Rozprawy doktorskie na temat „Tumours”

Neuroendocrine tumours(NETs) are heterogeneous with respect to biological behavior. Consequently, prognosis is variable and biomarkers predicting survival or tumour progression are required to inform clinical management. The best available biomarker, histological grade, is assigned using Ki-67 or mitotic count. Agreement between these two indices is implied but analysis of 131 pancreatic and 136 midgut NETs suggested discordances of 44% and 38% respectively. Ki-67 was the superior prognostic marker, making the additional value of mitotic count questionable. Detection of Circulating Tumour Cells(CTCs) using the Cellsearch™ platform requires expression of epithelial cell adhesion molecule(EpCAM). I demonstrated EpCAM expression by immunohistochemistry and detected CTCs in patients with metastatic NETs. In 175 patients, ≥1 CTC was detected in 51%(midgut) and 36%(pancreatic). ≥1 CTC was an independent poor prognostic factor, offering better prognostic value than grade or chromogranin A(CgA). Changes in CTCs 3-5 weeks after commencing therapy were predictive of response and survival, suggesting CTCs could provide an early assessment. Using chip-based capillary-electrophoresis, higher concentrations of circulating free DNA(cfDNA) were found in 88 patients with NETs compared to healthy controls with a correlation between cfDNA quantity and CTCs. Since cfDNA was detected in 25% of cases, more sensitive methods of detection are required before studies are conducted to validate cfDNA as a biomarker and to analyse mutations. The hypervascular nature of NETs suggested that circulating endothelial cells(CECs) might be informative. Using immunomagnetic separation and CD105 phenotyping, CECs were demonstrated in 55 patients. Although not significantly elevated, there was a wider range of CECs in NETs compared to controls. Further studies investigating changes with anti-angiogenic therapy could prove valuable. My research suggests circulating biomarkers, specifically CTCs, provide additional and better prognostic information than grade. Furthermore, detection of CTCs and cfDNA in NETs may allow future studies into molecular analysis, which may enhance understanding of NET pathogenesis.

The aim of this thesis was to identify genes which are important in the initiation and/or progression of sporadic ovarian cancer. A series of ovarian teratomas and carcinomas was collected and three candidate tumour suppressor genes were analysed. The c-mos gene is an ovarian teratoma susceptibility gene in mice; its absence causes the growth of these tumours. Twenty human ovarian teratomas were collected and the coding region of the c-mos gene was analysed for somatic and germline mutations. No disease-causing alterations were found. Germline mutations of the BRCA2 gene predispose individuals to breast and ovarian cancer. To determine whether mutations in BRCA2 are important in sporadic ovarian cancer, loss of heterozygosity studies and mutation analysis were carried out on BRCA2 in a series of sporadic epithelial ovarian tumours. Loss of heterozygosity was identified in 46% of tumours. Four truncating mutations were identified in 50 tumours, two of which were germline and two somatic. All four mutations were accompanied by loss of the second allele. These results suggest that BRCA2 behaves as a tumour suppressor gene but that somatic mutations are not a common even in sporadic ovarian cancer. The insulin-like growth factor II receptor gene (IGF2R) on chromosome 6q is in a region which is frequently lost in ovarian tumours. A loss of heterozygosity analysis of the IGF2R locus in 38 informative epithelial ovarian tumours demonstrated 55% with loss of one allele. To perform mutation analysis of IGF2R, the technique of fluorescent chemical cleavage of mismatch was established in the laboratory and used to analyse IGF2R cDNA from 18 tumours. No disease-causing alterations were identified. Antibodies were used to examine the expression of the IGF2R protein through immunohistochemical studies of 53 ovarian tumour tissue sections. Seven tumours were identified in which epithelial tumour cells stained negatively for IGF2R. No correlation could be found between immunohistochemical results and LOH and mutation analysis results, suggesting that IGF2R is probably down-regulated at the level of transcription or translation in those samples which showed negative staining.

Androgen deprivation therapy (ADT) such as bicalutamide (BCA) has become the mainstay of treatment for locally-advanced prostate cancer (PCa). Despite initial remissions, ADT resistance almost inevitably occurs with progression to metastatic castrate-resistant PCa. Hypoxia is a common hallmark of many solid tumours and is associated with treatment failure and malignant progression; androgen withdrawal has been shown to induce profound hypoxia in androgen-sensitive tissue. This prompted an investigation into the effect of ADT on tumour oxygenation and malignant progression in PCa. Androgen-dependent luciferase-expressing PCa xenografts (LNCaP-luc) were implanted in SCID mice. When tumours reached -150mm3 mice were treated daily with SCA (2 or 6mg/kg). A reduction in tumour oxygen was observed within 24hrs (Oxylite™ oxygenelectrode); this continued to a nadir of 0.1 % oxygen at day 3 or 7 respectively. Tumours remained profoundly hypoxic until day 14-21 when oxygen levels began to rise, concomitant to time-dependent remodelling of the tumour vasculature (dorsal skin fold model). By day 28, BCA-treated xenografts were more malignant and showed greater metastatic spread to the lungs. Gene expression changes during BCA treatment of LNCaP xenografts were investigated using qPCR arrays; significant differences were found in the expression many genes involved in angiogenesis, invasion and metastasis, apoptosis resistance and the PI3K1AktimTOR signalling pathway. Informed treatment regimens combining SCA with a unidirectional hypoxia activated prod rug (AQ4N and its novel analogue OCT1002; 50mg/kg, day 7) or an Akt inhibitor (30mg/kg t.i.w) resulted in a reduction in tumour growth and metastatic spread to the lungs. When the anti-angiogenic VEGF-inhibitor (B20.4.1.1; 5mg/kg, day 14) was combined with bicalutamide, this blocked the revascularisation associated with BCA alone. This study shows that BCA-induced hypoxia induces critical changes in the tumour microenvironment which cause modified gene expression and drives malignant progression. Targeted therapeutic regimens, informed by this knowledge, may improve treatment outcomes of androgen-dependent PCa.

The historical precedence for employing bacteria to target cancer stretches long back some 300 years. Despite of the advancements in chemotherapy, problems such as metastasis and tumour resistance to chemotherapy and radiotherapy have limited the scope of therapeutic effects of existing clinical treatments. The microenvironment of solid tumours provides an ideal environment for growth of facultative and strictly anaerobic bacteria. These bacteria can colonize such environments leading to tumour regression. Such investigations have given the concept of bacteriolytic therapy, in which live bacteria could be employed for the targeted delivery into tumours leading to its suppression. With this concept in mind it was decided to explore the above idea by using probiotic bacterium (Lactobacillus casei), which is considered to be non-pathogenic and also provides health benefits to the host. In order to minimize dispersion of the bacteria throughout the host and to facilitate its delivery into tumours, some kind of containment was desirable. To test this kind of approach it was decided to exploit immobilisation technology to microencapsulate live bacteria for later injection into tumour sites. To date no such approach has been employed to develop such a microencapsulation system that could be utilized as a delivery vehicle for live bacteria to deliver it through a hypodermic needle directly into tumours. In order to pursue the above objective, the microencapsulation method was also characterised with respect to stability and viability of the L.casei. Microencapsulated preparations of Lactobacillus casei NCDO 161 were developed and demonstrated that the culture supernatant of microencapsulated preparation inhibited growth of tumour cells (in vitro). Further investigation of a variety of bacterial preparations on tumour growth (in vivo) following intra-tumoural injection demonstrated that the live microencapsulated preparation had severe inhibitory effect on tumour growth when compared with non-encapsulated live and encapsulated heat killed preparations. Histological studies were performed to demonstrate the presence of live bacteria in tumours at early stages and to study the effects of the bacteria on tumour architecture during the treatment.

