Immune evasion is an emerging hallmark of cancer. Dysregulation of the major histocompatibility complex class I (MHC-I) is a frequent mechanism of immune evasion utilized by tumor cells and is particularly relevant to the pathogenesis of B-cell lymphomas, including diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (cHL). A better understanding of MHC-I dysregulation in B-cell lymphomas is necessary to identify factors related to the risk, development, and progression of these tumors. In this thesis, we investigate the role of MHC-I dysregulation in DLBCL and cHL through the application of computational approaches to study genomic data. First, we introduce some background information about the normal function of MHC-I in the immune response to cancer and viral infection as well as the phenomenon of MHC-I dysregulation in the context of cancer. We provide an overview of how factors such as germline zygosity of HLA class I (HLA-I) genes and somatic alterations in the genes B2M and HLA-I that encode the protein subunits of MHC-I contribute to the development of DLBCL and cHL. Second, we present a study of the effects of HLA-I allele zygosity on survival in a cohort of 519 DLBCL patients treated with R-CHOP immunochemotherapy stratified by molecular subtype. Homozygosity in HLA-I was associated with a worse overall survival in patients whose tumors were classified in the “EZB” subtype, associated with somatic mutation in the epigenetic regulator EZH2. We find an association between the zygosity of the genes HLA-B and -C specifically and overall survival in EZB-DLBCL. These findings indicate that HLA-I zygosity may be a risk factor for worse clinical prognosis in patients with the EZB subtype of DLBCL. Third, we present a study of the genetic landscape of cHL tumors that are associated with infection with Epstein-Barr virus (EBV). We analyze inherited HLA-I allele types, somatic mutations, copy number changes, and mutational signatures in a cohort of 57 cHL patients (15 EBV-positive). We find that EBV-positive cHL is genetically distinct from EBV-negative cHL and is characterized by lower somatic mutation load and different activities of mutation signatures. Further, we find that cHL tumors are characterized by different patterns of MHC-I dysregulation depending on the EBV infection status. Germline homozygosity in HLA-I was associated with the EBV-positive subtype of cHL, while somatic alterations in HLA-I were associated with the EBV-negative subtype of cHL. These results suggest that inherited HLA-I homozygosity may be a risk factor for the EBV-positive subtype of cHL. Fourth, we expand our study of HLA-I in virus-associated cHL to perform a comparative analysis of virus-positive and virus-negative tumors in nine cancers linked to five viruses. We find that virus-positive tumors occur more frequently in males and show geographical disparities in incidence. Genomic analysis of 1,658 tumors reveals virus-positive tumors exhibit distinct mutation signatures, recurrent mutations in the RNA helicases DDX3X and EIF4A1, and a lower somatic mutation burden compared to virus-negative tumors of the same cancer type. We find that germline homozygosity in HLA-I is a potential risk factor for the development of EBV-positive cHL, but not other common virus-associated solid or hematological malignancies. Finally, we present in the Appendix a study of the genomic characterization of plasmablastic lymphoma (PBL), a rare EBV-associated B-cell lymphoma that occurs in the context of immunodeficiency caused by human immunodeficiency virus (HIV) infection. We find that PBL is characterized by mutations leading to constitutive activation of the JAK-STAT pathway. We additionally identify recurrent mutations in immune-related genes, such as B2M. These findings indicate a potential role for MHC-I and immune dysregulation in the pathogenesis of other B-cell lymphomas.

