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Telomeres are nucleoprotein structures that contain non-coding (TTAGGG) tandem repeats and associated telomere binding proteins at the end of chromosomes. As a consequence of end-replication losses, telomeres undergo gradual erosion with ongoing cell division. It is hypothesised that in addition to the end-replication problem, mutational mechanisms may contribute to telomere erosion by generating large-scale telomeric deletion events. As short dysfunctional telomeres are capable of fusion to other chromosome ends, large-scale telomeric deletions can lead to genomic instability which in turn may drive tumour progression. The primary aim of this thesis was to investigate putative mutational mechanisms that could lead to large-scale telomeric deletion. The role of oxidative stress and it potential contribution to telomere dynamics was assessed. The induction of fragility and replication inhibition at telomeres was also examined. Furthermore, the role that G-quadruplex structure within telomere repeat sequences and the possible induction of replication fork stalling and resolution as single or double stranded breaks was also considered as a mutational mechanism that could lead to telomere deletion. High-resolution analysis of telomere dynamics using Single Telomere Length Analysis (STELA), following the induction of oxidative stress in IMR90 fibroblasts, revealed that oxidative damage does not appear to affect the rate of telomere erosion, or the frequency of large-scale telomeric deletion. The data are more consistent with the view that premature senescence does not arise as a consequence of accelerated telomere erosion, but instead more likely results from stochastic DNA damage across the rest of the genome. The analysis of telomere dynamics following the induction of chromosome fragility, showed that telomere length in Seckel cell (SCK) fibroblasts were significantly different from those of untreated cells following treatments with aphidicolin with an increase in stochastic telomeric deletion. Whilst in MRC5 fibroblasts, the induction of the telomere fragility impacted on the upper to lower allele ratio, with a loss of the longer telomere length distributions. The stabilisation of G-quadruplex structures using the G-quadruplex ligand (RHPS4), together with ATRX knockdown, showed that an absence of ATRX sensitised cells to the ligand, but that the stabilisation of G-quadruplexes, did not significantly affect the telomere dynamics as determined using STELA. Taken together, the data presented in this thesis are not consistent with a role for oxidative stress, or the formation of G-quadruplex structures, in generating large-scale telomeric deletion; however telomeric mutational events may occur following the induction of chromosome fragile sites, specifically in the context of an ATR deficiency.

Antigens (Ags) expressed by cancer cells can be specifically recognised by T cells contributing to anti-tumour immunity. For this reason, tumour Ag-specific T helper type 1 (Th1) cells can be detected in cancers and their infiltration is predictive of a prolonged disease-free survival. However, their activity is suppressed by T cells with inhibitory functions i.e. regulatory T cells (Tregs). High infiltration of tumours by these cells often correlates with poor survival. It is therefore crucial to understand their activity. Since Th1 and Treg cells are activated after Ag recognition, it is plausible that tumour Ag-specific Tregs inhibit Th1-mediated tumour immunity. Furthermore, vaccination, rather than inducing effector T cells, may be detrimental by activating Tregs. Increased numbers of CD25hi Treg cells are found in cancer patients, including CRC. However, in contrast to many cancers, the phenotype of these cells has not been studied in CRC patients. In this thesis, phenotypes of circulating, colon and tumour infiltrating Tregs are described. A high percentage of intratumoural CD4+CD25hi Tregs was found to express immunosuppressive molecules and these cells were enriched in late-stage CRC tumours. In addition, this thesis describes the measurement of Th1 and Treg cells recognising the oncofetal Ag, 5T4. Four immunogenic regions within the oncofoetal tumour Ag 5T4 and 14 peptides able to bind to most commonly found human HLA-DR alleles were identified. CD4+CD25hi cells mediated the control of tumour Ag 5T4-specific Th1 responses in CRC patients and their suppressive activity was not restricted to Th1 cells of the same Ag specificity. Also, responses to some, but importantly not all, immunogenic helper peptides discovered were influenced by Tregs. Thus Tregs may contribute to CRC progression by influencing tumour Ag- specific responses of Th1 cells. This body of work brings more information regarding the tumour Ag-specificity of Tregs and offers a future rationale to design peptide-based vaccines which exclude Treg peptides.

