Cancer immunotherapy has experienced remarkable advances in the last decades. Striking clinical responses have been achieved for several solid cancers, particularly cancer types with a high... Show moreCancer immunotherapy has experienced remarkable advances in the last decades. Striking clinical responses have been achieved for several solid cancers, particularly cancer types with a high mutation burden, which placed tumour-mutated antigens (neoantigens) centre stage as targets of tumour immunity and cancer immunotherapy. Neoantigens can be presented in complex with HLA molecules on the tumour cell surface, where T cells with the correct specificity can recognize the neoantigen as ‘non-self’ which will trigger killing of the tumour cell by the T cell. In theory, cancers with a low/moderate mutation burden that present neoantigens in complex with HLA class molecules could still be eligible for T cell-mediated immunotherapy. This thesis, describes the finding that neoantigen-specific T cells are present in mismatch-repair proficient (MMR-p) colorectal cancer patients, a low mutation burden cancer type. Moreover, CD39 and CD103 were found as cell surface markers that pinpoint the T cell population that contains the neoantigen-specific T cells. In addition, subsequent metastasis of a melanoma patient cohort were studied and revealed that also at advanced, late-stage disease, neoantigen-directed T cell therapy is, in theory, still applicable. Taken together, the studies reveal potential for the development of neoantigen-directed cancer immunotherapy for a broader patient population. Show less
To generate a successful novel therapy, a deep understanding of oncogenesis in combination with mechanistic understanding of anti-cancer compounds are needed. The work described in this thesis aims... Show moreTo generate a successful novel therapy, a deep understanding of oncogenesis in combination with mechanistic understanding of anti-cancer compounds are needed. The work described in this thesis aims to contribute to the knowledge on SUMO regulated oncogenesis, understanding the consequences of abolishment of SUMO signaling and exploiting the potential of SUMO E1 inhibitors. To this end, we describe SUMO as a potential biomarker for cancer aggressiveness and increase our understanding on SUMO’s role in cell cycle progression. We exploited the potential of SUMO E1 inhibition by combining with hypomethylating compound 5-Aza-2’ deoxycytidine, leading to increased cytostatic efficacy. Furthermore, we repurposed the SUMO E1 inhibitor TAK981 and hypomethylating drug 5-Aza-2’ deoxycytidine to improve engineered TCR (eTCR) T cell therapy and broaden our understanding of its immunomodulatory potential. Show less
This thesis aims to improve the treatment of patients with stage III melanoma. The first part describes different aspects of treatment with Talimogene Laherparepvec (T-VEC), a genetically modified... Show moreThis thesis aims to improve the treatment of patients with stage III melanoma. The first part describes different aspects of treatment with Talimogene Laherparepvec (T-VEC), a genetically modified herpes virus, which is used as oncolytic immunotherapy for skin and lymph node metastases in melanoma patients. We show that patients with a low tumor burden have the best outcomes, suggesting T-VEC should be used earlier on in the course of the disease. We present a prediction model, allowing a more accurate selection of patients for T-VEC monotherapy. Two studies focused on the use of T-VEC in clinical practice and the results allowed us to make recommendations on the use of PET/CT and dermoscopy during T-VEC treatment. Part two focuses on the value of surveillance and screening imaging in high-risk melanoma patients. We show that FDG-PET/CT is a valuable imaging tool to detect recurrence after complete resection of stage III disease, even shortly after surgery (before starting adjuvant therapy). Finally, we conclude that nodal staging with US as adjunct to SLNB is useful in the work- up of stage IIB/C melanoma, as it can lead to alterations in treatment and prevent unnecessary surgery. Show less
Monique Krystyna van der Kooij shows that a combination of treatments enhancing the immune system can conquer metastasized melanoma in heavily pre-treated patients. Immunotherapy is not a new... Show moreMonique Krystyna van der Kooij shows that a combination of treatments enhancing the immune system can conquer metastasized melanoma in heavily pre-treated patients. Immunotherapy is not a new concept. However, in Leiden a milder, and therefore better tolerated preconditioning regimen is used before the immune cells are administered. This milder preconditioning, in combination with the patient’s own immune cells and an immune checkpoint inhibitor is unique. This thesis shows that this combination is safe and preliminary data also show that some patients have (lasting) clinical responses. A second important finding described in this thesis is that treatment with immune checkpoint inhibitors can safely be prescribed to patients with common autoimmune diseases. Approximately 1 in 10 metastatic melanoma patients suffer from such an autoimmune disease. Until now clinicians were hesitant to prescribe these immune checkpoint inhibitors out of fear of unleashing the autoimmune disease. Showing that this is not the case made it possible for this large group of patients to gain access to this often-successful treatment. However, immune checkpoint inhibitor monotherapy is not indicated for all patients with metastatic melanoma. Patients with uveal melanoma do not benefit from this type of treatment, and do suffer from the adverse events. Show less
