Abstract 4354: An exosome-based ESR1 monitoring RT-qPCR technology that rapidly and accurately detects circulating tumor acquired resistance variants at ≤0.1% frequency in liquid biopsy samples

Introduction: Hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-) breast cancer is the most common type of breast cancer. Patients with HR+/HER2- metastatic breast cancer (mBC) often become resistant to aromatase inhibitors commonly used in endocrine therapy (ET). Estrogen receptor (ESR1) ligand binding domain mutations are frequently detected in HR+ mBC and have been reported to be associated with ET resistance, noting up to 60% patients with mBC will develop resistance to treatment via acquired resistance in ESR1. Recent studies have shown that monitoring of ESR1 mutations in plasma may serve as a predictive biomarker of acquired resistance to ET, showcasing a strong need for sensitive nucleic acid-based assays. We describe a comprehensive methodology for targeted clinical RT-qPCR monitoring of ESR1 mutations in plasma that utilizes both exosomal nucleic acids and cfDNA to report multiple mutations, provide a streamlined workflow, and accommodate a range of inputs from clinically relevant samples. Methods: Exosomal nucleic acids (exosomal DNA and RNA) and circulating cell-free DNA (cfDNA) were co-isolated using the ExoLution Plus Isolation Kit (Exosome Diagnostics). RT-qPCR-based target enrichment was performed using modified QuantideX reagents (Asuragen) and mutations were confirmed on the QuantStudio qPCR Platform (ThermoFisher). Bioinformatic analyses were conducted using custom software. Results: We developed novel technologies and tested greater than samples across key ET resistance mutations, utilizing contrived samples as needed due to availability. Preliminary studies demonstrated that ESR1 exosomal RNA + cfDNA ranged from approximately 9,500 copies to 64,000 copies in a set of 2mL female presumed normal plasma samples; these results suggest that even 2mL plasma could provide sufficient exosomal RNA/DNA + cfDNA for mutation detection at variant frequencies less than or equal to 0.01%. The ESR1 RT-qPCR showed greater than 90% analytical sensitivity (at less than or equal to 1% mutant allele frequency) and greater than90% analytical specificity. Further, the technology detected less than 10 mutant copies for most tested mutants, showing an LOD of at least 0.1% (5 mutant copies in a background of 5,000 WT copies). Conclusion: A fast, efficient, and sensitive ESR1 RT-qPCR panel approach was developed and evaluated, demonstrating the reliable and specific detection of rare variants in liquid biopsy specimens. Import