Cancer immunotherapies utilizing immune checkpoint blockade (ICB) therapy targeting CTLA-4 and PD-1/PD-L1 relieve tumor-induced immune suppression and induce durable tumor regression. The use of... Show moreCancer immunotherapies utilizing immune checkpoint blockade (ICB) therapy targeting CTLA-4 and PD-1/PD-L1 relieve tumor-induced immune suppression and induce durable tumor regression. The use of ICB therapy have demonstrated remarkable therapeutic efficacy in a proportion of patients with melanoma. However, still a substantial percentage of patients does not respond (durable) to ICB treatment and many questions remain. Therefore, in this thesis, the aim is to improve our understanding of ICB efficacy. We demonstrate the promise of neoadjuvant ICB therapy (approach in which ICB therapy is applied before surgery) and analyze different cohorts of melanoma patients. This results in the identification of several markers that are associated with prognosis, including IFN-y related gene signature score, Batf3 dendritic cell associated gene signature score, tumor mutational burden and systemic LRG1 expression. These markers can potentially be targeted and might facilitate rational combination therapies that can boost the efficacy of ICB therapy. For this purpose, we perform a repurposing compound screen that targets antigen cross-presentation. Togethers, this work increases our understanding of factors that determine ICB therapy efficacy and toxicity, with the goal to identify novel strategies to improve outcome of melanoma patients in a rationale and personal manner. Show less
Uveal melanoma (UM) is a rare ocular tumor. Up to 50% of the patients develop distant metastases predominantly targeting the liver. The median survival after diagnosis of patients with hepatic... Show moreUveal melanoma (UM) is a rare ocular tumor. Up to 50% of the patients develop distant metastases predominantly targeting the liver. The median survival after diagnosis of patients with hepatic metastases is approximately 4-6 months and hardly increased in the past decades due to lack of novel effective therapeutic options. Within the scope of this thesis we investigated the signaling landscape of metastatic UM and searched for novel avenues of therapy. In Chapter 2 we demonstrate that combinations of the multitarget drug Trabectedin with either the CK2/Clk double-inhibitor Silmitasertib or with the c-MET/TAM receptor inhibitors show synergistic growth inhibitory effects and induce apoptosis of UM cells in vitro. Chapter 3 describes the application of a CRISPR-Cas9 synthetic lethality screen for identification of molecular targets whose inhibition synergistically enhances the effect of the mTOR inhibitor everolimus in UM cells. In Chapter 4 we show that the combination of genetic depletion YAP1/TAZ together with Mcl-1 inhibition resulted in a synergistic inhibitory effect on the viability of UM cell lines. In Chapter 5 we analyzed the phospho-proteome of two UM metastatic cell lines and a primary tumor cell line from the same individual, and studied the role of MARK3 in YAP1/TAZ signaling. Show less
The aim of this thesis was to develop novel treatment strategies for different types of eye melanoma utilizing zebrafish models. We first establish orthotopic and ectopic xenograft models for uveal... Show moreThe aim of this thesis was to develop novel treatment strategies for different types of eye melanoma utilizing zebrafish models. We first establish orthotopic and ectopic xenograft models for uveal and conjunctival melanoma by engraftment of the immortalized cells derived from these tumors into zebrafish embryos. Next, we expanded these models with spheroids and zebrafish patient-derived xenografts for pre-clinical, personalized screening of anti-uveal melanoma drug responses. We demonstrated that these models can be harnessed to explore the in vivo interactions of the tumor cells with blood vessels and macrophages leading to angiogenic response. We finally apply the conjunctival melanoma model to clarify the inhibitory effects of ginsenosides and correlate their structures with potential antitumoral mechanisms. Show less
Ocular melanoma and colorectal carcinoma are two malignancies with a predilection for metastasizing to the liver. Patients with liver-only or liver-dominant metastatic disease might be eligible for... Show moreOcular melanoma and colorectal carcinoma are two malignancies with a predilection for metastasizing to the liver. Patients with liver-only or liver-dominant metastatic disease might be eligible for locoregional or so-called liver-directed therapy. Liver-directed therapies include surgery and thermal ablation, as well as various arterial therapies such as percutaneous hepatic perfusion with melphalan (M-PHP). Although M-PHP is well-tolerated by most patients, hematologic events due to bone marrow suppression were quite common in M-PHP using the first-generation filter. In an attempt to reduce bone marrow suppression by increasing the filter extraction rate, a new second-generation filter (GEN 2 filter) was developed and became commercially available in 2012. In this thesis, it was demonstrated that M-PHP using the GEN 2 filter has an acceptable safety and toxicity profile and it seems to reduce hematologic toxicity when compared to M-PHP with a first-generation filter. This thesis contributes to the scientific evidence showing that M-PHP using the GEN 2 filter is an effective treatment for liver metastases from ocular melanoma. In contrast, M-PHP seems to have no additional value in the current treatment of unresectable liver metastases from colorectal carcinoma. Show less
