The aim of this thesis was to study novel tools and biomarkers for improved detection of vulvar premalignant disease and aid the investigation of potential new drug targets for the indication of... Show moreThe aim of this thesis was to study novel tools and biomarkers for improved detection of vulvar premalignant disease and aid the investigation of potential new drug targets for the indication of vulvar and HPV-driven diseases.Firstly, this thesis focuses on the recognition and validation of imaging-based biomarkers for recognition of diseases of the skin and external genitalia. Clinical studies were performed using 3D photography, dermatoscopy optical coherence tomography (OCT) and reflectance confocal microscopy (RCM) on vulvar HSIL, lichen sclerosus, cutaneous warts and anogenital warts. These techniques were subsequently implemented in a clinical trial in which a novel small molecule was tested for safety and exploratory efficacy for the treatment of cutaneous warts.Furthermore, this thesis explores sequencing-based biomarkers for vulvar disease, facilitating insight into the aetiology of vulvar diseases and identifying potential new therapeutic targets. The currently available literature on the vulvar microbiome composition was investigated and expanded in an observational study characterising the vulvar microbiome composition of healthy volunteers, vulvar HSIL patients and lichen sclerosus patients. Lastly, a perspective on recent developments in the field of premalignant vulvar disease is provided, with recommendations for future applications of the biomarkers studied in this thesis. Show less
Pagan, L.; Huisman, B.W.; Wurff, M. van der; Naafs, R.G.C.; Schuren, F.H.J.; Sanders, I.M.J.G.; ... ; Poelgeest, M.I.E. van 2023
BackgroundThe role of the vulvar microbiome in the development of (pre)malignant vulvar disease is scarcely investigated. The aim of this exploratory study was to analyze vulvar microbiome... Show moreBackgroundThe role of the vulvar microbiome in the development of (pre)malignant vulvar disease is scarcely investigated. The aim of this exploratory study was to analyze vulvar microbiome composition in lichen sclerosus (LS) and vulvar high-grade squamous intraepithelial lesions (HSIL) compared to healthy controls.MethodsWomen with vulvar lichen sclerosus (n = 10), HSIL (n = 5) and healthy controls (n = 10) were included. Swabs were collected from the vulva, vagina and anal region for microbiome characterization by metagenomic shotgun sequencing. Both lesional and non-lesional sites were examined. Biophysical assessments included trans-epidermal water loss for evaluation of the vulvar skin barrier function and vulvar and vaginal pH measurements.ResultsHealthy vulvar skin resembled vaginal, anal and skin-like microbiome composition, including the genera Prevotella, Lactobacillus, Gardnerella, Staphylococcus, Cutibacterium, and Corynebacterium. Significant differences were observed in diversity between vulvar skin of healthy controls and LS patients. Compared to the healthy vulvar skin, vulvar microbiome composition of both LS and vulvar HSIL patients was characterized by significantly higher proportions of, respectively, Papillomaviridae (p = 0.045) and Alphapapillomavirus (p = 0.002). In contrast, the Prevotella genus (p = 0.031) and Bacteroidales orders (p = 0.038) were significantly less abundant in LS, as was the Actinobacteria class (p = 0.040) in vulvar HSIL. While bacteria and viruses were most abundant, fungal and archaeal taxa were scarcely observed. Trans-epidermal water loss was higher in vulvar HSIL compared to healthy vulvar skin (p = 0.043).ConclusionThis study is the first to examine the vulvar microbiome through metagenomic shotgun sequencing in LS and HSIL patients. Diseased vulvar skin presents a distinct signature compared to healthy vulvar skin with respect to bacterial and viral fractions of the microbiome. Key findings include the presence of papillomaviruses in LS as well as in vulvar HSIL, although LS is generally considered an HPV-independent risk factor for vulvar dysplasia. This exploratory study provides clues to the etiology of vulvar premalignancies and may act as a steppingstone for expanding the knowledge on potential drivers of disease progression. 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
This thesis contains a variety of information about the natural and vaccine induced immunity against the human papillomavirus. The spontaneously induced HPV-specific humoral response after... Show moreThis thesis contains a variety of information about the natural and vaccine induced immunity against the human papillomavirus. The spontaneously induced HPV-specific humoral response after infection was assessed in population-based studies. The vaccine-induced changes in HPV-seroprevalence among the HPV unvaccinated Dutch population aged 0-89 years, where we compared the HPV-seroprevalence before the introduction of the HPV vaccine with data of approximately six years post-implementation of the national HPV vaccination program. Also, the HPV immune status of the Dutch Caribbean population just after introduction of HPV vaccination was determined. Moreover, the longitudinal relation between the hr-HPV antibody levels and the prevalence of HPV infections in three-dose vaccinated girls were studied. And more insight was gained into humoral and cellular immune responses after just a one-dose of the HPV vaccine. At last, the kinetics of innate and adaptive immune responses directly after vaccination different HPV vaccines were investigated. In the coming years some important changes are expected regarding HPV screening and vaccination. The effectiveness of the one-dose schedule will become clear as clinical trials end. In the Netherlands, a sex-neutral vaccination will be implemented soon. These changes will need to be monitored to provide scientific answers about the effectiveness and immunogenicity. Show less
