Phase I/II Study of Human Chorionic Gonadotropin and Epidermal Growth Factor Supplementation (Pregnyl®) to Support Tolerance and Repair As Adjunct Therapy in High-Risk or Refractory Acute Graft-Versus-Host Disease
Phase: PHASE1
Conditions: Graft vs Host Disease
Sponsor: Masonic Cancer Center, University of Minnesota

Masonic Cancer Center, University of Minnesota 
Blood and Marrow Transplantation Program 
 
 
Phase I/II Study of Human Chorionic Gonadotropin and Epidermal 
Growth Factor Supplementation (Pregnyl®) to Support Tolerance and 
Repair As Adjunct Therapy in High-Risk or  
Refractory Acute Graft-Versus-Host Disease 
 
MT2014-12 
CPRC #2014LS020 
IND 127323 
 
 
University Of Minnesota 
Principal Investigator/IND Sponsor/Investigator: 
Shernan G. Holtan, MD 
 
 
Co-Investigators: 
Armin Rashidi, MD, PhD 
Margaret L. MacMillan, MD, MSc  
Daniel J. Weisdorf, MD 
Pamala Jacobson, PharmD* 
Angela Panoskaltsis-Mortari, PhD* 
Bruce R. Blazar, MD* 
 
*non-clinical - will not consent participants 
Affiliate Institution: 
Rush University, Chicago, IL 
Celalettin Ustun, MD 
 
Biostatistician: 
 Qing Cao, MS  
 
 
Version Date: 
May 27, 2020 
 
 
 
Confidential 
 
 

MT2014-12: hCG supplementation in high-risk or refractory aGVHD 
 
 
 
 
 
 
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CPRC #2014LS020 
Key Study Personnel Contact Information 
Site 
Contact Information 
Role 
Masonic Cancer 
Center, University of 
Minnesota 
Shernan G. Holtan, MD 
Hematology, Oncology and Transplantation 
Department of Medicine 
MMC 480  
420 Delaware Street SE 
Minneapolis, MN  55455 
612-301-1095 (phone) 
612-625-6919 (fax) 
sgholtan@umn.edu (email) 
IND 
sponsor/ 
Study PI 
Affiliate Manager 
Clinical Trials Office 
MMC 498 
420 Delaware Street SE 
Minneapolis, MN 55455 
Phone: 612 626-5174 
Email:  affiliates@umn.edu 
Fax: 612 625-6145 
Affiliate 
Sites 
Manager 
Rush University 
Celalettin Ustun, MD  
Associate Professor 
Department of Internal Medicine, Division of 
Hematology, Oncology and Cell Therapy, Rush 
Medical College 
Section Chief, Bone Marrow and Stem Cell Transplant 
1725 W. Harrison St. 
Suite 836 
Chicago, IL 60612 
Phone: (312) 942-7100 
Email: Celalettin_Ustun@rush.edu 
Fax: (312) 563-0059 
Site PI 
 
Refer to the Procedures Manual for Affiliate Sites for a complete list of study personnel and 
contact information.  

MT2014-12: hCG supplementation in high-risk or refractory aGVHD 
 
 
 
 
 
 
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CPRC #2014LS020 
Revision History 
 
Revision 
# 
Version 
Date 
Summary Of Changes 
Consent 
Changes 
 
04/08/2015 
original to CPRC 
 
 
05/11/2015 
response to CPRC initial review 
n/a 
 
07/07/2015 
clarify maintenance in schema and section 7 
minor edits/clarification in section 10 prior to IRB/FDA submission 
n/a 
 
08/05/2015 
a complete response to the FDA’s deficiency request: 
• Synopsis, section 5.2, appendix I – exclude patients unwilling or 
unable to stop supplemental sex hormone therapy (estrogen, 
progesterone, and/or testosterone preparations) 
• Schema, sections 4 and 8.1 – Add “Each lot of Pregnyl® will be 
tested for EGF content prior to administration so that the precise 
EGF dose administered is known.” 
• Schema, sections 4, 7.1, 7.2, 10.1, 10.2, 11.2, 13.1- revise DLT 
assessment period to “occurring by day 21 after the 1st dose of 
Pregnyl®”, previously was day 7 for males and day 14 for 
females  
• Sections 7.1, 7.2 and 7.4 – remove individual dose level 
reductions due to DLTs 
• Section 10.3 – delete reference to patients receiving supplement 
as now such patients are excluded 
• Section 13.1 – expand CRM section for clarity  
• Section 13.4 – recalculated early study stopping rule by lowering 
the expected toxicity rate 
• Section 15 – update reference 22 from abstract to full publication 
    
Parent  
 
08/14/2015 
A complete response to the FDA’s deficiency request (dated 
08/13/2015): 
• Synopsis, Sections 5.1, 10.1 and 10.2, appendix I – add 
baseline eGRF and LVEF requirements for eligibility 
• Synopsis, Section 5.2 and appendix I - expand exclusion 
criteria to exclude patients with a hormone responsive 
malignancy, an active or recent thrombus (irrespective of 
anticoagulation status) 
• Need to correct reference 
yes 
 
09/12/2016 
Section 5.1Clarification of inclusion/exclusion criteria, expansion to 
include patients with steroid-dependent aGVHD, late-onset 
aGVHD, and overlap syndrome 
 
 
 
11/30/2016 
Section 5, Clarification of eligibility criteria, evaluability, patient 
replacement, and Appendix 1 
No 
 
03/22/2017 
Corrected typo in Section 6.4 in response to CPRC stipulations on 
version 11/30/16 
No 
 
06/21/2017 
Section 5, Updated eligibility to include patients from 0-12 
Section 7.1 and Section 7.2, Separated Arm 2 into Arm 2A 
(steroid-dependent) and 2B (steroid-refractory), and expanded 
dosing cohorts for Arm 2B given preliminary laboratory analyses. 
Yes 
 
10/28/2017 
Section 13, Edited the statistical section due to CPRC stipulations 
on 6/21/17 amendment 
 
 
05/17/2018 
Fixed typographical error in study schema dose levels; 
Section 5,  updated eligibility based upon biomarkers 
no 

MT2014-12: hCG supplementation in high-risk or refractory aGVHD 
 
 
 
 
 
 
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CPRC #2014LS020 
 
01/22/2019 
Title page, Key personnel, Section 6.1, Section 11.3, Section 12.7: 
Added 2nd site (Rush University) – throughout document, protocol 
updated with information for affiliate institutions. 
Section 8.1, Added bioequivalent form of hCG/EGF from 
Fresenius Kabi should Merck product not be available. 
Section 11.2, Added relapse of primary malignancy to reportable 
events. 
Yes 
 
07/01/2019 
Minor administrative clarification – synopsis, sections 7.3 and 
Section 8.1 - for Arm 2B, dose level 5,to avoid using vials from 
more than one Lot of study product, the maximum dose is either 
(1) the contents of a single 10,000 IU vial, or (2) a maximum dose 
of 10,000 IU from multiple vials. All vials used should be from the 
same Lot.  
No 
 
05/27/2020 
Section 5.1: Arm 1, updated definition of high-risk aGVHD to 
include Minnesota standard with aGVHD with no response to 7 
days of sirolimus plus a calcineurin inhibitor 
Deleted eligibility checklist (Appendix I) as per SOP – this 
document is stored in Oncore 
 
 
 
 
 

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CPRC #2014LS020 
Table of Contents 
 
Key Study Personnel Contact Information .................................................................................. 2 
Protocol Synopsis ...................................................................................................................... 7 
1 
Study Objectives ................................................................................................................10 
1.1 
Primary Objective........................................................................................................10 
1.2 
Secondary Objectives .................................................................................................10 
1.3 
Correlative Objectives .................................................................................................10 
1.4 
Transplant Related Objectives ....................................................................................10 
2 
Background ........................................................................................................................11 
3 
Summary and Rationale .....................................................................................................16 
4 
Study Design .....................................................................................................................18 
5 
Patient Selection ................................................................................................................20 
5.1 
Inclusion Criteria .........................................................................................................20 
5.2 
Exclusion Criteria ........................................................................................................21 
6 
Patient Registration ............................................................................................................22 
6.1 
Registration with the Masonic Cancer Center Clinical Trials Office .............................22 
6.2 
Pregnyl® Dose Level Assignment ...............................................................................22 
6.3 
Patients Who Are Registered and Do Not Receive Study Treatment ..........................22 
6.4 
Patients Who Do Not Complete All Study Treatments .................................................23 
7 
Treatment Plan ..................................................................................................................23 
7.1 
Arm 1 – High-Risk aGVHD ..........................................................................................23 
7.2 
Arm 2A – Steroid-Dependent aGVHD .........................................................................24 
7.3 
Arm 2B – Steroid-Refractory aGVHD ..........................................................................26 
7.4 
Supportive Care ..........................................................................................................27 
7.5 
Duration of Study Therapy ..........................................................................................27 
7.6 
Duration of Study Participation ....................................................................................28 
8 
Study Drug Formulation, Preparation, Side Effects ............................................................28 
8.1 
Human Chorionic Gonadotropin (HCG) (Pregnyl® (Merck), or generic urinary-derived 
hCG from Fresenius Kabi) .....................................................................................................28 
9 
Disease Reassessment and Response Definitions ............................................................30 
10 
Schedule of Patient Activities .........................................................................................31 
10.1 Correlative Studies and Hypotheses ...........................................................................33 
11 
Adverse Event Reporting ................................................................................................34 

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CPRC #2014LS020 
11.1 Definitions ...................................................................................................................34 
11.2 Adverse Event Documentation ....................................................................................36 
11.3 Institutional Event Reporting Table .............................................................................37 
11.4 Required Reporting: FDA, IRB and Masonic Cancer Center’s SAE Coordinator .........38 
12 
Study Data Collection and Monitoring .............................................................................39 
12.1 Data Management ......................................................................................................39 
12.2 Case Report Forms .....................................................................................................39 
12.3 Data and Safety Monitoring Plan (DSMP) ...................................................................39 
12.4 IND Annual Reports ....................................................................................................40 
12.5 Monitoring ...................................................................................................................40 
12.6 Teleconferences – Lead Site and Affiliate Site ............................................................40 
12.7 Affiliate Site Monitoring ...............................................................................................40 
12.8 Record Retention ........................................................................................................41 
13 
Statistical Considerations ...............................................................................................41 
13.1 Study Design ..............................................................................................................41 
13.2 Sample Size Consideration .........................................................................................46 
13.3 Statistical Analysis ......................................................................................................47 
13.4 Early Stopping Rule ....................................................................................................48 
14 
Conduct of the Study ......................................................................................................48 
14.1 Good Clinical Practice .................................................................................................48 
14.2 Ethical Considerations ................................................................................................48 
14.3 Informed Consent .......................................................................................................48 
15 
References .....................................................................................................................49 
  
 
 
 
 
 

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CPRC #2014LS020 
Protocol Synopsis 
 
Study Design: 
This multi-center center phase I/II study will use Continual Reassessment Method (CRM) to 
establish a maximum tolerated dose (MTD) of human chorionic gonadotropin and epidermal 
growth factor supplementation (hCG) as an adjunct to facilitate mucosal healing and immune 
tolerance in conjunction with accepted, standard immunosuppressive therapy in the setting 
of high-risk (Arm 1) and steroid-dependent (Arm 2A) and steroid-refractory (Arm 2B) acute 
graft-versus-host disease (aGVHD).  Enrollment and dose escalation of Pregnyl® will be 
independent for each arm. 
 
