Mantle cell lymphoma (MCL) is a rare B cell neoplasm that accounts for approximately 4-8% of non-Hodgkin’s lymphomas (NHLs). The median age at diagnosis is 65 years with a male to female predominance of 3:1. It has also been demonstrated that female MCL patients have a greater response to therapy, especially immunomodulatory therapy compared to male MCL patients. The concept of cancer immunosurveillance is well described and it is perceived that females mount a greater immune response compared to males. In addition, although lymphomas are generally not perceived to be hormone controlled, epidemiological studies have demonstrated lower prevalence of lymphoma in females taking exogenous oestrogen. This aim of this thesis was to explore the gender difference observed in MCL. The study investigated the difference in the quantity of immune cells in the peripheral blood and lymph node biopsies of untreated male and female MCL patients. There was a significantly greater number of T cells in the peripheral blood of male MCL patients compared to the female MCL patients. Conversely, greater numbers of immune cells were observed in the lymph node biopsies of female MCL patients compared to male MCL patients. In addition, four NK cell activating receptors; NKp46, NKp44, NKp30 and NKG2D were examined to determine if their expression was different between the genders. The cell mediated cytotoxic function of the immune cells (PBMCs) from male and female MCL patients and healthy controls was also examined. Interestingly the healthy controls exhibited greater cytotoxicity compared to the MCL patients. PBMCs were incubated with oestrone (female hormone in postmenopausal women), lenalidomide and IL-2 to further investigate the effects of these on the immune cells from male and female MCL patients. Incubation with IL-2 resulted in a significant increase in the cytotoxicity activity of male MCL patients but not female MCL patients in this cohort. The lymph node biopsies from MCL patients were examined for the presence of oestrogen receptors. Oestrogen receptor β was predominantly expressed on MCL cells in all the biopsies examined. This is an area that warrants further studies. This thesis provides some insight into the mechanisms that may influence the gender difference observed in MCL, however further studies are needed.

Une meilleure compréhension des mécanismes moléculaires impliqués dans la malignité des lymphome B diffus à grande cellules (LBDGC) et dans leur résistance aux traitements devraient permettre de développer de nouvelles opportunités thérapeutiques innovantes et d’envisager dans un futur proche de nouveaux agents de thérapie ciblée. Jusqu’à ce jour, le traitement standard se solde encore par un échec chez un nombre significatif de patients. Les rechutes post traitement et les cas réfractaires restent le défi majeur pour améliorer le taux de survie global. Plusieurs méchanismes ont été décrits comme responsables de ces échecs, dont, pour une grande part, le dysfonctionnement de la machinerie apoptotique. Dès lors, des stratégies thérapeutiques activant des voies non-apoptotiques plus sélectivement dans les cellules malignes sont absolument nécessaires. La Thymoquinone (TQ), un principe actif isolé de la plante médicinale Nigell a Sativa, a démontré des propiétés anticancéreuses in vitro ainsi que dans des modèles animaux, en agissant via plusieurs mécanismes incluant principalement des effets antiprolifératifs et pro-apoptotiques. Malgré les nombreuses investigations, les mécanismes de destruction cellulaire sous-jacents à l’effet anti-cancéreux de la TQ restent ambigus et peu élucidés, notament sur les voies de mort cellulaire non-apoptotiques. L’objectif de notre travail vise l’évaluation de la sélectivité de la TQ vis-à-vis des lignées cellulaires de lymphomes B diffus à grandes cellules (LBDGC), et l’élucidation des méchanismes critiques responsables de la destruction cellulaire. Dans une première partie, nous avons démontré la capacité de la TQ à produire un effet cytotoxique plus prononcé dans des lignées cellulaires lymphomateuses (LBDGC) par rapport aux cellules normales provenant de donneurs sains. Le profil de sélectivité de la TQ s’est avéré intéressant par rapport aux agents de chimiothérapie conventionnelle dans le LBDGC. A l’échelle moléculaire, on a mis en évidence l’implication de voies de mort cellulaire non-apoptotiques, en supplément des voies apoptotiques, dans l’effet cytotoxique de la TQ dans la plupart des lignées étudiées. Nos investigations ont d’abord révélé un effet pro-apoptotique vraisemblablement sous-jacent à l’effet génotoxique de la TQ. L’importance de cette modalité de mort cellulaire dans l’effet cytotoxique de la TQ est toutefois limitée aux lignées les moins sensibles. Nos observations ont révélé la capacité de la TQ à activer la voix mitochondriale des caspases suite à la libération du cytochrome c. La mort cellulaire produite par la TQ n’est toutefois pas freinée par l’inhibition des caspases, et nos investigations ont également exclu l’implication des voies apoptotiques caspases-indépendantes. Paradoxalement, ces investigations ont montré le rôle