p53 and the malignant progression of Barrett's esophagus

Barrett's esophagus (BE) is a metaplastic disorder in which specialized columnar epithelium replaces healthy squamous epithelium (intestinal metaplasia). Even though its pathophysiology and the steps of its neoplastic progression are not completely understood, BE can be considered as a complication of gastroesophageal reflux disease (GERD). Given that esophageal adenocarcinoma, which is continually increasing in the Western world, still has a poor prognosis and suffers from late diagnosis, and because BE is a precancerous lesion, there is a strong need for good molecular markers of malignant progression in Barrett's metaplasia (BM). The aim of this review is to examine the published data regarding the role that assessment of p53 may play in the management of BE, trying to understand if it may be a useful marker to early diagnose BE malignant transformation.     

Karyometry in Barrett's esophagus

OBJECTIVE: To derive a progression curve for lesions in Barrett's esophagus based on karyometric features. STUDY DESIGN: High-resolution imagery of 900 nuclei from normal gastric tissue, Barrett's metaplasia, Barrett's high grade dysplasia and adenocarcinoma of the esophagus was recorded. Karyometric features were computed, and nuclear signatures and lesion signatures for these lesions were derived. A progression curve was defined. RESULTS: Esophageal lesions were distinctly different from the normal gastric fundus tissue, with nuclei from Barrett's metaplasia deviating from normal almost as much as nuclei from high grade dysplasia and adenocarcinoma. There was considerable case-to-case variability and overlap between lesions histologically assigned to different diagnostic categories. CONCLUSION: The karyometric data suggest that Barrett's metaplasia is a more developed lesion than previously assumed.     

Three-tiered risk stratification model to predict progression in Barrett's esophagus using epigenetic and clinical features

Background

Barrett''s esophagus predisposes to esophageal adenocarcinoma. However, the value of endoscopic surveillance in Barrett''s esophagus has been debated because of the low incidence of esophageal adenocarcinoma in Barrett''s esophagus. Moreover, high inter-observer and sampling-dependent variation in the histologic staging of dysplasia make clinical risk assessment problematic. In this study, we developed a 3-tiered risk stratification strategy, based on systematically selected epigenetic and clinical parameters, to improve Barrett''s esophagus surveillance efficiency.

Methods and Findings

We defined high-grade dysplasia as endpoint of progression, and Barrett''s esophagus progressor patients as Barrett''s esophagus patients with either no dysplasia or low-grade dysplasia who later developed high-grade dysplasia or esophageal adenocarcinoma. We analyzed 4 epigenetic and 3 clinical parameters in 118 Barrett''s esophagus tissues obtained from 35 progressor and 27 non-progressor Barrett''s esophagus patients from Baltimore Veterans Affairs Maryland Health Care Systems and Mayo Clinic. Based on 2-year and 4-year prediction models using linear discriminant analysis (area under the receiver-operator characteristic (ROC) curve: 0.8386 and 0.7910, respectively), Barrett''s esophagus specimens were stratified into high-risk (HR), intermediate-risk (IR), or low-risk (LR) groups. This 3-tiered stratification method retained both the high specificity of the 2-year model and the high sensitivity of the 4-year model. Progression-free survivals differed significantly among the 3 risk groups, with p = 0.0022 (HR vs. IR) and p<0.0001 (HR or IR vs. LR). Incremental value analyses demonstrated that the number of methylated genes contributed most influentially to prediction accuracy.

Conclusions

This 3-tiered risk stratification strategy has the potential to exert a profound impact on Barrett''s esophagus surveillance accuracy and efficiency.     

Gain of allelic gene expression for IGF-II occurs frequently in Barrett's esophagus

