Patients with rheumatoid arthritis (RA) are at increased risk of developing malignant lymphoma, although details about this association remain unclear. The aims of this thesis were to investigate risk factors for lymphoma in patients with RA and to characterize these lymphomas regarding subtype, presence of Epstein-Barr virus (EBV), clinical manifestations and prognosis. The Swedish hospital discharge register and the cancer register were used to identify RA patients with lymphoma. Two case-control studies were performed, one smaller including RA patients with lymphoma hospitalised in Uppsala health care region 1964-1983 (n=41) and one larger study of hospitalised RA patients with lymphoma in Sweden 1964-1995 (n=378). RA patients from the same cohorts, but without lymphoma, were matched as controls. Medical records for cases and controls were scrutinized for exposure information. The lymphoma tissues were reclassified according to the WHO classification, and presence of EBV was analysed by EBER in situ hybridisation. The most important risk factor for lymphoma development was high RA disease activity. No association was determined between treatment with traditional disease modifying drugs, non-steroidal anti-inflammatory drugs, aspirin, peroral and intra-articular corticosteroids and lymphoma risk. Diffuse large B-cell lymphoma (DLBCL) was more frequent in RA patients than in lymphoma patients in the general population and displayed stronger association with RA disease activity than other lymphoma subtypes. RA patients with DLBCL had increased extranodal involvement and more advanced lymphoma stage at presentation than DLBCL patients in general, and the prognosis was poor. A further subdivision of DLBCL into germinal centre (GC) and non-GC subtypes by the expression patterns of CD10, bcl-6 and IRF-4 showed a predominance of the non-GC subtype. This suggested peripheral activated B-cells as the cells of origin in these lymphomas. The presence of EBV was low in lymphomas in RA patients (12%).

Le virus Epstein-Barr (VEB) est un pathogène opportuniste qui a la capacité d’immortaliser les lymphocytes B et de provoquer une prolifération maligne, appelée syndrome lymphoprolifératif post-transplantation (SLP), chez les individus immunodéprimés. A l’intérieur de ce groupe, les personnes à plus haut risque sont les enfants, puisqu’ils sont à risque de développer une infection primaire par le VEB pendant leur régime d’immunosuppression post-greffe. Dans le but de développer un anticorps préventif, notre laboratoire s’est attardé au rôle du cycle lytique du VEB dans le développement du SLP. À cette fin, le premier objectif du présent projet vise à fournir la preuve expérimentale de l’existence ou non d’une phase réplicative productive pendant l’infection aiguë des lymphocytes B sanguins. Un examen des événements qui se déroulent au tout début de l’infection par le VEB tant au niveau de la réplication virale qu’au niveau de l’expression des gènes lytiques précoces et tardifs a révélé l’existence d’une phase réplicative productive pendant l’infection aiguë. Ceci a permis de justifier l’élaboration, dans notre laboratoire, d’un anticorps chimère (murin-humain) neutralisant, dirigé contre la protéine gp350 située sur l’enveloppe virale. Le deuxième objectif, quant à lui, vise à fournir la preuve expérimentale de la capacité neutralisante de cet anticorps chimère. Des essais de caractérisation in vitro ont démontré une capacité de reconnaissance de la protéine cible, notamment la gp350, et une capacité de neutralisation du virus par l’anticorps chimère. L’anticorps chimère anti-gp350 pourra faire l’objet d’essais précliniques in vivo en vue d’évaluer sa capacité à reconnaître le virus et à prévenir l’apparition de tumeurs de type SLP chez les souris SCID. Il pourrait être éventuellement utilisé, par la suite, comme traitement préemptif contre les tumeurs dans l’espoir de mieux gérer les patients à risque de développer un SLP. / Epstein-Barr virus (EBV) is an opportunistic pathogen in immunocompromised transplant patients. In these patients EBV infection can lead to malignant B-cell lymphoproliferation, called post-transplant lymphoproliferative disease (PTLD). This thesis project aimed to investigate the role of lytic EBV infection in the genesis of PTLD. The first experimental objective was to provide in vitro proof that EBV could induce productive replication upon acute in vitro infection of B cells. Data obtained through study of viral DNA replication and transcription during the first 96 hours post-infection indicate that lytic infection does occur. These results provided justification for proceeding to the second experimental objective which involved the characterization of an anti-gp350 human-mouse chimeric antibody for its capacity to recognize and neutralize EBV. Results showed that this antibody did possess neutralization activity. Further study of this anti-gp350 chimeric antibody in SCID mice is necessary in order to evaluate its in vivo efficacy against PTLD.

