title: Detection of mutations in the mitogen-activated protein kinase pathway in human melanoma.
abstract: OBJECTIVE
  Recent studies suggest that activating point mutations in B-RAF may commonly occur in melanoma. We devised a method to detect point mutations in heterogeneous tissues containing both wild-type and mutant B-RAF and N-RAS genes by using site-directed mutagenesis to introduce new restrictions sites in the cDNA sequence when the specific point mutations are present. We used this technique to determine the incidence of mitogen-activated protein kinase (MAPK) mutations in human melanoma.
  
  
  METHODS
  We screened 85 melanoma samples for the most common B-RAF and N-RAS mutations found in melanoma using a site-directed mutagenesis-based detection technique. Western blotting was used to evaluate downstream up-regulation of the mitogen-activated protein kinase pathway in these tissues.
  
  
  RESULTS
  Thirty-three samples (7 of 25 primaries, 15 of 25 regional metastases, 5 of 25 nodal metastases, and 6 of 10 distant metastases) harbored the V599E B-RAF mutation (39%), 12 contained a Q61R N-RAS mutation and 5 a Q61K N-RAS mutation. Western blotting with antiphosphorylated extracellular signal-regulated kinase 1/2 antibodies demonstrated up-regulation of the MAPK pathway in samples containing activating B-RAF or N-RAS mutations compared with wild-type samples. This method of detection was sensitive and specific with no false positives.
  
  
  CONCLUSIONS
  Activating mutations of the MAPK pathway were present in approximately 60% of samples tested and caused activation of this cellular pathway that appears to be important in the pathogenesis of melanoma.
authors.affiliation: Division of Surgical Oncology, The University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, The Cancer Institute of New Jersey, New Brunswick, New Jersey 08901, USA.
authors.forename: Janivette
authors.initials: J
authors.lastname: Alsina
authors.forename: David H
authors.initials: DH
authors.lastname: Gorsk
authors.forename: F Joseph
authors.initials: FJ
authors.lastname: Germino
authors.forename: Weichung
authors.initials: W
authors.lastname: Shih
authors.forename: Shou-En
authors.initials: SE
authors.lastname: Lu
authors.forename: Zhi-Gang
authors.initials: ZG
authors.lastname: Zhang
authors.forename: Jin-Ming
authors.initials: JM
authors.lastname: Yang
authors.forename: William N
authors.initials: WN
authors.lastname: Hait
authors.forename: James S
authors.initials: JS
authors.lastname: Goydos
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D003062:Codon; D017931:DNA Primers / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D006801:Humans; D020935:MAP Kinase Signaling System; D008545:Melanoma / Q000235:genetics* / Q000473:pathology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017354:Point Mutation; D012680:Sensitivity and Specificity; D015854:Up-Regulation
chemical_list: D003062:Codon; D017931:DNA Primers; D018076:DNA, Complementary; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases