Endemic Burkitt lymphoma (eBL) is the leading pediatric cancer in sub-Saharan Africa and is associated with Epstein-Barr virus (EBV) and Plasmodium falciparum malaria co-infections. Current treatment options in Africa are combination chemotherapy with a survival rate hovering around 50%. Relapsed or refractory eBL patients have failed to receive any targeted treatments in the clinic. Our focus was to delineate immune responses in eBL, interrogate the tumor variation in responses to targeted treatments and develop mouse models that can be used to target essential mediators of tumor pathogenesis. Immune-based treatments including immune checkpoint inhibition have recently become an effective therapeutic modality in oncology. However, some B cell lymphomas such as Hodgkin Lymphoma (HL), are more receptive to checkpoint inhibition than others suggesting a need to understand the efficacy of checkpoint inhibition on different lymphoma subtypes. Checkpoint inhibitors act by blocking inhibitory receptors on T cells and improving anti-tumor responses. One of the goals of this thesis was to characterize checkpoint inhibitors on Tumor-infiltrating lymphocytes (TILs) in eBL tumors and to identify T cell subsets that exhibit increased expression of inhibitory receptors, poor cytokine production, poor proliferation and express transcription factors associated with exhaustion. Using scRNA seq, we identified T cell clusters that co-expressed inhibitory receptors, poor proliferative markers but also sustained costimulatory signals, as well as cytokine expression suggesting a pre dysfunctional state and not terminally exhausted state. Furthermore, we quantified the dominant co-inhibitory receptors PD1 and TIGIT that are upregulated in the tumor microenvironment via immunohistochemistry (IHC) and in peripheral blood of eBL patients via flow cytometry. We compared eBL patients with healthy pediatric cohorts with a history of persistent malaria exposure to those who had little to no malaria infections, to understand uniquely T cell mediated responses in BL children. Tumors had high co-expression of PD1 and TIGIT but fewer PD1 only populations, suggesting that both ligands may play a role in restraining immune activation via IHC. Next, we investigated if PD1 ligands or TIGIT ligands were overexpressed in eBL tumors. Nectin-2, TIGIT ligand was highly expressed in eBL tumors but was not highly correlated with TIGIT expression. These studies provide insights for PD1/ TIGIT blockade in Burkitt lymphoma patients. Additionally, we established new patient-derived cell lines from eBL tumors to study tumor variation and to study targeted treatments. We established five new patient-derived eBL lines BL717, BL 719, BL720, BL725, and BL740 that were interrogated for their inter-patient variation by studying their gene expression profiles. Further, we developed a patient cell-line derived xenograft (CDX) mouse model by injecting newly patient-derived BL cell lines in immunodeficient mice (NSG BL) and studying BL tumorigenesis. Having successfully established NSG BL tumors, we observed differences in tumor growth sensitivity and survival. We tested rituximab efficacy, one of the most established treatments for B cell lymphomas in our mouse model. We also identified pathways associated with unfolded protein response (UPR) and the mammalian target of rapamycin (mTOR) signaling, as well as apoptosis in one of the cell line xenografts, BL740, in response to rituximab. BL717, BL720 cell line xenograft failed to control tumor growth and was enriched in IFN-ɑ signature genes. This mouse model will prove to be useful to study combination therapy against eBL tumors as well as mechanisms of resistance to drug targets. Collectively, these studies provide insights into intratumoral variation including subtypes during tumor progression and expression profiles of TILs in eBL tumors. This will be important in designing new therapeutic strategies as well as help pose novel therapeutic targets.

