Abstract Young women with primary breast cancer (40 years) often present with more aggressive disease and show lower estrogen receptor alpha (ESR1) expression than older women (≥60 years), but the underlying mechanisms remain unclear. We hypothesized that DNA methylation contributes to ESR1 repression in tumors from young women. We analyzed TCGA-BRCA HumanMethylation450 data from 108 primary tumors (54 young, 54 older) matched by PCAPAM50 subtype and race. After preprocessing, the dataset contained 363,870 CpG sites. Differential methylation was performed using limma, adjusting for subtype and race. Gene set enrichment analysis (GSEA) used preranked t-values for 21,582 genes mapped from CpGs. Matched TCGA RNA-seq data were used to evaluate correlations between ESR1 methylation and expression. Validation was conducted using the Lund cohort (GSE75067; n = 61); genome-wide modeling was not feasible due to smaller sample size, so Wilcoxon tests assessed ESR1-associated CpGs. We identified 3,012 differentially methylated CpGs (FDR 0.05, fold change 2), with 36 hypermethylated and 2,976 hypomethylated in tumors from young women, indicating a global trend toward higher methylation in older tumors. Among 53 ESR1-associated CpGs in TCGA, seven were significantly hypermethylated exclusively in young luminal tumors and all 7 are significantly hypermethylated in both luminal A/B. Notably, none of the ESR1 CpGs were hypermethylated in older women. ESR1 methylation of these 7 sites was inversely correlated with expression (ρ = −0.35 to −0.51), consistent with transcriptional silencing. In the Lund dataset, of 61 ESR1 CpGs, 2 were hypermethylated in young Luminal A and 16 in Luminal B tumors, whereas 1 showed hypomethylation in Luminal B. Of the 7 CpGs hypermethylated in TCGA, one each was significant in young LumA and LumB; others showed similar trends, though not statistically significant, likely due to small sample size. Methylation-based GSEA showed enrichment of hypermethylation within the early estrogen response pathway in the older group, consistent with previous studies reporting heightened estrogen pathway activity in younger women. We report consistent ESR1 promoter hypermethylation in young breast cancer, especially in Luminal A/B subtypes, supported by inverse correlation with transcript levels. These findings suggest that ESR1 silencing is a recurrent feature of young breast tumors and reinforce the potential utility of methylation-based biomarkers for age-stratified cancer profiling. Disclaimer: The contents of this publication are the sole responsibility of the author(s) and do not necessarily reflect the views, opinions or policies of USUHS, HJF, the DoW or the Departments of the Army, Navy or Air Force. Mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. Government. Citation Format: Praveen Kumar Raj Kumar, Anupama Praveen Kumar, Mariano Russo, Jianfang Liu, Craig D. Shriver, Hai Hu, Xiaoying Lin. Epigenetic silencing of ESR1 in primary breast tumors from young women compared to those from older women: An integrative methylation and expression analysis abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1945.
Kumar et al. (Fri,) studied this question.