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Introduction Magnetic sphincter augmentation (MSA) is an effective treatment for patients with gastroesophageal reflux disease. In early studies, patients with a hiatal hernia (HH) ≥ 3 cm were excluded from consideration for implantation and initially the FDA considered its use as “precautionary” in this context. This early approach has led to an attitude of hesitance among some surgeons to offer this therapy to patients with HH. This study was designed to evaluate the impact of HH status on the outcome of MSA and to report the rate of HH recurrence after MSA. Methods and procedures This is a retrospective review of prospectively collected data of patients who underwent MSA between June 2013 and August 2017. Baseline clinical and objective data were collected. Patients were divided into four groups based on HH status: no HH, small HH (

Extracellular matrix (ECsM) bioscaffolds have been successfully used to treat five esophageal adenocarcinoma (EAC) patients following resection of neoplastic mucosal tissue. The present study evaluated the in vitro effect of ECM harvested from nonmalignant, decellularized tissue on EAC cell phenotype to understand the molecular mechanisms underlying the clinical findings. Nonmalignant (Het-1A), metaplastic (CP-A), and neoplastic (SK-GT-4, OE33) esophageal epithelial cells were exposed to ECM degradation products (250 μg/mL) prepared from heterologous urinary bladder tissue or homologous esophageal mucosa tissue, and evaluated for cell morphology, cell function, and EAC signaling pathways. Both the ECM sources downregulated neoplastic cell phenotype, but had distinctive tissue-specific effects. Urinary bladder ECM decreased OE33 and SK-GT-4 metabolism and increased CP-A apoptosis. Esophageal ECM decreased SK-GT-4, CP-A, and Het-1A proliferation; robustly downregulated PI3K-Akt-mTOR, cell cycle/DNA replication signaling, and upregulated autophagy signaling in OE33 cells; and increased cell cycle/DNA replication signaling in Het-1A cells. Both ECM sources decreased OE33 proliferation and phosphorylated AKT in OE33 cells, and in contrast, increased phosphorylated AKT in Het-1A cells. The results support the concept that the biochemical signals in nonmalignant ECM can downregulate neoplastic cell phenotype with minimal, and sometimes opposite, effects on normal cells. PI3K-Akt signaling has been implicated in EAC progression and these ECM-mediated effects may be favorable for an esophageal therapy following cancer resection. IMPACT STATEMENT: Extracellular matrix (ECM) biomaterials were used to treat esophageal cancer patients after cancer resection and promoted regrowth of normal mucosa without recurrence of cancer. The present study investigates the mechanisms by which these materials were successful to prevent the cancerous phenotype. ECM downregulated neoplastic esophageal cell function (proliferation, metabolism), but normal esophageal epithelial cells were unaffected in vitro, and suggests a molecular basis (downregulation of PI3K-Akt, cell cycle) for the promising clinical results. The therapeutic effect appeared to be enhanced using homologous esophageal ECM. This study suggests that ECM can be further investigated to treat cancer patients after resection or in combination with targeted therapy.

