Study of a Germline Variant in Lymphotoxin Alpha (LTA) Gene in Colorectal Cancer
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2019-02-15
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O cancro colorretal (CRC) representa a quarta causa de morte relacionada com o cancro no mundo. Sendo causada por diversos fatores de risco como a dieta, história familiar, doenças inflamatórias, o cancro colorretal é influenciado por alterações em genes importantes para a função celular. A linfotoxina alfa (LTA) pertence à superfamília do fator de necrose tumoral. Esta citocina é expressa pelos linfócitos T e B, células dendríticas e linfócitos NK. LTA pode ligar-se ao recetor linfotoxina-beta e aos recetores fator de necrose tumoral. Após a ligação aos seus recetores nas células no microambiente tumoral, a proteína LTA pode regular a apoptose, proliferação, sobrevivência e a diferenciação. No cancro, a sua influência ainda não está bem explicada. Foram realizados alguns estudos, contudo os resultados não são conclusivos. A LTA pode estar associada com atividade anti-tumoral tendo efeitos citotóxicos nas células de cancro pelo recrutamento de células NK para a lesão. Contudo, outros estudos têm revelado que LTA pode promover o crescimento celular e a adesão das células de cancro. Desta forma, os objetivos deste estudo foram caracterizar e analisar se os genótipos do polimorfismo funcional rs1041981 do LTA, particularmente a sobrevivência global e a sobrevivência livre de progressão em pacientes com cancro colorretal e avaliar os macrófagos e linfócitos T em tecidos CRC, por imunohistoquímica, e a sua associação com os dados dos genótipos. Para realizar o estudo, foram recolhidas amostras sanguíneas em 172 pacientes sobreviventes com CRC no departamento de Oncologia, Centro Hospitalar de Trás-os-Montes e Alto Douro (CHTMAD) pelos clínicos. As amostras foram transportadas para o departamento de Genética e Biotecnologia na UTAD, para separação em soro, plasma e buffy coat. Foi otimizado um protocolo de extração de DNA, e foi realizado PCR em tempo-real para descriminação alélica usando sondas Taqman e subsequente confirmação por sequenciação de um amplicão específico. O alelo C e o alelo A apresentaram, respetivamente, frequências alélicas de 70 % e 30 %. As frequências genotípicas do CC, CA e AA foram 49%, 42% e 9%, respetivamente. Considerando os parâmetros clinico-patológicos e a associação com os dados genéticos, foi possível colocar em evidência que as variáveis que influenciam a sobrevivência dos pacientes com CRC nesta população foram a percentagem de linfócitos no sangue, a localização e o lado do tumor, terapia adjuvante e o genótipo CA/AA pelo modelo dominante. O alelo A parece ter um efeito protetor para os pacientes com cancro colorretal nesta população para o endpoints primário e secundário, respetivamente a sobrevivência global e a sobrevivência livre de progressão. Os pacientes com a asparagina na proteína LTA parecem ter um melhor prognóstico que os pacientes com a treonina na proteína LTA na mesma posição. Os resultados para a possível associação entre TAMs e TILs no cancro colorretal e a variante LTA rs1041981 revelaram diferenças significativas no rácio TILs/TAMs entre os indivíduos CC e CA no estudo. Este gene, como demostrado neste trabalho, parece ter uma importante função no cancro colorretal. A compreensão do impacto deste polimorfismo na evolução da doença pode trazer novas informações na regulação do microambiente tumoral.
