Bernard Le Floch

• 20 December 2019
• Supervisors: Prof L. Peremans, Prof H. Bastiaens, Prof T. Montier and Prof J.-Y. Le Reste
• Language: French

Laure Sorber​

• 20 December 2019
• Supervisors: Prof P. Pauwels, Prof C. Rolfo & Prof G. Roeyen
• Language: English

Abstract

Liquid biopsy, consisting of the analysis of several biomarkers in the blood, is a minimally invasive technique to assess the molecular landscape of the tumor. The development of highly sensitive analysis platforms has enabled the analysis of several of these biomarkers, such as circulating cell-free (tumor) DNA (cfDNA / ctDNA) and RNA (cfRNA / ctRNA). However, the lack of standard operating procedures has limited its implementation in a clinical setting.

In this PhD dissertation we have focused on identifying key steps to compose an optimized liquid biopsy workflow. As even highly sensitive techniques are dependent on the number of genome equivalents, we started by evaluating several cfDNA isolation kits. We selected the most optimal isolation kit for use throughout this dissertation. Next, we assessed the generation of cfDNA and cfRNA by several centrifugation protocols in both standard EDTA tubes and stabilizing blood collection (Streck) tubes. We demonstrated that the selected centrifugation protocol is dependent on the downstream analysis. Another important aspect of the liquid biopsy workflow is the time-to-processing of blood samples and the choice of blood collection tube (BCT). We performed a systematic review to evaluate the effect of delayed processing of blood samples collected in different (specialized) BCTs on cfDNA. Furthermore, we confirmed these findings in a small study. Next, we performed a multicenter study to investigate the influence of several (pre-) analytical variables on cfDNA-based liquid biopsy. In total, we analyzed 549 plasma samples of 234 non-small cell lung cancer (NSCLC) patients.

Based on these findings, we have composed an optimal liquid biopsy workflow to ensure successful cfDNA analysis. Lastly, we further highlighted the potential of liquid biopsy in a case report. We clearly demonstrated the advantage of combining clinical data with molecular information from tissue and liquid biopsy to provide patients with personalized treatment.
To conclude, in this PhD dissertation we constructed an optimized workflow for plasma-based liquid biopsy. The combination of several biomarkers is becoming more important. Hence, our work on both cfDNA and cfRNA will aid in the standardization and further implementation of liquid biopsy in routine clinical setting. Furthermore, future research will reveal whether this workflow is stringent enough to also enable urine- or sputum-based liquid biopsy. Thereby providing patients with a completely non-invasive tool for real-time follow-up.

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Patrice Nabbe​

• 20 December 2019
• Supervisors: Prof. P. Van Royen, Prof. H. Van Marwijk, Prof. T. Montier and Prof. J.-Y. Le Reste
• Language: French
  

Michael Nambozi​

• 20 December 2019
• Supervisors: Prof. J.-P. Van geertruyden and Prof. U. D'Alessandro
• Language: English

Timothi Van Mulder​

• 19 December 2019
• Supervisors: Prof. H. Theeten, Prof. P. Van Damme and Prof. V. Vankerckhoven
• Language: English

Abstract

This doctoral thesis aimed to investigate anatomical and physiological characteristics of the skin in children and adolescents to develop an intradermal injection device for this population. To meet regulatory requirements, the different clinical investigations were first performed in adults and subsequently in children.

To allow determining the maximum penetration depth and needle characteristics in adults and children, the skin thickness at the proximal ventral and dorsal forearm, deltoid and the lateral part of the upper leg (only in 8 weeks to 2 year-olds) was assessed and compared in an age and gender stratified sample using high-frequency ultrasound as highly accurate imaging. Both in adults and children, the overall mean skin thickness increased from the proximal ventral and dorsal forearm, the lateral part of the upper leg, to the deltoid region. A significant association between gender and adult skin thickness was shown in this thesis., whereas the skin thickness increases significantly with age in children and adolescents (8 weeks to 18 years) over the different body sites. An increase in BMI was associated with an increase in skin thickness at all investigated body sites for all ages, except for the lateral part of the upper leg. To guarantee accurate intradermal delivery, which includes no leakage to the hypodermal layer nor to the body surface, the stratification of the population to design different dimensions is not of interest, in case of emergencies, such as pandemics, where single solutions, meaning a device that is configured to be suited for the entire population independent of age, gender or other confounding factors,  are preferred. Therefore, the smallest skin thickness found at the different investigated body sites drove the development of the accurate drug delivery system VAX-ID®.

Based on the data in this doctoral thesis and according to the ease of use of the forearm (as done with the Mantoux technique), the proximal dorsal forearm is proposed as the designated injection site for an intradermal injection device in children and adults. A needle length of 1.0mm is the best option for adults and 0.7 mm is advised to guarantee intradermal injection in children (8 weeks to 18 years). It’s undeniable that a 1 size fits all is of more interest. Accordingly, an intradermal injection device having a maximum penetration depth of 0.65mm is advised to use in the entire population. VAX-ID®, designed by Novosanis, fits this purpose and has been designed, also based on this thesis, to allow for use with 27G, 32G or 33G needle.

To evaluate device and needle characteristics, different verification studies have been performed. Firstly, skin thickness of different animal species was mapped using histology and high-frequency ultrasound, resulting in pigs, piglets and lamas as most suitable verification models for intradermal injections with VAX-ID^®. Through various injection studies it was shown that VAX-ID^® facilitates in a proper injection into dermal layer of the skin of the used animal models. Secondly, technical verification tests were performed to evaluate the dose sparing potentiality (e.g. reducing the per injection-cost, by minimizing the overfill and correct amount of injected fluid), the impact of injection forces and shear stress, and biological safety.

The developed intradermal injection device, VAX-ID®, was validated on the following aspects: 1) functionality, 2) performance, 3) usability, and 4) clinical evaluation. An evaluation in healthy adults was performed twice. Firstly, an evaluation was performed on the acceptability and usability of the device. The new VAX-ID® device was shown to have a high degree of acceptability as well as usability. Secondly the device was evaluated on its efficacy (e.g. correct delivery of the fluid into the dermal layer by triggering an immunogenic reaction) and safety with a commercially available vaccine. VAX-ID® has been compared to the Mantoux technique and intramuscular injection in a booster vaccination study with recombinant hepatitis B vaccine. A dose of 10µg/1.0ml was injected intramuscularly, whereas a dose of 4µg/0.1ml was injected intradermally, meaning that the ID dose was in fact 2.5 times less compared to the IM dose. Higher anti-HBs levels were recorded for the intradermal groups compared to the intramuscular group, nevertheless those results were not statistically significant. The reported adverse events were similar to other trials. The frequency of local adverse events reported in the intradermal group was significantly higher, but they were rather mild.

               Building upon the successful performance results in adults, the acceptability and efficacy of utilising VAX-ID®, having a 33G needle, in children around 6 - 12 years of age is to be assessed. The injection of 0.1ml of sterile, pyrogen free 0.9% NaCl solution will be visualised using high-frequency ultrasound and the pain perception will be measured using the Wong-Baker Scale. Additionally, the participants are required to complete a diary for 5 days to report potential adverse events.

VAX-ID® device can bring an added value to intradermal drug delivery, as the dose-sparing effect of the device will result in a reduction of healthcare costs (especially in developing countries) and offers a solution for vaccine shortages. Additionally, an innovative solution for intradermal injection will offer the following benefits: 1) reduced cold-chain volumes; 2) decreased number of failed injections and loss of dose by increased usability and user-independent administration; 3) potential for self-administration. Furthermore, a painless injection could convince hesitant parents to vaccinate their children. Finally, an “invisible” needle provides additional benefits for people with needle-phobia.

               This doctoral thesis has been accomplished by the interdisciplinary conjunction of engineering, medical sciences, mathematics and product development. This thesis reflects on the pre-commercial trajectory of a new medical device. Despite the absence of validation data in children, the results are beneficial for the further expansion of the device platform VAX-ID®.

Dit proefschrift heeft als doelstelling een overzicht te bieden van het onderzoeks- en ontwikkelingsproces van een intradermaal injectie device geschikt voor kinderen en adolescenten. Om aan de wettelijke vereisten te voldoen, werden de verschillende klinische onderzoeken eerst uitgevoerd in volwassenen en vervolgens in kinderen.

Om de maximale loodrechte injectiediepte van een naald bij volwassenen en kinderen te bepalen, werd de huiddikte van de proximale ventrale en dorsale onderarm, deltoïd en het laterale deel van het bovenbeen (enkel bij kinderen vanaf 8 weken oud tot 2 jaar) onderzocht en vergeleken in een leeftijd-geslacht gestratificeerde steekproef met behulp van zeer nauwkeurige beeldvormingstechnologie, nl. hoogfrequentie echografie. Zowel bij volwassenen als bij kinderen (8 weken t.e.m. 18 jaar) nam de totale gemiddelde huiddikte toe van de onderarm, het laterale deel van het bovenbeen, naar de deltoïd. Dit onderzoek heeft aangetoond dat geslacht een significante invloed heeft op de huiddikte bij volwassenen, mannen hebben een dikkere huid dan vrouwen. Bij kinderen en adolescenten (8 weken t.e.m. 18 jaar) is deze invloed niet aanwezig, huiddikte wordt in deze groep bepaald door de leeftijd. Een toename van de BMI werd geassocieerd met een toename van de huiddikte voor alle leeftijden, op alle onderzochte lichaamslocaties, behalve voor het laterale deel van het bovenbeen. Om een correcte intradermale toediening te garanderen, waarbij lekkage naar de vetlaag of naar het lichaamsoppervlak wordt vermeden, is het aangewezen om eenzelfde device te hebben dat geschikt is voor iedereen. Vooral in noodgevallen, zoals pandemieën, waar ”one size fits all” de voorkeur geniet. Om hieraan tegemoet te komen werd de ontwikkeling van VAX-ID® dan ook gedreven door de lichaamslocatie met de dunste huid.

Op basis van de gegevens in dit proefschrift, alsook het gebruiksgemak van de onderarm (zoals uitgevoerd volgens de Mantoux-techniek), wordt de proximale dorsale onderarm voorgesteld als de aangewezen injectieplaats voor een intradermaal injectiedevice bij kinderen en volwassenen. Een naaldlengte van 1,0 mm is de beste optie voor volwassenen en 0,7 mm wordt geadviseerd om intradermale injectie bij kinderen te kunnen garanderen. Vermits 1 type device voor de hele populatie interessanter is, wordt geadviseerd om een intradermale injectiedevice met een maximale penetratiediepte van 0,65 mm te gebruiken. VAX-ID®, een intradermaal injectiedevice ontworpen door Novosanis, is gedeeltelijk gebaseerd op dit proefschrift en kan bestaan uit een 27G-, 32G- of 33G-naald.

Om device- en naaldkarakteristieken te evalueren zijn verschillende verificatiestudies uitgevoerd. Ten eerste werd de huiddikte van verschillende diersoorten in kaart gebracht met behulp van histologie en hoogfrequentie echografie. Varkens, biggen en lama’s zijn de meest geschikte verificatiemodellen voor intradermale injecties met VAX-ID®. Uit verscheidene injectiestudies kan worden afgeleid dat VAX-ID® een juiste injectie faciliteert in de dermale huidlaag van de gebruikte diermodellen. Ten tweede werden technische verificatietests uitgevoerd om de dosisbesparende potentialiteit, de impact van injectiekrachten en schuifspanning en biologische veiligheid te evalueren.

