Clinical trial NCT02610816
Conditions: Eosinophilic Esophagitis Eosinophilic Gastrointestinal Disorders (EGIDs)
Sponsor: Children's Hospital Medical Center, Cincinnati
Start date: 2016-03-21; Completion date: 2018-05-16
Enrollment: 67 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
The purpose of this interventional study is to test and compare the effectiveness of two elimination diets-the 1-food elimination diet (1FED, milk only) and the 4-food elimination diet (4FED, milk, egg, wheat, and soy) for eosinophilic esophagitis (EoE). The study will also test the effectiveness of swallowed glucocorticoid therapy in some of the study participants for whom diet therapy was not effective.

Detailed description:
This study will consist of two phases, plus a screening period. During the screening period, subject eligibility for the study will be determined. During Phase 1, qualified participants will be randomly assigned to one of two elimination diet therapies--the 1FED or the 4FED. Participants will remain on the assigned dietary therapy for 12 weeks. At the end of 12 weeks of therapy, esophageal biopsies from participant's standard of care (i.e. normal, routine care) endoscopy will be evaluated to determine disease status. Participants whose EoE is in remission (i.e. \<15 eos/hpf) will be done with the study.

Participants whose EoE is still active (i.e. ≥15 eos/hpf) will continue into Phase 2 of the study. During Phase 2, participants who were on 1FED in Phase 1 will receive 4FED therapy for 12 weeks, and participants who were on 4FED during Phase 1 will receive swallowed glucocorticoid therapy for 12 weeks. At the end of 12 weeks of therapy, esophageal biopsies from participant's standard of care (i.e. normal, routine care) endoscopy will be evaluated to determine disease status.

Arms: arm 1: 1-food elimination diet: Participants eliminate milk from the diet in Phase 1 arm 2: 4-food elimination diet: Participants eliminate milk, egg, wheat, soy from the diet in Phase 1 arm 3: Participants that fail to respond to 1FED in Phase 1 eliminate milk, egg, wheat, soy from the diet in Phase 2 arm 4: Participants that fail to respond to 4FED in Phase 1 administer swallowed glucocorticosteroids (Flovent HFA) 800 mcg twice daily in Phase 2
Interventions: intervention 1: 1 Food Elimination Diet intervention 2: 4 Food Elimination Diet intervention 3: 4 Food Elimination Diet (post 1FED failure) intervention 4: Fluticasone Propionate, 800 mcg twice daily (post 4FED failure)
Intervention descriptions: intervention 1: Participants eliminate milk from the diet for 12 weeks intervention 2: Participants eliminate milk, egg, wheat, soy from the diet for 12 weeks intervention 3: Participants that fail to respond to 1FED in phase 1 eliminate milk, egg, wheat, soy from the diet for 12 weeks in Phase 2 intervention 4: Participants that fail to respond to 4FED in phase 1 administer swallowed glucocorticosteroids (Flovent HFA) 800 mcg twice daily for 12 weeks in Phase 2

Protocol documents:
Prot_000.pdf: 
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PROTOCOL TITLE: 
Eosinophilic Esophagitis (EoE) Intervention Trial-Randomized 1 Food 
Elimination vs. 4 Food Elimination Diet followed by Swallowed Glucocorticoids 
PROTOCOL NUMBER: 
7805 
STUDY DESIGN: 
 
Prospective, non-blinded clinical trial 
AMENDMENT NUMBER: Not applicable 
SPONSOR:  
 
Marc E. Rothenberg, MD, PhD 
 
 
 
 
Cincinnati Children’s Hospital Medical Center 
 
 
 
 
Division of Allergy & Immunology 
 
 
 
 
3333 Burnet Avenue, MLC 7028 
 
 
 
 
Cincinnati, OH 45229 
 
 
 
 
Phone: (513) 636-6675 
 
OVERALL PRINCIPAL  Marc E. Rothenberg, MD, PhD 
INVESTIGATOR:  
Cincinnati Children’s Hospital Medical Center 
 
 
 
 
Division of Allergy & Immunology 
 
 
 
 
3333 Burnet Avenue, MLC 7028 
 
 
 
 
Cincinnati, OH 45229 
 
 
 
 
Phone: (513) 636-6675 
CCHMC PRINCIPAL 
INVESTIGATOR: 
 
Vince Mukkada, MD 
 
 
 
 
Cincinnati Children’s Hospital Medical Center 
 
 
 
 
Division of Allergy & Immunology 
 
 
 
 
3333 Burnet Avenue, MLC 2010 
 
 
 
 
Cincinnati, OH 45229 
 
 
 
 
Phone: (513) 803-4468 
 
 
Good Clinical Practice 
This study was conducted under Good Clinical Practices, in accordance with the Declaration of Helsinki, in 
compliance with the International Conference on Harmonisation (ICH) guidelines. 
 
 


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Confidentiality Statement 
This document contains confidential information of the Sponsor. This information is to be disclosed only to the 
recipient study staff and the Institutional Review Board or Board of Ethics Committee reviewing this protocol. 
This information can be used for no other purpose than evaluation or conduct of this study without prior written 
consent from the Sponsor. 
 
 


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CONTENTS 
1 
ABBREVIATIONS AND DEFINITIONS 
6 
2 
INTRODUCTION 
8 
3 
BACKGROUND AND RATIONALE 
9 
3.1 
Eosinophilic Esophagitis 
9 
3.2 
Previous Research On The Use Of Dietary Therapy In Eosinophilic Esophagitis 
9 
3.3 
Population To Be Studied 
10 
3.4 
Statement Of Compliance 
10 
4 
POTENTIAL RISKS, DISCOMFORTS, INCONVENIENCES AND PRECAUTIONS 
10 
4.1 
Esophageal Biopsies 
10 
4.2 
Treatment With Dietary Therapy 
11 
4.3 
Treatment With Topical Swallowed Steroids 
11 
4.4 
Blood Draws 
11 
4.5 
Skin Testing For Allergies 
11 
4.6 
Histology Scoring System (HSS) 
11 
4.7 
Study Survey 
12 
4.8 
Privacy And Confidentiality 
12 
4.9 
Standard of Care Procedures Associated with This Study 
13 
4.10 
Potential Benefits 
14 
4.11 
Risk/Benefit Analysis 
14 
5 
STUDY OBJECTIVES 
14 
5.1 
Primary Objective 
14 
5.2 
Secondary Objectives 
15 
6 
INVESTIGATIONAL PLAN / OVERALL STUDY AND PLAN 
15 
6.1 
Number Of Participants 
15 
6.2 
Study Design 
16 
6.3 
Screening Criteria 
16 
6.4 
Safety 
18 
6.5 
Efficacy 
18 
6.5.1 
AIM 1: To Determine The Efficacy Of A 1FED Vs. 4FED In EoE 
18 


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6.5.2 
AIM 2: Clinical Parameters & Biomarkers That Predict Therapeutic Response 
21 
6.6 
FIGURE 1: Esophageal Transcriptome Analysis 
24 
6.7 
End Of Therapy (EOT) And Follow-Up 
24 
6.8 
FIGURE 2: STUDY DESIGN FLOWCHART 
25 
6.9 
Minimization Of Bias And Method Of Treatment Assignment 
25 
6.10 
Expected Duration Of Study And Participant Participation 
26 
7 
STUDY VISITS AND ASSESSMENTS 
26 
7.1 
TABLE 1. STUDY DESIGN AND SCHEDULE OF ASSESSMENTS 
26 
7.2 
Screening (Weeks -12 To -1) 
27 
7.3 
Phase I: Enrollment & Dietary Treatment (Week 0) 
28 
7.4 
Phone Visits, Phase I 
29 
7.5 
End Of Treatment Phase I Or Early Withdrawal (Week 12) 
29 
7.6 
Phase II Visit 1 (Week 13) 
30 
7.7 
Phone Visits, Phase II 
31 
7.8 
End Of Treatment Phase II Or Early Withdrawal (Week 25) 
31 
7.9 
Pregnancy 
32 
7.10 
Unscheduled Visits 
32 
8 
SELECTION AND WITHDRAWAL OF PARTICIPANTS 
32 
8.1 
Participant Inclusion Criteria 
32 
8.2 
Participant Exclusion Criteria 
33 
9 
PARTICIPANT WITHDRAWAL CRITERIA 
33 
9.1 
Replacement Of Participants 
34 
9.2 
Study Site Termination Criteria 
34 
9.3 
Study Discontinuation 
34 
10 TREATMENT OF PARTICIPANTS AND DESCRIPTION OF STUDY INTERVENTION 
35 
10.1 
Concomitant Medications 
35 
10.2 
TABLE 2. Prohibited Medications And Washout Time 
35 
10.3 
Treatment Compliance 
36 
10.4 
Study Intervention Administration-Diet And Medication Instruction 
36 
10.5 
Efficacy Evaluation 
36 


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10.6 
Primary Efficacy Endpoint: 
36 
10.7 
Secondary Efficacy Endpoints: 
36 
11 ASSESSMENT OF SAFETY 
37 
11.1 
Safety Parameters 
37 
11.1.1 Data Safety Monitoring Board 
38 
11.2 
Adverse Events 
38 
11.3 
TABLE 3. Grading Of Adverse Events 
39 
11.4 
TABLE 4. Relationship To Study Treatment 
41 
11.5 
Serious Adverse Event 
41 
11.6 
Recording Adverse Events 
42 
11.7 
Reporting Adverse Events 
42 
11.8 
Reporting Serious Adverse Events To Sponsor 
43 
11.9 
DMCC Adverse Event Data Management System (AEDAMS) 
44 
12 STATISTICAL METHODS 
45 
12.1 
Sample Size Calculation 
45 
12.2 
Statistical Analyses: Aim 1 
46 
12.3 
Statistical Analyses: Aim 2 
47 
12.4 
Direct Access To Source Date/Documents 
47 
13 INVESTIGATOR REQUIREMENTS 
47 
13.1 
Protocol Adherence 
47 
13.2 
Case Report Forms 
47 
13.3 
Source Document Maintenance 
48 
13.4 
Study Completion 
48 
13.5 
Audits And Inspections 
48 
13.6 
Institutional Review Board Approval 
48 
14 ETHICS 
49 
14.1 
Ethics Review 
49 
14.2 
Ethical Conduct Of The Study 
49 
14.3 
Written Informed Consent And Assent 
50 
15 DATA HANDLING AND RECORDKEEPING 
50 


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15.1 
Inspection Of Records 
50 
15.2 
Retention Of Records 
50 
15.3 
Data Management 
50 
15.4 
Registration 
51 
15.5 
Data Entry 
51 
15.6 
Data Quality and Monitoring Measures 
51 
15.7 
Laboratory Data Flow 
52 
16 REFERENCES 
52 
 
1 ABBREVIATIONS AND DEFINITIONS 
Abbreviation 
Term 
EoE 
Eosinophilic Esophagitis 
hpf 
High Power Field 
6FED 
6-food elimination diet 
4FED 
4-food elimination diet 
3FED 
3-food elimination diet 
1FED 
1-food elimination diet 
IRB 
Institutional Review Board 
CI 
Confidence level 
EOT 
End of treatment 
EGD 
Esophagogastroduodenoscopy 
GI 
Gastroenterology 
HSS 
Histology Scoring System 
PI 
Principal Investigator 
PHI 
Protected health information 
CCHMC 
Cincinnati Children’s Hospital Medical Center 
GI 
Gastrointestinal 
PPI 
Proton pump inhibitor 
AE 
Adverse Event 
SAE 
Serious Adverse Event 


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CHOP 
Children’s Hospital of Philadelphia 
UNC 
University of North Carolina 
NW 
Northwestern University 
RHC 
Riley Hospital for Children 
UCSD 
University of California at San Diego 
EDP 
Eosinophil Diagnostic Panel 
PRO 
Patient Related Outcomes 
 
 


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2 INTRODUCTION 
The primary objective of this proposal is to conduct a prospective, non-blinded randomized trial comparing 
patient reported outcomes scores in novel empiric elimination dietary therapies in eosinophilic esophagitis 
(EoE) in order to assess the therapeutic viability of minimally restrictive empiric elimination diets as related to 
measures of patient’s symptoms, quality of life, and pathology. Moreover, we aim to assess the response to 
topical swallowed steroids in those who were non-responders to empiric dietary therapy regimens used during 
the initial phases of this study. Participants aged 6 to 17 years with active EoE will be enrolled and the primary 
efficacy outcome will be the change in the standardized and validated Pediatric Eosinophilic Esophagitis 
Symptom Score v2 (PEESS)1, 2 obtained following a 3 month randomized trial of one of two minimally 
restrictive empiric diets. During the screening process, active EoE will be confirmed by histologic evaluation of 
esophageal biopsies obtained during an esophagogastroduodenoscopy (EGD) in subjects with a history 
consistent with EoE. 
 
This trial is comprised of 2 phases with each phase lasting 3 months. In the first phase, all participants will be 
randomized 1:1 to either 1 food elimination (milk elimination alone, 1FED) or 4 food elimination (milk, egg, 
wheat, and soy elimination, 4FED) therapeutic diet. At the end of this phase, an EGD will be performed to 
assess histologic remission, which represents one of the primary current tools used in evaluating EoE disease 
activity during clinical trials and daily EoE care. Attainment of remission (esophageal eosinophil counts <15 
eosinophils/high powered field) after Phase I will result in study discontinuation and maintenance of the 
“successful” dietary therapy. Dietary therapy non-responders who were on 4FED during Phase I will receive 
topical swallowed steroids for 3 months while on an unrestricted diet (Phase 2) followed by EGD with 
esophageal biopsies.  Participants receiving SGC will return to an unrestricted diet (i.e. stop 4FED) prior to 
initiating SGC therapy. Dietary therapy non-responders who were on 1FED during Phase 1 will proceed to 
4FED for 3 months (Phase 2) followed by EGD with esophageal biopsies.  Unlike previous studies, the primary 
efficacy endpoint reported will be the change in the PEESS score following each dietary regimen. A variety of 
secondary endpoints, including pre- and post-therapy peak eosinophil counts per hpf and percentages of patients 
that attain histologic remission (<15 eosinophils/hpf), along with detailed histologic and endoscopic scores, 
novel biomarkers, and patient related outcome (PRO) measures. In addition, we will assess the possible value of 
a variety of clinical characteristics and biomarkers in terms of predicting response to therapy. 
 