Mammalian/mechanistic target of mTOR complex 1 (mTORC1) regulates multiple cellular processes, including de novo protein synthesis, autophagy and apoptosis. mTORC1 overactivation occurs in a range of cancers and benign tumour dispositions as a result of mutations which increase mitogenic stimulus or cause malfunction of the tuberous sclerosis complex, the prime regulator of mTORC1 activity. mTORC1 overactivation results in elevated endoplasmic reticulum (ER) stress which, at low levels, elicits a pro-survival response. However, prolonged or excessive ER stress causes cell death. The present study utilised clinically relevant drug combinations to simultaneously enhance levels of ER stress and inhibit compensatory survival pathways in in vitro models of mTORC1 overactivity in order to cause non-genotoxic cell death. The main drugs used in this study were nelfinavir, an ER stress-inducer, chloroquine, an autophagy inhibitor, and bortezomib, a proteasome inhibitor. The key findings of this study include identification of drug combinations nelfinavir and chloroquine, nelfinavir and mefloquine, or nelfinavir and bortezomib to induce significant and selective cell death in mTORC1-driven cells, as measured by flow cytometry with DRAQ7 staining and western blot analysis for cleavage of apoptotic markers. Cell death is likely mediated through ER stress signalling, as shown by increased ER stress markers at both the level of mRNA and protein. Of interest, this study found cell death as a result of combined treatment with nelfinavir was not dependent on proteasome inhibition by nelfinavir, or autophagy inhibition by chloroquine. Additionally, nelfinavir-chloroquine-mediated cell death was completely rescued by inhibition of the vacuolar ATPase by bafilomycin-A1. In conclusion, mTORC1 overactive cells have higher basal levels of ER stress which can be manipulated with drug treatment beyond a survivable threshold, whereas cells capable of reducing mTORC1 signalling are able to survive. This study ascertained a combination of nelfinavir and chloroquine, nelfinavir and mefloquine, or nelfinavir and bortezomib, to cause effective cytotoxicity in mTORC1-driven cells.

The reactive stroma in prostate cancer is predominantly composed of myofibroblasts, which are thought to be required for tumour progression. Bone-marrow derived mesenchymal stem cells (BM-MSCs) are known to migrate into the tumour and are one of the many potential precursors of myofibroblasts. The factors secreted by cancer cells which may drive myofibroblastic differentiation of MSC, however are poorly understood. The aim of this thesis was to explore for the first time, the impact of TGF-β1 expressing exosomes (nano-sized vesicles) secreted by prostate cancer cells in directing the differentiation of BM-MSCs and subsequently the functions of exosome differentiated BM-MSCs. Exosomes isolated from prostate cancer cells skewed BM-MSCs away from differentiating into adipocytes, and instead towards alpha-smooth muscle actin (α-SMA) positive myofibroblasts. BM-MSCs treated with exosomes exhibited enhanced secretion of VEGF-A, HGF and had an altered transcript profile with heightened matrix metalloproteinases (MMP-1, -3 and -13). Impairing the secretion of exosomes by Rab27a knockdown or depleting exosomes from prostate cancer cells culture media by high speed ultracentrifugation, attenuated myofibroblastic differentiation of BM-MSCs, demonstrating exosomes as the key driving factor for this. Furthermore, differentiation of BM-MSCs into myofibroblasts was dependent on exosomally tethered TGF-β1, however BM-MSCs treated with soluble TGF-β1 at the same dose, failed to obtain the same myofibroblastic phenotype. The exosome-differentiated MSCs enhanced endothelial and cancer cell proliferation and migration, supported endothelial vessel formation and promoted tumour cell invasion into peri-tumoural matrix in vitro. In conclusion, this study reports prostate cancer exosomes expressing TGF-β1, to dominantly modulate the fate of BM-MSCs, generating cells with tumour promoting myofibroblastic traits.

