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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

Abnormal DNA ploidy status has been suggested as a prognostic factor for Barrett's esophagus progression into esophageal adenocarcinoma (EAC). The aim of the study was to compare image cytometry DNA analysis (ICDA) and fluorescent in situ hybridization (FISH) in the assessment of DNA ploidy status in Barrett's esophagus (BE), and to determine the value of these abnormalities as an adjunct to conventional cytology in detection of dysplasia and EAC. Brush cytology specimens of 90 BE patients were examined using ICDA and FISH with peri-centromeric probes for chromosomes 7 and 17. The results of ICDA and FISH were compared with each other, and with dysplasia grade or EAC as determined by histology and cytology. FISH and ICDA detected abnormalities in 41% (37/90) and 22% (19/90) of the BE cases, respectively. Gains of chromosome 7 and/or 17 were present in 13% of nondysplasia cases, which further increased with dysplasia stage, while overall DNA content aneuploidy was detected predominantly in high grade dysplasia (HGD) and EAC. Using FISH results combined with cytology, we were able to identify IND/LGD (indefinite/ low grade dysplasia) with a sensitivity and specificity of 75 and 76%, respectively. FISH alone detected HGD/EAC with a high sensitivity and specificity of 85 and 84%, which was superior to that of cytology alone. Thus, FISH is more sensitive than ICDA to detect chromosomal abnormalities in BE brush cytology specimens. FISH detects chromosomal gains in early stages of BE and represents a valuable adjunct to conventional cytology to detect dysplasia or EAC. (C) 2008 Wiley-Liss, Inc.

Esophageal adenocarcinoma (EA) and esophageal squamous cell carcinoma (ESCC) are the two main types of esophageal cancer. Despite extensive research the exact molecular basis of these cancers is unclear. Therefore we evaluated the transcriptome of EA in comparison to non-dysplastic Barrett’s esophagus (BE), the metaplastic epithelium that predisposes for EA, and compared the transcriptome of ESCC to normal esophageal squamous epithelium. For obtaining the transcriptomes tissue biopsies were used and serial analysis of gene expression (SAGE) was applied. Validation of results by RT-PCR and immunoblotting was performed using tissues of an additional 23 EA and ESCC patients. Over 58,000 tags were sequenced. Between EA and BE 1013, and between ESCC and normal squamous epithelium 1235 tags were significantly differentially expressed (p < 0.05). The most up-regulated genes in EA compared to BE were SRY-box 4 and Lipocalin2, whereas the most down-regulated genes in EA were Trefoil factors and Annexin A10. The most up-regulated genes in ESCC compared to normal squamous epithelium were BMP4, E-Cadherin and TFF3. The results could suggest that the BE expression profile is closer related to normal squamous esophagus then to EA. In addition, several uniquely expressed genes are identified.

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.

An accurate evaluation of the Her-2 status has important prognostic and therapeutic implications in many carcinomas. The aim of the study was to correlate Her-2 locus (17q11.2) amplification and chromosome 17 gains as assessed by fluorescent in situ hybridization (FISH) with Her-2 protein overexpression by immunohistochemistry (IHC) in patients with Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). We analyzed 34 patients with Her-2 amplification and/or chromosome 17gains using FISH on brush cytology specimens. Seven patients (21%) showed high Her-2 locus amplification ( Her-2: Cep17 5 : 1), 5 (15%) showed low Her-2 locus amplification ( Her-2: Cep17 2 5 : 1), and 22 (65%) displayed gains of chromosome 17 only. Further, we confirmed Her-2 amplification on corresponding biopsies that were taken at the same occasion as the cytologybrushings. Then, we compared the FISH results with IHC data obtained from the corresponding biopsies and showed that low level of Her-2 amplification does not correlate with Her-2 protein overexpression (score / ; ), in contrast to the high amplification level ( ). Thus, in our population of BE and EAC patients, low level of Her-2 amplification does not result in detectable level of Her-2 protein as assessed by IHC.

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.

The progression of Barrett's esophagus to esophageal adenocarcinoma is often characterized by the accumulation of genetic abnormalities. The goal was to evaluate the copy number alterations of several oncogene loci, including 7p12 [epidermal growth factor receptor (EGFR)], 8q24 (c-myc), and 20q13 in the sequence of no dysplasia–dysplasia–adenocarcinoma of Barrett's esophagus. Fluorescence in situ hybridization with DNA probes for the centromeric region of chromosome 7 and the locus-specific regions of 7p12 (EGFR), 8q24 (c-myc), and 20q13 was applied on 99 brush cytology specimens of patients with Barrett's esophagus with different stages of dysplasia or esophageal adenocarcinoma. Gains (3-4 copies) of chromosome 17, 8q24 (c-myc), and 20q.13 loci were found in the low frequencies in nondysplastic Barrett's esophagus. Their frequencies increased with the stage of dysplasia and reached a high incidence in esophageal adenocarcinoma. Amplification (>4 copies) of at least 1 of the loci was observed in 14% of high-grade dysplasia and increased to 50% in esophageal adenocarcinoma (P = 0.015). The most frequently amplified locus was c-myc (18%), followed by 20q13 (13%) and EGFR (11%) in the high-grade dysplasia/esophageal adenocarcinoma cases. High amplification levels (>10 copies) of the loci were more frequent in esophageal adenocarcinoma (72%) compared with high-grade dysplasia (20%; P = 0.049). Amplifications of the c-myc, EGFR, and 20q12 loci may serve as diagnostic markers to identify patients with Barrett's esophagus with high-grade dysplasia or esophageal adenocarcinoma. Gains of the loci might be of value as prognostic markers because they are already present in nondysplasia cases and may precede the later event of the amplification as observed in high-grade dysplasia and esophageal adenocarcinoma. (Cancer Epidemiol Biomarkers Prev 2008;17(6):1380–5)

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

Epidemiological studies indicate that vitamin D exerts a protective effect on the development of various solid cancers. However, concerns have been raised regarding the potential deleterious role of high vitamin D levels in the development of esophageal adenocarcinoma (EAC). This study investigated genetic variation in the vitamin D receptor (VDR) in relation to its expression and risk of Barrett esophagus (BE) and EAC. VDR gene regulation was investigated by immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and gel shift assays. Fifteen haplotype tagging single-nucleotide polymorphisms (SNPs) of the VDR gene were analyzed in 858 patients with reflux esophagitis (RE), BE or EAC and 202 healthy controls. VDR mRNA expression was higher in BE compared with squamous epithelium. VDR protein was located in the nucleus in BE. An rs1989969T/rs2238135G haplotype was identified in the 5' regulatory region of the VDR gene. It was associated with an approximately two-fold reduced risk of RE, BE and EAC. Analysis of a replication cohort was done for BE that confirmed this. The rs1989969T allele causes a GATA-1 transcription factor binding site to appear. The signaling of GATA-1, which is regarded as a negative transcriptional regulator, could explain the findings for rs1989969. The rs2238135G allele was associated with a significantly reduced VDR expression in BE; for the rs1989969T allele, a trend in reduced VDR expression was observed. We identified a VDR haplotype associated with reduced esophageal VDR expression and a reduced incidence of RE, BE and EAC. This VDR haplotype could be useful in identifying individuals who benefit most from vitamin D chemoprevention.

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