title: High-resolution melting analysis as a sensitive prescreening diagnostic tool to detect KRAS , BRAF , PIK3CA , and AKT1 mutations in formalin-fixed, paraffin-embedded tissues.
abstract: BACKGROUND
  As the availability of targeted therapies for several tumor types increases, the need for rapid and sensitive mutation screening is growing. KRAS mutations constitutively activate the RAS/RAF/mitogen-activated protein kinase (MAPK) pathway and therefore play an important role in anti-epidermal growth factor receptor therapy for patients with colorectal cancers. Mutationally activated PIK3CA and AKT1 genes are promising therapeutic targets in breast cancer. In 60% to 70% of malignant melanomas, a mutation in BRAF can be found. Thus, the blocking of the oncogenic signaling induced by this mutation is now used as treatment approach.
  
  
  OBJECTIVE
  To establish high-resolution melting assays for routinely used predictive analyses of KRAS , AKT1 , PIK3CA , and BRAF mutations.
  
  
  METHODS
  High-resolution melting assays were developed by using specifically designed primers and genomic DNA isolated either from cell lines or formalin-fixed paraffin-embedded tissues, oligonucleotides, or plasmids. Melting curve analyses were performed on the LightCyler platform and mutation analyses were additionally confirmed by Sanger sequencing.
  
  
  RESULTS
  We developed high-resolution melting assays by using genomic DNA containing the desired mutation, which enabled us to detect percentages of mutated DNA (3.1% to 12.5%) mixed in a wild-type background. Assays were evaluated by hybridization probes and/or Sanger sequencing to exclude pseudogene amplification. The high-resolution melting assays were validated with genomic DNA from different tumor entities. The concordance between Sanger sequencing and high-resolution melting was 99% for KRAS exon 2 and PIK3CA exon 20 and 100% for the remaining assays.
  
  
  CONCLUSIONS
  High-resolution melting provides a valid and powerful tool for detecting genomic mutations efficiently.
authors.affiliation: Institute of Pathology, University of Bonn, Germany.
authors.forename: Jasmin Teresa
authors.initials: JT
authors.lastname: Ney
authors.forename: Stefanie
authors.initials: S
authors.lastname: Froehner
authors.forename: Angelika
authors.initials: A
authors.lastname: Roesler
authors.forename: Reinhard
authors.initials: R
authors.lastname: Buettner
authors.forename: Sabine
authors.initials: S
authors.lastname: Merkelbach-Bruse
mesh_terms: D018938:Caco-2 Cells; D045744:Cell Line, Tumor; D058534:Class I Phosphatidylinositol 3-Kinases; D004252:DNA Mutational Analysis / Q000379:methods; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D005091:Exons / Q000235:genetics; D005557:Formaldehyde; D045325:HCT116 Cells; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D016612:Paraffin Embedding / Q000379:methods; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics*; D016133:Polymerase Chain Reaction; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016707:Tissue Fixation / Q000379:methods; D018631:ras Proteins / Q000235:genetics*
chemical_list: D004273:DNA, Neoplasm; D005557:Formaldehyde; D019869:Phosphatidylinositol 3-Kinases; D058534:Class I Phosphatidylinositol 3-Kinases; C484760:PIK3CA protein, human; C494918:AKT1 protein, human; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D051057:Proto-Oncogene Proteins c-akt; D018631:ras Proteins