The study of this thesis is related to the previously established MYC/miR-150/MYB/ZDHHC11 network regulating Burkitt lymphoma (BL) growth. Our first aim was to study the role of this network in Hodgkin lymphoma (HL) and diffuse large B-cell lymphoma (DLBCL). We found a similar role of three of the network components, while miR-150 overexpression showed no or only very mild effects on HL and DLBCL growth. Therefore, we considered miR-150 as a BL-specific component. Our second aim was to study the specific roles of the protein coding and circular ZDHHC11 gene products. ZDHHC11 belongs to a family of 24 ZDHHC proteins that are characterized by a DHHC motif which mediates palmitoylation. Loss of palmitoylation induced apoptosis in three B-cell lymphoma subtypes indicating the overall importance of protein palmitoylation for B-cell lymphoma. Based on the observed expression patterns we pinpointed seven DHHC family members that might be relevant in B-cell lymphoma. Further studies to elucidate the role of the ZDHHC11 protein were hampered due to difficulties in detecting the protein but provided some evidence that this protein is not a critical factor in regulating BL growth. Lastly, we showed that circular ZDHHC11 RNA transcripts do play a critical role in growth of B cell lymphoma, but this effect is independent of binding miR-150, as deletion of the miR-150 binding site region did not alter the observed phenotype. In summary, we extended current knowledge on the role of the MYC/miR-150/MYB/ZDHHC11 network and addressed the function of ZDHHC11 transcripts and protein in B-cell lymphoma.

Classic Hodgkin lymphoma (cHL) is a cancer of the immune system common in adolescents and young adults. The chance to develop cHL is strongly influenced by genetically defined human leukocyte antigen (HLA) types. The main function of HLA is to present antigenic peptides and it is a critical player in the normal immune system. In this thesis we focused on the mechanisms of HLA-related associations with cHL. First, we showed that part of the cHL-associated HLA alleles are associated with loss of HLA expression on tumour cells. Second, we identified novel ERAP – HLA interactions that were specific for cHL patients and not observed in controls. Together these data show that antigen presentation is a critical component in defining the susceptibility to develop cHL. Lastly, we identified an association between cHL and killer cell immunoglobulin-like receptors (KIRs). These KIRs interact with HLA class I molecules expressed on target cells and play an important role in clearance of virus-infected or tumour cells. We found that KIR haplotype B protects against the development of a specific cHL subgroup (EBV+ nodular sclerosis). In addition, we observed presence of KIR2DL2 – HLA-C1 and KIR2DS2 – HLA-C1 at significantly lower carrier frequencies in this cHL subgroup compared to controls. Overall, our studies showed that multiple HLA associated molecules influence susceptibility to develop cHL. The results of this thesis further support the critical role of HLA and antigen presentation in cHL susceptibility and extends on the biological mechanisms underlying the development of cHL.

Hodgkin lymphoma (HL) is a common malignancy in young adults. It is characterized by a minority of tumor cells surrounded by an abundant inflammatory infiltrate, which mainly consists of CD4+ T cells. The current treatment strategy has a high cure-rate, but often results in long-term treatment related adverse events. Therefore, new treatment options such as immune checkpoint blockade (ICB) are being explored. In this project we characterized CD4+ T cells and their interactions with tumor cells, with a specific focus on the role of CD4+ T cells in ICB therapy. We discovered that CD4+ T cells are weakly activated by T cell receptor (TCR)-HLA class II and CD2-CD58 interactions with the tumor cells. These T cells that are in close proximity to the tumor cells have been activated before (antigen experienced) and are diverse (polyclonal). In addition, they specifically express transcription factors TOX and TOX2 that are associated with T cell exhaustion and can be induced by chronic or repeated TCR stimulation. This suggests that Hodgkin tumor cells actively inhibit cells in their surrounding to promote tumor cell survival. We developed a long-term co-culture model and found that blocking PD-1 with nivolumab results in pronounced CD4+ T cell activation and proliferation, confirming the potential of this treatment in HL. In addition, we identified soluble PD-L1 as a promising biomarker in HL patients. In summary, our studies provide novel insights into the function of CD4+ T cells in HL biology and treatment responses. This may help improve immunotherapy in HL patients.

Epstein-Barr virus (EBV) infects >90% of the world’s population and leads to the development of Hodgkin lymphoma (HL) and nasopharyngeal carcinoma (NPC) in a low percentage of  individuals. In addition, uncontrolled growth of EBV infected B cells can cause post-transplant lymphoproliferative disorder {PTLD) in patients treated with immunosuppressive drugs following organ transplant. The main aims of this thesis are to explore i) risk factors of HL and NPC, and ii) biomarkers in NPC, HL and PTLD.
Several human leukocyte antigen (HLA) alleles have been associated with susceptibility to HL and NPC. We identified novel NPC-associated HLA-alleles in the high-risk Bidayuh minority population from Malaysia. We also showed that loss or retention of HLA expression by tumor cells is associated with a subset of known protective or risk alleles in both HL and NPC. These data suggest that the susceptibility effects of HLA alleles are early events in pathogenesis, while the pressure on tumor cells to downregulate HLA is most likely related to emerging immune responses later on.
With plasma or nasal washing derived EBV DNA, we could detect NPC disease activity with high sensitivity. Chromosomal copy number variations were detected in cell-free DNA of >50% of HL and PTLD patients. Targeted sequencing of EBV demonstrated 100% sensitivity in detecting disease in EBV-positive HL and PTLD patients. These findings suggest that measuring  cell-free biomarkers in liquid biopsies has clinical value in detecting disease activity in NPC, HL and PTLD.

The aim of this research project was to improve diagnostic methods, explore resistance mechanisms to targeted therapy and establish prognostic value of molecular biomarkers in non-small cell lung cancer (NSCLC) and esophageal squamous cell carcinoma (ESCC). We set up and validated the performance of an RNA-based assay allowing simultaneous testing of multiple therapeutic targets. This effort was successful and can be applied on biopsies in clinical practice. However, tumor-educated platelets of patients with known driver variants and NSCLC showed no tumor-derived mRNA molecules, so this cannot be implemented in clinical practice. Circulating tumor DNA levels in low disease-stage ESCC patients were associated with tumor load in real-time and may be used to monitor disease load.
Personalized medicine facilitated by routine molecular diagnostics has markedly improved clinical management of cancer patients. We showed that high EGFR copy numbers as determined by amplicon-based NGS data predicts a worse overall survival in EGFR mutated patients treated with first-line EGFR-TKI, especially in those who developed a T790M mutation. To explore resistance mechanisms in BRAF p.(V600E) mutated patients treated with BRAF/MEK inhibitors we analyzed the presence of concurrent mutations of genes in the PI3K or MAPK pathways. This study revealed a comparable survival of patients with or without concurrent mutations. Finally, we described treatment response of two EGFR mutant patients who developed a BRAF V600E resistance mutation. Combined treatment with BRAF/MEK inhibitors and the EGFR-TKI osimertinib showed promising results, also in additional cases reported in the literature.