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Updated: Apr 29, 2026
Detection of Targetable Alterations in Non-small Cell Lung Cancer using Next-generation Sequencing
Published on: October 10, 2025
KRAS Mutation Detection in Non-small Cell Lung Cancer Using a Peptide Nucleic Acid-Mediated Polymerase Chain Reaction
Boram Lee1, Boin Lee1, Gangmin Han1
1Department of Pathology, Samsung Medical Center, Sungkyunkwan University College of Medicine, Seoul, Korea.
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.
Insights
Peptide nucleic acid (PNA)-mediated PCR clamping offers a sensitive method for detecting KRAS mutations in non-small cell lung cancer (NSCLC), outperforming direct sequencing for low-level mutations.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- KRAS mutations are key drivers in non-small cell lung cancers (NSCLCs).
- Patients with KRAS-mutated NSCLCs show limited benefit from adjuvant chemotherapy.
- Targeting KRAS mutations in NSCLC requires accurate detection methods.
Purpose of the Study:
- To compare the efficacy of direct Sanger sequencing and PNA-mediated PCR clamping for KRAS mutation detection in NSCLC.
- To explore associations between KRAS mutations and clinicopathological factors.
- To enhance the sensitivity of next-generation sequencing (NGS) for low-level KRAS mutations.
Main Methods:
- Direct Sanger sequencing and PNA-mediated PCR clamping were performed on 134 NSCLC samples.
- Next-generation sequencing (NGS) was used for validation and mutant enrichment.
- PNA-mediated PCR clamping enriched samples prior to NGS to improve resolution of low-level somatic mutations.
Main Results:
- KRAS mutations were detected in 15.7% of cases by direct sequencing, with PNA-mediated PCR clamping identifying two additional cases.
- Conventional NGS showed KRAS mutant allele frequencies of 2% and 4%, while mutant-enriched NGS increased detection to 90% and 89%.
- KRAS mutations were more frequent in smokers (p=.012) and stage IV tumors (p=.032).
Conclusions:
- Direct sequencing is accurate but may require larger tumor samples.
- PNA-mediated PCR clamping provides rapid and sensitive detection of KRAS mutations, even with limited sample volume.
- PNA-mediated PCR clamping enhances NGS sensitivity for detecting low-frequency KRAS mutations in NSCLC.