PURPOSE/OBJECTIVE(S) Esophageal adenocarcinoma is the sixth leading cause of cancer-related deaths in the United States. For locally advanced patients, neoadjuvant chemoradiotherapy followed by surgery is the standard of care. Risk stratification relies heavily on clinicopathologic features, particularly pathologic response, which is inadequate. This highlights the need for the development of biomarkers that can help improve outcomes. Herein, we have developed 2 distinct biomarker signatures to either predict response to neoadjuvant therapy or stratify patients into risk categories for survival. MATERIALS/METHODS We assembled a cohort of 34 patients with locally advanced esophageal adenocarcinoma, with the majority (21) receiving concurrent chemoradiotherapy with carboplatin/paclitaxel. A capture based targeted sequencing was performed in paired biopsy specimens obtained at baseline, 3- and 6-weeks post-treatment. Differentially mutated gene analysis between responders and non-responders of treatment was performed to determine predictors of response. A univariate Cox proportional hazard regression was used to examine associations between gene mutation status and overall survival. A risk score was then calculated for each patient using a linear combination of univariate coefficient and mutation status of gene. RESULTS 15 patients (71%) receiving the CROSS regimen were classified as responders. Among responders to chemotherapy, the most frequently mutated genes were MKI67, SYNE1, PCLO, MSH3, RECQL4, NOTCH2, ILR7, CIITA, LRRK2 and EML4. Tumor mutation count was significantly reduced for these genes in post-treatment samples (P = 5.89E-03). We have identified a 3 gene signature based on mutations in EPHA5, BCL6, and ERBB2 that robustly predicts response to carboplatin/paclitaxel based neoadjuvant therapy. For this model, sensitivity is 84.6% and specificity is 100%. In the overall cohort, a 9 gene signature using APC, MAP3K6, ETS1, CSF3R, PDGFRB, GATA2, ARID1A, and FGF6, was also created that significantly stratifies patients into risk categories, prognosticating for improved relapse-free (P = 0.005) and overall survival (P = 3.325E-06). Sensitivity for this model is 73.33% and specificity is 94.74%. CONCLUSION We have identified a 3-gene signature (EPHA5, BCL6 and ERBB2) that is predictive of response to neoadjuvant therapy and a separate 9 gene classifier which prognosticates for survival outcomes. These provide significant potential for personalized management of locally advanced esophageal cancer.

Improved methods of diagnosis of laryngopharyngeal reflux (LPR) would enable surgeons to better identify patients who may benefit from antireflux surgery (ARS). The objective of the present study was to assess if hypopharyngeal Pepsin and Sep70 expression combined with hypopharyngeal multichannel intraluminal impedance (HMII) has the potential to increase diagnostic sensitivity of LPR. This study was performed on patients who underwent unsedated transnasal endoscopy with hypopharyngeal biopsy and 24-h HMII to determine abnormal proximal exposure (APE) and DeMeester score (DMS) from 2013 to 2016. Pepsin and Sep70 protein expression was assessed by Western blots of biopsy specimens. The outcomes of ARS were assessed using reflux symptom index (RSI). HMII APE classification, Sep 70, and Pepsin protein levels were compared in normative and symptomatic LPR patients and further analyzed alongside quality of life changes following ARS. Of 30 subjects enrolled, 23 were excluded for abnormal HMII results or endoscopic evidence of esophagitis. Seven subjects and 105 patients were included in the normative and symptomatic groups, respectively. Compared to the normative group, only Pepsin expression was significantly higher in the symptomatic group [APE+/LPR+ (p = 0.000), APE+/LPR− (p = 0.001), and APE− (p = 0.047)]. Further, the ratio of Sep70/Pepsin was significantly lower in the symptomatic group [APE+/LPR+ (p = 0.008), APE+/LPR− (p = 0.000), and APE− (p = 0.050)], and a cutoff ratio for a diagnosis of LPR was established as

Esophageal adenocarcinoma (EAC) is a deadly disease with limited therapeutic options. In the present study, we determined the preclinical efficacy of CDK4/6 inhibitor abemaciclib for treatment of EAC. In vitro, apoptosis, proliferation, and pathway regulation were evaluated in OE19, OE33, and FLO1 EAC cell lines. In vivo, esophagojejunostomy was performed on rats to induce EAC. At 36 weeks post-surgery, MRI and endoscopic biopsy established baseline tumor volume and molecular correlates, respectively. Next, the study animals were randomized to 26mg/kg intraperitoneal abemaciclib treatment or vehicle control for 28 days. Pre and post treatment MRIs, histopathology, and qRT-PCR were utilized to determine response. Our results demonstrated treatment with abemaciclib lead to increased apoptosis, and decreased proliferation in OE19 (p=0.185), OE33 (p=0.048), and FLO1 (p=0.043) with anticipated downstream molecular inhibition. In vivo, 78.9% of treatment animals demonstrated >20% tumor volume decrease (placebo 0%). Mean tumor volume changed in the treatment arm by -65.5% (placebo +133.5%) (p