Het ontwikkelde intradermale injectiedevice, VAX-ID®, is gevalideerd op de volgende aspecten: 1) functionaliteit, 2) prestaties, 3) bruikbaarheid en 4) klinische performantie. Een eerste klinische studie omvatte de evaluatie van de aanvaardbaarheid en bruikbaarheid van het device. In de daaropvolgende klinische studie werd het apparaat geëvalueerd op zijn werkzaamheid en veiligheid met een commercieel verkrijgbaar vaccin. In een boostervaccinatiestudie werd VAX-ID® vergeleken met een intradermale injectie met de Mantoux-techniek en een intramusculaire injectie met een gereduceerde dosis van een recombinant hepatitis B-vaccin. Het nieuwe device werd als pijnloos een zeer gebruiksvriendelijk ervaren. Bijkomend leverde het hogere anti-HBs bloedwaarden op in vergelijking met een intramusculaire injectie. De gemelde lokale en systemische bijwerkingen na vaccinatie zijn vergelijkbaar aan die gerapporteerd in andere onderzoeken. De frequentie van lokale bijwerkingen bij personen die een intradermale injectie kregen is hoger in vergelijking met personen die een intramusculaire injectie kregen, maar ze werden als mild gerapporteerd.

De aanvaardbaarheid, gebruiksgemak en effectiviteit van VAX-ID® bij kinderen moet nog worden beoordeeld. Hiervoor is een studie voorzien waarbij een steriele zoutoplossing van 0,1 ml  intradermaal zal worden geïnjecteerd en gevisualiseerd met behulp van hoogfrequentie echografie. De pijnperceptie zal worden gemeten met behulp van de Wong-Baker-schaal. Via een dagboek zullen gedurende 5 dagen mogelijke bijwerkingen worden gerapporteerd.

VAX-ID® kan een toegevoegde waarde zijn voor de intradermale toediening van geneesmiddelen. Het dosisbesparende effect van het device kan leiden tot lagere zorgkosten (vooral in ontwikkelingslanden) en kan een oplossing bieden voor vaccintekorten. Noodzakelijk injecteerbare geneesmiddelen kunnen toegankelijker worden vanwege het gebruiksgemak en het potentieel van zelftoediening. Bovendien kan een pijnloze injectie aarzelende ouders overhalen om hun kinderen alsnog te laten vaccineren. Ten slotte biedt een "onzichtbare" naald extra voordelen voor mensen met een naaldfobie.

Dit proefschrift is tot stand gekomen dankzij de interdisciplinaire combinatie van ingenieurstechniek, medische wetenschappen, wiskunde en productontwikkeling. Het reflecteert op het pre-commerciële traject van een nieuw medisch hulpmiddel. In afwachting van validatiegegevens bij kinderen, zijn de beschikbare resultaten gunstig voor de verdere uitbreiding van het VAX-ID® platform.

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Lauren Kosten

• 18 December 2019
• Supervisors: Prof. S. Staelens and Prof. S. Stroobants
• Language: English

Vera Saldien​

• 16 December 2019
• Supervisors: Prof. T. Menovsky and Prof. M. Vercauteren
• Language: English

Abstract

The prevalence in the adult population of unruptured aneurysms varies between 3% and 5%. The most feared complication of a cerebral aneurysm is rupture, leading to a subarachnoid hemorrhage (SAH). The incidence of a SAH, caused by the rupture of an aneurysm is less than 0,02 % a year.

The therapy of a cerebral aneurysm is performed on a selective, case-by-case basis. The three main options are observation, endovascular coiling and microsurgical clipping.

Several techniques have been proposed to decrease the intra-aneurysmal pressure, to prevent its premature rupture or to reduce the intraoperative bleeding in case of a rupture during clipping. The most widely used technique for facilitation of the dissection and a better visualisation of the aneurysm, branches and perforators is the temporary clipping of the feeding arteries.

Our research project indicates that RVP is a technique well suited in those circumstances where short, repetitive flow arrests are adequate to allow the neurosurgeon to manipulate the aneurysm.

Rapid ventricular pacing (RVP) enforces ventricular tachycardia, ventricular filling is compromised because of the high rate and the absence of atrioventricular synchrony, leading to decreased blood pressure without causing cardiac arrest and vasodilatation.

We demonstrated that RVP is an effective blood pressure lowering technique operating as an on-off mechanism.  Blood pressure and CBF dropped with 50% after RVP initiation, remained at that level during the pacing and rebounded to pre-pacing values immediately after RVP cessation.

We analysed the effect of RVP on the heart.  Our study results indicated that troponin levels increased slightly following RVP and normalised to pre-operative levels 24h after surgery.

The intra- and post operatively impact of RVP on the brain was evaluated. We used P_btO₂ and rS_cO₂ intra-operatively as measures of cerebral oxygenation.  Whereas blood pressure reacts in an on-off mode to RVP, the impact on cerebral oxygenation is delayed and extends beyond the pacing period.

Postoperatively, we compared pre- and postoperative MRI to assess the impact of RVP on the brain.   No new areas of restricted diffusion were observed in the contralateral hemisphere or posterior fossa. 

The safe application of RVP requires a concerted effort of the neuro- surgeon and anesthesiologist. 

Following the conclusion of this research project, the use of RVP in neurosurgical surgery has been reinstated and has been included in the standard intra- operative management of neurovascular clipping in our centre.

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Jorrit De Waele​

• 16 December 2019
• Supervisors: Prof. E. Smits and Prof. A. Wouters
• Language: English

Abstract

Glioblastoma (GBM) is the most common malignant primary brain tumor and remains one of the most deadliest cancers to date. The long-term standstill in progression of the standard of care in combination with inevitable tumor recurrence and death underscore the urgency of efficacious treatment approaches. The past decade, the interest in immunotherapy for GBM has been invigorated by clinical successes in other solid tumors and a better understanding of immunological mechanisms in the central nervous system. The main objective of this doctoral thesis was to study approaches that could contribute to the unlocking of immunotherapy for GBM and its subsequent implementation in clinical practices.

The greatest immunotherapeutic breakthrough in cancer, i.e. the blockade of immune checkpoints (ICB), remains elusive for GBM. In the first part of this thesis, we sensitized GBM to ICB via polyriboinosinic-polyribocytidylic acid, or poly(I:C). By mimicking a viral infection, poly(I:C) acts as a danger signal to which immune cells become activated. While poly(I:C) contributes to the development of antitumor immunity, it also elicits a desirable safety profile without reported objective protumoral responses in GBM. Our work on primary patient-derived GBM cell cultures elucidated a poly(I:C)-driven pro-inflammatory secretome that resulted in T cell attraction and activation of lymphocytes. As a negative feedback mechanism, GBM cells upregulated programmed cell death 1 ligand 1 or PD-L1, whose blockade further strengthened the immune response.

In the second part of this thesis, we looked at novel approaches that might support immunotherapy in GBM. First, we aimed to target hypoxia via hypoxia-inducible factors (HIF), the primary mediators of the hypoxic response. Hypoxia indeed impaired immune cell-mediated cytotoxicity of GBM cells and modulated the expression of immunomodulatory molecules. However, we were unable to robustly and reproducibly investigate the targeting of HIF, underscoring the need for further optimization to study these intriguing targets for cancer (immuno)therapy. Finally, we investigated cold atmospheric plasma or ionized gas as a novel cancer treatment modality. GBM cell lines died following direct plasma therapy or plasma-activated medium. These results support further research to investigate the effects of plasma with established oncological treatment modalities, e.g. immunotherapy.
In conclusion, this doctoral thesis supports the use of poly(I:C) in ICB as a means to unlock the potential of this highly-investigated immunotherapeutic strategy in GBM. In addition, the foundation was laid for exploring HIF targeting and cold atmospheric plasma treatment appeared as a potentially promising candidate for the (immuno)therapeutic landscape of GBM.

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Els Gielis​

• 12 December 2019
• Supervisors: Prof. D. Abramowicz, Prof B. De Winter, K. Ledeganck (PhD) and M. Eikmans (PhD)
• Language: English

Maarten Lanclus ​

• 11 December 2019
• Supervisor: Prof. W. De Backer
• Language: English

Abstract

Current medical practice within pulmonology focuses on outdated spirometry methods such as pulmonary function tests. Seeing these tests are bulk measures, they don’t show any regional information of the lungs, and they are subject to significant intrinsic variability and uncertainty. This eventually leads to false diagnoses and non-optimized treatment in patients. Furthermore, disease progression and effects of medical interventions are usually studied on a population level, which makes it hard to draw conclusions for patient-specific cases.

FLUIDDA has developed Functional Respiratory Imaging (FRI), which combines computational fluid dynamics and biomedical imaging techniques. FLUIDDA's proprietary FRI technology allows for visualization and quantification of regional lung structures and lung function, enabling characterization of lung health with more sensitive endpoints. FRI has been used extensively to both map disease progression, as well as study effects of medical interventions on a population level, but extrapolation of those results to patient-specific strategies was not established yet.

In this work, the possibilities of combining FRI with artificial intelligence, and more specifically, machine learning applications were studied, to ensure more optimized patient-specific diagnoses, treatments and follow-up. FRI endpoints were first tested for their sensitivity, by studying the effects of bronchodilators in asthma and COPD patients with both FRI and standard spirometry endpoints. From the results it showed that FRI parameters were substantially more sensitive, which makes them ideal input parameters for machine learning applications.  

Two types of machine learning projects were established, i.e. prediction of patient-specific disease progression and responder phenotyping for medical treatments. In a first project, imminent exacerbations in COPD patients could be predicted with an accuracy of 80%. For a second project, responders to a new compound for IPF treatment could be identified with an accuracy of 86.5%. Finally, in the last machine learning project, lung transplant rejection could be predicted with an accuracy of 85%.

With this work it has been proven that in the respiratory space as well, artificial intelligence algorithms in combination with comprehensive imaging endpoints allow for the creation of very robust predictive models, enabling major potential for well-established, patient-specific diagnosis, follow-up and treatment.

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Laure Jacquemin ​

• 9 December 2019
• Supervisors: Prof. P. Van de Heyning, Prof. A. Gilles and Prof. V. Van Rompaey
• Language: Dutch

Elkanah Omenge Orang'o​

• 22 November 2019
• Supervisors: Prof. J.-P. Bogers and Prof. M. von Knebel Doeberitz
• Language: English
  

Jozef Michielsen ​

• 21 November 2019
• Supervisor: Prof. J. Somville
• Language: Dutch

Abstract

The objective of this prospective, randomized controlled trial was to assess both the clinical and radiographic effect of rhBMP-2 in patients treated with a single-level posterior lumbar interbody arthrodesis with polyetheretherketone cages and pedicular screw instrumentation.

Forty patients were randomized with a 1:1 ratio. Two patients were excluded. Interbody arthrodesis was performed using cages, which were filled with 8 mg of rhBMP-2 in the study group and 2,5 mL of autologous iliac crest bone in the control group. All patient completed the Oswestry Disability Index, the Short Form-36 and the visual analogue scale pre-operatively and postoperatively at three, six, twelve and twenty-four months. Thin-slice computed tomography scans with coronal and sagittal reconstructions were made in both groups at three, six, twelve, twenty-four and thirty-six months. All CT scans were read by a radiologist blinded to the randomization assignment. The status of the interbody fusion was quantified using the bridging trabecular bone scale. Bone density measurements were performed in three circular areas with a total surface of 30 mm².

There were no significant differences in clinical results between the groups at each postoperative visit.

Concerning efficacy, fusion was equally achieved when rhBMP-2 was used versus ICBG but bridging trabecular bone formation on the CT scan occurred at a slower rate and interbody bone density was lower within the first year after surgery in the study group. Bridging trabecular bone formation continued beyond 2 years, while bone density was maximal at two years post-surgery.

Regarding safety, certain complications were more frequently noted in the study group using rhBMP-2.

Vertebral endplate resorption was observed in all study group patients and was not observed in the control group.

Vertebral osteolysis was present in 36.8 % of the study group patients and in none of the control group patients.

Cage subsidence was seen in one patient with the use of rhBMP-2. No cage migration was notice in either groups.

Ectopic bone formation was present in 47.4 % of the study patients and was not observed in the control group.

The results of our study suggest there is no link between vertebral osteolysis and ectopic bone formation. Our results are in line with the available scientific literature.

Further research should focus on the least effective dose of rhBMP-2 to promote fusion, on optimizing surgical skills, on the idea of a “sealing barrier” and on the optimal behaviour of carrier systems.

