Assessment of Glucocorticoid Responsiveness and Mechanisms of Resistance in Hypereosinophilic Syndromes
Phase: PHASE4
Conditions: Eosinophilia, Hypereosinophilic Syndrome, Leukocyte Disorder, Hematologic Diseases
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

Assessment of Glucocorticoid Responsiveness and Mechanisms of Resistance in 
Hypereosinophilic Syndromes
NIH Protocol Number:
12-I-0026
Sponsored by: 
National Institute of Allergy and Infectious Diseases (NIAID)
Principal Investigator:
Paneez Khoury MD, LPD, NIAID, NIH
Laboratory of Parasitic Diseases
National Institute of Allergy and Infectious Diseases
Building 10, Rm 12C103
9000 Rockville Pike
Bethesda, MD 20892
Phone: (301) 402-3673
E-mail: paneez.khoury@nih.gov
Version Number: 
V13
Day Month Year
September 04, 2020
Investigational Agent:
Drug Name:
Prednisone (under 
multiple trade names)
IND Number:
N/A (exempt)
Sponsor:
N/A
Drug Supplied By:
NIH Pharmacy

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Table of Contents
STATEMENT OF COMPLIANCE .....................................................................................4
List of Abbreviations .........................................................................................................5
Protocol Summary ............................................................................................................6
Précis................................................................................................................................7
1.
Introduction & Background ........................................................................................8
1.1.
Rationale ............................................................................................................8
1.2.
Background ........................................................................................................8
1.2.1.
Inappropriate Absorption of Glucocorticoids ...............................................9
1.2.2.
Alterations in Glucocorticoid Receptor function ..........................................9
1.2.3.
Glucocorticoid effects on trafficking and eosinophil transcriptomics.........10
2.
Study Objectives......................................................................................................11
2.1.
Primary Objective.............................................................................................11
2.2.
Secondary Objectives ......................................................................................11
2.3.
Exploratory Objectives .....................................................................................11
3.
Study Design and Methods .....................................................................................11
3.1.
Description of the Study Design.......................................................................11
3.2.
Screening .........................................................................................................11
3.3.
Baseline Visit....................................................................................................12
3.4.
Procedures and Laboratory Testing.................................................................12
3.5.
Study Endpoints ...............................................................................................13
3.5.1.
Primary Endpoint.......................................................................................13
3.5.2.
Secondary Endpoint..................................................................................14
3.5.3.
Exploratory Endpoints...............................................................................14
4.
Inclusion and Exclusion Criteria ..............................................................................14
4.1.
Subject Inclusion Criteria..................................................................................14
4.2.
Subject Exclusion Criteria ................................................................................15
5.
Monitoring and Withdrawal......................................................................................15
5.1.
Clinical Evaluations ..........................................................................................15
5.2.
Laboratory Evaluations.....................................................................................15
5.3.
Follow-up..........................................................................................................15
5.4.
Early Termination Visit .....................................................................................16
5.5.
Re-contact of Subjects after Study Termination...............................................16
5.6.
Premature Withdrawal of a Subject..................................................................16
6.
Analysis Plan...........................................................................................................16
6.1.
Description of the Analyses..............................................................................16
6.1.1.
Appropriate Methods and Timing for Analyzing Outcome Measures........17
6.1.2.
Addressing Study Objectives ....................................................................17
6.2.
Sample Size Justification .................................................................................18
6.3.
Final Analysis Plan...........................................................................................18
6.4.
Interim Analysis Plan........................................................................................19
7.
Human Subjects Protection.....................................................................................20
7.1.
Study Population ..............................................................................................20
7.1.1.
Rationale for Subject Selection.................................................................20
7.1.2.
Recruitment Plan.......................................................................................20

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7.2.
Justification for Eligibility of Special Populations..............................................21
7.2.1.
Exclusion of Pregnant Women..................................................................21
7.2.2.
Exclusion of Children Weighing <48 kg ....................................................21
7.2.3.
Inclusion of Children Weighing ≥48 kg......................................................21
7.3.
Potential Risks..................................................................................................21
7.4.
Potential Benefits .............................................................................................22
7.5.
Informed Consent Process...............................................................................22
7.5.1.
Assent of Children.....................................................................................22
7.5.2.
Consent for minors when they reach the age of majority..........................23
7.5.3.
Telephone Consent Process.....................................................................23
7.6.
Subject Confidentiality......................................................................................24
8.
Adverse Event Reporting Plan ................................................................................24
8.1.
Specification of Safety Parameters ..................................................................24
8.2.
Reporting Procedures ......................................................................................24
8.3.
Type and Duration of the Follow-up of Subjects after Adverse Events............25
8.4.
Assessing Adverse Events...............................................................................25
8.5.
Relationship Assessment.................................................................................25
9.
Data and Safety Monitoring and Management Plan................................................26
9.1.
Study Records Retention .................................................................................26
10.
Protocol Monitoring Plan......................................................................................27
11.
Plan for Research Use and Storage of Human Samples ....................................27
11.1.
Intended Use ................................................................................................27
11.2.
Storage and Tracking ...................................................................................28
11.3.
Disposition at the Completion of the Protocol...............................................28
11.4.
Reporting Loss or Destruction of Samples/Specimens/Data to the IRB.......28
12.
Remuneration Plan for Subjects ..........................................................................28
Appendix A: Definition of Hypereosinophilic Syndrome .................................................29
Appendix B: Schedule of Procedures/Evaluations .........................................................30
Appendix C: Adult Blood Volumes for Specimen Collection...........................................31
Appendix D: Pediatric Blood Volumes for Specimen Collection.....................................32
Appendix E: Steroid Taper Schedule .............................................................................34
Appendix F: Exploratory Endpoints Timeline..................................................................35
Appendix G: Figures.......................................................................................................36
Scientific References......................................................................................................38

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STATEMENT OF COMPLIANCE
The trial will be carried out in accordance with International Council for Harmonisation 
Good Clinical Practice (ICH GCP) and the United States (US) Code of Federal 
Regulations (CFR) applicable to clinical studies (45 CFR Part 46, 21 CFR Part 50, 
21 CFR Part 56, 21 CFR Part 312, and/or 21 CFR Part 812) 
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who 
are responsible for the conduct, management, or oversight of NIH-funded clinical trials 
have completed Human Subjects Protection and ICH GCP Training.
The protocol, informed consent form(s), recruitment materials, and all participant 
materials will be submitted to the Institutional Review Board (IRB) for review and 
approval. Approval of both the protocol and the consent form must be obtained before 
any participant is enrolled. Any amendment to the protocol will require review and 
approval by the IRB before the changes are implemented to the study. In addition, all 
changes to the consent form will be IRB-approved; a determination will be made 
regarding whether a new consent needs to be obtained from participants who provided 
consent, using a previously approved consent form.

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List of Abbreviations
ACR
American College of Rheumatology
AE
Adverse Event/Adverse Experience
AEC
Absolute Eosinophil Count
AF
Activation Function (AF-1, AF-2)
CBC
Complete Blood Count
COPD
Chronic Obstructive Pulmonary Disease
CRF
Case Report Form
CRIMSON
Clinical Research Information Management System of the NIAID
ERR
Eosinophil Response Rate
EGID
Eosinophilic Gastrointestinal Disease
FIP1L1
FIP1-like1
F/P
FIP1L1-PDGFR
GC
Glucocorticoid
GR
Glucocorticoid Receptor
GRE
Glucocorticoid Response Element
HES
Hypereosinophilic Syndrome
IL
Interleukin
IRB
Institutional Review Board
LFT
Liver function test
N
Number (typically refers to the number subjects or the sample size)
OHSR
Office of Human Subjects Research 
OCRPRO
Office of Clinical Research Policy and Regulatory Operations
PBMC
Peripheral Blood Mononuclear Cells
PDGFR
Platelet-derived growth factor 
PHI
Protected Health Information
PI
Principal Investigator
RNA PAX
Ribonuclease PAXgene blood tube
SAE
Serious Adverse Event/Serious Adverse Experience

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Protocol Summary
Full Title: 
Assessment of Glucocorticoid Responsiveness and 
Mechanisms of Resistance in Hypereosinophilic Syndromes
Short Title:
Glucocorticoid responsiveness in HES
Principal Investigator: 
Paneez Khoury
Sample Size: 
N=40
Accrual Ceiling: 
40
Study Population:
100 children and adults, ages 7-100
Accrual Period: 
6-13 years
Study Design: 
Single blinded clinical trial
Study Duration: 
14 years
Start Date: 
Oct 2011 
End Date: 
Dec 2026
Primary Objective:
To develop a model to determine whether a single dose 
glucocorticoid (GC) challenge can be used to predict GC 
responsiveness in subjects with HES 
Secondary Objectives:
To delineate the mechanisms of decreased GC 
responsiveness in GC-resistant subjects.
Exploratory Objectives: To assess in-vitro correlates of therapeutic responsiveness 
in HES. 
Endpoints: 
Primary: GC responsiveness, defined as the lowest dose of 
prednisone at which eosinophils remain <1000/µL, for at 
least two consecutive weekly blood draws, and at which 
symptoms are controlled as determined by the investigator
Secondary: Identification of GC non-responders and/or 
suboptimal responders for studies of GC resistance in HES.
Exploratory: Delineation of the mechanisms of GC 
resistance and in-vitro correlates of response in subjects 
with HES.

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Précis
This study aims to develop a model to determine whether a single, oral, weight-based 
dose of glucocorticoid (GC) can predict clinical and biologic response to GC’s over the 
long term in subjects with hypereosinophilic syndrome (HES). Subjects with 
FIP1L1/PDGFRα-negative HES, who are symptomatic with eosinophil count >1500/µL 
and receiving ≤10 mg prednisone daily, will be enrolled. A single oral dose of 
prednisone (1 mg/kg rounded to the nearest 5mg) will be administered. Eosinophil count 
and various laboratory parameters will be assessed at 2 hours, 4 hours and 24 hours 
following prednisone administration (investigators will be blinded to the results of the 
eosinophil counts). The subjects will then begin GC therapy at 30 mg prednisone daily 
followed by a standardized taper. The lowest dose of GC at which symptoms and 
eosinophilia are controlled will be compared to the change in eosinophil count at 2, 4 
and 24 hours post-challenge. Mechanisms and in vitro correlates of GC resistance will 
also be explored. 

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1. Introduction & Background 
1.1.
Rationale
Although glucocorticoids (GC) are the mainstay of therapy for many autoimmune and 
inflammatory conditions, not all patients respond equally, and high doses of inhaled or 
systemic GCs are often used in non-responders.  Consequently, GC resistance is an 
active area of study in asthma, smokers with asthma, chronic obstructive pulmonary 
disease (COPD), and other inflammatory disorders.  GCs are considered standard first-
line therapy in patients with hypereosinophilic syndrome (HES) who lack the FIP1L1-
PDGFRα (F/P) mutation. In fact, more than 85% of patients with HES studied 
retrospectively had a complete or partial response to GC at 1 month(1).  Although 
decreased expression of the GC receptor on the surface of eosinophils was implicated 
in 3 out of 5 patients with GC-resistant HES(2), the mechanisms of decreased or absent 
GC response in diverse variants of patients with HES are largely unexplored.  
A single-dose prednisone challenge has been proposed as a way to assess steroid-
responsiveness and measure prognosis in patients with F/P negative HES. Although 
patients presenting with HES are typically treated with a large dose of GC followed by a 
steroid taper, the utility of this approach has not been validated. Studying the effects of 
a steroid challenge would be beneficial for 4 reasons.  First, an eosinopenic response 
after a 1 mg/kg dose could provide information about the efficacy of high dose GC in the 
event of rapidly progressive disease. Second, if the acute response to a single dose GC 
challenge were able to predict the maintenance dose after a taper, this might allow 
patients to spend less time on higher doses.  Third, the lack of response to a steroid 
challenge would identify patients for whom a trial of steroids would be unwarranted 
allowing faster institution of alternate therapies for HES. Finally, as subjects with GC-
resistant HES are systematically identified in a standardized fashion, further 
mechanistic work into causes of resistance, included elucidation of signaling pathways, 
glucocorticoid receptor function, and perhaps even genetic reasons for resistance may 
be explored. 
1.2.
Background 
Glucocorticoids act on a number of glucocorticoid-responsive genes relevant to 
inflammatory conditions via the glucocorticoid receptor (GR). The GR itself is composed 
of a DNA ligand and an immunogenic or N-terminal domain. Alternative splicing of the 
GR gene generates 2 receptor isoforms. The classically active form is GRα, which 
resides in the cytoplasm and is expressed in cells of most organs and tissues in the 
human body. GRβ, the second isoform, acts as a dominant negative receptor. 
Administration of GCs in the setting of inflammatory and allergic conditions results in 
expression of anti-inflammatory cytokines, as well as diminished expression of 
inflammatory or adhesion molecules on airway or vascular epithelium. A number of 
mechanisms have been proposed to explain decreased GC responsiveness. The 
following section describes some of these mechanisms and their potential relevance in 
HES (Appendix G, Figure 1) . 

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1.2.1. Inappropriate Absorption of Glucocorticoids 
While there have been no studies of GC absorption in HES, it is possible that patients 
with eosinophilic involvement of the GI tract might have difficulty with GC absorption 
(Appendix G, Figure 1). Although GCs are used for the treatment for eosinophilic 
gastrointestinal disease (EGID), especially in the setting of malabsorption, the 
mechanism of GI involvement in HES may be different (mucosal edema instead of 
eosinophilic inflammation, for example). Furthermore, it is not clear that oral dosing in 
the form of prednisone has similar bioavailability or pharmacokinetics of absorption to 
the compounded liquid forms of GC used most often in EGID.  
1.2.2. Alterations in Glucocorticoid Receptor function
GR function involves multiple steps, including 1) binding of GC to the receptor, 2) 
translocation of the GC-receptor complex into the nucleus, and 3) direct and indirect 
effects on GR response elements leading to modulation of the cytokine response 
(Appendix G, Figure 1). Abnormalities in any of these could potentially lead to 
decreased GC responsiveness. 
Increased expression of GRβ in place of the active GRα has been reported in steroid 
insensitive nasal polyps(3), fatal asthma(4), steroid resistant asthma(5), and other 
steroid insensitive inflammatory diseases(6); however, the degree to which differential 
expression of GR isoforms contributes to steroid resistance remains controversial. To 
complicate matters further, an additional cohort of receptor proteins produced by 
alternative translation initiation from a single GR mRNA species was recently described 
and results in an increased number of GR isoforms(7). GR isoform expression has not 
been evaluated in HES to date.
One of the ways that glucocorticoids exert their anti-inflammatory effects is through 
inhibition of transcription factors, which in turn alter cytokine expression profiles. 
Differential expression of the NFκB DNA binding unit (NFKB1), STAT-4 and IL-4R in 
stimulated PBMCs predicted the GC resistant phenotype in asthmatics(8) Furthermore, 
cytokine footprints of decreased GC responsiveness have been described in patients 
with eosinophilic esophagitis and asthma. In one study of patients with eosinophilic 
esophagitis, non-responders to budesonide therapy had elevated numbers of TGFβ1 
positive cells compared with responders(9). Studies in asthmatic patients have 
demonstrated increased number of cells expressing Th2 cytokines, including IL-4(5, 
10), IL-5(5), and IL-13(10), as well as increased IFNγ(5) mRNA in bronchoalveolar 
lavage (11) from GC resistant subjects. The cytokine phenotype of GC resistance in 
HES has not been studied.
Despite the complexity of GC signaling (Appendix G, Figure 2)(12), some progress has 
been made in evaluating kinetic phosphorylation events occurring at the N-terminus 
phosphorylation sites of the GR after binding of corticosteroids.  Phosphorylation events 
affect subcellular transport of the GR and therefore have effects on GR function in the 
setting of inflammatory conditions. Examples of assessment of phosphorylation events 
in asthma include: demonstration of elevated p38 MAPK and pERK1/2 in asthma 

