Candidate methylation sites associated with endocrine therapy resistance in ER+/HER2-breast cancer

Background: Estrogen receptor (ER) positive breast cancer is often effectively treated with drugs that inhibit ER signaling, i.e., tamoxifen (TAM) and aromatase inhibitors (AIs). However, 30% of ER+ breast cancer patients develop resistance to therapy leading to tumour recurrence. Changes in the methylation profile have been implicated as one of the mechanisms through which therapy resistance develops. Therefore, we aimed to identify methylation loci associated with endocrine therapy resistance.

Methods: We used genome-wide DNA methylation profiles of primary ER+/HER2- tumours from The Cancer Genome Atlas in combination with curated data on survival and treatment to predict development of endocrine resistance. Association of individual DNA methylation markers with survival was assessed using Cox proportional hazards models in a cohort of ER+/HER2- tumours (N = 552) and two sub-cohorts corresponding to the endocrine treatment (AI or TAM) that patients received (N = 210 and N = 172, respectively). We also identified multivariable methylation signatures associated with survival using Cox proportional hazards models with elastic net regularization. Individual markers and multivariable signatures were compared with DNA methylation profiles generated in a time course experiment using the T47D ER+ breast cancer cell line treated with tamoxifen or deprived from estrogen.

Results: We identified 134, 5 and 1 CpGs for which DNA methylation is significantly associated with survival in the ER+/HER2-, TAM and AI cohorts respectively. Multi-locus signatures consisted of 203, 36 and 178 CpGs and showed a large overlap with the corresponding single-locus signatures. The methylation signatures were associated with survival independently of tumour stage, age, AI treatment, and luminal status. The single-locus signature for the TAM cohort was conserved among the loci that were differentially methylated in endocrine-resistant T47D cells. Similarly, multi-locus signatures for the ER+/HER2- and AI cohorts were conserved in endocrine-resistant T47D cells. Also at the gene set level, several sets related to endocrine therapy and resistance were enriched in both survival and T47D signatures.

Conclusions: We identified individual and multivariable DNA methylation markers associated with therapy resistance independently of luminal status. Our results suggest that these markers identified from primary tumours prior to endocrine treatment are associated with development of endocrine resistance.