Aim To efficiently replicate the biology and pathogenesis of human esophageal adenocarcinoma (EAC) using the modified Levrat model of end-to-side esophagojejunostomy. Methods End-to-side esophagojejunostomy was performed on rats to induce gastroduodenoesophageal reflux to develop EAC. Animals were randomly selected and serially euthanized at 10 (n = 6), 17 (n = 8), 24 (n = 9), 31 (n = 6), 38 (n = 6), and 40 (n = 6) wk postoperatively. The esophagi were harvested for downstream histopathology and gene expression. Histological evaluation was completed to determine respective rates of carcinogenic development. Quantitative reverse transcription-polymerase chain reaction was performed to determine gene expression levels of MUC2, CK19, and CK20, and results were compared to determine significant differences throughout disease progression stages. Results The overall study mortality was 15%. Causes of mortality included anastomotic leak, gastrointestinal hemorrhage, stomach ulcer perforation, respiratory infection secondary to aspiration, and obstruction due to tumor or late anastomotic stricture. 10 wk following surgery, 100% of animals presented with esophagitis. Barrett's esophagus (BE) was first observed at 10 wk, and was present in 100% of animals by 17 wk. Dysplasia was confirmed in 87.5% of animals at 17 wk, and increased to 100% by 31 wk. EAC was first observed in 44.4% of animals at 24 wk and increased to 100% by 40 wk. In addition, two animals at 38-40 wk post-surgery had confirmed macro-metastases in the lung/liver and small intestine, respectively. MUC2 gene expression was progressively down-regulated from BE to dysplasia to EAC. Both CK19 and CK20 gene expression significantly increased in a stepwise manner from esophagitis to EAC. Conclusion Esophagojejunostomy was successfully replicated in rats with low mortality and a high tumor burden, which may facilitate broader adoption to study EAC development, progression, and therapeutics.

The purpose of the current study is to determine the efficacy of a PI3K/mTOR dual inhibitor, LY3023414, on established EAC in an in vivo model.Esophageal adenocarcinoma (EAC) is a highly lethal cancer with limited treatment options. The PI3K/mTOR pathway is upregulated in EAC and may be a target for novel therapies.Esophagojejunostomy was performed on Sprague-Dawley rats to induce carcinogenesis, and LY3023414 was cyclically administered intraperitoneally between 32 and 40 weeks postsurgery to treatment animals. Magnetic resonance imaging (MRI) and histology were used to determine clinical response. Immunohistochemistry, immunofluorescence, and Western blot were used to validate apoptosis by cleaved caspase-3, proliferation by Ki67, and pathway inhibition, respectively.Mean MRI tumor volume increased by 109.2% in controls (n = 32) and decreased by 56.8% in treatment animals (n=17) (P0.01). Treatment with LY3023414 demonstrated tumor volume increase in 0% (control = 46.4%) (P0.01), decrease in 58.8% (control = 7.1%) (P0.01), and stable volume in 41.2% (control = 46.4%) (P = 0.77). EAC prevalence in controls increased by 25%; whereas, prevalence in treatment animals decreased by 29.4% (P0.01). Approximately, 75% of treatment animals presenting with residual masses on MRI had a histological response50%. Increased apoptosis by cleaved caspase-3 (P = 0.03) and decreased proliferation by Ki67 (P0.01) were demonstrated in the treatment arm, when compared with the control arm. On Western blot analysis of pathway checkpoints, p-mTOR (p=0.03) and PI3K-α (P = 0.04) were downregulated in treatment responsive residual tumors, when compared with controls.LY3023414 demonstrates efficacy against EAC in a preclinical model, establishing the rationale for clinical testing.

