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.

Cancers of the upper gastrointestinal tract present at an advanced stage and carry a poor prognosis. Oesophageal and gastric tumours have a rich stroma composed of vascular cells, immune cells, and myofibroblasts, which promotes tumour growth, invasion and metastasis. In addition, mesenchymal stromal cells (MSCs) are recruited from the bone marrow to the tumour stroma; the mechanisms underpinning this have not yet been defined. Extracellular proteases play a role in cell migration, invasion, and cell signalling and are known to influence cancer growth in conflicting ways. Myofibroblasts present in normal tissue differ from those found in cancer. Cancer-associated myofibroblasts (CAMs) are known to modulate extracellular protease activity by secreting plasminogen activator inhibitor-1 (PAI-1), an inhibitor of the serine protease urokinase plasminogen activator, and matrix metalloproteinases (MMPs). This investigation studies the role of PAI-1 in gastric cancer and assesses the contribution of myofibroblast-derived MMPs to tumour growth. Finally, the role of chemerin in recruiting MSCs has been investigated. The expression of PAI-1 in myofibroblasts was found to be higher than in gastric cancer cells. Overexpression of PAI-1 in gastric cancer cells resulted in decreased cell adhesion and decreased tumour growth in an in vivo subcutaneous xenograft model of gastric tumour growth. The addition of gastric CAMs potentiated the growth of gastric cancer subcutaneous xenografts. This was not accounted for by differences in cell proliferation rate, apoptosis or final stromal content. Xenografts containing CAMs suppress the growth of a contralateral xenograft without CAMs, demonstrating that a long-range signal can be generated as a result of stromal-epithelial interactions. MMP and cathepsin activity was compared between xenografts containing myofibroblasts to those without. MMP activity is increased in xenografts injected with CAMs, compared to those injected with myofibroblasts taken from normal stomach or those with gastric cancer cells alone. In an organotypic co-culture system, MMP inhibition resulted in a decrease in gastric cancer cell invasion. The injection of fluorescently labelled MSCs injected resulted in homing of these cells to subcutaneous oesophageal tumours containing CAMs. Antagonism at the ChemR23 receptor inhibited this MSC homing to oesophageal xenografts containing CAMs. This work emphasises the importance of assessing the contribution of specific proteases and their inhibitors in gastric cancer. The stroma is an important contributor to extracellular protease activity and myofibroblasts contribute both proteases and their inhibitors to the tumour microenvironment, resulting in the modulation of tumour growth and cell adhesion. MSCs are recruited to oesophageal tumours via a novel signalling pathway.

This volume presents the research carried out in partial fulfilment of the Doctorate in Clinical Psychology at the University of Liverpool. It contains three papers, addressing the area of cancer patients‘ trust in care. In times of distress, patients engaged in a course of psychological therapy wish to feel safe, contained and to trust that the clinician is working in their best interests. The same holds true in a medical setting, where being diagnosed with a life-threatening illness such as cancer evokes feelings of vulnerability, helplessness and intense fear. Clinicians can be viewed as attachment figures in this time of stress, being the cancer patient‘s main hope for creating safety in the face of threat. Whilst a relationship with the clinician characterised by trust is beneficial at this time, unfortunately not all patients experience the relationship in this way. Before we can suggest means to improve cancer patients‘ sense of trust in the clinician, we need to understand the factors which prevent patients from developing trust in their clinician. Unlike the field of psychotherapy, where patient factors influencing the relationship with the therapist have been extensively researched, the medical field has focused its efforts on attempting to highlight the contribution of the clinician. This thesis aims to address this current dearth of literature, and focus on patient factors which could impede them from having trust in cancer care. Paper 1: Literature review The review paper provides the backdrop for the research. A specific sub-section of the background literature that informed the development of the research is considered here. A general review of current knowledge relating to all aspects of cancer patients‘ experiences highlighted an area of relative dearth in research. From this, a specific research question was developed: what is the role of patients‘ experiences in trust in cancer care? A focussed literature review was conducted in response to this pre-determined question, investigating whether patient factors of trauma, abuse and attachment style shape patients‘ trust in cancer care. The review adopted a structured approach to interrogating the evidence base, and the search terms and eligibility criteria for inclusion of papers are outlined. This section presents the identified papers, comparing and contrasting aims, design, methodology and findings, and explores the key themes that arose. The collective limitations, inconsistencies and gaps in this literature base are explored, and future directions to address these points are suggested. The review paper presents a picture of the current knowledge