Thesis (MSc)--Stellenbosch University, 2010. / ENGLISH ABSTRACT: Oesophageal cancer (OC) is a disease characterised by development of malignant tumours in the cell lining of the oesophagus. The disease is divided into two subtypes, which shows marked ethinic variation, with adenocarcinoma (ADC) being more prevalent in Caucasians and squamous cell carcinoma (SCC) more prevalent in the Black population. There are several factors that have been associated with OC development, including oesophageal injury and inflammation, as well as excess iron, which contributes to increased tumour growth. Investigation into OC development is essential due to the rapidly increasing incidence rates and the poor survival rate of this complex disease. The present study aimed to investigate nucleotide variation in the promoter region of SLC40A1, a gene implicated in iron metabolism, in a Black South African OC population. The patient group encompassed 80 (41 males and 39 females) unrelated patients presenting with SCC of the oesophagus. The control group consisted of 71 unrelated, healthy population-matched control individuals. The techniques applied for mutation detection in this study included polymerase chain reaction (PCR) amplification, heteroduplex single stranded conformation polymorphism (HEXSSCP) analysis, restriction fragment length polymorphism (RFLP) analysis and hybridisation probe analysis. Identified variants were confirmed by bi-directional semi-automated DNA sequencing analysis. Statistical analysis was performed to determine associations between identified variants and disease incidence, as well as between identified variants and various iron parameters. Mutation analysis of the promoter region of SLC40A1 resulted in the identification of nine previously described (-1470C/T, -1461T/C, -1399G/A, -1355G/C, -1098G/A, -750G/A, -501T/C, - 23A/G and -8C/G) and three novel, (-1087G/C, -663C/T and -637G/A) variants as well as a previously described trinucleotide repeat. Statistical analyses revealed statistically significant association between -501T/C and OC in this population (P = 0.004). Statistical investigation of the effect of the variants on iron parameters revealed various statistically significant associations. The survival rate of OC remains poor due to absence of early symptoms and therefore late diagnosis of the disease, after which treatment is highly ineffective. Treatment of OC would significantly improve with earlier detection and treatment. This can be achieved by establishing a screening programme in the populations of high risk areas, such as the Transkei area in Southern Africa. Therefore investigation into nucleotide variation of potential modifier genes is of great importance to improved diagnosis, treatment and counselling to individuals presenting with OC. To our knowledge, this is the first study investigating the promoter region of SLC40A1 and possible associations with iron dysregulation in the Black South African population with OC. / AFRIKAANSE OPSOMMING: Oesofageale kanker is ‘n siekte wat gekenmerk word deur die ontwikkeling van kwaadaardige gewasse in the selvoering van die oesofagus. Die siekte word verdeel in twee subtipes wat opvallende etniese variasie toon, met adenokarsinoom wat meer algemeen in die Kaukasiese populasie voorkom en plaveisel selkarsinoom wat meer algemeen in die Swart populasie is. Daar is verskeie faktore wat verbind word met die ontwikkeling van oesofageale kanker, insluitend oesofageale besering en ontsteking, asook ‘n oormaat yster wat bydra tot verhoogde gewasgroei. Ondersoek met betrekking tot die ontwikkeling van oesofageale kanker is noodsaaklik as gevolg van die verhoogde voorkoms-tempo en die swak oorlewingsyfers van hierdie komplekse siekte. Die huidige studie het beoog om die nukleotied variasie in die promoter area van SLC40A1, ‘n geen betrokke in yster metabolisme, in ‘n Swart Suid-Afrikaanse oesofageale kanker populasie te ondersoek. Die pasiënt-groep het bestaan uit 80 (41 mans en 39 vrouens) onverwante pasiënte by wie plaveisel selkarsinoom van die oesofagus voorgekom het. Die kontrole groep het bestaan uit 71 