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Zacharie Ndizeye​

• 21 November 2019
• Supervisors: Prof. J.-P. Van geertruyden & Prof. D. Vanden Broeck
• Language: English

Alex Mutombo Baleka

• 21 November 2019
• Supervisors: Prof. Y. Jacquemyn and Prof. J.-P. Van geertruyden
• Language: English

Abstract

Background and Rationale
Women in the Democratic Republic of the Congo (DRC), Cervical cancer (CC) remains the first most common cancer diagnosed in with an estimated number of 3839 cases per year. Vaccination against human papillomavirus (HPV), the main cause of CC, has not been introduced yet in DRC. Cytology screening is opportunistic due to high cost, lack of infrastructure and scarcity of cytotechnicians. Data on the epidemiology of circulating HPV types is limited and the strains involved in invasive CC are not known in the DRC.

Objectives
Main objective: to contribute to the improvement of cervical cancer prevention and control strategies in low-resource settings.

Specific objectives:

- to assess the clinical efficacy of the viricide AV2® in the topical treatment of HPV-associated lesions of the uterine cervix;

- to assess the prevalence and the distribution of HPV genotypes in a community of Kinshasa, DRC.

Design and methods
A phase 2 clinical trial first assessed the efficacy and safety of the antiviral AV2® in the treatment of cervical colposcopic lesions. Then a phase 3 randomized clinical trial (RCT) on the efficacy of AV2® in the treatment of HPV-associated precancerous lesions of the cervix was conducted. The main outcome was the regression of cervical lesions after the application of the topical drug AV2®.

Major findings

Efficacy of AV2®
In the phase 2 clinical trial AV2®, application of AV2® resulted in regression of lesions in 21 out of 28 (75%) women, compared to 0% (0 out of 5) in the placebo group. The difference between the two groups was statistically significant (p < 0.001).

During the phase 3 RCT, 327 women were enrolled. At 2 months, regression of lesions occurred in 127 (89.4%) out of 142 women in AV2® group compared to 120 (91.6%) out of 131 women in the placebo group (P = 0.708).

Prevalence and genotype distribution of HPV in a community of Kinshasa
The overall HPV prevalence was 28.2 %. Women younger than 30 years had the highest prevalence (42.2%). HPV68 was the most prevalent genotype. HPV genotypes distribution was different from other regions of the world.

Conclusion and perspectives for research
The high prevalence of HPV-related cervical diseases warrants improvement of the health system and the need for a nationwide and sustainable implementation of cervical cancer control strategies in DRC. Assessment of HPV genotypes involved in invasive CC will help in the design of future vaccines.

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Daniele Bertoglio ​

• 13 November 2019
• Supervisors: Prof. S. Staelens and Prof. J. Verhaeghe
• Language: English

Abstract

Huntington’s disease (HD) is a rare, autosomal dominant inherited neurodegenerative disorder caused by an expanded polyglutamine sequence in the huntingtin gene (HTT), which encodes for mutant huntingtin (mHTT), the causative agent of the disorder. The genetic mutation responsible for the disorder has been identified in 1993, recently marking the 25 years from its discovery. Since then, research has tremendously advanced in the characterization of the natural disease progression, identification of underlying molecular mechanisms, and assessment of potential endpoints for the evaluation of the efficacy of therapeutic interventions. Although a number of candidate therapeutic approaches aimed at lowering mHTT are underway, their development and evaluation are hindered by the lack of objective in vivo markers to assess the efficacy of these therapeutic strategies. In this context, positron emission tomography (PET) imaging could represent a prime non-invasive in vivo tool for direct assessment of alterations in a number of relevant proteins.

In this thesis we aimed to provide a substantial contribution in the selection of preclinically valuable non-invasive PET markers to investigate candidate biomarkers for monitoring of HD-related disease progression for future disease-modifying therapeutic interventions.

A total of six radioligands quantifying five different proteins were investigated in the knock-in Q175 mouse model of HD. Prior to applying each PET radioligand in extensive longitudinal studies, we performed a thorough methodological validation to assure application of the most suited imaging protocol in order to achieve accurate quantification.

All the investigated biomarkers were found to be affected during disease progression in HD mice. The assessment of phosphodiesterase 10A (PDE10A) with [¹⁸F]MNI-659 PET imaging resulted in a profound decline of the binding in HD mice. Quantification of the Group I metabotropic glutamate receptors type 1 (mGluR1) and type 5 (mGluR5) (using [¹¹C]ITDM and [¹¹C]ABP688, respectively) identified differential profiles in HD mice. Synaptic vesicle glycoprotein 2A (SV2A), measured with [¹¹C]UCB-J, provided non-invasive evidence for a progressive synaptic decline affecting mutation carrier mice. Finally, non-invasive quantification of mHTT accumulation was performed for the first time in the brain of HD mice using two different radioligands. Remarkably, both radioligands proved high capability in measuring mHTT accumulation during natural disease progression.

In conclusion, this work established a preclinical imaging platform and identified the most suited markers for assessment of disease-modifying therapies in order to aid the future translation from bench-to-bedside.

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Thirsa Van Dongen​

• 13 November 2019
• Supervisors: Prof. B. Sabbe and Prof. I. Glazemakers
• Language: Dutch

Stijn Servaes​

• 9 October 2019
• Supervisor: Prof. S. Staelens
• Language: English
  

Joséphine Behaegel​

• 4 October 2019
• Supervisors: Prof. M. Ten Tusscher, Prof. C. Koppen and Prof. S. Ni Dhubhgaill
• Language: English

Abstract

Limbal stem cell deficiency (LSCD) results from a loss of corneal epithelial stem cells and a breakdown of the limbal barrier, due to acquired or congenital diseases. This condition causes varying ocular surface morbidity, dependent on the degree of limbal stem cell damage. Severe LSCD is characterised by vascularisation and conjunctivalisation of the cornea, leading to pain, photophobia and blindness, and is considered to be one of the most challenging ocular surface conditions to manage.  Limbal stem cell grafts aim to restore the limbal barrier and reconstruct the ocular surface, improving rates of subsequent corneal graft survival. While the earliest techniques of stem cell transplantation required large sections of limbal donor tissue, the introduction of ex vivo cultivated limbal epithelial stem cell transplantations (CLET) offered an alternative to overcome the limited access to limbal tissue available for transplantation and the potential risks for the donor eye.

The core of this dissertation was the conduction of a multicentre, single-arm phase II clinical trial, to assess the safety and efficacy of transplanting standardized, non-xenogeneic limbal epithelial stem cell grafts for patients with LSCD. Since the start of the trial in April 2014, a total of 32 transplants have been performed on 26 severe limbal stem cell deficient eyes. Furthermore, in this thesis, we additionally considered aspects ‘beyond the limbal stem cell technique’. Doing so, we have made efforts to optimize the CLET procedure: from image-based clinical examination, grading of LSCD and the ethics of consenting, to targeted biopsy harvesting, performing a safer surgery, critically examining adverse events and implementing resolutions, reporting the results over the long-term and, finally, developing better tools for follow-up and grading results. The current available CLET techniques and latest optimizations offer a treatment modality to patients with otherwise very limited therapeutic alternatives.

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Elyssa Cannaerts

• 1 October 2019
• Supervisors: Prof. B. Loeys, Prof. A. Verstraeten and Prof. L. Van Laer
• Language: English
  

Lieselot Croes​

• 18 September 2019
• Supervisors: Prof. G. Van Camp and Prof. P. Pauwels
• Language: English
  

Samia Labassi​

• 16 September 2019
• Supervisors: Prof. P. Van de Heyning, Prof. M. De Bodt and Prof. V. Van Rompaey
• Language: English
  

Helena Van den Steene

• 16 September 2019
• Supervisors: Prof. D. van West and Prof. I. Glazemakers
• Language: English

Abstract

Increasing case complexity challenges health and social care. Collaboration is put forward as a strategy to address it. Adolescent girls with multiple and complex needs constitute an especially vulnerable population, whose difficulties put their own development at stake and have a negative impact on their relatives and at the level of society.  The Van Celst project is a collaboration between child and adolescent psychiatry and child welfare, aiming to address the needs of adolescent girls with multiple and complex needs. Both partners take joint responsibility for all of the girls enrolled, and for the whole care trajectory.

Starting from the practice needs, this innovative collaboration project and their vulnerable target population, a participatory action research was developed. In this research approach researchers, practitioners, and the community engage in the research process together, starting from practice needs. Participatory action research uses joint expertise to enrich knowledge, facilitate social change, and empower stakeholders from different backgrounds. The aims were to advance knowledge on the target population and the collaboration project Van Celst, and to optimize its practice.

This resulted in a description of the target population, including needs of youths and relatives, and a description of the collaboration project, its development course and benefits and pitfalls. In a second stage, a Delphi study constructed a definition of multiple and complex needs in children and adolescents, endorsed by local and international experts.

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Anke Van Dijck

• 9 July 2019
• Supervisors: Prof. F. Kooy and Prof. B. Ceulemans
• Language: English

Abstract

Intellectual disability (ID) affects approximately 1-2% of the general population. Until recently, the cause of ID remained unknown in 50-70% of the affected individuals. Knowing the genetic cause of ID in a relative can bring comfort to families and is of major importance in care and counselling. It provides insight in comorbidity, associated behavioral problems, prognosis and recurrence risk. The genetics of intellectual disability and autism is a highly dynamic area. Most notably, the next generation sequencing technologies have drastically increased our diagnostic toolbox and created unprecedented diagnostic opportunities.

The aims of this thesis were to monitor the progress in the diagnostic landscape of ID/ASD syndromes and to aid in the first steps into targeted therapy.
Starting from a large X-linked pedigree as well as patients with de novo mutations in strong candidate genes for ID our research group identified two new intellectual disability disorders using next-generation sequencing technologies. My main contribution to this work was the description of the clinical phenotype of 11 affected males in the pedigree, all carrier of a mutation in ZNF711. I also described the clinical features associated with de novo HIST1H1E mutations in 21, mostly novel, cases.

We further clinically delineated two ID syndromes. Our research group previously discovered ADNP mutations as a frequent cause of an hitherto ill-defined form of syndromic autism. The aim of this thesis was to collect a large patient-cohort of ADNP-Related ID/ASD syndrome to describe the phenotypic features of this syndrome in detail. In addition, we were able to find preliminary evidence of a genotype-phenotype correlation. We also describe five patients with a 1q21.1 triplication, an ultra-rare genetic abnormality.
In the last part of this thesis, we aimed to validate our encouraging pre-clinical studies in animal models of the fragile X syndrome, one of the most common inherited forms of ID. We carried out a randomized, double-blind, placebo-controlled, crossover trial of ganaxolone, a GABA(A) receptor positive allosteric modulator in an attempt to alleviate symptoms such as anxiety, hyperactivity, and attention deficits in children with fragile X syndrome. This is an example of how fundamental insights into pathophysiological mechanisms can be translated into clinical practice.

Emmanuel Jacobs​

• 9 July 2019
• Supervisors: Prof. N. Roussel and Prof. J. Gielen
• Language: Dutch

Joseph Ngonzi​

• 9 July 2019
• Supervisors: Prof. Y. Jacquemyn and Prof. J.-P. Van geertruyden
• Language: English

Abstract

This PhD thesis entitled “Postpartum Infection at Mbarara Regional Referral Hospital, South Western Uganda - Epidemiology, Microbiology and Control” assessed the burden, causes and risk factors for maternal deaths at Mbarara Regional Referral Hospital (MRRH) in south-Western Uganda. With puerperal sepsis as the leading cause of maternal deaths at MRRH and the drivers of mortality being late referrals, non-attendance of antenatal care, un-education and HIV sero-positivity, further work was done to describe the microbiology and epidemiology of sepsis.

