Can the Presence of Methylation of the Retinonic Acid Receptor-beta2 P2 Promoter in Random Periareolar Fine Needle Aspiration be Used to Risk-stratify Women at Risk for Breast Cancer?

Gregory Bean
Division of Medical Oncology; Duke University Medical Center

Background: Loss of RARbeta2 function in mammary epithelial cells is hypothesized to be the result of both genetic and epigenetic events. Methylation of the RARbeta2 P2 promoter is hypothesized to be an important mechanism for loss of RARbeta2 expression during early mammary carcinogenesis. Periareolar Random Breast Fine-Needle Aspiration (RPFNA) is a research technique developed to repeatedly sample mammary cells from the whole breast of asymptomatic high-risk patients to assess both 1) breast cancer risk and 2) response to chemoprevention. RARbeta P2 methylation is a highly sensitive marker that is detected in as few as 5 cells. Coupling RARbetaP2 methylation to rFNA has the potential to enhance the sensitivity and specificity of RPFNA.

Hypothesis: This study aims to test whether the presence of RARbeta2P2 promoter hypermethylation predicts the presence of cytological atypia in women at high-risk for breast cancer.

Methods: The frequency of RARbeta2 promoter methylation was tested in 1) sixteen early stage breast cancers and 2) sixty-seven Random Periareolar Fine Needle Aspiration (RPFNA) samples obtained from 38 asymptomatic women who were at increased risk for breast cancer. Risk was defined as either 1) 5 year Gail risk calculation > 1.7%, 2) prior biopsy exhibiting atypical hyperplasia, LCIS/DCIS, or 3) known BRCA1/2 mutation carrier. RARbeta2 promoter methylation was assessed at two regions, M3 (-51 to +162 bp) and M4 (+104 to +251 bp). In early stage cancers, M4 methylation was observed in 11/16 (69%) cases; in RPFNA aspirates, methylation was present at M3 and M4 in 28/56 (50%) and 19/56 (38%) cases, respectively. RPFNAs were stratified for cytologic atypia using the Masood cytology index. The distribution of RARbeta2 P2 promoter methylation was reported as a function of increased cytologic abnormality. Methylation at both M3 and M4 was observed in 1) 0/10 (0%) of RPFNAs with Masood scores of 10 or less (non-proliferative), 2) 3/20 (15%) with Masood scores of 11-12 (low-grade proliferative), 2) 3/10 (30%) with Masood scores of 13 (high-grade proliferative), and 3) 7/14 (50%) with Masood scores of 14-15 (atypia).

Conclusion: Results from this study indicate that the RARbeta2 P2 promoter is frequently methylated (69%) in primary breast cancers and demonstrates a positive association with increasing cytologic abnormality in RPFNA. The presence of RARbeta2 P2 methylation is currently being used in our high-risk breast cancer prevention cohort to 1) track response to chemoprevention agents and 2) test whether the presence of RARbeta2P2 methylation in RPFNA predicts cytological progressing in high-risk women.