Tumor Suppressor Gene Promoter Hypermethylation in Serum of Breast Cancer Patients

Abstract

Purpose: Breast cancer is the most common malignancy in American women and the second leading cause of death from cancer. The genetic and epigenetic alterations that initiate and drive cancer can be used as targets for detection of neoplasia in bodily fluids. Tumor cell-specific aberrant promoter hypermethylation can be detected in nipple aspirate and ductal lavage from breast cancer patients. In this study, we examine serum, a more readily accessible bodily fluid known to contain neoplastic DNA from individuals with cancer, for methylation-based detection of breast neoplasia.Experimental Design: We examined the promoter methylation status of three normally unmethylated biologically significant cancer genes, RAS association domain family protein 1A (RASSF1A), adenomatous polyposis coli (APC), and death-associated protein kinase (DAP-kinase), by sensitive methylation-specific PCR in 34 breast tumor and paired preoperative serum DNA. The 34 patients comprised 7 ductal carcinoma in situ (CIS), 3 lobular CIS, 5 stage I and 15 stage II to IV invasive ductal carcinomas, and 4 invasive lobular carcinomas. Normal and benign tissue and serum control DNA were also examined to determine the specificity of hypermethylation.Results: Hypermethylation of one or more genes was found in 32 of 34 (94%) breast tumor DNA. APC was hypermethylated in 15 of 34 (47%), RASSF1A in 22 of 34 (65%), and DAP-kinase in 17 of 34 (50%) tumors. Twentysix (76%) of the corresponding serum DNA were positive for promoter hypermethylation, including ductal CIS, lobular CIS, stage I disease, and lobular carcinoma patients. No hypermethylation of APC, RASSF1A, or DAP-kinase was observed in serum DNA from normal healthy women and patients with inflammatory breast disease or nonneoplastic breast tissue specimens. A gene unmethylated in the tumor DNA was always found to be unmethylated in the matched serum DNA (100% specificity).Conclusions: Tumor cell specific promoter hypermethylation of APC, RASSF1A, and DAP-kinase is present in ductal CIS, lobular CIS, and all grades and stages of invasive breast cancer. Hypermethylation can be detected by methylation-specific PCR analysis in serum DNA from patients with preinvasive and early-stage breast cancer amenable to cure. If confirmed in additional studies, hypermethylation-based screening of serum, a readily accessible bodily fluid, may enhance early detection of breast cancer.

Single CpG hypermethylation, allele methylation errors, and decreased expression of multiple tumor suppressor genes in normal body cells of mutation‐negative early‐onset and high‐risk breast cancer patients

“…In addition to allele methylation errors, we found single CpG hypermethylation in multiple TS genes in normal body cells of approximately one third of EO BC patients. Previous studies45, 46, 47 observed cumulative hypermethylation of TS genes in tumor tissue and/or serum (probably due to cell‐free tumor DNA). The epigenetic abnormalities of TS genes in whole blood DNA in our study are not derived from tumor cells.…”

Genome-wide identification of OTP gene as a novel methylation marker of breast cancer

“…Recently, several groups, using a panel of multiple genes which are well known to be tumorassociated, have demonstrated the prevalence of methylation positive genes in sera or plasma from breast cancer patients (20)(21)(22)(23)(24)(25)(26)(27). Nevertheless, current multiple gene panel assays still lack the necessary sensitivity for application in the clinical setting.…”

Clinicopathological significance of RASSF1A reduced expression and hypermethylation in hepatocellular carcinoma

“…It has been suggested that RASSF1A methylation is one of the most common aberrations so far identified in human cancers and that the loss of the functional protein may promote the development of many human tumors. Hypermethylation of the RASSF1A has been detected frequently in the tissues of different cancers [10][11][12][13][14][15][16][17], while in the body fluid, methylation of RASSF1A promoter has also been documented in 50% sputum and 84% serum from lung cancer [30], 35% urine from bladder cancer [15], 56-60% serum from breast caner [31][32][33], and 5% serum from undifferentiated nasopharyngeal carcinoma [34], which indicated the value of RASSF1A methylation in DNA for the early-stage diagnosis of tumors. Detection of methylated DNA has then been suggested as a potential biomarker for early detection of cancer.…”