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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• 作者：Alexandre Harlé (1) (2) (3)
  Benoit Busser (4)
  Marie Rouyer (1)
  Valentin Harter (5)
  Pascal Genin (1)
  Agnès Leroux (1) (2) (3)
  Jean-Louis Merlin (1) (2) (3)
  
• 关键词：KRAS ; TaqMan ; HRM ; COBAS ; Colorectal cancer ; FFPE
• 刊名：Virchows Archiv
• 出版年：2013
• 出版时间：March 2013
• 年：2013
• 卷：462
• 期：3
• 页码：329-335
• 全文大小：190KB
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• 作者单位：Alexandre Harlé (1) (2) (3)
  Benoit Busser (4)
  Marie Rouyer (1)
  Valentin Harter (5)
  Pascal Genin (1)
  Agnès Leroux (1) (2) (3)
  Jean-Louis Merlin (1) (2) (3)
  
  1. Service de Biopathologie, Institut de Cancérologie de Lorraine, 6 Avenue de Bourgogne -CS 30519, 54519, Vandoeuvre-lès-Nancy Cedex, France
  2. CNRS, UMR 7039 CRAN, Nancy, France
  3. Université de Lorraine, Nancy, France
  4. UJF Grenoble 1/Inserm U823, Grenoble, France
  5. Département de biostatistiques, Institut de Cancérologie de Lorraine, Vandoeuvre-lès-Nancy, France
  
• ISSN：1432-2307

文摘

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--.937; p-lt;-.001 -four discordant cases were found, mostly concerning codon 61 mutations and k--.891; p-lt;-.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--.7185; dol--9; α--?%). 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