Abstract 6109: Multi-site evaluation of novel BRCA1/2 reference materials including large genomic rearrangements
Authors: Lowe Dana Ruminski , Forson Benedicta , Cowen Maria , Butler Matthew G. , Konigshofer Yves , Berenger Melissa , Nahlik Krystyna , Xia Dianren , Huang Catherine , Garlick Russell , Anekella Bharathi 
Published: April 2023

Abstract
Mutations in the tumor suppressor BRCA1 and BRCA2 genes can cause cell damage that significantly increases the risk of developing breast, ovarian, prostate, and pancreatic cancers. Large genomic rearrangements (LGRs) are defined as deletions, duplications or insertions; often involving a significant portion of an exon. Usually pathogenic, they have been reported to account for up to 27% of the BRCA1 and 5% of BRCA2 disease-causing mutations with a strong founder effect accounting for about 1/3 of all cancer diagnoses in some populations. Accurate detection of a BRCA1/2 pathogenic variants has immense impact on clinical management of disease including eligibility for PARP inhibitor therapy. However, these LGRs are frequently missed by PCR-based methods and NGS assays that do not detect partial or complete exon losses or gains. Given the difficulty in detecting LGRs, there is a need for improvement of BRCA1/2 testing algorithms including reference materials that incorporate challenging LGRs to support NGS assays that analyze for these mutations.
Biosynthetic DNA constructs bearing clinically relevant BRCA1/2 variants including deletions up to 500 bp spanning 2 exons and duplications up to 170 bp, were mixed with purified genomic DNA from the GM24385 human reference cell line at 10% and 50% variant allele frequency (VAF) to represent somatic and inherited BRCA disease states, respectively. The same variants were engineered into GM24385 cells at the desired VAF (&gt;5%) and the cells were formalin fixed and paraffin embedded using a proprietary method to mimic preserved tumor biopsies. In both materials, VAF was measured using digital PCR. The prepared purified gDNA and FFPE materials were sent to multiple clinical laboratories and analyzed using various NGS assays to assess variant detection performance and ensure that the product was compatible with clinical testing workflows.
Digital PCR confirmed the presence of all 20 BRCA1 and BRCA2 variants within a 20% range of the target VAF in the purified gDNA material and between 11-23% in FFPE. NGS results were variable but confirmed the presence of most variants. A few variants were detected at very low levels but reported as not detected by the bioinformatic software due to limit of detection.
We have successfully developed a reference material to support both BRCA1 and BRCA2 inherited and somatic genetic testing, providing laboratories and assay developers with a tool to challenge and optimize their assay’s detection and quantitation of pathogenic variants by NGS
Provided as full process in FFPE format or in purified gDNA, these reference materials include 11 challenging LGRs, which are useful in evaluating the ability of different NGS platforms to detect such variants. This study has highlighted the need for reference materials that contain these challenging genomic alterations to improve genetic testing and ultimately, support PARP inhibitor treatment selection.
Citation Format: Dana Ruminski Lowe, Benedicta Forson, Maria Cowen, Matthew G. Butler, Yves Konigshofer, Melissa Berenger, Krystyna Nahlik, Dianren Xia, Catherine Huang, Russell Garlick, Bharathi Anekella. Multi-site evaluation of novel BRCA1/2 reference materials including large genomic rearrangements. [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 6109.