In this thesis I investigate new ways to use MRI (magnetic resonance imaging) for the diagnosis, treatment and follow-up of uveal melanoma (UM) patients, mainly in relation to the planning of... Show moreIn this thesis I investigate new ways to use MRI (magnetic resonance imaging) for the diagnosis, treatment and follow-up of uveal melanoma (UM) patients, mainly in relation to the planning of proton beam therapy. Proton beam therapy is performed while sitting whereas MRI scans are scanned in prone position. In chapter 2 I have shown that the shape of the eye and tumor are not affected by the change in position. During treatment planning the tumor shape needs to be determined. This can be done by drawing the tumor on MRI. In chapter 3 I have shown that the variance between segmentations performed by different doctors are on average smaller then 0.4mm. As MRI based planning is not yet available for UM patients we have developed an MRI protocol to support the current model based treatment planning software with MRI based measurements. In chapter 4 I compare these MRI based measurements with conventional measurements and show that MRI measurements are comparable and sometimes even better. A common side effect of UM is retinal detachment. This is sometimes treated with silicon oil. Unfortunately ultrasound imaging is not possible in these patients. In chapter 5 I describe and evaluate a MRI protocol to imaging these tumors with MRI. Finally, MRI can also provide information about tissue and functional characteristics. In chapter 6 I present a method to overcome eye specific challenges in the quantitative analysis of perfusion weighted MRI. Show less
Ocular melanoma is a rare disease that originates from melanocytes in the eye. It is the most prevalent primary ocular malignancy in adults, and has a high metastatic rate. Two important questions... Show moreOcular melanoma is a rare disease that originates from melanocytes in the eye. It is the most prevalent primary ocular malignancy in adults, and has a high metastatic rate. Two important questions for good patient care are: 1) How to differentiate between (benign) nevi, and (malignant) melanoma?, and 2) How to treat this tumor best, particularly in cases with metastases?This thesis addresses two types of ocular melanoma: melanoma of the internal parts of the eye (uveal melanoma) and melanoma of the mucous membrane covering the eye (conjunctival melanoma). This thesis combines patient-related projects with projects from the lab.With new imaging techniques we demonstrate that oxygen values differ in eyes with melanoma compared to other eyes including those with a nevus. We use OCT-angiography to depict tumour vessels non-invasively in conjunctival and iris lesions. These two techniques may be used in the future to differentiate lesions, and to monitor patients after treatment.With studies in the lab we show that new drugs (immunotherapy) that are recently used in cutaneous melanoma, can also be used to treat conjunctival melanoma. We show that vascular growth in uveal melanoma is related to other (genetic and immunologic) characteristics, providing new clues for therapy. Show less
Part I - Hepatic perfusion for the treatment of unresectable liver metastasesBecause the majority of metastasized uveal melanoma (UM) patients have unresectable liver only metastases, locoregional... Show morePart I - Hepatic perfusion for the treatment of unresectable liver metastasesBecause the majority of metastasized uveal melanoma (UM) patients have unresectable liver only metastases, locoregional therapy was developed. In this thesis percutaneous hepatic perfusion (PHP) is described as a treatment for these patients. During this procedure, the chemotherapeutic agent is infused in the hepatic artery and thereby delivered to the liver and metastases directly. Via a veno-venous filtration system, the chemotherapeutic agent is filtered before it reaches the systemic circulation. In this thesis a clinical study was described treating 20 UM patients with metastases confined to the liver with PHP. It was concluded that the results PHP outbalanced the (minimal) toxicity for patients with uveal melanoma metastases.Part II - Tailored care for patients with pancreatic cancerThe poor prognosis of pancreatic cancer did not change much over the last decades, despite the improvements in treatment modalities. Previous studies have reported variations in incidence and mortality in pancreatic cancer between countries worldwide and European countries. A collaboration was initiated across Europe to compare patterns of care and identify best practices for pancreatic cancer care. A core dataset was identified to identificate differences in age, gender, incidence, tumour stage and differences in treatment strategies. To identify any differences in treatment and/or survival of elderly patients with pancreatic cancer, a comparison was performed of data on geriatric pancreatic cancer care and survival with data from the Netherlands and a special ‘Senior Adult Oncology Program’. Show less
