Abstract 6199: Interlab study supporting hereditary cancer testing in an NGS-focused landscape
Authors: Dana Ruminski Lowe, Deepika Philkana, Krystyna Nahlik, Melissa Berenger, Andrew Anfora, Yves Konigshofer, Catherine Huang, Russell Garlick, Bharathi Anekella
Journal: Cancer Research (2025)

Abstract Up to 10% of all cancers may be caused by inherited genetic changes. Consequently, the increasing prevalence of cancer is driving the growth of the hereditary cancer testing as it is important to implement surveillance and risk-reduction strategies, detect cancer at an early, more treatable stage, and provide tailored treatment and therapies. Panels are often designed to cover hundreds of genes and performing validation and assessing performance requires samples with common mutations in each of those genes. Biosynethic reference materials can address these challenges. Here we describe the development and multisite evaluation of Seraseq® Inherited Cancer DNA Mix v2, which contains genes recommended for testing by the American College of Medical Genetics, the National Comprehensive Cancer Network, and the American Society of Clinical Oncology. Genomic DNA from the GM24385 cell line was blended with 61 biosynthetic DNA variants in 55 different genes including 21 deletions, 16 duplications, 3 insertions, 5 INDELs, 15 SNVs and 1 Inversion. Variant allele frequency (VAF) was targeted to 50% and verified using the Bio-Rad QX-200 Droplet Digital PCR (ddPCR) System. The mix was analyzed with the Axen Hereditary Cancer Panel v2 (Macrogen), the ViennaLab Hereditary Cancer Assay (ViennaLab) on the MiSeq System (Illumina), the Twist Library Preparation and AVITI 2x150 Sequencing Kit Cloudbreak Low Output on the Element Biosciences AVITI (Halo Precision Diagnostics), the GALEAS Hereditary Plus v2 Panel (Nonacus) on the NextSeq 2000 System (Illumina), and the Celemics Library Preparation Kit and Celemics Target Enrichment Kit and the NextSeq 500/550 Kits v2.5 (Illumina) on the NextSeq 500 System (Illumina).All biosynthetic variants were detected within 40-60% by ddPCR. Most NGS assays were not able to detect all variants as many that were included are difficult to detect. Identification of these complex variants required modifications to alignment, variant calling, and annotation settings. For example, RAD51D c.694_715delins is detected as two separate c.694C&amp;gt;T and c.715C&amp;gt;T variants (e.g. at ViennaLab), unless the variant caller is set to merge. At Nonacus, the Boland inversion and BRCA2 and STK11 large deletions were detected by a combination of short-range and long-range paired-end mapping and split-read analysis. An Alu-specific module using repeat masking allowed successful calling of the BRCA2 AluY insertion; both increased detection accuracy from 85.5% to 95.1%. The performance of the Seraseq Inherited Cancer DNA Mix v2 across NGS assays, supported by robust digital PCR data, demonstrated that these materials have broad compatibility and will allow for assessment of the ability of variant callers to detect complex variants. Citation Format: Dana Ruminski Lowe, Deepika Philkana, Krystyna Nahlik, Melissa Berenger, Andrew Anfora, Yves Konigshofer, Catherine Huang, Russell Garlick, Bharathi Anekella. Interlab study supporting hereditary cancer testing in an NGS-focused landscape [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 6199.