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Performance of amplicon-based next generation DNA sequencing for diagnostic gene mutation profiling in oncopathology

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Abstract

Purpose

Next generation DNA sequencing (NGS) holds promise for diagnostic applications, yet implementation in routine molecular pathology practice requires performance evaluation on DNA derived from routine formalin-fixed paraffin-embedded (FFPE) tissue specimens. The current study presents a comprehensive analysis of TruSeq Amplicon Cancer Panel-based NGS using a MiSeq Personal sequencer (TSACP-MiSeq-NGS) for somatic mutation profiling.

Methods

TSACP-MiSeq-NGS (testing 212 hotspot mutation amplicons of 48 genes) and a data analysis pipeline were evaluated in a retrospective learning/test set approach (n = 58/n = 45 FFPE-tumor DNA samples) against ‘gold standard’ high-resolution-melting (HRM)-sequencing for the genes KRAS, EGFR, BRAF and PIK3CA. Next, the performance of the validated test algorithm was assessed in an independent, prospective cohort of FFPE-tumor DNA samples (n = 75).

Results

In the learning set, a number of minimum parameter settings was defined to decide whether a FFPE-DNA sample is qualified for TSACP-MiSeq-NGS and for calling mutations. The resulting test algorithm revealed 82 % (37/45) compliance to the quality criteria and 95 % (35/37) concordant assay findings for KRAS, EGFR, BRAF and PIK3CA with HRM-sequencing (kappa = 0.92; 95 % CI = 0.81–1.03) in the test set. Subsequent application of the validated test algorithm to the prospective cohort yielded a success rate of 84 % (63/75), and a high concordance with HRM-sequencing (95 % (60/63); kappa = 0.92; 95 % CI = 0.84–1.01). TSACP-MiSeq-NGS detected 77 mutations in 29 additional genes.

Conclusion

TSACP-MiSeq-NGS is suitable for diagnostic gene mutation profiling in oncopathology.

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References

  1. C. Ferte, F. Andre, J.C. Soria, Molecular circuits of solid tumors: prognostic and predictive tools for bedside use. Nat Rev Clin Oncol 7, 367–380 (2010)

    Article  PubMed  CAS  Google Scholar 

  2. S.A. Aparicio, D.G. Huntsman, Does massively parallel DNA resequencing signify the end of histopathology as we know it? J Pathol 220, 307–315 (2010)

    PubMed  CAS  Google Scholar 

  3. R. Rosell, T.G. Bivona, N. Karachaliou, Genetics and biomarkers in personalisation of lung cancer treatment. Lancet 382, 720–731 (2013)

    Article  PubMed  CAS  Google Scholar 

  4. L.A. Garraway, J. Verweij, K.V. Ballman, Precision oncology: an overview. J Clin Oncol 31, 1803–1805 (2013)

    Article  PubMed  Google Scholar 

  5. P.B. Chapman, A. Hauschild, C. Robert, J.B. Haanen, P. Ascierto, J. Larkin, R. Dummer, C. Garbe, A. Testori, M. Maio, D. Hogg, P. Lorigan, C. Lebbe, T. Jouary, D. Schadendorf, A. Ribas, S.J. O’Day, J.A. Sosman, J.M. Kirkwood, A.M. Eggermont, B. Dreno, K. Nolop, J. Li, B. Nelson, J. Hou, R.J. Lee, K.T. Flaherty, G.A. McArthur, Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med 364, 2507–2516 (2011)

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  6. J.Y. Douillard, K.S. Oliner, S. Siena, J. Tabernero, R. Burkes, M. Barugel, Y. Humblet, G. Bodoky, D. Cunningham, J. Jassem, F. Rivera, I. Kocakova, P. Ruff, M. Blasinska-Morawiec, M. Smakal, J.L. Canon, M. Rother, R. Williams, A. Rong, J. Wiezorek, R. Sidhu, S.D. Patterson, Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer. N Engl J Med 369, 1023–1034 (2013)

