244 MICROBIOME ANALYSIS OF UPPER DIGESTIVE TRACT BIOPSY SAMPLES FROM INDIVIDUALS ALONG THE METAPLASIA-DYSPLASIA-ADENOCARCINOMA SEQUENCE.

The human microbiota, the collection of microbes that inhabit the human body, is increasingly being appreciated as playing a role in human health. A seminal example of this relationship is Helicobacter pylori and gastric cancer oncogenesis. The drop in H.pylori infections and non-cardia gastric cancer incidences has coincided with the rise in oesophageal adenocarcinoma (OAC) incidences. We sought to explore the relationship between the upper digestive tract microbiome and OAC oncogenesis. Methods Pinch biopsies were taken from individual’s oesophagus and stomach who were along the metaplasia-dysplasia-adenocarcinoma sequence (GERD, Barrett's oesophagus, dysplasia, OAC, metastatic OAC) as well as healthy controls. We carried out 16 s rRNA gene DNA sequencing protocols on these samples. DNA extraction and library preparation was performed with consideration to the low mass nature of oesophageal biopsies. Raw reads were processed and amplicon sequence variants (ASVs) were generated using the DADA2. We dissected ecological differences between sample site and clinical classification using a variety of approaches including examining differentially abundant taxa and inferred metabolic pathways, alpha diversity and beta-diversity. Results The upper digestive tract was found to be dominated by the genera Streptococcus, Prevotella, and Haemophilus. There was no statistically significant shift in beta diversity with respect to biopsy location. Alpha diversity was reduced in gastric biopsies compare to oesophageal biopsies. A slight yet significant shift was seen in beta diversity (Bray–Curtis Dissimilarity) with respect to clinical classification in biopsies derived from the gastroesophageal junction (GEJ) and stomach. Various taxa were found to be differentially abundant between biopsy site and with regard to clinical classification. Conclusion OAC primarily occurs at the GEJ. Community structure was shifted in samples derived from the GEJ and the stomach. Fusobacterium nucleatum was overrepresented in oesophageal biopsies from individuals with diseased oesophagus compared to individuals with a histologically normal oesophagus. This bacterium has been implicated in oncogenesis of various cancers most notably colorectal cancer. Serval ASVs assigned to the genus Prevotella were depleted in stomachs of individuals with metastatic OAC compared to all other groups.

Pathophysiology of reflux oesophagitis: role of Toll-like receptors 2 and 4 and Farnesoid X receptor

The pathogenesis of gastroesophageal reflux disease (GERD) is not fully understood. It involves the activation of mucosal immune-mediated and inflammatory responses. Toll-like receptors (TLR) 2 and TLR4 are pattern-recognition receptors of the innate immune system; they recognize microbial and endogenous ligands. Farnesoid X receptor (FXR) is a bile acid receptor that regulates the inflammatory response. We aimed to evaluate TLR2, TLR4 and FXR expression patterns in GERD. We re-evaluated 84 oesophageal biopsy samples according to the global severity (GS) score, including 26 cases with histologically normal oesophagus, 28 with histologically mild oesophagitis and 30 with severe oesophagitis. We used immunohistochemistry and in situ hybridization to assess the expression patterns of TLR2, TLR4 and FXR in oesophageal squamous cells. Immunohistochemistry showed that nuclear and cytoplasmic TLR2 was expressed predominantly in the basal layer of normal oesophageal epithelium. In oesophagitis, TLR2 expression increased throughout the epithelium, and the superficial expression was significantly more intensive compared to normal epithelium, p <0.01. Nuclear and cytoplasmic TLR4 was expressed throughout the thickness of squamous epithelium, with no change in oesophagitis. FXR was expressed in the nuclei of squamous cells, and the intensity of the expression increased significantly in oesophagitis (p <0.05). FXR expression correlated with basal TLR2. In situ hybridization confirmed the immunohistochemical expression patterns of TLR2 and TLR4. In GERD, TLR2, but not TLR4, expression was upregulated which indicates that innate immunity is activated according to a specific pattern in GERD. FXR expression was increased in GERD and might have a regulatory connection to TLR2.