in this area, enabling the reader to locate the research study in its broader context. Paper 2: Empirical paper Building upon paper one, the second paper describes the main features of the research study, presented according to author guidelines set out for the journal Psycho-Oncology (Appendix A). Whilst a trusting patient/clinician relationship is repeatedly highlighted as important in healthcare, there is a tendency in the research to neglect the patient‘s contribution to this interaction. Attempts to identify individual patient factors that contribute to the sense of clinical relationship are dwarfed by extensive research focussing upon the clinician‘s competence, skill, communication and interpersonal style. The little research that has been carried out in this area has predominantly sampled women with breast cancer, leaving unexplored questions about the generalisability to other populations. In an attempt to redress balance, this research investigated patient factors which affect trust in the clinician, in a sample of gynaecological cancer survivors. The process of developing and conducting the study took two and a half years, whilst recruitment commenced after ethical approval was sought, and spanned 52 weeks. The key features of the study are outlined in this section. Paper 3: Concluding discussion The third section draws out the main findings of the research, and discusses the wider relevance. Further attention is given to the relative strengths and weaknesses of the study, and the study‘s applicability to clinical practice is considered. A short lay summary is presented, which was written for dissemination of the research to the participants who had requested feedback. This discusses the key contributions of the research to the literature base, and states what will be done with the findings. Finally, a research proposal describing a possible follow up study is outlined, taking into account the limitations of this research and considering how it can be extended. This concludes the thesis.

Introduction: Colorectal cancer is the second most common UK cancer. Biomarkers which predict survival may be valuable for targeting adjuvant therapy and can provide insights into tumour biology. Small and early cancers are being diagnosed more commonly in the UK population due to the introduction of population-based colorectal cancer screening in 2005. Analysis of resected small (≤20mm across) tumours in Liverpool has established that flat and depressed morphology can predict advanced stage at presentation. Proteomic analysis of small cancers was conducted with the aim of generating biomarkers which correspond to morphology, stage and patient survival. Patients and Methods: Laser capture microdissection was used to procure enriched matched benign and malignant colorectal epithelial cell populations. Laser captured proteins were extracted into lysis buffer, normalised against a reference standard, separated using 2D SDS-PAGE and visualized with silver staining. Comparison was made between the tumour gels, (n=10) and matched normal colonic gels, (n=9) by two different observers and gel analysis software, Progenesis SameSpots. Differentially expressed proteins were identified using tandem mass spectrometry and included redox proteins peroxiredoxin 2, peroxiredoxin 6 and SH3 binding glutamic acid-rich protein-like 3; and cytoskeletal protein cofilin1. Also identified were the anti-apoptotic protein heat shock protein 27 and inflammatory protein S100A8, which had been previously identified in 2D gel analysis of undissected colorectal cancer in our Institution (n=12 gels) and previously validated in a small cohort of paraffin-embedded colorectal cancers (n=98). In this study, HSP27 was further evaluated in a large cohort of paraffin-embedded colorectal cancer tissue (n=404). S100A8 and related proteins S100A9 and Smad4 were similarly evaluated in a large cohort (n=313). Results: High HSP27 levels were strongly associated with poor cancer-specific survival in rectal cancer (n=205, P=0.0063) but not colon cancer; (n=199, P=0.7385). Multivariate Cox regression confirmed nodal metastases (P=0.0001) and HSP27 expression (P=0.0233) as independent markers of survival in rectal cancer. HSP27 levels remained unchanged in the majority of cases 65/80 (81%) between diagnostic biopsies and matched surgical samples, regardless of whether patients had undergone preoperative radiotherapy. S100A8 expression co-localised with a subset of S100A9-positive monocytes. S100A9 was co-expressed with CD14 in tumour-associated monocytes, but not with CD68 in tissue macrophages. Smad4 was expressed in the tumour cytoplasm of 262/304 (14%) tumours. Loss of Smad4 expression correlated with a reduction in the stromal S100A8-positive, but not S100A9-positive cell count, (P=0.034, Mann-Whitney U test) and was associated with a poorer overall survival in patients with stage I-II disease, but not stage III disease. Antibodies to cofilin1 and cofilin-phospho(ser3) were assessed in colorectal cancer cell and tissue lysate and found to be specific on 1D and 2D western blot. Conclusion: Elevated HSP27 is an independent marker of poor prognosis in rectal cancer whose expression is not altered by neo-adjuvant radiotherapy. Smad4-negative tumours are associated with fewer infiltrating S100A8 positive stromal monocytes. In node-negative tumours, loss of Smad4 expression in associated with a poorer prognosis. These findings provide a sound platform for further investigation of both S100A8 and HSP27 proteins in colorectal cancer.

