Barrett's Esophagus Epithelium Research Articles

652. EVOLUTION OF BARRETT’S ESOPHAGUS IN PATIENTS WHO UNDERWENT UPPER DIGESTIVE TRACT SURGERY: RETROSPECTIVE STUDY AT HIGH VOLUME CENTER

Abstract Continuous episodes of Gastroesophageal reflux (GER) attack the esophageal mucosa and may lead to the unusual replacement of the squamous cell epithelium for the glandular columnar cell epithelium, with goblet cells. The intestinal metaplasia, in the esophageal mucosa characterizes Barrett's Esophagus (BE). BE occurs in 10 to 15% of chronic and long-term reflux patients. In addition, BE's epithelium is susceptible to chronic inflammation with risks for dysplasia and adenocarcinoma. We analyzed the impact of unconventional techniques on the evolution of Barrett's esophagus extension, as: Antrectomy (n = 1), Roux-en-Y Bypass (n = 5), Cardiomyotomy (n = 6), Esophagectomy (n = 10), Gastrectomy (n = 12), Lind fundoplication (n = 12). For this purpose, a retrospective study with BE patients treated at our service was carried out, evaluating the extension of Barrett's epithelium between the first and the last endoscopy of each patient. The greatest mean absolute regression of the BE extension was seen in esophagectomy, equivalent to 2.87 cm; esophagectomy combined with gastrectomy, 1.15 cm; and gastrectomy, with 0.97 cm. Antrectomy and procedures involving Roux-en-Y, in our series, did not reduce the extent of the columnar epithelium, actually showing progression or formation of Barrett's Esophagus. It is worth mentioning that Lind fundoplication, once the main treatment for GERD, obtained a mean regression of 0.37 cm in the BE extension, however it was not the treatment with the best result, and 17% of the evaluated patients showed lesion progression. The majority of evaluated procedures as alternative procedures for the treatment of reflux in patients with BE can be effective in controlling the evolution and even contributing to its regression.

Tu1128 Stem Cells From Submucosal Glands as a Possible Origin for Barretts Esophagus Epithelium

Tu1128 Stem Cells From Submucosal Glands as a Possible Origin for Barretts Esophagus Epithelium

A statistical framework for multiparameter analysis at the single-cell level

Phenotypic characterization of individual cells provides crucial insights into intercellular heterogeneity and enables access to information that is unavailable from ensemble averaged, bulk cell analyses. Single-cell studies have attracted significant interest in recent years and spurred the development of a variety of commercially available and research-grade technologies. To quantify cell-to-cell variability of cell populations, we have developed an experimental platform for real-time measurements of oxygen consumption (OC) kinetics at the single-cell level. Unique challenges inherent to these single-cell measurements arise, and no existing data analysis methodology is available to address them. Here we present a data processing and analysis method that addresses challenges encountered with this unique type of data in order to extract biologically relevant information. We applied the method to analyze OC profiles obtained with single cells of two different cell lines derived from metaplastic and dysplastic human Barrett's esophageal epithelium. In terms of method development, three main challenges were considered for this heterogeneous dynamic system: (i) high levels of noise, (ii) the lack of a priori knowledge of single-cell dynamics, and (iii) the role of intercellular variability within and across cell types. Several strategies and solutions to address each of these three challenges are presented. The features such as slopes, intercepts, breakpoint or change-point were extracted for every OC profile and compared across individual cells and cell types. The results demonstrated that the extracted features facilitated exposition of subtle differences between individual cells and their responses to cell-cell interactions. With minor modifications, this method can be used to process and analyze data from other acquisition and experimental modalities at the single-cell level, providing a valuable statistical framework for single-cell analysis.

