Minimally-Invasive Detection of Barrett's Esophagus and Barrett's Esophagus Related Dysplasia/Carcinoma by a Sponge on String Device
Phase: NA
Conditions: Barrett Esophagus
Sponsor: Mayo Clinic

 
 
Minimally-invasive Detection of Barrett’s 
Esophagus and Barrett’s Esophagus Related 
Dysplasia/Carcinoma by a Sponge on String 
Device 
 
NCT02560623 
 
April 29, 2021 

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4/29/2021                                                                                         Protocol version 21 
 
Minimally-invasive Detection of Barrett’s Esophagus and Barrett’s Esophagus Related 
Dysplasia/Carcinoma by a Sponge on String Device  IRB 15-004540 
PI : Prasad G. Iyer MD MSc,  
Co-Investigators: David Ahlquist MD, David A Katzka MD, Herbert Wolfsen, MD, Cadman 
Leggett, MD, Eduardo Antpack, MD, Francisco Ramirez, MD, Allon Kahn, MD, John Kisiel, MD, 
Arvind Trindade, MD, Vani Konda, MD,  Grace Dosanjh, MD 
Michele Johnson, Ramona Lansing, Rachel Rhody, Erin Gibbons, Frances Cayer, Karen 
Doering, Seth Slettedahl, Douglas Mahoney, Kelli Burger, Mary Devens, William Taylor, Pooja 
Bhakta, Molly Stewart, Daniel Kim, Kristen Garcia, Amanda O’connor 
Background 
Barrett’s esophagus (BE) is the strongest risk factor for and only known precursor for 
esophageal adenocarcinoma (EAC), a lethal malignancy with poor survival (<20% at 5 years) 
when detected after the onset of symptoms. (1) The incidence of esophageal adenocarcinoma 
has increased by almost 600% in the last three decades in the population.(2) BE progresses to 
EAC through a step-wise pathway from no dysplasia, to low grade dysplasia (LGD) to high 
grade dysplasia (HGD) to carcinoma. 
This metaplasia to dysplasia to carcinoma sequence has prompted several national 
gastroenterology societies to recommend screening for BE in high risk subjects with multiple 
risk factors followed by endoscopic surveillance (depending on the grade of dysplasia) to detect 
the development of dysplasia or carcinoma at an early stage.(3-5) Endoscopic treatments of 
LGD, HGD and early carcinoma have been developed and shown to be effective in reducing the 
incidence of carcinoma and improving survival in BE subjects.(6-9) 
Screening for BE is currently performed using conventional sedated endoscopy (sEGD) which 
reveals the replacement of the normal squamous lining of the esophagus by metaplastic 
columnar epithelium in subjects with BE. However sedated endoscopy is expensive with both 
direct and indirect costs and not suitable for widespread application. It is also associated with 
potential complications.(10) Other techniques such as unsedated transnasal endoscopy (uTNE) 
have comparable accuracy to sEGD with lower cost, but continue to be poorly regarded as a 
widely applicable tool by providers.(11-13) Despite adequate access to the uTNE device the 
utilization of uTNE by referring physicians remains limited.(14) The absence of accurate risk 
stratification tools to determine BE risk and target screening efforts are additional limitations to a 
widely applicable BE screening.(10) 
Endoscopic detection of dysplasia is currently performed using four quadrant random biopsies 
every 1-2 cm of the BE segment in addition to careful inspection of the BE segment with high 
resolution white light imaging and advanced imaging techniques. While this has been 

