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Ph.D., Faculty of Health Sciences, University of the Witwatersrand, 2011 / Oesophageal squamous cell carcinoma (OSCC) has a high prevalence in the Asiatic belt and areas of Africa. In South Africa (SA), the incidence of this cancer in the Eastern Cape is one of the highest in the world. The molecular carcinogenesis of this disease remains unresolved. Single nucleotide polymorphism (SNP) array technology provides a high resolution technique to determine DNA copy number imbalances across the whole genome. DNA copy number changes can affect oncogenes and tumour suppressor genes, contributing to carcinogenesis. The aim of this study was to map common chromosomal break points previously identified in five SA OSCC cell lines by multi colour fluorescence in situ hybridisation (FISH) and to characterise copy number changes in these cell lines and OSCC patient’s specimens using SNP array technology. Genome wide copy number analysis was performed on the cell lines and 51 OSCC retrospective samples from the Eastern Cape region using Affymetrix® 500K SNP arrays. A number of genes were significantly affected by copy number changes across specimens. The copy number status of some of these candidate genes identified by arrays, were verified by (FISH) in a subset of the samples. Expression of the EPHA3, FGF3, FGF4, FGF19 and C-MYC candidate genes was assessed in the cell lines and four fresh samples. The common translocation break point previously detected in 5 cell lines involving chromosome 3p11.2 correlated with deletions affecting the EPHA3 gene in 4 of the 5 cell lines and was deleted in 74% of the OSCC cohort. EPHA3 is an ephrin A3 receptor tyrosine kinase that has been shown to have both oncogenic and tumour suppressor functionality. In addition, significant regions of amplification and deletion identified genes (CCND1, C-MYC, FHIT, SFRP1, SFRP2, FGF3, FGF4, FGF19, SMAD4, SMAD6 and FBXW7) involved in the Wnt, TGF-β and FGF. Deletion of the genes, WRN, ATM, RAD18 and XRCC4 involved in DNA repair pathways, may contribute to genetic instability that is characteristic of OSCC. This study has highlighted some molecular pathways that may contribute to better understanding carcinogenesis of OSCC in South Africa.

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Faculty of Science School of Pathology 9900713m Cell #: 083 718 9093 / Oesophageal cancer (OC) is the third most common malignancy in South Africa (SA), affecting 1 in 20 and 1 in 76 black males and females respectively. Squamous cell carcinoma (SSC) is an aggressive disease showing a poor prognosis due to late diagnosis. Identification of genetic changes associated with these tumours may shed light on its pathophysiology and aetiology in SA. The chromosomal status of five OC cell lines, established in SA, was assessed to identify possible common chromosomal alterations by M-FISH (multicolour fluorescence in situ hybridisation) and specifically the fragile site loci, FRA3B and FRA16D by FISH (Fluorescence in situ hybridisation). The genes at these loci, FHIT (Fragile Histidine Triad) and WWOX (WW domain containing oxidoreductase) respectively, were analysed by RT-PCR (Reverse transcriptase polymerase chain reaction). FHIT was aberrantly expressed in four of the five cell lines while WWOX expression was normal. The EGFR (epidermal growth factor receptor) locus is frequently amplified and this gene is also over-expressed in OC. Increased EGFR expression was previously found in three of the cell lines, for this reason, particular attention was paid to markers involving the EGFR locus on 7p. An interesting marker chromosome seven was identified in one of the cell lines and further analysis, using a specific EGFR probe, revealed an amplification unit involving EGFR in this cell line. Common translocations involving chromosomes 3 and 1 as well as 3 and 22 were identified in two cell lines; these may involve a locus involved in OC and warrants further investigation.

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.

