Zhengquan Yang1,2, Jialu Li1,2, Yujie Hu1,2, Meihua Chen1,2, Danli Peng1,2, Dan Zong1,2, Qingjuan Shang1,2, Lianqin Tao3, Yanling Zhao1,2, Yiyun Ni1,2, Jinyan Ye4, Yupeng Xie4, Li Yang4, Quan Lin4, Chang Cai4, Ning Xu5, Xiaoping Huang5, Xiaoting Dong6, Zhonghui Zhou7, Yali Yu7, Zongxiao Shangguan8, Yangyang Xu9, Weiping Ying9, Meiling Weng10, Zuguo Yuan11, Zhijun Dong12, Jifa Li13, Zhe Zheng14, Jiongwei Pan15, Lu Liu16, Junhui Ye17, Zhan Zhang18, Wenfeng Li19, Junfei Zhu20, Shengnan Jin21,22, Yuping Li23, Chunming Ding24,25. 1. School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China. 2. Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, Zhejiang, China. 3. Department of Pulmonary Medicine, Taizhou Central Hospital, Taizhou, Zhejiang, China. 4. Department of Pulmonary and Critical Care Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China. 5. Department of Respiratory Medicine, Ningbo First Hospital, Ningbo, Zhejiang, China. 6. Wenzhou Hospital of Traditional Chinese Medicine, Wenzhou, Zhejiang, China. 7. Ningbo Hospital of Traditional Chinese Medicine, Ningbo, Zhejiang, China. 8. The Third Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China. 9. Zhejiang Jinhua Guangfu Hospital, Jinhua, Zhejiang, China. 10. Department of Oncology, The People's Hospital of Quzhou, Quzhou, Zhejiang, China. 11. Department of Radiotherapy and Chemotherapy, Yinzhou Hospital Affiliated to Medical School of Ningbo University, Ningbo, Zhejiang, China. 12. ChinacoHealthcare Corporation International Hospital, Ningbo, Zhejiang, China. 13. Yue Qing General Hospital, Yue Qing, Zhejiang, China. 14. The Ping Yang Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China. 15. Department of Respiratory and Critical Care Medicine, Lishui People's Hospital, Lishui, Zhejiang, China. 16. Department of Interventional Radiology, Zhejiang University Lishui Hospital, Lishui, Zhejiang, China. 17. Department of Respiratory Medicine, Sanmen People's Hospital of Zhejiang, Taizhou, Zhejiang, China. 18. Huangyan Hospital of Wenzhou Medical University, Taizhou, Zhejiang, China. 19. Department of Radiotherapy and Chemotherapy, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China. 20. Department of Pulmonary Medicine, Taizhou Central Hospital, Taizhou, Zhejiang, China. zjf9609@126.com. 21. School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China. snjin1997@qq.com. 22. Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, Zhejiang, China. snjin1997@qq.com. 23. Department of Pulmonary and Critical Care Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China. wzliyp@163.com. 24. School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China. cmdingchina@qq.com. 25. Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, Zhejiang, China. cmdingchina@qq.com.
Abstract
BACKGROUND: 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. OBJECTIVE: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. TRIAL REGISTRATION: Clinical Trials.gov identifier: NCT02804100.
BACKGROUND: 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. OBJECTIVE: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. TRIAL REGISTRATION: Clinical Trials.gov identifier: NCT02804100.
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