Introduction Magnetic sphincter augmentation (MSA) results in less severe side effects compared with Nissen fundoplication, but dysphagia remains the most common side effect reported by patients after MSA. This study aimed to characterize and review the management of postoperative dysphagia and identify the preoperative factors that predict persistent dysphagia after MSA. Material and Methods This is a retrospective review of prospectively collected data of patients who underwent MSA between 2013 and 2018. Preoperative objective evaluation included upper endoscopy, esophagram, high-resolution impedance manometry (HRIM), and esophageal pH testing. Postoperative persistent dysphagia was defined as a postoperative score of > 3 for the dysphagia-specific item within the GERD-HRQL at a minimum of 3 months following MSA. A timeline of dysphagia and dilation rates was constructed and correlated with the evolution of our patient management practices and modifications in surgical technique. Results A total of 380 patients underwent MSA, at a mean (SD) follow up of 11.5 (8.7) months, 59 (15.5%) patients were experiencing persistent dysphagia. Thirty-one percent of patients required at least one dilation for dysphagia or chest pain and the overall response rate to this procedure was 67%, 7 (1.8%) patients required device removal specifically for dysphagia. Independent predictors of persistent dysphagia based on logistic regression model included (1) absence of a large hernia (OR 2.86 (95% CI 1.08–7.57, p = 0.035)); (2) the presence of preoperative dysphagia (OR 2.19 (95% CI 1.05–4.58, p = 0.037)); and (3) having less than 80% peristaltic contractions on HRIM (OR 2.50 (95% CI 1.09–5.73, p = 0.031)). Graded cutoffs of distal contractile integral (DCI), mean wave amplitude, DeMeester score, sex, and body mass index were evaluated within the model and did not predict postoperative dysphagia. Frequent eating after surgery, avoidance of early dilation, and increase in the size of the LINX device selected decreased the need for dilation. Conclusion In a large cohort of patients who underwent MSA, we report 15.5% rate of persistent postoperative dysphagia. The overall response rate to dilation therapy is 67%, and the efficacy of dilation with each subsequent procedure reduces. Patients with normal hiatal anatomy, significant preoperative dysphagia, and less than 80% peristaltic contractions of the smooth muscle portion of the esophagus should be counseled that they have an increased risk for persistent postoperative dysphagia.

Esophageal adenocarcinoma (EAC) is an aggressive cancer necessitating the development of improved risk stratification tools for personalized care. Previously, microRNAs have been shown to correlate with the progression and prognosis of various cancer types; however, the value in EAC remains largely unexplored. We performed global microRNA profiling on 32 formalin-fixed, paraffin-embedded EAC specimens to identify microRNAs associated with progression. Literature search and pathway analysis further refined output to five significantly deregulated candidate biomarkers. Four of the five microRNAs (miR-652-5p, miR-7-2-3p, miR-3925-3p, and miR-219-3p) were validated by qRT-PCR. Survival outcomes were evaluated in testing set of 26 stage II/III EAC patients to determine the prognostic relevance of the selected microRNAs. In the testing set, miR-652-5p and miR-7-2-3p expressions were significantly associated with progression-free survival (p-value = .00771 and p-value = .00293). The highest area under receiver operating characteristic (ROC) curve was 0.8212 for the combination of miR-652-5p and miR-7-2-3p. Collectively, our findings demonstrated that the miR-652-5p/miR-7-2-3p signature may serve as a promising prognostic marker in patients with locally advanced EAC.

A rat model of surgically induced reflux recapitulates the development and progression of human esophageal adenocarcinoma (EAC). In this study, reflux was induced in rats followed by postoperative endoscopy with biopsy, to diagnose and monitor disease progression. Overall, percentage agreement between visual endoscopy and gold standard histology was 95%, with disease-specific classification accuracies of 100% and 75% for Barrett's with dysplasia and EAC, respectively. Additionally, the percentage agreement for biopsy in tumors4 mm was 75%. Thereby, establishing endoscopic evaluation as a reliable tool to assess disease progression and provide biopsies for downstream correlates in a de novo EAC model.

