Office Based Screening Test for Barrett's Esophagus
Phase: NA
Conditions: Barrett Esophagus
Sponsor: Case Comprehensive Cancer Center

Study Title: Office Based Screening Test for Barrett's Esophagus 
ClinicalTrials.gov Identifier: NCT02451124  
Protocol Date: March 22, 2016 

OFFICE BASED SCREENING TEST FOR BARRETT’S ESOPHAGUS 
SPECIFIC AIMS: 
Prospectively collected Surveillance Epidemiology and End Results (SEER) data indicate that the incidence of 
esophageal adenocarcinoma (EAC) has increased more than five-fold in the past three decades.1-4 Over 10,000 cases 
are now diagnosed annually. The prognosis for patients with EAC is poor, with less than 20% of patients surviving 
beyond five years.4,5  
Barrett’s esophagus (BE), a pre-malignant metaplastic condition that is the only known precursor of EAC, is 
diagnosed only when patients undergo Esophagogastroduodenoscopy (aka Upper Endoscopy) (EGD). However, due 
to the high cost of EGD and the lack of a randomized controlled trial supporting its efficacy for preventing EAC 
associated mortality, endoscopy to screen for BE is not routinely recommended.6,7 Thus, less than 5% of EACs are 
diagnosed in individuals with previously detected BE.8 Most BE remains undetected. Clearly, to make an impact on 
the dismal prognosis of EAC, we need to first develop alternative methods for identifying BE that are less expensive 
than EGD and that can be widely accepted and adopted in a population at risk.  
Dr. Sanford Markowitz’s laboratory has discovered aberrant methylation of vimentin gene exon 1 DNA as a 
sensitive biomarker of gastrointestinal neoplasia.19,25 Aberrant methylation of vimentin (mVIM) DNA can be 
detected in DNA extracted from BE in at least 90% of subjects with BE, making BE the most methylated VIM 
(mVIM) linked tissue change in the GI tract.9 Moreover, in work featured on the cover of Cancer Epidemiology, 
Biomarkers, and Prevention, Markowitz and Chak showed that mVIM was easily detected in DNA from esophageal 
brushings of BE patients, suggesting the possibility of a non-endoscopic “molecular cytology” screening approach 
for BE screening and detection. The hypothesis of this proposal is that a sensitive molecular diagnostic test for BE, 
such as mVIM, in combination with a non-endoscopic brushing devices such as the capsule sponge proposed by 
Kadri et al,10 will be a cost-effective method for BE screening. Our supplemental research proposal will support a 
clinical trial led by Drs. Markowitz and Chak to determine whether we can detect mVIM in BE patients using a 
novel non-endoscopic balloon based brushing device designed by Drs. Markowitz, Chak and Willis in collaboration 
with the industrial design group at Nottingham Spirk. The Specific Aims of this proposal are:  
Specific Aim 1. To determine acceptability of non-endoscopic balloon brushing of esophagus. 
Specific Aim 2. To demonstrate successful performance of the mVIM assay in balloon brushings from subjects with 
and without BE.  
Specific Aim 3. To examine molecular and histological factors that might lead to false negative and false positive 
mVIM assays in BE screening.  
Specific Aim 4. To assay additional methylated DNA biomarkers that might improve the sensitivity and specificity of 
mVIM in balloon brushings.  
RESEARCH STRATEGY 
Overview 
To make an impact on the prevention and early detection of EAC the challenge is to first develop an alternative 
method for BE screening that is less costly and safer than EGD, while being more acceptable and accessible in the 
primary care setting. In preparation for a full scale screening study, we will first perform this pilot study at 
University Hospitals Case Medical Center and at the Cleveland Clinic. Together, these institutions see a large adult 
male population that is at increased risk for BE. A non-endoscopic method for detecting BE would allow screening 
in primary care clinics. We have demonstrated that aberrant mVIM can be detected in DNA extracted from 
endoscopic brushings in over 90% of subjects BE.9 We now propose a pilot study to determine whether we can 
detect mVIM non-endoscopically by testing for mVIM in samples obtained from BE patients using a novel 
esophageal sampling device.  

We wish to clarify that this pilot project only addresses the current challenges with BE screening. A non-endoscopic 
esophageal sampling device, such as the one proposed in this project, could potentially help with the surveillance and 
the assessment of BE recurrence after ablative therapies. These future applications will require the development of 
appropriate biomarkers that can be tested in prospective long-term full-scale studies.  
SIGNIFICANCE  
Early detection of BE requires the development of unsedated screening procedures that can be applied to an adult 
population at risk. -- The major limitations of the current guidelines that recommend sedated EGD in patients with 
multiple BE risk factors, refractory Gastroesophageal Reflux Disease (GERD), or alarm symptoms are clearly 
evident. This strategy fails to detect BE in asymptomatic subjects. It also fails to detect BE in patients whose 
symptoms are well controlled with either over the counter medications or physician-prescribed therapies. Thus, less 
than 5% of esophageal adenocarcinomas are diagnosed at an early stage in patients with previously detected BE.8 
Ablative non-surgical therapies that have been developed for BE with high grade dysplasia over the past 
decade11,12 will have little impact and the five year survival for esophageal adenocarcinomas will remain a dismal 
15%13 unless we develop more effective programs for identifying BE in the general population.  
Non-endoscopic detection of BE needs to improve. – Early studies of non-endoscopic balloon sampling of the 
esophagus relied on cytology, but were limited in their ability to obtain adequate material.14 More recently, 
investigators from the UK reported on a device that can non-invasively assess for BE: an expandable Cytosponge in 
a dissolvable capsule that is swallowed. This sponge is tethered to a string, released in the stomach, and then 
retrieved, thus sampling the esophagus with a sensitivity of 73% and specificity of 94% for diagnosing BE.10 This 
sponge method detects Barrett’s epithelial cells by immunochemical staining using antibodies directed against TFF-
3. It is not yet available in the United States. Robust molecular DNA marker(s) that could detect BE in esophageal 
brushings would be more preferable than current cytological techniques, because DNA tests can be automated and 
require far less material.  
INNOVATION  
Aberrant DNA methylation of vimentin (mVIM) is common in BE and Esophageal Adenocarcinoma – Aberrant 
methylation of CpG rich islands in DNA may lead to silencing of tumor suppressor genes and is a common event in 
many cancers. These methylated CpG islands can be identified in DNA extracted from various biospecimens (blood, 
urine, stool, etc.) and are potentially useful biomarkers for tumor detection. Aberrant methylation of the first exon of 
vimentin was initially discovered in Dr. Sanford Markowitz’s laboratory in colon cancers and advanced colonic 
adenomas.15 This soon led to the development of a robust stool DNA mVIM assay that was clinically adapted for 
colon cancer detection.16 Subsequently, we found that mVIM was present in an even higher proportion of epithelial 
upper gastrointestinal tract tumors.9 Specifically, mVIM was detected in 90% of non-dysplastic BE, dysplastic BE, 
and most esophageal adenocarcinomas (Figure 1).  
mVIM can help diagnose BE in esophageal brushings. More recent research 
in the laboratory (unpublished) has concentrated on developing mVIM as a 
biomarker for detecting BE. Brushings were obtained from the distal 
esophagus/gastroesophageal junction and proximal squamous esophagus from 
69 patients with normal esophagus or erosive esophagitis, 38 patients with 
non-dysplastic BE, 8 patients with low grade dysplasia (LGD), 10 with high 
grade dysplasia (HGD), and 63 patients with esophageal or junctional 
adenocarcinoma. mVIM > 1% was detected in DNA extracted from the distal 
esophagus brush in 88% of BE and adenocarcinoma patients and 14% of non-
BE patients. The area under the ROC curve was 0.92, highly comparable to 
the AUC of 0.93 of the Exact panel (Cologuard assay) for colon cancer 
detection that recently won FDA  
Fig. 2. ROC curve 
for methylated 
Vimentin positive 
assays in individuals 
with versus without 
BE and/or EAC. 
versus those without  

approval. Interestingly, mVIM was also detected in the proximal esophagus in 41/113 (36%) 
smokers vs. 11/76 (14%) non-smokers, p = 0.0009, indicating that the presence of mVIM in the 
proximal esophagus is related to smoking. These studies suggest that mVIM can be used to 
detect BE and esophageal/junctional with a high sensitivity, but it will be necessary to use a 
device that samples the gastroesophageal junction and distal esophagus while avoiding 
contamination from the proximal esophagus.  
Other methylation biomarkers are being developed. BETRNet Project 2 led by Dr. William 
Grady assayed genome wide methylation in a variety of BE and control tissues using the 
Illumina HM450 methylation array. Six promising candidate biomarkers were identified and 
validated by pyrosequencing assays. Technical and clinical validation studies were run with two 
of these markers (ZNF793 and B3GAT2), and Methylight assays for mZNF793 and mB3GAT2 
were developed. These assays showed >95% sensitivity and specificity for detecting BE in a 
training set of esophagus cytology samples. Furthermore, B3GAT2 and ZNF793 methylation 
were also detectable in endoscopic brushings from the distal esophagus in four out of five BE 
cases tested.  
Inflatable soft balloons can sample distal esophagus reliably and safely. – The concept of 
sampling the esophagus with an inflatable balloon was proposed nearly 20 years ago when Falk, 
et al. attempted to use a non-endoscopic inflatable balloon device to sample the esophagus.14 
Although the cytological material obtained in the Falk study was not able to identify dysplasia 
sensitively, the bio-material should be sufficiently adequate for assaying a molecular DNA 
marker such as mVIM. Nearly half the 63 patients in the Falk study found the balloon device as 
tolerable as regular EGD and there were no concerns regarding safety. The next step in the 
development of non-endoscopic esophageal sampling devices was the development of the 
Cytosponge in the UK. This tethered sponge inside a capsule has been found to be safe and 
tolerable in over 500 study participants.10 However, this device is still susceptible to 
contamination from the proximal esophagus. Our experiments demonstrate it is important to 
avoid sampling the proximal esophagus because smoking causes methylation of VIM in the 
proximal squamous epithelium.  
Figure 3. Balloon device  
Balloon Device: For the present study, we propose to use an inflatable balloon device that 
incorporates next generation improvements compared to the device used by Falk, et al.14 The 
new modified balloon device has been developed with the assistance of Nottingham Spirk, a 
nationally reputed industrial design firm. The balloon has been made thinner to be even more 
tolerable, and the surface has been modified to add soft bristles to improve cytological yield. In 
addition, the new balloon is also strategically designed to invert when it is deflated. This key 
feature will allow us to sample the distal esophagus tolerably, safely, and reliably while avoiding 
contamination from the proximal esophagus. The blow-molded balloon (Fig. 5) will be 
constructed of a typical biocompatible catheter material, which may be, but is not limited to, 
nylon or polyethylene. The size of the device is such that even if it is inadvertently swallowed it 
will pass safely through the digestive tract. The outer diameter of the delivery and inflation 
tubing will be approximately three mm, increasing to a maximum of approximately eight mm at 

the balloon. The maximum inflated size of the balloon will be 18 mm. The balloon will be 
compliant and the pressure in the balloon will be less than the pressure generated in current 
esophageal sizing balloons that are safely inflated to diameters > 30 mm at pressures less than 
0.25 atm. A pop-off relief valve will prevent over-pressurization of the balloon device during 
inflation. No esophageal perforations have been reported with high compliance low pressure 
esophageal balloon sizing in over 85,000 cases of esophageal ablation. The perforation rate is 
0.01% with ablation balloons that are of size > 18 mm and inflated at higher pressures 
(radiofrequency ablation registry database, barrx.com). On preliminary testing, the size of the 
capsule balloon device appears to be a size that can be swallowed comfortably and safely. The 
balloon and capsule will be made of biocompatible material and of a size where, even if they are 
inadvertently swallowed or break, they will pass safely through the gastrointestinal tract. The 
device will be smaller in diameter and length than the FDA-approved small bowel capsule device 
that is designed to be swallowed and transit through the gastrointestinal tract. The manufacturer 
of the prototypes is ISO 14385 and it is FDA GMP certified.  
 
Fig.4 Diagram of inverted balloon (left) and expanded balloon (right)  
Fig 5. blow-molded balloon in pig esophagus  
APPROACH  
We now propose to combine a non-endoscopic esophageal sampling balloon device with the 
mVIM assay as a screening test to diagnose BE. This pilot study will test this strategy in a group 
of patients undergoing EGD at either University Hospitals Case Medical Center (UHCMC) or at 
the Cleveland Clinic (CCF). This study is being conducted at two institutions to enable the 
necessary recruitment and also to establish the infrastructure for a multi-center screening study. 
We will send de-identified samples to the Fred Hutchinson Cancer Center in Seattle, 
Washington, to be analyzed. If this study confirms that this approach is able to detect BE in at 
least 7 of 10 subjects with known BE, then for the BETRNet renewal we will propose a full scale 
screening study in subjects at risk for BE who have not had prior EGD.  
We will recruit 40 subjects with BE who are undergoing EGD for surveillance and 80 subjects 
without BE who are undergoing EGD for other indications. Eligibility criteria for this study will 
be:  
1. Adult patients at least > =18 years old who are undergoing clinically indicated EGD and can 
provide informed consent  
2. No known coagulopathy, no known history of esophageal varices.  
 
The study coordinator or a study investigator will approach patients scheduled for EGD in the 
endoscopy suites at UHCMC and CCF. At UHCMC, Dr. Amitabh Chak will act as Principal 
Investigator, with Dr. Sanford Markowitz as the Principal Investigator at the CCF and Dr. Joseph 
E. Willis acting as a Co-Investigator. Dr. Prashanti Thota will act as the Co-Investigator at the 

Cleveland Clinic and will be assisted by Mary Oldenburgh, who will act as the RN Coordinator. 
The PI at Fred Hutchinson Cancer Center in Seattle, Washington, where de-identified samples 
will be analyzed, is Dr. William Grady. The study will be explained to the patient, who will then 
have the opportunity to ask questions. All subjects who consent to the research study will have 
esophageal balloon sampling prior to their EGD.  
The technique for esophageal sampling will be similar to that used by Falk, et al.14 The subject 
will be given topical pharyngeal anesthesia with benzocaine spray. The balloon will be 
swallowed and advanced to 50 cm. It will then be inflated fully with 15 cc air. It will be 
withdrawn until a tug is felt at the gastroesophageal junction (GEJ). Once the GEJ is located, 10 
cc of air will be removed and the 5 cc balloon will be pulled back five cm to sample the distal 
esophagus. The balloon will then be completely deflated to cause inversion into its protective 
capsule, and will then be withdrawn. The balloon will be re-inflated outside the patient, and the 
obtained sample will be clipped with scissors into DNA buffer. The collected biospecimen will 
be stored frozen for later DNA extraction and assay of mVIM and other methylation markers. 
The vials will be labeled with a coded sample number (1, 2, 3, etc.). After all samples have been 
accrued, the coded samples will be assayed for mVIM in Dr. Markowitz’s laboratory in Wolstein 
Building, CWRU, and other methylation markers will be assayed (mZNF793 and mB3GAT2) in 
Dr. Grady’s laboratory at the Fred Hutchinson Cancer Research Center.  
All patients will undergo standard EGD. Presence or absence of BE will be noted. All patients 
with suspected BE will have screen/surveillance biopsies to confirm diagnosis as per standard 
practice. These biopsy samples will also be tested for mVIM and other methylated markers. 
Endoscopic brushings will also be obtained from BE in cases and from the normal-appearing 
GEJ in controls.  
Specific Aim 1. To determine acceptability of non-endoscopic balloon brushing of esophagus.  
Subjects who agree to participate will complete a tolerability questionnaire that has been used in 
previous studies.17 This questionnaire evaluates anxiety, pain, gagging, and choking using a 
Likert scale. The questionnaire also determines the subject’s willingness to undergo the 
procedure in the future. All procedure-related complications will be recorded. A complication 
will be defined as an untoward and unplanned event that occurs during, or immediately after, the 
balloon procedure or administration of topical anesthetic. Data will also be collected regarding 
age, gender, race, indication for EGD, and findings on EGD. All patient identifiers will be stored 
by the study coordinator in a secure computer.  
Analysis and Definitions of Tolerance: Descriptive statistics will be calculated for all tolerance 
variables (anxiety, pain, choking, gagging, and overall tolerance). The threshold for tolerability 
will be a score of < 3 on 10 point scale for symptoms elicited on the Tolerance Survey. The 
balloon screening procedure will be considered tolerable if < 15% of subjects who undergo the 
screening procedure score all symptom questions < 3 and at least 80% are willing to undergo the 
procedure again if needed.  
Specific Aim 2. To assay mVIM in balloon brushings from subjects with and without BE.  

DNA from esophageal samples. Following esophageal sampling, balloons will be clipped into 
nuclease free 1.5ml cryo-safe tubes with buffer and immediately frozen. The tubes will be 
labeled with codes that are de-identified to the laboratory. DNA will be purified using QIAamp 
DNA micro kit (QIAGEN) according to the immediately for bisulfite conversion, or frozen at               
-80°C until use.  
Bisulfite conversion of the genomic DNA and real-time MS-PCR assay. To create a template 
for methylation-specific PCR, DNA samples will be subjected to bisulfite conversion and 
purified using an Epitect kit (QIAGEN), according to the manufacturer’s protocol. 1-100ng of 
bisulfite-converted DNA at a concentration of 0.2-the real-time MS-PCR assay. To normalize 
input DNA amounts, a companion real-time PCR assay will be designed against bisulfite 
converted Actin gene sequences that lack CpG dinucleotides and so are not affected by DNA 
methylation. The assay for Actin was designed to generate an amplification product of the same 
size as the assay for methylated VIM. For both ACTIN and VIM real-time PCR, a mixture of 
DNA from four colon cancer cell lines that are each fully methylated across the VIM CpG island 
will be used to generate a dilution standard curve that will be run with all real-time assays and 
used as part of data analysis in BioRad CFX manager software to convert the Ct values into ng 
DNA amounts. VIM methylation will be calculated as a percentage ratio of the amount of 
methylated DNA measured by VIM qPCR, divided by total bisulfite-converted DNA amount in 
the sample, as measured by Actin qPCR. The real-time MS-volume uses Light Cycler PCR 
master mix (Roche) with 400nM each primer and 200nM probe.9 Amplifications will be done in 
96-well plates in CFX96 Real-Time System (BioRad) under the following conditions: 95°C for 
10 min, followed by 50 cycles of 30sec at 95°C, and 60 sec at primer-specific 
annealing/extension temperature.  
Sample Size and Analysis: Descriptive statistics will be generated for demographic variables and 
other variables of interest. For this pilot study, we have selected to assay 40 subjects with BE and 
80 subjects without BE. We will compare patient endoscopic and pathologic findings relating to 
the diagnosis of BE with mVIM analyses of corresponding esophageal balloon derived samples. 
This is a sufficient sample size to give us a measure of tolerance and to also determine if mVIM 
is able to detect BE in at least 70% of subjects with known BE. The presence of at least 1% of 
methylated VIM in DNA extracted from the esophageal sampling balloon will be considered a 
positive assay. The sensitivity and specificity of the balloon mVIM assay for detecting BE will 
be calculated. Balloon device mVIM assays will be correlated with endoscopic brushing mVIM 
assays by calculating a Pearson’s correlation coefficient. This method will not be considered 
adequate if it is not tolerable or if it is not able to detect mVIM in at least 80% of subjects with 
BE with 90% specificity. The balloon device will be considered acceptable if the correlation 
coefficient with endoscopic brushings is at least 0.9.  
Specific Aim 3. To examine molecular and histological factors that might lead to false negative 
and false positive VIM assays in BE screening.  
In our preliminary study of DNA extracted from esophageal brushings obtained during 
endoscopy, mVIM had an 88% sensitivity and 86% specificity. To try and understand potential 
factors that lead to false negative mVIM tests in 12% of subjects with BE or EAC and false 

positive mVIM tests in 14% of subjects without BE, we will perform molecular and histological 
studies.  
We have formalin fixed paraffin embedded (FFPE) and snap frozen tissue available from 6 BE 
and 8 HGD/EAC cases that were mVIM negative on their esophageal brushings. We will select 
an additional 6 BE and 8 HGD/EAC samples that were mVIM positive as controls. Dr. Joseph E. 
Willis, an expert gastrointestinal pathologist at CWRU, will blindly grade the FFPE histology of 
these 14 mVIM brushing negative and 14 mVIM brushing positive samples for the presence of 
goblet cells (percent of specialized intestinal metaplasia – 0- <25%, 25 - <50%, 50 - < 75%, 75 – 
100%), percentage cardia type mucosa, intestinalization (cancer cases), and cancer grade (cancer 
cases only). Since the sample size is small, this will be a descriptive “hypothesis generating” 
analysis, where we will explore any differences in histo-morphology between BE and EAC that 
are mVIM negative and mVIM positive on brushings.  
To confirm that the mVIM negative brushings are indeed associated with low levels of VIM 
methylation, we will also extract DNA from the mVIM brushing negative BE and EAC tissues. 
mVIM will be assayed in the tissues and levels of VIM methylation in the biopsy tissue will be 
correlated with levels of VIM methylation in the DNA from esophageal brushings. A finding that 
low levels of mVIM in brushings are correlated with low levels of mVIM in biopsy material will 
indicate the need for additional methylation markers (see Aim 4) to improve the sensitivity of 
methylated markers for BE screening. A finding that low mVIM esophageal brushings can occur 
in the presence of high levels of mVIM methylation in tissues will reinforce the need for 
minimized contamination when performing balloon sampling.  
The esophageal brushing study we performed found mVIM > 1% in 10 of 69 subjects who had 
no evident BE. Three of these ten false positive cases occurred in the presence of high mVIM 
from the proximal esophagus in smokers. Our sampling device has been designed to invert the 
balloon into a protective hood to minimize proximal esophageal contamination. Other 
explanations for positive mVIM at the GE junction might be the presence of intestinal metaplasia 
in a normal appearing cardia or the presence of VIM methylation in gastric metaplasia of the 
esophagus.  
Indeed, the very definition of BE is debated. Glandular metaplasia of the distal esophagus is the 
primary etiology of most esophageal adenocarcinomas. In the US and as used in this proposal, 
the working definition of BE includes the presence of intestinal metaplasia (IM) occurring in 
variable proportion of gastric-type columnar mucosa in the distal esophagus. In the United 
Kingdom, glandular metaplasia of the esophagus linked to increased risk of EAC, i.e. BE, is 
defined solely by the presence of gastric-type columnar mucosa in the esophagus regardless of 
the presence or absence of IM. Previous studies have identified molecular features of intestinal 
differentiation in gastric-type columnar mucosa adjacent to IM.18 Another important glandular 
abnormality is IM of the cardia the presumed precursor lesion of gastroesophageal junctional 
adenocarcinoma. We propose to identify the presence or absence of mVIM in each of these 
glandular abnormalities using our extensive biorepository of clinically acquired formalin fixed 
paraffin embedded [FFPE] biopsies. We have previously demonstrated our ability to analyze for 
mVIM on FFPE specimens.9  

Dr. Willis will obtain 20 de-identified FFPE blocks from distal esophageal samples with only 
glandular metaplasia without IM; 20 samples of glandular metaplasia with extensive IM [defined 
as greater than 80% of cells are IM]; 20 samples of glandular metaplasia with small amounts of 
IM [defined as less than 10% of cells are IM]; 20 samples from the cardia without IM; and 20 
samples from the cardia with IM from the CWRU GI Cancer Tissue Archive. Histology will be 
reviewed to confirm the diagnosis. DNA will be extracted from FFPE tissues and assayed for 
level of mVIM as described above.  
We will determine the proportion of samples that show mVIM > 1% for each defined histology. 
These results will especially be useful in determining whether it will be necessary to obtain a 
biopsy from a normal appearing gastroesophageal junction when we design our full scale BE 
screening study.  
Specific Aim 4. To assay additional methylated DNA biomarkers that might improve the 
sensitivity and specificity of VIM in balloon brushings.  
Studies by Dr. Grady’s laboratory have already validated mB3GAT2 and mZNF793 as 
methylation markers for BE screening and found that these markers are also detectable in 
esophageal brushings. We will assay the methylation of these and any other screening markers 
that validate through our BETRNet pipeline (Drs. Markowitz’ and Grady’s laboratories) using 
DNA samples from the 40 BE and 80 non-BE cases that undergo balloon sampling in Aim 1.  
Analysis: Since mVIM is a robust biomarker with an AUC of 0.92 that has already been tested 
clinically, we will first determine if any of the new biomarkers increase the sensitivity and 
specificity for BE screening when combined with mVIM. We will then perform an independent 
analysis, where we will model an independent panel of biomarkers for BE screening. Using 
Receiver Operating Characteristics (ROC) analysis, we will rank biomarkers based on the 
highest Area Under the Curve (AUC) values. This type of analysis provides a continuum of 
sensitivity and specificity across different threshold settings for the biomarker. Rankings based 
on AUC values will be established for ability to discriminate BE from normal esophagus. 
Thirdly, a rank list will determined with respect to the overall prevalence of significant 
methylation in primary BE cases, and the absence of DNA methylation in normal cases. The 
rank lists will be cross-referenced to generate a panel of the most promising biomarkers, with an 
emphasis on those that have strong predictive ability (i.e. no detectable methylation in normal 
cases). We will select the panel with the highest sensitivity and specificity for further assessment 
in a full scale screening trial.  
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