Diffuse large B-cell lymphoma (DLBCL) is the most common and aggressive type of non-Hodgkin lymphoma. The current treatment for the disease currently cures only 60% of the patients, which means new therapies are needed to improve patient survival. In this project, we performed a large gene expression analysis in 1800 DLBCL patient samples to find new targets to improve the current treatment. Here we found high expression of the WEE1 protein, which is involved in cell cycle regulation and repair of DNA damage. Inhibition of the WEE1 protein in DLBCL cell lines induced disruption of the normal cell cycle and high levels of DNA damage, eventually causing cell death. In addition, WEE1 inhibition showed to enhance the currently used therapies for the treatment of DLBCL, which include rituximab (anti-CD20), radiation, CHOP (first line chemotherapy) and cytarabine (second line chemotherapy). Based on these findings, we also investigated the effect of WEE1 inhibition in combination with so-called “anti-apoptotic inhibitors”, which prevent cells from protecting themselves against cell death. These inhibitors are currently being tested in clinical trials for different types of non-Hodgkin lymphoma and leukemia’s, including DLBCL . We found that WEE1 inhibition worked very well together with the anti-apoptotic inhibitors, and that combination therapy significantly enhanced cell death in DLBCL. In total, our results demonstrate that inhibition of WEE1 is very successful in DLBCL, and would likely improve the current treatment available for DLBCL.

In classical Hodgkin lymphoma (cHL) there is an urgent need for biomarkers to determine prognosis or treatment response. In this thesis we summarized current knowledge on circulating biomarkers in cHL and studied a selection of these markers as treatment response or prognostic biomarkers. In the first part of this thesis we studied the application of Thymus and Activation Regulated Chemokine (TARC) as a biomarker for treatment response and compared TARC with sGal-1, sCD163 and sCD30 and interim FDG-PET imaging. We found that TARC at diagnosis correlates with metabolic tumor volume and that serial TARC measurements during and after treatment accurately reflect treatment response. In comparison to sGal-1, sCD163 and sCD30, TARC dynamics most accurately reflected treatment response. In addition, interim TARC had a higher positive predictive value for final treatment response compared to interim FDG-PET imaging. All together, we concluded that TARC is a cheap, non-invasive and highly accurate biomarker to determine treatment response in cHL patients.
In the second part of this thesis, we focused on microRNAs (miRNAs) as circulating biomarkers. We summarized the role of miRNAs in cHL and concluded that deregulation of miRNAs is a frequent event and an important factor in the pathobiology of cHL. Next, we summarized pre-analytical, analytical and post-analytical challenges in circulating miRNAs studies in general. Finally, we performed miRNA microarray profiling of serum of patients with cHL. Using this approach, we were unable to find circulating miRNAs with prognostic value in classical Hodgkin lymphoma.

The DLBCL microenvironment: A proteomics approach to identify novel players involved in the tumor cell – microenvironment interactions.
Diffuse large B-cell lymphoma (DLBCL) can present de novo or transform from a less aggressive lymphoma. The pathogenesis of DLBCL is a multistep process that involves genetic alterations, immune status of the patient and tumour microenvironment. The Human Leukocyte Antigen (HLA) system regulates the immune response and loss of HLA expression in the tumour cells leads to an altered host immunity and impairs effective anti-tumour responses. Our aim is to investigate the proteome of tumor cells to identify proteins that play a role in the cross talk between tumor cells and the microenvironment in DLBCL. The ultimate goal is to explain why patients react differently to therapy and to provide a tailored treatment plan in order to avoid over- and undertreatment.

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.