This study will provide significant novel information. This will be the first prospective dietary therapy trial 
using empiric elimination diets in pediatrics, and will be the first trial to assess these particular dietary 


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interventions prospectively, whilst monitoring dietary adherence and adequacy. This represents the first patient 
driven study of EoE, in which PROs are utilized as the primary outcome measure for the trial. Further, this 
study will contrast the current gold standard of EoE disease activity with the PROs. This will also be the first 
dietary study to determine whether diet failures can still be rescued with the use of topical steroids. As part of 
our secondary aims, we will also be utilizing a number of novel potential biomarkers to assess their ability in 
predicting and assessing response to therapy. 
3 BACKGROUND AND RATIONALE 
3.1 Eosinophilic Esophagitis 
EoE represents a complex clinical entity often requiring coordinated care within the fields of both allergy and 
gastroenterology (GI). EoE is diagnostically defined by revised consensus criteria, that in brief require 
esophageal-specific inflammation of ≥ 15 eosinophils per high power field (hpf) as obtained from two to four 
biopsies from either the proximal or distal esophagus that is not mitigated by the use of proton pump inhibitor 
(PPI) therapy3. Symptomatically, patients with EoE often complain of dysphagia, vomiting, reflux, food 
aversion, and food impaction3-5. These patients commonly have diagnoses associated with atopy and other 
allergic disorders such as IgE-mediated food allergy, asthma, allergic rhinoconjunctivitis, and eczema. Current 
treatment modalities focus on the use of the six food elimination diet (6FED), which consists of the empiric 
avoidance of the top 6 most common food allergens in the U.S. (milk, egg, wheat, soy, peanut/tree nuts, and 
fish/shellfish); implementation of a variety of directed elimination diets that rely on the use of allergy test 
results; the elimination of all antigenic proteins via the use of elemental formulas; or the use of swallowed 
steroid therapies (topical swallowed preparations of fluticasone or budesonide or oral systemic steroids) 6-15. 
While all of these treatments are efficacious, they require continuous use or the disease will recrudesce in the 
majority of patients. Due to concerns about the risk of chronic topical swallowed steroid, patients and/or their 
family frequently opt to initially implement dietary therapy. This study is designed in keeping with this 
preference; therefore one of two less-restrictive empiric dietary therapies will be implemented initially prior to 
the use of topical swallowed steroids in participants who do not respond to the 4FED dietary treatment. The use 
of dietary restrictions as a treatment modality requires family and/or personal commitment to eliminating food, 
which may lead to significant nutritional deficiencies and is often life-altering. 
3.2 Previous Research On The Use Of Dietary Therapy In Eosinophilic Esophagitis 
Eosinophilic esophagitis is a chronic esophageal inflammatory disease typically triggered by exposure to food 
antigens. Given this, dietary elimination of these antigens has been tested as a therapeutic option. Previous work 
has shown that the exclusive use of elemental formula resulted in a response rate from 88-100% when histology 
and symptoms are considered, but difficulties in compliance, cost, and formula palatability have led to attempts 


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to find alternative dietary approaches8, 15, 16. Use of allergy testing to direct dietary elimination has been 
advocated, and in studies response rates from 53-77% have been reported. However, in practice the utility of 
allergy testing is very dependent on local expertise and the response rate has been lower than hoped14, 15, 17. 
Given this, there has been an attempt to use an empiric 6FED, which removes milk, soy, wheat, egg, 
peanuts/tree nuts, and fish/shellfish from the diet. This intervention has shown response rates ranging from 53-
81% and is attractive due to the fact that no allergy testing is required. In all of these currently prescribed 
dietary therapies, the goal is to reintroduce foods systematically with subsequent endoscopy to try to identify 
specific causative antigens. A single small study of 17 children showed a partial or complete response rate of 
65% using milk elimination alone18. In published reports where causative antigens have been identified, milk, 
wheat, eggs and soy have consistently been reported to be the top 4 offending food antigens, and these four 
foods are therefore the basis of our less restrictive diet regimens14, 15, 19. Strictly adhering to any of these 
elimination diets is challenging for most children and families, and so any dietary therapy which achieves a 
similar response while minimizing the number of foods eliminated would represent a major advance in therapy. 
3.3 Population To Be Studied 
Participants aged 6 to 17 years with active EoE are eligible for entry into the study. 
3.4 Statement Of Compliance 
This study will be conducted in compliance with the protocol, Good Clinical Practices (GCP), and applicable 
local regulatory and Institutional Review Board (IRB) requirements. 
4 POTENTIAL RISKS, DISCOMFORTS, INCONVENIENCES AND PRECAUTIONS 
4.1 Esophageal Biopsies 
The risks associated with collecting additional esophageal biopsies (up to a total of four obtained from the 
proximal and/or distal esophagus) for research at the time of the endoscopy include: bleeding at the site of tissue 
(biopsy) collection, and a small chance of perforation (hole) of the stomach, duodenum, or esophagus.  
Perforation is the most severe gastrointestinal complication, but generally it is self-resolving and poses no life-
threatening risk.  To minimize the risks of collecting additional biopsies during endoscopy, the procedure will 
be performed or supervised by a skilled endoscopist, and additional biopsies will only be collected if the 
endoscopist feels it is appropriate to do so. Some samples that are collected during an endoscopy for research 
purposes may be frozen and shipped to other hospitals, institutions, and testing companies for analysis. Data 
may also be shared.  The data and/or samples will be de-identified per HIPPA and have no PHI associated with 
them. The data and/or samples will be used in a collaborative relationship between institutions, or testing 


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companies receiving the data and/or samples. All of these samples will be shared under a MTA, or other 
applicable agreement. 
4.2 Treatment With Dietary Therapy 
Minimal risk exists related to implementation of short-term dietary therapies in EoE. Dietary instruction 
regarding tenets of each dietary therapy and nutrition-related recommendations will be provided by a Registered 
Dietitian on how to implement and make appropriate substitutions for foods eliminated to ensure nutritional 
adequacy. A central dietitian will develop standardized instructional materials for use at all of the participating 
sites. 
4.3 Treatment With Topical Swallowed Steroids 
In the short term (such as the Phase II three month period steroid study interval), the primary risk is the 
development of oral thrush, which is a known side effect of topical swallowed steroid use11. Other reported 
concerns include behavior changes and difficulty sleeping. Such side effects are reversible with medication 
reduction or discontinuation. Risks from longer term use of topical swallowed corticosteroid are less well 
understood, but might include bone demineralization, trouble with elevated blood sugar or hypertension, and/or 
eye changes such as glaucoma or cataracts. However, these have not been commonly seen with use of inhaled 
steroids for other diseases such as asthma and have not yet been seen in any of the controlled studies with 
topical steroids for EoE. There is also a concern about the potential for adrenal suppression with chronic steroid 
use, and we will monitor for this with morning cortisol levels at the beginning and end of Phase II. 
4.4 Blood Draws 
Risks associated with the collection of blood are bleeding, bruising, and swelling, dizziness, fainting and 
infection at the site where the blood is drawn. In general, these procedures will be performed by individuals 
with expert skills in phlebotomy. To minimize the additional risks associated with phlebotomy, we obtain blood 
during the standard placement of intravenous lines, when possible. The amount of blood drawn will adhere to 
the institutional policy. 
4.5 Skin Testing For Allergies 
Skin testing for allergies may cause mild discomfort when the skin is pricked. Participants may experience 
symptoms such as itching, stuffy nose, red watery eyes, or a skin rash if they are allergic to the substance being 
tested. A rare but serious whole-body allergic reaction, known as anaphylaxis, may occur. Anaphylaxis can be 
life threatening, but this usually only occurs with intradermal testing, and the health care provider performing 
the skin testing will be prepared to treat this serious reaction. 
4.6 Histology Scoring System (HSS) 


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The histology scoring system is a method to evaluate esophageal biopsies containing eosinophils for various 
features (ex: basal layer hyperplasia, dilated intercellular spaces, surface epithelial alteration, apoptotic 
epithelial cells), eosinophilic inflammation (peak count, eosinophil abscess, eosinophil surface layering), and 
lamina propria (fibrosis). There is no risk associated with the utilization of the HSS. 
4.7 Study Survey 
Each participant between the ages of 6 to 17 years old will be asked to complete several age appropriate 
surveys, including Patient Related Outcome measures regarding both Health Related Quality of Life as well as 
disease specific symptom scores and QOL measures in order to address their EoE symptoms and problems/ 
feelings related to eating. The parent or legal guardian of participants will also be asked to complete 
questionnaires. The data gathered from these questionnaires will be correlated with several biomarkers of 
interest that have been associated with EoE. These surveys will be conducted at baseline and the end of each 
study phase. There are no foreseeable physical discomforts or significant risks related to answering the patient 
or parent-proxy reported outcomes or study questionnaires. However, some questions may be difficult to answer 
and may make the participants uncomfortable. The participants may also feel inconvenienced to complete the 
questionnaires. Participants may refuse to answer any questions asked on the survey. The participant 
questionnaires typically take approximately 15 to 20 minutes to complete. All participants will be given ample 
time to complete the survey and additional efforts will be made to have a complete survey of all questions, as 
this represents our primary outcome to be monitored. Please reference Table 1 for the guidelines on when the 
study questionnaires are completed. 
 
Some samples that are collected during an endoscopy for research purposes may be frozen and shipped to other 
hospitals, institutions, and testing companies for analysis. Data may also be shared. The data and/or samples 
will be de-identified per HIPPA and have no PHI associated with them. The data and/or samples will be used in 
a collaborative relationship between institutions, or testing company receiving the data and/or samples. All of 
these samples will be shared under a MTA, or other applicable agreement. 
4.8 Privacy And Confidentiality 
Although the study staff will do its best to adhere to strict privacy guidelines, the possible risk of a breach of 
confidentiality does exist. Every effort will be made to maintain the confidentiality of the participant’s medical 
and research information. The Investigator will take all necessary measures to keep all of the participants’ 
personal information private and confidential. Electronic study records will be protected with electronic 
safeguards (e.g., computer passwords, restricted access privileges). Paper-based private health information will 
be secured in locked cabinets. Access to study records will be limited only to research staff. For this study, the 


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research staff will collect and utilize data obtained from the participant’s medical and research records and 
observations. This information, along with the study survey forms, will be used by the Investigator as a part of 
the study data. The participants will be assigned and identified by a participant study identification number. The 
link to the participants’ study ID and their names and identifiers will be kept by the PI and/or the research staff 
in a secure location. Because the participant’s protected health information (PHI) will be collected and used for 
the study, the research staff will obtain a signed HIPAA authorization which is located in the informed consent 
form from the participant and/or legal guardian. The research staff will perform informed consent and all study 
procedures in a private setting away from the public. In addition, the research staff will only collect the 
minimum amount of personal information about the participant necessary for the research study. Storage and 
management of electronic data will be centralized at the Data Management and Coordinating Center (DMCC) at 
the University of South Florida (USF) Health Informatics Institute (HII) in Tampa, Florida, which supports 
clinical research trials from around the world. The USF HII is also the DMCC for the RDCRN, which includes 
the newly formed Consortium of Eosinophilic Gastrointestinal Disease Researchers (CEGIR, NIH U54 Grant 
U54AI117804-01, PI Marc Rothenberg) that is designed to delve into the entire spectrum of eosinophilic 
gastrointestinal disorders. Dr. Jeffrey P. Krischer is the principal investigator responsible for the DMCC, and 
has supported not only research work from the NIH, but is also involved in several studies supported by PCORI.  
 
This study will be a multi-site study in which CCHMC is the lead site. Each site will have access to the data 
collected at their site. Data from each site may be shared with the other sites. The data will be de-identified 
when possible, but PHI may also be disclosed to each study site if necessary for the research activities. The 
disclosure of PHI is outlined in the consent form. Each site will perform on-going quality checks to verify 
requirements of data collected and report outcomes to the CCHMC PI. 
 
The Sponsor, PI, members of the study’s research team, DMCC staff, CCHMC medical staff, CCHMC IRB and 
Office of Research Compliance and Regulatory Affairs may have access to the participant’s medical and 
research records related to this study. The data from the study may be published: however, the participant will 
not be identified by name. 
4.9 Standard of Care Procedures Associated with This Study 
Esophagogastroduodenoscopy (EGD) 
Endoscopy is a well-established procedure.  Few patients have unexpected or serious complications.  Since it is 
considered standard of care for patients with EoE to have an EGD at most every three months to monitor the 
disease when patients are trialing new therapies (foods or medications), the EGDs that are associated with this 


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study are not study procedures.  Endoscopies will be performed regardless of study participation as patients’ 
standard of care, and additional endoscopic procedures will not be performed because of participation in this 
study. 
 
There may be unknown or unforeseen risks associated with study participation. 
4.10 Potential Benefits 
As stated above, standard therapies for eosinophilic esophagitis consist of allergy test-directed and empiric 
elimination dietary therapies, complete elimination of all dietary antigens requiring the exclusive use of 
elemental formula, or the chronic use of topical swallowed steroids. These therapies are all associated with risks 
and benefits. Utilizing minimally restrictive dietary elimination regimens to control esophageal inflammation 
may permit earlier identification of a diet that controls symptoms and achieves remission compared with the 
standard strategy of starting with a more restrictive diet then reintroducing potentially causative antigens one-at-
a time. In addition, by limiting the number of potential reintroductions, these less restrictive diets have the 
potential to limit the need for endoscopies and attendant sedation and thus reduce risk to patients and costs to 
the health care system. This study would have the additional benefit of explicitly assessing the response to the 
studied therapies in terms of their effect on symptoms and quality of life, which has generally not been the case 
to date. 
 
There are no guaranteed benefits for any study participant. Potential benefits to the individual patient would 
include the use of a less restrictive diet compared to the standard 6FED, as well as a reduction in the time to 
identification of a minimally restrictive effective diet. In addition, the use of an effective less restrictive diet 
than the standard could potentially improve quality of life. 
4.11 Risk/Benefit Analysis 
This study poses minimal risk. The use of 6FED is currently a standard therapy used to treat EoE, with an 
expected histologic response rate of approximately 75%. The use of less restrictive diets may therefore increase 
the risk for treatment failure. This risk is mitigated by the short trial period and the fact that the patients will 
undergo follow-up endoscopy to assess their response, as well as telephone monitoring to make sure they are 
not experiencing excessive symptoms. 
5 STUDY OBJECTIVES 
The purpose of this study is to evaluate the following objectives: 
5.1 Primary Objective 


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To perform a prospective, randomized, non-blinded trial that determines patient reported outcomes scores 
following 1FED (milk elimination alone) vs. 4FED (milk, egg, wheat, and soy avoidance) and their relative 
efficacy (Phase I). 
 The primary endpoint for this study will be the change in the PEESS from baseline to post therapy 
assessed after the end of the 3 month Phase I. 
5.2 Secondary Objectives 
 To extend Phase I of this study with a prospective non-blinded trial that determines the rate of remission 
after treatment with 4FED (in participants failing 1FED in Phase I) or SGC (in participants failing 4FED 
in Phase I) (Phase II). 
 To evaluate the effect of each therapy on histological remission (defined as a post therapy eosinophil 
count of <15 eosinophils/HPF) and by a variety of changes in eosinophils, including (a) pre- and post-
therapy peak eosinophil counts; (b) partial remission (2-14 peak eosinophils/hpf); and (c) complete 
histological remission (≤ 1 peak eosinophils/hpf). 
 To evaluate the effect of each therapy by utilizing the histology scoring system (HSS) created to express 
the severity and extent of other abnormalities in the gastrointestinal (GI) tract that often accompany 
eosinophilic inflammation. 
 To determine the impact of each therapeutic intervention on biomarkers using the EoE Diagnostic Panel 
(EDP). 
 To evaluate the clinical and psychosocial effect of each therapy using the Peds Quality of Life Inventory 
Eosinophilic Esophagitis Module (PedsQL EoE) to assess EoE symptoms and problems/feelings related 
to eating. 
 To determine if any clinical parameters predict response to therapeutic intervention. 
 To determine if any biomarkers including component-resolved diagnostics (CRD) [serum IgE 
component testing] predict response to therapeutic intervention. 
 To determine if skin testing, in the form of prick and patch testing, predicts response to therapeutic 
intervention. 
6 INVESTIGATIONAL PLAN / OVERALL STUDY AND PLAN 
6.1 Number Of Participants 
We will be recruiting a total of 292 participants, approximately 50 participants between the ages of 6-7 years of 
age will be enrolled, approximately 120 participants between the ages of 8-12 years of age will be enrolled, and 
approximately120 participants between the ages of 13-17 years of age will be enrolled, to enter this study based 


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on the presence of active eosinophilic esophagitis and adherence to the inclusion and exclusion criteria. The 
DMCC at the University of South Florida Health Informatics Institute will serve as the central data management 
center, with CCHMC serving as the primary site and the central IRB. Additional sites include: Children’s 
Hospital of Colorado, Riley Hospital for Children, Children’s Hospital of Philadelphia, University of California 
at San Diego/Rady Children’s Hospital, Northwestern University/Lurie Children’s Hospital, Tufts Medical 
Center, Mt. Sinai Kravis Children’s Hospital, Arkansas Children’s Hospital, and the University of North 
Carolina. 
6.2 Study Design 
We intend to conduct a prospective non-blinded randomized trial that compares a 1-food elimination diet 
(1FED) versus a 4-food elimination diet (4FED) for three months (Phase I) in a 1:1 ratio, stratified by two age 
groups,  6-12 and 13-17 years of age. Subsequently, non-responders to 1FED dietary elimination therapy will be 
treated with 4FED and non-responders to 4FED will be treated with SGC (swallowed fluticasone at a dose of 
880 mcg twice a day) for 3 months (Phase II). Empiric dietary therapy interventions are as follows: 1-food 
elimination diet (1FED)—avoidance of milk vs. 4-food elimination diet (4FED)—avoidance of milk, egg, soy, 
and wheat for three months. Milk elimination for both 1FED and 4FED includes all mammal milk (i.e. goat, 
sheep, and cow’s milk must all be eliminated). The study design is shown schematically in Figure 2. 
 
