Barrett's metaplasia describes a condition in which the stratified squamous epithelium of the oesophagus switches to intestinal type columnar epithelium. The molecular mechanisms underlying the switch to intestinal epithelium is poorly understood but the transcription factor CDX2 has been implicated in the pathogenesis of Barrett's metaplasia and is sufficient to provoke an intestinal metaplasia in the stomach of transgenic mice. To address the molecular basis of Barrett’s, I developed an innovative explant system for adult mouse oesophageal epithelium in which the full repertoire of stratified squamous cell types is maintained for prolonged culture periods. In adult oesophageal cultures, cells expressing p63, K14, K4 and loricrin were detected. The ability of Cdx2 to induce intestinal genes in this model as well as in a human oesophageal cell line and foetal mouse oesophageal cultures was assessed. Cdx2 was sufficient to induce intestinal markers in Het-1A cells and foetal oesophageal epithelium but not in adult oesophageal explants. Following infection with Cdx2, Het-1A cells expressed four intestinal genes, Cdx1, Muc2, villin and K20. Embryonic oesophagus responds similarly and Muc2 and villin mRNA and Muc2 protein were detected. In contrast, infection of adult oesophageal explants did not provoke the expression of intestinal genes. These data suggest that additional factor(s) to Cdx2 are required in the conversion of adult oesophagus towards an intestinal phenotype as seen in Barrett's metaplasia. HNF4α is a candidate factor to cooperate with Cdx2 in intestinal development and therefore Barrett's metaplasia. Herein I demonstrate that HNF4α is sufficient to induce a columnar phenotype and the expression of intestinal genes within adult squamous oesophageal cells. The resultant phenotype is consistent with that seen in Barrett's metaplasia. Furthermore HNF4α and Cdx2 synergise to further enhance intestinalisation. This data suggests a hitherto unknown potential role for HNF4a in Barrett’s metaplasia.

Barrett's oesophagus (BO) is the major precursor of oesophageal adenocarcinoma (OA) and we do not understand the dynamics of the evolution of BO in order to identify patients at high risk of cancer. Studies have proposed that BO is a monoclonal lesion, however recent work has shown that there are multiple independent clones present. Project 1: Determines the evolution of polyclonal dysplasia through sequencing and mapping clones onto tissue sections. I show that several cases are polyclonal but in each case only one clone progresses to cancer, suggesting oesophageal cancers are monoclonal outgrows from polyclonal Barrett's dysplasia. Project 2: Aims to understand the clonal relationship between cells in glands displaying basal crypt dysplasia-like atypia (BCDA), as it is unclear whether those cells in the upper part of the gland arise from the same stem cell that generates the gland bases. Glands displaying BCDA show a common mutation between the dysplastic base and non-dysplastic surface suggesting a common cell of origin. Project 3: 50% of patients who undergo oesophagectomy for OA develop post-oesophagectomy Barrett's (neo- BO) within 3-5 years possibly due to a field effect, wherein pre-neoplastic cells remain post-resection in histologically normal areas of epithelium predisposing the patient to cancer recurrence. Here I show that no genetic link between the neo-BO and the cancer is present. Immunohistochemical analysis shows that neo- Barrett's glands are gastric in nature. Project 4: The stem cell dynamics and clonal expansion rates of BO are unknown. Here I employed diversity analysis of methylation patterns of CpG islands in the promoter regions of non-expressed genes as a molecular clock. My data suggests that 3-4 stem cells are found in each Barrett's gland. Methylation patterns within a gland were less diverse compared to adjacent and distant glands, suggesting BO is characterized by long periods of stasis followed by bursts of clonal expansions.