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that establishes a life-long latent infection of B cells. It is usually asymptomatic in healthy individuals; however, individuals with compromised immunity often develop EBV-induced lymphoma. EBV also encodes potential oncogenes that can contribute to tumorigenesis. Therefore, vaccine and immunotherapeutic strategies targeting EBV are desirable. Recent studies have shown that infusion of EBV-specific CD8+T cells can elicit remission of lymphomas arising after administration of immunosuppressive drugs during transplantation, suggesting an important role for T cells in the prevention of EBV-induced malignancy. A better understanding of the cellular immune components involved in the control of EBV will aid in the development of methods to prevent infection and/or treat EBV-associated disease. While EBV infection is usually acquired asymptomatically during childhood, primary infection of adolescents and young adults can result in an illness termed acute infectious mononucleosis (AIM). Because of the characteristic symptoms of the illness, individuals with AIM can be readily identified and diagnosed with acute EBV infection. Thus, primary CD4+ and CD8+ T cell responses against the virus can be evaluated. It has been previously found that there is a marked expansion of lytic EBV protein-specific CD8+ T cells early during AIM, with delayed detection of lower frequencies of latent EBV protein-specific CD8+ T cells. The magnitude and specificity of CD4+T cell responses during AIM has been less well characterized. This thesis dissertation presents data from both functional assays and direct staining experiments documenting the timing, magnitude, and antigen-specificity of CD4+ T cells over the course of primary EBV infection. Lytic and latent protein-specific CD4+ T cells were readily detected by intracellular IFN-γ production at presentation with AIM and declined rapidly thereafter. Blood EBV load was also quantitated and found to decrease over time following AIM. By contrast, CD8+T cell IFN-y responses remained high for several weeks following presentation with AIM. Direct staining of lytic epitope-specific CD4+ T cells during AIM revealed high frequencies of virus-specific cells with low proliferative and IFN-γ-producing potential. Blood EBV load in these patients was persistently high through 6 wk following AIM. These data suggest a relationship between high EBV load during acute infection and impaired EBV-specific CD4+ T cell responses, which are compatible with impaired CD4+ T cell responses reported during high viremia associated with other viral infections. This may represent a mechanism by which persistent viruses, such as EBV, are able to establish a life-long infection in their hosts.

No description available.

