Establishment of a novel mutant-enriched liquid chip technology for detecting EGFR mutations in plasma of patients with non-small cell lung cancer
10.3760/cma.j.issn.1009-9158.2012.11.006
- VernacularTitle:非小细胞肺癌患者外周血EGFR基因突变富集液相芯片检测方法的建立
- Author:
Lixia ZHENG
;
Chen HE
;
Ming LIU
;
Beixian ZHOU
;
Jun XU
- Publication Type:Journal Article
- Keywords:
Carcinoma,non-small-cell lung;
Receptor,epidermal growth factor;
Mutation;
Microchip analytical procedures
- From:
Chinese Journal of Laboratory Medicine
2012;(11):986-992
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a sensitive,specific,simple and high-throughput method for detection of the epidermal growth factor receptor (EGFR) mutation in the plasma samples of patients with non-small cell lung cancer (NSCLS) by the use of mutant-enriched liquid chip (MEL) assay.Methods The specific probes for the EGFR exon19 E746-750 deletion,exon 21 L858R mutation and wild-type sequence were designed and coupled to the microspheres coding with different fluorescent dye.The probe coupling efficiency was verified by crossing hybridization test with biotin-labeled reverse sequence.A blood-based MEL approach which integrates a sensitive mutant-enriched PCR and quantitative high throughput liquid chip assay for assessment of EGFR mutations was developed.The sensitivity and specificity of MEL was further evaluated using the mixture with different copy numbers of mutant and wild-type plasmids as template.The mutations of exon 19 and 21 of EGFR gene in plasma samples from 201 patients with stage ⅢB or Ⅳ NSCLC who enrolled in the First Affiliated Hospital of Guangzhou Medical College from September 2008 to April 2010 were analyzed by both the MEL and the mutant-enriched PCR assay.The result comparison was made between direct sequencing and MEL in 50 cases whose EGFR gene type had been tested by MEL and mutantenriched PCR.The correlation of EGFR gene mutation and the response to the Geftinib treatment was analyzed in 16 patients with lung adenocarcinoma as well.Results The probes were successfully coupled to the microspheres encoding with different fluorescent dye,and could be specifically recognized by the corresponding target sequence.The MEL was capable of detecting as few as 10 copies of EGFR mutants (sensitivity was 0.1％).Among the enrolled 201 cases of advanced NSCLC,the detection rate of the EGFR exon19 E746-750 del and exon 21 L858R was 55.7％ (112/201) by MEL assay.Conpared with mutantenriched PCR[58.2％ (117/201)],the coincidence rate was 97.5％ (196/201).There was no statistically significant difference between the results of mutant-enriched PCR and MEL (x2 =3.20,P ＞ 0.05).The mutations detection rate was 22.0％ (11/50) by directing sequencing,which was significantly lower than by MEL[50.0％ (25/50),x2 =12.07,P ＜ 0.05].Among the 16 patients treated with Gefitinib,9 cases who had EGFR mutation showed a higher response rate(P =0.041)and prolonged progression-free survival (x2 =6.76,P =0.009) after the treatment compared to those 7 who without EGFR mutation.Conclusions A new method of MEL with accuracy,specificity,fast and high-throughput is established for the detection of EGFR 19 E746-750 deletion and exon 21 L858R mutations in plasma from advanced NSCLC patients.It has the ability to provide the most direct and valuable guidance for clinicians to make decision on EGFR tyrosine kinase inhibitors therapy in the advanced NSCLC paticnts.
