Intragenic DNA methylation in ESR1 in exfoliated breast epithelial cells from expressed breast milk (EBM). (A) Flow cytometry of epithelial membrane antigen (EMA; median positive staining, 97.8%; 95.3%-99.4%; n = 60) and intracellular ESR1 (median, 90.5%; IQR, 84.4%-93.3%; n = 6). (B) qRT-PCR analysis of 11 EBM epithelial cell RNA samples showing high levels of ESR1 expression normalised against ER-pos MCF7 and ER-neg MDA-MB-231 cell lines, (range 2.2- 19-fold higher). (C) Methylation values from 450 K analysis of 36 DNA samples extracted from breast epithelial cells from expressed breast milk. The overall pattern matched those of ER-positive cancer cell lines, and regions of variability were found at regions that mapped to p2 and p7. (D) Validation of the methylation levels using pyrosequencing in a separate set of 53 EBM samples (black solid line). The pattern of methylation is very similar to that of the 450 K analysis of EBM samples and of ER-pos cancer cell lines (dashed line) with high levels of variability at p2 for both.