Made available in DSpace on 2015-07-22T20:49:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-24. Added 1 bitstream(s) on 2015-08-11T03:26:30Z : No. of bitstreams: 1 Publico-429a.pdf: 1596515 bytes, checksum: 2107c026687c3e2ecc62f0a5fffa1409 (MD5). Added 1 bitstream(s) on 2015-08-11T03:26:30Z : No. of bitstreams: 2 Publico-429a.pdf: 1596515 bytes, checksum: 2107c026687c3e2ecc62f0a5fffa1409 (MD5) Publico-429b.pdf: 1614171 bytes, checksum: d98715f7a666adc5ceb185cce978000e (MD5). Added 1 bitstream(s) on 2015-08-11T03:26:30Z : No. of bitstreams: 3 Publico-429a.pdf: 1596515 bytes, checksum: 2107c026687c3e2ecc62f0a5fffa1409 (MD5) Publico-429b.pdf: 1614171 bytes, checksum: d98715f7a666adc5ceb185cce978000e (MD5) Publico-429c.pdf: 1500700 bytes, checksum: 77791620b06c9404d28496c4f4ca3dc3 (MD5). Added 1 bitstream(s) on 2015-08-11T03:26:30Z : No. of bitstreams: 4 Publico-429a.pdf: 1596515 bytes, checksum: 2107c026687c3e2ecc62f0a5fffa1409 (MD5) Publico-429b.pdf: 1614171 bytes, checksum: d98715f7a666adc5ceb185cce978000e (MD5) Publico-429c.pdf: 1500700 bytes, checksum: 77791620b06c9404d28496c4f4ca3dc3 (MD5) Publico-429d.pdf: 1826591 bytes, checksum: 70c3dc706cb97372e3f26a7452c01208 (MD5) / O Linfoma de Hodgkin clássico (LHC) é caracterizado pela presença de uma pequena população de células grandes mono ou multinucleadas, denominadas células de Hodgkin-Reed-Sternberg (HRS), circundadas por uma grande massa inflamatória de células não neoplásicas. O vírus Epstein-Barr (EBV) está associado ao Linfoma de Hodgkin em cerca de 50% dos casos. O diagnóstico do LH EBV relacionado é possível por meio da identificação de proteínas virais nas células HRS. Os métodos considerados ideais para essa identificação são as reações de imuno-histoquímica utilizando anticorpos antiproteína latente de membrana (LMP1) e hibridação in situ com uma sonda para o RNA viral (EBER). A LMP-1 é considerada um oncogene clássico. Foi demonstrado que a LMP-1 pode controlar a expressão do gene da metaloproteinase 9 (MMP-9), em linhagem de células C33A. A MMP-9 é um membro da família das endopeptidases que facilita a invasão tumoral e metástases pela degradação do estroma extracelular. Objetivos: avaliar se a expressão da MMP-9 está relacionada ao status do EBV no tumor e se houve impacto na sobrevida livre de eventos (SLE) e sobrevida global (SG) em pacientes com LHC. Casuística e Métodos: foram examinados 97 pacientes com LHC. Todos os pacientes foram submetidos a protocolos de tratamentos equivalentes (MOPPABV ou ABVD). O diagnóstico histopatológico foi revisto e o subtipo classificado de acordo com a OMS. Reações de imuno-histoquímica para LMP-1 e MMP-9 e hibridação in situ para EBER foram realizadas. Resultados: A presença do EBV foi identificada em 52,5% dos casos. Houve uma maior prevalência do subtipo histológico celularidade mista em pacientes EBV positivos (P = 0,005). Não houve diferença na positividade do EBV em relação à faixa etária, sexo, estádio da doença ou pela presença de sintomas B. A presença do EBV no LHC não influenciou a SLE (P = 0,38) ou a SG (P = 0,80) com uma mediana de acompanhamento de 71 meses. A expressão da MMP-9 ocorreu em 87,6% dos casos estudados. Não houve diferença de casos positivos e negativos em relação ao status do EBV (P = 0,59). Quando avaliada a intensidade da expressão da MMP-9 nos casos positivos também não observamos correlação com a presença do EBV (P = 0,62). Não houve diferença entre o resultado da MMP-9 e os parâmetros: subtipo histológico, estádio, presença de sintomas B, idade e sexo. Não houve influência da MMP-9 na SLE (P = 0,98) e SG (P = 0,60). Conclusões: Demonstramos que a prevalência do LH relacionado ao EBV na população estudada é de 52,5%, e que a presença do vírus não altera a evolução clínica, SLE e SG de pacientes tratados uniformemente. Concluímos ainda que a MMP-9 é fortemente expressa nas células HRS. Não há correlação entre a expressão de MMP-9 e o status do EBV. Nem a SG nem a SLE foram influenciadas pela expressão dessa enzima. / Clinical and histological features of classical Hodgkin lymphoma (cHL) are primarily due to the effects of cytokines, enzymes and chemokines produced by Hodgkin-Reed-Sternberg (HRS) cells and