Résumé en français : Notre équipe étudie les mécanismes de l’apoptose induite par la nutline-3, une molécule capable de se fixer sur MDM2 et d’activer la p53, dans différents types de lymphomes associés au virus d’Epstein-Barr (EBV) comme le lymphome de Burkitt (LB) ou syndromes lymphoprolifératifs post-transplantation (PTLD). Nos résultats montrent que la nutline-3 induit l’apoptose des cellules de LB EBV (-) alors que les cellules EBV (+) en latence de type III sont résistantes. Mon travail de thèse a consisté à étudier les mécanismes impliqués dans ce phénomène de résistance afin de mettre en place des stratégies pour les contourner. Une première étude initiée par les résultats d’une analyse transcriptomique, effectuée après traitement avec la nutline-3 de deux lignées qui ne diffèrent que par leur statut EBV, nous a permis de montrer que : 1) l’autophagie est induite en réponse au traitement dans les cellules EBV (+) en latence de type III ; 2) ces cellules expriment fortement Bécline-1 et présentent une activation constitutive de l’autophagie ; 3) l’autophagie contribue à la résistance de ces cellules à l’apoptose. Par ailleurs, nos résultats indiquent que la protéine anti-apoptotique Bcl-2 est également impliquée dans la résistance de ces cellules et que l’utilisation d’ABT-737, un inhibiteur de Bcl-2, restaure leur sensibilité à la nutline-3. L’efficacité de ce composé a donc été évaluée in vivo, seul ou en combinaison avec des traitements conventionnels (Cyclophosphamide pour le LB et Rituximab pour les PTLD). Les résultats obtenus lors de ces études pré-cliniques montrent que : 1) ABT-737 réduit considérablement la croissance tumorale et augmente la survie de souris xénogreffées avec des cellules d’une lignée lymphoblastoïde (LCL, utilisées comme modèle pour les PTLD) alors qu’il n’a pas d’effets chez les souris xénogreffées avec une lignée de LB ; 2) la combinaison BT-737/Cyclophosphamide permet de limiter la croissance tumorale durant le traitement mais n’améliore pas la survie des souris xénogreffées avec une lignée de LB ; 3) l’association ABT-737/Rituximab est très efficace et induit une rémission complète chez 70% des souris xénogreffées avec la lignée de LCL / - Résumé en anglais : Our team is working on the mechanisms of apoptosis induced by nutlin-3, a small molecule which binds to MDM2 and activates p53, in different lymphomas associated with Epstein-Barr virus such as Burkitt lymphoma (BL) or Post-transplant lymphoproliferative disorder (PTLD). Our results show that nutlin-3 strongly induce apoptosis in EBV (-) cells whereas EBV (+) latency III cells are much more resistant. The aim of my PhD project was to study the mechanisms involved in the resistance of EBV (+) latency III cells to apoptosis and to develop new therapeutic strategies to bypass these mechanisms. A transcriptomic analysis was realized after treatment with nutlin-3 of two cell lines which only differs by their EBV status. Based on the results obtained, a study was performed which allow us to show that: 1) autophagy is induced after nutlin-3 treatment in EBV (+) latency III cells; 2) these cells strongly expressed beclin-1 and present a constitutively high level of autophagy; 3) autophagy is involved in the resistance of apoptosis observed in these cells. Furthermore, our results demonstrate that Bcl-2 also contributes to the resistance of EBV (+) latency III cells and that treatment with ABT-737, a Bcl-2 inhibitor, restores their susceptibility to nutlin-3 treatment. We thus assessed the efficiency of this compound in vivo, in monotherapy or associated with conventional treatments (Cyclophosphamide for BL and Rituximab for PTLD). Results obtained during these pre-clinical studies show that: 1) ABT-737 reduces tumor growth and increase the overall survival of mice xenografted with a lymphoblastoïd cell line (LCL, used as a model for PTLD studies) but has no effects on mice xenografed with BL cell lines; 2) the association ABT-737/Cyclophosphamide reduces tumor growth during treatment but doesn’t improve the overall survival of mice xenografed with BL cell lines; 3) the association ABT-737/Rituximab is very efficient and induces 70% of complete remission in mice xenografted with LCL.

Notre équipe étudie les mécanismes de l'apoptose induite par la nutline-3, une molécule capable de se fixer sur MDM2 et d'activer la p53, dans différents types de lymphomes associés au virus d'Epstein-Barr (EBV) comme le lymphome de Burkitt (LB) ou syndromes lymphoprolifératifs post-transplantation (PTLD). Nos résultats montrent que la nutline-3 induit l'apoptose des cellules de LB EBV (-) alors que les cellules EBV (+) en latence de type III sont résistantes. Mon travail de thèse a consisté à étudier les mécanismes impliqués dans ce phénomène de résistance afin de mettre en place des stratégies pour les contourner. Une première étude initiée par les résultats d'une analyse transcriptomique, effectuée après traitement avec la nutline-3 de deux lignées qui ne diffèrent que par leur statut EBV, nous a permis de montrer que : 1) l'autophagie est induite en réponse au traitement dans les cellules EBV (+) en latence de type III ; 2) ces cellules expriment fortement Bécline-1 et présentent une activation constitutive de l'autophagie ; 3) l'autophagie contribue à la résistance de ces cellules à l'apoptose. Par ailleurs, nos résultats indiquent que la protéine anti-apoptotique Bcl-2 est également impliquée dans la résistance de ces cellules et que l'utilisation d'ABT-737, un inhibiteur de Bcl-2, restaure leur sensibilité à la nutline-3. L'efficacité de ce composé a donc été évaluée in vivo, seul ou en combinaison avec des traitements conventionnels (Cyclophosphamide pour le LB et Rituximab pour les PTLD). Les résultats obtenus lors de ces études pré-cliniques montrent que : 1) ABT-737 réduit considérablement la croissance tumorale et augmente la survie de souris xénogreffées avec des cellules d'une lignée lymphoblastoïde (LCL, utilisées comme modèle pour les PTLD) alors qu'il n'a pas d'effets chez les souris xénogreffées avec une lignée de LB ; 2) la combinaison BT-737/Cyclophosphamide permet de limiter la croissance tumorale durant le traitement mais n'améliore pas la survie des souris xénogreffées avec une lignée de LB ; 3) l'association ABT-737/Rituximab est très efficace et induit une rémission complète chez 70% des souris xénogreffées avec la lignée de LCL