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patients compared to controls(11), and elevated p38 MAPK reduction of nuclear GR-
ligand binding affinity in steroid-dependent asthmatics compared with controls(13). It 
has also been proposed that phosphorylation of the GR might affect ligand affinity 
suggesting a role for tissue or cell specific responses. 
1.2.3. Glucocorticoid effects on trafficking and eosinophil transcriptomics
At the time of initiation of this study, early (<4h) kinetics of glucocorticoid action on 
eosinophils were not well described. Samples from this and other studies were 
subjected to transcriptomic evaluation to determine early kinetics of eosinophil decline. 
More recent knowledge suggests that there is a substantial decline of eosinophils in the 
peripheral circulation (at least in normally responsive eosinophils) by 3 hours. More 
striking, the major genes of interest in apoptosis and trafficking were noted to be up or 
down-regulated by 2 hours after GC administration. Of the 414 genes differentially 
expressed in eosinophils in the first 2 hours after prednisone administration, 12 are 
known to be involved in leukocyte migration: CXCR4, RIPOR2, PREX1, CCR1, 
PTGER4, SWAP70, IL16, PIK3CG, NLRP12, CD244, S1PR1, and CSF1.  Four of these 
genes were significantly differentially expressed prior to the initial decline in circulating 
eosinophils, which occurred in the interval between 60 and 120 minutes. In vitro and 
flow cytometric studies demonstrated that CXCR4 is upregulated in response to steroids 
and we hypothesize this might be important in trafficking of eosinophils from the 
peripheral blood into tissues (14). For this reason, we are splitting amount of research 
blood drawn in the 4 hour timepoint into a 2 and 4 hour timepoint in order to be able to 
study the exploratory endpoints without losing valuable data at the 4 hour timepoint. The 
primary clinical endpoint at 4 hours remains unchanged as it is not appropriate or 
possible to change the primary endpoint at this juncture. 
Ultimately, understanding the various processes that predict decreased GC 
responsiveness would assist in numerous ways in the care of patients with HES. First, it 
would allow tailoring of treatment regimens for patients with HES. Second, development 
of assays or biomarkers that can predict decreased responsiveness could reduce GC 
use in such patients by preventing unnecessary treatment trials in patients unlikely to 
respond and accelerating the time to maintenance dosing. Thirdly, understanding the 
underlying mechanisms of GC resistance might pave the way for more effective anti-
inflammatory treatments or adjuncts to GC, as has been demonstrated in COPD, where 
theophylline and selective PI3Kδ inhibitors have a synergistic effect with GC 
administration (15). Better understanding of transcriptional changes in eosinophils might 
provide additional insight into receptor ligands on eosinophils, their role in trafficking, 
and might pave the way for further research into therapeutic implications of these 
findings. Finally, insights into the causes of decreased responsiveness of eosinophils to 
GC and in HES may have implications for other inflammatory and allergic disorders.  

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2. Study Objectives
2.1.
Primary Objective
To develop a model to determine whether a single dose steroid challenge can be used 
to predict GC responsiveness in subjects with HES
2.2.
Secondary Objectives 
To define a group of GC non-responders and/or suboptimal responders for further study 
of the mechanisms and biologic correlates of GC resistance in HES 
2.3.
Exploratory Objectives 
To delineate some of the mechanisms of decreased GC responsiveness in GC-resistant 
subjects and to assess in-vitro correlates of therapeutic responsiveness in HES in a 
subset of subjects.
3. Study Design and Methods
3.1.
Description of the Study Design
The study is designed as a single center, open label, single-blinded observational study 
to develop a model to assess whether a single dose steroid challenge can predict GC 
responsiveness in subjects with HES. GC responsiveness is defined as the minimum 
dose of GC required to control eosinophilia and symptoms.
3.2.
Screening
Subjects with HES enrolled on Protocol #94-I-0079 (Eosinophil Characterization, 
Activation, and Function in Parasitic Infections and other Conditions with Increased 
Tissue or Peripheral Blood Eosinophilia in Humans) will be recruited for enrollment in 
this protocol. Since eligibility for this protocol will be determined on the basis of the 
standard clinical and laboratory evaluations performed as part of Protocol #94-I-0079, a 
separate screening visit will not be necessary for the GC challenge. Since the HES 
workup may need to take place over several visits, to determine eligibility, a CBC with 
differential will be performed at the NIH or at the patient’s local site within 14 days prior 
to enrolling on this protocol.
Informed consent will be obtained and subjects will be enrolled during a scheduled visit 
for protocol 94-I-0079 or via telephone prior to undergoing any procedures specific to 
this protocol. Telephone consents will be obtained according to current NIH policy 
(section 7.5.3).

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3.3.
Baseline Visit 
Baseline assessments and procedures will be performed up to 90 days prior to dosing 
with prednisone and will include (see Appendix B for complete list):

A complete history and physical examination, with emphasis on signs and 
symptoms associated with eosinophilia (i.e. fatigue, pruritus, skin involvement, 
neuropathy, arthralgias, myalgias etc.) 

Routine laboratory studies, including complete blood count with differential, 
routine chemistries including electrolytes, liver and kidney panels. 

Additional diagnostic testing, as clinically indicated, to characterize baseline 
clinical manifestations of eosinophilia. 

Blood -HCG at baseline visit. Urine -HCG will be performed in the morning 
prior to prednisone administration in women of childbearing potential. 

Assessment of eosinophil activation, additional research studies and storage of 
samples as outlined in Appendix C.
3.4.
Procedures and Laboratory Testing 
Subjects will receive a single dose of prednisone prescribed by the PI or designee to be 
taken by mouth at a dose of 1 mg/kg (rounded to the nearest 5 mg) between the hours 
of 7:30 and 9am on the day of study. Laboratory testing, including a baseline cortisol 
level, CBC with differential, and research specimens (see Appendix B) will be drawn 
prior to prednisone administration and at 2, 4 and 24 hours after prednisone 
administration. Investigators will be blinded to the 2-hour, 4-hour and 24-hour eosinophil 
counts (the results will not appear in the NIH Clinical Research Information System 
(CRIS), but will be available from the Clinical Pathology internal system for analysis 
once the trial is completed). All blood draws will be timed and will occur in outpatient 
phlebotomy, outpatient clinic or inpatient unit that is able to draw at specific times to 
prevent variability in draw times across the subject cohorts.  
In normal subjects, prednisone is rapidly absorbed and metabolized with peak levels at 
2-4 hours and a biologic half-life of 12-36 hours.  Serum will be stored from blood drawn 
at the 4-hour time point for assessment of synthetic glucocorticoid levels, including 
prednisone and its metabolite prednisolone. Subjects may be asked to undergo further 
workup for causes of poor absorption by their home physician or at NIH if indicated. 
After the 24-hour challenge, subjects will be started on a GC taper to determine the 
lowest dose of GC that is able to suppress signs and symptoms of HES and maintain 
the peripheral eosinophil count below 1000/µl. GC will be initiated at 30 mg in all 
subjects, and the taper will proceed according to an algorithm (Appendix E). GC will be 
tapered by 5 mg weekly until a dose of 15 mg is reached. The dose will then be tapered 
by 2.5 mg weekly until a dose of 5 mg is reached or the eosinophil count rises.
In some cases, an initial starting dose of 30 mg of prednisone will be insufficient to 
control eosinophilia. If AEC is >1000/µL 1 week after the initiation of GC, the GC dose 
will be increased to 60 mg and a taper initiated from this higher dose. A CBC with 

