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Rare RAS Mutations in Metastatic Colorectal Cancer Detected During Routine RAS Genotyping Using Next Generation Sequencing

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Abstract

Background

Overall survival of metastatic colorectal cancer (mCRC) patients has been improved with the addition of targeted therapy such as anti-epithelial growth factor receptor monoclonal antibodies (anti-EGFR mAbs) to standard chemotherapy. Retrospective studies and randomized trials showed that the presence of RAS mutations was linked to the absence of clinical response to anti-EGFR mAbs. Patients harboring KRAS and NRAS mutations on exons 2, 3 or 4 have little or no benefit from anti-EGFR therapies. Polymerase chain reaction (PCR)-based assays are routinely used to assess KRAS and NRAS status, whereas deep sequencing with next generation sequencing (NGS) currently represents an alternative method.

Objective

The objective of our study was to identify KRAS and NRAS non-hotspot mutations using NGS of mCRC tumor samples.

Method

DNA was extracted from 188 consecutive formalin-fixed paraffin embedded samples of histologically proven colorectal cancer tumor tissue from patients with mCRC. Following amplification, DNA was sequenced by ultra-deep pyrosequencing. Non-hotspot mutations identified by NGS (frequency of mutated allele range [1.8–70.6 %]) were confirmed by Sanger direct-sequencing when possible.

Results

NGS procedure was applicable in 94 % of the cases and detected mutations in 62 % of the samples. Nine uncommon mutational profiles were found with a frequency of mutated allele  > 1 %. Silent mutations were found in 3.6 % of the samples. Mutations at or near functional domains of RAS proteins, other than defined hotspots, were found in 3.6 %. NGS proved to be accurate, sensitive and suitable for routine RAS genotyping.

Conclusion

Clinical responses to anti-EGFR mAbs are potentially impaired in the presence of these uncommon RAS mutations.

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Author notes

    Authors and Affiliations

    1. Faculté de Pharmacie, Université de Lorraine, 54001, Nancy, France

      Alexandre Harlé & Jean-Louis Merlin

    2. CNRS UMR 7039 CRAN, 54506, Vandoeuvre-lès-Nancy, France

      Alexandre Harlé & Jean-Louis Merlin

    3. Service de Biopathologie, Institut de Cancérologie de Lorraine, 6 Avenue de Bourgogne, CS30519, 54519, Vandoeuvre les Nancy, France

      Alexandre Harlé, Pierre Filhine-Tresarrieu, Marie Husson, Marie Rouyer, Cindy Dubois, Agnès Leroux & Jean-Louis Merlin

    4. Department of Biology and Pathology of Tumors, Centre Georges-François Leclerc, 21000, Dijon, France

      Romain Boidot

    5. Platform of Transfer in Cancer Biology, Centre Georges-François Leclerc, 21000, Dijon, France

      Romain Boidot

    6. Inserm U866, 21000, Dijon, France

      Romain Boidot

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    1. Alexandre Harlé
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    Corresponding author

    Correspondence to Alexandre Harlé.

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    No funding has been received for the conduct of this study or preparation of this manuscript.

    Conflict of interest

    A Harlé, P Filhine-Tresarrieu, M Husson, R Boidot, M Rouyer, C Dubois, A Leroux and JL Merlin declare no conflict of interest.

    Additional information

    Alexandre Harlé and Pierre Filhine-Tresarrieu contributed equally to this work.

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    Supplementary Figure 1

    Sanger sequence electropherograms of samples bearing non-hotspot mutations of KRAS or NRAS (PPTX 135 kb)

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    Harlé, A., Filhine-Tresarrieu, P., Husson, M. et al. Rare RAS Mutations in Metastatic Colorectal Cancer Detected During Routine RAS Genotyping Using Next Generation Sequencing. Targ Oncol 11, 363–370 (2016). https://doi.org/10.1007/s11523-015-0404-7

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    • DOI: https://doi.org/10.1007/s11523-015-0404-7

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