Clostridium difficile is a spore-forming bacterium, the toxin-producing strains of which cause colitis. Risk factors are antibiotics, advanced age and severe comorbidity. C. difficile infection ... Show moreClostridium difficile is a spore-forming bacterium, the toxin-producing strains of which cause colitis. Risk factors are antibiotics, advanced age and severe comorbidity. C. difficile infection (CDI) has been regarded as mostly a hospital-acquired infection. Preventing relapses is considered the biggest challenge in CDI management. In this thesis, we show that CDI occurs in Dutch general practices, often in patients without contact with hospitals. Also, we show that the emerging virulent strain PCR ribotype 027 has not become dominant in European hospitals, but community-associated type 078 has become highly prevalent. Furthermore, we found that cystic fibrosis outpatients are frequently colonized with C. difficile, though mostly with nonpathogenic strains. Thus, acquisition of C. difficile in the community appears more important than previously thought. Next, we show that renal failure at the time of diagnosis predicts relapses. In addition, patients who fail to develop antibodies against C. difficile toxins have a higher chance of relapse. We describe an experimental product derived from the milk of cows vaccinated against C. difficile and its toxins, which might prevent relapses. The last part of the thesis consists of the European guideline for CDI treatment and its recent update Show less
Immunotherapies for cancer are an emerging class of therapeutic strategies which aim to treat cancer via augmentation of the immune system. Despite significant success of immunotherapies in the... Show moreImmunotherapies for cancer are an emerging class of therapeutic strategies which aim to treat cancer via augmentation of the immune system. Despite significant success of immunotherapies in the past decade, not all patients will respond to these treatments and the reasons why immunotherapies are successful in some patients, but not others, remain incompletely understood. The immune response to cancer is a complex, multistage process, and mathematical and computational models are a useful tool for understanding such complex systems. In this thesis, I develop mathematical and computational models of cytotoxic T lymphocytes (CTLs), who are key players in the immune system due to their ability to recognise, destroy, and provide long lasting protection against malignant or virally infected cells. Show less
This thesis focuses on using liposomes in two different treatment strategies; vaccination (or immunotherapy) and delivery of a small molecule, and in two different disease models; cancer and... Show moreThis thesis focuses on using liposomes in two different treatment strategies; vaccination (or immunotherapy) and delivery of a small molecule, and in two different disease models; cancer and atherosclerosis. For each of these treatment strategies, the liposomal formulation was tailored to obtain the desired therapeutic effect. Chapter 2 reviews some of the most important physicochemical properties (size, shape, and rigidity) that determine the immunological effects of liposomes and other nanoparticles. In chapter 3 we present a detailed study on the effect of the rigidity of anionic liposomes, as measured by atomic force microscopy, on antigen-specific regulatory T-cell (Treg) responses. In chapter 4, we show that our optimized anionic liposomes can induce potent antigen-specific Treg responses, and can be used to delay atherosclerosis progression in a mouse model. Chapter 5 also focuses on liposomal treatment of atherosclerosis, but here targeted liposomes were prepared to successfully deliver a small molecule to foam cells in atherosclerotic plaques. In Chapter 6, we used cationic liposomes in combination with an adjuvant for cancer immunotherapy in mice. Finally, we summarize the overall findings in chapter 7 and discuss perspectives of using liposomes for vaccination and targeted drug delivery. Show less
In this thesis, we report on various aspects of tumor - host (immune) interactions in Ewing sarcoma patients with the aim to obtain leads for immunotherapeutic or targeted treatment strategies. We... Show moreIn this thesis, we report on various aspects of tumor - host (immune) interactions in Ewing sarcoma patients with the aim to obtain leads for immunotherapeutic or targeted treatment strategies. We demonstrate a key role for interferon gamma (IFNg) in enhancing both Ewing sarcoma immunogenicity and susceptibility to cytotoxicity. An (endogenous) pro-inflammatory tumor microenvironment consisting of cytotoxic T-lymphocytes and IFNg-inducible chemokines provides prognostic benefit. Moreover, despite evidence for immunologic pressure in selection of HLA class I loss variants, intact IFNg-inducibility of the HLA class I antigen processing machinery emphasizes the significance of a pro-inflammatory microenvironment for initiation/ execution of adaptive anti-tumor immunity. Pre-clinical support for the potential of adoptive cell transfer therapies, in particular combinatorial natural killer cell-based therapy, is provide d. Sensitization of Ewing sarcoma by conventional or targeted therapies, including histone deacetylase inhibitors, combined with cytokine activation of natural killer cells enhances anti-tumor responses and overcomes both intrinsic functional natural killer cell defects as well as cross-resistance of chemotherapy-resistant Ewing sarcoma to natural killer cells. Finally, due to a crucial role of the CXCR4-CXCL12 axis in Ewing sarcoma progression, disruption of this axis by a CXCR4-specific antagonist may represent a promising treatment option for patients with Ewing sarcoma. Show less
