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Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing

Abstract

As more clinically relevant cancer genes are identified, comprehensive diagnostic approaches are needed to match patients to therapies, raising the challenge of optimization and analytical validation of assays that interrogate millions of bases of cancer genomes altered by multiple mechanisms. Here we describe a test based on massively parallel DNA sequencing to characterize base substitutions, short insertions and deletions (indels), copy number alterations and selected fusions across 287 cancer-related genes from routine formalin-fixed and paraffin-embedded (FFPE) clinical specimens. We implemented a practical validation strategy with reference samples of pooled cell lines that model key determinants of accuracy, including mutant allele frequency, indel length and amplitude of copy change. Test sensitivity achieved was 95–99% across alteration types, with high specificity (positive predictive value >99%). We confirmed accuracy using 249 FFPE cancer specimens characterized by established assays. Application of the test to 2,221 clinical cases revealed clinically actionable alterations in 76% of tumors, three times the number of actionable alterations detected by current diagnostic tests.

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Figure 1: NGS-based cancer genomic profiling test workflow.
Figure 2: Base substitution and indel detection performance.
Figure 3: CNA detection performance.
Figure 4: Concordance with clinical testing on FFPE specimens.
Figure 5: Reproducibility of mutation detection in FFPE specimens.
Figure 6: Clinically actionable alterations in patient samples.

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Acknowledgements

The authors would like to acknowledge L. Gay for advice and assistance with manuscript preparation. H.B. is the Damon Runyon-Gordon Family Clinical Investigator supported (in part) by the Damon Runyon Cancer Research Foundation (CI-67-13). M.L. was supported by a Wellcome Trust Fellowship (WT093855MA) and by the Austrian Science Fund (J2856).

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Authors

Contributions

G.M.F., D.L. and R.Y. designed the study, wrote the manuscript and developed and/or performed analyses. M.T.C. and P.J.S. designed the study and wrote the manuscript. V.A.M., J.S.R. and M.F.B. wrote the manuscript. G.A.O. designed the study. A.F., K.W., J.H., M.S.-L., J.W., E.M.S., P.A., J.S. and C.V. developed and/or performed analyses. G.A.O., S.R.D., K.B., F.J., V.B., S.B., J.B., A.D., L.G., K.I., A.M., K.M., T.R., S.T., E.W., M.Z., Z.Z., M.J., A.P., J.S.R. and J.C. planned and/or performed laboratory experiments. H.B., J.M.M., M.A.R., S.D., C.V.H., M.F.B., L.P., M.L. and C.B. planned and/or performed confirmatory experiments.

Corresponding authors

Correspondence to Doron Lipson or Roman Yelensky.

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Competing interests

All authors with a Foundation Medicine affiliation are current or former employees of and stockholders in Foundation Medicine. M.B. is a former consultant to Foundation Medicine.

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Frampton, G., Fichtenholtz, A., Otto, G. et al. Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing. Nat Biotechnol 31, 1023–1031 (2013). https://doi.org/10.1038/nbt.2696

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