The instructions for dietary phases will be standardized across sites by providing consensus documents that 
contain diet information, produced by a research Registered Dietitian who is an expert in dietary elimination in 
EoE. Similarly, standardized instructions for using SGC will be provided, based on our prior studies. We have 
chosen to use fluticasone proprionate (FP) rather than budesonide in this study, given our prior work in this 
area, and our recent finding that high-dose fluticasone is well tolerated and highly effective, and our preliminary 
findings concerning esophageal transcripts that predict responsiveness to FP therapy.  
 
The clinical trial information will be entered into a databank on www.clinicaltrials.gov maintained by the U.S. 
National Library of Medicine (NLM) at the National Institutes of Health (NIH). 
6.3 Screening Criteria 
Participants will be recruited at participating sites from the GI and Allergy clinics, or other clinics in which 
patients with EoE are often seen. In addition, potential participants will be identified through the EGID database 
(CCHMC IRB studies #2008-0090 and 2008-0098). The research information will be entered into a databank on 
www.clinicaltrials.gov, maintained by the U.S National Library of Medicine (NLM) at the National Institutes of 
Health (NIH), and may be advertised on other websites (CCHMC web pages, such as the Cincinnati Center for 


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Eosinophilic Disorders website, the RDCRN webpage, etc.). 
During screening (within twelve weeks of enrollment), a diagnosis of active EoE will be confirmed. Participants 
will be initially screened for the following: 
1. Age between 6 and 17 years 
2. An EGD with biopsies performed within twelve weeks prior to enrollment documenting active EoE (≥ 
15 eosinophils/hpf)  
3. PPI confirmed EoE. As a part of the diagnosis of active EoE, it must be demonstrated that acid reflux 
disease is not the primary cause of the participant’s symptoms by documentation that the participant has 
been on a high dose of PPI (at least one dose, once daily), for at least 6 - 8 weeks prior to a diagnostic 
endoscopy of EoE without histologic resolution (i.e., ≥ 15 eosinophils/hpf). (Due to the variety of doses 
and various proton pump inhibitors available, the dose and adherence will be confirmed adequate at the 
discretion of the principal investigator).  
Participants who meet the screening entry criteria of EoE will be enrolled in the study. An EGD and biopsy 
results will be accepted as baseline if done within twelve weeks prior to the enrollment visit and there have been 
no changes in either dietary or pharmaceutical treatment during that pre-screening interval. If the participant is 
on a current dose of acid reflux therapy (e.g., PPIs or histamine H2 receptor antagonists), the participant will 
continue his/her acid reflux therapy as long as the dose remains the same throughout the study. Screening 
exclusion criteria for patients entering the study include the presence of hypereosinophilic syndrome, parasitic 
infection, inflammatory bowel disease, Celiac disease, Helicobacter pylori infection, and patients who are 
currently on dietary therapy avoiding milk (i.e. on a 1FED) or milk, egg, soy, and wheat (i.e. on a 4FED).   
Ideally we would like to exclude patients with a history of diet and/or SGC therapy as knowing the results could 
bias selection of patients into the trial. However, in clinical practice most patients have already tried diet and/or 
steroid therapy by the time they are seen in EoE specialty clinics. In these situations, true compliance and 
efficacy of treatment are typically not clear. Narrowing the study to newly diagnosed patients is not practical. 
As such, we will exclude patients with known definitive response to diet and/or SGC, such as participation in 
prior clinical trials. In addition, patients cannot have been on topical swallowed steroids within the last 2 
months or systemic steroids within the last 3 months prior to enrollment. Participants may continue any inhaled 
steroids needed for asthma control as appropriate. We have selected the 6-year lower age range to minimize 
concern about compliance with and safety of the SGC in Phase II. The 17-year old upper age range was selected 
to align with the age ranges supported by our instruments measuring quality of life and patient symptom scores. 
 


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All protocol-required study procedures, including efficacy and safety assessments are summarized in Table 1 in 
the Study Visits and Assessments Section. 
6.4 Safety 
Safety will be monitored by physical exam findings at the screening visit, at the visit on the day of the 
endoscopy at the end of Phase I (12 weeks), and again at the end of Phase II (week 25) for those who fail to 
respond to the dietary therapy. In addition, for those who receive SGC during Phase II, morning cortisol will be 
checked at the beginning and end of Phase II to monitor for adrenal suppression. No additional endoscopies 
beyond the standard of care will be required for participation in this study, and no more than 4 additional 
biopsies will be obtained for research during endoscopy. Blood draws will occur whenever possible with the 
start of the IV for endoscopy. In addition, adverse events will be monitored from when informed consent is 
signed, at each visit, and throughout the study.  
 
The PI at each performance site will review all unexpected and study-related events and forward their 
assessments to the CCHMC PI and study sponsor for review. Finally, privacy and confidentiality will be 
maintained according to HIPAA guidelines. These and other risks are reviewed in the Informed Consent and 
approved by the central IRB. 
6.5 Efficacy 
6.5.1 AIM 1: To Determine The Efficacy Of A 1FED Vs. 4FED In EoE 
6.5.1.1 Primary End-Point 
The primary efficacy endpoint will be the change in PEESS v2 as measured prior to initiation of the study diet 
and at the end of Phases I & II1, 20. The PEESS v2, a validated PRO metric, measures clinical symptoms in EoE 
that broadly fall into four categories: dysphagia, gastrointestinal reflux disease, nausea/vomiting, and pain. We 
hypothesize that therapeutic interventions will improve clinical symptoms as measured by PRO metrics and 
4FED will be more effective than a 1FED with this measurement. This is an important issue to resolve as only a 
few prior studies have shown the clinical efficacy of therapeutic interventions, and these have been mainly in 
adults. In fact, several studies have shown dissociation between histology (eosinophil levels) and clinical 
improvement20, 21, but these studies have not used validated metrics for measuring symptoms.  We will therefore 
employ validated EoE specific measurement tools (Pediatric EoE Symptom Score (PEESS Version 2.0) (used 
as the primary endpoint) and Pediatric EoE Quality of Life (Ped EoE QL)) metrics using parent reported 
outcomes by proxy for patients1, 20, 22, 23 to measure the clinical efficacy of our therapeutic interventions and to 
correlate symptoms with histology. The measurement of pre/post diet symptoms using tools that have 


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undergone at least some validation in pediatric patients is a novel aspect of our study. Agreement about these 
measurement tools has significant bearing on the eventual FDA approval of therapeutic agents24 
6.5.1.2 Secondary End-Point Analysis: Histology And Endoscopy 
At the end of each phase, we will employ generic Health Related QOL metrics, such as PROMIS General 
Health Questionnaire and Peds-QL and eventually compare the changes in disease specific versus general health 
questionnaires. These PROs will be assessed at the screening visit and again at the end of each treatment phase 
to assess the effects of treatment. All participants will undergo EGD with biopsy and the percentage of patients 
who have <15 peak eosinophils/hpf will be determined. The slides will be reviewed by local pathologists for 
clinical purposes, who will be blinded to  the treatment group and patient study number. The local pathology 
review will be used to determine whether or not participants continue into Phase II of the study.  Participants 
with ≥15 peak eosinophils/hpf who were on 4FED during Phase I will continue into Phase II to receive SGC for 
three months while on an unrestricted diet.  Participants with ≥15 peak eosinophils/hpf who were on 1FED 
during Phase I will also continue into Phase II, but will maintain the 4FED therapeutic diet for 3 months. The 
Central Review Pathology Committee (CRPC), who will also be blind to the treatment group, will subsequently 
review the esophageal biopsy slides to determine other parameters (such as the Histology Scoring System 
(HSS)).  CRPC review will be completed, whenever possible, within 2 weeks of a subject’s endoscopy. . 
Participants who achieve histologic remission at the end of Phase I (as determined by the local pathology 
review) will have completed the study and will resume follow-up care with their primary gastroenterologist or 
allergist. Peak eosinophil change will be measured and we will also aim to determine the percentage of patients 
who achieve remission, defined as both complete remission (≤1 peak eosinophils/HPF) and partial remission 
(<10 and <6 peak eosinophils/HPF) as reported11; this will allow us to compare with other studies that have 
used 6FED16, 19, 25, 26. Furthermore, we aim to more broadly examine the impact of each therapeutic intervention 
on other histological parameters associated with EoE using a quantitative histology scoring system (HSS) that 
has been developed by our expert pathologist, Dr. Margaret Collins27. The HSS evaluates esophageal biopsies 
for various features (ex: basal layer hyperplasia, dilated intercellular spaces, surface epithelial alteration, 
apoptotic epithelial cells), eosinophilic inflammation (peak count, eosinophil abscess, eosinophil surface 
layering), and lamina propria (fibrosis). For central review, slides of relevant esophageal biopsies obtained 
during the course of the study will be scanned using an Aperio Imaging scanner and viewed by the CRPC using 
Aperio Imaging Software. Each member of the CRPC has access to an Aperio scanner in his or her pathology 
department and to the Aperio Imaging Software. Slides may be scanned into Aperio at the local site, or they 
may be sent to Cincinnati Children’s Hospital Medical Center (CCHMC) to be scanned into Aperio (if the local 
site does not have an Aperio scanner).  These slides will be returned to the local site once they have been 


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scanned in. A password-protected website housing the scanned images will be created at CCHMC, and the 
CRPC will have access to that site. A secure image repository may also be housed at the Data Management 
Coordinating Center of the Rare Diseases Clinical Research Network at the University of South Florida. The 
central review pathologists will establish interobserver and intraobserver variability in their evaluation of 
eosinophils and other pathologic features: The central pathologists will re-count eosinophils in the scanned 
images of biopsies that they previously reviewed at their microscope and compare the peak eosinophil counts 
that they obtained by viewing the scanned images to evaluate the variability in the counts. The CRPC 
pathologists will compare their results amongst themselves and resolve differences via teleconferences in which 
the scanned images are displayed (meetings hosted by GoToMeeting or equivalent sites). Ten percent 
intraobserver and interobserver variability will be considered optimum. Each review pathologist will review 
study slides from subjects enrolled at their own institution (Dr. Collins, CCHMC; Dr. Cappocelli, CHCO; Dr. 
Yang, NW). Relevant biopsy slides from the other participating sites will also be reviewed by the CRPC. The 
workload will be distributed as equally as possible among the CRPC pathologists with the assistance of the 
DMCC. Gastric and/or duodenal biopsies will only be reviewed by local clinical pathologists and will not be 
sent to the CRPC for review, except for cases where a participant’s diagnosis is unclear (for example, when it is 
unclear whether a potential participant does or does not have eosinophilic gastritis or colitis).  In such cases, the 
CRPC will review gastric and/or duodenal slides to confirm the appropriate diagnosis. Subjects may be 
included, without stomach or duodenal biopsies, if there is no clinical history of involvement of stomach or 
duodenum. We hypothesize that HSS will more accurately quantify changes associated with EoE and be 
reversible following each therapeutic intervention. Furthermore, we hypothesize that some patients may show 
partial responses, where a subset of the HSS may be improved earlier or in dissociation with eosinophil levels, 
for example. Finally, we will aim to determine the impact of the various therapeutic interventions on gross 
endoscopic changes, as determined by a recently developed endoscopy scoring system (EREFS) developed by 
Dr. Hirano and colleagues28. 
6.5.1.3 Secondary End-Point Analysis: Biomarkers 
In this sub-aim, we hypothesize that molecular biomarkers will change (improve) with each treatment phase, at 
least in part. Furthermore, we hypothesize that biomarkers may reveal (a) novel insight into therapeutic 
resistance and/or relapse; (b) reveal patient sub-groups not identified by histology; and (c) ultimately prove to 
be readily usable biomarkers in clinical settings and possess personalized medicine value. We will focus on 
molecular profiling of esophageal genes with the Eosinophil Diagnostic Panel (EDP), a set of 94 mRNA 
transcripts that have been established to differentiate EoE from controls (e.g. GERD and normal individuals), 
and to identify exposure and transcriptional signaling to SGCs29. The EDP is typically performed on an extra 


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biopsy taken from the distal esophagus29. If our hypotheses are proven to be correct, this has potential to 
transform monitoring of patients during therapeutic intervention, by identifying molecular markers that have 
therapeutic significance, and also by reducing the need for biopsies, particularly after therapy is initiated. 
Essentially, the associated EDP algorithm renders the raw Ct values of each embedded gene after real-time PCR 
in a way that the upregulated genes and down-regulated genes are summed up individually. A quantitative “EoE 
score” is derived from the ΔCt (normalized to GAPDH) summation to reflect disease severity and for statistical 
analysis. Utilizing different gene sets, EDP is also able to predict steroid exposure and remission status based on 
the same algorithm29. In this case, the EoE score will be calculated at baseline endoscopy and in the post-
treatment biopsies to assess change with treatment. 
6.5.2 AIM 2: Clinical Parameters & Biomarkers That Predict Therapeutic Response 
6.5.2.1 Clinical Phenotypes 
In this sub-aim, we hypothesize that certain patient phenotypes will correlate with response to each intervention 
in the trial. We will assess the relationship between response to 1FED, 4FED, and SGC on age, height, weight, 
BMI Z-score, race, ethnicity, allergic status, participant compliance (defined by diet and SGC usage diaries), 
and screening eosinophil levels as readouts. 
6.5.2.2 Skin Testing 
In this sub-aim, we hypothesize that skin test results (prick testing and atopy patch testing to the four food 
groups) will provide significant predictive value for outcomes in the diet trial phases. While a substantial body 
of work has already been done concerning skin testing, there still remains debate about its practice in managing 
EoE. As such, in this sub-aim, we will take several different approaches compared with prior work. In 
particular, we will be conducting a multi-site trial and have the advantage that we can standardize the skin 
testing procedure, by using agreed upon techniques, including food extracts, skin test reagents, and 
measurement parameters. We will thus be in a good position to dissect discrepant results between centers. 
Furthermore, our trial is unique in that half of the patients will undergo a milk elimination diet as part of the 
first phase. Thus, we will be in a position to focus on the value of skin testing for milk, which is particularly 
attractive as milk is the number one food trigger of EoE30. It is important to point out that although skin testing 
(including patch testing) is now readily performed in EoE, there have been no studies that have assessed the 
local response. For example, it has not yet even been determined if positive responses involve immunological 
reactions so basic cytokine and histological analyses are likely to be fundamentally informative. 
6.5.2.3 Component Testing 
Increased levels of food-specific IgE are present in patients with EoE yet their predictive value in guiding food 