    Article  PubMed  CAS  Google Scholar 

  7. N.I. Lindeman, P.T. Cagle, M.B. Beasley, D.A. Chitale, S. Dacic, G. Giaccone, R.B. Jenkins, D.J. Kwiatkowski, J.S. Saldivar, J. Squire, E. Thunnissen, M. Ladanyi, College of American pathologists international association for the study of lung cancer and association for molecular pathology, 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. J Mol Diagn 15, 415–453 (2013)

    Article  PubMed  CAS  Google Scholar 

  8. C.D. de Gonzalez, P.A. Clarke, B. Al-Lazikani, P. Workman, Personalized cancer medicine: molecular diagnostics, predictive biomarkers, and drug resistance. Clin Pharmacol Ther 93, 252–259 (2013)

    Article  Google Scholar 

  9. S.D. Boyd, Diagnostic applications of high-throughput DNA sequencing. Annu Rev Pathol 8, 381–410 (2013)

    Article  PubMed  CAS  Google Scholar 

  10. C.L. Corless, P.T. Spellman, Tackling formalin-fixed, paraffin-embedded tumor tissue with next-generation sequencing. Cancer Discov 2, 23–24 (2012)

    Article  PubMed  CAS  Google Scholar 

  11. S.E. Yost, E.N. Smith, R.B. Schwab, L. Bao, H. Jung, X. Wang, E. Voest, J.P. Pierce, K. Messer, B.A. Parker, O. Harismendy, K.A. Frazer, Identification of high-confidence somatic mutations in whole genome sequence of formalin-fixed breast cancer specimens. Nucleic Acids Res 40, e107 (2012)

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  12. N.J. Loman, R.V. Misra, T.J. Dallman, C. Constantinidou, S.E. Gharbia, J. Wain, M.J. Pallen, Performance comparison of benchtop high-throughput sequencing platforms. Nat Biotechnol 30, 434–439 (2012)

    Article  PubMed  CAS  Google Scholar 

  13. M.A. Quail, M. Smith, P. Coupland, T.D. Otto, S.R. Harris, T.R. Connor, A. Bertoni, H.P. Swerdlow, Y. Gu, A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. BMC Genomics 13, 341 (2012)

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  14. F. Chang, M.M. Li, Clinical application of amplicon-based next-generation sequencing in cancer. Cancer Genet 206, 413–419 (2013)

    Article  PubMed  CAS  Google Scholar 

  15. H.J. Dubbink, Z.C. Deans, B.B. Tops, F.J. van Kemenade, S. Koljenovic, H.J. van Krieken, W.A. Blokx, W.N. Dinjens, P.J. Groenen, Next generation diagnostic molecular pathology: Critical appraisal of quality assurance in Europe. Mol Oncol (2014). doi:10.1016/j.molonc.2014.03.004

    PubMed  Google Scholar 

  16. M. Salto-Tellez, D.G. de Castro, Next generation sequencing: a change of paradigm in molecular diagnostic validation. J Pathol (2014). doi:10.1002/path.4365

    PubMed  Google Scholar 

  17. FDA-approved Next-Generation sequencing system could expand clinical genomic testing: experts predict MiSeqDx system will make genetic testing more affordable for smaller labs. Am. J. Med. Genet. A 164A, x-xi (2014)

  18. V. Endris, R. Penzel, A. Warth, A. Muckenhuber, P. Schirmacher, A. Stenzinger, W. Weichert, Molecular diagnostic profiling of lung cancer specimens with a semiconductor-based massive parallel sequencing approach: feasibility, costs, and performance compared with conventional sequencing. J Mol Diagn 15, 765–775 (2013)

    Article  PubMed  CAS  Google Scholar 

  19. C.E. Cottrell, H. Al-Kateb, A.J. Bredemeyer, E.J. Duncavage, D.H. Spencer, H.J. Abel, C.M. Lockwood, I.S. Hagemann, S.M. O’Guin, L.C. Burcea, C.S. Sawyer, D.M. Oschwald, J.L. Stratman, D.A. Sher, M.R. Johnson, J.T. Brown, P.F. Cliften, B. George, L.D. McIntosh, S. Shrivastava, T.T. Nguyen, J.E. Payton, M.A. Watson, S.D. Crosby, R.D. Head, R.D. Mitra, R. Nagarajan, S. Kulkarni, K. Seibert, H.W. Virgin, J. Milbrandt, J.D. Pfeifer, Validation of a next-generation sequencing assay for clinical molecular oncology. J Mol Diagn 16, 89–105 (2014)

