title: Multiplex detection of KRAS mutations by a matrix-assisted laser desorption/ionization-time of flight mass spectrometry assay.
abstract: OBJECTIVE
  Mutations of the Kirsten rat sarcoma viral oncogene (KRAS) gene are known to be important in the pathogenesis of a variety of cancers. Patients with mutant KRAS tumors do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Testing for KRAS mutations is now being recommended as a tailored therapeutic strategy prior to anti-EGFR treatment; however, the low sensitivity of direct sequencing frequently leads to failure of detection of KRAS mutations in clinical samples.
  
  
  METHODS
  We developed restriction fragment mass polymorphism (RFMP) assays, to detect KRAS mutations in codons 12, 13, and 61. We performed RFMP analysis for KRAS on DNA isolated from eight different KRAS mutant cell lines and 34 formalin-fixed paraffin-embedded (FFPE) lung cancer tissues.
  
  
  RESULTS
  Overall, the RFMP assay was in good concordance with direct sequencing analysis in the detection of KRAS mutations. By using dilutions of KRAS mutant DNA in wild type DNA from mutation cell lines with a known KRAS status, we confirmed that the RFMP assay has a higher analytical sensitivity, requiring only 3% of cells in a sample to have mutant alleles, compared to direct sequencing, which had a detection threshold of ~25%. In the FFPE samples, RFMP successfully detected KRAS genotypes in codons 12, 13, and 61.
  
  
  CONCLUSIONS
  The RFMP might be efficiently applicable for the detection of KRAS mutations in a clinical setting, particularly for tumor samples containing abundant non-neoplastic cells.
authors.affiliation: Research and Development Center, GeneMatrix Inc., Seongnam, South Korea.
authors.forename: Hee Sook
authors.initials: HS
authors.lastname: Choi
authors.affiliation: Research and Development Center, GeneMatrix Inc., Seongnam, South Korea.
authors.forename: Kyung Tae
authors.initials: KT
authors.lastname: Min
authors.affiliation: Research and Development Center, GeneMatrix Inc., Seongnam, South Korea.
authors.forename: Yoon Seok
authors.initials: YS
authors.lastname: Cha
authors.affiliation: Research and Development Center, GeneMatrix Inc., Seongnam, South Korea. Electronic address: sunphong@genematrix.net.
authors.forename: Sun Pyo
authors.initials: SP
authors.lastname: Hong
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D017931:DNA Primers; D011905:Genes, ras; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction; D009154:Mutation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D017931:DNA Primers
keywords: EGFR; KRAS mutation; KRAS mutation testing; RFMP