In the clinic, several forms of immunotherapy are combined with the standard treatments, including chemotherapy. Translational studies trying to understand the different outcomes in patients have... Show moreIn the clinic, several forms of immunotherapy are combined with the standard treatments, including chemotherapy. Translational studies trying to understand the different outcomes in patients have led to new questions and hypotheses. The studies described in this thesis are to answer some of these questions. We revealed the immunostimulatory effect of the chemotherapy agent; cisplatin. Next, we studied the mechanism of relapse following immunotherapy with HPV16 SLP vaccination in mice. We demonstrated that unsuccessful immunotherapy results in immune editing and secondary resistance. To overcome this, the combination therapies are required. Moreover, we showed the importance of IL-6 producing by tumors in dampening anti-tumor response. To induce a long-term sustained effector T cell response, we examined the potency of mouse cytomegalovirus as a viral vector-based vaccine. We demonstrated that the demarcated thresholds of vaccine-specific T cells correlate to tumor protection. Recognizing the fact that at each phase of the antitumor immune response a different type of help might have to be provided to obtain maximal therapeutic efficacy, the correct timing of various types of chemotherapeutic agents or immune modulators when used in combination is discussed. Finally, we discussed the general aspects and relevance of the studies mentioned in this thesis. 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
Mucosal melanomas (MM) are malignant tumours arising from melanocytes located at the mucosal lining of the head and neck region or the respiratory, gastrointestinal, anorectal, or genital tract.... Show moreMucosal melanomas (MM) are malignant tumours arising from melanocytes located at the mucosal lining of the head and neck region or the respiratory, gastrointestinal, anorectal, or genital tract. Due to the low incidence the disease is still poorly understood and management is mostly based on guidelines of cutaneous melanoma (CM). Survival of patients with MM is poor and regardless of stage is worse than that of CM. This is explained by the advanced stage at diagnosis and high recurrence rates of MM. Moreover, whilst immunotherapeutic agents have revolutionized the therapeutic landscape in CM, in MM, the efficacy is low and survival has not improved since the introduction of these therapies.The MM located at the vulva (VMM) account for 60% of the female genital tract MM and together with the head and neck region are the most common locations of MM. In line with the MM located at all other locations, prognosis is poor. Whilst the majority of the patients is diagnosed with local disease, the aggressive course of disease is demonstrated by the high recurrence rates with short time to recurrence with a median overall survival of 33 months. To improve outcomes in MM, there is a critical need for clinical trials specifically designed for this disease and international collaboration. 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
High-grade osteosarcoma is a malignant bone tumor with the highest incidence in young patients. In chapter 2, we studied MSCs of osteosarcoma patients and found downregulation of HCLS1 in... Show moreHigh-grade osteosarcoma is a malignant bone tumor with the highest incidence in young patients. In chapter 2, we studied MSCs of osteosarcoma patients and found downregulation of HCLS1 in osteosarcoma patient derived MSCs as compared to healthy donor derived MSCs. Despite almost two years in culture, none of the samples underwent spontaneous transformation. An increase in binucleation was noted upon increasing passage in both osteosarcoma patient and healthy donor derived MSCs. In chapter 3, prognostic factors related to the survival of patients with pulmonary metastasized high-grade osteosarcoma were studied. Higher metastatic tumor burden (i.e. larger number of pulmonary nodules), presence of vital metastases upon resection and male sex were associated with an increased risk of death. In chapter 4 we show that osteosarcoma metastasis seems to be inhibited by the presence of macrophages in the tumor microenvironm ent. In chapters 5 and 6 we show that osteosarcoma cells are sensitive to lysis by both autologous and allogeneic NK cells. Patient derived NK cells can be adequately activated by cytokine treatment with IL-15 (chapter 5) or IFN-_ (chapter 6). Therefore, activation of autologous NK cells (either in vivo or ex vivo) may be efficacious. In conclusion, the activation of innate immune cells such as macrophages and NK cells is a promising new adjuvant treatment strategy to treat patients with high-grade osteosarcoma Show less
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
This thesis focuses on the outcome to immunotherapy in locoregional and metastatic urothelial cancer and biomarkers in the tumor-immune microenvironment that may inform outcome, ultimately... Show moreThis thesis focuses on the outcome to immunotherapy in locoregional and metastatic urothelial cancer and biomarkers in the tumor-immune microenvironment that may inform outcome, ultimately enhancing cancer immunotherapy. Firstly, we discuss the outcome of urothelial cancer patients treated with checkpoint immunotherapy in the metastatic (Part I) and preoperative setting (Part II). Next, we focus on the UC tumor immune microenvironment (Part III), as this may facilitate the discovery and development of novel cancer immunotherapy as well as predictive biomarkers for immunotherapy response in UC. A comprehensive framework based on tumor- and host-specific parameters to better understand immunotherapy response in UC is also provided (Part III). Show less
