Elimination of NF-κB signaling in Vimentin+ stromal cells attenuates tumorigenesis in a mouse model of Barrett's Esophagus.

Chronic inflammation induces Barrett's Esophagus (BE) which can advance to esophageal adenocarcinoma. Elevated levels of interleukin (IL)-1b, IL-6 and IL-8 together with activated nuclear factor-kappaB (NF-κB), have been identified as important mediators of tumorigenesis. The inflammatory milieu apart from cancer cells and infiltrating immune cells contains myofibroblasts (MFs) that express aSMA and Vimentin. As we observed that increased NF-κB activation and inflammation correlates with increased MF recruitment and an accelerated phenotype we here analyze the role of NF-κB in MF during esophageal carcinogenesis in our L2-IL-1B mouse model. To analyze the effect of NF-κB signaling in MFs, we crossed L2-IL-1B mice to tamoxifen inducible Vim-Cre (Vim-CreTm) mice and floxed RelA (p65fl/fl) mice to specifically eliminate NF-κB signaling in MF (IL-1b.Vim-CreTm.p65fl/fl). The interaction of epithelial cells and stromal cells was further analyzed in mouse BE organoids and patient-derived human organoids. Histological scoring of IL-1b.Vim-CreTm.p65fl/fl mice showed a significantly attenuated phenotype compared with L2-IL-1B mice, with mild inflammation, decreased metaplasia and no dysplasia. This correlated with decreased proliferation and increased differentiation in cardia tissue of IL-1b.Vim-CreTm.p65fl/fl compared with L2-IL-1B mice. Distinct changes of cytokines and chemokines within the local microenvironment in IL-1b.Vim-CreTm.p65fl/fl mice reflected the histopathological abrogated phenotype. Co-cultured NF-κB inhibitor treated MF with mouse BE organoids demonstrated NF-κB-dependent growth and migration. MFs are essential to form an inflammatory and procarcinogenic microenvironment and NF-κB signaling in stromal cells emerges as an important driver of esophageal carcinogenesis. Our data suggest anti-inflammatory approaches as preventive strategies during surveillance of BE patients.

Immune determinants of Barrett's progression to esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) develops from Barrett's esophagus (BE), a chronic inflammatory state that can progress through a series of transformative dysplastic states before tumor development. While molecular and genetic changes of EAC tumors have been studied, immune microenvironment changes during Barrett's progression to EAC remain poorly understood. In this study, we identify potential immunologic changes that can occur during BE-to-EAC progression. RNA sequencing (RNA-Seq) analysis on tissue samples from EAC patients undergoing surgical resection demonstrated that a subset of chemokines and cytokines, most notably IL6 and CXCL8, increased during BE progression to EAC. xCell deconvolution analysis investigating immune cell population changes demonstrated that the largest changes in expression during BE progression occurred in M2 macrophages, pro-B cells, and eosinophils. Multiplex immunohistochemical staining of tissue microarrays showed increased immune cell populations during Barrett's progression to high-grade dysplasia. In contrast, EAC tumor sections were relatively immune poor, with a rise in PD-L1 expression and loss of CD8+ T cells. These data demonstrate that the EAC microenvironment is characterized by poor cytotoxic effector cell infiltration and increased immune inhibitory signaling. These findings suggest an immunosuppressive microenvironment, highlighting the need for further studies to explore immune modulatory therapy in EAC.

Circulating Levels of Inflammatory and Metabolic Biomarkers and Risk of Esophageal Adenocarcinoma and Barrett Esophagus: Systematic Review and Meta-analysis.

Associations between circulating levels of obesity-related biomarkers and risk of esophageal adenocarcinoma and Barrett esophagus have been reported, but the results are inconsistent. A literature search until October 2018 in MEDLINE and EMBASE was performed. Pooled ORs with 95% confidence intervals (CI) were estimated for associations between 13 obesity-related inflammatory and metabolic biomarkers and risk of esophageal adenocarcinoma or Barrett esophagus using random effect meta-analyses. Among 7,641 studies, 19 were eligible for inclusion (12 cross-sectional, two nested case-control, and five cohort studies). Comparing the highest versus lowest categories of circulating biomarker levels, the pooled ORs were increased for leptin (OR, 1.68; 95% CI, 0.95-2.97 for Barrett esophagus), glucose (OR, 1.12; 95% CI, 1.03-1.22 for esophageal adenocarcinoma), insulin (OR, 1.47; 95% CI, 1.06-2.00 for Barrett esophagus), C-reactive protein (CRP; OR, 2.06; 95% CI, 1.28-3.31 for esophageal adenocarcinoma), IL6 (OR, 1.50; 95% CI, 1.03-2.19 for esophageal adenocarcinoma), and soluble TNF receptor 2 (sTNFR-2; OR, 3.16; 95% CI, 1.76-5.65 for esophageal adenocarcinoma). No associations were identified for adiponectin, ghrelin, insulin-like growth factor 1, insulin-like growth factor-binding protein 3, triglycerides, IL8, or TNFα. Higher circulating levels of leptin, glucose, insulin, CRP, IL6, and sTNFR-2 may be associated with an increased risk of esophageal adenocarcinoma or Barrett esophagus. More prospective studies are required to identify biomarkers that can help select high-risk individuals for targeted prevention and early detection.