The IGF-II gene normally exhibits genomic imprinting, a DNA modification that allows the expression of only one of the two inherited alleles. With loss of imprinting, there is a gain of allelic gene expression (GOAGE) due to IGF-II being expressed by both alleles. GOAGE for IGF-II has been demonstrated in a number of malignancies and in normal epithelia surrounding malignancies, but not in epithelia without associated neoplasia. We hypothesized that nonneoplastic Barrett's epithelium might have GOAGE for IGF-II that could facilitate its progression to neoplasia. Endoscopic biopsies were obtained from metaplastic esophageal, normal gastric, and normal duodenal epithelia from 43 patients with Barrett's esophagus. Genomic DNA were analyzed using PCR followed by ApaI restriction enzyme digestion or allele-specific PCR to identify an ApaI polymorphism of IGF-II. cDNA from patients with the ApaI polymorphism were analyzed for IGF-II GOAGE using exon connection PCR, followed by a secondary nested PCR and ApaI restriction enzyme digestion. We found that 13 (30%) of 43 samples of Barrett's metaplasia contained the ApaI polymorphism and were thus informative for IGF-II, and sufficient material was available for GOAGE analysis in 9 of those 13 cases. GOAGE for IGF-II was demonstrated in five (56%) of those nine cases. All patients with GOAGE in Barrett's metaplasia also demonstrated GOAGE in the gastric and duodenal epithelia. In contrast, patients without GOAGE in Barrett's metaplasia also had no GOAGE in their gastric and duodenal epithelia. We conclude that in patients with Barrett's esophagus, GOAGE for IGF-II is found frequently in the metaplastic esophageal epithelium as well as in normal gastric and duodenal epithelia.     

Helicobacter pylori in Barrett's esophagus.

Barrett's esophagus is an anatomicoclinical state in which, due to the prolonged action of gastroesophageal reflux, the squamous epithelium is replaced by columnar epithelium. Helicobacter pylori has been implicated in the pathogenesis of various gastrointestinal disorders and has occasionally been observed in Barrett's esophagus. The aim of this study is to determine the incidence of H. pylori in Barrett's esophagus and try to establish its role in the pathogenesis of this disorder. H. pylori was observed in 31 biopsies (44.3%) of the 70 studied, mainly when the epithelium is of the gastric atrophic-fundic type (p less than 0.01). Its presence shows no relation to the degree of inflammatory activity and does not seem, therefore, to play an important role in the pathogenesis of the lesion.     

Whole genome expression array profiling highlights differences in mucosal defense genes in Barrett's esophagus and esophageal adenocarcinoma

Esophageal adenocarcinoma (EAC) has become a major concern in Western countries due to rapid rises in incidence coupled with very poor survival rates. One of the key risk factors for the development of this cancer is the presence of Barrett's esophagus (BE), which is believed to form in response to repeated gastro-esophageal reflux. In this study we performed comparative, genome-wide expression profiling (using Illumina whole-genome Beadarrays) on total RNA extracted from esophageal biopsy tissues from individuals with EAC, BE (in the absence of EAC) and those with normal squamous epithelium. We combined these data with publically accessible raw data from three similar studies to investigate key gene and ontology differences between these three tissue states. The results support the deduction that BE is a tissue with enhanced glycoprotein synthesis machinery (DPP4, ATP2A3, AGR2) designed to provide strong mucosal defenses aimed at resisting gastro-esophageal reflux. EAC exhibits the enhanced extracellular matrix remodeling (collagens, IGFBP7, PLAU) effects expected in an aggressive form of cancer, as well as evidence of reduced expression of genes associated with mucosal (MUC6, CA2, TFF1) and xenobiotic (AKR1C2, AKR1B10) defenses. When our results are compared to previous whole-genome expression profiling studies keratin, mucin, annexin and trefoil factor gene groups are the most frequently represented differentially expressed gene families. Eleven genes identified here are also represented in at least 3 other profiling studies. We used these genes to discriminate between squamous epithelium, BE and EAC within the two largest cohorts using a support vector machine leave one out cross validation (LOOCV) analysis. While this method was satisfactory for discriminating squamous epithelium and BE, it demonstrates the need for more detailed investigations into profiling changes between BE and EAC.     

