Abstract 6635: Use of contrived multiplexed FFPE specimens to evaluate CGP from extraction to variant calling
Authors: Dana Ruminski Lowe, Richard Howard, Edward S. Davis, Andrew Anfora, Yves Konigshofer, Catherine Huang, Russell Garlick, Bharathi Anekella
Journal: Cancer Research (2025)

Formalin fixed and paraffin embedded (FFPE) samples are a common, yet challenging type of patient sample in comprehensive genomic profiling (CGP). DNA extracted from FFPE is often highly cross-linked, degraded, and fragmented. Yield from patient samples can also be limited, thus optimization of extraction in regard to amount and quality of DNA is crucial to the success of downstream analysis. Contrived reference materials are a convenient sample source to evaluate all steps in a CGP workflow. First, they can be used to optimize and validate extraction as they are readily available and consistent in yield and composition. In addition, they are highly multiplexed, making validation of the analytical part of CGP more efficient and cost effective. Finally, complex, difficult to call variants can be easily included to critically analyze the bioinformatics as well. Here we describe the development and evaluation of Seraseq® FFPE Solid Tumor Reference Material. GM24385 cells were engineered to carry 74 biosynthetic DNA variants across 62 genes including single nucleotide variants (SNVs), insertions and deletions (indels), structural variants (SVs), and mono- and dinucleotide biomarkers for microsatellite instability (MSI). Variant allele frequency (VAF) was verified using the Bio-Rad QX-200 Droplet Digital PCR (ddPCR) System. The cells were formalin fixed, embedded in paraffin blocks and sectioned at 10 micron thickness. Extraction was performed with various commercially available kits from QIAGEN, Promega, and AutoGen. Yields were analyzed using Qubit dsDNA HS and ddPCR. TapeStation Genomic DNA ScreenTape assay was used to assess fragment size and DNA integrity. The extracted DNA was analyzed with the Illumina TruSight™ Oncology 500 (TSO 500) and the ArcherDx VariantPlex (IDT) NGS assays as well as the Biocartis Idylla IDH1-2 Mutation Assay. Yields were variable between extraction kits; interestingly, yields determined using Qubit did not always correlate with those measured by ddPCR indicating some kits result in more amplifiable DNA than others. Quality and average fragment size also varied between kits but did not affect downstream analysis. The Idylla platform showed positive detection for all IDH1 and IDH2 variants in the FFPE sections. The average VAF was 26.7% ± 5.7% and 36.3% ± 7.3% as measured by the TSO 500 and VariantPlex assays, respectively. Variants that were originally missed by variant callers could often be seen in raw data files upon further investigation. This study indicates that use of a multiplexed FFPE reference material that provides a data rich readout of actionable variants can be valuable in the initial assessment, analytic validation, and performance monitoring of all parts of CGP workflows. Citation Format: Dana Ruminski Lowe, Richard Howard, Edward S. Davis, Andrew Anfora, Yves Konigshofer, Catherine Huang, Russell Garlick, Bharathi Anekella. Use of contrived multiplexed FFPE specimens to evaluate CGP from extraction to variant calling [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 6635.