T-cell lymphomas (TCLs) are a highly aggressive and heterogeneous group of non-Hodgkin lymphomas derived from post-thymic mature T- and NK-cells broadly classified peripheral (PTCL) or cutaneous (CTCL), indicating either a nodal or skin-homing disease, respectively. One of the main clinical challenges contributing to dismal outcomes for TCL patients is both the lack of curative treatment strategies and the high rate of relapse for currently approved therapies, underscoring the need for identification of novel targeted therapies for the treatment of TCL. While combination therapeutic strategies have been proposed and show great promise in pre-clinical and clinical trials for PTCL and CTCL, none are yet approved. Additional contributing factors toward the difficulty in studying TCL and the high rate of therapeutic failure is the highly heterogeneous genetic and molecular mechanisms driving TCL as well as the poorly understood role of non-tumor microenvironment cells in the pathogenesis of TCL. Indeed, while several studies have suggested that tumor associated macrophages play both a significant functional role in supporting tumor maintenance and are therapeutically targetable, less is known about potential tumor supporting roles of other cell microenvironment populations. Here, I used an unbiased and high-throughput approach to discover novel drug combinations in CTCL and to characterize at the single-cell level relevant molecular mechanisms driving T-cell lymphomagenesis. First, I demonstrate that the combination of romidepsin, a selective class I HDAC inhibitor, with afatinib, an inhibitor of the epidermal growth factor receptor (EGFR) family, produces strongly synergistic antitumor effects in CTCL models, both in vitro and in vivo, using mechanisms of action that involve down-regulation of the JAK-STAT signaling pathway. This result suggests a potential therapeutic role for the combination of HDAC inhibitors with afatinib in the treatment of CTCL that had not been previously recognized. Second, we developed single-nuclei analysis on a cohort of 30 TCL (PTCL-NOS, AITL, epstein barr virus positive PTCL) and 6 normal patient samples to identify and deconvolute genomic and functional mechanisms contributing to T-cell lymphomagenesis. Here, I implemented a comprehensive bioinformatics pipeline for the analysis of sparse single-nuclei transcriptomic data and characterized heterogeneous molecular mechanisms driving T-cell lymphomagenesis, such as the upregulation of the PI3K-AKT-mTOR and WNT signaling pathways in a subset of 5/9 PTCL-NOS and 7/19 AITL samples, respectively. Additionally, I identified the enrichment of both the macrophage compartment in PTCL-NOS and AITL, and the specific enrichment of CD8+ T cells in AITL. These results suggest a correlation between patient-specific characteristics, such as mutational status, and possibly therapeutically targetable molecular mechanisms driving neoplastic cell growth that warrants further investigation.

Ferroptosis is an iron-dependent form of cell death driven by peroxidation of phospholipids with polyunsaturated fatty acyl (PUFA) tails. Dietary factors, such as fatty acids and iron, regulate ferroptosis. Moreover, the incidence and progression of several cancers is correlated with diet; models of lymphoma have shown sensitivity to ferroptosis. We investigated the effects of altering dietary factors linked to ferroptosis on diffuse large B cell lymphomas (DLBCL). We found that DLBCL cells undergo ferroptosis in response to iron and PUFA treatments in vitro, and that their growth in xenograft models is substantially reduced. We observed that monounsaturated fatty acids (MUFAs), in contrast, suppress ferroptosis and promote growth in DLBCL cell and animal models. The inhibitory effect of the ferroptosis inducer imidazole ketone erastin (IKE) on DLBCL xenograft growth was lessened by dietary MUFA. Ferroptosis linked fatty acids and iron thus impact the growth and response to ferroptosis treatment of DLBCL tumors.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: In recent years, HIV-associated cancers have proven to be the bane of our time, since HIV is decimating humanity across the globe, even in the twilight of the last century. Cancer rates continue to rise in developing countries, where 95% of the world’s HIV-infected population lives, yet less than 1% have access to antiretroviral therapy. HIV-infected individuals have a higher proclivity to develop cancers, mainly from immunosuppression. An understanding of the immunopathogenesis of HIV-related cancers (HRC) is therefore a major prerequisite for rationally developing and/or improving therapeutic strategies, developing immunotherapeutics and proplylatic vaccines. In this study, we explore the pathology of HIV-related cancer malignancies, taking into account the pathogenic mechanisms and their potential for improving the treatment of management of these malignancies especially in developing countries. We mathematically model the dynamics of malignant tumors in an HIV-free environment, investigate the impact of cancer malignancies on HIV-positive patients and explore the benefits of various therapeutic intervention strategies in the management of HIV-related cancers. We present two deterministic models of infectious diseases to implement these, and they were analysed. We use HIV-related lymphomas in the Western Cape of South Africa as a case study. We validated the proposed models using lymphoma incidence data from the Tygerberg Lymphoma Study Group (TLSG), Tygerberg Hospital, Western Cape, South Africa. We show that the increasing prevalence of HIV increases lymphoma cases, and thus, other HIV-related cancers. Our models also suggests that an increase in the roll-out of the HAART program can reduce the number of lymphoma cases in the nearest future, while it averts many deaths. Furthermore, the results indicate that a highly crucial factor to consider in the prognosis of the incidence of lymphoma (and other cancer types) in HIV-infected patients is their CD4 cell count, irrespective of whether the patient has developed an HRC or not.

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

Les lymphomes T cutanés constituent un ensemble hétérogène de lymphomes non-hodkinniens. Les lymphomes T cutanés sont définis par une prolifération clonale de lymphocytes T malins et de cellules NK (natural killers) de localisation cutanée. Dans l'Union Européenne, l'indication du bexarotène par voie orale est le traitement des manifestations cutanées des lymphomes cutanés T épidermotropes (LCT), au stade avancé (ou dès un stade précoce aux États Unis). L'objectif de ce travail a été d'étudier les effets indésirables du bexarotène chez les patients atteints de lymphome cutané. Nous nous sommes plus particulièrement intéressés aux interactions du bexarotène avec le système de la coagulation et avec le métabolisme lipidique.Dans la première partie de nos travaux, nous avons étudié l'origine de la coagulopathie induite par le bexarotène. Nous avons montré que le bexarotène inhibe les facteurs IX et X de la coagulation, ce qui provoque le prolongement du temps de coagulation ; ces effets pourraient être à l'origine de la coagulopathie observée chez les patients traités au bexarotène.Dans la deuxième partie, nous avons montré que le bexarotène interagit également avec le métabolisme lipidique par l'activation du CETP et par l'inhibition de la lipoprotéine lipase, ce qui provoque une chlolésterémie et une triglyéridémie.Enfin, nous avons présenté les résultats préliminaires de l'essai clinique organisé au niveau de la région ouest de la France sur les effets indésirables du bexarotène.Les résultats de ce travail mettent en évidence l'importance de la structure chimique du bexarotène (effets de charges) dans la neutralisation des facteurs de coagulation d'une part et dans le mécanisme d'interaction avec le métabolisme lipidique (CETP et lipoprotéine lipase) d'autre part. La compréhension des mécanismes moléculaires conduisant à une coagulopathie et à la dyslipidémie représente un enjeu majeur pour prévenir la toxicité du bexarotène et pour permettre une meilleure prise en charge du patient. / The cutaneous T cell lymphomas are a heterogeneous group of non-lymphoma hodkinniens. The cutaneous T cell lymphomas are defined by a clonal proliferation of malignant T cells and NK (natural killer) cells from skin location. In the European Union, the indication of oral bexarotene is the treatment of cutaneous manifestations of cutaneous T-cell lymphomas epidermotropic (LCT) to advanced (or at an early stage in the United States). The objective of this work was to study the adverse effects of bexarotene in patients with cutaneous lymphoma. We are particularly interested in the interactions of bexarotene with the coagulation system and lipid metabolism.In the first part of our work, we studied the origin of the coagulopathy induced by bexarotene. We have shown that bexarotene inhibited factors IX and X of the coagulation causing the extension of clotting time and coagulopathy that was observed in bexarotene-treated patients. In a second part, we showed that Bexarotene induces in vitro cholesteryl ester transfer protein activity, and suppress lipoprotein lipase activity in human plasma.Finally, we presented the preliminary results of the clinical trial conducted at the western region of France on the adverse effects of bexarotene.The results of this study highlight the importance of the chemical structure of bexarotene (charge effects) in the neutralization of coagulation factors mechanisms on the one hand and in its interaction with lipid metabolism (CETP and lipoprotein lipase) on the other hand. Understanding the molecular mechanisms leading to coagulopathy and dyslipidemia is a major issue to prevent the toxicity of bexarotene and to elicit better management of the bexarotene-treated patient.

La réponse immunitaires est régulée par des molécules de co-signalisation, qui, en apportant des signaux inhibiteurs ou activateurs, modulent les fonctions lymphocytaires. Nous avons focalisé notre étude sur deux co-effecteurs et membres de la famille CD28/B7 : PD-1 (Programmed Death 1) et BTLA (B and T Lymphocyte Attenuator), tous deux décrits, à l’image de CTLA-4, comme inhibitrices de l’activation lymphocytaire T. Au cours de ma thèse, nous avons pu évaluer l’expression et le rôle de PD-1 et BTLA sur les lymphocytes B normaux et pathologiques. Dans une première partie, nos travaux ont montré que ces molécules sont exprimées à la surface des sous populations B du sang périphérique et des tissus et qu’elles sont également finement régulées au cours de l’activation B. Par la suite nous avons démontré pour la première fois que BTLA et PD-1, à l’image des cellules T, sont recrutées, après activation, au niveau du récepteur de la cellule B et qu’elles exercent un rôle inhibiteur sur l’activation de ces mêmes cellules. Dans une seconde partie, grâce au développement d’un outil en cytométrie multicouleurs, nous avons analysé, dans une cohorte de 72 lymphomes B, l’infiltrat immunitaire ainsi que l’expression de PD-1, BTLA et leurs ligands. Ce travail, par son analyse de la physiologie B d’une part et de pathologies tumorales B d’autre part, donne un nouvel éclairage sur les rôles complexes des co-récepteurs BTLA et PD-1. / The immune response is regulated by co-signaling molecules, which, by providing signals inhibitors or activators, modulate lymphocyte functions. We focused our study on two co-effectors and CD28/B7 family members: PD-1 (Programmed Death 1) and BTLA (B and T Lymphocyte Attenuator), both described, like CTLA-4, as inhibitors of T lymphocyte activation. This work allowed us to evaluate the expression and the role of BTLA and PD-1 on normal and tumoral B lymphocytes. In the first part, our work has shown that these molecules are expressed on B cell subsets of peripheral blood and tissues and are also finely regulated during the B cell activation. Subsequently, we have demonstrated for the first time that BTLA and PD-1, like T cells, are recruited after activation at the B cell receptor, and they exert an inhibitory role on the activation of these cells. In the second part, through the development of a multicolor cytometry tool, we analyzed the immune infiltrate and the expression of PD-1, BTLA and their ligands in a cohort of 72 B-cell lymphomas. Taken together, these results, by his analysis of the physiology of B cell from B cell malignancies, give a new insight into the complex roles of BTLA and PD-1 co-receptors.

Orientador: Júlio Lopes Sequeira / Banca: Renee Laufer Amorim / Banca: Sara Maria de Carvalho e Suzano / Resumo: Os linfomas não Hodgkin (LNHs) são as neoplasias hematopoiéticas mais comuns nos cães, sendo o Linfoma Difuso de Grandes Células B (DLBCL) o subtipo mais frequente. Os DLBCLs são neoplasias formadas por células linfoides B com padrão de crescimento difuso e podem apresentar pelo menos cinco variantes. O presente trabalho teve como objetivos traçar o perfil imunoistoquímico dos DLBCLs dos cães, classificá-los de acordo com os critérios estabelecidos pela WHO (2008), WHO veterinária (2002) e Classificação de Kiel e efetuar a avaliação dos anticorpos utilizados pelo método de microarranjo de tecido (TMA), comparando-o com o corte convencional. Foi avaliada a expressão imunoistoquímica de marcadores pan B (CD79a, CD20 e PAX-5); a determinação dos índices proliferativo (Ki-67) e apoptótico (caspase-3) e também da expressão de p53 mutante. Foram observados 24 linfomas centroblásticos monomórficos, 4 imunoblásticos B e 1 centroblástico polimórfico de acordo com a classificação de Kiel. Este perfil é de 25 Linfomas de Grandes Células B Difusos/ DLBCL, NOS variante centroblástica e 4 Imunoblásticos de Grandes Células /DLBCL, NOS variante imunoblástica, pela WHO de 2002 e 2008, respectivamente. Houve marcação em 100%, 75,8% e 58,6% dos casos para o CD79a, CD20 e PAX-5, respectivamente. A mediana de porcentagem de marcação para o Ki-67 e para a caspase-3 foi de 45,9% e 10%, respectivamente. Já a expressão da proteína p53 mutante foi verificada em 16 tumores (55,1%). A análise destes marcadores utilizando-se o TMA resultou em perfil imunofenotípico idêntico e medianas de caspase-3, Ki67 e p53 significativamente semelhantes quando comparadas com o corte convencional. Concluiu-se que em nossas amostras não houve diferença estatística entre os diferentes subtipos histológicos em relação aos índices proliferativo e apoptótico e expressão da p53. Ainda, o TMA é uma técnica adequada para avaliação do... / Abstract: Non Hodgkin lymphomas (LNHs) are the most common hematopoietic tumors of dogs, among which Diffuse Large B Cell Lymphoma (DLBCL) is the most frequent subtype. DLBCLs are tumors composed of lymphoid B cell with a diffuse growth pattern and may present at least five variants. The present work intended to delineate the immunohistochemical profile of canine DLBCL, classify them according to the criteria proposed by WHO (2008); veterinary WHO (2002) and Updated Kiel Classification and also perform the evaluation of the used antibodies on tissue microarray (TMA) method in comparison to conventional histological sections. The immunohistochemical expression of pan B markers (anti CD79a, anti CD20 and PAX-5) was assessed; as well as the determination of proliferation index (Ki-67) and apoptosis (caspase 3) and also the expression of the mutant p53. There were 24 monomorphic centroblastic lymphomas, 4 imunoblastic B and 1 polimorphic centroblastic according to Kiel. That profile is 25 Diffuse Large B Cell Lymphoma/ DLBCL, NOS centroblastic variant and 4 Large Cell Imunoblastic/ DLBCL, NOS imunoblastic variant according to WHO 2002 and 2008 respectively. Immunolabeling was seen in 100%, 75.8% and 58.6% of the cases for CD79a, CD20 and PAX-5 respectively. The immunolabeling median percentage of Ki67 was 45.9% and for caspase-3 it was 10%. The expression of mutant p53 was observed in 16 tumors (55.1%). The analysis of those markers using TMA resulted in identical imunophenotype and significantly similar medians of caspase-3, Ki67 e p53 when compared to conventional sections. In conclusion there was no statistical difference among the histological subtypes regarding proliferation and apoptotic indexes and p53 expression in our samples. Also, TMA is an adequate technique for evaluating imunophenotype, proliferation and apoptotic indexes as well as presence of mutant p53 / Mestre

