LAUSR

Introduction: Circulating cell-free DNA (ccfDNA) is used as an analyte in liquid biopsy-based cancer research with promising applications in the fields of cancer screening, therapeutic decision making and monitoring and minimal residual disease control. However, despite the multitude of future applications, challenges regarding the sensitivity of analytical tests remain, thereby limiting the routine use of ccfDNA within the above mentioned fields. In this study, we investigated the compatibility of the PAXgene® Blood ccfDNA Tube and the QIAcuity® Digital PCR System as an optimal “sample to insight” workflow with regard to the detection of EGFR T790M mutations spiked into apparently healthy human donor blood specimens. Methods: Blood from 22 apparently healthy consented donors was collected into PAXgene Blood ccfDNA Tubes* (PreAnalytiX). Within 2 h of blood draw, blood samples were either spiked with an EGFR-Multiplex 5% AF cfDNA standard (SensID) containing an EGFR T790M mutation or left unspiked as a control. Tubes were incubated for 3 d at room temperature to simulate transport over a weekend and plasma was generated by double centrifugation. Plasma pools were generated from spiked and unspiked samples and ccfDNA was isolated with the QIAamp® Circulating Nucleic Acid Kit (QIAGEN). CcfDNA quantity was measured by Qubit® dsDNA HS Assay (Thermo Fisher Scientific) and fragment size distribution was determined by TapeStation® Cell-free DNA ScreenTape® (Agilent Technologies). EGFR T790M mutation detection was performed using the dPCR Mutation Assay EGFR 6240 Human (QIAGEN) with a 26K 24-well Nanoplate (QIAGEN) on the QIAcuity Digital PCR System (QIAGEN). Data analysis was conducted using the QIAcuity Software Suite (QIsAGEN). Results: CcfDNA isolated from PAXgene Blood ccfDNA Tubes had a fragment size distribution profile with distinct mononucleosomal peaks at ~180 bps without high-molecular weight DNA, indicating successful nucleated cell stabilization over the simulated transport duration and extraction of high-quality ccfDNA. Digital PCR analysis of extracted ccfDNA revealed positive detection of EGFR T790M mutations in spiked blood samples at a relative mutation fraction of approximately 2% in the investigated samples. Conclusion: PAXgene Blood ccfDNA Tubes enabled high-quality ccfDNA isolation by stabilizing nucleated cells during simulated transport condition. Spiked EGFR T790M mutations were successfully detected by dPCR following sample storage. This proof of principle study shows the combined usability of both systems for mutation detection in human blood specimens, highlighting the potential of both technologies for a broader application in the liquid biopsy field to detect oncogenic driver as well as tumor suppressor gene mutations in a multitude of tumor types. *The PAXgene Blood ccfDNA Tube is for Research Use Only in the US. Not for use in diagnostic procedures. Citation Format: Christian Neander, Andrea Huxhold, Maike Schoenborn, Thorsten Voss. Digital PCR-based EGFR T790M mutation detection from human blood samples collected in PAXgene Blood ccfDNA Tubes [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 780.