Myeloid cells are a major component of most forms of malignant tumour. The plasticity of such cells means that they can alter their phenotype in response to changes in the tumour microenvironment, including the pronounced ones that take place after chemotherapy. Tumour cell death, as well as the various cytokines and chemokines released by cells in tumours after such treatments, are now known to alter both the recruitment and function of myeloid cells. Recent studies have shown that monocytes recruited into tumours during/following chemotherapy can promote tumour chemoresistance and metastasis. The data presented in this thesis suggest that, following chemotherapy, tumour-associated macrophages (TAMs) may increase their expression of the neutrophil-recruiting chemokines, CXCL1, CXCL2 and CXCL5, and possibly stimulate the intratumoural accumulation of neutrophils. Responding to these CXC chemokines (and possibly other secreted factors in chemotherapy-treated tumours), neutrophils may then upregulate their expression of such inflammatory cytokines as TNFα, CCL2 and CCL3. Furthermore, data from in vitro invasion assays suggest that neutrophil-derived TNFα is capable of inducing tumour cell invasiveness. Notably, the number of tumour-infiltrating neutrophils was significantly increased after chemotherapy in 3 of the 4 mouse tumour models used. Furthermore, in all 4 tumour models there were significantly more TNFα+ neutrophils after chemotherapy compared to control tumours. Combined treatment with chemotherapy and SB 265610, a CXCR2 antagonist that inhibits CXCL1, CXCL2 and CXCL5 signalling, successfully reduced both the number of these tumour-infiltrating, TNFα+ neutrophils, and the overall level of immunodetectable TNFα in tumours after chemotherapy. Although TNFα is known to be capable of supporting tumour growth, angiogenesis and metastasis, it remains to be seen whether such an increase in neutrophil TNFα expression contributes significantly to the post-chemotherapy regrowth of either primary or metastatic tumours. This could be achieved by giving chemotherapy to mice in which TNFα has been selectively knocked out/down in neutrophils. Data presented here suggest that combining chemotherapy with CXCR2 inhibitors like SB 265610 to inhibit the above neutrophil-mediated events could improve patient tumour responsiveness to chemotherapy and reduce tumour relapse.

Different steps forward have been made in the recent years to identify the molecular determinants in carcinogenesis and the evidence of a multistep process where cancer cells accumulate multiple and consecutive genetic alterations has been formulated. Recently, tumour progression has been recognized as the product of a complex crosstalk between tumour cells and their surrounding and supporting tissue, named tumour stroma. This stroma is known to influence the growth of cancer and it is composed by several types of cells, including endothelial cells of blood and lymphatic circulation, stromal fibroblasts and a variety of bone marrow-derived cells, such as macrophages, mast cells, neutrophils, lymphocytes and mesenchymal stem cells. The supportive microenvironment is generate and modulated by cancer cells through the production and activation of stroma growth factors including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta). Concomitant with altered growth-factor expressions, induced by their autocrine and paracrine effect on the tumour and stromal cells, cancer cells are able to produce proteolytic enzymes, such as Matrix metalloproteinases (MMPs), which operate the remodelling of extracellular matrix (ECM) and basement membrane, thus activating cell-surface and ECM-bound growth factors. All these processes are described to contribute to the extensive crosstalk between the microenvironment and the cancer cells. Therefore, the microenvironment is implicated in the regulation of cell growth, determining angiogenesis, tumour invasion and metastasis, and impacting the outcome. Even if stromal cells are not malignant, their role in supporting cancer growth is vital to the survival of the tumour. For this purpose, cells of microenvironment have become an attractive target for therapeutic agents. The present project has been divided in different tasks to identify the molecular mechanisms implicated in cell migration, angiogenesis and tumour growth led by stroma cells and their crosstalk with cancer cells in different neoplasia in dog. Canine mammary tumour, cutaneous mast cell tumour, lymphoid leukaemia and lymphoma were selected for the study and gene expression profiling and proteomic analysis of different growth factors (VEGF-TGF-beta-PDGF) and MMPs were analyzed in association with their possible prognostic and predictive role and crosstalk. Several important results have obtained highlighting the background of the tumour progression and the role of microenvironment in veterinary oncology. Selected results are shown below: - MMP-2, MT1-MMP, MMP-9 were significantly involved in canine mammary tumour and a significant role of the stromal compartment was described; - MMP-9 and VEGF-A were associated with the histological tumour grade in cutaneous mast cell tumour; - MMP-9, MT1-MMP, TIMP-1 and VEGF were correlated in T-cell lymphoma and in dogs with higher stage; - A potential role of MT1-MMP and TIMP-2 in the pathogenesis of canine acute lymphoblastic leukaemia has been discovered; - In chronic lymphocytic leukaemia, residual normal leukocytes have shown a significative influence in the expression of MMP-9, MT1-MMP, VEGF and TIMPs; - Lymphoma and leukaemia in vitro model exhibited a significative discrepancy that enhanced the importance of microenvironment in vivo; - PDGF-B mRNA expression was identified in canine T-cell lymphoma and cutaneous lymphomas. A functional autocrine and/or paracrine loop of growth stimulation was proposed due to the co-expression of PDGFs and PDGFRs at different time point during disease. Therefore, the obtained results may significantly improve the understanding of cancerogenesis of the most frequent tumours in dogs. The summarized data here show a primary role for the microenvironment during carcinogenesis. Development of novel cancer therapies that target the process of metastasis formation, tumour growth and differentiation, by interfering with the ability of cancer cells to transmigrate into blood and lymph vessels and to invade the connective tissue, is widely expected in veterinary oncology. Further data are necessary to indicate that the use of chemopreventive agents to control the function and behaviour of cells in the microenvironment might be an important approach to the overall control of cancer.
Negli ultimi anni nell’ambito dell’oncologia, diversi studi hanno identificato diverse molecole target implicate nella cancerogenesi e sono stati evidenziati numerosi processi attraverso cui le cellule tumorali sono in grado di accumulare alterazioni genetiche. Recentemente, la progressione del tumore è stata riconosciuta come il prodotto di un complesso crosstalk tra le cellule tumorali e il tessuto circostante, chiamato stroma tumorale. Questo stroma è noto per influenzare la crescita del tumore ed è composto da diverse tipologie cellulari, che comprendono cellule endoteliali della circolazione sanguigna e linfatica, fibroblasti stromali ed una varietà di cellule derivate dal midollo osseo, come macrofagi, mastociti, neutrofili, linfociti e cellule staminali mesenchimali. Ulteriormente, il microambiente di supporto è generato e modulato da cellule tumorali attraverso la produzione e attivazione di fattori di crescita prodotti dallo stroma stesso, come Vascular Endothelial Growth Factor (VEGF), Platelet-Derived Growth Factor (PDGF) e Transforming Growth Factor-beta (TGF). Concomitante all’alterata espressione di questi fattori e per il loro effetto autocrino e paracrino sulle cellule tumorali e su quelle stromali, le cellule neoplastiche iniziano a produrre enzimi proteolitici, come metalloproteasi di matrice (Matrix metalloproteinases - MMPs). Le MMPs operano il rimodellamento della matrice extra cellulare e della membrana basale, attivando così fattori di crescita legati alla superficie cellulare e alla matrice stessa. Tutti questi processi contribuiscono all’esteso crosstalk tra il microambiente e le cellule tumorali. Il microambiente quindi è implicato nella regolazione della crescita cellulare, determinando neoangiogenesi, invasione, metastasi tumorali e influenzando il risultato della terapia. Anche se le cellule stromali non sono considerabili fenotipicamente maligne, il loro ruolo nel sostenere la crescita della neoplasia è essenziale per la sopravvivenza del tumore. Con questo presupposto, le cellule del microambiente sono diventate un bersaglio attrattivo per diversi agenti terapeutici. Il progetto di ricerca è stato suddiviso in diverse fasi per identificare i meccanismi molecolari implicati nella migrazione cellulare, nell'angiogenesi e nella crescita neoplastica, da parte di cellule stromali e dal loro crosstalk con le cellule tumorali, in diverse neoplasie del cane. Per lo studio sono state selezionate le tipologie tumorali più frequenti nel cane: tumore mammario, mastocitoma cutaneo, leucemie linfoidi e linfoma, analizzando i profili di espressione genica e proteica di diversi fattori di crescita (VEGF-TGF-beta-PDGF) e delle MMPs, in associazione al loro crosstalk e ad un loro eventuale ruolo prognostico. Sono stati ottenuti importanti risultati evidenziando lo scenario della progressione tumorale e il ruolo del microambiente in oncologia veterinaria. E’ stato dimostrato che: - MMP-2, MT1-MMP, MMP-9 sono significativamente coinvolte nel tumore mammario ed è stato descritto un loro ruolo rilevante del compartimento stromale; - MMP-9 e VEGF-A sono associati al grado istologico nei mastocitomi cutanei; - MMP-9, MT1-MMP, TIMP-1 e VEGF sono correlate nel linfoma T e nei cani con linfoma con stadio clinico più alto; - MT1-MMP e TIMP-2 hanno un ruolo nella patogenesi nelle leucemie linfoblastiche acute; - Nella leucemia linfocitica cronica, i leucociti residui normali mostrano un'influenza significativa nell'espressione di MMP-9, MT1-MMP, VEGF e dei TIMPs; - Il linfoma e la leucemia nel modello in vitro mostrano una considerevole discrepanza per alcune MMPs e VEGF che avvalora l'importanza del microambiente in vivo; - L’espressione genica del PDGF-B è significativa nei linfomi T e nei linfomi cutanei. E’ stato inoltre proposto un loop funzionale autocrino e/o paracrino di stimolazione della crescita della neoplasia, dovuto alla co-espressione dei PDGFs e dei recettori in diversi tempi durante la malattia. I risultati ottenuti potrebbero migliorare significativamente la comprensione della cancerogenesi nei tumori più frequenti nel cane. I dati qui sintetizzati mostrano un ruolo primario del microambiente durante la carcinogenesi. Lo sviluppo di nuove terapie antitumorali che colpiscano il processo di formazione di metastasi, la crescita e la differenziazione della neoplasia, interferendo con la capacità delle cellule tumorali di trasmigrare nel sangue e nei vasi linfatici e di invadere il tessuto connettivo, sarà ampiamente perseguito in oncologia veterinaria nel futuro prossimo. Sono però necessari ulteriori studi per indicare se l'uso di agenti chemio-preventivi per controllare la funzione ed il comportamento delle cellule nel microambiente possa essere un importante approccio al controllo complessivo del cancro.

