Quantitative methylation of six genes in breast tumors among the Singapore population

B63

Breast cancer is the leading cause of cancer-related mortality in women worldwide. In Singapore, breast cancer remains one of the most common types of cancer diagnosed among woman, with a 1 in 15 risk of women developing breast cancer. Gene methylation is considered to be one of the most common mechanism for silencing cancer-related genes, resulting in the loss of gene expression. In contrast to genetic mutations, epigenetic alteration in DNA though heritable, does not alter the primary DNA sequence. DNA prompter hypermethylation has been implicated in the loss of gene expression via the inactivation of tumor suppressor genes that regulate cell cycle control, DNA repair, tumor susceptibility, carcinogen detoxification, cell adhesion and angiogenesis. In this study the quantitative multiplex methylation-specific PCR (QM-MSP) technique was used to detect the hypermethylation of 6 tumor suppressor genes in 31 breast cancer tumor specimens and their matching adjacent normal breast tissue. The QM-MSP is superior to the methylation specific PCR (MSP) and the quantitative MSP (Q-MSP) as it allows the co-amplification of many genes within a patient sample, when the quantity of DNA is limited. Results from this study demonstrated that the QM-MSP was able to provide 97% sensitivity in detecting methylation in breast tumors in at least one of the six genes. We observed that among the tumors, 77% were positive for RASSF1A, 67% for APC, 61% for TWIST, 45% for cyclin D2, 64% for RAR-ß, and 52% for HIN1 respectively. In contrast, the adjacent normal breast tissue showed low levels for positive methylation, RASSF1A (10%), APC (10%), TWIST (10%), cyclin D2 (14%), RAR-ß (10%) and HIN1 (8%) respectively. A significant difference in the frequency of positive methylation between tumor and normal tissues was also observed (RASSF1A, p<0.0001, APC, p<0.0001, TWIST, p=0.0002, cyclin D2, p= 0.0009, RAR-ß, p= 0.0019 and HIN1, p= 0.0018). Interestingly, although six genes were tested in this study, only one tumor sample demonstrated methylation in all 6 genes, while most tumor samples were positive for methylation in 2-4 genes (65%) in varies degree. When compared to the patients’ estrogen receptor (ER) status, we found that among the ER⁺ group, higher frequency of methylation was oberved in patients that were above 50 years of age, suggesting that gene hypermethylation increases with age in patients that are positive for ER. Current studies are underway to evaluate the quantitative characterization of genes hypermethylated in breast cancer in a larger tumor series. Epigenetic studies such as this study may be useful to determine the correlations between the methylation patterns with clinical findings, so as to provide relevant information for pathological assessment and clinical management of breast cancer.