This thesis examines factors influencing the outcomes of patients receiving multidisciplinary stage-directed treatment for oesophagogastric cancer. The hypotheses tested were: 1.The TNM7 staging system is a more accurate prognostic tool for oesophageal cancer (OC) than TNM6. 2.Use of CT-PET upstages a significant number of patients with occult metastases. 3.OC recurrence patterns differ following definitive chemoradiotherapy (dCRT) and surgery, but overall recurrence rates and survival are comparable for advanced stage disease. 4.An involved circumferential resection margin (CRM+) following oesophagectomy is associated with poorer survival and its incidence can be reduced with neoadjuvant chemoradiotherapy. 5.Early enteral nutrition improves clinical outcomes following upper GI cancer resection. 6.Centralisation of oesophagogastric cancer (OGC) surgery in S.E. Wales is feasible and associated with improved clinical outcomes. Reclassification with TNM7 resulted in stage re-categorisation of 11.9% of OC patients. Multivariate analysis indicated only TNM7 prognostic group to be independently and significantly associated with survival. CT-PET upstaged OC M stage in 24.0% of patients. Loco-regional OC recurrence was commoner after dCRT (p<0.0001) but distant recurrence commoner after surgery (p=0.001). Disease-free survival was better after surgery for stage I (p=0.069) and II (p=0.011) but comparable with dCRT for stage III (p=0.878) and IV (p=0.710). CRM+ occurred in 38.0% of all OC patients, and 62.4% of pT3 patients. Multivariate analysis revealed lymphovascular invasion (p<0.0001) and CRM+ (p=0.002) were independently and significantly associated with disease-free survival. Multivariate analysis revealed EUS T stage (p<0.0001) and neoadjuvant chemoradiotherapy (p<0.0001) were independently associated with CRM+. Early enteral nutrition (EEN) was associated with reduced hospital stay (p=0.023) and less operative morbidity (p=0.044) than control management, due to fewer wound infections (p=0.017), chest infections (p=0.036) and anastomotic leaks (p=0.055). Following centralisation, OGC critical care (p<0.0001) and total hospital stay (p=0.037) were significantly reduced. Serious operative morbidity (Dindo-Clavien grade III+) decreased from 33.3% to 16.7% (p=0.066).

CREB, cAMP responsive element binding protein is a positive regulatory protein transcriptional factor, for genes including aromatase, an enzyme that converts androgens to oestrogens, c-fos, tyrosine hydroxylase and neuropeptides like somatostatin and enkephalin. The expression of aromatase is highly aberrant in human breast cancer and has been implicated in the disease progression. Aromatase expression in breast cancer tissue is directed mainly by promoters 1.3 and II. CRE1 and CRE2 are essential for cAMP induced promoter II activity. CRE binding protein (CREB) bound to this element (CRE) and that this interaction was enhanced in the presence of cAMP. Despite the extensive work on aromatase, little information is available on the expression and role of CREB in human breast cancer. The aim of this study was to investigate the molecular impact of CREB family of proteins on the aggressive nature of breast cancer cells and to investigate the expression pattern in breast cancer tissues in relation to tumour histopathological grade, stage, nodal status and the clinical outcome of the patients. In this study we examined the expression of CREB1 and ATFs (Activating transcription factors) in breast cancer cell lines using RT-PCR, which allowed us to design the strategy of in vitro experiments. Ribozyme knockdown technology was used to target the expression of CREB1 in a breast cancer cell line MDA-MB-231. Knockdown of CREB1 using ribozyme transgenes resulted in decrease in in vitro cell growth and invasiveness in breast cancer cells. The results presented here demonstrate that the level of CREB-1 and ATFs in breast cancer patients was elevated. The study results presented here revealed a significant link between CREB and mortality, in that high levels are associated with shorter disease free survival and interestingly we found significantly low levels of ATFs in patients with poor prognosis, metastatic disease and nodal involvement. We conclude that the level of CREB-1 and ATFs are aberrantly expressed in human breast cancer which may be associated with disease progression in breast cancer patients and has significant bearing to the clinical outcome of the patients. Over-expression of aromatase in adipose tissue surrounding breast tumour could arise through increase in both CREB expression and CREB transcriptional activity. Inhibition of CREB activity could inhibit aromatase expression and hence decrease oestrogen production in breast tissue. An understanding of the molecular mechanisms of expression of CREB, together with aromatase between non-cancerous and cancerous breast tissue at both transcriptional and translational levels may help in the design of a therapy based on suppressing aromatase expression in breast cancer tissues.