After completion of the dose finding trial for each arm, the final doses will be carried forward 
into a two-stage phase II extension trial to confirm safety and make a preliminary 
determination of the activity level for Arm 1, Arm 2A and Arm 2B. If the phase I trial enrolls 
fewer than 13 patients at the MTD, we will employ Simon’s Minmax two-stage design with 
the possibility to discontinue after the 1st stage if the response rate is low.  
• Stage 1: Enroll a total of 12 patients (including all treated at the MTD in phase I). If 5 or 
more of these respond, the trial will continue to stage 2. 
• Stage 2: Enroll an additional 14 patients. If 14 or more out of 26 patients respond, hCG 
will be considered promising for further investigation.  
If phase I trial enrolls more than 12 patients at the MTD, we will adjust the futility rule above 
for the two stage design with the giving number of patients enrolled at the MTD. 
 
Primary Objective: 
Phase I:  To determine the maximum tolerated dose (MTD) of Pregnyl® when given in 
combination with standard immunosuppressive therapy in pediatric and adult patients with 
high-risk (Arm 1) or steroid-refractory/dependent (Arm 2) aGVHD 
 
Phase II:  To determine the proportions of complete, partial, mixed, and no response among 
surviving patients at days 28 after initiation of protocol therapy in pediatric and adult patients 
with high-risk (Arm 1) or steroid-refractory/dependent (Arm 2) aGVHD 
  
Secondary 
Objectives: 
• To determine the safety and feasibility of hCG supplementation with Pregnyl® in 
combination with standard immunosuppressive therapy in pediatric and adult patients with 
high-risk or refractory aGVHD 
• To determine the incidence of acute GVHD flare after CR/PR requiring increase of steroids 
or other systemic treatment at days 28 and 56 
• To compare the rate of treatment failure for acute GVHD at days 28 and 56 after initiation 
of protocol therapy to historical controls 
 
Transplant Related 
Objectives: 
• Disease-free survival, overall survival, and non-relapse mortality at day 28, 56, 180 and 1 
year of protocol treatment 
• Incidence of chronic GVHD at day 180 and 1 year 
 
Correlative 
Objectives: 
• Pharmacokinetics (clearance, half-life, volume of distribution) of the both the hCG and 
EGF components of urinary hCG when administered to patients with GVHD  
• Monitoring for hormonal effects via determination of serum concentrations of hCG, 
estradiol, progesterone, and testosterone on days 7, 14, 28, and 56, compared to baseline 
• Collection of blood samples for measurement of immune and tissue damage/repair 
biomarkers to determine change from pre-treatment levels to levels obtained on days 7, 
14, 28, and 56 post-treatment 
• Analysis of previously collected skin and/or gastrointestinal biopsies for biomarkers of 
refractory aGVHD and response to therapy 
 

MT2014-12: hCG supplementation in high-risk or refractory aGVHD 
 
 
 
 
 
 
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CPRC #2014LS020 
Patient Population: 
HCT recipients 0 to 76 years of age within the first 7 days of initial treatment of high-risk 
(Arm 1) or steroid-refractory (Arm 2) aGVHD at any time in their treatment course 
 
Inclusion Criteria: 
Renal:  Serum creatinine ≤2.5x upper limit of normal (ULN) 
Cardiac:  Left ventricular ejection fraction (LVEF) ≥ 35%  
 
Exclusion Criteria: 
• Progressive malignancy 
• Uncontrolled infection at initiation of protocol treatment 
• Unwilling or unable to stop supplemental sex hormone therapy (estrogen, progesterone, 
and/or testosterone preparations) 
• Diagnosis of a hormone responsive malignancy  
• Current thromboembolic disease requiring full-dose anticoagulation - patients receiving 
pharmacologic prophylaxis for thromboembolic disease will be eligible  
• Active or recent (within prior 3 months) thrombus, irrespective of anticoagulation status 
• Pregnancy 
• Women or men of childbearing potential unwilling to take adequate precautions to avoid 
unintended pregnancy from the start of protocol treatment through 28 days after the last 
treatment 
 
Enrollment Plan: 
Phase I:  Up to 20 patients in each arm with an estimated 2 years to accrue patients  
Phase II component (for each arm):   
Stage 1: Enroll a total of 12 patients (including all treated at the MTD in phase I). If 5 or 
more of these respond, the trial will continue to stage 2. 
 
Stage 2: Enroll an additional 14 patients. If 14 or more out of 26 patients respond, hCG 
will be considered promising for further investigation. 
 
 
 
*NOTE: Arm 2B, dose level 5: to avoid using vials from more than one Lot of study product, the maximum dose is either (1) 
the contents of a single 10,000 IU vial, or (2) a maximum dose of 10,000 IU from multiple vials. All vials used should be from 
the same Lot. 
 

MT2014-12: hCG supplementation in high-risk or refractory aGVHD 
 
 
 
 
 
 
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CPRC #2014LS020 
Arm 1, Arm 2A and Arm 2B will be treated as separate, independent cohorts.  Each lot of Pregnyl® will be 
tested for EGF content prior to administration so that the precise EGF dose administered is known. 
   
Phase I:   
For each arm, dose levels of Pregnyl® will be tested starting at dose level 1. Dose levels -1 and -2 will be 
used only if dose level 1 proves too toxic.   
 
The 1st 2 patients will be enrolled in dose level 1. The next cohort of 2 patients will not begin treatment 
until all patients in the current cohort have reached day 21.  Each new cohort of 2 patients will be 
assigned by the study statistician to the most appropriate dose level based on toxicity probabilities set for 
this study.  The MTD for each arm (high-risk and steroid-refractory aGVHD) will be identified at or before 
the total sample size of 20 patients per arm is reached.   
 
The dose limiting toxicity (DLT) is defined as any of the following occurring by day 21 after the 1st dose of 
Pregnyl®:  
• In males and females:  Grade 2-4 thromboembolic event (CTCAE v4) i.e., any thrombosis more 
severe than a superficial venous thrombosis where treatment is required 
• In males and females:  Grade 3-4 ascites (CTCAE v4)  
• Specific to females: Ovarian hyperstimulation syndrome, defined as the rapid development of 
enlarged, cystic, painful ovaries 
 
 
Phase II Extension:   
• Stage 1: Enroll a total of 12 patients (including all treated at the MTD in phase I). If 5 or more of these respond, 
the trial will continue to stage 2. 
• Stage 2: Enroll an additional 14 patients. If 14 or more out of 26 patients respond, hCG will be considered promising 
for further investigation.  
 

MT2014-12: hCG supplementation in high-risk or refractory aGVHD 
 
 
 
 
 
 
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CPRC #2014LS020 
1 
Study Objectives 
1.1 
Primary Objective 
Phase I:  To determine the maximum tolerated dose (MTD) of Pregnyl® 
when given with standard immunosuppressive therapy in pediatric and adult 
patients with high-risk or refractory acute graft-versus-host disease 
(aGVHD). 
 
Phase II:  To determine the proportions of complete, partial, mixed, and no 
response among surviving patients at days 28 after initiation of protocol 
therapy in pediatric and adult patients with high-risk (Arm 1) or refractory 
(Arm 2) aGVHD. 
1.2 
Secondary Objectives 
• To determine the safety and feasibility of hCG supplementation with 
Pregnyl® in combination with standard immunosuppressive therapy in 
pediatric and adult patients with high-risk or refractory aGVHD 
• To determine the incidence of acute GVHD flare after CR/PR requiring 
increase of steroids or other systemic treatment at days 28 and 56 
• To compare the rate of treatment failure for acute GVHD at days 28 
and 56 after initiation of protocol therapy to historical controls 
1.3 
Correlative Objectives 
• Pharmacokinetics (clearance, half-life, volume of distribution) of the both 
the hCG and EGF components of urinary hCG when administered to 
patients with GVHD  
• Monitoring 
for 
hormonal 
effects 
via 
determination 
of 
serum 
concentrations of hCG, estradiol, progesterone, and testosterone on 
days 7, 14, 28, and 56, compared to baseline 
• Collection of blood samples for measurement of immune and tissue 
damage/repair biomarkers to determine change from pre-treatment 
levels to levels obtained on days 7, 14, 28, and 56 post-treatment 
• Analysis of previously collected skin and/or gastrointestinal biopsies for 
biomarkers of refractory aGVHD and response to therapy 
1.4 
Transplant Related Objectives 
• To determine the disease-free survival, overall survival, and non-relapse 
mortality at day 28, 56, 180 and 1 year of protocol treatment  
• To determine the incidence of chronic GVHD at day 180 and 1 year  

MT2014-12: hCG supplementation in high-risk or refractory aGVHD 
 
 
 
 
 
 
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2 
Background 
 
Patients with high-risk acute graft-versus host disease (HR-aGVHD) have poor 
responses to initial therapy and an increased risk of death.  
 
In the US, nearly 7,000 patients undergo allogeneic hematopoietic cell transplantation 
(HCT) annually in an effort to cure hematologic malignancies and other bone marrow 
disorders.  The majority of allogeneic HCT recipients will experience acute graft-
versus-host 
disease 
(aGVHD), a 
complication 
in 
which cells from the 
immunocompetent donor graft attack the recipient’s organs and tissues [1].  Patients 
with severe skin or visceral involvement, or multi-organ aGVHD, have high-risk 
disease, characterized by a less than 50% complete or partial response to first-line, 
high-dose corticosteroids and a 2-3 fold risk of treatment-related mortality [2].  Before 
the recent ability to discriminate high-risk versus standard-risk aGVHD based upon 
organ staging at onset, it was noted that one-half of patients aGVHD did not sustain 
response to first-line therapy with corticosteroids (i.e., are steroid-refractory) [3].  
Damage to the gastrointestinal tract is the major cause of morbidity and mortality in 
patients with severe, refractory aGVHD.   
 
Figure 1 shows typical colonoscopic findings of a 
University of Minnesota patient with aGVHD with 
mucosal edema, erythema, loss of normal vascular 
patterns, and extensive ulcerations.  Clinically, such 
patients have anorexia, abdominal cramping and 
pain, malabsorption, and large volume (often >2 
liters) of diarrhea daily.  They become severely 
malnourished, require intravenous medications and 
nutrition due to poor oral absorption, and they 
consequently endure very prolonged hospital stays.  
80-100% of patients die within two years of the 
development 
of 
steroid-refractory 
[4] 
(and 
unpublished quality improvement data).  
Figure 1.  Appearance of transverse colon in a 
patient suffering from steroid-refractory aGVHD. 

