Toshiaki Miura

Methylation of multiple genes as molecular markers for diagnosis of a small, well-differentiated hepatocellular carcinoma.

The current study was conducted to identify robust methylation markers and their combinations that may prove useful for the diagnosis of early hepatocellular carcinoma (HCC). To achieve this, we performed in silico CpG mapping, direct sequencing and pyrosequencing after bisulfite treatment, and quantitative methylation‐specific PCR (MSP) in HCC and non‐HCC liver tissues. In the filtering group (25 HCCs), our direct sequencing analysis showed that, among the 12 methylation genes listed by in silico CpG mapping, 7 genes (RASSF1A, CCND2, SPINT2, RUNX3, GSTP1, APC and CFTR) were aberrantly methylated in stages I and II HCCs. In the validation group (20 pairs of HCCs and the corresponding non‐tumor liver tissues), pyrosequencing analysis confirmed that the 7 genes were aberrantly and strongly methylated in early HCCs, but not in any of the corresponding non‐ tumor liver tissues (p < 0.00001). The results obtained using our novel quantitative MSP assay correlated well with those observed using the pyrosequencing analysis. Notably, in MSP assay, RASSF1A showed the most robust performance for the discrimination of HCC and non‐HCC liver tissues. Furthermore, a combination of RASSF1A, CCND2 and SPINT2 showed 89–95% sensitivity, 91–100% specificity and 89–97% accuracy in discriminating between HCC and non‐HCC tissues, and correctly diagnosed all early HCCs. These results indicate that the combination of these 3 genes may aid in the accurate diagnosis of early HCC.

Circulating cell-free DNA as a predictive marker for distant metastasis of hepatitis C virus-related hepatocellular carcinoma.

In a previous study, we showed that levels of cell-free DNA (cfDNA) were significantly higher in sera of patients with hepatocellular carcinoma (HCC) associated with hepatitis C virus (HCV) than in sera of non-HCC patients with HCV. To confirm this finding, we analysed serum cfDNA levels in a cohort of 96 patients with HCV-related HCC and in 100 HCV carriers without known HCC. Again we found that serum cfDNA levels were significantly higher in HCC patients than in HCV carriers (115.9±98.3 vs 34.4±40.4 ng ml−1 (mean±s.d.), P<0.0001). Of 87 eligible patients who underwent curative hepatectomy, those with a high cfDNA level had a significantly shorter overall survival (OS) time than those in whom the cfDNA level was not high. Cox proportional hazards model showed the cfDNA level to be an independent prognostic factor for OS and cancer recurrence in distant organs. Our results suggest that the serum cfDNA level reflects the metastatic potential of HCV-related HCC and that it can be a useful predictive biomarker for distant metastasis after curative surgery.

Methylated cyclin D2 gene circulating in the blood as a prognosis predictor of hepatocellular carcinoma.

BACKGROUND Prognosis of hepatocellular carcinoma (HCC) remains poor because of high recurrence rate. We examined preoperatively the methylated CCND2 gene levels present in the serum following release from HCC cells as a prognosis predictor in patients undergoing curative hepatectomy. METHODS Quantitative real-time RT-PCR and quantitative methylation-specific PCR were used to measure methylated CCND2 gene and its mRNA levels. RESULTS The CCND2 mRNA levels were down-regulated in HCC with early intrahepatic recurrence (IHR) within 1year of curative hepatectomy. We also identified that this down-regulation was due to promoter hypermethylation. In 70 HCC patients who underwent curative hepatectomy, 39 patients sero-positive for the methylated CCND2 gene (>70pg/ml serum) exhibited a significantly shorter disease-free survival (DFS) period (P=0.02) than the 31 patients who were sero-negative for the methylated CCND2 gene. None of the sero-negative patients demonstrated early IHR, and this method of serum testing did not produce any false-negative predictions for early IHR. Multivariate analysis showed that the serum level of methylated CCND2 was an independent risk factor for DFS (hazard ratio of 1.866, 95% CI: 1.106-3.149). CONCLUSION Methylated CCND2 gene in the serum serves as a prognosis predictor of HCC after curative hepatectomy.

