Abstract PS4-02-29: Development of an ultra-sensitive droplet digital PCR test for monitoring ESR1 variants in liquid biopsies

Abstract Background: Estrogen receptor 1 (ESR1) mutations are a critical mechanism of acquired resistance to aromatase inhibitor (AI) therapy in hormone receptor-positive (HR+) breast cancer, occurring in approximately 20-40% of patients during treatment. The detection of ESR1 mutations in circulating tumor DNA (ctDNA) through liquid biopsy represents a new approach to real-time monitoring of treatment resistance and therapeutic decision-making. Recent interim results from the landmark SERENA-6 Phase III trial demonstrated the potential clinical utility of ctDNA-guided treatment switching, where median progression-free survival was 16.0 months in the camizestrant group versus 9.2 months in the aromatase-inhibitor group when treatment was switched based on ESR1 mutation detection. This study represented the first global Phase 3 trial to demonstrate the potential clinical utility of using ctDNA for treatment guidance and breast cancer management. Methods: We have developed a droplet digital PCR (ddPCR) test for the detection of ESR1 variants in plasma-derived cfDNA. The ESR1 ddPLEX kitted assay, in combination with a supplementary 4-plex Expert Design assay, targets the 11 most prevalent ESR1 variants (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%. 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, enabling detection of low-level ESR1 mutations present in early resistance development. 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. Conclusions: We have successfully developed a highly sensitive and specific ddPCR test for the detection of multiple ESR1 variants in plasma. The assay performed as designed and meets the analytical requirements required for further clinical development. The achieved sensitivity of 0.025-0.1% with as little as 10 ng of cfDNA positions this ddPCR assay as a potentially valuable tool for liquid biopsy monitoring applications in breast cancer. Clinical validation studies may further support the initial utility of ctDNA guided treatment selection demonstrated in SERENA-6. The availability of this test could enable detection of emerging resistance mutations from AI therapy in HR+ breast cancer in advance of traditional monitoring methods, e.g. imaging. Citation Format: L. JacksonG. A. Pestano. Development of an ultra-sensitive droplet digital PCR test for monitoring ESR1 variants in liquid biopsies abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-02-29.