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differential will be drawn after 2 days on this higher dose and if the AEC remains 
>1000/µL, the subject will be withdrawn and total GC dose used for the final analysis 
will be the weight-based challenge dose for that individual. If the subject is responsive at 
the higher dose, the subject will be kept at 60 mg for one week after which the GC dose 
will be tapered by 10 mg every week until a dose of 30 mg of prednisone is reached.  If 
AEC is still suppressed and they are able to continue the taper, they will then taper 
according to the standard taper schedule outlined above and shown in Appendix E. 
Weekly eosinophil counts will be obtained with the taper until the AEC rises above 
1000/µL or the subject experiences HES symptoms. HES symptoms will be defined by 
the investigator and will be based on data collected at the time of the baseline visit, as 
well as clinical experience in the treatment of HES. If either eosinophils rise or HES 
symptoms are present, prednisone will be increased to the previous dose, and a taper 
will be re-attempted.  If the AEC increases after an attempted decrease in dose, the rate 
of the tapering schedule will be decreased to 2.5 mg/week from 5 mg/week.  The taper 
will continue according to this schedule until 2 attempts to taper from a given dose fail or 
a dose of 5 mg of prednisone is reached. If a subject’s AEC continues to climb despite a 
dose increase, at the discretion of the investigator the dose may be increased by a 
larger dose than outlined in Appendix E. Attempts to capture the true effective dose of 
glucocorticoid that controls counts and symptoms will be attempted; however if not 
practical due to time or side effect profile of prednisone, subjects will return for an end of 
study visit and be removed from the study. If a subject is unable to be tapered, their 
responsiveness, or lack thereof, will be captured at that dose.  Depending on the dose 
and the clinical presentation they may be continued on prednisone however they will be 
withdrawn from the protocol at the dose at which they were no longer able to taper.
The GC taper will be individualized in subjects in whom a prolonged course of a higher 
dose is clinically indicated (e.g., Churg Strauss syndrome/EGPA) or a slower taper is 
needed to prevent steroid withdrawal. Investigators will not be blinded to the eosinophil 
counts obtained during the taper, as the primary endpoint is dependent on the initial 
results of the steroid challenge to which the investigators are blinded. 
3.5.
Study Endpoints
3.5.1. Primary Endpoint
The primary response variable is GC responsiveness, defined as the lowest dose of 
prednisone at which eosinophils remain <1000/µl, for at least two consecutive weekly 
blood draws, and at which symptoms are controlled as determined by the investigator; 
OR, dose of prednisone at which subject is unable to taper prednisone further without 
rebound of eosinophilia and/or symptoms after three attempts; OR, the challenge dose, 
if there is no drop in eosinophil counts within the first week of challenge or taper. 
GC responsiveness in each individual will be assessed using the percent change in 
eosinophil count (eosinophil response rate [ERR]) from baseline to 4h (or 24h) post-
steroid challenge.

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3.5.2. Secondary Endpoint 
Subjects will be classified into 3 categories on the basis of GC responsiveness: 
unresponsive, suboptimally responsive, and steroid-responsive. These categories are 
defined in Section 6.1 “Description of the analyses.”
Clinical factors associated with GC responsiveness (including HES variant, pattern of 
organ involvement and laboratory parameters) will be explored. 
3.5.3. Exploratory Endpoints 
The mechanisms of GC resistance will be explored (Appendix F) by dividing subjects 
into response categories for the following assessment in all subjects:  
a) GI absorption of prednisone by evaluation of plasma levels of 
prednisone/prednisolone 
b) intracellular cytokine profiles in lymphocytes using multiparameter flow cytometry
c) RNA expression profiles by microarray profiling of whole blood
d) GR isoform assessments:  GR isoforms will be assessed on all subjects using RT-
PCR to understand relationship between responsiveness and predominant isoforms
e) Proteomics: A collaboration with Dr. Konrad Pazdrak (Assistant Professor, 2.230 
Basic Science Building, 301 University Boulevard, Galveston, TX 77555-0635) has 
been initiated using samples from the screening protocol for eosinophilia (94-I-0079). 
If preliminary findings are promising, coded samples, without personal identifying 
information, from 12-I-0026 will be sent to him for further proteomic evaluation of 
steroid responsiveness. 
Not all studies will be performed for each subject due to time or sample constraints, 
although efforts will be made to proceed with exploratory investigations in a uniform 
manner. Additionally, investigations not outlined above may be performed based on 
preliminary findings and collaborations with other investigators in the field of GC 
responsiveness.
4. Inclusion and Exclusion Criteria
4.1.
Subject Inclusion Criteria 
Subjects on Protocol #94-I-0079 will be eligible for participation in the study only if all of 
the following criteria apply:
1. Subjects must be 7 years of age or older to enroll
2. Subject meets diagnostic criteria for HES (AEC >1500/µL, absence of a 
secondary cause and signs and/or symptoms attributable to the eosinophilia)
3. AEC >1500/µL obtained within 14 days prior to enrollment

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4. Willingness to perform the timed steroid challenge
5. Appropriate candidate for GC treatment after challenge
6. Willingness to have samples stored for future research
4.2.
Subject Exclusion Criteria 
A subject will not be eligible to participate in the study if any of the following apply:
1. Receiving >10 mg prednisone or equivalent at the time of enrollment.
2. Receiving ≤10 mg of prednisone or equivalent but have not been on a fixed dose 
for at least 3 weeks (subjects on a current corticosteroid taper will be excluded)
3. AEC ≤1500/µl on the day of the steroid challenge 
4. Use of immunomodulatory medications, (other than ≤10 mg/day prednisone) 
including but not limited to biologics, within the past 6 months  
5. Pregnant at the time of screening.  
6. Have a known mutation in the FIP1L1-PDGFR gene 
7. Any condition that, in the opinion of the investigator, places the subject at undue 
risk by participating in the protocol
8. Weight less than 48 kg (106 lbs) in subjects less than 18 years of age 
5. Monitoring and Withdrawal 
5.1.
Clinical Evaluations
Standard safety assessments for this study will include history and physical 
examinations, and vital signs at baseline and at 24 hours. Clinical assessments of SAEs 
and AEs will be performed at all study visits, as well as during phone encounters.
5.2.
Laboratory Evaluations
Standard laboratory evaluations will include clinical chemistry panel including renal, 
hepatic panels and electrolytes, CBC with differential, cortisol levels and stored samples 
as described in Appendix B and Appendix C. 
5.3.
Follow-up 
Follow-up will consist of phone-calls within a window of 7 ± 3 days to coordinate the 
tapering schedule of prednisone. All changes in prednisone doses and tapers will be 
appropriately documented with concurrent eosinophil counts as applicable.  Blood for 
RNA analysis will be obtained at 1 week to assess medium term effects of GC 
administration. The blood draw will be performed at the subject’s home institution (with 
sample sent to NIH in accordance with appropriate procedures), or may be performed at 
NIH if the subject lives locally. 
Subjects will be asked to return 2-4 weeks after the completion of their GC taper for 
their final visit on this protocol.   

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5.4.
Early Termination Visit
Subjects who withdraw from the study prematurely (see below) will continue to be 
followed on Protocol #94-I-0079.
5.5.
Re-contact of Subjects after Study Termination 
After study termination, subjects will be contacted yearly as part of the required study 
visits for Protocol #94-I-0079.  In settings where subjects request to withdraw consent 
for Protocol #94-I-0079, they will be asked whether they would be willing to be re-
contacted in the future.   
5.6.
Premature Withdrawal of a Subject
Subjects may be withdrawn for the following reasons:
1. Significant adverse events from GC therapy requiring withdrawal of subject 
and/or switching to alternate therapies
2. Determination of positive F/P gene status during steroid therapy that was 
previously unknown.  
NOTE: Most subjects referred to NIH with HES have known F/P gene status and 
are already on tyrosine kinase inhibitors if indicated.  In extremely rare cases 
where gene status is unknown, GC therapy might be initiated while F/P gene 
status is being investigated as part of Protocol #94-I-0079.  If a positive result 
were obtained, the subject would be withdrawn from the study and imatinib 
therapy would be recommended. Such subjects would be excluded from the 
analysis. 
3. Complete non-response to GC’s evidenced by lack of reduction of peripheral 
eosinophil count from baseline at the initial 30 mg dose or the increased 60 mg 
dose.  Subjects will be switched to an alternative therapy either by their primary 
care physician or at the follow-up visit. Although subjects will be withdrawn from 
further steroid tapering, evidence of non-response will be included in the 
analysis. 
4. AEC ≤1500/µL on the day of steroid challenge. 
5. Subject becomes pregnant (subjects who become pregnant after the first GC 
dose will be followed for safety on 94-I-0079).
6. Analysis Plan
6.1.
Description of the Analyses 
The primary objective of this study is to develop a model to predict GC responsiveness 
(see section 2) using the percent change in the eosinophil count at 4 or 24 hours 
following steroid challenge (ie, ERR). GC responsiveness is defined as the dose 
required to control disease manifestations and eosinophilia. Since this response is 
necessarily subject to many factors including physician expert opinion, it is important 
that the PI and Accountable Physician be blinded to the predictor variables. In order not 

