Abstract 4576: Sequential purification of cfDNA and cfRNA from blood stabilized in Nucleic Acid BCT using an automated workflow
Authors: Nicholas M. George, Lisa Bartron, Jordan LaRue
Journal: Cancer Research (2025)

Abstract Liquid biopsies, which interrogate body fluids in search of analytes released by healthy or diseased cells, have become increasingly common in oncology. Foremost among the technologies used are those that employ nucleic acids, such as cfDNA and cfRNA. While cfDNA remains the primary choice for mutation detection, cfRNA is also informative, as it is released by both live and dying cells and represents the true disease state-mediated transcriptional output. Further, gene fusion events, which may be expressed at high levels in tumors, are easier to detect using cfRNA. As such, combined analysis of both cfDNA and cfRNA could provide a more comprehensive analysis. However, if blood samples are not stable, cell death or activation could lead to spurious increases in both analytes. To address this issue, Streck developed Nucleic Acid BCT (NAC), a stabilizing blood collection tube formulated to minimize changes to cfDNA and cfRNA concentrations during storage and shipping. To determine whether a combined cfDNA and cfRNA isolation protocol is compatible with NAC, blood from self-proclaimed healthy donors was drawn into EDTA and NAC and stored at ambient temperature for up to 7 days, plasma was isolated, and cfDNA and cfRNA were sequentially isolated using the Promega Maxwell RSC System with applicable kits. To mimic mutational events observed in lung cancer, synthetic 170-mer oligonucleotides encoding the EGFRL858R or EGFRdel19 events were spiked to an anticipated 1% MAF. Likewise, an RNA Ultramer encoding the EML4-ALK fusion was spiked at a copy number representative of that observed in clinical samples. Samples were then analyzed using ddPCR to determine concentrations of EGFRL858R and EGFRdel19 relative to the wildtype allele and EML4-ALK copy number relative to the housekeeping gene UBB. Our data support the combined use of the Promega Maxwell system with applicable extraction kits with plasma isolated from NAC. As expected, samples collected into NAC had minimal increases in cfDNA and cfRNA concentrations during storage compared to blood samples stored in EDTA. Baseline MAF of the spiked mutant cfDNA alleles was completely lost in samples stored in EDTA, while equivalent blood samples stored in NAC maintained MAFs reflective of draw time levels. A similar trend was observed when plasma RNA expression was investigated where samples collected in NAC had similar concentrations of cfRNA at draw and after storage, but those collected into EDTA did not. Specifically, EML4-ALK copy number relative to the UBB housekeeping gene fell precipitously in EDTA but maintained draw-time frequencies in blood samples stored in NAC. Taken together, these data indicate that the combined use of NAC and the sequential nucleic acid extraction protocol described herein can provide the liquid biopsy community with a robust workflow for both blood sample stabilization and multianalyte extraction and downstream analysis. Citation Format: Nicholas M. George, Lisa Bartron, Jordan LaRue. Sequential purification of cfDNA and cfRNA from blood stabilized in Nucleic Acid BCT using an automated workflow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4576.