    Article  PubMed  CAS  Google Scholar 

  20. C.M. McCourt, D.G. McArt, K. Mills, M.A. Catherwood, P. Maxwell, D.J. Waugh, P. Hamilton, J.M. O’Sullivan, M. Salto-Tellez, Validation of next generation sequencing technologies in comparison to current diagnostic gold standards for BRAF, EGFR and KRAS mutational analysis. PLoS One 8, e69604 (2013)

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  21. H. Do, S.Q. Wong, J. Li, A. Dobrovic, Reducing sequence artifacts in amplicon-based massively parallel sequencing of formalin-fixed paraffin-embedded DNA by enzymatic depletion of uracil-containing templates. Clin Chem 59, 1376–1383 (2013)

    Article  PubMed  CAS  Google Scholar 

  22. L. Lacroix, A. Boichard, F. Andre, J.C. Soria, Genomes in the clinic: the Gustave Roussy cancer center experience. Curr Opin Genet Dev 24C, 99–106 (2014)

    Article  Google Scholar 

  23. S.Q. Wong, J. Li, A.Y. Tan, R. Vedururu, J.M. Pang, H. Do, J. Ellul, K. Doig, A. Bell, G.A. MacArthur, S.B. Fox, D.M. Thomas, A. Fellowes, J.P. Parisot, A. Dobrovic, Sequence artefacts in a prospective series of formalin-fixed tumours tested for mutations in hotspot regions by massively parallel sequencing. BMC Med Genomics 7, 23 (2014)

    Article  PubMed  PubMed Central  Google Scholar 

  24. S. Chevrier, L. Arnould, F. Ghiringhelli, B. Coudert, P. Fumoleau, R. Boidot, Next-generation sequencing analysis of lung and colon carcinomas reveals a variety of genetic alterations. Int J Oncol 45, 1167–1174 (2014)

    PubMed  CAS  Google Scholar 

  25. D. Kramer, F.B. Thunnissen, M.I. Gallegos-Ruiz, E.F. Smit, P.E. Postmus, C.J. Meijer, P.J. Snijders, D.A. Heideman, A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations. Cell Oncol 31, 161–167 (2009)

    PubMed  CAS  Google Scholar 

  26. D.A. Heideman, F.B. Thunnissen, M. Doeleman, D. Kramer, H.M. Verheul, E.F. Smit, P.E. Postmus, C.J. Meijer, G.A. Meijer, P.J. Snijders, A panel of high resolution melting (HRM) technology-based assays with direct sequencing possibility for effective mutation screening of EGFR and K-ras genes. Cell Oncol 31, 329–333 (2009)

    PubMed  CAS  Google Scholar 

  27. D.A. Heideman, I. Lurkin, M. Doeleman, E.F. Smit, H.M. Verheul, G.A. Meijer, P.J. Snijders, E. Thunnissen, E.C. Zwarthoff, KRAS and BRAF mutation analysis in routine molecular diagnostics: comparison of three testing methods on formalin-fixed, paraffin-embedded tumor-derived DNA. J Mol Diagn 14, 247–255 (2012)

    Article  PubMed  CAS  Google Scholar 

  28. Dutch Federation of Biomedical Scientific Societies, Human Tissue and Medical Research: Code of conduct for responsible use (2011)

  29. P.Fang, Z.Yan, W.Liu, A.Darwanto, K.Pelak, K.Anoe, C.Spittle, S.Sankar, C.Galderisi, J.Li, Validation of Illumina TruSeq Amplicon Cancer Panel with concordance testing using Ion AmpliSeq Cancer Panel and other methods. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6–10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 73, Abstract nr 3229 (2013)