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to compromise patient care, the 24 hour post-challenge CBC with differential will be 
assessed by an independent physician (Dr. Irina Maric or her designee) who will decide 
whether un-blinding is necessary for patient safety.
To allow for flexibility in building the predictive model, we will first try the class of linear 
models on the GC responsiveness and pick the best model in terms of a small sample 
adjusted Akaike Information Criterion(16).  If the fit of that model is not good (for 
example, the errors look far from normal), then transformations on the GC 
responsiveness may be tried. If none of those models have good fit, then we will 
consider a cumulative logit model where we partition the GC responsiveness into 3 
ordered categories: steroid responsive (<15 mg of prednisone or equivalent alternate 
corticosteroid daily), moderately responsive (requiring between 16-40 mg of prednisone 
or equivalent), or resistant (>41 mg of prednisone required daily with or without a 
response).
The primary objective of this study is exploratory, because we are looking for the best 
model for prediction and do not want to limit ourselves to 1 class of models.  In order to 
protect ourselves from creating a model that appears to predict the response when in 
fact there is no real relationship between the predictors and the response, we will first 
test for a significant Spearman correlation between GC responsiveness and ERR to 
make sure that there is a significant effect.  If there is a significant correlation, then we 
have some comfort that our final model is not just modeling noise. For our sample size 
justification (section 6.2), we assume that if we have a large enough sample size to 
show a significant correlation between GC responsiveness and 1 of the continuous 
predictive variables, then we would have a sample size large enough to explore these 
models. Because our objective is to find a reasonable model, and sample sizes are 
limited by availability of subjects, the sample size calculation is mostly to ensure that 
with such small sample sizes we would be able to detect moderate (i.e., correlations of 
about 0.5) effects.
6.1.1. Appropriate Methods and Timing for Analyzing Outcome Measures
Outcome measures will be measured using weekly or bi-weekly eosinophil count 
assessments at the subject’s home physician office.  Outcome measures will be 
assessed once 30% of subjects have been enrolled and challenged, and again at 
completion of the final subject enrollment. 
6.1.2. Addressing Study Objectives
Although the primary objective is an exploratory model building, we do consider the 
simple null hypothesis that there is no correlation between % change in eosinophil count 
at 4 and 24 hours after challenge and GC response.  Regardless of whether that test is 
significant or not, we will attempt to predict the GC response through the model building 
strategy described in the Section 6.1 on Description of the Analyses. 

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6.2.
Sample Size Justification 
The sample size of this study is practically limited by the number of subjects available 
for study. Currently, about 30-35 new hypereosinophilic subjects per year are seen at 
the NIH and enrolled on protocol 94-I-0079, of which approximately 10% would meet 
inclusion criteria for this protocol.  Consequently, we expect that we can enroll 30-40 
subjects in this study over a 13-year period.
To show that this number of subjects is not unreasonably small, we calculate the 
sample size for a Spearman’s correlation coefficient. If true correlation is 0.5 between a 
predictor variable (ERR at 4 or 24 hours) and the GC responsiveness (see Description 
of the Study, Section 3.1), the total number of subjects needed to show that the 
correlation is different from zero at the two-sided 0.05 level with 80% power is about 32 
subjects. We calculated this by simulating 10,000 data sets of 32 bivariate random 
normal variables with correlation 0.5 and seeing that the Spearman coefficient is 
significantly different from zero 79.5% of the time. This is similar to the usual sample 
size calculation for Pearson’s correlation(17) or (18) that gives a sample size of 29 for 
the same situation.
Because the predictor and outcome variables are both continuous, if the true change in 
the dependent variables is 0.5 standard deviations per 1 standard deviation change in 
the independent variable, a clinically important measurement of association between 
response to a steroid challenge and final required dose of GC will be possible.
6.3.
Final Analysis Plan
1. Study Endpoints: 
o
Primary:  GC responsiveness.

Definition: See primary endpoint for definition of GC responsiveness.

Validity and Reliability: This is a rare disease, and standard care for this 
disease is still being developed. There is no standard endpoint, and this 
endpoint has not been used before. One of the purposes of this study is 
to see if this endpoint can be reliably predicted. The endpoint has face 
validity because the dose that controls eosinophil concentration is useful 
for determining the extent of a subject’s disease and their responsiveness 
to steroid therapy. We will first try and predict GC responsiveness as 
described in section “Description of the Analyses” (Section 6.1), but may 
also try to predict the categorization of the GC responsiveness into 
3 ordered categories described in section “Description of the Analyses”. 

Predictor Variables: All predictor variables will be measured in the first 
24 hours after initial dose, and will be blinded to the PI and Accountable 
Physician. These variables will be used to predict the GC responsiveness: 

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percent change in eosinophil count from baseline to 4 and 24 hours and 
steroid absorption. 
o
Secondary:

Classification of subjects by GC response for assessment of clinical 
factors associated with GC responsiveness (including HES variant, 
pattern of organ involvement and laboratory parameters)
o
Exploratory

Exploratory analyses of potential mechanisms and in vitro correlates of 
GC resistance, including impaired steroid absorption, cell surface markers 
on lymphocytes, intracellular and serum cytokine profiles.
2. Description of the Statistical Methods to Be Employed: 

Spearman correlation of GC responsiveness and percent change in 
eosinophil from baseline to 4 and 24 hours, with confidence intervals and 
testing whether it is different from zero.

Model building as described in section “Description of the Analyses” 
(Section 6.1).
3. Level of Significance to be Used: Two-sided 0.05
4. Exploratory Analyses: See section 6.1 for primary objective. Similar model building 
strategies will be used for secondary endpoints and exploratory endpoints. 
5. Handling of Missing and Spurious Data: We have defined the primary endpoint so 
that there should be little missing data. For example, subjects that are unable to 
control their eosinophil counts with 30-60 mg prednisone will still have a defined 
endpoint and their results will be included in the analysis. Based on prior experience 
with this subject cohort, we anticipate an extremely low dropout rate (<5%/year). 
6.4.
Interim Analysis Plan
Due to the rare nature of the disease, various investigators have asked whether an 
interim analysis would be appropriate in this rare population. Since approximately two 
thirds of the projected subjects have completed the study, we are able to perform an 
interim analysis using ERR to predict GC responsiveness. Although this is an additional 
analysis, since the original final analysis plan allowed for an unspecified number of 
different models, this does not change the overall analysis plan in any substantial way. 
Nevertheless, trying many models may result in an overfit final result, therefore, for the 
interim analysis we will try only one flexible model.
We use a linear model to predict the GC responsiveness using either a first, second or 
third order polynomial of log(ERR) at 4 hours in the model and the final end-prednisone 
dose of each subject as the GC response as defined in section 3.5.1. The choice 
between the three models will be made by the one with minimum AIC (Akiake 

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information criteria). To evaluate the prediction, we will use a leave-one-out cross 
validated estimate of R squared, where for each fit with a point left out we use the 
minimum AIC model of the three. Because the entire model finding procedure is cross 
validated, the estimate of R squared will not be biased upward by using the lowest AIC 
from the three models. As in the final analysis plan, to test for significance between 
log(ERR) and GC responsiveness, we test whether the Spearman (i.e., rank) correlation 
is significantly different from zero, and calculate estimates and associated confidence 
intervals for Spearman’s correlation.
If the interim model appears to explain the variation of the GC responsiveness (i.e., has 
a large cross validated R squared), then we can use the interim model to predict the GC 
responsiveness of the subsequent subjects that enter the study as a validation cohort. 
Because we expect few additional subjects (<15), our validation would only be able to 
detect extreme departures from the model. We test for extreme departures from the 
model by creating a 95% prediction interval and testing whether we can show that 
significantly less than 95% of the subsequent subjects fall within that prediction interval. 
For all sample sizes 5 or greater, we have at least 80% power to show that the model 
fails (the 95% prediction interval covers less than 95% of subsequent responses) if the 
true proportion that fall within the 95% prediction interval is 50% or less.  It is possible 
that the prediction intervals from the model are so wide that they will not be clinically 
useful, perhaps as a result of insufficient subjects enrolled, in which case the study will 
continue since the secondary and exploratory outcomes of the study will continue to be 
useful for studies of GC effects on eosinophils.      
7. Human Subjects Protection
7.1.
Study Population
7.1.1. Rationale for Subject Selection 
New and returning subjects enrolled on Protocol #94-I-0079 who have documented 
HES, AEC >1500/µL on ≤ 10 mg prednisone equivalent daily and are candidates for GC 
therapy will be recruited for this protocol. Subjects receiving higher prednisone doses at 
baseline will be excluded to maximize the likelihood that the steroid challenge will have 
observable effects. Subjects with chronic eosinophilic leukemia, including those with 
PDGFR-associated disease, will also be excluded since imatinib is first line therapy for 
this condition. 
7.1.2. Recruitment Plan 
Individuals contacting the investigators for evaluation of HES as well as subjects 
already enrolled in Protocol #94-I-0079 (Eosinophil Activation and Function in Parasitic 
Infections and other Conditions with Increased Tissue or Blood Eosinophilia in Humans) 
will be considered for participation in this protocol. 

