DNA methylation profile analysis of patients with ovarian tumor

Abstract

Epithelial ovarian cancer has the highest mortality rate of cancers of the reproductive organs and is the fifth leading cause of cancer death in women in the U.S. Since there are no obvious symptoms in the early stage ovarian cancer, and due to the lack of early detection strategies, most patients are diagnosed in the advanced stages. Therefore, the development of useful molecular markers for screening and prognostic prediction is warranted to enable early detection.Aberrant methylation of CpG islands in promoter regions can permanently inactivate tumor-suppressor genes and therefore play an important role in the occurrence and development of cancer. The objective of this study was to evaluate aberrant promoter hypermethylation as a means to detect epigenetic alterations of genes specific to ovarian cancer, and investigate any difference in the DNA methylation status between benign and malignant ovarian tumor by comparing quantitative data produced by pyrosequencing. Five samples from stage IIIc or IV malignant epithelial ovarian cancer and 5 samples from benign ovarian tumor patients were obtained from the patients’ venous blood prior to surgical resection of ovarian tumor. Genomic DNA was extracted from the samples and methylated DNA immunoprecipitation (MeDIP) microarray chip analysis was employed to detect genes that showed significant hypermethylation of the promoter CpG islands. Seventeen genes were stratified2according to the MeDIP chip analysis, and the following genes were feasible for further pyrosequencing analysis: P2X3, CRTC1, MAD1L1, and HCCA2. Methylation status of 3-5 CpG sites per gene was determined by pyrosequencing. Significant CpG methylation of gene promoters within all samples was found. However, promoter methylation was significantly elevated in the cancer group only for MAD1L1 (p = .047) but not for P2X3 (p = .600), CRTC1 (p = .465), and HCCA2 (p = .175). The methylation level did not show any significant relationship with histological grade or follow up months after surgery in malignant ovarian cancer group. Promoter methylation analysis using pyrosequencing reveals valuable quantitative data; however our results did not reveal any significant quantitative differences in methylation between cancer and benign ovarian tumor group except for a single gene. Although numerous issues remain to be resolved, using combined methodology as in this study for the detection and quantitative measurement of methylated DNA in serum may be a promising tool for ovarian cancer diagnosis.