Angiogenesis, the growth of new blood vessels, is vital to tumour growth. Prevailing dogma has been that tumours cannot grow without angiogenesis. Based on this premise, anti-angiogenic drugs are used clinically. However, the principle of angiogenesis as an absolute requirement for tumour growth has been challenged with reports that many tumours are entirely or partially non-angiogenic. This study describes and quantifies characteristics of non-angiogenic non-small cell lung tumours, demonstrates non-angiogenic growth in small-cell/neuroendocrine lung tumours and investigates the underlying pathogenetic processes by comparison with angiogenic lung tumours. Hypoxia is an important stimulus for angiogenesis. Differences in response to hypoxia may determine whether a tumour produces new vessels. In order to test this, levels of. necrosis, often considered a surrogate marker of hypoxic stress, were quantified but no difference in quantity of necrosis was found Moreover, immunohistochemical investigation of hypoxia and angiogenesis factors provided no unambiguous explanation for the differences in angiogenesis. Significant differences were seen, however, in fibrosis and inflammation, which were both greater in angiogenic tumours. Differences were greater for lymphocytes rather than cells of the ‘innate’ immune system. This provided an alternative hypothesis: angiogenesis occurs during wound healing and in the growth of granulation tissue, so it is possible that tumour angiogenesis is a response to factors produced by immune cells rather than the tumour itself. A tumour’s angiogenic status may, therefore, be determined by the response it provokes from the immune system. Further work to test this theory would compare levels of immunogenic factors such as Tumour Necrosis Factor and tumour cell surface antigens such as the HLA class I molecules. The study concludes with an investigation into the molecular basis of non-angiogenic growth using the technique of comparative genomic hybridisation (CGH) which allows amplifications and deletions of areas of DNA to be calculated. High-resolution array CGH was evaluated against conventional CGH, and the results compared with previous RNA studies from our laboratory. These revealed a set of genes with consistent changes in both RNA and DNA, several of which form part of known angiogenic and inflammatory pathways.

Pharyngoesophageal (PE) tumours are tumours involving simultaneously the hypopharynx and the cervical oesophagus. The challenge in its surgical management lies in its deep-seated location behind the manubrium bone in the cervicothoracic region, in close proximity to great vessels in the lower neck and superior mediastinum. Classically curative surgery is in the form of total pharyngo-laryngo-oesophagectomy (PLO) and gastric pull-up (GPU) via a three-phase one-stage operation. However PLO and GPU is a major undertaking associated with high operative morbidity and reported in-hospital mortality rates of up to 10%. With a comprehensive preoperative work-up we demonstrated accurate tumour diagnosis and staging, with a 100% negative predictive rate. Together with vigilant postoperative surveillance and compliant follow-up, incidence of synchronous and metachronous tumours were low at 11.9% and 1.7% respectively. Manubrial resection (MR) provided access to PE tumours in the cervicothoracic region enabling resection under direct vision with adequate resection margins - pharyngo-laryngo-cervico-oesophagectomy (PLCO). The trachea was resected and re-sited as a mediastinal tracheostoma in case of posterior tracheal wall invasion. Paratracheal and paraoesophageal lymph node dissection was performed in case of nodal metastasis. MR provided ample space for reconstruction of the resultant defect. Furthermore, it enabled access to vessels in the superior mediastinum to support microvascular tissue transfer. Intra-thoracic volume changes on maximal inspiration and expiration measured using computed tomography scan did not show significant difference pre- and post- MR. With attention to operative details, MR proved to be safe with minimal functional disturbance. Free jejunal (FJ) flap was the preferred reconstructive modality as it offered the lowest pharyngocutaneous fistula and anastomotic stricture rates, and donor site morbidities. All patients resumed unrestricted oral diet postoperation. Videofluoroscopic swallowing studies (VFSS) and high resolution manometry (HRM) demonstrated significantly prolonged transit times for all bolus consistencies compared with normal subjects due to asynchronous contractions between the FJ and the oesophageal remnant, presence of retrograde propulsion and residue accumulation within the FJ. However, patients reported significant improvement in swallowing outcomes and associated quality of life (QOL) compared with preoperation (65.3% vs. 42.7%, p=0.02). Majority of patients were able to speak conveniently with a modality of their choice. MR, PLCO and FJ flap showed significantly lower operative morbidities (58.3% vs. 85.7%, p=0.05), shorter hospital stay (42.5 vs. 50.7 days, p=0.37), and lower in-hospital mortality (8.3% vs. 9.5%, p=0.52) compared with PLO and GPU. None required intensive care unit postoperation. In resecting less, oncological outcomes and survival were not inferior to PLO and GPU. FJ patients were able to resume oral diet sooner than GPU with a higher functional oral intake scale (FOIS) at 6 months (100.0% vs. 92.8%). Shorter transit times for all bolus consistencies were demonstrated in VFSS and HRM of GPU patients due to the lack of contractions within the gastric tube. Swallowing, speech and associated QOL outcomes were comparable between the 2 groups. In conclusion, MR, PLCO and FJ flap should be adopted in the surgical management of patients with isolated PE tumours.
published_or_final_version
Surgery
Master
Master of Surgery

Introduction: Most cancers show heterogeneity of response to chemotherapy. This may be due in part to the differential expression of drug resistance proteins and the molecular targets of the drugs concerned. Methods: An ex vivo ATP-based Tumour Chemosensitivity Assay (ATP-TCA), immunohistochemistry and quantitative RT-PCR have been used to assess the chemosensitivity and resistance of a variety of solid tumours and cell lines. Results: (a) Melanoma cell lines showed higher chemosensitivity than tumour-derived cells, partially reversible by lowering the serum concentration, and hence the proliferation rate of the cells. (b) Studies of retinoblastoma samples confirmed that this malignancy is susceptible to cytotoxic drugs of all types, though multidrug resistance may occur in some cases. (c) The ATP-TCA was used to study the activity of high-dose doxorubicin in combination with other cytotoxic agents in ovarian adenocarcinoma samples. The combination of liposomal doxorubicin + vinorelbine was selected for further development. (d) A number of experimental drugs with varying sensitivity to resistance mechanisms were also assessed. One drug, XR5944, has entered phase I/II clinical trials during the course of this project, and the data have provided clinical indications. (e) An inhibitor of multi-drug resistance, tariquidar, has been tested in combination with doxorubicin, vinorelbine or paclitaxel, and has been shown to reverse this resistance. (f) Molecular studies have determined the expression of topoisomerases and drug transporters in tumour cells before and after exposure to chemotherapeutic agents. P-gp expression has been found to be a determinant of sensitivity to a certain number of drugs. Conclusion: The results suggest that drug resistance contributes to heterogeneity of chemosensitivity in many solid tumour types, as well as other mechanisms. Reversal of such resistance may benefit a subset of patients undergoing chemotherapy.