their surrounding inflammatory cells in response to signals triggered by etiological factors such as Epstein-Barr virus (EBV). Matrix metalloproteinase-9 (MMP-9) has been associated with poorer survival in patients with aggressive non-Hodgkin lymphomas. In EBV-related cancers the expression of viral latent membrane protein 1 (LMP1) correlates with an increased MMP-9 expression. In this study, we evaluated the prognostic relevance of MMP-9 expression and EBV status in HRS cells in patients with cHL in Brazil. Material and Methods: We selected 97 patients with cHL. Patients were included if they had: 1) > 18 years, 2) Undergone similar chemotherapy protocols, 3) Paraffin blocks available for review and for EBV and MMP-9 detection and 4) Clinical, epidemiological and laboratorial parameters available. Results: EBV was detected in 52.5% of all cases. MMP-9 expression positivity was found in 87.6% of all cases. There was no correlation between MMP-9 expression and EBV status. Response to treatment and relapse rate were independent of MMP-9 expression and EBV status. When stratified according to chemotherapy protocol used or disease stage, we still did not find any difference. MMP-9 positivity did not influence overall survival and event free survival. Conclusion: MMP-9 are expressed in the majority of HRS cells and did not correlated with EBV status or survival. The consistent MMP-9 expression in HRS cells makes this enzyme a potential target for therapy. / TEDE / BV UNIFESP: Teses e dissertações

Le virus d’Epstein-Barr (EBV) est un herpèsvirus humain qui infecte plus de 90% de la population mondiale. Il est décrit comme associé à plusieurs pathologies cancéreuses humaines comme les carcinomes nasopharyngés et gastriques et divers lymphomes, comme le lymphome de Burkitt, les lymphomes NK/T et certains lymphomes de Hodgkin. Le lymphome T angio-immunoblastique (LTAI), un cancer des cellules T folliculaires helper TFH, contient souvent des cellules B porteuses de l’EBV. Mais jusqu’à présent le rôle de l’EBV dans la pathogenèse de cette maladie reste inconnu. Dans ce contexte, notre travail avait pour objectif de déterminer si l’EBV associé au LTAI présentait une particularité laissant envisager son rôle dans cette pathologie. Pour ce faire, nous avons étudié la séquence complète de l’EBV au sein d’échantillons de LTAI et comparé les résultats à ceux obtenus pour d’autres lymphomes (B, NK/T) ainsi qu’aux séquences publiées. Le séquençage a tout d’abord été réalisé sur 7 lignées cellulaires positives pour l’EBV, afin de valider la technique, et a ensuite été appliqué aux échantillons d’adénopathies de 40 patients atteints de syndrome lymphoprolifératif, parmi lesquels 20 souffraient de LTAI. L’enrichissement en génome viral a été réalisé par capture à l’aide de sondes spécifiques du génome de l’EBV. Ensuite les librairies ont été synthétisées et séquencées sur les plateformes Illumina MiSeq et NextSeq. Dans un deuxième temps, nous avons réalisé l’assemblage de novo des reads et déterminé la séquence complète du virus majoritaire dans chaque échantillon. Les données obtenues ont été analysées bioinformatiquement. D’une manière intéressante, le virus a été trouvé clonal ou quasi-clonal dans les LTAI alors que les lymphocytes B étaient dans la plupart des cas polyclonaux. En outre, le profil de mutations trouvé présentait des similitudes avec ce qui était trouvé pour les autres lymphomes associés à l’EBV, notamment au niveau des épitopes cibles des cellules de l’immunité suggérant un processus de sélection de la souche virale identique à celui d’une tumeur clonale associée à l’EBV. Ceci pourrait jouer un rôle important dans l’échappement au système immunitaire du virus dans ce contexte multicellulaire complexe. La présence de cellules B polyclonales avec un EBV clonal dans un compartiment T tumoral clonal pourrait relever d’une double sélection tumorale, endogène T et exogène EBV clonal, et pourrait suggérer l’existence de cross-talk entre les cellules B-T. / More than 90% of the world's population is infected by Epstein-Barr virus (EBV), a human herpesvirus. EBV is thought to be implicated in the pathogenesis of several human malignancies including epithelial