Análise mutacional do gene supressor de tumor TP53 e imunorreatividade da p53 em linfomas caninos
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Data
2009-09-18
Autores
Calazans, Sabryna Gouveia [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Sabendo-se da influência das mutações no gene TP53 no desenvolvimento das neoplasias e da discrepância entre os resultados obtidos pelas técnicas de sequenciamento e imunoistoquímica, esta pesquisa teve como objetivo avaliar a sequência do TP53 e a imunorreatividade da p53, relacionando-as com fatores clínicopatológicos em cães com linfoma. Foram avaliados 12 cães com linfoma e cinco cães sadios. Amostras de neoplasia/linfonodo e de bulbo piloso foram obtidas. O diagnóstico histopatológico foi determinado pela classificação de Kiel. O imunofenótipo e a imunomarcação da p53 foram determinados por imunoistoquímica. Para a imunomarcação com a p53, utilizou-se anticorpo policlonal anti-p53 (CM1) na diluição de 1:500. A região do gene TP53 compreendida entre os exons quatro e nove (incluindo íntrons) foi amplificada por PCR e submetida ao sequenciamento. A média da imunorreatividade da p53 nos cães com linfoma foi 27,33%±12,14%, diferente da média do grupo controle (0,75±0,5) (P<0,05). A ausência de marcação da p53 foi observada em apenas um caso de linfoma de baixo grau de malignidade, cujo cão apresentou um ano de sobrevida. O outro caso de linfoma de baixo grau de malignidade apresentou a menor marcação da p53 (14,12%). O linfoma com maior marcação da p53 (50,56%) consistiu em um linfoma alimentar cujo cão evoluiu para o óbito no dia da consulta. As maiores médias de marcação foram observadas nos linfomas cutâneos, estádio clínico V, subestádio “b”, imunofenótipo B e tempo de sobrevida de até trinta dias, sem diferença estatística (P>0,05). Apesar dos resultados obtidos pela imunoistoquímica, nenhuma mutação foi encontrada nas sequências analisadas...
TP53 mutations are usually involved in cancer, but sequencing and immunohistochemistry results are often controversial. Thus, the aim of this study was to analyse both TP53 sequence and p53 immunostaining and to associate their findings to clinical-pathological features in dogs with lymphoma. Twelve dogs with lymphoma and five healthy dogs were included in this study. Tumor and hair sample was collected. Histopathological diagnosis was performed according to Kiel classification. Immunohistochemistry was performed to identify immunophenotype as well as p53 expression. Polyclonal antibody anti-p53 (CM1) was used at a 1:500 dilution. The region that encompasses exons 4-9 was amplified by means of PCR reactions and sequencing was then performed. p53 immunolabeling mean was 27,33%±12,14 in canine lymphoma group. It was statistically different from control group mean (0,75±0,5) (P<0,05). Only one p53-negative case was observed in a dog with low grade lymphoma, which presented one-year survival. Other low grade lymphoma presented lowest p53 immunoreactivity (14,12%). Highest reactivity (50,56%) was observed in dog with alimentary lymphoma, which died in the day of diagnosis. The highest immunolabeling means were verified in cutaneous presentation, clinical stage V, substage “b”, immunophenotype B and survival time up to 30 days, but there was no significant difference (P>0,05). Nevertheless, gene mutations were not observed in any sequence. In conclusion, p53 immunostaining is not essentially related to mutation in the gene TP53, particularly to the region between exons 4-9
TP53 mutations are usually involved in cancer, but sequencing and immunohistochemistry results are often controversial. Thus, the aim of this study was to analyse both TP53 sequence and p53 immunostaining and to associate their findings to clinical-pathological features in dogs with lymphoma. Twelve dogs with lymphoma and five healthy dogs were included in this study. Tumor and hair sample was collected. Histopathological diagnosis was performed according to Kiel classification. Immunohistochemistry was performed to identify immunophenotype as well as p53 expression. Polyclonal antibody anti-p53 (CM1) was used at a 1:500 dilution. The region that encompasses exons 4-9 was amplified by means of PCR reactions and sequencing was then performed. p53 immunolabeling mean was 27,33%±12,14 in canine lymphoma group. It was statistically different from control group mean (0,75±0,5) (P<0,05). Only one p53-negative case was observed in a dog with low grade lymphoma, which presented one-year survival. Other low grade lymphoma presented lowest p53 immunoreactivity (14,12%). Highest reactivity (50,56%) was observed in dog with alimentary lymphoma, which died in the day of diagnosis. The highest immunolabeling means were verified in cutaneous presentation, clinical stage V, substage “b”, immunophenotype B and survival time up to 30 days, but there was no significant difference (P>0,05). Nevertheless, gene mutations were not observed in any sequence. In conclusion, p53 immunostaining is not essentially related to mutation in the gene TP53, particularly to the region between exons 4-9
Descrição
Palavras-chave
Dog, Lymphosarcoma, Cão, Cancer em cão, Linfossarcoma, p53
Como citar
CALAZANS, Sabryna Gouveia. Análise mutacional do gene supressor de tumor TP53 e imunorreatividade da p53 em linfomas caninos. 2009. xvii, 53 f. Tese (doutorado) - Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, 2009.