title: 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.
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.
authors: Service de Biopathologie, Institut de Cancérologie de Lorraine, 6 Avenue de Bourgogne - CS 30519, 54519 Vandoeuvre-lès-Nancy Cedex, France. a.harle@nancy.unicancer.fr
authors: Alexandre
authors: A
authors: Harlé
authors: Benoit
authors: B
authors: Busser
authors: Marie
authors: M
authors: Rouyer
authors: Valentin
authors: V
authors: Harter
authors: Pascal
authors: P
authors: Genin
authors: Agnès
authors: A
authors: Leroux
authors: Jean-Louis
authors: JL
authors: Merlin
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics*; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015179:Colorectal Neoplasms / Q000235:genetics*; D005260:Female; D005557:Formaldehyde; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D012680:Sensitivity and Specificity; D016707:Tissue Fixation; D018631:ras Proteins / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D005557:Formaldehyde; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins
references: Virchows Arch. 2012 Feb;460(2):141-9
references: 22173329
references: Genet Test Mol Biomarkers. 2011 Nov;15(11):831-4
references: 21699410
references: Ann Oncol. 2011 Jul;22(7):1535-1546
references: 21228335
references: Cancer Res. 2006 Apr 15;66(8):3992-5
references: 16618717
references: CA Cancer J Clin. 2011 Mar-Apr;61(2):69-90
references: 21296855
references: Genes Chromosomes Cancer. 2011 May;50(5):307-12
references: 21305640
references: J Clin Oncol. 2011 May 20;29(15):2011-9
references: 21502544
references: Br J Cancer. 2012 Aug 7;107(4):626-31
references: 22805329
references: JAMA. 2010 Oct 27;304(16):1812-20
references: 20978259
references: Crit Rev Oncol Hematol. 2013 Jan;85(1):45-81
references: 22647972
references: Pharmacogenomics. 2009 Jan;10(1):1-3
references: 19102707
references: Lancet Oncol. 2010 Aug;11(8):753-62
references: 20619739
references: N Engl J Med. 2008 Oct 23;359(17):1757-65
references: 18946061
references: Anal Biochem. 2009 Aug 15;391(2):166-8
references: 19464247
references: BMC Cancer. 2006 Dec 21;6:295
references: 17184525
references: J Clin Oncol. 2008 Apr 1;26(10):1626-34
references: 18316791
references: Clin Chim Acta. 2006 Jan;363(1-2):32-47
references: 16182268
references: Clin Colorectal Cancer. 2012 Dec;11(4):291-6
references: 22537608
references: Virchows Arch. 2011 Aug;459(2):155-60
references: 21701884
references: Biochem Biophys Res Commun. 2010 Apr 23;395(1):158-62
references: 20361930
references: Ann Oncol. 2008 Mar;19(3):508-15
references: 17998284
references: Eur J Cancer. 2012 Jul;48(10):1466-75
references: 22446022
references: J Clin Oncol. 2008 Jan 20;26(3):374-9
references: 18202412
references: Cell. 1986 Jan 17;44(1):167-76
references: 3510078
references: J Clin Oncol. 2009 Feb 10;27(5):663-71
references: 19114683
references: J Clin Oncol. 2010 Nov 1;28(31):4697-705
references: 20921465
references: Oncologist. 2011;16(4):467-78
references: 21441573
references: Br J Cancer. 2009 Aug 18;101(4):715-21
references: 19603018
references: J Clin Oncol. 2012 Oct 10;30(29):3570-7
references: 22734028