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elimination diets for disease treatment is limited31. Recently, it has been proposed that the inability of food 
specific IgE tests to predict clinical reactivity may related in part to the use of allergen extract reagents that do 
not contain the full set of relevant allergens. This diagnostic limitation has spurred the development of 
molecular diagnostic tests, referred to as component-resolved diagnostics (CRD), which utilize purified native 
or recombinant allergens to detect IgE sensitivity to individual allergen molecules; this also has the advantage 
as interactions between IgE sensitization and clinical reactivity can be examined, such as food-pollen cross-
reactivity32-35. One of the goals of the present study is to examine the potential value of CRD in predicting diet 
outcomes in EoE. To address this goal, we will subject a baseline serum sample to CRD ImmunoCap 
(ThermoFischer Scientific); includes 3 egg components (Gal d 1 [ovomucoid], Gal d 2 [ovalbumin], and Gal d 3 
[ovotransferrin]), and 4 milk components (Bos d 4 [alpha lactoglobulin], Bos d5 [beta lactoglobulin], Bos d8 
[casein] and Bos d lactoferrin) as well as other foods and pollens when indicated. We will initially focus on the 
correlation between the presence of positive milk component (>0.1 kUA/L) and responsiveness in the group 
doing 1FED (milk elimination diet) because (a) this will provide proof-of-principle for other foods; (b) milk is 
the most common food that shows positive IgE in EoE patients36; and (c) all patients in this group will undergo 
sole milk elimination and the results should be definitive concerning whether milk is indeed driving disease 
induction. With the wider range of CRD data, we will perform principal component analysis (PCA) to dissect 
the key allergens that are contributing to most of the variation. Similar to the way we process the EDP dataset, 
we also plan to derive personal heat-diagrams and present the CRD data in reviewable format by dimensionality 
reduction, such as multi-dimensional scaling (MDS) analysis. These approaches are expected to prioritize the 
key drivers in the component results and multiple statistical analyses can be subsequently investigated. For cost 
savings, serum samples from all cohorts will be analyzed in batches so we can determine if any trends are 
present before we analyze all samples. 
6.5.2.4 Predictive Biomarker-Analysis Of EoE Transcriptome 
In this sub-aim, we aim to identify biomarkers that may predict responsiveness to any or all of the treatment 
interventions associated with this trial. Determining such a biomarker would have a profound effect on disease 
management as patients could be quickly triaged into different therapies, e.g. diet versus SGC; thus saving time, 
reducing the number of endoscopies and potentially improving quality of life issues. As such, in this sub-aim, 
we will perform the EDP analysis on baseline biopsies, focused on identifying transcript levels that differentiate 
patients that respond to each of the interventions. In preliminary studies, we have conducted this type of 
analysis on patients that recently participated in the randomized controlled trial of high dose fluticasone (1760 
mcg per day) for the treatment of EoE. Forty-two participants were enrolled in this study (28 FP, 14 placebo). 
After 3 months, 65% of FP-treated and 0% of placebo-treated participants had complete remission (P = 0.0001); 


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12% of FP-treated and 8% of placebo-treated participants had partial remission. Of the FP-treated participants 
in complete remission, 73% continued in complete remission, and 20% were in partial remission after halving 
the daily FP dosage37. 
 
Baseline esophageal gene expression was compared between FP responders and non-responder patients by 
statistically screening two cohorts of samples before the FP exposure, namely the FP responders and FP-non-
responders. A total of 15 genes (Figure 1, p < 0.05, fold change > 2.0) on the EDP were identified with a 
tendency to predict subsequent FP efficacy with CR criteria (≤1 eosinophil/HPF in the distal esophagus). It is 
interesting to note that some of these transcripts include cardinal EoE transcriptome genes such as CCL26 
(eotaxin-3) and periostin and CLC (Charcot-Leyden crystals, a marker of eosinophils) suggesting that elevated 
production was a positive prognostic factor for FP responsiveness; these findings are consistent with a recent 
report suggesting that more severe tissue inflammation including eosinophil levels was associated with FP 
responsiveness38. This preliminary result highlights the potential ability of baseline molecular transcripts to 
differentiate patients but also importance of repeating this analysis with an independent cohort of FP treated 
patient samples, as preliminarily identified genes have not yet passed the false discovery rate (FDR) correction 
due to a relatively small n. It is expected that a replication cohort will bring in a higher power to pass the FDR 
filter. Meanwhile, multiple false-correction methods will be applied to the collective data set, such as the West-
Young and Bonferroni methods. 
 


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6.6 FIGURE 1: Esophageal Transcriptome Analysis 
 
Figure 1. Esophageal Transcriptome Analysis: Total RNA from separately acquired biopsy specimens was subjected to EDP signature analysis. 
(A) The heat diagram depicts the changes after FP exposure in the bidirectional gene dysregulation in each group with reference historical EoE and 
normal (NL) cohorts (n = 15 and 14, respectively), and the samples at screening (no FP exposure) on the left. At the end of phases 1 and 2, expression 
signatures from FP non-responders (NRs), FP responders with partial remission (PRs), and FP responders with complete remission (CRs) are shown 
in the heat diagram (yellow, increased expression; blue, decreased expression). (B) On the basis of the expression levels of the 77 diagnostic genes 
and a dimensionality reduction algorithm, an EoE score reflecting the EoE disease activity was calculated at the end of phase 1 (**P < .001, ***P < 
.0001, 2-tailed Student t-test). (C) To assess the prediction value of the EDP on FP responsiveness, samples at screening (i.e., pre-FP exposure) and 
the placebo samples at the end of phase 1 (i.e., pre-FP exposure) were screened by bioinformatics in the context of their subsequent FP 
responsiveness on the basis of the CR criteria of fewer than 2 eosinophils/HPF in the distal esophagus, where the biopsy specimens were acquired. 
The 15-gene cluster listed shows a potential for FP efficacy prediction with a P value of less than .05 and fold change greater than 2.0. 
 
6.7 End Of Therapy (EOT) And Follow-Up 
The primary efficacy endpoint (e.g. PEESS v2 scoring) will be determined at the end of Phase I & II (weeks 12 
& 25) at which time all participants will undergo EGD with biopsy. Histologic scoring of biopsy samples 
obtained during the end of Phase I endoscopy, will be used to determine the treatment pathway to be followed. 


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Participants who achieve histologic remission at the end of Phase I will have completed the study and will 
resume follow-up care with their primary GI and/or Allergy physician(s). Those whose disease remains active 
in terms of histologic disease activity (eosinophil count ≥15 per HPF) will go on to Phase II (twelve week 
treatment with 4FED for Phase I 1FED non-responders OR SGC for Phase I 4FED non-responders). This was 
modified from the original protocol on the basis of discussion with the PAGs, and their strong interest in 
potentially pursuing a more extensive dietary restriction as a means of achieving symptomatic and histologic 
improvement in disease activity. The study design is shown schematically in Figure 2 below. 
6.8 FIGURE 2: STUDY DESIGN FLOWCHART 
 
Figure 2. Study Design Flowchart: Diagram demonstrating the flow of subjects through the study. Following completion of the screening process 
and EGD #1, subjects undergo randomization into one of the two dietary arms of Phase I. Those subjects who respond histologically following either 
of the initial dietary therapies will exit the study, and the PEESS v2 will be used to score for the presence of symptomatic relief as the primary 
outcome. Those subjects, who histologically fail dietary therapy, will enter Phase II.  Phase I 1FED non-responders will go onto 4FED in Phase II.  
Phase 1 4FED non-responders will be treated with swallowed glucocorticoids. Upon completion of Phase II, EGD #3 would be performed and 
scoring of symptoms with PEESS, and histologic scoring would be performed as seen for those subjects who responded at the end of Phase I.
6.9 Minimization Of Bias And Method Of Treatment Assignment 
All eligible participants will be randomized 1:1 to either the 1FED or 4FED. Randomization will be stratified by 
parameters as determined by our statistician.  Following this 3 month phase I, all patients will undergo EGD 
with biopsy and all those whose disease remains active will move on to Phase II. The sponsor, investigator, 
select study staff, and participant will all have knowledge of the treatment being received. 


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6.10 Expected Duration Of Study And Participant Participation 
Each participant will participate in the study for a maximum of 36 weeks. This includes a maximum twelve 
week screening period, followed by 2 sequential 12-week treatment periods. At the end of each 12-week 
treatment phase there will be a clinic visit and EGD to assess treatment response. Participants whose disease 
goes into remission will complete the trial at the end of Phase I, and the remainder will move on to the next 
treatment phase with more extensive dietary restriction or SGC. Participants will have an initial screening visit, 
followed by clinic visit and EGD at week 12, and (if disease remains active at week 12), a visit to initiate Phase 
II at around week 13, followed by another clinic visit and EGD 12 weeks later at approximately week 25. It is 
estimated that the duration of the entire study—including enrollment, performance of study procedures, data 
collection, analysis, and report writing—will last approximately 2-3 years. 
7 STUDY VISITS AND ASSESSMENTS 
7.1 TABLE 1. STUDY DESIGN AND SCHEDULE OF ASSESSMENTS 
 
 
 


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7.2 Screening (Weeks -12 To -1) 
Participants will be screened as above in Table 1. The screening visit should occur at a maximum of twelve 
weeks prior to the enrollment visit. The research staff member will perform the informed consent process after 
obtaining informed consent, the following study procedures should be performed and documented up to twelve 
Phase
Pre-treatment
Visit Number
1
2
3
4
5
6
7
8
9
10
Visit Description
Screening
Enrollment/
Baseline
Phone 
Visit #1
Phone 
Visit #2
Phone 
Visit #3
EOT or Early 
Withdrawal
Phase 2 
Visit 1
Phone 
Visit #4
Phone 
Visit #5
EOT or Early 
Withdrawal
Week
-12 to -1
0
2
4
11
12
13
14
24
25
Perform Informed Consent
X
Assess Eligibility 
(inclusion/exclusion criteria)
X
X
Obtain medical & dietary history
X
Assess con-meds and/or con-
therapies
X
Vitals (height, w eight, BP, heart 
rate, respiratory rate, 
temperature)
X
X
X
X
X
Physical Exam
X
X
X
Research Biopsies (taken 
during SOC EGD)
Xa
X
Xb
Study Questionnaires
X
X
X
Dietary Questionnaires
X
X
X
X
X
X
Skin testing for allergies (prick 
and patch)
X
Research Lab Samples
EDP, otherc
Component 
Testing
EDP, 
component 
testing, 
otherc
EDP, otherc
Morning cortisol (serum)
X
X
CBC w ith differential
X
X
Pregnancy test (urine)
X
X
X
Assess AEs
X
X
X
X
X
X
X
X
Assess diet and/or con-med 
compliance
X
X
X
X
X
X
X
X
X
Provide food diary
X
X
X
X
X
Provide instructions for diet or 
medication
X
X
Determine compliance w ith 
treatment
X
X
X
X
X (SGC)
Dispense SGC
X
a First SOC EGD used to determine eligibility may be conducted within 12 weeks prior to enrollment visit.
bThis EOT SOC EGD pertains only to Phase 1 Non-Responders.
Each visit window (for visits 2-10) is ±7 days.
Treatment Phase 1
Treatment Phase 2
cOther refers to collection of blood or saliva for future genetic testing.  These samples will only be collected once, but may be collected at any of the visits 


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weeks prior to the enrollment (Week 0) visit: 
 Assess eligibility (inclusion and exclusion criteria) 
 Collect medical, surgical and diet history 
 Vitals (height, weight, blood pressure, heart rate, respiratory rate, temperature) 
 Demographics (age, gender, race) 
 Physical exam performed by a study clinician or qualified staff member 
 Obtain CBC with differential  
 Obtain up to 4 biopsies from the distal and proximal esophagus for research purposes (taken during 
standard of care EGD) 
o One of these esophageal biopsies will be used for EDP 
 Research lab samples (blood or saliva) for future genetic testing may be collected at this visit 
 Assessment of concomitant medications and therapies 
 Provide food diary 
 Pregnancy Test (urine) 
Esophageal biopsies for research may be taken during a participant’s standard of care EGD (SOC EGD). If the 
participant has had a SOC EGD and biopsies performed at a participating site or at an outside institution prior to 
the screening visit (and consent), the results of the EGD may be used to determine eligibility if it falls within the 
twelve-week window prior to enrollment. In these cases, research biopsies and EREFS evaluation will not be 
required at baseline. EGDs performed at an outside institution may be used as baseline if the corresponding 
pathology report and slides are available and the eosinophil counts meet the inclusion criterion of ≥15 
eosinophils/hpf.  In cases where the local pathologist has concerns regarding the eosinophil count, slides may be 
sent to the CRPC for rapid review in order to verify eligibility. These participants will have all other screening 
procedures completed at the in-office, screening visit. 
 
CBC results will be obtained from a participant’s standard of care lab. The amount of blood drawn (clinical plus 
research) will adhere to institutional policy limits. 
 
7.3 Phase I: Enrollment & Dietary Treatment (Week 0) 
All eligible participants will be enrolled into the study at Week 0 if they meet all entry criteria and have 


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completed the screening procedures. If a participant is screened and enrolled within a 5 day time frame study 
procedures that are repeated will only be performed once. At this visit, staff members will: 
 Assess eligibility (inclusion and exclusion criteria) 
 Measure vitals (blood pressure, heart rate, respiratory rate, temperature), height and weight 
 Administer patient and parent-reported outcome questionnaires, which will include a dietary 
questionnaire to measure intake of the food allergens 
 Review food diary (from screening) with subject 
 Assess concomitant medications and/or therapies 
 Serum collection for component-resolved diagnostics (CRD) [serum IgE component testing], IgG4 
 Skin testing for allergies including prick (milk, egg, wheat and soy) and patch testing (milk only). 
 Randomize patients to the 1FED or 4FED diet 
 Provide standardized dietary instructions for 1FED or 4FED 
The participants will be encouraged to call the PI and/or study coordinator if they have any questions, concerns, 
adverse events, or changes in medication. 
7.4 Phone Visits, Phase I 
A study staff member will conduct three phone visits (at weeks 2, 4, and 11) during Phase I in order to monitor 
participants’ progress throughout the trial. The following procedures will occur during these phone visits: 
 Assess con-med and/or con-therapy compliance 
 Assess AEs 
 Determine compliance with treatment 
o A dietary questionnaire to measure intake of the food allergens will be completed by the 
participants during weeks 2 and 11 for therapy compliance assessment 
o A food diary will be completed by participants during weeks 2 and 11 to measure adherence 
7.5 End Of Treatment Phase I Or Early Withdrawal (Week 12) 
For participants who have completed the 12 week Phase I or have withdrawn early from the study, the 
following procedures will occur: 
 Measure vitals (blood pressure, heart rate, respiratory rate, temperature), height and weight 


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 Physical exam performed by study clinician or qualified staff member 
 Administer patient and parent-reported outcome questionnaires 
 Assess AEs 
 Obtain up to 4 biopsies from the distal and proximal esophagus for research purposes (taken during 
standard of care EGD)Research lab samples: 
o Esophageal biopsy for EDP 
o Serum (~2 tsp, or 10 mL) for serum IgE/CRD/IgG4 
o Blood or saliva for future genetic testing may be collected at this visit 
 Obtain CBC with differential (SOC) 
 Assess participant compliance with diet and concomitant medications 
 Pregnancy Test (urine) 
If a participant withdraws from the study before completing 12 weeks of dietary therapy, the end of 
treatment/early withdrawal visit, should be conducted within one week of the participant’s stopping dietary 
therapy. Participants should stay on their dietary therapy until results of biopsies are communicated to them. 
Those participants who have resolution of their inflammation on endoscopy will complete the study at this time 
while those with continued active inflammation will move on to the next phase, which consists of either 4FED 
(for Phase I 1FED non-responders) or swallowed fluticasone therapy at 880 mcg twice a day while back on an 
unrestricted diet (for Phase I 4FED non-responders). 
7.6 Phase II Visit 1 (Week 13) 
Those participants who have completed the Phase I diet protocol but have continued active inflammation will 
return to clinic once their biopsies have been read, within 1 week of their EGD. At this visit, the following 
procedures will occur: 
 Measure vitals (blood pressure, heart rate, respiratory rate, temperature), height and weight 
 Assess AEs 
 Assess participant compliance with concomitant medications 
 Provide standardized instructions for administration of the fluticasone for those moving on to SGC 
 Provide standardized instructions for 4FED to participants who will be on 4FED 
 Provide diet or medication diary/log to participants 