Eosinophilic Esophagitis and Microbiota: State of the Art

Eosinophilic esophagitis (EoE) is a chronic, food-triggered, immune-mediated disease of the oesophagus, clinically characterized by symptoms referred to oesophagal dysfunction, and histologically defined by an eosinophil productive inflammation of the oesophagal mucosa, among other cell types. The involvement of an adaptive Th2-type response to food antigens in EoE was known since 2000; several cytokines and chemokines promote food-specific responses, during which local production of IgE, but also IgG4 derived from plasma cells in lamina propria of oesophagal mucosa might play an important role. Evidence pointing towards a possible role for the innate immunity in EoE has arisen recently. Together, this evidence gives rise to a potential role that the innate immune system in general, and also the microbial pattern recognition receptors (PRRs) might play in EoE pathogenesis. Among PRRs, Toll-like receptors (TLRs) are type-I transmembrane receptors expressed both on epithelial and lamina propria cells with the capacity to distinguish between pathogen and commensal microbes. As TLRs in the different intestinal epithelia represent the primary mechanism of epithelial recognition of bacteria, this evidence underlines that oesophagal TLR-dependent signaling pathways in EoE support the potential implication of microbiota and the innate immune system in the pathogenesis of this disease. The oesophagal mucosa hosts a resident microbiota, although in a smaller population as compared with other districts of the gastrointestinal tract. Few studies have focused on the composition of the microbiota of the normal oesophagus alone. Still, additional information has come from studies investigating the oesophagal microbiota in disease and including healthy patients as controls. Our review aims to describe all the evidence on the oesophagal and intestinal microbiota in patients with EoE to identify the specific features of dysbiosis in this condition.

Computational modelling suggests that Barrett’s oesophagus may be the precursor of all oesophageal adenocarcinomas

ObjectiveBarrett’s oesophagus (BE) is a known precursor to oesophageal adenocarcinoma (OAC) but current clinical data have not been consolidated to address whether BE is the origin of all incident OAC, which would reinforce evidence for BE screening efforts. We aimed to answer whether all expected prevalent BE, diagnosed and undiagnosed, could account for all incident OACs in the US cancer registry data.DesignWe used a multiscale computational model of OAC that includes the evolutionary process from normal oesophagus through BE in individuals from the US population. The model was previously calibrated to fit Surveillance, Epidemiology and End Results cancer incidence curves. Here, we also utilised age-specific and sex-specific US census data for numbers at-risk. The primary outcome for model validation was the expected number of OAC cases for a given calendar year. Secondary outcomes included the comparisons of resulting model-predicted prevalence of BE and BE-to-OAC progression to the observed prevalence and progression rates.ResultsThe model estimated the total number of OAC cases from BE in 2010 was 9970 (95% CI: 9140 to 11 980), which recapitulates nearly all OAC cases from population data. The model simultaneously predicted 8%–9% BE prevalence in high-risk males age 45–55, and 0.1%–0.2% non-dysplastic BE-to-OAC annual progression in males, consistent with clinical studies.ConclusionThere are likely few additional OAC cases arising in the US population outside those expected from individuals with BE. Effective screening of high-risk patients could capture the majority of population destined for OAC progression and potentially decrease mortality through early detection and curative removal of small (pre)cancers during surveillance.

Measuring the distribution of fitness effects in somatic evolution by combining clonal dynamics with dN/dS ratios

The distribution of fitness effects (DFE) defines how new mutations spread through an evolving population. The ratio of non-synonymous to synonymous mutations (dN/dS) has become a popular method to detect selection in somatic cells. However the link, in somatic evolution, between dN/dS values and fitness coefficients is missing. Here we present a quantitative model of somatic evolutionary dynamics that determines the selective coefficients of individual driver mutations from dN/dS estimates. We then measure the DFE for somatic mutant clones in ostensibly normal oesophagus and skin. We reveal a broad distribution of fitness effects, with the largest fitness increases found for TP53 and NOTCH1 mutants (proliferative bias 1–5%). This study provides the theoretical link between dN/dS values and selective coefficients in somatic evolution, and measures the DFE of mutations in human tissues.

