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

Establishment of a novel mutant-enriched liquid chip technology for detecting EGFR mutations in plasma of patients with non-small cell lung cancer

目的建立灵敏、特异、简便易行、高通量血浆检测非小细胞肺癌（NSCLC）表皮生长因子受体（EGFR）基因突变热点的突变富集液相芯片法（MEL）。方法设计EGFR外显子19E746-750缺失和外显子21L858R突变及野生型的特异探针，并偶联到不同荧光编码微球上，用生物素标记的探针反向序列交叉试验验证探针偶联效果后，与突变富集PCR产物互补配对杂交，然后经液相芯片仪检测分析，建立血浆检测EGFR突变的MEL技术。将不同拷贝数突变型与野生型质粒混合做模板，检测MEL的灵敏度与特异性。收集2008年9月至2010年4月在广州医学院第一附属医院住院的201例11IB或Ⅳ期NSCLC患者血浆标本，用突变富集PCR和MEL平行检测EGFR外显子19、21突变，同时筛选经2种方法鉴定过的50例标本的PCR产物，经直接测序验证MEL的灵敏度与特异性。并初步分析16例肺腺癌患者血浆EGFR基因突变与吉非替尼治疗疗效的关系。结果探针成功偶联到不同荧光编码的微球上，且可特异识别相应靶序列，MEL可检出低至10个拷贝的EGFR突变（灵敏度0．1％）。201例NSCLC患者中，MEL共检出EGFR外显子19E746-750缺失和外显子21L858R突变112例（55．7％），与突变富集PCR检出率[58．2％（117／201）]相比，差异无统计学意义（X2=3．20，P〉0．05），符合率为97．5％（196／201）；直接测序法从50例NSCLC患者中仅检出EGFR突变11例（22．0％），且低于MEL[50．0％（25／50），x2=12．07，P〈0．05]。经Fisher精确检验，接受吉非替尼治疗的肺腺癌患者中，9例血浆EGFR外显子19突变阳性患者较7例阴性患者具有较高的客观缓解率（P=0．041）和较长的疾病无进展生存期（x2=6．76，P=0．009）。结论成功构建了一种灵敏、特异、快速、高通量的NSCLC血浆诊断EGFR基因外显子19E746—750缺失和外显子21L858R突变的MEL检测平台，对指导晚期肺癌患者个体化治疗有重要价值。

[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 exonl9 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 m B or 1V 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 mutant- enriched 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 exonl9 E746-750 del and exon 21 L858R was 55.7% （ 112/201 ） by MEL assay. Compared with mutant- enriched 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. 0dl ）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 patients.