title: Validation of a Multiplex Allele-Specific Polymerase Chain Reaction Assay for Detection of KRAS Gene Mutations in Formalin-Fixed, Paraffin-Embedded Tissues from Colorectal Cancer Patients.
abstract: BACKGROUND
  Patients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC). The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR) assay to detect KRAS mutations.
  
  
  METHODS
  We developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D). We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay.
  
  
  RESULTS
  Overall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%), G12D (25.83%) and G12V (12.50%) in codon 12.
  
  
  CONCLUSIONS
  The MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues.
authors.affiliation: Department of Transfusion Medicine and Clinical Microbiology, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand.
authors.forename: Sirirat
authors.initials: S
authors.lastname: Seekhuntod
authors.affiliation: Department of Medical Services, Institute of Pathology, Ministry of Public Health, Bangkok, Thailand.
authors.forename: Paninee
authors.initials: P
authors.lastname: Thavarungkul
authors.affiliation: Department of Transfusion Medicine and Clinical Microbiology, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand.
authors.forename: Nuntaree
authors.initials: N
authors.lastname: Chaichanawongsaroj
mesh_terms: D000368:Aged; D000483:Alleles; D000595:Amino Acid Sequence; D003062:Codon; D015179:Colorectal Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005260:Female; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D008875:Middle Aged; D008969:Molecular Sequence Data; D060885:Multiplex Polymerase Chain Reaction / Q000592:standards*; D016612:Paraffin Embedding; D017354:Point Mutation / Q000235:genetics*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D018631:ras Proteins / Q000235:genetics*
chemical_list: D003062:Codon; D004273:DNA, Neoplasm; D005557:Formaldehyde; D018631:ras Proteins
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