Routine multiplex mutational profiling of melanomas enables enrollment in genotype-driven therapeutic trials

Abstract

Purpose: Knowledge of tumor mutation status is becoming increasingly important for the treatment of cancer, as mutation-specific inhibitors are being developed for clinical use that target only sub-populations of patients with particular tumor genotypes. Melanoma provides a recent example of this paradigm. We report here development, validation, and implementation of an assay designed to simultaneously detect 43 common somatic point mutations in 6 genes (BRAF, NRAS, KIT, GNAQ, GNA11, and CTNNB1) potentially relevant to existing and emerging targeted therapies specifically in melanoma.

Methods: The test utilizes the SNaPshot method (multiplex PCR, multiplex primer extension, and capillary electrophoresis) and can be performed rapidly with high sensitivity (requiring 5-10% mutant allele frequency) and minimal amounts of DNA (10-20 nanograms). The assay was validated using cell lines, fresh-frozen tissue, and formalin-fixed paraffin embedded tissue. Clinical characteristics and the impact on clinical trial enrollment were then assessed for the first 150 melanoma patients whose tumors were genotyped in the Vanderbilt molecular diagnostics lab.

Results: Directing this test to a single disease, 90 of 150 (60%) melanomas from sites throughout the body harbored a mutation tested, including 57, 23, 6, 3, and 2 mutations in BRAF, NRAS, GNAQ, KIT, and CTNNB1, respectively. Among BRAF V600 mutations, 79%, 12%, 5%, and 4% were V600E, V600K, V600R, and V600M, respectively. 23 of 54 (43%) patients with mutation harboring metastatic disease were subsequently enrolled in genotype-driven trials.

Conclusion: We present development of a simple mutational profiling screen for clinically relevant mutations in melanoma. Adoption of this genetically-informed approach to the treatment of melanoma has already had an impact on clinical trial enrollment and prioritization of therapy for patients with the disease.

Figure 1. Melanoma SNaPshot screen (v1.0).

A, five multiplexed panels can detect the mutational status of twenty gene loci. Each peak color represents a particular nucleotide at that locus. The gene name, amino acid, and nucleotide are labeled above each peak. An “(R)” after the nucleotide denotes a reverse extension primer. B, pan-positive control for melanoma SNaPshot screen. Peaks are labeled as described in A. C, SNaPshot sensitivity measurement using cell line DNA carrying known mutations. Numbers indicate the arbitrary fluorescence units of WT (panel 1: green, panels 2, 3: blue) and mutant (panel 1: blue, panels 2, 3: green) peaks. Solid arrows indicate mutant peaks and dotted arrows show background peaks. Background peaks in the negative controls (far right panel) are indicated by their peak height and a star (*).

Figure 2. Distribution of mutations in the first 150 tumors genotyped in the molecular diagnostic lab.

Left: distribution of all mutations. Right: distribution of V600 mutations. See Table S10 for more details.