title: High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer.
abstract: BACKGROUND
  The development of targeted therapies has created a pressing clinical need for the rapid and robust molecular characterisation of cancers. We describe here the application of high-resolution melting analysis (HRM) to screen for KRAS mutations in clinical cancer samples. In non-small cell lung cancer, KRAS mutations have been shown to identify a group of patients that do not respond to EGFR targeted therapies and the identification of these mutations is thus clinically important.
  
  
  METHODS
  We developed a high-resolution melting (HRM) assay to detect somatic mutations in exon 2, notably codons 12 and 13 of the KRAS gene using the intercalating dye SYTO 9. We tested 3 different cell lines with known KRAS mutations and then examined the sensitivity of mutation detection with the cell lines using 189 bp and 92 bp amplicons spanning codons 12 and 13. We then screened for KRAS mutations in 30 non-small cell lung cancer biopsies that had been previously sequenced for mutations in EGFR exons 18-21.
  
  
  RESULTS
  Known KRAS mutations in cell lines (A549, HCT116 and RPMI8226) were readily detectable using HRM. The shorter 92 bp amplicon was more sensitive in detecting mutations than the 189 bp amplicon and was able to reliably detect as little as 5-6% of each cell line DNA diluted in normal DNA. Nine of the 30 non-small cell lung cancer biopsies had KRAS mutations detected by HRM analysis. The results were confirmed by standard sequencing. Mutations in KRAS and EGFR were mutually exclusive.
  
  
  CONCLUSIONS
  HRM is a sensitive in-tube methodology to screen for mutations in clinical samples. HRM will enable high-throughput screening of gene mutations to allow appropriate therapeutic choices for patients and accelerate research aimed at identifying novel mutations in human cancer.
authors.affiliation: Molecular Pathology Research Laboratory, Peter MacCallum Cancer Centre, Locked Bag 1, A'Beckett St, Melbourne, Victoria 8006, Australia. michael.krypuy@petermac.org <michael.krypuy@petermac.org>
authors.forename: Michael
authors.initials: M
authors.lastname: Krypuy
authors.forename: Genni M
authors.initials: GM
authors.lastname: Newnham
authors.forename: David M
authors.initials: DM
authors.lastname: Thomas
authors.forename: Matthew
authors.initials: M
authors.lastname: Conron
authors.forename: Alexander
authors.initials: A
authors.lastname: Dobrovic
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics; D018287:Carcinoma, Large Cell / Q000235:genetics; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics*; D003062:Codon / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D009154:Mutation; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins
chemical_list: D003062:Codon; D004273:DNA, Neoplasm; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins