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1.  Beyond Field Effect: Analysis of Shrunken Centroids in Normal Esophageal Epithelia Detects Concomitant Esophageal Adenocarcinoma 
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.
Methods
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.
Results
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.
Conclusions
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.
PMCID: PMC2323355  PMID: 18425214
2.  Evaluation of a Minimally Invasive Cell Sampling Device Coupled with Assessment of Trefoil Factor 3 Expression for Diagnosing Barrett's Esophagus: A Multi-Center Case–Control Study 
PLoS Medicine  2015;12(1):e1001780.
Background
Barrett's esophagus (BE) is a commonly undiagnosed condition that predisposes to esophageal adenocarcinoma. Routine endoscopic screening for BE is not recommended because of the burden this would impose on the health care system. The objective of this study was to determine whether a novel approach using a minimally invasive cell sampling device, the Cytosponge, coupled with immunohistochemical staining for the biomarker Trefoil Factor 3 (TFF3), could be used to identify patients who warrant endoscopy to diagnose BE.
Methods and Findings
A case–control study was performed across 11 UK hospitals between July 2011 and December 2013. In total, 1,110 individuals comprising 463 controls with dyspepsia and reflux symptoms and 647 BE cases swallowed a Cytosponge prior to endoscopy. The primary outcome measures were to evaluate the safety, acceptability, and accuracy of the Cytosponge-TFF3 test compared with endoscopy and biopsy.
In all, 1,042 (93.9%) patients successfully swallowed the Cytosponge, and no serious adverse events were attributed to the device. The Cytosponge was rated favorably, using a visual analogue scale, compared with endoscopy (p < 0.001), and patients who were not sedated for endoscopy were more likely to rate the Cytosponge higher than endoscopy (Mann-Whitney test, p < 0.001). The overall sensitivity of the test was 79.9% (95% CI 76.4%–83.0%), increasing to 87.2% (95% CI 83.0%–90.6%) for patients with ≥3 cm of circumferential BE, known to confer a higher cancer risk. The sensitivity increased to 89.7% (95% CI 82.3%–94.8%) in 107 patients who swallowed the device twice during the study course. There was no loss of sensitivity in patients with dysplasia. The specificity for diagnosing BE was 92.4% (95% CI 89.5%–94.7%). The case–control design of the study means that the results are not generalizable to a primary care population. Another limitation is that the acceptability data were limited to a single measure.
Conclusions
The Cytosponge-TFF3 test is safe and acceptable, and has accuracy comparable to other screening tests. This test may be a simple and inexpensive approach to identify patients with reflux symptoms who warrant endoscopy to diagnose BE.
Editors' Summary
Background
Barrett's esophagus is a condition in which the cells lining the esophagus (the tube that transports food from the mouth to the stomach) change and begin to resemble the cells lining the intestines. Although some people with Barrett's esophagus complain of burning indigestion or acid reflux from the stomach into the esophagus, many people have no symptoms or do not seek medical advice, so the condition often remains undiagnosed. Long-term acid reflux (gastroesophageal reflux disease), obesity, and being male are all risk factors for Barrett's esophagus, but the condition's exact cause is unclear. Importantly, people with Barrett's esophagus are more likely to develop esophageal cancer than people with a normal esophagus, especially if a long length (segment) of the esophagus is affected or if the esophagus contains abnormally growing “dysplastic” cells. Although esophageal cancer is rare in the general population, 1%–5% of people with Barrett's esophagus develop this type of cancer; about half of people diagnosed with esophageal cancer die within a year of diagnosis.
Why Was This Study Done?
Early detection and treatment of esophageal cancer increases an affected individual's chances of survival. Thus, experts recommend that people with multiple risk factors for Barrett's esophagus undergo endoscopic screening—a procedure that uses a small camera attached to a long flexible tube to look for esophageal abnormalities. Once diagnosed, patients with Barrett's esophagus generally enter an endoscopic surveillance program so that dysplastic cells can be identified as soon as they appear and removed using endoscopic surgery or “radiofrequency ablation” to prevent cancer development. However, although endoscopic screening of everyone with reflux symptoms for Barrett's esophagus could potentially reduce deaths from esophageal cancer, such screening is not affordable for most health care systems. In this case–control study, the researchers investigate whether a cell sampling device called the Cytosponge coupled with immunohistochemical staining for Trefoil Factor 3 (TFF3, a biomarker of Barrett's esophagus) can be used to identify individuals who warrant endoscopic investigation. A case–control study compares the characteristics of patients with and without a specific disease. The Cytosponge is a small capsule-encased sponge that is attached to a string. The capsule rapidly dissolves in the stomach after being swallowed, and the sponge collects esophageal cells for TFF3 staining when it is retrieved by pulling on the string.
What Did the Researchers Do and Find?
The researchers enrolled 463 individuals attending 11 UK hospitals for investigational endoscopy for dyspepsia and reflux symptoms as controls, and 647 patients with Barrett's esophagus who were attending hospital for monitoring endoscopy. Before undergoing endoscopy, the study participants swallowed a Cytosponge so that the researchers could evaluate the safety, acceptability, and accuracy of the Cytosponge-TFF3 test for the diagnosis of Barrett's esophagus compared with endoscopy. Nearly 94% of the participants swallowed the Cytosponge successfully, there were no adverse effects attributed to the device, and those participants that swallowed the device generally rated the experience as acceptable. The overall sensitivity of the Cytosponge-TFF3 test (its ability to detect true positives) was 79.9%. That is, 79.9% of the individuals with endoscopically diagnosed Barrett's esophagus were identified as having the condition using the new test. The sensitivity of the test was greater among patients who had a longer length of affected esophagus and importantly was not reduced in patients with dysplasia. Compared to endoscopy, the specificity of the Cytosponge-TFF3 test (its ability to detect true negatives) was 92.4%. That is, 92.4% of people unaffected by Barrett's esophagus were correctly identified as being unaffected.
What Do These Findings Mean?
The case–control design of this study means that its results are not generalizable to a primary care population. Also, the study used only a single measure of the acceptability of the Cytosponge-TFF3 test, Nevertheless, these findings indicate that this minimally invasive test for Barrett's esophagus is safe and acceptable, and that its accuracy is similar to that of colorectal cancer and cervical cancer screening tests. The Cytosponge-TFF3 test might, therefore, provide a simple, inexpensive way to identify those patients with reflux symptoms who warrant endoscopy to diagnose Barrett's esophagus, although randomized controlled trials of the test are needed before its routine clinical implementation. Moreover, because most people with Barrett's esophagus never develop esophageal cancer, additional biomarkers ideally need to be added to the test before its routine implementation to identify those individuals who have the greatest risk of esophageal cancer, and thereby avoid overtreatment of Barrett's esophagus.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001780.
The US National Institute of Diabetes and Digestive and Kidney Diseases provides detailed information about Barrett's esophagus and gastroesophageal reflux disease
The US National Cancer Institute provides information for patients and health professionals about esophageal cancer (in English and Spanish)
Cancer Research UK (a non-profit organization) provides detailed information about Barrett's esophagus (including a video about having the Cytosponge test and further information about this study, the BEST2 Study) and about esophageal cancer
The UK National Health Service Choices website has pages on the complications of gastroesophageal reflux and on esophageal cancer (including a real story)
Heartburn Cancer Awareness Support is a non-profit organization that aims to improve public awareness and provides support for people affected by Barrett's esophagus; the organization's website explains the range of initiatives to promote education and awareness as well as highlighting personal stories of those affected by Barrett's esophagus and esophageal cancer
The British Society of Gastroenterology has published guidelines on the diagnosis and management of Barrett's esophagus
The UK National Institute for Health and Care Excellence has published guidelines for gastroesophageal reflux
The Barrett's Esophagus Campaign is a UK-based non-profit organization that supports research into the condition and provides support for people affected by Barrett's esophagus; its website includes personal stories about the condition
In a multi-center case-control study, Rebecca Fitzgerald and colleagues examine whether a minimally invasive cell sampling device could be used to identify patients who warrant endoscopy to diagnose Barrett's esophagus.
doi:10.1371/journal.pmed.1001780
PMCID: PMC4310596  PMID: 25634542
3.  NSAIDs Modulate CDKN2A, TP53, and DNA Content Risk for Progression to Esophageal Adenocarcinoma 
PLoS Medicine  2007;4(2):e67.
Background
Somatic genetic CDKN2A, TP53, and DNA content abnormalities are common in many human cancers and their precursors, including esophageal adenocarcinoma (EA) and Barrett's esophagus (BE), conditions for which aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) have been proposed as possible chemopreventive agents; however, little is known about the ability of a biomarker panel to predict progression to cancer nor how NSAID use may modulate progression. We aimed to evaluate somatic genetic abnormalities with NSAIDs as predictors of EA in a prospective cohort study of patients with BE.
Methods and Findings
Esophageal biopsies from 243 patients with BE were evaluated at baseline for TP53 and CDKN2A (p16) alterations, tetraploidy, and aneuploidy using sequencing; loss of heterozygosity (LOH); methylation-specific PCR; and flow cytometry. At 10 y, all abnormalities, except CDKN2A mutation and methylation, contributed to EA risk significantly by univariate analysis, ranging from 17p LOH (relative risk [RR] = 10.6; 95% confidence interval [CI] 5.2–21.3, p < 0.001) to 9p LOH (RR = 2.6; 95% CI 1.1–6.0, p = 0.03). A panel of abnormalities including 17p LOH, DNA content tetraploidy and aneuploidy, and 9p LOH was the best predictor of EA (RR = 38.7; 95% CI 10.8–138.5, p < 0.001). Patients with no baseline abnormality had a 12% 10-y cumulative EA incidence, whereas patients with 17p LOH, DNA content abnormalities, and 9p LOH had at least a 79.1% 10-y EA incidence. In patients with zero, one, two, or three baseline panel abnormalities, there was a significant trend toward EA risk reduction among NSAID users compared to nonusers (p = 0.01). The strongest protective effect was seen in participants with multiple genetic abnormalities, with NSAID nonusers having an observed 10-y EA risk of 79%, compared to 30% for NSAID users (p < 0.001).
Conclusions
A combination of 17p LOH, 9p LOH, and DNA content abnormalities provided better EA risk prediction than any single TP53, CDKN2A, or DNA content lesion alone. NSAIDs are associated with reduced EA risk, especially in patients with multiple high-risk molecular abnormalities.
In a ten-year study of people with Barrett's esophagus, nonsteroidal anti-inflamatory drugs were associated with reduced risk of esophageal adenocarcinoma, especially in patients with multiple high-risk molecular abnormalities.
Editors' Summary
Background.
Normally, the cells in the human body divide only when extra cells are needed, after an injury, for example. Sometimes, however, cells accumulate genetic changes (mutations) that allow them to divide uncontrollably to form a disorganized mass or tumor. If these altered cells also acquire mutations that allow them to spread around the body, a malignant tumor or cancer results. Scientists have identified numerous genetic changes that occur in tumors and are now investigating whether these molecular abnormalities can be used as “biomarkers” to choose the best treatments for patients, to identify who will benefit from cancer-prevention strategies, to detect cancer early, and to predict which cancers are most likely to become life-threatening. This last application is particularly important for cancers with a well-defined premalignant stage. Because the cells in premalignant tissues have acquired some of the genetic changes required for cancer development, they are more likely to become malignant than normal cells. Barrett's esophagus, for example, is a premalignant disorder of the muscular tube that takes food from the mouth to the stomach. People with Barrett's esophagus are much more likely to develop esophageal cancer than the general population.
Why Was This Study Done?
Esophageal cancer is often incurable by the time it is detected, so it would be helpful to know which people with Barrett's esophagus are most likely to develop esophageal cancer—only 1 in 200 of them develop cancer each year. In this study, the researchers evaluated whether a panel of genetic alterations could identify this subset of patients. They also investigated whether the regular use of aspirin or other nonsteroidal anti-inflammatory drugs (NSAIDs) affects the risk of developing esophageal cancer in people with Barrett's esophagus—other evidence suggests that NSAIDs may help to prevent several types of cancer, including esophageal cancer.
What Did the Researchers Do and Find?
The researchers took esophageal tissue samples from patients with Barrett's esophagus and looked for alterations in the genes encoding the tumor-suppressor proteins TP53 and CDKN2A. These proteins normally stop cells dividing but are often inactivated in cancer cells by mutation of one of the two gene copies that encode each of them and also loss of the other copy (so-called “loss of heterozygosity” or LOH). The researchers also looked for changes in the cellular DNA content of the samples (tumor cells often contain unusual amounts of DNA) and asked the study participants about their NSAID use before waiting to see which participants developed esophageal cancer. After 10 y, the participants whose tissue samples had LOH of the short arms (p) of Chromosome 17 or 9 (the sites of the genes encoding TP53 and CDKN2A, respectively), or an altered DNA content, were more likely to have developed esophageal cancer than those without these abnormalities; those whose samples contained all three abnormalities had the highest risk of developing esophageal cancer. Overall, just 12% of patients with no abnormalities but nearly 80% of patients with three abnormalities developed esophageal cancer. NSAID use reduced the risk of cancer development in all the participants, but its effect was greatest in those with three genetic abnormalities.