Introduction: The main risk factor for the development of oesophageal adenocarcinoma is Barrett’s oesophagus (BE). To diagnose those patients who will progress to cancer early to improve the dismal survival rate of oesophageal adenocarcinoma, patients with BE undergo regular endoscopic surveillance. The vast majority of patients, however, will never progress and are therefore monitored unnecessarily. Copy number changes have been shown to be important in the progression of BE to oesophageal adenocarcinoma (Li et al., 2014). Shallow whole genome sequencing (sWGS) has been established as a cost-effective method of investigating copy number changes in formalin fixed paraffin embedded (FFPE) tissue (Scheinin et al., 2014). We hypothesised that copy number alterations may be valuable markers in disease progression and aimed to characterise them in the progression of Barrett’s using sWGS in order to predict progression in patients from a point in time as close to baseline endoscopy as possible and to integrate p53 staining. Methods: To optimise sWGS we compared 50X WGS on frozen tissue with 0.1X WGS from FFPE tumour material from the same patient. To address poor cellularity in endoscopic biopsies, shallow WGS data from a 50% cellularity biopsy with a 90% frozen sample from a single patient were compared. Accounting for poor biopsy cellularity 0.4X coverage was used. We performed FFPE shallow WGS on 806 samples from an 89-patient cohort comprising a 1:1 ratio of patients who progressed to high grade dysplasia (HGD) and patients who never progressed. 1-31 samples per patient were collected over time and space throughout surveillance. Non-progressors had significantly longer follow-up (p-value = 0.0008). Data was processed based on published bioinformatic pipelines. Copy number analysis was carried out using a generalised linear model (GLM) in order to develop a predictive algorithm. Results: During optimisation, ˃85% of copy number changes were detected in both frozen and FFPE samples from spatially distinct regions of an individual tumour. We found 91% and 93% agreement in copy number calls using orthogonal platforms between 90% (frozen) and 50% (FFPE) cellularity samples from one tumour. In the 806 sample Barrett’s cohort, we observed larger copy number alterations in patients who progressed to cancer compared with non-progressors and significantly more CN alterations in progressor patients (p-value ˂ 0.001). More cancer-associated genes were affected in progressors and we observed significant heterogeneity between patients. There was also a greater level of complexity seen in the progressor patients when analysed using affinity propagation clustering. These data allowed us to develop a regression model to predict progression. Using the GLM model, we successfully classified samples as early as progressor or not with an AUC of 85.75% and a sensitivity and specificity of 84 and 79% respectively. At the patient level 94% progressor patients had at least one sample classified as at risk of progression and non-dysplastic progressor samples were classified as early as 13 years prior to HGD diagnosis. Depending on the classification threshold used, all samples over time and space were not classified as being at risk of progression in at least 60% patients who have not yet progressed to HGD/cancer. We observed 2 pathways to progression supporting previous observations. 90% of progressors had samples prior to their HGD or cancer diagnosis classified as being predisposed to progression suggestive of genetically unstable lesions from early on in surveillance that progressed to HGD over time. The remaining 10% appeared as non-progressors until their diagnosis of HGD. We investigated p53 expression in our patient cohort as the only biomarker to have successfully transitioned into the clinic for Barrett’s surveillance. Whilst we found our cohort to be representative in staining compared to other published cohorts, it did not contribute to the GLM and the copy number data out-performed the use of p53 IHC in the context of Barrett’s surveillance. Conclusions: We have optimised the use of shallow WGS in oesophageal adenocarcinoma and Barrett’s. Using these copy number data, we can confidently distinguish between patients who will progress to cancer and the majority of patients who will never progress. This approach has led to the development of a model for predicting progression in the clinical setting which is promising for further clinical validation.

No / Neoadjuvant chemoradiotherapy (neo-CRT) prior to surgery is the standard of care for oesophageal adenocarcinoma (OAC) patients. Unfortunately, most patients fail to respond to treatment. MiR-187 was previously shown to be downregulated in neo-CRT non-responders, whist in vitro miR-187 overexpression enhanced radiosensitivity and upregulated PTEN. This study evaluates the role of miR-187 and downstream PI3K signalling in radiation response in OAC. The effect of miR-187 overexpression on downstream PI3K signalling was evaluated in OAC cell lines by qPCR and Western blotting. PTEN expression was analysed in OAC pre-treatment biopsies of neo-CRT responders and non-responders. Pharmacological inhibition of PI3K using GDC-0941 was evaluated in combination with radiotherapy in two-dimensional and three-dimensional OAC models in vitro and as a single agent in vivo. Radiation response in vitro was assessed via clonogenic assay. PTEN expression was significantly decreased in neo-CRT non-responders. MiR-187 overexpression significantly upregulated PTEN expression and inhibited downstream PI3K signalling in vitro. GDC-0941 significantly reduced viability and enhanced radiation response in vitro and led to tumour growth inhibition as a single agent in vivo. Targeting of PI3K signalling is a promising therapeutic strategy for OAC patients who have repressed miR-187 expression and do not respond to conventional neo-CRT. This is the first study evaluating the effect of PI3K inhibition on radiosensitivity in OAC, with a particular focus on patients that do not respond to neo-CRT. We have shown for the first time that targeting of PI3K signalling is a promising alternative therapeutic strategy for OAC patients who do not respond to conventional neo-CRT.