BACKGROUND: Neuroendocrine tumours (NETs) are fairly rare neoplasms that present many clinical challenges. The complexity, heterogeneity, and rarity of NETs have contributed to their limited therapeutic options. To improve the outcome from NETs, a better understanding of their biology is needed. Several receptor targets exist, against whom agents such as antibodies or tyrosine kinase inhibitors are being developed and tested in clinical trials. Several of these agents have been used in combination with chemotherapy or radiation with promising results. These receptors include the tyrosine kinases EGFR and C-KIT, and the somatostatin receptor SSTR2. However, their role in neuroendocrine tumour growth remains unclear. METHODS: We investigated the anti-proliferative effect of EGFR inhibitor gefitinib, as single agent or in combination with widely used chemotherapeutic agents. The chemotherapeutic agents were also examined for their effect on EGFR activity. Cisplatin and radiation were studied for their effect in EGFR activity and localisation in combination with gefitinib and the anti-EGFR antibody cetuximab by immunoblotting and immunofluorescence, while the comet assay for quantitation of DNA damage was used to examine modulation of DNA repair by radiotherapy. The cytotoxic efficacy of agents against SSTR2 was also examined, while the expression of C-KIT was analysed in 95 NET patients by immunohistochemistry. RESULTS: Gefitinib demonstrated anti-proliferative effect associated with induction of apoptosis but no cell cycle arrest. Cisplatin induced a transient activation of EGFR and nuclear translocation, which was mediated through the Ras/MAPK and PI-3K/Akt signalling cascades. Cisplatin and radiation-induced EGFR translocation was blocked by gefitinib and cetuximab, and this was associated with a delay in the repair of radiation-induced DNA strand breaks. Nuclear translocation was mediated by nuclear pore complex importins and exportins, and utilized the EGFR nuclear localisation sequence. C-KIT was identified in a number of NET patients. The drugs against SSTR2 had no effect on the growth of cells. CONCLUSIONS: Targeting EGFR in combination with radiation may provide therapeutic potential in neuroendocrine tumours patients.

This thesis considers aspects of modelling solid tumours. We begin by considering the common assumption that nutrient or drug concentrations in avascular tumour spheroids are radially symmetric. We derive a simple Poisson equation for biomolecular diffusion into an avascular tumour, but with highly oscillatory boundary conditions due to the surrounding capillary network. We find that the assumption of radial symmetry is legitimate for biomolecules that are taken up in sufficient quantities by proliferating cancer cells; however radially symmetric profiles need not be observed otherwise. We then investigate how the gap between an avascular tumour and the neighbouring vasculature varies as the tumour grows. This is explored by (i) using scaling arguments based on ordinary differential equations, (ii) coupling the rate of oxygen flux from the vasculature to oxygen evolution within the tumour, and (iii) deriving a system of six coupled non-linear partial differential equations modelling the tumour evolution. It is found that as the tumour grows any initial gap between the tumour and neighbouring vasculature closes since there is no mechanism which would sufficiently up-regulate non-cancerous cell proliferation. This is in contrast to the intra-cornea implantation observations, upon which several mathematical models are based. Finally, we study the growth and treatment of a vascular tumour subjected to chemotherapies, particularly when the therapies can exhibit an anti-angiogenic effect and resistance to the therapy is incorporated. A multi-compartment model is derived for the evolution of a tumour undergoing treatment and parameters are estimated, with extensions to incorporate numerous different therapy protocols in the literature. We find that anti-angiogens can be effective, though the appropriate scheduling is counter-intuative and contradicts many standard therapy rules. We conclude that chemotherapy protocol design is very sensitive to the mode of action of the drug and simple general strategies will, in many cases, not be the most effective.

