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.

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.

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

Mantle cell lymphoma (MCL) is a rare lymphoproliferative disorder (LPD) that has very poor survival. Like other LPDs, the neoplastic cells of MCL have an intimate dependence upon accessory cells within haematopoietic tissues. Understanding and exploiting the tissue-relationships of the mantle cells may therefore lead to further new approaches to treatment. This study work has set out to construct an in vitro system to model relevant aspects of the tissue-dependent behaviour of the neoplastic mantle cells, seeking to establish the link between in vitro behaviour and clinical phenotype, and establishing the feasibility of this system to study the effects of different therapeutic interventions. The first experimental chapter employs relevant mouse and human stromal models to mirror the tissue environment of MCL in vivo. Testing relevant agents, the work establishes that the system can identify different behaviour between indolent and aggressive forms of MCL, and demonstrates a particular importance for CD40 ligand both in the proliferation and survival of the neoplastic mantle cells, but shows also how these effects are modulated by the soluble factors interleukin-4 (IL-4) and the toll-like receptor-9 ligand (TLR9-L). The second experimental chapter examines the adhesion molecules expressed on MCL cells. Considerable variation in the level of expression is observed between cases, but overall the cases express particularly high levels of the integrin receptors LFA-1 (detected by alpha chain CD11a) and VLA-4 (detected by alpha chain CD49d). Cases also showed a significant difference in overall adhesion and chemokine-receptor expression between cases that had either a nodal or leukaemic clinical pattern, although no single adhesion molecule was characteristic of clinical phenotype. The final experimental chapter looked at 3-D culture of MCL. Within tissues MCL grows in a 3-D rather than 2-D matrix and it is recognised that cells employ different forms of adhesion and migration within the different spatial environments. This chapter establishes the feasibility of growing cells in 3-D systems and looks at optimal conditions to preserve and examine the cellular characteristics of cells within a 3-D environment. Overall, this thesis demonstrates the feasibility and pathobiological relevance of ex vivo culture of MCL cells giving insights into the factors that drive MCL survival and proliferation and the correlation between in vitro behaviour and clinical phenotype. It is proposed that this work can be expanded to examine therapeutic interventions in the disorder.

Existing treatments for mantle cell lymphoma (MCL) are non-curative, demonstrating a need for a refined treatment approach. Recent clinical trials have shown promising results with the use of mammalian target of rapamycin inhibitors. I hypothesize that the anti-tumor effect of mTOR inhibitors in mantle cell lymphoma is mediated by an increase in manganese superoxide dismutase (MnSOD) protein expression and accumulation of hydrogen peroxide (H₂O₂). Findings indicate that the rapamycin-induced cytostatic effect is characterized by increased levels of MnSOD and H₂O₂, and is necessary for the full growth inhibitory effect of rapamycin. Furthermore, over-expression of MnSOD elevated the level of H₂O₂ and increased sensitivity to MnSOD. Treatment with rapamycin resulted in a loss of serine 473 phosphorylation of AKT and increased levels of MnSOD were found to be due to inhibition of the mTORC2 complex. These results are the first to suggest that long term treatment of MCL cells with rapamycin inhibits the mTORC2 complex. By understanding the key signaling molecules and affected pathways in the anti-tumor effects of mTOR inhibitors, we may be able to identify additional predictive markers to improve the therapeutic value, or study drug combinations that will enhance the effect of ROSinduced cytotoxicity. A retrospective study utilizing samples from lymphoma patients receiving standard anthracycline-based therapies, identified single nucleotide polymorphisms in oxidative stressrelated genes associated with survival. Individuals carrying minor allele SNPs in myeloperoxidase (MPO) and an aldo-keto reductase (AKR1C3) were found to be associated with shorter time to disease progression and death. This data suggest that some patients may benefit from a different therapy than the current standard of care and that regulation of the redox environment plays a role in aggressive lymphoma treatment response.

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.

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.

No description available.

Mantle cell lymphoma (MCL) is one of several sub-types of B-cell lymphomas. The malignancy is very aggressive and average survival time is short. The hallmark of MCL is over expression of cyclin D1, however about 15% of all MCL cases do not display this over expression and are easily misdiagnosed. Recently the transcription factor Sox11 has been shown to be specifically over expressed in the nucleus of MCL-tumour cells, and polyclonal rabbit anti-Sox11 antibodies have been used to successfully identify MCL in both cyclin D1 positive and negative cases. Howev-er, human recombinant MCL-specific antibodies as have several advantages over these polyclonal rabbit antibodies; they can easily be produced in large quantities in vitro, their specificity is constant from batch to batch and they can possibly be used for therapeutic purposes. Because of this, it is desirable to produce human recombinant antibodies against proteins over expressed in MCL. In this study human recombinant IgGs have been produced towards two pro-teins over expressed in MCL, Sox11 and KIAA0882. This was done by cloning of single chain variable fragments (scFvs), previously selected from a large scFv library through phage display selection against Sox11- and KIAA0882-protein epitope signature tag (PrEST), into vectors containing human IgG constant regions followed by expression of human IgG antibodies in human embryonic kidney (HEK) 293 cells. One IgG clone for each antigen was shown to be functional and specific. Both clones were shown to have overlapping binding epitopes with their polyclonal rabbit antibody counterpart (rabbit anti-Sox11/KIAA0882) through competitive ELISA. The anti-Sox11 IgG was able to detect two bands in cell lysate in Western blot, of which one probably is Sox11 while the other band possibly could be Sox4. However, this needs to be confirmed in future experiments. The affinity of the anti-Sox11 IgG was measured in Biacore and compared to the affinity of its original scFv. This gave a rough estimation of the affinities, but the values are unreliable and the measurements need to be redone. Although more work has to be put into evaluating the potential of the produced IgGs, they compose a promising starting point to an improved understanding and improved diagnosis of MCL.

<p>Mantle cell lymphoma (MCL) is one of several sub-types of B-cell lymphomas. The malignancy is very aggressive and average survival time is short. The hallmark of MCL is over expression of cyclin D1, however about 15% of all MCL cases do not display this over expression and are easily misdiagnosed. Recently the transcription factor Sox11 has been shown to be specifically over expressed in the nucleus of MCL-tumour cells, and polyclonal rabbit anti-Sox11 antibodies have been used to successfully identify MCL in both cyclin D1 positive and negative cases. Howev-er, human recombinant MCL-specific antibodies as have several advantages over these polyclonal rabbit antibodies; they can easily be produced in large quantities in vitro, their specificity is constant from batch to batch and they can possibly be used for therapeutic purposes. Because of this, it is desirable to produce human recombinant antibodies against proteins over expressed in MCL. In this study human recombinant IgGs have been produced towards two pro-teins over expressed in MCL, Sox11 and KIAA0882. This was done by cloning of single chain variable fragments (scFvs), previously selected from a large scFv library through phage display selection against Sox11- and KIAA0882-protein epitope signature tag (PrEST), into vectors containing human IgG constant regions followed by expression of human IgG antibodies in human embryonic kidney (HEK) 293 cells. One IgG clone for each antigen was shown to be functional and specific. Both clones were shown to have overlapping binding epitopes with their polyclonal rabbit antibody counterpart (rabbit anti-Sox11/KIAA0882) through competitive ELISA. The anti-Sox11 IgG was able to detect two bands in cell lysate in Western blot, of which one probably is Sox11 while the other band possibly could be Sox4. However, this needs to be confirmed in future experiments. The affinity of the anti-Sox11 IgG was measured in Biacore and compared to the affinity of its original scFv. This gave a rough estimation of the affinities, but the values are unreliable and the measurements need to be redone. Although more work has to be put into evaluating the potential of the produced IgGs, they compose a promising starting point to an improved understanding and improved diagnosis of MCL.</p>