Highly sensitive detection of driver mutations from cytological samples and cfDNA in lung cancer

Abstract 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 DN...

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Autores principales:	Kazutaka Fujita, Masayuki Nakayama, Masafumi Sata, Yoshiaki Nagai, Shu Hisata, Naoko Mato, Takuji Suzuki, Masashi Bando, Nobuyuki Hizawa, Koichi Hagiwara
Formato:	article
Lenguaje:	EN
Publicado:	Wiley 2021
Materias:	cell‐free DNA cytological samples EGFR mutation lung cancer secondary mutation Neoplasms. Tumors. Oncology. Including cancer and carcinogens RC254-282
Acceso en línea:	https://doaj.org/article/d401f2ddbd7f445f8908f840d1ed0e76
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spelling	oai:doaj.org-article:d401f2ddbd7f445f8908f840d1ed0e762021-12-01T04:49:16ZHighly sensitive detection of driver mutations from cytological samples and cfDNA in lung cancer2045-763410.1002/cam4.4330https://doaj.org/article/d401f2ddbd7f445f8908f840d1ed0e762021-12-01T00:00:00Zhttps://doi.org/10.1002/cam4.4330https://doaj.org/toc/2045-7634Abstract 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.Kazutaka FujitaMasayuki NakayamaMasafumi SataYoshiaki NagaiShu HisataNaoko MatoTakuji SuzukiMasashi BandoNobuyuki HizawaKoichi HagiwaraWileyarticlecell‐free DNAcytological samplesEGFR mutationlung cancersecondary mutationNeoplasms. Tumors. Oncology. Including cancer and carcinogensRC254-282ENCancer Medicine, Vol 10, Iss 23, Pp 8595-8603 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	cell‐free DNA cytological samples EGFR mutation lung cancer secondary mutation Neoplasms. Tumors. Oncology. Including cancer and carcinogens RC254-282
spellingShingle	cell‐free DNA cytological samples EGFR mutation lung cancer secondary mutation Neoplasms. Tumors. Oncology. Including cancer and carcinogens RC254-282 Kazutaka Fujita Masayuki Nakayama Masafumi Sata Yoshiaki Nagai Shu Hisata Naoko Mato Takuji Suzuki Masashi Bando Nobuyuki Hizawa Koichi Hagiwara Highly sensitive detection of driver mutations from cytological samples and cfDNA in lung cancer
description	Abstract 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.
format	article
author	Kazutaka Fujita Masayuki Nakayama Masafumi Sata Yoshiaki Nagai Shu Hisata Naoko Mato Takuji Suzuki Masashi Bando Nobuyuki Hizawa Koichi Hagiwara
author_facet	Kazutaka Fujita Masayuki Nakayama Masafumi Sata Yoshiaki Nagai Shu Hisata Naoko Mato Takuji Suzuki Masashi Bando Nobuyuki Hizawa Koichi Hagiwara
author_sort	Kazutaka Fujita
title	Highly sensitive detection of driver mutations from cytological samples and cfDNA in lung cancer
title_short	Highly sensitive detection of driver mutations from cytological samples and cfDNA in lung cancer
title_full	Highly sensitive detection of driver mutations from cytological samples and cfDNA in lung cancer
title_fullStr	Highly sensitive detection of driver mutations from cytological samples and cfDNA in lung cancer
title_full_unstemmed	Highly sensitive detection of driver mutations from cytological samples and cfDNA in lung cancer
title_sort	highly sensitive detection of driver mutations from cytological samples and cfdna in lung cancer
publisher	Wiley
publishDate	2021
url	https://doaj.org/article/d401f2ddbd7f445f8908f840d1ed0e76
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Highly sensitive detection of driver mutations from cytological samples and cfDNA in lung cancer

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