tumors such as nasopharyngeal and gastric carcinomas as well as lymphoproliferative diseases such as Burkitt's lymphoma, NK/T lymphomas and some Hodgkin lymphomas. In angioimmunoblastic T-cell lymphoma (AITL), a peripheral neoplasm of follicular helper T (TFH) cells, a recurrent finding is the presence of EBV-positive B lymphocytes at the beginning of the disease. However, whether this EBV infection of B cells plays a role in AITL pathogenesis remains unclear. In this context, our work aimed to determine if the EBV associated with the AITL presented an oncogenic profile allowing us to consider its role in this pathology. To do this, we sequenced the whole EBV genomes in AIL samples and compared the results to those obtained for other lymphomas (B, NK / T) as well as to previously published sequences. Sequencing was first performed on 7 EBV-positive cell lines to validate the technique, and then was applied to lymphadenopathy specimens from 40 patients with lymphoproliferative disease, of whom 20 had AITL. Enrichment of the viral genome was performed by capture using specific EBV genome probes. The libraries were synthesized and sequenced on Illumina MiSeq and NextSeq platforms. In a second step, we performed de novo assembly and determined the sequence of the virus in each sample. The data obtained were analyzed bioinformatically. Interestingly, the virus was found to be clonal or quasi-clonal in AITL, while B cells were in some cases polyclonal. In addition, the mutational pattern was similar to other EBV-associated lymphomas, especially at the level of the target epitopes of immune cells suggesting a process of selection of the viral strain identical to that of a clone tumor associated with EBV. This could play an important role in the virus escape from the immune system in this context. The presence of polyclonal B cells with clonal EBV in a clonal tumor T cell compartment could be a dual tumor selection; or that is endogenous T and exogenous clonal EBV, and could therefore suggest the existence of a cross-talk between B-T cells.

Introdução: Pacientes com infecção pelo HIV têm risco aumentado para o desenvolvimento de linfomas não-Hodgkin de células B comparado à população geral. Dentre os mecanismos que podem estar relacionados a esta patologia, encontra-se a reativação do vírus de Epstein-Barr secundária a imunossupressão. O papel do sistema imune para desenvolvimento de tumores é citado há longa data, e seu equilíbrio é mantido pelos linfócitos T regulatórios, cujo principal regulador e marcador é o fator de transcrição FOXP3. Neste estudo, avaliamos a presença de EBER e FOXP3 em amostras diagnósticas, além da carga viral para o vírus de Epstein-Barr em pacientes com linfomas relacionados à Aids a fim de avaliar e correlacionar os resultados como marcadores prognósticos nesta população. Métodos: Análise prospectiva da carga viral para Epstein-Barr no plasma e em células mononucleares do sangue periférico em 15 pacientes com linfomas relacionados à Aids acompanhados no Serviço de Hematologia do Instituto de Infectologia Emílio Ribas e do Hospital das Clínicas/Instituto do Câncer do Estado de São Paulo da Faculdade de Medicina da Universidade de São Paulo. As mensurações foram realizadas para cada paciente por reação da cadeia de polimerase em tempo real ao diagnóstico, término do tratamento e três meses após o término do tratamento. Dois grupos controles constituídos de 26 pacientes infectados pelo HIV em uso de anti-retroviral e sem diagnóstico de linfoma ou infecção oportunista e 30 indivíduos saudáveis também foram analisados para comparação da carga viral para o vírus de Epstein-Barr. Amostras coletadas por biópsia para o diagnóstico de linfoma foram submetidas a análise imuno-histoquímica para FOXP3 e para EBER por hibridização in situ. Resultados: 13 pacientes eram do sexo masculino e dois do sexo feminino, dos quais 14 foram tratados com quimioterapia e um com radioterapia de sistema nervoso central. Nove de 15 pacientes (60%) completaram o tratamento proposto e obtiveram remissão completa. A mediana da carga viral para o vírus de Epstein-Barr antes do tratamento foi 13 cópias/106 nas células mononucleares do sangue periférico (1-1472 cópias/106) e 70 cópias/mL (0-24900 cópias/mL) no plasma. Após o tratamento foi de 0,5/106 (0-109,5) e indetectável no plasma, com diminuição significativa da carga viral em células mononucleares do sangue periférico (p=0,022) e no plasma (p=0,003) ao término do tratamento em comparação ao diagnóstico. Nos pacientes em remissão completa, a carga viral para o vírus de Epstein-Barr diminuiu tanto no plasma como em células mononucleares do sangue periférico na maioria dos casos. A hibridização in situ para EBER resultou positiva em 7/15 (46,7%) casos, sendo significativamente superior no grupo de pacientes com linfomas relacionados a AIDS com mais de um sítio extralinfonodal comprometido (p=0,041) e com linfócitos T CD4 <100 células/L (p=0,026). A expressão de FOXP3 foi negativa em 15/15 (100%) dos pacientes com ARL. Conclusões: A expressão de EBER foi positiva em 7/15 (46,7%) dos pacientes com linfomas relacionados à Aids e superior de forma significativa nos pacientes com estádio mais avançado do linfoma e maior grau de imunossupressão. Observou-se diminuição estatisticamente significativa da mediana de carga viral para o vírus de Epstein-Barr em células mononucleares do sangue periférico (p=0,022) e plasma (p=0,003) após o tratamento do linfoma em comparação aos valores do diagnóstico em pacientes que atingiram remissão completa, o que poderia ser considerado um marcador prognóstico de resposta a terapia / Introduction: Patients with HIV infection have increased risk for development of non-Hodgkins lymphoma compared to general population. Among mechanisms that could be related to this disease is the reactivation of Epstein-Barr virus infection secondary to immunosuppression. The role of immune system in development of tumors was reported a long time ago, and balance of this system is maintained by regulatory T cells; FOXP3 transcription factor is the main regulator and marker of these cells. In this study we evaluated the presence of EBER and FOXP3 in diagnostic samples, and also viral load of Epstein-Barr virus in patients with Aids-related lymphoma to evaluate and correlate the results as prognostic markers in this population. Methods: Prospective analysis of viral load of Epstein-Barr virus in plasma and peripheral blood mononuclear cells from 15 patients with Aids-related lymphoma treated at Instituto de Infectologia Emílio Ribas and Hospital das Clínicas/Instituto do Câncer do Estado de São Paulo da Faculdade de Medicina da Universidade de São Paulo. Viral load measures were performed by real time polymerase chain reaction at diagnosis of lymphoma, completion of treatment and three months afterwards. Two control groups composed by 26 HIV-positive patients in use of HAART and without diagnosis of lymphoma or opportunistic infection and 30 healthy persons were also analyzed for viral load comparison. Biopsy samples performed to lymphoma diagnosis were submitted to immunohistochemistry for FOXP3 and in situ hybridization to EBER. Results: 13 patients were male and two females, 14 were treated with chemotherapy and one with radiotherapy of central nervous system. Nine of 15 patients (60%) completed treatment achieving complete remission. Median viral load of Epstein-Barr virus before treatment was 13 copies/106 in peripheral blood mononuclear cells (1-1472 copies/106) and 70 copies/mL (0-24900 copies/mL) in plasma. After treatment it was 0,5/106 (0-109,5) and not detectable in plasma, with significant decrease of viral load in peripheral blood mononuclear cells (p=0,022) and in plasma (p=0,003) after treatment compared to diagnosis. In patients with complete remission, viral load decreased in the majority of cases. In situ hybridization for EBER was positive in 7/15 (46,7%), and significant higher in the group of patients with Aids-related lymphoma with more than one extra nodal site (p=0,041) and CD4 T-cells <100 cells/L (p=0,026). FOXP3 expression was negative in 15/15 (100%) of patients with ARL. Conclusions: EBER expression was positive in 7/15 (46,7%) of patients with Aids-related lymphoma and significantly higher in patients with advanced stages of lymphoma and higher degree of immunosuppression. Significant decrease in median viral load of Epstein-Barr virus was observed in peripheral blood mononuclear cells (p=0,022) and plasma (p=0,003) after lymphoma treatment compared to diagnosis in patients that achieved complete remission, what could be considered a prognostic marker of response to therapy

