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

Abstract

<b>Purpose:</b> 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). <i>ESR1</i> epigenetic silencing potentially affects response to endocrine treatment. We evaluated <i>ESR1</i> methylation in CTCs and paired plasma ctDNA. We evaluated <i>ESR1</i> methylation in CTCs and paired plasma ctDNA as a potential biomarker for response to everolimus/exemestane treatment.<b>Experimental Design:</b> A highly sensitive and specific real-time MSP assay for <i>ESR1</i> 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. <i>ESR1</i> methylation status was investigated in CTCs isolated from serial peripheral blood samples of 19 patients with ER⁺/HER2^- advanced breast cancer receiving everolimus/exemestane.<b>Results:</b><i>ESR1</i> methylation was detected in: (i) 25/65 (38.5%) FFPEs, (ii) <i>EpCAM⁺ CTC fractions</i>: 26/112 (23.3%) patients and 1/30 (3.3%) HD, and (iii) <i>plasma ctDNA:</i> 8/108 (7.4%) patients and 1/30 (3.3%) HD. <i>ESR1</i> methylation was highly concordant in 58 paired DNA samples, isolated from CTCs (CellSearch) and corresponding plasma. In serial peripheral blood samples of patients treated with everolimus/exemestane, <i>ESR1</i> methylation was observed in 10/36 (27.8%) CTC-positive samples, and was associated with lack of response to treatment (<i>P</i> = 0.023, Fisher exact test).<b>Conclusions:</b> We report for the first time the detection of <i>ESR1</i> methylation in CTCs and a high concordance with paired plasma ctDNA. <i>ESR1</i> methylation in CTCs was associated with lack of response to everolimus/exemestane regimen. <i>ESR1</i> methylation should be further evaluated as a potential liquid biopsy-based biomarker. <i>Clin Cancer Res; 24(6); 1500-10. ©2017 AACR</i>.