What Do These Findings Mean?
These findings suggest that the combined measurement of 17pLOH, 9pLOH, and cellular DNA content might be a powerful way to identify those patients with Barrett's esophagus who are most likely to develop esophageal cancer. They also suggest that NSAID use is associated with a reduced risk of esophageal cancer, particularly in patients with multiple genetic abnormalities. Because very few participants developed cancer during the study, these results need confirming in more patients. Also, the ability of NSAIDs to prevent the progression of Barrett's esophagus to esophageal cancer needs testing in multicenter randomized trials; the use of the panel of abnormalities described here to identify the people with Barrett's esophagus most at risk of developing esophageal cancer should facilitate such studies.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040067.
CancerQuest information from Emory University (Atlanta, Georgia, United States) on cancer biology, including the role of tumor suppressor proteins
US National Cancer Institute patient and physician information on esophageal cancer and its prevention
MedlinePlus encyclopedia pages on Barrett's esophagus and esophageal cancer
Cancerbackup (UK charity) patient information on esophageal cancer and Barrett's esophagus
doi:10.1371/journal.pmed.0040067
PMCID: PMC1808095  PMID: 17326708
4.  Gene Expression Profiling Reveals Stromal Genes Expressed in Common Between Barrett’s Esophagus and Adenocarcinoma 
Gastroenterology  2006;131(3):925-933.
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1053/j.gastro.2006.04.026
PMCID: PMC2575112  PMID: 16952561
5.  Alterations of glutathione S-transferase and matrix metalloproteinase-9 expressions are early events in esophageal carcinogenesis 
AIM: To investigate the role of glutathione S-transferase (GST) and matrix metalloproteinase-9 (MMP-9) expressions in the development and progression of reflux esophagitis-Barrett’s metaplasia-dysplasia-adenocarcinoma sequence in the esophagus.
METHODS: GST and MMP-9 expressions were analyzed in 51 paraffin-embedded tissue samples by immunohistochemistry including patients with reflux esophagitis (n = 7), Barrett’s metaplasia (n = 14), Barrett and esophagitis (n = 8), Barrett and dysplasia (n = 7), esophageal adenocarcinoma (n = 8) and a control group without any histological changes (n = 7). Immunostaining was determined semiquantitatively. Statistical analysis with one-way ANOVA, LSD test and correlation analysis were performed. P value of < 0.05 was considered significant.
RESULTS: GST expression was significantly higher while MMP-9 expression was significantly lower in control group compared to Barrett’s metaplasia and the other groups. No major changes were observed between Barrett, esophagitis, and Barrett and concomitant esophagitis. Barrett and concomitant dysplasia, and adenocarcinoma revealed a significant lower expression of GST and higher levels of MMP-9 compared to all other groups. Adenocarcinoma showed almost no expression of GST and significantly higher levels of MMP-9 than Barrett and concomitant dysplasia. Alterations of GST and MMP-9 were inversely correlated (r = - 0.82).
CONCLUSION: Decreased GST and increased expression of MMP-9 in Barrett’s metaplasia-dysplasia-adenocarcinoma sequence as compared to normal tissue suggest their association with esophageal tumorigenesis. Loss of GST and gain of MMP-9 in Barrett with dysplasia compared to non-dysplastic metaplasia indicate that these alterations may be early events in carcinogenesis. Quantification of these parameters in Barrett’s esophagus might be useful to identify patients at higher risk for progression to cancer.
doi:10.3748/wjg.v13.i5.676
PMCID: PMC4065999  PMID: 17278189
Glutathione S-transferase; Matrix metallo-proteinase-9; Barrett’s metaplasia; Esophagus; Adenocarcinoma; Dysplasia
6.  Gene expression in Barrett’s esophagus and reflux esophagitis induced by gastroduodenoesophageal reflux in rats 
AIM: To investigate the difference of gene expression profiles between Barrett’s esophagus and reflux eso-phagitis induced by gastroduodenoesophageal reflux in rats.
METHODS: Eight-week-old Sprague-Dawley rats were treated esophagoduodenostomy to produce gastroduode-noesophageal reflux, and another group received sham operation as control. Esophageal epithelial tissues were dissected and frozen in liquid nitrogen immediately for pathology 40 wk after surgery. The expression profiles of 4096 genes in reflux esophagitis and Barrett’s esophagus tissues were compared with normal esophageal epithelium by cDNA microarray.
RESULTS: Four hundred and forty-eight genes in Barrett’s esophagus were more than three times different from those in normal esophageal epithelium, including 312 up-regulated and 136 down-regulated genes. Two hundred and thirty-two genes in RE were more than three times different from those in normal esophageal epithelium, 90 up-regulated and 142 down-regulated genes. Compared to reflux esophagitis, there were 214 up-regulated and 142 down-regulated genes in Barrett’s esophagus.
CONCLUSION: Esophageal epithelium exposed excessively to harmful ingredients of duodenal and gastric reflux can develop esophagitis and Barrett’s esophagus gradually. The gene expression level is different between reflux esophagitis and Barrett’s esophagus and the differentially expressed genes might be related to the occurrence and development of Barrett’s esophagus and the promotion or progression in adenocarcinoma.
doi:10.3748/wjg.v11.i21.3277
PMCID: PMC4316063  PMID: 15929182
Gastroduodenoesophageal reflux; Esophagitis; Barrett’s esophagus; Gene expression
7.  Recent advances in oesophageal diseases 
Abstract
Dong Y, Qi B, Feng XY, Jiang CM. Meta-analysis of Barrett's esophagus in China. World J Gastroenterol 2013;19(46):8770-8779
The disease pattern of Barrett's esophagus (BE) in China is poorly characterised particularly in comparison with other developed countries. This meta-analysis of 3873 cases of BE collated from 69 clinical studies conducted in 25 provinces between 2000 and 2011 investigated the epidemiology and characteristics of BE in China compared to Western countries. The total endoscopic detection rate of BE was 1.0% (95%CI: 0.1%-1.8%) with an average patient age of 49.07 ± 5.09 years, lower than many Western countries.The authors postulate this may be attributed to environmental risk factor variation, distinct genetics and different medical practice including diagnostic criteria for BE and expertise in endoscopy. This study identified a 1.781 male predominancefor BE in China, consistent with Western reports. Short-segment BE accounted for 80.3% of cases with island type and cardiac type the most common endoscopic (44.8%) and histological (40.0%) manifestations respectively. Of the 1283 BE cases followed up for three to 36 months the incidence of esophageal cancer was 1.418 per 1000 person-years, lower than the incidence reported in Western countries.
Lee HS, Jeon SW. Barrett esophagus in Asia: same disease with different pattern. ClinEndosc 2014;47(1):15-22
Barrett's esophagus (BE) is a common, pre-cancerous condition characterised by intestinal metaplasia of squamous esophageal epithelium usually attributed to chronic gastric acid exposure. This review article explores important differences in the disease pattern of BE between Asian and the Western countries.
Overall the prevalence of BE is lower in Asia compared to the West with a greater proportion of short-segment type. The authors identify great variability in the endoscopic and pathologic diagnostic criteria for BE. Many of the studies in Asian countries did not use a standardised four-quadrant biopsy protocol which may have led to an underestimation of BE prevalence. The review highlights an increasing incidence of esophageal adenocarcinoma in the West but unclear disease trend in Asia with inter-country variability. Similarly in Asian and Western countries BE is associated with the presence of hiatus hernia, advancing age, male gender, alcohol consumption, smoking, abdominal obesity and longer duration of gastro-esophageal reflux disease. The authors postulate that Helicobacter pylori infection, more prevalent in Asia than the West, may have a protective effect on BE.
There is a need for larger, prospective studies to further clarify the disease pattern of BE in Asian countries. Clearly standardisation of the diagnostic process for BE is important to validate the differences in disease trends between Asian and Western countries.
Kiadaliri AA. Gender and social disparities in esophagus cancer incidence in Iran, 2003-2009: a time trend province-level study.Asian Pac J Cancer Prev 2014;15(2):623-7
Esophageal cancer (EC) is a major cause of morbidity and mortality particuarly in Iran where the incidence rate exceeds the global average. An understanding of the factors influencing the province-specific incidence of EC in Iran is important to inform disease-prevention strategies and address health inequalities. This ecological study used cancer registry data to investigate the relationship between gender and social class and the incidence of EC in Iran at province-level between 2003 and 2009. The age standardised incidence rates (ASIR) of EC were greatest in the Northern provinces of Iran, specifically Razavi Khorasan in males and Kordestan in females. Overall the EC incidence did not significantly differ according to gender.
Interestingly, during the study period the ASIR increased by 4.6% per year in females (p=0.08) and 6.5% per year in males (p=0.02). This may reflect increasing rates of establised risk factors for EC including obsesity and gastro-esophageal reflux disease alongside more vigilant recording of new cases. Social class was inversely associated with the ASIR of EC regardless of gender which may be attributed to class differences in risk factor distribution particularly smoking, diet and obesity. An appreciation for the limitations of an epidemiological study is important when interpreting results which should be further evaluated in future studies.
Islami F et al.Determinants of gastroesophageal reflux disease, including hookah smoking and opium use- A cross-sectional analysis of 50,000 individuals. PLoS One 2014;9(2):e89256
Gastroesophageal reflux disease (GERD) is a highly prevalent cause of gastrointestinal symptoms worldwide incurring great cost to the primary and secondary healthcare sectors. An improved understanding of the factors which influence GERD symptoms in low- to medium- income countries may inform public health initiatives. This study analysed prospective data from the Golestan cohort study, primarily established to investigate determinants of upper gastrointestinal cancers, toexplore the risk factors influencing GERD symptoms (regurgitation and/or heartburn) in 50,045 individuals aged 40-75 years in Golestan Province, Iran enrolled between 01/2004 and 06/2008.Of note, 39.12% of individuals denied ever experiencing GERD symptoms. A further 19.89% reported at least once weekly GERD symptoms with 11.83% experiencing daily symptoms. Severe symptoms, defined as disturbing daily work or sleep, were recorded by 11.33% of individuals.
Separately the occurrence of daily GERD symptoms and severe symptoms were inversely associated with male gender (OR 0.36, 95% CI 0.33-0.39 both), level of formal education (p=0.01 and p=0.001 respectively), wealth score (p<0.001 both) and regular nass chewing (OR 0.86, 95% CI 0.75-0.98 and OR 0.87, 95% CI 0.76-0.99 respectively)and were positively associated with body mass index (p<0.001 both), intensity of physical activity (p=0.04 both), cigarette pack years (p<0.001 both), alcohol consumption (OR 1.36, 95% CI 1.13-1.64 and OR 1.53, 95% CI 1.28-1.83 respectively) and opium use (OR 1.82, 95% CI 1.67-1.99 and OR 1.70, 95% CI 1.55-1.87 respectively).In addition hookah smoking had a borderline significant correlation with mild and moderate severity GERD symptoms in individuals who had never smoked cigarettes (OR 1.41, 95% CI 1.00-1.99 and OR 1.25, 95% CI 0.99-1.57 respectively).
Overall this large study contributes useful data to inform the prevention and management of GERD symptoms particularly regarding the use of hookah, opium and nass which was previously unclear.
Barbera M et al. The human squamous oesophagus has widespread capacity for clonal expansion from cells at diverse stages of differentiation. Gut 2014;0:1–9. doi:10.1136/gutjnl-2013-306171
Current knowledge on human esophageal tissue homeostasis and injury repair is derived predominantly from murine models and hence may be inaccurate due to cellular and architectural differences. This study used 3D imaging in conjunction withstaining for cell lineage markers to investigate the cellular mechanisms involved in homeostasis of the normal human squamous esophagus in 10 participants undergoing esophagectomy for esophageal cancer. The self-renewal potential of cell subpopulations was also assessed using in vitro and in vivo assays.
A decreasing gradient of cell proliferation was observed from the inter-papillary basal layer to the tip of the papilla where there was no evidence of mitosis. The expression ofβ1-integrin, a putative stem cell marker, was consistent throughout the basal layer and therefore the entire basal layer can be considered undifferentiated. Quiescent β1-integrin/CD34-positive cells which failed to stain for CD45, S-100 or F4-80were identified at the tip of the papilla suggesting this is an extension of the basal layer. Contrary to previous data, this study found progenitor cells widely distributed in human esophageal tissue and included already differentiated epithelial cells. This insight into esophageal homeostasis may inform future studies exploring the pathological mechanisms underpinning homeostatic disruption in disease states such as Barrett's esophagus.
