Abstract 5437: Sensitive detection of rare cfDNA variants utilizing molecular technologies and a novel informatics platform: A combined genomic and proteomic MRD application.

Abstract Advances in next-generation sequencing (NGS) are producing massive amounts of data at rapidly declining costs. Effective processing of millions of high-depth sequencing reads while maintaining sensitivity and specificity is a growing challenge in the design of new molecular tests. One critical application is detection and subsequent monitoring of rare variants in plasma for molecular residual disease (MRD). Our MRD testing strategy utilizes a preliminary proteomic risk of recurrence mass spectrometry assay to stratify high-risk baseline samples as likely to need further monitoring, followed by design of bespoke ddPCR assays to mark recurrence in the follow-up plasma of the high-risk population. To qualify assays for genomic monitoring, we developed a novel informatics pipeline utilizing approximately 24 million reads and 1 million structural variant calls to profile a cohort of 14 metastatic castration resistant prostate cancer patients (mCRPCa). Tissue-derived NGS data were systematically analyzed to identify a MRD fingerprint for monitoring molecular recurrence in post-treatment plasma specimens. We achieved sequencing depths of 64-367x across the cohort. Data filtering strategies included quality score (≥20), identical homology (≤5), structural variant size, and intersection of multiple gold-standard bioinformatics tools, Delly and Manta. Analytical turnaround time was 16.5 - 72 hours from raw NGS data to designed primers. Bespoke primers were successfully designed or available off-the-shelf for all evaluated patients. Initial studies identified tissue-informed variants derived from four mCRPCa patients with matched baseline plasma. 6/6 of the tested variants were found in the matched patient plasma specimens. Significantly, longitudinal ctDNA analyses identified clearance of AR p.L702H from circulation in a patient with evidence of radiographic and biochemical response to docetaxel after progression on multiple lines of AR signaling inhibitors (ARSI). AR p.L702H is an acquired resistance mechanism to ARSIs, and the elimination of sub-clonal cell populations harboring the alteration could indicate restored sensitivity to these agents. Additionally, declines in ctDNA coincided with declines in serum PSA. This report showcases the design of a novel MRD test combining proteomics for early and impactful risk of recurrence stratification with cost-effective and rapid genomic PCR monitoring in the follow-up plasma of patients at a high-risk of recurrence, for utility in real-world clinical diagnostics laboratories Citation Format: Emma Kaitlyn Longshore, Ethan Barnett, Ingrid T. Erazo, Leisa Jackson, Helen Halpin, Reis Pestano, Patricia Schnepp, Adam Corner, Howard I. Scher, Gary A. Pestano. Sensitive detection of rare cfDNA variants utilizing molecular technologies and a novel informatics platform: A combined genomic and proteomic MRD application abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5437.