Background Esophageal adenocarcinoma (EAC) is a lethal disease with poor prognosis due to the limited treatment options. STING is a transmembrane protein that activates transcription of type I IFN genes, resulting in stimulation of APCs and enhanced CD8+ T cell infiltration. Recently, STING agonists have demonstrated not only potent efficacy against the primary tumor but also in the distant metastasis and recurrences. Interestingly, combining the STING agonist with radiotherapy and immune checkpoint inhibitors has demonstrated durable anticancer activity in solid tumors. Hence, the aim of the study was to specifically evaluate the efficacy and immunologic regulatory effects of STING agonist +/- radiation in an established EAC model. Methods Esophagojejunostomy was performed on rats to induce gastroduodenojejunal reflux leading to the development EAC. At 32 weeks post operatively, rats received either 50μg STING (ADU-S100) +/- 16Gy radiation or placebo (PBS) +/- 16Gy radiation. Drug or placebo control was delivered intratumorally twice via endoscopy, 3 weeks apart. Drug efficacy was evaluated by pre- and post- treatment MRI, serial biopsies, histology and RT-PCR. Additionally, IHC was performed using CD8 and PD-L1 antibodies. Results There was no difference in observed mortality among the groups (p = 0.3). Mean MRI tumor volume decreased by 19.7% and 18.6% in ADU-S100 and ADU-S100 + radiation animals and increased by 34.7% and 90.3% in placebo and placebo + radiation animals, respectively (P = 0.0006). Downstream gene expression, pre- to on- treatment, demonstrated upregulation of IFNγ (p = 0.0003) and TNFα (p = 0.02) in the treatment groups vs. placebo. The most remarkable change was observed in ADU-S100 group (5.5X IFNγ and 10.9X TNFα; p Conclusions ADU-S100 exhibits potent antitumor efficacy and a promising immunomodulatory profile in a de novo EAC model providing the rationale for clinical strategies, likely in combination with immune checkpoint inhibitors. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure A.H. Zaidi: Shareholder / Stockholder / Stock options: Array Biopharma; Research grant / Funding (institution): Eli Lilly & Co. All other authors have declared no conflicts of interest.

Background Toll-like receptors (TLRs) recognize known molecules from microbes and have an established role in tumorigenesis. Using a rat model of esophageal adenocarcinoma, and human clinical samples, we investigated genes central to TLR-mediated signal transduction and characterized the esophageal microbiome across the spectrum of esophageal adenocarcinoma carcinogenesis. Methods We surgically induced bile/acid reflux in rats and their esophagi were harvested at 40 weeks post-surgery. Tissue samples from the model were selected for gene expression profiling. Additionally, for rat and human samples microbiome analysis was performed using PCR-ESI-MS-TOF technology with validation by fluorescence in situ hybridization. Results Gene expression results in the rat model indicated a significant upregulation of TLRs 1-3, 6, 7 and 9 in EAC compared to normal epithelium. PCR-ESI-MS-TOF analysis revealed a prevalence of Escherichia coli in Barrett’s esophagus (60 %) and esophageal adenocarcinoma (100 %), which was validated by fluorescence in situ hybridization. In the human clinical samples, Streptococcus pneumonia was detected in high abundance in gastroesophageal reflux disease and Barrett’s esophagus (50–70 %) in comparison to tumor adjacent normal epithelium, dysplasia, and esophageal adenocarcinoma (20–30 %). E. coli was detected in the Barrett’s esophagus and esophageal adenocarcinoma groups but was absent in the tumor adjacent normal epithelium, dysplasia, and the gastroesophageal reflux disease groups. Conclusions We demonstrated an association between the TLR signaling pathway and E. coli hinting towards possible early molecular changes being mediated by microbes in the rat model of esophageal adenocarcinoma carcinogenesis. Studies on human clinical samples also corroborate results to some extent; however, a study with larger sample size is needed to further explore this association. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2093-8) contains supplementary material, which is available to authorized users.

To assess the efficacy of heat-shock protein 90 (Hsp90) inhibitor, NVP-AUY922-AG (AUY922), in the treatment of esophageal adenocarcinoma (EAC) in vitro and in vivo.EAC is a leading cause of cancer death, and current treatment options are limited. Hsp90, a chaperone protein that regulates several oncoproteins, is upregulated in EAC, and may be a novel target for therapy.In vitro, EAC cell lines were utilized to evaluate AUY922, alone and in combination with 5-fluorouracil (5-FU) and cisplatin. BrdU ELISA and flow cytometry were used to assess proliferation and measure apoptosis, respectively. Western blot and RT-PCR were performed to quantitate Hsp90 pathway expression. In vivo, esophagojejunostomy was performed on rats and treatment animals received AUY922 32 to 40 weeks postoperatively. Drug efficacy was evaluated with magnetic resonance imaging (MRI), endoscopic biopsy, gross histological evaluation, and Hsp90 pathway expression.In vitro, AUY922 demonstrated antiproliferative activity in both cell lines and showed enhanced efficacy with cisplatin and 5-FU. Western Blot and RT-PCR demonstrated downregulation of CDK1 and CDK4 and upregulation of Hsp72. In vivo, AUY922 showed decrease in tumor volume in 36.4% of rats (control = 9.4%), increase in 9.1% (control = 37.5%), and stable disease in 54.5% (control = 43.7%). Necropsy confirmed the presence of EAC in 50% of treatment animals and 75% of control animals. mRNA expression, pre- and posttreatment, demonstrated significant downregulation of MIF, Hsp70, Hsp90β, and CDK4, and upregulation of Hsp72.AUY922 exhibits antitumor efficacy in vitro and in vivo for EAC, suggesting the need for human clinical trials.

4060 Background: PD-1 inhibitors in metastatic gastroesophageal cancer have demonstrated response rates of approximately 25%. Unfortunately, the majority of patients do not respond. Therefore, a rationale strategy of combining immunotherapeutic agents with CRT in earlier stage esophageal cancer may prevent metastatic disease in a greater proportion of patients. This study assessed the impact of CRT on the immune microenvironment and the expression patterns of multiple immune checkpoints to optimally design neoadjuvant clinical trials. Methods: To determine the effects of CRT on resected esophageal adenocarcinomas (EAC), we examined the immune microenvironment pre and post CRT using IHC, LCM followed by qRT-PCR, and functional analysis of tumor-infiltrating lymphocytes. Additionally, to assess the duration and dependency of radiation-induced PD-L1 sensitization, esophagojejunostomy were performed on rats to induce gastroduodenoesophageal reflux and EAC formation. First, tumor bearing animals were dosed with single fraction 13 Gy or 16 Gy radiation to determine safety, dose correlation, and PD-L1 sensitization using qRT-PCR post-radiation. Next, longitudinal PD-L1 expression levels within individual tumor bearing animals were determined using serial endoscopic biopsies at baseline, 1, 5 and 9 weeks post 16 Gy radiation. Results: The majority of cancers displayed enhanced interferon γ and activated CD8+ T lymphocytes at the tumor stroma interface. These tumors also demonstrated enhanced upregulation of PD-L1 and multiple other immune checkpoints including – TIM3, GITR, IDO1, LAG3, CD137, OX40 and KIR. The animal model results indicated PD-L1 upregulation is dose dependent and transiently elevated post radiation exposure. Conclusions: Collectively, these findings provide insights into the evolving immune landscape after CRT and have significant implications for future neoadjuvant trial designs that will combine radiotherapy with immune checkpoint inhibitors. Currently, we are conducting a neoadjuvant trial assessing Nivolumab or Nivolumab/Ipilimumab in combination with CRT in stage II/III operable esophageal cancer.