Oesophageal tumours are very common worldwide. This thesis aims to delineate the clinicopathological features and molecular biology of oesophageal tumours in Hong Kong Chinese. Over a 30-year study period, oesophageal tumours were obtained in the pathology files of the Queen Mary Hospital, Hong Kong. The tumours were prevalent in males and had a modal peak occurrence in the 7th decade. Patients often presented at an advanced stages. At autopsy, the prevalence of incidental oesophageal cancers and early-stage cancers were low. Many histological subtypes of oesophageal cancers were noted and were different from the Western populations. The most common histological subtype was squamous cell carcinoma, often moderately-differentiated. Besides the classical squamous cell carcinomas, variants like mucoepidermoid carcinoma/adenosquamous carcinoma, basaloid squamous carcinoma and sarcomatoid carcinoma were noted. The prognosis of squamous cell carcinoma with a mucin-secreting component (mucoepidermoid carcinoma and adenosquamous carcinoma) was not significantly different from that of patients with pure squamous cell carcinoma or adenocarcinoma. The glandular component of this group of tumours histochemically differentiated in the direction of oesophageal glands. Basaloid squamous cell carcinoma had distinctive clinicopathological features and its long-term prognosis was no worse than squamous cell carcinoma. Glomerulonephritis could be a para-neoplastic manifestation of basaloid squamous carcinoma of oesophagus. Sarcomatoid carcinomas were also found, and rarely double sarcomatoid carcinomas could be noted in the same patient. The other carcinomas noted in the oesophagus were small cell carcinoma and adenocarcinoma. Oesophageal small cell carcinoma was an aggressive tumour. The high proliferative index correlates with aggressive behaviour and high sensitivity to chemotherapy and radiotherapy. Oesophageal adenocarcinoma was uncommon in Hong Kong. On the other hand, intestinal metaplasia, known to be associated with adenocarcinoma, was prevalent at the gastroesophageal junction in Chinese patients undergoing endoscopy. The non-epithelial tumours in the oesophagus comprised melanoma and mesenchymal tumours. Melanoma of the oesophagus was an aggressive tumour. All patients with the tumour had short survival. Mesenchymal tumours consisted of leiomyoma, undifferentiated stromal tumour and autonomic nerve tumour. Intramural metastasis and multiple tumours were frequently observed in oesophageal cancer. This implies that wide excision with wide margins should be considered for local control of the disease. Pre-operative chemotherapy was commonly employed for the treatment of oesophageal cancer. High-grade nuclear pleomorphism in oesophageal carcinomas was correlated to chemo-responsiveness of the tumour. Four cancer cell lines were established from patients with oesophageal squamous cell carcinomas. These newly established cell lines serve as a useful model for studying the molecular pathogenesis, and testing new therapeutic reagents for oesophageal squamous cell carcinoma. Proliferative activity, as defined by the MIB-1 labelling index, was related to tumour differentiation in oesophageal squamous cell carcinoma. The activity was high in poorly-differentiated squamous cell carcinoma, basaloid squamous carcinoma and small cell carcinoma of the oesophagus. MIB-1 labelling index was found to be valuable as an independent prognostic marker in addition to tumour stage and size. Image analysis could assist h~ counting of the proliferative activity. Human papilloma virus was detected in a small proportion of oesophageal squamous cell carcinomas. There was no correlation between the prevalence of HPV and p53 mutation in these tumours. Epstein Barr virus was not detected in squamous cell carcinomas and mesenchymal tumours. The pattern of expression of cytokeratins in oesophageal carcinomas is different from that in normal oesophageal epithelia and varies with differentiation. Cancer-related genes studied in oesophageal cancers were p53, p21, c-erbB-2, PTEN and telomerase activity. p53 mutations were common in oesophageal squamous cell carcinomas and small cell carcinomas. The distribution of p53 mutations in oesophageal cancers suggests that the gene has complex exogenous and endogenous interactions. p53 mutations also appear to play a role in predicting the survival of patients with stage III oesophageal squamous cell carcinomas. The pattern of p21 and p53 expression predicts an aggressive clinical course of oesophageal squamous cell carcinomas. c-erbB-2 (Her-2) oncoprotein was expressed in a portion of oesophageal squamous cell carcinomas and precursor lesions. This suggests that c-erbB-2 activation plays a certain role, mostly probably during the early stages, in carcinogenesis. PTEN/MMAC1 mutations were not detected in oesophageal squamous cell carcinoma. Telomerase activation was common in small cell carcinoma and basaloid squamous cell carcinoma of the oesophagus. The level of telomerase activity had a prognostic role in oesophageal cancer, suggesting a possible therapeutic role of anti-telomerase treatment for this aggressive tumour. Multiple genetic mutations in oesophageal squamous cell carcinomas could be mapped by gene arrays and comparative genomic hybridization. These different newly discovered genetic alterations were analysed both in laboratory and in relationship with the clinical prognosis. Multiple mutations could be detected in dysplasia as well as carcinoma. The techniques identify the roles of some new cancer-related genes like Fra-1, Neogenin, Id-i, CDC2SB and MET in oesophageal squamous cell carcinomas. Chromosomal aberrations were common in oesophageal squamous cell carcinoma. Gain in l2p was found to be indicative of aggressive behaviour and poor prognosis. In summary, identification of the different histological types of oesophageal tumours and their characteristic molecular profiles is essential for both in-depth research and clinical management.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medicine
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[Truncated abstract] The design of effective immunotherapies, using tumour antigens to stimulate a functional effector cytotoxic T cell (CTL) response in a tumour bearing host, requires an understanding of the 'real time' in vivo relationship between the host immune system and antigens expressed by the developing tumour. However, effector function of endogenous anti-tumour CTLs generated during tumour progression has largely been assessed by indirect ex vivo assays and often focused on a single antigen. Therefore, studies in this thesis evaluated the endogenous in vivo CTL response to multiple tumour antigenic epitopes in murine tumour models using Lewis lung carcinoma cells transfected with ovalbumin (an antigen that contains several intra-molecular MHC class I epitopes with a defined hierarchy) or a polyepitope (that contains a string of immunodominant MHC class I epitopes). Potent effector CTLs were generated to multiple dominant tumour antigenic epioptes early in tumour progression. However, in general, these CTL effectors only transiently retarded tumour growth, and at the later time points of tumour growth they were no longer generated in tumour draining lymph nodes. This coincided with diminished tumour antigen presentation in the same nodes which was found to be due to antigen loss. In both models antigen loss was the result of two processes; immuno-editing of the tumour by the host immune response and genetic instability resulting in antigen loss variants that could evade immune surveillance. A third model was generated that maintained low level tumour antigen expression throughout tumour progression. ... The impact of pre-existing endogenous dominant-epitope specific CTLs on tumour expressing the same epitope was also assessed, and resulted in a reduced tumour incidence and a CTL response restricted to a single antigen of the same MHC allele. Finally, the effects of two different immunotherapy regimens were examined. Intratumoural IL-2 treatment enhanced pre-existing CTL responses to the dominant epitopes leading to tumour regression. In addition, use of a multiple peptide vaccination regimen that avoided T cells competing for peptide-MHC complexes on APC was far more likely to be effective than one that did not. These results demonstrate that immunotherapies targeting tumours that express several dominant neo antigenic epitopes can be effective. The caveat for this approach is that it will only be effective in tumours that have generated an endogenous CTL response and must be used before antigen loss variants emerge.

The investigation of an abdominal mass in a child is a common problem in the radiology department of the Red Cross Children's Hospital. The majority of these masses involve the urinary tract. The commonest neoplasm is a Wilms' tumour of the kidney. Against a pathological and clinical background, the investigation of Wilms' tumour by diagnostic imaging is presented. The imaging modalities currently utilised are the intravenous urogram (IVU), ultrasound (US), computed tomography (CT) and magnetic resonance (MR). Using the material available in the last decade, the principles, techniques and imaging characteristics of these modalities are investigated and compared. These results are reflected against those reported in the medical literature. This literature is not yet extensive as the current technology has only been available for the last six to seven years. The IVU has in the past been the main imaging modality and we still use it extensively. Its strengths and weaknesses are discussed. In the last five years US has taken its place as the primary method of diagnostic imaging. We have found that with our increasing experience that this is justified. The use of US and IVU in a practiced hand is a powerful diagnostic combination. CT as a primary investigation is not readily available at our institution. We have used it for comparative purposes in about 20% of our recent cases. CT has not added greatly to our initial diagnostic impression. However, it has been most useful for follow up of metastasis and for assessing the normality of the lungs before ceasing chemotherapy. Our experience with MRI is limited and confined to unusual presentations in the last year. Other modalities such as arteriography and nuclear medicine have special indications which are to be discussed. The remaining tumours of the upper urinary tracts are all rare, but are reported and the literature researched. In the lower urinary tract the main pelvic lesion is a rhabdomyosarcoma. The comparative advantages of the IVU, US, CT and MRI are also noted. In the pelvis, US has also become the primary imaging modality, and is replacing contrast medium cystography. However, examples of the latter are included as it still has a place, particularly in the less sophisticated institutes. CT and MRI, when available, have imaging advantages in the pelvis and are becoming the methods of choice for follow up. The main objective of this document has been to investigate the available imaging techniques, but, against this overall theme, the clinical care of the child is most important. With this in mind the treatment protocols that are used at our hospital are noted in the appendices to the thesis.

Malignant tumours of the small intestine are rare. Age-standardised incidence in Europe is between 0.5-1.5 per 100 000. As the small intestine represents more than 90 % of the gastrointestinal mucosal surface, it is surprising that it gives rise to less than 2 % of gastrointestinal malignancies. The dominating histological subtypes are carcinoids and adenocarcinomas.

We used three population-based registries in Sweden to study survival, second malignant tumours, causes of death, and Crohn’s disease as a risk factor for small intestinal adenocarcinoma and carcinoid.

We evaluated tumour site, sex, age, and year of diagnosis as prognostic factors. For adenocarcinomas there was no difference in survival between duodenal and jejunal/ileal tumours. Women with jejunal/ileal adenocarcinomas showed higher probabilities of survival than men, while no such relation was found for duodenal tumours. Old age correlated with poor survival for duodenal tumours, and prognosis has improved in later years. For carcinoids, duodenal tumours had a more favourable prognosis than jejunal/ileal tumours. There was no difference in survival between sexes. Old age correlated with poor survival, and survival has improved in recent years.

Female patients with adenocarcinoma had increased risk of acquiring cancer in the genital organs and breasts, and both sexes had increased risks of second tumours in the gastrointestinal tract and skin. Men with carcinoid tumours had increased risk of prostate cancer. Both sexes had increased risk of malignant melanoma and malignancies of endocrine organs.

Patients with adenocarcinoma had increased risk of dying from malignant diseases other than the primary small intestinal cancer and from gastrointestinal disease. The cohort with carcinoid had higher than expected risk of dying from malignant disease, gastrointestinal disease, and cardiovascular disease.

Patients with Crohn’s disease had increased risk of small intestinal adenocarcinoma and carcinoid, and the risk has increased for patients diagnosed in recent years.

