改良Blocker PCR检测EGFR-TKI耐药后非小细胞肺癌血浆EGFR T790M突变的价值
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摘要
目的探讨改良PCR(Blocker PCR)方法在晚期非小细胞肺癌(non-small-cell lung cancer,NSCLC)患者血浆游离DNA(cell free DNA,cfDNA)中检测继发EGFR T790 M突变的应用价值。方法采用Blocker PCR方法对127例表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptor-tyrosine kinase inhibitor,EGFR-TKI)耐药后肺癌患者的血浆标本行EGFR敏感突变和T790 M突变检测,统计T790 M突变的检出率;为验证Blocker PCR血浆检测的可靠性,部分患者血浆标本行二代测序(next generation sequencing,NGS)和Blocker PCR配对比较;获得配对血浆和组织的病例行Blocker PCR配对检测。结果在127例采用Blocker PCR方法检测的EGFR-TKI耐药NSCLC患者中,T790 M耐药突变的检出率为40.15%(51/127),其中21.56%(11/51)为单纯T790 M耐药突变,78.44%(40/51)为T790 M合并原有EGFR敏感突变。组织与血浆配对检测中,EGFR-TKI耐药后二次活检组织标本T790 M的检出率为54.54%(6/11),血浆T790 M的检出率为43.75%(14/32)。在同时行Blocker PCR和二代测序基因检测的18例患者中,敏感突变位点及T790 M突变位点在两种检测方法中的一致率均为100%。结论在EGFR-TKI耐药后的NSCLC患者中使用Blocker PCR检测血浆T790 M突变是对组织活检的重要补充。
Objective To evaluate the feasibility of Blocker PCR assays in monitoring T790 M mutations in plasma of non-small-cell lung cancer(NSCLC)patients with epidermal growth factor receptor-tyrosine kinase inhibitor(EGFR-TKI)acquired resistance. Methods Blocker PCR assays were employed to identify mutations in plasma for 127 advanced NSCLC with acquired EGFR-TKI resistance.In addition,the paired tumor re-biopsy or PE samples were obtained to analyze EGFR mutations.Meanwhile,we evaluated the detection accuracy of Blocker PCR assays in comparison with the next generation sequencing(NGS). Results Among the 127 patients,40.15%(51/127)EGFR T790 M was detected in the plasma,78.44%(40/51)coexisted with an EGFRactivating mutation.Additionally,54.54%(6/11)EGFR T790 M was identified in re-biopsy tissues,while 43.75%(14/32)were detected in the plasma.Furthermore,the concordance rate of Blocker PCR and NGS in identifying EGFRsensitizing mutations and EGFR T790 M mutations was 100%. Conclusions Blocker PCR is a highly sensitive and reliable method in monitoring EGFR T790 M mutations in the plasma of NSCLC patients with EGFR-TKI acquired resistance.
Objective To evaluate the feasibility of Blocker PCR assays in monitoring T790 M mutations in plasma of non-small-cell lung cancer(NSCLC)patients with epidermal growth factor receptor-tyrosine kinase inhibitor(EGFR-TKI)acquired resistance. Methods Blocker PCR assays were employed to identify mutations in plasma for 127 advanced NSCLC with acquired EGFR-TKI resistance.In addition,the paired tumor re-biopsy or PE samples were obtained to analyze EGFR mutations.Meanwhile,we evaluated the detection accuracy of Blocker PCR assays in comparison with the next generation sequencing(NGS). Results Among the 127 patients,40.15%(51/127)EGFR T790 M was detected in the plasma,78.44%(40/51)coexisted with an EGFRactivating mutation.Additionally,54.54%(6/11)EGFR T790 M was identified in re-biopsy tissues,while 43.75%(14/32)were detected in the plasma.Furthermore,the concordance rate of Blocker PCR and NGS in identifying EGFRsensitizing mutations and EGFR T790 M mutations was 100%. Conclusions Blocker PCR is a highly sensitive and reliable method in monitoring EGFR T790 M mutations in the plasma of NSCLC patients with EGFR-TKI acquired resistance.
