Mutation profiling of KRAS, BRAF, and PIK3CA genes in colorectal cancer tissue
| Autor: | Martin Crockard, Helena Murray, M. P. Beaney, John Victor Lamont, Mark J Latten, Stephen Peter Fitzgerald, J. E. Doherty, S. McKeown |
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| Rok vydání: | 2012 |
| Předmět: | |
| Zdroj: | Journal of Clinical Oncology. 30:e14022-e14022 |
| ISSN: | 1527-7755 0732-183X |
| Popis: | e14022 Background: Personalised cancer medicine based on mutation profiling of individual tumours is now a reality in the treatment of metastatic colorectal cancer (CRC) since the discovery of mutant KRAS status and resistance to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies. However, up to 65% of patients with KRAS wildtype tumours are resistant to anti-EGFR therapy. Mutations in other downstream effectors of the EGFR signaling pathway may also have a negative impact on patient response to anti-EGFR therapy. This study reportsthe analytical performance of a biochip array for the rapid simultaneous detection of 20 mutations within KRAS (including codons 12, 13, 61 and 146), BRAF and PIK3CA genes in CRC tissue samples. Methods: DNA from frozen CRC tissue samples was analysed using the Evidence Investigator KRAS/BRAF/PIK3CA Array. The assay is based on a combination of multiplex PCR and biochip array hybridisation, which enables high multiplexing detection. Innovative PCR priming technology permits high discrimination between multiple wildtype and mutant DNA regions. Providing there are enough copies of DNA present, approximately 1% of mutant can readily be detected in a background of wildtype genomic DNA. Analysis can be completed, from template DNA, through PCR (single reaction) to data readout in less than 3 hours. Confirmation of assay results was performed using Sanger sequencing and pyrosequencing. Results: Mutations were detected in 56% (14/25) of CRC tissue samples assessed. 6 samples were positive for KRAS, 3 for BRAF and 6 for PIK3CA with one sample harbouring both a KRAS and PIK3CA mutation. KRAS targets detected also included much rarer codon 61 and 13 mutations. Sanger sequencing confirmed all positive results apart from two, which were assessed via pyrosequencing. Conclusions: These findings demonstrate the applicability of the KRAS/BRAF/PIK3CA Array to rapidly simultaneously detect mutations within these genes in CRC tissue. The detection of additional markers besides KRAS and extending the profile of this gene to include other codons beyond 12 and 13 may aid in the selection of candidate patients to receive anti-EGFR therapy thereby maximising drug efficacy and minimising adverse patient effects. |
| Databáze: | OpenAIRE |
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