MT2014-12: hCG supplementation in high-risk or refractory aGVHD 
 
 
 
 
 
 
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CPRC #2014LS020 
Current standard treatment of aGVHD depletes circulating and tissue immune 
effector cells. The reason for poor outcomes in high-risk and steroid-refractory 
aGVHD is likely not solely due to poor efficacy of immunosuppressant medications.  
High-dose steroids, calcineurin inhibitors, anti-thymocyte globulin, and anti-cytokine 
therapies effectively deplete the circulating pool of cytokine-producing cells 
responsible for GVHD effects.  Figure 2 
shows an example of the effects of 
steroid-refractory 
aGVHD 
and 
its 
treatment 
on 
peripheral 
blood 
leukocytes (PBL) in humans (Holtan, 
unpublished data).  The scatterplot on 
the left depicts the distribution of normal 
PBL, 
including 
TNFα-producing 
monocytes, 
neutrophils, 
and 
lymphocytes in a healthy donor.  The 
scatterplot on the right depicts PBL of a 
patient treated for steroid-refractory 
aGVHD 
with 
high-dose 
steroids, 
cyclosporine, and TNFα inhibition. It is 
apparent that TNFα-producing monocytes and lymphocytes, the cell subsets 
responsible for aGVHD effects, are substantially depleted from the peripheral blood 
of the steroid-refractory aGVHD patient.   
 
These inflammatory cytokine-producing cells are also depleted from colonic tissue in 
patients with refractory aGVHD.  Figure 3 shows that the lamina propria in a patient 
with refractory aGVHD (right) is relatively devoid of hematopoietic cells (CD45 staining 
in brown) compared to that of a normal donor (left, Holtan, unpublished data).  Based 
upon 
these 
preliminary 
findings 
as 
well 
as 
>20 
years 
of 
intensified 
immunosuppression as the sole paradigm for high-
risk or refractory aGVHD, it is very unlikely that 
investigational drugs that exert their effects by 
intensification of immune suppression will be able to 
facilitate healing or improve organ function.  New 
paradigms aimed at supporting immunologic 
tolerance as well as restitution of damaged tissues 
such as the methods proposed in this trial are 
necessary. 
 
 
Figure 2.  Spectrum of TNFα-producing and non-TNFα-producing 
peripheral blood leukocytes after stimulation with lipopolysaccharide in 
a normal donor (left) versus a patient treated for steroid-refractory 
aGVHD (right). 
Figure 3.  Fewer CD45+ (brown) hematopoietic 
cells in colon tissue (lamina propria) normal donor 
(left) versus a patient treated for steroid-refractory 
aGVHD (right).

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Mucosal healing capacity may be reduced in high-risk or refractory GVHD due 
to deficiency of trophic growth factors.   
Markers of endothelial damage (angiopoietin-2) and angiogenesis (vascular 
endothelial growth factor, VEGF) have been shown to be prognostic in steroid-
refractory acute GVHD [5].  A recent broader study of angiogenic factors in allogeneic 
HCT has been completed.  In that pilot study, epidermal growth factor (EGF) levels 
were over 10-fold lower in 17 patients experiencing severe (grade III or IV) aGVHD 
compared to 17 allogeneic HCT recipients at 3-months post-HCT who did not 
experience aGVHD (median 11.5 vs 141.4 pg/ml, p=0.004). The finding of low EGF 
levels at the onset of aGVHD was 
subsequently 
validated 
in 
two 
independent cohorts of patients enrolled 
on multicenter treatment trials, Blood and 
Marrow Transplant Clinical Trials Network 
(BMT CTN) 0302 and 0802 compared to a 
separate cohort of patients without 
aGVHD [[6], and Holtan et al, manuscript 
accepted].  Furthermore, in BMT CTN 
0802, where plasma (as opposed to 
serum, which can have EGF levels 
increase due to release from activated 
platelets) was analyzed, EGF levels 
significantly decreased at day 28 after 
initial aGVHD therapy (p=0.01, Wilcoxon 
signed rank) in patients who had no 
response.  This was particularly apparent 
in patients who had high-risk aGVHD at 
onset (Figure 4, top), where day 28 
median EGF was 6-fold lower (3.8 versus 
23.2 pg/mL) in patients with no response 
to initial therapy.  In contrast, patients with 
complete responses to initial therapy 
demonstrated increases in plasma EGF 
by day 28 of treatment.   
Decreased circulating EGF levels have been identified in inflammatory bowel disease 
[7], which shares many manifestations with GVHD.  EGF has been shown to enhance 
gut epithelial restitution after radiation injury [8] and protect against the development 
of colitis in a rat model [9].  Furthermore, EGF treatment can improve ion transport 
capabilities, especially sodium reabsorption, in inflamed colonic mucosa [10].  In 
Figure 4.  Plasma EGF levels at aGVHD onset and day 28 of 
initial therapy in patients enrolled in BMT CTN 0802, stratified by 
high-risk aGVHD and standard risk aGVHD.  Patients with high-
risk (top) and standard risk aGVHD (bottom) and no response 
(NR) to therapy show a decline in EGF levels at day 28 of 
therapy, with the decrease even more dramatic in high-risk 
aGVHD.

MT2014-12: hCG supplementation in high-risk or refractory aGVHD 
 
 
 
 
 
 
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CPRC #2014LS020 
addition to providing mitogenic signals for intestinal epithelial stem cells, EGF also 
has been shown to regulate inflammation, intestinal epithelial apoptosis, and 
autophagy in the setting of necrotizing enterocolitis [11].  Chronic administration of 
intraluminal EGF enhanced colonic mucosal growth in both a rodent model [12] and 
in humans [13].  However, intraluminal administration is not practical or likely to be 
efficacious in steroid-refractory aGVHD due to the typical widespread gastrointestinal 
involvement.  Feasibility of systemic treatment with EGF has recently been 
demonstrated.  In a randomized, placebo-controlled trial of intravenous recombinant 
EGF in premature infants with necrotizing enterocolitis, 6-day continuous IV 
administration of EGF at 100 ng/kg/hour improved gut mucosal thickness by 54% 
over baseline as early as day 4 of therapy [14].  No significant infusional or other 
systemic side effects from EGF administration were noted in the trial.  Whether similar 
responses could be elicited in steroid-refractory aGVHD has not been tested.  
Unfortunately, pharmaceutical grade recombinant EGF is not currently available for 
clinical testing in humans, making alternate sources of EGF necessary. 
 
Circulating EGF levels are high and excreted in the urine during pregnancy.   
 
In recent studies of the immune and growth factor milieu of early pregnancy, EGF 
levels have been identified as being approximately 20-fold elevated in the first 
trimester of pregnancy, while several inflammatory biomarkers were significantly 
decreased (Table 1) relative to non-pregnant controls (Holtan et al, manuscript 
accepted).  EGF is critical for the initiation and maintenance of pregnancy by 
stimulating hCG production in the early developing placenta [15].  As pregnancy 
progresses, deficiency of EGF leads to intrauterine growth retardation [16] and can 
predispose to necrotizing enterocolitis [17].  Considering its importance in placentation 
and fetal growth, it is not a surprise that EGF is a biologically functional component of 
commercial urinary-derived hCG preparations.  With testing by the Cytokine 
Reference Laboratory at the University of Minnesota, the concentration of EGF in 
Pregnyl® was determined to be 7,037 pg/mL (Panoskaltsis-Mortari, unpublished 
data). 
 
 
 
 
Analyte 
Non-pregnant 
Median (N=11) 
Min 
Max 
Pregnant Median (N=16) 
Min 
Max 
p 
EGF 
24.8 
8.6 
178.2 
489.4 
14.6 
1506 
0.003 
sCD40L 
424.7 
132.7 
1423 
29.2 
0 
1583 
0.004 
L-17 
14.8 
2.2 
161.6 
5.5 
<LLD 
46.9 
0.03 
IL-6 
7.9 
<LLD 
82 
<LLD 
<LLD 
34.3 
0.03 
Table 1.  Differences in serum growth factors and markers of inflammation between pregnant (first 
trimester) and non-pregnant individuals. LLD=lower limit of detection. 

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Tolerance induction during pregnancy:  central role of hCG.   
 
HCG is widely studied for its immune tolerance-inducing effects.  Recently described 
immunomodulatory properties of hCG include stimulating an increase in regulatory T 
cells with a concomitant reduction of inflammatory cytokines [18], maintenance of 
dendritic cells in a tolerogenic state [19], and modulation of innate immunity [20].  The 
molecular mechanisms of hCG-induced immune modulation are not completely 
known but involve inhibition of nuclear transcription factor-κB and activator protein-1 
gene transcription driven by TNFα [21].  Recently, chronic administration of low-dose 
hCG was piloted as a therapy for refractory chronic GVHD and found to be tolerable 
with suggestion of clinical efficacy [22].  In this study, an increase in indoleamine 2,3-
dioxygenase (IDO) and IL-10 blood levels were observed, both beneficial for runaway 
GVHD immune responses.  It is possible that some of the results, including the gut 
epithelial restitution depicted in this report, were also due to supplementation of EGF. 
 
Potential complementary roles of EGF and hCG in high-risk and steroid-
refractory aGVHD.  
 
It is possible that both the hCG and EGF components of Pregnyl® will contribute to 
improvement of clinical outcomes in high-risk and refractory aGVHD.  HCG is well 
known for its ability to induce immunologic tolerance, and EGF is well known for its 
role in repair of damaged tissues.  Considering the gut trophic properties of EGF and 
the immune modulatory properties of hCG, our central hypothesis is that 
administration of an EGF-containing urinary preparation of hCG to patients with 
steroid-refractory aGVHD may help restore organ function, support the development 
of immunologic tolerance, and improve quality of life, and without adding significant 
toxicity to standard immunosuppressive therapy.  Before large scale application of this 
approach, the safety, feasibility, pharmacokinetic, immunologic and other 
pharmacodynamic correlates of its administration in a limited number of patients must 
first be determined.   
 