Cancers trigger angiogenesis in neighbouring tissue and recruit new blood vessels to grow and metastasise. Therefore, therapeutic and diagnostic strategies have been moving towards targeting tumour neoangiogenesis as a key factor in the progress and assessment of the disease. High-frequency ultrasound (HF-US) offers a low-cost and non invasive approach for studying the efficacy of antiangiogenic therapies in the pre-clinical setting. However, there is little information in the literature about the performance of HF-US in vivo in regards of evaluating tumour vascularity and its correlation with microvessel density (MVD). The aim of this thesis was to optimise a HF-US system to investigate its ability to visualise and to quantify tumour parameters, particularly tumour blood flow, in colorectal cancer (CRC) mouse model. The performance of a HF~US (~ 25 MHz) system was evaluated in vitro using test objects in order to assess the ability of the system to study the normal mouse colon and to optimise its scanning parameters for imaging tumour blood flow. This was followed by two in vivo approaches. Firstly, the feasibility of using HF-US in depicting the mouse colon and measuring its thickness accurately and reproducibly as a potential model for CRC was established. Secondly, the performance of different HF-US blood flow imaging techniques was compared in a CRC xenograft model and correlations between the different ultrasonic vascular parameters and MVD determined.

96 Oligodendroglial gliomas were first studied by a genome-wide 1Mb array-CGH followed by analysis using chromosome 7 and 10 tile-path and regional tile-path arrays to more exactly define the extent of copy number aberrations. This strategy allowed the identification of several novel amplifications and homozygous deletions (HD). A comparative survival analysis for the groups of patients defined by unsupervised hierarchical clustering of the 1Mb array data as well as histopathological diagnosis, 1p/19q status and Tp53 mutation were performed. At least four distinct subgroups were identified: two groups with 1p/19q total loss and two groups without, exhibiting distinct genetic profiles and survival. Some individual genomic region abnormalities also correlated with survival. A number of these are novel. While total loss of 19q was the most predominant pattern in oligodendroglial tumours, astrocytic tumours had complex patterns of partial deletions, grains, trisomy or monosomy of chromosome 19. This suggests that there may be several astrocytoma relevant regions on chromosome 19. A novel candidate tumour suppressor gene (TSG) region at 19q13.41 was delineated in the astrocytic tumours. A rare novel somatic homozygous deletion in the kallikrein gene cluster region was identified in one oligodendroglioma. To explore the potential breakpoints of the t(1;19) translocation on 19q, gliomas with 1p/19q total losses were screened. The findings indicate the existence of several recurrent breakpoints in the gene-depleted pericentrometric region of 19q. Other novel findings include 3 tumours with 1p/19q deletions that had HDS of PTPRD on 9p23-9p24.1. These deletions affected only the 5’UTR of the long isoforms of PTPRD. No somatic mutations affecting the coding sequence, aberrant methylation of the gene or significantly altered mRNA expression levels were observed.

Assessment of disease status in fish is used as an indicator of the biological effects of contaminants in the marine environment. At some UK offshore sites the prevalence of liver tumours in Limanda limanda (dab) exceeds 20%. However, the molecular mechanisms of tumour formation and the causative agents are not known. The contribution of epigenetic mechanisms, although well-established in human tumourigenesis, is under-studied in tumours of aquatic species. In this thesis, alteration in the DNA methylation patterns in tumours of two fish species, the model species zebrafish (Danio rerio) and the un-sequenced marine flatfish dab, were investigated. The data presented provided a comprehensive characterisation of DNA methylation pattern in zebrafish liver and the first evidence of alterations in DNA methylation profiles of key genes in tumourigenesis pathways in any aquatic species. A statistically significant lower level of global DNA methylation was demonstrated in hepatocellular adenoma (HCA) and non-cancerous surrounding liver tissue (ST) compared to liver of non-cancer bearing dab. The evidence presented in this thesis suggests that chronic exposure to a mixture of pollutants contribute to global DNA hypomethylation followed by further epigenetic and genomic changes, leading to the development of tumours in dab. These findings suggest a link between the environment, epigenome and cancer in fish tumours in the wild.

Small intestinal neuroendocrine tumours (SI NETs) are the most common malignancy of the small intestine, however they remain poorly characterised and the underlying pathogenic mechanisms driving disease development have yet to be elucidated. Whole genome and exome sequencing has suggested SI NETs to be mutationally quiet, with the most frequent mutation in Cyclin Dependent Kinase 1B occurring in only 8% of tumours, suggesting mechanisms other than genetic mutations may be responsible for driving SI NET tumourigenesis. Using integrated genomic and epigenomic analysis three distinct somatic copy number alteration (SCNA) profiles of SI NET were identified. The largest subgroup characterised by loss of heterozygosity at chromosome 18, negative CpG island methylator phenotype (CIMP) status, and the presence of CDKN1B mutations, is associated with improved clinical outcomes. A novel Multiple-SCNA signature has been described which defines a smaller subgroup of SI NETs and is characterized by significantly (p=0.04) reduced progression-free survival. A panel of 21 recurrently epigenetically dysregulated genes has been identified, and these represent putative novel pathogenic drivers for SI NET tumourigenesis and candidate novel biomarkers. Epigenetically dysregulated genes identified at a recurrence rate of 80-100% include gastric inhibitory polypeptide receptor (GIPR)(73.5%) – a target for novel imaging techniques in NETs, CDX1 (85.7%), CELSR3 (83.7%), FBP1 (83.7%), PCSK1 (67.3%) and TRIM15 (63.3%). The utility of methylated circulating tumour DNA analysis, and molecular profiling of circulating tumour cells as novel non-invasive biomarkers in SI NETs have been demonstrated. This is the first comprehensive integrated molecular analysis of SI NETs, providing evidence for epigenetic rather than mutational events in addition to SCNAs as drivers of SI NET development. These findings will facilitate improved patient management, treatment selection and prognostication.

Capillaries in tumours are often severely buckled (in a plane perpendicular to the axis) and / or chaotic in their direction. We develop a model of these phenomena using nonlinear solid mechanics. Our model focusses on the immediate surrounding of a capillary. The vessel and surrounding tissue are modelled as concentric annulii. The growth is dependent on the concentration of a nutrient (oxygen) diffusing from the vessel into the tumour interstitium. The stress is modelled using a multiplicative decomposition of the deformation gradient F=F_e F_g. The stress is determined by substituting the elastic deformation gradient F_e (which gives the deformation gradient from the hypothetical configuration to the current configuration) into a hyperelastic constitutive model as per classical solid mechanics. We use a Blatz-Ko model, parameterised using uniaxial compression experiments. The entire system is in quasi-static equilibrium, with the divergence of the stress tensor equal to zero. We determine the onset of buckling using a linear stability analysis. We then investigate the postbuckling behaviour by introducing higher order perturbations in the deformation and growth before using the Fredholm Alternative to obtain the magnitude of the buckle. Our results demonstrate that the growth-induced stresses are sufficient for the capillary to buckle in the absence of external loading and / or constraints. Planar buckling usually occurs after 2-5 times the cellular proliferation timescale. Buckles with axial variation almost always go unstable after planar buckles. Buckles of fine wavelength are initially preferred by the system, but over time buckles of large wavelength become energetically more favourable. The tumoural hoop stress T_{ThetaTheta} is the most invariant (Eulerian) variable at the time of buckling: it is typically of the order of the tumoural Young's Modulus when this occurs.

This thesis analyses and expands upon an existing multi-physicsagent-based model for avascular tumour growth, in which cell behaviours are determined by a cellular pressure distribution andan oxygen concentration. Two models are developed and included within the avascular tumour model and their effects on the tumour growth patterns are analysed. The development of the two models favours fundamental principles over directly including macro-scalephenomena in order to preserve the simplicity and low computational cost of the original model. The first model includes effects of cell-cell attraction (referred to as the simpleattraction model) and the second model includes effects from the resistance of the surrounding tissue which the tumour expands into(referred to as the external tissue pressure model). Results from the simple attraction model shows that the overall tumour growth is not inhibited, yet the growth pattern is notably altered. Results from the external tissue pressure model suggests that the growth is inhibited, at least for a comparatively very large time scale. Furthermore, computational artefacts from the discretization of the computational domain are found in the longterm growth pattern of the tumour. However, higher values of the model stiffness parameter resulted in an earlier growth inhibition as well as the absence of said computational artefacts. In addition, a useful relation between the final steady state tumour size and an oxygen threshold parameter for cell proliferation was discovered. Finally, the two models are included within a more complex model for tumour growth within the same computational framework. The resulting growth patterns were notably different for the two models, but growth was not inhibited in any of the experiments conducted here.

