Please use this identifier to cite or link to this item: http://hdl.handle.net/10668/362
Título : Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing
Autor : Borràs, Emma
Jurado, Ismael
Hernan, Imma
Gamundi, María José
Dias, Miguel
Martí, Isabel
Mañé, Begoña
Arcusa, Àngels
Agúndez, José AG
Blanca, Miguel
Carballo, Miguel
Filiación: [Borràs, E; Hernan,I; Gamundi,MJ; Dias,M; Martí,I; Mañé,B; Carballo,M] Molecular Genetics Unit, Hospital de Terrassa. [Jurado,I] Pathology Service, Hospital de Terrassa. [Arcusa,A] Oncology Service, Hospital de Terrassa. [Agúndez, JAG] Department of Pharmacology, Universidad de Extremadura. [Blanca,M] Allergy Service, Hospital Carlos Haya.
Palabras clave : Neoplasias
Secuencia de Bases
Análisis Mutacional de ADN
Terapia Molecular Dirigida
Factores Biológicos
Alineación de Secuencia
MeSH: Medical Subject Headings::Phenomena and Processes::Genetic Phenomena::Genetic Structures::Base Sequence
Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Genetic Techniques::Sequence Analysis::Sequence Analysis, DNA::DNA Mutational Analysis
Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Genetic Techniques::Sequence Analysis::High-Throughput Nucleotide Sequencing
Medical Subject Headings::Information Science::Information Science::Information Services::Documentation::Molecular Sequence Data
Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Therapeutics::Drug Therapy::Molecular Targeted Therapy
Medical Subject Headings::Diseases::Neoplasms
Medical Subject Headings::Chemicals and Drugs::Biological Factors
Medical 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-raf
Medical Subject Headings::Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Membrane Proteins::Receptors, Cell Surface::Receptors, Peptide::Receptors, Growth Factor::Receptor, Epidermal Growth Factor
Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Evaluation Studies as Topic::Reproducibility of Results
Medical Subject Headings::Phenomena and Processes::Mathematical Concepts::Sensitivity and Specificity
Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Genetic Techniques::Sequence Alignment
Medical Subject Headings::Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Intracellular Signaling Peptides and Proteins::Monomeric GTP-Binding Proteins::ras Proteins
Fecha de publicación : Sep-2011
Editorial : BioMed Central
Cita Bibliográfica: Borràs 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;11:406
Abstract: BACKGROUND: 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.
URI: http://hdl.handle.net/10668/362
Versión del editor : http://www.biomedcentral.com/1471-2407/11/406
DOI: 10.1186/1471-2407-11-406
ISSN : 1471-2407
Appears in Collections:01- Artículos - Hospital Regional de Málaga

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