Ultra-deep massively parallel sequencing with unique molecular identifier tagging achieves comparable performance to droplet digital PCR for detection and quantification of circulating tumor DNA from lung cancer patients

Autoři: Le Son Tran aff001;  Hong-Anh Thi Pham aff001;  Vu-Uyen Tran aff001;  Thanh-Truong Tran aff001;  Anh-Thu Huynh Dang aff004;  Dinh-Thong Le aff005;  Son-Lam Nguyen aff005;  Ngoc-Vu Nguyen aff005;  Trieu-Vu Nguyen aff006;  Binh Thanh Vo aff001;  Hong-Thuy Thi Dao aff001;  Nguyen Huu Nguyen aff001;  Tam Huu Tran aff007;  Chu Van Nguyen aff008;  Phuong Cam Pham aff009;  Anh Tuan Dang-Mai aff010;  Thien Kim Dinh-Nguyen aff011;  Van Hieu Phan aff010;  Thanh-Thuy Thi Do aff004;  Kiet Truong Dinh aff002;  Han Ngoc Do aff001;  Minh-Duy Phan aff001;  Hoa Giang aff001;  Hoai-Nghia Nguyen aff004
Působiště autorů: Gene Solutions, Ho Chi Minh, Vietnam aff001;  Medical Genetics Institute, Ho Chi Minh City, Vietnam aff002;  Graduate program of Genetics, Ho Chi Minh city University of Science, Ho Chi Minh city, Vietnam aff003;  University of Medicine and Pharmacy, Ho Chi Minh city, Vietnam aff004;  Pham Ngoc Thach Hospital, Ho Chi Minh city, Vietnam aff005;  Thu Duc Hospital, Ho Chi Minh city, Vietnam aff006;  Center for Standardization and QC in Medical Lab of Ho Chi Minh City, Ho Chi Minh City, Vietnam aff007;  Vietnam National Cancer Hospital, Ha Noi, Vietnam aff008;  Bach Mai Hospital, Ha Noi, Vietnam aff009;  Center for Forensic Science, Ho Chi Minh City, Vietnam aff010;  Tan Hung General Hospital, Ho Chi Minh City, Vietnam aff011;  School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Australia aff012
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: 10.1371/journal.pone.0226193


The identification and quantification of actionable mutations are of critical importance for effective genotype-directed therapies, prognosis and drug response monitoring in patients with non-small-cell lung cancer (NSCLC). Although tumor tissue biopsy remains the gold standard for diagnosis of NSCLC, the analysis of circulating tumor DNA (ctDNA) in plasma, known as liquid biopsy, has recently emerged as an alternative and noninvasive approach for exploring tumor genetic constitution. In this study, we developed a protocol for liquid biopsy using ultra-deep massively parallel sequencing (MPS) with unique molecular identifier tagging and evaluated its performance for the identification and quantification of tumor-derived mutations from plasma of patients with advanced NSCLC. Paired plasma and tumor tissue samples were used to evaluate mutation profiles detected by ultra-deep MPS, which showed 87.5% concordance. Cross-platform comparison with droplet digital PCR demonstrated comparable detection performance (91.4% concordance, Cohen’s kappa coefficient of 0.85 with 95% CI = 0.72–0.97) and great reliability in quantification of mutation allele frequency (Intraclass correlation coefficient of 0.96 with 95% CI = 0.90–0.98). Our results highlight the potential application of liquid biopsy using ultra-deep MPS as a routine assay in clinical practice for both detection and quantification of actionable mutation landscape in NSCLC patients.

Klíčová slova:

Amplification-refractory mutation system analysis – Biopsy – Blood plasma – Circulating tumor DNA – Mutation detection – Non-small cell lung cancer – Point mutation – Polymerase chain reaction


1. 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: a cancer journal for clinicians 2018;68:394–424.