引文
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[24]UCHIDA J,KATO K,KUKITA Y,et al.Diagnostic accuracy of noninvasive genotyping of EGFR in lung cancer patients by deep sequencing of plasma cell-free DNA[J].Clin Chem,2015,61(9):1191-1196.
[25]JIN Y,SHAO Y,SHI X,et al.Mutational profiling of non-small-cell lung cancer patients resistant to firstgeneration EGFR tyrosine kinase inhibitors using next generation sequencing[J].Oncotarget,2016,7(38):61755-61763.
[26]OXNARD GR,ARCILA ME,SIMA CS,et al.Acquired resistance to EGFR tyrosine kinase inhibitors in EGFRmutant lung cancer:distinct natural history of patients with tumors harboring the T790M mutation[J].Clin Cancer Res,2011,17(6):1616-1622.
[27]HATA A,KATAKAMI N,YOSHIOKA H,et al.Rebiopsy of non-small cell lung cancer patients with acquired resistance to epidermal growth factor receptortyrosine kinase inhibitor:comparison between T790M mutation-positive and mutation-negative populations[J].Cancer,2013,119(24):4325-4332.
[28]XU S,LOU F,WU Y,et al.Circulating tumor DNA identified by targeted sequencing in advanced-stage nonsmall cell lung cancer patients[J].Cancer Lett,2016,370(2):324-331.
[29]HATA AN,NIEDERST MJ,ARCHIBALD HL,et al.Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition[J].Nat Med,2016,22(3):262-269.
[2]MIYAUCHI E,INOUE A,KOBAYASHI K,et al.Efficacy of chemotherapy after first-line gefitinib therapy in EGFR mutation-positive advanced non-small cell lung cancer-data from a randomized PhaseⅢstudy comparing gefitinib with carboplatin plus paclitaxel(NEJ002)[J].Jpn J Clin Oncol,2015,45(7):670-676.
[3]BAI H,WANG Z,CHEN K,et al.Influence of chemotherapy on EGFR mutation status among patients with non-small-cell lung cancer[J].J Clin Oncol,2012,30(5):3077-3083.
[4]GERLINGER M,ROWAN AJ,HORSWELL S,et al.Intratumor heterogeneity and branched evolution revealed by multiregion sequencing[J].N Engl J Med,2012,366(10):883-892.
[5]ZHANG J,FUJIMOTO J,ZHANG J,et al.Intratumor heterogeneity in localized lung adenocarcinomas delineated by multiregion sequencing[J].Science,2014,346(6206):256-259.
[6]MOK T,WU Y,LEE JS,et al.Detection and dynamic changes of EGFR mutations from circulating tumor DNA as a predictor of survival outcomes in NSCLC patients treated with first-line intercalated erlotinib and chemotherapy[J].Clin Cancer Res,2015,21(14):3196-3203.
[7]OLSSON E,WINTER C,GEORGE A,et al.Serial monitoring of circulating tumor DNA in patients with primary breast cancer for detection of occult metastatic disease[J].EMBO Mol Med,2015,7(8):1034-1047.
[8]ZHENG D,YE X,ZHANG MZ,et al.Plasma EGFR T790MctDNA status is associated with clinical outcome in advanced NSCLC patients with acquired EGFR-TKI resistance[J].Sci Rep,2016,6:20913.
[9]KOSAKA T,YATABE Y,ENDOH H,et al.Analysis of epidermal growth factor receptor gene mutation in patients with non-small cell lung cancer and acquired resistance to gefitinib[J].Clin Cancer Res,2006,12(19):5764-5769.
[10]NEWMAN AM,BRATMAN SV,To J,et al.An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage[J].Nature Med,2014,20(5):548-554.
[11]KOBOLDT DC,ZHANG Q,LARSON DE,et al.VarScan2:somatic mutation and copy number alteration discovery in cancer by exome sequencing[J].Genome Res,2012,22(3):568-576.
[12]ETTINGER DS,WOOD DE,AKERLEY W,et al.NCCN guidelines insights:non-small cell lung cancer,Version 4.2016[J].J Natl Compr Canc Netw,2016,14(3):255-264.