Glucocorticoids, key hormonal regulators of the stress response, powerfully influence inflammation and metabolism. Reducing excessive glucocorticoid exposure is beneficial in treating metabolic and cognitive disorders, but manipulating systemic endogenous glucocorticoids risks compromising their beneficial effects. The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activates glucocorticoids in target tissues and thus inhibition of this enzyme presents a clinical opportunity to reduce tissue-specific glucocorticoid action. Active glucocorticoids also exert potent angiostatic effects by binding the glucocorticoid receptor (GR), and 11β-HSD1 inhibitors have proven beneficial in models of myocardial infarction by promoting angiogenesis. The possibility that 11β-HSD1 inhibitors may increase pathological angiogenesis, such as that seen in solid tumours, remains unaddressed. This project tested the hypothesis that 11β-HSD1 inhibition promotes tumour growth as a result of increased angiogenesis, using murine models of squamous cell carcinoma (SCC) and pancreatic ductal adenocarcinoma (PDAC). Murine SCC or PDAC cells were injected (1x106 cells/flank) into WT female mice fed either standard diet, or diet containing the 11β-HSD1 inhibitor UE2316 (175 mg/kg, N=6/group), or into 11β-HSD1 knockout (Del1) mice fed standard diet. Developing tumours were measured by callipers over several weeks, before animals were culled and tissues collected. SCC tumours grew more rapidly in UE2316-treated mice to reach a significantly (P < 0.01) larger final volume (0.158 ± 0.037 cm3) than in control mice (0.051 ± 0.007 cm3). PDA tumours were unaffected by 11β-HSD1 inhibition or deletion. Immunofluorescent co-staining of tumour sections for CD31/α-smooth muscle actin revealed no differences in vessel density, and RT-qPCR showed no difference in angiogenic factor expression, after 11β-HSD1 inhibition/deletion in either tumour type. GR and 11β-HSD1 RNA expression were greater in SCC vs PDAC tumours (P < 0.001), as was 11β-HSD1 activity (P < 0.0001). In studies using the aortic ring assay of ex vivo angiogenesis, 11β-HSD1 deletion, but not inhibition with UE2316, was shown to prevent glucocorticoid-mediated angiostasis. The growth/viability of tumour cell lines was not affected by UE2316 or corticosterone, as assessed by live cell imaging using the Incucyte imaging system. RNA-sequencing of SCC tumours revealed that multiple factors involved in the innate immune/inflammatory response were reduced in UE2316-treated tumours, and that extracellular matrix regulation was also altered by UE2316. Imaging of tumour sections using Second Harmonic Generation microscopy confirmed that UE2316 altered Type I collagen deposition in SCC (P < 0.001) but not PDAC. 11β-HSD1 inhibition can increase tumour growth, possibly via suppression of inflammatory/immune cell signalling and alteration of the extracellular matrix, and tumours with higher GR and 11β-HSD1 content, such as SCC, may be more at risk. Interestingly this investigation found no evidence of increased angiogenesis in vivo or ex vivo after UE2316 treatment, suggesting that 11β-HSD1 inhibition does not promote angiogenesis in all ischaemic environments. Future work must focus on the effects of 11β-HSD1 inhibition on the immune and extracellular matrix component of the tumour microenvironment. While promotion of pathological angiogenesis does not appear to pose a major threat, 11β-HSD1 inhibitors may still interact with the immune and inflammatory environment in tumours to the detriment of health.

The electrochemical treatment (EChT) of tumours entails thattumour tissue is treated with a continuous direct currentthrough two or more electrodes placed in or near the tumour.Promising results have been reported from clinical trials inChina, where more than ten thousand patients have been treatedwith EChT during the past ten years. Before clinical trials canbe conducted outside of China, the underlying destructionmechanism behind EChT must be clarified and a reliabledose-planning strategy has to be developed. One approach inachieving this is through mathematical modelling.

Mathematical models, describing the physicochemical reactionand transport processes of species dissolved in tissuesurrounding platinum anodes and cathodes, during EChT, aredeveloped and visualised in this thesis. The consideredelectrochemical reactions are oxygen and chlorine evolution, atthe anode, and hydrogen evolution at the cathode. Concentrationprofiles of substances dissolved in tissue, and the potentialprofile within the tissue itself, are simulated as functions oftime. In addition to the modelling work, the thesis includes anexperimental EChT study on healthy mammary tissue in rats. Theresults from the experimental study enable an investigation ofthe validity of the mathematical models, as well as of theirapplicability for dose planning.

The studies presented in this thesis have given a strongindication of the destruction mechanism involved in EChT. It isshown by the modelling work, in combination with theexperiments, that the most probable cause of tissue destructionis acidification at the anode and alkalisation at the cathode.The pH profiles obtained from the theoretical models have showngood correlation with the experimentally measured destructionzones, assuming that a pH above and below certain values causetissue destruction. This implies that the models presented inthis thesis could be of use in predicting the tumourdestruction produced through EChT, and thereby provide a basisfor a systematic dose planning of clinical treatments.Moreover, the models can serve as valuable tools in optimisingthe operating conditions of EChT.

Modelling work of theanode processes has explained the roleof chlorine in the underlying destruction mechanism behindEChT. It is found that the reactions of chlorine with tissueplay important roles as generators of hydrogen ions. Thecontribution of these reactions to the acidification of tissue,surrounding the anode, is strongly dependent on the appliedcurrent density and increases with decreasing currentdensity.

Keywords:cancer, direct current, dose planning,electrochemical treatment (EChT), electrotherapy, mathematicalmodelling, tumour.

This study was carried out to determine the phenotype of special dog mammary gland tumours that were grown in nude mice. 26 tumours were examined by the immunohistochemical ABC-Elite protocol. The tumour tissues were labelled with following anti-human antibodies:

- AE1/AE3 (pankeratin antibody) labelled epithelial and myoepithelial cells

- CD 31 labelled endothelial cells

- desmin labelled cross-striated and smooth muscle cells

- myosin labelled cross striated muscle cells

- neurofilament (NF) labelled nerve cells

- osteopontin labelled preosteoblasts, osteoblasts and osteocytes

- p63 labelled nuclei of the myoepithelial cells

- smooth muscle actin (SMA) labelled the cytoplasm of myoepithelial cells

- type I collagen labelled the extracellular matrix in connective tissue and bone

- type II collagen labelled the extracellular matrix in cartilage

- vimentin labelled fibroblasts, fibrocytes, lipocytes, smooth muscle cells, endothelial cells, nerve cells, macrophages and myoepithelial cells

The tumours were also submitted to a double immunolabelling study using p63 and SMA.

The study could not give a final conclusion about the origin the tumours. There was still need for more research to answer that question. However, the immunohistochemical technique was analysed in detail, in order to obtain perfect labelings.

Initially, all the antibodies were tested on normal dog tissue, to acquire the best working dilutions with the lowest background problems. In the tumours, good results were obtained with these dilutions for the antibodies p63, SMA, vimentin, desmin, NF, AE1/AE3 and CD 31. Except for type I collagen, type II collagen and osteopontin that gave too much unspecific labelling of the mouse connective tissue. Even, when using the Vector® M.O.M. blocking kit, the results were still very difficult to interpretate.

The antigen retrieval methods were evaluated for all the antibodies. The antibodies p63, SMA, vimentin, desmin, AE1/AE3, myosin, neurofilament and CD 31 needed the antigen retrieval treatment. The antibodies type I collagen and type II collagen needed the treatment with the enzyme pepsin, while osteopontin did not need any pretreatment at all.

The double immunolabelling with p63 and SMA gave excellent results. Different combinations were tried out with different substrates, namely Vector® Nova RED, Vector® DAB and Vector® SG. Vector® methyl green was used as counterstaining, but it interfered with the other substrates, and better results were obtained without this counterstaining.