2. Sher T, Dy GK, Adjei AA. Small cell lung cancer. Mayo Clinic proceedings 2008;83:355–67. doi: 10.4065/83.3.355 18316005

3. Birring SS, Peake MD. Symptoms and the early diagnosis of lung cancer. Thorax 2005;60:268–9. doi: 10.1136/thx.2004.032698 15790977

4. Liang H, Huang J, Wang B, Liu Z, He J, Liang W. The role of liquid biopsy in predicting post-operative recurrence of non-small cell lung cancer. Journal of thoracic disease 2018;10:S838–s45. doi: 10.21037/jtd.2018.04.08 29780630

5. Absenger G, Terzic J, Bezan A. ASCO update: lung cancer. Memo 2017;10:224–7. doi: 10.1007/s12254-017-0373-x 29250201

6. Tafe LJ, Pierce KJ, Peterson JD, de Abreu F, Memoli VA, Black CC, et al. Clinical Genotyping of Non-Small Cell Lung Cancers Using Targeted Next-Generation Sequencing: Utility of Identifying Rare and Co-mutations in Oncogenic Driver Genes. Neoplasia (New York, N.Y.) 2016;18:577–83.

7. Lindeman NI, Cagle PT, Beasley MB, Chitale DA, Dacic S, Giaccone G, et al. Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer 2013;8:823–59.

8. 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. The New England journal of medicine 2010;362:2380–8. doi: 10.1056/NEJMoa0909530 20573926

9. Mok TS, Wu YL, Thongprasert S, Yang CH, Chu DT, Saijo N, et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. The New England journal of medicine 2009;361:947–57. doi: 10.1056/NEJMoa0810699 19692680

10. Douillard JY, Ostoros G, Cobo M, Ciuleanu T, McCormack R, Webster A, et al. First-line gefitinib in Caucasian EGFR mutation-positive NSCLC patients: a phase-IV, open-label, single-arm study. British journal of cancer 2014;110:55–62. doi: 10.1038/bjc.2013.721 24263064

11. Sequist LV, Yang JC, Yamamoto N, O’Byrne K, Hirsh V, Mok T, et al. Phase III study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 2013;31:3327–34.

12. 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. Clinical cancer research: an official journal of the American Association for Cancer Research 2013;19:2240–7.

13. Soria JC, Ohe Y, Vansteenkiste J, Reungwetwattana T, Chewaskulyong B, Lee KH, et al. Osimertinib in Untreated EGFR-Mutated Advanced Non-Small-Cell Lung Cancer. The New England journal of medicine 2018;378:113–25. doi: 10.1056/NEJMoa1713137 29151359

14. Brose MS, Volpe P, Feldman M, Kumar M, Rishi I, Gerrero R, et al. BRAF and RAS mutations in human lung cancer and melanoma. Cancer research 2002;62:6997–7000. 12460918

15. Ding L, Getz G, Wheeler DA, Mardis ER, McLellan MD, Cibulskis K, et al. Somatic mutations affect key pathways in lung adenocarcinoma. Nature 2008;455:1069–75. doi: 10.1038/nature07423 18948947

16. Kinno T, Tsuta K, Shiraishi K, Mizukami T, Suzuki M, Yoshida A, et al. Clinicopathological features of nonsmall cell lung carcinomas with BRAF mutations. Annals of oncology: official journal of the European Society for Medical Oncology 2014;25:138–42.

17. Kalemkerian GP, Narula N, Kennedy EB, Biermann WA, Donington J, Leighl NB, et al. Molecular Testing Guideline for the Selection of Patients With Lung Cancer for Treatment With Targeted Tyrosine Kinase Inhibitors: American Society of Clinical Oncology Endorsement of the College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology Clinical Practice Guideline Update. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 2018;36:911–9.