critique des voies de mort cellulaire non-apoptotiques, en particulier dans les lignées sensibles à l’effet de la TQ. En étudiant l'origine de cette voie alternative de mort cellulaire, on a constaté que la TQ provoque le dysfonctionnement réticulaire qui se manifeste par l’activation des voies de transduction UPR et se produit de façon plus significative dans les lignées sensibles. En effet, dans cette dernière catégorie nos investigations ont démontré que la TQ produit un effet important par l’augmentation du calcium cytosolique principalement via la dépletion de celui-ci à partir des compartiments réticulaires. En accord avec ces observations, nous avons demontré que l’augmentation du calcium cytosolique joue un rôle critique, plus particulièrement dans l’effet non-apoptotique de la TQ opérant principalement dans les lignées fort sensibles aux effets de la TQ. Afin de mieux caractériser les mécanismes de susceptibilité et de résistance des lignées LBDGC, la deuxième partie de ce travail a été consacrée à l’évaluation de l'implication de l’histone déacétylase SIRT1 ( silent information regulator 1) dans la sensibilité des lignées aux effets de la TQ. SIRT1 intervient notament dans la résistance au stress oxydatif et à l’apoptose. SIRT1 serait également protectrice contre les dommages à l'ADN. Rappelons que l’expression de SIRT1 est considérée comme un facteur de mauvais pronostic pour les patients atteints de LBDGC. En focalisant nos investigations sur les lignées résistantes aux traitementx standard et moins sensibles à la TQ, nous avons démontré l'effet protecteur de SIRT1 vis-à-vis de l'effet pro-apoptotique et génotoxique de la TQ. En résumé, la capacité de la TQ à déclencher des modalités de mort cellulaire non apoptotiques pourrait constituer une stratégie prometteuse pour surmonter les mécanismes anti-apoptotiques responsables en partie de l’échec du traitement standard. Tenant compte du rôle critique de l’effet génotoxique dans le traitement des LBDGC, notre travail suggère que l'inhibition de SIRT1 pourrait être une stratégie préventive pour surmonter la résistance native ou acquise. D’autre part, nos résultats contribuent à une compréhension plus précise des mécanismes critiques responsables de l’effet cytotoxique de la TQ. Nos travaux pourraient d’autre part permettre l’étude de combinaisons innovantes de médicaments incluant la TQ in vivo. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

The post-translational modification of histones and their interaction with transcription factors is essential to gene regulation. Furthermore, these targets would greatly benefit from probe molecules to fully elucidate their biological actions and to potentially lead to therapeutics. However, these protein-protein interactions have been considered difficult to inhibit and few high-quality chemical probes currently exist for the study of epigenetic biological systems in particular.

The prognosis for multiple myeloma (MM) and the activated B-cell subtype of diffuse large B-cell lymphoma (ABC DLBCL) is poor. Gene expression profiling studies have identified that the transcription factor, nuclear factor kappa B (NF-κB) is overexpressed and confers a poor prognosis in MM and ABC DLBCL. NF-κB regulates the transcription of genes involved in cell proliferation and survival. Thus, several groups have tried to identify and/or develop agents that target NF-κB to improve therapy and patient prognosis for MM and ABC DLBCL. Our laboratory has shown that the manganese porphyrin MnTE-2-PyP5+ inhibits NF-κB in a murine lymphoma cell culture model and enhances tumor cell death in combination with dexamethasone and cyclophosphamide, two agents that are routinely used to treat these neoplasms. MnTE-2-PyP5+ inhibits NF-κB by glutathionylating p65, a member of the NF-κB family. The objective of the following studies was to determine whether MnTE-2-PyP5+ enhances the chemotherapeutic response in human MM and ABC DLBCL cells that overexpress and depend on NF-κB for survival. The following studies demonstrate that MnTE-2-PyP5+ glutathionylates and inhibits NF-κB in human MM and ABC DLBCL cells. MnTE-2-PyP5+ also synergizes with several MM and DLBCL chemotherapeutics, including dexamethasone, cyclophosphamide, vincristine and bortezomib to enhance cell death. The data from these human cell lines will provide the basis for future studies to test MnTE-2-PyP5+ in animal models and for translating MnTE-2-PyP5+ to the clinic.