Abstract 2745: Evaluation of a new system for collection, stabilization, and purification of circulating tumor DNA
Authors: Fleischhacker Michael , Schmidt Bernd , Baker Rohan , Pedersen Susanne , Cant Natasha , Zahedi-Nejad Maryam , Voss Thorsten , Ullius Andrea , Groelz Daniel 
Published: July 2017

Abstract
Introduction:
The current preanalytical workflows for circulating tumor DNA (ctDNA) analysis have limitations that affect the accurate detection and quantification of these plasma cancer biomarkers. Release of genomic DNA (gDNA) from white blood cells (WBCs) due to cell lysis or apoptosis during whole blood storage in EDTA tubes creates higher gDNA background levels, affecting the sensitivity of ctDNA assays. Current tubes that stabilize WBCs often contain crosslinking reagents, which have negative effects on sensitive downstream assays, including methylation-based assays.
Using ctDNA assays, the new PAXgene® Blood ccfDNA System*, consisting of a blood collection tube with unique, non-crosslinking chemistry and an automated circulating cell-free DNA (ccfDNA) extraction kit, was evaluated in three research studies.
Methods:
Blood samples were collected into paired PAXgene and EDTA tubes and stored for 7 days at room temperature (RT). ccfDNA was isolated from plasma using the PAXgene kit or the QIAGEN QIAamp® Circulating Nucleic Acid Kit.
Study 1: The ccfDNA from lung cancer patients was quantified by real-time PCR for the amount of the ERV sequence (as a measure of the total plasma DNA quantity) and after bisulfte treatment for mSHOX2 as a marker for ctDNA.
Study 2: Blood from healthy donors was spiked with fragmented, fully-methylated CpGenome DNA. During storage, tubes were intermittently inverted to simulate tubes in transit. Subsequent to bisulfite conversion, PCR assays targeting ACTB and methylated BCAT1 and IKZF1 DNA were used to determine the yields of ccfDNA and ctDNA, respectively.
Study 3: Restriction enzyme treated EGFR DNA containing exon 19 deletions and exon 20 and 21 substitutions (T790M, L858R) were spiked into healthy donors’ blood. ccfDNA was tested with the QIAGEN therascreen® EGFR Plasma RGQ PCR Kit*.
Results:
Both study 1 and 2 demonstrated constant levels of the methylation ctDNA markers, SHOX2, BCAT1 and IKZF1, over the investigated time course. There was no significant release of gDNA in the PAXgene tube whereas a significant release of gDNA was detected in EDTA samples. Likewise, study 3 showed constant EGFR Ct values in the PAXgene system with reliable mutation detection, whereas the high DNA concentration from the EDTA system resulted in false-positive callings.
Conclusions:
The new system allows researchers to accurately detect and quantify plasma cancer biomarkers from blood samples that have been stored in the tube for up to 7 days at RT. This includes challenging assays based on methylated ctDNA. The system provides the required assay sensitivity to allow the correct assay interpretation beyond the typical 3–6 hour storage limit for EDTA tubes.
*For Research Use Only. Not for use in diagnostic procedures.
Citation Format: Michael Fleischhacker, Bernd Schmidt, Rohan Baker, Susanne Pedersen, Natasha Cant, Maryam Zahedi-Nejad, Thorsten Voss, Andrea Ullius, Daniel Groelz. Evaluation of a new system for collection, stabilization, and purification of circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2745. doi:10.1158/1538-7445.AM2017-2745