The generally poor prognosis of patients with epithelial ovarian cancer patients treated with curative intent, calls for additional treatment modalities and possible success might lie in a... Show moreThe generally poor prognosis of patients with epithelial ovarian cancer patients treated with curative intent, calls for additional treatment modalities and possible success might lie in a combination of chemotherapy and immunotherapy. This thesis focuses on the interaction of chemotherapy with the immune system and describes new combined chemo-immunotherapy treatment strategies. This thesis has explored new strategies, immune-modulation of the IL-6 pathway and a vaccine against p53, to enhance immune surveillance and to disable tumour immune evasion in ovarian cancer patients. The future challenge for immunotherapy against ovarian cancer is a tailored combinatorial approach to test the rationale of potentially synergistic therapies that can induce efficient antitumour immunity and prolong patients’ survival. Show less
Most lymphomas and leukemias are neo¬plasms of B cells. Due to the many different B cell development stages from which these neoplasms arise, the resulting diseases are quite heterogeneous, which... Show moreMost lymphomas and leukemias are neo¬plasms of B cells. Due to the many different B cell development stages from which these neoplasms arise, the resulting diseases are quite heterogeneous, which amongst other things is manifested in different tumor growth location, proliferation potential and surface antigen repertoire. Neverthe¬less, some population characteristics are found in almost all B cell malignancies as the cell-of-origin is identical. One of these is the cell surface antigen CD20. Originally used as a marker to distinguishing B cells from other lymphocytes, it quickly became a target for immunotherapy. Immuno-therapy is a treatment that makes use of immune system components to fight cancer, in this case by the injection of a monoclonal antibody specifically targeting one protein: CD20. The addition of CD20-targeting an¬tibodies to an anti-tumor treatment allows your immune system to recognize CD20-ex¬pressing B cells (diseased and healthy), and dispose of them. Overall, after several decades of research and therapeutic experience with antibodies targeting CD20, new functional discoveries as well as therapeutic advances are still being made, and CD20 therefore remains a highly attractive and fruitful target for the therapy of B cell malignancies as well as certain B-cell mediated autoimmune diseases. Show less
Vulvar intraepithelial neoplasia is a chronic premalignant disease caused by a persistent human papillomavirus infection for which conventional surgical therapies are only partially successful with... Show moreVulvar intraepithelial neoplasia is a chronic premalignant disease caused by a persistent human papillomavirus infection for which conventional surgical therapies are only partially successful with high recurrence rates and psychosexual problems. Immunotherapy is a new form of therapy that stimulates the body__s own immune system to resolve infections and cancers. uVIN is the first HPV-induced disease successfully treated by immunotherapy, stressing the capacity of the immune system to deal with disease. Despite these considerable successes of immunotherapy, there is a need to identify parameters of the immune system which allow to select patients most likely to respond to treatment as well as to understand why others do not respond. The studies in this thesis resulted in the identification of a number of immuneparameters that positively or negatively predict the course of disease. These may be of great use as new prognostic biomarkers to identify patients most likely to respond to current successful immune therapeuties or identify patients at risk to the recurrent or progressive course of the disease. Moreover the knowledge of the immune profile may help to understand the non-responsiveness to immunotherapy of some patients. This can be used to optimize these therapies and to foster individualised (immune) therapies. Show less
Radiotherapy is intriguing as it not only eliminates tumor cells but also triggers a response from cytotoxic T cells, which attack the tumor. Thus, radiotherapy and immunotherapy are being combined... Show moreRadiotherapy is intriguing as it not only eliminates tumor cells but also triggers a response from cytotoxic T cells, which attack the tumor. Thus, radiotherapy and immunotherapy are being combined in clinical studies, although their success has been limited. We used mouse tumor models to understand how radiotherapy induces T cell priming and subsequent anti-tumor immunity. In a model resembling lymphocyte-depleted cancer, we identified obstacles to systemic radiotherapy-induced T cell responses and proposed interventions to overcome them. Additionally, we explored strategies to counter local T cell suppression in the tumor microenvironment. In poorly immunogenic tumors, radiotherapy can provoke a T cell response, but this is counteracted by the generation of immunosuppressive Tregs. Combining radiotherapy with checkpoint immunotherapy, despite its success in humans, unexpectedly amplified the Treg response, further hindering cytotoxic T-cell activity. Our findings suggest this immunotherapy may not benefit these cancers. We discovered that molecules like CD80 and CD86, capable of stimulating T cells via the CD28 receptor, have distinct roles in promoting cytotoxic and Treg cells. Blocking CD86 enhanced cytotoxic T cell responses post-radiotherapy, leading to tumor rejection. Our study elucidates how tumor characteristics shape T-cell responses, how radiotherapy can evoke both favorable and unfavorable responses, and how targeted antibody immunotherapy can influence this interplay. Show less
