This thesis follows the research portfolio format and is carried out in part fulfilment of the academic component of the Doctorate in Clinical Psychology at the University of Edinburgh. An abstract provides an overview of the entire portfolio thesis. Chapter One contains a systematic review of published research investigating the experience of living with incurable forms of haematological malignancy. Chapter Two is an empirical study exploring adults’ experiences of living with follicular lymphoma while being maintained under the ‘watch and wait’ protocol. Both chapters are prepared for submission to the European Journal of Cancer Care, and follow their author guidelines.

Tumour-associated macrophages (TAM) are a major component of the inflammatory infiltrate that typifies most malignancies. Among them, Burkitt’s lymphoma (BL), a high-grade non Hodgkin lymphoma (NHL) of B-cell origin, represents a characteristic example. Studies from our group have shown that TAM in BL exert pivotal roles that are mainly supportive of tumourigenesis such as maintaining an immunosuppressive microenvironment. In order to unravel the molecular mechanisms underlying TAM functions in BL, solid tumours from a mouse xenograft model of BL have been used to obtain TAM and assess their activation status in vivo. Laser-capture microdissection has been successfully used to procure intact macrophage sections from the tumour site, allowing the production of a pure, in situ gene expression signature of TAM in BL. Tingible-body Mφ from lymph node germinal-centres and resident tissue Mφ from resting lymph nodes of non-tumour bearing mice were chosen for direct comparison with TAM. Whole-genome microarray technology has revealed a distinct TAM gene expression profile, with 454 genes being significantly up-regulated (fc ≥ 2, p<0.05) and 1293 genes being significantly down-regulated (fc ≤ -2, p<0.05) between TAM and either of the two normal Mφ populations. Further bioinformatics analysis of gene functions has highlighted matrix remodeling, phagocytosis, and immune response among the processes most highly enriched in TAM. Importantly, mRNA and tissue expression of selected differentially expressed genes relevant to these processes was validated by real-time qPCR and immunofluorescence labeling respectively. Following the generation of the TAM profile in situ, in vitro experimental approaches were undertaken in order to investigate how specific elements of the BL microenvironment drive the observed TAM signatures. Specifically, the direct role of apoptotic tumour cells, a key component of the BL microenvironment, versus that of viable tumour cells in driving TAM matrix remodelling gene expression was assessed in short-term mouse and human Mφ-NHL cell co-cultures. From the aforementioned cluster, emphasis was given to MMP12 and MMP2 transcripts: mRNA and protein expression of these MMPs was found to be up-regulated in Mφ following viable tumour cell co-cultures and this effect was further enhanced following apoptotic tumour cell co-cultures, implying that apoptotic NHL cells could directly shape TAM matrix remodeling phenotype in BL in vivo. Whereas the mRNA of both MMPs was solely Mφ-derived in this system, MMP12 and MMP2 protein was surprisingly found also to be increased in NHL cells in the apparent absence of increased mRNA. Detailed examination of MMP12 production by NHL cells revealed that it is most likely an apoptosis-dependent process, since apoptotic NHL cells generated through different apoptosis stimuli, as well as apoptotic cell-derived microparticles, showed markedly increased MMP12 protein levels. In conclusion, the data presented in this thesis, provide the first insight into the in vivo activation status of TAM in high-grade NHL, through generation of the TAM gene signature in situ. Upon further in vitro studies, apoptotic NHL cells were shown to directly modulate the matrix remodelling component of the TAM signature as well as to actively produce matrix remodelling mediators themselves, suggesting distinct roles for tumour cell apoptosis within the NHL microenvironment that can profoundly influence the disease outcome.

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.

