Abstract 2505: The Oncomine BRCA expanded next-generation sequencing assay: Development and analytical validation of a new panel for detection of SNV, insertions, deletions and copy number variants in a panel of 15 genes involved in homologous recombination repair of double-strand break DNA damage
Authors: Scafe Charles , Zhu Yun , Yang Chenchen , Tseng Yu-Ting , McKnight Brooke , Farfan Fernando , Bandla Santoshi , Sadis Seth , Roman Steven , Hyland Fiona C. 
Published: September 2020

Introduction: Many tumors display phenotypic similarity across tissue types, characterized by defects in biological pathways leading to susceptibility to particular therapies. Deficiencies in one such pathway, homologous recombination DNA damage repair (HR DDR), affect the response of breast and ovarian cancers to agents that inhibit PARP function. The Oncomine BRCA Expanded panel targets 15 genes (BRCA1 and 2, ATM, BARD1, BRIP1, CDK12, CHEK2, FANCD2, MRE11, NBN, PALB2, PPP2R2A, RAD51B, RAD54L, and TP53) that play various roles in the HR DDR pathway. The assay includes 1011 amplicons covering both hotspot variants and coding regions of these genes. An additional set of pre-qualified genes is available for assay customization, including a recommended set of 25 genes of particular relevance to HR DDR pathway.

Methods: Targeted regions were selected, and amplicons were designed, optimized, and experimentally verified to perform well with semi-conductor based sequencing. Measures used included gene-base and hotspot-base coverage uniformity, variant calling accuracy, and detection of known CNV gains in both FFPE and cell line DNA samples. Large gene rearrangements in BRCA1 and BRCA2, including exon deletions and duplications, could be detected by using a custom exon deletion pipeline. Analysis was performed on an integrated bioinformatics platform, Ion Reporter, which includes variant calling, identification and annotation of the likely deleterious mutations, and production of configurable reports, including visualization of the extent of copy number changes.

Results: The assay's performance was evaluated using a variety of sample types, including commercially available controls and FFPE samples for breast, ovarian, prostate, and other tumor types. Sensitivity and positive predictive value for small variant detection was &gt;95% in Acrometrix Hotspot Controls, and &gt;90% of CNV gains greater than 6 were detected in cell line samples. Examples of all detected variant classes were displayed based on the Ion Reporter user interface.

Conclusions: We introduce an expanded BRCA panel for researching genetic changes influencing homologous recombination DNA damage repair. The option to customize the gene content of the panel is enabled via a web-based interface to inventoried, pre-qualified targets. This approach allows identifying a broad set of mutation types in a single assay with high accuracy, enabling interrogation of HR DDR genes in FFPE preparations.

For research use only. Not for use in diagnostic procedures.

Citation Format: Charles Scafe, Yun Zhu, Chenchen Yang, Yu-Ting Tseng, Brooke McKnight, Fernando Farfan, Santoshi Bandla, Seth Sadis, Steven Roman, Fiona C. Hyland. The Oncomine BRCA expanded next-generation sequencing assay: Development and analytical validation of a new panel for detection of SNV, insertions, deletions and copy number variants in a panel of 15 genes involved in homologous recombination repair of double-strand break DNA damage [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 2505.