In this thesis, we start with a general introduction in Chapter 1 to briefly present the state of PDT, immune therapies, and nanotechnology in the field of cancer. PDT is a well-established... Show moreIn this thesis, we start with a general introduction in Chapter 1 to briefly present the state of PDT, immune therapies, and nanotechnology in the field of cancer. PDT is a well-established approach in superficial cancer treatment. The aim of my Ph.D. research work has been to improve therapeutic responses in solid tumors by novel combinatorial strategies based on PDT and the utilization of nanotechnology. Insights and concepts in these works are expected to help to design personalized therapeutic interventions in cancer progression. In Chapter 2, we focused on the combination of PDT with a stimulator of interferon genes (STING) agonist: ADU-S100. We investigated the anti-tumor efficiency and survival time after this combined treatment in colon tumor mice models. We found that ADU-S100 post-PDT treatment could enhance PDT-induced inflammation and immune responses, which lead to abscopal effects in a distal untreated tumor. The combination also protected cured mice from tumor recurrence through memory T cell anti-tumor immune responses with high probability. In Chapter 3, we found that PDT in combination with viral core particles could prime systematic immune responses and serum antibody intensity to against colon cancer process in MC38 tumor-bearing mice. In Chapter 4, we reviewed the current challenges facing the combination of PDT and multiple cancer treatment options based on current published literature. We highlighted the opportunities of nanoparticle-based PDT in cancer therapies. In Chapter 5, we investigated how hydrogel-supported near-infrared (NIR) -PDT with improved therapy potential in tumor-bearing mice by combining it with immune checkpoint inhibitors. In addition to the improved tumor growth inhibitory effects and prolonged survival time, immune mechanisms were also studied. We found that hydrogel-supported NIR-PDT by multi-stimulation could induce a higher level of lymphocytes in the circulating blood and increased lymphocytes infiltration into tumor site. A general discussion of overall data observed in this work, and clinical and research prospects related to this thesis are provided in Chapter 6. Show less
The immune system plays a dual role in cancer development. Besides the potential to eliminate cancer cells, immunoregulatory mechanisms exist that counteract anti-tumor immunity.Research in this... Show moreThe immune system plays a dual role in cancer development. Besides the potential to eliminate cancer cells, immunoregulatory mechanisms exist that counteract anti-tumor immunity.Research in this thesis focusses on the role of regulatory T cells (Tregs), a type of adaptive immune cell that plays a major role in tumor-associated immunosuppression. Specifically, the role of Tregs was investigated during the development of primary- and metastatic breast cancer, and in the context of novel immunotherapeutics. This was done by using advanced genetically engineered mouse models that recapitulate human breast cancer.The results in this thesis describe that breast tumors are, already early in their development, able to mobilise Tregs in the tumor-draining lymph nodes, thereby creating a local immunosuppressive niche leading to increased lymph node metastasis. In addition, it was found that the immunotherapeutic treatments anti-PD1 and anti-CTLA4 inadvertently activate Tregs, resulting in a diminished efficacy of this treatment in mice bearing breast tumors. Finally, we describe a mechanism by which intratumoral macrophages are critical promote the intratumoral accumulation of Tregs in breast tumors.Insights from this thesis may eventually contribute to the development of therapeutic applications that are aimed at overcoming immunoregulatory mechanisms in breast cancer. Show less