MicroRNA-196a & microRNA-101 expression in Barrett's oesophagus in patients with medically and surgically treated gastro-oesophageal reflux

BackgroundProton pump inhibitor (PPI) medication and surgical fundoplication are used for the control of gastro-oesophageal reflux in patients with Barrett's oesophagus, but differ in their effectiveness for both acid and bile reflux. This might impact on the inflammatory processes that are associated with progression of Barrett's oesophagus to cancer, and this may be evident in the gene expression profile and microRNA expression pattern in Barrett's oesophagus mucosa. We hypothesised that two miRNAs with inflammatory and oncogenic roles, miR-101 and miR-196a, are differentially expressed in Barrett's oesophagus epithelium in patients with reflux treated medically vs. surgically.FindingsMucosal tissue was obtained at endoscopy from patients with Barrett's oesophagus whose reflux was controlled by proton pump inhibitor (PPI) therapy (n = 20) or by fundoplication (n = 19). RNA was extracted and the expression of miR-101 and miR-196a was measured using real-time reverse transcription - polymerase chain reaction. There were no significant differences in miR-101 and miR-196a expression in Barrett's oesophagus epithelium in patients treated by PPI vs. fundoplication (p = 0.768 and 0.211 respectively). Secondary analysis showed a correlation between miR-196a expression and Barrett's oesophagus segment length (p = 0.014).ConclusionThe method of reflux treatment did not influence the expression of miR-101 and miR-196a in Barrett's oesophagus. This data does not provide support to the hypothesis that surgical treatment of reflux better prevents cancer development in Barrett's oesophagus. The association between miR-196a expression and Barrett's oesophagus length is consistent with a tumour promoting role for miR-196a in Barrett's oesophagus.

A novel mechanism of acid and bile acid-induced DNA damage involving Na+/H+ exchanger: implication for Barrett's oesophagus

ObjectiveBarrett's oesophagus is a premalignant disease associated with oesophageal adenocarcinoma. The major goal of this study was to determine the mechanism responsible for bile acid-induced alteration in intracellular pH (pHi)...

Endoscopy of Barrett's Esophagus Guided By Fluorescence Imaging Using An Overlay Imaging Technique

Background: White light endoscopy suffers from differentiation between Barrett's mucosa with and without dysplasias. Time gated detection of protoporphyrin IX (PpIX) fluorescence allows the simultaneous performance of endoscopy and fluorescence imaging. In a previous study (Ortner et al., GUT 2003;52:28-33) we were able to demonstrate, that with time gated fluorescence spectroscopy the detection of dysplasias can be improved significantly. The aim of this study was to improve the diagnostic capability of fluorescence guided endoscopy by presenting the information of fluorescence intensity as an overlay on the endoscopic image and to shorten the time for endoscopic investigation. Methods: In 16 patients (70% male,age 64 [48-77 y]) with known or suspected Barrett's esophagus fluorescence endoscopy was performed about 2 hours after topical application of 500 mg 5-aminolaevulinic acid. Biopsies were taken at positions with high fluorescence intensity by using the novel overlay technique and for control at locations with no intensity from areas with columnar lined epithelium. Subsequently the biopsy samples were analyzed by fluorescence spectroscopy immediately after the examination. Moreover, all samples were sent to pathology for routine histologic examination. Results: A total of 84 biopsies were investigated. Samples from Barrett's mucosa showed significantly higher fluorescence intensity of (PpIX) as compared to regular columnar lined epithelium (median 0.23 for columnar lined mucosa vs. 0.99 for Barrett's mucosa, p < 0.0009 (two sample t test). Moreover, samples with low grade dysplasia (LGD) showed a higher fluorescence intensity than Barrett's biopsies without dysplasias. However, this trend was not statistically significant, most probably due to the small number of biopsies with LGD. Conclusion: Fluorescence spectroscopy seems to be more specific than wide field illumination most likely due to focal inspection and less bleaching of fluorescence. In patients with known or suspected Barrett's esophagus time gated fluorescence overlay imaging can improve the detection of Barrett's esophagus or dysplastic epithelium by identifying the regions with high fluorescence intensity. The detection rate for dysplastic lesions may therefore be increased and time for endoscopic investigation could be shortened compared to other enhanced techniques.

