a higher degree of reduction in platelet counts at week 4 (P < 0.0001), as well as protection against the reduction in hemoglobin, whereas the CC genotype had significantly less reduction in the mean platelet counts compared with the AA/CA genotype (P < 0.0001 for weeks 2, 4, 8, 12), due to a reactive increase of the platelet count through weeks 1-4. Our present results may provide a valuable pharmacogenetic diagnostic tool for tailoring PEG-IFN/RBV dosing to minimize drug-induced adverse events.

Phase I/II study of immunotherapy using tumor antigen-pulsed dendritic cells in patients with hepatocellular carcinoma

Dendritic cells (DCs) are increasingly used as adjuvants for vaccination strategies; however, there has been very little development in DC vaccines for patients with hepatocellular carcinoma (HCC). In this study, we assessed the safety, feasibility and efficacy of a multiple tumor-associated antigen (TAA)-pulsed DC vaccine in 5 patients with advanced HCC. DCs were generated by culturing blood monocytes in the presence of granulocyte macrophage-colony stimulating factor and interleukin-4 for 5 days. The DC vaccine was prepared by pulsing DCs with cytoplasmic transduction peptide-attached α-fetoprotein, glypican-3 and MAGE-1 recombinant fusion proteins and cultivating them in the presence of maturation cocktail. DCs were injected subcutaneously near the inguinal lymph nodes, followed by topical application of toll-like receptor-7 agonist around the injection site. We showed that our DC vaccine was safe and well-tolerated over 6 vaccinations in 5 patients. All 5 patients showed T cell responses against TAAs. Clinical benefit was observed in one of the 5 patients. In conclusion, the feasibility, safety and immune activity of DCs pulsed with TAAs were confirmed in HCC patients. However, clinical response was detected only in one patient. Future trials may consider applying this therapy in a less advanced stage to obtain better clinical responses.

Real‐time tissue elastography for evaluation of hepatic fibrosis and portal hypertension in nonalcoholic fatty liver diseases

The aim of this study was to prospectively measure liver stiffness with real‐time tissue elastography in patients with nonalcoholic fatty liver diseases (NAFLD) and to compare the result with the clinical assessment of fibrosis using histological stage. One hundred and eighty‐one prospectively enrolled patients underwent real‐time tissue elastography, with the first 106 being analyzed as the training set and the remaining 75 being evaluated as the validation set. Hepatic and splenic elastic ratios were calculated and compared with stage of histological fibrosis. Portal hypertension (PH) was assessed. Real‐time tissue elastography cut‐off values by stage in the training set were 2.47 for F1, 2.67 for F2, 3.02 for F3, and 3.36 for F4. Using these cut‐off values, the diagnostic accuracy of hepatic fibrosis in the validation set was 82.6%‐96.0% in all stages. Only portal fibrosis correlated with the hepatic elastic ratio by multivariate analysis. The area under the receiver operating characteristic curve of elastic ratio better correlated than serum fibrosis markers in both early and advanced fibrosis stages. Patients with PH, defined by splenic elasticity, had early fibrosis. Patients with severe PH were found only in the group with cirrhosis. Conclusion: Real‐time tissue elastography is useful in evaluating hepatic fibrosis and PH in patients with NAFLD. (HEPATOLOGY 2012)

Splenic Elasticity Measured with Real-time Tissue Elastography Is a Marker of Portal Hypertension

PURPOSE To prospectively correlate spleen elasticity and degree of portal hypertension estimated with the hepatic venous pressure gradient (HVPG) and to evaluate splenic elasticity as a predictor of gastroesophageal varices. MATERIALS AND METHODS The institutional review board approved this study, and patients provided written informed consent. In a pilot study of 60 patients with chronic liver damage, the authors measured liver and spleen elasticity with real-time tissue elastography (RTE), obtained serum markers related to fibrosis, examined hepatic and splenic blood flow with duplex Doppler ultrasonography, estimated HVPG, and performed upper gastrointestinal endoscopy. Then, with use of thresholds determined in the pilot study, the authors conducted a validation trial with another 210 patients, performing all studies except the measurement of HPVG. The relationship between HVPG and the other parameters was analyzed. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) in the diagnosis of gastroesophageal varices were calculated by using cutoff values obtained from receiver operating characteristic curves. RESULTS Among the parameters associated with HVPG, correlation was closest with splenic elasticity (R = 0.854, P < .0001). When 8.24 was selected as the cutoff of splenic elasticity for predicting HVPG of more than 10 mm Hg, the accuracy of diagnosing gastroesophageal varix was 90% (sensitivity, 96%; specificity, 85%; PPV, 83%; NPV, 97%). The results of the validation trial showed that the 8.24 cutoff for splenic elasticity was associated with a diagnostic accuracy of 94.8% (sensitivity, 98%; specificity, 93.8%; PPV, 82.1%; NPV, 99.4%) for gastroesophageal varices. CONCLUSION Splenic elasticity determined with RTE is the most closely associated parameter for evaluating HVPG and is useful as a clinical marker of portal hypertension and a predictive marker of gastroesophageal varices.

