Abstract LB-443: Methylation in exfoliated epithelial cells from lactating women at increased risk for developing breast cancer
Authors: Browne Eva P. , Punska Elizabeth C. , Anderton Douglas L. , Lenington Sarah , Arcaro Kathleen F. 
Published: July 2012

Abstract
Breast cancer is the second most commonly diagnosed cancer among women, and is difficult to diagnose during pregnancy and lactation. Frequent methylation of the promoter region of key tumor suppressor genes occurs in the early etiology of breast cancer. We aimed to evaluate the feasibility of using DNA extracted from the epithelial cells exfoliated in breast milk as a biomarker for 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. Breast milk samples from both the biopsied and non-biopsied breasts of women scheduled for or who had had a breast biopsy were collected and processed within 24 hours of expression. Epithelial enriched cell fractions were isolated via immunomagnetic separation and DNA subsequently isolated and bisulfite treated for RASSF1, GSTP1, and SFRP1 promoter methylation analysis via pyrosequencing. When available, pathology reports from each participant were also obtained to categorize the outcome of the biopsy.
Over the past two years, we have collected breast milk samples from 230 women who were scheduled for or had a breast biopsy. Of those 230 participants, we obtained the biopsy reports from 85% of participants (196 biopsy reports). Using pyrosequencing, we analyzed 30 individual CpG sites on 3 genes (RASSF1, GSTP1 and SFRP1) in epithelial enriched cell fractions of the biopsied and non-biopsied breast. In women whose biopsy resulted in a non-proliferative lesion (n=104), there was no significant difference in the average epithelial DNA methylation of their biopsied breast compared to their non-biopsied breast for RASSF1 (p=0.15) and GSTP1 (p=0.92), but for SFRP1 the average methylation was significantly higher in the biopsied breast (p = 0.02). In women whose biopsy result revealed cancer (n=10), there was a significant increase in average RASSF1 methylation of their biopsied breast compared to their non-biopsied breast (p=0.05).
This study is the first to collect breast milk and obtain epithelial cells and DNA from a population of women at increased risk of breast cancer. Utilizing this unique population, we have been able to assess promoter methylation in 3 genes known to be methylated in breast cancer. Long term follow-up is ongoing for women enrolled in this study to determine breast health outcomes. Knowledge of promoter methylation along with long term follow-up may help us to better assess individual breast cancer risk. Given the high percentage of women that give birth and therefore produce milk, if even for just a few days, a breast cancer risk screen based on epithelial cells in milk has the potential to affect a significant portion of all women.
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 LB-443. doi:10.1158/1538-7445.AM2011-LB-443