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recommended by GI societies (3-5), the compliance with these recommendations amongst 
practicing gastroenterologists remains poor.(15) Indeed compliance decreases with increasing 
BE segment length leading to increasing rates of missed dysplasia. Other challenges with 
dysplasia detection in BE include the spotty distribution of dysplasia in BE(16) which leads to 
sampling error, poor inter-observer agreement amongst pathologists while grading dysplasia 
and the relatively poor sensitivity of current surveillance strategies in detecting prevalent 
dysplasia or carcinoma.(17) The utility of advanced imaging techniques in the community 
remains unclear with only a third of practicing gastroenterologists reporting use routinely in BE 
surveillance.(18) 
Recently a sponge on a string device has been studied in BE screening.(19) This device 
consists of a polyurethane foam sponge compressed in a vegetable material derived capsule, 
attached to a string. The capsule is swallowed by the patient. The shell of the capsule dissolves 
in the gastric fluid releasing the foam device as a sphere which is then pulled out with the 
attached string, providing brushing/cytology samples of the proximal stomach and esophagus. 
Biomarker studies can then be performed on these samples to detect BE. Two large multicenter 
studies have been performed in the United Kingdom with such a device using trefoil factor 3 (a 
protein specific to BE epithelium) detected on immunohistochemistry as a BE marker, 
demonstrating the feasibility, safety and accuracy of this approach. (19, 20) The sensitivity and 
specificity of this marker in the detection of BE has been reported to be 73% and 94% for BE 
segments of > 1 cm in circumferential length. Additionally this capsule sponge device has been 
used safely in a study conducted at Mayo Clinic Rochester in subjects with eosinophilic 
esophagitis.(21) 
Methylated DNA markers specific to BE epithelium (with and without dysplasia) have been 
described by us and others.(22)  We have identified several methylated DNA markers which are 
highly specific and sensitive for the detection of BE and BE related dysplasia/carcinoma (see 
preliminary data section). 
An investigational capsule sponge device (SOS: EsophaCap) is currently available in the United 
States in two sizes (25 mm and 30 mm size) and two porosity configurations (10 pores per inch 
[ppi] and 20 pores per inch). There is currently no data on the optimal size and pore 
configuration in terms of DNA yield, patient tolerability and safety on this device. 
The aims of our Phase 1 pilot study are: 
1. Compare patient acceptance and tolerability with the 25 mm 10 ppi and 25 mm 20 ppi 
configurations capsule sponge devices. 
2. Compare presence and degree of mucosal abrasions following swallowing and 
withdrawal of capsule sponge, as determined on endoscopy with the 25 mm 10 ppi and 
25 mm 20 ppi configurations) capsule sponge devices 
3. Compare DNA yield obtained from esophageal cytology specimens obtained with the 25 
mm 10 ppi and 25 mm 20 ppi and capsule sponge devices. 
4. Explore sensitivity and specificity of DNA methylation markers in detecting BE and BE 
related dysplasia. 

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Phase 2 Aims: 
Following successful completion of Phase 1, we will proceed with Phase 2 of the study. The 
aims of this phase will be: 
1. Assess the sensitivity and specificity of a panel of DNA methylation markers in the non-
endoscopic detection of BE using a capsule sponge device. 
2. Assess the sensitivity and specificity of a panel of DNA methylation markers in the non-
endoscopic detection of BE related dysplasia/carcinoma using a capsule sponge device. 
3. Estimate the degree of missed dysplasia during routine surveillance endoscopy with 
random endoscopic biopsies, by utilizing follow up intensive endoscopic surveillance and 
capsule sponge examination.   
4. Explore the utility of demographic, anthropometric and circulating biomarker risk factors 
in predicting the presence of BE and BE related dysplasia. 
The inclusion and exclusion criteria, study procedures and biomarker assays will be identical to 
those described in Phase 1. All subjects will undergo the capsule sponge assessment followed 
by the clinically indicated endoscopy as described in Phase 1. All studies will be conducted 
using a single size sponge with the same porosity configuration selected from Phase 1. Study 
procedures, testing and follow up will be similar to that in Phase 1.  Recruitment will be 
monitored and a future interim analysis may be conducted. 
In addition to Mayo Clinic Rochester, subjects will also be recruited at Mayo Clinic Jacksonville; 
Mayo Clinic Arizona; Mayo Clinic Health Systems – Austin; Mayo Clinic Health Systems – 
Mankato; Northwell Health in Manhasset, NY; and Baylor University Medical Center in Dallas, 
TX.  Enrollment is closed at University of Colorado, Denver – Anschutz Medical Campus in 
Aurora, CO. 
Dr. Herbert Wolfsen will be site PI at Mayo Clinic Jacksonville.  Dr. Francisco Ramirez will be 
site PI at Mayo Clinic Arizona.  Dr. Eduardo Antpack, MD will be the site PI at Mayo Clinic 
Health Systems – Austin.  Dr. Grace Dosanjh will be the site PI at Mayo Clinic Health Systems – 
Mankato. Dr. Arvind Trindade will be site PI at Northwell Health.  Dr. Vani Konda will be the site 
PI at Baylor University Medical Center.   
 
