Abstract Background: Activating mutations in the ESR1 gene represent a well-known mechanism of acquired resistance to aromatase inhibitors (AIs) in patients with hormone receptor-positive (HR+) metastatic breast cancer. Liquid biopsy, through the analysis of circulating free DNA (cfDNA), provides a non-invasive and dynamic approach to detect ESR1 mutations, enabling real-time monitoring of tumor evolution. This is especially relevant in the context of novel oral selective estrogen receptor degraders (SERDs), where ESR1 mutation profiling will inform treatment selection and act as a biomarker of resistance and response. As these agents become part of routine clinical care, the ability to accurately detect and monitor ESR1 mutations in plasma is critical. As several next-generation sequencing (NGS)-based assays are commercially available, studies comparing their analytical concordance in detecting ESR1 mutations in real-life plasma samples are needed. Methods: This study aims to assess the analytical concordance between two amplicon-based NGS assays (Oncomine™ Precision Assay and Pillar Biosciences™ OncoReveal Essential LBx) and a hybrid-capture based NGS test, VHIO360 (tech transfer to VHIO of the Guardant360® assay) for the detection of ESR1 mutations in cfDNA. VHIO360, with established clinical validation is considered as the reference gold standard. One hundred plasma samples from patients with known ESR1 mutation genotypes as per VHIO360 testing (from the VHIO Molecular Prescreening Program), containing 64 pathogenic/ likely pathogenic variants in ESR1 (59 corresponding to hotspot Y537S/N/C, D538G, and E380Q/K) will be additionally tested with the aforementioned amplicon-seq based approaches. Mutant variant frequencies in the sample set are present at a wide range (0.02-69%). Concordance is measured using metrics such as overall agreement, positive and negative percent agreement, and analytical sensitivity across variant allele frequencies. Discordant results will be further investigated to identify assay-specific limitations or systematic biases, including variant dropout or differential coverage. Results: Results provide a comparative assessment of analytical performance across platforms, focusing on concordance in detection of clinically relevant ESR1 mutations and determination of assay-specific limits of detection. Particular attention will be paid to samples with low variant allele frequency (VAF) mutations, where differences in platform sensitivity may impact clinical interpretation. Conclusions: This study will provide critical evidence for the analytical performance and clinical reliability of commonly used NGS-based assays for detecting ESR1 mutations in plasma. As SERDs and other ESR1-targeted therapies become integrated into standard-of-care for HR+ metastatic breast cancer, accurate and validated mutation testing is essential in order to guide clinicians in selecting appropriate local testing approaches. Citation Format: A. Yarunin, A. Martin, M. Gomez-Rey, A. Baizan, M. Sesé, S. Clavé, L. Camacho, C. Saura, J. Longshore, J. Hernández-Losa, B. Bellosillo, A. Vivancos. Analytical concordance of NGS-based tests for ESR1 mutation detection in plasma 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 PS4-04-03.
Yarunin et al. (Tue,) studied this question.