Diss. (sammanfattning) Stockholm : Karol. inst., 1999. / Härtill 7 uppsatser.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [128]-143).

Thesis (doctoral)--Ludwig Maximilians-Universität zu München, 1979.

Mantle cell lymphoma (MCL) is characterized by the presence of the t(11 ;14)(g13 ;g32) translocation which results in cyclin Dl over-expression. MCL is one of the most difficult lymphoproliferative disorders to manage with a median survival rate of 43 months from diagnosis. The poor prognosis associated with MCL is due in large part to its late classification as a separate clinical entity leading to a dearth in available pre-clinical models. The specific objectives of the research described in this thesis were (1) to establish MCL preclinical models of disease and (2) to evaluate deregulated cell signaling pathways in MCL that can impact treatment response. Pre-clinical models of MCL were established from pre-existing cell lines containing the t(11 ;14)(g13 ;g32). These cell lines were previously misclassified because they were developed prior to the classification of MCL as a distinct lymphoma subtype. With the establishment of MCL models, deregulated cell signaling pathways in MCL and response to different treatment strategies were investigated. These included an investigation of the cell signaling pathways activated in bcl-2 over-expressing MCL cells that were treated with oblimersen; a molecular gene silencing strategy that effectively suppresses bcl-2 in vitro and in vivo. Silencing bcl-2 provided insight into which pathways were influenced by bcl-2 over-expression in MCL. More specifically loss of cyclin D1, NF-KB, p53, bax and p27 were observed following bcl-2 silencing. Additional studies investigated how abnormal expression of CD40/CD40L and Fas/FasL along with bcl-2 family members contributes to B cell clonal expansion and influences Rituximab-mediated cell death in MCL models. Rituximab is a chimeric monoclonal antibody targeted against B cells and both Rituximab-sensitive and insensitive MCL models were defined. An abnormally high expression of bcl-2, bcl-x L, mcl-1, CD40/CD40L and Fas were observed in all MCL cells, as well as high levels of soluble FasL, capable of blocking Fas-mediated apoptosis. These deregulated pathways were associated with response to Rituximab treatment in a sensitive MCL model. These studies demonstrated some of the key pathways associated with treatment response in MCL, and the establishment of well characterized MCL models enables us to continue to explore new treatment strategies currently being studied in other lymphomas. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

CD40 is an important antigen involved in immune regulation and anti-CD40 antibody therapies against human CD40 expressing tumours may be particularly advantageous. The antibody could induce tumour response in two ways. Firstly it might have a direct anti-tumour effect and secondly it could 'boost' the immune system to provide a heightened immune response that evades tumour tolerance. This project has explored the effects of CD40 ligation of a variety of CD40 expressing tumours including transformed human B cell lines (RL and Daudi), human epithelial cell lines (MG79 [ovarian] and Caski [cervical]) and primary human B cell non-Hodgkin's lymphomas obtained, with consent, from patients undergoing excision lymph node biopsy or splenectomy. The ligation of CD40, on transformed human B cell lines, using chinese hamster ovary cells transfected to express human CD40L and human soluble CD40L has shown significant cellular growth inhibition (p<O.001). Ligation of CD40 on human epithelial cell lines using human soluble CD40L has also resulted in significant growth inhibition. In primary B cell human non-Hodgkin's lymphomas ligation of CD40 with both CD40L expressing CHO cells and human soluble CD40L in the presence of human IL4 has caused significant cellular proliferation (p<O.001) and induced upregulation of cell surface and costimulatory molecules including CD80, CD86, CD58, CD54. Anti-tumour activity has been identified in a xenograft model of a transformed human B cell line (Daudi) treated with both a mouse anti-human CD40 antibody and a chimeric human anti-CD40 antibody. I have performed important preclinical toxicology studies testing mouse anti -CD40 (3/23) by injecting the antibody intraperitoneally or intravenously in to BALB/c mice. The mice have been culled following treatment and a reversible dose dependent transaminitis associated with microscopic evidence of a reversible Iympho-granulomatous hepatitis, that at the highest dose levels is acutely necrotising, has been identified. These antiCD40 antibody toxicology studies are essential before a protocol for a phase I trial of human anti-CD40 antibody against CD40 expressing human tumours is developed. I have developed a new chimeric human anti -CD40 antibody containing human constant regions and mouse variable regions. This antibody is currently undergoing large scale production for a proposed trial to treat patients with CD40 expressing tumours (excluding low-grade non-Hodgkin's lymphoma) who have failed conventional therapies.