Subcutaneously-delivered urinary hCG is has safely been given to both men and 
women for endocrinologic indications, with data regarding safety and efficacy 
spanning over 4 decades [23, 24, 25, 26, 27].  Severe reactions including arterial 
thromboembolism and ovarian hyperstimulation syndrome occur in <1% of patients.  
The doses administered on this protocol are within the range of established standard 
of care dosing for endocrinologic indications today. Similar low doses of urinary hCG  
given to a cohort of patients with steroid-refractory chronic GVHD was well tolerated.  
Therefore, we believe the safety risks of this study are very minimal.  However, the 

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doses required to significantly increase plasma and tissue EGF levels and exert 
immunologic effects are not known, making the studies proposed herein necessary 
for further repurposing of the drug. 
3 
Summary and Rationale 
High-risk and steroid-refractory acute graft-versus-host disease (aGVHD) are frequent 
and often fatal complications of allogeneic hematopoietic cell transplantation (HCT).  
The current treatment paradigm, intensification of immunosuppression, does not yield 
durable improvement of organ (especially gastrointestinal) function or long-term 
survival in most patients.  Many patients die of consequences of severe mucosal 
damage 
of 
the 
gastrointestinal 
tract 
and 
complications 
of 
profound 
immunosuppression.  This study takes an entirely different approach to the treatment 
of otherwise fatal aGVHD by hastening mucosal healing and inducing immune 
tolerance with the hormonal supplement, Pregnyl®.  We recently identified that 
circulating epidermal growth factor (EGF) levels are very low in patients with severe 
aGVHD compared to allogeneic HCT recipients without aGVHD.  EGF is an important 
protein involved in the proliferation of epidermal and epithelial cells, and it is involved 
in healing of damaged mucosal tissues.  Extensive preclinical and clinical data has 
demonstrated that administration of EGF induces remission of other life-threatening 
forms of intestinal inflammation, such as necrotizing enterocolitis and inflammatory 
bowel disease, but this approach has never been tested in aGVHD.  A readily available 
and inexpensive source of EGF is urinary-derived human chorionic gonadotropin 
(hCG) (e.g., Pregnyl® and other brands) which is commonly used for reproductive 
endocrinology indications.  Pregnyl® is isolated from the urine of pregnant women 
which contains both hCG and EGF.  Although the EGF component contained in 
Pregnyl® is not essential for reproductive endocrinology indications, it may be highly 
effective in providing EGF for repair of tissues damaged by aGVHD.   
 
HCG is a hormone critical for the development of maternal immune tolerance towards 
a fetus during pregnancy, arguably the most commonly encountered and effective 
example of induced immune tolerance in nature.  HCG induces immune tolerance in 
pregnancy by mechanisms including induction of indoleamine 2,3 dioxygenase (IDO) 
and expansion of endogenous regulatory T cells (Tregs), both of which are protective 
against GVHD.  Therefore, hCG may be effective in promoting immune tolerance in 
aGVHD.  Importantly, a urinary-derived preparation of hCG has already shown early 
promise in inducing tolerance and reversing organ damage without untoward toxicity 
in 12 of 20 (60%) of patients with heavily pre-treated patients with steroid-refractory 
or intolerant chronic GVHD [22].  
 

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The objective of this study is to test for the first time the use of supplemental EGF and 
hCG (Pregnyl®) as an adjunct to facilitate improved mucosal healing and immune 
tolerance in addition to accepted, standard immunosuppressive therapy in the setting 
of high-risk (Arm 1) and steroid-refractory (Arm 2) aGVHD.  Our central hypothesis is 
that administration of urinary hCG and EGF will provide (a) EGF for facilitation of 
gastrointestinal healing and (b) hCG for induction of immune tolerance, while not 
worsening the rates of infectious or other complications, in patients with severe 
aGVHD.  However, the optimal supplemental doses of EGF necessary to achieve an 
epithelial restitution effect are not known.  Therefore, this study will use the Continuous 
Reassessment Method (CRM) to test up to five dose levels of Pregnyl® in order to 
identify the maximum tolerated dose (MTD) by each patient cohort (high-risk (arm 1), 
steroid dependent (arm 2A) and steroid-refractory (arm 2B)).  We will determine 
pharmacokinetics of both the EGF and hCG components of urinary HCG and identify 
correlative biomarkers of aGVHD response.  The pharmacokinetic and correlative 
studies would not be possible without intercollegiate collaborations.  The knowledge 
gained from these aims will provide critical feasibility, preliminary safety, and 
correlative data for efficacious dose selection and identification of leading biomarkers 
for the phase II expansion of this trial.  These data will be necessary to successfully 
compete for National Institutes of Health or similar funding for a larger scale, 
multicenter trial, if the approach proves to be feasible, tolerable, and efficacious in this 
initial study.   
 
Urinary HCG is an inexpensive, commercially-available hormone with a long track 
record of safety and tolerability.  Therefore, if effective, we anticipate this could quickly 
be repurposed into a standard adjunctive treatment for high-risk or refractory aGVHD.   
 
The first goal of this study is to find the dose of Pregnyl® needed to at least double 
patients’ plasma EGF levels from baseline levels while not exceeding the maximum 
tolerated dose.  Our preliminary data indicate that baseline plasma EGF levels are in 
the range of 10-30 pg/ml, whereas the median plasma EGF level of a normal 
allogeneic HCT recipient without aGVHD is over 10-fold higher at 140 pg/mL. 
Dose justification:  The doses selected for testing in this trial are within the range of 
doses of Pregnyl® used for endocrinology purposes today and do not represent “high” 
doses.  The standard dose for ovulation trigger is 10,000 USP of the hCG component, 
which is more than double the amount that patients on the highest dose level on this 
protocol will receive.  The starting doses for this protocol are estimated to be the 
minimum sufficient to determine an increase in plasma EGF with therapy.  The 1-week 
cumulative EGF dose obtained from Pregnyl® at dose level 1 is expected to be 
equivalent to approximately 50% of the circulating EGF in a typical patient with severe 
aGVHD.  The 1-week cumulative EGF dose at dose level 2 is expected to be 

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approximately 100% of (effectively doubling) circulating EGF in patients with severe 
aGVHD.  Each lot of Pregnyl® will be tested for EGF content prior to administration 
so that the precise EGF dose administered is known. 
4 
Study Design 
This multi-center phase I/II study will use Continual Reassessment Method (CRM) to 
establish a maximum tolerated dose (MTD) of human chorionic gonadotropin and 
epidermal growth factor supplementation (hCG) as an adjunct to facilitate improved 
mucosal healing and immune tolerance in conjunction with accepted, standard 
immunosuppressive therapy in the setting of high-risk (Arm 1), steroid dependent (Arm 
2A), and steroid-refractory (Arm 2B) acute graft-versus-host disease (aGVHD).   
Enrollment and dose escalation of Pregnyl® will be independent for each arm.   
Arm 1, Phase I:  For each arm up to 5 dose levels of Pregnyl® will be tested; however 
dose levels -1 and -2 will be used only if dose level 1 proves too toxic.  In additional 
to Pregnyl®, patients will continue on their current standard of care aGVHD treatment. 
 
The 1st 2 patients will be enrolled in dose level 1. The next cohort of 2 patients will not 
begin treatment until all patients in the current cohort have reached day 21.  Each new 
cohort of 2 patients will be assigned by the study statistician to the most appropriate 
dose level based on toxicity probabilities set for this study.  The MTD for each arm 
(high-risk and steroid-refractory aGVHD) will be identified when the total sample size 
of 20 patients within an arm is reached.  Enrollment on phase I is expected to take 2 
years based on an enrollment of approximately 20 patients per year for each arm. 
 
Phase II Extension:  After completion of the dose finding trial for each arm, the final 
doses will be carried forward into a two-stage phase II extension trial to confirm safety 
and make a preliminary determination of the activity level for Arm 1 and Arm 2. If the 
phase I trial enrolls fewer than 13 patients at the MTD, we will employ Simon’s Minmax 
two-stage design with the possibility to discontinue after the 1st stage if the response 
rate is low.  
• Stage 1: Enroll a total of 12 patients (including all treated at the MTD in phase I). If 
5 or more of these respond, the trial will continue to stage 2. 
• Stage 2: Enroll an additional 14 patients. If 14 or more out of 26 patients respond, 
hCG will be considered promising for further investigation.  
If phase I trial enrolls more than 12 patients at the MTD, we will adjust the futility rule 
above for the two stage design with the giving number of patients enrolled at the 
MTD.  

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Arm 2A (steroid-dependent), Phase I:  For each arm up to 5 dose levels of Pregnyl® 
will be tested; however dose levels -1 and -2 will be used only if dose level 1 proves 
too toxic.  In additional to Pregnyl®, patients will continue on their current standard of 
care aGVHD treatment. 
 
The 1st 2 patients will be enrolled in dose level 1. The next cohort of 2 patients will not 
begin treatment until all patients in the current cohort have reached day 21.  Each new 
cohort of 2 patients will be assigned by the study statistician to the most appropriate 
dose level based on toxicity probabilities set for this study.  The MTD for each arm 
(high-risk and steroid-refractory aGVHD) will be identified when the total sample size 
of 20 patients within an arm is reached.  Enrollment on phase I is expected to take 2 
years based on an enrollment of approximately 20 patients per year for each arm. 
 
Phase II Extension:  After completion of the dose finding trial for each arm, the final 
doses will be carried forward into a two-stage phase II extension trial to confirm safety 
and make a preliminary determination of the activity level for Arm 1 and Arm 2. If the 
phase I trial enrolls fewer than 13 patients at the MTD, we will employ Simon’s Minmax 
two-stage design with the possibility to discontinue after the 1st stage if the response 
rate is low.  
• Stage 1: Enroll a total of 12 patients (including all treated at the MTD in phase I). If 
5 or more of these respond, the trial will continue to stage 2. 
• Stage 2: Enroll an additional 14 patients. If 14 or more out of 26 patients respond, 
hCG will be considered promising for further investigation.  
If phase I trial enrolls more than 12 patients at the MTD, we will adjust the futility rule 
above for the two stage design with the giving number of patients enrolled at the 
MTD.  
Arm 2B (steroid-refractory), Phase I:  For each arm up to 7 dose levels of Pregnyl® 
will be tested; however dose levels -1 and -2 will be used only if dose level 1 proves 
too toxic.  In additional to Pregnyl®, patients will continue on their current standard of 
care aGVHD treatment. 
 
The 1st 2 patients will be enrolled in dose level 1. The next cohort of 2 patients will not 
begin treatment until all patients in the current cohort have reached day 21.  Each new 
cohort of 2 patients will be assigned by the study statistician to the most appropriate 
dose level based on toxicity probabilities set for this study.  The MTD for each arm 
(high-risk and steroid-refractory aGVHD) will be identified when the total sample size 
of 20 patients within an arm is reached.  Enrollment on phase I is expected to take 2 
years based on an enrollment of approximately 20 patients per year for each arm. 