As HLA Class I expression is an important target for cytotoxic T cells but an in inhibitor of NKcells, we were interested in the regulation of its expression.We review HLA expression in UM, how it... Show moreAs HLA Class I expression is an important target for cytotoxic T cells but an in inhibitor of NKcells, we were interested in the regulation of its expression.We review HLA expression in UM, how it is involved in the inflammatory phenotype, how it is regulated and how putative treatments might be effective in its expression.We investigate the potential role of the NFkB pathway in the regulation of inflammation in UM and its potential association with HLA Class I expression.In order to increase our understanding for the reason behind the elevated HLA Class I expression in UM tumours, we investigate the involvement of epigenetics. We focus on a set of epigenetic enzymes called histone deacetylases and report that these regulators are highly expressed in Monosomy 3 UM.We wonder whether HDAC expression is influenced by the presence of infiltrating lymphocytes and macrophages.We focus on miRNA’s as another set of epigenetic regulators of inflammation. We investigate the potential relation of a set of 125 miRNA’s with HLA Class I expression and the presence of an infiltrate in UM and report two patterns of miRNA expression.We study the LAG3 immune checkpoint in UM tumours. As immune checkpoints might be responsible for the T cell exhaustion which is observed in UM, we investigate the involvement of LAG in prognostication and study how LAG3 and its ligands are distributed among different UM tumours. Show less
We used the corneal immunosuppressive environment to test the possibility of inserting a Fish Scale-Derived Collagen Matrix in a corneal pocket, and the excellent results and lack of primary... Show moreWe used the corneal immunosuppressive environment to test the possibility of inserting a Fish Scale-Derived Collagen Matrix in a corneal pocket, and the excellent results and lack of primary and secondary immune responses led to a clinical trial, which is currently underway. The immunosuppressive environment of the eye allows the growth of malignant melanocytes which leads to the formation of uveal melanoma. The association between increased numbers of macrophages and lymphocytes in uveal melanoma and an increased development of uveal melanoma metastases suggest an influence of pro-angiogenic macrophages, The relation between loss of BAP1 expression and a very bad prognosis and the influx of leukocytes into the primary tumor, suggests that BAP1 functions as a regulator of inflammation. Further research will focus on the role of BAP1 in the regulation of inflammation in the uveal melanoma microenvironment. Show less
The focus of this thesis is uveal melanoma (UM) which, once metastasized, is lethal due to lack of effective treatment options. To repress p53 activity approximately 65% of UM tumors express high... Show moreThe focus of this thesis is uveal melanoma (UM) which, once metastasized, is lethal due to lack of effective treatment options. To repress p53 activity approximately 65% of UM tumors express high levels of the p53 inhibitory proteins MDMX or MDM2. The aim of this thesis is to unravel the oncogenic function of MDMX and provide new treatment options for patients with metastasized UM. Chapter 2 describes the regulation of the transcriptome by MDMX in UM and proposes novel p53-independent effects of MDMX, i.e. FOXO inhibition. In chapter 3 the opportunities of a combined targeting of two common signaling pathways as therapeutic intervention for metastasized UM patients is investigated. Genetic interference with either MDMX or PKC δ expression or activity showed that beneficial effects can already be achieved by a more specific targeting, which is presumable less toxic to the patient. In chapter 4 it is described, opposed to what has been reported before, that enhancer of zeste homolog 2 (EZH2) inhibition poses a valuable novel therapeutic invention for UM. In chapter 5 it is shown that combining two clinically approved drugs, Quisinostat and Flavopiridol, could serve as an effective therapeutic intervention for UM patients. Show less
