ABSTRACT
O câncer de bexiga está entre os dez tipos de câncer mais freqüentes em todo o mundo. Os mecanismos de carcinogênese e o aparecimento de recorrências no câncer de bexiga não estão esclarecidos, portanto o desenvolvimento de marcadores moleculares para melhorar o diagnóstico ou prever a ocorrência de recidivas levaria a um controle mais efetivo deste tipo tumoral... Quase 91% das amostras apresentaram um ou mais genes metilados. Como a metilação de 14-3-3sigma ainda não havia sido demonstrada em tumores de bexiga, linhagens celulares derivadas deste tipo tumoral que tinham seu padrão de metilação conhecido para este gene foram submetidas ao tratamento com o agente desmetilante 5'-aza-dCR e após este tratamento, a expressão deste gene foi re-estabelecida... Os sete genes (CALCA, PGP9.5, CCND2, AIM1, MINT1, CRBP e CCNA1) foram então testados em um conjunto de validação composto de 93 amostras tumorais e 26 normais. Nenhuma metilação foi observada em amostras normais para os genes MINT1 e CCNA1. Em amostras de câncer de bexiga, encontrarmos 57% de metilação em CCNA1 e 31,2% em MINT1. Em tumores, a freqüência de hipermetilação de CRBP foi de 38,7% e em normais de 3,9%. O gene CALCA apresentou-se metilado em 64,5 dos tumores e 15,4% dos normais. Em 19,2% dos normais foi observada hipermetilação em PGP9.5 e CCND2 e nos tumores 71% e 57%, respectivamente. Metilação em AIM1 estava presente em 83,9% dos tumores e amostras normais... Nossos resultados também demonstram que QMSP utilizando os genes aqui selecionados podem apresentar especificidades e sensibilidades adequadas para o uso clínico destes testes na rotina. Estudos mais amplos com maior número de amostras e seguimento longitudinal e o teste destes marcadores em DNA extraído de urina vão ajudar a definir a sua utilidade para a detecção precoce de câncer de bexiga e o acompanhamento desta doença...
Bladder cancer is among the ten most frequent cancer types worldwide. Mechanisms of bladder carcinogenesis and the development of recurrent bladder cancer remain unclear; therefore, the development of molecular markers to improve diagnosis or tumor recurrence prediction would facilitate more effective management of this cancer type. Tumor markers based on the detection of aberrant promoter methylation of several known or putative tumor suppressor genes offer a potentially powerful approach for cancer detection. We selected four genes (E-CAD, RARß, 14-3-3σ, and APC), frequently silenced by aberrant methylation in different tumor types, and investigated their aberrant methylation profile in 73 bladder tumors. DNA was extracted from formalin-fixed, paraffin-embedded sections and the DNA was subjected to MSP. Methylation frequencies of the tested genes were 71.2% for 14-3-3σ, 58.9% for APC, 49.3% for RARß, and 28.8% for E-CAD. Almost 91% of the samples studied showed one or more of these genes methylated. Because 14-3-3σ hypermethylation was not described in bladder cancer, cell lines known to be methylated and unmethylated for this gene were submitted to the treatment with the demethylating agent 5'-aza-2´-deoxycytidine and the expression of this gene was restored. We then selected additional 21 genes to study their methylation status (CTNNB1, CALCA, hMLH1, PGP9.5, DAPK, CCND2, HIC1, AIM1, DCC, MINT1, MINT31, FANC-F, TGF-ß, ATM, CRBP, THBS1, 14-3-3σ, CCNA1, ESR, FHIT, and MT1G) by quantitative fluorogenic real-time methylation specific PCR (QMSP) in an evaluation set consisting of 25 bladder tumor samples and 5 bladder normal samples. Based on the frequency of methylation observed for these genes in the evaluation set, we selected 7 candidate genes (CALCA, PGP9.5, CCND2, AIM1, MINT1, CRBP, and CCNA1) for further analysis in a set of 93 tumor samples and 26 normal controls. We found no methylation in normal samples for MINT1 and CCNA1. In primary bladder tumors, we found CCNA1 hypermethylation in 57% and MINT1 hypermethylation in 31,2%. CRBP hypermethylation was observed in 38.7% of the tumors samples and 3.9% of the normal samples. CALCA hypermethylation was observed in 64,5% of the tumors and 15,4% of the normal samples. 19,2% of normal samples showed methylation for PGP9.5 and CCND2, and 71% and 57% of the tumors, respectively. AIM1 hypermethylation was present in 83.9% of tumor samples and 27% of the normal ones. In general, methylation levels were higher in tumors compared with normal samples. Almost 95% of the tumor samples showed one or more of these genes methylated. The high frequencies of hypermethylation found both by MSP and QMSP assay for the genes selected in tumor samples and the very low frequency observed in normal tissues (considering the seven genes studied in normal samples) suggest that these epigenetic alterations are important in bladder cancer development. Our results also demonstrate that the QMSP using the genes selected in this study may have the desired specificity and sensitivity for routine clinical use. Further studies using large cohorts with longitudinal follow up and testing these markers in urine DNA will help defining their utility for early bladder cancer detection and disease monitoring (AU)