Análise do perfil de expressão gênica em amostras de linfoma de Hodgkin clássico: estudo da patogênese com ênfase no papel da infecção pelo vírus de Epstein-Barr / Gene expression analysis in classical Hodgkin lymphoma: a pathogenetic study emphasizing the role of Epstein-Barr virus infection

RESUMO

O linfoma de Hodgkin clássico é uma neoplasia linfóide monoclonal caracterizada pela presença de raras células de Hodgkin e Reed-Sternberg em meio a um infiltrado inflamatório abundante constituído por linfócitos, eosinófilos, plasmócitos, macrófagos e neutrófilos. Aspectos específicos da patogênese desta neoplasia, particularmente as alterações que impedem a entrada das células neoplásicas em apoptose, não são ainda totalmente conhecidos... Material e métodos. Foram utilizadas 3 linhagens celulares negativas para o EBV (L428, L1236 e KM-H2) e uma linhagem positiva (L591), gentilmente cedidas pelo Prof. Dr. Harald Stein, e 10 amostras de tecido fresco congelado envolvidas por linfoma de Hodgkin clássico, fornecidas pelo Banco de Tumores do Hospital A C Camargo... Blocos de parafina contendo material conservado em formalina de 148 casos de LHC foram selecionados do arquivo do Departamento de Patologia do Centro de Tratamento e Pesquisa Hospital A C Camargo, São Paulo, Brasil, no período de 1970 a 2005... Resultados. Foi observada expressão diferencial de 756 genes, que após análise funcional se agruparam em diversos grupos relevantes, incluindo os grupos de genes relacionados à sinalização célula-célula, ao desenvolvimento do sistema imune, envolvidos na regulação da via de NFkB, e quimiotaxia... Conclusões. Os resultados do presente estudo sugerem a capacidade das CHRS de explorarem diversas vias de sinalização para alterar seu ciclo-celular e controle mitótico, assim como evadir ao sistema de imunovigilância do organismo...

ABSTRACT

Background. Classical Hodgkin lymphoma is a monoclonal lymphoid neoplasm whose hallmark is the Reed-Sternberg cell and its variants, which are surrounded by an inflamatory background. Although extensively studied, many aspects of its pathogenesis, especially those involving the programmed cell death pathway, are still not understood. The role of Epstein-Barr virus (EBV), which is detected in approximately 50% of Hodgkin lymphomas, is also not well established. Although data on EBV association with clinical outcome of Hodgkin lymphoma patients are controversial, some studies suggest that the cases in which EBV is detected follow a different pathway leading to apoptosis blockage.This can stimulate the search for new treatments based on the presence or absence of EBV. Studies employing differential gene expression techniques can add new information to help solving these questions. Objective. This study analyzed the gene expression pattern of Hodgkin cell lines and tissues involved by classical Hodgkin lymphoma, comparing the differences related to the presence or absence of Epstein-Barr virus infection. Additionally, a Tissue Microarray containing cases of classical Hodgkin lymphoma was constructed to validate the protein expression of some of the genes observed in the analysis, comparing the results with Epstein-Barr status and clinical outcome of the patients. Materials and methods. Three EBV-negative Hodgkin cell lines (L428, L1236 e KM-H2), one EBV-positive Hodgkin cell line (L591), and 10 classical Hodgkin lymphoma frozen tissue samples were used in the gene expression analysis study. Following total RNA extraction, cRNA probes were hybridized in oligoarray glass slides. Data from the image acquisition were submitted to quality control pre-analysis prior to bioinformatic statistical analysis for gene clustering experiments and functional analysis. Paraffin-blocks from 148 retrospective cases of classical Hodgkin Lymphoma, diagnosed between 1970 and 2005, were retrieved from the archives of the Department of Pathology. Cases without enough formalin-fixed and paraffin-embedded tissue to run the immunohistochemical (IHC) reactions, relapse biopsies and HIVassociated HL were excluded. Histological diagnosis was revised, with the use of immunostains when necessary. A tissue microarray was built and immunostains performed with antibodies against Aurora-B, Caspase-1, Caveolin-1, CCL20, CDC2, MMP9 and LMP-1. Cases were also tested for EBV using "in situ" hybridization for "EBV early RNAs" (EBER-1). The reactions were analyzed and the results submitted to statistical analysis. Results. We observed that 756 genes are differentially expressed between EBV-positive and EBV-negative Hodgkin cell lines. These genes are functionally related to many relevant groups, such as cell-cell signaling, immune system development, NFkB regulation and chemotaxis. Some genes belonging to these groups were selected for immunoistochemical validation (Aurora-B, Caspase-1, Caveolin-1, CCL20, CDC2 and MMP9), whose protein expression was detected in 58,78%, 38,51%, 25,68%, 49,32%, 75,68% e 52,03% of the cases, respectively. CCL20 protein expression was specifically associated with EBV-infection (p<0,0001). Disease-specific survival rates of patients between 15 and 45 years who expressed Caspase-1 and MMP9 in neoplastic cells were significantly lower than those who did not express these markers. The expression of MMP9 by neoplastic cells was an independent prognostic factor is this group of patients. Conclusions. The results of this study suggest the ability of Hodgkin-ReedSternberg cells to explore different signaling pathways to control their cell-cycle and mitotic activity, as well as evade immunosurveillance, regulating different genes according to EBV infection status. CCL20 protein expression is associated with EBV infection in Hodgkin lymphoma cases. We also observed the expression of new proteins by Hodgkin-Reed-Sternberg cells, such as Caspase-1 and Caveolin-1. The expression of Caspase-1 and MMP9 by Hodgkin-Reed-Sternberg cells associates with a poor outcome in Hodgkin lymphoma patients between 15 and 45 years. MMP9 expression by neoplastic cells is an independent prognostic factor in this group of patients.