Background and Aims
Because of the extremely low neoplastic progression rate in Barrett’s esophagus, it is difficult to diagnose patients with concomitant adenocarcinoma early in their disease course. If biomarkers existed in normal squamous esophageal epithelium to identify patients with concomitant esophageal adenocarcinoma, potential applications would be far-reaching. The aim of the current study was to identify global gene expression patterns in normal esophageal epithelium capable of revealing simultaneous esophageal adenocarcinoma, even located remotely in the esophagus.
Tissues comprised normal esophageal epithelia from 9 patients with esophageal adenocarcinoma, 8 patients lacking esophageal adenocarcinoma or Barrett’s, and 6 patients with Barrett’s esophagus alone. cDNA microarrays were performed, and pattern recognition in each of these subgroups was achieved using shrunken nearest centroid predictors.
Our method accurately discriminated normal esophageal epithelia of 8/8 patients without esophageal adenocarcinoma or Barrett’s esophagus and of 6/6 patients with Barrett’s esophagus alone from normal esophageal epithelia of 9/9 patients with Barrett’s esophagus and concomitant esophageal adenocarcinoma. Moreover, we identified genes differentially expressed between the above subgroups. Thus, based on their corresponding normal esophageal epithelia alone, our method accurately diagnosed patients who had concomitant esophageal adenocarcinoma.
These global gene expression patterns, along with individual genes culled from them, represent potential biomarkers for the early diagnosis of esophageal adenocarcinoma from normal esophageal epithelia. Genes discovered in normal esophagus that are differentially expressed in patients with vs. without esophageal adenocarcinoma merit further pursuit in molecular genetic, functional, and therapeutic interventional studies.
Barrett’s esophagus, a pre-malignant condition that can lead to esophageal adenocarcinoma, is characterized by histological changes in the normal squamous epithelium of the esophagus. Numerous molecular changes occur during the multistage conversion of Barrett’s metaplasia to dysplasia and frank adenocarcinoma. Epigenetic changes, especially changes in DNA methylation are widespread during this process. Aberrant DNA methylation has been shown to occur at promoters of tumor suppressor genes, adhesion molecules and DNA repair genes during Barrett’s esophagus. These epigenetic alterations can be used as molecular biomarkers for risk stratification and early detection of esophageal adenocarcinoma. We also show that genome wide analysis of methylation surprisingly reveals that global hypomethylation and not hypermethylation is the dominant change during Barrett’s metaplasia. The transformation of Barrett’s esophagus to frank adenocarcinoma is in turn characterized by much smaller wave of selective promoter hypermethylation. These studies reveal many novel, potential targets for new therapies and illustrate the utility of incorporating these epigenetic changes as biomarkers during endoscopic surveillance interval for patients with Barrett’s esophagus.
Barrett’s esophagus; DNA methylation; esophageal adenocarcinoma; global hypomethylation
Background & Aims
Barrett’s esophagus is a precursor of esophageal adenocarcinoma. DNA microarrays that enable a genome-wide assessment of gene expression enhance the identification of specific genes as well as gene expression patterns that are expressed by Barrett’s esophagus and adenocarcinoma compared to normal tissues. Barrett's esophagus length has also been identified as a risk factor for progression to adenocarcinoma, but whether there are intrinsic biological differences between short and long-segment Barrett's esophagus can be explored with microarrays.
Gene expression profiles for endoscopically obtained biopsies of Barrett’s esophagus or esophageal adenocarcinoma, and associated normal esophagus and duodenum were identified for 17 patients using DNA microarrays. Unsupervised and supervised approaches for data analysis defined similarities and differences between the tissues as well as correlations with clinical phenotypes.
Each tissue displays a unique expression profile that distinguishes it from each other. Barrett’s esophagus and esophageal adenocarcinoma express a unique set of stromal genes that is distinct from normal tissues, but similar to other cancers. Adenocarcinoma also showed lower and higher expression for many genes compared to Barrett's esophagus. No difference in gene expression was found between short and long-segment Barrett's esophagus.
The genome-wide assessment provided by current DNA microarrays reveals many candidate genes and patterns not previously identified. Stromal gene expression in Barrett’s esophagus and adenocarcinoma are similar, indicating that these changes precede neoplasia.
Barrett’s esophagus is a metaplastic alteration of the normal esophageal epithelium that is detected on endoscopic examination and pathologically confirmed by the presence of intestinal metaplasia on biopsy. Its major significance is as a predisposing factor for esophageal adenocarcinoma, which carries a high mortality rate and a rapidly growing incidence in the United States. Detection of Barrett’s esophagus allows for endoscopic surveillance in order to detect the potential development of dysplasia and early cancer before symptoms develop, and thereby significantly increases treatment options and may lower mortality from esophageal adenocarcinoma. Much current work in the field is aimed at reducing the risk of progression from Barrett’s esophagus to cancer, and in the identification of biomarkers that may predict progression towards cancer. Barrett’s esophagus is present in 10%–20% of patients with gastroesophageal reflux disease (GERD) and has also been detected in patients who deny classic GERD symptoms and are undergoing endoscopy for other indications. We used an evidence-based approach to describe treatment options for patients with Barrett’s esophagus.
