Analytical Validation of a High-Definition Tumor-Informed MRD Assay Demonstrates Robust Detection at Low Tumor Fractions Common in Breast Cancer

Background: Molecular residual disease (MRD) refers to the small number of tumor cells remaining in the body during or after cancer treatment. These tumor cells shed DNA into the bloodstream, resulting in circulating tumor DNA (ctDNA). ctDNA can be identified in plasma-derived cell-free DNA (cfDNA) via the presence of tumor-specific somatic variants. We have developed a second-generation tumor-informed MRD assay to detect the presence and quantity of ctDNA in the plasma of breast-cancer patients with residual disease or tumor recurrence. Our assay detects the presence of ctDNA using a hybrid-capture based sequencing panel, targeting tumor-specific somatic variants. Each panel is designed with up to 1000 carefully selected somatic variant targets identified based on tumor profiling by matched tumor and normal whole genome sequencing and is optimized to provide high sensitivity and specificity at low tumor fraction, which is particularly important because many breast tumors have low shedding of ctDNA. Targeted sequence data is fit to a statistical model that incorporates panel-specific and sample-specific parameters to provide a robust estimate of the ctDNA fraction with a rigorous assessment of confidence for each test result. Here, we evaluated the performance of our assay at low ctDNA fraction. Methods: Contrived mixtures of matched tumor and normal breast cancer cell lines and healthy donor plasma were were evaluated over a range of tumor DNA concentrations from 1 part per million (ppm) to 10,000 ppm, and were processed across multiple days, reagent lots, operators and instruments. Results: Analysis of healthy donor samples paired with non-patient matched panels demonstrated the Limit of Blank (LoB) at a 95% specificity threshold—i.e., the threshold at which 95% of negative samples return a negative result—to be 0.3 ppm. When the LoB was used as the detection threshold, we found the Limit of Detection with 95% sensitivity (LoD95) to be <5ppm. Using a more stringent detection threshold of 1.4 ppm—corresponding to a specificity of 99.615%—we found the LoD95 to be 10 ppm. Both PPV and NPV were 100% when assessed with 56 positive samples at 50 ppm and 126 negative samples. Additionally, quantification of ctDNA fraction was linear over the entire range of tumor DNA concentrations assessed. Conclusions: Overall, our assay demonstrated high sensitivity, specificity and measurement accuracy, which, together, will facilitate improved resolution in residual-disease detection and extend lead times in recurrence detection. Citation Format: Ashley Acevedo, Kyle Trettin, Nafei Xu, Matt LaBella, CJ Battey, Ravi Patel, Kiefer Haug, Elise Buser, Shalee Carlson, Thanh Tran, Britney Sadler, Abby Tucker, Genevieve Gould, Dale Muzzey. Analytical validation of a high-definition tumor-informed MRD assay demonstrates robust detection at low tumor fractions common in breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P2-04-23.