title: KRAS Mutation Detection in Non-small Cell Lung Cancer Using a Peptide Nucleic Acid-Mediated Polymerase Chain Reaction Clamping Method and Comparative Validation with Next-Generation Sequencing.
abstract: BACKGROUND
  KRAS is one of commonly mutated genetic "drivers" in non-small cell lung cancers (NSCLCs). Recent studies indicate that patients with KRAS-mutated tumors do not benefit from adjuvant chemotherapy, so there is now a focus on targeting KRAS-mutated NSCLCs. A feasible mutation detection method is required in order to accurately test for KRAS status.
  
  
  METHODS
  We compared direct Sanger sequencing and the peptide nucleic acid (PNA)-mediated polymerase chain reaction (PCR) clamping method in 134 NSCLCs and explored associations with clinicopathological factors. Next-generation sequencing (NGS) was used to validate the results of discordant cases. To increase the resolution of low-level somatic mutant molecules, PNA-mediated PCR clamping was used for mutant enrichment prior to NGS.
  
  
  RESULTS
  Twenty-one (15.7%) cases were found to have the KRAS mutations using direct sequencing, with two additional cases by the PNA-mediated PCR clamping method. The frequencies of KRAS mutant alleles were 2% and 4%, respectively, using conventional NGS, increasing up to 90% and 89%, using mutant-enriched NGS. The KRAS mutation occurs more frequently in the tumors of smokers (p=.012) and in stage IV tumors (p=.032).
  
  
  CONCLUSIONS
  Direct sequencing can accurately detect mutations, but, it is not always possible to obtain a tumor sample with sufficient volume. The PNA-mediated PCR clamping can rapidly provide results with sufficient sensitivity.
authors.affiliation: Department of Pathology, Samsung Medical Center, Sungkyunkwan University College of Medicine, Seoul, Korea.
authors.forename: Boram
authors.initials: B
authors.lastname: Lee
authors.affiliation: Department of Pathology, Samsung Medical Center, Sungkyunkwan University College of Medicine, Seoul, Korea.
authors.forename: Boin
authors.initials: B
authors.lastname: Lee
authors.affiliation: Department of Pathology, Samsung Medical Center, Sungkyunkwan University College of Medicine, Seoul, Korea.
authors.forename: Gangmin
authors.initials: G
authors.lastname: Han
authors.affiliation: Department of Pathology, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang, Korea.
authors.forename: Mi Jung
authors.initials: MJ
authors.lastname: Kwon
authors.affiliation: Department of Pathology, Samsung Medical Center, Sungkyunkwan University College of Medicine, Seoul, Korea.
authors.forename: Joungho
authors.initials: J
authors.lastname: Han
authors.affiliation: Department of Pathology, Samsung Medical Center, Sungkyunkwan University College of Medicine, Seoul, Korea.
authors.forename: Yoon-La
authors.initials: YL
authors.lastname: Choi
keywords: KRAS; Lung neoplasms; Mutation; Peptide nucleic acids