Published in:
01-10-2014 | Original Paper
Performance of amplicon-based next generation DNA sequencing for diagnostic gene mutation profiling in oncopathology
Authors:
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, Henk M. Verheul, Egbert F. Smit, Bauke Ylstra, Daniëlle A. M. Heideman
Published in:
Cellular Oncology
|
Issue 5/2014
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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.