Validation of a droplet digital PCR test for monitoring ESR1 ctDNA variants in blood.

e13066 Background: Estrogen receptor 1 (ESR1) mutations represent a critical mechanism of acquired resistance to aromatase inhibitor (AI) therapy in hormone receptor-positive (HR+) breast cancers. These mutations may occur in 20-40% of patients during treatment. Recent results from the landmark SERENA-6 Phase III trial demonstrated the potential clinical utility of ctDNA-guided treatment switching. This study represented the first global Phase 3 trial to demonstrate the potential clinical utility of using ESR1 ctDNA monitoring for treatment guidance and management of breast cancers. Methods: We have validated a droplet digital PCR (ddPCR) test for the detection of ESR1 variants in plasma-derived cfDNA. The ESR1 ddPCR test targets the 11 most prevalent ESR1 variants, approximately 97% coverage of ESR1 mutations in breast cancer. Analytical sensitivity studies were performed using synthetic DNA controls diluted into wild-type background DNA at varying input concentrations. Serial dilutions were tested to establish the limit of detection (LOD) across different DNA input amounts, with variant allele frequencies (VAF) ranging from 0.025% to 0.2%. Clinical validation is being conducted in a blinded study comparing performance of the new assay versus two standard of care NGS panels. Results: Our studies demonstrated robust analytic performance of the ESR1 ddPCR assay across multiple input concentrations. With 30 ng cfDNA input, we achieved analytical sensitivity ranging from 0.025-0.5% VAF, potentially enabling sensitive detection of low-level ESR1 mutations that may be present in early resistance. When DNA input was reduced to 10 ng, reflecting real-world clinical sample limitations, the assay maintained excellent sensitivity of 0.025-0.1% VAF. The assay also demonstrated high specificity with no false positive calls in wild-type controls. We observed assay sensitivity (0.2%-9.9%) and specificity (100%) in interim analysis of clinical specimens (n = 31). Conclusions: We have successfully developed a highly sensitive and specific ddPCR test for the detection of multiple ESR1 variants in plasma. With sensitivity as low as 0.025% VAF using 10 ng of cfDNA and 100% clinical concordance with reference NGS assays, this approach represents a robust technology for monitoring cfDNA in liquid biopsies. Importantly, the affordability of PCR-based testing and its suitability for decentralized implementation offer a practical path for broader global adoption of ctDNA-guided treatment strategies, as highlighted by emerging clinical utility data.