Preliminary Data: 
• 
Identification and initial feasibility testing of methylated DNA markers for BE diagnosis. 
We aimed to prospectively assess the accuracy of methylated BMP3 and NDRG4 to identify BE 
using endoscopic biopsies (Phase 1) and brushings from the whole esophagus and cardia to 
simulate non-endoscopic sampling devices (Phase 2).  
Methods: Cases with and controls without BE were recruited prior to endoscopy. BE cases had 
>1cm of circumferential columnar mucosa with confirmed intestinal metaplasia; controls had no 
BE endoscopically. In Phase 1, biopsies were obtained in cases from BE, gastric cardia ((GC); 1 

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cm below Z-line) and squamous epithelium ((SE); >2 cm above BE) and in controls from GC (as 
for BE) and SE (5 cm above Z-line); then promptly frozen. Biopsy samples were processed as a 
batch, and assayed in blinded fashion. In Phase 2, specimens were obtained using a high capacity 
endoscopic cytology brush (Hobbs Medical, Stafford Springs CT); the cardia, BE (in cases), and 
full esophageal length were brushed to simulate a swallowed sponge sampling device. Following 
DNA extraction and bisulfite treatment, methylation on target genes was assayed by quantitative 
allele-specific real-time target and signal amplification. β-actin was also quantified as a marker for 
total human DNA.  
Results: We prospectively studied 100 subjects. Phase 1: Among 40 BE cases and 40 controls: 
median age was 65 (quartiles 55-77) and 54 (37-69) and men comprised 78% and 48%, 
respectively. Median BE length was 6 cm (range 3-10). Median levels of methylated markers were 
substantially higher (34-600 times) in BE than in adjacent SE and GC or than in normal SE and 
GC (Table 1). In contrast to methylated markers, β-actin distributions were similar across tissue 
groups.  Both marker levels increased with BE length and age, p<0.001 whereas only NDRG4 
increased significantly with presence of dysplasia (none (19), low grade (9), high grade (11); 
p=0.003). Factors not significantly affecting marker levels included sex and inflammation. Phase 
2: Among 10 BE cases and 10 controls, median age was 64 (59-70) and 66 (49, 71) and men 
comprised 80 and 30% respectively. Median BE length was 2 cm (range 1-4). Discrimination of 
BE by markers was extraordinary with AUC of 1.0 for NDRG4 and 0.99 for BMP3; levels were 
>100 times higher in cases than controls (Figure 1).  
Conclusions: Selected methylated DNA markers highly discriminate BE from normal GC and SE, 
both in biopsy and brushed specimens, and hold promise for non-endoscopic screening 
applications.  
Table 1: Marker levels (copy numbers of markers adjusted for beta actin) for BMP3 and NDRG4 
biopsies from BE cases (cardia, Barrett’s, squamous) and controls (cardia, squamous). 
 
BMP3 
NDRG4 
Normal 
controls 
Barrett’s 
cases 
Normal 
controls 
Barrett’s 
cases 
Squamous 
Q1, Q3 
P90, P95 
0.8 
0.3, 2.2 
7.0, 23.0 
 
5.6 
0.7, 14.8 
25.5, 50.3 
 
1.0 
0.5, 2.7 
5.0, 13.7 
 
4.9 
1.5, 10.9 
32.0, 64.1 
 
BE  
Q1, Q3 
P90, P95 
 
300.2 
137.1, 659.5 
1083.1, 1219.0  
 
390.6 
146.6, 763.5 
921.8, 1006.6 
 
Cardia  
Q1, Q3 
P90, P95 
0.5 
0.3, 1.9 
10.3, 16.4 
 
8.2 
2.8, 40.3 
190.7, 431.5 
 
2.3 
1.0, 6.3 
13.1, 15.4 
 
11.5 
5.0, 48.3 
116.7, 345.0 
 
Composite  
Q1, Q3 
P90, P95 
1.3 
0.4, 3.8 
10.0, 15.3 
 
131.4 
67.1, 242.7 
402.9, 417.9 
 
2.3 
1.1, 5.3 
8.1, 12.5 
 
136.5 
68.9, 272.3 
344.0, 383.3 
 

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Pvalue 
<0.0001 
<0.0001 
 
Figure 1:  Marker levels of BMP3 and NDRG4 in brushings (cardia + whole esophagus) in BE 
cases and controls. 
 
