Abstract 5041: Next generation liquid biopsy reference material performance across NGS assays and platforms

Abstract Liquid biopsy testing has grown to support early disease diagnosis, therapy selection, and disease and treatment surveillance in cancer survivors. Recent advances in next generation sequencing (NGS) have enabled larger panel sizes, pan-cancer target sets, and wider ranges of genomic alterations. More inclusive analytical validation materials are needed to assess assay sensitivity, specificity, and limit of detection of all variant types to serve the expanded utility of liquid biopsy testing. Here we describe the development and multisite evaluation of expanded Seraseq® ctDNA Mutation Mix v4 reference materials. Genomic DNA from the GM24385 cell line was blended with 93 clinically relevant biosynthetic DNA variants including single nucleotide variants (SNVs), insertions and deletions (indels), structural variants (SVs), copy number variations (CNVs), and mono- and dinucleotide biomarkers for microsatellite instability (MSI). Variant allele frequency (VAF) was targeted to 0.5% and total copy number for CNVs was targeted to 2.5 and verified using the Bio-Rad QX-200 Droplet Digital PCR (ddPCR) System. The DNA mix was fragmented and purified to create ctDNA-like size profiles. Wild-type (WT) material was prepared for comparison and identification of background variants. Agilent Bioanalyzer High Sensitivity DNA and TapeStation Cell-free DNA ScreenTape analyses were used to assess fragment size. The ctDNA mixes were analyzed with the Illumina TruSight™ Oncology 500 (TSO 500) ctDNA assay, the Thermo Oncomine™ Precision assay, and additional assays. All biosynthetic SNVs, indels, SVs, and CNVs in the 0.5% ctDNA mix were detected near target values. The average fragment size was about 190 bp, similar to native ctDNA. The VAFs and CNVs observed in the TSO 500 data correlated well with those that were obtained by dPCR. Variants reported by all evaluation site assays showed good concordance of VAF values. The two sites that tested the materials with TSO 500 revealed consistency in both VAF and total copies with an average CV of 16.1% and 2.5%, respectively. The sequencing performance of the reference material was similar to patient samples and showed comparable levels of background noise. The performance of the Seraseq ctDNA v4 mutation mix across NGS assays, supported by robust digital PCR data, demonstrated that these materials will allow for assessment of complex and sensitive liquid biopsy tests. Comparing data measured at different sites highlights the variability in liquid biopsy assay performance and the importance of highly multiplexed and consistent reference materials. Remnant samples are typically not an option due to limited yields from a standard blood draw, thus contrived standards are especially needed. Expanded variant content and improvements in the methods used to produce the Seraseq ctDNA Mutation Mix v4 materials will enable advancements in the blood-based NGS clinical diagnostics space. Citation Format: Dana Ruminski Lowe, Sanchita Jamindar, Yves Konigshofer, Andrew Anfora, Krystyna Nahilk, Dianren Xia, Edward S. Davis, David Merriam, Catherine Huang, Russell Garlick, Bharathi Anekella. Next generation liquid biopsy reference material performance across NGS assays and platforms [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 5041.