RESUMO
The 1p/19q codeletion is the result of a translocation between chromosome 1 (Chr1p) and chromosome 19 (Chr19q) with the loss of derivative (1;19)(p10;q10) chromosome. The 1p/19q codeletion has predictive and prognostic significance, and it is essential for the classification of gliomas. In routine practice, the fluorescence in situ hybridization (FISH) diagnosis of 1p/19q codeletion is sometimes unexpected. This study aimed to develop a next-generation sequencing panel for the concurrent definition of the 1p/19q codeletion and IDH1/IDH2 mutation status to resolve these equivocal cases. A total of 65 glioma samples were investigated using a 1p/19q-single-nucleotide polymorphism (SNP)-IDH panel. The panel consists of 192 amplicons, including SNPs mapping to Chr1 and Chr19 and amplicons for IDH1/IDH2 analysis. The 1p/19q SNP-IDH panel consistently identified IDH1/IDH2 mutations. In 49 of 60 cases (81.7%), it provided the same 1p/19q results obtained by FISH. In the remaining 11 cases, the 1p/19q SNP-IDH panel uncovered partial chromosome imbalances as a result of interstitial amplification or deletion of the regions where the FISH probes map, leading to a mistaken overdiagnosis of 1p/19q codeletion by FISH. The 1p/19q SNP-IDH next-generation sequencing panel allows reliable analysis of the 1p/19q codeletion and IDH1/IDH2 mutation at the same time. The panel not only allows resolution of difficult cases but also represents a cost-effective alternative to standard molecular diagnostics procedures.
Assuntos
Neoplasias Encefálicas/genética , Cromossomos Humanos Par 19/genética , Cromossomos Humanos Par 1/genética , Deleção de Genes , Glioma/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Hibridização in Situ Fluorescente/métodos , Isocitrato Desidrogenase/genética , Sobrediagnóstico , Translocação Genética/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/patologia , Estudos de Coortes , Análise Custo-Benefício , Análise Mutacional de DNA/economia , Análise Mutacional de DNA/métodos , Feminino , Glioma/patologia , Sequenciamento de Nucleotídeos em Larga Escala/economia , Humanos , Hibridização in Situ Fluorescente/economia , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/economia , Técnicas de Diagnóstico Molecular/métodos , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos Testes , Adulto JovemRESUMO
The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes.