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Clinical validation of the detection of KRAS and BRAF mutations from circulating tumor DNA

Nature Medicine volume 20, pages 430–435 (2014)Cite this article

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Abstract

Assessment of KRAS status is mandatory in patients with metastatic colorectal cancer (mCRC) before applying targeted therapy. We describe here a blinded prospective study to compare KRAS and BRAF mutation status data obtained from the analysis of tumor tissue by routine gold-standard methods and of plasma DNA using a quantitative PCR–based method specifically designed to analyze circulating cell-free DNA (cfDNA). The mutation status was determined by both methods from 106 patient samples. cfDNA analysis showed 100% specificity and sensitivity for the BRAF V600E mutation. For the seven tested KRAS point mutations, the method exhibited 98% specificity and 92% sensitivity with a concordance value of 96%. Mutation load, expressed as the proportion of mutant alleles in cfDNA, was highly variable (0.5–64.1%, median 10.5%) among mutated samples. CfDNA was detected in 100% of patients with mCRC. This study shows that liquid biopsy through cfDNA analysis could advantageously replace tumor-section analysis and expand the scope of personalized medicine for patients with cancer.

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Figure 1: Schematics of the methodology and the analysis of point mutation detection from plasma.
Figure 2: Quantitative analysis of the plasma from patients with mutant tumors, as determined by Intplex.

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Acknowledgements

We thank J. Robert, J. Dubois, A. Lievre, C. Theillet, S. Du Manoir, C. Sardet, D. Tousch and R. Gimbaud for helpful discussions. We would like to thank L. Kamraoui, A. Laybats, J.-Y. Cance and M. Cavalier for excellent technical assistance. F. Mouliere is supported by a grant from CNRS and the Region of Languedoc-Roussillon (CNRS044406). The study was funded by grants from the Fondation ARC pour la Recherche sur le Cancer and the Association GEFLUC (DCMLP10-184, France). We thank Bio-Rad for the qPCR thermocycler free loan. A.R.T. is supported by INSERM.

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Authors and Affiliations

  1. U896 INSERM, Institut Recherche en Cancérologie de Montpellier, Montpellier, France

    Alain R Thierry, Florent Mouliere, Safia El Messaoudi, Celine Gongora, Bruno Robert & Maguy Del Rio

  2. Sysdiag UMR3145 CNRS, CAP DELTA, Montpellier, France

    Alain R Thierry, Florent Mouliere, Safia El Messaoudi, Fanny Rolet & Franck Molina

  3. Unité de Biostatistique, Institut du Cancer de Montpellier, Montpellier, France

    Caroline Mollevi

  4. Laboratoire de Biologie Spécialisée, Institut du Cancer de Montpellier, Montpellier, France

    Evelyne Lopez-Crapez & Pierre-Jean Lamy

  5. Centre Hospitalier Universitaire de Clermont-Ferrand, Service de Chirurgie Digestive Unité d'Oncologie Digestive, UMR Unité Inserm/Université d'Auvergne U1071, Clermont-Ferrand, France

    Brigitte Gillet & Denis Pezet

  6. Centre Hospitalier Universitaire de Clermont-Ferrand, Anatomie Pathologique, Unité d'Oncologie moléculaire, Clermont-Ferrand, France

    Pierre Dechelotte & Anne-Sophie Jarrousse

  7. Service de Pathologie, Unité de Biopathologie Institut du Cancer de Montpellier, Montpellier, France

    Frederic Bibeau

  8. Centre Hospitalier Universitaire de Limoges, Service de Chirurgie Digestive, Centre d'Investigation Clinique, INSERM 0801, Limoges, France

    Michelle Nouaille & Muriel Mathonnet

  9. Service de Chirurgie Digestive, Institut du Cancer de Montpellier, Montpellier, France

    Virginie Loriot & Marc Ychou

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  1. Alain R Thierry
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Contributions

A.R.T. obtained funding, designed the study, interpreted experiments and wrote the manuscript. M.M., D.P., F.B., E.L.-C., M.D.R., P.-J.L., F. Mouliere and M.Y. designed the study. F. Mouliere, S.E.M. and F.R. performed experiments and analyzed the data. F. Mouliere, S.E.M. and A.R.T. prepared the manuscript. C.G., B.R., S.E.M., E.L.-C., M.D.R., M.M., D.P. and M.Y. critically revised the manuscript. C.G., B.R. and F. Molina gave conceptual advice and assisted in the design of experiments. C.M. designed the study, acquired all of the clinical data and performed the statistical analysis. E.L.-C., B.G., P.D., M.N., V.L. and A.-S.J. collected blood samples and acquired the clinical data. F. Mouliere, S.E.M. and A.R.T. developed the Intplex assay. C.G., B.R., F. Mouliere, F. Molina, S.E.M., E.L.-C., M.D.R., M.M., D.P., F.B., P.-J.L., C.M. and M.Y. interpreted the experiments. All authors approved the content of the manuscript.

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Correspondence to Alain R Thierry.

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Thierry, A., Mouliere, F., El Messaoudi, S. et al. Clinical validation of the detection of KRAS and BRAF mutations from circulating tumor DNA. Nat Med 20, 430–435 (2014). https://doi.org/10.1038/nm.3511

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