Next-generation sequencing for tumor mutation quantification using liquid biopsies
-
Mariano Provencio
Abstract
Background
Non-small cell lung cancer (NSCLC) patients benefit from targeted therapies both in first- and second-line treatment. Nevertheless, molecular profiling of lung cancer tumors after first disease progression is seldom performed. The analysis of circulating tumor DNA (ctDNA) enables not only non-invasive biomarker testing but also monitoring tumor response to treatment. Digital PCR (dPCR), although a robust approach, only enables the analysis of a limited number of mutations. Next-generation sequencing (NGS), on the other hand, enables the analysis of significantly greater numbers of mutations.
Methods
A total of 54 circulating free DNA (cfDNA) samples from 52 NSCLC patients and two healthy donors were analyzed by NGS using the Oncomine™ Lung cfDNA Assay kit and dPCR.
Results
Lin’s concordance correlation coefficient and Pearson’s correlation coefficient between mutant allele frequencies (MAFs) assessed by NGS and dPCR revealed a positive and linear relationship between the two data sets (ρc = 0.986; 95% confidence interval [CI] = 0.975–0.991; r = 0.987; p < 0.0001, respectively), indicating an excellent concordance between both measurements. Similarly, the agreement between NGS and dPCR for the detection of the resistance mutation p.T790M was almost perfect (K = 0.81; 95% CI = 0.62–0.99), with an excellent correlation in terms of MAFs (ρc = 0.991; 95% CI = 0.981–0.992 and Pearson’s r = 0.998; p < 0.0001). Importantly, cfDNA sequencing was successful using as low as 10 ng cfDNA input.
Conclusions
MAFs assessed by NGS were highly correlated with MAFs assessed by dPCR, demonstrating that NGS is a robust technique for ctDNA quantification using clinical samples, thereby allowing for dynamic genomic surveillance in the era of precision medicine.
Acknowledgments
We would like to thank the patients for their participation in the study.
Author contributions: AR conceived and coordinated the study. CBP, AR, MB and MM contributed to experimental design and data analysis. CBP, MB, ES, RS, RLB, ACB, PM and PM carried out the experiments. AR, MB and CPB performed the statistical analyses. MM, VC, JCS and FF selected the study population. AR and CPB contributed to manuscript preparation. All authors read and approved the final manuscript. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: This study was supported by the Carlos III Institute of Health, the Spanish Ministry of Science and Innovation and the European Regional Development Fund (grant numbers: PI16/01818 and PIE17/01977). MB was financed by i-PFIS predoctoral fellowship (grant number IFI18/00051) from the ISCIII-MINECO-AES-FEDER (Plan Estatal I+D+I 2013-2016). ES was financed by the Consejería de Educación, Juventud y Deporte of Comunidad de Madrid and by the Fondo Social Europeo (Programa Operativo de Empleo Juvenil, and Iniciativa de Empleo Juvenil, PEJ-2017-AI/SAL-6478).
