| Summary: | <p>Abstract</p> <p>Background</p> <p>The development of targeted therapies has created a pressing clinical need for the rapid and robust molecular characterisation of cancers. We describe here the application of high-resolution melting analysis (HRM) to screen for <it>KRAS </it>mutations in clinical cancer samples. In non-small cell lung cancer, <it>KRAS </it>mutations have been shown to identify a group of patients that do not respond to EGFR targeted therapies and the identification of these mutations is thus clinically important.</p> <p>Methods</p> <p>We developed a high-resolution melting (HRM) assay to detect somatic mutations in exon 2, notably codons 12 and 13 of the <it>KRAS </it>gene using the intercalating dye SYTO 9. We tested 3 different cell lines with known <it>KRAS </it>mutations and then examined the sensitivity of mutation detection with the cell lines using 189 bp and 92 bp amplicons spanning codons 12 and 13. We then screened for <it>KRAS </it>mutations in 30 non-small cell lung cancer biopsies that had been previously sequenced for mutations in <it>EGFR </it>exons 18–21.</p> <p>Results</p> <p>Known <it>KRAS </it>mutations in cell lines (A549, HCT116 and RPMI8226) were readily detectable using HRM. The shorter 92 bp amplicon was more sensitive in detecting mutations than the 189 bp amplicon and was able to reliably detect as little as 5–6% of each cell line DNA diluted in normal DNA. Nine of the 30 non-small cell lung cancer biopsies had <it>KRAS </it>mutations detected by HRM analysis. The results were confirmed by standard sequencing. Mutations in <it>KRAS </it>and <it>EGFR </it>were mutually exclusive.</p> <p>Conclusion</p> <p>HRM is a sensitive in-tube methodology to screen for mutations in clinical samples. HRM will enable high-throughput screening of gene mutations to allow appropriate therapeutic choices for patients and accelerate research aimed at identifying novel mutations in human cancer.</p>
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