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7.2.
Justification for Eligibility of Special Populations
7.2.1. Exclusion of Pregnant Women
Subjects who are pregnant, including those for whom GC are clinically indicated, will be 
excluded from participation in this study because of the increased risk of gestational 
diabetes in the setting of prolonged GC exposure in pregnancy. These subjects will be 
referred to their referring/treating physician for management. 
7.2.2. Exclusion of Children Weighing <48 kg
Children younger than 18 years of age weighing <48 kg will be excluded from the study 
due to limitations of maximal blood draw over time for research purposes.
7.2.3. Inclusion of Children Weighing ≥48 kg
Children younger than 18 years of age weighing ≥48 kg will be eligible to enroll in this 
study because HES is a genetic disease that can affect children. Therefore, there is 
important information about this disease and the use of GCs as treatment to be learned 
from pediatric participants. Additionally, children stand to benefit from this study the 
same as adults.
7.3.
Potential Risks
Venipuncture
The drawing of blood may be associated with discomfort, occasional bruising at the site 
of puncture, and rarely, fainting.  All blood will be drawn from arm veins.
IV line insertion
The risks of having an IV inserted including pain, bruising, swelling, and in rare 
instances, fainting, thrombosis, or infection.
Prednisone
Prednisone (or its equivalent) is commonly used to treat a wide variety of disorders, 
including HES. The common side effects of prednisone are well-described and include 
increased appetite, insomnia, fluid retention, indigestion, headache, facial swelling, 
menstrual irregularities, and visual changes. When used over extended periods of time, 
GCs (such as prednisone) may cause weight gain, cataracts, myopathy, osteoporosis, 
glucose intolerance, increased risk of infections such as P. jiroveci, and can affect 
mood. Abrupt cessation of prednisone may cause headaches, vomiting, hypotension, 
fatigue, and in severe cases, may result in loss of consciousness and even death. If 
participants become severely ill or need to undergo surgery during the prednisone 
taper, their doses may need to be increased termporarily. There is evidence that 
participants who take high doses of prednisolone may secrete the steroid in breast milk. 
Waiting to breastfeed for 3-4 hours after receipt of prednisolone reduces this risk. In any 

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case, there are no known effects of corticosteroids in breastfed infants. Participants on 
long-term dosing will be monitored closely for these side effects.  In addition, for 
subjects that will be on greater than 30-mg prednisone for 3 weeks or longer (ie those 
on the increased 60-mg dose), standard P. jiroveci prophylaxis will be instituted.  
Similarly, evidence-based approaches for GC bone loss prevention or treatment will be 
followed according to the latest ACR 2010 recommendations for prevention of GC 
induced osteoporosis(19, 20).
7.4.
Potential Benefits
Since the study is designed to evaluate the degree of response to GC’s in the treatment 
of hypereosinophilic syndrome, subjects may or may not benefit from therapy. The 
information learned from this study may improve understanding of how GCs can be 
used to treat HES, which may lead to improvements in clinical care of these patients.
7.5.
Informed Consent Process
Informed consent is a process where information is presented to enable persons to 
voluntarily decide whether or not to participate as a research subject. It is an on-going 
conversation between the human research subject and the researchers that begins 
before consent is given and continues until the end of the subject's involvement in the 
research. Discussions about the research will provide essential information about the 
study and include: purpose, duration, experimental procedures, alternatives, risks and 
benefits.  Subjects will be given the opportunity to ask questions and have them 
answered. 
Subjects will sign the informed consent document prior to undergoing any research 
procedures. The subjects may withdraw consent at any time throughout the course of 
the trial.  A copy of the informed consent document will be given to the subjects for their 
records. The researcher will document the signing of the consent form in the subject’s 
medical record. The informed consent process will emphasize that the quality of 
subjects medical care will not be adversely affected if they decline to participate in this 
study. 
7.5.1. Assent of Children
Where deemed appropriate by the clinician and the child’s parent(s) or guardian, the 
child will also be included in all discussions about the trial and age-appropriate 
language will be used to describe the procedures and tests involved in this study, along 
with the risks, discomforts and benefits of participation. Verbal assent will be obtained 
as appropriate for children ages 8 years and older. Children under the age of 18, but 
who are age 8 or older will be asked to sign an age appropriate assent form. Children 
under the age of 8 will not be required to provide assent as they typically do not have 
the ability to fully understand the nature of research. The consent/assent process will be 
documented in the child’s medical record, including the assessment of the child’s ability 
to provide assent (verbal versus written) as applicable. All children will be contacted 

Glucocorticoid Responsiveness in HES v13.0
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after they have reached the age of 18 to determine whether they wish to continue on the 
trial and informed consent will be obtained from them at that time.
7.5.2. Consent for minors when they reach the age of majority 
When a pediatric subject reaches age 18, continued participation (including ongoing 
interactions with the subject or continued analysis of identifiable data) will require 
consenting of the now adult with the standard protocol consent document to ensure 
legally effective informed consent has been obtained.
If reconsent is not feasible, we request waiver of informed consent to continue to use 
data and/or specimens for those individuals who become lost to follow up or who have 
been taken off study prior to reaching the age of majority.
Requirements for Waiver of Consent consistent with pre-2018 45 CFR 46.116 (d):
(1) The research involves no more than minimal risk to the subjects.
a. Analysis of samples and data from this study involves no additional 
risks to subjects.
(2) The research could not practicably be carried out without the waiver or 
alteration.
a. Considering the length of time between the minor’s last contact with 
the research team and their age of majority, it will likely be very difficult 
to locate them again. A significant reduction in the number of samples 
analyzed is likely to impact the quality of the research. 
(3) The waiver or alteration will not adversely affect the rights and welfare of the 
subjects.
a. Retention of these samples or data does not affect the welfare of 
subjects. 
(4) Whenever appropriate, the subjects will be provided with additional pertinent 
information after participation.
a. We only request a waiver of consent for those subjects who have been 
lost to follow-up or who have been taken off study prior to reaching the 
age of majority.
7.5.3. Telephone Consent Process
Subjects with HES will be screened for this study on Protocol 94-I-0079.  If for any 
reason a subject chooses not to or is unable to sign the Informed Consent during a 
scheduled visit on Protocol 94-I-0079 but he/she chooses to enroll on this protocol, 
permission to consent subjects over the telephone may be permitted.  In these 
instances, permission to email, mail or fax the consent document will be obtained from 
the subject.  Once consent is received, the subject is instructed to call the investigator 
for discussion of the study and consent document.  The study will be thoroughly 
explained with ample time for questions or concerns related to participation. Informed 
consent will be obtained by the principal investigator or a designated associate 

