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This thesis is primarily concerned with oesophageal motility, but certain aspects of the mechanism of deglutition were also studied in detail, either deliberately or by chance. · All dissections, experiments and microscopical and other examinations were conducted by the author and some specimens personally blocked, Out and stained. The liberal use of illustrations clarifies many points which otherwise would not have been so convincingly described; they also ,emphasise strongly the wide variation of normal, which unfortunately we only too often lose sight of in medicine. The literature on this wide subject is so vast that it cannot be completely covered or referred to in a thesis of this nature. The references at the end of this thesis are mainly recent and should, give the·reader·some indication of the divergence of opinion and current concepts on this complex subject.

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Introduction: Barrett’s oesophagus (BO) is a metaplastic premalignant disease which can undergo a metaplasia-­‐dysplasia-­‐adenocarcinoma pathway. It represents an example of field cancerization by which an area occupied by BO can undergo molecular and genetic changes associated with carcinogenesis without being phenotypically cancerous. Previous work suggested that non-­‐cancerous BO contains a monoclonal population. More recent work demonstrated that premalignant Barrett’s fields are polyclonal suggesting that clonal interactions may be important in carcinogenesis. It is the aim of this thesis to further investigate clonal interactions in BO by understanding the effects of therapy in altering the relationships of clonal populations in BO, by assessing the relationship of clonal populations in dysplasia as compared with the associated cancer, and by attempting to elucidate a potential molecular mechanism of clonal interactions. Results: The overall results can be summarised as follows: 1.Premalignant clonal populations are well mixed allowing for clonal interactions. However, the adenocarcinoma associated with high grade dysplasia is monoclonal and derived from clonal populations found in the dysplasia, indicating possible clonal interactions during carcinogenesis. 2. Patients with persistent disease after endoscopy retain the same clonal populations. However, the clonal populations of recurrent disease changes such that new clonal populations arise or may benefit from the extinction of others. 3. These clonal populations may be derived from deep submucosal glands or may be found in phenotypically normal squamous epithelium indicating a common stem cell origin. 4. A possible mechanism of clonal interaction may be the senescence associated secretory phenotype: senescence is abundant in BO and can cause proliferation in neighbouring cells in vitro. Conclusion: This thesis has investigated the implications of clonal interactions in BO. The demonstration of temporal clonal heterogeneity as a result of endoscopic therapy, as well as spatial clonal heterogeneity possibly resulting in carcinogenesis, asks for a mechanistic explanation of clonal interactions. The consequences of senescence may well provide one such mechanism.

p21 C/P 1/"El and p 2 7K/P 1 are cyclin-dependant kinase inhibitors that fonn an integral part of the cell cycle process. These proteins function as cell-cycle inhibitors, and are able to induce cell cycle arrest by binding to cyclin complexes at key stages. p21 and p27 have been found to be down-regulated in various cancers. This study investigated aberrations at microsatellite markers linked to the p21 and p27 cell cycle genes, in a large cohort of oesophageal squamous cell carcinomas in South Africa. Fluorescent-based PCR were performed on markers linked to both the p21 and p27. The products were run with a 50-500hp marker on 6% denaturing polyacrylamide gels, on the ALFexpresstm' DNA sequencer. The detection and analysis of PCR products was achieved using the AL F e xp res sT M and Fragment M an a aerTm software programmes. Our findings indicate that markers linked to p27 display infrequent aberrations, with loss of heterozygosity ranging from 19% to 37%, and microsatellite instability at 3% to 7%. However, significant relationships between decreased survival time, and aberrations in markers DI2S391 and Dl2S364, were found to exist. Marker D6S1575 linked to p21 displayed frequent allelic loss at 47%, and was comparable to similar studies on the 6p region Further, LOH-Al in this marker was found to be significantly associated with poorly differentiated tumours. The findings from our study indicate that microsatellite aberrations occur infrequently at the p21 and p27 loci in oesophageal cancer. with the exception of marker D6S1575. In addition,this study clearly demonstrates the accuracy and sensitivity of the technology employed. This is the first microsatellite-based investigation of the p21/p27 gene loci in oesophageal cancer in South Africa, using a fluorescent-based PCR assay. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2008.

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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.