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.

Tumor cell survival and immune escape mechanisms in classical Hodgkin lymphoma

The nature of classical Hodgkin lymphoma (HL), a minority of tumor cells in a reactive background and loss of B cell phenotype, decides its dependence on the microenvironment for signals to contribute to survival and proliferation while at the same time trying to escape an anti-tumor response especially fired up because of the presence of Epstein Barr virus (EBV) in the tumor cells.
The data in this thesis show involvement of new players in the survival of the tumor cells in HL and their escape from anti-tumor immune responses. In addition to known pathways that are aberrantly activated in the tumor cells, aberrant expression of Insulin-like growth factor-1 receptor (IGF-1R), Ephrin family members and Toll-like receptors (TLRs) might provide additional signals leading to the survival and growth of the tumor cells. Despite the oncogenic effect of IGF-1R in cell lines, IGF-1R expression in the tumor cells of primary cases predicts a favorable outcome. The wide expression of Ephrin family members both in the tumor cells and the microenvironment suggests that there is a role for the Ephrin family in the pathogenesis of HL. Similarly, the expression of TLRs by the tumor cells supports a role in HL pathogenesis, although the effect of triggering of TLRs on HL cell lines was limited.
In terms of immune escape, potential mechanisms involve lack of functional antigen expressing, such as downregulation of HLA class I and II, and retention of CLIP, by loss of HLA-DM, in HLA class II molecules impairing antigen presentation in HL. In undifferentiated nasopharyngeal carcinoma, another EBV associated cancer, HLA class I and II downregulation was observed in part of the cases. The expression of HLA class II was strongly correlated to HLA-DM and HLA-DO expression, suggesting interference of HLA-DO with the function of HLA-DM, leading to expression of CLIP and impaired antigen presentation.