Determinação do perfil lipidômico de soro humano para rastreamentode potenciais biomarcadores de carcinoma hepatocelular associado às hepatites virais
Data
2015-08-27
Autores
Passos-Castilho, Ana Maria 
Orientadores
Granato, Celso Francisco Hernandes
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: A lipidômica é a ciência que visa identificar e quantificar o perfil global ou parcial de lipídios presentes em um sistema biológico em um determinado tempo. O carcinoma hepatocelular é o sexto câncer mais comum em todo o mundo e as hepatites crônicas B e C representam a sua principal causa. Biomarcadores para o diagnóstico do carcinoma hepatocelular ainda são escassos. Objetivos: Avaliar a aplicabilidade da lipidômica como ferramenta para rastreamento de biomarcadores de carcinoma hepatocelular, desenvolver e padronizar um método de lipidômica eficaz para detecção global e quantificação relativa de lipídios em amostras de sangue periférico humano, e determinar o perfil lipidômico de soro humano no carcinoma hepatocelular associado às hepatites virais B e C para rastrear biomarcadores com potencial diagnóstico. Métodos: Primeiramente realizou-se um estudo piloto em que o perfil lipidômico de 25 pacientes com carcinoma hepatocelular associado à hepatite C obtido por espectrometria de massas por dessorção/ionização a laser assistida por matriz foi comparado ao de 15 pacientes com cirrose hepática e 25 pacientes com hepatite crônica. Em seguida, desenvolveu-se e padronizou-se um método de lipidômica utilizando cromatografia líquida de ultra eficiência acoplada à espectrometria de massas por ionização por spray de elétrons. A seguir, realizou-se um estudo de aplicação do método padronizado com pacientes com hepatite B crônica em que foram incluídos 32 pacientes com carcinoma hepatocelular, 30 com cirrose hepática, 25 com hepatite B crônica e 34 controles saudáveis. Por fim, realizou-se um estudo de aplicação do método padronizado em pacientes com hepatite C crônica, com 56 pacientes com carcinoma hepatocelular, 59 com cirrose hepática, 62 com hepatite C xx crônica e 58 controles saudáveis. Candidatos a potenciais biomarcadores foram selecionados por análise estatística e curvas de característica de operação do receptor foram geradas para comparar a acurácia dos biomarcadores selecionados e níveis de alfafetoproteína no diagnóstico do carcinoma hepatocelular. As bases The Human Metabolome Database e The LIPID MAPS Lipidomics Gateway foram pesquisadas para a identificação dos candidatos por meio de sua relação massa/carga. Resultados: O estudo-piloto inicial resultou em um algoritmo de 7 picos que foi capaz de distinguir carcinoma hepatocelular de cirrose hepática com sensibilidade de 96% e especificidade de 87%. Em seguida, o método desenvolvido de cromatografia líquida de ultra eficiência acoplada à espectrometria de massas por ionização por spray de elétrons apresentou boa performance na separação de diferentes grupos estudados e na detecção de diferentes espécies de lipídios de várias ordens de grandeza. A seguir, o perfil lipidômico distinguiu os pacientes com câncer e forneceu um diagnóstico mais preciso para os pacientes com cirrose hepática que a detecção convencional de alfafetoproteína. Foi possível distinguir carcinoma hepatocelular associado à hepatite B com sensibilidade de 78% e especificidade de 64%. Por fim, pôde-se distinguir carcinoma hepatocelular associado à hepatite C de cirrose hepática com 100% de sensibilidade e especificidade utilizando 13 candidatos, incluindo a PC(32:1). Conclusões: O presente estudo confirmou a possibilidade de aplicação de um método lipidômico por cromatografia líquida de ultra eficiência acoplada à espectrometria de massas para a distinção entre carcinoma hepatocelular e cirrose hepática e identificou a PC(32:1) como um potencial novo biomarcador eficaz para a detecção precoce do carcinoma hepatocelular associado à hepatite C crônica.