  30. M. Martin, Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. Journal 17, 10–12 (2011)

    Google Scholar 

  31. Hannon Lab, FASTX-Toolkit. http://hannonlab.cshl.edu/fastx_toolkit/. Electronic Citation (2010)

  32. T. Magoc, S.L. Salzberg, FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27, 2957–2963 (2011)

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  33. H. Li, R. Durbin, Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589–595 (2010)

    Article  PubMed  PubMed Central  Google Scholar 

  34. P. Cingolani, V.M. Patel, M. Coon, T. Nguyen, S.J. Land, D.M. Ruden, X. Lu, Using Drosophila melanogaster as a model for genotoxic chemical mutational studies with a new program, SnpSift. Front Genet 3, 35 (2012)

    Article  PubMed  PubMed Central  Google Scholar 

  35. J.T. Robinson, H. Thorvaldsdottir, W. Winckler, M. Guttman, E.S. Lander, G. Getz, J.P. Mesirov, Integrative genomics viewer. Nat Biotechnol 29, 24–26 (2011)

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  36. G.R. Abecasis, D. Altshuler, A. Auton, L.D. Brooks, R.M. Durbin, R.A. Gibbs, M.E. Hurles, G.A. McVean, A map of human genome variation from population-scale sequencing. Nature 467, 1061–1073 (2010)

    Article  PubMed  Google Scholar 

  37. P.J. Groenen, W.A. Blokx, C. Diepenbroek, L. Burgers, F. Visinoni, P. Wesseling, J.H. van Krieken, Preparing pathology for personalized medicine: possibilities for improvement of the pre-analytical phase. Histopathology 59, 1–7 (2011)

    Article  PubMed  Google Scholar 

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Acknowledgments

We thank Debby Boon, Marjolein Bekker-Lettink, Divera Pronk, René Pol, Douwe Buma, Martijn Bogaarts, Francois Rustenburg, Sandra Mongera, and Marianne Tijssen for helpful technical assistance. The work was supported by the Center for Translational Molecular Medicine (CTMM) projects 03O-101 DeCoDe and 05 T-401 ICT TraIT, the Health Insurance Company (LPT/AGIS grant), an unrestricted grant from AMGEN, and the research program of the VUmc Cancer Center Amsterdam.

Statement of author contributions

DAMH was the project leader and designed the study with DS, PJFS, GAM and BY. DS and DAMH drafted the manuscript. MWD and MIHvM were responsible for molecular testing. EFS and HMV were responsible for clinical management. KG, NCTvG and ET performed histopathological evaluations and guided macro-dissections. BY, HFvE and PPE facilitated NGS analyses. DS was responsible for the bioinformatics pipeline. All authors had full access to the data of the study, take responsibility for the integrity and accuracy of data analysis, critically reviewed the manuscript and approved the final version.

Ethical standards

All samples were used in compliance with the respective institutional ethical regulations for surplus material [28].

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Departments of Pathology, VU University Medical Center, PO Box 7057, 1007, MB, Amsterdam, The Netherlands

    Daoud Sie, Peter J.F. Snijders, Gerrit A. Meijer, Marije W. Doeleman, Marinda I. H. van Moorsel, Hendrik F. van Essen, Paul P. Eijk, Katrien Grünberg, Nicole C. T. van Grieken, Erik Thunnissen, Bauke Ylstra & Daniëlle A. M. Heideman

  2. Medical Oncology, VU University Medical Center, PO Box 7057, 1007, MB, Amsterdam, The Netherlands

    Henk M. Verheul

  3. Pulmonary Diseases, VU University Medical Center, PO Box 7057, 1007, MB, Amsterdam, The Netherlands

    Egbert F. Smit

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  1. Daoud Sie
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Correspondence to Daniëlle A. M. Heideman.

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Sie, D., Snijders, P.J., Meijer, G.A. et al. Performance of amplicon-based next generation DNA sequencing for diagnostic gene mutation profiling in oncopathology. Cell Oncol. 37, 353–361 (2014). https://doi.org/10.1007/s13402-014-0196-2

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