The Peptide Nucleic Acid-Locked Nucleic Acid Polymerase Chain Reaction Clamp-Based Test for Epidermal Growth Factor Receptor Mutations in Bronchoscopic Cytological Specimens of Non-Small Cell Lung Cancer

Yamada, Noriyuki; Oizumi, Satoshi; Asahina, Hajime; Shinagawa, Naofumi; Kikuchi, Eiki; Kikuchi, Junko; Sakakibara‐Konishi, Jun; Tanaka, Tomoaki; Kobayashi, Kunihiko; Hagiwara, Koichi; Nishimura, Masaharu

doi:10.1159/000338327

Cited by 11 publications

(17 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In our study, the PNA–LNA PCR clamp technique, which inhibits wt gene amplification and simultaneously enhances amplification of the mutated allele, achieved very high sensitivity (1 % of tumour cells for both exons), in accordance with other reports [6, 9, 24]. In an experimental setting, PNA–LNA PCR clamp not only clearly identified mutated alleles intermixed as 1 % of the normal human diploid genome, but also detected one mutant allele in 1,000 diploid human genomes (i.e.…”

Section: Discussionsupporting

confidence: 92%

“…The reliability of PNA–LNA PCR clamp has been also confirmed in clinical settings, with high sensitivity (97 %) and specificity (100 %) demonstrated in variety of cytological specimens (bronchoscopy samples, sputum, pleural and pericardial effusion) in addition to paraffin-embedded tissues [1, 24, 26]. Accordingly, Yamada et al [24], demonstrated that the PNA–LNA PCR clamp method allowed positive diagnosis in 33.6 % of 122 cytological samples from Asian NSCLC patients . Studies by Ikeda et al [26], compared the effectiveness of several highly sensitive PCR methods (ME-PCR, PNA–LNA PCR clamp and PCR invader) to detect EGFR mutations in paraffin-embedded tumour sections, frozen cytology specimens obtained by bronchoscopy (washing and brushing) or from malignant pleural effusions.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

EGFR activating mutations detected by different PCR techniques in Caucasian NSCLC patients with CNS metastases: short report

Wojas‐Krawczyk

Sternschuss²,

Krawczyk

et al. 2013

Clin Exp Metastasis

View full text Add to dashboard Cite

EGFR mutation testing has become an essential determination to decide treatment options for NSCLC. The mutation analysis is often conducted in samples with low percentage of tumour cells from primary tumour biopsies. There is very little evidence that samples from metastatic tissues are suitable for EGFR testing. We had evaluated the frequency of EGFR mutations with three highly sensitive PCR techniques in formalin-fixed, paraffin-embedded samples of 143 NSCLC patients with central nervous system (CNS) metastases. 32 corresponding primary tumours were also examined. We used PCR followed by DNA fragments length analysis (FLA), ASP–PCR and PNA–LNA PCR clamp techniques. We found 9 (6.29 %) EGFR gene mutations in CNS samples: 3 (2.1 %) in exon 19 and 6 (4.2 %) in exon 21. The full concordance between CNS metastases and primary tumour samples was observed. PCR followed by DNA–FLA and PNA–LNA PCR clamp were sensitive enough to detect exon 19 deletions. Two mutations in exon 21 were detected by ASP–PCR only, one L858R substitution was detected only by PNA–LNA PCR clamp. With respect to sensitivity, PCR followed by DNA–FLA achieved a level of detection of at least 10 % of mutated DNA for exon 19 deletion, as for ASP–PCR it was at least 5 % of mutated DNA for L858R substitution. Higher sensitivity of 1 % of mutated DNA was achieved by PNA–LNA PCR clamp technique for both mutations. The use of different methodological techniques authenticates the negative result of molecular tests.

show abstract

Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

EGFR activating mutations detected by different PCR techniques in Caucasian NSCLC patients with CNS metastases: short report

Wojas‐Krawczyk

Sternschuss²,

Krawczyk

et al. 2013

Clin Exp Metastasis

View full text Add to dashboard Cite

show abstract

“…). In total, 4999 samples were analyzed for EGFR mutation from all articles (Table ), and information on the number of enrolled patients was available for 19 studies . In 1 of these studies, information was available for all enrolled patients, including those who had histologic samples .…”

Section: Resultsmentioning

confidence: 99%

“…In 3 studies, information on the number of patients was not available. Eleven studies included only cytologic samples, and 11 also reported the results from surgical/core‐needle biopsy specimens . The oldest study with more than 100 specimens was published in 2007, and the most recent series retrieved were published online in 2014 .…”

Section: Resultsmentioning

confidence: 99%

“…Other high‐sensitivity, PCR‐based methods, such as high‐resolution melting analysis (HRMA), ARMS, peptide nucleic acid‐locked nucleic acid, and coamplification at lower denaturation temperature (COLD)‐PCR, were the main methods used in 4 studies, two studies, two studies, and 1 study, respectively. Three of the studies that used HRMA had confirmation of mutated or abnormal samples performed by Sanger sequencing, and the other study combined HRMA with mutant‐enriched PCR and sequencing .…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Preanalytic parameters in epidermal growth factor receptor mutation testing for non–small cell lung carcinoma: A review of cytologic series

Santos

Saieg²

2015

Cancer Cytopathology

View full text Add to dashboard Cite

The results from molecular assays can be affected significantly by the preanalytic condition of cytologic samples. The authors review current knowledge on the use of cytologic samples for epidermal growth factor receptor (EGFR) mutation testing in non–small cell lung cancer with a focus on preanalytic parameters. A systematic electronic search of the MEDLINE database was performed to identify original articles that reported the use of cytologic samples for EGFR molecular analysis and included a minimum of 100 samples. The information collected included author(s), journal, and year of publication; number of patients and samples; sampling method; type of preparation; type of fixative; staining techniques; mutation analysis techniques; tumor cellularity; the percentage of tumor cells; data on DNA quantity, quality, and concentration; failed assays; and the mutation rate. EGFR mutation analysis was conducted on 4999 cytologic samples from 22 studies that fulfilled the inclusion criteria. Fine‐needle aspirates and pleural effusions were the most common types of specimens used. DNA was mainly extracted from cell blocks and smears, and the most commonly reported fixatives included formalin, ethanol, and CytoLyt. Cellularity assessments and DNA yields were available from 5 studies each. The average success rate for the assays that used cytologic specimens was 95.87% (range, 85.2%‐100%). The mutation rate ranged from 6% to 50.46%, and a higher mutation detection rate and lower numbers of insufficient cases were reported for pleural effusions and lymph node samples from endobronchial ultrasound‐guided transbronchial needle aspiration compared with histologic specimens. Low cellularity and a low percentage of tumor cells were associated with higher test failure rates. Future guidelines should consider the current data for specific recommendations regarding cytologic samples. Cancer (Cancer Cytopathol) 2015;123:633–643. © 2015 American Cancer Society.

show abstract

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

et al. 2021

Self Cite

View full text Add to dashboard Cite

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.

show abstract

The Peptide Nucleic Acid-Locked Nucleic Acid Polymerase Chain Reaction Clamp-Based Test for Epidermal Growth Factor Receptor Mutations in Bronchoscopic Cytological Specimens of Non-Small Cell Lung Cancer

Cited by 11 publications

References 50 publications

EGFR activating mutations detected by different PCR techniques in Caucasian NSCLC patients with CNS metastases: short report

EGFR activating mutations detected by different PCR techniques in Caucasian NSCLC patients with CNS metastases: short report

Preanalytic parameters in epidermal growth factor receptor mutation testing for non–small cell lung carcinoma: A review of cytologic series

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

Contact Info

Product

Resources

About