Peptide Nucleic Acid Clamping Versus Direct Sequencing for the Detection of EGFR Gene Mutation in Patients with Non-small Cell Lung Cancer
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Purpose Direct sequencing (DS) is the standard method for detection of epidermal growth factorreceptor (EGFR) gene mutation in non-small cell lung cancer (NSCLC); however, low detectionsensitivity is a problem. The aim of this study is to demonstrate higher detection rateof EGFR gene mutation with peptide nucleic acid (PNA) clamping compared with DS. Materials and MethodsThis is a single arm, prospective study for patients with stage IIIB/IV or relapsed NSCLC. Using tumor DNA from 138 patients, both DS and PNA clamping for EGFR gene in exon 18,19, 20, and 21 were performed. Discrepant results between the two methods were verifiedusing Cobas and a mutant enrichment based next generation sequencing (NGS). Patientswith activating mutations were treated with EGFR tyrosine kinase inhibitor (EGFR-TKI, gefitinib,or erlotinib) as first line treatment. ResultsOf 138 paired test sets, 24 (17.4%) and 45 (32.6%) cases with activating mutations weredetected by DS and PNA clamping, respectively. The difference of detection rate betweenthe two methods was 15.2% (95% confidence interval, 8.7% to 17.8%; p < 0.001). Betweenthe two methods, 25 cases showed discrepant results (n=23, PNA+/DS–; n=2, PNA–/DS+). Mutations were confirmed by Cobas or NGS in 22 of 23 PNA+/DS– cases. The responserates to EGFR-TKI were 72.2% in the PNA+/DS+ group and 85.0% in the PNA+/DS– group. ConclusionPNA clamping showed a significantly higher detection rate of EGFR gene mutation comparedwith DS. Higher sensitivity of PNA clamping was not compromised by the loss of predictivepower of response to EGFR-TKI.
Abstract
Purpose Direct sequencing (DS) is the standard method for detection of epidermal growth factorreceptor (EGFR) gene mutation in non-small cell lung cancer (NSCLC); however, low detectionsensitivity is a problem. The aim of this study is to demonstrate higher detection rateof EGFR gene mutation with peptide nucleic acid (PNA) clamping compared with DS. Materials and MethodsThis is a single arm, prospective study for patients with stage IIIB/IV or relapsed NSCLC. Using tumor DNA from 138 patients, both DS and PNA clamping for EGFR gene in exon 18,19, 20, and 21 were performed. Discrepant results between the two methods were verifiedusing Cobas and a mutant enrichment based next generation sequencing (NGS). Patientswith activating mutations were treated with EGFR tyrosine kinase inhibitor (EGFR-TKI, gefitinib,or erlotinib) as first line treatment. ResultsOf 138 paired test sets, 24 (17.4%) and 45 (32.6%) cases with activating mutations weredetected by DS and PNA clamping, respectively. The difference of detection rate betweenthe two methods was 15.2% (95% confidence interval, 8.7% to 17.8%; p < 0.001). Betweenthe two methods, 25 cases showed discrepant results (n=23, PNA+/DS–; n=2, PNA–/DS+). Mutations were confirmed by Cobas or NGS in 22 of 23 PNA+/DS– cases. The responserates to EGFR-TKI were 72.2% in the PNA+/DS+ group and 85.0% in the PNA+/DS– group. ConclusionPNA clamping showed a significantly higher detection rate of EGFR gene mutation comparedwith DS. Higher sensitivity of PNA clamping was not compromised by the loss of predictivepower of response to EGFR-TKI.
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