Characterizing caspase-1 involvement during esophageal disease progression.

Barrett's esophagus (BE) is an inflammatory condition and a neoplastic precursor to esophageal adenocarcinoma (EAC). Inflammasome signaling, which contributes to acute and chronic inflammation, results in caspase-1 activation leading to the secretion of IL-1ß and IL-18, and inflammatory cell death (pyroptosis). This study aimed to characterize caspase-1 expression, and its functional importance, during disease progression to BE and EAC. Three models of disease progression (Normal-BE-EAC) were employed to profile caspase-1 expression: (1) a human esophageal cell line model; (2) a murine model of BE; and (3) resected tissue from BE-associated EAC patients. BE patient biopsies and murine BE organoids were cultured ex vivo in the presence of a caspase-1 inhibitor, to determine the importance of caspase-1 for inflammatory cytokine and chemokine secretion.Epithelial caspase-1 expression levels were significantly enhanced in BE (p < 0.01). In contrast, stromal caspase-1 levels correlated with histological inflammation scores during disease progression (p < 0.05). Elevated secretion of IL-1ß from BE explanted tissue, compared to adjacent normal tissue (p < 0.01), confirmed enhanced activity of caspase-1 in BE tissue. Caspase-1 inhibition in LPS-stimulated murine BE organoids caused a significant reduction in IL-1ß (p < 0.01) and CXCL1 (p < 0.05) secretion, confirming the importance of caspase-1 in the production of cytokines and chemokines associated with disease progression from BE to EAC. Targeting caspase-1 activity in BE patients should therefore be tested as a novel strategy to prevent inflammatory complications associated with disease progression.

Immune microenvironment in Barrett's esophagus adjacent to esophageal adenocarcinoma: possible influence of adjacent mucosa on cancer development and progression.

The immune microenvironment plays a pivotal role in cancer development and progression. Therefore, we studied the status of immune cells in esophageal adenocarcinoma (EAC) and adjacent Barrett's esophagus (BE) and their association with the clinical course of patients. We included 87 patients with EAC who underwent surgical resection or endoscopic submucosal dissection. CD3, CD8, Foxp3, p53, and Ki-67 were immunolocalized in EAC and adjacent BE (N = 87) and BE without EAC (N = 13). BE adjacent to EAC exhibited higher CD3+ lamina propria lymphocyte (LPL) numbers than BE without EAC. Abundant Foxp3+ LPLs in BE were associated with dysplasia and increased Ki-67 labeling index (LI) in BE glandular cells and tended to link to aberrant p53 expression. Abundant CD8+ LPLs in adjacent BE were associated with worse prognosis of EAC patients (P = 0.019). Results of our present study firstly revealed the potential influence of the tissue immune microenvironment of BE adjacent to EAC on cancer development and eventual clinical outcome of EAC patients. T cell infiltration could play pivotal roles in facilitating the dysplasia-adenocarcinoma sequence in BE. The number of Foxp3+ T cells is increased at the early stage of carcinogenesis and could help identify patients harboring dysplastic and highly proliferating cells. CD8+ T cells could reflect unfavorable inflammatory response in adjacent tissue microenvironment and help predict worse prognosis of EAC patients.

Antibodies against human papillomavirus proteins in Barrett's dysplasia and intramucosal esophageal adenocarcinoma.

