Abstract 5597: Enhanced detection and classification of cell-free DNA alterations through matched normal analyses with PGDx elioTM plasma complete
Authors: Watkins Tonya N. , McGregor Paul , Riley David , Ayazseven Tolga , Waskiewicz Kristy , Gerding Kelly M. , Verner Ellen L. , Greer Amy , Karandikar Aanavi , Zahr Rami , Valkenburg Kenneth C. , Platt Jamie , Angiuoli Samuel V. , Sausen Mark 
Published: June 2023

Abstract
Liquid biopsies represent a transformation in the management of cancer as they have the potential to detect, characterize, and monitor cancers earlier than can be achieved with conventional diagnostic modalities. However, cell-free DNA (cfDNA)-based alterations can be derived from the tumor, germline, or may be associated with clonal hematopoiesis (CH), which can confound non-invasive tumor profiling, molecular response assessment, and clonal evolution analyses through inaccurate variant classification. To facilitate global access to a decentralized liquid biopsy solution to address this, we developed and validated the 521 gene PGDx elio plasma complete test for paired analysis of cfDNA and matched leukocyte DNA. PGDx elio plasma complete enables detection of single nucleotide variants, insertions and deletions, copy number amplifications, translocations, microsatellite instability, blood tumor mutation burden, and loss of heterozygosity. We first optimized the assay workflow to incorporate genomic DNA derived from leukocytes to facilitate direct detection and characterization of germline alterations as well as those that may be associated with CH, resulting in de-duplicated, error-corrected sequencing coverage of approximately 1,750-fold. A fully automated bioinformatics algorithm was then developed and validated to perform integrated analyses of cfDNA-derived alterations to assign the appropriate biological source of these variants. To assess the impact of these paired sample analyses, we analyzed the blood samples obtained from 24 patients representing seven different solid tumor types (breast, colorectal, gastric, gastro-esophageal junction, lung, and melanoma). Across this cohort, of the alterations detected in cfDNA (n=322), 87.3% were correctly classified as somatic, germline or CH without the patient-matched normal blood sample. Specifically, of the variants that were determined to be associated with CH (n=26), only 35% were appropriately assigned without the paired comparison. Additional sources of discordance for somatic and germline alterations were primarily attributed to patients with high levels of ctDNA where differentiation of these variant sources can be challenging through solely computational-based techniques. Taken together, these data demonstrate that through the integrated analysis of cell-free DNA and matched leukocyte DNA, classification of the source of cfDNA-derived alterations can be achieved, which may improve the accuracy of non-invasive tumor profiling, molecular response assessment, and clonal evolution analyses.
Citation Format: Tonya N. Watkins, Paul McGregor, David Riley, Tolga Ayazseven, Kristy Waskiewicz, Kelly M. Gerding, Ellen L. Verner, Amy Greer, Aanavi Karandikar, Rami Zahr, Kenneth C. Valkenburg, Jamie Platt, Samuel V. Angiuoli, Mark Sausen. Enhanced detection and classification of cell-free DNA alterations through matched normal analyses with PGDx elioTM plasma complete. [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 5597.