ESR1 Methylation: A Liquid Biopsy-Based Epigenetic Assay for the Follow-up of Patients with Metastatic Breast Cancer Receiving Endocrine Treatment

Purpose: Liquid biopsy provides real-time monitoring of tumor evolution and response to therapy through analysis of circulating tumor cells (CTCs) and plasma-circulating tumor DNA (ctDNA). ESR1 epigenetic silencing potentially affects response to endocrine treatment. We evaluated ESR1 methylation in CTCs and paired plasma ctDNA. We evaluated ESR1 methylation in CTCs and paired plasma ctDNA as a potential biomarker for response to everolimus/exemestane treatment. Experimental Design: A highly sensitive and specific real-time MSP assay for ESR1 methylation was developed and validated in (i) 65 primary breast tumors formalin-fixed paraffin-embedded (FFPE), (ii) EpCAM(+) CTC fractions (122 patients and 30 healthy donors; HD), (iii) plasma ctDNA (108 patients and 30HD), and (iv) in CTCs (CellSearch) and in paired plasma ctDNA for 58 patients with breast cancer. ESR1 methylation status was investigated in CTCs isolated from serial peripheral blood samples of 19 patients with ER+/HER2(-) advanced breast cancer receiving everolimus/exemestane. Results: ESR1 methylation was detected in: (i) 25/65 (38.5%) FFPEs, (ii) EpCAM(+) CTC fractions: 26/112 (23.3%) patients and 1/30 (3.3%) HD, and (iii) plasma ctDNA: 8/108 (7.4%) patients and 1/30 (3.3%) HD. ESR1 methylation was highly concordant in 58 paired DNA samples, isolated from CTCs (Cell-Search) and corresponding plasma. In serial peripheral blood samples of patients treated with everolimus/exemestane, ESR1 methylation was observed in 10/36 (27.8%) CTC-positive samples, and was associated with lack of response to treatment (P = 0.023, Fisher exact test). Conclusions: We report for the first time the detection of ESR1 methylation in CTCs and a high concordance with paired plasma ctDNA. ESR1 methylation in CTCs was associated with lack of response to everolimus/exemestane regimen. ESR1 methylation should be further evaluated as a potential liquid biopsy-based biomarker. 2017 AACR.