title: Methylation of multiple genes as molecular markers for diagnosis of a small, well-differentiated hepatocellular carcinoma.
abstract: 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.
authors.affiliation: Research Group, Molecular Diagnostics R&D Department, Roche Diagnostics K.K., Minato-ku, Tokyo, Japan.
authors.forename: Toyoki
authors.initials: T
authors.lastname: Moribe
authors.forename: Norio
authors.initials: N
authors.lastname: Iizuka
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Miura
authors.forename: Naoki
authors.initials: N
authors.lastname: Kimura
authors.forename: Shigeru
authors.initials: S
authors.lastname: Tamatsukuri
authors.forename: Hideo
authors.initials: H
authors.lastname: Ishitsuka
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Hamamoto
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Sakamoto
authors.forename: Takao
authors.initials: T
authors.lastname: Tamesa
authors.forename: Masaaki
authors.initials: M
authors.lastname: Oka
mesh_terms: D001483:Base Sequence; D014408:Biomarkers, Tumor / Q000097:blood*; D006528:Carcinoma, Hepatocellular / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D016022:Case-Control Studies; D002454:Cell Differentiation; D018899:CpG Islands; D019175:DNA Methylation; D017931:DNA Primers; D006801:Humans; D008113:Liver Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D016133:Polymerase Chain Reaction
chemical_list: D014408:Biomarkers, Tumor; D017931:DNA Primers