Immune evasion is an emerging hallmark of cancer. Dysregulation of the major histocompatibility complex class I (MHC-I) is a frequent mechanism of immune evasion utilized by tumor cells and is particularly relevant to the pathogenesis of B-cell lymphomas, including diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (cHL). A better understanding of MHC-I dysregulation in B-cell lymphomas is necessary to identify factors related to the risk, development, and progression of these tumors. In this thesis, we investigate the role of MHC-I dysregulation in DLBCL and cHL through the application of computational approaches to study genomic data. First, we introduce some background information about the normal function of MHC-I in the immune response to cancer and viral infection as well as the phenomenon of MHC-I dysregulation in the context of cancer. We provide an overview of how factors such as germline zygosity of HLA class I (HLA-I) genes and somatic alterations in the genes B2M and HLA-I that encode the protein subunits of MHC-I contribute to the development of DLBCL and cHL. Second, we present a study of the effects of HLA-I allele zygosity on survival in a cohort of 519 DLBCL patients treated with R-CHOP immunochemotherapy stratified by molecular subtype. Homozygosity in HLA-I was associated with a worse overall survival in patients whose tumors were classified in the “EZB” subtype, associated with somatic mutation in the epigenetic regulator EZH2. We find an association between the zygosity of the genes HLA-B and -C specifically and overall survival in EZB-DLBCL. These findings indicate that HLA-I zygosity may be a risk factor for worse clinical prognosis in patients with the EZB subtype of DLBCL. Third, we present a study of the genetic landscape of cHL tumors that are associated with infection with Epstein-Barr virus (EBV). We analyze inherited HLA-I allele types, somatic mutations, copy number changes, and mutational signatures in a cohort of 57 cHL patients (15 EBV-positive). We find that EBV-positive cHL is genetically distinct from EBV-negative cHL and is characterized by lower somatic mutation load and different activities of mutation signatures. Further, we find that cHL tumors are characterized by different patterns of MHC-I dysregulation depending on the EBV infection status. Germline homozygosity in HLA-I was associated with the EBV-positive subtype of cHL, while somatic alterations in HLA-I were associated with the EBV-negative subtype of cHL. These results suggest that inherited HLA-I homozygosity may be a risk factor for the EBV-positive subtype of cHL. Fourth, we expand our study of HLA-I in virus-associated cHL to perform a comparative analysis of virus-positive and virus-negative tumors in nine cancers linked to five viruses. We find that virus-positive tumors occur more frequently in males and show geographical disparities in incidence. Genomic analysis of 1,658 tumors reveals virus-positive tumors exhibit distinct mutation signatures, recurrent mutations in the RNA helicases DDX3X and EIF4A1, and a lower somatic mutation burden compared to virus-negative tumors of the same cancer type. We find that germline homozygosity in HLA-I is a potential risk factor for the development of EBV-positive cHL, but not other common virus-associated solid or hematological malignancies. Finally, we present in the Appendix a study of the genomic characterization of plasmablastic lymphoma (PBL), a rare EBV-associated B-cell lymphoma that occurs in the context of immunodeficiency caused by human immunodeficiency virus (HIV) infection. We find that PBL is characterized by mutations leading to constitutive activation of the JAK-STAT pathway. We additionally identify recurrent mutations in immune-related genes, such as B2M. These findings indicate a potential role for MHC-I and immune dysregulation in the pathogenesis of other B-cell lymphomas.

