Published online before print October 1, 2009

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Journal of Molecular Diagnostics 2009, Vol. 11, No. 6
Copyright © 2009 American Society for Investigative Pathology & Association for Molecular Pathology
DOI: 10.2353/jmoldx.2009.090022

Technical Advances

Sensitive Detection of KRAS Mutations in Archived Formalin-Fixed Paraffin-Embedded Tissue Using Mutant-Enriched PCR and Reverse-Hybridization

Christoph Ausch^*, Veronika Buxhofer-Ausch, Christian Oberkanins, Barbara Holzer, Michael Minai-Pour^¶, Stephan Jahn^¶, Nadia Dandachi^||, Robert Zeillinger and Gernot Kriegshäuser

From the Department of Surgery, * 2^nd Department of Medicine, the Division of Oncology, and the Department of Pathology, ^¶ Donauspital SMZ Ost, Vienna; the Cluster for Translational Oncology, Ludwig Boltzmann Research Society, Medical University of Vienna, Vienna; ViennaLab Diagnostics GmbH, Vienna; and the Department of Oncology, ^|| University Clinic of Internal Medicine, Medical University of Graz, Graz, Austria

Recently, evidence has emerged indicating that assessment of KRAS mutations before anti-epidermal growth factor receptor therapy improves outcome in patients with metastatic colorectal cancer (CRC). We report here a novel reverse-hybridization (RH) assay to screen for KRAS mutations in formalin-fixed paraffin-embedded colorectal tissue samples. We combined mutant-enriched PCR based on peptide nucleic acid clamping and RH of amplification products to nitrocellulose test strips that contained a parallel array of oligonucleotide probes targeting 10 frequent mutations in codons 12 and 13 of the KRAS gene. DNA mixing experiments, which included eight different tumor cell lines with known KRAS mutations, were performed to examine the sensitivity of mutation detection. All KRAS mutations present in tumor cell lines were unambiguously identified by the RH assay with 1% of each cell line DNA diluted in normal DNA. RH was then used to screen for KRAS mutations in 74 colorectal tumor and 4 normal control samples. Twenty-six (35%) of the 74 tumor samples showed KRAS mutations. No mutation was found in the four samples of normal colorectal tissue. DNA sequencing without previous mutant enrichment, however, failed to detect four (15%) out of 26 KRAS-positive formalin-fixed paraffin-embedded samples (FFPE). This finding suggests that even after microdissection, mutant sequences in a given DNA isolate can be rare and more sensitive methods are needed for mutation analysis.