Sensitive multiplex detection of KRAS codons 12 and 13 mutations in paraffin-embedded tissue specimens. Issue 1 (28th October 2010)

• Record Type: Journal Article
• Authors: Laosinchai-Wolf, Walairat; Ye, Fei; Tran, Vu; Yang, Zhe; White, Roxanna; Bloom, Kenneth; Choppa, Paul; Labourier, Emmanuel
• Is Part Of: Journal of clinical pathology. Volume 64:Issue 1(2011)
• Page Start: 30
• Page End: 36
• Publication Date: 2010-10-28
• Subjects: KRAS -- mutation -- EGFR -- colorectal cancer -- FFPE -- diagnostics -- molecular pathology; Pathology -- Periodicals; Pathology, Molecular -- Periodicals; 616.0705
• DOI: 10.1136/jcp.2010.081539
• Languages: English
• ISSNs: 0021-9746

Abstract

Abstract : Background: Colorectal cancer patients harbouring KRAS mutations in codon 12 or 13 do not benefit from current anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapies. Efficient and robust methods are therefore required for routine clinical testing of KRAS mutation status. Aims: To evaluate a novel multiplex assay for the rapid detection of common KRAS mutations in formalin-fixed paraffin-embedded (FFPE) tissues. Methods: Genomic DNA was amplified by multiplex PCR using primers targeting the KRAS codon 12/13 region and an internal control gene. PCR products were hybridised on a liquid bead array containing target-specific probes and detected by particle flow cytometry. Results: Analytical performance assessed with plasmid DNA and genomic DNA extracted from cell lines or model FFPE cell line dilutions showed specific detection of seven distinct KRAS mutations with a limit of detection equivalent to 1% tumour. The assay was evaluated at two independent sites with a total of 140 clinical specimens. At site 1, about 45% of the specimens from a set of 86 archived FFPE blocks with unknown KRAS mutation status were found positive for a KRAS mutation. At site 2, each of the seven mutations was detected in at least five independent specimens from a selected set of 54 residual genomic DNAs previously tested with an ARMS/Scorpion laboratory-developed test. Conclusions: This novel single-well assay is a sensitive tool compatible with the clinical laboratory workflow for the rapid assessment of KRAS mutations in solid tumour specimens. Its performance and multiplex format warrant the development of broader panels including other relevant mutations in the EGFR pathway.