Abstract 3646: Promoter methylation in epithelial cells isolated from breast milk of women from Istanbul, Turkey.
Authors: Browne Eva P. , Punska Elizabeth C. , Dinc Signem E. , Vitrinel Ayca , Erdag Gulay C. , Anderton Douglas L. , Arcaro Kathleen F. , Yilmaz Bayram 
Published: August 2014

Abstract
Current models of assessing breast cancer risk do not adequately predict whether an individual woman will develop breast cancer. Assessment of healthy tissue for epigenetic changes that result in silencing of tumor suppressor genes may provide a more accurate assessment of individual breast cancer risk. Breast milk is an underutilized source of epithelial cells from non-symptomatic women that can be used for risk assessment.
In the present pilot study, we examined promoter methylation of three tumor suppressor genes in epithelial cells isolated from breast milk of Turkish women. Forty-three women living in Istanbul, Turkey were recruited to complete a health and reproductive history questionnaire and to donate a single milk sample. Women ranged in age from 19 to 43 years old, had an average of 2 children (range: 1-5), a mean age at first birth of 25 years (range: 16-36), and a mean BMI of 26 (range 19.3-33.6). Nine percent reported smoking and thirty two percent reported exposure to second-hand smoke. Three of the 43 women had a family history of breast cancer, while four women reported having a family history of ovarian cancer. Each woman donated a single breast milk sample, with a mean volume of 17 ml (S.D. 11.6 ml, range: 2-49 ml), which was processed within 12 hours of donation. Epithelial cells were isolated from the milk via immunomagnetic beads. The breast milk samples yielded an average of 320,000 epithelial cells (S.D. 480,000, range: 10,000-2,600,000), from which DNA was extracted.
Utilizing the extracted epithelial DNA, promoter methylation of three tumor suppressor genes; Rass association domain family 1(RASSF1), secreted frizzle related protein 1(SFRP1), and glutathione-S-transferase class pi 1 (GSTP1), was determined by pyrosequencing. For RASSF1 the average promoter methylation was 6.3% (n= 33); there were two outliers whose average methylation was 54.48%. For SFRP1 the average promoter methylation was 5.7 % (n= 17) with one outlier at 53.4% methylation. For GSTP1, the mean methylation was 4.4% (n= 29), with two outliers whose average methylation was 16.9%. Methylation scores were not correlated with any of the demographic factors, such as second hand smoke exposure, age or BMI. As expected of non-symptomatic breast tissue, promoter methylation scores were in general low; however, outlier scores suggest that promoter methylation may be useful in assessing risk. Long-term follow-up is required to evaluate the predictive value of promoter methylation in tumor suppressor genes.
Citation Format: Eva P. Browne, Elizabeth C. Punska, Signem E. Dinc, Ayca Vitrinel, Gulay C. Erdag, Douglas L. Anderton, Kathleen F. Arcaro, Bayram Yilmaz. Promoter methylation in epithelial cells isolated from breast milk of women from Istanbul, Turkey. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3646. doi:10.1158/1538-7445.AM2013-3646