Glucocorticoid Responsiveness in HES v13.0
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investigator on this protocol.  The subject will identify him/herself by name and date of 
birth, and state that he/she gives consent to participate in this study.  The staff member 
will verify the subject’s name and the verbal consent to participate.  Upon completion of 
the telephone call, the manner of obtaining consent and the names of the person 
administering and providing consent will be documented in the medical record, signed 
and dated.  The subject must sign and date the consent in his/her possession. This 
signed and dated consent must be sent to the investigator.  No testing will occur without 
this consent.  Once received, the consent will be checked for accuracy and signed by 
the NIH investigator/designee.  The signed consent will then be filed in the medical 
record.
7.6.
Subject Confidentiality
All records will be maintained in a locked file in the Clinical Center and/or in the Medical 
Records Department of the NIH where they are accessible only to authorized personnel.
All records will be kept confidential to the extent provided by federal, state, and local 
law. The study monitors and other authorized individuals may inspect all documents and 
records required to be maintained by the investigator, including but not limited to, 
medical records. Records will be kept locked and data will be coded. Any personally 
identifiable information maintained for this study will be kept on restricted-access 
computers and networks. Personally identifiable information will only be shared with 
individuals authorized to receive it under this protocol. Individuals not authorized to 
receive personally identifiable information will be provided with coded information only, 
as needed. Clinical information will not be released without written permission of the 
participant, except as necessary for monitoring by the IRB, NIAID, and OHRP.
8. Adverse Event Reporting Plan
8.1.
Specification of Safety Parameters
HES is a complex multisystem disorder. Exacerbations of symptoms or signs related to 
eosinophilia are expected to occur during this protocol and will be recorded, but not 
reported as AEs. Similarly, side effects of GC are well-described (see section 7.3) and 
are expected to occur during this protocol. Although GC administration at the doses to 
be used in this study is considered standard of care for HES, we will record but not 
report disease-related AEs, and record and report all research-related AEs (including 
expected events from the GCs) per NIH Human Research Protections Program (HRPP) 
Policy 801 described in section 8.3.
8.2.
Reporting Procedures
Unanticipated problems, non-compliance, and other reportable events will be reported 
to the IRB according to HRPP Policy 801. 
The principal investigator will report UPs, major protocol deviations, and deaths to the 
NIAID clinical director according to institutional timelines.

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8.3.
Type and Duration of the Follow-up of Subjects after Adverse Events
Subjects who have AEs or SAEs will be followed per standard clinical practice until their 
condition stabilizes or the AE resolves.  AEs and other reported events are defined in 
HRPP Policy 801. 
Subjects who withdraw from the protocol as a result of an AE or SAE will be followed on 
Protocol #94-I-0079.
8.4.
Assessing Adverse Events 
All laboratory and clinical AE’s that occur in a subject will be assessed for severity and 
classified into one of the categories below:

Graded 1 (Mild): Event requires minimal or no treatment and does not interfere 
with the patient’s daily activities. 

Grade 2 (Moderate): Event results in a low level of inconvenience or concern 
with the therapeutic measures. Moderate events may cause some interference 
with functioning. 

Grade 3 (Severe): Event interrupts a patient’s usual daily activity and may 
require systemic drug therapy or other treatment. Severe events are usually 
incapacitating

Grade 4 (Life Threatening): Any adverse drug experience that places the 
patient or 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. 

Grade 5: (Death) 
8.5.
Relationship Assessment
For all collected AE’s the clinician who examines and evaluates the subject will 
determine the adverse event’s causality based on the temporal relationship and his/her 
clinical judgment. The degree of certainty about causality will be graded using the 
categories below. 
Unrelated: Clearly not related to research 
Unlikely: Doubtfully related to research 
Possible: May be related to the research 
Probable: Likely related to the research 
Definite: Clearly related to the research. 

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9. Data and Safety Monitoring and Management Plan
Study data will be collected at the study site(s) and maintained on an electronic data 
system (CRIMSON). These forms or systems are to be completed on an ongoing basis 
during the study.  Data entered into electronic data systems shall be performed by 
authorized individuals.  Corrections to electronic data systems shall be tracked 
electronically (password protected or through an audit trail) with time, date, individual 
making the correction, and what was changed.  
The Investigator is responsible for assuring that the data collected is complete, 
accurate, and recorded in a timely manner.  Source documentation (the point of initial 
recording of information) should support the data collected and must be signed and 
dated by the person recording and/or reviewing the data.  Source documents include all 
recordings of observations or notations of clinical activities, and all reports and records 
necessary for the evaluation and reconstruction of the clinical trial.  Source documents 
include, but are not limited to, the subject’s medical records, laboratory reports, ECG 
tracings, x-rays, radiologist’s reports, subject’s diaries, biopsy reports, ultrasound 
photographs, progress notes, pharmacy records, and any other similar reports or 
records of procedures performed during the subject’s participation in the study.  Data 
from CRIMSON Data System will be collected directly from subjects during study visits 
and telephone calls or will be abstracted from subjects’ diaries/memory cards and 
medical records.  The subject’s medical record must record his/her participation in the 
clinical trial and, study treatment/vaccination (with doses and frequency) or other 
medical interventions or treatments administered, as well as any adverse reactions 
experienced during the trial.
Clinical data will be collected during the initial evaluation and at subsequent follow-up 
visits and will be stored using the CRIMSON database using a pre-specified Case 
Report Form (CRF).  
9.1.
Study Records Retention  
The investigator is responsible for retaining all essential documents listed in the 
International Council for Harmonisation Good Clinical Practice Guideline.  All essential 
documentation for all study subjects are to be maintained by the investigators in a 
secure storage facility for a minimum of three years per NIAID policies. These records 
are also to be maintained in compliance with IRB, state, and federal medical records 
retention requirements, whichever is longest. All stored records are to be kept 
confidential to the extent required by federal, state, and local law. 
Should the investigator wish to assign the study records to another party and/or move 
them to another location, the investigator must provide written notification of such intent 
to OCRPRO/NIAID with the name of the person who will accept responsibility for the 
transferred records and/or their new location. NIAID must be notified in writing and 
written NIAID/OCRPRO permission must be received by the site prior to destruction or 
relocation of research records.

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10.
Protocol Monitoring Plan
Monitors under contract to the NIAID/OCRPRO will visit the clinical research site to 
monitor several aspects of the study in accordance with the appropriate regulations and 
the approved protocol.  Only pediatric subjects will be monitored and the objectives of a 
monitoring visit will be: 1) to verify the existence of signed informed consent documents 
and documentation of the IC process for each monitored pediatric subject; 2) to verify 
AEs and SAEs, including the prompt reporting of all SAEs; 3) to compare applicable 
CRIMSON data abstracts with individual subjects’ records and source documents 
(subjects’ charts, laboratory analyses and test results, physicians’ progress notes, 
nurses’ notes, and any other relevant original subject information); and 4) to help ensure 
investigators are in compliance with the protocol. 
The investigator (and/or designee) will make study documents (e.g., consent forms, 
CRIMSON data abstracts and pertinent hospital or clinical records) readily available for 
inspection by the local IRB, the site monitors, and the NIAID staff for confirmation of the 
study data. 
A specific protocol monitoring plan will be discussed with the Principal Investigator and 
study staff prior to enrollment. The plan will outline the frequency of monitoring visits 
based on such factors as study enrollment, data collection status and regulatory 
obligations. 
The study team is responsible for monitoring data collected from our adult population.
11.
Plan for Research Use and Storage of Human Samples
11.1. Intended Use
Samples and data collected under this protocol may be used to study mechanisms of 
responses to GCs. 
11.2. Storage and Tracking
Serum and plasma will be stored in a –80oC freezer, cells in liquid nitrogen, and tissue 
samples at room temperature or frozen as appropriate.  Access will be limited using 
locked rooms or freezers depending on the type of sample. Stored research samples 
will be labeled with a code that only the study team can link to the participant.  The 
coded samples may be sent for research purposes to other investigators.  Data will be 
kept in password-protected computers. Only investigators or their designee(s) will have 
access to the samples and data. 
Samples will be tracked using BSI-II (Biological Specimen Inventory) v.7.2.  Samples 
sent to the NCI at Frederick Central Repository for processing after blood draw will be 
stored in the same freezers after receipt and assignment of a unique code.

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11.3. Disposition at the Completion of the Protocol
In the future, other investigators (both at NIH and outside) may wish to study these 
samples and/or data. If the planned research falls within the category of “human 
subjects research” on the part of the NIH researchers, IRB review and approval will be 
obtained. This includes the NIH researchers sending out coded and linked samples or 
data and getting results that they can link back to their subjects. 
11.4. Reporting Loss or Destruction of Samples/Specimens/Data to the IRB
Any loss or unanticipated destruction of samples (for example, due to freezer 
malfunction) or data (for example, misplacing a printout of data with identifiers) that 
meets the definition of a Protocol Deviation, unanticipated problem, and/or 
compromises the scientific integrity of the data collected for the study, will be reported to 
the NIH IRB. 
12.
Remuneration Plan for Subjects
No remuneration will be provided for participating in the study. 