High-risk human papillomavirus (HPV) types 16/18 have been associated with Barrett's dysplasia (BD)/esophageal adenocarcinoma (EAC). Nevertheless, no data exist in relation to serological analysis for HPV antibodies in BD/EAC with site-specific viral DNA status. We prospectively examined antibodies to multiple HPV types in 438 patients representing hospital/reflux controls and Barrett's metaplasia (BM)/BD/intramucosal EAC. Antibody responses to HPV6/11/16/18/31/33/45/52/58 were analyzed using multiplex serology, including antibodies to E6/E7/E1/E2 and L1 antigens. Seropositivity for individual HPV proteins was infrequent in both cases and controls and was ≤10.2%. There was no difference in the seroprevalence of antibodies to any HPV antigen/antibody combination between reclassified cases (BD/EAC) and controls (hospital/reflux/BM) or between HPV16 or HPV18 DNA cases and controls, respectively. Among HPV16 DNA-positive BD/EAC cases, antibodies to HPV16 E7 were significantly more prevalent (3/26, 11.5%) than in hospital and reflux controls plus BM (5/328, 1.5%) (adjusted OR = 10.12, 95% CI: 1.61-63.73, P = 0.014). Among HPV18 DNA-positive cases, antibodies to HPV18 E1 were present in 3/6 (50%) cases versus 5/328 (1.5%) controls (adjusted OR = 44.28, 95% CI: 6.10-321.47, P = 0.0002). Although antibodies against HPV were generally uncommon in cases and controls, immune responses against two early proteins of HPV16/18 were significantly more frequent in viral DNA-positive BD/intramucosal EAC.

Novel biomarkers for risk stratification of Barrett's oesophagus associated neoplastic progression-epithelial HMGB1 expression and stromal lymphocytic phenotype.

BACKGROUND: The incidence of oesophageal adenocarcinoma is increasing globally. Barrett's oesophagus (BO) is a pre-malignant condition with no biomarker to risk stratify those at highest risk of dysplasia and malignant transformation. METHODS: Subcellular epithelial protein (HMGB1, p53, RUNX3) expression, alongside expression of CD20, CD4, CD8 and Foxp3 to characterise stromal B lymphocyte, and helper, cytotoxic and regulatory T-lymphocyte cell infiltrate, respectively, was assessed by immunohistochemistry in 218 human tissue samples including normal oesophageal/gastric biopsies (n = 39), BO (non-dysplasia, dysplasia, non-dysplastic background from progressors to dysplasia or cancer, n = 121) and oesophageal adenocarcinoma (n = 58). RESULTS: There is a dynamic subcellular epithelial expression of HMGB1 (loss of nuclear, emergence of cytoplasmic), associated with epithelial p53 expression and differential immune cell phenotype in oesophageal neoplastic progression. We identify a protein signature and lymphocyte infiltrate in non-dysplastic BO when progressive disease (dysplasia or adenocarcinoma) is present but not histologically represented in the biopsied field. There is a dynamic stromal lymphocytic infiltrate in oesophageal neoplastic progression. CONCLUSIONS: This data reveals novel insights into the microenvironment of BO and progression towards cancer and identifies a novel high-risk biomarker of disease progression to aid surveillance strategies to identify early progression and impact future incidence of oesophageal cancer.

Achalasia and associated esophageal cancer risk: What lessons can we learn from the molecular analysis of Barrett's-associated adenocarcinoma?

Idiopathic achalasia and Barrett's esophagus (BE) are preneoplastic conditions of the esophagus. BE increases the risk of esophageal adenocarcinoma (EAC), while achalasia is associated with both EAC and esophageal squamous cell carcinoma (ESCC). However, while the molecular mechanisms underlying the transformation of esophageal epithelial cells in BE are relatively well characterized, less is known regarding these processes in achalasia. Nevertheless, both conditions are associated with chronic inflammation and BE can occur in achalasia patients, and it is likely that similar processes underlie cancer risk in both diseases. The present review will discuss possible lessons that we can learn from the molecular analysis of BE for the study of achalasia-associated cancer and contrast findings in BE with those in achalasia. First, we will describe cellular fate during development of BE, EAC, and ESCC, and consider the inflammatory status of the epithelial barrier in BE and achalasia in terms of its contribution to carcinogenesis. Next, we will summarize current data on genetic alterations and molecular pathways involved in these processes. Lastly, the plausible role of the microbiota in achalasia-associated carcinogenesis and its contribution to abnormal lower esophageal sphincter (LES) functioning, the maintenance of chronic inflammatory status and influence on the esophageal mucosa through carcinogenic by-products, will be discussed.

High-Fat Diet Accelerates Carcinogenesis in a Mouse Model of Barrett's Esophagus via Interleukin 8 and Alterations to the Gut Microbiome.

