Comparison of Direct Sequencing and PNA Clamping Methods to Examine EGFR Mutation in Non-Small Cell Lung Carcinoma

Abstract

Background: Mutations of the epidermal growth factor receptor (EGFR) tyrosine kinase have been reported in non–small cell lung carcinoma (NSCLC) patients with dramatic responses to EGFR tyrosine kinase inhibitors (TKIs). Therefore, it is necessary to establish a rapid and sensitive method to detect mutant EGFR in the NSCLC tissue specimens. The aim of this study is to evaluate the efficacy of peptide nucleic acid (PNA) clamping method compared with direct sequencing (DS) in the formalin-fixed paraffin embedded (FFPE) tissue of NSCLC and to clarify the relationship between the EGFR mutation status and clinical manifestations including survivals and the responsiveness to EGFR TKIs.

Methods: Mutation analysis of EGFR exons 18~21 was performed on 103 cases of NSCLC using the DS and PNA clamping. The results were correlated with clinicopathologic features and response to EGFR TKIs.

Results: Mutations in the EGFR gene were detected in 65 of 103 patients (63%) by DS and in 58 of 103 patients (56%) by PNA clamping method. The two methods showed good concordance rate (78%) in detecting EGFR mutation (Kappa coefficient: 0.672). In 14 cases, mutations were detected only by DS and not by PNA clamping and in 7 cases mutations were detected by PNA clamping method only. Four out of 14 cases that EGFR mutations were detected by DS only harbored undesignated EGFR mutations by PNA clamping method. Regarding tumor response to EGFR TKIs, there were no significant differences between the two detection methods. The objective response rate (OR) was significantly higher in the patients with EGFR mutation than those with wild type EGFR (31% vs. 12%; p=0.028 by DS and p=0.038 by PNA clamping). The median progression-free survival was 19 months in the patients with EGFR mutation and 11 months in those with wild type EGFR by both methods (p<0.001 in DS and p=0.017 in PNA clamping).

Conclusions: DS showed higher rates for the detection of EGFR mutations than PNA clamping (63% vs. 56%), but DS and PNA clamping had no significant differences in the responses to EGFR TKIs. Presence of some discordant cases raises the needs for standardization and validation of these two methods.