Abstract Analysis of promoter methylation patterns in genes frequently downregulated in breast cancer may serve as an early indicator of breast cancer risk. To date, studies of methylation patterns in breast epithelial cells from asymptomatic women have been limited to cells obtained from fine nipple aspirate and ductal lavage. Analysis of promoter hypermethylation in exfoliated epithelial cells isolated from breast milk provides a non-invasive opportunity to assess breast cancer risk. Our preliminary data indicate that we can detect a cancer methylation phenotype in the milk of women at high risk. We propose that epithelial cells isolated from breast milk of lactating women scheduled to have a breast biopsy provide a resource for assessing early changes in DNA related to increased breast cancer risk. Two hundred and fifty breast milk samples from women who are scheduled to have, or have had a breast biopsy will be collected. Of these women, we expect that roughly ten percent will be diagnosed with breast cancer. Breast milk will be collected from both the biopsied and contralateral breast of each participant. To date, 153 participants have donated breast milk, yielding 300 breast milk samples with an average size of 50 ml (±30 ml) per breast. After collection, the epithelial enriched and epithelial depleted cells from each breast were isolated. Subsequent DNA isolation of both the epithelial enriched and depleted fraction of cells yielded an average of 372 ng (range 12-5000 ng) of high quality DNA. Using the DNA extracted from epithelial enriched and depleted fractions of cells isolated from breast milk, the promoter methylation level of 9 genes known to be methylated in breast cancer will be examined using pyrosequencing technology. We will assess the promoter methylation levels of: CDH1, DAP-K, ER-α, GSTP1, HIN1, P16, RAR-ß, RASSF1A, and SFRP1 in the epithelial enriched and depleted bisulfite treated DNA of each breast milk sample. Preliminary pyrosequencing assays conducted in our lab have shown that there is a difference in RASSF1A methylation levels between the epithelial enriched DNA from the cancerous breast compared to the epithelial depleted DNA from the non-cancerous breast of the same participant. Further methylation analysis may prove that breast milk can be used as non-invasive alternative to ductal lavage or fine needle aspirate, which may yield accurate breast cancer risk assessment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3764.