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Phase II Extension:  After completion of the dose finding trial for each arm, the final 
doses will be carried forward into a two-stage phase II extension trial to confirm safety 
and make a preliminary determination of the activity level for Arm 1 and Arm 2. If the 
phase I trial enrolls fewer than 13 patients at the MTD, we will employ Simon’s Minmax 
two-stage design with the possibility to discontinue after the 1st stage if the response 
rate is low.  
• Stage 1: Enroll a total of 12 patients (including all treated at the MTD in phase I). If 
5 or more of these respond, the trial will continue to stage 2. 
• Stage 2: Enroll an additional 14 patients. If 14 or more out of 26 patients respond, 
hCG will be considered promising for further investigation.  
If phase I trial enrolls more than 12 patients at the MTD, we will adjust the futility rule 
above for the two stage design with the giving number of patients enrolled at the 
MTD.  
5 
Patient Selection 
Study entry is open to persons 0 to 76 years of age regardless of gender or ethnic 
background.  While there will be every effort to seek out and include women and 
minority patients, the patient population is dependent upon the transplant population 
at the University of Minnesota.  
5.1 
Inclusion Criteria 
• Acute graft versus host disease (GVHD) fitting one of the following 
categories: 
 
High-Risk aGVHD (ARM 1) 
Pediatric or adult (ages 0-76 years) HCT recipients with: 
o high-risk acute GVHD, as determined either by: 
o  the 
refined 
MN 
acute 
GVHD 
risk 
score 
[28]: 
http://z.umn.edu/MNAcuteGVHDRiskScore  
o OR high risk on the basis of blood biomarkers (Ann Arbor 
Score 3 or amphiregulin ≥ 33 pg/ml) [[29, 30]] 
o OR 
 
 
Minnesota 
standard 
(http://z.umn.edu/MNAcuteGVHDRiskScore) with aGVHD with 
no response to 7 days of sirolimus plus a calcineurin inhibitor.   
Patients in this arm must start treatment within the first 7 days after onset 
of high-risk aGVHD. 
 
or 

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Steroid- Dependent aGVHD (ARM 2A) 
Pediatric or adult (ages 0-76 years) HCT recipient with grade II-IV steroid-
dependent acute GVHD, defined as any one of the following: 
o Flare of acute GVHD of at least grade II/IV severity within 8 weeks of 
tapering down or (off of) immunosuppression for acute GVHD, with the 
flare occurring on ≤0.5 mg/kg prednisone. This can include late-onset 
aGVHD and overlap syndrome. 
 
or 
 
 
Steroid-Refractory aGVHD (ARM 2B) 
Pediatric or adult (ages 0-76 years) HCT recipient with grade II-IV steroid 
refractory acute GVHD, defined as any one of the following: 
o No response of acute GVHD after at least 4 days of systemic 
corticosteroids of at least 2 mg/kg prednisone or equivalent 
o Progression of acute GVHD within 3 days of systemic corticosteroids 
of at least 2 mg/kg prednisone or equivalent 
o Failure to improve to at least grade II acute GVHD after 14 days of 
systemic corticosteroids, with initial doses of at least 2 mg/kg 
prednisone or equivalent 
o Flare of acute GVHD of at least grade II/IV severity while on steroids 
at a dose >0.5/mg/kg/day. This can include late-onset aGVHD and 
overlap syndrome. 
 
• Adequate organ function at study enrollment defined as: 
Renal: Serum creatinine ≤2.5x ULN (within 7 days) 
Cardiac:  LVEF ≥ 35% (within 42 days) 
 
• Voluntary written consent (adult or parent/guardian with minor assent for 
12 through 17 year olds) 
 
5.2 
Exclusion Criteria 
• 
Progressive malignancy 
• 
Diagnosis of a hormone responsive malignancy 
• 
Uncontrolled infection at initiation of protocol treatment 
• 
Current thromboembolic disease requiring full-dose anticoagulation - 
patients receiving pharmacologic prophylaxis for thromboembolic 
disease will be eligible 

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• 
Active or recent (within prior 3 months) thrombus, irrespective of 
anticoagulation status  
• 
Pregnancy as assessed on baseline blood hCG level 
• 
Unwilling or unable to stop supplemental sex hormone therapy 
(estrogen, progesterone, and/or testosterone preparations) 
• 
Women or men of childbearing potential unwilling to take adequate 
precautions to avoid pregnancy from the start of protocol treatment 
through 28 days after the last treatment 
6 
Patient Registration 
Registration will occur after the patient has signed the subject consent and 
eligibility is confirmed, but before any treatment has been administered.  To be 
eligible for registration to this study, the patient must meet each criteria listed on 
the eligibility checklist based on the eligibility assessment documented in the 
patient’s medical record.  A copy of the eligibility checklist is under attachments 
within the study in OnCore. 
6.1 
Registration with the Masonic Cancer Center Clinical Trials Office 
Upon completion of the screening evaluation, eligibility confirmation and 
obtaining written consent, the study coordinator or designee will enroll the 
patient into OnCore.  At the time of registration the patient will be assigned 
to either Arm 1 (high-risk aGVHD) or Arm 2A (steroid-dependent aGVHD) 
or Arm 2B (steroid-refractory aGVHD) in OnCore. 
Complete registration information is found in the study’s Procedures Manual 
for Affiliate Sites. 
Affiliate sites only:  At the time of registration, the signed consents will be 
uploaded into OnCore as an attachment under the appropriate record.  
Affiliates are responsible for fulfilling any local registration requirements. 
6.2 
Pregnyl® Dose Level Assignment  
The dose level of Pregnyl® will be assigned based on the currently enrolling 
dose for the patient specific aGVHD status (Arm 1, Arm 2A or Arm 2B) and 
Phase. 
6.3 
Patients Who Are Registered and Do Not Receive Study Treatment 
If a patient is registered to the study, and is later found not able to begin 
study treatment, for whatever reason, the patient will be removed from study 
and treated at the physician’s discretion.  The study coordinator or designee 
will update OnCore of the patient’s non-treatment status and notify the 

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Principal Investigator.  The reason for removal from study will be clearly 
documented in OnCore.  Such patients will be replaced within their assigned 
arm (Arm 1 – high-risk aGVHD or Arm 2A (steroid-dependent aGVHD) or 
Arm 2B steroid-refractory aGVHD). 
6.4 
Patients Who Do Not Complete All Study Treatments 
A patient must receive at least 3 doses of Pregnyl to be evaluable for 
safety/tolerability in the phase I portion of the study.  If a patient receives 
fewer doses and discontinues study therapy for a reason unrelated to study 
drug toxicity, the patient will be replaced within their assigned arm, however 
these patients may re-consent and enroll on the study again if they are 
eligible at a later time.   
In the phase II portion of the study, a patient must receive at least 3 doses 
of Pregnyl to be assessed for response in Arm 1.  In Arm 2A and 2B, a 
patient must receive at least 6 doses to be assessed for response.  If a 
patient does not complete the number of doses necessary for response 
assessment, the patient will be replaced within their assigned arm, however 
these patients may re-consent and enroll on the study again if they are 
eligible at a later time. 
7 
Treatment Plan 
Patients may remain on their standard of care current acute GVHD therapy, 
including calcineurin inhibitors, sirolimus, and systemic corticosteroids at the start 
of study treatment, unless any of these agents are determined to unsafe due to an 
unforeseen complication. 
7.1 
Arm 1 – High-Risk aGVHD 
 
Standard 
of 
care 
immunosuppression, 
plus 
Pregnyl® 
(hCG 
supplementation) at assigned dose subcutaneously every other day for 4 
doses 
 
If a patient has a toxicity that fits the definition of dose limiting as defined in 
the phase I section below, the patient will discontinue study treatment. 
 
Continue protocol treatment through day 7.  Assess response at day 7: 
• If a complete or partial response, the patient is eligible to receive 
Pregnyl maintenance at the same dose twice weekly for 10 doses 
beginning day 9 to 12.  Refer to section 9 for response definitions. 
• If no response, the patient will be taken off study treatment 

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Pregnyl® dose level assignment will be done by treatment arm, with Arm 1 
independent of Arms 2A and 2B.   
For phase I, up to 5 dose levels of Pregnyl® will be tested; however dose 
levels -1 and -2 will be used only if dose level 1 proves too toxic.   
 
Dose Level 
Dose 
-2 
125 USP hCG/875 pg EGF/m2 
-1 
250 USP hCG/1,750 pg EGF/m2 
1 (start) 
500 USP hCG/3,500 pg EGF/m2 
2 
1,000 USP hCG/7,000 pg EGF/m2 
3 
2,000 USP hCG/14,000 pg EGF/m2 
 
The 1st 2 patients will be enrolled in dose level 1. The next cohort of 2 
patients will not begin treatment until all patients in the current cohort have 
reached day 21 in order to assess for dose limiting toxicity (DLT).   
 
Dose limiting toxicity (DLT) is defined as any of the following occurring by 
day 21 after the 1st dose of Pregnyl® (phase I component only):  
• In males and females:  Grade 2-4 thromboembolic event (CTCAE v4) 
i.e., any thrombosis more severe than a superficial venous thrombosis 
develops and treatment with anticoagulation is required 
• In males and females:  Grade 3-4 ascites (CTCAE v4)  
• Specific to females: Grade 3-4 ovarian hyperstimulation syndrome, 
defined as the rapid development of enlarged, cystic, painful ovaries - 
refer to section 11.2 for CTCAE grading guidance 
 
Each new cohort of 2 patients will be assigned by the study statistician to 
the most appropriate dose level based on toxicity probabilities set for this 
study.  The MTD will be identified when the total sample size of 20 patients 
is reached. 
 
For the phase II the dose of Pregnyl will be that identified as the MTD during 
phase I.   
7.2 
Arm 2A – Steroid-Dependent aGVHD 
 
Standard of care immunosuppression, plus Pregnyl® (hCG 
supplementation) at assigned dose subcutaneously every other day for 7 
doses. 

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If a patient has a toxicity that fits the definition of dose limiting as defined in 
the phase I section below, the patient will discontinue study treatment. 
 
Continue protocol treatment through day 14.  Assess response at day 14: 
• If a complete or partial response, the patient is eligible to receive 
Pregnyl maintenance at the same dose twice weekly for 10 doses 
beginning day 15 to 17.  Refer to section 9 for response definitions. 
• If no response, the patient will be taken off study treatment 
 
Pregnyl® dose level assignment will be done by treatment arm, with Arm 2 
independent of Arm 1.   
 
For phase I, up to 5 dose levels of Pregnyl® will be tested; however dose 
levels -1 and -2 will be used only if dose level 1 proves too toxic.   
Dose Level 
Dose 
-2 
125 USP hCG/875 pg EGF/m2 
-1 
250 USP hCG/1,750 pg EGF/m2 
1 (start) 
500 USP hCG/3,500 pg EGF/m2 
2 
1,000 USP hCG/7,000 pg EGF/m2 
3 
2,000 USP hCG/14,000 pg EGF/m2 
 
The 1st 2 patients will be enrolled in dose level 1. The next cohort of 2 
patients will not begin treatment until all patients in the current cohort has 
reached day 21 to assess for dose limiting toxicity (DLT).   
 
DLT is defined as any of the following occurring by day 21 after the 1st dose 
of Pregnyl® (phase I component only):  
• 
In males and females:  Grade 2-4 thromboembolic event (CTCAE v4) 
i.e., any thrombosis more severe than a superficial venous thrombosis 
develops and treatment with anticoagulation is required 
• 
In males and females:  Grade 3-4 ascites (CTCAE v4)  
• 
Specific to females: Grade 3-4 ovarian hyperstimulation syndrome, 
defined as the rapid development of enlarged, cystic, painful ovaries– 
refer to section 11.2 for CTCAE grading guidance 
 
Each new cohort of 2 patients will be assigned by the study statistician to 
the most appropriate dose level based on toxicity probabilities set for this 
study.  The MTD will be identified when the total sample size of 20 patients 
is reached. 

