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Contains fulltext : 225282.pdf (Publisher’s version ) (Open Access) Barrett's esophagus (BE) is a metaplastic condition of the distal esophagus, resulting from longstanding gastroesophageal reflux disease (GERD). BE predisposes for the highly malignant esophageal adenocarcinoma (EAC). Both BE and EAC have the highest frequencies in white males. Only a subset of patients with GERD develop BE, while

Background & Aims: Barrett's esophagus (BE) is a metaplastic condition in which normal squamous esophageal epithelium is replaced by columnar epithelium. It is proposed that one of the possible mechanisms is dedifferentiation of squamous epithelium into columnar epithelium. The pathophysiology through which this metaplasia occurs is unknown. A recent study by serial analysis of gene expression showed that bone morphogenetic protein 4 (BMP-4) is uniquely expressed in BE. In this study, the role of the BMP pathway in the metaplastic transformation of normal squamous cells into columnar cells was examined. Methods: Tissues from patients with esophagitis and BE and in an esophagitis-BE rat model were examined for the activation of the BMP pathway. Short-term cultures of primary normal squamous esophageal cells were treated with BMP-4, and cell biological changes were examined by Western blot analysis, immunohistochemistry, and microarrays. Results: In both human and rat tissues, the BMP pathway proved to be activated in esophagitis and BE. Upon incubation of squamous cell cultures with BMP-4, the cytokeratin expression pattern showed a shift that was consistent with columnar epithelium. Involvement of the BMP pathway was suggested by up-regulation of Phosphorylated-Smad 1/5/8 (P-Smad 1/5/8) that was effectively blocked by Noggin, a BMP antagonist. Comparison of the gene expression profiles of squamous cells, BMP-4-treated squamous cells, and BE cells showed a significant shift in the profile of the BMP-4-treated squamous cells toward that of the cultured BE cells. Conclusions: These results suggest that the BMP pathway could play a role in the transformation of normal esophageal squamous cells into columnar cells.

BACKGROUND: Neoplastic progression of BE towards EAC is associated with increased expression of COX-2. Increased COX-2 expression and enzyme activity is linked to the COX-2 CA haplotype, which consists of two gene polymorphisms in the COX-2 promoter. AIM: To study the impact of COX-2 haplotypes on the risk of developing EAC in patients with different forms of gastroesophageal reflux disease including BE. METHODS: DNA was obtained from a total of 635 Dutch white patients comprised of 140 patients with EAC, 255 with BE, and 240 with reflux esophagitis. COX-2 haplotypes were based on the gene polymorphisms at -765C/G and -1195A/G, as determined by PCR-RFLP. RESULTS: The tested population contained 170 (14%) CA- (-765C and -1195A) haplotypes, 829 (65%) GA and 271 (21%) GG-haplotypes, and no GC-haplotypes. The haplotype distribution in patients with reflux esophagitis and BE was similar (CA 12%, GA 68%, GG 21%), but differed significantly from that in patients with EAC (CA 21%, GA 58%, GG 20%). Particularly, the CA-haplotype was more common (P < 0.001) in EAC patients. CA-carriership was associated with EAC (OR 2.8, 95% CI 1.3-6.2, P = 0.008), with homozygosity for the CA-allele being statistically most significantly associated (OR 6.1, 95% CI 1.6-24.2, P = 0.01). CONCLUSION: The COX-2 CA-haplotype is more frequently observed in patients with EAC than in patients with BE and reflux esophagitis. These data suggest a direct link between COX-2 activity and neoplastic progression in patients with BE and reflux esophagitis.

