Abstract B03: Urine cell-free DNA (cfDNA) concentration and stability test for future clinical use
Authors: Bracht Jillian W. P. , Karachaliou Niki , Berenguer Jordi , Fernandez-Bruno Manuel , Garzón Mónica , Gimenez-Capitan Ana , Mayo-de-las-Casas Clara , Molina-Vila Miguel Angel , Rosell Rafael 
Published: June 2020

Abstract
Introduction: Urine was shown to be a liquid biopsy source with high sensitivity for cfDNA mutation detection in both early- and late-stage cancer patients, outperforming plasma cfDNA and tissue samples in some patients. In addition, obtaining a urine sample is completely noninvasive. Limitations of using urine samples in the clinical setting include their unknown nucleic acid stability and the large total volume of one deposit, and therefore the presence of highly diluted cfDNA. Urine volume concentration, cfDNA stability, cfDNA quantity, and detection of wild-type (wt) EGFR alleles were explored to determine the future suitability of urine cfDNA as a diagnostic or prognostic biosource.
Methods: First-void urine samples, with a higher fraction of nucleic acids due to overnight concentration, were collected from 5 healthy donors and 5 cancer patients. Cell-free urine samples were concentrated using Amicon filter tubes to determine differences in cfDNA concentration. In addition, we tested the ability to detect wild-type EGFR exon 19 in cfDNA from concentrated and unconcentrated urine samples using a peptide nucleic acid (PNA) Taqman Assay. Hereafter, stability of urine cfDNA was explored using variable storage temperatures and times and addition of a urine cfDNA preservative directly after sample collection. cfDNA was extracted using the automated QIAsymphony system with the DSP virus/pathogen kit and quantified using the Qubit dsDNA HS Assay in all experiments.
Results: Amicon filter-based concentration of urine samples from 15 mL to 1.2 mL yielded a two-fold increase in cfDNA concentration. Importantly, while wild-type EGFR could not be detected in first-void urine using a PNA Taqman Assay, detection was possible using concentrated urine samples. Therefore, in all following experiments, samples were concentrated unless specified otherwise. Stability tests indicated that storage temperature matters when samples are not processed within two hours, with nearly doubled cfDNA concentrations found in samples stored at four degrees for 26 hours, compared to room temperature (RT). Addition of a cfDNA preservative yielded a six-fold higher cfDNA concentration after 26 hours of storage at RT, compared to samples without a preservative added. For this reason, we tested the use of only cfDNA preservative without concentrating the sample and found a four-fold increase in cfDNA concentration when adding only preservative, versus a 15-fold increase when using both after sample storage at RT for 24 hours.
Conclusion: Patient urine samples should be either concentrated and processed within two hours, or a urine cfDNA preservative should be added directly after sample collection to prevent cfDNA degradation. Depending on the downstream application, samples where preservative was added may be concentrated to enhance the cfDNA yield, for example, when downstream NGS analysis will be performed.
Citation Format: Jillian W. P. Bracht, Niki Karachaliou, Jordi Berenguer, Manuel Fernandez-Bruno, Mónica Garzón, Ana Gimenez-Capitan, Clara Mayo-de-las-Casas, Miguel Angel Molina-Vila, Rafael Rosell. Urine cell-free DNA (cfDNA) concentration and stability test for future clinical use [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B03.