Persistent expression of the full genome of hepatitis C virus in B cells induces spontaneous development of B-cell lymphomas in vivo

Extrahepatic manifestations of hepatitis C virus (HCV) infection occur in 40%-70% of HCV-infected patients. B-cell non-Hodgkin lymphoma is a typical extrahepatic manifestation frequently associated with HCV infection. The mechanism by which HCV infection of B cells leads to lymphoma remains unclear. Here we established HCV transgenic mice that express the full HCV genome in B cells (RzCD19Cre mice) and observed a 25.0% incidence of diffuse large B-cell non-Hodgkin lymphomas (22.2% in males and 29.6% in females) within 600 days after birth. Expression levels of aspartate aminotransferase and alanine aminotransferase, as well as 32 different cytokines, chemokines and growth factors, were examined. The incidence of B-cell lymphoma was significantly correlated with only the level of soluble interleukin-2 receptor α subunit (sIL-2Rα) in RzCD19Cre mouse serum. All RzCD19Cre mice with substantially elevated serum sIL-2Rα levels (> 1000 pg/mL) developed B-cell lymphomas. Moreover, compared with tissues from control animals, the B-cell lymphoma tissues of RzCD19Cre mice expressed significantly higher levels of IL-2Rα. We show that the expression of HCV in B cells promotes non-Hodgkin-type diffuse B-cell lymphoma, and therefore, the RzCD19Cre mouse is a powerful model to study the mechanisms related to the development of HCV-associated B-cell lymphoma.

Association of IL28B variants with response to pegylated-interferon alpha plus ribavirin combination therapy reveals intersubgenotypic differences between genotypes 2a and 2b†

Genetic polymorphisms of the interleukin 28B (IL28B) locus are associated closely with outcomes of pegylated‐interferon (PEG‐IFN) plus ribavirin (RBV) combination therapy. The aim of this study was to investigate the relationship between IL28B polymorphism and responses to therapy in patients infected with genotype 2. One hundred twenty‐nine chronic hepatitis C patients infected with genotype 2, 77 patients with genotype 2a and 52 patients with genotype 2b, were analyzed. Clinical and laboratory parameters, including genetic variation near the IL28B gene (rs8099917), were assessed. Drug adherence was monitored in each patient. Univariate and multivariate statistical analyses of these parameters and clinical responses were carried out. Univariate analyses showed that a sustained virological response was correlated significantly with IL28B polymorphism, as well as age, white blood cell and neutrophil counts, adherence to RBV, and rapid virological response. Subgroup analysis revealed that patients infected with genotype 2b achieved significantly lower rapid virological response rates than those with genotype 2a. Patients with the IL28B‐major allele showed higher virus clearance rates at each time point than those with the IL28B‐minor allele, and the differences were more profound in patients infected with genotype 2b than those with genotype 2a. Furthermore, both rapid and sustained virological responses were associated significantly with IL28B alleles in patients with genotype 2b. IL28B polymorphism was predictive of PEG‐IFN plus RBV combination treatment outcomes in patients infected with genotype 2 and, especially, with genotype 2b. In conclusion, IL‐28B polymorphism affects responses to PEG‐IFN‐based treatment in difficult‐to‐treat HCV patients. J. Med. Virol. 83:871–878, 2011.

B Cell-Activating Factor Controls the Production of Adipokines and Induces Insulin Resistance

Visceral adipose tissue (VAT) inflammation has been linked to the pathogenesis of insulin resistance and metabolic syndrome. VAT has recently been established as a new component of the immune system and is involved in the production of various adipokines and cytokines. These molecules contribute to inducing and accelerating systemic insulin resistance. In this report, we investigated the role of B cell‐activating factor (BAFF) in the induction of insulin resistance. We investigated BAFF levels in the sera and VAT of obese mice. In obese mice, the BAFF levels were preferentially increased in VAT and sera compared to these levels in normal control mice. Next, we treated mice with BAFF to analyze its influence on insulin sensitivity. BAFF impaired insulin sensitivity in normal mice. Finally, we investigated the mechanisms underlying insulin resistance induced by BAFF in adipocytes. BAFF also induced alterations in the expression levels of genes related to insulin resistance in adipocytes. In addition, BAFF directly affected the glucose uptake and phosphorylation of insulin receptor substrate‐1 in adipocytes. We propose that autocrine or paracrine BAFF and BAFF‐receptor (BAFF‐R) interaction in VAT leads to impaired insulin sensitivity via inhibition of insulin signaling pathways and alterations in adipokine production.