Borràs, EmmaJurado, IsmaelHernan, ImmaGamundi, María JoséDias, MiguelMartí, IsabelMañé, BegoñaArcusa, ÀngelsAgúndez, José AGBlanca, MiguelCarballo, Miguel2012-03-192012-03-192011-09Borràs E, Jurado I, Hernan I, Gamundi MJ, Dias M, Martí I, et al. Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing. BMC Cancer. 2011;11:406http://hdl.handle.net/10668/362BACKGROUND: Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF 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. METHODS: We describe a high resolution melting (HRM) assay for mutation detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform. RESULTS: We tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF activating mutations. Regarding EGFR, 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 KRAS and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while EGFR 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 KRAS somatic mutations. CONCLUSIONS: 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.enNeoplasiasSecuencia de BasesAnálisis Mutacional de ADNTerapia Molecular DirigidaFactores BiológicosAlineación de SecuenciaMedical Subject Headings::Phenomena and Processes::Genetic Phenomena::Genetic Structures::Base SequenceMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Genetic Techniques::Sequence Analysis::Sequence Analysis, DNA::DNA Mutational AnalysisMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Genetic Techniques::Sequence Analysis::High-Throughput Nucleotide SequencingMedical Subject Headings::Information Science::Information Science::Information Services::Documentation::Molecular Sequence DataMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Therapeutics::Drug Therapy::Molecular Targeted TherapyMedical Subject Headings::Diseases::NeoplasmsMedical Subject Headings::Chemicals and Drugs::Biological FactorsMedical Subject Headings::Chemicals and Drugs::Enzymes and Coenzymes::Enzymes::Transferases::Phosphotransferases::Phosphotransferases (Alcohol Group Acceptor)::Protein Kinases::Protein-Serine-Threonine Kinases::MAP Kinase Kinase Kinases::raf Kinases::Proto-Oncogene Proteins B-rafMedical Subject Headings::Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Membrane Proteins::Receptors, Cell Surface::Receptors, Peptide::Receptors, Growth Factor::Receptor, Epidermal Growth FactorMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Evaluation Studies as Topic::Reproducibility of ResultsMedical Subject Headings::Phenomena and Processes::Mathematical Concepts::Sensitivity and SpecificityMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Genetic Techniques::Sequence AlignmentMedical Subject Headings::Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Intracellular Signaling Peptides and Proteins::Monomeric GTP-Binding Proteins::ras Proteinsresearch article21943394open access10.1186/1471-2407-11-4061471-2407PMC3192787