In this thesis, the relationships among oesophageal motility, bolus clearance and sensory perception of oesophageal stimuli in patients with several dysphagia syndromes were investigated. The work is divided into the following major sections: 1) Current advances in the application of impedance and its utility in distinguishing clearance characteristics between primary and secondary peristalsis; 2) The advances in our understanding of peristaltic motor characteristics, oesophageal bolus clearance and symptom perception in dysphagia syndromes; 3) Peristaltic dysfunction, impaired bolus clearance and symptom perception in gastro-oesophageal reflux disease (GORD) and in patients with globus; 4) TRPV1 expression in oesophageal mucosa in patients with GORD. The main findings from this work are: 1) Secondary peristalsis is less effective as primary peristalsis regarding esophageal transit and clearance of a liquid bolus. 2) In patients with non-obstructive dysphagia (NOD), bolus clearance by both morphologically normal and aberrant secondary peristaltic sequences is impaired. 3) Although, when compared with healthy controls, patients with NOD have a higher prevalence of non-specifically abnormal motor patterns, there is a poor correlation between dysphagia and oesophageal dysmotility. 4) Whereas manometry identified motility abnormalities in one quarter of patients with GORD, impedance demonstrated that the majority of these patients, as well as some patients with normal manometry, had defective bolus clearance. 5) Although patients with erosive GORD have delayed oesophageal bolus clearance, manometric characteristics in these patients are comparable to those seen in non-erosive reflux disease (NERD). These findings are compatible with the hypothesis that abnormal oesophageal bolus clearance may reflect a continuum of dysfunction secondary to increasing oesophageal mucosal damage. 6) Patients with globus are characterized by oesophageal visceral hypersensitivity and aberrant viscerosomatic referral of mechanical and electrical stimuli to the oesophagus. These findings support the hypothesis that oesophageal hypersensitivity with associated viscerosomatic referral patterns are an important pathogenetic mechanism for globus. 7) Patients with erosive GORD exhibit greater gene expression of TRPV1 in oesophageal mucosa when compared with NERD or healthy controls. These findings support the hypothesis that chronic inflammation may lead to the release of mediators which may modulate function of primary sensory neurons.

In this thesis, the relationships among oesophageal motility, bolus clearance and sensory perception of oesophageal stimuli in patients with several dysphagia syndromes were investigated. The work is divided into the following major sections: 1) Current advances in the application of impedance and its utility in distinguishing clearance characteristics between primary and secondary peristalsis; 2) The advances in our understanding of peristaltic motor characteristics, oesophageal bolus clearance and symptom perception in dysphagia syndromes; 3) Peristaltic dysfunction, impaired bolus clearance and symptom perception in gastro-oesophageal reflux disease (GORD) and in patients with globus; 4) TRPV1 expression in oesophageal mucosa in patients with GORD. The main findings from this work are: 1) Secondary peristalsis is less effective as primary peristalsis regarding esophageal transit and clearance of a liquid bolus. 2) In patients with non-obstructive dysphagia (NOD), bolus clearance by both morphologically normal and aberrant secondary peristaltic sequences is impaired. 3) Although, when compared with healthy controls, patients with NOD have a higher prevalence of non-specifically abnormal motor patterns, there is a poor correlation between dysphagia and oesophageal dysmotility. 4) Whereas manometry identified motility abnormalities in one quarter of patients with GORD, impedance demonstrated that the majority of these patients, as well as some patients with normal manometry, had defective bolus clearance. 5) Although patients with erosive GORD have delayed oesophageal bolus clearance, manometric characteristics in these patients are comparable to those seen in non-erosive reflux disease (NERD). These findings are compatible with the hypothesis that abnormal oesophageal bolus clearance may reflect a continuum of dysfunction secondary to increasing oesophageal mucosal damage. 6) Patients with globus are characterized by oesophageal visceral hypersensitivity and aberrant viscerosomatic referral of mechanical and electrical stimuli to the oesophagus. These findings support the hypothesis that oesophageal hypersensitivity with associated viscerosomatic referral patterns are an important pathogenetic mechanism for globus. 7) Patients with erosive GORD exhibit greater gene expression of TRPV1 in oesophageal mucosa when compared with NERD or healthy controls. These findings support the hypothesis that chronic inflammation may lead to the release of mediators which may modulate function of primary sensory neurons.

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.