BACKGROUND & AIMS: Barrett's esophagus (BE) is a precursor to esophageal adenocarcinoma (EAC). Progression from BE to cancer is associated with obesity, possibly due to increased abdominal pressure and gastroesophageal reflux disease, although this pathogenic mechanism has not been proven. We investigated whether environmental or dietary factors associated with obesity contribute to the progression of BE to EAC in mice. METHODS: Tg(ED-L2-IL1RN/IL1B)#Tcw mice (a model of BE, called L2-IL1B mice) were fed a chow (control) or high-fat diet (HFD) or were crossbred with mice that express human interleukin (IL) 8 (L2-IL1B/IL8 mice). Esophageal tissues were collected and analyzed for gene expression profiles and by quantitative polymerase chain reaction, immunohistochemistry, and flow cytometry. Organoids were established from BE tissue of mice and cultured with serum from lean or obese individuals or with neutrophils from L2-IL1B mice. Feces from mice were analyzed by 16s ribosomal RNA sequencing and compared to 16s sequencing data from patients with dysplasia or BE. L2-IL1B were mice raised in germ-free conditions. RESULTS: L2-IL1B mice fed an HFD developed esophageal dysplasia and tumors more rapidly than mice fed the control diet; the speed of tumor development was independent of body weight. The acceleration of dysplasia by the HFD in the L2-IL1B mice was associated with a shift in the gut microbiota and an increased ratio of neutrophils to natural killer cells in esophageal tissues compared with mice fed a control diet. We observed similar differences in the microbiomes from patients with BE that progressed to EAC vs patients with BE that did not develop into cancer. Tissues from dysplasias of L2-IL1B mice fed the HFD contained increased levels of cytokines that are produced in response to CXCL1 (the functional mouse homolog of IL8, also called KC). Serum from obese patients caused organoids from L2-IL1B/IL8 mice to produce IL8. BE tissues from L2-IL1B mice fed the HFD and from L2-IL1B/IL8 mice contained increased numbers of myeloid cells and cells expressing Cxcr2 and Lgr5 messenger RNAs (epithelial progenitors) compared with mice fed control diets. BE tissues from L2-IL1B mice raised in germ-free housing had fewer progenitor cells and developed less dysplasia than in L2-IL1 mice raised under standard conditions; exposure of fecal microbiota from L2-IL1B mice fed the HFD to L2-IL1B mice fed the control diet accelerated tumor development. CONCLUSIONS: In a mouse model of BE, we found that an HFD promoted dysplasia by altering the esophageal microenvironment and gut microbiome, thereby inducing inflammation and stem cell expansion, independent of obesity.

CXCR4 Is a Potential Target for Diagnostic PET/CT Imaging in Barrett's Dysplasia and Esophageal Adenocarcinoma.

Purpose: Barrett's esophagus represents an early stage in carcinogenesis leading to esophageal adenocarcinoma. Considerable evidence supports a major role for chronic inflammation and diverse chemokine pathways in the development of Barrett's esophagus and esophageal adenocarcinoma.Experimental Design: Here we utilized an IL1ß transgenic mouse model of Barrett's esophagus and esophageal adenocarcinoma and human patient imaging to analyze the importance of CXCR4-expressing cells during esophageal carcinogenesis.Results: IL1ß overexpression induces chronic esophageal inflammation and recapitulates the progression to Barrett's esophagus and esophageal adenocarcinoma. CXCR4 expression is increased in both epithelial and immune cells during disease progression in pL2-IL1ß mice and also elevated in esophageal adenocarcinoma patient biopsy samples. Specific recruitment of CXCR4-positive (CXCR4+) immune cells correlated with dysplasia progression, suggesting that this immune population may be a key contributor to esophageal carcinogenesis. Similarly, with progression to dysplasia, there were increased numbers of CXCR4+ columnar epithelial cells at the squamocolumnar junction (SCJ). These findings were supported by stronger CXCR4-related signal intensity in ex vivo fluorescence imaging and autoradiography with advanced dysplasia. Pilot CXCR4-directed PET/CT imaging studies in patients with esophageal cancer demonstrate the potential utility of CXCR4 imaging for the diagnosis and staging of esophageal cancer.Conclusion: In conclusion, the recruitment of CXCR4+ immune cells and expansion of CXCR4+ epithelial cells in esophageal dysplasia and cancer highlight the potential of CXCR4 as a biomarker and molecular target for diagnostic imaging of the tumor microenvironment in esophageal adenocarcinoma. Clin Cancer Res; 24(5); 1048-61. ©2017 AACR.