18. Pao W, Wang TY, Riely GJ, Miller VA, Pan Q, Ladanyi M, et al. KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib. PLoS medicine 2005;2:e17. doi: 10.1371/journal.pmed.0020017 15696205

19. Loriot Y, Mordant P, Deutsch E, Olaussen KA, Soria JC. Are RAS mutations predictive markers of resistance to standard chemotherapy? Nature reviews. Clinical oncology 2009;6:528–34. doi: 10.1038/nrclinonc.2009.106 19597509

20. Roberts PJ, Stinchcombe TE. KRAS mutation: should we test for it, and does it matter? Journal of clinical oncology: official journal of the American Society of Clinical Oncology 2013;31:1112–21.

21. Pesek M, Benesova L, Belsanova B, Mukensnabl P, Bruha F, Minarik M. Dominance of EGFR and insignificant KRAS mutations in prediction of tyrosine-kinase therapy for NSCLC patients stratified by tumor subtype and smoking status. Anticancer research 2009;29:2767–73. 19596959

22. Melosky B. Current Treatment Algorithms for Patients with Metastatic Non-Small Cell, Non-Squamous Lung Cancer. Frontiers in oncology 2017;7:38. doi: 10.3389/fonc.2017.00038 28373963

23. Kim SS, Choi HJ, Kim JJ, Kim MS, Lee IS, Byun B, et al. Droplet digital PCR-based EGFR mutation detection with an internal quality control index to determine the quality of DNA. Scientific reports 2018;8:543. doi: 10.1038/s41598-017-18642-x 29323170

24. Crowley E, Di Nicolantonio F, Loupakis F, Bardelli A. Liquid biopsy: monitoring cancer-genetics in the blood. Nature reviews. Clinical oncology 2013;10:472–84. doi: 10.1038/nrclinonc.2013.110 23836314

25. NN S, JA P, H M, A Z, D Y, J T, et al.—Plasma DNA-based molecular diagnosis, prognostication, and monitoring of patients. D—101705370 T—ppublish.

26. Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, et al. High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Analytical chemistry 2011;83:8604–10. doi: 10.1021/ac202028g 22035192

27. Gu J, Zang W, Liu B, Li L, Huang L, Li S, et al. Evaluation of digital PCR for detecting low-level EGFR mutations in advanced lung adenocarcinoma patients: a cross-platform comparison study. Oncotarget 2017;8:67810–20. doi: 10.18632/oncotarget.18866 28978074

28. Zhu Y, Guo Z, Liu Y, Zheng X, Yang G, Zheng G. A novel ARMS-based assay for the quantification of EGFR mutations in patients with lung adenocarcinoma. Oncology letters 2018;15:2905–12. doi: 10.3892/ol.2017.7679 29435017

29. Gyawali B, West HJ. Plasma vs Tissue Next-Generation Sequencing in Non-Small Cell Lung Cancer-Either, Both, or Neither? JAMA oncology 2018.

30. Aggarwal C, Thompson JC, Black TA, Katz SI, Fan R, Yee SS, et al. Clinical Implications of Plasma-Based Genotyping With the Delivery of Personalized Therapy in Metastatic Non-Small Cell Lung Cancer. JAMA oncology 2018.

31. Feng WN, Gu WQ, Zhao N, Pan YM, Luo W, Zhang H, et al. Comparison of the SuperARMS and Droplet Digital PCR for Detecting EGFR Mutation in ctDNA From NSCLC Patients. Translational oncology 2018;11:542–5. doi: 10.1016/j.tranon.2018.02.007 29525631

32. Ma L, Zhang F, Xu Q, Tang X, Mao M. 2016;- 11.

33. Taylor SC, Laperriere G, Germain H. Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data. Scientific reports 2017;7:2409. doi: 10.1038/s41598-017-02217-x 28546538

34. Phallen J, Sausen M, Adleff V, Leal A, Hruban C, White J, et al. Direct detection of early-stage cancers using circulating tumor DNA. Science translational medicine 2017;9.