onverwante, gesonde bevolkings-soortgelyke individue. Die tegnieke wat gebruik is vir mutasie opsporing in hierdie studie sluit in: polimerase kettingreaksie amplifikasie, heterodupleks enkelstring konformasie polimorfisme (HEX-SSCP) analise, restriksie fragment lengte polimorfisme (RFLP) analise en hibridisasie peiler analise. Geïdentifiseerde variante is bevestig deur tweerigting semi-geoutomatiseerde DNS volgorde-bepalingsanalise. Statistiese analise is uitgevoer om moontlike assosiasies tussen geïdentifiseerde variante en siekte voorkoms, sowel as tussen geïdentifiseerde variante en verskeie yster parameters te bepaal. Mutasie analise van die promoter area van SLC40A1 het gelei tot die identifikasie van nege voorheen bekende (-1470C/T, -1461T/C, -1399G/A, -1355G/C, -1098G/A, -750G/A, -501T/C, - 23A/G and -8C/G) en drie nuwe (-1087G/C, -663C/T and -637G/A) variante, sowel as ‘n bekende trinukleotied herhaling. Statistiese analise het getoon dat daar ‘n statistiese betekenisvolle assosiasie tussen -501T/C en oesofageale kanker in hierdie populasie voorkom (P = 0.004). Statistiese ondersoek van die effek van die geïdentifiseerde variante op yster parameters het verskeie statisties betekenisvolle assosiasies getoon. Die oorlewingsyfers van oesofageale kanker bly laag as gevolg van die afwesigheid van vroeë simptome en dus word die siekte eers op ‘n laat stadium gediagnoseer, waarna behandeling hoogs oneffektief is. Behandeling van oesofageale kanker sou betekenisvol verbeter met vroegtydige identifikasie en behandeling. Dit is bereikbaar deur die vestiging van ‘n siftingsprogram vir die populasies van hoë risiko areas, soos die Transkei area in Suidelike Afrika. Ondersoeke na die nukleotied variasie van potensiële modifiserende gene kan daarom van groot belang wees vir verbeterde diagnose, behandeling en berading van individue met oesofageale kanker. Sover as wat ons kennis strek, is hierdie die eerste studie wat die promoter area van die SLC40A1 geen en die moontlike effek op yster disregulasie in ‘n Swart Suid-Afrikaanse populasie met oesofageale kanker ondersoek.

Introduction The prognosis in oesophago-gastric cancer is poor with less than 15% patients surviving beyond 5 years after diagnosis. The addition of neoadjuvant therapy has been shown to increase survival in patients suitable for curative surgery. However, the additional gains are modest and the majority of patients do not respond sufficiently from therapy to gain any benefit. There is an urgent need to identify markers that can predict response to neoadjuvant therapy in order provide safer, more effective, individualised treatment regimes. Methods A prospective, multi-centre, collaborative study was undertaken in patients with oesophago-gastric cancer undergoing neoadjuvant therapy and potentially curative surgery. Levels of circulating biomarkers M2-Pyruvate kinase, alkaline phosphatase, CA19-9, CEA and CA 72-4 were measured in patients before and after administering the first cycle of chemotherapy. Binary logistic regression analysis was performed to assess the ability of biomarkers to predict histological response to therapy. Results 165 patients were recruited to the main study. 105 patients had complete histopathological data for analysis. There were 27 responders and 78 non-responders to neoadjuvant therapy. There were no differences in pre-therapy demographic, pathological or treatment factors between the two groups. Responders had less post-operative lymphovascular invasion (P= 0.004) and higher R0 resection rates (P=0.03). Pre-therapy M2-Pyruvate kinase levels were lower in responders compared to non-responders (P=0.037) and levels were able to predict response with each unit increase in the biomarker level being associated with a 4.1% decrease in the likelihood of response (P=0.027). M2-PK levels were not associated with any pre-operative demographic, clinical or pathological factors. Conclusions Pre-therapy dimeric M2-PK levels can predict response to neoadjuvant therapy in patients with oesophago-gastric cancer. The test could be of clinical value for 1 in every 8 patients undergoing the test.