Tumour-associated macrophages (TAMs) have been shown to play an important role in tumour survival and progression. Thus, high numbers of macrophages in the tumour tissue are often associated with a poor prognosis. Identification of factors responsible for recruiting macrophages to the sites of different types of tumours might help to develop more effective cancer treatment. Burkitt's lymphoma (BL) is characterised by uncontrolled cell proliferation, high rate of spontaneous apoptosis and significant macrophage infiltration. Although BL cells undergo extensive apoptosis, in situ their corpses are cleared very effectively by macrophages infiltrating the tumour. It is now widely believed that dying cells are themselves able to release chemotactic molecules to ensure macrophage chemotaxis and subsequent clearance of their site of death. Previous work carried out in this laboratory identified fractalkine/CX3CL1 (FKN) released from dying BL cells to be an important player in macrophage chemotaxis to BL. Yet, these results have also indicated that FKN may not be the only chemokine involved in this process. Following from those observations, the first part of this work focused on examination of the potential role of monocyte chemoattractant protein-1 (MCP-1) in macrophage recruitment to BL. Despite the initial promising results, careful analysis of the data obtained by various techniques led to the conclusion that MCP-1 is, probably, not expressed by BL cells. Subsequently, effort was concentrated on understanding mechanisms regulating FKN processing during cell death. The studies performed before in this laboratory identified a new form of FKN to be present in apoptotic BL cells and showed that this is the form that is, most likely, responsible for mediating macrophage migration. Here, this apoptosis-related 60 kDa FKN was found to be a likely caspase-3 cleavage product. Moreover, it was demonstrated that FKN and active caspase-3 are released together in apoptotic BL cell-derived microparticles, suggesting that the proteolytic events could take place also extracellularly. In the final results chapter the differences between BL cell lines in the way they process FKN during cell death were revealed and a new cell death-associated 55 kDa FKN was observed. Through several lines of evidence, this new form was identified to be a possible product of calpain-mediated proteolysis. To conclude, this work provides the first evidence for a possible direct participation of the two major cell death executioner proteases – caspases and calpains, in production of ‘find me’ signals for macrophages and thus, ensuring effective clearance of dying cells. These results indicate that FKN cleavage and release might be of key importance during cell death. Moreover, the studies presented here contribute to better understanding of the process of FKN secretion.

Mantle cell lymphoma (MCL) is an extremely difficult to treat subtype of non-Hodgkin lymphoma (NHL) with a low patient survival rate compared to most common cancers. Recently, nanoparticle delivery systems have received a great deal of interest for treating NHL. One of the more promising cargo options for these systems is short interfering RNA (siRNA). siRNA is a 18-23 nucleotide long double stranded RNA that is used to inhibit the protein expression of target mRNAs in a sequence specific manner. MCLs have several commonly overexpressed genes compared to normal cells making it an ideal candidate for siRNA therapies. For RNA interference to occur, A delivery vehicle is needed for the siRNA to reach the cytoplasm of the cell. In this thesis, ionizable lipid-like materials termed lipidoids are formulated into lipid nanoparticles (LNPs) to deliver siRNA. A new library of lipidoids is constructed to gain a better understanding of how the lipidoid tail-structure affects the silencing ability of LNPs. A novel tail precursor is identified as conferring potency to LNPs. Then, LNPs are used to silence genes within difficult to transfect MCL cells. LNPs targeting the anti-apoptotic protein Mcl-1 exhibit potent gene silencing and cause an increase in the fraction of cells undergoing apoptosis. This is important because there is no therapeutic that is FDA approved that targets this commonly overexpressed protein. Because of this LNP’s potency, siRNAs targeting multiple genes can be encapsulated into LNPs without causing unwanted toxicity. LNPs targeting several genes in multiple pathways cause a larger fraction of MCL cells to undergo apoptosis compared to cells treated with LNPs targeting only one gene. A major issue in cancer therapeutics is that the majority of nanoparticles accumulate in the liver. In an effort to improve the delivery of LNPs to target cells, changes to their formulations and administration methods are investigated as a means to improve LNP circulation time, biodistribution, and silencing ability. Overall, this work identifies lipidoid nanoparticles as potent siRNA delivery systems to treat MCL and investigates key properties for further improvement in LNP siRNA delivery to target cells.

