ABSTRACT Background Oesophageal cancer has a high mortality; it is the 6th most common cause of death due to cancer worldwide. Of the common subtypes of oesophageal cancer, it is the adenocarcinomas that have been rising rapidly in incidence throughout the western world. The incidence of adenocarcinomas now exceeds the previously common squamous cell carcinoma. These recent changes in the incidence patterns of oesophageal cancer suggests that the environmental risk factors associated with these subtypes differ, and that changes in the prevalence of these exposures over time are the most likely explanation for the observed shifts in the incidence. However, due to its low incidence until a few decades ago, the adenocarcinoma subtype has been less studied compared to squamous cell carcinoma, and the environmental factors associated with this cancer have not been so clearly defined. Smoking and alcohol have been the strongest environmental risk factors reported for oesophageal squamous cell carcinoma (OSCC) whereas for oesophageal adenocarcinoma (OAC), the effect of smoking appears to be weaker, and the evidence for an effect of alcohol is scant and inconsistent. However, epidemiologic studies consistently identify people with frequent symptoms of gastro-oesophageal reflux (GOR) as having the highest risk of OAC, but the effect of GOR on OSCC has been negligible. Furthermore, it has been argued that adenocarcinoma occurring at the gastro-oesophageal junction (GOJAC) may have different aetiology again. Together, these reports suggest the three subtypes of oesophageal cancers (OAC, GOJAC and OSCC) may arise through different mechanisms with different strengths in the impact of risk factors. This thesis investigated the independent associations of smoking, alcohol and gastro-oesophageal reflux on cancers of the oesophagus by considering the possibility of variation in the risks due to differences in the dose effect patterns of various measures such as smoking, alcohol and GOR. Method Data from a population-based case-control study of oesophageal and ovarian cancers in Australia were used. Study participants comprised histologically confirmed cases of OSCC (n=308), OAC (n=367) and GOJAC (n=426) who were frequency matched to 1580 controls from the general population. Exposure history for both cases and controls were derived from health and lifestyle questionnaires. Unconditional multivariate logistic regression was used to calculate the odds ratios and 95% confidence intervals for the risk factors analysed. In addition, generalised additive model with a logit link was also used to explore and present the non-linearity in the dose effect pattern for continuous exposures adjusting for other confounding factors. The effects of two exposures combined on these cancers were assessed by obtaining synergy index. Results Smokers were at significantly higher risk of all three subtypes of oesophageal cancer with the risk greatest for OSCC. The effect of smoking was greater for adenocarcinoma occurring at the gastro-oesophageal junction compared to that of the oesophagus. Of the various measures of smoking, duration was significantly associated with all three subtypes of cancer whereas intensity was associated with only OSCC and GOJAC and the dose effect was non-linear. Time since quitting was associated with a steady decline in risk of all three cancers emphasising the health benefits of quitting among smokers. Alcohol was not associated with OAC or GOJAC but was significantly associated with OSCC among those drinking in excess of 170g/week. The association between alcohol and OSCC was modified by smoking; the association with alcohol was significantly greater among current smokers with effect. Low to moderate wine consumption was associated with significant risk reduction for all three cancers compared to non-drinkers. Increased frequency of GOR symptoms was associated with increased risks of OAC and GOJAC, although the risk of OSCC was constrained to frequent GOR symptoms only. The effect of GOR symptoms were exacerbated by smoking whereas it was weakened by regular NSAID use. Lastly, the sensitivity analysis that assessed the effect of non-participation among controls in the estimated effect of smoking and BMI (the two risk factors most likely to be affected by non-participation) showed a slight overestimation of effect of smoking assuming higher exposure rate among non-participants but not BMI while the effect remained strong and statistically significant. Conclusion Smoking, alcohol and GOR symptoms were the environmental factors strongly associated with all subtypes of oesophageal cancers. However, the dose effect patterns of these exposures varied by cancer subtypes. Smoking and alcohol were the larger contributing factors for OSCC whereas smoking and GOR symptoms had greater impact on OAC and GOJAC. Low to moderate wine consumption and regular NSAID use reduced the risk of all three subtypes significantly. While selection bias may have led to mildly inflated risks for smoking, the effects persisted even when modelled under extreme scenarios of biased participation amongst controls, and there was no evidence that selection bias materially affected the other associations.

