| Summary: | <p>Abstract</p> <p>Background</p> <p>Epidermal growth factor receptor (<it>EGFR</it>) and its downstream factors <it>KRAS </it>and <it>BRAF </it>are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the <it>EGFR </it>kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in <it>KRAS </it>and <it>BRAF </it>confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices.</p> <p>Methods</p> <p>We describe a high resolution melting (HRM) assay for mutation detection in <it>EGFR </it>exons 19-21, <it>KRAS </it>codon 12/13 and <it>BRAF </it>V600 using formalin-fixed paraffin-embedded samples. Somatic variation of <it>KRAS </it>exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform.</p> <p>Results</p> <p>We tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in <it>KRAS</it>, <it>BRAF </it>and <it>EGFR </it>were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For <it>KRAS</it>, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the <it>BRAF </it>activating mutations. Regarding <it>EGFR</it>, two cases showed exon 19 deletions (delE746-A750 and delE746-T751insA) and another two substitutions in exon 21 (one showed L858R with the resistance mutation T590M in exon 20, and the other had P848L mutation). Consistent with earlier reports, our results show that <it>KRAS </it>and <it>BRAF </it>mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while <it>EGFR </it>mutations were detected in 11.1% of the lung cancer specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM results and allowed detection and quantitation of <it>KRAS </it>somatic mutations.</p> <p>Conclusions</p> <p>HRM is a rapid and sensitive method for moderate-throughput cost-effective screening of oncogene mutations in clinical samples. Rather than Sanger sequence validation, next-generation sequencing technology results in more accurate quantitative results in somatic variation and can be achieved at a higher throughput scale.</p>
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