<p>Lymphomas are a heterogeneous group of hematological malignancies composed of diseases with diverse molecular origins and clinical outcomes. Derived from immune cells of lymphoid origin, lymphoma can arise from lymphoid cells present anywhere in the body, from the spleen and lymph nodes to peripheral sites like the liver and intestines. Current strategies for lymphoma diagnosis involve primarily histopathological examinations of the tumor biopsy, including cytogenetics and immunophenotyping. As more data becomes available, diagnoses may increasingly depend on genomic features that define each disease. Classification of lymphoid neoplasms is generally based on the cell of origin, or the lineage of the normal cell that the cancer is thought to arise from. Lymphomas can be classified into dozens of distinct diagnostic entities, though any two patients with the same diagnosis may have very different outcomes and molecular underpinnings, so we need to understand both the commonalities of patients with the same disease and the unique features that may require personalized treatment strategies. Patient prognosis in lymphoma depends greatly on the type of lymphoma, ranging from nearly curable diseases with over 90% five-year survival rates, to most patients dying in the first year in the worse entities. Greater clarity is needed in the role of the underlying genomics that contribute to these variable treatment responses and clinical outcomes. </p><p>Next-generation sequencing approaches allow us to delve into the molecular underpinnings of lymphomas, in order to gain insight about the origin and evolution of these diseases. High-throughput sequencing protocols allow us to examine the whole genome, exome, epigenome, or transcriptome of cancer cells in tens to hundreds of patients for each disease. As cost of sequencing is reduced, and the ability to generate more data increases, we face increasing computational challenges to both process and interpret the wealth of data available in cancer genomics. Developing efficient and effective bioinformatics tools is necessary to transform billions of sequencing reads into actionable hypotheses on the role of certain genes or biological pathways in a specific cancer type or patient. </p><p>In this dissertation, I present several strategies and applications of integrative computational genomics in lymphoma, with contributions throughout the research process, from development of initial assays and quality control strategies for the sequencing data, to joint analysis of clinical and genomic data, and finally through follow-up experimental models for lymphoma. </p><p>First, I focus on two rare T cell lymphomas, hepatosplenic T cell lymphoma (HSTL) and enteropathy associated T cell lymphoma (EATL), which are both diseases with very poor clinical outcomes and a previous dearth of knowledge on the genetic basis of the diseases. We define the somatic mutation landscape of HSTL, through application of exome sequencing and find SETD2 to be the most highly mutated gene. We further utilize the exome sequencing data to investigate copy number alterations and show a significant survival difference between cases with and without certain arm-level copy number alterations. Knockdown of SETD2 in an HSTL cell line, followed by RNA sequencing, demonstrates the role of SETD2 loss in proliferation and cell cycle changes, linking the SETD2 mutations to a potential oncogenic mechanism. Furthermore, we investigate the potentially targetable mutations in the JAK-STAT pathway and demonstrate oncogenic downstream molecular phenotypes and potential druggability of these mutations. In the enteropathy associated T cell lymphoma study, we apply exome and RNA sequencing to a large EATL cohort. Our findings show a significant role for loss of function mutations in chromatin modifiers and JAK-STAT signaling genes. EATL can be separated into two subtypes, Type I and Type II, which we show to have convergent genomic features, in the face of divergent gene expression. RNA sequencing data defines a distinct separation between the two subtypes. Delving further into the role of SETD2 in these T cell lymphomas, we generate a mouse model with a conditional knockout of SETD2 in T cells and demonstrate a role for SETD2 in altering the lineage development of T cells. </p><p>To understand more about why certain genetic abnormalities are recurrent in some disease entities and not others, we turn to the cell of origin for clues. We pair two different lymphomas, Burkitt lymphoma and mantle cell lymphoma, with their associated cells of origin, germinal center B cells and naive B cells. These closely related cell types have much in common as B cells, but from studies of their transcriptomes, we know that there are many molecular differences that distinguish the two. In this work, after looking more closely at mantle cell lymphoma genomics, we look at the underlying chromatin markers that define the epigenomes of these B cells. We test the association between chromatin markers and mutation rates of genes between these two cell types and lymphomas, and find that genes with more open chromatin may have a higher mutation rate, when comparing closely related cells and lymphomas. Finally, I present my work on developing an RNA sequencing based strategy for defining the complete transcriptome of diffuse large B cell lymphoma (DLBCL). Gene expression profiling with microarray has shown the existence of two subtypes in DLBCL, activated B cell like (ABC) and germinal center B cell like (GCB). However, the role for non-coding RNAs, alternative splicing, and mutations, in these two subtypes and the larger group is previously not well understood. We develop a strand-specific RNA sequencing strategy that will allow the investigation of the total RNA transcriptome in DLBCL, including microRNAs, lncRNAs, and other important non-coding RNAs. Furthermore, we show that RNA sequencing can be used to distinguish the two subtypes, including through RNA sequencing based mutation calls, as well as through differentially expressed lncRNAs that we define for the first time in DLBCL.</p><p>Broadly, this dissertation contributes novel findings in the field of lymphoma genomics, as well as presenting a framework for computational integrative genomics that can guide future studies. The heterogeneity of lymphoma across cases requires us to dive deep into individual diseases, even rare ones, as well as appreciate the similarities and differences across lymphomas. To improve diagnoses, prognoses, and treatment options, we need to understand the molecular origins of lymphoma. Using a range of molecular and computational approaches, we can move closer to true personalized medicine at the genomic level.</p> / Dissertation

No description available.

No description available.