Wnt signalling components are reported to be deregulated in breast cancer but the contribution of this pathway in endocrine resistance is less clearly defined. Endocrine resistance is an important clinical challenge affecting up to a quarter of all breast cancer patients and is associated with a poorer clinical prognosis. This project focussed on exploring the role of Wnt signalling in endocrine resistant breast cancer cell models. Wnt pathway elements were deregulated in the acquired tamoxifen resistant cell line (Tam-R) compared to tamoxifen sensitive parental cells (MCF-7), with changes supportive of Wnt signalling activation in this tamoxifen resistant model apparent from Affymetrix HGU-133A gene microarray data and Western blot analysis. In contrast, Wnt signalling appeared to be suppressed based on Affymetrix data for MCF-7 cells treated with oestradiol for 10 days, with equivocal changes in MCF-7 cells treated with tamoxifen for 10 days or a faslodex resistant cell model (Fas-R). Excitingly, Tam-R cells were also more sensitive than MCF-7 cells to pharmacological manipulation of Wnt signalling. While Wnt activation using Wnt3a and LiCl did not affect cell growth or migration, inhibition of Wnt signalling usingIWP2, PNU 74654 and iCRT14 suppressed Tam-R cell growth and migration. There is mounting evidence of cross talk between Wnt and EGFR signalling in breast cancer, and EGFR activity is upregulated in Tam-R cells. The project’s findings tentatively supported cross-talk between the two signalling pathways in this model. Thus, targeting of the Wnt pathway alongside EGFR blockade was superior in suppressing cell growth and migration in Tam-R cells. The effect appeared to be more pronounced when Wnt signalling was inhibited at the nuclear level using iCRT14. Collectively, these data suggest that Wnt signalling may play an important role in tamoxifen resistance where it may offer an opportunity for more effective therapeutic intervention to control relapse and associated tumour aggressiveness.

DNA methylation helps regulate transcriptional activity and is widely studied in cancer biology. This investigation aimed to establish the significance of Human Papillomavirus (HPV) DNA methylation in HPV-associated disease both in terms of basic biology and as a potential biomarker. Assays to assess DNA methylation and gene expression were developed and evaluated. Pyrosequencing was used to assess DNA methylation of four regions of the HPV16 genome (E2, L1/L2, enhancer, promoter). Gene expression was assessed using quantitative PCR with assays for E2, E6 and E7. HPV integration was assessed using Detection of Integrated Papillomavirus Sequences (DIPS). The relationship between HPV methylation, gene expression and integration was explored in vitro and in vivo using cell cultures and clinical cohorts. A variety of sample materials were used including short term and immortal cell lines, cervical cancer biopsies, cytology samples and Vulval Intraepithelial Neoplasia (VIN) biopsies. In general, hypermethylation of the HPV genome was associated with low HPV gene expression and the presence of integrated HPV genomes. To better understand the potential clinical utility of HPV DNA methylation, the relationship between HPV DNA methylation and various stages of cervical disease was determined. The HPV genome was progressively hypermethylated with increasing severity of cervical disease and certain regions of the HPV genome were more affected than others. A longitudinal study was also performed in order to determine a relationship between HPV methylation and clinical outcome. Differences in HPV methylation among patients who had persistent HPV infection and low grade disease, persistent infection and high grade disease and patients that cleared HPV infections were observed. Throughout the study the potential application of a HPV biomarker was considered and the correct biomarker design procedures were referred to. Several of the early biomarker development steps were successfully achieved.