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 Administer dietary questionnaire to all participants to measure intake of the food allergens. Review food 
diary (from Visit #5) for those who will be on 4FED 
 Dispense the fluticasone to the participant for those moving on to SGC 
 Measure morning cortisol (serum, ~1 tsp or 5 mL) for those moving on to SGC 
7.7 Phone Visits, Phase II 
A study staff member will conduct two phone visits (at weeks 14 and 24) during Phase II in order to monitor 
participants’ progress throughout the trial. The following procedures will occur during these phone visits: 
 Assess con-med and/or con-therapy compliance 
 Assess AEs 
 Determine compliance with treatment 
o A dietary questionnaire to measure intake of the food allergens will be completed by the all 
participants during week 14 and 24. The questionnaires will be used  for therapy compliance 
assessment for those on 4FED.  
o A food diary will be completed by participants who are on 4FED during weeks 14 and 24 of the 
trial to measure adherence 
7.8 End Of Treatment Phase II Or Early Withdrawal (Week 25) 
For those participants who have completed the Phase II therapy or have withdrawn early from the study, 
the following procedures will occur: 
 Measure vitals (blood pressure, heart rate, respiratory rate, temperature), height and weight 
 Physical exam performed by a study clinician or qualified staff member 
 Administer patient and parent-reported outcome questionnaires 
 Assess AEs 
 Obtain up to 4 biopsies from the distal and proximal esophagus for research purposes (taken during SOC 
EGD) 
 Assess participant compliance with concomitant medications/therapies 
 Assess participant compliance with therapy (SGC) 
 Pregnancy Test (urine) 
 Measure morning cortisol (serum, ~1 tsp, or 5 mL) for those treated with SGC 
 Research lab samples 


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o Esophageal biopsy for EDP 
o Blood or saliva for future genetic testing may be collected at this visit 
If a participant withdraws from the study before completing this 12 weeks of SGC or 4FED therapy, the end of 
treatment/early withdrawal visit should be conducted within one week of the participant’s stopping therapy. 
Participants should stay on SGC or 4FED until results of biopsies are communicated to them. 
Following the receipt of their biopsy results, the patients will return to standard clinical care. 
7.9 Pregnancy 
Should pregnancy occur during either phase of the study, dietary therapy and/or dosing of study medication will 
be discontinued and the participant will be withdrawn from the study. The pregnancy will be followed clinically 
to term. 
7.10 Unscheduled Visits 
Unscheduled visits will take place for any complication or AE/SAE that requires an extra visit. The procedures 
to be performed at the unscheduled visits will be at the discretion of the PI. Procedures that may be performed 
at an unscheduled visit include: 
 Vital signs (blood pressure, heart rate, respiratory rate, temperature), height and weight 
 Physical exam 
 Assess AEs 
 Lab tests 
 Assess/address problems with dietary adherence 
These visits will be documented in the unscheduled visit case report. 
8 SELECTION AND WITHDRAWAL OF PARTICIPANTS 
8.1 Participant Inclusion Criteria 
At study enrollment, subjects will be eligible if they: 
 Have diagnosis of EoE (based on consensus criteria)3 
 Are aged 6 to 17 years 
 Have histologically confirmed active disease >15 eosinophils/hpf in either distal or proximal esophagus 
within 12 weeks of enrollment visit 
 PPI confirmation 


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 Symptomatic (have experienced symptoms within the last month prior to enrollment) 
 Has a negative urine pregnancy test at screening if of childbearing potential. Females of childbearing 
potential must have a negative urine pregnancy test (β-hCG) prior to enrollment into the study (i.e., at 
screening). Subsequently, these participants must agree to use adequate birth control measures (e.g., 
condom, oral/injectable/subcutaneous contraceptives, intrauterine device, or sexual abstinence) during 
the study and for at least one month after the last dose of study drug which will be documented in the 
source documents. 
8.2 Participant Exclusion Criteria 
Subjects will be excluded if they: 
 Have been treated with topical swallowed steroids within the last 2 months or systemic steroids within 
the past 3 months 
 Have eosinophilia in segments of the GI tract other than the esophagus 
 Have been diagnosed with a GI malabsorption disorder (i.e., Inflammatory bowel disease, Crohn’s 
disease) or Celiac disease 
 Are currently on dietary therapy avoiding milk (i.e. on a 1FED), or milk, egg, soy, and wheat (i.e. on a 
4FED) 
 Have concurrent H pylori gastritis or parasitic infection 
 Are unable to obtain EGD with esophageal biopsies at CCHMC or other participating institution within 
4 weeks of study completion 
 Have previously failed (in a clinical trial setting) dietary therapy with one of these regimens or topical 
steroid treatment with fluticasone at a total dose of 1760 mcg per day. 
 Have definitely responded (in a clinical trial setting) to either dietary therapy avoiding these antigens or 
to swallowed fluticasone at a total dose of 1760 mcg per day 
 Are concurrently receiving any of the prohibited medications listed in Table 2 
 On immunotherapy for pollen (if not on maintenance therapy) or IgE-mediated food allergy. 
 Not fluent in English 
9 PARTICIPANT WITHDRAWAL CRITERIA 
An intent to treat approach will be used. All data acquired prior to termination for the reasons outlined below 
will be included in the primary analysis unless patient withdraws consent. Every effort will be made to conduct 


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a final study visit with the participant and participants will be followed clinically until, if applicable, all adverse 
events resolve.  
• 
Withdrawal of consent 
• 
Withdrawal by the participant 
• 
Withdrawal by the investigator 
• 
Intercurrent illness or event that precludes further visits to the study site or ability to evaluate disease 
(e.g.-mental status change, large pleural effusion). 
• 
Participants will be discontinued from the study if they are unable to comply with the dietary 
restrictions or develop severe complications from their EoE (e.g., esophageal strictures).  
9.1 Replacement Of Participants 
Enrolled participants who start on the initial dietary protocol and prematurely discontinue/withdraw from the 
study will be replaced if they do not reach Week 12 of the study including the EGD. Additional participants will 
be enrolled in the same manner as all other participants. Participant numbers will not be re-used. Any enrolled 
participant will be included into any intent to treat analysis. 
9.2 Study Site Termination Criteria 
The study can be terminated or stopped at the site for reasons including but not limited to: 
1. Investigator or sponsor request to withdraw from study participation. 
2. Serious and/or persistent noncompliance by the investigator with the protocol or other local applicable 
regulatory guidelines in conducting the study. 
3. IRB or DSMB decision to terminate or suspend approval for the investigation or the investigator. 
The DSMB will promptly inform the Principal Investigator and Sponsor conducting the study if the study is 
suspended or terminated for safety reasons, and will also inform the regulatory authorities of the suspension or 
termination of the study and the reason(s) for the action.  The investigator must inform the IRB promptly and 
provide the reason for the suspension or termination. 
 
9.3 Study Discontinuation 
The sponsor and local IRBs (at their local site) have the authority to stop or suspend this trial at any time. This 
study may be suspended or closed if: 


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 Early stopping rules have been met  
 Accrual has been met 
 The study objectives have been met 
 The Study Chair / Study Investigators believe it is not safe for the study to continue 
 The sponsor suspends or closes the trial 
 The FDA suspends or closes the trial 
 
10 TREATMENT OF PARTICIPANTS AND DESCRIPTION OF STUDY INTERVENTION 
We intend to conduct a 2-phase randomized prospective, non-blinded trial (each phase is 3 months in duration) 
to assess changes in patient reported outcomes in pediatric patients with EoE that utilizes one of two minimally 
restrictive dietary therapy regimens followed by topical swallowed steroid therapy (for 4FED non-responders), 
which is intended to provide data that will inform clinical management practices. 
 
Empiric dietary therapy interventions for Phase I will consist of either a 1-food elimination (milk elimination 
alone) or a 4-food elimination (milk, egg, soy, and wheat) diet. Phase I 1FED non-responders will receive 4FED 
in Phase II, whereas Phase I 4FED therapy non-responders will receive topical swallowed steroid therapy 
(swallowed fluticasone at a dose of 880 mcg twice a day) for 3 months in Phase II. 
10.1 Concomitant Medications 
Throughout the study duration, all participants are expected to maintain medications such as a PPI, oral or nasal 
allergy medications such as antihistamines and any asthma-related medications that were prescribed prior to 
study entry. 
10.2 TABLE 2. Prohibited Medications And Washout Time 
Medication 
Washout Time Prior to Screening Visit 
(i.e., Study Entry) 


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Anti-immunoglobulin E [IgE] mAb 
Methotrexate, cyclosporine, interferon-α 
Anti-tumor necrosis factor [TNF] mAb) 
Other systemic immunosuppressive or 
immunomodulation agents 
Oral or intravenous systemic steroids 
Topical swallowed steroids 
Other investigative biologic 
Anti-IL 5 agents 
Anti-IL 13 agents 
Other investigative drugs or device 
6 months 
3 months 
6 months 
3 months 
 
3 months 
2 months 
1 month 
3 months 
6 months 
1 month 
 
10.3 Treatment Compliance 
A participant will be considered compliant if he/she has remained adherent to the study dietary 
recommendations. Adherence to the fluticasone phase will be based on receiving at least 80% of prescribed 
doses. All non-compliance issues will be documented. 
 
10.4 Study Intervention Administration-Diet And Medication Instruction 
All participants will receive standardized written dietary instructions with regards to the study dietary 
interventions. In addition, study dietitians will be available to answer questions from participants and their 
families as they arise. Similarly, all patients entering Phase II who will take SGC will receive standardized 
instruction on fluticasone administration. 
10.5 Efficacy Evaluation 
We will calculate and compare efficacy for each dietary therapy (1FED or 4FED) as well as for fluticasone 
therapy for dietary non-responders. 
10.6 Primary Efficacy Endpoint: 
 Pediatric EoE Symptom Score (PEESS, version 2). The PEESS total score change from pre-treatment to 
post-treatment is the primary efficacy endpoint. 
10.7 Secondary Efficacy Endpoints: 


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 Patient Related Outcome (PRO) metrics pre- and post-treatment 
o PedsQL EoE Module 
o PROMIS General Health Questionnaire and PedsQL 
o Individual domain PEESS scores 
 Histology and endoscopy measures 
o Percent of participants who achieve histologic remission (<15 peak eosinophils/HPF) post therapy 
assessed after the end of the 3 months 
o Division of patients into complete (peak eosinophils≤1/HPF) or partial (≤6 or ≤10) remission 
o Histology Scoring system 
o Endoscopic scoring system (EREFS) 
 Biomarkers pre- and post-treatment 
o Assessment of Eosinophil Diagnostic Panel EoE Score 
 Clinical parameters and biomarkers to predict responsiveness to diet and SGC therapy 
o Correlation of clinical characteristics to response to therapy 
o Assessment of predictive value of skin prick and patch allergy skin testing 
o Correlation between presence of positive milk component allergy testing to response to 1FED 
o Predictive biomarker-analysis of EoE transcriptome from EDP analysis on baseline biopsies in 
correlation to response to treatment. 
11 ASSESSMENT OF SAFETY 
11.1 Safety Parameters 
The Study Chair has primary oversight responsibility of this clinical trial and the Data Safety Monitoring Plan 
(DSMP) for this clinical trial. The Study Chair will review accrual, patterns and frequencies of all adverse 
events, and protocol compliance every 6 months.  
Safety will be assessed through documentation of AEs, adverse drug reactions, physical examination findings 
and vital signs, and pre- and post-treatment morning cortisol for those patients on SGC in Phase II. Each site’s 
Principal Investigator and their research team (co-Investigators, research nurses, clinical trial coordinators, and 
data managers) are responsible for identifying adverse events. Aggregate reports - detailed by severity, 
attribution (expected or unexpected), and relationship to the study drug/study procedures – will be available 
from the DMCC for site review.  Adverse events will be reviewed twice a year by the research team.   A 
separate report detailing protocol compliance will also be available from the DMCC for site review on a 


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monthly basis. The research team will then evaluate whether the protocol or informed consent document 
requires revision based on the reports. 
11.1.1 Data Safety Monitoring Board 
The Data Safety Monitoring Board (DSMB) in conjunction with the Sponsor will develop and adopt a Charter 
which will delineate: 
 Monitoring the safety of study participants by reviewing the incidence of clinically significant AEs; 
 Monitoring compliance with the study protocol, especially as it relates to participant  safety and the 
integrity of the study; 
 Study stopping criteria as they relate to study drug-related AEs; 
 Reporting the committee’s deliberations to the Sponsor following scheduled and ad hoc meetings; 
The DSMB members will review and approve the DSMB charter which defines the membership of the board, 
frequency of meetings, and roles and responsibilities. 
The DSMB will meet: 
 Twice during the conduction of the study (in person or via conference call), at a minimum; and 
 On an ad hoc basis for safety concerns, per the DSMB Chair  
The DSMB will be provided access to the safety data including AEs, study withdrawals, and SAEs.  The DSMB 
may also review data on study progress including participant enrollment and disposition, especially as related to 
discontinuation at study site. 
11.2 Adverse Events 
An adverse event is defined as any adverse experience (an unfavorable and unintended sign, symptom or 
disease) —including side effect, injury, toxicity, sensitivity reaction, intercurrent illness, or sudden death—
whether or not it is considered related to the use of the study agent, that occurs any time after the participant 
signs informed consent/assent (Screening or Enrollment study visit) and will end after the cessation of the study 
agent (Week 29 [30 days after the last dose of the study agent]). This includes the onset of new illness and the 
exacerbation of pre-existing conditions other than the indication under study (i.e., EoE). A pre-existing 
condition is one that is present at the start of the study and is reported as part of the participant’s medical 
history. A pre-existing condition should be reported as an AE only if its frequency, intensity or character 
worsens during study treatment. Participants (parents or legal guardians) should be instructed to report all AEs 


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to the investigator. All AEs must be appropriately documented in the participant’s original source documents 
and on the CRF. Investigators should report syndromes rather than list symptoms. All AEs will be assessed for 
severity and relationship to the study agent. Expected EoE symptoms, including vomiting, regurgitation, 
abdominal pain, chest pain/heartburn, or dysphagia are not to be considered as AEs, except if they show a clear 
temporal relationship to the study agent administration or result in hospitalization. Hospitalization is to be 
reported as an SAE. 
 
Serious adverse events include those events that:  “result in death; are life-threatening; require inpatient 
hospitalization or prolongation of existing hospitalization; create persistent or significant disability/incapacity, 
or a congenital anomaly/birth defects.”  
 
An unexpected adverse event is defined as any adverse experience…the specificity or severity of which is not 
consistent with the risks of information described in the protocol.  
 
Expected adverse events are those that are identified in the research protocol as having been previously 
associated with or having the potential to arise as a consequence of participation in the study 
 
All reported adverse events will be classified using the current version of the Common Terminology Criteria for 
Adverse Events (CTCAE) developed and maintained by CTEP at National Cancer Institute. 
11.3 TABLE 3. Grading Of Adverse Events 
The severity of each AE will be categorized using the following criteria: 
Mild  
(Grade 1) 
Awareness of sign, symptom or event, but easily tolerated; requires no special 
treatment and does not interfere with the participant’s daily activities. 
Moderate 
(Grade 2) 
Discomfort enough to cause interference with usual activity and may warrant 
intervention. 
Severe  
(Grade 3) 
Incapacitating with inability to do usual activities or significantly affects 
clinical status, and warrants intervention. 
Life-threatening 
(Grade 4) 
Places the participant, in the view of the investigator, at immediate risk of 
death from the reaction as it occurred, (i.e. it does not include a reaction that, 
had it occurred in a more severe form, might have caused death). 


