Can RARbeta p2 promoter methylation in breast random periareolar fine-needle aspiration serve to risk stratify women at high risk for breast cancer?

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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 rFNA. Hypothesis: We tested the hypothesis that RARbeta P2 methylation is frequently observed in both primary breast cancers and in rFNA from high-risk women. Methods/Observations: The frequency of RARbeta2 P2 promoter methylation was tested in 1) twenty low-risk, primary breast cancers with 5 year clinical follow up and 2) fifty rpFNA samples performed in women 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. We observe RARbeta2 P2 methylation in 16/20 (80%) primary breast tumors and in 25/55 rpFNA aspirates tested. rpFNA were stratified for cytologic atypia using the Masood cytology index. The distribution of RARbeta2 P2 methylation was reported as a function of increased cytologic abnormality. RARbeta2 P2 methylation was present in 1) <10% of rpFNA with Masood scores of 10 (benign), 2) 15% with Masood score of 11-12 (low-grade proliferative), 2) 33% with Massood score of 13 (high-grade proliferative), and 3) 50% with Masood scores of 14-15 (atypia). Conclusions: RARbeta2 P2 methylation is observed in 80% of low-risk breast cancers and demonstrates a positive association with increasing cytologic atypia. These observations provide strong rationale for using RARbeta2 P2 promoter methylation in rFNA as a sensitive marker and readily detectable marker of early breast cancer risk.