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-06-01T12:31:29Z No. of bitstreams: 1 grazielatoledocostamayrink.pdf: 5985830 bytes, checksum: 06ca10d53dd3ecb4c31ad3500ea80e8c (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-06-02T15:13:59Z (GMT) No. of bitstreams: 1 grazielatoledocostamayrink.pdf: 5985830 bytes, checksum: 06ca10d53dd3ecb4c31ad3500ea80e8c (MD5) / Made available in DSpace on 2017-06-02T15:13:59Z (GMT). No. of bitstreams: 1 grazielatoledocostamayrink.pdf: 5985830 bytes, checksum: 06ca10d53dd3ecb4c31ad3500ea80e8c (MD5) Previous issue date: 2016-08-10 / Introdução: A associação entre Linfoma de Hodgkin clássico e o status tumoral do vírus Epstein- Barr é bem definida. Entretanto, a expressão da positividade do vírus Epstein-Barr nas células de Reed-Sternberg/Hodgkin e o impacto dessa relação na sobrevida do Linfoma de Hodgkin clássico permanecem controversos e apresentam resultados conflitantes em estudos de diversas regiões do mundo. Considera-se essencial o entendimento fisiopatogênico desse vírus no prognóstico dos pacientes com Linfoma de Hodgkin clássico. Objetivo: Correlacionar o status do vírus Epstein Barr com os fatores de risco desfavoráveis e fatores prognósticos do Linfoma de Hodgkin clássico em uma população brasileira. Métodos: A positividade do vírus Epstein-Barr foi determinada pelo método de Hibridização in situ para o ácido ribonucleico viral e pela imuno-histoquímica para proteína de membrana latente viral 1. A revisão histopatológica das amostras e a análise dos testes de identificação foram realizadas por uma hematopatologista experiente. Avaliou-se o impacto prognóstico do status do vírus Epstein-Barr em 29 pacientes com Linfoma de Hodgkin clássico. Os fatores prognósticos do Escore Prognóstico Internacional para estadio avançado e os fatores de risco desfavoráveis instituídos pelo Grupo Alemão de Estudos em Hodgkin para estadio limitado foram correlacionados com o status viral nas células tumorais. Para as associações entre presença do vírus Epstein-Barr e outras variáveis categóricas, aplicaram-se os testes de Qui-quadrado ou exato de Fisher. A Sobrevida Global e a Sobrevida Livre de Eventos foram analisadas pelo método de Kaplain-Meier e Modelo de Regressão Proporcional de Cox. Resultados: A média de idade ao diagnóstico foi 33 anos. O status do vírus Epstein-Barr nas células tumorais foi positivo em 37,9%. As células tumorais positivas para o vírus foram mais frequentes em pacientes com idade maior que 45 anos, sem diferença estatística. O subtipo celularidade mista foi o mais frequente (p = 0,02) e o tamanho de efeito desse teste foi de moderada magnitude. Na análise univariada, as sobrevidas Livre de Eventos e Global não apresentaram significância estatística para idade, sexo, estadio clínico, hemoglobina, leucocitose, linfocitopenia, albumina, envolvimento nodal, sintomas B, doença extranodal e doença Bulky entre os pacientes positivos e negativos para o vírus Epstein-Barr (p > 0,05). Os pacientes positivos apresentaram maior Sobrevida Livre de Eventos quando comparados aos pacientes negativos, embora a diferença não apresentasse significância (p = 0,07). Na análise multivariada, a positividade ao vírus Epstein-Barr não demonstrou fator prognóstico significante. Conclusões: Apesar do status do vírus Epstein-Barr nas células tumorais não ter revelado associação com fatores prognósticos adversos e não ter influenciado a Sobrevida Global e a Sobrevida Livre de Eventos, observou-se uma associação positiva entre a presença desse vírus e o subtipo celularidade mista, demonstrando uma relação com o subtipo histológico de pior prognóstico. / Introduction: The association between classical Hodgkin’s Lymphoma and tumor Epstein-Barr virus status is well established. However, the expression of Epstein-Barr virus presence in Hodgkin/Reed-Sternberg cells and its prognosis remains controversial and presentes conflicting results in studies