Abstract
Background
In metastatic melanoma, 40%–50% of patients harbor a BRAF V600E mutation and are thereby eligible to receive a combined BRAF/MEK inhibitor therapy. Compared to standard-of-care tissue-based genetic testing, analysis of circulating tumor DNA (ctDNA) from blood enables a comprehensive assessment of tumor mutational status in real-time and can be used for monitoring response to therapy. The aim of our study was to directly compare the performance of two highly sensitive methodologies, droplet digital PCR (ddPCR) and a combination of ARMS/asymmetric-rapid PCR/melting curve analysis, for the detection of BRAF V600E in plasma from melanoma patients.
Methods
Cell-free DNA (cfDNA) was isolated from 120 plasma samples of stage I to IV melanoma patients. Identical plasma-cfDNA samples were subjected to BRAF V600E mutational analysis using in parallel, ddPCR and the combination of ARMS/asymmetric-rapid PCR/melting curve analysis.
Results
BRAF V600E mutation was detected in 9/117 (7.7%) ctDNA samples by ddPCR and in 22/117 (18.8%) ctDNA samples by the combination of ARMS/asymmetric- rapid PCR/melting curve analysis. The concordance between these two methodologies was 85.5% (100/117). The comparison of plasma-ctDNA analysis using ddPCR and tissue testing revealed an overall agreement of 79.4% (27/34), while the corresponding agreement using the combination of ARMS/asymmetric-rapid PCR/melting curve analysis was 73.5% (25/34). Moreover, comparing the detection of BRAF-mutant ctDNA with the clinics, overall agreement of 87.2% (48/55) for ddPCR and 79.2% (42/53) was demonstrated. Remarkably, the duration of sample storage was negatively correlated with correctness of genotyping results highlighting the importance of pre-analytical factors.
Conclusions
Our direct comparison study has shown a high level of concordance between ddPCR and the combination of ARMS/asymmetric-rapid PCR/melting curve analysis for the detection of BRAF V600E mutations in plasma.
Acknowledgments
The authors thank RomyEichner for helping to collect samples and Victor Costina for critically reading the manuscript.
Author contributions: Eleni Tzanikou: experiments, manuscript writing and editing, Verena Haselmann: study design, supervising experimental procedures, statistical analysis, manuscript writing and editing, Athina Markou: experiments, Angelika Duda: experiments, Michael Neumaier: study design, Jochen Utikal: patient enrollment, Evi Lianidou: study design, supervising experimental procedures, manuscript writing and editing. All authors contributed to revision of the manuscript and approved it for submission. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. The ethical approval was requires for this study.
Research funding: This research has been co-financed by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH–CREATE–INNOVATE (project code: T1RCI-02935).
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. Edge S, Compton C, Fritz A, Greene F, Trotti A. Melanoma of the skin. In: Edge S, editor. Skin (part VI). New York, NY: Springer, 2010:648.Search in Google Scholar
2. Domingues B, Lopes JM, Soares P, Pópulo H. Melanoma treatment in review. Immunotargets Ther 2018;7:35–49.10.2147/ITT.S134842Search in Google Scholar PubMed PubMed Central
3. Dummer R, Hauschild A, Lindenblatt N, Pentheroudakis G, Keilholz U, ESMO Guidelines Committee. Cutaneous melanoma: ESMO clinical practice guidelines for diagnosis, treatment and follow-up. Ann Oncol 2015;26:v126–32.10.1093/annonc/mdv297Search in Google Scholar PubMed
4. Gaiser MR, von Bubnoff N, Gebhardt C, Utikal JS. Liquid biopsy to monitor melanoma patients. J Dtsch Dermatol Ges 2018;16:405–14.10.1111/ddg.13461Search in Google Scholar PubMed
5. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, et al. Mutations of the BRAF gene in human cancer. Nature 2002;417:949–54.10.1038/nature00766Search in Google Scholar PubMed
