Droplet digital polymerase chain reaction (ddPCR) is an emerging technology for quantitative cell-free DNA oncology applications. However, a ddPCR assay for the epidermal growth factor receptor (EGFR) p.Thr790Met (T790M) mutation suitable for clinical use remains to be established with analytical and clinical validations.
We aimed to develop and validate a new ddPCR assay to quantify the T790M mutation in plasma for monitoring and predicting the progression of advanced non-small-cell lung cancer (NSCLC).
Specificity of the ddPCR assay was evaluated with genomic DNA samples from healthy individuals. The inter- and intraday variations of the assay were evaluated using mixtures of plasmid DNA containing wild-type EGFR and T790M mutation sequences. We assessed the clinical utility of the T790M assay in a multicenter prospective study in patients with advanced NSCLC receiving tyrosine kinase inhibitor (TKI) treatment by analyzing longitudinal plasma DNA samples.
We set the criteria for a positive call when the following conditions were satisfied: (1) T790M mutation frequency > 0.098% (3 standard deviations above the background signal); (2) at least two positive droplets in duplicate ddPCR reactions. Among the 62 patients with advanced NSCLC exhibiting resistance to TKI treatment, 15 had one or more serial plasma samples that tested positive for T790M. T790M mutation was detected in the plasma as early as 205 days (median 95 days) before disease progression, determined by imaging analysis. Plasma T790M concentrations also correlated with intervention after disease progression.
We developed a ddPCR assay to quantify the T790M mutation in plasma. Quantification of longitudinal plasma T790M mutation may allow noninvasive assessment of drug resistance and guide follow-up treatment in TKI-treated patients with NSCLC.
Clinical Trials.gov identifier: NCT02804100.
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Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68(6):394–424. https://doi.org/10.3322/caac.21492.
Rosell R, Carcereny E, Gervais R, Vergnenegre A, Massuti B, Felip E, et al. Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial. Lancet Oncol. 2012;13(3):239–46. https://doi.org/10.1016/s1470-2045(11)70393-x.
Mok TS, Wu YL, Thongprasert S, Yang CH, Chu DT, Saijo N, et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med. 2009;361(10):947–57. https://doi.org/10.1056/NEJMoa0810699.
Maemondo M, Inoue A, Kobayashi K, Sugawara S, Oizumi S, Isobe H, et al. Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N Engl J Med. 2010;362(25):2380–8. https://doi.org/10.1056/NEJMoa0909530.
Mitsudomi T, Morita S, Yatabe Y, Negoro S, Okamoto I, Tsurutani J, et al. Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial. Lancet Oncol. 2010;11(2):121–8. https://doi.org/10.1016/s1470-2045(09)70364-x.
Yu HA, Arcila ME, Rekhtman N, Sima CS, Zakowski MF, Pao W, et al. Analysis of tumor specimens at the time of acquired resistance to EGFR-TKI therapy in 155 patients with EGFR-mutant lung cancers. Clin Cancer Res. 2013;19(8):2240–7. https://doi.org/10.1158/1078-0432.ccr-12-2246.
Kobayashi S, Boggon TJ, Dayaram T, Janne PA, Kocher O, Meyerson M, et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med. 2005;352(8):786–92. https://doi.org/10.1056/NEJMoa044238.
Ohashi K, Maruvka YE, Michor F, Pao W. Epidermal growth factor receptor tyrosine kinase inhibitor-resistant disease. J Clin Oncol. 2013;31(8):1070–80. https://doi.org/10.1200/jco.2012.43.3912.
Ettinger DS, Aisner DL, Wood DE, Akerley W, Bauman J, Chang JY, et al. NCCN guidelines insights: non-small cell lung cancer, version 5.2018. J Natl Compr Cancer Netw JNCCN. 2018;16(7):807–21. https://doi.org/10.6004/jnccn.2018.0062.
Goto K, Ichinose Y, Ohe Y, Yamamoto N, Negoro S, Nishio K, et al. Epidermal growth factor receptor mutation status in circulating free DNA in serum: from IPASS, a phase III study of gefitinib or carboplatin/paclitaxel in non-small cell lung cancer. J Thorac Oncol. 2012;7(1):115–21. https://doi.org/10.1097/JTO.0b013e3182307f98.
de Bruin EC, McGranahan N, Mitter R, Salm M, Wedge DC, Yates L, et al. Spatial and temporal diversity in genomic instability processes defines lung cancer evolution. Science. 2014;346(6206):251–6. https://doi.org/10.1126/science.1253462.
