Abstract 6600: The nRichDX ® revolution sample prep system enables recovery of more amplifiable copies and more cancer fraction detection from cfDNA as evaluated on the MassARRAY ® System
Authors: Jafari Nafiseh , Saidian Mayer , Irwin Darryl 
Published: April 2023

Abstract
Introduction Liquid biopsies utilize cell-free DNA (cfDNA) in blood plasma or urine across various applications, including cancer detection, treatment selection, and treatment/minimal residual disease monitoring. Higher amplifiable copies recovered from liquid biopsies can lead to better cancer detection and monitoring. In this study, we evaluated the recovery of cfDNA amplifiable copies and circulating tumor DNA (ctDNA) dynamics using the nRichDX extraction method, which can extract cfDNA from a broad range of biological fluids (1mL - 20mL). cfDNA quality and ctDNA dynamics were assessed using Liquid IQ and UltraSEEK on the MassARRAY System.
Methods Whole blood from healthy donors was collected in K2EDTA tubes, and plasma was isolated and then pooled in preparation for cfDNA extraction. Plasma aliquots at 5mL (6) and 20mL (6) were extracted using the Revolution Max20 cfDNA Extraction Kit. (12) replicate samples were extracted using QiAmp Circulating Nucleic Acid Kit. Half of the samples were spiked with a cfDNA standard containing a KRAS p.G12V mutation. The quantity and quality of extracted cfDNA were determined using the Liquid IQ panel on the MassARRAY system, which measures amplifiable cfDNA copies, high/low molecular weight dynamics, white blood cell (WBC) contamination along with a molecular barcode for sample matching. Recovery and frequency of the spiked ctDNA standard were performed with the UltraSEEK Lung V2 Panel, capable of detecting variants as low as 0.1% allele frequency.
Results This study compared the amplifiable copies recovered from cfDNA extracted using the nRichDX and Qiagen kit. The amplifiable copies for the nRichDX and Qiagen methods, respectively, were: 5mL non-spiked 517+/-36 Vs. 398+/-17, 5mL spiked 946+/-99 Vs. 732+/-73; 20mL non-spiked 1526+/-26 Vs. 1071+/-55 and 20mL spiked 3004+/-278 Vs. 1777+/-207. All results were statistically different by conventional criteria, evaluated by a two-tailed t-test. Other cfDNA quality characteristics, such as low/high molecular weight DNA and WBC contamination, were concordant between the 2 methods. The spiked KRAS mutation was recovered consistently by both methods with a mutation frequency (5.3+/-0.3) and mutation significance z-score (296+/-19).
Conclusion The nRichDX Revolution System extracts cfDNA and ctDNA with consistently high yields. Two sample T-Test analysis shows that nRichDX, at all levels, performed significantly better than Qiagen at p &lt; 0.05. The results demonstrate the capability of the nRichDX system to recover more amplifiable copies with consistent cfDNA quality and ctDNA frequency and hence offer the opportunity to increase the sensitivity of detecting lower frequency mutations in cfDNA and a wider variety of biomarkers.
Citation Format: Nafiseh Jafari, Mayer Saidian, Darryl Irwin. The nRichDX ® revolution sample prep system enables recovery of more amplifiable copies and more cancer fraction detection from cfDNA as evaluated on the MassARRAY ® System [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6600.