Characterization of tissue autofluorescence in Barrett's esophagus by confocal fluorescence microscopy

High grade dysplasia and early cancer in Barrett's esophagus can be distinguished in vivo by endoscopic autofluorescence point spectroscopy and imaging from non-dysplastic Barrett's mucosa. We used confocal fluorescence microscopy for ex vivo comparison of autofluorescence in non-dysplastic and dysplastic Barrett's esophagus. Unstained frozen sections were obtained from snap-frozen Barrett's esophagus biopsy samples and scanned with confocal fluorescence microscopy (458 nm excitation; 505-550 nm [green] and > 560 nm [red] emission). Digital micrographs were taken from areas with homogenous and specific histopathology. Visual inspection and statistical analysis were used to evaluate the image datasets. Dysplastic and non-dysplastic Barrett's esophagus epithelia fluoresced mainly in the green spectrum and the main sources of autofluorescence were the cytoplasm and lamina propria. High-grade dysplasia was differentiated from non-dysplastic Barrett's esophagus by microstructural tissue changes. However, there were no specific changes in either the locations or average intensities of intrinsic green and red autofluorescence at the epithelial level that could differentiate between dysplastic and non-dysplastic Barrett's esophagus epithelia, ex vivo. Detectable differences in autofluorescence between BE and dysplasia/cancer in vivo are probably not caused by specific changes in epithelial fluorophores but are likely due to other inherent changes (e.g. mucosal thickening and increased microvascularity) attenuating autofluorescence from the collagen-rich submucosa. Furthermore, confocal fluorescence microscopy provides 'histology-like' imaging of Barrett's tissues and may offer a unique opportunity to exploit microstructural tissue changes occurring during neoplastic transformation for in vivo detection of high-grade dysplasia in Barrett's patients using newly developed confocal fluorescence microendoscopy devices.

Bone Morphogenetic Protein 4 Expressed in Esophagitis Induces a Columnar Phenotype in Esophageal Squamous Cells

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. 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. 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. These results suggest that the BMP pathway could play a role in the transformation of normal esophageal squamous cells into columnar cells.

Chromosomal Instability in Barrett's Esophagus Is Related to Telomere Shortening

Barrett's esophagus is a useful model for the study of carcinogenesis, as the metaplastic columnar epithelium that replaces squamous esophageal epithelium is at elevated risk for development of adenocarcinoma. We examined telomere length and chromosomal instability (CIN) in Barrett's esophagus biopsies using fluorescence in situ hybridization. To study CIN, we selected centromere and locus-specific arm probes to chromosomes 17/17p (p53), 11/11q (cyclin D1), and 9/9p (p16 INK4A), loci reported to be involved in early stages of Barrett's esophagus neoplasia. Telomere shortening was observed in Barrett's esophagus epithelium at all histologic grades, whereas CIN was highest in biopsies with dysplastic changes; there was, however, considerable heterogeneity between patients in each variable. Alterations on chromosome 17 were strongly correlated with telomere length (r = 0.55; P < 0.0001) and loss of the 17p arm signal was the most common event. CIN on chromosome 11 was also associated with telomere shortening (r =0.3; P = 0.05), although 11q arm gains were most common. On chromosome 9p, arm losses were the most common finding, but chromosome 9 CIN was not strongly correlated with telomere length. We conclude that CIN is related to telomere shortening in Barrett's esophagus but varies by chromosome. Whether instability is manifested as loss or gain seems to be influenced by the chromosomal loci involved. Because telomere shortening and CIN are early events in Barrett's esophagus neoplastic progression and are highly variable among patients, it will be important to determine whether they identify a subset of patients that is at risk for more rapid neoplastic evolution.

Difference of gene expression profiles between Barrett’s esophagus and cardia intestinal metaplasia by gene chip

The difference of gene expression profile changes in Barrett's esophagus (BE) and cardia intestinal metaplasia (CIM) epithelium was studied and the novel associated genes were screened in the early stage by cDNA microarray. The cDNA retro-transcribed from equal quantity mRNA from BE and CIM epithelial tissues were labeled with Cy3 and Cy5 fluorescence as probes. The mixed probe was hybridized with three pieces BiostarH-40s double dot human whole gene chip. The chips were scanned with a ScanArray 4000. The acquired images were analyzed using GenePix Pro 3.0 software. It was found a total of 141 genes were screened out that exhibited differentially expression more than 2 times in all three chips. It was identified that in gene expression profiles of BE, 74 genes were up-regulated and 67 down-regulated as compared with CIM. The comparison between the difference of gene expression profile changes in BE and CIM epithelia revealed that there existed the difference between BE and CIM at gene level. 141 genes with the expression more than two time were probably related to the occurrence and development of BE and the promotion or progress in adenocarcinoma.