Impact of the inflammatory microenvironment on T-cell phenotype in the progression from reflux oesophagitis to Barrett oesophagus and oesophageal adenocarcinoma.

The incidence of oesophageal adenocarcinoma (OAC), arising from reflux-induced Barrett oesophagus (BO), is increasing dramatically. T-cells have recently been implicated in the initiation of oesophagitis; however, their role in the progression from oesophagitis to BO and OAC has not been fully elucidated. Previous studies have examined the secreted cytokines from oesophageal tissue during disease progression but this study is the first to examine the activation phenotype and the inflammatory profile of CD4(+) and CD8(+) T-cells in human oesophagitis, BO and OAC tissue. Results demonstrated significantly higher levels of IL-4 producing CD4(+) T-cells and secreted levels of IL-6, confirming a Th2 phenotype in BO. In OAC tissue, both pro- and anti-inflammatory cytokines were secreted, with significantly higher levels of IL-6, IL-1ß, TNF-α, IFN-γ, IL-2 and IL-10 compared with normal oesophageal tissue. In addition, CD4(+) T-cells infiltrating OAC tissue displayed a decreased activation profile, with significantly lower CD45RO and CD69 expression compared with normal tissue. Data from this study suggest that factors in the tissue microenvironment may alter T-cell phenotype and function early during oesophageal disease progression and may represent targets for immune intervention.

Dendritic Cells in Esophageal Adenocarcinoma: The Currently Available Information and Possibilities to use Dendritic Cells for Immunotherapeutic Approaches.

Esophageal adenocarcinoma (EAC) is the second frequent cancer of the esophagus. Barrett's esophagus (BE) takes precedence over EAC. BE is a metaplastic change of the stratified squamous epithelium to the intestinal columnar epithelium due to the acidic gastrointestinal reflux. Further, the disease takes the hyperplastic stage followed by EAC. An initial immune response is an essential reaction of a body to an occurrence of alien/modified cells to be removed. It has been appreciated that an inflammatory reaction occurs in the early stages of EAC or even in BE. Dendritic cells (DCs) play a key role in a frontier of an immune response due to their advanced ability to recognize foreign antigens and mobilize naive T cells to effectors. However, in a cancer condition, tumor-delivered immunosuppression occurs in a variety of mechanisms that alter/switch the functionality of DCs from immune activating to immune suppressive cells. In this brief review, we consider tumor-induced paths of a capacity of tumor cells to down-regulate DCs, with a focus on EAC, and also discuss a possibility to use DCs for immunotherapeutic approaches. Indeed, DCs represent a promising tool for developing new immunotherapeutic approaches for cancer treatment including EAC. It has been reported to achieve effective DC-mediated immune responses by raising anti-tumor cytotoxic T cell responses against multiple cancer antigens through loading DCs with total tumor RNA. However, more studies should be performed in order to understand a precise role in tumor-induced mechanisms of DC suppression in BE/EAC. Likely, these mechanisms should involve general carcinogenic and EAC-specific pathways.

Squamous tissue lymphocytes in the esophagus of controls and patients with reflux esophagitis and Barrett's esophagus are characterized by a non-inflammatory phenotype.

BACKGROUND AND OBJECTIVE: Reflux esophagitis (RE) is characterized by inflammation of the squamous epithelium (SQ) of the esophagus and may progress to Barrett's esophagus (BE) characterized by intestinal metaplasia. The role of inflammation in this transition has been postulated but lacks experimental evidence. Here, the inflammatory responses in the esophagus of these patients were investigated. PATIENTS AND METHODS: Fifty-one esophageal biopsies from with patients BE (n = 19), RE (n = 8) and controls (n = 23) were analyzed. T-cells were analyzed before and after ex vivo expansion (14 days) by multicolor flow cytometric analysis. The following markers were studied: CD3, CD4, CD8 (T-cell markers), Granzyme B (marker of cytotoxicity), CD103 (αE/epithelial integrin) and NKg2a (inhibitory receptor on T-cells and NK-cells). RESULTS: Analysis of ex vivo cultures from normal looking SQ from controls, RE patients, and BE patients revealed no significant differences in the number and phenotypes of T-cells. In contrast, tissue from RE was different to normal SQ in four aspects: 1) higher percentages of CD3+ CD4+-cells (72±7% vs 48±6%, p = 0.01) and 2) CD8+ GranzymeB+-cells (53±11% vs 26±4%, p<0.05), while 3) lower percentages of CD4+ CD103+-cells (45±19% vs 80±3%, p = 0.02) and 4) CD8+ NKg2a+-cells (31±12% vs 44±5%). CONCLUSION: Despite the fact that both tissues are exposed to the same reflux associated inflammatory triggers, the immune response observed in RE is clearly distinct from that in SQ of BE. The differences in immune responses in BE tissue might contribute to its susceptibility for transformation into intestinal metaplasia.