Papers were prepared by:
Drs Ishfaq Ahmad and Luke Materacki, Department of Medicine, Alexandra Hospital, Redditch, UK
PMCID: PMC4129572  PMID: 25120902
8.  Math1/Atoh1 contributes to intestinalization of esophageal keratinocytes by inducing the expression of Muc2 and Keratin-20 
Digestive diseases and sciences  2011;57(4):845-857.
Background
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.
Aims
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.
Methods
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.
Results
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.
Conclusions
We conclude that ectopic Math1/Atoh1 expression makes unique contributions to the intestinalization of esophageal epithelium in Barrett’s esophagus.
doi:10.1007/s10620-011-1998-y
PMCID: PMC3407817  PMID: 22147253
Barrett’s esophagus; Math1/Atoh1; Keratin-20; metaplasia; Mucin-2; organotypic culture
9.  Immunohistochemical assessment of NY-ESO-1 expression in esophageal adenocarcinoma resection specimens 
AIM: To assess NY-ESO-1 expression in a cohort of esophageal adenocarcinomas.
METHODS: A retrospective search of our tissue archive for esophageal resection specimens containing esophageal adenocarcinoma was performed, for cases which had previously been reported for diagnostic purposes, using the systematised nomenclature of human and veterinary medicine coding system. Original haematoxylin and eosin stained sections were reviewed, using light microscopy, to confirm classification and tumour differentiation. A total of 27 adenocarcinoma resection specimens were then assessed using immunohistochemistry for NY-ESO-1 expression: 4 well differentiated, 14 moderately differentiated, 4 moderate-poorly differentiated, and 5 poorly differentiated.
RESULTS: Four out of a total of 27 cases of esophageal adenocarcinoma examined (15%) displayed diffuse cytoplasmic and nuclear expression for NY-ESO-1. They displayed a heterogeneous and mosaic-type pattern of diffuse staining. Diffuse cytoplasmic staining was not identified in any of these structures: stroma, normal squamous epithelium, normal submucosal gland and duct, Barrett’s esophagus (goblet cell), Barrett’s esophagus (non-goblet cell) and high grade glandular dysplasia. All adenocarcinomas showed an unexpected dot-type pattern of staining at nuclear, paranuclear and cytoplasmic locations. Similar dot-type staining, with varying frequency and size of dots, was observed on examination of Barrett’s metaplasia, esophageal submucosal gland acini and the large bowel negative control, predominantly at the crypt base. Furthermore, a prominent pattern of apical (luminal) cytoplasmic dot-type staining was observed in some cases of Barrett’s metaplasia and also adenocarcinoma. A further morphological finding of interest was noted on examination of haematoxylin and eosin stained sections, as aggregates of lymphocytes were consistently noted to surround submucosal glands.
CONCLUSION: We have demonstrated for the first time NY-ESO-1 expression by esophageal adenocarcinomas, Barrett’s metaplasia and normal tissues other than germ cells.
doi:10.3748/wjg.v20.i14.4011
PMCID: PMC3983456  PMID: 24744590
Esophageal adenocarcinoma; Immunohistochemistry; NY-ESO-1; Stem cells; Vesicle trafficking
10.  Molecular Pathways: Pathogenesis and clinical implications of microbiome alteration in esophagitis and Barrett’s esophagus 
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.
doi:10.1158/1078-0432.CCR-11-0934
PMCID: PMC3725293  PMID: 22344232
Barrett's Esophagus; esophagitis; adenocarcinoma; microbiome; inflammation
11.  Characterization of bacterial biota in the distal esophagus of Japanese patients with reflux esophagitis and Barrett’s esophagus 
BMC Infectious Diseases  2013;13:130.
Background
The distal esophagus harbors a complex bacterial population. We hypothesized that a better understanding of bacterial communities in the esophagus would facilitate understanding of the role of bacteria in esophageal disease. Here, we investigated bacterial composition in the distal esophagus in subjects with a normal esophagus, reflux esophagitis, and Barrett’s esophagus.
Methods
Two biopsy specimens were obtained from the distal esophagus at 1 cm above the gastroesophageal junction under endoscopic examination in 18 patients (6 each with normal esophagus, reflux esophagitis, and Barrett’s esophagus) and used for histological examination and DNA extraction. Fragments of 16S rDNA genes were amplified by PCR using general bacterial primers, and bacterial populations were examined. A third biopsy specimen was taken from the patients with Barrett’s esophagus to histologically confirm the replacement of squamous epithelium with columnar epithelium in the distal esophagus.
Results
Endoscopic diagnoses of normal esophagus, esophagitis, and Barrett’s esophagus were confirmed by histological findings. The total amount of bacterial DNA detected did not significantly differ among groups (p > 0.1). On average, each of the 18 subjects yielded about 350 clones, of which 40 were randomly picked and sequenced. Analysis of 147 16S rDNA sequences from 240 clones of 6 subjects with normal esophagus yielded four phyla, Proteobacteria (49%), Firmicutes (40%), Bacteroidetes (8%), and Actinobacteria (3%). Similar analysis of 139 16S rDNA sequences from 240 clones of 6 patients with reflux esophagitis yielded 6 phyla, Proteobacteria (43%), Firmicutes (33%), Bacteroidetes (10%), Fusobacteria (10%), Actinobacteria (2%), and TM7 (2%). while that of 138 16S rDNA sequences from 240 clones of 6 cases of Barrett’s esophagus yielded 5 phyla, Firmicutes (55%), Proteobacteria (20%), Bacteroidetes (14%), Fusobacteria (9%), and Actinobacteria (2%). Thus, microbial communities differed among patients with a normal esophagus, reflux esophagitis and Barrett’s esophagus.
Conclusions
Esophageal bacterial composition differs under conditions of normal esophagus, reflux esophagitis, and Barrett’s esophagus. Diverse bacterial communities may be associated with esophageal disease.
doi:10.1186/1471-2334-13-130
PMCID: PMC3599685  PMID: 23496929
Bacterial biota; 16S rDNA; Esophagitis; Barrett’s esophagus
12.  Role of epigenetic alterations in the pathogenesis of Barrett’s esophagus and esophageal adenocarcinoma 
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.
PMCID: PMC3396065  PMID: 22808291
Barrett’s esophagus; DNA methylation; esophageal adenocarcinoma; global hypomethylation
13.  Hedgehog signaling regulates FOXA2 in esophageal embryogenesis and Barrett’s metaplasia 
The Journal of Clinical Investigation  2014;124(9):3767-3780.
