Detection of EGFR gene mutation in lung cancer by mutant-enriched polymerase chain reaction assay.
OBJECTIVE
Mutations in the epidermal growth factor receptor (EGFR) gene have been reported to be present in non-small cell lung cancer (NSCLC) and related to the responsiveness of tumors to EGFR tyrosine kinase inhibitors, suggesting its usefulness as a biomarker. Because clinical samples contain tumor and normal cells or genes, a highly sensitive assay for detecting mutation is critical for clinical applications.


METHODS
The mutant-enriched PCR is a rapid and sensitive assay with selective restriction enzyme digestion. We developed the mutant-enriched PCR assay targeting exons 19 and 21 of EGFR and applied the developed assay to detect mutations in 108 cases of surgically resected specimens of NSCLCs, 18 samples of computed tomography (CT)-guided needle lung biopsies, and 20 samples of pleural fluid. In addition, results were then compared with those from direct sequencing and a nonenriched PCR assay.


RESULTS
The mutant-enriched PCR that was proved to enrich one mutant of 2 x 10(3) normal genes detected mutations in 37 cases of 108 resected tumors, seven samples of CT-guided lung biopsies, and seven samples of pleural fluid. Among mutant cases, four resected tumors, two CT-guided lung biopsies, and two pleural fluid were identified as additional mutant cases by the mutant-enriched PCR, which were considered normal based on nonenriched assays.


CONCLUSIONS
Our results indicate that EGFR mutations are readily detectable by mutant-enriched PCR in various clinical samples. Thus, mutant-enriched PCR may provide a valuable method of potentially detecting a small fraction of mutant genes in heterogeneous specimens, indicating its possible use in clinical application for NSCLC.
Asano
Hiroaki
H
Department of Cancer and Thoracic Surgery, Graduate School of Medicine and Dentistry, Okayama University, Okayama, Japan.
Toyooka
Shinichi
S
Tokumo
Masaki
M
Ichimura
Kouichi
K
Aoe
Keisuke
K
Ito
Sachio
S
Tsukuda
Kazunori
K
Ouchida
Mamoru
M
Aoe
Motoi
M
Katayama
Hideki
H
Hiraki
Akio
A
Sugi
Kazuro
K
Kiura
Katsuyuki
K
Date
Hiroshi
H
Shimizu
Nobuyoshi
N
D001706:Biopsy; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000473:pathology; D004252:DNA Mutational Analysis; D017931:DNA Primers; D005260:Female; D018773:Genes, erbB-1 / Q000235:genetics*; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D009154:Mutation; D010996:Pleural Effusion; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers