The efficacy of therapeutic modalities for non-Hodgkin’s lymphoma have been tested and improved throughout the 19th century through various series of drug trials aimed at eliminating cellular malignancies, first through chemotherapy treatment, and more recently through immunotherapy. While to an extent successful in eliminating cancerous lesions and affected cells, chemotherapy treatments have shown to influence the induction of new malignancies, through genetic mutation, as well as unwanted toxic effects of systemic poisoning. The purpose of this thesis is to compare treatment methods in terms of their biomolecular activity, precision of intended results, and possible drawbacks, as well as their application to specific populations of Non-Hodgkin lymphoma diagnoses, including Follicular and Diffuse Large B-Cell lymphomas. In the following sections on contributing factors specific to Diffuse Large B-Cell lymphomas and Follicular lymphoma, elements of disease prognosis will be analyzed from a molecular and clinical point of view. This includes a focus on the impact of genetic mutation, the immunohistochemical evidence these changes present, as well as the variances in immune cell functionality, and finally a description of symptoms with direction to specific underlying causes. An analysis of standard of care chemotherapy, and monoclonal antibody treatments will then be provided for each occurrence. The second segment will discuss novel techniques being developed for the treatment of lymphoma including but not limited to new monoclonal antibodies, synthetic lethality modulation, inhibition of selected chemokine receptors, DNA vector immunization for production of internal host antibodies, concepts of cell mediated bispecific antibody induced destruction, and new generations of Immunomodulatory drugs. With the recent development of cost effective sequencing technology, included is a discussion of the shift towards personalized medicine treatments, targeting appropriate phenotypic specific populations for optimal results, as it relates to therapies for Diffuse Large B-Cell lymphoma and Follicular lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma (NHL). The current standard of care is the combination of rituximab with cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP), but this only results in a 60% over-all 5-year survival rate, thus highlighting a need for new therapeutic approaches. Histone deacetylase inhibitors (HDACi) are novel therapeutics that is being clinically evaluated for combination therapy. Rational selection of companion therapeutics for HDACi is difficult due to their poorly understood, cell-type specific mechanisms of action. To understand these mechanisms better, we developed a pre-clinical model system of response to the HDACi belinostat. Using this model system, we identified two major responses. Resistance, consisting of a reversible G1 cell cycle arrest with little induction of apoptosis; or sensitivity, consisting of mitotic arrest and high levels of apoptosis. In this dissertation, we determine that the induction of G1 cell cycle arrest is due to the increased expression of cyclin dependent kinase inhibitors (CDKi) that bind to and inhibit the cyclin E/CDK2 complex thereby blocking the final repressive phosphorylation steps of Rb protein. Repression of transcriptional elongation blocked CDKi upregulation and prevented G1 cell cycle arrest in belinostat-resistant cells. Additionally, we identified that belinostat arrests sensitive cells prior to metaphase and belinostat-resistant cells slow-down in mitosis but complete the process prior to arresting in G1. The combination of belinostat with the microtubule-targeting agent, vincristine resulted in strong synergistic induction of apoptosis by targeting mitotic progression. Furthermore, this combination prevents polyploidy, a key mechanism of resistance to microtubule targeting agents. Finally, we utilized selective class one HDAC inhibitors to identify the individual contributions of HDACs in the eliciting the responses observed with belinostat treatment. HDAC1&2 inhibition recapitulated the belinostat-resistant phenotype of G1 cell cycle arrest with little apoptosis, in both belinostat-resistant and sensitive cell lines. HDAC3 inhibition resulted in the induction of DNA damage, increased S phase and the induction of apoptosis in belinostat-resistant cells. Belinostat-resistant cells did not have observable effects to HDAC3 inhibitor alone but when combined with vincristine had significantly increased G2/M population at early time points. This suggests that HDAC3 maintains roles in DNA replication and also in mitotic progression. HDAC3 inhibition combined with vincristine resulted in a significant increase in polyploidy, suggesting that HDAC3 might not regulate the expression of apoptotic regulating factors as belinostat does.

No description available.

Thesis (M.A.) PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / High dose chemotherapy followed by autologous stem cell transplantation (ASCT) remains the standard of care for patients with relapsed or refractory (R/R) diffuse large B cell lymphoma (DLBCL) who are chemosensitive to salvage therapy. There is now evidence that the achievement of complete remission by PET scan (PET-CR) after salvage therapy is a favorable determinant of ASCT outcome, implying that PET response should be part of the prognostic assessment for patients considering ASCT. However, it is unclear whether other prognostic factors are still relevant in patients getting post-salvage PET scanning. Moreover, while ASCT is often also used for patients with R/R transformed indolent lymphoma (TIL), there are no data on whether prognostic factors that are important for DLBCL patients, especially PET response to salvage, are similarly prognostic in this population. We conducted a retrospective study of 143 patients with R/R DLBCL and TIL who were transplanted in the last decade and had a post-salvage PET scan prior to ASCT. We examined prognostic factors in both groups, and constructed a prognostic score for DLBCL patients. For patients with DLBCL, post-salvage PET response was an important prognostic factor. Advanced age and symptomatic relapse were also significantly associated with inferior outcome. A simple score could stratify patients into 3 risk groups with 4-year post-ASCT overall survival of 84%, 59%, and 10%, and 4-year progression-free survival of 67%, 41% and 0% (p<0.0001 for both). However, none of those factors (including PET response to salvage) could be demonstrated for TIL, likely because of the limited sample size. Our novel prognostic score for DLBCL patients undergoing ASCT may be useful for prognostication, for stratification in clinical trials, and to motivate the design of new strategies for patients in the highrisk group, who may not derive benefit from standard ASCT. Those factors, however, do not apply to patients with TIL, which has important implications for their treatment and inclusion in ASCT clinical trials with larger sample sizes. / 2031-01-01

Philosophiae Doctor - PhD / The incidence of Diffuse large B cell Lymphoma has been increasing lately at an alarming rate especially, in developing countries like Sudan. The standard therapy in Sudan is based solely on the R-CHOP chemotherapy regimen, yet it has been noticed that Diffuse Large B cell Lymphoma prognosis remains unfavorable. The late diagnosis and the consequent side-effects of the therapy directly affected the disease’s poor outcome. There is a scarcity of scientific publications regarding DLBCL in Sudan, but the increased burden necessitates the need for further research.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20260号 / 医博第4219号 / 新制||医||1020(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 前川 平, 教授 木原 正博, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease with multiple distinct molecular subtypes. Increased NF-κB activity and expression of the microRNA miR-155 (product of the BIC gene) are associated with one subtype, called the activated B-cell (ABC) subtype. It is shown here that induction of NF-κB activity leads to increased miR-155 expression, the levels of miR-155 in a panel of B-lymphoma cell lines correlate with increased NF-κB activity, and the NF-κB p50/p65 heterodimer binds to a specific DNA site in the BIC promoter. Also described is a regulatory network wherein NF-κB-dependent up-regulation of miR-155 leads to reduced PU.1 transcription factor expression and consequently reduced PU.1-driven expression of B-lymphoma marker CD10 in the human B-lymphoma cell line BJAB. Genetic variation in DLBCL can be used to explain the response of individual patients to chemotherapy. One cancer therapeutic approach currently in clinical trials uses histone deacetylase inhibitors (HDACi's) as a monotherapy or in combination with other vi agents. It is shown here that two pan-HDACi's, trichostatin A and vorinostat, induce apoptosis in seven of eight human DLBCL cell lines. Ectopic over-expression of antiapoptotic proteins BCL-2 and BCL-XL or the pro-apoptotic protein BIM in select DLBCL cell lines can confer further resistance or sensitivity, respectively, to HDACi treatment. Additionally, the BCL-2 family antagonist ABT-737 can increase the sensitivity of several DLBCL cell lines to vorinostat-induced apoptosis, including the HDACi-resistant SUDHL6 cell line. Moreover, one vorinostat-resistant variant of the HDACi-sensitive cell line SUDHL4 has increased expression of anti-apoptotic proteins BCL-XL and MCL-1 and decreased sensitivity to ABT-737, and a second such variant cell line has increased expression of anti-apoptotic protein MCL-1. These results suggest that the balance of anti- to pro-apoptotic BCL-2 family protein expression is important in determining the sensitivity of DLBCL cell lines to HDACi-induced apoptosis. Thus, the sensitivity of DLBCL cell lines to treatment with HDACi's appears to depend on the complex regulation of BCL-2 family members, suggesting that the response of a subset of DLBCL patients to HDACi treatment may benefit from co-treatment with BCL-2 antagonists.

<p dir="ltr">Lymphoma is a prevalent malignancy in dogs. Diffuse large B-cell Lymphoma (DLBCL) is the common subtype that represents about 50% of the clinically seen lymphoma cases. DLBCL diagnosis relies on cytological examination of a fine needle aspirate and histological evaluation by immunohistochemistry (IHC) in most common practices. This workflow is sufficient to confirm the diagnosis; however, it may be challenging to differentiate reactive and neoplastic forms in some controversial cases. In such cases, PCR-based clonality assays and flow cytometry (FC) can help with more conclusive diagnoses. So, finding more biomarkers that can detect and track DLBCL early and over time is a must for a final diagnosis and helps us learn more about how DLBCL starts at the molecular level. MicroRNAs (miRNAs), the small non-coding RNAs, regulate gene expression by binding to the 3'-untranslated region of protein-coding RNAs, leading to either RNA degradation or translational repression. They can switch on and off genes to regulate physiological and pathological processes. MicroRNA stability features and tissue availability make them promising biomarkers for identifying and sub-classifying patients and sequentially evaluating the disease status or the response toward a specific medicine. This dissertation investigates the small RNA sequence analysis, the differentially expressed miRNAs between healthy and DLBCL-affected lymph nodes, and the miRNA profile in DLBCL cases with different outcomes.</p>

A família paraoxonase (PON1, PON2 e PON3) tem sido objeto de grande interesse por prevenir o estresse oxidativo e o processo inflamatório, condições importantes na carcinogênese. O Linfoma Difuso de Grandes Células B (LDGCB) consiste no subtipo histológico mais comum dentre os linfomas, doenças que se originam a partir das células do tecido linfoide e exibem distintos comportamentos clínicos, fatores patológicos e características epidemiológicas. Há escassez de dados sobre a atuação das paraoxonases na susceptibilidade diferencial ao risco de linfomas. Deste modo, o objetivo do presente estudo foi investigar a frequência alélica e genotípica dos polimorfismos 192QR e 55LM, no gene da PON1, e 148AG e 311SC, no gene da PON2 e o efeito desses polimorfismos sobre as atividades da enzima PON e perfil lipídico em 182 indivíduos (78 pacientes com LDGCB e 104 indivíduos saudáveis). O sangue foi coletado, em 4 momentos, para a determinação do perfil lipídico e das atividades arilesterase e paraoxonase da PON. O DNA foi extraído de leucócitos do sangue periférico pelo método de extração salina. A análise dos polimorfismos foi realizada por PCR/RFLP. Não houve diferença estatística na distribuição de genótipos e frequência de alelos dos polimorfismos nos genes da PON1 e PON2. A atividade sérica da arilesterase apresentou valores significativamente maiores apenas entre os indivíduos saudáveis (p=0,001). As variantes 55MM e 192QQ, do gene da PON1, influenciaram as atividades arilesterase (p=0,011) e paraoxonase (0,001). O polimorfismo PON2 311SS associou-se a atividade arilesterase (p=0,021). A concentração de autoanticorpos oxLDL foi alterada, pela presença do genótipo 55LM (p=0,037) nos indivíduos com LDGCB / The paraoxonase family (PON1, PON2 and PON3) have been the subject of great interest, since they are responsible for preventing oxidative stress and inflammation, conditions important in carcinogenesis. The Diffuse Large B Cell Lymphoma (DLBCL) is the most common histological subtype among lymphomas, diseases that originate from cells of the lymphoid tissue and exhibit clinically distinct behaviors and pathological and epidemiological factors. There are paucity of data on the activity of paraoxonase in the differential susceptibility to the risk of lymphoma. Thus, the objective of this study was to investigate the genotypic and allelic frequency of polymorphisms 192QR and 55LM, in the PON1 gene and 148AG and 311SC, in the PON2 gene and the effect of these polymorphisms on PON enzyme activities and lipid profile in 182 subjects (78 patients with DLBCL and 104 healthy subjects). Blood was collected in four moments for the determination of lipid profile and paraoxonase and arylesterase activities of PON. The DNA was extracted from peripheral blood leukocytes by salt extraction method. The analysis of polymorphisms was performed by PCR/RFLP. There was no statistical difference in the distribution of genotypes and allele frequencies of polymorphisms in the PON1 and PON2 genes. The serum arylesterase activity was significantly higher only among healthy subjects (p=0.001). 192QQ and 55MM variants of the PON1 gene, influenced arylesterase (p=0.011) and paraoxonase (0.001) activities. The PON2 polymorphism was associated with 311SS arylesterase activity (p = 0.021). The concentration of oxLDL autoantibodies was altered by the presence of 55LM genotype (p = 0.037) in patients with DLBCL

This study consists of two pieces of work investigating haematological malignancies; Acute Lymphoblastic Leukaemia (ALL) and Diffuse Large B Cell Lymphoma (DLBCL). Firstly, Pre-B ALL represents the most common paediatric malignancy and despite increasingly improved outcomes for patients, ~ 20% of all patients diagnosed with ALL relapse. Activating mutations in the RAS pathway are common (~50%) and result in hyperactivation of the MAPK pathway. I identified Erk negative feedback control via DUSP6 to be crucial for NRASG12D-mediated pre-B cell transformation and investigated its potential as a therapeutic target. I showed that a small molecule inhibitor of DUSP6 (BCI) selectively induced cell death in patient-derived pre-B ALL cells; with a higher sensitivity observed in relapse pre-B ALL. I also discovered that a high level of Erk activity is required for proliferation of normal pre-B cells, but dispensable in leukemic pre-B ALL cells. In addition, I found that human B cell malignancies can be grouped into three categories that fundamentally differ in their ability to control Erk signalling strength. Secondly, DLBCL is the most common haematological malignancy and although potentially curable with chemotherapy, 40% of patients still succumb from their disease. Recent exome sequencing studies have identified hundreds of genetic alterations but, for most, their contribution to disease, or their importance as therapeutic targets, remains uncertain. I optimised a novel approach to screen the functional importance of these mutations. This was achieved by reconstituting non-malignant, primary, human germinal centre B cells (GC B cells) with combinations of wildtype and mutant genes to recapitulate the genetic events of DLBCL. When injected into immunodeficient mice, these oncogene-transduced GC B cells gave rise to tumours that closely resemble human DLBCL, reinforcing the biological relevance of this system. To screen potential tumour suppressor mutations in this system in a high throughput fashion, I developed a lymphoma-focused CRISPR library of 692 genes recurrently altered in B cell lymphomas. These experiments identified GNA13 as an unexpectedly potent tumour suppressor in human GC B cells and provided new understanding to its mechanism of action. These findings provide novel understanding of the complexity of oncogenic mechanisms in human B cell malignancies.