T cell exhaustion is a state of T cell hypofunction arising during persistent viral infections and cancer. Recent advances in the field of immunology uncover the roles of cytokines in regulating T... Show moreT cell exhaustion is a state of T cell hypofunction arising during persistent viral infections and cancer. Recent advances in the field of immunology uncover the roles of cytokines in regulating T cell responses. Using LCMV Clone-13 as a model of persistent viral infection, this thesis investigates the roles of IL-27 and IFN-I in regulating T cells during infection. In addition, the thesis explores the potential of JAK inhibitor in rescuing T cell exhaustion during persistent viral infection and cancer. Show less
Gemcitabine was examined as switch maintenance therapy after standard first line chemotherapy in the NVALT19 trial, which provided the first conformation of gemcitabine activity in malignant... Show moreGemcitabine was examined as switch maintenance therapy after standard first line chemotherapy in the NVALT19 trial, which provided the first conformation of gemcitabine activity in malignant mesothelioma (MM). We describe additional analyses to confirm the prognostic value of CYFRA 21-1 and examine the prognostic value of CYFRA 21-1 in MM patients treated with gemcitabine. We also searched how gemcitabine could improve antitumor immune responses by positively modulating the immune system.In this thesis we describe a cohort of 107 malignant mesothelioma patients treated with nivolumab in the Netherlands. The real- world data of single agent PD-1 blocking were disappointing compared to previous reported single arm phase II trials. To examine clinical and peripheral blood biomarkers univariable and multivariable analyses were performed.As malignant peritoneal mesothelioma is even rarer then pleural mesothelioma, we hypothesized that centralization of care for peritoneal mesothelioma could benefit patients as they would likely receive treatment more often.Prognosis has a significant influence on the treatment preferences of patients, but prognosis of individual MM patients is variable and is hard to predict. We presented the MESOPRO score for patient with MM which are about the start with any systemic treatment. Show less
Antibodies, the cardinal effector molecules of the immune system, are being leveraged to enormous success as biotherapeutic drugs. Adaptive immune responses consist of epitope-diverse polyclonal... Show moreAntibodies, the cardinal effector molecules of the immune system, are being leveraged to enormous success as biotherapeutic drugs. Adaptive immune responses consist of epitope-diverse polyclonal antibody mixtures that are capable of neutralizing their targets via binding interference and by mediating humoral and cellular effector functions. A mechanistic theme fundamental to virtually all aspects of antibody biology, including antibody-antigen binding, clonal selection and effector functions, is the utilization of avidity to drive and tune functional responses. Manipulating antibody avidity has since emerged as an important design principle for enhancing or engineering novel properties in antibody biotherapeutics. In the context of ‘classical’ effector functions, complement-dependent cytotoxicity (CDC) can be improved by single point mutations in the IgG Fc domain that increase intermolecular Fc-Fc interactions upon binding to membrane-bound targets, thereby facilitating enhanced IgG hexamer formation and C1q binding. Such engineering approaches illustrate the relevance of promoting avidity interactions such as antibody clustering to enhance effector functions. The aim of this thesis was to explore the role of antibody avidity interactions, and more specifically the importance of ‘ordered clustering’, in antibody mechanisms of action and to apply the knowledge obtained in designing novel and improved antibody-based therapeutics Show less
The TME has increasingly been recognized as an important player in tumor progression and metastasis and a possible target for therapy. The TME consists of multiple cell types secreting growth... Show moreThe TME has increasingly been recognized as an important player in tumor progression and metastasis and a possible target for therapy. The TME consists of multiple cell types secreting growth factors and cytokines that exert either pro- or anti-tumor effects. This thesis mainly focuses on studies of the TME, especially the effects of Endoglin, on several cell types within the TME, including endothelial cells, fibroblasts, and immune cells.The TME has increasingly been recognized as an important player in tumor progression and metastasis and a possible target for therapy. The TME consists of multiple cell types secreting growth factors and cytokines that exert either pro- or anti-tumor effects. This thesis mainly focusses on studies of the TME, especially the effects of Endoglin, on several cell types within the TME, including endothelial cells, fibroblasts, and immune cells. This thesis aims to unravel the role of Endoglin as a possible target on various cell types within the TME of solid tumors. Endoglin is known for its role during angiogenesis, however, an increasing number of studies have shown the importance of Endoglin expression on several other cell types (e.g., immune cells, CAFs, tumor cells). 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