Barrett’s esophagus; esophageal adenocarcinoma; evidence-based approach; endoscopic surveillance
Esophageal intestinal metaplasia, also known as Barrett’s esophagus, is the replacement of the normal epithelium with one that resembles the intestine morphologically. Generally, this includes intestinal mucin-secreting goblet cells. Barrett’s esophagus is an important risk factor for adenocarcinoma development. In vitro models for Barrett’s esophagus have not, to date, focused on the induction of goblet cells in Barrett’s epithelium.
To explore the contribution of Math1/Atoh1 in the induction of Barrett’s esophagus and intestinal mucin-secreting goblet cells from normal human esophageal epithelium.
We explored the level and pattern of Math1/Atoh1 mRNA and protein expression in human Barrett’s esophagus. Then, using retroviral-mediated gene expression, we induced Math1 mRNA and protein expression in a human esophageal keratinocyte cell line. We evaluated the effects of this ectopic Math1 expression upon cell proliferation and gene expression patterns in cells cultured under 2-dimensional and 3-dimensional tissue engineering conditions.
Math1/Atoh1 mRNA and protein are detected in human Barrett’s esophagus specimens, but the mRNA levels vary considerable. In the keratinocyte expression studies, we observed that Math1/Atoh1 ectopic expression significantly reduced cell proliferation and altered cell morphology. Moreover, Math1/Atoh1 expression is associated with a more intestinalized gene expression pattern that is distinct from prior published studies using other intestinal transcription factors. Most significantly we observe the induction of the Barrett’s esophagus markers Mucin-2 and Keratin-20 with Math1/Atoh1 expression.
We conclude that ectopic Math1/Atoh1 expression makes unique contributions to the intestinalization of esophageal epithelium in Barrett’s esophagus.
Barrett’s esophagus; Math1/Atoh1; Keratin-20; metaplasia; Mucin-2; organotypic culture
Barrett's esophagus, a risk factor for esophageal adenocarcinoma, is associated with reflux disease. The aim of this study was to assess the expression of bile acid receptors in the esophagus (normal, esophagitis, Barrett's esophagus and adenocarcinoma) and to investigate their possible function.
the expression of the bile acid receptors FXR and VDR in esophageal biopsies from patients with a normal mucosa, esophagitis, Barrett's esophagus or adenocarcinoma (n = 6 per group) and in cell lines derived from Barrett's esophagus and esophageal adenocarcinoma, was assessed by real time Q-PCR and immunohistochemistry. The effect of guggulsterone, an antagonist of bile acid receptors, on apoptosis of Barrett's esophagus-derived cells was assessed morphologically, by flow cytometry and by measuring caspase 3 activity.
The expression of FXR was increased in esophagitis, Barrett's esophagus and adenocarcinoma compared to normal mucosa by a mean of 44, 84 and 16, respectively. Immunohistochemistry showed a weak expression in normal esophagus, a strong focal reactivity in Barrett's esophagus, and was negative in adenocarcinoma. VDR expression did not significantly differ between groups. In cell cultures, the expression of FXR was high in Barrett's esophagus-derived cells and almost undetectable in adenocarcinoma-derived cells, whereas VDR expression in these cell lines was not significantly different. In vitro treatment with guggulsterone was associated with a significant increase in the percentage of apoptotic cells and of the caspase 3 activity.
the bile acid receptor FXR is significantly overexpressed in Barrett's esophagus compared to normal mucosa, esophagitis and esophageal adenocarcinoma. The induction of apoptosis by guggulsterone in a Barrett's esophagus-derived cell line suggests that FXR may contribute to the regulation of apoptosis.
In Barrett’s esophagus, the stratified squamous epithelium lining the esophagus is replaced by specialized intestinal-type columnar epithelium. The prevalence of Barrett’s esophagus has ranged from 0.9% to 4.5%. The rate of progression from Barrett’s esophagus to esophageal adenocarcinoma is 0.5% per patient-year. Proton-pump inhibitors are the mainstay of symptom control in Barrett’s patients. Nondysplastic Barrett’s and Barrett’s with low-grade dysplasia (LGD) are typically managed by periodic surveillance. Radiofrequency ablation is being evaluated as a modality for managing nondysplastic Barrett’s and Barrett’s with LGD. The options for the management of Barrett’s patients with high-grade dysplasia (HGD) include endoscopic therapy, surgery, and intensive surveillance until biopsy reveals adenocarcinoma. Endoscopic therapy involves endoscopic mucosal resection (EMR) and ablation. More aggressive techniques such as endoscopic submucosal dissection and larger segment endoscopic mucosal resection are under study. In this review, we discuss the diagnosis and management of Barrett’s esophagus. The recommendations from the major gastroenterologic societies and the current and investigational endoscopic modalities for the management of Barrett’s esophagus with and without dysplasia are reviewed.
