Patient segmentation assay for MAPK pathway mutations.

Abstract

11059 Background: Mutational status of solid tumors is increasingly important for identifying the best treatment options. Tumors harboring activating mutations in RAS and RAF result in constitutive activation of the RAS/MAPK signal transduction pathway and do not respond well to MAPK pathway inhibitors. Scientific attention is mostly focused on the major mutational hotspots in these genes (e.g., KRAS codons 12, 13 and 61, BRAF codon 600). However, there is increasing evidence that other mutations can be tumorigenic. Thus, there is a need for highly sensitive and specific assays to detect these mutations. To address this need, we developed and analytically validated an assay that detects 33 mutations that activate the MAPK pathway. Methods: We constructed a set of multiplexed single nucleotide primer extension (SNPE) assays that detect 33 activating mutations in KRAS, NRAS, or BRAF. The assays were analytically validated using a set of 60 formalin fixed paraffin embedded (FFPE) tissue samples from various tumor types, cell lines, and oligos with specific mutations. Performance was compared to dideoxy sequencing. Discordant calls were resolved with next generation sequencing (NGS). The limit of detection was determined by serial dilution of mutant DNA into wildtype DNA. Results: The developed multiplexed assay requires only 15 ng genomic DNA, relies on established technology, is cost effective, is amenable to high throughput, and can yield a patient eligibility decision in a CLIA lab in 4 days, making it a practical alternative to a NGS-based assay. Since low allele frequency mutations may be critical to patient survival, we devised a replicate strategy to increase the specificity and sensitivity of the assay. We showed that analysis of technical triplicates relative to no replicates increased the sensitivity from 97 to 100% and the specificity from 91 to 100%. Limit of detection varied from 2 -12%, depending on the mutation. Conclusions: We developed a selective and sensitive SNPE assay capable of detecting 33 hotspots in the MAPK pathway. Our approach reduced false positive and false negative calls of low allele frequency samples. The assay has clinical applicability for the selection of patients for early phase clinical studies.