• 
Detection of Barrett’s Dysplasia by Assay of Methylated DNA Markers on Whole 
Esophageal Brushings: A Prospective Feasibility Study 
Molecular markers may aid in detection of Barrett’s esophagus (BE) and surveillance of BE-
related dysplasia by either endoscopic or non-endoscopic methods. Assay of methylated DNA 
markers accurately detects BE on whole esophageal brushings (Gastroenterology 
2014;146:S148), but it is less clear whether  later methylation events occur and can be targeted 
to discriminate emergent dysplasia. We aimed to (1) identify and validate novel methylated DNA 
markers for BE dysplasia, (2) test the feasibility of candidate markers for detection of BE 
dysplasia from whole-esophageal brushings.  
Methods: Discovery & Validation. Using whole methylome bisulfite sequencing on DNA from BE 
tissues with no dysplasia, low grade dysplasia (LGD), high grade dysplasia (HGD) or 
adenocarcinoma (EAC) (18 specimens per group), we identified candidate markers to separate 
BE with from BE without dysplasia. The top 63 candidate markers were validated by methylation-
specific PCR assay in independent tissues including BE without dysplasia, BE- LGD, and BE-
HGD (30-36 specimens per group). The best 12 validated markers were selected for blinded 
analyses in the subsequent clinical feasibility study. Feasibility Testing on Esophagus Brushings: 
Consenting BE subjects scheduled for endoscopic BE surveillance or endoscopic assessment of 
BE related cancers underwent whole esophageal brushings using a high capacity cytology brush 

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(Hobbs Medical, Stafford Springs, CT) with circumferential sampling from the cardia through the 
full esophageal length (BE + squamous mucosa) to simulate a swallowed sponge-on-string 
device. Following DNA extraction and bisulfite treatment, methylation on target genes was 
assayed by methylation-specific PCR or quantitative allele-specific real-time target and signal 
amplification. Marker levels were normalized to β-actin (marker for total human DNA).  
Results: Discovery & Validation Study. The 12 aberrantly methylated genes that best 
discriminated BE with from BE without dysplasia (e.g. areas under ROC curve 0.86-0.97) were 
selected for feasibility testing, including DIO3, MAX20.218, CD1D, T-SPYL5, ZNF568, ST8SIA1, 
ELMO1, ELOVL2, BMP3, NDRG4, HUNK, and CDKN2A;. Feasibility Study on Brushings. 39 
subjects were studied with a median age was 69 (28-94) years, 74% were males, and median BE 
length was 4 (1-14) cm; 18 had no dysplasia and 21 had dysplasia (9 LGD, 7 HGD, and 5 EAC 
(4 asymptomatic early stage). A 3 marker set (DIO3, MAX20.218, NDRG4) at 95% specificity 
detected 78% of LGD, 71% of HGD, 100% of EAC and 81% of all dysplasia (Figure 2). This study 
demonstrates that selected methylated DNA markers discriminate BE without from BE with 
dysplasia.  
Conclusions: Combinations of such markers appear feasible for detection of BE dysplasia when 
assayed from brushed samples of the whole esophagus. Test optimization and further clinical 
studies are warranted. 
 
 
Figure 2 : Positivity rates of a 3-marker panel (DIO3, MAX20.218, NDRG4)  in tissue DNA 
from BE subgroups without dysplasia and with different severities of dysplasia.  
 
Methods: 
Study Design. 