139 Background: PD-L1 expression has been reported in 40% of gastroesophageal cancers, but little data exists with regards to expression of other immune checkpoints. Here, we examined baseline expression levels for multiple immune modulators in 31 resected esophageal adenocarcinomas (EAC) and investigated the change in expression levels post-chemoradiation. Methods: Slides were stained on a Ventana BenchMark ULTRA automated stainer for CTLA-4 (Santa Cruz Clone F-8, dilutions 1:100) and PD-L1 (Dako clone 22C3 prediluted). Tonsil tissue was used as a control for lymphocytes, and a provided tumor control was used for PD-L1. Positivity was classified as > 1%, and the stained cell type was recorded (lymphocytes or tumor). Additionally, laser capture microdissection was performed, and RNA was isolated and pre-amplified to determine expression levels of TIM-3, GITR, IDO-1, LAG-3, CD-137, OX-40, and KIR-3 using RT-PCR. Gene expression was calculated using the ΔΔ -Ct method, and intergroup comparisons were performed and normalized against a cohort of 15 pathologically confirmed normal esophageal epithelium cases. Results: Post-chemoradiation cases demonstrated increased expression for PD-L1, when compared to matched pre-chemoradiation controls [p-value = 0.0098]. Similarly, all immune modulators profiled by gene expression were significantly upregulated in matched post vs. pre samples, except for CD-137. There was no significant difference in percentage of 4-fold or more upregulation based on PD-L1 status for IDO-1, OX-40, CD-137 and KIR-1, indicating that these markers may function independently of PD-L1 positivity. Conclusions: PD-1 inhibiton in both the neoadjuvant and adjuvant setting is currently being investigated in EAC, but future studies may look to select patients according to personalized checkpoint expression patterns. [Table: see text]

e15554Background: Esophageal adenocarcinoma (EAC) is a lethal cancer with limited treatment options. The PI3K/mTOR pathway is upregulated in EAC, and overexpression of p-mTOR is linked to poor prog...

28 Background: The discovery of the link between H. pylori and gastric cancer may be the most direct proof that bacterial signaling and host response can result in carcinogenesis. Accumulating evidence supports that activation of the Toll-like receptor (TLR) signaling pathway by microbes is associated with the development of GI malignancies. Using the modified Levrat model of gastroduodenojejunal reflux which mimics the physiological and molecular sequence of human EAC in the rat, this study profiles the expression of genes central to TLR-mediated signal transduction as well as characterizes the esophageal microbiome across the spectrum of EAC development. Methods: Modified Levrat’s surgery induced chronic acid reflux in Sprague-Dawley’s with harvest of esophagus 40 weeks post-surgery. Macordissection of normal adjacent epithelium, Barrett’s esophagus (BE), dysplasia and EAC tumor was performed followed by RNA/DNA isolation. Five samples per group were selected for gene expression profiling on the Qiagen TLR Signaling Pathway PCR Array as well as microbiome analysis by IBIS technology. Validation of IBIS was performed by fluorescence in situ hybridization (FISH). Results: Gene expression analysis identified TLRs 1-3 and 6, 7, 9 as significantly upregulated in EAC compared to normal esophagus. TLR 1 and 5 were significantly upregulated in dysplasia. TLR 1 was significantly upregulated in BE and normal adjacent epithelium. Thirty seven genes involved in the TLR signaling pathway were dysregulated in EAC, 30 in dysplasia, 21 in BE and 23 in normal adjacent. IBIS analysis revealed a prevalence of E. coli in BE and EAC which was validated by FISH. Conclusions: Toll-like receptor (TLR) signaling pathway responses to E. coli may participate in the development of EAC. E. coli may be a potential risk factor for EAC requiring further clinical validation.