Cervical cancer is the second most common cancer among women worldwide. Infection with Human Papillomavirus (HPV) is essential but not the only contributing factor in cervical cancer development. HPV integration is reported to be present in over 80% of cervical cancers and disruption of HPV genome through integration leads to high levels of HPV oncogene expression. DNA damage and repair pathways are thought to induce HPV integration since HPV is detected at fragile sites in the human genome. There is controversy as to whether integration is an early or late event in cervical oncogenesis and there are no published studies to date that have investigated HPV integration using sensitive, DNA based, techniques at the nucleotide level in cervical precancers. This study aimed to test the hypothesis that integration is an early event in cervical neoplasia and episomal loss causes malignant transformation through transcription of integrated HPV. Also, this study served to pilot whether HPV integration can predict high-grade cervical disease in women with cytological abnormalities with an aim to improve current cervical screening methods. Assays to detect integration and E2 as a marker of episomal state were developed for HPV16, HPV18 and HPV45 and applied to cervical smears and biopsies from women with varying disease grades. The data presented in this thesis highlight that integration may not be essential for cervical cancer progression and different modes of disease progression may exist between young women and older women. Integration was detected at chromosome fragile sites but was more prevalent at SINE or LINE repeat elements; this implies a role for retroelements in the mechanism of integration. Finally, the data here suggest that integration induces a unique selective process in each individual and clonal selection may arise due to altered HPV oncogene expression and/or disruption to human gene expression.

LKB1 has been implicated in a wide range of cellular functions and is associated with many potential substrates in in vitro studies, however the in vivo role of LKB1 remains unclear and its precise contribution to the prevention of intestinal tumours in the hereditary Peutz-Jegers syndrome is as yet uncharacterised. Conditional deletion of LKB1 in the murine small intestine resulted in significant disruption of intestinal homeostasis, particularly that of the differentiation process, suggesting LKB1 plays a key role in intestinal differentiation and it is loss of this function that predisposes to tumourigenesis

Psychology uses theory supported by empirical evidence to accumulate generalisable knowledge and learn from various contexts. Two commonly used theories are the Theory of Planned Behaviour (TPB) and Common-Sense Self Regulation Model (CS-SRM). Possible limitations in previous studies using these theories have been identified: (1) TPB studies focus upon one specified behaviour and ignore behavioural alternatives; (2) the CS-SRM is routinely not fully operationalised. This research aimed to develop an ‘extended TPB’ assessing ‘intention choice’ (where participants report their intended course of action regarding one or more specified behavioural alternatives) and to operationalise the CS-SRM more fully than is usually reported. The context was people with urological cancer. Methods: Three studies were conducted: (1) CS-SRM-based longitudinal study of people with urological cancer (n=172) to predict anxiety and depression; (2) Extended-TPB-based prospective study of men with localised prostate cancer (n=35) using both between- and within-person approaches to explore intention choice with respect to treatment; (3) Extended-TPB-based before-after study of medical students (n=93) to evaluate the effects of teaching on simulated communication behaviour. Results: (1) Anxiety and depression at Time 2 were predicted by number of information sources reported at Time 1 after controlling for baseline anxiety and depression; (2) TPB constructs were consistent with intention choice and intention choice was consistent with actual treatment. Within-persons, the model could not identify men who later reported poor outcomes; (3) All TPB-related cognitions changed between Time 1 and Time 2 but simulated behaviour scores did not change. Discussion: Relationships between the TPB constructs both between- and within-persons were consistent with actual treatment. The extended TPB measures were sensitive to change when behavioural alternatives were assessed. The CS-SRM was operationalised beyond illness representations. Conclusions: It was feasible to apply both the extended TPB (between- and within-persons) and CS-SRM in this context.

Automatic segmentation of tumours using Positron Emission Tomography (PET) was recommended for radiotherapy treatment (RT) planning of head and neck (H&N) cancer patients, and investigated in the scientific literature without reaching a consensus on the optimal process. This project aimed at evaluating the performance of PETCbased automatic segmentation (PETCAS) methods and developing an optimal PETC AS process to be used at Velindre Cancer Centre (VCC). For this purpose, ten algorithms were implemented to represent the most promising PETCAS approaches from a systematic review of the literature. The algorithms’ performance was evaluated on filled phantom inserts with variable size, geometry, tumour intensity and image noise. The impact of thick insert plastic walls on both image quantification and segmentation was thoroughly assessed. The PETCAS methods were further applied to realistic H&N tumours, modelled using a printed subresolution sandwich phantom developed and calibrated in house. Results showed that different PETCAS performed best for different types of target objects. An Advanced decision TreeCbased Learning Algorithm for Automatic Segmentation (ATLAAS) was therefore developed and validated for the selection of the optimal PETCAS approach according to the target object characteristics. Finally, a protocol was designed for the use of PETCAS within RT planning at VCC. The protocol was used retrospectively on a group of 10 oropharyngeal cancer patients, and the results highlighted the additional information brought by PET beyond anatomical imaging. In a prospective study on 10 additional patients, PETCAS replaced manual PET/CT delineation, and accounted for up to 33% of the modifications of manually drawn CT/MRI contours to derive the final planning contour. This study demonstrated the usefulness and reliability of the PETCAS method in RT planning, and led to modifying the clinical workflow for H&N patients at VCC. This work has the potential to be extended to other tumour sites and institutions.