Aberrant methylation of secreted frizzled‐related protein genes in esophageal adenocarcinoma and Barrett's esophagus

Hypermethylation of secreted frizzled-related proteins (SFRP) genes frequently occurs with several cancers but has not been studied in esophageal adenocarcinoma or its precursor-Barrett's esophagus. To explore the role of SFRP methylation in the neoplastic progression of Barrett's esophagus and to evaluate methylated SFRP genes as biomarkers for Barrett's esophagus and cancer, methylation of SFRP genes was determined in esophageal adenocarcinomas, Barrett's esophagus and normal epithelia using methylation-specific PCR. Protein expression of SFRP genes was then assessed in these tissues by immunohistochemistry. The mRNA expression of SFRP genes was quantified by real-time reverse-transcription PCR in esophageal adenocarcinoma cell lines with and without demethylation by 5-aza-2'deoxycytidine and inhibition of deacetylation by trichostatin A treatment. Hypermethylation of SFRP1, 2, 4 and 5 was detected in 93%, 83%, 73% and 85% of 40 cancers; 81%, 89%, 78% and 73% of 37 Barrett's epithelia; 25%, 64%, 32% and 21% of 28 adjacent normal epithelia from Barrett's patients; and 10%, 67%, 0% and 13% of 30 normal esophagogastric epithelia from healthy individuals, respectively (p < 0.001 for SFRP1, 4 and 5; p < 0.05 for SFRP2). Protein expression of SFRP1, 2 and 4 was downregulated in 87%, 67% and 90% of cancers, and expression correlated inversely with grade and stage of cancers and with grade of dysplasia. Expression of SFRP2 and SFRP4 proteins was lower in cancers with corresponding gene methylation (p < 0.05). Demethylation treatment effectively re-expressed SFRP mRNA in cancer cell lines. Thus, hypermethylation of SFRP genes is a common early event in the evolution of esophageal adenocarcinoma, and methylation of SFRP1, 4 and 5 might serve as biomarkers for Barrett's neoplasia. Aberrant promoter methylation appears to functionally silence SFRP gene expression in esophageal adenocarcinoma.

Cell Cycle Phase Abnormalities Do Not Account for Disordered Proliferation in Barrett's Carcinogenesis

Barrett's esophagus (BE) epithelium is the precursor lesion for esophageal adenocarcinoma. Cell cycle proteins have been advocated as biomarkers to predict the malignant potential in BE. However, whether disruption of the cell cycle plays a causal role in Barrett's carcinogenesis is not clear. Specimens from the Barrett's dysplasia—carcinoma sequence were immunostained for cell cycle phase markers (cyclin D1 for G1; cyclin A for S, G2, and M; cytoplasmic cyclin B1 for G2; and phosphorylated histone 3 for M phase) and expressed as a proportion of proliferating cells. Flow cytometric analysis of the cell cycle phase of prospective biopsies was also performed. The proliferation status of nondysplastic BE was similar to gastric antrum and D2, but the proliferative compartment extended to the luminal surface. In dysplastic samples, the number of proliferating cells correlated with the degree of dysplasia (P < .001). The overall levels of cyclins A and B1 correlated with the degree of dysplasia (P < .001). However, the cell cycle phase distribution measured with both immunostaining and flow cytometry was conserved during all stages of BE, dysplasia, and cancer. Hence, the increased proliferation seen in Barrett's carcinogenesis is due to abnormal cell cycle entry or exit, rather than a primary abnormality within the cell cycle.