Background: Oesophagogastric cancers generally present late in life with advanced disease and carry a poor prognosis. Few patients receive curative treatment. Polo-like Kinase 1 (PLK1) is a mitotic kinase with regulatory functions at the G2/M cell cycle phase transition. In mammalian cells, PLK1 phosphorylates and activates FOXM1, a forkhead transcription factor at the G2/M cell cycle phase transition. FOXM1 then promotes transcription of multiple gene products, including PLK1 and CCNB1, which then act individually or in complexes to further phosphorylate FOXM1 generating a positive feedback loop driving the cell into M phase. Aims: We aimed to assess the expression of PLK1 and FOXM1 in oesophageal and gastric cancer patients. Secondly we aimed to investigate the expression and inter- relationship of PLK1 and FOXM1 in oesophageal cell lines during the cell cycle. Results: FOXM1 and PLK1 are expressed in oesophageal cell lines and demonstrate cross-regulatory interactions. Inhibition of PLK1 leads to the decreased expression of FOXM1 and it’s target gene in oesophageal cell lines. FOXM1 and PLK1 are also concomitantly overexpressed in a large proportion of oesophageal and gastric carcinoma’s at both the protein and mRNA level. Other FOXM1 target genes including, CCBN1, AURKB and CKS1 are co-expressed in a similar manner. In a homogenous cohort of patients who underwent surgery, the expression of PLK1 and AURKB was prognostic for overall survival. Conclusions: This study has demonstrated that FOXM1 and a number of target genes including PLK1 are coordinately expressed in a proportion of oesophageal and gastric carcinomas. This suggests that chemotherapeutic treatments that target this pathway may be of clinical utility.

Purpose: In patients with advanced oesophageal carcinoma palliation of dysphagia is important to maintaining a reasonable quality of life. The primary aim of this study was to determine the dysphagia progression-free survival (DPFS) in patients with advanced oesophageal carcinoma treated with palliative radiotherapy (RT). Methods: The medical records of all patients with oesophageal carcinoma presenting to Groote Schuur Hospital, Cape Town between January 2015-December 2016 were reviewed and patients who were not candidates for curative treatment and received palliative RT were selected. For these patients, the dysphagia score (DS) was recorded prior to RT, 6 weeks after RT and at each follow-up visit. The DPFS was calculated as the time from completion of RT to worsening in DS by ≥1 point or until death. Other outcomes measured were objective change in DS and survival post RT. Results: The study population comprised 84 patients. Squamous cell cancer was the primary histological subtype (93%). The median duration of DPFS after RT was 73 days, with approximately two-thirds of patients remaining able to swallow at least liquids and soft diet until death. The difference in median duration of DPFS was not statistically significant in stented versus non-stented patients (54 days vs 83 days; p =0.224). The mean change in DS was 0.45 ± 0.89 points following RT and the post RT survival was significantly shorter in patients with stent insertion (81 days vs 123 days; p=0.042). Conclusion: Palliative RT can be used successfully to prolong DPFS in patients with locally advanced and metastatic squamous cell cancer of the oesophagus.

Estimates for 2017 indicate that 20% of cancers globally are gastrointestinal tract (GIT) cancers, with oesophageal cancer being the 8th most common cancer. Oesophageal squamous cell carcinoma (OSCC) occurs in the upper to mid oesophagus and is present at high incidence in developing countries including South Africa. There are no early symptoms, resulting in late diagnosis and poor prognosis. In this study, tumour and blood DNA was obtained from 35 OSCC patients and subjected to whole genome sequencing (WGS). Bioinformatics analysis pipelines were designed to identify the possibility of novel viral insertions, investigating Human Endogenous Retroviruses (HERV's) insertions alongside the presence of somatic mutations in patient samples. The aims being to identify integration of any foreign DNA, to investigate if there is any linkage between HERV insertion and somatic mutations, and to identify any somatic mutations of potential interest in the OSCC cohort. The novel virus investigations however, proved to be inconclusive and there appeared to be no link between HERV insertions and somatic mutations present in the patients. Very significantly, it was determined that numerous somatic mutations were present in the MUC3A gene of the patient cohort, an interesting observation as no such previous association with OSCC has been recorded. MUC3A is a membrane-bound glycoprotein component of mucous gels, and its aberrant expression has been correlated with invasion and metastasis in a variety of other cancers. However, due to the complexity of the particular gene sequence and the known inconsistencies of variant calling performed on complex data sets, these mutations should be viewed with extreme caution as they are likely to be false positives. Analysis of RNA-seq data showed a 4.6 log2 fold increase in MUC3A expression in the tumour samples of these OSCC patients, with a P-adjusted value of 7.05e-06, suggesting highly significant differential gene expression. Functional enrichment analysis further showed that MUC3A was significantly associated with one of the top 5 gene ontologies (extracellular matrix structural constituent) for molecular function ontology class together with a number of collagen (COL) and MMP genes known to play a role in oncogenic progression and membrane stiffness. GSEA and KEGG analysis indicated predominantly chemokine/cytokine pro-inflammatory enriched pathways. Immunohistochemistry staining showed 10 out of 13 of the samples had no detectable levels of MUC3A protein, suggesting that the production of a non-functional truncated protein may lead to the upregulation of MUC3A expression that could possibly play a role in downstream pro-oncogenic signalling.