Metaplasia can result when injury reactivates latent developmental signaling pathways that determine cell phenotype. Barrett’s esophagus is a squamous-to-columnar epithelial metaplasia caused by reflux esophagitis. Hedgehog (Hh) signaling is active in columnar-lined, embryonic esophagus and inactive in squamous-lined, adult esophagus. We showed previously that Hh signaling is reactivated in Barrett’s metaplasia and overexpression of Sonic hedgehog (SHH) in mouse esophageal squamous epithelium leads to a columnar phenotype. Here, our objective was to identify Hh target genes involved in Barrett’s pathogenesis. By microarray analysis, we found that the transcription factor Foxa2 is more highly expressed in murine embryonic esophagus compared with postnatal esophagus. Conditional activation of Shh in mouse esophageal epithelium induced FOXA2, while FOXA2 expression was reduced in Shh knockout embryos, establishing Foxa2 as an esophageal Hh target gene. Evaluation of patient samples revealed FOXA2 expression in Barrett’s metaplasia, dysplasia, and adenocarcinoma but not in esophageal squamous epithelium or squamous cell carcinoma. In esophageal squamous cell lines, Hh signaling upregulated FOXA2, which induced expression of MUC2, an intestinal mucin found in Barrett’s esophagus, and the MUC2-processing protein AGR2. Together, these data indicate that Hh signaling induces expression of genes that determine an intestinal phenotype in esophageal squamous epithelial cells and may contribute to the development of Barrett’s metaplasia.
doi:10.1172/JCI66603
PMCID: PMC4151220  PMID: 25083987
14.  Urinary metabolomic signature of esophageal cancer and Barrett’s esophagus 
Background
Esophageal adenocarcinoma (EAC) often presents at a late, incurable stage, and mortality has increased substantially, due to an increase in incidence of EAC arising out of Barrett’s esophagus. When diagnosed early, however, the combination of surgery and adjuvant therapies is associated with high cure rates. Metabolomics provides a means for non- invasive screening of early tumor-associated perturbations in cellular metabolism.
Methods
Urine samples from patients with esophageal carcinoma (n = 44), Barrett’s esophagus (n = 31), and healthy controls (n = 75) were examined using 1H-NMR spectroscopy. Targeted profiling of spectra using Chenomx software permitted quantification of 66 distinct metabolites. Unsupervised (principal component analysis) and supervised (orthogonal partial least-squares discriminant analysis OPLS-DA) multivariate pattern recognition techniques were applied to discriminate between samples using SIMCA-P+ software. Model specificity was also confirmed through comparison with a pancreatic cancer cohort (n = 32).
Results
Clear distinctions between esophageal cancer, Barrett’s esophagus and healthy controls were noted when OPLS-DA was applied. Model validity was confirmed using two established methods of internal validation, cross-validation and response permutation. Sensitivity and specificity of the multivariate OPLS-DA models were summarized using a receiver operating characteristic curve analysis and revealed excellent predictive power (area under the curve = 0.9810 and 0.9627 for esophageal cancer and Barrett’s esophagus, respectively). The metabolite expression profiles of esophageal cancer and pancreatic cancer were also clearly distinguishable with an area under the receiver operating characteristics curve (AUROC) = 0.8954.
Conclusions
Urinary metabolomics identified discrete metabolic signatures that clearly distinguished both Barrett’s esophagus and esophageal cancer from controls. The metabolite expression profile of esophageal cancer was also discrete from its precursor lesion, Barrett’s esophagus. The cancer-specific nature of this profile was confirmed through comparison with pancreatic cancer. These preliminary results suggest that urinary metabolomics may have a future potential role in non-invasive screening in these conditions.
doi:10.1186/1477-7819-10-271
PMCID: PMC3579706  PMID: 23241138
Metabolomics; Urine; Esophageal cancer; Barrett’s esophagus; Diagnosis; Screening and surveillance
15.  Biomarkers in the molecular pathogenesis of esophageal (Barrett) adenocarcinoma 
Current Oncology  2006;13(1):33-43.
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.
PMCID: PMC1891165  PMID: 17576439
Barrett esophagus; esophageal adenocarcinoma; molecular pathogenesis; biomarkers
16.  Clonal Expansion and Loss of Heterozygosity at Chromosomes 9p and 17p in Premalignant Esophageal (Barrett’s) Tissue 
Background: Abnormalities involving the p16 (also known as cyclin-dependent kinase N2 [CDKN2], p16 [INK4a], or MTS1) and p53 (also known as TP53) tumor suppressor genes are highly prevalent in esophageal adenocarcinomas. Loss of heterozygosity (LOH) at 9p21 and 17p13 chromosomes (locations for p16 and p53 genes, respectively) is frequently observed in the premalignant condition, Barrett’s esophagus. We studied extensively the distribution and heterogeneity of LOH at 9p and 17p chromosomes throughout the Barrett’s segment in patients who have not yet developed esophageal adenocarcinoma. Methods: We evaluated 404 samples from 61 consecutive patients enrolled in the Seattle Barrett’s Esophagus Study from February 1995 through September 1998. All patients had high-grade dysplasia but no diagnosis of cancer. The samples were assayed for LOH at 9p and 17p chromosomes after amplification of genomic DNA by use of polymerase chain reaction and DNA genotyping. The cell fractions were purified by flow cytometry on the basis of DNA content and proliferation-associated antigen labeling. Association between LOH at 9p and LOH at 17p with flow cytometric abnormalities was determined by chi-squared test, and logistic regression models were used to model and test for the extent to which a particular genotype was found in 2-cm intervals. Results and Conclusions: LOH at 9p and 17p chromosomes are highly prevalent somatic genetic lesions in premalignant Barrett’s tissue. LOH at 9p is more common than LOH at 17p in diploid samples and can be detected over greater regions of Barrett’s epithelium. In most patients with high-grade dysplasia, the Barrett’s mucosa contains a mosaic of clones and subclones with different patterns of LOH. Some clones had expanded to involve extensive regions of Barrett’s epithelium. LOH at 9p and 17p chromosomes may be useful biomarkers to stratify patients’ risk of progression to esophageal cancer.
PMCID: PMC1559996  PMID: 10601379
17.  Expression of the bile acid receptor FXR in Barrett's esophagus and enhancement of apoptosis by guggulsterone in vitro 
Molecular Cancer  2006;5:48.