6. Chapman PB, Hauschild A, Robert C, Haane JB, Ascierto P, Larkin J, et al. BRIM-3 Study Group. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med 2011;364:2507–16.10.1056/NEJMoa1103782Search in Google Scholar PubMed PubMed Central
7. Lianidou E, Hoon D. Circulating tumor cells and circulating tumor DNA. In: Nader R, Horrath A, Wittwer C, editors. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 6th ed. Philadelphia, PA, USA: Elsevier Ltd, 2017:1111–44.Search in Google Scholar
8. Huang SK, Hoon DS. Liquid biopsy utility for the surveillance of cutaneous malignant melanoma patients. Mol Oncol 2016;10:450–63.10.1016/j.molonc.2015.12.008Search in Google Scholar PubMed PubMed Central
9. Perakis S, Auer M, Belic J, Heitzer E. Advances in circulating tumor DNA analysis. Adv Clin Chem 2017;80:73–153.10.1016/bs.acc.2016.11.005Search in Google Scholar PubMed
10. Malentacchi F, Pizzamiglio S, Verderio P, Pazzagli M, Orlando C, Ciniselli CM, et al. Influence of storage conditions and extraction methods on the quantity and quality of circulating cell-free DNA (ccfDNA): the SPIDIA-DNAplas external quality assessment experience. Clin Chem Lab Med 2015;53:1935–42.10.1515/cclm-2014-1161Search in Google Scholar PubMed
11. Haselmann V, Ahmad-Nejad P, Geilenkeuser WJ, Duda A, Gabor M, Eichner R, et al. Results of the first external quality assessment scheme (EQA) for isolation and analysis of circulating tumour DNA (ctDNA). Clin Chem Lab Med 2018;56: 220–8.10.1515/cclm-2017-0283Search in Google Scholar PubMed
12. Keppens C, Dequeker EM, Patton SJ, Normanno N, Fenizia F, Butler R, et al. International pilot external quality assessment scheme for analysis and reporting of circulating tumour DNA. BMC Cancer 2018;18:804.10.1186/s12885-018-4694-xSearch in Google Scholar PubMed PubMed Central
13. Zhou L, Wang Y, Wittwer CT. Rare allele enrichment and detection by allele-specific PCR, competitive probe blocking, and melting analysis. BioTechniques 2011;50:311–8.10.2144/000113668Search in Google Scholar
14. Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 2009;45:228–47.10.1016/j.ejca.2008.10.026Search in Google Scholar PubMed
15. Milosevic D, Mills JR, Campion MB, Vidal-Folch N, Voss JS, Halling KC, et al. Applying standard clinical chemistry assay validation to droplet digital PCR quantitative liquid biopsy testing. Clin Chem 2018;64:1732–42.10.1373/clinchem.2018.291278Search in Google Scholar PubMed
16. Shehata HR, Ragupathy S, Shanmughanandhan D, Kesanakurti P, Ehlinger TM, Newmaster SG. Guidelines for validation of qualitative real-time PCR methods for molecular diagnostic identification of probiotics. J AOAC Int 2019;102:1774–8.10.5740/jaoacint.18-0320Search in Google Scholar PubMed
17. Overman MJ, Modak J, Kopetz S, Murthy R, Yao JC, Hicks ME, et al. Use of research biopsies in clinical trials: are risks and benefits adequately discussed? J Clin Oncol 2013;31:17–22.10.1200/JCO.2012.43.1718Search in Google Scholar PubMed PubMed Central
18. Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med 2014;6:224ra24.10.1158/1538-7445.AM2014-5606Search in Google Scholar
19. Heitzer E, Ulz P, Geigl JB. Circulating tumor DNA as a liquid biopsy for cancer. Clin Chem 2015;61:112–23.10.1373/clinchem.2014.222679Search in Google Scholar PubMed
20. Yancovitz M, Litterman A, Yoon J, Ng E, Shapiro RL, Berman RS, et al. Intra- and inter-tumor heterogeneity of BRAFV600E mutations in primary and metastatic melanoma. PLoS One 2012;7:e29336.10.1371/journal.pone.0029336Search in Google Scholar PubMed PubMed Central
21. Lin J, Goto Y, Murata H, Sakaizawa K, Uchiyama A, Saida T, et al. Polyclonality of BRAF mutations in primary melanoma and the selection of mutant alleles during progression. Br J Cancer 2011;104:464–8.10.1038/sj.bjc.6606072Search in Google Scholar PubMed PubMed Central