Background & Aims: The metaplastic process in which the normal squamous epithelium of the distal esophagus is replaced by columnar-lined epithelium, known as Barrett’s esophagus (BE), is poorly understood. The aim of this study was to define, analyze, and compare transcription profiles of BE, normal cardia epithelium, and squamous epithelium to gain more insight into the process of metaplasia and to identify uniquely expressed genes in these epithelia. Methods: Serial analysis of gene expression was applied for obtaining transcription libraries of biopsy specimens taken from a BE-affected patient with intestinal type of metaplasia and from normal squamous and gastric cardia epithelia. Validation of results by reverse-transcription polymerase chain reaction and immunoblotting was performed using tissues of 20 patients with BE. Results: More than 120,000 tags were sequenced. Between BE and squamous 776, and between BE and gastric cardia 534 tags were significantly differentially expressed (P < .05, pairwise comparison). In contrast, squamous compared with gastric cardia epithelia showed significant differential expression of 1316 tags. The most up-regulated genes in BE compared with squamous epithelium were trefoil factors, annexin A10, and galectin-4. Each of the epithelia showed a unique cytokeratin expression profile. Conclusions: This study provides a comparison of the transcriptomes of BE, squamous epithelium, and gastric cardia epithelium. BE proves to be an incompletely differentiated type of epithelium that shows similarities to both normal squamous and gastric cardia epithelia. In addition, several uniquely expressed genes are identified. These results are a major advancement in understanding the process of metaplasia that leads to BE.

OBJECTIVES: Endoscopic radiofrequency ablation (RFA) eradicates intestinal metaplasia and intraepithelial neoplasia associated with Barrett's esophagus (BE), restoring an endoscopically normal neosquamous epithelium (NSE). We evaluated the post-RFA NSE for genetic abnormalities and buried glandular mucosa. METHODS: Eligible patients underwent RFA for BE containing early cancer and/or high-grade intraepithelial neoplasia with subsequent complete histological reversion to normal NSE. At baseline, the BE was sampled by brush cytology and biopsies. At least 2 months after RFA, the NSE was sampled by brush cytology, keyhole biopsies, and endoscopic resection. The untreated squamous epithelium was biopsied as a control. The baseline BE and post-RFA NSE were evaluated for immunohistochemical expression of Ki-67 and p53, and genetic abnormalities (DNA-fluorescent in situ hybridization: chromosome 1 and 9, p16 and p53). In addition, biopsy depth was compared for biopsies from the NSE and untreated squamous epithelium. The presence of buried glandular mucosa in NSE was assessed with primary and keyhole biopsy, and endoscopic resection. RESULTS: All pretreatment specimens from all 22 patients showed abnormalities on immunohistochemical staining and fluorescent in situ hybridization, whereas all post-RFA NSE specimens were normal. All the post-RFA biopsies from the NSE contained full epithelia, whereas 37% contained lamina propria, a finding no different from biopsies from untreated squamous epithelium (36% lamina propria). Deeper keyhole biopsies contained lamina propria in 51%. All endoscopic resection specimens contained submucosa, whereas no biopsy or endoscopic resection specimen contained buried glandular mucosa. CONCLUSIONS: Rigorous evaluation of the post-RFA NSE in patients who, at baseline, had BE containing early cancer high-grade intraepithelial neoplasia, showed neither persistent genetic abnormalities nor buried glandular mucosa

Objective. Barrett's esophagus (BE) is a premalignant condition of the distal esophagus. For diagnostic purposes it is important to find biomarkers that can specifically identify BE, for instance to differentiate BE epithelial cells from gastric cardia epithelial cells in brush cytology specimens. The objective of this study was to determine the specificity of CDX-2 and a set of cytokeratins (CKs) as specific markers for BE as compared with normal squamous esophageal and gastric cardia tissue. Material and methods. Immunohistochemistry (IHC) with specific antibodies against CDX-2, and a set of CKs was performed on fresh frozen consecutive tissue sections of normal squamous, gastric cardia and non-dysplastic BE of 80 patients. Results. IHC results showed CK8, CK18 and CK20 expression in both BE and gastric cardia, while CK7 was seen in all BE but also in 26% of gastric cardia biopsies. CK10/13 was only expressed in normal squamous epithelium. CDX-2 nuclear staining was found in 87.5% of the BE biopsies, whereas normal squamous esophagus and cardia biopsies were negative. Conclusions. CDX-2 in combination with a set of CKs can be used as biomarkers to distinguish between BE and normal squamous esophagus. In order to distinguish BE from cardia tissue, a combination of CDX-2 and CK7 is most informative.