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For the phase II the dose of Pregnyl will be that identified as the MTD during 
phase I.   
 
7.3 
Arm 2B – Steroid-Refractory aGVHD 
 
Standard of care immunosuppression, plus Pregnyl® (hCG 
supplementation) at assigned dose subcutaneously every other day for 7 
doses. 
 
If a patient has a toxicity that fits the definition of dose limiting as defined in 
the phase I section below, the patient will discontinue study treatment. 
 
Continue protocol treatment through day 14.  Assess response at day 14: 
• If a complete or partial response, the patient is eligible to receive 
Pregnyl maintenance at the same dose twice weekly for 10 doses 
beginning day 15 to 17.  Refer to section 9 for response definitions. 
• If no response, the patient will be taken off study treatment 
 
Pregnyl® dose level assignment will be done by treatment arm, with Arm 2 
independent of Arm 1.   
 
For phase I, up to 7 dose levels of Pregnyl® will be tested; however dose 
levels -1 and -2 will be used only if dose level 1 proves too toxic. The reason 
for this expansion is lack of rise in plasma EGF levels with Pregnyl therapy 
in patients with the most severe aGVHD in the first 6 patients in phase 1. 
   
Dose Level 
Dose 
-2 
125 USP hCG/875 pg EGF/m2 
-1 
250 USP hCG/1,750 pg EGF/m2 
1 (start) 
500 USP hCG/3,500 pg EGF/m2 
2 
1,000 USP hCG/7,000 pg EGF/m2 
3 
2,000 USP hCG/14,000 pg EGF/m2 
4 
3,500 USP hCG/24,500 pg EGF/m2 
5* 
5,000 USP hCG/35,000 pg EGF/m2 
 
*NOTE: To avoid using vials from more than one Lot of study product, the maximum dose is either (1) the 
contents of a single 10,000 IU vial, or (2) a maximum dose of 10,000 IU from multiple vials. All vials used 
should be from the same Lot. 

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The 1st 2 patients will be enrolled in dose level 1. The next cohort of 2 
patients will not begin treatment until all patients in the current cohort has 
reached day 21 to assess for dose limiting toxicity (DLT).   
 
DLT is defined as any of the following occurring by day 21 after the 1st dose 
of Pregnyl® (phase I component only):  
• 
In males and females:  Grade 2-4 thromboembolic event (CTCAE v4) 
i.e., any thrombosis more severe than a superficial venous thrombosis 
develops and treatment with anticoagulation is required 
• 
In males and females:  Grade 3-4 ascites (CTCAE v4)  
• 
Specific to females: Grade 3-4 ovarian hyperstimulation syndrome, 
defined as the rapid development of enlarged, cystic, painful ovaries– 
refer to section 11.2 for CTCAE grading guidance 
 
Each new cohort of 2 patients will be assigned by the study statistician to 
the most appropriate dose level based on toxicity probabilities set for this 
study.  The MTD will be identified when the total sample size of 20 patients 
is reached. 
 
For the phase II the dose of Pregnyl will be that identified as the MTD during 
phase I.   
7.4 
Supportive Care 
Patients will receive transfusions, infection prophylaxis (including 
appropriate viral, fungal, bacterial prophylaxis and preemptive viral 
monitoring per standard of care), growth factor support, and nutritional 
support according to the current University of Minnesota supportive care 
guidelines. 
7.5 
Duration of Study Therapy 
Arm 1 (high-risk aGVHD):  Study treatment will be for a minimum of 7 days 
with study therapy continuing for an additional 5 weeks if a complete or 
partial response is documented at the initial response evaluation.  Non-
responders at 7 days will be taken off study therapy and treated at the 
discretion of the treating physician.   The treating physician may remove 
patients from study participation sooner if concerns regarding safety 
emerge prior to completing 7 days on protocol.   
Arm 2A and 2B (steroid-refractory/dependent aGVHD):  Study treatment will 
be for a minimum of 14 days with study therapy continuing for an additional 
5 weeks if a complete or partial response is documented at the initial 
response evaluation.  Non-responders at 14 days will be taken off study 

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therapy and treated at the discretion of the treating physician.  The treating 
physician may remove patients from study participation sooner if concerns 
regarding safety emerge prior to completing 14 days on protocol. 
An exception to the above is if a patient experiences an event that fits the 
definition of a dose limiting toxicity (Grade 2-4 thromboembolic event, Grade 
3-4 ascites or Grade 3-4 ovarian hyperstimulation syndrome).  Study 
treatment will be permanently discontinued in this situation.   
7.6 
Duration of Study Participation 
All participants will be followed for adverse events through day 56, or if a 
non-responder at initial assessment; for 28 days after the last dose of 
Pregnyl® to rule-out early thromboembolic events.  The remainder of follow 
up data for study endpoints will coincide with routine transplant related 
follow-up (chart review database query). 
8 
Study Drug Formulation, Preparation, Side Effects 
8.1 
Human Chorionic Gonadotropin (HCG) (Pregnyl® (Merck), or 
generic urinary-derived hCG from Fresenius Kabi)  
Pregnyl® or its generic equivalent (chorionic gonadotropin for injection 
USP) is a highly purified pyrogen-free preparation obtained from the urine 
of pregnant females. 
Complete prescribing information for urinary HCG can be located at the 
following internet address: 
http://www.accessdata.fda.gov/drugsatfda_docs/label/2011/017692s021lbl
.pdf 
PROCUREMENT:  Pregnyl® or its generic equivalent will be purchased 
from a commercial source for the purposes of this study. 
 
DOSAGE AND ADMINISTRATION:  This drug is currently licensed by the 
FDA for intramuscular use only.  However, in clinical practice, it is commonly 
given subcutaneously.  Because of the risk of bleeding due to IM injections, 
and because of the otherwise off-label use for this drug, we have sought an 
IND from the FDA to administer Pregnyl® or its generic equivalent via 
subcutaneous route during this clinical trial.   
PREPARATION:  The drug is supplied in two-vial package containing:  

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1. 1-10 mL lyophilized multiple dose vial containing: 10,000 USP Units 
chorionic gonadotropin per vial, NDC 0052-0315-10 (Pregnyl) or NDC 
63323 (Fresenius Kabi).  
2. 1-10 mL vial of solvent containing: water for injection with sodium 
chloride 0.56% and benzyl alcohol 0.9%, NDC 0052-0325-10 (Pregnyl) 
or NDC 63323 (Fresenius Kabi).  
 
Each lot of Pregnyl® or its generic equivalent will be tested for EGF content 
prior to administration so that the precise EGF dose administered is known. 
   
When reconstituted, each 10 mL vial contains:  
• Chorionic gonadotropin 10,000 USP Units  
• Monobasic sodium phosphate 5 mg 
• Dibasic sodium phosphate 4.4 mg 
• Sodium chloride 0.56%  
• Benzyl alcohol 0.9% 
 
If required pH adjusted with sodium hydroxide and/or phosphoric acid.  
 
NOTE: For Arm 2B, Dose level 5, in order to avoid using vials from more 
than one Lot of study product, the maximum dose is either (1) the contents 
of a single 10,000 IU vial, or (2) a maximum dose of 10,000 IU from multiple 
vials. All vials used should be from the same Lot. 
 
IMPORTANT:  RECONSTITUTED SOLUTION IS STABLE FOR 60 DAYS 
WHEN REFRIGERATED.    
 
PRECAUTIONS  
1. General:  Since androgens may cause fluid retention, hCG should be 
used with caution in patients with cardiac or renal disease, epilepsy, 
migraine, or asthma.  
2. Pediatric Use:  Induction of androgen secretion by hCG may induce 
precocious puberty in pediatric patients treated for cryptorchidism. 
Therapy should be discontinued if signs of precocious puberty occur.  
3. Geriatric Use:  Clinical studies of PREGNYL® (chorionic gonadotropin 
for injection, USP) and its generic equivalent did not include subjects 
aged 65 and over.  
4. ADVERSE 
REACTIONS: 
 
Headache, 
irritability, 
restlessness, 
depression, fatigue, edema, precocious puberty, gynecomastia, pain at 

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the site of injection.  Hypersensitivity reactions, both localized and 
systemic in nature, have been reported.   
9 
Disease Reassessment and Response Definitions 
Participants will be staged according to the Minnesota grading schema below: 
 
 
Overall 
Grade 
Organ Stage 
Skin 
Liver 
Lower GI 
Upper GI 
   I 
1-2 
0 
0 
0 
   II 
3 
1 
1 
1 
   III 
- 
2-4 
2-3 
  
   IV 
4 
- 
4 
  
 
Response will be based on the following definitions: 
• Complete Response (CR): a Stage of 0 for all organs with no additional 
intervening therapy for their GVHD. 
• Partial Response (PR): improvement by at least 1 stage in all involved organs 
without progression in others with no additional intervening therapy for their 
GVHD. 
• Mixed Response (MR): improvement in one organ with deterioration in another 
organ manifesting symptoms of GVHD or development of symptoms of GVHD 
in a new organ. 
• Progression: deterioration of any organ involved. 

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• No Response (NR): absence of any improvement or progression as defined 
above. 
10 
Schedule of Patient Activities 
Scheduled evaluations after screening and through day 19 of the last treatment 
cycle may be performed +/-2 days from the targeted date; assessments performed 
after day 28 and through day 56 may be done +/-5 days of the targeted date.  After 
day 56, assessments will coincide with the post-transplant follow-up schedule (i.e., 
additional clinic visits specifically for this study will not be required).  In addition, 
targeted days may be altered as clinically appropriate, and will not constitute a 
protocol deviation.  
Schedule of Treatment, Tests and Procedures – Arm 1 
 
Baseline 
Treatment Days 
+/- 2 days 
Phase 
I only 
Follow 
Up 
+/- 5 
days*** 
Procedures, evaluations, 
and treatment 
 
1 
3 
5 
7 
9 
11 
13 
14 
15 
17 
19 
21 
28 
56 
Standard of Care 
Vital signs and weight 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
Performance status 
(Karnofsky or Lansky) 
X 
 
 
 
X 
 
 
 
X 
 
 
X 
 
X 
X 
Acute GVHD staging 
X 
 
 
 
X 
 
 
 
X 
 
 
 
 
X 
X 
Complete blood count 
X 
 
 
 
X 
 
 
 
X 
 
 
X 
 
X 
X 
Complete metabolic panel 
X 
 
 
 
X 
 
 
 
X 
 
 
X 
 
X 
X 
Serum creatinine 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Ejection Fraction by 
echocardiogram or MUGA 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Research 
Pregnyl® dosing* 
 
X 
X 
X 
X 
* 
 
 
 
 
 
 
 
 
 
Assess for toxicity  
 
beginning with the 1st dose of Pregnyl® and continuing for 4 weeks after the last 
dose per section 11.2 (if phase I assess for DLT at day 21) 
Pharmacokinetics** 
 
X 
 
 
X 
 
 
 
 
 
 
 
 
 
 
Serum hCG 
X^ 
 
 
 
X 
 
 
 
X 
 
 
 
 
X 
X 
Estradiol, progesterone, and 
testosterone 
X 
 
 
 
X 
 
 
 
X 
 
 
 
 
X 
X 
Collection of blood samples 
for immune and tissue 
damage/repair biomarkers 
(refer to section 10.3 for 
blood volume and tube 
type)s 
X 
 
 
 
X 
 
 
 
X 
 
 
 
 
X 
X 
 

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Research related blood samples will go to Translational Therapy Lab (TTL) for processing and storage until study related testing. 
 
