ESR1 intragenic DNA methylation in breast tumour DNA. (A) Pyrosequencing based methylation data across the ESR1 gene in fresh frozen (FF) human breast tumour samples (n = 10 ER-negative, n = 10 ER-positive [n = 5 grade 2 vs. grade 3]). At regions p2 and p3 (approx. 2–8 kb downstream of the TSS), methylation levels were variable and significantly more methylated in ER-negative tumours, *p < 0.05. (B) In ER-positive FF tumours, methylation levels were very similar between grades. (C) 450 K Beadchip methylation analysis of ESR1 methylation from TCGA breast tumours (blue line = ER-positive tumours, n = 365; red line = ER-negative tumours, n = 109). The methylation pattern is very similar to the data shown from breast tissue biopsy material shown in (A), with the only differences appearing within the first intron (equivalent to regions p2 and p3 of the pyrosequenced assays in (A), Wilcoxon rank sum test, *p < 1.0 x 10−7.