Barrett esophagus; endoscopic surgical procedures; esophageal neoplasms
Barrett’s esophagus is a condition in which the normal stratified squamous epithelium of the distal esophagus is replaced by intestinal metaplasia. For more than three decades the prevailing clinical paradigm has been that Barrett’s esophagus is a complication of symptomatic reflux disease that predisposes to esophageal adenocarcinoma, yet no clinical strategy for cancer prevention or early detection based on this paradigm has been proven to reduce esophageal adenocarcinoma mortality in a randomized clinical trial in part because only about 5-10% of individuals with Barrett’s esophagus develop esophageal adenocarcinoma. Recent research indicates that Barrett’s metaplasia is an adaptation for mucosal defense in response to chronic reflux in most individuals. The risk of progressing to esophageal adenocarcinoma is determined by development of genomic instability and dynamic clonal evolution in the distal esophagus modulated by host and environmental risk and protective factors, including inherited genotype. The challenge in Barrett’s esophagus lies in integrating knowledge about genomic instability and clonal evolution into clinical management to increase the lifespans and quality of life of individuals with this condition.
Since the early 1970s, a dramatic change has occurred in the epidemiology of esophageal malignancy in both North America and Europe: the incidence of adenocarcinomas of the lower esophagus and esophagogastric junction is increasing. Several lifestyle factors are implicated in this change, including gastroesophageal reflux disease (gerd). Primary esophageal adenocarcinomas are thought to arise from Barrett esophagus, an acquired condition in which the normal esophageal squamous epithelium is replaced by a specialized metaplastic columnar-cell-lined epithelium.
Today, gerd is recognized as an important risk factor in Barrett esophagus. Progression of Barrett esophagus to invasive adenocarcinoma is reflected histologically by the metaplasia–dysplasia–carcinoma sequence. Although several molecular alterations associated with progression of Barrett esophagus to invasive adenocarcinoma have been identified, relatively few will ultimately have clinical application. Currently, the histologic finding of high-grade dysplasia remains the most reliable predictor of progression to invasive esophageal adenocarcinoma. However other promising molecular biomarkers include aneuploidy; 17p loss of heterozygosity, which implicates the TP53 tumour suppressor gene; cyclin D1 protein overexpression; and p16 alterations. It is anticipated that models incorporating combinations of objective scores of sociodemographic and lifestyle risk factors (that is, age, sex, body mass index), severity of gerd, endoscopic and histologic findings, and a panel of biomarkers will be developed to better identify patients with Barrett esophagus at increased risk for malignant progression, leading to more rational endoscopic surveillance and screening programs.
Barrett esophagus; esophageal adenocarcinoma; molecular pathogenesis; biomarkers
Barrett’s esophagus is a well-known premalignant lesion of the lower esophagus that is characterized by intestinal metaplasia of the squamous epithelium. It is clinically important due to the increased risk (0.5% per annum) of progression to esophageal adenocarcinoma (EA), which has a poor outcome unless diagnosed early. The current clinical management of Barrett’s esophagus is hampered by the lack of accurate predictors of progression. In addition, when patients develop EA, the current staging modalities are limited in stratifying patients into different prognostic groups in order to guide the optimal therapy for an individual patient. Biomarkers have the potential to improve radically the clinical management of patients with Barrett’s esophagus and EA but have not yet entered mainstream clinical practice. This is in contrast to other cancers like breast and prostate for which biomarkers are utilized routinely to inform clinical decisions. This review aims to highlight the most promising predictive and prognostic biomarkers in Barrett’s esophagus and EA and to discuss what is required to move the field forward towards clinical application.
Barrett’s esophagus; Esophageal adenocarcinoma; Esophageal dysplasia; Prognosis
Background & Aims
Patients with Barrett’s esophagus (BE) show increased risk for developing esophageal adenocarcinoma and are routinely examined using upper endoscopy with biopsy to search for neoplastic changes. Angle-resolved low coherence interferometry (a/LCI) uses in vivo depth-resolved nuclear morphology measurements to detect dysplasia. We assessed the clinical utility of a/LCI in the endoscopic surveillance of BE patients.