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This thesis is the construct of a computational system for studying the nasopharyngeal carcinoma (NPC) using high-throughput sequencing data. The system involves several components, including discovery of gene fusion in NPC cell line, construction of Esptein-Barr virus (EBV) genome, and evaluation on contaminated sequencing data alignment approaches. We successfully discovered a gene fusion (UBR5-ZNF423) in a NPC cell line (C666-1) which was verified by lab experiments and found in 8.3% of primary tumors. It was discovered the regulation of this gene affect the growth of cancer cell. We constructed the EBV genome in C666-1. It serves as an important reference for studying this important NPC cell line, which was the only NPC cell line in the world for a long time. We also evaluated three mapping approaches. Two of them are designed to filter out potential mouse contamination reads on human sequencing data, which can originate from NPC human-in-mouse xenografts. We found that special care should always be applied to contaminated data. Although direct mapping can give acceptable results if in most cases, the combined-based approached is suggested. It can effectively reduce false positive variants and maintain good enough numbers of true positive variants. Filtering approach is an alternative to the combined-based approach that can also effectively reduce contamination when memory is not sufficient. / 本論文利用電腦有系統地研究鼻咽癌，當中的數據利用了高通量測序技術來定序。其中章節包括在鼻咽癌胞系中尋找融合基因、組建潛藏於人體可引致鼻咽癌的EB病毒基因組、還有評價幾種可處理受污染序列的序列排列方法。我們成功地在鼻咽癌胞系（C666-1）中發現出一個融合基因（UBR5-ZNF423），並在實驗中確定此成果，其中發現在原發腫瘤中有8.3%的樣本中找出此融合基因。此外，也發現這融合基因調控會影響到癌細胞的生長。C666-1鼻咽癌胞系在過往有一段很長的時間裡，都是全世界唯一的鼻咽癌胞系，因此它有非常重要的參考價值，在此研究，我們組建了在C666-1裡的EB病毒基因組，使它作為研究C666-1的參考樣本。另外，我們評價了三種處理排列的方法，其中兩種的設計能過濾部分人類序列數據當中老鼠基因組的污染，老鼠基因組的污染可以來自於異種移植，即把人類癌細腫瘤移植於老鼠身上種植，我們建議在情況許可下都使用特殊的處理方法而不是直接作序列排列。直接作序列排列數據雖然已有合理的表現，但相比之下組合基因組式序列排列方法能有效減少錯誤肯定的遺傳變異，並同時保留足夠多正確肯定的遺傳變異，所以組合基因組式序列排列方法應在情況許可下都使用它。過濾式序列排列方法也是一種特殊的處理方法，它也能有效減少錯誤肯定的遺傳變異，它對記憶體的需求比組合基因組式序列排列方法少，可在電腦的記憶體不足時使用它。 / Tso, Kai Yuen. / Thesis M.Phil. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 112-120). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016). / Detailed summary in vernacular field only.

Based on matching analysis between different EBV strains, we found two nucleotide variations in miR-BART21 and four nucleotide changes in miR-BART22. Interestingly, two nucleotide variations upstream of mature miR-BART22 likely favor its biogenesis by Drosha/DGCR8 processing and we experimentally confirmed this augmentation by in-vitro Drosha digestion, and thus may underline the high and consistent expression of miR-BART22 in NPC tumors. / Infection with the Epstein-Barr virus (EBV) is a strong predisposing factor in the development of nasopharyngeal carcinoma (NPC). Many viral gene products including EBNA1, LMP1 and LMP2 have been implicated in NPC tumorigenesis, although the de novo control of these viral oncoproteins remain largely unclear. / MicroRNAs (miRNAs) are a class of small, non-coding RNAs with a size around 18--24 nucleotides with significant roles in regulating gene expression by either transcriptional silencing or translational suppression. As gene regulators, recent miRNA studies have emphasized the contribution of aberrant miRNA expression in cancer development. The recent discovery of EBV encoded viral miRNAs (ebv-miRNAs) in lymphoid malignancies has prompted us to examine the NPC-associated EBV miRNAs. In this study, we have systematically examined the NPC associated EBV genome for viral-encoded miRNA expression. By constructing small cDNA libraries from a native EBV positive NPC cell line (C666-1) and a xenograft (X2117), we screened about 3000 clones and detected several small EBV fragments, within which two novel ebv-miRNAs in the BARTs region were identified. These two newly identified miRNAs, now named miR-BART21 and miR-BART22, were proven to be abundantly expressed in most NPC samples by both Northern blot and QRT-PCR analysis. / Taken together, this thesis shows that two newly identified EBV-encoded miRNAs are highly expressed in latent EBV infection in NPC. Frequent expression of miR-BART22 can be explained partially by a specific EBV strain that is associated with NPC in our locality. Our findings emphasize the role of miR-BART22 in modulating LMP-2A expression. Because LMP-2A is a potent immunogenic viral antigen that is recognized by the cytotoxic T cells (CTLs), down-modulation of LMP-2A expression by mir-BART22 may permit escape of EBV-infected cells from host immune surveillance. / We attempted to predict the potential viral and cellular targets of miR-BART21 and miR-BART22 by public available computer programs, miRanda and RNAhybrid. A number of potential cellular