This thesis describes investigations into the genetic basis and pathophysiology of three distinct inherited diseases in humans, two of which are strongly associated to the function of the ectodomain sheddase enzyme ADAM17. The first of these is a novel inherited syndrome of neonatal onset inflammatory skin and bowel disease, which is associated in a consanguineous family with homozygous loss-offunction mutations in ADAM17. This thesis describes investigations of the expression and function of ADAM17 – and downstream proteins it regulates – in an individual affected by this disease. This is accompanied by genetic investigations into other individuals suspected of suffering from the same syndrome. The second investigated disease is Tylosis with Oesophageal Cancer (TOC), an inherited cutaneous disease which represents the only known syndrome of familial oesophageal cancer susceptibility. This disease was associated to dominantly inherited mutations in the Rhomboid protein iRHOM2. This work describes investigations of immortalised keratinocyte cell lines and tissues derived from TOC-affected individuals, and illustrates that the pathogenesis of TOC is characterised by increased iRHOM2-dependent activation and activity of ADAM17, and upregulation of the shedding of ADAM17 substrates, particularly in the EGFR ligand family, accompanied by increased desmosome turnover and transglutaminase 1 activity. This pattern of upregulation results in attendant increases in growth factor signalling, proliferation and motility in TOC keratinocytes, dependent on ADAM17. The third focus of this thesis is a life-threatening inherited gastrointestinal disease (accompanied by severe extraintestinal complications) whose symptoms correspond to Cryptogenic Multifocal Ulcerative Stenosing Enteritis. This work describes the identification of mutations in cytosolic phospholipase A2-α (cPLA2α) – an enzyme responsible for arachidonic acid production, the first step in the eicosanoid synthesis pathway – as associated with this condition in a single affected family. The expression and function of cPLA2α in this disease was investigated, using platelet aggregation stimulated by a downstream product of cPLA2α (Thromboxane A2) as a model.

The high incidence of gastrointestinal cancers in the general population and the presence of premalignant dysplastic precursor lesions in the gastrointestinal tract make the gastrointestinal tract an ideal environment to study cancer clonality and clonal expansion. Background: Intestinal metaplastic (IM) glands in the human stomach are clonal, contain multiple stem cells and spread by fission. This mechanism of gland fission causes field cancerisation. We hypothesised that gastric adenocarcinoma (GA) progresses through a series of genetic events arising from a founder mutation. A process analogous to niche succession may also take place in the normal oesophagus. We hypothesise that oesophageal squamous cell cancer occurs by a process of field cancerisation of the oesophagus. RHBDF2 has been identified as the gene responsible for tylosis with oesophageal carcinoma (TOC). We hypothesise that RHBDF2 germline gain of function mutations might be lost during tumour progression in TOC and this might affect iRhom2 localisation in the cell. Methods and results: A cohort of 23 patients with dysplasia and a cohort of 51 GA patients were screened for genes accounting for 75% of all somatic mutations previously reported in GA. Only 13% of dysplastic patients and 31.4% of GA patients had mutations. Three dysplastic patients and six GA patients were analysed by microdissection. Small gastric cancer foci in a cohort of hereditary diffuse gastric cancer (HDGC) patients (n=5) were also screened by laser-capture microdissection (LCM) for mutations in TP53. A cohort of 30 patients was screened for common mutations in OSCC and for RHBDF2 mutations. 36.36% of the patients presented mutations. Three patients with mutations were randomly selected and areas of oesophageal squamous cell dysplasia and OSCC were analysed by LCM. Three TOC patients were also analysed by LCM and immunohistochemistry was performed for iRhom2 and ADAM17. Conclusions: The usual mutational events established for GA development during the metaplasiadysplasia- carcinoma sequence (MCS) do not fit the results from either of our two LCM mutation studies in the human stomach. Dysplasia was shown to be clonal and GA demonstrates genetic heterogeneity through clonal evolution. Field cancerisation could not be detected in HDGC using TP53 as a clonal marker. The low incidence of OSCC patients with mutations implies that other genes may be involved in the premalignant pathway leading to OSCC. Oesophageal squamous cell dysplasia and OSCC demonstrate clonal expansion through tumour progression. RHBDF2 mutations do not occur in sporadic OSCC but germline RHBDF2 mutations can be lost during tumour progression in TOC patients with LOH in 17q. Overall, the somatic mutation theory of carcinogenesis seems to hold true for both the progression to GA and OSCC, as both carcinomas seem to evolve from a single mutated stem cell and acquire genetic heterogeneity as the tumours evolve.