This investigation studied the occurrence, distribution and morphology; and the transplantation, ultrastructural and in vitro characteristics of histiocytic lymphoma in the BALB/c and C57BL/6 strains of mice. The disease was more common in aged females of both strains. The liver was the major organ involved in the males and the liver and uterus were commonly involved in the females. Subcutaneous transplants readily metastasized to the lungs. Ultrastructurally the neoplastic cells resembled histiocytes and did not contain microfilaments or a basement membrane. The results suggest that this neoplasm is histiocytic in origin.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoma subtype with heterogeneous clinical outcome and a significant number of patients still die of their disease. Characterising lymphoma patients’ autoantibody repertoires represent one approach to improve the understanding of their disease biology. Our hypothesis was that characterisation of antigens eliciting humoural immune responses in lymphoma patients may provide insights into mechanisms of lymphomagenesis and identify novel diagnostic/therapeutic targets. HIP1R was validated as a novel B-NHL autoantigen by immunoblotting with patients’ sera. No response was identified to the related HIP1 protein. Consistent with this finding, more widespread expression of HIP1R, compared to HIP1, was observed in lymphoma cell lines. Expression studies, at both transcript (qRT-PCR and analysis of microarray datasets) and protein levels (blotting and immunolabelling), identified abundant HIP1R in normal B cells and low level expression in a poor prognosis activated B-cell (ABC)-like DLBCL subtype. Upregulation of HIP1R expression was observed in activated non-malignant B cells at both transcript and protein levels, suggesting that downregulation of HIP1R expression in ABC-DLBCL might be a disease-related process. Despite its potential for DLBCL subtyping, HIP1R protein expression was not statistically significantly associated with patients’ survival in a series of 256 DLBCL. Short FOXP1 isoforms were identified as one mechanism repressing HIP1R transcription in an ABC-DLBCL cell line. Phenotypic analysis of HIP1R-depleted B-cell lymphoma cells indicated that HIP1R silencing could increase the surface levels of B-cell receptor (BCR) components such as IgM and CD79b. HIP1R represents a novel lymphoma autoantigen and cell-of-origin marker for distinguishing germinal centre- versus ABC-DLBCL subtypes. As ABC-DLBCL survival is dependent on chronic BCR signalling, future studies will address whether HIP1R silencing plays a fundamental role in disease pathogenesis by promoting BCR signalling.

Les neuroblastomes, même de haut risque répondent bien à la chimiothérapie initiale mais deviendront fréquemment résistants au traitement. Les inhibiteurs de topoisomérase I représentent un outil thérapeutique important dans la prise en charge des neuroblastomes réfractaires. Pour étudier la résistance aux inhibiteurs de topoisomérase I acquise dans un contexte thérapeutique, un modèle murin de neuroblastome résistant au CPT-11 a été développé. La chimiorésistance est connue comme un phénomène multifacoriel. Nous avons donc utilisé plusieurs approches pour mieux caractériser les mécanismes à l'origine de la résistance dans notre modèle. Une approche génomique a permis d'identifier la dérégulation de la voie de signalisation formée du récepteur ALK et de deux ligands PTN et MDK. Alors que ALK est décrit comme gène majeur de prédisposition au neuroblastome, principalement par le biais de mutations activatrices, nous avons démontré que l'activation du récepteur survenait par des mécanismes alternatifs aux mutations dans une large majorité de cas et participerait à l'initiation de la maladie. En revanche, nous n'avons pas pu prouver l'implication de ce récepteur dans la progression de la maladie ou dans sa réponse au traitement. Il semble que la régulation de ALK soit complexe et le rôle exact de ce récepteur dans la progression du neuroblastome reste à établir. En revanche, nous avons démontré l'importance du ligand MDK dans la régulation de l'expression et de l'activation de ALK ainsi que dans le contrôle de la survie des cellules neuroblastiques. Inhiber cette cytokine représente une stratégie thérapeutique intéressante, complémentaire des thérapies anti-ALK, actuellement en développement clinique dans le neuroblastome. D’autre part, la caractérisation phénotypique du modèle a permis de mettre en évidence une signalisation altérée des dommages à l'ADN associée à une instabilité génétique accrue dans les tumeurs résistantes. Celles-ci présentent également une modification de progression dans le cycle cellulaire et une proportion plus importante de cellules quiescentes. Au final, ce travail a permis d'identifier différents mécanismes de résistance qui représentent des marqueurs de réponse au traitement et des cibles thérapeutiques intéressantes dans le neuroblastome. / Neuroblastoma, including high-risk cases, show a good initial response to chemotherapy but will frequently become resistant to treatment. Topoisomerase I inhibitors represent an important therapeutic option for refractory neuroblastoma. To study the reisitance to topoisomerase I inhibitors acquired in a therapeutic setting, we developed in vivo a resistant model to irinotecan (CPT-11). Chemoresistance is known as a multifactorial phenomenon. We have therefore used several approaches to better characterize mechanisms leading to resistance in our model. A genomic approach enabled us to identify the deregulation of a signaling pathway, constituted with a receptor (ALK) and two lignads (PTN and MDK). While ALK is decsribed as a major neuroblastoma predisposition gene, mainly through activating mutations, we demonstrated that the activation of ALK occurs via mechanisms others than mutation in a large majority of cases. Moreover ALK activation is an important event in the initiation of the disease. However, we couldn’t prouve the implication of the receptor in the progression of the disease or in its response to treatment. It seems that the regulation of ALK is complex and its precise role in the progression of neuroblastoma remains to be precisely defined. Nevertheless, we have demonstrated the importance of MDK, one of ALK ligands in the regulation of the expression and activation of ALK as well as in the control of the neuroblastoma cells survival. The inhibition of the cytokine, MDK represents an interesting therapeutic strategy, complementary to anti-ALK therapies, currently in clinical development in neuroblastoma. On another hand, the phenotypic characterization of the model, showed an alteration of the signaling of DNA damage and an increased genomic instability in the resistant tumors. Those tumors also harbor a modification in the cell cycle progression, particularly an increased proportion of quiescent cells. Finally, this work enables us to identify several resistance mechanism that represent markers of response to chemotherapy and relevant therapeutic targets in neuroblastoma.

Mantle Cell Lymphoma (MCL) is a rare but aggressive Non-Hodgkin Lymphoma (NHL). Although t(11;14) is a hallmark of MCL, it is insufficient for lymphomagenesis. The phosphoinositide-3 kinase (PI3K) pathway is thought to play an important role in MCL pathogenesis and the PI3K p110δ isoform is enriched in leucocytes making it an attractive target. Early phase trials evaluating the p110δ selective inhibitor GS-1101 however demonstrate inferior responses in MCL compared to chronic lymphocytic leukaemia and indolent NHL. The relative contribution of the class Ia PI3K isoforms p110α (PIK3CA), p110β (PIK3CB) and p110δ (PIK3CD) was therefore evaluated in MCL. Immunohistochemistry on MCL tissue microarrays revealed that while p110δ was highly expressed, p110α showed wide variation and p110β expression was the weakest. A significant increase in p110α expression was found with disease progression. Although GS-1101 was sufficient to abolish B-cell receptor mediated PI3K activation, additional p110α inhibition was necessary to abolish constitutive PI3K activation in MCL exhibiting high p110α expression. Compared to GS-1101, GDC-0941 (p110α and p110δ inhibitor) had greater in vitro toxicity and a high PIK3CA/PIK3CD mRNA ratio (> twice ratio in healthy B-cells) was able to identify primary MCL samples that were resistant to GS-1101 but significantly more sensitive to GDC—0941. This ratio also increased with disease progression. No PIK3CA or PIK3R1 activating mutations were found. In summary, blockade of both p110α and p110δ appears to be necessary for effective PI3K inhibition in MCL, particularly with relapse. The PIK3CA/PIK3CD mRNA ratio may help identify those patients that are most likely to respond. Finally, a disseminated xenograft model of human primary MCL was established in NSG mice. Engraftment of primary MCL was demonstrated by peripheral blood flow cytometry, tissue immunohistochemistry and FISH for t(11;14). This model is potentially valuable for pre-clinical in vivo testing of novel drugs for this incurable disease.

Magister Scientiae Dentium - MSc(Dent) / The aim of the study was to investigate and describe the incidence of Plasmablastic Lymphoma (PBL) diagnosed at the Divisions of Anatomical Pathology and Haematopathology at Tygerberg Hospital from 2005 to 2017, and to ascertain a possible correlation with HIV infection, by identifying the number of HIV positive and negative patients diagnosed with Plasmablastic Lymphoma. Method: This was a retrospective study using the case records of all newly diagnosed PBL patients from 2005 to 2017. Results: Fifty-seven cases of PBL were diagnosed from 2005-2017. The overall result shows an increasing incidence of PBL in the intended period with the maximum incidence occurring in 2017. Most of the cases, 40.4%, were diagnosed in the age range 40-49-years. Forty-five patients were HIV-positive (78.9%) with (P value 0.011) and the majority of the patients were males (66.7%).