P98 UNSATURATED LIPIDS ARE SIGNIFICANTLY UPREGULATED IN OESOPHAGEAL ADENOCARCINOMA

Aim This work aims to determine the glycerophospholipid (GPL) abundance between Oesophageal adenocarcinoma (OA) and proximal non-cancerous tissue at multiple levels using Desorption Electrospray Ionisation Mass spectrometry (DESI-MS) tissue imaging. Background & Methods The prevalence of Oesophageal cancer in the United Kingdom has steadily risen since the 1970s. The prognosis for most patients is poor, with two thirds presenting with incurable disease. The abundance of lipids in OA has been shown to be atypical of normal cellular metabolism.1 This work utilises mass spectrometry imaging to identify variations in lipid abundance between OA and normal oesophagus (NO). Tissue was sampled from specimens dissected immediately post-resection from the tumour and at 5-centimetre intervals in the proximal oesophagus. DESI-MS tissue imaging, an ambient form of Mass Spectrometry, was performed to identify GPLs.2 DESI-MS images were correlated with Hematoxylin and Eosin staining (current gold standard). Principle component analysis with a supervised model classified tissue types by sensitivity and specificity. Results Tissue analysis confirmed unsaturated Phosphatidylglycerols and Phosphatidic acids were most abundant in OA compared to NO (p<0.001). Tissue analysis by a Partial Least Square (PLS) supervised linear regression model classified cancer and non-cancer tissue with 85% sensitivity and 88% specificity. The abundance of several unsaturated GPL classes decreased proportionally in tissue sections at 5 and 10 centimetres from the primary tumour. Gene set enrichment has demonstrated potential dysregulation of this pathway in the literature. Conclusions This work highlights an important variant of lipid metabolism in OA, identified by DESI-MS. There is a significant overabundance of unsaturated Phosphatidylglycerol’s in OA compared to proximal NO. In addition, the unsaturated GPL classes found in the cancer become increasingly saturated at 5cm and 10cm from the tumour site. This variation in lipid molecular structure suggests a novel pathway for potential therapeutic modulation.

Measuring the distribution of fitness effects in somatic evolution by combining clonal dynamics with dN/dS ratios

The distribution of fitness effects (DFE) defines how new mutations spread through an evolving population. The ratio of non-synonymous to synonymous mutations (dN/dS) has become a popular method to detect selection in somatic cells, however the link, in somatic evolution, between dN/dS values and fitness coefficients is missing. Here we present a quantitative model of somatic evolutionary dynamics that yields the selective coefficients from individual driver mutations from dN/dS estimates, and then measure the DFE for somatic mutant clones in ostensibly normal oesophagus and skin. We reveal a broad distribution of fitness effects, with the largest fitness increases found for TP53 and NOTCH1 mutants (proliferative bias 1-5%). Accurate measurement of the per-gene DFE in cancer evolution is precluded by the quality of currently available sequencing data. This study provides the theoretical link between dN/dS values and selective coefficients in somatic evolution, and reveals the DFE for mutations in human tissues.

Modelling Barrett's oesophagus

Barrett's oesophagus is the replacement of normal squamous oesophageal epithelium with an intestinalized columnar epithelium. Although some insight has been gained as to what Barrett's oesophagus is, how this columnar epithelium emerges from within a stratified squamous epithelium remains an unanswered question. We have sought to determine whether oesophageal keratinocytes can be trans-differentiated into Barrett's oesophagus cells. Using an Affymetrix microarray, we found unexpectedly that gene-expression patterns in the Barrett's oesophagus were only slightly more similar to the normal small intestine than they were to the normal oesophagus. Thus gene-expression patterns suggest significant molecular similarities remain between Barrett's oesophagus cells and normal squamous oesophageal epithelium, despite their histological resemblance with intestine. We next determined whether directed expression of intestine-specific transcription factors could induce intestinalization of keratinocytes. Retroviral-mediated Cdx2 (Caudal-type homeobox 2) expression in immortalized human oesophageal keratinocytes engineered with human telomerase reverse transcriptase (EPC2-hTERT cells) could be established transiently, but not maintained, and was associated with a reduction in cell proliferation. Co-expression of cyclin D1 rescued proliferation in the Cdx2-expressing cells, but co-expression of dominant-negative p53 did not. Cdx2 expression in the EPC2-hTERT.D1 cells did not induce intestinalization. However, when combined with treatments that induce chromatin remodelling, there was a significant induction of Barrett's oesophagus-associated genes. Studies are ongoing to determine whether other intestinal transcription factors, either alone or in combination, can provoke greater intestinalization of oesophageal keratinocytes. We conclude that, on the basis of gene-expression patterns, Barrett's oesophagus epithelial cells may represent an intermediate between oesophageal keratinocytes and intestinal epithelial cells. Moreover, our findings suggest that it may be possible to induce Barrett's oesophagus epithelial cells from oesophageal keratinocytes by altering the expression of certain critical genes.