Gastric-type mucin and TFF-peptide expression in Barrett's oesophagus is disturbed during increased expression of MUC2.

Barrett's oesophagus constitutes metaplastic epithelium, often diagnosed by mucin histochemistry. We determined the mucins and trefoil factor family (TFF)-peptides that were expressed in Barrett's oesophagus, in order to study changes in protein expression in early stages of Barrett's oesophagus development. Biopsy specimens of 71 Barrett's oesophagus patients were collected, and sections were stained for secretory mucins by histochemistry. Immunohistochemistry was performed for secretory mucins (MUC2, MUC5AC, MUC5B, MUC6), TFFs (TFF1, TFF2, TFF3), and proliferation (Ki67). Protein expression in the tissue was measured semiquantitatively. MUC5AC and TFF1 showed high levels and strong colocalization in the surface epithelium, whereas MUC6, MUC5B and TFF3 were found in the deeper glandular structures. TFF2 was found in both surface and glandular epithelium. The co-ordinate expression patterns of these six markers were similar to gastric antrum epithelium. MUC2 expression was ubiquitously associated with goblet cells within intestinal metaplasia, occurring in 68% of patients, and was correlated with increasing proliferation in the epithelium. Virtually all cells in Barrett's oesophagus epithelium displayed a secretory phenotype, demonstrating a co-ordinate gastric-type MUC and TFF expression. When MUC2 expression was more pronounced, the expression patterns of the other MUCs and the TFFs were increasingly disturbed. MUC2 expression may constitute a marker for early change in the phenotype of Barrett's oesophagus as a precancerous lesion.

Extended lifespan of Barrett's esophagus epithelium transduced with the human telomerase catalytic subunit: a useful in vitro model.

As there has been no previous information on the consequences of telomerase expression in genetically altered, mortal cells derived from pre-malignant tissue, we sought to determine the effect of hTERT (human catalytic subunit of telomerase reverse transcriptase) transduction of pre-malignant cell strains from Barrett's esophagus that do not contain telomerase activity and possess a finite lifespan. Primary cultures of Barrett's esophageal epithelium transduced with a retrovirus containing hTERT were characterized by growth factor requirements, cytogenetics and flow cytometry. Expression of telomerase lengthened telomeres and greatly extended the lifespan of hTERT transduced (hTERT+) Barrett's esophagus cells. Growth factor dependency of the hTERT+ cultures remained largely similar to the parental cultures, although there was a modest increase in the ability to grow in agar. Chromosomal instability, measured by both karyotypic and FISH (fluorescence in situ hybridization) analyses, was reduced but not abrogated by hTERT transduction, suggesting that telomerase expression can enhance genomic stability. However, the persistence of residual instability gave rise to new clonal and non-clonal genetic variants, and in one hTERT+ culture a new DNA aneuploid population was observed, the only time such a ploidy shift has been seen in Barrett's cell strains in vitro. These in vitro observations are analogous to the clinical progression to aneuploidy that often precedes cancer in Barrett's esophagus, and suggest that reactivation of telomerase may be permissive for continued genetic evolution to cancer. Long-lived Barrett's esophagus epithelial cultures should provide a useful in vitro model for studies of neoplastic evolution and chemopreventive therapies.

The effect of selective cyclooxygenase-2 inhibition in Barrett's esophagus epithelium: an in vitro study.