Inflammation and oxidative stress markers and esophageal adenocarcinoma incidence in a Barrett's esophagus cohort.

BACKGROUND: Persons with Barrett's esophagus experience increased risk of esophageal adenocarcinoma. Prediagnostic inflammation markers predict several cancers, but their role in predicting esophageal adenocarcinoma is unknown. METHODS: We investigated whether biomarkers of inflammation [C-reactive protein (CRP), interleukin-6 (IL6), soluble tumor necrosis factor (sTNF) receptors I and II], and of oxidative stress (F2-isoprostanes) predicted progression to esophageal adenocarcinoma in a prospective cohort of 397 patients with Barrett's esophagus, 45 of whom developed esophageal adenocarcinoma. Biomarkers were measured in stored plasma samples from two time points during follow-up, the mean of which served as the primary predictor. Adjusted hazard ratios (HR) and 95% confidence intervals (CI) were estimated using Cox regression. RESULTS: CRP level above the median was associated with an 80% increased risk of esophageal adenocarcinoma. The HR and 95% CI adjusted for age, gender, and further adjusted for waist-hip ratio and smoking were 1.98 (1.05-3.73) and 1.77 (0.93-3.37), respectively, with Ptrend for continuous CRP = 0.04. Persons with IL6 levels above the median also had almost 2-fold increased risk [HR and 95% CI adjusted for age and gender, and further adjusted for waist-hip ratio and smoking were 1.95 (1.03-3.72) and 1.79 (0.93-3.43), respectively, but no evidence of a trend was observed]. Concentrations of TNF receptors and F2-isoprostanes were not associated with esophageal adenocarcinoma risk. CONCLUSIONS: Further research is needed to evaluate the role of inflammation and associated markers in esophageal adenocarcinoma development in persons with Barrett's esophagus. IMPACT: This prospective study suggests that inflammation markers, particularly CRP and IL6, may help identify persons at higher risk of progression to esophageal adenocarcinoma.

Evaluation of Barrett esophagus by multiphoton microscopy.

CONTEXT: Multiphoton microscopy (MPM) based on 2-photon excitation fluorescence and second-harmonic generation allows simultaneous visualization of cellular details and extracellular matrix components of fresh, unfixed, and unstained tissue. Portable multiphoton microscopes, which could be placed in endoscopy suites, and multiphoton endomicroscopes are in development, but their clinical utility is unknown. OBJECTIVE: To examine fresh, unfixed endoscopic biopsies obtained from the distal esophagus and gastroesophageal junction to (1) define the MPM characteristics of normal esophageal squamous mucosa and gastric columnar mucosa, and (2) evaluate whether diagnosis of intestinal metaplasia/Barrett esophagus (BE) could be made reliably with MPM. DESIGN: The study examined 35 untreated, fresh biopsy specimens from 25 patients who underwent routine upper endoscopy. A Zeiss LSM 710 Duo microscope (Carl Zeiss, Thornwood, New York) coupled to a Spectra-Physics (Mountain View, California) Tsunami Ti:sapphire laser was used to obtain a MPM image within 4 hours of fresh specimen collection. After obtaining MPM images, the biopsy specimens were placed in 10% buffered formalin and submitted for routine histopathologic examination. Then, the MPM images were compared with the findings in the hematoxylin-eosin-stained, formalin-fixed, paraffin-embedded sections. The MPM characteristics of the squamous, gastric-type columnar and intestinal-type columnar epithelium were analyzed. In biopsies with discrepancy between MPM imaging and hematoxylin-eosin-stained sections, the entire tissue block was serially sectioned and reevaluated. A diagnosis of BE was made when endoscopic and histologic criteria were satisfied. RESULTS: Based on effective 2-photon excitation fluorescence of cellular reduced pyridine nucleotides and flavin adenine dinucleotide and lack of 2-photon excitation fluorescence of mucin and cellular nuclei, MPM could readily identify and distinguish among squamous epithelial cells, goblet cells, gastric foveolar-type mucous cells, and parietal cells in the area of gastroesophageal junction. Based on the cell types identified, the mucosa was defined as squamous, columnar gastric type (cardia/fundic-type), and metaplastic columnar intestinal-type/BE. Various types of mucosa seen in the study of 35 biopsies included normal squamous mucosa only (n = 14; 40%), gastric cardia-type mucosa only (n = 2; 6%), gastric fundic mucosa (n = 6; 17%), and both squamous and gastric mucosa (n = 13; 37%). Intestinal metaplasia was identified by the presence of goblet cells in 10 of 25 cases (40%) leading to a diagnosis of BE on MPM imaging and only in 7 cases (28%) by histopathology. In 3 of 35 biopsies (9%), clear-cut goblet cells were seen by MPM imaging but not by histopathology, even after the entire tissue block was sectioned. Based on effective 2-photon excitation fluorescence of elastin and second-harmonic generation of collagen, connective tissue in the lamina propria and the basement membrane was also visualized with MPM. CONCLUSIONS: Multiphoton microscopy has the ability to accurately distinguish squamous epithelium and different cellular elements of the columnar mucosa obtained from biopsies around the gastroesophageal junction, including goblet cells that are important for the diagnosis of BE. Thus, use of MPM in the endoscopy suite might provide immediate microscopic images during endoscopy, improving screening and surveillance of patients with BE.