Bioinformatic analyses of microRNA-targeted genes and microarray-identified genes correlated with Barrett's esophagus

Barrett's esophagus (BE) is defined as a metaplasia condition in the distal esophagus, in which the native squamous epithelium lining is replaced by a columnar epithelium with or without intestinal metaplasia. It is commonly accepted that BE is a precancerous lesion for esophageal adenocarcinoma. The aim of this study was to investigate the aberrant microRNAs (miRNAs) and differentially expressed genes (DEGs) associated with BE based on online microarray datasets. One miRNA and five gene expression profiling datasets were retrieved from the Gene Expression Omnibus Database. Aberrant microRNAs and DEGs were obtained using R/Bioconductor statistical analysis language and software. 23 dysregulated miRNAs and 632 DEGs demonstrating consistent expression tendencies in the five gene microarrays were identified in BE. Moreover, 1962 target genes of aberrant miRNAs were predicted using three bioinformatic tools, namely TargetScan, RNA22-HSA and miRDB. Ultimately, 93 target DEGs were obtained, after which functional annotation was performed on DAVID Bioinformatics Resources. Among Gene Ontology (GO) biological processes, digestive tract development and epithelial cell differentiation have demonstrated significant associations with BE pathogenesis. In addition, analysis of the KEGG pathways has revealed associations with cancer. To enable further study, one miRNA-target DEGs regulatory network was constructed using Cytoscape. 6 target DEGs demonstrated higher-degree distributions in the network, and ROC analysis indicated that FNDC3B may be the best potential biomarker for BE diagnosis. The data presented herein may provide new perspectives for exploring BE pathogenesis and may offer hits with regard to potential biomarkers in BE diagnosis, prediction and therapeutic evaluation.     

Ciliated cells in papillary adenocarcinomas of Barrett's esophagus.

Eighteen surgical specimens having an adenocarcinoma arising in Barrett's esophagus were reviewed, and special attention was paid to the presence of ciliated cells. The tumors were classified as glandular (9), papillary (4), diffuse (3) and mixed (2) types. Ciliated cells were observed in one specimen, in cystically dilated glands in Barrett's mucosa adjacent to a papillary adenocarcinoma. Ciliated tumor cells were found in three of the four papillary adenocarcinomas. The fourth papillary tumor, 1 mixed papillary-diffuse adenocarcinoma and the remaining 13 adenocarcinomas had no ciliated cells. Thus, the presence of cilia in exfoliated tumor cells from the esophagus should raise the suspicion of a papillary adenocarcinoma arising in Barrett's mucosa.     

Development and characterization of an organotypic model of Barrett's esophagus

Understanding the molecular and cellular processes underlying the development, maintenance, and progression of Barrett's esophagus (BE) presents an empirical challenge because there are no simple animal models and standard 2D cell culture can distort cellular processes. Here we describe a three-dimensional (3D) cell culture system to study BE. BE cell lines (CP-A, CP-B, CP-C, and CP-D) and esophageal squamous keratinocytes (EPC2) were cultured on a matrix consisting of esophageal fibroblasts and collagen. Comparison of growth and cytokeratin expression in the presence of all-trans retinoic acid or hydrochloric acid was made by immunohistochemistry and Alcian Blue staining to determine which treatments produced a BE phenotype of columnar cytokeratin expression in 3D culture. All-trans retinoic acid differentially affected the growth of BE cell lines in 3D culture. Notably, the non-dyplastic metaplasia-derived cell line (CP-A) expressed reduced squamous cytokeratins and enhanced columnar cytokeratins upon ATRA treatment. ATRA altered the EPC2 squamous cytokeratin profile towards a more columnar expression pattern. Cell lines derived from patients with high-grade dysplasia already expressed columnar cytokeratins and therefore did not show a systematic shift toward a more columnar phenotype with ATRA treatment. ATRA treatment, however, did reduce the squamoid-like multilayer stratification observed in all cell lines. As the first study to demonstrate long-term 3D growth of BE cell lines, we have determined that BE cells can be cultured for at least 3 weeks on a fibroblast/collagen matrix and that the use of ATRA causes a general reduction in squamous-like multilayered growth and an increase in columnar phenotype with the specific effects cell-line dependent.     