Cell penetrating peptides (CPP) are short amino acid sequences with the potential to be used as vectors for delivering macromolecular therapeutics into cells. Five CPPs [R8, FFR8, (RXR)4, TP10 and PFV] were studied in primary human chronic lymphocytic leukaemia (CLL) cells using fluorescence-labelled CPPs. Uptake, sub-cellular localisation and toxicity were studied by confocal microscopy and flow cytometry. Two of the CPPs were selected, based on their cellular uptake and intracellular distribution characteristics, and used as delivery vectors for peptide-based NF-κB inhibitors. Four novel NF-κB inhibitory CPPs directed against p50 and p65 subunits were tested in primary CLL cells. Apoptosis was measured using AnnexinV/PI labelling and a caspase-3 activity assay by flow cytometry. Apoptosis was evident after one hour in cells treated with TP10-p50i and TP10-p65i and the LC50 of TP10-p50i and TP10-p65i was 6 μM and 10 μM respectively at 24 hours. This represents a ten-fold increase in toxicity when compared to the commercially available CPP NF-κB-inhibitors. Western blot analysis of NF-κB subunit translocation revealed NF-κB inhibition in some of the samples treated with TP10-p50i. However, the effects of the peptide varied from sample to sample. Studies using EMSA to measure NF-κB DNA binding revealed similar inconsistencies, even when CLL cells were stimulated with CD40L or CpG. Flow cytometic analysis of cell surface makers in CLL cells demonstrated that TP10-p50i did not alter the expression of CD69, a cell surface molecule regulated by NF-κB, indicating that the variations seen previously by EMSA and western blotting did not result from direct NF- κB inhibition. Although the exact mechanism of action of TP10-p50i was not determined, the cytotoxic effects observed with TP10-p50i are not likely to be related to a modulation of NF-κB activity.

Class of 2012 Abstract / Specific Aims: To evaluate disease- and patient-related characteristics, mortality, and charges associated with non-AIDS defining malignancies (NADM) among inpatient settings in the United States from 2005 to 2009. Methods: This retrospective cohort investigation utilized nationally-representative hospital discharge records from the Agency for Healthcare Research and Quality (AHRQ) Healthcare Cost and Utilization Project (H-CUP) Nationwide Inpatient Sample. Inclusion criteria included adult inpatients ≥18 years with a diagnosis of HIV or AIDS and malignant neoplasms. Multivariate regression analyses were used to assess inpatient mortality and charges. Main Results: Overall, 104,488 were included. Average age associated with each case was 46.9 years (±10.66), with 21.9% cases being female (n=22,868). The mean length of stay was 8.6 days (±10.5) and inpatient mortality occurred in 7.7% of cases (n=8,035). The mean number of procedures performed was 2.3 (±2.5) and the mean number of diagnoses on record was 9.5 (±4.4). Charges for each episode of care averaged $59,483 (±85,748), summing to a national bill of $6.14 billion (2011 dollars) over the five-year course. A higher number of cases were associated with teaching hospitals (74.1%), the south (42%), large metropolitan areas (75.1%), median household income in the 0-25th percentile (41.2%), and Medicaid payers (34.3%). Increased mortality was associated with increased age, increased number of diagnoses and procedures, and the comorbidities of anemia, coagulopathy, lymphoma, and fluid and electrolyte disorders. Conclusions: This investigation of NADMs suggest a considerable clinical and economic burden of illness, summing to a 7.7% inpatient death rate and $1.3 billion in charges per year.