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22742号 / 医博第4660号 / 新制||医||1046(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武田 俊一, 教授 武藤 学, 教授 滝田 順子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Le lymphome de Hodgkin est caractérisé, d’un point de vue histologique, par la présence de rares cellules tumorales nommées cellules de Reed et Sternberg, au sein d’un infiltrat cellulaire polymorphe, inflammatoire et réactionnel. Cette dernière résulte de la transformation tumorale de cellules lymphocytaires B qui acquièrent des propriétés d’échappement au système immun, de prolifération, de résistance à l’apoptose et une instabilité chromosomique. Néanmoins, la rareté des cellules tumorales, impliquant des problèmes techniques mais aussi de caractérisation des évènements primaires dans l’initiation de cette instabilité chromosomique, a été bien débattue dans la littérature. Mais les mécanismes impliqués dans l’instabilité chromosomique dans le lymphome de Hodgkin demeurent obscurs.La première partie de cette thèse a été consacrée à l’étude des mécanismes impliqués dans l’instabilité génomique du lymphome de Hodgkin via l’instabilité des microsatellites et l’instabilité chromosomique en utilisant 7 lignées de lymphome de Hodgkin. Nous avons montré pour la première fois l’implication des microsatellites dans l’instabilité génomique des lymphomes de Hodgkin (MSI-H (microsatellite instability-high) dans 3/7 lignées). De plus, nous avons montré que deux mécanismes favorisent l’émergence d’une instabilité chromosomique : le premier implique une instabilité télomérique qui est présente essentiellement dans les petites cellules tumorales induisant la formation des chromosomes dicentriques, des amplifications des gènes (Jak2 comme exemple) et des arrangements chromosomiques complexes. Le deuxième mécanisme est lié essentiellement à un défaut de réparation des cassures double-brin avec l’apparition des chromosomes dicentriques sporadiques et une fréquence importante des micronoyaux avec la formation des ponts anaphasiques.La deuxième partie de cette thèse a été consacrée à l’étude des mécanismes de maintenance des télomères dans les ganglions tumoraux du lymphome de Hodgkin (50 patients) comme dans les lignées tumorales. Nous avons montré qu’il existe une cohabitation entre les deux mécanismes importants de maintenance des télomères, l’activation de la télomérase d’une part et le mécanisme ALT (alternative lengthening of telomeres) d’autre part. Nous avons identifié la présence de petites cellules dans les ganglions hodgkiniens comme dans les lignées tumorales avec une forte activité de la télomérase par contre la cellule de Reed Sternberg est caractérisée par un profil ALT avec la présence des corps PML et une très faible activité de télomérase. La fréquence des cellules télomérase ou ALT varie d’un ganglion à un autre et d’une lignée à une autre. Un drastique raccourcissement télomérique a été observé dans les cellules exprimant la télomérase. Pour les cellules ALT, une grande hétérogénéité de la taille des télomères ainsi que la présence des chromosomes dicentriques sporadiques ont été détectées. Le suivi des patients à long terme pendant 10 ans, nous a permis d’établir une corrélation entre le profil ALT et la survenue de mortalités et de morbidités. De plus, l’étude de la radiosensibilité des lignées tumorales a montré que les lignées ALT sont plus résistantes que les lignées télomérases.La troisième partie de cette thèse a été consacrée à la validation de ces deux concepts d’instabilité chromosomique via l’instabilité télomérique et à celle des mécanismes de maintenance des télomères, en utilisant un modèle de lymphome de Hodgkin établi dans le laboratoire à partir de la lignée L428.Ces données auront une retombée clinique importante non seulement dans la compréhension et le traitement des lymphomes de Hodgkin mais aussi dans d’autres pathologies malignes. / The study of Hodgkin lymphoma (HL), with its unique microenvironment and long clinical outcomes, has provided exceptional insights into several areas of tumour biology. Findings in HL have not only improved our understanding of human carcinogenesis, but have also pioneered its translation into the clinic.Tumoral cells in HL, called Hodgkin and reed Sternberg cells (HRS), are characterized by a highly altered genomic landscape with a wide spectrum of genomic alterations, including somatic mutations, copy number alterations, complex chromosomal rearrangements, and aneuploidy. Moreover, the scarcity of HRS cells and the resulting technical problems of their in situ characterization, the primary cytogenetic events and the clonality of these possible aberrations has been a matter of debate in the past. As a consequence, a few accepted and established HL cell lines are widely used in the majority of research projects conducted worldwide.In this thesis, first we have first investigated the possible mechanisms underlying genomic instability including microsatellite and chromosomal instability in HL cell lines. We provide the first evidence that the genomic instability observed in HL is related to microsatellite instability and chromosomal instability related to two major mechanisms: first, telomere fusion leading to dicentric chromosomes formation and breakage/fusion/bridge (B/F/B) cycles involving the repeated fusion and breakage of chromosomes following the loss of telomeres in small cells associated with the lower expression of TRF2, as well as an elevated copy number of the Jak2 gene and the presence of nucleoplasmic bridges containing telomere and centromere sequences. The second mechanism is related to defective DNA repair via non homologous end-joining (NHEJ) repair with the presence of nucleoplasmic bridges without telomere or centromere sequences, accompanied by the micronucleus without centromere