We described the incidence of postpartum infection among women with postpartum fever or hypothermia presenting for delivery or postpartum care, compared clinical outcomes and examined the risk factors associated with incident fever/hypothermia. A composite infection outcome sepsis among mothers delivering at MRRH was done. Cesarean section was independently associated with in-hospital postpartum infection while antenatal clinic attendance was associated with reduced odds of postpartum infection. With Cesarean section being the commonest operation done on the Maternity ward at MRRH, there’s need to explore factors contributing to the high Cesarean section rates at MRRH and other factors that contribute to the occurrence of post-operative infection before delivery, during delivery and post-operatively.

We further looked at whether vaginal colonization with GBS, E. coli, or Enterococcus was associated with increased morbidity among women with obstructed labor and determined the risk factors for colonization. There was no difference in maternal or neonatal morbidity between women with vaginal colonization with E. coli, GBS, and Enterococcus and those who were not colonized. Assessment of microbiology of infections is challenging especially in cases where the numbers are few. There are however contributors to infection and colonization such as poor hygiene and also nosocomial infections. Duration of labor was associated with increased risk of vaginal colonization in women with obstructed labor. This would mean that there are other factors that drive poor clinical maternal and neonatal outcomes in women who are colonized with potential pathogens and not necessarily the long labor duration.

An intervention of the WHO Surgical Safety Checklist and pre-operative antibiotics for Cesarean section was introduced there was an increase of 77.6% and 69% in the use of WHO checklist and pre-operative antibiotics for Cesarean respectively. There was also a drop in surgical site infection by 6.9%.

Deze PhD thesis getiteld "Postpartum infecties in Mbarara regional Referral Hospital, Zuid-westen van Oeganda - epidemiologie, microbiologie en controle"  bekeek de problematiek, en de oorzaken en risicofactoren op moedersterfte in Mbarara regional Referral Hospital (MRRH) in Zuidwest-Oeganda. Puerperal sepsis was de belangrijkste oorzaak van de moedersterfte in MRRH en de redenen van sterfte waren late doorverwijzingen, niet-participatie in prenatale zorg, gebrek aan onderwijs en HIV besmetting. Wij bekeken verder ook nog de microbiologische aspecten en de epidemiologie van sepsis.

We beschreven de incidentie van postpartum infecties bij vrouwen met postpartum koorts of hypothermie die zich aanboden voor bevalling of postpartum zorg, vergeleken de klinische resultaten en onderzochten de risicofactoren op koorts /hypothermie.  Een composiet  infectieresultaat sepsis bij moeders bevallen in MRRH werd uitgevoerd. Bleek dat keizersnede onafhankelijk geassocieerd was met ziekenhuis postpartum infectie terwijl prenatale zorg werd geassocieerd met verminderde kansen op postpartum infectie. Daar keizersnede de meest voorkomende operatie is op de kraamafdeling op MRRH, is het nodig om de factoren te analyseren die leiden tot het hoog aantal keizersnedes op MRRH en de andere factoren die bijdragen aan post-operatieve infecties voor en tijdens de bevalling en post-operatief.

Verder keken we of vaginale kolonisatie met GBS, E. coli en Enterococcus geassocieerd werd met verhoogde morbiditeit bij vrouwen met geblokkeerde arbeid en bepaalde de risicofactoren voor kolonisatie. Er was geen verschil in de moeder- of neonatale morbiditeit tussen vrouwen met vaginale kolonisatie met GBS, E.coli en Enterococcus en degenen die niet werden gekoloniseerd. Beoordeling van de microbiologie van infecties is niet evident vooral indien er maar weinig gevallen zijn. Er zijn echter riscofactoren voor infectie en kolonisatie zoals slechte hygiëne en ook nosocomiale infecties. Arbeidsduur werd geassocieerd met verhoogd risico op vaginale kolonisatie bij vrouwen met geblokkeerde arbeid. Dit betekende dat er nog andere factoren zijn dat slechte klinische maternale en neonatale consequenties veroorzaken bij vrouwen die gekoloniseerd zijn met potentiële ziekteverwekkers en niet noodzakelijkerwijs de lange arbeidsduur.

Een interventie met de WGO chirurgische checklijst en pre-operatieve antibiotica gebruik voor keizersnede werd geïntroduceerd en er was een stijging van 77,6% en 69% in het gebruik van respectievelijk WGO checklijst en preoperatieve antibiotica voor keizersnede. Er was ook een daling van de chirurgische site infectie met 6,9%.

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Bieke Dobbels​

• 28 June 2019
• Supervisors: Prof. V. Van Rompaey, Prof. P. Van de Heyning and Prof. G. Mertens
• Language: Dutch

Ines De Pauw

• 24 June 2019
• Supervisors: Prof. A. Wouters, Prof. F. Lardon and Prof. M. Peeters
• Language: Dutch

Abstract

During the past decades, important advances have been made in the understanding of the molecular biology of cancer. This has led to the development of targeted therapies and a shift towards precision medicine for cancer patients. Deregulated or increased signaling of the epidermal growth factor receptor (EGFR) plays an integral role in the development of various cancer types, including colorectal cancer (CRC) and head and neck squamous cell carcinoma (HNSCC), making it a compelling drug target. After initially promising results of EGFR-targeted therapies, such as cetuximab, it became clear that both intrinsic and acquired therapeutic resistance are major roadblocks in the field of cancer medicine that compromise the efficacy of available treatment regimens in the clinic. Therefore, understanding these resistance mechanisms is an area of extreme importance and novel therapeutic strategies are needed to overcome this drug resistance. Moreover, as an increasing number of patients are currently considered as candidates for treatment with EGFR-targeted therapy, identification of predictive biomarkers is extremely important.

The objective of this doctoral thesis was to unravel and overcome resistance to the EGFR-targeting agent cetuximab in CRC and HNSCC. Hereby, we focused on the identification of drug resistance mechanisms, novel drug targets and therapeutic strategies as well as predictive biomarkers.

The present study demonstrated that afatinib, a second-generation irreversible inhibitor of EGFR, HER2 and HER4, has the potential to overcome cetuximab resistance in CRC and HNSCC cell lines. Therefore, these data support the hypothesis that afatinib may be a promising therapeutic agent to treat CRC and HNSCC patients experiencing intrinsic or acquired cetuximab resistance. Furthermore, we found that increased phosphorylation of Akt seems to be characteristic for acquired cetuximab resistance in HNSCC. Although further confirmation in tumor samples of HNSCC patients is imperative, Akt appears a novel drug target to improve outcome after cetuximab treatment as well as a potential predictive biomarker for EGFR-targeted therapies in HNSCC patients. In this view, we encourage further studies that focus on targeting Akt in combination with cetuximab, as this may be a promising strategy to overcome drug resistance in HNSCC patients. As such, these findings can form a solid basis for further experiments with advanced in vitro and in vivo models using patient-derived tumor material. We are hopeful that additional research will lead to the start-up of clinical studies and ultimately an improved treatment of CRC and HNSCC patients.

In de afgelopen jaren werden binnen het kankeronderzoek belangrijke vooruitgangen geboekt in het ontrafelen van de moleculair-biologische eigenschappen van tumorcellen. Dit heeft aanleiding gegeven tot de ontwikkeling van doelgerichte therapieën die momenteel een actueel thema vormen in de zoektocht naar een selectieve en geïndividualiseerde behandeling van kankerpatiënten. Afwijkende activatie van de epidermale groeifactor receptor (EGFR) speelt een belangrijke rol in de ontwikkeling van verscheidene tumortypes, met inbegrip van colorectaalkanker (CRC) en hoofdhalskanker van het plaveiselceltype (HNSCC), waardoor EGFR als een aantrekkelijk doelwit voor doelgerichte therapieën beschouwd wordt. Na de initieel beloftevolle resultaten van doelgerichte therapieën in het kankeronderzoek duikt nu echter de problematiek van therapieresistentie op, een belangrijke belemmering in de verdere ontwikkeling van gepersonaliseerde kankerbehandelingen. De precieze oorzaken van intrinsieke en verworven resistentie tegen EGFR-inhibitoren blijven tot op heden echter onduidelijk. Bijgevolg kan het ontrafelen van deze moleculaire mechanismen leiden tot de ontwikkeling van nieuwe behandelingsmodaliteiten om resistentie te omzeilen, met een verbeterde response op EGFR-blokkade tot gevolg.

Het doel van deze doctoraatsstudie was het ontrafelen en therapeutisch omzeilen van resistentiemechanismen tegen EGFR-gerichte agentia, meer bepaald intrinsieke en verworven resistentie tegen het monoklonale antilichaam cetuximab, in CRC en HNSCC. De focus lag hierbij op de identificatie van resistentiemechanismen, bijkomende doelwitten voor behandeling, nieuwe therapeutische combinatiestrategieën en predictieve biomerkers.

In de huidige studie toonden we aan dat afatinib, een irreversibel inhibitor van EGFR, HER2 en HER4, in staat is om cetuximabresistentie te overwinnen in CRC en HNSCC cellijnen. Bovendien kwamen we tot ontdekking dat toegenomen fosforylatie van Akt karakteristiek bleek te zijn voor verworven cetuximabresistentie bij HNSCC. Hoewel bevestiging in tumorstalen van HNSCC patiënten nog essentieel is, doen onze resultaten sterk vermoeden dat Akt beschouwd kan worden als zowel een nieuw potentieel doelwit voor een verbeterde behandeling van HNSCC patiënten, evenzeer als een potentiële predictieve biomerker voor EGFR-gerichte agentia.  Zodanig moedigen we bijkomende studies aan om het potentieel effect van de combinatie van een Akt-gerichte inhibitor met cetuximab te bestuderen, aangezien deze combinatiebehandeling een veelbelovende strategie vormt om cetuximabresistentie in HNSCC te doorbreken.

Onze bevindingen vormen een uitstekende basis voor toekomstige experimenten met geavanceerde in vitro en in vivo modellen, gebruik makend van tumormateriaal afkomstig van patiënten. We zijn ervan overtuigd dat bijkomstig onderzoek op termijn zal leiden tot het opstarten van innovatieve klinische studies, met als uiteindelijk doel vooruitgang te boeken bij de behandeling van patiënten met colorectaal- of hoofdhalskanker.

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Linda Van Diermen​

• 18 June 2019
• Supervisors: Prof. B. Sabbe, Prof. D. Schrijvers and Prof. T. Birkenhager
• Language: Dutch

Annemiek Snoeckx​

• 12 June 2019
• Supervisors: Prof. P. Parizel and Prof. J. Van Meerbeeck
• Language: Dutch

Abstract

For decades, Computed Tomography imaging has had a key role in diagnosis and management of patients with lung cancer, unfortunately often presenting in advanced disease stages. Early detection is however crucial to improve outcome and survival. Where imaging of advanced disease is often straightforward, diagnosis of early lung cancers – often presenting as pulmonary nodules- as well as uncommon manifestations and mimickers, is far more challenging. The first part of this thesis tries to answer the following questions: what are the morphological features that are important to differentiate benign from malignant pulmonary nodules? What are the imaging manifestations of primary lung cancer mimicking benign diseases and what are the imaging and clinical characteristics of the uncommon entity ‘lung cancer associated with cystic airspaces’? Imaging technology has made radical changes over the past decades.

The (r)evolution of multidetector CT and transition to PACS caused an increase in images to read. This triggered the idea of merging multiple conventional window settings into a single ‘All-In-One’ (AIO) window. The AIO-window is a novel technique that fuses multiple conventional window settings such as mediastinal, lung and bone, into a single window to read. To investigate the possible clinical value, two studies were performed in the second part of this thesis, trying to answer the following questions: first, is lesion detection on a combined AIO-window as good as on conventional window settings? Secondly, what is the effect on intra-and interobserver variability on lesion measurement on this novel window?

Currently, AI is reshaping the imaging world with radiologists on the frontline of AI innovation in the medical sector. It will play a role in many aspects of lung cancer imaging and will force us to come out of the dark. The last part of this thesis deals with two projects that are related to the role of radiologists as clinician in an imaging world that is changing at high speed. In this regard, we investigated two topics: the role for radiologists in communication, focused on the imaging features of urgencies and emergencies in the lung cancer patient and secondly, we explored if the presence of a radiologist as co-author in case reports containing radiological images influences quality.

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Sakib Burza​

• 11 June 2019
• Supervisors: Prof. J.-P. Van geertruyden and Prof. M. Boelaert
• Language: English

Abstract

Leishmaniasis is a neglected tropical disease that affects over 1 million people worldwide. In India, it remains one of the major neglected conditions, affecting the poorest and most vulnerable in society, and has been marked for elimination as a public health problem. One of the pillars of the elimination strategy is the use of effective treatment for patients.

The first chapter is a comprehensive literature review covering the last 10 years of evidence covering all aspects of leishmaniasis worldwide, with a focus on the clinical management. Major advances, as well as remaining lacunae are identified for both cutaneous and visceral forms of the disease.

The second chapter focuses on the use of liposomal amphotericin B (LAMB) as a treatment for visceral leishmaniasis in Bihar, the most endemic state in India. It analyses the characteristics and outcomes of 8749 patients treated under programme conditions with 20mg/kg LAMB, including the use of the regimen at PHC level through task shifting. It then describes the risk factors for relapse and also the characteristics of post-kala azar dermal leishmaniasis (PKDL) following treatment.

The third chapter examines a much under-stated aspect of treatment related studies – how patients perceive the treatments that they are receiving. It describes a qualitative study on cohorts of patients whom have received different regimens of treatment to determine the psychosocial determinants and perceptions of treatment, and how policy makers should consider these drivers when making rational treatment policy guidelines.

The fourth chapter focuses on an area that was previously considered to be an issue confined to East Africa – VL in patients co-infected with HIV. It describes the prevalence of co-infection in a cross-sectional cohort study of 2077 patients presenting with VL, and then treatment outcomes using two different treatment regimens. It then describes through a qualitative study the quality of life perceptions in patients with VL-HIV co-infection, and suggest how their experiences and perceptions can be leveraged by policy makers to improve quality, access and standards of care.

The final section of the thesis describes emerging issues in India and the sub-continent related to increasing knowledge and experience of immunosuppression in leishmaniasis.

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Barbara Versyck​

• 7 June 2019
• Supervisors: Prof. R. Slappendel, Prof. G.-J. Heffen, Prof. Van Houwe and Prof. G. Hubens
• Language: Dutch

Abstract

Breast cancer is the most common female cancer comprising about 25% of all worldwide cases. Particularly in Belgium, breast cancer and its sequelae are a clinically significant problem. Belgium has both the highest incidence rate of female breast cancer as well as the highest rate of breast cancer survivors. The treatment of breast cancer is multidisciplinary and involves surgical –, radiation – and medical oncology treatment. For the majority of breast cancer cases, surgery is the primary and most effective therapeutic intervention. Even minimal breast surgery can result in significant acute pain and up to two thirds of female breast cancer surgery patients develop persistent pain and sensory disturbances in the months after treatment. This pain may impair women’s professional lives, sleep and other activities.

Anesthesiologists have developed and introduced many strategies to manage pain during and after breast cancer surgery. These strategies include multimodal systemic analgesia and the introduction of regional anesthesia techniques such as the thoracic epidural or thoracic paravertebral block. However, there has not yet been a widespread clinical adoption of any regional anesthesia technique.

The goal of this doctoral project has been to investigate the analgesic characteristics of a novel regional anesthesia technique in breast cancer surgery: the Pecs block. We concluded that the Pecs block is a easy and reliable technique that significantly improves the quality of analgesia and reduces opioid consumption following breast cancer surgery compared to systematic analgesia alone. Given that its analgesic efficacy is similar to that of a thoracic paravertebral blockade, the Pecs block warrants consideration as a first-line option for regional anesthesia in breast cancer surgery. To achieve an optimal effect, we recommend to aim the needle just medial of the thoraco-acromial artery and use a high volume injection.

Borstkanker is de meest voorkomende kanker bij vrouwen en omvat ongeveer 25% van alle gevallen wereldwijd. Zeker in België zijn borstkanker en de gevolgen ervan een klinisch significant probleem. België heeft zowel de hoogste incidentie van borstkanker bij vrouwen alsook het hoogste aantal overlevenden van borstkanker. De behandeling van borstkanker is multidisciplinair en omvat chirurgie, bestraling en medische oncologische behandelingen. Meestal is chirurgie de primaire en meest effectieve therapeutische interventie. Zelfs minimale borstoperaties kunnen resulteren in aanzienlijke acute pijn en tot twee derde van de vrouwelijke borstkankerpatiënten ontwikkelt aanhoudende pijn en sensorische stoornissen in de maanden na de behandeling. Deze pijn kan het professionele leven, de slaap en andere activiteiten van deze vrouwen schaden.

Anesthesiologen hebben reeds meerdere strategieën ontwikkeld en geïntroduceerd om pijn tijdens en na borstkankeroperaties te behandelen. Deze strategieën omvatten multimodale systemische pijnstilling en de introductie van regionale pijnstillingstechnieken zoals de thoracale epidurale of het thoracaal paravertebraal blok. Er is echter nog geen enkele van deze regionale pijnstillingstechnieken die wijdverspreid gebruikt wordt. 

Het doel van dit doctoraatsproject was om de pijnstillende kenmerken van een nieuwe regionale pijnstillingstechniek bij borstkankerchirurgie te onderzoeken: het Pecs blok. We concludeerden dat het Pecs blok een eenvoudige en betrouwbare techniek is die de kwaliteit van pijnstilling aanzienlijk verbetert en het morfinegebruik na borstkankerchirurgie vermindert in vergelijking met systematische pijnstilling alleen. Aangezien de pijnstillende werking vergelijkbaar is met die van een thoracaal paravertebraal blok, dient het Pecs blok overwogen te worden als eerstelijnsoptie voor regionale pijnstilling bij borstkankerchirurgie. Om een optimaal effect te bereiken, raden we aan om de naald net mediaal van de thoraco-acromiale slagader te richten en een injectie met hoog volume te gebruiken.

Arash Khojasteh​

• 6 June 2019
• Supervisor: Prof. N. Nadjmi
• Language: English

Severien Van Keer​

• 29 May 2019
• Supervisors: Prof. P. Van Damme, Prof. X. van Ostad and Prof. A. Vorsters
• Language: Dutch

Abstract

Within this thesis we aimed to investigate the opportunities of first-void urine as a liquid biopsy, harbouring biomarkers for cervical cancer prevention. First-void urine is a particularly interesting self-sample, as it non-invasively captures mucus and debris from exfoliated cells originating from the female genital organs, including the cervix. These cervicovaginal secretions accumulate around the urethra opening and small labia, and hence are washed away with the initial urine stream (first-void). As the value of using first-void urine for detection of Human Papillomavirus (HPV) DNA – the aetiological agent for cervical cancer – has been demonstrated, we hypothesised that also biomarkers could be present for (i) monitoring primary prevention (through vaccine-induced antibodies against HPV) and (ii) secondary cervical cancer prevention (specific biomarkers for cervical cancer screening and triage of high-grade precancerous lesions).

First evidence was presented that HPV vaccine-induced antibodies are detectable in first-void urine, and able to discriminate vaccinees from unvaccinated women (chapter 2). Based on a review of the literature (chapter 3), interesting candidate biomarkers for screening were identified, followed by detection thereof in first-void urine samples. Good and excellent HR-HPV DNA and HPV16/18 DNA agreements between paired first-void urine and cervical samples were observed, as well as significant positive correlations in HPV copies on genotype level (chapter 4/A). Moreover, first-void urine sampling is well accepted among screening-eligible women. Detection of HPV E6/E7 mRNA is also feasible, yet, requires optimizations to improve sensitivity (chapter 4/B). Furthermore, we have demonstrated elevated host cell methylation marker levels in first-void urine with increasing severity of underlying disease (chapter 5). At last, a protocol was designed to test the accuracy of different HPV test/self-sampling combinations (chapter 6), allowing us to validate both HR-HPV primary screening and molecular triage compared to clinician- and self-collected cervical/cervicovaginal samples in the near future.

In summary, this dissertation provided information and data, contributing to a better understanding of first-void urine as a resourceful liquid biopsy, containing biomarkers for primary as well as secondary cervical cancer prevention.

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Sven De Bruycker​

• 24 May 2019
• Supervisors: Prof. S. Stroobants and dr C. Vangestel
• Language: English

Abstract

Hypoxia, a frequent phenomenon in solid tumors, is associated with chemoradiotherapy resistance. However, most efforts to target or overcome tumor hypoxia have yielded equivocal results due to the lack of biomarkers for appropriate patient selection. The use of non-invasive molecular imaging such as positron emission tomography (PET) may have some advantages over traditional tissue-based biomarkers, such as the capacity to follow up the entire tumor mass longitudinally and evaluate drug efficacy early in the course of therapy. Thus, radiotracers that specifically accumulate in hypoxic cells have been developed, such as [¹⁸F]fluoromisonidazole ([¹⁸F]FMISO) and [¹⁸F]flortanidazole ([¹⁸F]HX4). A hypoxia PET scan could not only be used to image hypoxia and assess a patient’s prognosis, it also holds potential as a predictive marker for patient selection and therapy response, thereby enabling personalized medicine. Therefore, our aim is to validate [¹⁸F]FMISO and [¹⁸F]HX4 PET as imaging biomarkers for hypoxia-targeting therapy combinations in preclinical studies that simulate the concept of precision medicine.

We investigated [¹⁸F]FMISO PET as a biomarker for a combination therapy of the hypoxia-inducible factor 1 (HIF-1) inhibitor PX-12 and standard 5-fluorouracil chemotherapy in a colorectal cancer xenograft model. In this study, [¹⁸F]FMISO could predict that more hypoxic tumors were resistant to chemotherapy and that this resistance could be alleviated by PX-12. We also investigated metformin as a radiosensitizer in a lung cancer xenograft model. Using [¹⁸F]HX4 PET, we showed that metformin could decrease tumor hypoxia with 10%, thereby sensitizing tumors to the effects of radiotherapy. These radiosensitizing effects depended on the baseline degree of hypoxia as assessed with [¹⁸F]HX4, which validated this hypoxia tracer as a predictive biomarker within this setup. Remarkably, this predictive potential of [¹⁸F]HX4 could not be observed in a similar study in a colorectal cancer model, which is most probably due to metformin’s cell-line dependent working mechanisms. Importantly however, regardless of its predictive properties, [¹⁸F]HX4 PET could be validated as a prognostic biomarker in both the colorectal cancer and lung cancer model.

Our translational experimental results add to the ongoing validation of hypoxia PET as a predictive and prognostic biomarker. Although further research is warranted, we are optimistic that the investigated molecular imaging tools, which are currently also under investigation in different clinical trials, have the potential to eventually become a standard part of drug research or even routine clinical practice, in this way leading to precision medicine and improved quality of life for cancer patients.

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Elly Marcq​

• 24 May 2019
• Supervisors: Prof. E. Smits, Prof. J. Van Meerbeeck and Prof. P. Pauwels
• Language: English

Abstract

Malignant pleural mesothelioma (MPM) is a highly aggressive and nearly always fatal cancer that is in most afflicted patients causally associated with a previous, mostly professional, asbestos exposure.1 The highest incidence rates, around 30 cases per million inhabitants, are reported for Australia, Belgium and the UK2,3. Although preventive measures to limit asbestos use and exposure have been around for several decades, the incidence of MPM is still expected to increase over the next decade due to the long latency (20 up to 40 years) between asbestos exposure and MPM development and because asbestos is still being used in developing countries4 4.

