Highly sensitive detection of driver mutations from cytological samples and cfDNA in lung cancer.
Bronchoscopy
/ methods
Cell-Free Nucleic Acids
ErbB Receptors
/ genetics
Humans
Lung Neoplasms
/ genetics
Mutation
Neoplasm Staging
Nucleic Acid Amplification Techniques
Peptide Nucleic Acids
/ analysis
Polymerase Chain Reaction
/ methods
Proto-Oncogene Proteins B-raf
/ genetics
Proto-Oncogene Proteins p21(ras)
/ genetics
Sensitivity and Specificity
Sequence Analysis, DNA
EGFR mutation
cell-free DNA
cytological samples
lung cancer
secondary mutation
Journal
Cancer medicine
ISSN: 2045-7634
Titre abrégé: Cancer Med
Pays: United States
ID NLM: 101595310
Informations de publication
Date de publication:
12 2021
12 2021
Historique:
revised:
28
08
2021
received:
27
06
2021
accepted:
29
08
2021
pubmed:
8
10
2021
medline:
23
3
2022
entrez:
7
10
2021
Statut:
ppublish
Résumé
Bronchoscopy is a minimally invasive procedure for establishing the diagnosis of lung cancer. It sometimes fails to obtain tissue samples but readily collects cytological samples. We developed PNA-LNA dual-PCR (PLDP), which amplified mutant sequences by a high-fidelity DNA polymerase in the presence of a peptide nucleic acid (PNA) oligomer having a wild-type sequence. Mutations are detected either by locked nucleic acid (LNA) probes for quick detection of a limited number of mutations, which are EGFR, KRAS, and BRAF mutations in the current study, or by direct sequencing for a comprehensive screening. In a total of 233 lung cancer samples, the results for cytological samples by PLDP were compared with those for tissue samples by cobas® EGFR mutation test (cobas) or by the PNA-LNA PCR clamp method (P-LPC). Moreover, the performance of PLDP using cell-free DNA (cfDNA) was investigated. Peptide nucleic acid-LNA dual-PCR was able to detect each synthesized mutant sequence with high sensitivity. PLDP detected EGFR mutations in 80 out of 149 clinical samples, while the cobas or the P-LPC detected in 66 matched. The correctness of PLDP was confirmed both by clinical response and by the results of sequencing using a next-generation sequencer. PLDP detected mutations from cfDNA in approximately 70% of patients who harbors mutations in the tumor. Peptide nucleic acid-LNA dual-PCR exhibited an excellent performance, even using cytological samples. PLDP is applicable for the investigation of cfDNA. The combination of bronchoscopy and PLDP is attractive and will expand the utility of bronchoscopy in clinical practice.
Sections du résumé
BACKGROUND
Bronchoscopy is a minimally invasive procedure for establishing the diagnosis of lung cancer. It sometimes fails to obtain tissue samples but readily collects cytological samples.
METHODS
We developed PNA-LNA dual-PCR (PLDP), which amplified mutant sequences by a high-fidelity DNA polymerase in the presence of a peptide nucleic acid (PNA) oligomer having a wild-type sequence. Mutations are detected either by locked nucleic acid (LNA) probes for quick detection of a limited number of mutations, which are EGFR, KRAS, and BRAF mutations in the current study, or by direct sequencing for a comprehensive screening. In a total of 233 lung cancer samples, the results for cytological samples by PLDP were compared with those for tissue samples by cobas® EGFR mutation test (cobas) or by the PNA-LNA PCR clamp method (P-LPC). Moreover, the performance of PLDP using cell-free DNA (cfDNA) was investigated.
RESULTS
Peptide nucleic acid-LNA dual-PCR was able to detect each synthesized mutant sequence with high sensitivity. PLDP detected EGFR mutations in 80 out of 149 clinical samples, while the cobas or the P-LPC detected in 66 matched. The correctness of PLDP was confirmed both by clinical response and by the results of sequencing using a next-generation sequencer. PLDP detected mutations from cfDNA in approximately 70% of patients who harbors mutations in the tumor.
CONCLUSIONS
Peptide nucleic acid-LNA dual-PCR exhibited an excellent performance, even using cytological samples. PLDP is applicable for the investigation of cfDNA. The combination of bronchoscopy and PLDP is attractive and will expand the utility of bronchoscopy in clinical practice.
Identifiants
pubmed: 34617674
doi: 10.1002/cam4.4330
pmc: PMC8633228
doi:
Substances chimiques
Cell-Free Nucleic Acids
0
KRAS protein, human
0
Peptide Nucleic Acids
0
EGFR protein, human
EC 2.7.10.1
ErbB Receptors
EC 2.7.10.1
BRAF protein, human
EC 2.7.11.1
Proto-Oncogene Proteins B-raf
EC 2.7.11.1
Proto-Oncogene Proteins p21(ras)
EC 3.6.5.2
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
8595-8603Informations de copyright
© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.
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Cancer Med. 2021 Dec;10(23):8595-8603
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