The role of non-coding RNAs in B-cells lymphoma
In this thesis we explored the role of long non-coding (lnc)RNAs in B cell lymphoma. This very large family of non-coding RNA transcripts are mostly uncharacterized and their role in disease states such as cancer and B cell lymphoma is mainly unknown. The overall aim of this thesis was to characterize expression profiles of normal B cell subsets and to identify and functionally study lncRNAs relevant to the pathogenesis of Burkitt lymphoma and Hodgkin lymphoma, two germinal center B-cell-derived malignancies. The studies described in this thesis showed that lncRNA expression is actively regulated during normal B-cell maturation. In Burkitt lymphoma we showed that knockdown of MYC significantly altered expression of a substantial number of lncRNAs. In Hodgkin lymphoma we showed altered expression of lncRNAs compared to their normal counterparts. For part of the deregulated lncRNAs we have preliminary data to support a regulation in cis. In addition, we observed that a substantial proportion of the lncRNAs interacted with miRNAs, although the overall percentage was lower than the percentage of protein-coding genes interacting with miRNAs. For three lncRNAs we showed a role in growth of B-cell lymphoma cells. No eQTL effects were observed for lncRNAs mapping at Hodgkin lymphoma susceptibility loci, whereas we did find eQTL effects for some of the protein-coding genes at these loci. Together, our data provides comprehensive overviews on the lncRNAs that are expressed and deregulated in normal and malignant B-cells and pinpoints several lncRNAs that contribute to lymphomagenesis.

The tumor cells in Hodgkin lymphoma (HL) are characterized by constitutive activation of several signaling pathways. This characteristic phenotype is partly caused by mutations in genes of these pathways. Most of these mutations were identified as part of a targeted sequencing approach on cell lines or microdissected HL tumor cells, i.e. the so-called Hodgkin and Reed-Sternberg (HRS) cells.
In this thesis, we determined the mutational landscape of HL cell lines by whole exome sequencing (WES). We identified 463 recurrently mutated genes in HL. Among them are several genes related to functionality of the immune system (B2M, CIITA, CD58, CSF2RB). Inactivating mutations in B2M were common and explain the loss of HLA class I expression commonly observed in HL. Inactivation of CIITA might explain downregulation of the HLA class II expression in cHL cases. Loss of CD58 expression is common in HL cell lines and possibly a late event in HL pathogenesis. In tissue samples loss of CD58 is associated with relapse of disease. CSF2RB mutations are common in HL, with mutations in more than half of the HL cell lines. These mutations may all contribute to tumor cell escape from immune responses. Mutations in the oncogenic transcription factor MYB were also found in part of the HL cell lines. Strong oncogenic effects of wild type and truncated MYB were observed in HL cell lines. This knowledge might contribute to a better understanding of HL pathogenesis.

Kidney cancer of the clear cell type is often lethal and causes more than 100,000 deaths worldwide every year. Understanding the biology of this cancer type may help to develop better ways to diagnose and treat it. Damage in DNA (genes) is present in all cancer cells and clear cell kidney cancer is no exception. Exactly how these DNA changes contribute to the development of kidney cancer is still mostly unclear. The PhD study undertaken by Jun Li (Genetics department, UMCG) looked at the effects of damage in 2 genes that appear to be important in kidney cancer: SETD2 and PBRM1. Normal kidney cells cannot be grown in culture for many days, but after silencing the SETD2 gene the growth potential of these cells in became abnormally large. This suggests that damage to SETD2 can contribute to the change from normal kidney cells into cancer cells. In contrast, silencing the PBRM1 gene did not show this effect. However, it did cause changes in the activity of other genes, especially of some involved in immune response. These changes were, more strongly, also observed in kidney cancer cells. Together, these findings contribute to our further understanding of the biology of kidney cancer and may help in improving diagnostics and treatment of this aggressive cancer type.

Hodgkin lymphoma (HL) as a type of lymphoma with two subtypes including classical Hodgkin lymphoma (cHL) and nodular lymphocyte predominant Hodgkin lymphoma (NLPHL). HL is a unique type of lymphoma with a population of neoplastic cells which consist less than1% of the total cell population- in a background of immune cells mainly consist of T-cells, B cells, so-called microenvironment. The cells of microenvironment do not react against the neoplastic cells and their interaction with neoplastic cells, provide survival signals and protection against immune system, for neoplastic cells. A comprehensive view of combination of the cell populations of the microenvironment, would provide a chance to obtain a deeper understanding of the interaction of neoplastic cells and microenvironment.

In this thesis we determined the cell populations of microenvironment of cHL and NLPHL in general and with a specific focus on the cells in close vicinity of the neoplastic cells. Current thesis indicates that the combination of microenvironment of cHL in Epstein Barr virus infected cHL cases (EBV+ cHL) is different compared to EBV- cHL cases, which might be due specific reaction of immune system against this virus. On the other hand, the combination of microenvironment of NLPHL indicates a significant population of TFH cells. Subsequent study also suggests that the TFH cells form rosette around neoplastic cells of the NLPHL. In addition, comparison of the cell populations of cHL and NLPHL indicate differences in combination of microenvironments of two subtypes. This fact suggests that each sub-type, applies different mechanisms for survive.

B cell lymphoma is the most common hematologic malignancy. Besides alterations in the malignant lymphoma cells, also the interaction with the microenvironment has gained much interest in recent years. The aim of this thesis is to characterize the malignant cells as well as the surrounding microenvironment by proteomics and by flow analysis.
In the first part we investigated changes in the expression levels of membrane proteins of non Hodgkin lymphoma (NHL) cell lines compared with a lymphoblastoid cell line. We found differential expression of peptidylprolyl isomerase A which can be considered as a potential target for treatment of lymphoma patients. Next, we profiled the secretome of NHL cell lines and identified potential novel disease biomarkers such as macrophage inhibition factor and CD70.
In the second part, we analyzed the composition of tonsil and reactive lymph node (RLN) to select the best normal counterpart for the subsequent analysis of the B cell lymphoma samples. Then, we compared the composition of the microenvironment of different NHL as well as Hodgkin lymphoma (HL) with RLN. We found more cytotoxic T cells in more aggressive lymphomas, these cells are probably in an hypo-immune response status since they showed high expression of an exhaustion marker. Converting the exhaustive phenotype of the T cells might present a novel treatment strategy to induce an effective immune response in B cell lymphomas. In HL we found that the percentage of T regulatory cells is high in the tumor areas and that these cells most likely suppress the immune response.