The prognosis of patients diagnosed with MPM remains dismal with an untreated median overall survival of only 9-12 months and a 5-year survival rate of less than 5%4,5, due to its aggressive nature, its diagnostic delay and extended stage at presentation4,6. Chemotherapy consisting of a combination of a platinum compound and a folate antimetabolite, either pemetrexed or raltitrexed, have a significant but limited impact on overall survival7,8. Palliative platinum-antifolate chemotherapy results in a median overall survival of about 1 y compared with the 8– 10 months observed for chemotherapy-naïve patients7,9,10. Therefore, new therapeutic strategies are needed to complement the limited armamentarium against MPM6. The observation that the immune system can recognize and eliminate tumors is the impetus of the fast-growing research domain of tumor immunology and immunotherapy. For MPM specifically, the development of immunotherapy is supported by the recent knowledge that our immune system plays a critical role in its pathogenesis and in protection against MPM6,11,12. Immunotherapy of cancer has entered a new and exciting phase because of the discovery of immune checkpoint (ICP) receptors. In this doctoral thesis we investigated immune checkpoint blockade (ICPB) as novel treatment strategy for MPM. First of all, I focused on the tumor microenvironment and the ICP expression profile of tissue samples and effusions from MPM patients. The obtained results showed that ICPs are expressed in mesothelioma patient samples and identified TIM-3 and LAG-3 as promising new targets13,14. In vitro, we showed that (combined) ICPB might offer new opportunities to improve mesothelioma patient prognosis which is confirmed by preliminary in vivo data that show a survival benefit for mice treated with PD-L1 monotherapy and its combination with LAG-3 blockade. Further research will aim to unveil the working mechanisms of ICPB and the investigation of other treatments for advantageous combination strategies.

References

• 1. Robinson, B.W., Musk, A.W. & Lake, R.A. Malignant mesothelioma. Lancet 366, 397-408 (2005).
• 2. Bianchi, C. & Bianchi, T. Malignant mesothelioma: global incidence and relationship with asbestos. Ind Health 45, 379-387 (2007).
• 3. Bianchi, C., Brollo, A., Ramani, L. & Bianchi, T. Malignant mesothelioma in central and Eastern Europe. Acta Med Croatica 54, 161-164 (2000).
• 4. Robinson, B.W. & Lake, R.A. Advances in malignant mesothelioma. N Engl J Med 353, 1591-1603 (2005).
• 5. Musk, A.W., et al. Predicting survival in malignant mesothelioma. Eur Respir J 38, 1420-1424 (2011).
• 6. Bagia, M. & Nowak, A.K. Novel targeted therapies and vaccination strategies for mesothelioma. Curr Treat Options Oncol 12, 149-162 (2011).
• 7. Vogelzang, N.J., et al. Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma. J Clin Oncol 21, 2636-2644 (2003).
• 8. van Meerbeeck, J.P., et al. Randomized phase III study of cisplatin with or without raltitrexed in patients with malignant pleural mesothelioma: an intergroup study of the European Organisation for Research and Treatment of Cancer Lung Cancer Group and the National Cancer Institute of Canada. J Clin Oncol 23, 6881-6889 (2005).
• 9. van Meerbeeck, J.P., et al. Randomized phase III study of cisplatin with or without raltitrexed in patients with malignant pleural mesothelioma: an intergroup study of the European Organisation for Research and Treatment of Cancer Lung Cancer Group and the National Cancer Institute of Canada. J.Clin.Oncol. 23, 6881-6889 (2005).
• 10. Muers, M.F., et al. Active symptom control with or without chemotherapy in the treatment of patients with malignant pleural mesothelioma (MS01): a multicentre randomised trial. Lancet 371, 1685-1694 (2008).
• 11. Izzi, V., et al. Immunity and malignant mesothelioma: from mesothelial cell damage to tumor development and immune response-based therapies. Cancer Lett 322, 18-34 (2012).
• 12. Cornelissen, R., et al. New roads open up for implementing immunotherapy in mesothelioma. Clin Dev Immunol 2012, 927240 (2012).
• 13. Marcq, E., et al. Abundant expression of TIM-3, LAG-3, PD-1 and PD-L1 as immunotherapy checkpoint targets in effusions of mesothelioma patients. Oncotarget 8, 89722-89735 (2017).
• 14. Marcq, E., et al. Prognostic and predictive aspects of the tumor immune microenvironment and immune checkpoints in malignant pleural mesothelioma. Oncoimmunology 6, e1261241 (2017).

​

Sarah De Backer​

• 16 May 2019
• Supervisors: Prof. S. Malhotra and Prof. H. Goossens
• Language: English

Abstract

In addition to multidrug resistance (methicillin-resistant Staphylococcus aureus, MRSA) and presence of virulence-specific factors, the success of S. aureus is also attributable to prolonged persistence of infections linked to the formation of sessile matrix-encased microcolonies termed biofilms. These biofilms are notoriously difficult to eradicate, with sessile populations being up to 1000-fold more resistant than their planktonic counterparts. Despite the important role of S. aureus biofilms in disease, our comprehension of the molecular mechanisms promoting biofilm formation is incomplete. Recent studies of S. aureus biofilms hint that the extracellular matrix consists of proteins, DNA and/or polysaccharide (polysaccharide intercellular adhesin, PIA/PNAG). However, it has become evident that clinical MRSA isolates are not only dependent on PIA/PNAG for biofilm formation. Protein-mediated biofilm formation has emerged as another option to PIA/PNAG, and many surface adhesins, such as Bap, Spa, FnBPA, FnBPB, and SasG have been implicated in this divergent biofilm mechanism.

We aim to dissect the fundamental aspects underlying the pathogenic success of S. aureus/MRSA. Three highly pathogenic, globally successful and prolific biofilm forming MRSA clones, USA300-UAS391, EMRSA-15 H-EMRSA-15 and ST239-SCCmecIII EU_ST239_16, were used as a model to look for commonalities in global MRSA biofilm formation. MRSA transcriptional signatures associated with in vitro planktonic and biofilm phenotypes were identified by means of differential gene expression analysis. Additionally, in order not to overlook other important genes with non-significant up-regulation in the transcriptome analysis, screening of a high-throughput USA300-UAS391 transposon library revealed a large number of mutants displaying changes in biofilm formation capacity. Interesting ‘hits’ were functionally validated for biofilm formation in both static and shear flow in vitro biofilm assays. The relevance of these mutations was also further confirmed by transduction experiments to look for similar phenotypic effects and complementation to demonstrate restoration of the wild type phenotype when a functional protein was present. As such, fatty acid kinase A (fakA), intermediates of the TCA-cycle (fumC, sdhA and sdhB), and staphylococcal surface protein X (sasX) were marked as important determinants contributing to the pathogenic success of MRSA.

The delineation of factors that define virulence in the entire range of infectious MRSA strains will permit the rational design of anti-biofilm drugs that can be combined with conventional antibiotics, thus restoring efficacy the latter show to staphylococci in a non-biofilm status. Interestingly, this research opens up the possibility of a single target affecting two key aspects of MRSA pathogenesis; β-lactam resistance and biofilm formation.

Seline Van den Ameele​

• 13 May 2019
• Supervisors: Prof. B. Sabbe and Prof. M. Morrens
• Language: Dutch

Abstract​

Pathophysiological mechanisms underlying bipolar disorder (BD) are still largely unknown. Although the finding of an increased pro-inflammatory state in patients with BD is rather robust, cautious interpretation of results remains warranted because of many confounding factors. In our systematic literature review, we assessed cytokine levels in medication-free patients with BD and the effect of single mood-stabilizing drugs on these levels. We found evidence for a mood state-related increase in pro-inflammatory cytokines (TNF-α and IL-6) with normalization during euthymia in medication-free patients. Euthymic lithium users had cytokine levels similar to healthy controls.

Cytokines are able to contribute to the development of psychiatric symptoms by stimulating key enzymes in both kynurenine metabolism and BH₄ dependent monoamine synthesis. Kynurenine metabolism gives rise to several neuroactive metabolites, some with neurotoxic and others with rather neuroprotective properties. Objectivation of this interaction in clinical populations is limited.

In our study, we aimed for a longitudinal evaluation of mood, as well as inflammation, kynurenine metabolism and BH₄ dependent monoamine synthesis in patients with BD versus healthy controls. We included patient with BD during an acute mood episode (depression or (hypo)mania) and had 6 test moments over 8 months follow-up. No differences in inflammatory markers (CRP, IFN-y, TNF-α and IL-6) were found between patients in different mood states and controls. Similar to a pro-inflammatory status accompanying healthy aging, we saw increasing pro-inflammatory markers with age, but with a premature onset in patients. Independent of mood state, patients had low KYNA levels, a putative neuroprotective kynurenine metabolite. In patients with chronic subsyndromal depressive symptoms, we found a decreased neuroprotective ratio KYNA/3-HK during the whole follow-up. In presence of mood symptoms, i.e. acute manic as well as chronic subsyndromal depressive symptoms, we saw a strong association between inflammation and a neurotoxic shift in kynurenine metabolism. During mania, TNF-α was strongly correlated to neurotoxic kynurenine metabolites (3-HK and QA), while during chronic depressive symptoms, IFN-y was related to increased neurotoxic QA and activation of IDO-1, the key enzyme in activation of kynurenine metabolism in response to pro-inflammatory cytokines.

Regarding BH₄ dependent monoamine synthesis, monoamines levels (TYR, TRP and PHE), were lower in patients and this most pronounced during depression. Furthermore, we saw an increased correlation between neopterin and PHE/TYR in patients overall, typical for situations of chronic inflammation and suggesting increased neopterin formation at the expense of BH₄ synthesis and compromised monoamine synthesis.

In normal situations, pro-inflammatory and neurotoxic processes balance with neurotrophic processes. We assessed trophic markers BDNF, VEGF and soluble VEGF receptor at baseline and 2 months follow-up. Despite rigorous methodology, we could not identify differences in levels of these markers between patients and controls. Better understanding of methodological issues are needed to guide future research.

To conclude, our research shows evidence for increased neurotoxicity along the illness trajectory of BD with more pronounced toxicity when mood symptoms were present. Our data should be interpreted with caution and need replication.

​

Christopher Delgado Ratto

• 10 May 2019
• Supervisors: Prof. J.-P. Van geertruyden, Prof. D. Gamboa Vilela and Prof. U. D'Alessandro
• Language: English

Hadis Shakeri​

• 9 May 2019
• Supervisors: Prof. V. Segers and Prof. P.-J. Guns
• Language: English

Hannah Stamberger

• 7 May 2019
• Supervisors: Prof. P. De Jonghe, Prof. P. De Witte and Prof. S. Weckhuysen
• Language: English

Abstract

The epilepsies are a heterogeneous group of disorders characterized by an enduring predisposition to epileptic seizures. Epilepsy is ranked fifth of all neurological disorders in terms of global burden of disease, underscoring the critical socio-economic impact of the disorder. The developmental and/or epileptic encephalopathies (DEEs) are a group of rare epilepsy syndromes that are distinguished by their early onset therapy-resistant seizures and development delay that often leads to severe cognitive impairment. It is hypothesized that genetic factors play a role in up to 70 % of epilepsies, both as a causal factor or as a risk or modifier allele. Based on current knowledge, DEEs with a genetic aetiology are regarded as monogenic disorders mainly, and the culprit gene mutation is identified in 30-40 % of patients.

The general purpose of this PhD thesis was to obtain a better understanding of the genetic architecture of rare epilepsy syndromes, with special focus on the DEEs. This was done through  [1] the identification of novel molecular genetic causes of epilepsy, both on a structural and single gene level, with the identification of the valyl-tRNA synthestase gene, VARS, as a novel epilepsy gene; [2] the delineation of the phenotypic spectrum and description of genotype-phenotype correlations of two known epilepsy genes, STXBP1 and NEXMIF; and [3] generation and characterization of  a vars knock-out zebrafish model.

The research presented in this thesis can aid clinicians and geneticists in formulating a genetic diagnosis and in counseling parents about prognosis, comorbidities and recurrence risk of the DEEs covered. Furthermore, the research brings more insight into the complexities of the underlying neurobiology of DEEs, highlighting the role of tRNA synthetase encoding genes. In time, a better understanding of the genetic architecture of DEEs and the underlying neurobiology may accelerate the development of novel precision medicine approaches to treat this severe group of disorders.

