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

D.L.S. Sie; P.J.F. Snijders; G.A. Meijer; M.W. Doeleman; M.I.H. van Moorsel; H.F. van Essen; P.P. Eijk; K. Grunberg; N.C.T. van Grieken; E. Thunnissen; H.M. Verheul; E.F. Smit; B. Ylstra; D.A.M. Heideman

doi:10.1007/s13402-014-0196-2

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

D.L.S. Sie, P.J.F. Snijders, G.A. Meijer, M.W. Doeleman, M.I.H. van Moorsel, H.F. van Essen, P.P. Eijk, K. Grunberg, N.C.T. van Grieken, E. Thunnissen, H.M. Verheul, E.F. Smit, B. Ylstra, D.A.M. Heideman

Research output: Contribution to journal › Article › Academic › peer-review

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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.

Original language	English
Pages (from-to)	353-361
Number of pages	9
Journal	Cellular oncology
Volume	37
Issue number	5
DOIs	https://doi.org/10.1007/s13402-014-0196-2
Publication status	Published - 11-Sept-2014
Externally published	Yes

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Sie, D. L. S., Snijders, P. J. F., Meijer, G. A., Doeleman, M. W., van Moorsel, M. I. H., van Essen, H. F., Eijk, P. P., Grunberg, K., van Grieken, N. C. T., Thunnissen, E., Verheul, H. M., Smit, E. F., Ylstra, B., & Heideman, D. A. M. (2014). Performance of amplicon-based next generation DNA sequencing for diagnostic gene mutation profiling in oncopathology. Cellular oncology, 37(5), 353-361. https://doi.org/10.1007/s13402-014-0196-2

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

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 {\textquoteleft}gold standard{\textquoteright} 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.",

author = "D.L.S. Sie and P.J.F. Snijders and G.A. Meijer and M.W. Doeleman and {van Moorsel}, M.I.H. and {van Essen}, H.F. and P.P. Eijk and K. Grunberg and {van Grieken}, N.C.T. and E. Thunnissen and H.M. Verheul and E.F. Smit and B. Ylstra and D.A.M. Heideman",

year = "2014",

month = sep,

day = "11",

doi = "10.1007/s13402-014-0196-2",

language = "English",

volume = "37",

pages = "353--361",

journal = "Cellular oncology",

issn = "2211-3428",

publisher = "Springer",

number = "5",

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Sie, DLS, Snijders, PJF, Meijer, GA, Doeleman, MW, van Moorsel, MIH, van Essen, HF, Eijk, PP, Grunberg, K, van Grieken, NCT, Thunnissen, E, Verheul, HM, Smit, EF, Ylstra, B & Heideman, DAM 2014, 'Performance of amplicon-based next generation DNA sequencing for diagnostic gene mutation profiling in oncopathology', Cellular oncology, vol. 37, no. 5, pp. 353-361. https://doi.org/10.1007/s13402-014-0196-2

TY - JOUR

T1 - Performance of amplicon-based next generation DNA sequencing for diagnostic gene mutation profiling in oncopathology

AU - Sie, D.L.S.

AU - Snijders, P.J.F.

AU - Meijer, G.A.

AU - Doeleman, M.W.

AU - van Moorsel, M.I.H.

AU - van Essen, H.F.

AU - Eijk, P.P.

AU - Grunberg, K.

AU - van Grieken, N.C.T.

AU - Thunnissen, E.

AU - Verheul, H.M.

AU - Smit, E.F.

AU - Ylstra, B.

AU - Heideman, D.A.M.

PY - 2014/9/11

Y1 - 2014/9/11

N2 - 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.

AB - 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.

U2 - 10.1007/s13402-014-0196-2

DO - 10.1007/s13402-014-0196-2

M3 - Article

SN - 2211-3428

VL - 37

SP - 353

EP - 361

JO - Cellular oncology

JF - Cellular oncology

IS - 5

ER -

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

Abstract

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