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Ultra-deep sequencing confirms immunohistochemistry as a highly sensitive and specific method for detecting BRAF V600E mutations in colorectal carcinoma

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Abstract

The activating BRAF V600 mutation is a well-established negative prognostic biomarker in metastatic colorectal carcinoma (CRC). A recently developed monoclonal mouse antibody (clone VE1) has been shown to detect reliably BRAF V600E mutated protein by immunohistochemistry (IHC). In this study, we aimed to compare the detection of BRAF V600E mutations by IHC, Sanger sequencing (SaS), and ultra-deep sequencing (UDS) in CRC. VE1-IHC was established in a cohort of 68 KRAS wild-type CRCs. The VE1-IHC was only positive in the three patients with a known BRAF V600E mutation as assessed by SaS and UDS. The test cohort consisted of 265 non-selected, consecutive CRC samples. Thirty-nine out of 265 cases (14.7 %) were positive by VE1-IHC. SaS of 20 randomly selected IHC negative tumors showed BRAF wild-type (20/20). Twenty-four IHC-positive cases were confirmed by SaS (24/39; 61.5 %) and 15 IHC-positive cases (15/39; 38.5 %) showed a BRAF wild-type by SaS. UDS detected a BRAF V600E mutation in 13 of these 15 discordant cases. In one tumor, the mutation frequency was below our threshold for UDS positivity, while in another case, UDS could not be performed due to low DNA amount. Statistical analysis showed sensitivities of 100 % and 63 % and specificities of 95 and 100 % for VE1-IHC and SaS, respectively, compared to combined results of SaS and UDS. Our data suggests that there is high concordance between UDS and IHC using the anti-BRAFV600E (VE1) antibody. Thus, VE1 immunohistochemistry is a highly sensitive and specific method in detecting BRAF V600E mutations in colorectal carcinoma.

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Acknowledgements

We thank Dr. Adriana von Teichman, Marion Bawohl, and Sonja Brun-Schmid for their excellent technical assistance. We are very grateful to Prof. Dieter Zimmermann for his critical proof-reading of the manuscript.

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The authors declare that they have no conflicts of interest.

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Authors and Affiliations

  1. Institute of Surgical Pathology, University Hospital Zurich, Schmelzbergstrasse 12, 8091, Zurich, Switzerland

    Matthias Rössle, Michèle Sigg, Jan H. Rüschoff, Peter J. Wild, Holger Moch, Achim Weber & Markus P. Rechsteiner

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  1. Matthias Rössle
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  2. Michèle Sigg
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Correspondence to Matthias Rössle.

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Matthias Rössle and Michèle Sigg contributed equally to this study.

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Rössle, M., Sigg, M., Rüschoff, J.H. et al. Ultra-deep sequencing confirms immunohistochemistry as a highly sensitive and specific method for detecting BRAF V600E mutations in colorectal carcinoma. Virchows Arch 463, 623–631 (2013). https://doi.org/10.1007/s00428-013-1492-3

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