Multiplex Mutation Screening by Mass Spectrometry

Abstract

There is an immediate and critical need for a rapid, broad-based genotyping method that can evaluate multiple mutations simultaneously in clinical cancer specimens and identify patients most likely to benefit from targeted agents now in use or in late-stage clinical development. We have implemented a prospective genotyping approach to characterize the frequency and spectrum of mutations amenable to drug targeting present in urothelial, colorectal, endometrioid, and thyroid carcinomas and in melanoma. Cancer patients were enrolled in a Personalized Cancer Medicine Registry that houses both clinical information and genotyping data, and mutation screening was performed using a multiplexed assay panel with mass spectrometry-based analysis to detect 390 mutations across 30 cancer genes. Formalin fixed, paraffin-embedded specimens were evaluated from 820 Registry patients. The genes most frequently mutated across multiple cancer types were BRAF, PIK3CA, KRAS, and NRAS. Less common mutations were also observed in AKT1, CTNNB1, FGFR2, FGFR3, GNAQ, HRAS, and MAP2K1. Notably, 48 of 77 PIK3CA-mutant cases (62%) harbored at least one additional mutation in another gene, most often KRAS. Among melanomas, only 54 of 73 BRAF mutations (74%) were the V600E substitution. These findings demonstrate the diversity and complexity of mutations in druggable targets among the different cancer types and underscore the need for a broadspectrum, prospective genotyping approach to personalized cancer medicine. The identification of somatic mutations that cause aberrant activation of intracellular signaling pathways has transformed the diagnosis and treatment of cancer. Mutations in specific genes define distinct subtypes of cancer, and provide invaluable markers for disease diagnosis and prognosis. Many of the mutated proteins also represent targets for novel therapeutic agents that are more specific, more efficacious, and less toxic than broad-based chemotherapeutic regimens.1-5 Indeed, matching the right drug to the right cancer genotype is a proven model for improving treatment and outcome in patients with chronic myelogenous leukemia (CML), nonsmall-cell lung carcinoma (NSCLC), gastrointestinal stromal tumor (GIST), colorectal carcinoma, and, most recently, malignant melanoma. 2,5-16The major successes from this therapeutic approach have been in diseases in which there is limited molecular heterogeneity, with all or most cases having a drug-sensitive mutation. Prime examples include BCR-ABL in CML 9 and KIT in GIST. 3,7 There is increasing evidence that many common cancers similarly harbor potentially druggable targets, albeit at relatively lower frequencies. For example, subsets of NSCLC have oncogenic mutations in EGFR, KRAS, BRAF, PIK3CA, or HER2 or a translocation involving the ALK gene. [17][18][19] In the more molecularly heterogeneous cancers, mutations in proteins other than the intended therapeutic target can profoundly affect response to therapy. Thus, in the case of EGFR inhibitor therapy in lung and colon cancer, KRAS and B...

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