Barrett's metaplasia is the only known morphological precursor to oesophageal adenocarcinoma and is characterized by replacement of stratified squamous epithelium by columnar epithelium. The cell of origin is uncertain and the molecular mechanisms responsible for the change in cellular phenotype are poorly understood. We therefore explored the role of two transcription factors, Cdx2 and HNF4α in the conversion using primary organ cultures. Biopsy samples from cases of human Barrett's metaplasia were analysed for the presence of CDX2 and HNF4α. A new organ culture system for adult murine oesophagus is described. Using this, Cdx2 and HNF4α were ectopically expressed by adenoviral infection. The phenotype following infection was determined by a combination of PCR, immunohistochemical and morphological analyses. We demonstrate the expression of CDX2 and HNF4α in human biopsy samples. Our oesophageal organ culture system expressed markers characteristic of the normal SSQE: p63, K14, K4 and loricrin. Ectopic expression of HNF4α, but not of Cdx2 induced expression of Tff3, villin, K8 and E-cadherin. HNF4α is sufficient to induce a columnar-like phenotype in adult mouse oesophageal epithelium and is present in the human condition. These data suggest that induction of HNF4α is a key early step in the formation of Barrett's metaplasia and are consistent with an origin of Barrett's metaplasia from the oesophageal epithelium.

Introduction Barrett’s oesophagus is the condition whereby the normal stratified squamous epithelium at the lower oesophagus is replaced with simple columnar epithelium in the context of gastro-oesophageal reflux disease. This increases the risk of oesophageal adenocarcinoma (OA) ten-fold, and the majority of people diagnosed with OA do not survive for 12 months. Our understanding of the molecular and cellular mechanisms underlying Barrett’s oesophagus is hindered due to lack of at a suitable model. We set out to develop a model from explant tissue biopsies of adult human oesophagus, as opposed to murine models (mice have a keratinised oesophageal epithelium and do not get Barrett’s oesophagus) or the use of cell lines (which do not recapitulate the normal tissue). Method We devised a study protocol and secured ethical approval (REC reference number 13/YH/0197) to obtain forceps biopsy specimens from consenting adults attending for routine diagnostic gastroscopies at the Royal United Hospital Bath. Specimens were then transported to the University of Bath where we have tested multiple culture conditions. These include choice of culture media, substrate (eg glass, plastic – coated or uncoated, semi-permeable membrane at air/liquid interface), cell adhesion factors (eg collagen I, collagen IV, fibronectin, Matrigel®), choice of tissue preparation (enzyme treatment with dispase in the absence or presence of trypsin, dissection with scissors or minced with a blade), plating density, media volume and use of a cell feeder layer. Results We have recruited 42 participants to date and attempted tissue culture on 36 of these. (Biopsies from 6 patients have been processed for RNA analysis.) Over time we have gradually optimised culture conditions. Of the most recent 6 participants, 5 have cultured successfully with cell survival for at least 14 days. Explant colony growth had been particularly vigorous (>12 mm) when plated on Matrigel® and a feeder layer cell line (mitomycin C-inactivated mouse embryonic fibroblast (iMEF) cell line 3T3-Swiss albino, ATCC® CCL-92™) on plastic coverslips. Explants have also adhered and cultured successfully on collagen IV-coated (sc-29010, Santa Cruz Biotech) plastic coverslips without a feeder layer. Explant colonies were fixed/permeabalised before immunofluorescent staining was performed for cytokeratin 14 (K14), smooth muscle actin (SMA), p63, E-cadherin and SLUG antigens. iMEFs are clearly delineated with SMA staining and the explant outgrowths are positive for K14, p63, E-cadherin and SLUG. Conclusion It is possible to culture adult human oesophageal tissue in vitro from standard pinch-biopsy forceps specimens at gastroscopy and we are in the final stages of optimising conditions, confirming characterisation by repeating experiments and devising a protocol. Disclosure of interest None Declared.

