1174P - Detection of NRAS, KRAS and BRAF mutations in FFPE derived DNA with a novel targeted resequencing-based diagnostics assay

Abstract

Identification of cancer-associated mutations has become standard care for cancer treatment. Somatic mutations in NRAS, KRAS and BRAF genes are important to decide the treatment options for patients with cancer. Mutations in NRAS, KRAS or BRAF help to determine the treatment options for patients when EGFR inhibitor or BRAF inhibitor treatment is considered. The novel SOMATIC 1 MASTRTM Plus Dx® assay has been developed to allow analysis of the hotspot mutations or of the full coding region of KRAS, NRAS and BRAF genes in combination with Next Generation Sequencing (NGS). To assess the performance characteristics of the SOMATIC 1 MASTRTM Plus Dx® assay, a multicenter study was performed. The performance of SOMATIC 1 MASTR Plus Dx to detect single nucleotide variants (SNV) and small insertions and/or deletions (indels) in the NRAS, KRAS and BRAF genes at a variant allele frequency (VAF) as 5% in the entire coding region and for specific hotspot mutations was evaluated. The sample population comprised 252 formalin-fixed, paraffin-embedded (FFPE) clinical samples derived from a variety of different cancer types and 4 proficiency samples (Tru-Q Reference Standards, Horizon Discovery). Amplicon libraries were analysed using Illumina MiSeq platform. Data analysis was performed with the SeqNext module (JSI Medical Systems) and the Sophia DDM platform (ILL1MR1S5_somatic1_v2). SOMATIC 1 MASTRTM Plus Dx® showed amplification uniformity of 97.8% (% of amplicons covered at 0.2x of the mean coverage) and a target specificity of 99.1%. The overall limit of detection (LOD) of the assay was determined as 2% VAF, and showed to be as low as 1% for hotspot mutations. Diagnostically, the assay showed 99.96% (95 % CI ≥ 99.90 %) accuracy, 99.96% (95 % CI ≥ 99.90 %) specificity and 100% (95 % CI ≥ 98.54 %) sensitivity. Furthermore, Somatic 1 MASTRTM Plus Dx® assay showed to be 99.99% (95 % CI ≥ 99.97 %) repeatable and 99.96% (95 % CI ≥ 99.91%) reproducible. We have established a targeted and cost-effective NGS assay for the detection of KRAS, NRAS, and BRAF somatic mutations in clinical FFPE samples that can be routinely incorporated into clinical practice.