ABSTRACT Background Oesophageal cancer has a high mortality; it is the 6th most common cause of death due to cancer worldwide. Of the common subtypes of oesophageal cancer, it is the adenocarcinomas that have been rising rapidly in incidence throughout the western world. The incidence of adenocarcinomas now exceeds the previously common squamous cell carcinoma. These recent changes in the incidence patterns of oesophageal cancer suggests that the environmental risk factors associated with these subtypes differ, and that changes in the prevalence of these exposures over time are the most likely explanation for the observed shifts in the incidence. However, due to its low incidence until a few decades ago, the adenocarcinoma subtype has been less studied compared to squamous cell carcinoma, and the environmental factors associated with this cancer have not been so clearly defined. Smoking and alcohol have been the strongest environmental risk factors reported for oesophageal squamous cell carcinoma (OSCC) whereas for oesophageal adenocarcinoma (OAC), the effect of smoking appears to be weaker, and the evidence for an effect of alcohol is scant and inconsistent. However, epidemiologic studies consistently identify people with frequent symptoms of gastro-oesophageal reflux (GOR) as having the highest risk of OAC, but the effect of GOR on OSCC has been negligible. Furthermore, it has been argued that adenocarcinoma occurring at the gastro-oesophageal junction (GOJAC) may have different aetiology again. Together, these reports suggest the three subtypes of oesophageal cancers (OAC, GOJAC and OSCC) may arise through different mechanisms with different strengths in the impact of risk factors. This thesis investigated the independent associations of smoking, alcohol and gastro-oesophageal reflux on cancers of the oesophagus by considering the possibility of variation in the risks due to differences in the dose effect patterns of various measures such as smoking, alcohol and GOR. Method Data from a population-based case-control study of oesophageal and ovarian cancers in Australia were used. Study participants comprised histologically confirmed cases of OSCC (n=308), OAC (n=367) and GOJAC (n=426) who were frequency matched to 1580 controls from the general population. Exposure history for both cases and controls were derived from health and lifestyle questionnaires. Unconditional multivariate logistic regression was used to calculate the odds ratios and 95% confidence intervals for the risk factors analysed. In addition, generalised additive model with a logit link was also used to explore and present the non-linearity in the dose effect pattern for continuous exposures adjusting for other confounding factors. The effects of two exposures combined on these cancers were assessed by obtaining synergy index. Results Smokers were at significantly higher risk of all three subtypes of oesophageal cancer with the risk greatest for OSCC. The effect of smoking was greater for adenocarcinoma occurring at the gastro-oesophageal junction compared to that of the oesophagus. Of the various measures of smoking, duration was significantly associated with all three subtypes of cancer whereas intensity was associated with only OSCC and GOJAC and the dose effect was non-linear. Time since quitting was associated with a steady decline in risk of all three cancers emphasising the health benefits of quitting among smokers. Alcohol was not associated with OAC or GOJAC but was significantly associated with OSCC among those drinking in excess of 170g/week. The association between alcohol and OSCC was modified by smoking; the association with alcohol was significantly greater among current smokers with effect. Low to moderate wine consumption was associated with significant risk reduction for all three cancers compared to non-drinkers. Increased frequency of GOR symptoms was associated with increased risks of OAC and GOJAC, although the risk of OSCC was constrained to frequent GOR symptoms only. The effect of GOR symptoms were exacerbated by smoking whereas it was weakened by regular NSAID use. Lastly, the sensitivity analysis that assessed the effect of non-participation among controls in the estimated effect of smoking and BMI (the two risk factors most likely to be affected by non-participation) showed a slight overestimation of effect of smoking assuming higher exposure rate among non-participants but not BMI while the effect remained strong and statistically significant. Conclusion Smoking, alcohol and GOR symptoms were the environmental factors strongly associated with all subtypes of oesophageal cancers. However, the dose effect patterns of these exposures varied by cancer subtypes. Smoking and alcohol were the larger contributing factors for OSCC whereas smoking and GOR symptoms had greater impact on OAC and GOJAC. Low to moderate wine consumption and regular NSAID use reduced the risk of all three subtypes significantly. While selection bias may have led to mildly inflated risks for smoking, the effects persisted even when modelled under extreme scenarios of biased participation amongst controls, and there was no evidence that selection bias materially affected the other associations.

