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.
- Author:
Boram LEE
1
;
Boin LEE
;
Gangmin HAN
;
Mi Jung KWON
;
Joungho HAN
;
Yoon La CHOI
Author Information
1. Department of Pathology, Samsung Medical Center, Sungkyunkwan University College of Medicine, Seoul, Korea. ylachoi@skku.edu
- Publication Type:Original Article
- Keywords:
Lung neoplasms;
KRAS;
Mutation;
Peptide nucleic acids
- MeSH:
Alleles;
Carcinoma, Non-Small-Cell Lung*;
Chemotherapy, Adjuvant;
Constriction*;
Humans;
Lung Neoplasms;
Peptide Nucleic Acids;
Polymerase Chain Reaction*
- From:Korean Journal of Pathology
2014;48(2):100-107
- CountryRepublic of Korea
- Language:English
-
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.
