In this thesis we studied genetic alterations in lung cancer associated with COPD, tumor progression and resistance to targeted therapy. We found no significant difference in the percentage of KRAS mutations in 325 lung cancer patients with or without COPD, whereas EGFR mutations were significantly more common in lung cancer patients without COPD. Mutation analysis of all protein-coding exons in three non-small cell lung and two small cell primary lung tumors with their corresponding metastases showed that only half of the mutations were shared between the tumors from different locations in the non-small cell lung cancer patients. In contrast, more than 95% of all mutations in small cell lung cancer patients were shared between the primary and metastatic tumors at different locations. Based on single cell whole genome sequencing of one of the two small cell lung cancer patients, we showed that two out of 83 examined cells of the primary tumor had chromosomal abnormalities that were consistent with the genomic aberrations in the liver metastasis. Finally, we examined three patients using paired-end RNA sequencing and focused on treatment-induced fusion genes as potential mechanisms of resistance to crizotinib. Only one of the four fusion genes could be potentially translated into protein and this fusion was identified in both pre- and post-treatment samples. In summary, we provide new insights into the genomic abnormalities of lung tumors in relation to COPD subtype and resistance to treatment.

Altered T cell subset composition and functionality occurs during ageing. MicroRNAs (microRNA) impact T-cell physiology by gene expression changes. In this thesis we reviewed the literature with respect to involvement of miRNAs in specific T cell subsets during maturation and upon ageing. We described a complex pattern of miRNA expression regulation upon T-cell activation, which reflects activation kinetics and may play a role downstream of T-cell signaling. We showed that the most pronounced changes in miRNA expression are related to population doublings (PD’s) of T-cell clones regardless of the age of the donors. In addition, we provided an evidence for functional relevance of miR-9 in T cell response and survival. We observed age-related differences in miRNA expression predominantly within the CD45RO- and not in CD45RO+ T-cell compartment, which can be explained by the changes in cellular composition, activation and cellular ageing. We showed involvement of the miR-23a~24-2 cluster in the IL-15-induced downregulation of T-cell co-stimulation. We dissected a role of miR-21 in preventing T-cell apoptosis and identified novel target genes, including the pro-apoptotic LATST1, via which miR-21 may exert this function.
Overall, this thesis describes T-cell activation and age-related induced changes in miRNA expression with the role of specific miRNAs in the age-related alterations of T-cell function.

Esophageal adenocarcinoma is a deadly cancer of the esophagus. Barrett’s esophagus is a precursor lesion of esophageal adenocarcinoma. Currently, the development of Barrett’s esophagus and esophageal adenocarcinoma is poorly understood. In this thesis, we show that signaling pathways and transcription factors that are involved in the embryological development of the esophagus also play a central role in the development of Barrett’s esophagus and oesophageal adenocarcinoma. Barrett’s esophagus is treatable, while oesophageal adenocarcinoma is often diagnosed too late. In a pilot study we show that miRNA’s, a group of small nucleotide molecules that are remarkably stable in patiënt’s blood, can be usefull to identify patients with Barrett’s esophagus. Currently, curative treatment for oesophageal adenocarcinoma consists of chemo-and radiotherapy followed by surgery. Some patients with esophageal adenocarcinoma do well after treatment, while others respond poorly. We show that patients whose tumors do not express the proteins CD44 and SOX2 have a better survival compared with patients whose cancer does express these two proteins.

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.

Classical Hodgkin lymphoma (cHL) is a complex cancer, the incidence and its association with Epstein-Barr virus (EBV) varies significantly with age, sex, histological subtype, ethnicity, geography and socio-economic status. CHL is characterized by minority tumor cells in a large background of reactive immune cells, showing strong involvement of immune system. Both genome wide and targeted gene association studies show strong association of human leukocyte antigen (HLA) with cHL susceptibility, however, mostly lacking cHL subgroup analysis stratified based on age, EBV and subtype. In this thesis, we systematically reviewed genetic polymorphisms in the cHL and concluded HLA region is strongly associated with cHL susceptibility. Several non-HLA gene associations are found to be important for the immunopathology of the disease. Genetic associations for cHL prognosis and secondary malignancies and toxicities are limited. Next, we determined the associations between classical HLA class I and class II alleles and susceptibility to cHL overall and specific cHL subgroups stratified based on age and tumor cell EBV status. HLA class I alleles are associated with EBV+ cHL and HLA class II alleles with EBV- cHL susceptibility. The alleles of the HLA-DQB1*06-DRB1*15 haplotype are associated with risk of cHL at younger age (≤45 years), whereas the alleles of the HLA-B*08-DRB1*03 haplotype are risk factors for cHL at older age (>45 years. In a Brazilian cHL population showed that HLA-A*01 is a risk allele and HLA-A*02 is a protective allele for developing EBV+ cHL. This susceptibility pattern is similar to the pattern observed in the Western Europe population. The findings of this thesis support the relevance of HLA alleles in susceptibility of cHL. The observed HLA associations in cHL are consistent with the proposed immunological nature of the disease and the importance of the infiltrating T cells. The findings contribute to a better understanding of the susceptibility mechanisms in cHL.