Quantitative Analysis Of Methylated CpG Islands Of P16 In Gastric Carcinomas

Purpose: Gastric carcinogenesis is a multistep process of genetic and epigenetic alterations involving many genes. Methylation of CpG islands around promotor region is an important epigenetic changes, another pathway of inactivation of the genes in addition to mutations. In gastric carcinoma tissues, high frequency of inactivation of the tumor suppressor gene p16 by methylation was detectable. Aberrant p16 methylation was observed frequently even in gastric premalignant lesion dysplasia. Hence, p16 aberrant methylation might involve in progression of gastric carcinomas. Most current analytical assays for detection of CpG methylation are qualitative methods. Because of lack of convenient quantitative assay, the amount of methylation of CpG islands in the tested samples was not reported in most of cases. The aim of the present study is to setup a quantitative method for detection of the methylated p16 CpG islands with denatureing high-performance liquid chromatography (DHPLC) and subsequently to study the possible role of p16 methylation in progression of gastric carcinomas quantitatively. Method: Genomic DNA samples were extracted from 56 surgical primary gastric carcinoma samples for which the information of pathological and clinical characteristics was available. The methylation status of p16 CpG islands of each sample was detected by methylation-specific PCR (MSP) first. Then, universal primers were used to amplify both methylated and unmethylated templates of p16 CpG islands in methylated p16 MSP-positive detection sample (MSP-positive). A DHPLC assay was setup for quantification of the methylated p16 CpG islands. And then, the amplicons was analyzed by DHPLC to detect amount of methylated and unmethylated p16. The percentage of methylated p16 in the testing MSP-positive sample was calculated. The relationship between amout of p16 methylation and pathological stages of gastric carcinomas was analyzed further. Human carcinoma cell line RKO and MGC803 were used as p16 methylated and unmethylated control, respectively. Result: A DHPLC quantitative assay for detection of p16 methylation was established. The retention time for methylated p16 peak(s) was from 5.3~6.0 minute; unmethylated one, 4.0~5.2 minute on chromatogram. The area of methylated p16 peak is positively correlated with the content of methylated p16 templates from RKO cell line. Aberrant p16 methylation was observed in 19 of 56 samples by MSP. The remarkable methylated p16 peak was detected in 5 of 19 MSP-positive samples by DHPLC (DHPLC-positive). Higher pathological stage was observed among these 5 DHPLC-positive samples than DHPLC-negative and MSP-positive samples significantly (p=0.03). Conclusions: Frequence of p16 methylation is not significant associated with the pathological stage,gender,age,differentiation and the metastasis of lymph node of gastric carcinomas. DHPLC can be used to analyze amount of p16 methylation quantitatively. There is remarkable different amount of p16 methylation among those MSP-positive samples : few tissues (26%) exist affluent p16 methylated cells (20~80%), while a majority of samples (73%) hold a small quantity of p16 methylated cells (1~20%). Amount of p16 methylation is correlated with the pathological stage of gastric carcinomas significantly,but no significant association with gender,age,differentiation and the metastasis of lymph node of gastric carcinomas.