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Master of Science - Biology / á-catenin plays a crucial role in cell adhesion. Expression levels of á-catenin have been shown to be decreased in almost all tumours studied. The levels of the epidermal growth factor receptor (EGFR) were shown to be increased in oesophageal squamous cell carcinoma (OSCC) cell lines. á-catenin therefore, may play a part in linking the EGF pathway or other signal transduction pathways, bringing about some of the changes in the OSCC cell lines. The á-catenin gene from five OSCC cell lines was sequenced. Three out of five OSCC cell lines studied were found to harbour mutations. One of the mutations resulted in a change in the amino acid sequence of á-catenin. It was concluded that this alteration may not have affected the functioning of á-catenin. á-catenin was largely expressed at the plasma membrane with some weaker cytoplasm/nuclear expression occurring in all of the OSCC cell lines. Treatment of the OSCC cells with EGF for a 12 hour period resulted in no noticeable change in the expression levels of á- catenin. The results obtained from this study indicated that á-catenin could play a role in signal transduction pathways in the OSCC cell lines.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Medicine in the branch of Radiation Oncology Johannesburg, 2016 / Purpose: To assess the improvement in swallowing status, overall survival and treatment related complications in patients with Carcinoma of the Oesophagus treated with palliative radiotherapy. Methods: A retrospective analysis of patients with advanced squamous cell carcinoma of the oesophagus who were treated for palliation from May 2007 to June 2012 at the Charlotte Maxeke Johannesburg Academic Hospital (CMJAH) was done. Ninety- nine patients received palliative radiation therapy during this period, 63% were male and 37% female with a mean age of 60, 6 years. The predominant site of lesion was middle 3rd (56%) and 86, 9% of patients had lesions more than 5cm in length. Patients received palliative External beam irradiation (EBRT) with or without High dose rate brachytherapy (HDRBT) as per the CMJAH, Department of Radiation Oncology protocol. Results: There was an overall significant improvement in swallowing status (p<0,001). Eighty –four patients (85%) had an improvement in swallowing score after treatment. The effect of treatment was not significant in the relationship between the change in swallowing status and treatment group. Overall mean time to progression was 3, 7 months. The median overall survival was 7, 7 months. The type of treatment did not affect survival significantly, unadjusted (p=0, 31) or adjusted for prognostic parameters (age, sex, length of lesion, site of lesion, and pre-treatment swallowing status) (p=0.29). There were treatment related complications in 32% of cases, consisting of ulcerations (24%), tracheo- oesophageal fistula (5%) and strictures (3%). Conclusion: In patients with advanced squamous cell oesophageal carcinoma, palliative radiotherapy is an effective modality in improving a patient’s dysphagia and thus quality of life. / MT2016

Epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase which activates, upon EGFR binding, a number of signaling pathways including the mitogenic protein kinase pathway (MAPK) and phosphatidylinositol 3-kinase cascade (PI3K). Over expression of EGFR is a common feature in variety of human cancers including lung, colorectal, breast, pancreatic and oesophageal cancers and results in autonomous cell growth, enhanced metastatic potential, tissue invasion and increased resistance to current cancer therapeutics. Thus EGFR has been identified as a potential target in cancer therapeutics. Using the RNA interference (RNAi) pathway, the aim was to specifically knockdown expression levels of endogeneous EGFR in human oesophageal squamous carcinoma cell (HOSCC) lines. The RNAi pathway was initiated through the transfection of three specifically designed short hairpin RNAs (shRNAs) against human EGFR. The shRNAs were specifically designed using bioinformatics tools and their individual knockdown efficacy determined through the introduction of an exogeneous based target reporter systems, psiCHECK and pcieGFP. Expression levels of EGFR were determined using Western blot analysis followed by densitometry. Knockdown of EGFR was achieved by all three EGFR shRNAs in the three HOSCC cell lines (WHCO1, WHCO5 and WHCO6) despite low transfection levels of ~10%. Greastest knockdown of EGFR (85%) was achieved by EGFR sh2 in WHCO5. EGFR sh2 and sh1 resulted in average knockdown of EGFR of ~ 65% in WHCO1 and WHCO5 respectively. Weakest knockdown of EGFR (~ 20%) was obtained by all three EGFR shRNAs following transfection of WHCO6. RNAi-based approaches therefore show substantial potential for the specific and efficient targeting of EGFR in human cancer cells.

ABSTRACT MUC1, a mucin-like transmembrane glycoprotein, is highly overexpressed and aberrantly localized in several invasive carcinomas. MUC1 is proposed to play numerous roles in the transformed behaviour of cells in which it is expressed. A number of these roles are facilitated by the interaction of MUC1 with β-catenin, a protein that is central to both cellular adhesion as well as Wnt-responsive gene transcription. The aim of this study was to investigate MUC1 expression, localization, and interaction with β-catenin, as a means of providing insight into the behaviour of human oesophageal squamous cell carcinoma. This cancer-type is exceptionally aggressive and is a major cause of cancer-related morbidity and mortality in South Africa. MUC1 is expressed and aberrantly localized in oesophageal squamous cell carcinoma cell lines, as demonstrated by RT-PCR, western blotting and indirect immunofluorescence. Moreover, evidence from coimmunoprecipitation assays shows that the MUC1 cytoplasmic tail and β-catenin form a complex both at the cell membrane and importantly, within the nucleus of these cell lines. This is the first demonstration of such a complex in the nucleus of a carcinoma derived from stratified, as opposed to simple, epithelia. Data presented here further indicates that activation of the epidermal growth factor receptor results in modulation of the association between MUC1 and β-catenin at the cell membrane. MUC1 membrane-localization, and interaction with β-catenin, may modulate cellular adhesion through steric interference of cell surface adhesion molecules as well as through sequestration of β-catenin away from adherens junctions. On the other hand, MUC1 association with β-catenin may enhance β- catenin signalling either through the stabilization of β-catenin, or as an essential functional component of the β-catenin/LEF/TCF transcription factor complex. Furthermore, results presented in this study identify oesophageal squamous cell carcinoma as a prime candidate for MUC1-specific immunotherapy. This finding is of substantial importance considering the ineffectual nature of existing therapies used in the treatment of oesophageal carcinoma.

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.