Abstract PS2-12-13: Breast Cancer Mutation Detection via Liquid Biopsy Using cfDNA from 50 mL Urine Samples

Abstract Background: Urine cfDNA offers a promising, non-invasive option for molecular profiling in breast cancer. However, challenges with cfDNA stability, yield, and compatibility with preservatives have limited its widespread clinical adoption. This study evaluated the recovery and mutation detection performance of large-volume (50 mL) urine samples preserved with three common urine stabilizers and analyzed after storage for 0 and 3 days at room temperature. Methods: Pooled healthy donor urine was contrived with Anchor Molecular’s cfDNA reference standards containing four clinically actionable breast cancer mutations: IDH1 (R132H), PIK3CA (E545K), PTEN (R130G), and TP53 (R248Q). Three preservatives—Streck Urine preservative, UAS urine analyte stabilizer, and Tris-EDTA (TE) buffer pH (8.0)—were evaluated at time zero (T0) and after 3 days (T3) of room temperature storage. For each condition, three technical replicates were extracted from 50 mL urine samples using the nRichDX Revolution Max 50 cfDNA Kit. cfDNA yield and fragment integrity were measured using the Agilent cfDNA High Sensitivity ScreenTape assay. Mutation detection was performed using a multiplex qPCR assay targeting the four hotspot mutations. Results: cfDNA was successfully recovered in all conditions, with consistent detection of all four mutations across replicates, time points, and preservatives. The cfDNA fragment size profile was preserved from T0 to T3 across all preservatives, indicating minimal degradation during storage. Quantitative results showed that cfDNA yield varied by preservative, with average recovery of 88% (Streck), 75% (UAS), and 62% (TE) relative to input. Despite differences in yield, mutation detection was robust across all conditions, with no statistically significant shift in Ct values between T0 and T3 (p > 0.05). Inter-replicate variation remained low (CV < 5%), and all replicates consistently showed reliable detection of IDH1, PIK3CA, PTEN, and TP53 mutations. Conclusion: This study demonstrates that 50 mL urine inputs allow for consistent recovery and detection of clinically relevant breast cancer mutations across multiple preservative chemistries, even after 3 days at room temperature. Mutation standards from Anchor Molecular were reliably detected using qPCR in all conditions, and the nRichDX Revolution Max 50 cfDNA Kit was compatible with all three preservatives tested, showing variable but sufficient recovery to support downstream mutation detection. These findings support the feasibility of urine as a stable and scalable liquid biopsy sample type for breast cancer genotyping, longitudinal monitoring, and remote sample collection workflows. Citation Format: N. Jafari, Mayer Saidian, Jason Saenz, Carlos Hernandez, Daniel Cedeno, Cameron Van-Dieren, Y. Lu. Breast Cancer Mutation Detection via Liquid Biopsy Using cfDNA from 50 mL Urine Samples [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 PS2-12-13.