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

No description available.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Endemic Burkitt lymphoma (eBL) is the most common pediatric cancer in malaria-endemic equatorial Africa and nearly always contains Epstein-Barr virus (EBV), unlike sporadic Burkitt Lymphoma (sBL) that occurs with a lower incidence in developed countries. Despite this increased burden the study of eBL has lagged. Additionally, while EBV was isolated from an African Burkitt lymphoma tumor 50 years ago, however, the impact of viral variation in oncogenesis is just beginning to be fully explored. In my thesis research, I focused on investigating molecular genetics of the endemic form of this lymphoma with a particular emphasis on the role of the virus and its variation in pathogenesis using novel sequencing and bioinformatic strategies. First, we sought to understand pathogenesis by investigating transcriptomes using RNA sequencing (RNAseq) from 30 primary eBL tumors and compared to sBL tumors. BL tumor samples were prospectively obtained from 2009 until 2012 in Kenya. Within eBL tumors, minimal expression differences were found based on anatomical presentation site, in-hospital survival rates, and EBV genome type; suggesting that eBL tumors are homogeneous without marked subtypes. The outstanding difference detected using surrogate variable analysis was the significantly decreased expression of key genes in the immunoproteasome complex in eBL tumors carrying type 2 EBV compared to type 1 EBV. Secondly, in comparison to previously published pediatric sBL specimens, the majority of the expression and pathway differences were related to the PTEN/PI3K/mTOR signaling pathway and was correlated most strongly with EBV status rather than the geographic designation. Moreover, the common mutations were observed significantly less frequently in eBL tumors harboring EBV type 1, with mutation frequencies similar between tumors with EBV type 2 and without EBV. In addition to the previously reported genes, we identified a set of new genes mutated in BL. Overall, these suggested that EBV, particularly EBV type 1, supports BL oncogenesis alleviating the need for particular driver mutations in the human genome. Second, we sought to comprehensively define sequence variations of EBV across the viral genome in eBL tumor cells and normal infections, and correlate variations with clinical phenotypes and disease risk. We investigated the whole genome sequence of EBV from primary tumors (N=41) and plasma from eBL patients (N=21) as well as EBV in the blood of healthy children (N=29) within the same malaria endemic region. We conducted a genome wide association analysis study with viral genomes of healthy kids and BL kids. Furthermore, we found that the frequencies of EBV types among healthy kids were at equal levels while they were skewed in favor of type 1 (70%) among eBL kids. To pinpoint the fundamental divergence between viral genome subtypes, type 1 and type 2, we constructed phylogenetic trees comparing to all public EBV genomes. The pattern of variation defined the substructures correlated with the subtypes. This investigation not only deciphers the puzzling pathogenic differences between subtypes but also helps to understand how these two EBV types persist in the population at the same time. Overall, this research provides insight into the molecular underpinning of eBL and the role of EBV. It further provides the groundwork and means to unravel the complexity of EBV population structure and provide insight into the viral variation that may influence oncogenesis and outcomes in eBL and other EBV-associated diseases. In addition, genomic and mutational analyses of Burkitt lymphoma tumors identify key differences based on viral content and clinical outcomes suggesting new avenues for the development of prognostic molecular biomarkers and therapeutic interventions.

Burkitt lymphoma is a highly aggressive B cell non-Hodgkin's lymphoma characterised by translocation of gene on chromosome 8. This translocation is usually detected by fluorescent in-situ hybridisation (FISH) studies as part of routine diagnostic work-up and prognostication. FISH testing is commonly done with the break-apart probe (BAP). This case illustrates how this testing can be falsely negative. This patient is a young male diagnosed with Stage I low-risk Burkitt with FISH negative for translocation initially on BAP testing. Additional testing with dual FISH probe detected translocation. FISH testing using BAPs alone may be falsely negative for translocations creating a diagnostic challenge and compromising the treatment approach and assessment of prognosis.