RESUMO
The cancer genome is moulded by the dual processes of somatic mutation and selection. Homozygous deletions in cancer genomes occur over recessive cancer genes, where they can confer selective growth advantage, and over fragile sites, where they are thought to reflect an increased local rate of DNA breakage. However, most homozygous deletions in cancer genomes are unexplained. Here we identified 2,428 somatic homozygous deletions in 746 cancer cell lines. These overlie 11% of protein-coding genes that, therefore, are not mandatory for survival of human cells. We derived structural signatures that distinguish between homozygous deletions over recessive cancer genes and fragile sites. Application to clusters of unexplained homozygous deletions suggests that many are in regions of inherent fragility, whereas a small subset overlies recessive cancer genes. The results illustrate how structural signatures can be used to distinguish between the influences of mutation and selection in cancer genomes. The extensive copy number, genotyping, sequence and expression data available for this large series of publicly available cancer cell lines renders them informative reagents for future studies of cancer biology and drug discovery.
Assuntos
Sítios Frágeis do Cromossomo/genética , Deleção de Genes , Genes Neoplásicos/genética , Genes Recessivos/genética , Genoma Humano/genética , Homozigoto , Neoplasias/genética , Seleção Genética/genética , Linhagem Celular Tumoral , Cromossomos Humanos/genética , Variações do Número de Cópias de DNA/genética , Análise Mutacional de DNA , Dosagem de Genes/genética , Humanos , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Mapeamento Físico do Cromossomo , Reprodutibilidade dos TestesRESUMO
The development of tyrosine kinase inhibitor treatments has made it important to test cancer patients for clinically significant gene mutations that influence the benefit of treatment. Targeted next-generation sequencing (NGS) provides a promising method for diagnostic purposes by enabling the simultaneous detection of multiple mutations in various genes in a single test. The aim of our study was to screen EGFR, KRAS, and BRAF mutations by targeted NGS and commonly used real-time polymerase chain reaction (PCR) methods to evaluate the feasibility of targeted NGS for the detection of the mutations. Furthermore, we aimed to identify potential novel mutations by targeted NGS. We analyzed formalin-fixed, paraffin-embedded (FFPE) tumor tissue specimens from 81 non-small cell lung carcinoma patients. We observed a significant concordance (from 96.3 to 100%) of the EGFR, KRAS, and BRAF mutation detection results between targeted NGS and real-time PCR. Moreover, targeted NGS revealed seven nonsynonymous single-nucleotide variations and one insertion-deletion variation in EGFR not detectable by the real-time PCR methods. The potential clinical significance of these variants requires elucidation in future studies. Our results support the use of targeted NGS in the screening of EGFR, KRAS, and BRAF mutations in FFPE tissue material.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise Mutacional de DNA/métodos , Feminino , Fixadores/química , Formaldeído/química , Estudos de Associação Genética , Testes Genéticos/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação INDEL , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular , Inclusão em Parafina , Proteínas Proto-Oncogênicas p21(ras) , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Recent EUCAST advice asserts that, with low breakpoints, susceptibility results for cephalosporins and carbapenems can be reported 'as found', even for strains with extended-spectrum ß-lactamases (ESBLs) and carbapenemases. The CLSI has similar advice, but with higher ceftazidime and cefepime breakpoints than those of EUCAST. Pharmacodynamic and animal data are used to support these views, along with some analysis of clinical case series. We contend that such advice is misguided on three counts. First, whilst there are cases on record where cephalosporins and carbapenems have proved effective against infections due to low-MIC ESBL producers and low-MIC carbapenemase producers, respectively, there are similar numbers of cases where such therapy has failed. Second, routine susceptibility testing is less precise than in research analyses, meaning that ESBL and carbapenemase producers with 'real' MICs of 1-8 mg/L will oscillate between susceptibility categories according to who tests them and how. Third, although EUCAST continues to advocate ESBL and carbapenemase detection for epidemiological purposes, the likely consequence of not seeking these enzymes for treatment purposes is that some laboratories will not seek them at all, leading to a loss of critical infection control information. In short, it is prudent to continue to seek ESBLs and carbapenemases directly and, where they are found, generally to avoid substrate drugs as therapy.