Development of a clinical-grade ultrasensitive droplet digital PCR test for monitoring of relevant

Breast cancer is the second most prevalent cancer-type globally. The current standard of care for hormone receptor-positive (HR+), human epidermal growth factor 2-negative (HER2-) advanced breast cancer (ABC) is endocrine therapy (ET) plus cyclin-dependent kinase 4/6 inhibitor (CDK4/6i). Most ABC patients receiving ET + CDK4/6i eventually develop resistance, leading to disease progression. In approximately 20-40% of HR + cases, resistance is driven by acquired activating mutations in the ligand binding domain (LBD) of the estrogen receptor gene ( ESR1 ). The next-generation oral selective estrogen receptor degraders (SERDs) comprise an advanced class of ETs intended for use in ER+/HER2- ABC with ESR1 mutation(s) that has progressed on prior ET. We have developed and validated a New York State Clinical Laboratory Evaluation Program (CLEP)-approved laboratory developed test (LDT) for detection of clinically relevant, and the most prevalent ESR1 LBD mutations in ABC. The LDT uses advanced multiplexing on the QX600 ddPCR system to identify the presence of ESR1 E380Q, V422del, S463P, L536 H/P/R, Y537C/D/N/S, and D538G in plasma. Our test was clinically validated for accuracy and found to be 100% concordant when compared to two NGS reference tests (11/11 eligible ESR1 variants and 18/18 ESR1 WT samples were correctly called). One variant was detected by the ddPCR test at 0.01% VAF that was not present in the reference dataset, likely due to sample input differences between our test and the reference. Analytic specificity was 100%, and the 95% analytic limit of detection (LoD95) was as low as 0.065% VAF. The Limit of Blank (LOB) was 0 - 3.4 copies depending on the assay. These findings support the use of our multiplexed ddPCR test as a rapid, sensitive, and scalable liquid biopsy approach for identifying emerging ESR1 mutations and may inform endocrine therapy selection in the treatment of HR+/HER2− advanced breast cancer.