*If a patient achieves a complete or partial response, he/she may continue to receive maintenance Pregnyl® doses twice per week, 
in the dose level he or she was already assigned, for another 10 doses, beginning during protocol day 9-12. 
**Blood samples (2 ml plasma) collected 30 (± 10) minutes pre-dose, and at post-dose 1 hour (± 5 min), 3 hours (± 5 min), 6 hours 
(± 10 min), and 24 hours (± 30 min). 
***After day 56 endpoint assessments will coincide with the post-transplant anniversary visits and the information may be abstracted 
from the medical record and/or the BMT database. 
^ testing to rule-out pregnancy will be performed on females for eligibility screening  
 
Schedule of Treatment, Tests and Procedures – Arm 2 
 
Baseline 
 
Treatment Days  
+/- 2 days 
 
Phase I 
only 
Follow 
Up 
+/- 5 
days*** 
Procedures, evaluations, 
and treatment 
 
1 
3 
5 
7 
9 
11 
13 
14 
15 
17 
19 
21 
28 
56 
Standard of Care 
Vital signs and weight 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
Performance status 
(Karnofsky or Lansky) 
X 
 
 
 
X 
 
 
 
X 
 
 
X 
 
X 
X 
Acute GVHD staging 
X 
 
 
 
X 
 
 
 
X 
 
 
X 
 
X 
X 
Complete blood count 
X 
 
 
 
X 
 
 
 
X 
 
 
X 
 
X 
X 
Complete metabolic panel 
X 
 
 
 
X 
 
 
 
X 
 
 
X 
 
X 
X 
Serum creatinine 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Ejection Fraction by 
echocardiogram or MUGA 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Research 
Pregnyl® dosing* 
 
X 
X 
X 
X 
X 
X 
X 
 
* 
 
 
 
 
 
Assess for toxicity  
 
beginning with the 1st dose of Pregnyl® and continuing for 4 weeks after the last 
dose per section 11.2 (if phase I assess for DLT at day 21) 
Pharmacokinetics** 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
 
Serum hCG 
X^ 
 
 
 
X 
 
 
 
X 
 
 
 
 
X 
X 
Estradiol, progesterone, 
and testosterone 
X 
 
 
 
X 
 
 
 
X 
 
 
 
 
X 
X 
Collection of blood samples 
for immune and tissue 
damage/repair biomarkers  
(refer to section 10.1 for 
blood volume and tube 
type) 
X 
 
 
 
X 
 
 
 
X 
 
 
 
 
X 
X 
 
Research related blood samples will go to Translational Therapy Lab (TTL) for processing and storage until study related testing. 
 
*If a patient achieves a complete or partial response, he/she may continue to receive maintenance Pregnyl® doses twice per week, 
in the dose level he or she was already assigned, for another 10 doses, beginning during protocol day 15-17. 
**Blood samples (plasma) collected 30 (± 10) minutes pre-dose, and at post-dose 1 hour  (± 5 min), 3 hours (± 5 min), 6 hours (± 10 
min), and 24 hours  (± 30 min). 
***After day 56 endpoint assessments will coincide with the post-transplant anniversary visits and the information may be abstracted 
from the medical record and/or the BMT database. 
^ testing to rule-out pregnancy will be performed on females for eligibility screening 

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10.1 Correlative Studies and Hypotheses 
1. Pharmacokinetics:  Clearance, half-life, and volume of distribution of the 
both the hCG and EGF components of urinary hCG when administered 
to patients with GVHD.  This objective tests the hypothesis that as 
enteropathy improves, the PK profile of both hCG and EGF will change. 
2. Hormonal effects:  concentrations of hCG, estradiol and progesterone, 
and testosterone at baseline, and on days 7, 14, 28, and 56 of protocol 
treatment.  This objective tests the hypothesis that any increase in sex 
hormones induced by the hCG will be reversible and associated with no 
adverse events.   
3. Biomarkers of response in routine clinical laboratory parameters:  This 
objective tests the hypothesis that those receiving HCG will have (a) 
higher circulating numbers of total lymphocytes, monocyte, platelets, 
and albumin levels as measured by routine clinical laboratory testing and 
that all will correlate positively, with HCG treatment and plasma levels 
of both EGF and hCG. 
The following studies detailed below in number 4-7 will be exploratory 
laboratory studies performed on blood samples collected for the study 
protocol.  At each time point, the following samples will be collected: 
For patients ≥ 40 kg 
• 60 ml of heparinized blood (green top tubes) for PBMCs, and 
plasma stored in 15 x 1 ml aliquots. 
 
For patients < 40 kg 
• Collect up to 2 cc/kg to a maximum of 65 ml total blood volume for 
placed in green top tubes.  Plasma will be stored in 1 ml aliquots.  
  
4. Angiogenic biomarkers of response:  This objective tests the hypothesis 
that higher plasma levels of EGF and VEGF-A and lower levels of 
follistatin will be observed in those receiving HCG treatment and will 
correlate with response to treatment. 
 
5. Suppressive/regulatory markers of response:  This objective tests the 
hypothesis that higher blood levels of suppressive/regulatory cytokines 
cells (IL-10, IL-22 – measured by multiplex array, and regulatory T cells 
– measured by flow cytometry) will be observed in those receiving HCG 
treatment and correlate with response to therapy. 

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6. Inflammatory markers of response:  This objective tests the hypothesis 
that lower blood levels of inflammatory markers (TNFα, IL-6, IL-17, IL-
33,– measured by multiplex array) will be observed in those receiving 
HCG treatment and correlate with response to therapy. 
7. Accepted biomarkers of GVHD response:  This objective tests the 
hypothesis that reduced levels of validated biomarkers of GVHD (e.g., 
TNFR1, REG3α, ST2, where elevated levels are indicative of poor 
response – measured by ELISA) will be observed in those receiving 
HCG treatment and will correlate with response to therapy. 
8. Tissue expression of markers of regenerative capacity:  This objective 
tests the hypothesis that tissue expression of EGF, IL-22, IDO, Ki-67 will 
be higher in patients with complete response to therapy.  Analytes will 
be measured by immunohistochemistry (IHC) performed on slides 
prepared from gastrointestinal specimens previously collected for 
clinical purposes.  No additional biopsies are required for protocol 
participation. 
Separation of PBMCs and plasma, and enumeration of regulatory T cells by flow 
cytometry, will be performed by the Translational Therapy Laboratory.  ELISAS 
and multiplex arrays will be performed by the Cytokine Reference Laboratory.  IHC 
will be performed by the Histology core/AHC Bionet. 
11 
Adverse Event Reporting 
Toxicity and adverse events will be classified according to NCI's Common 
Terminology Criteria for Adverse Events V 4.0 (CTCAE).  A copy of the CTCAE 
can 
be 
downloaded 
from 
the 
CTEP 
home 
page 
http://evs.nci.nih.gov/ftp1/CTCAE/About.html  
11.1 Definitions 
Note:  throughout this section the generic term “drug” refers to the Pregnyl®, 
or its generic equivalent, injection. 
The following definitions are based on the Code of Federal Regulations Title 
21 Part 312.32 (21CFR312.32(a)). 
Adverse Event:  Any untoward medical occurrence associated with the use 
of a drug in humans, whether or not considered drug related.  
Suspected Adverse Reaction:  Any adverse event for which there is a 
reasonable possibility that the drug caused the adverse event.  

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Life-Threatening Adverse Event Or Life-Threatening Suspected 
Adverse Reaction: An adverse event or suspected adverse reaction is 
considered “life-threatening” if, in the view of either the investigator or 
sponsor, its occurrence places the patient or subject at immediate risk of 
death. 
Serious Adverse Event Or Serious Suspected Adverse Reaction:  An 
adverse event or suspected adverse reaction is considered “serious” if, in 
the view of either the investigator or sponsor, it results in any of the following 
outcomes:  
• Death 
• A life-threatening adverse event 
• Inpatient hospitalization or prolongation of existing hospitalization 
• A persistent or significant incapacity or substantial disruption of the 
ability to conduct normal life functions 
• A congenital anomaly/birth defect.  
Important medical events that may not result in death, be life-threatening, 
or require hospitalization may be considered serious when, based upon 
appropriate medical judgment, they may jeopardize the patient or subject 
and may require medical or surgical intervention to prevent one of the 
outcomes listed in this definition.  
If either the IND sponsor or the investigator believes the event is life-
threatening or serious, the event must be evaluated by the sponsor for 
expedited reporting (21CRF 312.32(a)).  
Unexpected adverse event or unexpected suspected adverse 
reaction: An adverse event or suspected adverse reaction is considered 
“unexpected” if it is not listed in the investigator brochure or is not listed at 
the specificity or severity that has been observed; or, if an investigator 
brochure is not required or available, is not consistent with the risk 
information described in the general investigational plan or elsewhere in the 
current application, as amended.  Thus, adverse events that occur as part 
of the disease process or underlying medical conditions are considered 
unexpected; however, they will not be reportable per section 11.4. 
Major Deviation: A deviation or violation that impacts the risks and benefits 
of the research; may impact subject safety, affect the integrity of research 
data and/or affect a subject’s willingness to participate in the research. 