worldwide. Understanding the pathophysiological role of this virus in the prognosis of patients with classical Hodgkin’s Lymphoma is essential. Objective: The aim of this study is to correlate the clinical outcome with Epstein-Barr virus status in a Brazilian population. Methods: Epstein-Barr virus positivity was determined by in situ hybridization for Epstein-Barr virus-encoded ribonucleic acid and immunohistochemistry for viral latent membrane protein-1. The histopathology review and the analysis of identification tests were performed by an hematopathologist expert. The prognostic impact of Epstein-Barr virus status in 29 patients with classical Hodgkin’s Lymphoma was evaluated. Prognostic factors from International Prognostic Score to advanced stage and risk factors from German Hodgkin Study Group to limited stage were correlated with tumor cells Epstein-Barr virus status. In order to determine associations between the presence of Epstein-Barr virus and other categorical variables, Chi-square or Fisher's exact tests were applied. Overall and event-free survivals were analyzed with Kaplan-Meier method and Cox proportional hazards regression models. Results: The mean age at diagnosis was 33 years. Tumor cells Epstein-Barr virus status was positive in 37.9%. Epstein-Barr virus-positive classical Hodgkin’s Lymphoma was more frequent in patients older than 45 years, with no statistical difference. Mixed cellularity histological subtype was more common in Epstein-Barr virus-related tumor cells (p = 0.02) and its effect-size index was medium. Univariate analysis, event-free survival and overall survival were not significantly associated to age, sex, clinical stage, hemoglobin, leukocytes, lymphocytes, albumin, nodal involvement, B symptoms, extranodal disease and Bulky disease in Epstein-Barr virus-positive and negative patients (p > 0.05). Epstein-Barr virus-positive patients had longer event free survival when compared to Epstein-Barr virus-negative ones, even though the difference was not statistically significant (p = 0.07). In multivariate analysis, Epstein Barr virus positivity was not a significant prognostic factor. Conclusions: Although the Epstein-Barr virus status in tumor cells was not associated with adverse prognostic factors and did not influence the overall and event-free survivals, a positive association between the presence of Epstein-Barr virus and Mixed-cellularity subtype was noticed.

Epstein-Barr Virus (EBV) is a ubiquitous human virus linked to several diseases, including cancers. CD8 T cells are important for controlling EBV replication. Generation and maintenance of virus-specific CD8 T cells is dependent on specific interaction between MHC-peptide complexes on the infected cell and the CD8 T cell receptor (TCR). Several lines of evidence suggest that the TCR repertoire is an essential component of the CD8 T-cell immune response. The current work focuses on delineating the features of the TCR repertoire that drive the selection of EBV-specific CD8 T cells into the memory phase. We used bulk and single-cell TCRαβ sequencing to analyze the TCR repertoire of human CD8 T cells specific for two immunodominant HLA-A02:01-restricted EBV-derived epitopes: BRLF1109-117 (YVLDHLIVV) and BMLF1280-288 (GLCTLVAML) during the acute and memory phases of primary EBV infection in humans. We showed that persistent EBV-specific clonotypes accounted for only 9% of unique clonotypes but were highly expanded in acute EBV infection and more commonly expressed identifiable features than non-persistent clonotypes. The other 91% of highly diverse unique clonotypes disappeared and were replaced in convalescence by equally diverse “de-novo” clonotypes. We provide evidence suggesting that recognition of BRLF1109-117may be driven by the TCRα. We identified a highly dominant and degenerate BRLF1109-117-specific TCRα sequence, AV8.1-CAVKDTDKLIF-AJ34, that was shared by all donors studied and identified conserved residues within this sequence that were important for antigen recognition. These findings are relevant to current efforts to develop or optimize the efficacy of T cell based therapies or vaccines.

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