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We will conduct this study in two phases: Phase 1 and Phase 2. Phase 1 will consist of the 
currently submitted study wherein the most optimal sponge on string (SOS) design will be 
chosen based on patient acceptance, tolerability, mucosal irritation and DNA yield, using a 
randomized factorial design pilot trial. Patients will first undergo the SOS test followed by clinical 
endoscopy. Following completion of the trial, outcomes (specified above) will be assessed and 
the most optimal capsule sponge (SOS) configuration will be chosen for a larger Phase 2.  
Phase 1 (pilot) and Phase 2:  
Participants: Patients with and without known BE will be recruited from the Esophageal, 
Barrett’s esophagus and general gastroenterology clinics in Mayo Clinic, Rochester (Phase I 
and 2), Mayo Clinic Health Systems – Austin, Mayo Clinic Health Systems – Mankato, Mayo 
Clinic Florida, Mayo Clinic Arizona, Northwell Health, and Baylor University Medical Center,. 
(Phase 2 only). Patients with known BE will be recruited to enrich the sample with BE subjects 
and those with BE related dysplasia/carcinoma.  Potential cases declining to participate in the 
capsule sponge procedure or those not eligible due to exclusion criteria b.3. below will have the 
option to participate in the esophageal brushings portion of the study.   
Inclusion criteria: 
a. Subjects with known BE. 
1. Patient between the ages 18 – 90. 
2. Patients with a BE segment ≥ 1cm in maximal extent endoscopically. 
3. Histology showing evidence of intestinal metaplasia with or without presence of 
dysplasia. 
4. Undergoing clinically indicated endoscopy. 
b. Subjects without known evidence of BE 
1. Undergoing clinically indicated diagnostic endoscopy. 
Exclusion criteria: 
a. Subjects with known BE. 
1. Patients with prior history of ablation (photodynamic therapy, radiofrequency 
ablation, cryotherapy, argon plasma coagulation). Patients with history of endoscopic 
mucosal resection alone will not be excluded. 
2. Patients with history of esophageal resection for esophageal carcinoma.   
b. Subjects with or without known evidence of BE (on history or review of medical records). 
1. Pregnant or lactating females. 
2. Patients who are unable to consent. 
3. Patients with current history of uninvestigated dysphagia (this does not apply to the 
brushings only portion of the study). 
4. History of eosinophilic esophagitis, achalasia. 
5. Patients on oral anticoagulation including Coumadin, Warfarin. 

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6. Patients on antiplatelet agents including Clopidogrel, unless discontinued for at least 
three days prior to the sponge procedure. 
7. Patients on oral thrombin inhibitors including Dabigatran and oral factor X a inhibitors 
such as rivaroxaban, apixaban and edoxaban, unless discontinued for at least three 
days prior to the sponge procedure. 
8. Patients with history of known varices or cirrhosis. 
9. Patients with history of esophageal resection for esophageal carcinoma.   
10. Patients with congenital or acquired bleeding diatheses. 
11. Patients with a history of esophageal squamous dysplasia. 
12. Patient has known carcinoma of the foregut (pancreatic, bile duct, ampullary, 
stomach, or duodenum) within 5 years prior to study enrollment. 
 
Study procedures: 
Participants will report fasting for ≥ 6 hours for the study. 
A. Pre-procedure assessment:  
a. Baseline height, weight, hip, and waist circumference will be measured by the 
research coordinator. 
b. A validated questionnaire assessing reflux 
symptoms (RSQ) will be completed by the 
subject. 
c. At the Mayo Clinic sites only, 10 ml of blood 
will be obtained via venipuncture for 
measurement of circulating biomarkers 
(cytokines, adipokines and methylated DNA 
markers) of BE risk. Blood will be 
processed and separated into serum, 
plasma, and buffy coat for subsequent 
analysis for BE biomarkers.  
d. Pregnancy test will be obtained for women of child bearing potential. 
 
B. Capsule sponge assessment: 
For phase 1 subjects will be randomized using 
concealed allocation to one of two capsule 
sponges: 25 mm 10 ppi, 25 mm 20 ppi. All phase 2 
subjects will receive the same single size and 
porosity sponge (porosity to be determined after 
analysis of Phase 1). 
a. Participants will be seated upright and 
asked to swallow the capsule sponge to 
which they have been randomized to, with 
sips of water. The pharynx will then be anesthetized with Benzocaine 20% aerosol 
Figure 1 : 25 mm sponge, intact and expanded 
Figure 2 : 30 mm sponge, intact and expanded 

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spray (Topex). After an interval of 5-10 min, the capsule sponge will be pulled out 
using steady traction using the string attached to the capsule sponge. As detailed in 
another Mayo IRB approved protocol (IRB 11-006429). The sponge will then be 
placed in a preservative solution and transferred to the laboratory for storage and 
subsequent analysis. 
b. Following completion of the capsule sponge study participants will fill out a tolerability 
questionnaire which allows patients to grade of discomfort during the procedure on a 
Likert scale of 0-10. 10 representing the “worst experience” and 0 the “best 
experience.” This scale allows a comprehensive and individual assessment of the 
degree of pain, choking, gagging, and anxiety experienced during the procedure. 
Participants will also be asked if they would have the procedure again (yes/no). 
 