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
References
1. Keedy VL, Temin S, Somerfield MR, Beasley MB, Johnson DH, McShane LM, et al. American Society of Clinical Oncology provisional clinical opinion: epidermal growth factor receptor (EGFR) mutation testing for patients with advanced non-small-cell lung cancer considering first-line EGFR tyrosine kinase inhibitor therapy. J Clin Oncol 2011;29:2121–7.10.1200/JCO.2010.31.8923Suche in Google Scholar PubMed
2. Novello S, Barlesi F, Califano R, Cufer T, Ekman S, Levra MG, et al. Metastatic non-small-cell lung cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2016;27(suppl_5):v1–27.10.1093/annonc/mdw326Suche in Google Scholar PubMed
3. 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 OncologyEndorsement of the College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology Clinical Practice Guideline Update. J Clin Oncol 2018;36:911–9.10.1200/JCO.2017.76.7293Suche in Google Scholar PubMed
4. Provencio M, Torrente M, Calvo V, Pérez-Callejo D, Gutiérrez L, Franco F, et al. Prognostic value of quantitative ctDNA levels in non small cell lung cancer patients. Oncotarget 2018;9:488–94.10.18632/oncotarget.22470Suche in Google Scholar PubMed PubMed Central
5. Provencio M, Torrente M, Calvo V, Gutiérrez L, Pérez-Callejo D, Pérez-Barrios C, et al. Dynamic circulating tumor DNA quantificaton for the individualization of non-small-cell lung cancer patients treatment. Oncotarget 2017;8:60291–8.10.18632/oncotarget.20016Suche in Google Scholar PubMed PubMed Central
6. Perez-Barrios C, Nieto-Alcolado I, Torrente M, Jimenez-Sanchez C, Calvo V, Gutierrez-Sanz L, et al. Comparison of methods for circulating cell-free DNA isolation using blood from cancer patients: impact on biomarker testing. Transl Lung Cancer Res 2016;5:665–72.10.21037/tlcr.2016.12.03Suche in Google Scholar PubMed PubMed Central
7. García-Saenz JA, Ayllón P, Laig M, Acosta-Eyzaguirre D, García-Esquinas M, Montes M, et al. Tumor burden monitoring using cell-free tumor DNA could be limited by tumor heterogeneity in advanced breast cancer and should be evaluated together with radiographic imaging. BMC Cancer 2017;17:210.10.1186/s12885-017-3185-9Suche in Google Scholar PubMed PubMed Central
8. Oxnard GR, Thress KS, Alden RS, Lawrance R, Paweletz CP, Cantarini M, et al. Association between plasma genotyping and outcomes of treatment with osimertinib (AZD9291) in advanced non-small-cell lung cancer. J Clin Oncol 2016;34:3375–82.10.1200/JCO.2016.66.7162Suche in Google Scholar PubMed PubMed Central
9. Kivioja T, Vähärautio A, Karlsson K, Bonke M, Enge M, Linnarsson S, et al. Counting absolute numbers of molecules using unique molecular identifiers. Nat Methods 2012;9:72–4.10.1038/nmeth.1778Suche in Google Scholar PubMed
10. Schmitt MW, Kennedy SR, Salk JJ, Fox EJ, Hiatt JB, Loeb LA. Detection of ultra-rare mutations by next-generation sequencing. Proc Natl Acad Sci USA 2012;109:14508–13.10.1073/pnas.1208715109Suche in Google Scholar PubMed PubMed Central
11. Passing H, Bablok, W. A new biometrical procedure for testing the equality of measurements from two different analytical methods. Application of linear regression procedures for method comparison studies in clinical chemistry, Part I. J Clin Chem Clin Biochem 1983;21:709–20.10.1515/cclm.1983.21.11.709Suche in Google Scholar PubMed
12. Iwama E, Sakai K, Azuma K, Harada T, Harada D, Nosaki K, et al. Monitoring of somatic mutations in circulating cell-free DNA by digital PCR and next-generation sequencing during afatinib treatment in patients with lung adenocarcinoma positive for EGFR activating mutations. Ann Oncol 2017;28:136–41.10.1093/annonc/mdw531Suche in Google Scholar PubMed
13. Chaudhuri AA, Chabon JJ, Lovejoy AF, Newman AM, Stehr H, Azad TD, et al. Early detection of molecular residual disease in localized lung cancer by circulating tumor DNA profiling. Cancer Discov 2017;7:1394–403.10.1158/2159-8290.CD-17-0716Suche in Google Scholar PubMed PubMed Central
14. Kim T-O, Oh I-J, Kho BG, Park HY, Chang JS, Park C-K, et al. Feasibility of re-biopsy and EGFR mutation analysis in patients with non-small cell lung cancer. Thorac Cancer 2018;9:856–64.10.1111/1759-7714.12762Suche in Google Scholar PubMed PubMed Central
15. Katayama R, Lovly CM, Shaw AT. Therapeutic targeting of anaplastic lymphoma kinase in lung cancer: a paradigm for precision cancer medicine. Clin Cancer Res 2015;21:2227–35.10.1158/1078-0432.CCR-14-2791Suche in Google Scholar PubMed PubMed Central
16. Friboulet L, Li N, Katayama R, Lee CC, Gainor JF, Crystal AS, et al. The ALK inhibitor ceritinib overcomes crizotinib resistance in non-small cell lung cancer. Cancer Discov 2014;4:662–73.10.1158/2159-8290.CD-13-0846Suche in Google Scholar PubMed PubMed Central