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Appendix A: Definition of Hypereosinophilic Syndrome 
Historically, HES has been defined according to the original Chusid’s criteria (21): 
sustained peripheral blood eosinophilia of unknown origin, exceeding 1500/µl for more 
than 6 consecutive months, and responsible for the development of organ dysfunction 
and/or damage.
More recently the definition of HES has been refined as follows, and will be used for the 
purposes of this protocol (22).
1. Blood eosinophilia of greater than 1500/µL on at least 2 occasions or evidence of 
prominent tissue eosinophilia associated with symptoms and marked blood 
eosinophilia
2. Exclusion of secondary causes of eosinophilia, such as parasitic or viral infections, 
allergic diseases, drug-induced or chemical-induced eosinophilia, hypoadrenalism, 
and neoplasms

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Appendix B: Schedule of Procedures/Evaluations 
* Symptom evaluation to be performed at baseline/H&P or prior to dosing of steroids
#  Weekly taper for assessments not completed at NIH will not include vital signs and weight.
GC Challenge
Evaluation
Baseline/
H&P
     0 hour    
2 hour
4 hour
24 hour
GC Taper
Weekly
End of Study Visit
Informed Consent
X
Demographics (update CRIS and CRIMSON)
X
H&P and Medication History
X
X
Clinical Assessment (Vital signs and weight, Symptom 
evaluation (HES and steroid related)
*X
*X
X
#X
X
Acute care, mineral and hepatic panels
X
X
X
X
CBC with differential
X
X
X
X
X
X
X
PT/PTT, INR
X
Flow cytometry
X
X
Urinalysis
X
Pregnancy Testing – Serum Beta Hcg
X
Pregnancy Testing – Urine Hcg
X
Cortisol
X
Research blood
X
X
X
X
X
X

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Appendix C: Adult Blood Volumes for Specimen Collection 
Evaluations
Study Schedule
GC Challenge
(1 )Visit
Baseline
H&P
0 hr
2 hr
4 hr
24 h
Weekly draws
Weekly 
draws
Approx. 
max # of 
weeks
End of Study
Follow-up (2)
Week of Study
1
W1
W 1
W 1
W 1
W 2-8
W 9-16
W 16-24
W 16-24
Day of Study
X
D 1
D1
D 1
D 2
Clinical
 
CBC, differential, platelets
3 mL
3 mL
3mL
3 mL
3 mL
5 mL
5 mL
5 mL
5 mL
3 mL
PT, aPTT, INR
4.5 mL
-HCG 
4 mL
4 mL
Acute care, mineral and hepatic 
l
4 mL
4 mL
4 mL
4 mL
Flow cytometry
6 mL
6 mL
Cortisol
4 mL
Research
PBMC Eosinophil & Plasma Storage
60 mL
50 mL
30 mL
30 mL
60 mL
60 mL
Serum Storage 
8 mL
8 mL
8 mL
8 mL
8 mL
RNA PAX
3 mL
3 mL
3 mL
3 mL
3 mL
Volume (mL)**
89.5 mL
76 mL
33 mL
44 mL
78 mL
(177.5)**
43
(5 mL x 8 
wks)+3
40
(5 mL 
x 8 
wks)
45
(5 mL x 9 
wks)
120
(45 mL + 
follow up)
(3)Cumulative 8 week Volumes (mL)
363.5
40
45
165
1 Visit Windows: Screening may be completed over 2 visits. Other visits and permitted windows are as follows: (2) Baseline within 90 days of GC challenge. Follow up 2-4 weeks 
after completion of taper (window +/- 1 week). Weekly calls/draws within 3 days of timepoint 3Per NIH MEC Policy M95-9, maximum blood volumes drawn for research purposes 
for an adult subject (aged 18 years or older) may not exceed: 10.5 mL/kg or 550 mL, whichever is smaller, over any eight-week period. Exceptions to this policy shall be 
approved by the IRB. ** Denotes 24 hour volume
4 Day X evaluations are the baseline for subsequent safety assessments.  Day X evaluations must take place within 90 days prior to GC challenge. A CBC with differential must 
be available within 14 days of the enrollment. 

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Appendix D: Pediatric Blood Volumes for Specimen Collection 
Evaluations
Study Schedule
GC Challenge
Weekly draws
Weekly 
draws
Approx. 
max # of 
weeks
Follow-up (2)
(1 )Visit
Baseline
H&P
0 hr
4 hr
24 hr
Week of Study
-1
1
W 1
W 1
W 1 
W 2-8
W 9-16
W 16-24
W 16-24
Day of Study
X
D 1
D 1
D 2
Clinical
 
CBC, differential, platelets
3 mL
3 mL
3 mL
3 mL
5 mL
5 mL
5 mL
5 mL
3 mL
PT, aPTT, INR
4.5 mL
-HCG 
4 mL
4 mL
Acute care, mineral and hepatic 
l
4 mL
4 mL
4 mL
4 mL
Flow cytometry
6 mL
6 mL
Cortisol
4 mL
Research
PBMC Eosinophil & Plasma Storage
30 mL
20 mL
30 mL
30 mL
30 mL
Serum Storage 
4 mL
4 mL
4 mL
4 mL
4 mL
RNA PAX
3 mL
3 mL
3 mL
3mL
3 mL
Volume (mL)**
55.5
42
40 mL
44 mL
(163)**
43
(5 mL x 8 
wks)+3
40
(5 mL 
x 8 
wks)
45
(5 mL x 9 
wks)
95
(45 mL + 
follow up)
(3)Cumulative 8 week Volume (mL)
260.5
40
45
105
1 Visit Windows: Screening may be completed over 2 visits. Other visits and permitted windows are as follows: (2) Baseline within 90 days of GC challenge. Follow 
up 2-4 weeks after completion of taper (window +/- 1 week). Weekly calls/draws within 3 days of timepoint 3Per NIH MEC Policy M95-9, maximum blood volumes 
drawn for research purposes for pediatric subjects: no more than 5 mL/kg in a single day, and no more than 9.5 mL/kg may be drawn over any eight-week period. 
Exceptions to this policy shall be approved by the IRB. ** Denotes 24 hour volume
4 Day X evaluations are the baseline for subsequent safety assessments.  Day X evaluations must take place within 90 days prior to GC challenge

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Appendix E: Steroid Taper Schedule 
 

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Appendix F: Exploratory Endpoints Timeline 
1) All subjects will be evaluated for GC absorption 4 hours after GC dosing by evaluation 
of blood prednisone and prednisolone levels and assessment of pharmacokinetics.
2) All subjects will have RNA extracted from whole blood and frozen for evaluation of 
changes in expression after dosing with GC. Results will be evaluated using microarray 
technology once all subjects have been enrolled.
3).GR isoforms will be performed by RT-PCR
4) Additionally, plasma, PBMC’s and eosinophil RNA will be stored for further studies not 
described above. 

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Appendix G: Figures 
Figure 1: Glucocorticoid Action
1 – GC absorption through the gastrointestinal tract
2a –Cytoplasmic GR becomes ligand bound and enters the nucleus where it undergoes homo- 
or heterodimerization. The GC/GR complex binds to glucocorticoid response elements (GREs) 
leading to activation or repression of cytokine transcription 
2b – Alternative splicing of the GR transcript into GR and GR as well as alternate transcript 
forms. GR is primary localized in the nucleus, whereas GR is cytoplasmic before it binds 
ligand.
2c – The formation of the transcriptional complex: involvement and interaction of transcription 
factors, co-activators, and chromatin modulators modulate the AF-1 and AF-2 portions of the GR 
complex. 
2d – Post-transcriptional regulation mechanisms including GC/GR complex binding to 
transcription factors AP-1, NFκB, and cyclic AMP response element binding protein (CREB) and 
GR binding to mRNA(23)
3 – Cytokine expression profile/footprint in GC resistance

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Figure 2: Altered GC receptor signaling
From: Kino, T, Chrousos GP. Intracellular Glucocorticoid Signaling: A Formerly Simple System 
Turns Stochastic. Science Oct 2005(12).

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