C. Endoscopic assessment: 
a. All participants will undergo a diagnostic clinically indicated sedated endoscopy within 
24 hours of the Capsule sponge test. This will be performed in one of the clinical 
endoscopy units at Mayo Clinic Rochester, Mayo Clinic Florida, Mayo Clinic Arizona,  
Mayo Clinic Health Systems – Austin, Mayo Clinic Health Systems - Mankato, 
Northwell Health, and Baylor University Medical Center. 
b. Patients will receive sedation (using a combination of intravenous midazolam and 
Demerol) or monitored anesthesia care as clinically appropriate and undergo clinically 
indicated endoscopy with standard endoscopic equipment.  
i. The following landmarks will be identified, recorded (in centimeters from the incisors) 
and photographed: squamo-columnar junction, gastroesophageal junction (GEJ)  
(defined as top of the gastric folds with the esophagus partially deflated)  and the 
diaphragmatic hiatus. Endoscopically suspected BE will be defined as the presence 
of ≥ 1cm of columnar mucosa above the GEJ. Targeted clinical biopsies will be 
obtained to confirm a diagnosis of BE. Presence of intestinal metaplasia with goblet 
cells will be a pre-requisite for the diagnosis of BE (histologically confirmed BE).  
ii. The presence or absence of esophagitis will be recorded. If present, esophagitis will 
be graded as per the LA classification. 
iii. The presence of any trauma from the passage of the sponge will be assessed and 
recorded photographically. This will be defined as: 
1. No evidence of trauma  
2. Superficial mucosal abrasion without bleeding 
3. Superficial mucosal tear abrasion with minimal oozing similar to that from biopsy. 
4. Deep mucosal abrasion without bleeding. 
5. Deep mucosal abrasion with greater than minimal oozing. 
6. Bleeding requiring endoscopic therapy. 
iv. In those with known BE, clinical surveillance biopsies (with or without endoscopic 
mucosal resection if clinically indicated for visible lesions) will be obtained as per 
current clinical recommendations: four quadrant biopsies every 1-2 cm. All clinical 
histology will be read by GI pathologists as per standard practice at Mayo Clinic 
Rochester, Mayo Clinic Florida, Mayo Clinic Arizona, or Mayo Clinic Health Systems.  
Slides or scanned slides from Northwell Health and Baylor University Medical Center 