Emerging evidence suggests the immune system has a role in preventing cancer, and in advanced cancer evidence of immune dysfunction is widespread. This project focused on cytotoxic T lymphocyte antigen 4 (CTLA4), a key negative regulator of T cell activation found on dedicated regulatory T cells (Treg) and activated T lymphocytes, and asked whether modulation of immune control with anti-CTLA4 blockade led to significant anti-tumour activity. Clinical and laboratory investigation of anti-CTLA4 blockade using tremelimumab in a phase II trial of second-line therapy in advanced oesophageal and gastric adenocarcinomas was combined with an attempt to establish a suitable pre-clinical model based on therapeutic vaccination against the tumour associated antigen (TAA) 5T4.Eighteen patients received tremelimumab. Most drug-related toxicity was mild but there was a single death due to bowel perforation. Four patients had stable disease with clinical benefit; one achieved a partial response after eight cycles (25.4 months) and remains well on study after four years. Markers of regulatory phenotype, forkhead box protein 3 (FoxP3) and CTLA4, doubled transiently in CD4+CD25high lymphocytes in the first month after tremelimumab before returning to baseline. In contrast, CTLA4 increased in CD4+CD25low/negative lymphocytes throughout the cycle of treatment. Post-treatment expanded Treg expressed FoxP3 without interleukin-2 and their defining suppressive function was not abolished despite prolonged anti-CTLA4 blockade. De novo proliferative responses to TAA 5T4 (8 of 18 patients) and carcinoembryonic antigen (CEA; 5 of 15) were detected. Patients with a post-treatment CEA proliferative response had median survival of 17.1 months compared to 4.6 months for non-responders (p=0.002). Baseline interleukin-2 release after T lymphocyte activation was higher in patients with clinical benefit and toxicity. Heterologous mouse 5T4 (m5T4) vaccination showed some evidence of weak therapeutic benefit, but all tumour models investigated had rapidly lethal kinetics. Specific m5T4 immune responses could be detected by serum antibody ELISA and IFN-gamma ELISPOT assays in naive animals but were lower frequency than published responses to h5T4, and were further attenuated in tumour-bearing animals. The addition of anti-CTLA4 blockade did not result in significant augmentation of m5T4 specific immunity after vaccination in non tumour-bearing animals and combination treatment was ineffective as therapy in this autologous model. Results are discussed in the context of emerging immunotherapeutics in melanoma and prostate cancer. In the absence of supportive data from the model system it would not be appropriate to pursue combination heterologous 5T4 vaccine with anti-CTLA4 blockade, but in view of the unusual durability of the best response to tremelimumab, and in vitro evidence of enhanced proliferative responses to relevant TAA, further investigation of drug activity may be warranted in metastatic gastric and oesophageal second-line treatment.