sequences and a higher frequency of sporadic dicentric chromosomes.The second part of this thesis has focused on investigating telomere maintenance mechanisms (TMMs) not only in HL cell lines but also in lymph nodes of HL patients. A telomerase-independent mechanism for TMM in HL has been proposed in the absence of detectable telomerase activity (TA) in some cases. The major finding of this work has been the demonstration of the presence of both telomerase and ALT mechanism in lymph nodes of HL patients as well as in HL cell lines. We have identified a subset of tumors with some small cells expressing telomerase and Reed Sternberg cells containing ALT-associated PML bodies. A significant correlation was observed between telomere length and TMMs. Drastic telomere shortening was observed in cells with telomerase expression and elevated heterogeneity of telomere length was found in ALT profile cells. Interestingly, complex chromosomal rearrangements, included sporadic dicentric formation, were observed in ALT profile cell lines. Interestingly, the relationship between TMMs and all-cause mortality and morbidity during 10 years of follow-up of HL patients using cox proportion hazard models demonstrated a poor clinical outcome for HL patients exhibiting primarily ALT mechanisms. Similarly, higher radiation sensitivity was observed for cell lines with high telomerase activity compared to cell lines with the ALT profile.

This study examines the risk of cancer in children of pesticide applicators from the Agricultural Health Study. The study includes 36,537 children of Iowa participants who were evaluated for cancer incidence during 1975 through 2013 from birth through the age of seventeen. Standard incidence rates for any cancer and specific groups of cancers classified using the International Classification of Childhood Cancer was calculated using rates from the general population of Iowa controlling for year of follow, age, sex, and race. Hazard ratios for Group I-III cancers and paternal exposure to specific pesticides were calculated using exposure information collected on 50 pesticides during phase 1 and 2 of the Agricultural Health Study. The exposure information allowed for calculation of intensity-weighted days of exposure to pesticides using the Agricultural Health Study exposure algorithm. Additionally, maternal ever exposure to specific pesticides was used to evaluate risk of childhood cancer. There were 118 cancers identified in children of Agricultural Health Study participants. The all-cancer standardized incidence ratio was significantly elevated (SIR = 1.27 95% CI: 1.04-1.50). The most common groups of cancers were Group I leukemia, myeloproliferative disease, and myelodysplastic disease (n=34) followed by Group III central nervous system (CNS) and miscellaneous intracranial and intraspinal neoplasms (n=25). For paternal intensity-weighted days of exposure, there were 31 of 50 specific pesticides that had sufficient cases of cancer to investigate using Cox proportional hazard models. The herbicide trifluralin significantly increased the risk for Group I childhood cancers for any parental pesticide exposure 2 years before birth through birth when compared to children with no paternal exposure (HR = 2.72 95% CI: 1.15, 6.44). This was consistent with results found from analyzing exposure split into two quantiles based on median exposure of exposed children with a Group I cancer. Parental use of the herbicide S-Ethyl-dipropylthiocarbamate (EPTC) did not result in a sufficient number of Group III cancer cases to look at levels of exposure to EPTC, but ever exposure showed an increased hazard ratio when compared to children with unexposed fathers (HR = 2.56 95% CI: 1.06, 6.20). Other pesticides (dicamba, cyanazine, and terbufos) showed mixed evidence of an association with specific childhood cancers, but were either under powered to evaluate with sensitivity analysis or showed inconsistent risk across exposure levels. Less extensive exposure information was available for mothers of children of the Agricultural Health Study, so analysis was restricted to ever or never exposure to pesticides during a mother’s lifetime. Additionally, there were a limited number of cases of cancer for which maternal exposure to specific pesticides was reported resulting in only 4 pesticides being evaluated for childhood cancer risk (glyphosate, 2,4-dichlorophenoxyacetic acid (2,4-D), carbaryl, and malathion). For these four pesticides, this study did not detect any increased risk of childhood cancer from maternal exposure. In summary, this study provides the first epidemiological evidence of an increased risk of childhood cancer for trifluralin and EPTC. Since this study provides the first evidence of this increased risk, additional analysis is needed to validate the results. This study demonstrates how pesticide exposure information from participants of the AHS can be used in the evaluation of their children’s cancer risk. Additional follow-up and analysis of this cohort beyond the age of 17 would provide further insight into cancer risk during early adulthood from early life pesticide exposure.

