A large number of aberrantly expressed microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) have been reported in B-cell lymphomas. However, their role in the pathogenesis of B-cell lymphoma remains largely unknown. In this thesis, the functions of non-coding RNAs were explored in two distinct subtypes of germinal center B-cell (GC-B) derived lymphomas, i.e. classical Hodgkin lymphoma (cHL) and Burkitt lymphoma (BL).
In a loss-of-function screen in three cHL cell lines we identified four miRNAs with an effect on cell growth. Follow-up experiments for miR-21-5p showed that this miRNA was upregulated in cHL compared to GC-B cells and protects cHL cells from apoptosis possibly via targeting BTG2 and PELI1. Small RNA sequencing in BL and GC-B cells revealed a clearly aberrant expression profile. In subsequent miRNA loss- and gain-of-function screens we identified 18 miRNAs that affected growth of BL, including some previously reported oncogenic miRNAs. Functional follow-up studies revealed promising target genes for miR-378a-3p and miR-26b-5p. Focusing on MYC-induced lncRNAs revealed 18 candidates that were consistently higher expressed in BL cell lines compared to GC-B cells. Three of these lncRNAs showed effects on BL cell growth in a loss-of-function screen. Further validation experiments of MAFG-AS1 confirmed its effect on growth in BL cell lines.
In summary, we identified several aberrantly expressed and MYC-regulated non-coding RNAs, with clear effects on growth of cHL and BL cells, and unveiled their underlying mechanisms in cell growth regulation. Our findings add to the current knowledge about the roles of non-coding RNAs in B-cell lymphomas.

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.

The aim of this thesis is to improve diagnostic methods to identify genomic aberrations with clinical significance in non-small cell lung cancer (NSCLC) patients. We tested the feasibility of an all-in-one transcriptome-based assay to simultaneously identify different types of genetic variants with clinical significance in those patients in Chapter 2. An accuracy of 100% can be reached by using criteria for quality control e.g. RNA integrity and total unique reads. Our study showed the potential of application in diagnostic settings. In chapter 3, we analyzed EGFR mutation and amplification status in advanced NSCLC patients using targeted DNA sequencing data available from the routine molecular diagnostics. We showed the feasibility of using the generation sequencing (NGS) data to identify amplifications in genes relevant for daily diagnostics. Amplification of EGFR in patients with EGFR mutations were associated with poor tumor response to EGFR TKI. In chapter 4, we aimed to identify potential novel crizotinib-induced resistance mechanisms in ALK-break positive NSCLC patients. By analyzing whole exome sequencing data on paired pre- and post TKI tissue samples, we found the enrichment of mutations in genes associated with EMT-related pathways, indicating that loss of epithelial differentiation represents a resistance mechanism for crizotinib. In chapter 5, using a cohort of esophageal squamous cell carcinoma patients, we showed that somatic mutations can be detected in pre-surgery cfDNA in early stage patients, and at a lower frequency in post-surgery cfDNA, indicating that cfDNA could potentially be used to monitor disease load.

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.

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.