Introduction Barrett’s metaplasia (BM) is the substitution of stratified squamous epithelium with intestinal-type columnar epithelium in the distal oesophagus. It is a condition with malignant potential and yet we do not have any satisfactory treatments to reverse it. The cellular and molecular mechanisms are poorly understood, although several transcription factors are implicated including CDX2. We examined expression of CDX2 along the gastrointestinal tract epithelium, as well as more novel transcription factors ISX and HNF4a. Methods Immunohistochemistry protocols were refined for all three transcription factors for formalin-fixed paraffin-embedded sections of forceps biopsies of human tissue from normal and Barrett’s oesophagus, and from the gastro-oesophageal junction, stomach, small and large bowel. Additionally, the oesophagi of 8 patients were biopsied; 5 with a normal oesophagus and 3 with BM, where biopsies were taken from both the mid-oesophagus and the BM segment. These biopsies were processed for RT-PCR to see whether the immunohistochemistry findings could be confirmed at mRNA level. Results Slide section immunohistochemistry demonstrates that ISX, HNF4a and CDX2 all express nuclear antibody staining in small and large bowel epithelium. ISX and HNF4a are additionally expressed in stomach mucosa epithelium. None of the transcription factors are seen in normal oesophagus aside from very sparse cells with ISX, but all three are demonstrated in BM. RT-PCR of all 3 transcription factors for all 8 patients from normal oesophagus samples was negative. All 3 BM samples were positive for HNF4a, and 2 of the BM samples were positive for ISX and CDX2. However, one BM sample was negative for both ISX and CDX2. Figure legend: Both plates demonstrate nuclear ISX (brown), left = ileum, right = Barrett’s metaplasia. Conclusion CDX2 has been intensively studied in BM, but we also show robust expression of ISX and HNF4a. ISX may be a novel key transcription factor in BM, and our findings suggest that the transcription factor profile in BM echoes that found in intestinal epithelium. Our theory is that HNF4a induction occurs earlier than CDX2 in the genesis of BM; it is possible this is an example of an intermediate stage in this process. Disclosure of Interest None Declared

Introduction Barrett’s metaplasia (BM) is the substitution of stratified squamous epithelium with intestinal-type columnar epithelium in the distal oesophagus. It is a condition with malignant potential and yet we do not have any satisfactory treatments to reverse it. The cellular and molecular mechanisms are poorly understood, although several transcription factors are implicated including CDX2. We examined expression of CDX2 along the gastrointestinal tract epithelium, as well as more novel transcription factors ISX and HNF4a. Methods Immunohistochemistry protocols were refined for all three transcription factors for formalin-fixed paraffin-embedded sections of forceps biopsies of human tissue from normal and Barrett’s oesophagus, and from the gastro-oesophageal junction, stomach, small and large bowel. Additionally, the oesophagi of 8 patients were biopsied; 5 with a normal oesophagus and 3 with BM, where biopsies were taken from both the mid-oesophagus and the BM segment. These biopsies were processed for RT-PCR to see whether the immunohistochemistry findings could be confirmed at mRNA level. Results Slide section immunohistochemistry demonstrates that ISX, HNF4a and CDX2 all express nuclear antibody staining in small and large bowel epithelium. ISX and HNF4a are additionally expressed in stomach mucosa epithelium. None of the transcription factors are seen in normal oesophagus aside from very sparse cells with ISX, but all three are demonstrated in BM. RT-PCR of all 3 transcription factors for all 8 patients from normal oesophagus samples was negative. All 3 BM samples were positive for HNF4a, and 2 of the BM samples were positive for ISX and CDX2. However, one BM sample was negative for both ISX and CDX2. Figure legend: Both plates demonstrate nuclear ISX (brown), left = ileum, right = Barrett’s metaplasia. Conclusion CDX2 has been intensively studied in BM, but we also show robust expression of ISX and HNF4a. ISX may be a novel key transcription factor in BM, and our findings suggest that the transcription factor profile in BM echoes that found in intestinal epithelium. Our theory is that HNF4a induction occurs earlier than CDX2 in the genesis of BM; it is possible this is an example of an intermediate stage in this process. Disclosure of Interest None Declared

