Abstract 2430: Enhancing urine-based cfDNA detection for prostate cancer diagnosis through increased urine input volume
Authors: Jafari Nafiseh , Dorn Annika , Saenz Jason , Lee Lauren , Hernandez Carlos , Dunnigan Andrew , Saidian Mayer 
Published: March 2024

Abstract
Introduction: Cell-free DNA (cfDNA) extracted from urine has created new opportunities in non-invasive cancer diagnostics, particularly for prostate cancer. Detecting cfDNA in urine can be challenging due to its inherently low concentration. Increasing the input volume of urine addresses this limitation, enhancing the sensitivity of cfDNA detection. This study explores the benefit of collecting larger volumes of urine spiked with a TP53 mutation and the corresponding increase in assay sensitivity for early prostate cancer diagnosis and monitoring.
Methods: Pooled urine from healthy donors was collected, preserved, processed, and extracted from 10 mL, 20 mL, 30 mL, 40 mL, and 50 mL inputs (with four replicates each) using the Revolution Max 20 cfDNA Extraction kit. Replicate samples were also extracted using a MagMAX cfDNA Isolation kit from Thermo Fisher Scientific. All samples were spiked with a cfDNA standard containing TP53 R58L mutation. The quantity and quality of extracted cfDNA were determined using the Agilent TapeStation system with the Cell-free DNA ScreenTape assay and qPCR mutation detection assay.
Results: The Revolution Max 20 cfDNA kit consistently delivered a higher yield of cfDNA, demonstrating excellent extraction efficiency and quality, as shown by the Agilent Cell-free DNA ScreenTape assay. Notably, the Revolution Max 20 cfDNA kit exhibited a linear increase in yield as the sample volume grew from 10 mL to 50 mL. The MagMax kit did not provide this yield linearity. Furthermore, both methods successfully maintained the integrity of cfDNA fragmentation, which was confirmed through the Agilent Cell-free DNA ScreenTape assay.
Conclusion: Compared to the MagMax kit, the Revolution Max 20 cfDNA kit outperforms in extracting cfDNA with high efficiency, as shown by the Agilent Cell-free DNA ScreenTape and the qPCR assay. The Revolution Max 20 cfDNA kit offers the unique advantage of accommodating up to 50 mL of urine in a single extraction. As input volume increases, it consistently produces high cfDNA yields while maintaining quality. In contrast, the MagMax kit is limited to an input volume of only 10 mL, necessitating the cumbersome process of dividing a 50 mL urine sample into five separate extractions and pooling the eluates. This scheme resulted in lower yields. The Agilent Cell-free DNA ScreenTape assay is a valuable tool for quickly and accurately assessing the %cfDNA quality metric. Its minimal sample volume requirement makes it an efficient quality control method for determining cfDNA samples before performing downstream testings. The enhanced capability of the Revolution Max 20 kit to yield more significant quantities of high-quality cfDNA from urine samples can profoundly impact liquid biopsy test sensitivity and the early detection of prostate cancer, potentially revolutionizing the diagnostics process.
Citation Format: Nafiseh Jafari, Annika Dorn, Jason Saenz, Lauren Lee, Carlos Hernandez, Andrew Dunnigan, Mayer Saidian. Enhancing urine-based cfDNA detection for prostate cancer diagnosis through increased urine input volume [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2430.