RESUMO
Several studies described a role for the E2F/Rb pathway in ovarian serous carcinomas (SCAs). Since E2F/Rb pathway deregulation is a general hallmark of human cancer, it remains unclear whether this deregulation is of particular importance in SCAs or whether it reflects a common oncological feature. Here, we have clarified this issue by the examination of microarray expression profiles of SCAs and particularly by the comparison with another, less malignant, ovarian cancer type, serous borderline tumours (SBTs). Results were validated by quantitative RT-PCR, both on the microarray samples and on an independent panel, and TP53 mutation analysis was performed. This integrated analysis revealed a significant increase in the expression of the transcription factors E2F1 and E2F3 in SCAs, when compared to SBTs. This was associated with vast overexpression of E2F target genes in SCAs compared to SBTs. High-grade SCAs in particular exhibited a major deregulated E2F target expression pattern. Generally, overexpression of E2F targets in SCAs appeared to be well structured since those targets considered negative regulators of the cell cycle or promoters of apoptosis were usually not overexpressed in SCAs. Similar to E2F target deregulation, TP53 mutations were identified in SCA3s, to a lesser extent in SCA1s, and not in SBTs. These results suggest that a structured, generally up-regulated E2F transcription factor activity is associated with a global cell-cycle disturbance in high-grade SCAs and exceeds typical E2F/Rb pathway disruption in tumours, at least compared with SBTs.
Assuntos
Cistadenocarcinoma Seroso/genética , Fator de Transcrição E2F1/genética , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/genética , Ciclo Celular , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Progressão da Doença , Fator de Transcrição E2F1/fisiologia , Feminino , Perfilação da Expressão Gênica/métodos , Genes p53 , Humanos , Mutação , Proteínas de Neoplasias/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transdução de Sinais , Regulação para CimaRESUMO
Serous borderline tumours (SBTs) of the ovary were originally classified as such because the vast majority behave in a remarkably indolent manner, even in the presence of widespread tumour deposits, termed implants, and/or lymph node involvement. The pathogenesis of the implants is currently unknown. Two major hypotheses have been proposed: the first favours a monoclonal origin, arguing that the peritoneal lesions derive from neoplastic cells that are shed from the primary ovarian tumour. The second hypothesis favours a polyclonal origin as a result of a field defect of susceptible Müllerian cells from which multiple independent tumours arise. To test both hypotheses, genome-wide allelotyping and B-RAF/K-RAS mutation analyses were employed to assess clonality in 25 metachronous or synchronous tumours from ten SBT patients. Loss of heterozygosity (LOH) profiling and K-RAS/B-RAF mutation analysis showed concordance of the genetic changes in all sites in 21 tumours from eight patients who were informative. These results favour a common origin, underscored by a likelihood ratio (probability of common origin/probability of independent origin) ranging from 2.43 to 7,662,850. In conclusion, this study strongly supports the hypothesis that both non-invasive and invasive implants arise as a consequence of spread from a single ovarian site.
Assuntos
Neoplasias Ovarianas/genética , Análise Mutacional de DNA/métodos , Feminino , Genes ras/genética , Genótipo , Humanos , Perda de Heterozigosidade/genética , Repetições de Microssatélites/genética , Mutação , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Proteínas de Neoplasias/genética , Estadiamento de Neoplasias , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/patologia , Peritônio/patologia , Proteínas Proto-Oncogênicas B-raf/genéticaRESUMO
Polymerase chain reaction (PCR) analysis to study loss of heterozygosity (LOH) and microsatellite instability (MSI) in tumors is widely used. Microdissection techniques are applied to obtain tumor-specific tissue cells. By microdissection, however, the amount of template DNA extracted may vary considerably and interfere with optimal PCR amplification. To circumvent LOH and MSI misinterpretations due to low DNA input, we have assessed the critical level of DNA input for reliable PCR analysis. PCR analysis was performed by using 18 polymorphic markers (mono-, di-, tri-, and tetranucleotide) on DNA derived from both paraffin-embedded, formalin-fixed, and fresh frozen tumor specimens at template input levels ranging from 0.05 to 25.0 ng. We show a highly significant relation between DNA input and the occurrence of LOH and MSI artifacts. Furthermore, for DNA extracted from paraffin-embedded material, the percentage of LOH artifacts is significantly higher compared with DNA extracted from frozen tissue. For reliable PCR analyses using a mono-, di-, tri-, or tetranucleotide marker, a minimum of 10.0 ng DNA is required when DNA is isolated from formalin-fixed, paraffin-embedded tissue and 5.0 ng when isolated from fresh frozen tissue. HUM PATHOL 31:1414-1419.
