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

Abstract

35Background: Estrogen receptor (ER) positive breast cancer is often effectively treated with 36 drugs that inhibit ER signaling, i.e., tamoxifen (TAM) and aromatase inhibitors (AIs). 37However, about 30% of ER+ breast cancer patients develop resistance to therapy leading to 38 tumour recurrence. Changes in the methylation profile have been implicated as one of the 39 mechanisms through which therapy resistance develops. Therefore, we aimed to identify 40 methylation loci associated with endocrine therapy resistance. 41Methods: We used genome-wide DNA methylation profiles of primary ER+ tumors from The 42Cancer Genome Atlas in combination with curated data on survival and treatment to predict 43 development of endocrine resistance. Association of individual DNA methylation markers 44 with survival was assessed using Cox proportional hazards models in a cohort of 45 ER+/HER2-tumours (N=552) and two sub-cohorts corresponding to the endocrine treatment 46 (AIs or TAM) that patients received (N=210 and N=172, respectively). Models were adjusted 47 for clinical variables tumour stage, age, AI treatment and luminal subtype. We also identified 48 signatures of multiple methylation loci associated with survival using Cox proportional 49 hazards models with elastic net regularization. Individual markers and multivariable 50 signatures were compared with DNA methylation profiles generated in a time course 51 experiment using the T47D ER+ breast cancer cell line treated with tamoxifen or deprived 52 from estrogen. 53 Results:We identified 132, 9 and 1 CpGs for which DNA methylation is significantly 54 associated with survival in the ER+/HER2-, TAM and AI cohorts respectively. Corresponding 55 multi-locus signatures consisted of 171, 50 and 160 CpGs and showed a large overlap with 56 the corresponding single-locus signatures. Single-locus signatures for the ER+/HER2-and 57 TAM cohorts were conserved among the loci that were differentially methylated in endocrine-58 resistant T47D cells. Similarly, multi-locus signatures for the ER+/HER2-and AI cohorts were 59 conserved in endocrine-resistant T47D cells. 60 Conclusions:We identified individual and multivariable DNA methylation markers 61 associated with therapy resistance independently of luminal status. Our results suggest that 62 these markers identified from primary tumours and prior to any endocrine treatment are 63 associated with development of endocrine resistance. 64 65 Keywords (three to ten keywords representing the main content of the article): breast 66 cancer, DNA methylation, endocrine therapy, resistance, survival, T47D 67 68