Abstract 1995: Promoter methylation analysis of separate cell populations in human breast milk is required for assessing breast cancer risk
Authors: Williams Kristin E. , Browne Eva P. , Anderton Douglas L. , Poulin Matthew , Yan Liying , Arcaro Kathleen F. 
Published: August 2014

Abstract
Gene specific promoter methylation in breast epithelial cells is considered one of the most promising biomarkers for assessing breast cancer risk. Unfortunately, obtaining breast epithelial cells is difficult. We recently demonstrated that human breast milk provides a source of epithelial cells useful for assessing breast cancer risk through analysis of DNA promoter methylation patterns. However, it has not been determined whether separation of the epithelial cells from the total cell population is necessary to obtain reliable promoter methylation patterns useful in assessing this risk.
The present study determines the extent to which separation of the cell populations in breast milk alters the observed methylation patterns. A fresh breast milk sample (average size of 118 mL ± 29 mL) was collected from six lactating women between 25 and 43 years of age. For each milk sample, three groups of cells were collected: an unseparated (or total) cell population, an epithelial-enriched cell fraction and an epithelial-depleted cell fraction. The depleted and enriched cell fractions were obtained by immunomagnetic separation. Cell fractions were frozen and shipped to EpigenDX for DNA isolation, bisulfite treatment and pyrosequencing. Promoter methylation of five tumor suppressor genes (APC, CyclinD2, p16, RARβ, and RASSF1A) was examined.
Cell separation resulted in distinct methylation patterns. Overall, the highest methylation scores were detected in the epithelial-enriched cell fractions: mean methylation scores were significantly higher in the epithelial-enriched fraction as compared to the depleted fraction or total cell population for APC, CyclinD2, RARβ, and RASSF1A. Only p16 showed very low methylation: &lt;5% for all genes in all cell fractions from all women. Interestingly, mean methylation scores of two women of comparable age, both with a previous biopsy, revealed strikingly different methylation patterns that might be associated with age at first pregnancy and parity. The woman who had her first pregnancy at age 16 years followed by an additional seven live births had the lowest mean methylation score for all genes (2.67), while the woman who had a first and only birth at age 37 years had the highest mean methylation score for all genes (25.86). Repeat pyrosequencing analysis completed using the bisulfite-modified DNA stored at -80°C for two years resulted in remarkably similar methylation values. These data show that cell separation is necessary to see distinct changes in promoter methylation patterns. Further methylation analysis on a larger cohort of women is needed to determine the association of age at first pregnancy and promoter methylation.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1995. doi:10.1158/1538-7445.AM2011-1995