Shigeru Tamatsukuri

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.

Spontaneous negativation of serum hepatitis C virus RNA is a rare event in type C chronic liver diseases: analysis of HCV RNA in 320 patients who were followed for more than 3 years

BACKGROUND/AIMS The natural course of hepatitis C virus (HCV) replication in type C liver diseases has not yet been elucidated. The aim of the study was to investigate the spontaneous outcome of the viremia by examining the changes in HCV RNA in patients with chronic type C liver diseases. METHODS Among patients who visited our liver clinic between June 1981 and December 1993, 320 patients with chronic type C liver diseases were followed for at least 3 years and had no history of interferon treatment. HCV RNA was examined by a highly specific reverse transcription-polymerase chain reaction method in paired serum samples obtained from these patients at the beginning and end of follow-up. RESULTS Among the 320 cases, HCV RNA was seropositive in 310 (97%) cases at the beginning of follow-up. Of these 310, HCV RNA remained seropositive in 304 (98%) and became seronegative in six (2%) cases by the end of follow-up. All of these six patients had liver cancer. HCV RNA became seronegative after the patients entered the state of liver failure because of the development of tumors or portal thrombosis. The remaining 10 cases who were seronegative for HCV RNA at the beginning were seropositive at the end of follow-up. Among the 320 cases, serum alanine aminotransferase normalized and remained normal for more than 12 months until the end of follow-up in 11 cases (0.6%/year/case), but none became negative for HCV RNA. CONCLUSIONS Thus, during the natural course of chronic HCV infection, spontaneous negativation of serum HCV RNA seems extremely rare, at least in patients with chronic active hepatitis or cirrhosis of the liver, and may occur primarily at the terminal stage when tumors cause liver failure.

Titration of Hepatitis C Virus in Chimpanzees for Determining the Copy Number Required for Transmission

Objective: To determine the copy number of hepatitis C virus (HCV) RNA, determined by nucleic acid amplification test (NAT) for screening blood units in Japan, that can transmit infection to chimpanzees. Methods: Fresh-frozen plasma with markers of HCV infection, as well as inocula pedigreed from 1 of them, were evaluated for the infectious activity in chimpanzees. Results: One unit each (273–282 ml) of fresh-frozen plasma from 2 blood donors or a pool from 13 donors to make a unit, which contained high-titered antibody to HCV but without HCV RNA detectable by NAT, did not infect any of 3 chimpanzees. Two chimpanzees were infected, however, when they were inoculated with 1 ml of serum from a blood donor in the ‘window period’ of HCV infection and containing 7.0 × 106 copies/ml of HCV RNA. The preacute phase serum from 1 of them harvested 7 weeks after the inoculation was titrated in 2 chimpanzees, and an inoculum containing approximately 2 × 101 copies of HCV RNA could transmit infection to both of them. Conclusion: Approximately 20 copies of HCV can transmit infection to recipients, which needs to be taken into consideration in planning the screening of blood units for HCV RNA by NAT. Although the sensitivity of present NAT could be improved further, there would be a limit of it in detecting a low-level HCV RNA in the window period of donors with the infectious capacity in recipients.

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.

Application of oligo(dT)30-latex for rapid purification of poly(A)+ mRNA and for hybrid subtraction with the in situ reverse transcribed cDNA

The carboxyl groups on the surface of latex beads were linked to amino moiety of cytidine residue of oligo(dC)10(dT)30. The resultant latex beads-(dC)10(dT)30 showed a very stable suspension and yet is precipitable to a small pellet by centrifugation. These properties merits the oligomer-linked beads to be applied for experiments in which poly(A)+ mRNAs are involved. An efficient (> 95%) hybridization to poly(A)+ mRNA occurred in a short reaction period (10 min), and more than 95% of bound mRNAs were recovered from the beads by heating (65 degrees C, 5 min) followed by centrifugation. Interestingly, the poly(A)+ mRNAs could be transcribed to cDNAs in situ by reverse transcriptase, with the covalently linked oligo(dT)30 as primers. These properties allowed the oligo(dT)30-latex to prepare the cDNA covalently bound to latex which was used for mRNA hybrid subtraction. In a model experiment with the mixture of vaccinia virus and HeLa mRNAs, about 200-fold enrichment of vaccinia mRNA species was obtained after four cycles of hybrid subtraction with HeLa cDNA-latex.

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.