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Death 
(Grade 5) 
Death related to AE. 
 
 
 


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11.4 TABLE 4. Relationship To Study Treatment 
The investigator must assess the relationship of any AE to the use of the study agent, based on the available 
information, using the following guidelines: 
Definitely not 
related 
The event is definitely not associated with the study agent administration, and is 
judged clearly due to causes other than the study agent. 
Probably not related 
An event that follows a temporal sequence from administration of the study agent 
such that a relationship is not likely, and could be reasonably explained by the 
participant’s clinical state or other modes of therapy administered to the participant . 
Possibly related 
An event that follows a reasonable temporal sequence from administration of the 
study agent, but that may be due to another cause and could also be reasonably 
explained by the participant’s clinical state or other modes of therapy administered 
to the participant. 
Probably related 
An event that follows a reasonable temporal sequence from 
administration of the study agent, that is not easily explained by another cause such 
as known characteristics of the participant’s clinical state or other treatment, and is 
confirmed by improvement on stopping or slowing administration of the study agent 
(de-challenge), if applicable. 
Definitely related 
 
In the event of death, the cause of death should be recorded as the AE. “Death” is an outcome and is NOT the 
AE. The only exception is “sudden death” when the cause is unknown. 
11.5 Serious Adverse Event 
The investigator is required to determine if each AE is a SAE. An SAE is any AE occurring between signing 
informed consent/assent and within 30 days after administration of the last dose of the study agent, that results 
in any of the following outcomes 
(International Conference on Harmonization [ICH] Guide (E6) for GCP, 1996): 
 Death; 
 A life-threatening adverse drug experience; 
 Inpatient hospitalization or prolongation of existing hospitalization, or 
 A persistent or significant disability/incapacity; 
 Congenital anomaly/birth defect. 
Important medical events that may not result in death, be life-threatening, or require hospitalization may be 


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considered SAEs when, based upon appropriate medical judgment, they may jeopardize the participant and may 
require medical or surgical intervention to prevent one of the outcomes listed in this definition. Examples of 
such medical events include allergic bronchospasm requiring intensive treatment in an emergency room or at 
home, blood dyscrasias or convulsions that do not result in inpatient hospitalization, or the development of drug 
dependency or drug abuse. Reports of all SAEs will be communicated to the appropriate IRB and reported in 
accordance with local laws and regulations. 
11.6 Recording Adverse Events 
All AEs will be recorded in the participant’s source documents and CRFs. The onset and end dates, severity, 
and relationship to the study agent will be recorded for each AE. Any action or outcome (e.g., hospitalization, 
discontinuation of the study agent, etc.) will be recorded for each AE. 
11.7 Reporting Adverse Events 
Participants (or parents or legal guardians) will be questioned and/or examined by the investigator or designee 
for evidence of AEs. The questioning of participants (or parents or legal guardians) with regard to the possible 
occurrence of AEs should be generalized such as: “How have you (or how has your child) been feeling since 
your last visit?” 
 
Additional sources for AEs include, but are not limited to: direct observation, voluntary, spontaneous comments 
from the participant (parent or legal guardian), and clinically significant laboratory, physical examination, vital 
sign findings, or other test results. The investigator will exercise his/her medical and scientific judgment in 
deciding whether an abnormal clinical laboratory finding or abnormal assessment is clinically significant. 
The presence or absence of specific AEs will not be solicited. Participants who experience AEs will be 
monitored with relevant clinical assessments and clinical laboratory tests as determined by the investigator. All 
AEs will be followed to satisfactory resolution or stabilization. Any actions taken and follow-up results will be 
recorded on the appropriate CRFs, as well as in the participant’s source documentation. Follow-up laboratory 
results will be filed with the participant’s source documentation. Non-serious AEs that occur from the time of 
consent/assent until 30 days after administration of the last dose of study agent will be reported. Any non-
serious AEs will be considered as “continuing” if not resolved at this time. SAEs that occur from the time of 
informed consent/assent until 30 days after administration of the last dose of study agent will be reported. All 
SAEs should be followed until resolution or stabilization. For the purpose of reporting AEs, reporting will begin 
after informed consent/assent and will end after the cessation of the study agent [30 days after the last dose of 


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study agent]). Occasionally, AEs that occur during a study are unresolved at the time the participant’s study 
participation ends. AEs are to be followed to resolution; until they resolve, disappear, or become stable up to 30 
days. Note that any events which occur prior to the start of study agent are considered as medical history and 
should be recorded as such on the medical history screen of the CRF. 
 
SAEs will be reported to regulatory authorities per 21 CFR 312.32 and institutional policy. Adverse events will 
be reported, in summary form, at the time of continuing annual review to the IRB, FDA, and DSMB. 
11.8 Reporting Serious Adverse Events To Sponsor 
 
Within 24 hours (of learning of the event), investigators must report any reportable Serious Adverse Event 
(SAE) that: 
 Is considered life-threatening/disabling or results in death of subject 
-OR- 
 Is Unexpected/Unanticipated  
 
Investigators must report all other reportable SAEs within 5 working days (of learning of the event).  All other 
(suspected) reportable AEs must be reported to the RDCRN within 20 working days of the notification of the 
event or of the site becoming aware of the event. Local institutional reporting requirements to IRBs, any GCRC 
oversight committee and the FDA, if appropriate, remain the responsibility of the treating physician and the 
Study Chair. 
 
The research personnel should document the SAE on the source documents and CRFs. 
The SAE information will include the following (as available): 
 Participant identification number, site, investigator name; date site was notified of event 
 SAE information: event term, onset date, event SOC, severity, local of event treatment, and casual 
relationship to study agent; 
 Basic demographic information (e.g., age, sex, weight); 
 The outcome(s) attributable to the event (death, a life-threatening adverse drug experience, inpatient 
hospitalization or prolongation of existing hospitalization, a persistent or significant 


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disability/incapacity, congenital anomaly/birth defect, or other important medical event); 
 A summary of relevant test results, pertinent clinical laboratory data, and any other relevant medical 
history; 
 The first and last dates of the study agent administration; 
 A statement whether the study agent was discontinued or the study agent administration was modified; 
 A statement whether or not the SAE recurred after reintroduction of the study agent if administration 
had been discontinued or withheld; 
 Supplemental information may include the following hospital records: clinical laboratory results, 
radiology reports, progress notes, admission and emergency room notes, holding/observation notes, 
discharge summaries, autopsy reports, and death certificate. 
The SAE should be reported to the Sponsor within the expected reporting period with as much of the above 
information as available at that time. All initial and follow-up SAEs should be reported to the Sponsor via the 
DMCC AE reporting system. For the purpose of reporting SAEs, reporting will begin after informed 
consent/assent is signed and will end after the cessation of the study agent (Week 29 [30 days after the last dose 
of study agent]). Occasionally, SAEs that occur during a study are unresolved at the time the participant’s study 
participation ends. SAEs are to be followed to resolution; until they resolve, disappear, or become stable. It is 
important for the investigator to realize that SAEs may occur in a study participant after the study follow-up 
period is complete, but which still merit reporting to the sponsor. The investigator together with the sponsor will 
try to determine if the SAE is related to the study agent. 
11.9 DMCC Adverse Event Data Management System (AEDAMS) 
 
Upon entry of a serious adverse event, the DMCC created Adverse Event Data Management System 
(AEDAMS) will immediately notify the Study Chair, site PIs, the  Medical Review Officer, and any additional 
agencies (if applicable- industry sponsor, CTEP, etc) of any reported adverse events via email.  
 
Serious adverse events: The Medical Review Officer (MRO) determines causality (definitely not related, 
probably not related, possibly related, probably related, definitely related) of the adverse event. The MRO and, 
if applicable, sponsor may request further information if necessary and possibly request changes to the protocol 
or consent form as a consequence of the adverse event. A back-up notification system is in place so that any 


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delays in review by the MRO beyond a specified period of time are forwarded to a secondary reviewer. The 
Adverse Event Data Management System (AEDAMS) maintains audit trails and stores data (and data updated) 
and communication related to any adverse event in the study.  
 
Non-serious expected adverse events: Except those listed above as immediately reportable, non-serious 
expected adverse events  that are reported to or observed by the investigator or a member of his/her research 
team will be submitted to the DMCC in a timely fashion (within 20 working days). The events will be presented 
in tabular form and given to the MRO on a bi-annual basis. Local site investigators are also required to fulfill all 
reporting requirements of their local institutions. 
 
The DMCC will post aggregate reports of all reported adverse events for site investigators and IRBs. 
 
12 STATISTICAL METHODS 
12.1 Sample Size Calculation 
Sample size was determined based on the primary endpoint of the trial: The overall PEESS score change from 
pre-treatment. Based on a preliminary report18 we estimate that 40-50% of patients may respond to 1FED as our 
study is prospective compared with 65% response in this preliminary retrospective report and based on food 
reintroduction trial results30. Likewise, we would expect 75% of patients to respond to 6FED based on the 
previously reported studies, and from this we extrapolated a projected response of 65% to 4FED, given that the 
4 eliminated foods are consistently identified as the most common triggering antigens30. As such, we estimate 
that 45% of patients will respond to 1FED and wish to detect an additional 20% increase for the 4FED (i.e. 65% 
response rate). A non-parametric Wilcoxon rank sum test will be applied for the primary efficacy endpoint 
comparison. We are assuming a difference in response rate of 45% vs 65% between the two treatment groups, 
and we further assume that those who respond to 1FED will naturally respond to 4FED. We also assume that 
those who responded will have a higher PEESS score change than those who do not respond. Then the 
estimated probability of 4FED scores higher than 1FED is about 0.60 ( = 0.5 x prob. of response to both or 
neither + 1.0 x incremental response rate from 1FED to 4FED). This will require 131 patients per group to reach 
80% power at 0.05 test level. By accounting for a 10% early dropout rate, we propose a sample size of 146 per 
group, or 292 total patients to be enrolled. 
 


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12.2 Statistical Analyses: Aim 1 
Demographic and patient characteristics will be summarized for the two treatment groups using mean ± 
standard deviation or median [interquartile range] for continuous variables and frequency and percentages for 
categorical variables. Treatment comparisons will be performed using the two-sample t-test or Wilcoxon Rank 
sum test for continuous variables and the Fisher’s Exact test for categorical variables. All tests will be 
conducted at α=0.05, including the primary and secondary analyses. 
 
There will be two sets of analysis populations in terms of efficacy assessment, the intent-to-treat and per 
protocol. All patients randomized to Phase I of the study who have at least one clinical observation post 
randomization will be considered in intent-to-treat set.  Patients who are at least 80% compliant to dietary 
assignment during Phase I, and who do not have any major protocol violations will be considered in per 
protocol set. If there is more than 10% difference in patient numbers between these two populations, the 
primary efficacy analysis will be performed on both intent-to-treat analysis and per protocol populations. 
Otherwise, all analyses will be based on intent-to-treat population.  Patients who do drop out of the trial before 
the end of Phase I will have their PEESS done at the time of dropout.  
 
The primary endpoint is overall PEESS score change from pre-treatment to the end of Phase I. If normality 
assumption is approximately valid, a linear mixed effect model will be applied to the primary efficacy variable, 
account for site random effect, and other potentially important covariates, e.g. gender, race, and PPI use etc. 
Pertaining to the specific aim, the comparison of response (PEESS change from baseline) will be made between 
1FED and 4FED groups via the above model.  Because of non-normal nature of the scores observed in previous 
research, a Wilcoxon rank sum test may be applied to compare between two treatment groups when normality is 
violated. 
 
As a secondary endpoint analysis, remission rates (as defined by the percentage of participants achieving 
histologic remission (eosinophils less than 15/HPF) at the end of Phase I will be compared between the two 
treatment groups using the generalized linear mixed effects model with the logit link (for binary outcome) that 
accounts for the clustering within sites. Important covariates will be analyzed in this model including gender, 
race, PPI use, atopy, age, and anthropomorphic features. Treatment group comparisons for secondary endpoints 
will be performed using the generalized linear mixed effects model using the link that is appropriate for the 
endpoint (i.e. logit for binary, log for Poisson count, identity for continuous). These models will account for the 


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clustering within sites and will include the same covariates as for the primary analysis. The remission rate for 
non-responders who proceed to Phase II will be summarized separately depending on the treatment patients 
received during Phase I and Phase II. The 95% confidence interval for the rates will be provided. 
12.3 Statistical Analyses: Aim 2 
To evaluate the ability of biomarkers and phenotypic characteristics to predict response to therapeutic 
intervention, multivariable logistic regression analysis will be conducted with histological evaluation of 
remission/no remission as the dependent variable and biomarker levels as the independent variables. Multi-
collinearity of the independent variables will be assessed. The area under the receiver operating characteristic 
curve (AUROC) will be estimated along with 95% confidence interval. This analysis will be conducted based 
on the remission status at the end of Phase I. It will be repeated at the end of Phase II by including those patients 
who also responded to SGC. 
 
A sample size of 292 total patients will provide at least 80% power to detect an AUROC of 0.60. This 
calculation is based on the assumption that 161 patients (66 1FED, 95 4FED) will be in remission at the end of 
Phase I (and therefore not move on to phase II) and 131 patients will not be in remission (with an estimated 
10% dropping out and not continuing on to Phase II). 
 