Uveal melanoma (UM) is an aggressive intraocular tumor with a high propensity to metastasize. Accurate prognostication is relevant for patient counselling, planning of follow-up and... Show moreUveal melanoma (UM) is an aggressive intraocular tumor with a high propensity to metastasize. Accurate prognostication is relevant for patient counselling, planning of follow-up and stratification of patients in clinical trials. Discoveries of prognostically relevant genetic markers in the last few decades have fuelled the advancement of prognostication in UM considerably. In this thesis, we explored ways of improving genetic prognostication in UM, evaluated the effect of irradiation on chromosome testing, and investigated the association of DNA repair genes and epigenetic regulators with prognosis. We reveal that chromosome markers of high malignancy such as monosomy 3 and chromosome 8q gain are less often observed in patients who die due to metastases at a late stage. We demonstrate that combining the AJCC staging and chromosome 3 and 8q status results in enhanced risk stratification. We provide evidence that supports the taking of biopsies before radiotherapy is applied since chromosome testing seems to fail more often in irradiated tumors. Furthermore, we show that evaluating the role of DNA repair and epigenetics in uveal melanoma can help in unraveling the biology of uveal melanoma and identifying new markers for prognostication. Show less
Uveal melanoma is a highly malignant intraocular tumor with quite homogeneous tumor tissue and a diffuse leukocytic infiltration. In contrast with many other malignancies, the presence of... Show moreUveal melanoma is a highly malignant intraocular tumor with quite homogeneous tumor tissue and a diffuse leukocytic infiltration. In contrast with many other malignancies, the presence of infiltrating macrophages and T cells is associated with a poor prognosis rather than a good one. The clear link between inflammation and this malignancy provides a paradigm for macrophage plasticity and function. Macrophages in uveal melanoma have an M2-like phenotype and are associated with the loss of one specific chromosome - monosomy 3. The central players involved in this process and discussed include macrophages, T lymphocytes, chemokines and cytokines, including the macrophage-attraction molecules. When a tumor acquires the ability to release significant amounts of macrophage-attraction molecules it causes the expansion of a population of myeloid immature cells that may not only help the tumor to suppress immune reactions but also aid in the construction of new blood vessels for tumor growth. A better understanding of the molecular basis of a local myelomonocytic cell population will bring a better understanding of the immunopathology of this disease and will lead to therapeutic interventions in uveal melanoma. This thesis focuses on the roles of the local inflammatory microenvironment in the development and progression of uveal melanoma. Show less
Uveal melanoma (UM) is the most common malignancy of the eye in adults and it is the second most common form of melanoma after cutaneous melanoma (CM). The identification of patients who have a... Show moreUveal melanoma (UM) is the most common malignancy of the eye in adults and it is the second most common form of melanoma after cutaneous melanoma (CM). The identification of patients who have a high risk of developing metastases would allow the possibility of providing adjuvant therapies to prevent metastases. The application of FISH on transvitreal fine-needle aspiration biopsies is thought to be a reliable method for assaying genetic parameters such as chromosome 3 loss. However, this is based on the assumption that this chromosomal abnormality is distributed homogeneously throughout the tumor. We show that UM can be heterogeneous for the number of copies of chromosome 3 and investigated whether any evidence can be found for heterogeneity in the regulation of tumor-suppressor genes (TSG). Recently, a segregation study identified a potential locus harboring a TSG. One of the genes in this area, RASEF, was analyzed whether the RASEF gene was affected by mutations or gene silencing due to promoter methylation. The MAPK pathway is involved in the balance between melanocyte proliferation and differentiation. Whereas mutant B-RAF and N-RAS are responsible for the activation of the MAPK pathway in most CM, mutations in these genes are usually absent in UM. Nowadays, an assay with increased potential to identify mutations is available and we set out to reanalyze UM cell lines and primary UM for B-RAF mutations. We set out to explore the MAPK pathway by using MAPK profiling and tyrosine kinase arrays. Finally, conclusions drawn from above mentioned studies are summarized and put into perspective. Show less
Uveal melanoma (UM) is a disease with many faces: ophthalmologists treat the primary tumor, but the patient faces the problem of developing metastases, which are often deadly after a short period.... Show moreUveal melanoma (UM) is a disease with many faces: ophthalmologists treat the primary tumor, but the patient faces the problem of developing metastases, which are often deadly after a short period. Recent insight, indicates the need for knowledge-based treatment of UM. The __pseudohypoxic__ and tumor promoting effects of bevacizumab as described in this thesis is especially relevant. Bevacizumab is frequently used off-label to treat macular edema in UM patients suffering of radiation retinopathy, without the knowledge of possible effect on still viable UM cells. We further observed that specific peptides, such as UMAP1, which can successfully be internalized by targeted UM cells, have demonstrated potential for UM-targeted treatment. These peptides have to be investigated in vivo, to ascertain whether they are a viable clinical tool. Labeling the peptides with radionuclides, and demonstrating specificity for UM cells are some of the challenges which have to be overcome. Another aspect in the patient-specific treatment of UM is the in vitro analysis of primary UM samples to predict treatment responses. In case of Dasatinib, we describe treatment responses associated with monosomy 3 and __kinase__ activity in different primary UM samples. Show less
