ESR1 methylation and ESR1 mutations in circulating tumor cells (CTCs) and paired plasma-cfDNA of advanced breast cancer patients: A feasibility proof-of-concept study.

Endocrine resistance is a complex phenomenon, including alterations of the ESR1 gene. The aim of this study was to simultaneously analyze ESR1 promoter methylation and ESR1 hotspot mutations in circulating tumor cells (CTCs) and paired plasma-circulating tumor DNA (ctDNA) from patients with estrogen receptor-positive (ER+) advanced breast cancer (BC). We retrospectively analyzed samples from 42 ER+ advanced BC patients characterized for CTCs and ctDNA at Northwestern University. CTCs were enumerated using the CellSearch® system, while ctDNA was analyzed with the Guardant360 NGS platform. Genomic DNA from CellSearch-enriched CTC fractions was amplified and analyzed using the ESR1-NAPA assay. ESR1 methylation analysis was performed in 34 samples. ESR1 mutations were detected in 59.5% CTC-derived samples, a significantly higher proportion than in paired plasma ctDNA (29.6%). ESR1 methylation was observed in 26.5% patients. Concurrent ESR1 mutations and methylation were identified in six cases, suggesting combined genetic and epigenetic mechanisms of endocrine resistance. Overall, CTC-derived genomic DNA showed higher sensitivity for detecting ESR1 mutations than plasma ctDNA, supporting the potential value of CTC analysis for characterizing endocrine resistance in advanced BC.