Les lymphomes T périphériques (ou PTCL) sont des lymphomes malins non Hodgkiniens ayant pour cellules d’origine des lymphocytes T (LT) ou Natural Killer matures. Ces lymphomes sont rares, hétérogènes et méconnus. Des arguments issus de la littérature suggérant l’implication de la stimulation chronique du récepteur T à l’antigène (TCR) dans la transformation des LT, nous ont conduits à développer un modèle murin basé sur la stimulation chronique du TCR pour adresser spécifiquement cette question. Dans ce modèle, le transfert de LT p53-/- dans des souris CD3e-/- entraine l’apparition de lymphomes T périphériques (PTCL) clonaux dans 60% des cas avec une médiane de survenue de 230 jours alors que les souris transférées avec des LT wt ne développent pas de lymphomes. Ces PTCL présentent un phénotype T effecteur-mémoire CD62LLo-CD44hi-CD122lo-CD25lo ainsi qu’une profonde downrégulation de l’expression des gènes impliqués dans la voie du TCR illustrant l’impact de la stimulation chronique dans la lymphomagénèse. L’étude de ces lymphomes a révélé qu’ils ne dépendent plus, pour la plupart, de l’engagement du TCR pour leur survie et qu’ils acquièrent des caractéristiques « innate-like » avec notamment l’expression de récepteurs NK inhibiteurs (NKiR) et de récepteurs NK activateurs (NKaR) ainsi que des protéines adaptatrices DAP12 et FceRIg. Cette expression est associée à celle de Syk et PLC?2, impliquées dans la signalisation des NKaR. Nous montrons que les NKaR et leurs voies de signalisation associées sont fonctionnelles et participent à la survie des cellules lymphomateuses, le blocage de certains NKaR retardant notamment le développement lymphomateux in vivo. Nous avons par la suite exploré l’expression des NKR, de Syk et de PLCg2 au sein des PTCL humains et nous montrons ou confirmons que certaines entités expriment des panels variés de NKR ainsi que les effecteurs Syk et PLCg2 suggérant l’existence de mécanismes de lymphomagénèse similaires à ceux identifiés dans notre modèle au sein d’un certain nombres de PTCL humains / Peripheral T-cell lymphomas (PTCL) are rare non Hodgkin malignant lymphomas emerging from mature T or NK cells. PTCL are highly heterogeneous and mainly misunderstood. As several evidences pointed the potential role of TCR chronic stimulation in human T-cell lymphomagenesis, we developed a murine model based on chronic TCR stimulation to address this question. In this model, transfer of p53-/- T-cells into T-cell deficient mice (CD3e-/-) triggered PTCL development in 60% of cases with a median survival of 230 days while transfer of wt T-cells in CD3e-/- mice did not lead to PTCL development. These PTCL exhibited an effector-memory phenotype CD62LLo-CD44hi-CD122lo-CD25lo associated with a dramatic downregulation of TCR pathway genes expression consistent with a chronic TCR stimulation highlighting it’s implication in lymphomagenesis. The analysis of these PTCL revealed that a large majority of cases (80%) do not depend anymore on TCR stimulation for their growth and survival and that they acquire innate-like features with expression of inhibitory NKR (NKiR) and activating NK receptors (NKaR) as well as the adaptor proteins DAP12 or FceRIg. Expression of these receptors is associated with the expression of SYK and PLC?2, which are classical key effectors downstream of NKaR. We show that these NKaR are functional and can mediate TCR-independent activation in mPTCL and that this signaling is involved in cell survival/proliferation as in vivo blockade of NKG2D and NKp46 delays PTCL development in PTCL transplantation experiments. In parallel, we studied NKR, Syk and PLCg2 expression in human PTCL and found that some entities express a large range of these receptors as well as Syk and PLCg2, suggesting similar lymphomagenesis mechanisms in some human PTCL