Abstract 5020: Pre-analytical characterization of cell-free DNA to enable liquid biopsy for solid tumors
Authors: Summersgill Robert J. , Fox Jesse M. , Dickey Jennifer S. , Cox Liam T. , Palumbo Cal D. , Valkenburg Kenneth C. , Caveney Brian J. , Ramkissoon Shakti , Jackson Jennifer B. 
Published: March 2024

Abstract
Background: The integration of liquid biopsy in oncology has transformed clinical management of patients with cancer. Cell-free DNA (cfDNA) represents extracellular nucleic acid fragments shed during cellular apoptosis, necrosis, or secretion, and can be extracted from whole blood. PGDx elio plasma focus analyzes cfDNA to enable non-invasive genomic profiling with next-generation sequencing. In this study, we characterized the cfDNA fraction (%) and yield (ng) across multiple tumor types and stages.
Methods: cfDNA fraction was obtained using automated electrophoresis (Agilent 4200 TapeStation System) to assess a region of 50-700 bp across 293 clinical plasma samples (tumor stages I - IV). The fraction of cfDNA to total DNA present was then calculated to determine overall cfDNA percentage. The overall DNA yield (ng) was quantified via Qubit fluorometer immediately following extraction from plasma and normalized to volume of plasma (ng of cfDNA/mL of plasma). In addition, data was evaluated from 100 clinical plasma cases with strict extraction conditions allowing for yield normalization to plasma volume. Sequencing metrics were evaluated for all clinical cases with a recommended input of 25 ng DNA using PGDx elio plasma focus.
Results: Average cfDNA fraction for individual tumor types ranged from 78.0% - 91.4%, with a mean (n=293) of 84.8%. When examining the yield normalized cohort (n=206), the average yield was 23.4 ng of cfDNA/mL of plasma. cfDNA yield and fraction varied across tumor types and increased with cancer stage. Despite these differences, the majority of cases (99.7%) yielded sufficient cfDNA to proceed with PGDx elio plasma focus testing. No statistical differences in sequencing performance (variants reported and coverage for each tumor type) were observed and the PGDx elio plasma focus assay success rate was 97%. There was a slightly higher failure rate (17.6% (3/17)) for samples below 60% cfDNA, indicating that high amounts of contaminating genomic DNA from white blood cells may obscure the number of available cfDNA fragments for analysis.
Conclusions: These data characterize pre-analytical factors that impact performance of PGDx elio plasma focus using cfDNA isolated from plasma of patients with solid tumors. We showed cfDNA yield and fraction varied across tumor types and increased with cancer stage. The overall success rate for testing was 97% while failed samples were associated with higher levels of genomic DNA contamination.
Citation Format: Robert J. Summersgill, Jesse M. Fox, Jennifer S. Dickey, Liam T. Cox, Cal D. Palumbo, Kenneth C. Valkenburg, Brian J. Caveney, Shakti Ramkissoon, Jennifer B. Jackson. Pre-analytical characterization of cell-free DNA to enable liquid biopsy for solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5020.