Abstract 4268: Technical validation of a NGS-based analysis pipeline for BRCA1/2 variants and large genomic rearrangements detection
Authors: Ma Xiangyuan , Bao Hua , Chang Zhili , Wu Yong , Du Changwan , Gao Rong , Song Yu , Robetyore Ryan S. , Wu Xue , Shao Yang W. 
Published: September 2020

Abstract
Background
Individual carrying germline mutations within BRCA1 or BRCA2 gene confers well-established increased risk of developing breast and ovarian cancers in the life-time than the general population. Recent advancement in clinical oncology and pharmacology discovered that poly ADP ribose polymerase (PARP) inhibitors are particularly effective in treating cancer that is homologous recombination deficient (HRD) through synthetic lethality, irrespective of the primary tumor site or the type of the BRCA1/2 inactivation variant.
Methods
A dedicated next-generation sequencing (NGS) panel was designed to interrogate the mutational status of BRCA1/2 and 18 other HRD-associated genes. Using a cohort of 522 clinical samples and artificially engineered cell line samples, we evaluated the mutation detection proficiency for single nucleotide variant (SNV) and small insertion or deletion (Indel) variants. BRCA1/2 inactivation is often the consequence of large genomic rearrangement (LGR) within the gene and phenotypically shown as exon-level copy number variant (CNV). Given the technical challenge of accurate exon-level CNV detection within a small panel, we developed a noise-reduction model based on a pool of normal (PON) samples for CNV result analysis. We further designed droplet digital polymerase chain reaction (ddPCR) for each exon of BRCA1/2 to verify the exonic CNV findings.
Results
Using a comparator assay as the gold standard, the BRCA1/2 panel achieved an accuracy of 96.7%, a precision of 98.9%, and a lower limit of detection of 3% variant allelic frequency (VAF) for SNV and Indel. The CNV pipeline achieved 97.8% sensitivity and 99.7% specificity for exonic CNV events that were supported by at least one piece of secondary evidence, out-performing publicly available software. The ddPCR verification further enhances assay accuracy and provides a significantly improved statistically confidence for the final CNV calls unparalleled by any other experimental methodologies.
Conclusions
Using a large cohort of clinical samples, the authors demonstrated the proficiency of a targeted NGS approach for variant and LGR detection of BRCA1/2 and the other genes of the DNA damage repair pathway. An effective method was developed to suppress the noise within the NGS-derived CNV profile, which is exaggerated by the small panel size and short targeted regions. The authors would also like to call the researcher's attention to a critical caveat within the CNV testing results generated from NGS-based methods.
Citation Format: Xiangyuan Ma, Hua Bao, Zhili Chang, Yong Wu, Changwan Du, Rong Gao, Yu Song, Ryan S. Robetyore, Xue Wu, Yang W. Shao. Technical validation of a NGS-based analysis pipeline for BRCA1/2 variants and large genomic rearrangements detection [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4268.