MicroRNAs (miRNAs) are small RNAs of ~22 nucleotides in length that regulate gene expression post-transcriptionally. MicroRNAs regulate many biological processes, and may contribute to cancer initiation and progression. In the normal gastrointestinal mucosa, myofibroblasts are involved in wound healing, and in tumours, they play a role in cancer progression. The present study aimed to determine the miRNA expression profiles in different populations of myofibroblasts from gastric and oesophageal tissues, namely, cancer-associated myofibroblasts (CAMs), adjacent non-tumour tissue myofibroblasts (ATMs) and normal tissue myofibroblasts (NTMs), and to investigate the processes that are influenced by differential miRNA abundance in different myofibroblast populations. In addition, miRNA profiling of a putative myofibroblast precursor cell type, mesenchymal stromal cells (MSCs), was studied. Global miRNA expression profiling of a panel of previously characterised gastric and oesophageal myofibroblasts was determined using locked nucleic acid (LNA) miRNA arrays. The microarray data for hsa-miR-29b, hsa-miR-181d, hsa-miR-214 and hsa-miR-424 were validated by quantitative RT-PCR. Using unsupervised miRNA datasets, principal component analysis (PCA) segregated gastric and oesophageal NTMs, and CAMs from gastric and oesophageal carcinomas. Analysis of global miRNA expression showed distinct profile of MSCs in comparison to gastric and oesophageal NTMs. Hierarchical clustering analysis of miRNAs exhibiting significantly different abundance revealed distinct clusters between CAMs and ATMs, CAMs and NTMs and between ATMs and NTMs. In 12 paired samples of gastric CAMs and their corresponding ATMs, 12 miRNAs were significantly different with hsa-miR-181d being the most up-regulated miRNA in CAMs. Using already validated targets of 10 of these miRNAs, an analysis using MetaCore® indicated that the regulation of cell cycle progression and Wnt signalling were differentially affected. In a comparison of each CAM and its corresponding ATM, Wnt signalling was the only process that was significantly targeted by miRNAs that were different in abundance, in all 12 pairs. Using previously determined gene expression data, pairwise analysis of 20 transcripts associated with Wnt signalling showed a significant increase in WNT5A and TGFB2 mRNAs in CAMs compared to their ATMs. Additionally, Western blot analysis showed increased expression of Wnt-5a in CAMs. The results of migration and EdU incorporation assays indicated that Wnt-5a induced the migration and proliferation of both CAMs and ATMs. The abundance of hsa-miR-181d was significantly increased in CAMs by Wnt-5a. Migration of AGS (gastric cancer) cells was stimulated by Wnt-5a and was inhibited in the presence of TIMP-3, a validated target of the miR-181 family. After hsa-miR-181d knockdown, the migration and proliferation of CAMs were significantly decreased, and stimulation with Wnt-5a reversed the migratory inhibition of hsa-miR-181d knockdown CAMs. Conditioned medium from hsa-miR-181d knockdown CAMs inhibited the migration of AGS cells. The findings suggest that differential miRNA abundance is a feature of normal myofibroblasts from the stomach and oesophagus, and myofibroblasts from normal and cancer tissues. One consequence is increased Wnt-5a in gastric CAMs that is implicated in the regulation of cell migration, in health and disease, at least partly through modulation of hsa-miR-181d.

The last three decades have seen a fundamental change in the profile of oropharyngeal squamous cell carcinoma (OPSCC) within the developed world. The incidence of OPSCC attributable to tobacco and alcohol exposure has been gradually declining whilst Human papillomavirus (HPV)-related OPSCC has seen a rapid increase. Detection of High Risk HPV has profound prognostic significance as it correlates with both a disease-specific and an overall survival advantage. The stringency of testing, both in terms of diagnostic and prognostic capacity is therefore of increasing importance. This study sought to define the relative abilities of the diagnostic tests presently available in clinical practice and to explore the potential of a novel test in reaching the improved stringency called for by the clinical community. Diagnostic biomarkers with prognostic capacity, such as those utilised in defining HPV status in this research have been well described, however, despite HPV positive OPSCC being biologically distinct from HPV negative malignancy, predictive biomarkers defining the transition from persistent to transforming infection are yet to be forthcoming. A lack of an apparent premalignant state, akin to that seen in HPV-mediated cervical malignancy has restricted biomarker recognition. This research aimed to better define the epigenetic state and clarify the impact of viral integration for the virus and host in HPV positive OPSCC. Although detectable epigenetic alterations, within the genome of the virus and that of the host, were capable of providing an improved description of this burgeoning disease state, they fell short of providing clinically relevant biomarkers. It was however demonstrated that the previously held concept of preferential E2 cleavage during viral integration as a means to disrupt gene expression, is overstated and the model persists to the exclusion of other viral and host genome disruptions. A paradigm shift may be necessary in HPV positive OPSCC to an understanding of obligatory viral integration, the significance of which however, is yet to be fully elucidated.

