Detection of RASSF1A and RARβ Hypermethylation in Serum DNA from Breast Cancer Patients

Breast cancer is fast emerging as the leading cancer amongst females, especially in young females in metropolitan cities in India. The epigenetic alterations involved in the onset and progression of breast cancer may serve as biomarkers for early detection and prognosis of the disease. Furthermore, using body fluids such as serum offers a noninvasive method to procure multiple samples for such analyses. In this study, we examined methylation status of two normally unmethylated but biologically significant cancer genes, RAS association domain family protein 1A (RASSF1A) and retionic acid receptor beta (RAR beta) by methylation specific PCR (MSP) in invasive ductal carcinomas of the breast and paired serum DNA. RASSF1A was found to be methylated in 17 of 20 (85%) breast tumors; while sera from 15 of 20 (75%) of the patients showed concordant methylated RASSF1A, with a sensitivity of 88%. RAR beta was methylated in 2/20 (10%) breast tumors. A gene unmethylated in the tumor DNA was always found to be unmethylated in the matched serum DNA for both RASSF1A and RAR beta genes; hence specificity was 100%. Immunohistochemical analysis of RAR beta protein in 15 breast carcinoma patients harboring unmethylated RAR beta in tumors and serum DNA showed the expression of RAR beta protein in tumors and paired normal breast tissues, confirming the MSP findings, suggesting that RAR beta promoter is functional in these cases. This study underscores the potential utility of DNA methylation based screening of serum, a readily accessible body fluid, as a surrogate marker for early detection of breast cancer.