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will be sent to Mayo Clinic Rochester GI study Pathologist to confirm Barrett’s 
Esophagus and dysplasia grade. 
v. Endoscopic brushings will be obtained from the Barrett’s mucosa in BE cases, and 
from the squamous mucosa in control subjects. Brushings will be obtained using a 
high capacity cytology brush (Hobbs Medical) to enable detection of methylation 
biomarkers of dysplasia.  1 brush will be used for every 5 cm of Barrett’s epithelium in 
BE case subjects.  In control subjects, 1 brush will be used to sample the cardia and 
the distal 5cm of the esophageal squamous mucosa. These brushings will be stored 
at -80oC for identification of additional biomarkers. 
vi. Two research biopsies will be obtained from the gastroesophageal junction in controls 
for identification of methylated DNA biomarkers from the GEJ.  One will be frozen and 
stored at -80oC and one will be formalin fixed, paraffin embedded. 
vii. The diagnostic part of the procedure will be video recorded and stored in a de-
identified fashion for subsequent review. 
viii. Patients who are biomarker positive (for dysplasia related DNA methylation markers) 
but negative for dysplasia, as well as those biomarker negative but positive for 
dysplasia on clinical surveillance histology will be offered a research endoscopy 
(funded by the study) after biomarker analysis is complete, for additional endoscopic 
surveillance to detect prevalent dysplasia which may have been missed at the initial 
study endoscopy as well as assess for sensitivity of the marker panel.  In addition to 
surveillance biopsies (paid for by the study), sponge procedure will be repeated and 
research esophageal brushings using a high capacity endoscopic brush (Hobbs 
Medical) will be obtained for detection of methylated biomarkers of dysplasia.   
ix. An equal number of biomarker negative (for dysplasia related DNA methylation 
markers) who are negative for dysplasia on clinical surveillance histology will also be 
offered a research endoscopy (funded by the study) In addition to surveillance 
biopsies (paid for by the study), sponge procedure will be repeated, and research 
esophageal brushings using a high capacity endoscopic brush (Hobbs Medical) will 
be obtained for detection of methylated DNA biomarkers of dysplasia.  
x. If the visit 2 research procedure coincides with a participants clinically indicated upper 
EGD and procedures that are ordered cannot be done on the Clinical Research Unit, 
(i.e. radiofrequency ablation, endoscopic mucosal resection, esophageal dilation, 
endoscopic ultrasound) the research portion of the procedure will be done during the 
clinically indicated upper endoscopy.  These research procedures will include the 
sponge on a string procedure, esophageal brushings, and surveillance biopsies (all 
paid for by research) as stated above.  
D. Post procedure assessment. 
a. Patients will be called by the research coordinator at 7 days following the study 
(capsule sponge and endoscopy) to assess for any complications or adverse 
effects including bleeding, pain, dysphagia, need for seeking medical care and 
loss of work.  
 
Assessment of Safety: 

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The Principal Investigator will monitor all adverse events and serious adverse events and they 
will be reported in IRB continuing reviews or immediately as appropriate.   All complications and 
the endoscopy complication score will be reviewed every month by the PIs and continuation of 
the study will be assessed on this basis. 
Biomarker assays:  
a. Methylated DNA marker assessment:  
Following histologic confirmation, tissue DNA will be extracted in usual fashion and bisulfite-
treated.  Methylation on target genes shown in our previous study to have >150-fold higher 
tissue levels in Barrett’s Esophagus compared to esophageal squamous mucosa (BMP3, 
NDGR4, TFPI2, and HPP1) will then be quantified in blinded fashion by methylation-specific 
PCR.  Novel markers for BE-related dysplasia and cancer identified by whole methylome 
sequencing will be quantified in blinded fashion on DNA extracted from cytologic brushings 
using bisulfite treatment and assay methods we have described previously. (23)  
b. Levels of circulating biomarkers: Serum levels of Leptin, cytokines : IL6, 8, 10, 12p7,and 
methylated DNA markers will be measured. Assays will be performed using commercially 
available enzyme immunoassay kits. Unutilized samples will be stored for subsequent 
biomarker analysis. 
Sample size and Statistical analysis: 
Phase1: Detect ideal size and porosity for tolerability and DNA yield  
Table 1 
Sponge Diameter 
25mm 
30mm 
Pores Per Inch 
10 
20 
10 
20 
BE 
5 
5 
5 
5 
No BE 
5 
5 
5 
5 
 
A factorial design will be used to compare porosity of sponge 25 mm 10 pores per inch (PPI) vs. 
25 mm 20 PPI, and Barrett’s Esophagus vs. normal esophagus. Tolerability will be measured 
using a tolerability score as well as level of mucosal abrasion (scale 0-6, 6=worst). The 
assumed standard deviation for the tolerability score of 2.2 was obtained from Sami, et al.. 
Using a two-sided test of significance of 5% and 80% power, we can detect a minimal shift in 
tolerability score of at least 2. For mucosal abrasion score, a rough estimate of 1 standard 
deviation (s.d. ≈ range of data / 4) will be used and assuming a two-sided test of significance of 
5% we have 86% power to detect a difference in mean mucosal abrasion score of at least 1.  
DNA yield will be measured using wild type Beta-actin.  Using a two-sided test of significance of 
5% we have 82% power to detect a fold change difference of 50%. All above power calculations 
are based on detection of main effect comparisons with n=20 per group. The ability to detect 
interactions between main effects will be very limited with the given sample size. Recruitment 
will continue until 40 individuals have successfully completed the sponge procedure using the 

