Accumulation of DNA methylation is associated with tumor stage in gastric cancer.

The authors purpose in this study was to clarify the difference in terms of clinicopathologic features between gastric cancer (GC) with high numbers of DNA methylated genes and CpG island methylator phenotype (CIMP)-positive GC as originally defined. We analyzed DNA methylation of 12 tumor-related genes (hMLH1, MGMT, p16(INK4a), CDH1, RAR-beta, HLTF, RIZ1, TM, FLNc, LOX, HRASLS, HAND1) in 75 samples of GC from 75 patients, 25 samples of corresponding nonneoplastic mucosa from 25 patients, and 10 samples of normal gastric mucosa from 10 healthy young individuals by methylation-specific polymerase chain reaction (PCR) and bisulfite PCR. We also investigated CIMP status by examining the methylation of five MINT loci and p53 mutation status by PCR single-strand conformation polymorphism. We measured levels of expression of mRNAs for these 12 genes by quantitative reverse transcription PCR in 50 GC specimens. The average number of methylated genes per tumor was 4.83. DNA methylation of each gene was correlated with low expression of the respective mRNA. High methylation (GC with 5 or more methylated genes) was detected in 39 (52.0%) of 75 GCs. Twenty-nine (37.8%) of 75 GCs were CIMP-positive. DNA methylation of each of the 12 genes was observed more frequently in the high-methylation group than in the low-methylation group. Methylation of 6 specific genes occurred more frequently in CIMP-positive GC than in CIMP-negative GC. Methylation of the remaining 6 genes was not correlated with CIMP-status. High methylation was found more frequently in Stage III/IV GC (26 of 40 cases, 65.0%) than in Stage I/II GC (13 of 35 cases, 37.1%, P = 0.029).CONCLUSIONS.These findings indicate that GCs with higher numbers of methylated genes have more distinct DNA methylation profiles than the originally defined CIMP-positive GCs. DNA methylation of tumor-related genes accumulates in conjunction with tumor progression.

Oue N ，Mitani Y ，Motoshita J ，Matsumura S ，Yoshida K ，Kuniyasu H ，Nakayama H ，Yasui W ... -  《CANCER》

DNA methylation of genes linked with retinoid signaling in gastric carcinoma: expression of the retinoid acid receptor beta, cellular retinol-binding protein 1, and tazarotene-induced gene 1 genes is associated with DNA methylation.

Hypermethylation of CpG islands has been associated with silencing of various tumor suppressor genes, and the retinoid acid receptor beta (RARbeta), cellular retinol-binding protein 1 (CRBP1), and tazarotene-induced gene 1 (TIG1) genes have been associated with retinoic acid signaling. To the authors' knowledge, little is known regarding the involvement of these three genes in gastric carcinoma (GC). In this study, the authors investigated the methylation status of these genes and analyzed the role of their DNA methylation in GC. DNA methylation of 3 retinoic acid-associated genes was analyzed in 42 samples of GC from 42 patients and in 8 GC cell lines by methylation-specific polymerase chain reaction (PCR) analysis. The mRNA expression levels for these three genes were measured by quantitative reverse transcription-PCR. In 7 of 8 GC cell lines, the CRBP1 gene was hypermethylated, and CRBP1 transcription was inactive. In 6 of 8 GC cell lines, the TIG1 gene was hypermethylated, and TIG1 transcription was inactive. Treatment with demethylating agent 5-aza-2'-deoxycytidine restored both CRBP1 and TIG1 transcription. DNA methylation of the RARbeta, CRBP1, and TIG1 genes was detected in 15 of 42 GC samples (36%), 14 of 42 GC samples (33%), and 4 of 42 GC samples (10%), respectively, and in 6 of 30 samples (20%), 0 of 30 samples (0%), and 1 of 30 samples (3%) of corresponding nonneoplastic mucosa. None of the 10 normal gastric mucosa samples from young, healthy individuals demonstrated hypermethylation of any of these genes. DNA methylation of each gene was associated significantly with low mRNA expression of the respective gene. Twenty-four of 42 GC samples (57%) demonstrated hypermethylation of at least 1 of the 3 genes. However, no significant, concordant hypermethylation of these genes was observed. The results suggested that gastric carcinogenesis involves transcriptional inactivation by aberrant DNA methylation of genes related to retinoid signaling.

Shutoh M ，Oue N ，Aung PP ，Noguchi T ，Kuraoka K ，Nakayama H ，Kawahara K ，Yasui W ... -  《CANCER》

CpG island methylation status in gastric carcinoma with and without infection of Epstein-Barr virus.