17. Garcia-Murillas I, Schiavon G, Weigelt B, Ng C, Hrebien S, CuttsRJ, et al. Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer. Sci Transl Med 2015;7:302ra133.10.1126/scitranslmed.aab0021Suche in Google Scholar PubMed
18. 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:e0193802.10.1371/journal.pone.0193802Suche in Google Scholar PubMed PubMed Central
19. Beije N, Helmijr JC, Weerts MJ, Beaufort CM, Wiggin M, Marziali A, et al. Somatic mutation detection using various targeted detection assays in paired samples of circulating tumor DNA, primary tumor and metastases from patients undergoing resection of colorectal liver metastases. Mol Oncol 2016;10:1575–84.10.1016/j.molonc.2016.10.001Suche in Google Scholar PubMed PubMed Central
20. Garcia J, Dusserre E, Cheynet V, Bringuier PP, Brengle-Pesce K, Wozny A-S, et al. Evaluation of pre-analytical conditions and comparison of the performance of several digital PCR assays for the detection of majorEGFRmutations in circulating DNA from non-small cell lung cancers: the CIRCAN_0 study. Oncotarget 2017;8:87980–96.10.18632/oncotarget.21256Suche in Google Scholar PubMed PubMed Central
21. Steensma DP, Bejar R, Jaiswal S, Lindsley RC, Sekeres MA, Hasserjian RP, et al. Clonal hematopoiesis of indeterminate potential and its distinction from myelodysplastic syndromes. Blood 2015;126:9–16.10.1182/blood-2015-03-631747Suche in Google Scholar PubMed PubMed Central
22. Hu Y, Ulrich B, Supplee J, Kuang Y, Lizotte PH, Feeney N, et al. False positive plasma genotyping due to clonal hematopoiesis. Clin Cancer Res 2018;24:4437–43.10.1158/1078-0432.CCR-18-0143Suche in Google Scholar PubMed
Supplementary Material
The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2019-0745).
©2019 Walter de Gruyter GmbH, Berlin/Boston
Artikel in diesem Heft
- Frontmatter
- Editorial
- Towards a personalized assessment of vitamin D status
- Reviews
- Circulating tumor DNA and their added value in molecular oncology
- Telomere length determinants in childhood
- Opinion Papers
- Serum or plasma? An old question looking for new answers
- Evaluating sample stability in the clinical laboratory with the help of linear and non-linear regression analysis
- General Clinical Chemistry and Laboratory Medicine
- Simultaneous measurement of 25(OH)-vitamin D and 24,25(OH)2-vitamin D to define cut-offs for CYP24A1 mutation and vitamin D deficiency in a population of 1200 young subjects
- How well do Croatian laboratories adhere to national recommendations for laboratory diagnostics of chronic kidney disease (CKD)?
- Underfilling of vacuum blood collection tubes leads to increased lactate dehydrogenase activity in serum and heparin plasma samples
- Calcium state estimation by total calcium: the evidence to end the never-ending story
- The use of faecal immunochemical testing in the decision-making process for the endoscopic investigation of iron deficiency anaemia
- Measurement uncertainty of β-lactam antibiotics results: estimation and clinical impact on therapeutic drug monitoring
- Practical approach to method verification in plasma and validation in cerebrospinal fluid under accreditation using a flexible scope in molecular virology: setting up the HIV, HBV and HCV Aptima™ Quant Dx assays
- Plasma neurofilament light chain is associated with mortality after spontaneous intracerebral hemorrhage
- Comparison of the diagnostic performance of two automated urine sediment analyzers with manual phase-contrast microscopy
- Development and validation of LC-MS/MS methods to measure tobramycin and lincomycin in plasma, microdialysis fluid and urine: application to a pilot pharmacokinetic research study
- Reference Values and Biological Variations
- Cord blood S100B: reference ranges and interest for early identification of newborns with brain injury
- Hematology and Coagulation
- Validation and standardization of the ETP-based activated protein C resistance test for the clinical investigation of steroid contraceptives in women: an unmet clinical and regulatory need
- Cancer Diagnostics
- Next-generation sequencing for tumor mutation quantification using liquid biopsies
- Cardiovascular Diseases
- Evolution of the slopes of ST2 and galectin-3 during marathon and ultratrail running compared to a control group
- Letters to the Editor
- Biological variation of two serum markers for preeclampsia prediction
- Daily monitoring of a control material with a concentration near the limit of detection improves the measurement accuracy of highly sensitive troponin assays
- Are patients adequately informed about procedures for 24-h urine collection?