Page 12 of 14 
 
25 mm sponge.   We anticipate 25% will decline swallowing sponges after they have consented, 
therefore the total number of people we anticipate recruiting will be set at 60. 
Phase 2:  For the detection of Barrett’s Esophagus, sample size justification will be based on 
minimizing the width of the 95% confidence interval of the sensitivity for detecting Barrett’s 
Esophagus with a fixed specificity of 90%. From table 2 below, based on various assumed 
sensitivities, the largest confidence interval width for the proposed sample size is less than ±9%. 
Similarly for detection of dysplasia among Barrett’s Esophagus patients, the largest width for the 
proposed sample size is less than ±12. These confidence widths are reasonable for this initial 
feasibility study. We also intend to perform multivariable modeling using machine learning 
techniques to evaluate a minimum of 14 methylation markers. Using the rule of thumb for 
machine learning of 10-15 events per marker, a minimum of 140 to 210 cases and an equal 
number of controls is required (total 280 to 420 patients) to account for assay failures and 
indeterminate histological findings on endoscopic exam.    We anticipate enrolling up to 1000 
participants.    
In addition, clinically archived FFPE tissue from up to 10 cases will be used to assess 
methylated DNA marker levels in biopsies versus the sponge. 
Due to the known dysplasia miss rate with routine surveillance endoscopy, we hypothesize that 
there will be undetected or missed dysplasia in BE patients with a negative surveillance 
histology. This imperfection in the endoscopic “gold” standard will bias the resulting accuracy 
estimates of methylation markers in a multitude of ways. There are several approaches in the 
literature to address this issue; all with their own caveats. The three approaches that will be 
investigated are latent class analysis, Bayesian estimation, and discrepant resolution. We have 
hence inflated the sample size estimates in table 2 to correspond with a 30% prevalence of 
undetected dysplasia in those with no dysplasia on conventional surveillance endoscopy to 
ensure that we have 50 true BE subjects without dysplasia. 
Table 2 
 
BE vs. No BE 
BE no dysplasia vs. BE w/dysplasia 
Confidence Level 
0.95 
0.95 
0.95 
0.95 
0.95 
0.95 
0.95 
0.95 
Sensitivity 
0.75 
0.80 
0.85 
0.90 
0.75 
0.80 
0.85 
0.90 
Confidence bands 
0.085 
0.078 
0.070 
0.059 
0.105 
0.097 
0.087 
0.073 
N no BE/BE without 
dys/BE with dys 
100 
100 
100 
100 
65 
65 
65 
65 
 
Statistical analysis 
Phase 1: Tolerability score and level of mucosal abrasion will be summarized as median and 
interquartile range. Analysis of variance will be used to detect statistically significant differences 
in tolerability score and level of mucosal abrasion between different levels of sponge porosity 
and size. Failure/complication rate will be summarized as the percentage of the group total. 
Comparisons between sponge porosity and size for failure/complication rate will be analyzed 
using the Chi-squared test. 

Page 13 of 14 
 
Phase 1 and 2: Marker specificity will be defined as the 90th percentile value in subjects without 
BE. Several markers will be combined based on the coefficients from a logistic regression 
model using subjects with BE vs subjects without BE as the response.  This approach is used 
as they represent the broadest spectrum of disease and any marker combination that fails to 
discriminate between these two subgroups would not be of future value.  The specificity of the 
combined markers will then be set equal to the 90th percentile in non-BE subjects.  The 
corresponding sensitivity of individual markers as well as combined markers will be estimated 
with 95% confidence intervals. 
Marker specificity will be defined as the 90th percentile value in subjects with BE and no 
dysplasia. Several markers will be combined based on the coefficients from a logistic regression 
model using BE subjects with dysplasia vs subjects without dysplasia as the response.  This 
approach is used as they represent the broadest spectrum of disease and any marker 
combination that fails to discriminate between these two subgroups would not be of future value.  
The specificity of the combined markers will then be set equal to the 90th percentile in BE 
subjects without dysplasia.  The corresponding sensitivity of individual markers as well as 
combined markers will be estimated with 95% confidence intervals.  
The association of demographic (age, gender, race), anthropometric (body mass index, waist 
hip ratio) and circulating biomarker levels (leptin, interleukins) with a BE diagnosis will be 
assessed using univariate and multivariable logistic regression analysis. 
 
 
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