Jamal-Hanjani M, Wilson GA, McGranahan N, Birkbak NJ, Watkins TBK, Veeriah S, et al. Tracking the evolution of non-small-cell lung cancer. N Engl J Med. 2017;376(22):2109–21. https://doi.org/10.1056/NEJMoa1616288.
Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA Jr, Kinzler KW. Cancer genome landscapes. Science. 2013;339(6127):1546–58. https://doi.org/10.1126/science.1235122.
Yang K, Li J, Zhao J, Ren P, Wang Z, Wei B, et al. Developing ultrasensitive library-aliquot-based droplet digital PCR for detecting T790M in plasma-circulating tumor DNA of non-small-cell-lung-cancer patients. Anal Chem. 2018;90(19):11203–9. https://doi.org/10.1021/acs.analchem.8b01776.
Marchetti A, Palma JF, Felicioni L, De Pas TM, Chiari R, Del Grammastro M, et al. Early prediction of response to tyrosine kinase inhibitors by quantification of EGFR mutations in plasma of NSCLC patients. J Thorac Oncol. 2015;10(10):1437–43. https://doi.org/10.1097/jto.0000000000000643.
Wan JCM, Massie C, Garcia-Corbacho J, Mouliere F, Brenton JD, Caldas C, et al. Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat Rev Cancer. 2017;17(4):223–38. https://doi.org/10.1038/nrc.2017.7.
Diaz LA Jr, Bardelli A. Liquid biopsies: genotyping circulating tumor DNA. J Clin Oncol. 2014;32(6):579–86. https://doi.org/10.1200/jco.2012.45.2011.
Xu T, Kang X, You X, Dai L, Tian D, Yan W, et al. Cross-platform comparison of four leading technologies for detecting EGFR mutations in circulating tumor DNA from non-small cell lung carcinoma patient plasma. Theranostics. 2017;7(6):1437–46. https://doi.org/10.7150/thno.16558.
Takahama T, Sakai K, Takeda M, Azuma K, Hida T, Hirabayashi M, et al. Detection of the T790M mutation of EGFR in plasma of advanced non-small cell lung cancer patients with acquired resistance to tyrosine kinase inhibitors (West Japan oncology group 8014LTR study). Oncotarget. 2016;7(36):58492–9. https://doi.org/10.18632/oncotarget.11303.
Thress KS, Brant R, Carr TH, Dearden S, Jenkins S, Brown H, 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. Lung cancer (Amsterdam, Netherlands). 2015;90(3):509–15. https://doi.org/10.1016/j.lungcan.2015.10.004.
Zheng D, Ye X, Zhang MZ, Sun Y, Wang JY, Ni J, et al. Plasma EGFR T790M ctDNA status is associated with clinical outcome in advanced NSCLC patients with acquired EGFR-TKI resistance. Sci Rep. 2016;6:20913. https://doi.org/10.1038/srep20913.
Lee JY, Qing X, Xiumin W, Yali B, Chi S, Bak SH, et al. Longitudinal monitoring of EGFR mutations in plasma predicts outcomes of NSCLC patients treated with EGFR TKIs: Korean Lung Cancer Consortium (KLCC-12-02). Oncotarget. 2016;7(6):6984–93. https://doi.org/10.18632/oncotarget.6874.
Yu Q, Huang F, Zhang M, Ji H, Wu S, Zhao Y, et al. Multiplex picoliter-droplet digital PCR for quantitative assessment of EGFR mutations in circulating cell-free DNA derived from advanced non-small cell lung cancer patients. Mol Med Rep. 2017;16(2):1157–66. https://doi.org/10.3892/mmr.2017.6712.
Suzawa K, Yamamoto H, Ohashi K, Hashida S, Tomida S, Kubo T, et al. Optimal method for quantitative detection of plasma EGFR T790M mutation using droplet digital PCR system. Oncol Rep. 2017;37(5):3100–6. https://doi.org/10.3892/or.2017.5567.