- Interferences in free thyroxine concentration using the Roche analytical platform: improvement of the third generation?
- Procedures for the diagnosis of macro-follicle stimulating hormone (FSH) in a patient with high serum FSH concentrations
- False-positive result of immunochromatographic (IC) strip test for the diagnosis of α-thalassemia in samples with autoantibodies
- Sample dilution for soluble interleukin-2 receptor α measurement: comparison of two different matrices
- The growing problem of predatory publishing: a case report
- Cerebrospinal fluid lactate levels according to the site of puncture
Artikel in diesem Heft
- Frontmatter
- Editorial
- Towards a personalized assessment of vitamin D status
- Reviews
- Circulating tumor DNA and their added value in molecular oncology
- Telomere length determinants in childhood
- Opinion Papers
- Serum or plasma? An old question looking for new answers
- Evaluating sample stability in the clinical laboratory with the help of linear and non-linear regression analysis
- General Clinical Chemistry and Laboratory Medicine
- Simultaneous measurement of 25(OH)-vitamin D and 24,25(OH)2-vitamin D to define cut-offs for CYP24A1 mutation and vitamin D deficiency in a population of 1200 young subjects
- How well do Croatian laboratories adhere to national recommendations for laboratory diagnostics of chronic kidney disease (CKD)?
- Underfilling of vacuum blood collection tubes leads to increased lactate dehydrogenase activity in serum and heparin plasma samples
- Calcium state estimation by total calcium: the evidence to end the never-ending story
- The use of faecal immunochemical testing in the decision-making process for the endoscopic investigation of iron deficiency anaemia
- Measurement uncertainty of β-lactam antibiotics results: estimation and clinical impact on therapeutic drug monitoring
- Practical approach to method verification in plasma and validation in cerebrospinal fluid under accreditation using a flexible scope in molecular virology: setting up the HIV, HBV and HCV Aptima™ Quant Dx assays
- Plasma neurofilament light chain is associated with mortality after spontaneous intracerebral hemorrhage
- Comparison of the diagnostic performance of two automated urine sediment analyzers with manual phase-contrast microscopy
- Development and validation of LC-MS/MS methods to measure tobramycin and lincomycin in plasma, microdialysis fluid and urine: application to a pilot pharmacokinetic research study
- Reference Values and Biological Variations
- Cord blood S100B: reference ranges and interest for early identification of newborns with brain injury
- Hematology and Coagulation
- Validation and standardization of the ETP-based activated protein C resistance test for the clinical investigation of steroid contraceptives in women: an unmet clinical and regulatory need
- Cancer Diagnostics
- Next-generation sequencing for tumor mutation quantification using liquid biopsies
- Cardiovascular Diseases
- Evolution of the slopes of ST2 and galectin-3 during marathon and ultratrail running compared to a control group
- Letters to the Editor
- Biological variation of two serum markers for preeclampsia prediction
- Daily monitoring of a control material with a concentration near the limit of detection improves the measurement accuracy of highly sensitive troponin assays
- Are patients adequately informed about procedures for 24-h urine collection?
- Interferences in free thyroxine concentration using the Roche analytical platform: improvement of the third generation?
- Procedures for the diagnosis of macro-follicle stimulating hormone (FSH) in a patient with high serum FSH concentrations
- False-positive result of immunochromatographic (IC) strip test for the diagnosis of α-thalassemia in samples with autoantibodies
- Sample dilution for soluble interleukin-2 receptor α measurement: comparison of two different matrices
- The growing problem of predatory publishing: a case report
- Cerebrospinal fluid lactate levels according to the site of puncture
