ESR1 analysis of liquid biopsy in breast cancer, one-year routine experience of an Italian clinical referral center

Background: Activating mutations in the ESR1 gene are a known mechanism of secondary resistance to endocrine therapy in metastatic estrogen receptor-positive (ER+)/human epidermal growth factor receptor 2-negative (HER2-) breast cancers. Liquid biopsy has become a non-invasive tool for molecularly characterizing these neoplasms and allows for dynamic monitoring through the analysis of circulating tumor DNA (ctDNA). Methods: We analyzed 161 plasma samples from patients with metastatic ER+/HER2- breast cancer who had previously undergone treatment with endocrine therapy and CDK4/6 inhibitors. ESR1 mutation analysis was performed using two NGS panels: OncomineTM Breast cfDNA Assay v2 (n = 102) and OncomineTM Precision Assay GX (n = 59). The sensitivity threshold (Limit of Detection - LOD) for variant detection was set at <= 0.5 %. Results: Twenty-one samples (12.4 %) did not meet the quality criteria for ESR1 analysis. ESR1 mutations were identified in 29.1 % (n = 41) of the remaining 141 cases. The most frequent ESR1 variant was the p.Asp538Gly (53.7 %). Multiple ESR1 mutations were observed in 29.3 % of mutated cases, and co-mutations were detected in 61 % of cases, mainly with PIK3CA (36.6 %) and TP53 (12.2 %). The median variant allele frequency (VAF) of ESR1 mutations was 1.46 %. No statistically significant difference in mutation frequency emerged between the two panels (p = 0.6993). Conclusions: ESR1 mutations are detectable in approximately one-third of ER+/HER2- metastatic patients undergoing liquid biopsy. NGS platforms allow for sensitive and in-depth analysis, highlighting co-mutations of potential clinical and therapeutic relevance.