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1476-4598-5-48
PMCID: PMC1624849  PMID: 17054793
18.  Diagnosing Barrett’s esophagus: reliability of clinical and pathologic diagnoses 
Gastrointestinal endoscopy  2009;69(6):1004-1010.
Background
The accuracy of a Barrett’s esophagus diagnosis is not well studied.
Objective
Our purpose was to evaluate the accuracy of a clinical Barrett’s esophagus diagnosis and the reproducibility of an esophageal intestinal metaplasia diagnosis.
Methods
All patients with a Barrett’s esophagus diagnosis between 1994 and 2005 were identified by use of International Classification of Disease (ICD) and Systematized Nomenclature of Medicine (SNOMED) coding. Subsets received manual record review (endoscopy/pathology reports), slide review by a referral pathologist (interrater reliability), and 2 blinded reviews by the same pathologist (intrarater reliability).
Setting
An integrated health services delivery system.
Main Outcome Measurements
Accuracy of electronic clinical diagnosis and reproducibility of esophageal intestinal metaplasia diagnosis.
Results
A total of 2470 patients coded with Barrett’s esophagus underwent record review; a subgroup (616) received manual pathology slide review. Review confirmed a Barrett’s esophagus diagnosis for 1533 (61.9%) patients: 437 of 798 subjects (54.8%) with a SNOMED diagnosis alone, 153 of 671 subjects (26.8%) with an ICD diagnosis alone, and 940 of 1101 subjects (85%) who had both a SNOMED and an ICD diagnosis. The same metaplasia diagnosis occurred with 88.3% of subjects (original vs referral pathologist, interrater reliability; κ =.42, 95% CI, 0.34–0.48). The referral pathologist made the same metaplasia diagnosis twice for a given patient for 88.6% of subjects (intrarater reliability, 2 reviews by same pathologist; κ = 0.65, 95% CI, 0.35–0.93).
Limitations
The accuracy of a Barrett’s esophagus diagnosis likely represents the minimum number, given the strict criteria.
Conclusions
A community pathologist’s diagnosis of esophageal intestinal metaplasia is likely to be confirmed by a referral pathologist. Electronic diagnoses of Barrett’s esophagus overestimate the prevalence, although they are usually confirmed in patients with both a SNOMED and ICD diagnosis of Barrett’s esophagus.
doi:10.1016/j.gie.2008.07.035
PMCID: PMC2677140  PMID: 19152897
19.  MicroRNA Expression can be a Promising Strategy for the Detection of Barrett's Esophagus: A Pilot Study 
Objectives:
Patient outcomes for esophageal adenocarcinoma (EAC) have not improved despite huge advances in endoscopic therapy because cancers are being diagnosed late. Barrett's esophagus (BE) is the primary precursor lesion for EAC, and thus the non-endoscopic molecular diagnosis of BE can be an important approach to improve EAC outcomes if robust biomarkers for timely diagnosis are identified. MicroRNAs (miRNAs) are tissue-specific novel biomarkers that regulate gene expression and may satisfy this requirement.
Methods:
Patients with gastroesophageal reflux disease (GERD) and BE were selected from an ongoing tissue and serum repository. BE was defined by the presence of intestinal metaplasia. Previously published miRNA sequencing profiles of GERD and BE patients allowed us to select three miRNAs, miR-192-5p, -215-5p, and -194-5p, for further testing in a discovery cohort and an independent validation cohort. Receiver operating curves were generated to calculate the diagnostic accuracy of these miRNAs for BE diagnosis. To test specificity, the miRNA signature was compared with those of the gastric cardia epithelium and the non-intestinal-type columnar epithelium (another definition of BE). In addition, to gain insights into BE origin (intestinal vs non-intestinal), global BE miRNA profiles were compared with the published miRNA profiles of other columnar epithelia in the gastrointestinal tract, that is, normal stomach and small and large intestine.
Results:
The discovery cohort included 67 white male patients (40 with GERD and 27 with BE). The validation cohort included 28 patients (19 with GERD and 11 with BE). In the discovery cohort, the sensitivity, specificity and area under the curve (AUC) of the three mRNAs for BE diagnosis were 92–100%, 94–95%, and 0.96–0.97, respectively. During validation, the sensitivity and specificity of miRNAs for BE diagnosis were as follows: miR-192-5p, 92% and 94%, AUC 0.94 (0.80–0.99, P=0.0004); miR-215-5p, 100% and 94%, AUC 0.98 (0.84–1, P=0.0004); and miR-194-5p, 91% and 94%, AUC 0.96 (0.80–0.99, P=0.0001), respectively. The tested miRNAs identified all BE patients in both the discovery and the validation cohorts. When compared with non intestinal-type columnar and gastric cardia epithelia, the miRNA signature was specific to the intestinal-type columnar epithelium. Comparisons of BE miRNA sequencing data to published data sets for the normal stomach, small intestine and large intestine confirmed that two of the three miRNAs (miR-215-5p and -194-5p) were specific to the intestinal-type epithelium.
Conclusions:
MicroRNAs are highly accurate for detecting intestinal-type BE epithelia and should be tested further for the non-endoscopic molecular diagnosis of BE.
doi:10.1038/ctg.2014.17
PMCID: PMC4274369  PMID: 25502391
20.  Clinicopathologic characteristics of high expression of Bmi-1 in esophageal adenocarcinoma and squamous cell carcinoma 
BMC Gastroenterology  2012;12:146.
Background
High expression of Bmi-1, a key regulatory component of the polycomb repressive complex-1, has been associated with many solid and hematologic malignancies including esophageal squamous cell carcinoma. However, little is known about the role of Bmi-1 in esophageal adenocarcinoma. The aim of this study is to investigate the amplification and high expression of Bmi-1 and the associated clinicopathologic characteristics in esophageal adenocarcinoma and squamous cell carcinoma.
Methods
The protein expression level of Bmi-1 was detected by immunohistochemistry (IHC) from tissue microarrays (TMA) constructed at the University of Rochester from using tissues accrued between 1997 and 2005. Types of tissues included adenocarcinoma, squamous cell carcinoma and precancerous lesions. Patients’ survival data, demographics, histologic diagnoses and tumor staging data were collected. The intensity (0–3) and percentage of Bmi-1 expression on TMA slides were scored by two pathologists. Genomic DNA from 116 esophageal adenocarcinoma was analyzed for copy number aberrations using Affymetrix SNP 6.0 arrays. Fisher exact tests and Kaplan-Meier methods were used to analyze data.