mRNA targets were suggested, although many failed to be validated by luciferase reporter assay. However, we found a putative miR-BART22 binding site in the LMP2A-3'UTR. Although the LMP-2A transcript is consistently detected in NPC, only 6 out of 26 (23%) primary NPC tumors show weak LMP-2A expression by immunohistochemistry (IHC). The expression levels of miR-BART22 and LMP-2A mRNA have also been determined in eleven of these tumors. Interestingly, the LMP-2A mRNA expression level did not directly correlate with protein expression, and relatively low expression levels of miR-BART22 miRNA were observed in all 3 LMP-2A positive-primary tumors. The suppressive effect of miR-BART22 on LMP-2A was also experimentally validated by a series of dual luciferase reporter assays using reporter constructs containing the putative or mutated recognition site at the LMP-2A 3'UTR. By co-transfection of different amounts of miR-BART22 with the LMP-2A-3'UTR expression vector in reporter assay, we confirmed that miR-BART22 suppressed the LMP-2A protein level in a dose-dependent manner. Furthermore, transfection of miR-BART22 into HEK293 cells that had been stably transfected with pcDNA3.1-LMP-2A, which contains a complete LMP-2A ORF and 3'UTR, readily suppressed levels of the LMP-2A protein. / Lung, Wai Ming Raymond. / Adviser: To Ka Fai. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 197-226). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2008. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 6 uppsatser. - Titel från omslaget.

Les proprotéines convertases (PCs) sont des enzymes impliquées de nombreux processus pathologiques. Nous avons étudié deux substrats des PCs: l'apeline et la glycoprotéine B (gB) du virus d'Epstein Barr (EBV), et le rôle de cette maturation protéolytique dans la médiation de leurs fonctions. L'apeline est surexprimée dans plusieurs cancers dont le cancer colorectal (CCR), et nous avons montré que la furine, un membre des PCs, clive l’apeline. Pour déterminer le rôle de ce clivage dans le CCR métastatique, nous avons généré un mutant non clivable (apeline-DM). In vitro, ce mutant inhibe la croissance de cellules cancéreuses du côlonet ne les protège pas de l'apoptose, contrairement à l'apeline sauvage. In vivo, l'apeline-DM diminue drastiquement la croissance tumorale et la formation de métastases hépatiques chez la souris. Les mêmes résultats sont obtenus dans des modèles de souris déficientes pour l’apeline, démontrant l'intérêt d'utiliser l'apeline-DM ou des dérivés comme potentiels agents anticancéreux dans le traitement des CCR métastatiques. La gB du virus EBV, virus impliqué dans certains cancers lymphoïdes et épithéliaux chez l'homme, permet l'entrée du virus dans la cellule lors de l'infection. Nous avons montré que les PCs, et notamment la furine, clivent la gB. In vitro, l'induction de la protéine virale LMP1 augmente l'expression de la furine, qui se traduit par une augmentation de l'infection par EBV. Ces résultats suggèrent l'existence d'une boucle de régulation entre la furine et LMP1 permettant d'améliorer la propagation cellulaire du virus. L'utilisation d'inhibiteurs de l'activité des PCs permettrait donc de bloquer l'infection par EBV. / Proprotein convertases (PCs) are enzymes involved in many pathological processes. We have identified two novel substrates of the PCs: apelin and glycoprotein B (gB) of Epstein Barr Virus (EBV), and studied the role of PC-mediated proteolytic maturation in their functions. Apelin is overexpressed in some cancers including colorectal cancer (CRC), and we demonstrated that furin, one of the PCs member, cleaves apelin in two peptides. To determine the role of apelin cleavage by furin in the metastatic CRC, we generated a non-cleavable mutant (apelin-DM). This mutant inhibited the growth of colon cancer cells in vitro and could not protect against apoptosis, unlike the wild-type apelin. In vivo, apelin-DM drastically reduces tumor growth and the formation of hepatic metastases in mice. These results were confirmed in apelin deficient mouse models thus demonstrating the potential interest of using apelin-DM, or its derivatives, as anticancer agents in the treatment of metastatic CRC. The gB of EBV, a virus involved in some lymphoid and epithelial cancers in humans, is involved in the entry of the virus into the cell during infection. We have shown that PCs, especially furin, cleave gB. In vitro,induction of the LMP1 viral protein increases furin expression, which results in an increase in EBV infection. These results suggest the existence of a regulatory loop between furin and LMP1 to improve the cellular propagation of the virus. The use of inhibitors of PCs activity would thus block EBV infection, a virus against which there is no treatment nowadays.