Chronic lymphocytic leukaemia (CLL) is a common type of adult leukaemia that accounts for approximately 30% of all mature B-lymphocyte malignancies. An important contributor to CLL pathogenesis is B-cell receptor (BCR) signalling which promotes survival of the malignant clone. BCR engagement on CLL cells provides survival signals by activating the NFκB pathway. Previous work to this thesis showed that CLL cells overexpress PKC-betaII and c-Abl, kinases which have been implicated in mediating NF-kappaB pathway activation in normal B cells. In particular, PKC-beta mediates activation of the CARMA1-Bcl10-MALT1 (CBM) complex leading to eventual activation of I-κB kinases (IKKs) in normal B cells responding to BCR engagement. Considering the importance of the BCR in providing pro-survival signals to CLL cells, the initial aim of this thesis was to characterise any potential role of PKCII and c-Abl in BCR-mediated activation of the NFκB pathway. We addressed this question in Chapter 3 and showed that inhibition of PKC-beta had no effect on BCR-induced activation of IKK, likely because Bcl10 is expressed at low levels in CLL cells. Investigation of the role of c-Abl using the inhibitor imatinib showed that the presence of this compound partially inhibited IKK phosphorylation in BCR-stimulated CLL cells, but the observed effect was variable between CLL patients, and this variability was unrelated to c-Abl expression. Imatinib can also inhibit Lck, a T cell-specific Src-family tyrosine kinase involved in antigen receptor signalling that is also expressed by CLL cells. A further aim of this thesis, answered in Chapter 4, is to define a possible role for Lck in BCR signalling in CLL cells. We showed that inhibition of this Src family kinase (SFK) with the specific inhibitor [4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[3,2d] pyrimidin -7-yl-cyclopentane (Lck-i)], or reduction of its expression with siRNA blocked BCR-stimulated induction of CD79a, Syk, IKK, Akt and ERK phosphorylation in CLL cells. Furthermore, we demonstrated that CLL cells with high levels of Lck expression had higher levels of BCR-mediated IKK, Akt and ERK phosphorylation as well as cell survival than did CLL cells with low levels of Lck expression. These data demonstrated a major role for Lck in proximal and distal BCR signalling in CLL cells. Importantly, these data suggested that Lck expression levels may be linked to disease prognosis. In Chapter 5 we investigated this possibility and showed that high Lck expression was associated with good disease outcome. We hypothesized that this may be because of a dual role played by Lck in promoting and suppressing BCR-induced signalling. We provided data to support this hypothesis and showed that CLL cells bearing low levels of Lck expressed a significantly higher proportion of mannosylated BCR than did CLL cells bearing high levels of Lck, a finding which was consistent with the presence of in vivo constitutive BCR signals. Furthermore, we found that Lck mediated the phosphorylation of ITIMs within CD22 during BCR stimulation of CLL cells. Taken together, these data suggest that high levels of Lck expression within CLL cells may function to interact with phosphatases and set an activation threshold to limit in vivo BCR signalling.