22. Sanmamed MF, Fernandez-Landazuri S, Rodriguez C, Zarate R, Lozano MD, Zubiri L, et al. Quantitative cell-free circulating BRAFV600E mutation analysis by use of droplet digital PCR in the follow-up of patients with melanoma being treated with BRAF inhibitors. Clin Chem 2015;61:297–304.10.1373/clinchem.2014.230235Search in Google Scholar PubMed
23. Gray ES, Rizos H, Reid AL, Boyd SC, Pereira MR, Lo J, et al. Circulating tumor DNA to monitor treatment response and detect acquired resistance in patients with metastatic melanoma. Oncotarget 2015;6:42008–18.10.18632/oncotarget.5788Search in Google Scholar PubMed PubMed Central
24. Gonzalez-Cao M, Mayo-De-Las-Casas C, Molina-Vila MA, De Mattos-Arruda L, Muñoz-Couselo E, Manzano JL, et al. BRAF mutation analysis in circulating free tumor DNA of melanoma patients treated with BRAF inhibitors. Melanoma Res 2015;25:486–95.10.1097/CMR.0000000000000187Search in Google Scholar PubMed
25. Aung KL, Donald E, Ellison G, Bujac S, Fletcher L, Cantarini M, et al. Analytical validation of BRAF mutation testing from circulating free DNA using the amplification refractory mutation testing system. J Mol Diagn 2014;16:343–9.10.1016/j.jmoldx.2013.12.004Search in Google Scholar PubMed
26. Haselmann V, Gebhardt C, Brechtel I, Duda A, Czerwinski C, Sucker A, et al. Liquid profiling of circulating tumor DNA in plasma of melanoma patients for companion diagnostics and monitoring of BRAF inhibitor therapy. Clin Chem 2018;64: 830–42.10.1373/clinchem.2017.281543Search in Google Scholar PubMed
27. Lipson EJ, Velculescu VE, Pritchard TS, Sausen M, Pardoll DM, Topalian SL, et al. Circulating tumor DNA analysis as a real-time method for monitoring tumor burden in melanoma patients undergoing treatment with immune checkpoint blockade. J Immunother Cancer 2014;2:42.10.1186/s40425-014-0042-0Search in Google Scholar PubMed PubMed Central
28. Santiago-Walker A, Gagnon R, Mazumdar J, Casey M, Long GV, Schadendorf D, et al. Correlation of BRAF mutation status in circulating-free DNA and tumor and association with clinical outcome across four BRAFi and MEKi clinical trials. Clin Cancer Res 2016;22:567–74.10.1158/1078-0432.CCR-15-0321Search in Google Scholar PubMed
29. Girotti MR, Gremel G, Lee R, Galvani E, Rothwell D, Viros A, et al. Application of sequencing, liquid biopsies, and patient-derived xenografts for personalized medicine in melanoma. Cancer Discov 2016;6:286–99.10.1158/2159-8290.CD-15-1336Search in Google Scholar PubMed
30. Tsao SC, Weiss J, Hudson C, Christophi C, Cebon J, Behren A, et al. Monitoring response to therapy in melanoma by quantifying circulating tumour DNA with droplet digital PCR for BRAF and NRAS mutations. Sci Rep 2015;5:11198.10.1038/srep11198Search in Google Scholar PubMed PubMed Central
31. Schreuer M, Meersseman G, Van Den Herrewegen S, Jansen Y, Chevolet I, Bott A, et al. Quantitative assessment of BRAF v600 mutant circulating cell-free tumor DNA as a tool for therapeutic monitoring in metastatic melanoma patients treated with BRAF/MEK inhibitors. J Transl Med 2016;14:95.10.1186/s12967-016-0852-6Search in Google Scholar PubMed PubMed Central
32. Knol AC, Vallée A, Herbreteau G, Nguyen JM, Varey E, Gaultier A, et al. Clinical significance of BRAF mutation status in circulating tumor DNA of metastatic melanoma patients at baseline. Exp Dermatol 2016;25:783–8.10.1111/exd.13065Search in Google Scholar PubMed
33. Pinzani P, Santucci C, Mancini I, Simi L, Salvianti F, Pratesi N, et al. BRAFV600E detection in melanoma is highly improved by COLD-PCR. Clin Chim Acta 2011;412:901–5.10.1016/j.cca.2011.01.014Search in Google Scholar PubMed
34. Zhang Y, Qu S, Zhao J, Yu T, Guo L, Yin S, et al. A novel RFLP-ARMS TaqMan PCR-based method for detecting the BRAF V600E mutation in melanoma. Oncol Lett 2018;16:1615–21.10.3892/ol.2018.8844Search in Google Scholar PubMed PubMed Central
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