Hoes, J.; Pasmans, H.; Knol, M.J.; Donken, R.; Marm-Wattimena, N. van; Schepp, R.M.; ... ; Melker, H.E. de 2020
The bivalent human papillomavirus (HPV) vaccine is highly effective and induces robust serological responses. Using a Dutch prospective cohort initiated in 2009, including 744 vaccinated and 294... Show moreThe bivalent human papillomavirus (HPV) vaccine is highly effective and induces robust serological responses. Using a Dutch prospective cohort initiated in 2009, including 744 vaccinated and 294 unvaccinated girls (1993-1994) who provide a vaginal self-swab sample, serum sample, and questionnaire yearly, we report a high, persisting antibody response up to 9 years after vaccination for vaccine types HPV-16 or HPV-18. Antibodies against nonvaccine HPV types 31, 33, 45, 52, and 58 were lower but still significantly higher than in unvaccinated individuals. This was also reflected in the seroprevalence. We compared participant characteristics and antibody levels between vaccinated women with and those without HPV infections 1 year before infection (204 incident and 64 persistent infections), but we observed no consistent difference in type-specific antibody levels. Having a high-risk HPV infection was associated with sexual risk behavior and smoking 1 year before infection. Although high antibody levels are necessary for protection, our study suggests that on the individual level other factors such as HPV exposure or antibody avidity could be important. Show less
Vos, R.A.; Pasmans, H.; Tymchenko, L.; Janga-Jansen, A.V.A.; Baboe-Kalpoe, S.; Hulshof, K.; ... ; Klis, F.R.M. van der 2020
Background: Incidence and mortality of human papillomavirus (HPV)-related cancers differs geographically, with high rates in Caribbean countries. Seroepidemiological data provide information on... Show moreBackground: Incidence and mortality of human papillomavirus (HPV)-related cancers differs geographically, with high rates in Caribbean countries. Seroepidemiological data provide information on lifetime cumulative HPV exposure and contributing risk factors, but has not been available yet for Caribbean Netherlands (CN), comprising the islands Bonaire, St. Eustatius and Saba. Therefore, a cross-sectional population-based serosurveillance study was performed in this (recently girls-only HPV-vaccinated) population in 2017.Methods: Blood samples from participants (n = 1,823, 0-90 years) were tested for seven high-risk (hr)-HPV-specific IgG-antibodies using a VLP-based multiplex-immunoassay. Risk factors for HPV-seropositivity were analysed among persons unvaccinated aged >= 15 years who ever had sex (n = 1,080).Results: Among unvaccinated individuals aged >= 15 years, overall seropositivity was high (34%), with over half of them being seropositive for >= 2 hr-HPV types, and HPV16 and 52 being most prevalent (13%). Seroprevalence was substantial higher in unvaccinated women (51%) than men (18%), predominantly peaking in women aged 20-59 years, and was highest on St. Eustatius (38%). Besides age and sex, sexual risk factors were associated with HPV-seropositivity.Conclusions: In accordance with the Caribbean region, seroprevalence of multiple hr-HPV types was high in CN. These data corroborate the decision regarding introduction of a sex-neutral HPV-vaccination program and the relevance for considering a population-based cervical cancer screening program. (C) 2020 The Authors. Published by Elsevier Ltd. Show less
This thesis provides an overview of the application of clinical pharmacology to the field of gynecology in general and early phase clinical trials in particular. The first part describes the... Show moreThis thesis provides an overview of the application of clinical pharmacology to the field of gynecology in general and early phase clinical trials in particular. The first part describes the development of novel tools and biomarkers for intervention trials in HPV-induced disorders, i.e. common and plantar warts, anogenital warts and vulvar high grade intraepithelial lesion is described. The second part of the thesis focuses on the application and implementation of the tools and biomarkers into intervention trials with different investigational medicinal products. Show less
The risk of cutaneous squamous cell carcinoma (cSCC) is highly increased in organ transplant recipients (OTR) and these tumours carry a major health burden for these patients. This thesis dealt... Show moreThe risk of cutaneous squamous cell carcinoma (cSCC) is highly increased in organ transplant recipients (OTR) and these tumours carry a major health burden for these patients. This thesis dealt with several aspects of cSCC in OTR. The chapters 2 and 3 review the role of HPV in cSCC and study HPV as a prognostic factor in the aetiology of cSCC. Subsequently, diagnostic factors were studied. In chapter 4 we intended to identify p16 as a possible histopathological marker for cSCC and in chapter 5 we studied the clinical implication of pain as a symptom to differentiate cSCC from other keratinocyte lesions. In chapter 6 we reviewed the literature on metastasis risk in immunocompetent versus OTR population. We studied in chapter 7 in our own population the risk for metastasis, and subsequently risk factors, of cSCC in OTR compared to immunocompetent patients. Show less