Geometric uncertainties are inevitable in radiotherapy. These uncertainties in tumour position are classified as systematic (Ε) and random (δ) errors. To account for these uncertainties, a margin is added to the clinical target volume (CTV) to create the planning target volume (PTV). The size of the PTV is critical for obtaining an optimal treatment plan. Dose-based (i.e., physical) margin recipes as a function of systematic and random errors based on coverage probability of a certain level of dose (90% or 95% of the prescription dose) have been published and widely used. However, with a TCP-based margin it is possible to consider fractionation and the radiobiological characteristics, especially the dose-response slope (50) of the tumour. Studies have shown that the density of the clonogens decrease from the boundary of the gross tumour volume (GTV). In such a scenario, dose that is lower than in the GTV should be sufficient to eradicate these clonogens. Thus a smaller PTV margin with a gradual dose fall off can be used if the clonogen density in the GTV-CTV region is found to be lower than in GTV. Studies have reported tiny tumour islets outside the CTV region. These tiny tumour islets can be eradicated in some cases by the incidental dose outside the PTV due to the nature of the photon beam irradiation, but if they are not in the beam path the treatment outcome is compromised. In this thesis, a Monte Carlo approach is used to simulate the effect of geometric uncertainties, number of fractions and dose-response slope (gamma50) using the 'enhanced Marsden' TCP model on the treatment outcome. Systematic and random errors were drawn from a pseudo-random number generator. The dose variations caused by tumour displacements due to geometric uncertainties in the CTV are accumulated each fraction on a voxel-by-voxel basis. Required margins for ≤ 1% mean population TCP (TCPpop) for four-field (4F) brick and a highly conformal spherical dose distribution for varying number of fractions, different γ50 and different combinations of Ε and δ are investigated. It is found that TCP-based margins are considerably smaller than dose-based recipes in most cases except for tumours with a steep dose-response slope (high γ50) and a small number of fractions for both 4F and spherical dose distributions. For smaller geometric uncertainties (Ε = δ = 1 mm) margins can be close to zero for the 4F technique due to high incidental dose outside the PTV. It is evident from the analyses that margins depend on the number of fractions, γ50, the degree of dose conformality in addition to Ε and δ. Ideally margins should be anisotropic and individualized, taking into account γ50, number of fractions, and the dose distribution, as well as estimates of Ε and γ. No single 'recipe' can adequately account for all these variables. Using an exponential clonogen distribution in the GTV-CTV region, possible PTV margin reduction is demonstrated. Moreover, the effect of extra-CTV tumour islets is studied using a prostate IMRT plan. The islets were randomly distributed around the CTV with in a radius of 3 cm to represent different patients. The doses were rescaled up to 102 Gy to obtain the dose-response curve (DRC). Interestingly, the obtained DRC showed a biphasic response where 100% TCP could not be achieved just by escalating the dose. Another potential problem encountered in intensity-modulated radiotherapy (IMRT) is the problems caused by the 'interplay' effect between the respiration-induced tumour motion and the multileaf collimator (MLC) leaves movement during treatment. Several dosimetric studies in the literature have shown that 'interplay' effects blur the dose distribution by producing 'hot' and 'cold' dose inside the tumour. Most of these studies were done in a phantom with ion chambers or films, which provide only 1D or 2D dose information. If 3D dose information is available, a TCP based analysis would provide a direct estimate of interplay on the clinical outcome. In this thesis, an in-house developed dose model enabled us to calculate the 3D time-resolved dose contribution to each voxel in the target volume considering the change in segment shapes and position of the target volume. Using the model, delivered dose is accumulated in a voxel-by-voxel basis inclusive of tumour motion over the course of treatment. The effect of interplay on dose and TCP is studied for conventionally and hypofractionated treatments using DICOM datasets. Moreover, the effect of dose rate on interplay is also studied for single-fraction treatments. Simulations were repeated several times to obtain mean population TCP (TCPpop) for each plan. The average variation observed in mean dose to the target volumes were -0.76 ± 0.36% for the 20 fraction treatment and -0.26 ± 0.68%, -1.05 ± 0.98% for the 3- and single-fraction treatments respectively. For the 20-fraction treatment, the drop in TCPpop was -1.05 ± 0.39%, whereas for the 3 and single fraction treatments it was -2.8 ± 1.68% and -4.0 ± 2.84% respectively. By reducing the dose rate from 600 to 300 MU/min for the single-fraction treatments, the drop in TCPpop was reduced by ~ 1:5%. In summary, the effect of interplay on treatment outcome is negligible for conventionally fractionated treatments, whereas a considerable drop in TCP is observed for the 3- and single-fraction treatments. Where no motion management techniques such as tracking or gating are available for hypo-fractionated treatments, reduced dose rate could be used to reduce the interplay effect.