Primary hyperparathyroidism (pHPT) due to parathyroid tumours with hypersecretion of parathyroid hormone and hypercalcaemia is a common disease with incompletely understood etiology affecting more than 1 % of the population, primarily postmenopausal women. In secondary hyperparathyroidism (sHPT), parathyroid tumours develop in response to calcium and vitamin D deficiency generally in patients with uraemia. HPT is usually treated by surgical removal of enlarged parathyroid glands.

The aim of this thesis was to examine the Wnt/β-catenin signalling pathway in parathyroid tumours.

Aberrantly accumulated β-catenin was found in all analysed pHPT and sHPT tumours, with a stabilising homozygous mutation (Ser37Ala) in 7.3% of the pHPT tumours. Truncation of the APC protein was not found. MYC, a β-catenin target gene was overexpressed in a substantial fraction of pHPT and sHPT parathyroid tumours.

A parathyroid tumour cell line (sHPT-1) was established from a hyperplastic gland removed at operation of a patient with sHPT. The cells produced parathyroid hormone and grew with a doubling time of approximately 72 hours. Stabilised nonphosphorylated transcriptionally active β-catenin was expressed. Efficient transfection of siRNA against β-catenin decreased expression of cyclin D1 and MYC, and inhibited cell growth with ensuring cell death.

The Wnt coreceptor LRP5 was found expressed with an internal deletion of 142 amino acids (LRP5Δ) in 86% and 100% of pHPT and sHPT tumours, respectively. Stabilising mutation of β-catenin and expression of LRP5Δ was mutually exclusive. Expression of LRP5Δ was required to maintain the nonphosphorylated transcriptionally active ß-catenin level, MYC expression, parathyroid cell growth in vitro, and tumour growth in transplanted SCID mice. Wnt3 ligand and LRP5Δ strongly activated transcription, and LRP5Δ was insensitive to inhibition by DKK1.

Aberrant accumulation of β-catenin by stabilising mutation or expression of LRP5Δ appears as a common pathogenic pathway for hyperparathyroid disease. LRP5Δ in particular presents a potential target for therapeutic intervention.

Abstract: The default mode network of the brain is a set of functionally connected regions associated with introspection and daydreaming. Recent fMRI studies have discovered that the default mode network is often perturbed in the diseased brain. For example, the default mode network is known to be modulated in dementia, ADHD, depression, and schizophrenia, among others. This has led many into believing that this network could have a role in the physiopathology of nervous system disease, or could be a useful marker of brain function. However, very few studies have yet been done which investigate how surgical lesions such as brain tumours affect the default mode network. Consequently, the goal of this project was to characterise the effect of brain tumours on the default mode network based on their location, histological type, and other parameters.
Le mode de fonctionement par défaut du cerveau est un réseau cérébral associé à la rêverie et à l’introspection. Des études récentes sur ce réseau ont découvert qu’il est perturbé dans plusieurs pathologies cérébrales. Par example, le mode de fonctionnement par défaut est modulé en démence, TDAH, dépression, schizophrénie et plusieurs autres maladies liés au cerveau. Ceci a mené à l’hypothèse que le mode de fonctionnement par défaut pourrait avoir un rôle dans la physiopathologie des maladies du système nerveux, ou pourrait être un marqueur utile du fonctionnement cérébral. Par contre, très peu d’études ont investigué l’effet de lésions chirurgicaux comme les tumeurs cérébrales sur le mode de fonctionnement par défaut. Par conséquent, le but de ce projet était de caractériser l’importance de l’histologie, de la localisation et de plusieurs autres paramètres de l’effet d’une tumeur cérébrale sur le mode de fonctionnement par défaut.

Germ cell tumours (GeTs)affect both paediatric and adult populations, and can occur either in gonadal or extragonadal regions along the body's ventral midline. These tumours can be broadly categorized into two subgroups, seminomatous (SEM) or nonseminomatous (N-SEM). The latter can be further subcategorized into embryonal carcinoma (EC), teratoma, yolk sac tumour (YST) and choriocarcinoma (eC) according to their differentiation. As in many other tumours, DNA methylation has been proposed to be involved in GCTdevelopment. However to date, most studies were performed using adult testicular GCTs. Furthermore, these studies only include a handful of genes in their analysis. Thus, the roles of DNA methylation in paediatric and extragonadal GeTs have not been explored. Therefore, this project attempted to fill this gap in knowledge by performing methylation analysis in a cohort of paediatric GCT samples and GCTcell lines. Although paediatric GCTsmostly consist of teratomas, seminomas or YSTs, only the latter two were included in the methylation analysis as they were the only samples in the available tumour bank. Using the methylation level of L1NE-l repeat elements as a measurement of global genome methylation, we found that both paediatric seminoma and YST samples displayed global hypomethylation as compared to somatic controls. However, when methylation at gene promoter regions was investigated using Illumina Golden Gate methylation arrays, seminoma and YST exhibited very different methylation features. YSTs were found to be highly methylated at many of the sites investigated. Surprlslnglv, we found that the methylation features in seminoma were similar to the somatic controls. From this analysis, we identified 85 genes that were differentially methylated in the VSTs. However, by correlating our methylation data with the expression array data performed by our collaborators on the same samples, only eight of these genes (PYCARO, CASPB, C02, HOAC9, TFAP2C, ETV1, EV/2A, HLA-F) were differentially expressed. As in previous GCTstudies, our analysis was focused on the methylation at epG islands. During the course of this project technological advancement led to the creation of new methylation arrays that offer wider genome coverage. One example is the Infinium Methylation 450K array that covers more than 450,000 CpG sites and includes regions flanking the CpG islands such as the CpG shores and CpG shelves. Since no previous GCT studies have attempted to investigate methylation in those regions, we utilized this methylation array on four GCTcell lines; TCAM2 (seminoma), NT2Dl (teratocarcinoma), GCT27 (embryonal carcinoma) and GCT44 (yolk sactumour). Similar to previous GCT studies, we found that nonseminomatous GCT cell lines displayed higher methylation at the CpG islands as compared to the seminoma cell lines. Strikingly, expanding our analysis to other regions (CpG shores and shelves etc.) revealed that each GCT subtype exhibited distinct methylation features. Both ECand teratoma cell lines displayed higher methylation than the seminoma and YST cell lines at all regions. Interestingly, the YST cell line only showed higher methylation than the seminoma cell line at the CpG islands and to a lesser extent at the CpG shores while the seminoma cell line exhibited higher methylation at the CpG shelves as compared to the YST cell line. This is the first time such features have been reported for GCTs. From this Infinium methylation data, we have also identified a high number of hypermethylated genes including those that are uniquely methylated for each cell line. By correlating this methylation data with Affymetrix gene expression data, 98 genes that were differentially methylated and differentially expressed in the YST cell line have been identified. However, further analysis needs to be performed to understand the role of these genes in YST development. As in other types of tumour, the hypermethylation observed in the YST cell line might be caused by many epigenetic modifiers. Using real-time RT-PCR on three epigenetic modifiers (DNMT38, EZH2, SUZ12), we found that DNMT38 was highly expressed in the YST samples and cell line as compared to the seminoma samples and cell line. This suggests that DNMT38 might contribute to YST hypermethylation and resulting differences in their biology. However, knockdown of DNA methyltransferases (DNMTs) and DNMT38 using 5-azadeoxycytidine and microRNA-29b respectively, did not seem to have any effect on the response of all four GCT cell lines towards cisplatin. On the other hand, both knockdowns only caused little effect on cell migration; affecting only the seminoma and YST cell lines. Nonetheless, further analysis is still needed to fully assess the role of DNA methylation in regulating cell behaviour. In summary, paediatric YSTs displayed hypermethylation at many promoter regions as compared to seminomas. Meanwhile, methylation analysis at regions outside of CpG islands in GCTcell lines revealed unique methylation features for each GCT subtype which might indicate different underlying mechanisms in their development. Further analysis on genes found to be differentially methylated and differentially expressed in both paediatric and GCTcell lines are now needed to fully establish their role in GCT development.