Oesophageal adenocarcinoma (OAC) is a leading cause of cancer death in the UK and current systemic therapies are ineffective for the majority of patients. The central aim of this work was to explore strategies to identify novel therapeutic targets. Research has failed, thus far, to identify a dominant oncogene in OAC, although the tumour suppressor p53 is frequently mutated. Inhibiting the mitotic kinase, polo-like kinase 1 (PLK-1), was proposed as a synthetic lethal strategy. PLK-1 was demonstrated to be over-expressed in both verified OAC cell lines and human OAC tissue compared to non-transformed cells and epithelium. Mutation of p53 was associated with over-expression of PLK-1 in both OAC and ovarian cancer tissue. Using a carefully validated viability assay, both an established and novel PLK-1 inhibitor were demonstrated to induce a G2/M arrest and reduce OAC cell proliferation. Relative selectivity was demonstrated for OAC compared to non-transformed cells. This therapeutic window could be enhanced with the induction of cancer cell cytotoxicity by pulsed administration of a short half-life inhibitor. Immunotherapeutics offer potential tumour-selectivity but no OAC-specific proteins have been defined. A comparative proteomic approach was employed to identify OAC-specific proteins as potential therapeutic targets. A tissue resource was established and methods to lyse fresh frozen biopsies optimised. An isobaric quantitative proteomic workflow was applied to OAC and matched normal biopsies and quantitative accuracy confirmed for 6 candidate proteins by immunohistochemistry. Proteome coverage and quantitative dynamic range were compared between isobaric and label-free systematic sequencing proteomic strategies applied to further patients’ tissues. The challenges of combining incomplete datasets were approached with a Bayesian framework to estimate the probability that a protein was missed during an experiment compared to not being present in the sample. This method was applied to generate a complete set of protein identifications and relative tissue expression. To gain insight into the dysregulated cellular processes in human OAC tissue, a network analysis was applied to the quantitative proteomic data. Enriched functional clusters were identified suggesting deranged glucose metabolism, potentially due to the Warburg effect. These findings were duplicated and candidate tumour-specific proteins identified in a further set of biopsies using the optimised quantitative proteomic method. The combined quantitative oesophageal proteomic dataset represents the largest in OAC to date. This thesis demonstrates a hypothesis-driven, synthetic lethal approach can yield cancer-selective therapeutic effects. Novel candidate therapeutic targets are also revealed through the development of quantitative proteomic methods and the application of network analysis.

The UK has experienced an increase in the incidence of oesophageal adenocarcinoma (OAC) in recent years. The prognosis for patients with OAC remains poor with currently available treatments prompting a search for alternative ‘chemopreventive’ treatments that inhibit oesophageal carcinogenesis. Both non-steroidal anti-inflammatory drugs (NSAIDS) and flavonoids are associated with a significant risk reduction for developing OAC in epidemiological studies. The aim of this study was to validate Levrat’s surgical model of OAC in the rat, and assess the chemopreventive effects of the NSAID aspirin, and the flavonoid quercetin on the development of OAC in the validated rat model. METHODS: Levrat’s model was validated in a time course experiment. Morphological and molecular events occurring in the distal oesophagus during disease progression were determined and compared to human disease. The effect of aspirin and quercetin on disease initiation and progression was determined by commencing treatment either before the onset of reflux, or 4-weeks afterwards. The incidence of Barrett’s oesophagus (BO) and OAC within each group was determined, along with methylation levels of the ESR-1, p16 and HPP1 gene promoter regions. RESULTS: The morphological and molecular changes in the distal oesophagus of the rat model are broadly consistent with those reported in human disease. The incidence of OAC was significantly lower in aspirin treated rats. A non-significant reduction in incidence of OAC was observed with quercetin treatment. Timing of treatment with regard to onset of reflux had no significant effect on OAC development in either treatment group. Neither treatment significantly effected methylation levels within the gene promoters examined. CONCLUSION: Use of Levrat’s model as a model of human OAC seems justified. Aspirin inhibits development of oesophageal adenocarcinoma induced by reflux in this rat model. No additional reduction in cancer incidence is observed if treatment is commenced prior to inception of reflux disease.

Barrett's oesophagus (BO) arises from chronic gastro-oesophageal reflux disease(GORD). Patients have an increased risk of adenocarcinoma (ADC), which is the sixth most common cause of cancer mortality in the UK. All ADC develop from BO, and over the last twenty years there has been a marked increase in both conditions. The reasons for this are not known, however, as with some forms of gastric cancer, it is possible that there may be a bacterial aetiology. This study employed both culturebased and molecular techniques to characterise microbial communities colonising the distal oesophageal mucosae in individuals with GORD, BO and ADC, together with healthy controls. Furthermore, in vitro models were designed to create an oral microbiota, from which an oesophageal community could develop. Microbial analysis identified a shift in oesophageal population composition with disease progression, with an incremental increase in total eubacterial scores related to the metaplasia-dysplasia sequence. Additionally, an increased proportion of Gram negative species and potentially pathogenic organisms, such as Peptostreptococcus were identified. Campylobacter spp. were isolated from 75%, 50% and 60% of GORD, BO and ADC patients, respectively, compared with 20% of controls. Helicobacter pylori, which has been proposed to be protective in oesophageal disease, was significantly reduced in disease, especially in ADC patients. In vitro models were successful, with a simple oral microbiota leading to the development of unique, varied oesophageal populations representative of those found in vivo. Additionally, after exposure of this community to bile acid, population dynamics were altered, with an increase in Gram negative species, associated with a rise in haemolytic and mucinolytic activities. Exposure of oesophageal cell lines to these stressed biofilms resulted in increased cell death, and in some cases, amplified expression of p53 and COX-2. In conclusion, this research proved an association between bacterial composition and oesophageal disease. With progression to adenocarcinoma, the community becomes increasingly diversified and Gram negative in character, and therefore, is proposed to be more pathogenic. Further research is required to investigate causal relationships, through which mechanisms for disease initiation and/or maintenance can be understood.