The Wnt/β-catenin signalling pathway is involved in regulating cellular transcription of numerous target genes in chronic myeloid leukaemia (CML). A previous report using granulocyte-macrophage progenitors (GMPs) showed that elevated levels of unphosphorylated (active) β-catenin, alongside mis-splicing of glycogen synthase kinase 3β (GSK3β), correlated with disease progression. It was of interest to determine if using the more readily available peripheral blood mononuclear cells (MNCs) also showed any correlation with patient outcome when β-catenin and GSK3β were measured in this source material. Further investigation in these cells addressed GSK3β activity regulation, possible regulation of c-Myc degradation by GSK3β, and investigation of Wnt transcription factors in CML. Results indicated that levels of β-catenin (and its phosphorylated variants) could not be used to distinguish patient treatment outcomes using CML MNCs. However the levels of BCR-ABL1 induced β-catenin-Tyr654 phosphorylation showed a significant decrease in patients in blast crisis compared to chronic phase (P=0.012), as did the levels of GSK3β – which demonstrated significantly decreased activity in blast crisis patients via a significant increase in Ser9 phosphorylation and a significant decrease in Tyr216 phosphorylation in blast crisis compared to chronic phase (P=0.026 and <0.001 respectively). This decrease in GSK3β activity was not reflected by changes in the levels of β-catenin and, as such, this led to the question of whether alternative mechanisms were influencing regulation of GSK3β activity other than Wnt/β-catenin signalling. Results showed that protein phosphatase 2A (PP2A) was regulating the inhibition of GSK3β in CML. This is a dual mechanism with GSK3β also affecting PP2A inhibition. GSK3β is known to have numerous substrates, which include c-Myc. Due to the role of c-Myc as an oncogene in CML and the role of GSK3β in c-Myc degradation, it was of interest to determine if the changes in GSK3β activity were reflected in the degradation of c-Myc. Results showed that neither GSK3β nor PP2A (also involved in the mechanism) were the rate limiting components of c-Myc degradation. Analysis of the Wnt signalling pathway revealed up regulation of WNT1, WNT8A, WNT9A and FZD7 mRNAs in chronic phase MNCs (P=0.011, 0.045, 0.037 and 0.037 respectively) - which was inconsistent with β-catenin findings. These unexpected results can be explained by internal regulation of the pathway by the negative regulator NKD1, which is also significantly higher in samples from chronic phase patients (P=0.013). Finally, investigation into Wnt transcription factors showed that factors TCF1, TCF4 and LEF1 mRNA levels were significantly higher in blast crisis samples compared to chronic phase (P=0.001, 0.016, and 0.003 respectively). This suggests a possible mechanism for Wnt-independent activation of these transcription factors in the blast crisis phase of the disease. In conclusion, it was determined that β-catenin levels cannot be used to either; distinguish between patient response cohorts, or be a factor of disease progression in CML MNCs. The activity of GSK3β is significantly down-regulated in blast crisis. PP2A is involved in this regulation. However when investigating c-Myc degradation, neither GSK3β nor PP2A act as the regulating factors in CML. There may be an internal regulation of the pathway by which NKD1 prevents an upregulation of pathway activity via WNT1, WNT8A, WNT9A and FZD7 reaching β-catenin (CTNNB1). Finally, the transcription factors mRNA expression is significantly upregulated in blast crisis, but they could be acting independently of the Wnt pathway.