Selenium, selenoenzymes, oxidative stress and risk of neoplastic progression from Barrett's esophagus: results from biomarkers and genetic variants

Clinical trials have suggested a protective effect of selenium supplementation on the risk of esophageal cancer, which may be mediated through the antioxidant activity of selenoenzymes. We investigated whether serum selenium concentrations, selenoenzyme activity, oxidative stress and genetic variation in selenoenzymes were associated with the risk of neoplastic progression to esophageal adenocarcinoma (EA) and two intermediate endpoints, aneuploidy and tetraploidy. In this prospective cohort study, during an average follow-up of 7.3 years, 47 EA cases, 41 aneuploidy cases and 51 tetraploidy cases accrued among 361 participants from the Seattle Barrett's Esophagus Research Study who were free of EA at the time of blood draw and had at least one follow-up visit. Development to EA was assessed histologically and aneuploidy and tetraploidy by DNA content flow cytometry. Serum selenium concentrations were measured using atomic absorption spectrometry, activity of glutathione peroxidase (GPX) 1 and GPX3 by substrate-specific coupled test procedures, selenoprotein P (SEPP1) concentrations and protein carbonyl content by ELISA method and malondialdehyde concentrations by HPLC. Genetic variants in GPX1-4 and SEPP1 were genotyped. Serum selenium was not associated with the risk of neoplastic progression to EA, aneuploidy or tetraploidy (P for trend?=?0.25 to 0.85). SEPP1 concentrations were positively associated with the risk of EA [hazard ratio (HR)?=?3.95, 95% confidence intervals (CI)?=?1.42-10.97 comparing the third tertile with the first] and with aneuploidy (HR?=?6.53, 95% CI?=?1.31-32.58), but not selenoenzyme activity or oxidative stress markers. No genetic variants, overall, were associated with the risk of neoplastic progression to EA (global p?=?0.12-0.69). Our results do not support a protective effect of selenium on risk of neoplastic progression to EA. Our study is the first to report positive associations of plasma SEPP1 concentrations with the risk of EA and aneuploidy, which warrants further investigation.     

Elevation of hepatic levels of metallothionein during experimental carcinogenesis

From the early stage of pancreatic adenocarcinoma in hamsters and also of hepatocellular carcinoma in rats, induced by treatment withN-nitrosobis (2-oxopropyl)amine and 3′-methyl-dimethylaminoazobenzene, respectively, hepatic levels of metallothionein (MT) were found to be continuously elevated. In the hepatoma-induced rats, this elevation preceded that of serum γ-glutamyl transpeptidase activity, a marker enzyme for hepatocellular carcinoma. These results indicate that, in the course of chemical carcinogenesis, the elevation of hepatic MT level occurred and continued from the early stage of carcinogenesis. This type of elevation of hepatic MT level was also observed in lung metastasis-induced mice. On the other hand, in rats with pancreatitis caused by the administration of deoxycholate, the hepatic level of MT rose only transiently.     

Review: Experimental models for Barrett's esophagus and esophageal adenocarcinoma

Several different cell culture systems and laboratory animal models have been used over the years to study Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). Most of the existing models have key differences with the human esophagus and complex pathogenesis of disease. None of the models offers an ideal system for the complex study of environmental exposure, genetic risk, and prevention strategies. In fact, different model systems may be required to answer different specific research questions about the pathogenesis of BE and EAC. Given the high mortality associated with EAC and the fact that current screening strategies miss most cases of EAC, advances in basic and translational science related to esophageal injury, repair, and carcinogenesis are clearly needed. This review describes several of the existing and potential model systems for BE and EAC with their benefits and disadvantages.     

Toward improved endoscopic surveillance with multidiameter single fiber reflectance spectroscopy in patients with Barrett's esophagus

Patients with Barrett's esophagus are at an increased risk to develop esophageal cancer and, therefore, undergo regular endoscopic surveillance. Early detection of neoplasia enables endoscopic treatment, which improves outcomes. However, early Barrett's neoplasia is easily missed during endoscopic surveillance. This study investigates multidiameter single fiber reflectance spectroscopy (MDSFR) to improve Barrett's surveillance. Based on the concept of field cancerization, it may be possible to identify the presence of a neoplastic lesion from measurements elsewhere in the esophagus or even the oral cavity. In this study, MDSFR measurements are performed on non-dysplastic Barrett's mucosa, squamous mucosa, oral mucosa, and the neoplastic lesion (if present). Based on logistic regression analysis on the scattering parameters measured by MDSFR, a classifier is developed that can predict the presence of neoplasia elsewhere in the Barrett's segment from measurements on the non-dysplastic Barrett's mucosa (sensitivity 91%, specificity 71%, AUC = 0.77). Classifiers obtained from logistic regression analysis for the squamous and oral mucosa do not result in an AUC significantly different from 0.5.