title: Detection of B-RAF and N-RAS mutations in human melanoma.
abstract: BACKGROUND
  It is now known that activating point mutations in components of the mitogen-activated protein kinase pathway commonly occur in melanoma. We previously described a method to detect point mutations in heterogenous 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 modified this technique to improve sensitivity and used it to determine the incidence of B-RAF and N-RAS mutations in human melanoma.
  
  
  METHODS
  We screened 115 melanoma samples for the most common B-RAF and N-RAS mutations found in melanoma using a site-directed mutagenesis-based detection technique. Southern blotting was used to increase sensitivity of the basic system. We also tested this method of genetic mutation detection in fine-needle aspiration specimens and paraffin-embedded tissues.
  
  
  RESULTS
  Sixty-eight samples (20 of 36 primaries, 18 of 27 regional metastases, 16 of 40 nodal metastases, and 9 of 12 distant metastases) harbored the V599E B-RAF mutation (59%), 17 contained a Q61R N-RAS mutation, and 4 contained a Q61K N-RAS mutation. We were able to detect the V599E mutation in genomic DNA from paraffin-embedded melanoma samples and could routinely detect this mutation in fine-needle aspirations of melanoma tumors. This method of detection was sensitive and specific with no false positives.
  
  
  CONCLUSIONS
  Activating mutations of B-RAF and N-RAS were present in approximately 60% and 18%, respectively, of samples tested. The site-directed mutagenesis system of mutation detection was both sensitive and specific in detecting these mutations and will likely prove very clinically useful in future studies.
authors.affiliation: Division of Surgical Oncology, UMDNJ-Robert Wood Johnson Medical School, The Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA.
authors.forename: James S
authors.initials: JS
authors.lastname: Goydos
authors.forename: Barbara
authors.initials: B
authors.lastname: Mann
authors.forename: Hyunjin J
authors.initials: HJ
authors.lastname: Kim
authors.forename: Emmanuel M
authors.initials: EM
authors.lastname: Gabriel
authors.forename: Janivette
authors.initials: J
authors.lastname: Alsina
authors.forename: F Joseph
authors.initials: FJ
authors.lastname: Germino
authors.forename: Weichung
authors.initials: W
authors.lastname: Shih
authors.forename: David H
authors.initials: DH
authors.lastname: Gorski
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics; D044963:Biopsy, Fine-Needle; D015139:Blotting, Southern; D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D008545:Melanoma / Q000235:genetics* / Q000473:pathology; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics; D016612:Paraffin Embedding; D017354:Point Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D012878:Skin Neoplasms / Q000235:genetics* / Q000473:pathology
chemical_list: D014408:Biomarkers, Tumor; D018076:DNA, Complementary; D012333:RNA, Messenger; D012334:RNA, Neoplasm; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D020928:Mitogen-Activated Protein Kinases