Classic Hodgkin lymphoma (cHL) is a B-cell malignancy marked by Hodgkin and Reed/Sternberg (HRS) cells that highly express CD30. We explored the roles of microRNAs (miRNAs) in cHL pathogenesis and CD30-targeting therapy using Brentuximab Vedotin (BV) through high-throughput screening techniques. Review of miRNA-related studies on known and potential miRNA-target gene interactions identified multiple miRNA-gene pairs with potential relevance for cHL pathogenesis. This highlighted the relevance of miRNAs in cHL, warranting further studies to deepen our understanding.
A microRNA overexpression library screen revealed 11 miRNA-target gene pairs implicated in cHL pathogenesis and 66 miRNA constructs that regulate BV treatment response. Among these, miR-590 was found to inhibit CD30 expression and induce BV resistance. Additionally, miR-191-5p not only conferred BV resistance but also promoted cell growth. By using AGO2-immunoprecipitation, we identified a network of involved target genes, including PTEN.
To investigate endogenous miRNAs involved in BV resistance mechanisms, we established two BV-resistant cHL cell lines and observed dynamic changes in CD30 expression. Moreover, we utilized a miRNA-focused CRISPR-Cas9 screen to identify endogenous miRNAs that regulate CD30 expression and/or BV sensitivity. To this end, we generated two inducible Cas9 monoclonal cell lines and optimized the entire screening process. These screens await further data analysis.
In summary, this thesis highlights the functional importance of specific miRNAs in cHL and provides detailed procedures of the miRNA-focused screening approaches, from initial setup to optimization and validation of results.

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.

Aggressive B-cell lymphomas are a heterogeneous group of mature B-cell neoplasms with morphological similarities, but phenotypic and genetic differences. This thesis investigated the aggressive lymphomas from bench-to-bedside aiming to enhance insight into its pathobiology, to investigate mechanisms underlying therapy resistance, and to explore novel treatment combinations. We focused on the role of MYC gene rearrangements, impact of DNA mutations, and altered protein expression. We observed that MYC rearrangements functioned as drivers of transformation in one third of cases in transformed follicular lymphoma. Next, we described loss of human leukocyte antigen (HLA) DM with loss of antigen expression by HLA class II as a novel mechanism of immune escape in Hodgkin lymphoma and diffuse large B-cell lymphoma (DLBCL). Mutational analysis of paired DLBCL tumor samples indicated variable mutational heterogeneity at relapse, with PIM1 and SOCS1 potentially related to resistance. Focusing on treatment, we observed that necrosis as assessed by 18F-FDG PET-scans was associated with metabolic active DLBCL irrespectively of MYC rearrangements and this might identify patients at increased risk of relapse. The combination of the JAK2 inhibitor ruxolitinib and the checkpoint inhibitor pembrolizumab showed a remarkable response in a patient with a primary chemo- and radiotherapy refractory primary mediastinal B-cell lymphoma. Finally, we studied the treatment outcomes of patient groups with rare poor risk lymphoma (relapsed stage I DLBCL and central nervous system DLBCL). The results and considerations from this thesis form the basis for ongoing Dutch HOVON trials, in several of which the PhD candidate plays a central role.