Consecutive patients undergoing routine surveillance upper endoscopy for Barrett’s esophagus were recruited at two endoscopy centers. A novel, endoscope compatible a/LCI system was used to measure the mean diameter and refractive index of cell nuclei in esophageal epithelium at 172 biopsy sites in 46 patients. At each site, an a/LCI measurement was taken and correlated with a concurrent endoscopic biopsy. Each biopsy was assessed histologically and classified as normal, non-dysplastic BE (NDBE), indeterminate for dysplasia, low-grade dysplasia (LGD), or high-grade dysplasia (HGD). The a/LCI data from multiple depths were analyzed to evaluate its ability to differentiate dysplastic from non-dysplastic tissue.
Pathology characterized five of the scanned sites as HGD, eight as LGD, seventy-five as NDBE, seventy as normal tissue types and fourteen as indeterminate for dysplasia. The a/LCI nuclear size measurements separated dysplastic from non-dysplastic tissue at a statistically significant (P < .001) level for the tissue segment 200-300μm beneath the surface with an accuracy of 86% (147/172). A receiver operator characteristic (ROC) analysis indicated an area under the curve (AUC) of 0.91, and an optimized decision point gave 100% (13/13) sensitivity and 84% (134/159) specificity.
These preliminary data suggest a/LCI is accurate in detecting dysplasia in vivo in BE patients.
Barrett’s esophagus; optical techniques; light scattering
Gastroesophageal reflux disease (GERD) affects an estimated 20% of the population in the United States. About 10%-15% of patients with GERD develop Barrett’s esophagus, which can progress to adenocarcinoma, currently the most prevalent type of esophageal cancer. The esophagus is normally lined by squamous mucosa, therefore, it is clear that for adenocarcinoma to develop, there must be a sequence of events that result in transformation of the normal squamous mucosa into columnar epithelium. This sequence begins with gastroesophageal reflux, and with continued injury metaplastic columnar epithelium develops. This article reviews the pathophysiology of Barrett’s esophagus and implications for its treatment. The effect of medical and surgical therapy of Barrett’s esophagus is compared.
Gastroesophageal reflux disease; Barrett’s esophagus; Lower esophageal sphincter; Esophageal motility; Proton pump inhibitors; Antireflux surgery
Specialized intestinal metaplasia (SIM) in Barrett’s esophagus is a risk factor of esophageal adenocarcinoma. It often occurs focally and cannot be distinguished from surrounding columnar epithelium with conventional endoscopy.
The purpose of this study was evaluation of methylene blue (MB) staining and magnification endoscopy with comparison of pit-pattern classifications according to Endo and Guelrud, in detection of SIM in Barrett’s esophagus.
Twenty-five patients, aged 33–77 years (average 57 years), with displacement of Z line were prospectively enrolled and underwent gastroscopy with the use of magnification up to 115 times (Olympus GIF Q160Z). Biopsy for histopathologic examination was taken from sites stained with MB and/or places with particular pit patterns. A control group consisted of ten patients with normal gastro-esophageal junction.
SIM was proved in nine patients, and significantly more frequently in patients with hiatal hernia and Barrett’s segment longer than 3 cm. Round or thin linear pit patterns according to Guelrud’s and small round and straight pit patterns according to Endo’s classification were coupled with columnar epithelium. SIM was associated with deep linear and foveolar pit patterns in Guelrud’s classification. Other pit patterns were less characteristic. Both classifications had high sensitivity (Endo’s 85.7%, Guelrud’s 92.8%) but poor specificity (respectively, 21.15 and 28.4%) in detection of SIM. Sensitivity and specificity of MB staining were, respectively, 71.4 and 40.6%.
Despite existing association between mucosal surface structure and histology, we find no convincing data indicating that pit-pattern evaluation may replace multiple biopsies taken according to recommendations from Seattle for detection of SIM in Barrett’s esophagus.
Magnification endoscopy; Barrett’s esophagus; Pit pattern; Methylene blue; Intestinal metaplasia
AIM: To investigate the microsatellite alterations in phenotypically normal esophageal squamous epithelium and metaplasia-dysplasia-adenocarcinoma sequence.
METHODS: Forty-one specimens were obtained from esophageal cancer (EC) patients. Histopathological assessment identified 23 squamous cell carcinomas (SCC) and 18 adenocarcinomas (ADC), including only 8 ADC with Barrett esophageal columnar epithelium (metaplasia) and dysplasia adjacent to ADC. Paraffin-embedded normal squamous epithelium, Barrett esophageal columnar epithelium (metaplasia), dysplasia and esophageal tumor tissues were dissected from the surrounding tissues under microscopic guidance. DNA was extracted using proteinase K digestion buffer, and DNA was diluted at 1:100, 1:1000, 1:5000, 1:10 000 and 1:50 000, respectively. Seven microsatellite markers (D2S123, D3S1616, D3S1300, D5S346, D17S787, D18S58 and BATRII loci) were used in this study. Un-dilution and dilution polymerase chain reactions (PCR) were performed, and microsatellite analysis was carried out.