Kasahara N, Kenmotsu H, Serizawa M, Umehara R, Ono A, Hisamatsu Y, et al. Plasma epidermal growth factor receptor mutation testing with a chip-based digital PCR system in patients with advanced non-small cell lung cancer. Lung Cancer (Amsterdam, Netherlands). 2017;106:138–44. https://doi.org/10.1016/j.lungcan.2017.02.001.
Watanabe M, Kawaguchi T, Isa S, Ando M, Tamiya A, Kubo A, et al. Ultra-sensitive detection of the pretreatment EGFR T790M mutation in non-small cell lung cancer patients with an EGFR-activating mutation using droplet digital PCR. Clin Cancer Res. 2015;21(15):3552–60. https://doi.org/10.1158/1078-0432.ccr-14-2151.
Hu Y, Ulrich BC, Supplee J, Kuang Y, Lizotte PH, Feeney NB, et al. False-positive plasma genotyping due to clonal hematopoiesis. Clin Cancer Res. 2018;24(18):4437–43. https://doi.org/10.1158/1078-0432.ccr-18-0143.
Oxnard GR, Paweletz CP, Kuang Y, Mach SL, O’Connell A, Messineo MM, et al. Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA. Clin Cancer Res. 2014;20(6):1698–705. https://doi.org/10.1158/1078-0432.ccr-13-2482.
Yung TK, Chan KC, Mok TS, Tong J, To KF, Lo YM. Single-molecule detection of epidermal growth factor receptor mutations in plasma by microfluidics digital PCR in non-small cell lung cancer patients. Clin Cancer Res. 2009;15(6):2076–84. https://doi.org/10.1158/1078-0432.ccr-08-2622.
Ma C, Wei S, Song Y. T790M and acquired resistance of EGFR TKI: a literature review of clinical reports. J Thorac Dis. 2011;3(1):10–8. https://doi.org/10.3978/j.issn.2072-1439.2010.12.02.
Lin MT, Mosier SL, Thiess M, Beierl KF, Debeljak M, Tseng LH, et al. Clinical validation of KRAS, BRAF, and EGFR mutation detection using next-generation sequencing. Am J Clin Pathol. 2014;141(6):856–66. https://doi.org/10.1309/ajcpmwgwgo34egod.
Seki Y, Fujiwara Y, Kohno T, Takai E, Sunami K, Goto Y, et al. Picoliter-droplet digital polymerase chain reaction-based analysis of cell-free plasma DNA to assess EGFR mutations in lung adenocarcinoma that confer resistance to tyrosine-kinase inhibitors. Oncologist. 2016;21(2):156–64. https://doi.org/10.1634/theoncologist.2015-0288.
The authors thank all the patients who participated in the study and all the support from the doctors and nurses for the collection of samples from 16 centers throughout Zhejiang Province.
The study was funded by Innovation Team in Zhejiang Province Universities, Innovation Discipline of Zhejiang Province in Nucleic Acid Molecular Diagnostics (437201702G), Key Discipline of Zhejiang Province in Medical Technology (First Class, Category A) (No. 437601607) and Zhejiang Provincial Natural Science Foundation (No. LY18H200007).
Conflict of Interest
Zhengquan Yang, Jialu Li, Yujie Hu, Meihua Chen, Danli Peng, Dan Zong, Qingjuan Shang, Lianqin Tao, Yanling Zhao, Yiyun Ni, Jinyan Ye, Yupeng Xie, Li Yang, Quan Lin, Chang Cai, Ning Xu, Xiaoping Huang, Xiaoting Dong, Zhonghui Zhou, Yali Yu, Zongxiao Shangguan, Yangyang Xu, Weiping Ying, Meiling Weng, Zuguo Yuan, Zhijun Dong, Jifa Li, Zhe Zheng, Jiongwei Pan, Lu Liu, Junhui Ye, Zhan Zhang, Wenfeng Li, Junfei Zhu, Shengnan Jin, Yuping Li and Chunming Ding have no conflicts of interest that might be relevant to the contents of this manuscript.
Ethics Approval and Consent to Participate
The study was approved by the Clinical Research Ethics Committee of Wenzhou Medical University. Written informed consent was obtained from participants.
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Yang, Z., Li, J., Hu, Y. et al. Dynamics of Plasma EGFR T790M Mutation in Advanced NSCLC: A Multicenter Study. Targ Oncol 14, 719–728 (2019). https://doi.org/10.1007/s11523-019-00682-0