Efficient detection of hepatocellular carcinoma by a hybrid blood test of epigenetic and classical protein markers

BACKGROUND There are few blood tests for an efficient detection of hepatocellular carcinoma (HCC) associated with hepatitis C virus (HCV) infection. METHODS The abilities of quantitative analyses of 7 genes hypermethylation in serum DNA, α-fetoprotein (AFP) and prothrombin-induced vitamin K absence II (PIVKA-II), and various combinations to detect HCC were evaluated in a training cohort of 164 HCV-infected patients (108 HCCs; 56 non-HCCs). An optimal hybrid detector, built using data for 2 methylated genes (SPINT2 and SRD5A2), AFP, and PIVKA-II, achieved the most satisfactory ability to detect HCC in the training cohort. We evaluated the ability of the optimal hybrid detector to detect HCC in an independent validation cohort of 258 consecutive HCV-infected patients (112 HCCs; 146 non-HCCs) who were newly enrolled in 4 distinct institutes. RESULTS In the validation cohort of 258 patients, accuracy, sensitivity, and specificity of the hybrid detector for detection of HCC were 81.4%, 73.2%, and 87.7%, respectively. Notably, even when detecting HCC ≤ 2 cm in diameter, the hybrid detector maintained markedly high abilities (84.6% accuracy, 72.2% sensitivity, 87.7% specificity). Youdens index (sensitivity+specificity - 1) for HCC ≤ 2cm was 0.60, vastly much superior to the 0.39 for AFP at a cut-off value of 20 ng/ml and the 0.28 for PIVKA-II at a cut-off value of 40 mAU/ml. CONCLUSIONS These results show that the optimal hybrid blood detector can detect HCV-related HCC more accurately.

Rapid and quantitative detection of CpG-methylation status using TaqMan PCR combined with methyl-binding-domain polypeptide

OBJECTIVES To assess the medical applicability of CpG methylation as molecular markers for cancer diagnosis, we established a new system to determine DNA methylation based on TaqMan PCR combined with a methyl-binding-domain polypeptide 2. DESIGN AND METHODS We evaluated the diagnostic applicability of this approach by examining the methylation status of two tumor suppressor genes, RASSF1A and APC, in 10 paired hepatocellular carcinoma (HCC) and the corresponding non-tumor liver tissues. RESULTS Methylation levels of total 20 clinical samples measured by the TaqMan PCR assay showed a significantly positive correlation (R=0.814, P<0.0005 for RASSF1A, R=0.736, P<0.00001 for APC) with those calculated by bisulfite sequencing. The methylated DNA amount measured by our TaqMan PCR system precisely replicated the methylation status estimated by direct sequencing. CONCLUSIONS This suggests our method may serve as a reliable and easy-to-use tool for cancer diagnosis using methylated genes as biomarkers.

Quantitative Assay of Hepatitis C Virus RNA Using an Automated Extraction System for Specific Capture with Probes and Paramagnetic Particle Separation

