title: Routine multiplex mutational profiling of melanomas enables enrollment in genotype-driven therapeutic trials.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Division of Hematology-Oncology, Department of Medicine, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Christine M
authors.initials: CM
authors.lastname: Lovly
authors.forename: Kimberly Brown
authors.initials: KB
authors.lastname: Dahlman
authors.forename: Laurel E
authors.initials: LE
authors.lastname: Fohn
authors.forename: Zengliu
authors.initials: Z
authors.lastname: Su
authors.forename: Dora
authors.initials: D
authors.lastname: Dias-Santagata
authors.forename: Donna J
authors.initials: DJ
authors.lastname: Hicks
authors.forename: Donald
authors.initials: D
authors.lastname: Hucks
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Berry
authors.forename: Charles
authors.initials: C
authors.lastname: Terry
authors.forename: MarKeesa
authors.initials: M
authors.lastname: Duke
authors.forename: Yingjun
authors.initials: Y
authors.lastname: Su
authors.forename: Tammy
authors.initials: T
authors.lastname: Sobolik-Delmaire
authors.forename: Ann
authors.initials: A
authors.lastname: Richmond
authors.forename: Mark C
authors.initials: MC
authors.lastname: Kelley
authors.forename: Cindy L
authors.initials: CL
authors.lastname: Vnencak-Jones
authors.forename: A John
authors.initials: AJ
authors.lastname: Iafrate
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Sosman
authors.forename: William
authors.initials: W
authors.lastname: Pao
mesh_terms: D045744:Cell Line, Tumor; D002986:Clinical Trials as Topic; D004252:DNA Mutational Analysis; D004608:Eligibility Determination; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D005820:Genetic Testing; D006801:Humans; D008297:Male; D008545:Melanoma / Q000175:diagnosis / Q000188:drug therapy / Q000235:genetics*; D025202:Molecular Diagnostic Techniques; D009419:Nerve Tissue Proteins / Q000235:genetics; D017354:Point Mutation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D012878:Skin Neoplasms / Q000175:diagnosis / Q000188:drug therapy / Q000235:genetics*
chemical_list: C554062:GNA11 protein, human; C554061:GNAQ protein, human; D044385:GTP-Binding Protein alpha Subunits; D009419:Nerve Tissue Proteins; C122162:SHANK2 protein, human; D019009:Proto-Oncogene Proteins c-kit; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D016283:Proto-Oncogene Proteins p21(ras)