A remote control sampling technique was developed for the collection of oesophageal fistula samples from goats. Number and size of samples can be varied and collected throughout the day without disturbing the animal's normal feeding behaviour. The equipment developed and tested in this study consists of an oesophageal fistula valve which allows the fistula to be opened and closed, a rechargeable battery pack and motor to operate the valve, a portable radio and receiver to activate the valve motor, and a harness to attach the equipment to the body of the animal In addition, a closing device to effectively seal large oesophageal fistulae (> 1 050 mm²), which in turn is required to accommodate the valve, was developed. During field tests with the fistula valve, 10 % of I 027 sampling attempts failed due to blockage of the valve, and an average of 1.3 incidences of equipment failure were recorded per animal per sampling day, from an average of 9.9 extrusa collections per day. Observed feeding behaviour (grazing/browsing) as well as grass / bush ratio of fistula valve and standard fistula bag samples of four goats, formed the basis for the evaluation of this technique. In addition, extrusa recovery rates, measured under controlled conditions, were used in the evaluation of this sampling method Differences in extrusa composition between the fistula valve and fistula bag techniques varied substantially during the browsing period within a camp and also between camps. Furthermore, during high frequencies of observed grazing, there were large differences between the fistula valve and fistula bag methods. During this study, the fistula valve technique provided a more realistic estimate (R²=.91) of the observed feeding behaviour of goats than the fistula bag technique (R²=.63). Under controlled conditions, the large oesophageal fistula, with or without the valve, enables high and consistent extrusa recovery rates (87 % recovery; SD 7.5). / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1993.

L’incidence de l’adénocarcinome de l’œsophage augmente depuis 30 ans dans les pays occidentaux et son pronostic est sombre. Le reflux gastro-œsophagien et les acides biliaires ont été incriminés dans l’apparition de la muqueuse de Barrett et sa dégénérescence en adénocarcinome, selon la séquence métaplasie/ dysplasie/ adénocarcinome. Au cours de cette séquence carcinogénétique, a été observée, à partir de tissus humain, une augmentation de l’expression des mucines MUC1 et MUC4. MUC1 et MUC4 sont des O-glycoprotéines membranaires impliquées dans les phénomènes de reconnaissance cellulaire et de signalisation intracellulaire. Les mécanismes du reflux gastro-œsophagien conduisant à l’adénocarcinome de l’œsophage (AO) demeurent mal compris. Tout d’abord, nous avons effectué une approche critique d’un modèle de reflux œsophagien induit chirurgicalement chez le rat. Pour cela, nous avons randomisé 108 rats Sprague-Dawley en deux groupes expérimentaux ; ainsi était réalisée une anastomose oesophagoduodenale avec ou sans gastrectomie afin d’induire respectivement un reflux duodéno-oesophagien (groupe RDO, n=63), et un reflux duodéno-gastro-oesophagien (groupe RDGO ; n=45). Les groupes contrôles comprenaient : (i) la réalisation d’une anastomose oesophago-jéjunale sur anse-en-Y (groupe chirurgical contrôle sans reflux), (ii) une laparotomie blanche, (iii) une gastrectomie subtotale pour induire un reflux duodéno-gastrique (groupe RDG), et enfin (iv) la même procédure que dans le groupe RDGO avec administration d’inhibiteurs de la pompe à protons (groupe IPP). Des analyses histologiques et moléculaires sont réalisées sur l’œsophage. La prévalence de l’œsophage de Barrett (OB) de la dysplasie et de l’AO dans les groupes expérimentaux étaient de 41%, 7% et 11% respectivement. Les analyses histologiques et moléculaires dans les groupes RDO, RDGO et RDG suggèrent que l’OB survient selon le schéma d’une métaplasie intestinale de novo et le migration proximale de cellules duodénales. Aucune métastase à distance n’a été identifiée. Les caractéristiques moléculaires de l’OB identifié chez le rat étaient similaires à celles de l’homme. L’OB était plus fréquent, la dysplasie et l’AO moins fréquent dans le groupe RDO que dans le groupe RDGO(44% vs 24% [ P = 0,038] et 7% vs 25% [ P =0,012], respectivement). Des élements de la séquence carcinogénétique survenait de façon moins fréquente dans le groupe IPP que dans le groupe RDGO [P = .019].Il a été mis en évidence une surexpression des gènes de mucines Muc1 et Muc4, de gènes impliqués dans la prolifération, l’invasion et les métastases, l’adhésion