The p53 tumor suppressor protein plays a key role in cancer and its direct or indirect inactivation is an almost universal feature of human tumors. P53 has a central position in the prevention of... Show moreThe p53 tumor suppressor protein plays a key role in cancer and its direct or indirect inactivation is an almost universal feature of human tumors. P53 has a central position in the prevention of genomic instability and protection of tumorigenesis. This thesis presents novel studies regarding the role of Hdmx in p53 inactivation during tumorigenesis, as well as the use of specific drugs for p53 reactivation as cancer treatment. Chapter 2 shows that constitutive Hdmx overexpression contributes to the neoplastic transformation of human fibroblasts and embryonic retinoblasts, thereby functionally resembling loss of p53. Chapter 3 establishes the importance of Hdmx as an oncogene in a subset of uveal melanomas. Importantly, the results described in this chapter extend the function of Hdmx beyond p53 inhibition. Chapter 4 evaluates the use of the specific p53 activating drugs Nutlin-3 and RITA in synergy studies as potential therapy for uveal melanoma. Chapter 5 is a more detailed analysis of the cellular responses to RITA. In particular, Chk2 is shown to be an essential mediator of the RITA-induced effects. Chapter 6 is a general discussion of the results presented in this thesis, and their implications for clinical exploitation and future research. Show less
This thesis describes the role of the immune system as an important phenomenon in the most frequently occurring form of eye cancer in adults, namely in uveal melanoma. We show that the immune... Show moreThis thesis describes the role of the immune system as an important phenomenon in the most frequently occurring form of eye cancer in adults, namely in uveal melanoma. We show that the immune system can be the cause of the tumor, and also plays a role in the development of a tumor, and may be an entry for therapy. In the first chapters of this thesis, we describe the phenomenon of "an inflammatory phenotype" in uveal melanoma. It appears that when this type of cancer shows more inflammation, the survival of patients decreases. A possible explanation for this observation is that one of the key players among the immune cells, the macrophage, plays an essential role in intra-ocular tumor growth. We demonstrate this in patient material, but also in experimental studies; we are able to inhibit tumor growth when we modulate the presence of macrophages. In this thesis, we also show that the immune system can be used effectively to eradicate eye melanomas in experimental models. T cell vaccination in combination with monoclonal antibodies gave promising results for treating eye cancer. Further research has to be performed to translate this into the clinic. Show less
For a long time enucleation has been the treatment of choice for uveal melanoma. New treatment modalities have been developed e.g. transscleral thermotherapy (TTT), proton beam radiation,... Show moreFor a long time enucleation has been the treatment of choice for uveal melanoma. New treatment modalities have been developed e.g. transscleral thermotherapy (TTT), proton beam radiation, stereotactic radiotherapy and ruthenium application 1-3 . These treatment options offer a better chance to spare the eye. Despite new treatment options, the overall survival of patients treated for uveal melanoma did not improve. Ultimately, most patients die of metastatic disease. Therefore, there is need for an effective treatment for distant metastases. Identification of genes, proteins and pathways has started to provide some insight in the dissemination of uveal melanoma. For development of novel anti-cancer strategies, more sensitive and less invasive cancer models are required, to detect and monitor tumor growth and metastatic disease in vivo. Main objective of this thesis is the development of a new tumor model to study local tumor growth and metastatic disease and to explore tumor growth mechanisms of uveal melanoma. Show less
This thesis describes the results of genomic and proteomic analyses of uveal melanoma, aiming to define prognostic markers and/or profiles to categorize uveal melanoma and to indentify the... Show moreThis thesis describes the results of genomic and proteomic analyses of uveal melanoma, aiming to define prognostic markers and/or profiles to categorize uveal melanoma and to indentify the biological mechanism of metastatic disease of uveal melanomas. Show less