Additionally, a correlation analysis between PEESS scores and biomarkers will be performed.  Spearman’s 
correlation coefficient and its confidence intervals will be computed to further assess predictive utility for the 
biomarkers of interest. 
12.4 Direct Access To Source Date/Documents 
Data generated by this study must be available for inspection by any regulatory authorities, by the Sponsor, 
DSMB, and the IRB as appropriate. 
13 INVESTIGATOR REQUIREMENTS 
13.1 Protocol Adherence 
The investigator must adhere to the protocol as detailed in this document and agree that the Sponsor and the 
IRB must approve any change to the protocol. The investigator will be responsible for enrolling only those 
participants who have met the protocol screening and study entry criteria. 
13.2 Case Report Forms 


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The CRFs will be used for the recording of all information and study data as specified by this protocol. The 
CRFs must be completed by the research personnel. The principal investigator is responsible for ensuring that 
accurate CRFs are completed in a timely manner. 
13.3 Source Document Maintenance 
Source documents are defined as the results of original observations and activities of a clinical investigation. 
Source documents may include, but are not limited to, study progress notes, e-mail correspondence, computer 
printouts, clinical laboratory data, and recorded data from automated instruments. All source documents 
produced in this study will be maintained by the investigator and made available for inspection by regulatory 
authorities. The original signed dated informed consent form for each participating participant shall be filed 
with records kept by the investigator and a copy given to the participant (parent or legal guardian, as 
appropriate). 
The patient and parent-reported outcome questionnaires will be recorded directly on the paper forms and will be 
considered as source data. 
13.4 Study Completion 
Before a study can be considered completed or terminated, the investigator must have the following data and 
materials: 
 Clinical laboratory findings, clinical data, and all special test results from screening through the EOT 
visit (to 30 days after the last dose of study agent). 
 CRFs properly completed by appropriate study personnel and reviewed and approved by the 
investigator. 
 Copies of protocol amendment(s) and IRB approval/notification if appropriate. 
 A summary of the study prepared by the principal investigator (an IRB/IEC summary letter is 
acceptable). 
13.5 Audits And Inspections 
The principal investigator will permit study-related monitoring, audits, IRB review, and regulatory inspections 
by providing direct access to CRFs and source data/documents. 
13.6 Institutional Review Board Approval 
This protocol, the informed consent document, and all relevant supporting data must be submitted to the IRB 
for approval. IRB approval of the protocol, informed consent document, study materials and any advertisement 


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(if applicable) used to recruit study participants must be obtained before the study may be initiated. 
The principal investigator (PI) is responsible for keeping the IRB advised of the progress of the study and of 
any changes made to the protocol as deemed appropriate, but in any case, at least once a year. The PI is also 
responsible for notifying the IRB of any unanticipated AEs that occur during the study in accordance with local 
IRB policies. Recent guidance from the USA FDA suggests that the following AEs should be reported to the 
IRB/IEC as “unanticipated problems:” 
 Any AE that, even without detailed analysis, represents a serious unexpected AE that is rare in the 
absence of drug exposure (such as agranulocytosis, hepatic necrosis, Stevens-Johnson syndrome). 
 A series of AEs that, on analysis, is both unanticipated and a problem for the study. There would be a 
determination that the series of AEs represents a signal that the AEs were not just isolated occurrences 
and were significant to the rights and welfare of participants. 
 An AE that is described or addressed in the IB/package insert, protocol, or informed consent documents, 
or is expected to occur in study participants at an anticipated rate (e.g., expected progression of disease, 
occurrence of events consistent with background rate in participant population), but occurs at a greater 
frequency or at greater severity than expected. 
 Any other AE that would cause the sponsor to modify the investigator brochure, study protocol, or 
informed consent form, or would prompt other action by the IRB to assure protection of human 
participants. 
It will be the responsibility of the investigator to assure that the essential documents are available at the 
investigator site. Any or all of these documents may be subject to, and should be available for, audit by 
CCHMC or Sponsor auditor and inspection by the regulatory authorities as defined in the monitoring plan. 
14 ETHICS 
14.1 Ethics Review 
The investigator will obtain approval to conduct the study from the appropriate regulatory agency in accordance 
with any applicable country and local-specific regulatory requirements prior to initiating the study. 
14.2 Ethical Conduct Of The Study 
This study will be conducted in compliance with the ethical principles of the Declaration of Helsinki, and any 
additional national or IRB-required procedures. 
The investigator is responsible for ensuring that this protocol, the site’s informed consent form, and any other 


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information that will be presented to potential participants/parents or legal guardians (e.g. advertisements or 
information that supports or supplements the informed consent) are reviewed and approved by the IRB. The 
investigator agrees to allow the IRB direct access to all relevant documents. The IRB must be constituted in 
accordance with all applicable regulatory requirements. The investigator will provide the IRB with relevant 
document(s)/data that are needed for approval of the study. 
14.3 Written Informed Consent And Assent 
This study will be conducted in compliance with ICH E6 GCP: Consolidated Guidelines pertaining to informed 
consent and assent. At the first visit, prior to initiation of any study-related procedures, participants (parents or 
legal guardians, as appropriate) will give their written consent to participate in the study after having been 
informed about the nature and purpose of the study, participation/termination conditions, and risks and benefits. 
If the participant is unable to provide written informed consent, the participant’s legally acceptable 
representative may provide written consent as approved by institutional specific guidelines. The informed 
consent document must be signed and dated by the participant, or the participant’s legally authorized 
representative, prior to study participation. A copy of the informed consent document must be provided to the 
participant or the participant’s legally authorized representative. Signed consent forms must remain in the 
participant’s study file and be available for verification by the monitor, IRB, and/or regulatory authorities at any 
time. If participant’s legally acceptable representative provides written consent, participants will also give their 
written assent to participate in the study, in accordance with institutional requirements. 
15 DATA HANDLING AND RECORDKEEPING 
15.1 Inspection Of Records 
Data generated by this study must be available for inspection by any regulatory authorities, and by the IRB as 
appropriate. At a participant’s/parent’s or legal guardian’s request, medical information may be given to his or 
her (child’s) personal physician or other appropriate medical personnel responsible for his or her (child’s) 
welfare. Participant medical information obtained during the course of this study is confidential and disclosure 
to third parties other than those noted above is prohibited. 
15.2 Retention Of Records 
The investigator shall retain records required to be maintained under this part for a period of 2 years. 
15.3 Data Management 


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All study data will be collected via systems created in collaboration with the Data Management and 
Coordinating Center and will comply with all applicable guidelines regarding patient confidentiality and data 
integrity. 
 
15.4 Registration 
Registration of participants on this protocol will employ an interactive data system in which the clinical site will 
attest to the participant’s eligibility as per protocol criteria and obtain appropriate informed consent. IRB 
approval for the protocol must be on file at the DMCC before accrual can occur from the clinical site. 
 
The DMCC will use a system of coded identifiers to protect participant confidentiality and safety.  Each 
participant enrolled will be assigned a local identifier by the enrollment site.  This number can be a combination 
of the site identifier (location code) and a serial accession number.  Only the registering site will have access to 
the linkage between this number and the personal identifier of the subject.  When the participant is registered to 
participate in the study, using the DMCC provided web-based registration system, the system will assign a 
participant ID number.  Thus each participant will have two codes: the local one that can be used by the 
registering site to obtain personal identifiers and a second code assigned by the DMCC.  For all data transfers to 
the DMCC both numbers will be required to uniquely identify the subject.  In this fashion, it is possible to 
protect against data keying errors, digit transposition or other mistakes when identifying a participant for data 
entry since the numbers should match to properly identify the participant.  In this fashion, no personal 
identifiers would be accessible to the DMCC. 
15.5 Data Entry 
Data collection for this study will be accomplished with online electronic case report forms.  Using encrypted 
communication links, on-line forms will be developed that contain the requisite data fields.   
15.6 Data Quality and Monitoring Measures 
As much as possible data quality is assessed at the data entry point using intelligent on-line data entry via visual 
basic designed screen forms. Data element constraints, whether independent range and/or format limitations or 
‘relative’ referential integrity limitations, can be enforced by all methods employed for data input. QA reports 
assess data quality post-data entry. As we note, data quality begins with the design of the data collection forms 
and procedures and incorporates reasonable checks to minimize transcription and omission errors.  Of the more 
important quality assurance measures are the internal validity checks for reasonableness and consistency. 


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• 
Data Monitoring: The DMCC identifies missing or unclear data and generates a data query to the 
consortium administrator contact.  
• 
Data Delinquency Tracking: The DMCC will monitor data delinquency on an ongoing basis.   
 
15.7 Laboratory Data Flow 
The DMCC will provide laboratories with on-line forms and/or electronic data exchange mechanisms - 
depending on their capabilities and needs - to enter, update and obtain relevant data.  On-line forms exist to 
verify specimen receipt, report specimen issues and submit test results for specimens.  The preferred method to 
exchange data electronically is through the Specimen Management System Web Service.  The Web Service 
allows laboratories to obtain specimen shipment information, receive individual specimens or specimen 
shipments, report specimen issues and communicate specimen aliquots in a secure manner (test result 
submission is planned).  The DMCC will also support uploading of files electronically.  All transactions are 
logged and validated for both methods. 
16 REFERENCES 
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2. Martin LJ, Franciosi JP, Collins MH, Abonia JP, Lee JJ, Hommel KA, et al. Pediatric Eosinophilic 
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4. Cheung KM, Oliver MR, Cameron DJ, Catto-Smith AG, Chow CW. Esophageal eosinophilia in children 
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5. Straumann A. The natural history and complications of eosinophilic esophagitis. Gastrointest Endosc Clin 
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6. Faubion WA, Jr., Perrault J, Burgart LJ, Zein NN, Clawson M, Freese DK. Treatment of eosinophilic 
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9. Liacouras CA, Spergel JM, Ruchelli E, Verma R, Mascarenhas M, Semeao E, et al. Eosinophilic 
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10. Aceves SS, Dohil R, Newbury RO, Bastian JF. Topical viscous budesonide suspension for treatment of 
eosinophilic esophagitis. J Allergy Clin Immunol 2005; 116:705-6. 
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blind, placebo-controlled trial of fluticasone propionate for pediatric eosinophilic esophagitis. 
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12. Schaefer ET, Fitzgerald JF, Molleston JP, Croffie JM, Pfefferkorn MD, Corkins MR, et al. Comparison of 
oral prednisone and topical fluticasone in the treatment of eosinophilic esophagitis: a randomized trial in 
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13. Straumann A, Conus S, Degen L, Frei C, Bussmann C, Beglinger C, et al. Long-term budesonide 
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14. Spergel JM, Brown-Whitehorn TF, Cianferoni A, Shuker M, Wang ML, Verma R, et al. Identification of 
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15. Henderson CJ, Abonia JP, King EC, Putnam PE, Collins MH, Franciosi JP, et al. Comparative dietary 
therapy effectiveness in remission of pediatric eosinophilic esophagitis. J Allergy Clin Immunol 2012; 
129:1570-8. 
16. Kagalwalla AF, Sentongo TA, Ritz S, Hess T, Nelson SP, Emerick KM, et al. Effect of six-food 
elimination diet on clinical and histologic outcomes in eosinophilic esophagitis. Clin Gastroenterol Hepatol 
2006; 4:1097-102. 
17. Spergel JM, Andrews T, Brown-Whitehorn TF, Beausoleil JL, Liacouras CA. Treatment of eosinophilic 
esophagitis with specific food elimination diet directed by a combination of skin prick and patch tests. Ann 
Allergy Asthma Immunol 2005; 95:336-43. 
18. Kagalwalla AF, Amsden K, Shah A, Ritz S, Manuel-Rubio M, Dunne K, et al. Cow's milk elimination: a 
novel dietary approach to treat eosinophilic esophagitis. J Pediatr Gastroenterol Nutr 2012; 55:711-6. 
19. Kagalwalla AF, Shah A, Li BU, Sentongo TA, Ritz S, Manuel-Rubio M, et al. Identification of specific 
foods responsible for inflammation in children with eosinophilic esophagitis successfully treated with 
empiric elimination diet. J Pediatr Gastroenterol Nutr 2011; 53:145-9. 
20. Pentiuk S, Putnam PE, Collins MH, Rothenberg ME. Dissociation between symptoms and histological 
severity in pediatric eosinophilic esophagitis. J Pediatr Gastroenterol Nutr 2009; 48:152-60. 
21. Alexander JA, Jung KW, Arora AS, Enders F, Katzka DA, Kephardt GM, et al. Swallowed fluticasone 
improves histologic but not symptomatic response of adults with eosinophilic esophagitis. Clin 
Gastroenterol Hepatol 2012; 10:742-9 e1. 
22. Franciosi JP, Hommel KA, Greenberg AB, DeBrosse CW, Greenler AJ, Abonia JP, et al. Development of 
the Pediatric Quality of Life Inventory Eosinophilic Esophagitis module items: qualitative methods. BMC 
Gastroenterol 2012; 12:135. 
23. Franciosi JP, Hommel KA, Bendo CB, King EC, Collins MH, Eby MD, et al. PedsQL eosinophilic 
esophagitis module: feasibility, reliability, and validity. J Pediatr Gastroenterol Nutr 2013; 57:57-66. 
24. Fiorentino R, Liu G, Pariser AR, Mulberg AE. Cross-sector sponsorship of research in eosinophilic 
esophagitis: a collaborative model for rational drug development in rare diseases. J Allergy Clin Immunol 
2012; 130:613-6. 
25. Gonsalves N, Yang GY, Doerfler B, Ritz S, Ditto AM, Hirano I. Elimination diet effectively treats 
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26. Lucendo AJ, Arias A, Gonzalez-Cervera J, Yague-Compadre JL, Guagnozzi D, Angueira T, et al. Empiric 
6-food elimination diet induced and maintained prolonged remission in patients with adult eosinophilic 
esophagitis: a prospective study on the food cause of the disease. J Allergy Clin Immunol 2013; 131:797-
804. 
27. Rothenberg ME, Aceves S, Bonis PA, Collins MH, Gonsalves N, Gupta SK, et al. Working with the US 
Food and Drug Administration: progress and timelines in understanding and treating patients with 
eosinophilic esophagitis. J Allergy Clin Immunol 2012; 130:617-9. 
28. Hirano I, Moy N, Heckman MG, Thomas CS, Gonsalves N, Achem SR. Endoscopic assessment of the 
oesophageal features of eosinophilic oesophagitis: validation of a novel classification and grading system. 
Gut 2013; 62:489-95. 
29. Wen T, Stucke EM, Grotjan TM, Kemme KA, Abonia JP, Putnam PE, et al. Molecular diagnosis of 
eosinophilic esophagitis by gene expression profiling. Gastroenterology 2013; 145:1289-99. 
30. Greenhawt M, Aceves SS, Spergel JM, Rothenberg ME. The management of eosinophilic esophagitis. J 
Allergy Clin Immunol Pract 2013; 1:332-40; quiz 41-2. 
31. Ishimura N, Furuta K, Sato S, Ishihara S, Kinoshita Y. Limited role of allergy testing in patients with 
eosinophilic gastrointestinal disorders. J Gastroenterol Hepatol 2013; 28:1306-13. 
32. Cox L. Overview of serological-specific IgE antibody testing in children. Curr Allergy Asthma Rep 2011; 
11:447-53. 
33. Simons FE, Ardusso LR, Bilo MB, Dimov V, Ebisawa M, El-Gamal YM, et al. 2012 Update: World 
Allergy Organization Guidelines for the assessment and management of anaphylaxis. Curr Opin Allergy 
Clin Immunol 2012; 12:389-99. 
34. Fung I, Kim JS, Spergel JM. Relating microarray component testing and reported food allergy and food-
triggered atopic dermatitis: a real-world analysis. Ann Allergy Asthma Immunol 2013; 110:173-7 e1. 
35. Treudler R, Simon JC. Overview of component resolved diagnostics. Curr Allergy Asthma Rep 2013; 
13:110-7. 
36. Erwin EA, James HR, Gutekunst HM, Russo JM, Kelleher KJ, Platts-Mills TA. Serum IgE measurement 
and detection of food allergy in pediatric patients with eosinophilic esophagitis. Ann Allergy Asthma 
Immunol 2010; 104:496-502. 
37. Butz BK, Wen T, Gleich GJ, Furuta GT, Spergel J, King E, et al. Efficacy, dose reduction, and resistance to 
high-dose fluticasone in patients with eosinophilic esophagitis. Gastroenterology 2014; 147:324-33 e5. 
38. Boldorini R, Mercalli F, Oderda G. Eosinophilic oesophagitis in children: responders and non-responders to 
swallowed fluticasone. J Clin Pathol 2013; 66:399-402. 
 

SAP_001.pdf: 
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Statistical Analysis Plan 
Eosinophilic Esophagitis (EoE) Intervention Trial-Randomized 1 Food Elimination vs. 4 Food Elimination Diet 
followed by Swallowed Glucocorticoids 
PROTOCOL NUMBER: 
7805 
STUDY DESIGN: 
 
Prospective, non-blinded clinical trial 
 
PRINCIPAL   
 
Marc E. Rothenberg, MD, PhD 
INVESTIGATOR:  
Cincinnati Children’s Hospital Medical Center 
 
 
 
 
Division of Allergy & Immunology 
 
 
 
 
3333 Burnet Avenue, MLC 7028 
 
 
 
 
Cincinnati, OH 45229 
 
 
 
 
Phone: (513) 636-6675 
 
Biostatistician:  
 
Lin Fei, PhD 
 
 
 
 
Division of Biostatistics and Epidemiology 
1 
INTRODUCTION 
 
Pertaining to the objectives of the protocol, this statistical analysis plan (SAP) specifies, prior to finalization of 
study database, a comprehensive description of strategies and statistical techniques that are planned to assess 
clinical efficacy and safety of 1 food elimination (milk elimination alone, 1FED) versus 4 food elimination 
(milk, egg, wheat, and soy elimination, 4FED) diet intervention of Eosinophilic Esophagitis (EoE), and the 
following Glucocorticoids treatment. 
 
