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Comparison of COBAS 4800 KRAS, TaqMan PCR and High Resolution Melting PCR assays for the detection of KRAS somatic mutations in formalin-fixed paraffin embedded colorectal carcinomas

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Abstract

Many studies documented the influence of KRAS mutation status on the response of patients with metastatic colorectal cancer (mCRC) to anti-EGFR monoclonal antibodies. The COBAS 4800 KRAS is an assay using real time PCR and TaqMelt technology, CE-IVD validated, for the detection of 19 KRAS somatic mutations in exons 2 and 3. We compared COBAS with previously validated PCR TaqMan and High Resolution Melting (HRM) assays on 156 formalin-fixed paraffin embedded (FFPE) specimens of colorectal carcinoma. DNA extraction procedures, using the Qiagen QiAMP kit and the Roche COBAS DNA kit, were also compared. Of the 156 samples, 132 were interpretable using COBAS and TaqMan and 92 using COBAS and HRM. No statistically significant difference was found between COBAS/TaqMan and COBAS/HRM (k = 0.937; p < 0.001 — four discordant cases were found, mostly concerning codon 61 mutations and k = 0.891; p < 0.001 — five discordant cases were found, three regarding codon 61 and two on codon 12/13, respectively). No difference was found between the two DNA extraction methods (t = 1.7185; dol = 39; α = 5 %). The three assays were found suitable to detect accurately KRAS mutations in colon FFPE specimens. COBAS and TaqMan were found to be more robust than HRM, as they yielded fewer non-interpretable results. DNA extraction kits were found to provide equivalent results. The present study shows that pre-screening using COBAS with further TaqMan mutation characterization constitutes an easy and reliable approach for routine diagnostic purposes

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Acknowledgments

The authors are grateful to the “Ligue contre le cancer“ for supporting this work, to Dr. Hélène Blons (Hopital Européen Georges Pompidou, Paris, France) for kindly providing KRAS mutated positive controls.

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Correspondence to Alexandre Harlé.

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Harlé, A., Busser, B., Rouyer, M. et al. Comparison of COBAS 4800 KRAS, TaqMan PCR and High Resolution Melting PCR assays for the detection of KRAS somatic mutations in formalin-fixed paraffin embedded colorectal carcinomas. Virchows Arch 462, 329–335 (2013). https://doi.org/10.1007/s00428-013-1380-x

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Keywords

  • KRAS
  • TaqMan
  • HRM
  • COBAS
  • Colorectal cancer
  • FFPE