Nach aktuellem Kenntnisstand zur Pathogenese der Multiplen Sklerose (MS) wird der Entzündungsprozeß im Zentralnervensystem (ZNS) durch autoreaktive, Myelin-spezifische T-Zellen aufrechterhalten, wobei virale Infektionen bekannte Auslöser klinischer Schübe darstellen. Wir zeigten, dass eine Infektion mit dem Epstein-Barr Virus (EBV) eine notwendige aber nicht hinreichende Voraussetzung für die Entwicklung einer MS darstellt, deren potentielle Bedeutung sich aus der Korrelation zwischen Reaktivierung einer latenten EBV-Infektion mit Krankheitsaktivität bei MS-Patienten ergibt, die eine Aktivierung autoreaktiver T-Zellen im Rahmen der antiviralen Immunantwort nahe legt. Als biologische Marker der Krankheitsaktivität identifizierten wir die Hochregulation der ß2-Kette des Interleukin-12 Rezeptors sowie des Chemokinrezeptors CCR5 in peripheren Blutlymphozyten von MS-Patienten. Ferner konnten wir erstmals direkt nachweisen, dass die Aktivierung und klonale Expansion Myelin-spezifischer T-Zellen in peripherem Blut direkt mit Krankheitsaktivität korreliert. Der Wirkmechanismus der immunmodulatorischen Therapie der MS mit Interferon (IFN)-ß ist weitestgehend unverstanden. Unverstanden ist auch, warum die Therapie nur in einer Subpopulation von Patienten ihre Wirksamkeit entfaltet. Mit dem Molekül TRAIL gelang uns die Identifizierung des ersten klinischen Response Markers einer IFN-ß Therapie bei MS, der zugleich als prädiktiver Marker eine prognostische Aussage über den individuellen Behandlungserfolg erlaubt. Auf Grund seiner funktionellen Relevanz für den Therapieerfolg einer IFN-ß Behandlung stellt TRAIL zudem ein vielversprechendes Zielmolekül für neue therapeutische Strategien bei der MS dar. / Multiple sclerosis (MS) is considered a T-cell mediated demyelinating disorder of the central nervous system (CNS). Furthermore, it is well established that viral infections might trigger disease relapses. We demonstrate that infection with the Epstein-Barr virus (EBV) represents a necessary but not alone sufficient prerequisite for developing MS. The potential significance of EBV infection in MS arises from the correlation between viral reactivation and disease activity, indicating an activation of autoreactive T cells in the course of EBV reactivation and rendering EBV one important trigger of MS relapses. As biological markers for monitoring disease activity, we identified expression and upregulation of the chemokine receptor CCR5 and of the interleukin-12 receptor b2-subunit on potential effector cells in the peripheral blood of MS patients. In addition, we demonstrate for the first time that activation and clonal expansion of autoreactive T cells in the peripheral blood is clearly correlated with MS activity. Despite the well documented efficacy of interferon-b (IFN-b) therapy in a subgroup of patients, it remains unclear how IFN-b alters the clinical course of MS. We identified TRAIL as the first clinical response marker of IFN-b therapy in MS that might even be used as prognostic marker of therapy response in individual patients. Furthermore, our data imply that the immunoregulatory potential of TRAIL represents a novel and promising target for future therapeutic strategies in MS.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.