Abstract 4355: Altered DNA methylation in breast milk from women with breast cancer
Authors: Salas Lucas A. , Lundgren Sara N. , Browne Eva P. , Punska Elizabeth C. , Anderton Douglas L. , Arcaro Kathleen F. , Christensen Brock C. 
Published: July 2017

Abstract
Prior candidate gene studies have shown that methylation-induced silencing of tumor suppressor genes in breast milk is related with history of breast biopsy, an established risk factor for breast cancer. To further establish the utility of breast milk as a tissue-specific biospecimen for investigations of breast carcinogenesis we measured genome-wide DNA methylation in breast milk from women with and without a diagnosis of breast cancer. Breast milk provides a source of cells without invasive procedure that is available during a window of increased breast cancer risk. Actively lactating women were enrolled and consented through the University of Massachusetts Amherst breast milk study targeting high risk subjects. Participants completed a questionnaire to provide demographic and breast cancer risk factor data. Genome-wide DNA methylation levels were quantified using the Illumina 450K HumanMethylation array. Data was processed in R using RnBeads. Beta-values were normalized (FUNnorm), low quality probes were filtered and transformed to M-values. Linear mixed effects models were fit including a random effect for subject and adjusted for history of breast biopsy, age, body mass index, age of the baby, and estimated cell type proportions, an interaction term for time of diagnosis was included. DNA methylation in women with breast cancer (n=9), was compared with women without a history of breast cancer (n=22). Among women with a diagnosis of breast cancer there were two groups: those diagnosed within one year of donating breast milk, and those diagnosed more than one year before sample collection. Four women in the control and five women in the cancer group provided bilateral breast milk samples at recruitment and both samples were included in the analyses. CpGs with significant differential methylation were tested for enrichment in gene pathways using the KEGG database. We identified 215 differentially methylated CpG sites in women diagnosed within one year of sample donation compared with women without a diagnosis of breast cancer (Q&lt;0.10). The top sites were in ATF3 (an adaptive-response gene linked to epithelial mesenchymal transition), and in COL5A3 (an extracellular matrix gene related to metastasis potential). In addition, among women with a breast cancer diagnosis more than one year before sample donation we identified 8148 differentially methylated CpG sites compared with women without a breast cancer diagnosis (Q&lt;0.05). Five sites were in OTOF a calcium-sensing protein in cancer. These differentially methylated sites were significantly enriched in relevant pathways such metabolic (Q= 3.12E-116), and cancer pathways (Q= 2.08E-58), PI3K-Akt signaling pathway (Q=7.05E-40), and transcriptional misregulation in cancer (Q=1.41E-29). Future studies are needed to validate our findings in independent subjects and to investigate the utility of breast milk as a biospecimen for understanding the molecular basis of disease risk and prospective risk assessment.
Citation Format: Lucas A. Salas, Sara N. Lundgren, Eva P. Browne, Elizabeth C. Punska, Douglas L. Anderton, Kathleen F. Arcaro, Brock C. Christensen. Altered DNA methylation in breast milk from women with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4355. doi:10.1158/1538-7445.AM2017-4355