The esophagitis to adenocarcinoma sequence; the role of inflammation.

Esophageal adenocarcinoma (EAC) is the eighth most common cancer worldwide, and approximately 15% of patients survive 5years. Reflux disease (GERD) and Barrett's esophagus (BE) are major risk factors for the development of EAC, and epidemiologic studies highlight a strong association with obesity. The immune, inflammatory and intracellular signaling changes resulting from chronic inflammation of the esophageal squamous epithelium are increasingly well characterized. In GERD and Barrett's, an essential role for T-cells in the initiation of inflammation in the esophagus has been identified, and a balance between T-cell responses and phenotype may play an important role in disease progression. Obesity is a chronic low-grade inflammatory state, fueled by adipose tissue derived- inflammatory mediators such as IL-6, TNF-α and leptin, representing a novel area for targeted research. Additionally, reactive oxygen species (ROS) and receptor tyrosine kinase (RTK) activation may drive progression from esophagitis to EAC, and downstream signaling pathways employed by these molecules may be important. This review will explain the diverse range of mechanisms potentially driving and maintaining inflammation within the esophagus and explore both existing and future therapeutic strategies targeting the process.

Haplotypes of the IL-1 gene cluster are associated with gastroesophageal reflux disease and Barrett's esophagus.

OBJECTIVES: Gastroesophageal reflux (GERD) is a one of the major public health problem that can lead to reflux esophagitis (RE), Barrett's esophagus (BE), and esophageal adenocarcinoma (EAC). The aim of our study was to determine the impact of IL-1 gene polymorphisms on the development of GERD, RE and BE. METHODS: Three hundred and thirty-three Czech patients with gastroesophageal reflux and 165 healthy controls were included in this case-control study. Four polymorphisms in the genes of the IL-1 cluster [IL-1A(-889C/T), IL-1B(-511C/T), IL-1B(+3953C/T), and IL-1RN(VNTR)] were analyzed. RESULTS: Significant differences were found in IL-1RN 1/2 genotype between patients with GERD/RE and controls and in IL-1B+3953 T allele between patients with BE and healthy subjects. In addition, complex analysis revealed differences in IL-1 haplotype frequencies between the groups. Specifically, the haplotype TCCL was significantly more frequent (p = 0.016) in GERD patients than in controls and the haplotype CCCL more frequent (p = 0.008) in RE patients than in controls. However, in patients with BE, frequency of haplotype TCTL was lower (p = 0.05) and haplotypes CTCL and TCCL were higher (p = 0.03 and p = 0.02) in comparison with the controls. CONCLUSIONS: Our results suggest that IL-1 haplotypes may be associated with susceptibility to GERD, RE and BE.

1,25-dihydroxyvitamin D in the pathogenesis of Barrett's esophagus and esophageal adenocarcinoma.