GRP78 is a known cyto-protective gene which is induced in microenvironments typical of tumours with low glucose and hypoxia. Furthermore up-regulation of GRP78 has been linked to chemo- and radioresistance as well as poor survival outcome. This study is the first to confirm that GRP78 is up-regulated in tumours of the oropharynx, larynx and hypopharynx compared with matched histologically normal tissue (p(Χ2)<0.001). Up-regulation of GRP78 may be important for tumour development and therefore we have investigated the consequences of inhibition of GRP78 in cells derived from laryngeal squamous cell carcinomas (LSCC). EGF-SubA is a novel drug consisting of EGF covalently attached to the A subunit of an E.coli derived AB5 toxin which can cleave GRP78. EGF-SubA was able to induce EGFR-dependent cytotoxicity in a panel of seven laryngeal SCC cells with an IC50 range of between 4-100pM. EGF-SubA treatment induced G1 cell cycle arrest as well as apoptosis. Therefore apoptosis may be the mechanism by which EGF-SubA causes cell death. In vitro studies demonstrated that EGF-SubA enhances the effects of clinically relevant genotoxic agents. Two Gy survival fractions (SF2) were significantly reduced with EGF-SubA pre-treatment (p<0.03). In addition EGF-SubA in combination with the primary head and neck cancer chemo-therapeutic agent cisplatin, resulted in IC50 drug combination indexes (CI) as low as 0.542, which is suggestive of a synergistic effect. The potency of EGF-SubA appears to be substantially dependent on EGFR membrane expression since cells expressing higher EGFR levels were associated with increased sensitivity to EGF-SubA where Spearman’s rank correlation coefficient = 0.919 (p=0.003). Furthermore pre-incubation of LSCC cells with sub-toxic doses of cetuximab, a therapeutic monoclonal antibody to EGFR, completely rescued cells from the cytotoxic effects of EGF-SubA (p(t test)≤0.005). This is an important proof of principal for a proposed combined toxin-protectant therapeutic strategy that would permit topical use of EGF-SubA peri- or post-operatively after tumour resection in order to kill any remaining tumour cells, or as an oral rinse in patients who present with pre-malignant lesions of the oral cavity, with any potential systemic toxicity being abrogated by cetuximab. In summary this study has found that GRP78 is up-regulated in head and neck cancers suggesting that this protein may be important for tumour development and survival. Thus inhibition of GRP78 through EGF-SubA may offer a novel approach to cancer therapy. In addition to suppressing the growth of LSCC cell, EGF-SubA was found to enhance the effects of relevant genotoxic agents in vitro of cisplatin and radiation. Further work is now warranted in order to assess the efficacy and toxicity of EGF-SubA, in vivo, before phase I clinical trials can commence.

Neuroblastoma is a paediatric cancer that arises from the sympathetic ganglia and adrenal medulla. Tumours with MYCN amplification have the worst prognosis, and make up around 30% of neuroblastoma diagnoses. Neuroblastoma exhibits an unusually high propensity for spontaneous regression, which occurs most frequently in the youngest patients (under 18 months of age), perhaps because developmental cues still present prompt belated differentiation of the tumour cells. This led to the hypothesis that factors from an appropriate embryonic environment may be capable of activating the correct molecular switches to encourage the differentiation and/or apoptosis of neuroblastoma cells, reprogramming the tumour to a benign phenotype. To test this hypothesis, EGFP-labelled MYCN-amplified Kelly cells were injected into the extra-embryonic vitelline veins of embryonic day 3 (E3) and E6 chick embryos, and the responses of injected cells were analysed at E10 and E14. Kelly cells injected at E3 respond to neural crest migratory cues and integrate into neural crest-derived tissues: some neural, notably the sympathetic ganglia and enteric nervous system, although never the adrenal gland; and others non-neural, such as the meninges and tail. Cells injected at E6 do not show such targeting, integrating into various tissues such as the liver, kidney and meninges. The cells respond to their respective microenvironments, and in sympathetic ganglia some cells differentiate, show reduced cell division, and crucially such cells have undetectable MYCN expression by E10. In non-neural locations, cells form more rapidly dividing clumps and continue to express MYCN. The downregulation of MYCN is dependent on continuous and direct interaction with the sympathetic ganglion environment. Kelly cells’ morphology, behaviour and gene expression are altered by the sympathetic ganglia microenvironment. Taking these key observations, we speculate that the Kelly cells’ MYCN amplicon may likely contain the required DNA regulatory sequences to enable MYCN expression to be altered in response to the embryonic environment. If the factors responsible for MYCN repression can be elucidated, they may represent effective new therapeutic targets.