Introduction Barrett’s oesophagus is the condition whereby the normal stratified squamous epithelium at the lower oesophagus is replaced with simple columnar epithelium in the context of gastro-oesophageal reflux disease. This increases the risk of oesophageal adenocarcinoma (OA) ten-fold, and the majority of people diagnosed with OA do not survive for 12 months. Our understanding of the molecular and cellular mechanisms underlying Barrett’s oesophagus is hindered due to lack of at a suitable model. We set out to develop a model from explant tissue biopsies of adult human oesophagus, as opposed to murine models (mice have a keratinised oesophageal epithelium and do not get Barrett’s oesophagus) or the use of cell lines (which do not recapitulate the normal tissue). Method We devised a study protocol and secured ethical approval (REC reference number 13/YH/0197) to obtain forceps biopsy specimens from consenting adults attending for routine diagnostic gastroscopies at the Royal United Hospital Bath. Specimens were then transported to the University of Bath where we have tested multiple culture conditions. These include choice of culture media, substrate (eg glass, plastic – coated or uncoated, semi-permeable membrane at air/liquid interface), cell adhesion factors (eg collagen I, collagen IV, fibronectin, Matrigel®), choice of tissue preparation (enzyme treatment with dispase in the absence or presence of trypsin, dissection with scissors or minced with a blade), plating density, media volume and use of a cell feeder layer. Results We have recruited 42 participants to date and attempted tissue culture on 36 of these. (Biopsies from 6 patients have been processed for RNA analysis.) Over time we have gradually optimised culture conditions. Of the most recent 6 participants, 5 have cultured successfully with cell survival for at least 14 days. Explant colony growth had been particularly vigorous (>12 mm) when plated on Matrigel® and a feeder layer cell line (mitomycin C-inactivated mouse embryonic fibroblast (iMEF) cell line 3T3-Swiss albino, ATCC® CCL-92™) on plastic coverslips. Explants have also adhered and cultured successfully on collagen IV-coated (sc-29010, Santa Cruz Biotech) plastic coverslips without a feeder layer. Explant colonies were fixed/permeabalised before immunofluorescent staining was performed for cytokeratin 14 (K14), smooth muscle actin (SMA), p63, E-cadherin and SLUG antigens. iMEFs are clearly delineated with SMA staining and the explant outgrowths are positive for K14, p63, E-cadherin and SLUG. Conclusion It is possible to culture adult human oesophageal tissue in vitrofrom standard pinch-biopsy forceps specimens at gastroscopy and we are in the final stages of optimising conditions, confirming characterisation by repeating experiments and devising a protocol. Disclosure of interest None Declared.

Introduction Barrett’s metaplasia (BM) is the main risk factor for oesophageal adenocarcinoma, a cancer which carries a mortality of >50% at 12 months. Refluxate containing gastric and bile acids seems to be causative for inflammation at the lower oesophagus, but it is not known how this induces replacement of stratified squamous epithelium (SSQE) with columnar epithelium at a molecular level.There is likely to be a progenitor cell population replacing denuded epithelium, although the origin of these cells has not been proven. Genes that play a role in gut tissue patterning during embryogenesis have received attention. One such ‘master switch’ our laboratory is investigating encodes the hepatocyte nuclear factor 4 alpha (HNF4α) transcription factor. Methods We optimised an immunohistochemistry protocol for demonstrating HFN4α on formalin-fixed paraffin-embedded slides of human tissue. This protocol was applied to forceps biopsy specimens of normal oesophagus, gastro-oesophageal junction (GOJ), stomach, ileum, colon and BM (UK REC reference: 13/YH/0197). Tissues were examined from at least 3 different patients per anatomical site. Results In healthy tissues, nuclear HNF4α positive immunostaining was demonstrated in stomach, ileum and colonic epithelium, but not in normal SSQE in the oesophagus. At the GOJ, there was clear delineation between HNF4α positive nuclei in the columnar gastric cardia mucosa, and negative HNF4α staining of SSQE. In contrast, the columnar epithelial nuclei in BM were consistently positive. Conclusion HNF4α transcription factor is demonstrable in BM, but not SSQE. We are not aware that this HNF4α gastrointestinal distribution has been previously published. HNF4α is likely to be a key transcription factor in the pathogenesis of BM. Previous work in our laboratory with a mouse explant tissue culture model has shown that another candidate transcription factor responsible for BM (Cdx2) was insufficient to induce an intestinal phenotype, whereas HNF4α induced villin, K18, trefoil factor 3 and mucin 5AC. We propose a 2-hit hypothesis for the development of BM: induction of HNF4α (which initially converts the oesophageal SSQE to columnar epithelium) and Cdx2 (which causes intestinalisation of the columnar epithelium). Demonstration of HNF4α in BM but not SSQE is supportive of this theory. Disclosure of Interest None Declared.