RESULTS: No statistically significant difference was found in microsatellite instability (MSI) and loss of heterozygosity (LOH) of un-diluted DNA between SCC and ADC. The levels of MSI and LOH were high in the metaplasia-dysplasia-adenocarcinoma sequence of diluted DNA. The more the diluted DNA was, the higher the rates of MSI and LOH were at the above 7 loci, especially at D3S1616, D5S346, D2S123, D3S1300 and D18S58 loci.
CONCLUSION: The sequence of metaplasia-dysplasia-adenocarcinoma is associated with microsatellite alterations, including MSI and LOH. The MSI and LOH may be the early genetic events during esophageal carcinogenesis, and genetic alterations at the D3S1616, D5S346 and D3S123 loci may play a role in the progress of microsatellite alterations.
Microsatellite alteration; Dilution PCR; Metaplasia-dysplasia-adenocarcinoma sequence; Esophageal squamous epithelium; Squamous cell carcinoma
Barrett's esophagus develops as a result of chronic injury of esophagus epithelium from gastroesophageal reflux disease. It is defined when metaplastic columnar epithelium replaces the stratified squamous epithelium which normally lies in the distal esophagus. The condition represents a risk factor for esophageal adenocarcinoma. The aim of the radiofrequency ablation (RFA) method is to destroy metaplastic epithelium with radiofrequency electric current and to stimulate reappearance of the flat multilayer epithelium in the distal esophagus.
To evaluate the efficiency and safety of the RFA technique, newly introduced in Poland, in the management of Barrett's esophagus.
Material and methods
Twelve patients were treated with the RFA method. Patients with Barrett's esophagus confirmed in the histopathological report were qualified for treatment. Two RFA techniques were applied using a BARRX® device: circular based on the balloon HALO360 system or focal based on the HALO90 system mounted to the endoscopic ending. The procedures were performed at 2-month intervals. The macroscopic and microscopic effects of RFA therapy, the patients’ treatment tolerance as well as potential complications were evaluated.
In the group of 12 patients subjected to RFA therapy, 10 completed the therapeutic cycle. A total of 37 procedures were performed: 5 HALO360 and 32 HALO90. In all patients eradication of the abnormal metaplastic esophageal epithelium was achieved, as confirmed in both endoscopic and histopathological evaluation. In 2 patients with ongoing therapy progressive eradication of metaplastic epithelium was observed. No significant RFA-related complications were reported.
Based on our preliminary results we consider this method to be promising, free of significant complications and well tolerated by patients. In most patients it results in successful eradication of metaplastic epithelium in the distal esophagus.
Barrett's esophagus; metaplasia; radiofrequency ablation
Bile reflux contributes to the development of esophageal injury and neoplasia. MUC5AC mucin is absent in the normal squamous epithelium of the esophagus but strongly expressed in Barrett’s esophagus (BE). The aim of this study was to determine whether and how bile acids influence the expression of MUC5AC in the esophagus.
MUC5AC expression was studied by immunohistochemistry and immunoblotting in human tissues, tissues from a rat model of BE, and in SKGT-4 cultured esophageal epithelial cells. MUC5AC transcription was studied by real-time PCR and transient transfection assays.
MUC5AC was absent from normal squamous epithelium but present in 100% of Barrett’s specimens and in 61.5% of human esophageal adenocarcinoma tissues examined. MUC5AC protein expression was induced to a greater degree by conjugated bile acids than by unconjugated bile acids, and this occurred at the transcriptional level. In the rat reflux model, MUC5AC mucin was abundantly expressed in tissues of BE stimulatesd by duodenoesophageal reflux. Conjugated bile acids induced AKT phosphorylation in SKGT-4 cells, but had no effects on ERK1/2, JNK, and P-38 kinase phosphorylation. The PI3K inhibitor LY294002 and a dominant-negative AKT construct prevented the induction of MUC5AC by conjugated bile acids. Transactivation of AP-1 by conjugated bile acids coincided with MUC5AC induction, and co-transfection with a dominant-negative AP-1 vector decreased MUC5AC transcription and its induction.
Conjugated bile acids in the bile refluxate contribute to MUC5AC induction in the esophagus. This occurs at the level of transcription, and involves activation of the PI3K/AKT/AP-1 pathway.
MUC5AC mucin; bile reflux; PI3K pathway; AP-1; Barrett’s Esophagus
In Barrett’s esophagus (BE), the normal esophageal squamous epithelium is replaced by specialized metaplastic columnar epithelium. BE is a premalignant lesion which can progress to esophageal adenocarcinoma (EAC). Currently there are no early molecular indicators that would predict progression from BE to EAC. As the only permanent residents of the epithelium, stem cells have been implicated in this metaplastic progression.