Cancer vaccines are aimed at raising an immune responses aganist defined tumor antigens. The formulation of a vaccine has a direct impact on the potency of the immune response generated. Cancer... Show moreCancer vaccines are aimed at raising an immune responses aganist defined tumor antigens. The formulation of a vaccine has a direct impact on the potency of the immune response generated. Cancer vaccines require particular effort in their formulation, as the immune system has to overcome immune suppression exerted by the tumor. In this thesis, different formulations to enhance cancer vaccine efficacy were explored. In the first part it is shown how chemical conjugation of antigen and adjuvant in form synthetic Toll-like receptor ligands represent a strategy to enahnce vaccine potency. Next, it is presented how the loading of peptide antigens onto dextran nanogel contributes to increase the breadth of the immune response induced. Lastly, a new DNA vaccine containing multiple tumor neoantigens was explored as a platform for personalized cancer vaccines. Together, these studies display how the formulation of cancer vaccines can be modified and optimized to achieve stronger therapeutic efficacy in the clinic. Show less
In this thesis, the effects of a new form of immunotherapy was investigated and studied how it can be used against different types of cancers. The investigated immunotherapy is based on injecting... Show moreIn this thesis, the effects of a new form of immunotherapy was investigated and studied how it can be used against different types of cancers. The investigated immunotherapy is based on injecting nanoparticles loaded with various immunologically active molecules (immunomodulatory nanoparticles) that can modulate the immune system to attack cancer cells more efficiently. The effectiveness of this immunomodulatory nanoparticles have been studied on different mouse cancer models and investigated as a single treatment or in combination with other known therapies, such as chemotherapy, therapeutic cancer vaccination, or photodynamic therapy. Show less
Immunotherapy approach to cancer is only benefiting to a minority of patients. In this study, we approach cancer solutions by studying the microenvironment and its immunological signature... Show moreImmunotherapy approach to cancer is only benefiting to a minority of patients. In this study, we approach cancer solutions by studying the microenvironment and its immunological signature throughout the body by focusing on the systemic immunity with new technology like mass cytometry. By highlighting specific immunological patterns in cancer, we were able to associate responsive immune cells and positive outcome, therefore paving the way to improve immunotherapy in cancer. Show less
The first part of this thesis provides insight in prognostic markers in VSCC to refine clinicopathological risk assessment. One of the most frequently described risk factors for recurrent disease... Show moreThe first part of this thesis provides insight in prognostic markers in VSCC to refine clinicopathological risk assessment. One of the most frequently described risk factors for recurrent disease is the minimal peripheral surgical margin. In order to improve the quality of future studies and clinical recommendations, we provided a practical guideline on how to uniformly measure this margin in chapter 2. We also determined the clinical relevance of the molecular classification of VSCC based on immunohistochemical staining for p16 and p53. In chapter 3 we described the immunohistochemical characterization of these molecular subtypes to aid their detection in routine clinical practice. We utilized this approach to show the difference in clinical outcome between the three distinct molecular subtypes of VSCC in chapter 4.The second part of this thesis contains studies on the tumor microenvironment as a first step towards immunotherapy for VSCC. An overview of the literature concerning immunity in VSCC at the start of our studies is provided in chapter 5. Subsequently, we interrogated the TME of different VSCC subtypes in chapter 6, and showed that high infiltration of CD4+ T cells is important for clinical outcome, irrespective of the molecular subtype of VSCC. In chapter 7 we performed an in-depth analysis on the TME based on RNA profiles and showed that highly T cell infiltrated VSCC are potentially eligible candidates for immunotherapy. In chapter 8 we exploited the expression of CD39 by CD4+ and CD8+ T cells as a marker to identify tumor specific T cells. Finally, in chapter 9 the general aspects and relevance of the studies mentioned in this thesis are combined, discussed, and placed in a broader perspective with suggestions for future research. Show less
