Prediagnostic breast milk DNA methylation alterations in women who develop breast cancer

ABSTRACT Background Prior candidate gene studies have shown tumor suppressor DNA methylation in breast milk 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 in two independent cohorts. Methods DNA methylation was assessed using Illumina HumanMethylation450k in 87 breast milk samples. After quality control, 368,171 autosomal CpG loci were analyzed. Cell type proportion estimates from RefFreeCellMix were calculated and adjusted for in this Epigenome Wide Association Study using linear mixed effects models adjusted for history of breast biopsy, age, time of delivery, cell type proportion estimates, array chip, and subject as random effect. Results Epigenome-wide analyses identified 58 differentially methylated CpG sites associated with a breast cancer diagnosis in the prospectively collected milk samples from the breast that would develop cancer compared with women without a diagnosis of breast cancer ( q -value < 0.05). Nearly all CpG sites associated with a breast cancer diagnosis were hypomethylated in cases compared with controls, and were enriched for CpG islands. In addition, inferred repeat element methylation was lower in breast milk DNA from cases compared to controls, and cases exhibited increased estimated epigenetic mitotic tick rate as well as DNA methylation age compared with controls. Conclusion Breast milk has utility as a biospecimen for prospective assessment of disease risk, for understanding the underlying molecular basis of breast cancer risk factors, and improving primary and secondary prevention of breast cancer.