The colorectal cancer (CRC) represents the fourth cause of cancer death in the world. Being caused by several risk factors as diet, family history, inflammatory diseases, the colorectal cancer can be influenced by alterations in important genes to cell function. Lymphotoxin alpha (LTA) belongs to tumor necrosis factor superfamily. This cytokine is expressed by T and B lymphocytes, dendritic cells and NK lymphocytes. LTA can bind to lymphotoxin-beta receptor and tumor necrosis factor receptors. After binding to its receptors in tumor microenvironment cells, LTA protein can regulate apoptosis, proliferation, survival and differentiation. In cancer, its influence is not well explained. Some studies were already performed; however, the results are not conclusive. The LTA may be associated with anti-tumor activity having cytotoxic effects on cancer cells by recruitment of NK cells to lesion. However, other studies have revealed that LTA can promote the cell growth and adhesion of cancer cells. Thus, the objectives of this study were to characterize and analyze whether genotype of LTA functional polymorphism rs1041981 influences clinicopathological parameters, particularly overall and progression-free survival in colorectal cancer patients and evaluate the macrophage and T lymphocytes in the CRC tissues, by immunohistochemistry, and its association with genotype data. To perform the study, blood samples of 172 survivor patients with CRC were collected in the Department of Oncology, Centro Hospitalar de Trás-os-Montes e Alto Douro (CHTMAD) by clinical researchers. The samples were transported to the Department of Genetics and Biotechnology at UTAD, to separate in serum, plasma and buffy coat. A DNA extraction protocol was optimized, and the allele discrimination was performed by real time-PCR using Taqman probes and subsequent confirmation by sequencing of a specific LTA amplicon. The C and A alleles presented, respectively, allelic frequency of 70 % 30 %. CC, CA and AA genotypic frequencies were 49%, 42% and 9%, respectively. Considering the clinicopathological parameters and the association with the genetic data, it was possible to put in evidence that the variables that influence the survival of CRC patients under study were blood lymphocytes percentage, tumor side and localization, adjuvant chemotherapy and the genotype CA/AA, by a dominant model. The A allele appears to be a protective factor for patients with colorectal cancer in this population for primary and secondary endpoints, respectively overall survival and progression-free survival. The patients with asparagine in LTA protein appear to have a better prognosis than patients that have the threonine in the same position. The results of possible association between TAMs and TILs in colorectal tumors and rs1041981 LTA variant revealed statistical differences in TILs/TAMs ratio between CC and CA individuals under study. This gene, as evidenced in this work, appears to have an important role in colorectal cancer. The understanding of the impact of this polymorphism on the evolution of the disease can bring new information on tumor microenvironment regulation.
The colorectal cancer (CRC) represents the fourth cause of cancer death in the world. Being caused by several risk factors as diet, family history, inflammatory diseases, the colorectal cancer can be influenced by alterations in important genes to cell function. Lymphotoxin alpha (LTA) belongs to tumor necrosis factor superfamily. This cytokine is expressed by T and B lymphocytes, dendritic cells and NK lymphocytes. LTA can bind to lymphotoxin-beta receptor and tumor necrosis factor receptors. After binding to its receptors in tumor microenvironment cells, LTA protein can regulate apoptosis, proliferation, survival and differentiation. In cancer, its influence is not well explained. Some studies were already performed; however, the results are not conclusive. The LTA may be associated with anti-tumor activity having cytotoxic effects on cancer cells by recruitment of NK cells to lesion. However, other studies have revealed that LTA can promote the cell growth and adhesion of cancer cells. Thus, the objectives of this study were to characterize and analyze whether genotype of LTA functional polymorphism rs1041981 influences clinicopathological parameters, particularly overall and progression-free survival in colorectal cancer patients and evaluate the macrophage and T lymphocytes in the CRC tissues, by immunohistochemistry, and its association with genotype data. To perform the study, blood samples of 172 survivor patients with CRC were collected in the Department of Oncology, Centro Hospitalar de Trás-os-Montes e Alto Douro (CHTMAD) by clinical researchers. The samples were transported to the Department of Genetics and Biotechnology at UTAD, to separate in serum, plasma and buffy coat. A DNA extraction protocol was optimized, and the allele discrimination was performed by real time-PCR using Taqman probes and subsequent confirmation by sequencing of a specific LTA amplicon. The C and A alleles presented, respectively, allelic frequency of 70 % 30 %. CC, CA and AA genotypic frequencies were 49%, 42% and 9%, respectively. Considering the clinicopathological parameters and the association with the genetic data, it was possible to put in evidence that the variables that influence the survival of CRC patients under study were blood lymphocytes percentage, tumor side and localization, adjuvant chemotherapy and the genotype CA/AA, by a dominant model. The A allele appears to be a protective factor for patients with colorectal cancer in this population for primary and secondary endpoints, respectively overall survival and progression-free survival. The patients with asparagine in LTA protein appear to have a better prognosis than patients that have the threonine in the same position. The results of possible association between TAMs and TILs in colorectal tumors and rs1041981 LTA variant revealed statistical differences in TILs/TAMs ratio between CC and CA individuals under study. This gene, as evidenced in this work, appears to have an important role in colorectal cancer. The understanding of the impact of this polymorphism on the evolution of the disease can bring new information on tumor microenvironment regulation.
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Master’s degree in Biotechnology for Health Sciences
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cancro colorretal , LTA , SNP , células inflamatórias