Abstract 1382: A complete workflow for high-throughput isolation and analysis of cell-free DNA from urine

Abstract

Abstract Circulating cell-free DNA (cfDNA) shed from tumors has gained considerable attention as a source of nucleic acid for testing cancer biomarkers. Critical to finding and implementing a diagnostic biomarker for cfDNA is the consistent and efficient isolation of the nucleic acid from blood. Sample preparation technologies are now commercially available for cfDNA isolation from plasma and serum, making these sample types used for most applications. However, plasma and serum require blood drawn by trained phlebotomist and only limited amounts can be obtained; additionally, individuals with advanced disease usually require routine monitoring and may not be able to spare additional blood drawn for cfDNA testing. Recently, it has been appreciated that urine may also serve as a valuable source for cfDNA. DNA from tissues and organs of the genitourinary system may be shed directly into urine and cfDNA circulating in blood can filter through the glomeruli in the kidneys to end up in urine. Compared to plasma and serum, urine is much easier to obtain, does not require a needle stick, and it can be collected in larger volumes making longitudinal studies more accessible. Urine presents a number of new challenges for the preparation of cfDNA that need to be overcome before this sample source can truly be utilized. The objective of this project was to develop reagents and workflows optimized for analysis of cfDNA from urine. Through our studies, we found that the slightly shorter cfDNA in urine requires optimized chemistry to maximize yield, a larger volume of urine may be necessary to isolate sufficient cfDNA compared to plasma/serum and urine must be treated with stabilization agents to minimize further degradation of cfDNA after collection. Using the MagMAX™ cell-free DNA isolation kit, we have developed a magnetic bead-based sample preparation protocol specific for isolating cfDNA from urine. Workflows for preparing cfDNA from urine through manual processing or by automated high throughput sample processing on the KingFisher™ instruments were developed. A small cohort of healthy donors was used to demonstrate compatibility of the cfDNA with qPCR, dPCR and next generation sequencing platforms. The effectiveness of this fast and easy workflow will be further tested on cfDNA from urine samples from donors with and without metastatic disease. We will analyze cfDNA from paired urine and plasma to understand the applicability to different tumor types. Citation Format: Alex J. Rai, Robert A. Setterquist, Xingwang Fang, Hannah E. Saunders, Matthew Carter, Charmaine San Jose Hinahon, Sarah E. Larocca, Susan M. Magdaleno. A complete workflow for high-throughput isolation and analysis of cell-free DNA from urine. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1382.