Chang MS ，Uozaki H ，Chong JM ，Ushiku T ，Sakuma K ，Ishikawa S ，Hino R ，Barua RR ，Iwasaki Y ，Arai K ，Fujii H ，Nagai H ，Fukayama M ... -  《CLINICAL CANCER RESEARCH》

Genetic, epigenetic, and clinicopathologic features of gastric carcinomas with the CpG island methylator phenotype and an association with Epstein-Barr virus.

The CpG island methylator phenotype (CIMP), which is characterized by simultaneous methylation of the CpG islands of multiple genes, has been recognized as one of the important mechanisms in gastrointestinal carcinogenesis. Methylation of the 5 methylated-in-tumors (MINT) loci and 12 tumor-related genes in 78 primary gastric carcinomas was examined using combined bisulfite-restriction analysis. Epstein-Barr virus (EBV)-associated gastric tumors were detected using real-time polymerase chain reaction analysis followed by an evaluation of the correlations between CIMP status, EBV-association, and genetic alteration of p53 and K-ras. The authors compared the clinicopathologic features of gastric carcinomas that had high CIMP methylation (CIMP-H) with tumors that had low CIMP methylation (CIMP-L) or negative CIMP methylation (CIMP-N). The methylation profiles of 12 genes showed nonrandom methylation, supporting the presence of CIMP in gastric carcinoma. No p53 mutations were detected among CIMP-H tumors, and no EBV association was detected in tumors that showed mutation of p53 and K-ras. In a multiple logistic regression model with CIMP-H as the dependent variable, proximal location (P = .011), diffuse type (P = .019), and less advanced pathologic TNM status (P = .043) contributed significantly to CIMP-H. Patients who had CIMP-N gastric tumors had a significantly worse survival than patients who had CIMP-H tumors (P = .004) or CIMP-L tumors (P = .012). EBV-associated tumors were associated strongly with CIMP-H, hypermethylation of tumor-related genes, and no p53 or K-ras mutation. CIMP status appeared to be associated with distinct genetic, epigenetic, and clinicopathologic features in gastric carcinomas. The finding that gastric carcinomas arose through different molecular pathways may affect not only tumor characteristics but also patient prognosis.

Kusano M ，Toyota M ，Suzuki H ，Akino K ，Aoki F ，Fujita M ，Hosokawa M ，Shinomura Y ，Imai K ，Tokino T ... -  《CANCER》

Distinct promoter hypermethylation of p16INK4a, CDH1, and RAR-beta in intestinal, diffuse-adherent, and diffuse-scattered type gastric carcinomas.

Hypermethylation of CpG islands in gene promoters is associated with silencing of various tumour suppressor genes. Recent studies of colorectal and gastric carcinomas have defined a CpG island methylator phenotype (CIMP), which involves the targeting of multiple genes by promoter hypermethylation. In this study, methylation-specific polymerase chain reaction (PCR) was performed to study methylation of CpG islands in the promoters of the p16(INK4a), cadherin 1 (CDH1), and retinoic acid receptor-beta (RAR-beta) genes in 45 gastric carcinomas and to investigate whether CDH1 and RAR-beta promoter hypermethylation is associated with CIMP-positive gastric carcinoma. CpG island hypermethylation of the p16(INK4a), CDH1, and RAR-beta promoters was detected in 12 (27%), 26 (58%), and 24 (53%) of the 45 gastric carcinomas, respectively. Hypermethylation of the p16(INK4a) promoter was more common in intestinal type than in diffuse type gastric carcinomas (p = 0.0023; Fisher's exact test) and was inversely associated with p53 mutations (p = 0.0225; Fisher's exact test). However, CDH1 and RAR-beta promoter hypermethylation was observed more frequently in diffuse-scattered type gastric carcinoma than in other types (intestinal and diffuse-adherent types) (p = 0.0175 and p = 0.0335, respectively; Fisher's exact test) and was not associated with p53 mutation status. Moreover, hypermethylation of the CDH1 and RAR-beta promoters occurred concordantly (p < 0.0001; Fisher's exact test). These results suggest that at least two types of promoter methylation status are involved in the development of the intestinal (p16(INK4a) promoter hypermethylation) and diffuse-scattered types (CDH1 and RAR-beta promoter hypermethylation) of gastric carcinoma.

Oue N ，Motoshita J ，Yokozaki H ，Hayashi K ，Tahara E ，Taniyama K ，Matsusaki K ，Yasui W ... -  《JOURNAL OF PATHOLOGY》