Results
By IHC, Bmi-1 was focally expressed in the basal layers of almost all esophageal squamous mucosa, which was similar to previous reports in other organs related to stem cells. High Bmi-1 expression significantly increased from squamous epithelium (7%), columnar cell metaplasia (22%), Barrett’s esophagus (22%), to low- (45%) and high-grade dysplasia (43%) and adenocarcinoma (37%). The expression level of Bmi-1 was significantly associated with esophageal adenocarcinoma differentiation. In esophageal adenocarcinoma, Bmi-1 amplification was detected by DNA microarray in a low percentage (3%). However, high Bmi-1 expression did not show an association with overall survival in both esophageal adenocarcinoma and squamous cell carcinoma.
Conclusions
This study demonstrates that high expression Bmi-1 is associated with esophageal adenocarcinoma and precancerous lesions, which implies that Bmi-1 plays an important role in early carcinogenesis in esophageal adenocarcinoma.
doi:10.1186/1471-230X-12-146
PMCID: PMC3544684  PMID: 23078618
Esophageal adenocarcinoma; Bmi-1; Squamous cell carcinoma; Barrett’s esophagus; Dysplasia; High expression; Biomarker; Overall survival
21.  Secondary Chemoprevention of Barrett’s Esophagus With Celecoxib: Results of a Randomized Trial 
Background
Barrett’s esophagus is a premalignant condition that is a risk factor for the development of esophageal adenocarcinoma, a disease whose incidence is rapidly increasing. Because aspirin and other nonsteroidal antiinflammatory drugs, such as celecoxib, may decrease the risk of developing esophageal cancer, we investigated the effect of long-term administration of celecoxib in patients with Barrett’s esophagus with dysplasia.
Methods
Chemoprevention for Barrett’s Esophagus Trial (CBET) is a phase IIb multicenter randomized placebo-controlled trial of celecoxib in patients with Barrett’s esophagus and low- or high-grade dysplasia. Patients were randomly assigned to treatment with 200 mg of celecoxib or placebo, both administered orally twice daily, and then stratified by grade of dysplasia. The primary outcome was the change from baseline to 48 weeks of treatment in the proportion of biopsy samples with dysplasia between the celecoxib and placebo arms. Secondary and tertiary outcomes included evaluation of changes in histology and expression levels of relevant biomarkers. All statistical tests were two-sided.
Results
From April 1, 2000, through June 30, 2003, 222 patients were registered into CBET, and 100 of them with low- or high-grade Barrett’s dysplasia were randomly assigned to treatment (49 to celecoxib and 51 to placebo). After 48 weeks of treatment, no difference was observed in the median change in the proportion of biopsy samples with dysplasia or cancer between treatment groups in either the low-grade (median change with celecoxib = − 0.09, interquartile range [IQR] = − 0.32 to 0.14 and with placebo = − 0.07, IQR = − 0.26 to 0.12; P = .64) or high-grade (median change with celecoxib = 0.12, IQR = − 0.31 to 0.55, and with placebo = 0.02, IQR = − 0.24 to 0.28; P = .88) stratum. No statistically significant differences in total surface area of the Barrett’s esophagus; in prostaglandin levels; in cyclooxygenase-1/2 mRNA levels; or in methylation of tumor suppressor genes p16, adenomatous polyposis coli, and E-cadherin were found with celecoxib compared with placebo.
Conclusions
Administration of 200 mg of celecoxib twice daily for 48 weeks of treatment does not appear to prevent progression of Barrett’s dysplasia to cancer.
doi:10.1093/jnci/djk112
PMCID: PMC3755596  PMID: 17405999
22.  Acid and Bile Salt Induced CDX2 Expression Differs in Squamous Cells from Patients with and without Barrett’s Esophagus 
Gastroenterology  2010;139(1):194-203.e1.
Introduction
It is not clear why only a minority of patients with gastroesophageal reflux disease (GERD) develop Barrett’s esophagus. We hypothesized that differences among individuals in molecular pathways activated when esophageal squamous epithelium is exposed to reflux underlie the development of Barrett’s metaplasia.
Methods
We used esophageal squamous cell lines from patients who had GERD with Barrett’s esophagus (NES-B3T & NES-B10T) and without Barrett’s esophagus (NES-G2T & NES-G4T) to study effects of acid and bile salts on expression of the CDX2 gene. Bay 11-705, Ad5IκBα-SR, and site-directed mutagenesis were used to explore effects of NF-κB inhibition on CDX2 promoter activity; DNA binding of the NF-κB subunits p50 and p65 was assessed by ChIP.
Results
Acid and bile salts increased CDX2 mRNA, protein, and promoter activity in NES-B3T and NES-B10T cells, but not in NES-G2T or NES-G4T cells. Inhibition of NF-κB abolished the increase in CDX2 promoter activity. Increased CDX2 promoter activity was associated with nuclear translocation of p50, which bound to the promoter. We also found CDX2 mRNA in 7 of 10 esophageal squamous biopsy specimens from patients with Barrett’s esophagus, but in only 1 of 10 such specimens from patients who had GERD without Barrett’s esophagus.
Conclusions
Acid and bile salts induce CDX2 mRNA and protein expression in esophageal squamous cells from patients with Barrett’s esophagus, but not from GERD patients without Barrett’s esophagus. We speculate that these differences in acid- and bile salt-induced activation of molecular pathways may underlie the development of Barrett’s metaplasia.
doi:10.1053/j.gastro.2010.03.035
PMCID: PMC2902607  PMID: 20303354
Barrett’s esophagus; gastroesophageal reflux; CDX2; NF-κB
23.  Expression, localization and polymorphisms of the nuclear receptor PXR in Barrett's esophagus and esophageal adenocarcinoma 
BMC Gastroenterology  2011;11:108.
Background
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.
Methods
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.
Results
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).
Conclusions
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.
doi:10.1186/1471-230X-11-108
PMCID: PMC3204292  PMID: 21977915
24.  Barrett’s esophagus: Incidence, etiology, pathophysiology, prevention and treatment 
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.
PMCID: PMC2387291  PMID: 18516262
Barrett’s esophagus; esophageal adenocarcinoma; evidence-based approach; endoscopic surveillance
25.  Dietary Factors and the Risks of Esophageal Adenocarcinoma and Barrett’s Esophagus 
Nutrition research reviews  2010;23(2):230-246.
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.
doi:10.1017/S0954422410000132
PMCID: PMC3062915  PMID: 20624335

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