​

Julie Ottoy​

• 30 April 2019
• Supervisors: Prof. S. Staelens, Prof. C. Van Broeckhoven and Prof. J. Verhaeghe
• Language: English

Abstract​

In recent years, it has become clear that brain damage from Alzheimer’s disease (AD) can occur decades before clinical symptoms arise. Pathological changes in the brain include accumulation of amyloid-β (Aβ) plaques and neurofibrillary tangles, leading to reduced cerebral blood flow (CBF), neuronal cell death, and cognitive decline. Thanks to the advances in non-invasive medical imaging, the individual hallmarks of AD can now be visualized in-vivo using positron emission tomography (PET). Moreover, following its quantitative nature, PET paved the way to more precise evaluation of brain changes caused by disease progression and drug therapy trials.

However, the role of PET in precision medicine has been challenged by the wide application of ‘simplified’ semi-quantitative metrics such as the standardized uptake value ratio (SUVR). SUVR is obtained as the ratio of PET-tracer uptake in an Aβ-rich region (target region) versus an Aβ-free region (reference region), and is derived from a short static scan. Although useful in clinical practice, SUVR has been widely implemented for various PET tracers without a thorough validation. Therefore, this thesis validated SUVR against gold standard techniques to quantify Aβ plaque deposition and CBF in AD.

To this end, we performed 60min dynamic [¹⁸F]AV45-PET with continuous arterial sampling and pharmacokinetic modeling in 10 controls, 18 mild cognitive impairment, and 10 AD dementia subjects. From this, the gold standard measurements of Aβ plaque deposition and CBF were derived as the volume of distribution (V_T) and the plasma-to-tissue tracer delivery rate (R1), respectively, for each brain region. First, by validating Aβ SUVR against V_T, we found that SUVR was dependent on reference region selection and CBF variations. The subcortical white matter was found to be the preferred reference region for SUVR (instead of the widely used cerebellum), but also made the SUVR most vulnerable to (therapy-induced) changes in global CBF. Second, by validating CBF SUVR against R1, we found that the latter was more sensitive to detect the disease severity.

In conclusion, due to the SUVR shortcomings, we suggest one dynamic [¹⁸F]AV45-PET protocol to quantify both Aβ deposition and CBF for studies in which high accuracy is needed (e.g. therapy-monitoring studies). However, the ultimate goal would be the development of a clinically feasible amyloid-PET protocol with short scanning time and without the need for continuous sampling. Therefore, we set up a bolus-plus-constant-infusion [¹⁸F]AV45 protocol in which the patient is sent to the waiting room while being constantly infused, after which a short static scan and one blood sample is taken. From this, the gold standard V_T is calculated as a simple ratio of Aβ in tissue versus blood at equilibrium - independent from CBF and reference region selection.

Steffi Matthyssen​

• 8 April 2019
• Supervisors: Prof. C. Koppen and Prof. N. Zakaria
• Language: English

Abstract

Sight is the most valuable sense in life. It allows us to observe, interpret and connect with our surroundings. The eye is an intricate sensory organ, a harmonious collaboration of multiple parts but it all starts with the incoming light through the cornea. When damaged, the transparent cornea becomes opaque through irreversible scar formation, which leads to visual impairment or even blindness. While most cases of corneal blindness could be either prevented or cured by a corneal transplantation, severe donor shortages lead to long waiting lists and ten million patients untreated.

Alternatives to transplanting a donor cornea include artificial cornea, but these are not biomimetic and therefore do not provide good outcomes. In the field of tissue engineering and regenerative medicine in ophthalmology the focus has shifted to restoration of the cornea using biocompatible materials. The stroma accounts for 90% of the corneal thickness and is extremely difficult to recreate in a laboratory due to the specific layering and orientation of collagen type I fibrils, which is crucial for corneal transparency. Based on these prerequisites, our approach towards the development of a biomimetic corneal stroma started from the use of high-resolution 3D printing using collagen as a bio-ink (in collaboration with KU Leuven) and investigated the characteristics, optimal expansion protocols and applicability of corneal stromal cells. We aimed at developing a scaffold that could not only host native stromal cells but also restore functionality. We have developed a method to produce thin collagen type III 3D printed films, which can support the growth of corneal stromal cells, and we hope to further advance this technique to produce functional corneal substitutes.

The cornea has been suggested as a source of mesenchymal stem cells (MSCs), a multipotent cell type typically found in the bone marrow. In vitro expansion of these cells involves fetal bovine serum. The risks associated with animal-derived serum include disease transmission and variability between lots. Animal-free culture protocols are indispensable for clinical translation. We investigated phenotypical MSC-characteristics of the cells and found that, except chondrogenesis, all criteria for MSC-characterization were met, even when using an animal-free protocol. To further complete the comparison, we investigated expression of genes specific for or associated with MSCs, stemness and differentiation and show that there are both considerable similarities and differences in expression of MSC-related genes, concluding that functional assessments are imperative to determine whether these cells are (derived from) stem cells, or not.

Het gezichtsvermogen is het meest waardevolle zintuig voor de mens. Het stelt ons in staat om te observeren, interpreteren en aansluiting te zoeken bij onze omgeving. Het oog is een ingewikkeld sensorisch orgaan, een harmonieuze aaneenschakeling van meerdere onderdelen maar het begint allemaal bij het inkomende licht doorheen het hoornvlies. Wanneer het hoornvlies beschadigd geraakt, ontstaat er onomkeerbare littekenvorming, wat leidt tot verminderd zicht of zelfs blindheid. Hoewel de meeste gevallen van hoornvliesblindheid kunnen worden verholpen met een hoornvliestransplantatie, zijn er grote tekorten aan donoren met als gevolg tien miljoen onbehandelde patiënten op de wachtlijst.

Alternatieven voor hoornvliestransplantaties zijn artificiële cornea maar deze zijn niet biomimetisch en daarom suboptimaal. In de regeneratieve oogheelkunde is de focus verschoven naar de ontwikkeling van biocompatibele hoornvliezen. De moeilijkheid bestaat erin dat het hoornvlies voor 90% uit stromale collageen type I-fibrillen die op een specifieke manier gerangschikt en georiënteerd moeten zijn voor de transparantie van het hoornvlies. Om een biomimetisch hoornvlies na te maken, gebruikten we een 3D-printer met zeer hoge resolutie en lichaamseigen collageen als bio-inkt (in samenwerking met de KU Leuven). Tegelijkertijd onderzochten we de karakteristieken, optimale kweekomstandigheden en toepasbaarheid van de cellen. We wilden een hoornvlies namaken dat niet enkel de cellen kan ondersteunen, maar dat ook de functionaliteit van het oog kan herstellen. We ontwikkelden een methode om dunne collageen type III-laagjes te 3D-printen. De collageenlaagjes kunnen de groei van de hoornvliescellen ondersteunen en we willen deze techniek verder ontwikkelen om een functioneel hoornvlies te creëren.

Het hoornvlies werd beschouwd als een bron van mesenchymale stamcellen (MSC), een multipotent celtype dat ook in het beenmerg voorkomt. Om deze cellen te kweken is er dierlijk serum nodig, hieraan zijn risico’s verbonden zoals de kans op ziekteoverdracht en grote verschillen tussen loten. Celkweekprocedures zonder dierlijke producten zijn noodzakelijk alvorens er kan worden overgegaan naar toepassingen in patiënten. We onderzochten de fenotypische MSC-karakteristieken van stromale hoornvliescellen. Uit de resultaten blijkt dat, behalve kraakbeendifferentiatie, aan alle criteria voor MSC-karakterisatie wordt voldaan, zelfs wanneer hoornvliescellen worden gekweekt zonder dierlijke producten. Om de vergelijking te vervolledigen, werd de expressie van genen specifiek voor of geassocieerd met MSCs, of differentiatie onderzocht. De resultaten tonen dat er zowel belangrijke overeenkomsten als verschillen zijn tussen beide celtypes. We concluderen dat functioneel onderzoek absoluut nodig is om een antwoord te kunnen bieden op de vraag of de cellen (afgeleid zijn van) stamcellen zijn, of niet.

Wendy Hens

• 25 March 2019
• Supervisors: Prof. D. Vissers, Prof. L. Van Gaal and Prof. J. Gielen
• Language: Dutch

Anne-Marie De Cock

• 18 March 2019
• Supervisors: Prof. M. Vandewoude, Prof. R. Remmen and Prof. V. Verhoeven
• Language: Dutch

Elisabeth Gillis​

• 22 March 2019
• Supervisors: Prof. B. Loeys, Prof. L. Van Laer and Prof. A. Verstraeten
• Language: Dutch

Abstract

The aorta is the largest artery in the human body. It starts from the heart and supplies the rest of the body with oxygen-carrying blood. The aorta can abnormally enlarge in diameter, this is called an aortic aneurysm. The aneurysm can then lead to dissection or rupture which is often deadly, accounting for 1-2% of all deaths in Western Europe. There are two kinds of aortic aneurysms depending on the position: thoracic aortic aneurysms (TAA) at the level of the chest cavity and abdominal aortic aneuryms in the abdomen. In this thesis, we focused on TAA, because it has a strong genetic component. About 20% of patients have a positive family history. The goal of this thesis was to contribute to the elucidation of the genetic aetiology of TAA, because many patients remain genetically undiagnosed. A genetic diagnosis is beneficial for the patient's treatment, follow-up and counselling. To better understand the genetic cause and underlying mechanism, researchers have focused on syndromic TAA, such as Marfan syndrome (MFS), Loeys-Dietz syndrome and Shprintzen-Goldberg syndrome. MFS is caused by mutations in the FBN1 gene.

Although this gene was already discovered in 1991, Chapter 1 tells the scientific story of a family with MFS and a mutation in FBN1 only diagnosed during this thesis. Even though the affected members showed clear symptoms of MFS, they did not have a molecular genetic confirmation for 10 years. By using a myriad of techniques, such as whole exome sequencing and cDNA sequencing, we were able to finally identify the causal intronic FBN1 mutation. The contribution of the TGF-β pathway to the pathomechanism of MFS was discovered in 2003. Since then, numerous TGF-β genes, such as TGFBR1/2, TGFB2, etc. have been linked to TAA-related syndromes. An extra gene was added to this list during this thesis; Chapter 2 describes the discovery of TGFB3 as an additional TAA gene. The fourth chapter describes the finding and preliminary functional experiments of a possible new TAA gene, KLF15. This gene encodes a transcription factor which might affect the TGF-β pathway as well. Chapter 3 focusses on bicuspid aortic valve (BAV) associated TAA. Between the heart and the aorta, the aortic valve prevents blood owing back into the heart. Normally the aortic valve has three leaflets, but the aortic valve of 1-2% of the general population only contains two aortic valve leaflets. This condition is often accompanied by an increased risk for the development of TAA. Unfortunately, it has been very difficult to pinpoint causal genes for isolated BAV. Because TAA and BAV most possibly share a genetic cause, we focused on BAV/TAV patients to unravel more genetic determinants. A targeted gene panel helped to identify the SMAD6 gene as a new causal gene for BAV/TAA.

​

Ine Decuyper​

• 15 March 2019
• Supervisors: Prof. D. Ebo, Prof. M. Haagendorens and M. Faber (MD)
• Language: English

Abstract

This thesis describes the largest cannabis allergic population up to now (n=120) confirming that cannabis allergy can manifest itself with a wide variety of clinical symptoms ranging from mild to severe anaphylaxis. Symptoms can be induced by cannabis smoking, cutaneous contact and/or ingestion. Moreover, a considerable number of patients report symptoms on mere passive smoke exposure and nearly half report systemic reactions to plant-derived foods.  
This dissertation revealed that Can s 3 (the nsLTP of Cannabis sativa) is a major cannabis allergen in our regions. Although the significance of OEEP2 and RuBisCO proteins as candidate cannabis allergens was also explored preliminary, they could not be identified as important allergens in our region.