The primary objective of this protocol is to conduct a prospective, non-blinded randomized trial comparing 
patient reported outcomes scores in novel empiric elimination dietary therapies in eosinophilic esophagitis 
(EoE) in order to assess the therapeutic viability of minimally restrictive empiric elimination diets as related to 
measures of patient’s symptoms, quality of life, and pathology.  The primary efficacy outcome will be the 
change in the standardized and validated Pediatric Eosinophilic Esophagitis Symptom Score v2 (PEESS). 
 


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Moreover, we also aim to assess the response to topical swallowed steroids in those who were non-responders 
to empiric dietary therapy regimens used during the initial phases of this study.  
A variety of secondary endpoints, including pre- and post-therapy peak eosinophil counts per hpf and 
percentages of patients that attain histologic remission (<15 eosinophils/hpf), along with detailed histologic and 
endoscopic scores, novel biomarkers, and patient related outcome (PRO) measures will be analyzed between 
treatment groups.  
 
In addition, we will assess the possible value of a variety of clinical characteristics and biomarkers in terms of 
predicting response to therapy.  Relationship between PEESS response and histologic response will be 
investigated as well to better understand PRO measures in predicting disease status. 
 
 
2 
STUDY DESIGN 
 
This trial is comprised of 2 phases with each phase lasting 3 months. In the first phase, all participants will be 
randomized 1:1 to either 1FED or 4FED therapeutic diet. At the end of this phase, an EGD will be performed to 
assess histologic remission, which represents one of the primary current tools used in evaluating EoE disease 
activity during clinical trials and daily EoE care. Attainment of remission (esophageal eosinophil counts <15 
eosinophils/high powered field) after Phase I will result in study discontinuation and maintenance of the 
“successful” dietary therapy. Dietary therapy non-responders who were on 4FED during Phase I will receive 
topical swallowed steroids for 3 months while on an unrestricted diet (Phase 2) followed by EGD with 
esophageal biopsies.  Participants receiving SGC will return to an unrestricted diet (i.e. stop 4FED) prior to 
initiating SGC therapy. Dietary therapy non-responders who were on 1FED during Phase 1 will proceed to 
4FED for 3 months (Phase 2) followed by EGD with esophageal biopsies.   
 
3 
STUDY OBJECTIVES 
The purpose of this study is to evaluate the following objectives: 
1.1 Primary Objective 
To perform a prospective, randomized, non-blinded trial that determines patient reported outcomes scores 


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following 1FED (milk elimination alone) vs. 4FED (milk, egg, wheat, and soy avoidance) and their relative 
efficacy (Phase I). 
 The primary endpoint for this study will be the change in PEESS scores from baseline to post therapy 
assessed after the end of the 3 month Phase I. 
1.2 Secondary Objectives 
 To extend Phase I of this study with a prospective non-blinded trial that determines the rate of remission 
after treatment with 4FED (in participants failing 1FED in Phase I) or SGC (in participants failing 4FED 
in Phase I) (Phase II). 
 To evaluate the effect of each therapy on histological remission (defined as a post therapy eosinophil 
count of <15 eosinophils/HPF) and by a variety of changes in eosinophils, including (a) pre- and post-
therapy peak eosinophil counts; (b) partial remission (2-14 peak eosinophils/hpf); and (c) complete 
histological remission (≤ 1 peak eosinophils/hpf). 
 To evaluate the effect of each therapy by utilizing the histology scoring system (HSS) created to express 
the severity and extent of other abnormalities in the gastrointestinal (GI) tract that often accompany 
eosinophilic inflammation. 
 To determine the impact of each therapeutic intervention on biomarkers using the EoE Diagnostic Panel 
(EDP). 
 To evaluate the clinical and psychosocial effect of each therapy using the Peds Quality of Life Inventory 
Eosinophilic Esophagitis Module (PedsQL EoE) to assess EoE symptoms and problems/feelings related 
to eating. 
 To determine if any clinical parameters predict response to therapeutic intervention. 
 To determine if any biomarkers including component-resolved diagnostics (CRD) [serum IgE 
component testing] predict response to therapeutic intervention. 
 To determine if skin testing, in the form of prick and patch testing, predicts response to therapeutic 
intervention. 
 Also to assess relationship between primary PEESS score changes and histological scores (HSS). 
4 
STUDY PARTICIPANTS 
4.1 Number of Participants 
A total of 292 participants between the ages of 6-17 years were to be enrolled.  Because of unexpectedly slow 


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enrollment, the actual number of participants will be around 50 total.  The DMCC at the University of South 
Florida Health Informatics Institute will serve as the central data management center, with CCHMC serving as 
the primary site and the central IRB. Additional sites include: Children’s Hospital of Colorado, Riley Hospital 
for Children, Children’s Hospital of Philadelphia, University of California at San Diego/Rady Children’s 
Hospital, Northwestern University/Lurie Children’s Hospital, Tufts Medical Center, Mt. Sinai Kravis 
Children’s Hospital, Arkansas Children’s Hospital, and the University of North Carolina. 
 
5 
ANALYSIS VARIABLES 
 
5.1 Primary Efficacy Endpoint: 
 Pediatric EoE Symptom Score (PEESS, version 2). The PEESS total score change from pre-treatment to 
post-treatment is the primary efficacy endpoint. The Parent Report for Children and Teens PEESS will 
be used as the primary. 
The PEESS total score comprises of two types: frequency and severity. A total of 11 frequency questions 
and 9 severity questions are there respectively.  The raw scores of 0 to 4 will be converted to 0 to 100 by 
multiplying each score by 25.  The total metric score is calculated as the mean of non-missing items from 
the 20 total possible. If more than 50% (10 items) are missing for a particular patient on a visit, the score 
will not be calculated, rather set as missing.   
There are four domains: Dysphagia, GERD, Nausea/vomiting, and Pain. Domain specific scores are 
calculated similarly. 
Dysphagia:  
How often does your child have trouble swallowing? 
How bad is your child’s trouble swallowing? 
How often does your child feel like food gets stuck in his/her throat or chest? 
How bad is it when your child gets food stuck in his/her throat or chest? 
How often does your child need to drink a lot to help swallow food? 
How bad is it when your child needs to drink a lot to help swallow food? 
How often does your child eat less than others? 
How often does your child need more time to eat than others? 
GERD: 


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How often does your child have heartburn (burning in the chest, mouth, or throat)? 
How bad is your child’s heartburn (burning in the chest, mouth, or throat)? 
How often does your child have food come back up in his/her throat when eating? 
How bad is it when food comes back up in your child’s throat? 
Nausea/vomiting: 
How often does your child vomit (throw up)? 
How bad is your child’s vomiting (throwing up)? 
How often does your child feel nauseous (feel like throwing up but does not)? 
How bad is your child’s nausea (feeling like throwing up, but does not)? 
Pain: 
How often does your child have chest pain, ache, or hurt? 
How bad is your child’s chest pain, ache, or hurt? 
How often does your child have stomach aches or belly aches? 
How bad are your child’s stomach aches or belly aches? 
5.2 Secondary Efficacy Endpoints: 
 Patient Related Outcome (PRO) metrics pre- and post-treatment 
o PedsQL EoE Module 
o Generic Health Related Quality of Life measures 
o PROMIS General Health Questionnaire and PedsQL 
o Individual domain PEESS scores 
 Histology and endoscopy measures 
o Percent of participants who achieve histologic remission (<15 peak eosinophils/HPF) post therapy 
assessed after the end of the 3 months 
o Division of patients into complete (all peak eosinophils≤1/HPF) or partial (the worse of 3 scores is 
from 2 to 14) remission, and non-remission (at least one > 14/HPF) 
o Histology Scoring system 
o Endoscopic scoring system (EREFS) 
 Biomarkers pre- and post-treatment 
o Assessment of Eosinophil Diagnostic Panel EoE Score 
 Clinical parameters and biomarkers to predict responsiveness to diet and SGC therapy 


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o Correlation of clinical characteristics to response to therapy 
o Assessment of predictive value of skin prick and patch allergy skin testing 
o Correlation between presence of positive milk component allergy testing to response to 1FED 
o Predictive biomarker-analysis of EoE transcriptome from EDP analysis on baseline biopsies in 
correlation to response to treatment. 
5.2.1.1 Secondary End-Point Analysis: Biomarkers 
In this sub-aim, we hypothesize that molecular biomarkers will change (improve) with each treatment phase, at 
least in part. Furthermore, we hypothesize that biomarkers may reveal (a) novel insight into therapeutic 
resistance and/or relapse; (b) reveal patient sub-groups not identified by histology; and (c) ultimately prove to 
be readily usable biomarkers in clinical settings and possess personalized medicine value. We will focus on 
molecular profiling of esophageal genes with the Eosinophil Diagnostic Panel (EDP), a set of 94 mRNA 
transcripts that have been established to differentiate EoE from controls (e.g. GERD and normal individuals), 
and to identify exposure and transcriptional signaling to SGCs28. Essentially, the associated EDP algorithm 
renders the raw Ct values of each embedded gene after real-time PCR in a way that the upregulated genes and 
down-regulated genes are summed up individually. A quantitative “EoE score” is derived from the ΔCt 
(normalized to GAPDH) summation to reflect disease severity and for statistical analysis. Utilizing different 
gene sets, EDP is also able to predict steroid exposure and remission status based on the same algorithm28. In 
this case, the EoE score will be calculated at baseline endoscopy and in the post-treatment biopsies to assess 
change with treatment. 
 
The change in “EoE score” will be analyzed between responders and non-responders based on histology 
assessment (<15 <15 peak eosinophils/HPF) at the end of Phase I via an ANOVA model, after proper 
transformation of the score, e.g. logarithmic if needed.  If normality assumption is seriously violated, a non-
parametric method of Wilcoxon test will be applied. 
 
 
5.2.2 AIM 2: Clinical Parameters & Biomarkers That Predict Therapeutic Response 
5.2.2.1 Predictive Biomarker-Analysis Of EoE Transcriptome 
In this sub-aim, we aim to identify biomarkers that may predict responsiveness to any or all of the treatment 


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interventions associated with this trial. As such, in this sub-aim, we will perform the EDP analysis on baseline 
biopsies, focused on identifying transcript levels that differentiate patients that respond to each of the 
interventions. 
 
Baseline esophageal gene expression was compared between responders and non-responder patients by 
statistically screening two cohorts. A list of genes with p < 0.05 (or FDR<0.05), fold change > 2.0 will be 
identified. 
 


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5.3 End of Therapy (EOT) And Follow-Up 
 
The primary efficacy endpoint (e.g. PEESS v2 scoring) will be determined at the end of Phase I & II (weeks 12 
& 25) at which time all participants will undergo EGD with biopsy. Histologic scoring of biopsy samples 
obtained during the end of Phase I endoscopy, will be used to determine the treatment pathway to be followed. 
Participants who achieve histologic remission at the end of Phase I will have completed the study and will 
resume follow-up care with their primary GI and/or Allergy physician(s). Those whose disease remains active 
in terms of histologic disease activity (eosinophil count ≥15 per HPF) will go on to Phase II (twelve week 
treatment with 4FED for Phase I 1FED non-responders OR SGC for Phase I 4FED non-responders).  
 
 
6 
STATISTICAL METHODS 
 
6.1 Statistical Analyses: Aim 1 
Demographic and patient characteristics will be summarized for the two treatment groups using mean ± 
standard deviation or median [interquartile range] for continuous variables and frequency and percentages for 
categorical variables. Treatment comparisons will be performed using the two-sample t-test or Wilcoxon Rank 
sum test for continuous variables and the Fisher’s Exact test for categorical variables. All tests will be 
conducted at α=0.05, including the primary and secondary analyses. 
 
There was going to be two sets of analysis populations in terms of efficacy assessment, the intent-to-treat and 
per protocol. However, since the total enrollment has been reduced to about 50, all patients randomized to Phase 
I of the study who have at least one clinical observation post randomization will be considered in intent-to-treat 
set.  Only this set of population will be included in the statistical analyses.  Originally, we plan to define 
patients who are at least 80% compliant to dietary assignment during Phase I, and who do not have any major 
protocol violations as in per protocol set. Now all analyses will be based on intent-to-treat population.  Patients 
who do drop out of the trial before the end of Phase I will have their PEESS done at the time of dropout. The 
primary endpoint is overall PEESS score change from pre-treatment to the end of Phase I. If normality 
assumption is approximately valid, a linear mixed effect model will be applied to the primary efficacy variable, 


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account for site random effect, and other potentially important covariates, e.g. gender, race, and PPI use etc. 
Because of non-normal nature of the scores in previous research, a Wilcoxon rank sum test may be applied to 
compare between two treatment groups when normality is violated. 
 
As a secondary endpoint analysis, remission rates (as defined by the percentage of participants achieving 
histologic remission (eosinophils less than 15/HPF) at the end of Phase I will be analyzed in the following 3 
steps. 
(1) Remission rates will be computed along with their 95% CIs for each diet group separately;  
(2) Remission rates will be compared between the two treatment groups using the generalized linear mixed 
effects model with the logit link (for binary outcome) that accounts for the clustering within sites. Important 
covariates will be analyzed in this model including gender, race, PPI use, atopy, age, and anthropomorphic 
features. Treatment group comparisons for secondary endpoints will be performed using the generalized linear 
mixed effects model using the link that is appropriate for the endpoint (i.e. logit for binary, log for Poisson 
count, identity for continuous). These models will account for the clustering within sites and will include the 
same covariates as for the primary analysis.  
(3) The remission rate for non-responders who proceed to Phase II will be summarized separately depending on 
the treatment patients received during Phase I and Phase II. The 95% confidence interval for the rates will also 
be provided. 
 
6.2 Statistical Analyses: Aim 2 
 
To evaluate the ability of biomarkers and phenotypic characteristics to predict response to therapeutic 
intervention, multivariable logistic regression analysis may be conducted with histological evaluation of 
remission/no remission as the dependent variable and biomarker levels as the independent variables. Multi-
collinearity of the independent variables will be assessed. The area under the receiver operating characteristic 
curve (AUROC) will be estimated along with 95% confidence interval. This analysis will be conducted based 
on the remission status at the end of Phase I. It may also be repeated at the end of Phase II by including those 
patients who also responded to SGC. 
 
 


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Additionally, a correlation analysis between PEESS scores and biomarkers will be performed.  Spearman’s 
correlation coefficient and its confidence intervals will be computed to further assess predictive utility for the 
biomarkers of interest. 
 
Missing data will be handled in the following manner.  For the primary efficacy measure of PEESS, each 
patient is expected to complete the questionnaire; even if the patient terminates participation early (dropout) for 
any reason.  The total PEESS score is the average of 20 item scores, as long as there are at least 10 item 
response non-missing, as described previously.  No imputation of missing total PEESS will be performed.  The 
early dropout reasons will be recorded, and rates will be assessed between treatment groups for potential 
patterns.  
For all other secondary efficacy and demographic variables, missing data will not be imputed.  However, if a 
participant’s biomarker panel is partially missing, multiple imputation may be applied for missing markers in 
predictive modeling analyses.  
 
Heterogeneity of treatment effect is referred to as the variability in the direction and magnitude of individual or 
subgroup treatment effects that are non-random. Descriptive heterogeneity of treatment effect analyses will first 
be carried out for subgroups by gender, by study site, and by age of subject. These analyses are included in the 
analysis plan above for primary and secondary efficacy analyses, where proper estimates of treatment effect will 
be extracted. Because of the small sample size in this study, any hypothesis testing of treatment heterogeneity is 
considered exploratory. P-values generated from multiple comparisons are not adjusted. The goal is to identify 
possible heterogeneity among subgroups for further confirmation. The interaction of demographic and baseline 
characteristics with treatment group effects may be tested in the generalized linear mixed effects models to 
assess heterogeneity.