Tumors are complex ecosystems containing not just cancer cells, but a large variety of cell types, including immune cells. Moreover, tumors have a systemic influence: they can signal long distances... Show moreTumors are complex ecosystems containing not just cancer cells, but a large variety of cell types, including immune cells. Moreover, tumors have a systemic influence: they can signal long distances using soluble molecules and hijack non-neoplastic cells (such as immune cells) in distant organs for their own benefit, thus maximising their metastatic potential. The phenotype of immune cells in tumors and in systemic environments is therefore a key determinant of cancer progression and response to therapy.This thesis aims to understand what governs the tumor-immune ecosystem. We argue that cancer-intrinsic genetic aberrations have a dominant role in determining the tumor immune contexture, as well as systemic inflammatory activation. Understanding the intricate connection between the genetics of breast cancer and anti-tumor immune responses will help develop personalised immune intervention strategies for cancer, tailored to the genetic makeup of a patient’s tumor. Furthermore, we examine in detail the role of neutrophils in cancer-induced systemic inflammation, and how they influence the progression and spread of breast cancer. While tumors can be highly heterogeneous in nature, we show that neutrophils themselves also have a tremendous phenotypic diversity. Mapping this heterogeneity in neutrophil phenotypes may help to utilise these cells in cancer immunotherapy. Show less
A type I immune response is crucial for adequate tumor eradication by the immune system. However, tumors often gain evasion mechanisms that create barriers to the generation or effectiveness of a... Show moreA type I immune response is crucial for adequate tumor eradication by the immune system. However, tumors often gain evasion mechanisms that create barriers to the generation or effectiveness of a type I immune response. Among these barriers is the suppression of effective T cell priming and the inhibition of proper T cell infiltration and function in tumors. At present, the only therapies to target these barriers are focused on direct inhibition of T cell function by the tumor, through checkpoint molecules. These therapies are thus dependent on an existing type I response, and are generally not successful when tumors have insufficient T cells primed or infiltrated. This thesis has revealed ways to improve T cell priming and the infiltration of T cells in tumors. Show less
This thesis sought to obtain a better understanding of the composition of the immune microenvironment in NSCLC and how to modulate this tumor immune microenvironment by RT to induce amplified... Show moreThis thesis sought to obtain a better understanding of the composition of the immune microenvironment in NSCLC and how to modulate this tumor immune microenvironment by RT to induce amplified antitumor immune responses to ICIs in advanced NSCLC patients. In the first part of this thesis, a multiangular approach of a combination of protein and mRNA expression with clinicopathological characteristics in a large cohort of early stage, resected NSCLC samples will be discussed. The second part focusses on the immune modulating effects of RT, in particular when combined with immunotherapy treatment in metastatic NSCLC. Show less
Malignant pleural mesothelioma (MPM) is a cancer of the pleura with few treatment options and an infaust prognosis. We developed a short-term primary tumor culture model from tumor cells derived... Show moreMalignant pleural mesothelioma (MPM) is a cancer of the pleura with few treatment options and an infaust prognosis. We developed a short-term primary tumor culture model from tumor cells derived from pleural fluid of MPM patients and performed drug screening on these cultures to guide treatment decisions of corresponding patients. We observed a high concordance between in vitro results and clinical outcomes and defined three subgroups responding differently to the anti-cancer drugs tested. Gene expression profiling yielded distinct signatures that segregated these subgroups and demonstrated that the fibroblast growth factor pathway was most prominently involved. Pharmacogenomic profiling revealed a subgroup of immortalized and primary MPM lines that appeared highly sensitive to FGFR inhibition. BAP1 protein loss was associated with enhanced sensitivity to FGFR inhibition. Gene expression analyses revealed an association between BAP1 loss and increased expression of the receptors FGFR1/3 and ligands FGF9/18. BAP1 loss was associated with activation of MAPK signaling. These associations were confirmed in a cohort of MPM patient samples. Furthermore, 34 patients were treated in a clinical trial with nivolumab which demonstrated meaningful clinical efficacy and a manageable safety profile in pretreated patients with mesothelioma. PD-L1 expression did not predict for response in this population. Show less
