Abstract PR11: Multimodal analysis of circulating tumor cell RNA, circulating cell-free DNA, and genomic DNA from a single blood sample collected into a PAXgene Blood ccfDNA Tube*
Authors: Provencher Eric , Remmel Maike , Hauch Siegfried , Otte Michael , Ullius Andrea , Groelz Daniel , Babayan Anna 
Published: June 2020

Abstract
Background: High demand for liquid biopsy tests in cancer research increases the need for combined approaches that allow multimodal testing from a limited blood volume. Although crosslinking-based fixatives allow for efficient preservation of the circulating cell-free DNA (ccfDNA) profile in whole blood, analysis of mRNA from circulating tumor cells (CTCs) is hampered. Currently, there are no blood collection tubes available that allow RNA analysis from CTCs after prolonged (&gt;48 hours) storage. In this research study, we present a newly developed combined workflow for blood stabilization and subsequent analysis of CTC RNA, ccfDNA, and gDNA after prolonged (≥72 hours) storage.
Methods: To establish the multimodal workflow, blood was collected from healthy donors into PAXgene Blood ccfDNA Tubes*, aliquoted and manually spiked with 20 individual LNCaP95 cells/5 mL blood as a CTC model or 20 µL PBS as a control. Blood samples were spiked and stored at 2–8°C for 3, 24, 48, and 72 hours before processing. CTCs were enriched and detected using the AdnaTest ProstateCancerPanel AR-V7† (QIAGEN). The plasma and cellular fraction of the CTC-depleted blood was then used to extract ccfDNA and gDNA, respectively. As a control for ccfDNA and gDNA yield measurements, plasma and the cellular fraction were also generated from whole samples (not CTC-depleted) spiked with 20 LNCaP95 cells/5 mL. The resulting multimodal workflow was tested: 1) in comparison to Streck Cell-Free DNA BCT; 2) to assess the impact of room temperature.
Results: Tumor cells spiked into PAXgene Blood ccfDNA Tubes could be enriched and detected at experimental time points 3, 24, 48, and 72 hours after spiking with 100% test sensitivity within 24 hours storage and 91% test sensitivity after 72 hours storage at 2–8°C. In unspiked samples, the PAXgene stabilization solution did not cause false positive signals at any experimental time point. Yields of ccfDNA and gDNA were not statistically significantly affected by CTC capture (p&gt;0.05). RNA from spiked tumor cells was detected in blood samples collected and stored in Streck Cell-Free DNA BCTs for 3 hours, but not at later time points. Storage of PAXgene stabilization solution-treated samples at room temperature is feasible, with 100% test sensitivity for CTC detection within 24 hours storage (similar to storage at 2–8°C) and 80% after 72 hours storage.
Conclusions: Blood collected into PAXgene Blood ccfDNA Tubes and stored for up to 72 hours at 2–8°C or at room temperature can be used for multimodal analysis of CTC RNA, ccfDNA, and gDNA from a single blood sample.
*For research use only. Not for use in diagnostic procedures.
†For molecular biology applications. Not intended for the diagnosis, prevention, or treatment of a disease.
This abstract is also being presented as Poster B18.
Citation Format: Eric Provencher, Maike Remmel, Siegfried Hauch, Michael Otte, Andrea Ullius, Daniel Groelz, Anna Babayan. Multimodal analysis of circulating tumor cell RNA, circulating cell-free DNA, and genomic DNA from a single blood sample collected into a PAXgene Blood ccfDNA Tube* [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 PR11.