To determine the expression of DCAMKL-1 and other putative gastrointestinal stem cell markers in normal esophageal mucosa (NEM), BE and EAC.
Human NEM, BE, EAC and multi-tissue microarrays were analyzed for DCAMKL-1 and immunohistochemically scored based on staining intensity and tissue involvement, with epithelia and stroma scored separately. Total RNA isolated from BE and paired NEM was subjected to real-time RT-PCR analysis for DCAMKL-1, LGR5 and Musashi-1 mRNA expression.
DCAMKL-1 is minimally expressed in squamous NEM, but increased in BE (with and without dysplasia) and EAC tissues. In EAC, we found increased stromal DCAMKL-1 staining compared to adjacent epithelia. Within the sub-mucosa of dysplastic BE tissues, an increase in endothelial cell expression of DCAMKL-1 was observed. Finally, an upregulation of DCAMKL-1, LGR5 and Musashi-1 mRNA was seen in BE compared to squamous NEM.
Here we report progressive increase of DCAMKL-1 expression in BE from dysplasia to EAC. Furthermore, there was an increase in putative stem cell markers DCAMKL-1, LGR5 and Msi-1 mRNA. These data taken together suggest that regulation of resident stem cells may play an important role in progression of BE to EAC.
Stem Cell Marker; DCAMKL-1; Dclk-1; Cancer Stem Cell Marker; Esophagus; Barrett’s Esophagus; LGR5; Musashi-1
The continuous exposure of esophageal epithelium to refluxate may induce ectopic expression of bile-responsive genes and contribute to the development of Barrett's esophagus (BE) and esophageal adenocarcinoma. In normal physiology of the gut and liver, the nuclear receptor Pregnane × Receptor (PXR) is an important factor in the detoxification of xenobiotics and bile acid homeostasis. This study aimed to investigate the expression and genetic variation of PXR in reflux esophagitis (RE), Barrett's esophagus (BE) and esophageal adenocarcinoma.
PXR mRNA levels and protein expression were determined in biopsies from patients with adenocarcinoma, BE, or RE, and healthy controls. Esophageal cell lines were stimulated with lithocholic acid and rifampicin. PXR polymorphisms 25385C/T, 7635A/G, and 8055C/T were genotyped in 249 BE patients, 233 RE patients, and 201 controls matched for age and gender.
PXR mRNA levels were significantly higher in adenocarcinoma tissue and columnar Barrett's epithelium, compared to squamous epithelium of these BE patients (P < 0.001), and RE patients (P = 0.003). Immunohistochemical staining of PXR showed predominantly cytoplasmic expression in BE tissue, whereas nuclear expression was found in adenocarcinoma tissue. In cell lines, stimulation with lithocholic acid did not increase PXR mRNA levels, but did induce nuclear translocation of PXR protein. Genotyping of the PXR 7635A/G polymorphism revealed that the G allele was significantly more prevalent in BE than in RE or controls (P = 0.037).
PXR expresses in BE and adenocarcinoma tissue, and showed nuclear localization in adenocarcinoma tissue. Upon stimulation with lithocholic acid, PXR translocates to the nuclei of OE19 adenocarcinoma cells. Together with the observed association of a PXR polymorphism and BE, this data implies that PXR may have a function in prediction and treatment of esophageal disease.
Prediction of progression to cancer in patients with Barrett’s esophagus is difficult using current techniques. We determined whether DNA promoter hypermethylation of genes frequently methylated in esophageal adenocarcinoma (p16 and APC) could be used as predictors of progression in Barrett’s esophagus.
We first performed a cross-sectional study to evaluate the prevalence of gene hypermethylation in biopsies from patients with normal esophagus (n=17), Barrett’s esophagus (n=102), and adenocarcinoma (n=42). We then performed a nested case-control study comparing gene hypermethylation in Barrett’s esophagus patients who progressed from baseline pathology to high-grade dysplasia or cancer (n=7) versus patients who did not progress (n=50).
None of the patients with normal esophagus had p16 or APC hypermethylation. Hypermethylation was prevalent in Barrett’s esophagus without dysplasia or low-grade dysplasia (p16=31% and APC=50%; p<0.01) and high-grade dysplasia or adenocarcinoma (p16=54% and APC=68%; p<0.001) compared to normal esophagus (not detected). Patients who progressed from baseline pathology to high-grade dysplasia or cancer had higher prevalence of hypermethylation in their initial esophagus biopsies compared to those who did not progress for both p16 (100% vs. 33%; p=0.008) and APC (86% vs. 40%; p=0.02). Hypermethylation of both p16 and APC was a strong predictor of subsequent progression to cancer during a mean follow-up time of 4.1 years (adjusted OR [95% CI]=14.97 [1.73,inf], p=0.01). Among patients who were negative for both p16 and APC hypermethylation, none progressed from baseline pathology to high-grade dysplasia or cancer.