Individuals with Barrett's esophagus, in which the normal squamous esophageal epithelium is replaced with a columnar mucosa, are at increased risk for esophageal adenocarcinoma. Mucosal injury may be involved in the progression to neoplasia via the synthesis of prostaglandins and other mediators of inflammation. Cyclooxygenase (COX)-2 is the rate-limiting enzyme involved in prostaglandin synthesis. We examined the effect of inhibiting COX-2 activity in Barrett's esophageal cells. Primary esophageal epithelial and fibroblast cell cultures were established from endoscopic biopsy specimens from 20 consecutive patients with Barrett's esophagus. COX-2 expression and activity were determined on pooled cell cultures by reverse transcription-polymerase chain reaction and prostaglandin E(2) (PGE(2)) enzyme immunoassay, respectively. Proliferation was measured by Ki-67 staining. PGE(2) levels were determined in supernatants from epithelial cells treated with the selective COX-2 inhibitor NS-398, proinflammatory cytokines (interleukin 1beta and tumor necrosis factor-alpha), and conditioned medium from fibroblast cultures (both unstimulated and stimulated with proinflammatory cytokines). Esophageal epithelial cells and fibroblasts expressed COX-2 messenger RNA. Compared with control-treated cells, NS-398 decreased proliferation of Barrett's esophageal epithelial cells by 55% (95% confidence interval = 47.1% to 63.8%; P<.001) and decreased COX-2 activity. The addition of exogenous PGE(2) reversed the antiproliferative effect of NS-398 on Barrett's esophageal epithelial cells. Proinflammatory cytokines did not affect COX-2 activity in esophageal epithelial cells but stimulated COX-2 activity in fibroblasts. However, conditioned medium from unstimulated and stimulated fibroblasts increased COX-2 activity in esophageal epithelial cells. COX-2 is functionally active in Barrett's esophagus because treatment with the COX-2 inhibitor hinders proliferation of Barrett's esophageal epithelial cells in culture, but proliferation is restored by treatment with prostaglandin. These results raise the possibility that inhibition of COX-2 may have chemopreventive potential for Barrett's esophagus.

Restoration of squamous mucosa after ablation of Barrett's esophageal epithelium

Restoration of squamous mucosa after ablation of Barrett's esophageal epithelium

Restoration of squamous mucosa after ablation of Barrett's esophageal epithelium

Background: Antireflux therapy has generally failed to induce regression of Barrett's epithelium. It was hypothesized that squamous epithelium could be restored if the columnar tissue was ablated while gastric acid secretion was suppressed. Methods: Ten white men with Barrett's esophagus received 40 mg of omeprazole daily. Thereafter, every 2–5 weeks they underwent videotaped endoscopies to argon laser photoablate columnar tissue, obtain biopsy specimens, and assess results. Squamous re-epithelialization was assessed by correlation of videotapes and directed biopsies. Results: Patients had one to eight areas ablated, totaling 0.5–12.0 cm2. Videotape assessments were corroborated by biopsy in all but one instance. Thirty-eight of 40 treatment locations partially or completely re-epithelialized with squamous tissue. Squamous regrowth appeared to occur by spread from contiguous squamous borders and de novo from glandular tissue. Regrowth was influenced by the extent of squamous borders and completeness of ablations. Nonablated glandular tissue persisted beneath squamous epithelium. Conclusions: Ablation of Barrett's epithelium and suppression of acid secretion facilitated squamous re-epithelialization. A progenitor cell within the metaplastic tissue has the potential to differentiate normally.

Glutathione and glutathione S-transferases in Barrett's epithelium

Glutathione content, enzyme activity and isoenzyme composition of glutathione S-transferases were assayed in normal and Barrett's esophageal epithelium of ten patients with Barrett's esophagus. In addition, gastric and duodenal specimens from the same patients were also investigated. Glutathione content, glutathione S-transferase enzyme activity as well as glutathione S-transferase pi content were all significantly lower in Barrett's epithelium as compared to normal esophageal mucosa. In contrast, glutathione S-transferase class alpha enzymes are markedly expressed in Barrett's epithelium, whereas only low amounts are present in normal esophageal epithelium. Glutathione and glutathione S-transferase composition in Barrett's epithelium show striking similarities with gastric epithelium, whereas duodenal epithelium is provided with considerable higher amounts of glutathione and glutathione S-transferases, except for levels of glutathione S-transferase class pi, which are lower. A significant negative correlation exists between glutathione S-transferase enzyme activity in the mucosa along the gastrointestinal tract, and the tumour incidence. Since glutathione and glutathione S-transferase are correlated with protection against cellular or cytogenetic damage, the low content of glutathione and glutathione S-transferases in the Barrett's esophagus may be a factor of relevance for the increased tumour risk in this tissue.