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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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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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