This thesis focused on different aspects of melanoma treatment with immunotherapy and targeted therapy. In chapter 2 we search for biomarkers that could be associated with overall survival in... Show moreThis thesis focused on different aspects of melanoma treatment with immunotherapy and targeted therapy. In chapter 2 we search for biomarkers that could be associated with overall survival in patients treated with ipilimumab. In chapter 3 we describe diarrea, a commonly seen side effect of immunotherapy. Here we show that there is no significant correlation between grade of diarrhea and severity of colitis as seen during endoscopy. Chapters 4 and 5 describe patients with brain metastases and/or leptomeningeal metastases. In chapter 4 we show the difference in overall survival in patients treated with vemurafenib, dabrafenib or the combination of dabrafenib + trametinib. Chapter 5 focusses on the treatment of leptomeningeal metastases. Here a significant difference in overall survival was noted between treated and untreated patients. Furthermore LDH was a predictive biomarker for overall survival. In chapter 6 we show that treating patients with vemurafenib beyond progression of disease has a significant impact on overall survival. Lastly in chapter 7 we review the past, present and future of treating patients with different kinds of cancer with tumor-infiltrating lymphocytes. 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
Human leukocyte antigen (HLA) matched allogeneic stem cell transplantation (SCT) is an established curative treatment for hematopoietic malignancies and an investigative immunotherapeutic approach... Show moreHuman leukocyte antigen (HLA) matched allogeneic stem cell transplantation (SCT) is an established curative treatment for hematopoietic malignancies and an investigative immunotherapeutic approach for solid tumors. The curative effect of allogeneic SCT is based on so called graft versus-tumor (GvT) responses. The GvT effect in the HLA-matched setting is mainly driven by donor immune responses against so called minor histocompatibility antigens (mHags). Emerging data suggest that immunotherapy targeting mHags may eradicate cancer without severe side effects. This thesis investigated 1) the proof of concept for the efficacy of mHag specific immunotherapy in vitro and in vivo, 2) the optimal circumstances under which mHag specific cytotoxic T-cells (CTLs) are most effective against established leukemia and solid tumors, 3) new immune escape mechanisms in cancer that need to be considered when targeting mHags, 4) the optimal protocols for the in vitro expansion of mHags CTLs, 5) the optimal design of mHag peptide vaccines and 6) the optimal parameters for the clinical guidance of mHags specific immunotherapy of cancer. The data acquired in this thesis have provided relevant knowledge that is already partially implemented in the design of new HA-1 vaccination studies aiming at curing leukemia and solid tumors. 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 an important role in the balance between viral clearance and viral persistence in HPV related (pre)malignant lesions. In this thesis, we analyzed HPV clade A9-specific T... Show moreThe immune system plays an important role in the balance between viral clearance and viral persistence in HPV related (pre)malignant lesions. In this thesis, we analyzed HPV clade A9-specific T-cell responses in relation to virological and clinical outcome to gain further insight into HPV-specific cellular immunity in relation to the natural course of disease. In depth analysis of cellular immune responses against the E6 antigen of HPV16 and the closely related members of clade A9 (HPV31, 33, 35, 52 and 58) showed us that HPV-specific cross-reactive CD4+ T cells are rare and unlikely to mediate cross protection (chapter 2). The clinical course of cervical HPV infection and HPV-specific immune responses in prospective studies are described in chapter 4 and 5. In those chapters, a strong correlation is observed between a persistent HPV infection or progressive disease and the lack or failure of a type-specific immune response (>90% of the cases). No correlation is detected between HPV type-specific cellular immune responses and virological clearance of the infection and HPV type-specific immunity may be associated with clearance of a cervical HPV induced lesion (chapter 4 and 5). Interestingly, a statistically significant trend could be detected between the presence of a type-specific immunity (HPV16E2) and regression of a low-grade lesion (chapter 5). A similar, but - due to small number of patients __ not statistically significant trend was observed in chapter 4. Together, this suggests that the local innate immune system might play a role in the clearance of transient infections, whereas the cellular immune response can play a role in regression of histologically proven HPV induced lesions. Detection of HPV16 type-specific cellular immune responses in vivo, by the use of a DTH skin test, confirmed that cellular immune responses in healthy subjects consist of both HPV-specific CD4+ Th1/Th2 and CD8+ T cells (chapter 3). Show less