Background: Few studies have investigated the relationship between physician and patient-assessed performance status (PS) in blood cancers. Methods: Retrospective analysis among 1418 patients with haematologic malignancies seen at Dana-Farber Cancer Institute between 2007 and 2014. We analysed physician-patient agreement of Eastern Cooperative Oncology Group PS using weighted kappa-statistics and survival analysis. Results: Mean age was 58.6 years and average follow-up was 38 months. Agreement in PS was fair/moderate (weighted kappa = 0.41, 95% CI 0.37-0.44). Physicians assigned a better functional status (lower score) than patients (mean 0.60 vs 0.81), particularly when patients were young and the disease was aggressive. Both scores independently predicted survival, but physician scores were more accurate. Disagreements in score were associated with poorer survival when physicians rated PS better than patients, and were modified by age, sex and severity of disease. Conclusions: Physician-patient disagreements in PS score are common and have prognostic significance.

Les cytokines sont d’importants médiateurs dans la physiopathologie des lymphomes hodgkiniens (LH). A partir d’une cohorte de 464 patients, nous avons évalué l’impact pronostique de onze SNPs parmi les gènes de cytokines : IL10 (rs1800890, rs1800896, rs1800871, rs1800872), TNFA (rs1800629) ; IL6 (rs1800795) ; IL1B (rs16944) ; ILRN (rs419598) ; INFG (rs2430561) ; IL12 (rs3212227) ; CCL17 (rs223828). Le génotypage du SNP de l’IL12 montre une distribution différente de celle attendue dans la population générale selon le test de Hardy-Weinberg. Ce résultat suggère que les variations génétiques de l’IL12 pourraient être impliquées dans la susceptibilité au LH. Les patients porteurs du génotype IL10-1082AA présentent un taux de rémission complète au traitement initial supérieur aux patients présentant un autre génotype (95% vs. 88% P = .02). Pour les patients de stade avancé III-IV, le taux de survie globale à 6 ans est statistiquement différent entre les génotypes IL10-592AA/CC/AC et IL10-819TT/CC/CT (100%, 94%, 78%, P = .03). Ce résultat est retrouvé pour les patients porteurs de LH n’exprimant pas l’EBV. Pour les LH EBV négatif, le taux de survie sans progression à 6 ans est différent en fonction du génotype du TNFA-308AA/GG/AG (100%, 84%, 68%, P = .03). Il n’a été pas retrouvé de corrélation entre les génotypes et les dosages plasmatiques de l’IL-10, TNFA, IL-1RA, IL-6. Cette étude montre que le ‘‘fond génétique immun’’ est important à prendre en considération pour définir le pronostic des patients. Le rôle des SNPs de l’IL10 et du TNFA dans les LH EBV négatif devra être confirmé ainsi que l’influence des variations génétiques de l’IL12 dans la susceptibilité au LH. / Cytokines are important immune mediators implicated in Hodgkin lymphoma (HL) pathogenesis but little is known on the role of immune gene variations. We assessed prospectively the prognostic role of cytokine gene single nucleotide polymorphisms (SNPs) in HL patients (pts) : IL10 (rs1800890, 448 pts; rs1800896, 459 pts ; rs1800871, 446 pts ; rs1800872, 447 pts), TNFA (rs1800629, 464 pts) ; IL6 (rs1800795, 201 pts) ; IL1B (rs16944, 198 pts) ; ILRN (rs419598, 199 pts) ; INFG (rs2430561, 200 pts) ; IL12 (rs3212227, 259 pts) ; CCL17 (rs223828, 198 pts). IL12 genotype distribution appears significantly different from what observed in general population according to Hardy-Weinberg test which was already observed in another published study. IL10-1082AA genotype was associated with better complete response than other IL10 genotypes (95% vs. 88% P = .02). For patients with stage III-IV HL, the 6-year overall survival was statistically different between IL10-592AA/CC/AC and IL10-819TT/CC/CT genotypes (100%, 94%, 78%, P = .03). This prognostic effect was observed in EBV-negative but not in EBV-positive HL. In EBV-negative HL, TNFA-308AA/GG/AG genotypes had a different 6-year progression-free survival (100%, 84%, 68%, P = .03). No correlation was observed between genotypes and IL-10, TNFA, IL-1RA, IL-6 cytokine levels. This exploratory study suggests an effect of IL10 and TNFA SNPs in predicting HL outcome but other studies are needed to decipher the role of the host immunogenetic background, in particular the relation with EBV. Regarding the IL12 genotyping results, whether IL12 polymorphism is implicated in HL susceptibility needs also to be clarify.