The incidence of reflux-related esophageal disease - Barrett's esophagus and esophageal adenocarcinoma - is rising, and the prognosis remains poor. Evidence exists that 1,25-dihydroxyvitamin D may augment the course of colon, breast and prostate cancer but little knowledge exists regarding its impact on disease of the esophagus. Important immune cells involved in reflux-related esophageal disease include CD4(+) T cells, macrophages and dendritic cells, and key signaling pathways include Wnt, Hedgehog, NFκ-B and IL-6-JAK-STAT. There is an inter-relationship between these entities and 1,25-dihydroxyvitamin D, which has been described in animal models and some human tissue. Despite this, there is an incomplete understanding of how the immune cell population and signaling pathways contribute to the course and prognosis of Barrett's esophagus and esophageal adenocarcinoma. More investigation with a focus on the clinical outcomes of patients with Barrett's esophagus and esophageal adenocarcinoma and the immune cell population and cell signaling activity in the diseased esophagus is necessary to determine the immunomodulatory role of 1,25-dihydroxyvitamin D in the pathogenesis of esophageal diseases.

Dendritic cells in Barrett's esophagus carcinogenesis: an inadequate microenvironment for antitumor immunity?

Barrett's esophagus corresponds to the replacement of the normal esophageal squamous epithelium by a columnar epithelium through a metaplastic process. This tissue remodeling is associated with chronic gastroesophageal reflux and constitutes a premalignant lesion leading to a 30- to 60-fold increase in the risk to evolve into esophageal adenocarcinoma. The present study aimed to investigate a possible immune evasion in Barrett's esophagus favoring esophageal adenocarcinoma development. We demonstrated that myeloid and plasmacytoid dendritic cells are recruited during the esophageal metaplasia-dysplasia-carcinoma sequence, through the action of their chemoattractants, macrophage inflammatory protein 3α and chemerin. Next, we showed that, in contrast to plasmacytoid dendritic cells, myeloid dendritic cells, co-cultured with Barrett's esophagus and esophageal adenocarcinoma cell lines, display a tolerogenic phenotype. Accordingly, myeloid dendritic cells co-cultured with esophageal adenocarcinoma cell lines stimulated regulatory T cell differentiation from naïve CD4(+) T cells. In agreement with those results, we observed that both metaplastic areas and (pre)malignant lesions of the esophagus are infiltrated by regulatory T cells. In conclusion, soluble factors secreted by epithelial cells during the esophageal metaplasia-dysplasia-carcinoma sequence influence dendritic cell distribution and promote tumor progression by rendering them tolerogenic.

The immune cell composition in Barrett's metaplastic tissue resembles that in normal duodenal tissue.

BACKGROUND AND OBJECTIVE: Barrett's esophagus (BE) is characterized by the transition of squamous epithelium into columnar epithelium with intestinal metaplasia. The increased number and types of immune cells in BE have been indicated to be due to a Th2-type inflammatory process. We tested the alternative hypothesis that the abundance of T-cells in BE is caused by a homing mechanism that is found in the duodenum. PATIENTS AND METHODS: Biopsies from BE and duodenal tissue from 30 BE patients and duodenal tissue from 18 controls were characterized by immmunohistochemistry for the presence of T-cells and eosinophils(eos). Ex vivo expanded T-cells were further phenotyped by multicolor analysis using flowcytometry. RESULTS: The high percentage of CD4(+)-T cells (69±3% (mean±SEM/n = 17, by flowcytometry)), measured by flowcytometry and immunohistochemistry, and the presence of non-activated eosinophils found in BE by immunohistochemical staining, were not different from that found in duodenal tissue. Expanded lymphocytes from these tissues had a similar phenotype, characterized by a comparable but low percentage of αE(CD103) positive CD4(+)cells (44±5% in BE, 43±4% in duodenum of BE and 34±7% in duodenum of controls) and a similar percentage of granzyme-B(+)CD8(+) cells(44±5% in BE, 33±6% in duodenum of BE and 36±7% in duodenum of controls). In addition, a similar percentage of α4ß7(+) T-lymphocytes (63±5% in BE, 58±5% in duodenum of BE and 62±8% in duodenum of controls) was found. Finally, mRNA expression of the ligand for α4ß7, MAdCAM-1, was also similar in BE and duodenal tissue. No evidence for a Th2-response was found as almost no IL-4(+)-T-cells were seen. CONCLUSION: The immune cell composition (lymphocytes and eosinophils) and expression of intestinal adhesion molecule MAdCAM-1 is similar in BE and duodenum. This supports the hypothesis that homing of lymphocytes to BE tissue is mainly caused by intestinal homing signals rather than to an active inflammatory response.