HPV has evolved mechanisms to resist immune attack. I showed that HPV impaired necroptosis induced by IFNγ and TNFα by down-regulation of RIPK3 expression. Methyltransferase inhibitor DZNeP... Show moreHPV has evolved mechanisms to resist immune attack. I showed that HPV impaired necroptosis induced by IFNγ and TNFα by down-regulation of RIPK3 expression. Methyltransferase inhibitor DZNeP restored the expression of RIPK3 in HPV+ keratinocytes, and increased the necroptosis in HPV+ keratinocytes. Simultaneously, hrHPV effectively inhibited IFNγ/TNFα-mediated arrest of cell growth by down-regulating IFITM1. Then I studied HPV associated oropharyngeal squamous cell cancer (OPSCC). The OPSCC infiltrating HPV specific CD4+ Tcells produce cytokines such as IFNγ and TNFα. I found that HPV16-positive tumor cells lacked expression of RIPK3, which were not sensitive to the necroptosis induced by IFNγ /TNFα. I also showed that a combination of IFNγ and TNFα with cisplatin synergized in killing tumor cells. Last but not least I showed that combination of the clinically applied EGFR inhibitor Cetuximab and IFNγ/TNFα increased gene expression of multiple cytokines, including CXCL9, CXCL10 and CCL5, which may attract more lymphocytes to the tumor site. By using the inhibitors of the targets downstream of EGFR, I found that JNK and MEK1 played major role in mediating the suppression of CCL5, CXCL9 and CXCL10 expression. Show less
Clinical responses to immunotherapy can be complete and sustained; however, compared to responses to conventional cancer therapies such as chemotherapy they are often delayed. It has become clear... Show more Clinical responses to immunotherapy can be complete and sustained; however, compared to responses to conventional cancer therapies such as chemotherapy they are often delayed. It has become clear that when different cancer therapies are combined they can potentially synergize. Understanding how individual cancer therapeutics work enables the development of the most optimal combinations of cancer therapies. In this thesis, we have used interesting combinations of chemotherapy, cancer vaccines and immune checkpoint blockade and obtained insights in the mechanisms involved in these combinations. For example, we combined chemotherapy with vaccination and observed that none of the tested chemotherapeutics had a negative effect on vaccination, while only a few displayed synergy with vaccination in tumor eradication. We found that vaccine-induced T cells produce Tumor Necrosis Factor-α which could sensitize tumor cells for chemotherapy-induced cell death. Furthermore, we have shown that different cancer therapies modulate intratumoral myeloid cells in a unique manner which can critically affect the therapeutic efficacy of the therapy. Understanding how different therapies affect the local tumor environment will help to improve clinical responses and decrease toxicity in patients. Show less
Persistent infections with high-risk type human papillomaviruses (hrHPVs) can progress to cancer. HrHPVs infect keratinocytes (KCs) and successfully suppress host immunity for up to two years... Show morePersistent infections with high-risk type human papillomaviruses (hrHPVs) can progress to cancer. HrHPVs infect keratinocytes (KCs) and successfully suppress host immunity for up to two years despite the fact that KCs are well equipped to detect and initiate immune responses to invading pathogens. HrHPV interferes with the innate immune response by affecting several signaling pathways that otherwise would prompt anti-viral mechanisms in the host cell. Furthermore, hrHPV interferes with the production of cytokines that are involved in the attraction of immune cells to the infected epithelium. In addition, hrHPV hides itself from the immune system by suppressing the antigen presentation machinery and employs means to hamper the response of KC__s to signals from adaptive immune cells. In this thesis we show that hrHPV attenuates innate immune signaling (Chapter 2) and CD40-mediated (Chapter 3) and IFN_ and/or TNF_-induced (Chapter 4) adaptive immune signaling. HrHPV exploits the cellular proteins UCHL1 (Chapter 2) and IFRD1 (Chapter 4) that act on multiple points in the IRF and NF_B signaling pathways. Moreover, hrHPV downregulates cellular IFITM1 to resist the growth inhibitory effects of IFN_ and/or TNF_ (Chapter 5). Our data provide important new insights on how hrHPV can persist in the face of host immunity. 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
Monsjou, H.S. van; Schaapveld, M.; Brekel, M.W.M. van den; Balm, A.J.M. 2015
Cervical cancer is caused by the human papillomavirus (HPV). The immune system plays an important role in the protection against HPV and failure of the immune system can lead to the development of... Show moreCervical cancer is caused by the human papillomavirus (HPV). The immune system plays an important role in the protection against HPV and failure of the immune system can lead to the development of cervical cancer. Immunotherapy aims at the restoration of an effective anti-tumour immunity. This thesis has led to new insights into the role of the immune system in HPV induced disease and forms a vital contribution to our understanding of tumor inducted immunesuppresion in patients with (pre-) cancerous lesions of the cervix. Furthermore it describes two clinical trails in which patients with pre-cancerous lesions of the cervix are vaccinated with an HPV16 E6/E7 synthetic overlapping long-peptide vaccine (HPV16-SLP). Multiple mechanisms restrain the host__s immune system to rise to the challenge of combating the tumour and favourable immune profiles need boosting in order to keep the balance in favour of tumour eradication. Future therapy should combine various synergistic approaches, and old and new therapies should be used side by side in order to enhance vaccination efficacy and counteract tumour suppression. Show less