Secondly, new cannabis diagnostics were developed and validated as up to now, diagnosis was mainly based on unstandardized prick prick tests. It was evidenced that the in-lab developed Can s 3 based diagnostics (BAT rCan s 3, specific (s)IgE rCan s 3 and the skinprick test with an nCan s 3 rich extract) are equally reliable and have the best performance compared to sIgE hemp and BAT with a crude cannabis extract. Because Can s 3 based diagnostics are commercially unavailable, we advise the sIgE hemp assay where there’s an unequivocal history of cannabis-related symptoms, as a negative result indicates a negligible risk of cannabis allergy. Nevertheless, a positive result needs further diagnostic exploration.

Concerning the risk of cannabis allergy following occupational cannabis exposure, our pilot study was not able to identify any causative allergenic factors for the reported symptoms on cannabis exposure in this population. However, as the reported symptoms are plentiful, extensive protective clothing was recommended during occupational cannabis exposure.

Finally, to be able to diagnose Can s 3 related plant-food allergies more efficiently, a diagnostic exploration of Pru p 3 and/or Mal d 3 sensitized patients (nsLTPs from peach and apple respectively and responsible for some of the cannabis related plant-food reactions) was performed. Patients from Antwerp and Barcelona were compared showing that there are important geographical differences both in allergy profile and in the ability of diagnostics to discriminate clinically relevant from irrelevant nsLTP-sensitizations.

In conclusion, this dissertation ascertains that cannabis allergy merits a place as a genuine allergy entity causing a variety of symptoms and cross-reactive plant-derived food allergies. We have identified reliable standardized diagnostics for cannabis allergy with Can s 3 playing an important role as cannabis allergen in our region.

​

Maria Slaats

• 1 March 2019
• Supervisors: Prof. S. Verhulst and Prof. W. De Backer
• Language: Dutch

Abstract

Pediatric obstructive sleep apnea (OSA) is a manifestation of sleep-disordered breathing. OSA is associated with a number of significant complications such as daytime neurobehavioral problems, learning deficits, growth retardation and cardiovascular complications. Therefore, OSA needs to be treated correctly. Adenotonsillar hypertrophy is the most important predisposing factor in children with OSA. The pathophysiology is likely to be multifactorial because of the high incidence of residual OSA after adenotonsillectomy (ATE). There are several risk factors for OSA such as obesity, Down syndrome (DS), and ethnicity.

The purpose of this thesis was to investigate whether upper airway (UA) imaging could provide more information about UA characteristics in children with OSA mainly to predict treatment outcome. In this thesis, we investigated the use of (functional) imaging in normal-weight children, obese children, and children with DS.

Firstly, we investigated whether functional respiratory imaging could provide more information about UA characteristics in normal-weight children with OSA mainly to predict treatment outcome. In this retrospective study, we concluded that both UA conductance of the functional imaging and the tonsil score by clinical examination predicted treatment response in 91 children.

Secondly, we characterized treatment outcome in 33 young children with DS and OSA by UA imaging. We concluded that children with a less favorable response to surgery had a smaller volume of the zones below the tonsils which could be due to enlargement of the lingual tonsils, glossoptosis or macroglossia which is not treated by ATE.

Thirdly, we evaluated the UA by imaging and drug induced sedation endoscopy (DISE), and compared the results after ATE in 27 obese children with OSA. We concluded that subjects with more central obesity and a more concave shape of the UA had significantly more oxygen desaturations during sleep. We concluded that a straighter shape of the UA and more complex OSA (a higher total score during DISE) correlated with a less improvement after treatment.

Finally, we investigated whether ethnicity could influence UA morphology, OSA severity or treatment response in European children by UA imaging. In our study, black African (bA) children had more severe OSA compared to Caucasian children. Furthermore, there was a difference in UA morphology between bA and Caucasian children: the UA volume of the tongue base and hypopharynx was significantly smaller in children from Africa.

​

Jasmine Coppens​

• 20 February 2019
• Supervisors: Prof. H. Goossens and Prof. S. Malhotra
• Language: English

Abstract

Infections caused by Gram-positive bacteria such as Staphylococcus aureus, Streptococus pyogenes, and Enterococcus faecium represent an enormous health burden in the community as well as in hospitals. While S. aureus and S. pyogenes are known to be highly virulent pathogens causing severe infections, associated with high morbidity and mortality, the past decades have also seen remarkable increases in antibiotic resistance in S. aureus (methicillin-resistant S. aureus, MRSA) and in E. faecium (vancomycin-resistant E. faecium, VRE) that have severely restricted therapeutic options against these pathogens. With the antibiotic pipeline yet to be rejuvenated, these outstanding issues have necessitated the development of alternate solutions, such as development of rapid diagnostic tests and of virulence/antibody- based preventive or therapeutic approaches. Remarkably, in each of these three pathogens, there are a few ‘pandemic’ clones that have spread worldwide and are recognized as the ‘disease-causing’ clones. However, the advent of next-generation sequencing has allowed to differentiate and dissect the microevolution among these pathogenic, and mostly multi-drug resistant clones.

In this thesis, we firstly address the epidemiology of the three major Gram-positive pathogens utilizing whole-genome sequencing. We studied CC239 MRSA, one of the most widely disseminated hospital-associated MRSA causing multiple epidemics around the world in recent decades, in two European (Serbia and Denmark) and in an Indian hospital to understand population structure differences in these three geographically distinct regions. These studies helped us to highlight the dynamic evolution of ST239 in India, the role of international travel in introducing several ST239 clades in Denmark, and the within-patient microevolutionary changes in the ST239 genome upon the switch from a colonizing to an infecting strain.

Furthermore, studying the genomic evolution of invasive and non-invasive S. pyogenes and of VRE in Belgian hospitals showed high genomic heterogeneity among CC17 E. faecium, in contrast to a remarkable genomic stability observed among emm1S. pyogenes strains collected over 19 years.

Further focussing on S. aureus in the clinical context, we studied S. aureus as an important causative agent of ventilator-associated pneumonia (VAP). These studies were primarily focused on comparison of novel, rapid diagnostics with the currently-used culture-based approaches to accurately detect respiratory tract colonization with S. aureus in mechanically ventilated patients, allowing for early and targeted antibiotic treatment or even an initiation of a novel alpha-toxin-targeting, antibody-based eradication strategy. We performed a literature review to understand the role of alpha-toxin in S. aureus pneumonia pathogenesis. This leads to the observational trial studying the occurrence of S. aureus, and a Phase II study investigating antibody treatment against S. aureus among ICU pneumonia in Europe.

Infecties veroorzaakt door Gram-positieve bacteriën, zoals Staphylococcus aureus, Streptococus pyogenes en Enterococcus faecium, vormen een enorm gezondheidsprobleem in de maatschappij en in ziekenhuizen. Hoewel bekend is dat S. aureus en S. pyogenes erg virulente pathogenen zijn, die vaak ernstige infecties veroorzaken en geassocieerd zijn met een hoge morbiditeit en mortaliteit, hebben we de afgelopen decennia ook een opmerkelijke toename gezien in de antibioticaresistentie van S. aureus (methicilline-resistente S. aureus, MRSA) en E. faecium (vancomycine-resistente E. faecium, VRE) waardoor de therapeutische opties beperkt werden. Vermits de pijplijn van antibiotica verouderd is, hebben bovenstaande onopgeloste problemen de ontwikkeling van alternatieve oplossingen noodzakelijk gemaakt; zoals de ontwikkeling van snelle diagnostische tests en van virulentie- en/of antilichaam- gebaseerde preventieve of therapeutische toepassingen.

Opmerkelijk genoeg bevat elk van deze drie pathogenen 'pandemische' klonen die zich wereldwijd verspreid hebben en worden deze herkend als 'ziekte-veroorzakende' klonen. Door de komst van next-generation sequencing is het echter ook mogelijk om de micro-evolutie van deze pathogenen, meestal ook multidrug-resistentie klonen, te definiëren en te ontleden.

In deze thesis wordt de epidemiologie van de drie belangrijkste Gram-positieve pathogenen besproken op basis van whole-genome sequencing analyses. Hiervoor werd CC239 MRSA, één van de meest verspreide ziekenhuis gerelateerde MRSA’s die wereldwijd meerdere epidemieën veroorzaakten in de afgelopen decennia, bestudeerd. We hebben deze analyses in twee Europese ziekenhuizen (Servië en Denemarken) en in één Indisch ziekenhuis onderzocht om zo de verschillen in de drie geografisch regio’s te begrijpen. Deze studies hebben ons geholpen om de dynamische evolutie van ST239 in India te begrijpen, alsook de rol van internationaal reizen waarbij de verschillende ST239-clades in Denemarken werden geïntroduceerd en de micro-evolutionaire veranderingen binnen eenzelfde patiënt, waarbij ST239 de overgang van een koloniserende naar een infecterende stam onderging.

Bovendien toont de studie de genomische evolutie van invasieve en niet-invasieve S. pyogenes en van VRE in Belgische ziekenhuizen. Een hoge heterogeniteit werd vastgesteld bij CC17 E. faecium op basis van genomische studies, in tegenstelling tot een opmerkelijke genomische stabiliteit die werd waargenomen bij emm1S. pyogenes-stammen tijdens een 19-jarige studie.

Met de focus op S. aureus in de klinische context hebben we S. aureus bestudeerd als belangrijke oorzaak van ICU pneumonie (ventilator geassocieerde pneumonie of VAP). Deze studies waren gericht op een vergelijking van nieuwe en snelle diagnostiek met de momenteel veel gebruikte cultuur gebaseerde methodes. Zo trachten we te werken naar een nauwkeurige detectie van respiratoire kolonisatie met S. aureus bij mechanisch geventileerde patiënten. Daarnaast is het ook belangrijk om een vroege en gerichte antibiotica-behandeling of zelfs een nieuwe behandeling met een antilichaam gebaseerde strategie te onderzoeken. Hiervoor hebben we literatuur-onderzoek uitgevoerd om de rol van alfa-toxine in de pathogenese van pneumonie beter te begrijpen. Dit leidt tot een observationele studie die het voorkomen van S. aureus ICU pneumonie bestudeert en een fase II-studie die een behandeling met antilichamen tegen S. aureus bij ICU-pneumonie in Europa onderzoekt.

​Andreas Gevaert

• 23 January 2019
  
• Supervisors: Prof C. Vrints and Prof E. Van Craenenbroeck
• Language: English
  

Abstract 

Over half of patients with heart failure now present with a normal contractile function, a condition which is called heart failure with preserved ejection fraction (HFpEF). There is currently no evidence-based treatment to improve prognosis in HFpEF, partly because the pathophysiology of this complex syndrome is incompletely understood. The prevailing hypothesis states that comorbidities such as hypertension, diabetes and obesity impair the normal function of the innermost layer of our blood vessels, the endothelium. This has adverse effects throughout the whole body, including the heart.

In this doctoral thesis, we evaluated endothelial function in HFpEF patients, and its relation with associated comorbidities. We hypothesized that exercise could improve endothelial dysfunction, and we postulated that the effect of exercise training could be predicted by biomarkers analysed prior to training.

We demonstrate that HFpEF patients have reduced microvascular endothelial function compared to healthy volunteers. Neither a single maximal exercise bout, nor a 12-week exercise training program had an effect on vascular function in HFpEF patients. Cells involved in the repair of the endothelium circulated in lower amounts in the blood of HFpEF patients. Acute exercise increased the amount of these cells, but exercise training did not.

We validated a novel animal model of HFpEF. In this model, we found that molecular changes associated with ageing contribute to the development of endothelial inflammation and HFpEF. Also, a machine learning analysis showed that large heterogeneity exists among HFpEF patients with regard to their comorbidity profile, which is reflected in differences in endothelial function. Iron deficiency was highly prevalent in HFpEF patients, especially in women. Iron deficiency even was the most frequent comorbidity in a subgroup of relatively young, female HFpEF patients with few cardiovascular risk factors and preserved endothelial function.

We found that not all HFpEF patients show a favourable response to training. We identified two biomarkers characterizing these ‘low responders’ These markers can aid in identifying ‘low responders’ prior to training, providing the possibility for individualised management. Moreover, they may help unravel HFpEF pathophysiology and open avenues for new interventions.