Accurate and precise targeted NGS with BRCA MASTR Plus Dx for BRCA1 and BRCA2 mutation detection in formalin-fixed paraffin-embedded tumor tissue-derived DNA.

Abstract

e23116 Background: Targeted next-generation sequencing (NGS) has tremendous potential in clinical diagnostics as it allows oncogenetic profiling to steer therapy. Inhibitors of poly-(ADP-ribose) polymerase (PARPi) have emerged as a new class of targeted anti-cancer drugs, specifically for tumors showing homologous recombination repair deficiency, including BRCA1- and BRCA2-mutated ovarian and breast cancers. This multicentre study evaluated the performance of BRCA Tumor MASTR Plus Dx* (Multiplicom) to routinely diagnose somatic and germline BRCA mutations in formalin-fixed paraffin-embedded (FFPE) tumor tissue-derived DNA. Methods: Three genetic centres participated in this performance evaluation study (PES) to detect single nucleotide variants (SNV) and small indels in the BRCA genes at a variant allele frequency down to 5%. The sample population comprised 54 FFPE-derived DNA extracts from 51 clinical and 3 reference samples. DNA extracts were subjected to quality control using Multiplicom’s QC plex assay. The clinical samples were characterized using an independent targeted NGS method and Integrative Genomics Viewer (IGV) analysis of the mapped raw reads. SNV calling was performed using third-party bioinformatics platforms in the BRCA coding regions +/- 2 intronic bp. Results: BRCA MASTR Plus Dx*showed a uniformity of 93.9%, i.e. the percentage of amplicons with at least 0.2x the mean amplicon coverage, and a target specificity of 99.1%. The limit of detection (LOD) proved to be as low as 1%. The diagnostic accuracy was ≥ 99.99% [95% CI ≥ 99.98%] (100% sensitivity [95% CI ≥ 99.02%] and ≥ 99.99% specificity [95% CI ≥ 99.98%]). Both repeatability and reproducibility were ≥ 99.99% [95% CI ≥ 99.98%]. Lot equivalence was 100% [95% CI ≥ 99.99%]. Conclusions: This multicentre study demonstrated that BRCA MASTR Plus Dx* can be routinely applied as an accurate and precise method with an LOD of 1%. The assay can be used to direct patients with somatic or germline BRCA mutations to PARPi therapy. Currently, a PES for BRCA MASTR Plus Dx* and Multiplicom’s MASTR Reporter software is ongoing. *Products described above are CE-IVD and not available for sale in the US