Monoclonal antibody-based immunotherapy has been widely used as a strategy to treat cancer. Successful treatment of B-cell lymphoma with the monoclonal antibody (mAb) Rituximab (RTX) in combination with chemotherapy has increased the survival of patients and minimized the side effects of the treatment. However, many patients do not react to the treatment with RTX or gain resistance quickly. Thus, strategies to enhance the tumor cell killing and improve the response rates of mAb-based immunotherapy are a fundamental goal. In this study, I use four different B-cell lymphoma cell lines grown into 3D structures, called spheroids, as a model organism. Those spheroids, which are closer to the in vivo situation of B-cell lymphoma in patients compared to conventional in vitro 2D cell cultures, in combination with RTX, are tested for the activation of effector functions to eliminate tumor cells and compared to experiments conducted in the same cell lines in 2D cell cultures. Moreover, the therapeutic mAb RTX is only approved by the FDA in an IgG1 isotype form. Here, I test different isotype forms of RTX on their efficacy to kill cancer cells by the complement system and also by the activation of monocytes to engulf them in the process of phagocytosis. Interestingly, the IgG3 isotype form of RTX can induce both effector functions most efficiently while the IgG1 isotype form, used in clinical approaches, is only second most efficient in eradicating cancer cells. In addition, when grown into spheroids, the efficacy of both effector functions is reduced compared to 2D cell cultures. Furthermore, the efficacy of the complement system to kill the different B-cell lymphoma cell lines was directly correlated with the expression of the complement regulatory surface protein CD59. By blocking CD59, the efficacy of the complement system could be partially enhanced when cells were treated in 2D cell cultures but not when grown into 3D spheroids. In addition, the antibody-dependent phagocytosis (ADP) of cancer cells by monocytes might correlate with the expression of the RTX target surface protein CD20. Also, the previous incubation of B-cell lymphoma cells with a chemotherapy agent can enhance the efficacy of ADP by presumably providing an “eat me” signal to the effector cells.  In summary, this work shows that the outcome of a treatment with RTX in B-cell lymphoma patients could be improved by the detection of the specific features of the cancer cells, for example the expression of CD59 and CD20 and the structure of the tumor. Moreover, the different isotypes of RTX can activate effector functions in different intensities. The IgG3 isotype form might be able to overcome resistance or lack of reaction to the treatment in B-cell lymphoma patients but further experiments will be needed to investigate these possibilities.

A dissertation submitted to the Faculty of Health Sciences, University of Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in the branch of Anatomical Pathology. 21 July 2017. / Aims and objectives The study design aimed to assess and validate various laboratory techniques in the detection of EBV in HIV positive patients with diffuse large B cell lymphoma. The sensitivity and specificity of each technique was determined, as was the presence of an asymptomatic (latent) or lytic phase infection and the viral strain. DLBL samples occurring in HIV seropositive patients were used as a vehicle for these laboratory procedures which included chromogenic in situ hybridisation (EBER), immunohistochemistry (EBNA 2, LMP 1), real time PCR, (EBNA 1, LMP 2 and BZLF 1) and nested PCR (EBNA 2). Materials and Methods 46 cases of previously diagnosed DLBL from HIV positive individuals were identified and retrieved from the archives of the Department of Anatomical Pathology of the University of Witwatersrand and NHLS. All in-situ hybridisation, immunohistochemical and PCR laboratory procedures were carried out in accordance with the Standard Operating Procedures of the Anatomical Pathology Molecular Laboratory, using appropriate negative and positive controls throughout. Ethical clearance was obtained (M140273). Results/Conclusion A 20% frequency of EBV in HIV positive DLBL cases was established. All EBV infections were found to be in the lytic phase, with an almost equal distribution of latency patterns II and III and an equal distribution of EBV strains 1 and 2. EBER in situ hybridisation was confirmed to be the most sensitive and reliable method of viral detection, and the presence of the BZLF 1 gene determined by real time PCR was found to be a reliable indicator of a lytic infection. / LG2018

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