ABSTRACT A commercially available automated specimen preparation instrument for specific probe capture and paramagnetic separation has been developed (AmpliCap/GT-12; Roche Molecular Systems). We evaluated assay performance of the AmpliCap/GT-12 in the quantitative assay for hepatitis C virus (HCV) RNA with the AMPLICOR HCV MONITOR Test (version 2.0). Assay linearity using serial dilutions from a serum panel was observed in the range of 500 to 850,000 IU/ml, with a slightly compromised slope in the higher viral titers. The overall within-run and between-run reproducibility of the entire detection process for 3 and 5 log10 (IU/ml) of HCV RNA in samples had a standard deviation of <0.2, which was comparable to a manual method based on organic extraction and isopropanol precipitation (Roche Molecular Systems). Comparison of the test results with those obtained by the manual method showed a good correlation (R2 = 0.972, n = 86). Using heparin (3, 6.5, and 13 U/ml), dextran sulfate (0.1, 1, and 5 mM), hemoglobin (1.13, 2.25, and 4.5 g/liter), conjugated or unconjugated bilirubin (7.5, 15, and 30 mg/dl), and ATP (1.25, 2.5, and 5.0 mM) as known inhibitors, inhibition was only detected at a dextran sulfate concentration of 1 mM with the manual method but not with the AmpliCap/GT-12 extraction. In summary, the AmpliCap/GT-12 system was shown to permit a stable extraction process and accurate results for the quantitative assay of HCV RNA, successfully eliminating the inhibitory effect of dextran sulfate. This automated extraction system provides reliable and reproducible test results and saves labor; thus, it is suitable for routine diagnostic PCR.

Second generation amplicor-HCV monitor assay: clinical features and predictors of the response to interferon.

The aims of this study were to compare the amplicor-HCV monitor assay versions 1.0 and 2.0, and to investigate the clinical usefulness of this assay in patients with chronic hepatitis C. We retrospectively analyzed 154 patients, and 133 of these patients received interferon therapy. Sixty-nine patients were complete responders (CR), and 64 were non-responders. Serum HCV RNA levels of version 1.0 and version 2.0 and HCV genotypes were determined in all patients. There was a good correlation between versions 1.0 and 2.0 in both genotype 1b and 2a, 2b (r=0.907 and 0.726, respectively). In genotype 1b, the mean HCV RNA level obtained by version 1.0 was 384+/-547 kcopies/ml and that obtained by version 2.0 was 488+/-825 kI.U./ml. In genotype 2a/2b, the mean level obtained by version 1.0 was 170+/-369 kcopies/ml and that obtained by version 2.0 was 340+/-402 kI.U./ml. Discriminant analysis revealed that the discriminating points of IFN response were 168 kcopies/ml (genotype 1b, version 1.0), 106 kcopies/ml (genotype 2a and 2b, version 1.0), 102 kI.U./ml (genotype 1b, version 2.0), and 277 kI.U./ml (genotype 2a and 2b, version 2.0). When the patients were stratified according to the discriminating points, the CR rate below the discriminating points were 73.8 and 86.2% in versions 1.0 and 2.0, respectively, in genotype 1b, and the rates were 73.2 and 82.3% in genotype 2a/2b. In addition, receiver-operating characteristic analysis revealed that version 2.0 had significantly better discriminative ability in patients with genotype 1b. We conclude that the second version of the amplicor-HCV monitor assay measures HCV RNA levels with the same precision as version 1.0 and is more useful for the prediction of interferon response than version 1.0.

Mutations to the probe of Cobas TaqMan HIV-1 ver. 1.0 assay causing undetectable viral load in a patient with acute HIV-1 infection

We encountered a human immunodeficiency virus (HIV)-1 in which the viral load was undetectable with the Cobas TaqMan HIV-1 ver. 1.0 (CTM v.1.0) in a patient with acute HIV-1 infection. The CTM v.1.0 assay showed more than 1,000-fold underestimation compared with the subsequent Cobas Amplicor Monitor v.1.5 assay. Because five mismatches to the CTM v.1.0 assay probe in the HIV-1 virus in the patient were disclosed by the manufacturer, partial gag regions of the HIV genome were directly sequenced from the patient’s plasma viral RNA. The detected single nucleotide point mutations were located near the 5′-end of the Cobas Amplicor Monitor probe. Clinicians should be very careful in making interpretations when indeterminate Western blot analysis results and a low or even undetectable HIV-1 viral load are encountered with the CTM HIV-1 ver. 1.0 assay in patients with suspected acute HIV infection. Repeating Western blot analysis is essential before considering a low HIV-1 viral load to be a false-positive result.