Hypermethylation of both p16 and APC strongly predicts progression to high-grade dysplasia or cancer in patients with Barrett’s esophagus. Absence of p16 and APC hypermethylation is associated with a benign course.
Patients with Barrett's esophagus have a significantly increased risk of esophageal adenocarcinoma, 40-125 times higher than the general population. Since only a small fraction of Barrett's esophagus patients will actually progress to esophageal adenocarcinoma, there is a need to develop markers that may accurately predict which patients with Barrett's esophagus are likely to have aggressive disease and progress to cancer versus patients who will remain histologically stable and have a benign course. This would allow for better risk stratification of patients with Barrett's esophagus in order to target aggressive surveillance and intervention towards only those patients at highest risk for neoplastic progression. Predictive biomarkers may thus have significant clinical utility in the management of Barrett's esophagus patients. The detection of dysplasia in esophageal biopsies is currently the only standard method used in clinical practice as a marker for increased risk of cancer. However, dysplasia has not been a accurate or reliable marker for predicting malignant progression and suffers from poor interobserver agreement among pathologists and sampling error. A multitude of potential biomarkers have been studied over the years. It is likely that the best model for predicting progression to esophageal adenocarcinoma in Barrett's esophagus patients will ultimately involve a combination of biomarkers, dysplasia grade and other pathological characteristics, as well as clinical and demographic attributes. In this review, we will discuss the most promising biomarkers that have been studied thus far.
Incidence rates for esophageal adenocarcinoma have increased by over 500% during the past few decades without clear reasons. Gastroesophageal reflux disease (GERD), obesity, and smoking have been identified as risk factors, although the demographic distribution of these risk factors is not consistent with the demographic distribution of esophageal adenocarcinoma, which is substantially more common among whites and males than any other demographic groups. Numerous epidemiological studies have suggested associations between dietary factors and the risks of esophageal adenocarcinoma and its precursor, Barrett’s esophagus, though a comprehensive review is lacking. The main aim of the present review is to consider the evidence linking dietary factors with the risks of esophageal adenocarcinoma, Barrett’s esophagus, and the progression from Barrett’s esophagus to esophageal adenocarcinoma. The existing epidemiological evidence is strongest for an inverse relationship between intake of vitamin C, β-carotene, fruits and vegetables, particularly raw fruits and vegetables and dark-green, leafy and cruciferous vegetables, carbohydrates, fiber and iron and the risk of esophageal adenocarcinoma and Barrett’s esophagus. Patients at higher risk for Barrett’s esophagus and esophageal adenocarcinoma may benefit from increasing their consumption of fruits and vegetables and reducing their intake of red meat and other processed food items. Further research is needed to evaluate the relationship between diet and the progression of Barrett’s esophagus to esophageal adenocarcinoma. Evidence from cohort studies will help determine whether randomized chemoprevention trials are warranted for the primary prevention of Barrett’s esophagus or its progression to cancer.
Although a combination of genomic and epigenetic alterations are implicated in the multistep transformation of normal squamous esophageal epithelium to Barrett esophagus, dysplasia, and adenocarcinoma, the combinatorial effect of these changes is unknown. By integrating genome-wide DNA methylation, copy number, and transcriptomic datasets obtained from endoscopic biopsies of neoplastic progression within the same individual, we are uniquely able to define the molecular events associated progression of Barrett esophagus. We find that the previously reported global hypomethylation phenomenon in cancer has its origins at the earliest stages of epithelial carcinogenesis. Promoter hypomethylation synergizes with gene amplification and leads to significant upregulation of a chr4q21 chemokine cluster and other transcripts during Barrett neoplasia. In contrast, gene-specific hypermethylation is observed at a restricted number of loci and, in combination with hemi-allelic deletions, leads to downregulatation of selected transcripts during multistep progression. We also observe that epigenetic regulation during epithelial carcinogenesis is not restricted to traditionally defined “CpG islands,” but may also occur through a mechanism of differential methylation outside of these regions. Finally, validation of novel upregulated targets (CXCL1 and 3, GATA6, and DMBT1) in a larger independent panel of samples confirms the utility of integrative analysis in cancer biomarker discovery.