Introduction: Lipidomics is the science that aims to identify and quantify the lipid profile of a biological system in a given time. Hepatocellular carcinoma is the sixth most common cancer worldwide and chronic hepatitis B and C represent its main cause. Biomarkers for hepatocellular carcinoma are still lacking. Objectives: To evaluate the applicability of lipidomics as a screening tool for hepatocellular carcinoma biomarkers, to develop and standardize an efficient lipidomics method capable of global detection and relative quantification of lipids in human peripheral blood samples, and to determine the human serum lipidomic profile of viral hepatitis-related hepatocellular carcinoma to screen for biomarkers with diagnostic potential. Methods: First a pilot study was conducted through the lipidomic profiling of 25 patients with hepatitis C-related hepatocellular carcinoma using matrix-assisted laser desorption/ionization, in comparison to 15 patients with liver cirrhosis and 25 patients with chronic hepatitis. Subsequently, an ultraperformance liquid chromatography electrospray ionization mass spectrometry lipidomic method was developed and standardized. Afterwards, a study applying the standardized method on hepatitis B patients was conducted with 32 patients with hepatocellular carcinoma, 30 with liver cirrhosis, 25 with chronic hepatitis B and 34 healthy controls. Finally, a study applying the standardized method on hepatitis C patients was conducted with 56 patients with hepatocellular carcinoma, 59 with liver cirrhosis, 62 with chronic hepatitis C and 58 healthy controls. Potential biomarkers were selected using statistical analysis and receiver operating characteristic curves were performed to compare the accuracy of selected biomarkers and alpha-fetoprotein levels in hepatocellular carcinoma diagnosis. The Human Metabolome Database e The LIPID MAPS Lipidomics Gateway were queried with the mass to charge ratio to identify the candidates. Results: The initial pilot study generated a 7-ion algorithm capable of distinguishing hepatocellular carcinoma from liver cirrhosis with 96% sensitivity and 87% specificity. Then, the developed ultraperformance liquid chromatography electrospray ionization mass spectrometry method presented good performance in the separation of the different study groups and in the detection of different lipids species of several orders of magnitude. Subsequently, the lipidomic profile distinguished cancer patients and provided more accurate diagnosis for liver cirrhosis patients than conventional alphafetoprotein detection. Hepatitis B-related hepatocellular carcinoma was distinguished from liver cirrhosis with up to 78% sensitivity and 64% specificity. Finally, hepatitis Crelated hepatocellular carcinoma was distinguished from liver cirrhosis with 100% sensitivity and specificity using 13 candidates, including PC(32:1). Conclusions: This study confirmed the feasibility of using a lipidomic method for differentiating hepatocellular carcinoma and liver cirrhosis and identified PC(32:1) as an effective new potential biomarker for early detection of hepatocellular carcinoma.
Introduction: Lipidomics is the science that aims to identify and quantify the lipid profile of a biological system in a given time. Hepatocellular carcinoma is the sixth most common cancer worldwide and chronic hepatitis B and C represent its main cause. Biomarkers for hepatocellular carcinoma are still lacking. Objectives: To evaluate the applicability of lipidomics as a screening tool for hepatocellular carcinoma biomarkers, to develop and standardize an efficient lipidomics method capable of global detection and relative quantification of lipids in human peripheral blood samples, and to determine the human serum lipidomic profile of viral hepatitis-related hepatocellular carcinoma to screen for biomarkers with diagnostic potential. Methods: First a pilot study was conducted through the lipidomic profiling of 25 patients with hepatitis C-related hepatocellular carcinoma using matrix-assisted laser desorption/ionization, in comparison to 15 patients with liver cirrhosis and 25 patients with chronic hepatitis. Subsequently, an ultraperformance liquid chromatography electrospray ionization mass spectrometry lipidomic method was developed and standardized. Afterwards, a study applying the standardized method on hepatitis B patients was conducted with 32 patients with hepatocellular carcinoma, 30 with liver cirrhosis, 25 with chronic hepatitis B and 34 healthy controls. Finally, a study applying the standardized method on hepatitis C patients was conducted with 56 patients with hepatocellular carcinoma, 59 with liver cirrhosis, 62 with chronic hepatitis C and 58 healthy controls. Potential biomarkers were selected using statistical analysis and receiver operating characteristic curves were performed to compare the accuracy of selected biomarkers and alpha-fetoprotein levels in hepatocellular carcinoma diagnosis. The Human Metabolome Database e The LIPID MAPS Lipidomics Gateway were queried with the mass to charge ratio to identify the candidates. Results: The initial pilot study generated a 7-ion algorithm capable of distinguishing hepatocellular carcinoma from liver cirrhosis with 96% sensitivity and 87% specificity. Then, the developed ultraperformance liquid chromatography electrospray ionization mass spectrometry method presented good performance in the separation of the different study groups and in the detection of different lipids species of several orders of magnitude. Subsequently, the lipidomic profile distinguished cancer patients and provided more accurate diagnosis for liver cirrhosis patients than conventional alphafetoprotein detection. Hepatitis B-related hepatocellular carcinoma was distinguished from liver cirrhosis with up to 78% sensitivity and 64% specificity. Finally, hepatitis Crelated hepatocellular carcinoma was distinguished from liver cirrhosis with 100% sensitivity and specificity using 13 candidates, including PC(32:1). Conclusions: This study confirmed the feasibility of using a lipidomic method for differentiating hepatocellular carcinoma and liver cirrhosis and identified PC(32:1) as an effective new potential biomarker for early detection of hepatocellular carcinoma.
Descrição
Citação
PASSOS, Ana Maria. Determinação do perfil lipidômico de soro humano para rastreamentode potenciais biomarcadores de carcinoma hepatocelular associado às hepatites virais. 2015. 190 f. Tese (Doutorado em Infectologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2015.