35. MacConaill LE, Burns RT, Nag A, Coleman HA, Slevin MK, Giorda K, et al. Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing. BMC genomics 2018;19:30. doi: 10.1186/s12864-017-4428-5 29310587

36. Newman AM, Bratman SV, To J, Wynne JF, Eclov NC, Modlin LA, et al. An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nature medicine 2014;20:548–54. doi: 10.1038/nm.3519 24705333

37. JG S, MSD M, LP L, KT P, LM S, GR O, et al.—Sensitivity of next-generation sequencing assays detecting oncogenic fusions in. D—8800805–96–9.

38. Zhang H, Liu R, Yan C, Liu L, Tong Z, Jiang W, et al. Advantage of Next-Generation Sequencing in Dynamic Monitoring of Circulating Tumor DNA over Droplet Digital PCR in Cetuximab Treated Colorectal Cancer Patients. Translational oncology 2019;12:426–31. doi: 10.1016/j.tranon.2018.11.015 30562681

39. Bio-Rad Laboratories I. Rare Mutation Detection Best Practices Guidelines.

40. L D, P C, AM K, S M, R BH, S T, et al.—Validation of a digital PCR method for quantification of DNA copy number. D—101667177–29–39.

41. Li JY, Ho JC, Wong KH. T790M mutant copy number quantified via ddPCR predicts outcome after osimertinib treatment in lung cancer. Oncotarget 2018;9:27929–39. doi: 10.18632/oncotarget.25332 29963252

42. Meador C, Hu Y, Yang JC-H, Mok T, Laus G, Hovey T, et al. Refining the sensitivity of plasma cell-free DNA (cfDNA) genotyping by controlling for plasma tumor content. Journal of Clinical Oncology 36:9071-.

43. TA C, JH C, M K, JD H, N C, DS L, et al.—Analytical Validation of a Hybrid Capture-Based Next-Generation Sequencing. D—100893612–686–702.

44. Kohsaka S, Petronczki M, Solca F, Maemondo M. Tumor clonality and resistance mechanisms in EGFR mutation-positive non-small-cell lung cancer: implications for therapeutic sequencing. Future oncology (London, England) 2019;15:637–52.

45. Suda K, Tomizawa K, Mitsudomi T. Biological and clinical significance of KRAS mutations in lung cancer: an oncogenic driver that contrasts with EGFR mutation. Cancer metastasis reviews 2010;29:49–60. doi: 10.1007/s10555-010-9209-4 20108024

46. Gainor JF, Varghese AM, Ou SH, Kabraji S, Awad MM, Katayama R, et al. ALK rearrangements are mutually exclusive with mutations in EGFR or KRAS: an analysis of 1,683 patients with non-small cell lung cancer. Clinical cancer research: an official journal of the American Association for Cancer Research 2013;19:4273–81.

47. Bai H, Mao L, Wang HS, Zhao J, Yang L, An TT, et al. Epidermal growth factor receptor mutations in plasma DNA samples predict tumor response in Chinese patients with stages IIIB to IV non-small-cell lung cancer. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 2009;27:2653–9.

48. Couraud S, Vaca-Paniagua F, Villar S, Oliver J, Schuster T, Blanche H, et al. Noninvasive diagnosis of actionable mutations by deep sequencing of circulating free DNA in lung cancer from never-smokers: a proof-of-concept study from BioCAST/IFCT-1002. Clinical cancer research: an official journal of the American Association for Cancer Research 2014;20:4613–24.

49. 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. Clinical cancer research: an official journal of the American Association for Cancer Research 2014;20:1698–705.

50. Plagnol V, Woodhouse S, Howarth K, Lensing S, Smith M, Epstein M, et al. Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling. PloS one 2018;13:e0193802. doi: 10.1371/journal.pone.0193802 29543828

51. Yang X, Zhuo M, Ye X, Bai H, Wang Z, Sun Y, et al. Quantification of mutant alleles in circulating tumor DNA can predict survival in lung cancer. Oncotarget 2016;7:20810–24. doi: 10.18632/oncotarget.8021 26989078

Článek vyšel v časopise


2019 Číslo 12