The following paper on the molecular biology of Barrett's esophagus (BE) includes commentaries on signaling pathways central to the development of BE including Hh, NF‐κB, and IL‐6/STAT3; surgical approaches for esophagectomy and classification of lesions by appropriate therapy; the debate over the merits of minimally invasive esophagectomy versus open surgery; outcomes for patients with pharyngolaryngoesophagectomy; the applications of neoadjuvant chemotherapy and chemoradiotherapy; animal models examining the surgical models of BE and esophageal adenocarcinoma; the roles of various morphogens and Cdx2 in BE; and the use of in vitro BE models for chemoprevention studies.

Introduction Barrett9s metaplasia, a precursor of oesophageal adenocarcinoma, is characterised by a change of the distal squamous oesophagus to a columnar/intestinal phenotype. The molecular mechanisms responsible for the phenotypic change seen in Barrett9s metaplasia are not known. Hepatocyte nuclear factor 4α (HNF4α) is a transcription factor originally identified in hepatoma cells, but with essential functions in gastrointestinal tract development and function. HNF4a is not normally found in squamous epithelium but is identified in Barrett9s metaplasia. The objectives of this research was to examine the utility of HNF4a in inducing a columnar/intestinal phenotypic change in squamous oesophageal epithelium following ectopic expression. Methods Ectopic HNF4a expression was induced in a mouse model of squamous epithelium using adenoviral vectors. The intestinal markers, cytokeratin 8 (K8), villin, trefoil factor 3, alkaline phosphatase and mucin2, along with the squamous epithelial proteins p63 and K14 were examined using immunohistochemistry or PCR following HNF4a expression. Results Three days of ectopic HNF4a is sufficient to provoke the expression of intestinal markers villin, trefoil factor 3 and K8 in squamous oesophageal cells. The expression of the squamous transcription factor p63 is down regulated following HNF4a expression. Interestingly ectopic HNF4a provokes the expression of E-cadherin in squamous oesophageal cells grown under low calcium conditions. Conclusion HNF4a is a new candidate factor for the induction of Barrett9s metaplasia. Ectopic expression is sufficient to provoke a change in the phenotype of squamous cells consistent to that seen in Barrett9s metaplasia. These experiments demonstrate for the first time the expression of columnar intestinal markers (K8, villin and trefoil factor 3) in squamous oesophageal cells following forced HNF4a induction. Understanding the molecular steps that regulate the induction of HNF4a in squamous epithelium will provide further insight to the early initiation of Barrett9s metaplasia and may provide therapeutic opportunities.

Introduction In September 2009, the JAG Endoscopy Training System (JETS) e-portfolio was launched. Since its release, over 75% of UK trusts have set the system up and over 70 000 procedures logged by trainees. Within the JETS e-portfolio, trainees record their data under pre-defined lists. These are dedicated service lists (reduced points and/or tailored case mix), ad-hoc training lists (training occurring on a standard service list) or service lists. With the European Working Time Directive, trainees have less opportunity for endoscopy training. Furthermore, in a cash-strapped NHS, training may become a low priority over service delivery. We studied the proportion of endoscopies performed on each of the list types by trainees to assess when trainees start to provide service lists within the NHS. Methods All endoscopy data from the JETS e-portfolio from September 2009 to 30 October 2010 were analysed. Results There were 70 499 procedures from 16 433 procedure lists. All non-trainee roles were then excluded, leaving 60 124 endoscopies performed by trainees, and of these, 22 358 (37%) were on service lists. 34 186 endoscopies were performed by registrar-level doctors and 9726 (28%) of these were on service lists. Of these, gastroenterology registrars did 29 876, of which 8686 on service lists (29%) and GI surgical registrars did 4310, of which 1040 were on service lists (24%) (Figure 1). The remaining 25 938 endoscopies, of which 12 632 were service (47%), were performed by others such as associate specialists and nurse endoscopists. Figure 1 PTU-126 100% stacked column of registrar-level endoscopy procedure type expressed proportionally, split by year of training and specialty, with total endoscopy numbers below. Conclusion UK trainee endoscopists contribute towards a large volume of service provision; in fact by year 5, gastroenterology registrars perform the majority of their endoscopies on service lists. This data shows that hospital trusts should prioritise endoscopy training. High quality training will result in competent, experienced, independent trainee endoscopists who can support the service of the trust in which they work and gain further valuable experience.