title: BRAF mutations in melanocytic lesions and papillary thyroid carcinoma samples identified using melting curve analysis of polymerase chain reaction products.
abstract: BACKGROUND
  Mutations of the proto-oncogene B-raf (BRAF) have been detected in melanocytic lesions and papillary carcinomas of the thyroid, and identification of these mutations could be useful in resolving some diagnostic problems.
  
  
  OBJECTIVE
  To develop a method to evaluate mutations of BRAF that could provide results much more rapidly than conventional polymerase chain reaction and DNA sequencing assays.
  
  
  METHODS
  An assay using a LightCycler was developed to evaluate DNA sequences encoding amino acids within the activation loop of BRAF.
  
  
  RESULTS
  Using this real-time polymerase chain reaction method, we analyzed 55 paraffin-embedded melanoma or nevus samples. The V600E mutation was found in 0 (0%) of 13 samples diagnosed histologically as Spitz nevi, 9 (24.3%) of 37 invasive melanomas, and 5 (100%) of 5 other melanocytic nevi. Two additional mutations, V600K and VK600-1E, also were identified in cases of invasive melanoma. We analyzed 14 paraffin-embedded papillary thyroid cancer (PTC) samples, 6 of which showed the V600E mutation. We found that our test worked efficiently with fine-needle aspirate specimens, and it identified 6 V600E mutations in 10 fine-needle aspirate specimens diagnosed as PTC. We also identified 4 V600E mutations in 6 specimens of PTC metastatic to lymph node. Unlike the melanocytic lesions, the PTC specimens yielded only V600E mutations. Comparison of our real-time polymerase chain reaction results with conventional polymerase chain reaction and DNA sequencing demonstrated 100% concordance. Surprisingly, we did not identify the previously reported VK600-1E or K601E mutations in our PTC specimens.
  
  
  CONCLUSIONS
  Our results show that the real-time polymerase chain reaction method is a rapid and accurate method for identifying BRAF mutations, such as V600E, in both paraffin-embedded tissue and fine-needle aspirate specimens.
authors.affiliation: Calgary Laboratory Services, University of Calgary, Alberta.
authors.forename: Robert
authors.initials: R
authors.lastname: Hay
authors.forename: Erin
authors.initials: E
authors.lastname: MacRae
authors.forename: Duane
authors.initials: D
authors.lastname: Barber
authors.forename: Moosa
authors.initials: M
authors.lastname: Khalil
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Demetrick
mesh_terms: D001483:Base Sequence; D044963:Biopsy, Fine-Needle; D002291:Carcinoma, Papillary / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D004273:DNA, Neoplasm / Q000235:genetics; D006801:Humans; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008969:Molecular Sequence Data; D009154:Mutation; D009508:Nevus, Pigmented / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D016133:Polymerase Chain Reaction / Q000379:methods; D000090063:Proto-Oncogene Mas; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics* / Q000378:metabolism; D012878:Skin Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D013964:Thyroid Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology