Abstract 6636: Multi-site evaluation of expanded solid tumor, pan-cancer reference material

Abstract Next generation sequencing (NGS) of biopsy-extracted material remains the gold standard for tumor profiling. Panels designed for this type of testing often target 500+ genes, creating a challenge for analytic validation in a clinical setting. Use of patient-like multiplexed commercial reference materials can be a more cost effective and reliable option versus relying on remnant samples with mutations of interest, especially with more stringent requirements from the FDA and other regulators in the early stages of implementation. Here we describe the development and multisite evaluation of expanded Seraseq® Tumor Mutation DNA Mix v3 reference materials, which contain an expanded variant list to meet the growing needs of NGS tumor profiling assay developers. Genomic DNA from the GM24385 cell line was blended with 113 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). Three mixes were made: AF7% in which all variants were targeted at variant allele frequency (VAF) of 7% and CNVs at 6 additional copies, AF10% in which all variants were targeted at VAF of 10% and CNVs at 12 additional copies, and Tri-Level in which certain variants were targeted at 4%, 7%, or 10% and CNVs at 3, 6, or 12 additional copies. All VAFs and CNVs were measured using the Bio-Rad QX-200 Droplet Digital PCR (ddPCR) System. DNA quality was analyzed with the Agilent TapeStation Genomic DNA ScreenTape assay. The mixes were analyzed with the Illumina TruSight™ Oncology 500 (TSO 500) assay and additional assays. All biosynthetic SNVs, indels, SVs, and CNVs were detected near target values and DIN (DNA integrity number) values were >7. 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. Even when variant callers did not report an expected variant, raw data files were investigated and reads with evidence of those variants were discovered. The performance of the Seraseq Mutation DNA Mix v3 materials across NGS assays, supported by robust digital PCR data, demonstrated that these materials will allow for assessment of more complex and tumor profiling tests using a single reference sample. Citation Format: Dana Ruminski Lowe, Patricia Helsel, Praveena Kamineni, Edward S. Davis, Andrew Anfora, Yves Konigshofer, Catherine Huang, Russell Garlick, Bharathi Anekella. Multi-site evaluation of expanded solid tumor, pan-cancer reference material [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 6636.