[13]OHASHI K,MARUVKA YE,MICHOR F,et al.Epidermal growth factor receptor tyrosine kinase inhibitor-resistant disease[J].J Clin Oncol,2013,31(8):1070-1080.
[14]GOU LY,LI AN,YANG JJ,et al.The coexistence of MET over-expression and an EGFR T790M mutation is related to acquired resistance to EGFR tyrosine kinase inhibitors in advanced non-small cell lung cancer[J].Oncotarget,2016,7(32):51311-51319.
[15]OXNARD GR,THRESS KS,ALDEN RS,et al.Association between plasma genotyping and outcomes of treatment with Osimertinib(AZD9291)in advanced nonsmall-cell lung cancer[J].J Clin Oncol,2016,34(28):3375-3382.
[16]CROSS DA,ASHTON SE,GHIORGHIU S,et al.AZD9291,an irreversible EGFR TKI,overcomes T790Mmediated resistance to EGFR inhibitors in lung cancer[J].Cancer Discov,2014,4(9):1046-1061.
[17]MCGRANHAN N,SWANTON C.Biological and therapeutic impact of intratumor heterogeneity in cancer evolution[J].Cancer Cell,2015,27(1):15-26.
[18]WANG Z,CHEN R,WANG S,et al.Quantification and dynamic monitoring of EGFR T790Min plasma cell-free DNA by digital PCR for prognosis of EGFR-TKI treatment in advanced NSCLC[J].PLoS One,2014,9(11):e110780.
[19]DIAZ LA JR,BARDELLI A.Liquid biopsies:genotyping circulating tumor DNA[J].J Clin Oncol,2014,32(6):579-586.
[20]LIU X,LU Y,ZHU G,et al.The diagnostic accuracy of pleural effusion and plasma samples versus tumour tissue for detection of EGFR mutation in patients with advanced non-small cell lung cancer:comparison of methodologies[J].J Clin Pathol,2013,66(12):1065-1069.
[21]THRESS KS,BRANT R,CARR TH,et al.EGFR mutation detection in ctDNA from NSCLC patient plasma:a cross-platform comparison of leading technologies to support the clinical development of AZD9291[J].Lung Cancer,2015,90(2015):509-515.
[22]YUNG TKF,CHAN KCA,MOK TSK,et al.Singlemolecule detection of epidermal growth factor receptor mutations in plasma by microfluidics digital PCR in nonsmall cell lung cancer patients[J].Clin Cancer Res,2009,15(6):2076-2084.
[23]ZHU G,YE X,DONG Z,et al.Highly sensitive droplet digital PCR method for detection of EGFR-activating mutations in plasma cell-free DNA from patients with advanced non-small cell lung cancer[J].J Mol Diagn,2015,17(3):265-272.
[24]UCHIDA J,KATO K,KUKITA Y,et al.Diagnostic accuracy of noninvasive genotyping of EGFR in lung cancer patients by deep sequencing of plasma cell-free DNA[J].Clin Chem,2015,61(9):1191-1196.
[25]JIN Y,SHAO Y,SHI X,et al.Mutational profiling of non-small-cell lung cancer patients resistant to firstgeneration EGFR tyrosine kinase inhibitors using next generation sequencing[J].Oncotarget,2016,7(38):61755-61763.
[26]OXNARD GR,ARCILA ME,SIMA CS,et al.Acquired resistance to EGFR tyrosine kinase inhibitors in EGFRmutant lung cancer:distinct natural history of patients with tumors harboring the T790M mutation[J].Clin Cancer Res,2011,17(6):1616-1622.
[27]HATA A,KATAKAMI N,YOSHIOKA H,et al.Rebiopsy of non-small cell lung cancer patients with acquired resistance to epidermal growth factor receptortyrosine kinase inhibitor:comparison between T790M mutation-positive and mutation-negative populations[J].Cancer,2013,119(24):4325-4332.
[28]XU S,LOU F,WU Y,et al.Circulating tumor DNA identified by targeted sequencing in advanced-stage nonsmall cell lung cancer patients[J].Cancer Lett,2016,370(2):324-331.
[29]HATA AN,NIEDERST MJ,ARCHIBALD HL,et al.Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition[J].Nat Med,2016,22(3):262-269.