Introduction: Oesophago-gastric (OG) cancers remain a worldwide challenge with little sign of major improvements in survival rates. Modern guidelines focus on alarm (or ‘red flag’) symptoms as key triggers for gastroscopy and recommend empirical symptomatic treatment and non-invasive H. pylori testing in those with simple dyspepsia. However, the early symptoms of OG cancer are very common and non-specific, and the traditional alarm symptoms have poor sensitivity or specificity for malignancy. Diagnosis therefore necessitates investigation of symptoms though upper GI endoscopy in a relatively large group of patients, most of whom do not have malignant disease. This has fuelled considerable controversy regarding the role for gastroscopy in detecting cancer at a treatable stage. Objectives: Firstly, to develop data extraction and linkage methods for studying OG cancer outcome, and General Practice population rates of elective diagnostic gastroscopy, using administrative data for English hospitals (Hospital Episode Statistics). Secondly, to confirm the face-validity of the methodology using external sources of information and local audit data. Thirdly, to test the hypothesis that variations in rates of gastroscopy in English General Practice (GP) populations are associated with inequalities in OG cancer outcome. Fourthly, to explore whether practices with lower rates of gastroscopy exhibit a higher yield of serious pathology, consistent with more selective referral practice. Fifthly, to confirm the existence of wide variation in gastroscopy rate between practices in close geographical proximity, Design and methods: Analysis of Hospital Episode Statistics (2006-8) linked to death registry and practice population data. General practices with new cases of OG cancer were included, grouped into tertiles according to standardised elective gastroscopy rate per capita (low, medium or high). Outcome measures for cancer cases were: emergency admission during diagnostic pathway; major surgical resection and mortality at 1 year. Co-variates were age, gender, co-morbidity, and deprivation. Associations between the gastroscopy rate at the patient’s general practice and cancer outcomes were tested in binary logistic regression models, with extensive sensitivity testing of gastroscopy rate ‘exposure’ variable. An algorithm was developed to analyse coded diagnoses for all first elective gastroscopies, using both national and local audit data. Practices were mapped based on postal code. Results: 22,488 incident cases of OG cancer from 6,513 general practices. Mean OGD rate for Low, Middle, High practices: 4.4 vs 8.1 vs 12.9 per 1,000. Mean age of patients undergoing OGD was highest for low tertile practices (60.2 vs 59.5 vs 58.4 yrs; p<0.001). OG cancer cases registered with practices in the lowest tertile had the lowest rate of surgery (15.4% v 16.3% v 17.4%; p=0.004) with the highest rate of emergency admission (34% v 26% v 25%; p<0.001), and the highest mortality (61.2% v 58.9% v 58.0%; p<0.001). After adjustment for co-variates in logistic regression, the gastroscopy rate at the patient’s general practice was an independent predictor of all three outcomes. Practices with low rates of gastroscopy tend to have a higher “diagnostic yield” of serious disease: (15.3% vs 13.9% vs 13.1%; p<0.001). Low tertile practices also showed a relatively lower referral rate for suspected cancer in general based on analysis of rates of ‘fast-track’ referrals under the two week wait pathway (17.3 vs 17.9 vs 19.3 per 1,000). Conclusions: Wide variation exists in gastroscopy rate among general practice populations in England. OG cancer patients belonging to practices with the lowest gastroscopy rates are at greater risk of poor outcome. Low referring practices achieve a higher yield of serious disease but may have increased risk of referral at a later stage in the disease process. This association is more apparent among the most socially deprived practices. These findings suggest that initiatives or current guidelines aimed at limiting the use of gastroscopy may adversely affect cancer outcomes.