[Detection of epidermal growth factor receptor mutations in non-small cell lung cancer by real-time PCR using TaqMan-MGB probes].
OBJECTIVE
To investigate mutations of EGFR TK gene in non-small cell lung cancer (NSCLC) and the diagnostic value of the mutations assayed by real-time PCR using TaqMan-MGB probes.


METHODS
Tyrosine kinase genes of EGFR (exons 18, 19 and 21) were amplified by PCR technology, and sequenced and analyzed by Chromas software in 80 NSCLC patients. Based on the results of sequencing, TaqMan-MGB probes were designed and used to detect the mutations of EGFR by real-time PCR. The results of detected mutations were compared between real-time PCR and direct sequencing. The sensitivity and specificity of real-time PCR using TaqMan-MGB probes were analyzed by adding different number of PC-9 cells (exon 19 deleted EGFR) into A549 cells (Wild-type EGFR).


RESULTS
Somatic mutations were identified in the tyrosine kinase domain of the EGFR gene in 21 of 80 NSCLC patients with an incidence rate of 26.3%. In-frame deletions of exon 19 occurred in 13 patients and point mutation occurred in codon 858 (exon 21) in 8 patients. Real-time PCR with the TaqMan MGB probes could detect all the mutations of EGFR found by sequencing. The sensitivity and specificity of the detection of EGFR mutations were both 100%. Real-time PCR with TagMan MGB probes could detect EGFR mutation in as rare as 50 EGFR mutant cells and in a proportion of 10% of mutant cells in a cell population. The mutations were significantly higher in the adenocarcinoma than in non-adenocarcinoma (16/38 vs. 5/42, chi2 = 9.702, P <0.01), in the female patients than in the male patients (14/29 vs. 7/51, chi2 = 11.4, P <0.01) and in non-smokers than in smokers (16/40 vs. 5/40, chi2 = 7.812, P < 0.01). The mutations were not related to patients'age, TNM staging, etc.


CONCLUSIONS
Somatic mutations of EGFR gene develop in NSCLC and are more common in female, non-smoker and adenocarcinoma patients. Real-time PCR using TaqMan-MGB, which can be used to detect the EGFR gene mutations, is easy to operate and deserves widespread application.
Zhou
Cai-Cun
CC
Department of Oncology , Shanghai Pulmonary Hospital, Tongii University, Shanghai 200433, China. caicunzhou@yahoo.com.cn
Zhou
Song-Wen
SW
Pan
Hong
H
Su
Bo
B
Gao
Zhi-Qiang
ZQ
D000230:Adenocarcinoma / Q000235:genetics / Q000473:pathology; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D016009:Chi-Square Distribution; D003062:Codon; D004252:DNA Mutational Analysis; D015342:DNA Probes / Q000235:genetics; D066246:ErbB Receptors / Q000235:genetics*; D005091:Exons; D005260:Female; D017353:Gene Deletion; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009367:Neoplasm Staging; D016133:Polymerase Chain Reaction / Q000379:methods*; D012737:Sex Factors; D012907:Smoking
D003062:Codon; D015342:DNA Probes; D066246:ErbB Receptors