cellulaire et dans la voie PI3K , une diminution de l’expression de gènes suppresseur de tumeur dans les lésions de métaplasie et d’adénocarcinome, dans les zones les plus exposées au reflux.Ensuite, nous avons étudié les propriétés biologiques de cellules issues d’un adénocarcinome de l’œsophage humain et mis en évidence que la perte d’expression de MUC1 dans les cellules OE33 (i) diminue fortement leurs propriétés de prolifération, migration et invasion in vitro et la croissance tumorale in vivo, suggérant un rôle majeur de MUC1 dans la tumorigenèse épithéliale si MUC1 est surexprimée (ii) est corrélée avec une diminution de l’expression de Nfκb et PI3K et peut-être corrélé à l’altération des propriétés biologiques, notemment de prolifération (iii) avec une diminution de l’expression de TSG 101 et Mcm6 potentiels marqueurs mis en évidence dans le modèle murin.En conclusion malgré des différences pathophysiologiques avec l’homme, le modèle de reflux oeso-gastro-duodénal reproduit les lésions histologiques et moléculaires de la séquence carcinogénétique de Barrett et a permis d’identifier des protéines associées à la tumorigenèse et dont l’impact sur les propriétés biologiques des cellules tumorales a été confirmé in vitro et qui pourraient être de potentiels biomarqueurs et cibles thérapeutiques dans l’adénocarcinome de l’œsophage. / Incidence of oesophageal adenocarcinoma developed on Barrett oesophagus increases since 30 years and its prognosis remains poor. Gastro esophageal reflux and biliary acid have been incriminated in Barrett oesophagus metaplasia and its degeneration in adenocarcinoma according to the sequence metaplasia/dysplasia/adencrcinoma. During the carcinogenetic sequence, was observed an increase of expression of the mucins MUC1 and MUC4. MUC1 and MUC4 are membrane bound O-glycoprotein implicated in cell recognition and intracellular signaling. The mechanisms of esophageal reflux leading to esophageal adenocarcinoma (EA) remain poorly understood. In a first part we appraised critically an operatively induced chronic reflux rat model.We randomized 108 Sprague-Dawley rats into 2 experimental groups; one was performing esophagoduodenal (ED) anastomosis with or without gastrectomy to induce duodeno-esophageal reflux (DER group; n = 63), and the other involved duodeno-gastro-esophageal reflux (DGER group; n = 45). Control groups included (i) Roux-en-Y esophagojejunal anastomosis, (ii) laparotomy alone, (iii) subtotal gastrectomy to induce duodenogastric reflux (DGR group), and (iv) the same procedure as in the DGER group plus proton pump inhibition (PPI group). The esophagus underwent histologic and molecular analyses.The prevalence of Barrett’s esophagus (BE), dysplasia, and EA in the experimental groups was 41%, 7%, and 11%, respectively. Histologic and molecular analyses in groups DER, DGER, and DGR suggested that BE occurred through de novo intestinal metaplasia and proximal migration of duodenal cells. No distant metastases were identified. The molecular characteristics of both BE and EA were similar to humans. BE was more common, and dysplasia and EA less frequent in the DER groupwhen compared with the DGER group (44% vs 24% [ P = .038] and 7% vs 25% [ P = .012], respectively). Compared with the DGER group, carcinogenic sequence occurred less frequently in the PPI-treated group (P = .019). It was highlighted an overexpression of MUC1 and MUC4 genes, of genes implicated in proliferation, invasion, metastasis, cell adhesion , in PI3K pathways, a diminution of the expression of tumors suppressor genes in metaplasia, adenocarcinoma lesions in the areas most exposed to reflux. Then cell biological properties studies were carried out in vitro in OE33 oesophageal adenocarcinomatous cells. We showed that loss of expression of MUC1 in OE33 cells (i) induced a reduction of their proliferation, migration and invasion in vitro properties , tumor growth in vivo suggesting a major role of MUC1in epithelial tumorigenesis si MUC1 is overexpressed, (ii) is linked with a diminution of the exprssion of nfκb and PI3K and can be correlated with an alteration of the biological properties especially proliferation (iii) is lined with e diminution of expression of TSG101 and MCM6 , potential biomarkers highlighted in the rat model.In conclusion, despite pathophysiologic differences with humans, the rat model of esophagoduodenostomy reproduces accurately histologic and molecular lesions in the carcinogenetic sequence of BE and allowed us to identify novel, tumor-associated proteins that may be potential biomarkers and new therapeutic targets in EA.