The incidence of esophageal adenocarcinoma (EA) is increasing at an alarming pace in the United States. Distinct pathological stages of Barrett's metaplasia and low- and high-grade dysplasia can be seen preceding malignant transformation. These precursor lesions provide a unique in vivo model for deepening our understanding the early steps in human neoplasia. By integrating genome-wide DNA methylation, copy number, and transcriptomic datasets obtained from endoscopic biopsies of neoplastic progression within the same individual, we are uniquely able to define the molecular events associated progression of Barrett esophagus. We show that the predominant change during this process is loss of DNA methylation. We show that this global hypomethylation occurs very early during the process and is seen even in preinvasive lesions. This loss of DNA methylation drives carcinogenesis by cooperating with gene amplifications in upregulating proteins during this process. Finally we uncovered proteins that upregulated by loss of methylation or gene amplification (CXCL1 and 3, GATA6, and DMBT1) and show their relevance by validating their levels in larger independent panel of samples, thus confirming the utility of integrative analysis in cancer biomarker discovery.
To identify the bacterial flora in conditions such as Barrett’s esophagus and reflux esophagitis to determine if they are similar to normal esophageal flora.
Using broad-range 16S rDNA PCR, esophageal biopsies were examined from 24 patients [9 with normal esophageal mucosa, 12 with gastroesophageal reflux disease (GERD), and 3 with Barrett’s esophagus]. Two separate broad-range PCR reactions were performed for each patient, and the resulting products were cloned. In one patient with Barrett’s esophagus, 99 PCR clones were analyzed.
Two separate clones were recovered from each patient (total = 48), representing 24 different species, with 14 species homologous to known bacteria, 5 homologous to unidentified bacteria, and 5 were not homologous (<97% identity) to any known bacterial 16S rDNA sequences. Seventeen species were found in the reflux esophagitis patients, 5 in the Barrett’s esophagus patients, and 10 in normal esophagus patients. Further analysis concentrating on a single biopsy from an individual with Barrett’s esophagus revealed the presence of 21 distinct bacterial species. Members of four phyla were represented, including Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria. Microscopic examination of each biopsy demonstrated bacteria in intimate association with the distal esophageal epithelium, suggesting that the presence of these bacteria is not transitory.
These findings provide evidence for a complex, residential bacterial population in esophageal reflux-related disorders. While much of this biota is present in the normal esophagus, more detailed comparisons may help identify potential disease associations.
Bacterial biota; Esophagus; 16S rDNA PCR
Purpose of review
Our article discusses the current understanding of screening and surveillance options for Barrett’s esophagus and emerging concepts that have the potential to improve the effectiveness and cost-effectiveness of surveillance.
Although endoscopic surveillance of patients with Barrett’s esophagus is commonly practiced in order to detect high-grade dysplasia and early esophageal adenocarcinoma (EAC), the reported incidence of EAC in Barrett’s esophagus patients varies widely. Recent studies found the risk of progression from Barrett’s esophagus to EAC to be significantly lower than previously reported, raising concerns regarding the limitations of current surveillance strategies. Advances in imaging techniques and their enhanced diagnostic accuracy may improve the value of endoscopic surveillance. Additionally, various efforts are ongoing to identify biomarkers that identify individuals at higher risk of cancer, possibly allowing for individual risk stratification.
These new data highlight some of the opportunities to revise and improve surveillance in patients with Barrett’s esophagus. The incorporation of new advances such as imaging techniques and biomarkers has the potential to improve the effectiveness and cost-effectiveness of new surveillance regimens.
Barrett’s esophagus; cost-effectiveness; esophageal adenocarcinoma; surveillance
Esophageal adenocarcinoma is preceded by the development of reflux-related intestinal metaplasia or Barrett’s esophagus which is a response to inflammation of the esophageal squamous mucosa, reflux esophagitis. Gastroesophageal reflux impairs the mucosal barrier in the distal esophagus, allowing chronic exposure of the squamous epithelium to the diverse microbial ecosystem or microbiome, and inducing chronic inflammation. The esophageal microbiome is altered in both esophagitis and Barrett's esophagus, characterized by a significant decrease in Gram-positive bacteria and an increase in Gram-negative bacteria in esophagitis and Barrett's esophagus. Lipopolysaccharides (LPS), a major structure of the outer membrane in Gram-negative bacteria, can up-regulate gene expression of proinflammatory cytokines via activation of the TLR4 and NF-kB pathway. The potential impact of LPS on reflux esophagitis may be through relaxation of the lower esophageal sphincter via iNOS and by delaying gastric emptying via COX-2. Chronic inflammation may be play a critical role in the progression from benign to malignant esophageal disease. Therefore analysis of the pathways leading to chronic inflammation in the esophagus may help to identify biomarkers in Barrett's esophagus patients for neoplastic progression and provide insight into molecular events suitable for therapeutic intervention in prevention of esophageal adenocarcinoma development in patients with reflux esophagitis and Barrett's esophagus.
Barrett's Esophagus; esophagitis; adenocarcinoma; microbiome; inflammation