Historically, therapeutic decisions for the treatment of malignant melanoma have been based upon stage and histology (ulceration and depth or volume of tumor), with the choice of systemic anti-cancer therapies guided mostly by empiric data leading to generally dismal outcomes 
. However, basic and translational research has uncovered molecular abnormalities in melanomas that not only drive and sustain the cancer but can also serve as attractive therapeutic targets. For example, mutant-specific inhibitors induce a >50% response rate in patients with BRAF V600-mutant tumors 
, and nearly 50% of tumors harboring certain KIT
mutations are highly sensitive to imatinib 
Here, we present development, validation, and clinical implementation of a disease-specific SNaPshot-based screen 
to assess melanoma tumor samples simultaneously for 43 somatic recurrent point mutations in 6 genes with relevance to targeted therapy. The SNaPshot assay can be performed rapidly with minimal amounts of starting FFPE-derived DNA material (20 nanograms) and high sensitivity 
, detecting mutations in samples when mutant DNA comprises <10% of the total DNA (see supplemental material). By comparison, direct dideoxynucleotide sequencing, used currently in many clinical molecular labs, requires that mutant DNA comprise >20–25% of the total DNA for mutation detection.
In its present form, the SNaPshot assay can detect mutations that occur in the majority of melanomas. Of the first 150 tumor samples tested in the clinical lab, 90 (60%) had an identifiable mutation, which were 38% BRAF
, 15% NRAS
, 4% GNAQ
, 2% KIT
, and ~1% CTNNB1
. Now with additional prospective testing for over 15 months, the numbers remain similar in their breakdown. The frequency of these mutations and the anatomic sites of origin for the primary tumor were consistent with previously published results. Of the 90 mutations detected, 57 mutations were identified that involved the BRAF V600 position. The percent of BRAF mutations that were V600E (79%) () is also consistent with what has been reported in the literature 
. Since our assay was designed to distinguish among various mutations that affect V600, our data further show that allele-specific molecular diagnostic assays designed to detect only the most common V600E mutation will miss ~20% of the total number of V600 mutations in melanoma.
Importantly, our results demonstrate the impact of tumor mutation assessment on directing melanoma patients to the most appropriate clinical trials with the therapeutic agents most likely to provide a benefit. Of the 54 patients with metastatic disease and a detected tumor mutation, 23 (43%) were subsequently enrolled onto genotype-driven trials based upon the results from their tumor mutational profiling. This is a dramatic advantage over a simple allele specific PCR for BRAF V600E. In addition to BRAF inhibitors, patients are directed to trials for KIT mutations, GNAQ/11 mutations in uveal melanoma, and even NRAS mutant melanoma. In addition, studies in patients who have disease progression following initial response to BRAF inhibitor therapy have revealed a secondary mutation in NRAS as the mechanism of resistance in nearly a quarter of this patient population 
. Therefore, mutational profiling of resistant disease after BRAF inhibitors may provide insight into selecting secondary therapy.
This prospective approach to mutation analysis has multiple advantages in melanoma. First and foremost, it allows prospective patient selection to the best available therapies or most relevant clinical trials based on tumor mutational status. Given the increasing number of clinically relevant genotypes in melanoma and the expanding repertoire of targeted inhibitors (Table S11
), clinical characteristics or tumor histology are no longer the most effective way to select and prioritize treatment options for patients with this disease. A single comprehensive tumor genotyping panel in the form of the SNaPshot test will allow patients and physicians to understand and incorporate complex tumor-gene-mutation information into their treatment algorithms. Second, because the disease can quickly progress, determining tumor mutation status as part of routine care enables faster treatment prioritization 
. Third, prospective genotyping allows for the determination of an accurate portrait of the genomic profile of patients who are routinely referred to this institution as opposed to retrospective studies reported from large databases (e.g. COSMIC) or other institutions. Finally, prospective tumor profiling may allow us to make previously unknown associations between a tumor mutation and clinical features and/or clinical activity of new drug combinations. Of greatest importance, this assay has proven benefit in directing patients to the most appropriate therapies and clinical trials, which will ultimately lead to improved outcomes for patients with melanoma.