OBJECTIVES: Malignant transformation of Barrett's mucosa is associated with the accumulation of genetic alterations. Stepwise radical endoscopic resection of the Barrett's segment with early neoplasia is a promising new treatment resulting in complete re-epithelialization of the esophagus with neosquamous epithelium. It is unknown whether radical resection also eradicates genetic abnormalities. The aim of this study was to prospectively evaluate whether genetic abnormalities as found in the Barrett's segment before radical resection are effectively eradicated and absent in the neosquamous epithelium. METHODS: Nine patients with early neoplasia who successfully underwent radical resection were included. Immunohistochemistry (IHC) was performed to assess p53 protein overexpression. DNA fluorescent in-situ hybridization was (DNA-FISH) performed for evaluation of numerical abnormalities of chromosomes 1 and 9, and losses of p16 and p53. Immunohistochemistry and DNA-FISH were performed on endoscopic resection specimens of the neoplasia and on follow-up biopsies of the neosquamous epithelium. RESULTS: DNA-FISH and IHC showed alterations in the pretreatment samples of all patients. All showed aneusomy of chromosome 1 and 9. Loss of p16 and p53 were seen in 6 and 8 patients. IHC showed intense p53 nuclear staining in seven patients. Post-treatment biopsies showed neosquamous epithelium with a normal diploid signal count for all DNA-FISH probes and normal IHC stainings in all patients. CONCLUSIONS: Radical resection of Barrett's esophagus with early neoplasia successfully eradicates pre-existing genetic abnormalities and results in neosquamous epithelium without these genetic abnormalities

examine the effect of acid on Wnt/β-catenin signaling activation and on regulating the expression of Dickkopf1 (DKK1), an inhibitor of the Wnt. Methods: Normal esophageal squamous cells lines (EPC1-hTERT, EPC2-hTERT), a non-dysplastic Barrett's esophageal cell line (CP-A) and an esophageal adenocarcinoma cell line (OE33) were exposed to acidic media (pH 4.0) in a pulsive manner. Human esophageal mucosal biopsies in triplicate from healthy (n=1), NERD (n=1), GERD (n=1) and Barrett's (n=1) patients were obtained and cultured in acidic (pH 4.0) or non acidic media. Localization and levels of β-catenin were determined by Immunofluorescence staining and Western blot. Wnt-activity was assessed by Luciferase assay following transfection with β-catenin-LEF/TCF-sensitive (TOP) or βcatenin-LEF/TCF insensitive (FOP) reporter vector. Immunofluorescence was used for βcatenin and E-Cadherin co-localization. DKK1 and β-catenin gene expression was resolved by qRT-PCR. DKK1 secretion in cells culture media and organ culture media was quantified by ELISA assay.Results: Acid destabilized E-cadherin/β-catenin complex in cell-cell junctions and resulted in β-catenin translocation to nucleus. Wnt-activity correlated with nuclear translocation of β-catenin. Cytosolic shuttling of β-catenin occurred in a rapid and transient manner after acid withdrawal. Chronic pulsed acid exposure increased DKK1 expression in normal squamous cells but not in metaplastic columnar cells. DKK1 overexpression correlated with a significant degradation of β-catenin. Mucosal biopsies from patients with NERD/ GERD secreted significantly higher levels of Dkk1 than healthy and metaplastic mucosa biopsies. Conclusions: These findings suggest that acid reflux induces the activation of Wnt-signaling pathway and that the overexpression of DKK1 is the long term response in the normal squamous esophageal tissue but not in the Barrett's esophagus. These findings also suggest a homeostatic role for DKK1 in GERD and its dysfunction to be a potential mechanism for progression to Barrett's metaplasia.

Detection of High-Grade Dysplasia and Esophageal Adenocarcinoma Using Endoscopic Mucosal Resection in Combination with Fluorescence in Situ Hybridization Wytske Westra, Ganapathy A. Prasad, Kevin C. Halling, Shannon M. Brankley, Emily Barr Fritcher, Trynda N. Oberg, Jesse S. Voss, Michael B. Campion, Navtej Buttar, LouisMichel Wong Kee Song, Lori S. Lutzke, Kelly T. Dunagan, Lynn S. Borkenhagen, Agnieszka M. Rygiel, Kausilia K. Krishnadath, Kenneth K. Wang