Estudo de alterações epigenéticas como biomarcadores no diagnóstico não invasivo do câncer colorretal
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Data
2014-09-30
Autores
Silva, Tiago Donizetti da [UNIFESP]
Orientadores
Forones, Nora Manoukian [UNIFESP]
Tipo
Tese de doutorado
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Objetivo: Analisar a metilação do DNA em 24 genes selecionando 5 candidatos a biomarcadores para uso diagnóstico não invasivo em câncer colorretal (CCR) comparando as alterações epigenéticas encontradas no tecido tumoral com as do tecido normal do cólon bem como o DNA livre circulante (FDNA) do sangue, avaliando o potencial destes marcadores na detecção do CCR. Métodos: O perfil de metilação dos 24 genes candidatos foi realizado utilizando o ensaio Methyl-ProfilerTM PCR Array System (SABiosciences); na segunda fase do estudo foi utilizado a técnica de HRM (High Resolution Melting) para os 5 genes selecionados. Resultados: Os 5 genes selecionados na primeira fase do estudo foram APC, DKK2, SFRP5, MLH1 e RUNX3. Entre os 68 indivíduos do grupo controle 42 eram mulheres e (DP) a média de idade foi de 59,9 anos (13,4). Entre os 46 indivíduos com CCR, 25 eram mulheres e a média de idade foi de 64,6 anos (13,6). No grupo caso 28 pacientes tinham tumor localizado no cólon (60,8%) e 18 pacientes com câncer retal (39,2%). De acordo com a classificação TNM, 2 pacientes (4,3%) eram estádio I, 20 pacientes (43,4%) eram estádio II, 18 pacientes (39,1%) eram estádio III, e 2 pacientes (4,3%) estágio IV. Os genes APC, DKK2 e SFRP5 mostraram perfil de metilação mais frequentes no grupo caso (p<0,001, p <0,001 e p<0,001). Os genes RUNX3 e MLH1, não apresentaram diferenças nos perfis de metilação entre os grupos (p=0,98 e p= 0,08, respectivamente). O perfil de metilação do FDNA do gene APC não mostrou diferença estatisticamente significativa entre os grupos (p = 1,000), enquanto o FDNA do DKK2 foi mais frequente no grupo caso com p = 0,025. Conclusões: Nossos resultados permitiram concluir o potencial do DKK2, APC e SFRP5 como marcadores epigenéticos para o diagnóstico de CCR no tecido e do DKK2 no plasma. Um possível painel de marcadores epigenéticos pode ser uma ferramenta de rastreio para a detecção de doentes com CCR. Indivíduos com hipermetilação do DNA livre circulante do DKK2 no plasma poderiam ser candidatos a colonoscopia.
Epigenetic changes can be detected in precancerous lesions in colorectal cancer (CRC), suggesting a potential involvement in the initial process of carcinogenesis. The potential use of molecular markers in plasma has been researched to detect epigenetic changes in free circulating tumor DNA (FDNA) in blood. Our study compared the changes in the level of DNA methylation found in normal and tumor tissues with blood seeking association of epigenetic markers in the detection of CRC. The candidate genes was performed using the technique Methyl-ProfilerTM PCR Array System (SABiosciences) were selected 5 markers that through the technique of High Resolution Melting (HRM). The 5 genes selected in the first phase of the study were APC, DKK2, SFRP5, MLH1 and RUNX3. Among the 68 controls, 42 were women and the mean (SD) age was 59.9 years (13.4). Among the 46 individuals with CRC, 25 were women and the mean age was 64.6 years (13.6). In the case group 28 patients had colon cancer (60.8%) and 18 (39.2%) had rectal cancer. According to the TNM classification, 2 patients (4.3%) were stage I, 20 patients (43.4%) stage II, 18 patients (39.1%) stage III, and 2 patients (4 3%) stage IV. The APC, DKK2 and SFRP5 genes showed more frequent methylation profile in the case group (p<0.001, p<0.001 and p<0.001 respectively). The RUNX3 and MLH1 gene had not differences in methylation profiles between the groups (p=0.98 and p=0.08 respectively). The qualitative methylation profile of FDNA showed no statistically significant difference between groups (p =1.000) in the APC gene, while the FDNA DKK2 methylation profile was more frequent in the case group with p=0.025. Our results indicated the potential of DKK2, SFRP5 and APC as epigenetic markers for the combined diagnosis of CRC in tissue and DKK2 in plasma. A possible marker panel could also be a screening tool cost effective for the detection of patients with CRC. Patients with hypermethylation of FDNA DKK2gene would be possible candidates for colonoscopy.
Epigenetic changes can be detected in precancerous lesions in colorectal cancer (CRC), suggesting a potential involvement in the initial process of carcinogenesis. The potential use of molecular markers in plasma has been researched to detect epigenetic changes in free circulating tumor DNA (FDNA) in blood. Our study compared the changes in the level of DNA methylation found in normal and tumor tissues with blood seeking association of epigenetic markers in the detection of CRC. The candidate genes was performed using the technique Methyl-ProfilerTM PCR Array System (SABiosciences) were selected 5 markers that through the technique of High Resolution Melting (HRM). The 5 genes selected in the first phase of the study were APC, DKK2, SFRP5, MLH1 and RUNX3. Among the 68 controls, 42 were women and the mean (SD) age was 59.9 years (13.4). Among the 46 individuals with CRC, 25 were women and the mean age was 64.6 years (13.6). In the case group 28 patients had colon cancer (60.8%) and 18 (39.2%) had rectal cancer. According to the TNM classification, 2 patients (4.3%) were stage I, 20 patients (43.4%) stage II, 18 patients (39.1%) stage III, and 2 patients (4 3%) stage IV. The APC, DKK2 and SFRP5 genes showed more frequent methylation profile in the case group (p<0.001, p<0.001 and p<0.001 respectively). The RUNX3 and MLH1 gene had not differences in methylation profiles between the groups (p=0.98 and p=0.08 respectively). The qualitative methylation profile of FDNA showed no statistically significant difference between groups (p =1.000) in the APC gene, while the FDNA DKK2 methylation profile was more frequent in the case group with p=0.025. Our results indicated the potential of DKK2, SFRP5 and APC as epigenetic markers for the combined diagnosis of CRC in tissue and DKK2 in plasma. A possible marker panel could also be a screening tool cost effective for the detection of patients with CRC. Patients with hypermethylation of FDNA DKK2gene would be possible candidates for colonoscopy.
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Citação
SILVA, Tiago Donizetti da. Estudo de alterações epigenéticas como biomarcadores no diagnóstico não invasivo do câncer colorretal. 2014. Tese (Doutorado) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2014.