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 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.

EGFR signaling pathway as therapeutic target in human cancers

“…World-wide screenings reported an intrinsic T790M frequency ranging ~3-80% [248][249][250][251][252][253], that was attributed to potential sequencing artifacts caused by tissue processing [254]. In a paired experiment, comparing mass spectrometry with standard direct sequencing, the frequency of T790M shifted from 31.5% to 2.7% [255], implying detection methods must be carefully standardized.…”

Overview and update on molecular testing in non‐small cell lung carcinoma utilizing endobronchial ultrasound‐guided transbronchial needle aspiration (EBUS‐TBNA) samples

“…In respect of the use of cytological material, several authors have detected BRAF mutations in cytological samples by PCR-based methods. 64,65 Kaya et al observed an adequacy rate of 100% for BRAF testing in EBUS-TBNA specimens. 66 However, other authors have reported lower adequacy rates for BRAF analysis.…”

“…However, these assays do not detect rare mutations, which may influence treatment response. In respect of the use of cytological material, several authors have detected BRAF mutations in cytological samples by PCR‐based methods 64,65 . Kaya et al observed an adequacy rate of 100% for BRAF testing in EBUS‐TBNA specimens 66 .…”