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Deviations that place a subject at risk, but do not result in harm are 
considered to be major deviations. 
Minor Deviation: A deviation or violation that does not impact subject 
safety, compromise the integrity of research data and/or affect a subject’s 
willingness to participate in the research. 
Expedited (Rapid) Reporting: Certain events may require rapid 
notification to entities providing patient safety oversight (e.g. IRB, FDA) as 
detailed in section 11.4.  For the IRB this is 5 business days from discovery.  
For studies under an IND, it is 7 or 15 calendar days. 
11.2 Adverse Event Documentation 
Due to the complex medical condition of the post-transplant patient, 
monitoring for adverse events will focus on the following events beginning 
with the 1st dose of Pregnyl® and continuing for 4 weeks after the last dose.  
Any of these events will be documented on the study specific case report 
form: 
• 
death 
• 
anaphylaxis 
• 
thromboembolic event 
• 
Grade 3-4 skin injection site reaction 
• 
Grade 3 headache 
• 
Grade 3 gynecomastia 
• 
Grade 3-4 ascites 
 
In addition, the following events occurring by day 21 after the 1st dose of 
Pregnyl® are considered a dose limiting toxicity (DLT) and should be 
reported as such for patients enrolled in the Phase I component per section 
11.4:  
• In males and females:  Grade 2-4 thromboembolic event (CTCAE v4)  
• In males and females:  Grade 3-4 ascites (CTCAE v4)  
• Specific to females:  Grade 3-4 ovarian hyperstimulation syndrome, 
defined as the rapid development of enlarged, cystic, painful ovaries, 
Note: as there is no CTCAE grading for this event, the generic grading 
will apply as follows:   
 
 
Grade 
Description 
1 
mild; asymptomatic or mild symptoms; clinical or diagnostic 
observations 
2 
moderate; minimal or non-invasive intervention indicated 

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Grade 
Description 
3 
severe or medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated 
4 
life-threatening consequences; urgent intervention indicated 
 
For the Phase II expansion cohort, an early study stopping rule was 
developed for excessive Grade 4/5 non-hematologic, non-infectious 
treatment probably or definitely attributed to the study drug occurring by day 
28 after the last dose of Pregnyl® and is reportable per section 11.4.  Refer 
to section 13.4 for details. 
 
For both cohorts, relapse of the patient's malignancy and/or development 
of a new one occurring 4 weeks after the last dose is reportable per section 
11.4. 
 
11.3 Institutional Event Reporting Table 
Individual institutional sites will be responsible for reporting any event meeting local 
reporting requirements to their institutional IRB and/or other research oversight 
committees. 
Additionally institutional sites will be responsible for reporting events detailed in 
sections 11.1 and 11.2 within the following time frames to the MCC Affiliate Sites 
Manager 
 
 
Event Type 
Reporting 
Timeframe 
Form in OnCore 
to Use 
Report to 
Any event meeting the 
definition of a SAE within 
4 weeks after last dose of 
pregnyl 
 
Within 24 hours 
of knowledge 
SAE Report 
Form 
Masonic Cancer Center (MCC) 
Affiliate Sites Manager  
affiliates@umn.edu 
 
Local institutional IRB or other entities 
per institutional policies and 
guidelines. 
 
 
 
Event counting toward the 
safety stopping rule as 
defined in Section 13.4 
Within 24 hours 
of knowledge 
Stopping Rule 
Event Form 
Major Deviations, as 
defined in Section 11.1. 
Within 5 working 
days of 
knowledge 
Deviation 
Report Form 
Minor Deviations, as 
defined in Section 11.1. 
Per Institutional 
Policy  
n/a (record in 
Deviations Tab) 
but it is not 
necessary to 
report the event 
to MCC 
For UMN MCC only: minor deviations 
are reported to the UMN IRB by the 
study’s regulatory specialist per IRB 
reporting requirements. 
 
For Affiliate Sites: minor deviations are 
not reportable to the Masonic Cancer 
Center. Report to local institutional IRB 
or other entities per institutional 
policies and guidelines. 

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11.4 Required Reporting: FDA, IRB and Masonic Cancer Center’s SAE 
Coordinator 
 
As the study sponsor, the Masonic Cancer Center has the following expedited reporting 
responsibilities for local events and events reported in Section 11.3. 
 
In each IND safety report, the sponsor must identify all IND safety reports 
previously submitted to the FDA concerning a similar suspected adverse reaction 
and must analyze the significance of the suspected adverse reaction in light of the 
previous, similar reports. 
The SAE Coordinator will provide the Masonic Cancer Center’s Data and Safety 
Monitoring Council (DSMC) with the SAE in an appropriate format depending on 
the individual SAE (as reported or in a summary format). 
 
 
Agency 
Criteria for reporting 
Timeframe 
Form to 
Use 
Submission 
address/ fax 
numbers 
U of MN 
IRB 
Events requiring prompt reporting including, but not 
limited to unanticipated death of a locally enrolled 
subject(s); new or increased risk; any adverse event  
that require a change to the protocol or consent form or 
any protocol deviation that resulting in harm  
refer to 
http://www.research.umn.edu/irb/guidance/ae.html#.VC
7xraI0-sh  
Within 5 
business days of 
event discovery 
IRB’s 
Report 
Form  
irb@umn.edu 
FDA 
 
Unexpected and fatal or life threatening suspected 
adverse reaction 
As soon as 
possible but no 
later than 7 
Calendar-Days 
MCC 
SAE 
Submit to 
CBER as an 
amendment to 
IND 
1) Serious and unexpected suspected adverse reaction 
or 
2)  increased occurrence of serious suspected adverse 
reactions over that listed in the protocol or investigator 
brochure or 
3) findings from other sources (other studies, animal or 
in vitro testing) 
As soon as 
possible but no 
later than 15 
Calendar-Days 
All other events per CRF 312.33 
At time of IND 
annual report 
Summary 
format 
Submit as part 
of the IND 
annual report 
Masonic 
Cancer 
Center SAE 
Coordinator 
Events that meet the criteria of dose limiting toxicity 
(DLT) or an early study stopping rule 
At time of 
reporting 
Event 
Form 
SAE 
Coordinator 
mcc-
saes@umn.edu 

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12 
Study Data Collection and Monitoring 
12.1 Data Management 
This study will report clinical data using The Online Enterprise Research 
Management Environment (OnCoreTM), a web based Oracle® database 
utilizing study specific electronic case report forms.  Key study personnel 
are trained on the use of OnCore and will comply with protocol specific 
instructions embedded within the OnCore.  Patient demographics, patient 
specific study treatment calendars, targeted adverse events and other 
information required for IND annual reporting will be placed in OnCore and 
other databases maintained by the Cancer Center. 
12.2 Case Report Forms 
Participant data will be collected using protocol specific electronic case 
report forms (e-CRF) developed within OnCore based on its library of 
standardized forms.  The e-CRF will be approved by study’s Principal 
Investigator and the Biostatistician prior to release for use.  The Study 
Coordinator or designee will be responsible for registering the patient into 
OnCore at time of study entry, completing e-CRF based on the patient 
specific calendar, and updating the patient record until patient death or end 
of required study participation.   
12.3 Data and Safety Monitoring Plan (DSMP) 
The study’s Data and Safety Monitoring Plan will be in compliance with the 
University of Minnesota Masonic Cancer Center's Data & Safety Monitoring 
Plan (DSMP), which can be accessed at http://z.umn.edu/dmsp. 
For the purposes of data and safety monitoring, this study is classified as 
high-risk (phase I).  Therefore the following requirements will be fulfilled: 
 The PI will complete and submit a quarterly Trial Progress Report to the 
Masonic Cancer Center Data and Safety Monitoring Council (DSMC) 
with the understanding the Cancer Protocol Review Committee (CPRC) 
may require more frequent reporting. 
 The PI will comply with at least twice yearly monitoring of the project by 
the Masonic Cancer Center monitoring services. 
 The PI will oversee the submission of all reportable adverse events per 
the definition of reportable in section 11.1 to the Masonic Cancer 
Center’s SAE Coordinator, the University of Minnesota IRB, and the 
FDA.   
 The PI with the CTO has oversight responsibility for trial monitoring at 
affiliate sites. 

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In addition, at the time of the continuing review with the University of 
Minnesota IRB, a copy of the report with any attachments will be submitted 
to the Cancer Protocol Review Committee (CPRC). 
12.4 IND Annual Reports 
In accordance with regulation 21 CFR § 312.33, the sponsor-investigator 
with assistance from the MCC Clinical Trials Office (CTO) will submit a 
progress report annually.  The report will be submitted within 60 days of the 
anniversary date that the IND went into effect.    
12.5 Monitoring 
The investigator will permit study-related monitoring, audits, and 
inspections by the sponsor-investigator and/or sponsor/investigator 
designee, IRB, government regulatory bodies, and University of Minnesota 
compliance groups.   The investigator will make available all study related 
documents (e.g. source documents, regulatory documents, data collection 
instruments, study data, etc.).  The investigator will ensure the capability for 
inspections of applicable study-related facilities (e.g. pharmacy, diagnostic 
laboratory, etc.) will be available for trial related monitoring, audits, or 
regulatory inspections. 
12.6 Teleconferences – Lead Site and Affiliate Site  
Regular teleconferences to facilitate communication between participating 
sites regarding the study’s progress, patient updates, summary of safety 
reports, case report form completion, and other issues for discussion. The 
University of Minnesota Affiliate Manager will be responsible for arranging 
these teleconferences and preparing the agenda. Meetings will occur at 
least every 3 weeks; however, these may be scheduled more or less 
frequently at the discretion of the lead institution. Participation of a minimum 
of one representative from each affiliate site will be required. These 
teleconferences are in addition to other previously described site 
interactions including centralized patient registration, institutional and MCC 
required reporting of safety related issues, case report form completion in 
the study’s central database (OnCore) and affiliate oversight through self-
monitoring in compliance with the Masonic Cancer Center’s Data and 
Safety Monitoring plan.  
12.7 Affiliate Site Monitoring 
The PI (Dr. Holtan) with the CTO has oversight responsibility for trial 
monitoring at affiliate sites. Affiliate sites must self-monitor following the 
University of Minnesota Masonic Cancer Center Data and Safety Monitoring 

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Plan (DSMP - http://z.umn.edu/dmsp) and the CTO Affiliate and Satellite Site 
Monitoring SOPs.  
The investigator will permit study-related monitoring, audits, and 
inspections by the study’s Principal Investigator or any designees, the local 
IRB, government regulatory bodies, and University of Minnesota 
compliance groups. The investigator will make available all study related 
documents (e.g. source documents, regulatory documents, data collection 
instruments, study data, etc.). The investigator will ensure the capability for 
inspections of applicable study-related facilities (e.g. pharmacy, diagnostic 
laboratory, etc.) will be available for trial related monitoring, audits, or 
regulatory inspections. 
12.8 Record Retention 
The investigator will retain study records including source data, copies of 
case report form, consent forms, HIPAA authorizations, and all study 
correspondence in a secured facility for at 6 years after the study file is 
closed with the IRB and FDA.   
In addition, the Clinical Trials Office (CTO) will keep a master log of all 
patients participating in the study with sufficient information to allow retrieval 
of the medical records for that patient.  
Please contact the CTO before destroying any study related records. 
13 
Statistical Considerations 
13.1 Study Design 
This is a multi-center phase I/II study of supplemental hCG as an adjunct to 
facilitate improved mucosal healing and immune tolerance in conjunction 
with accepted, standard immunosuppressive therapy in the setting of high-
risk (Arm 1), steroid-dependent (Arm 2A) aGVHD and steroid-refractory 
(Arm 2B). Once the MTD is identified, the objective will be to confirm safety 
and obtain preliminary estimates of the primary trial endpoint defined as 
complete response (CR) and partial response (PR) at day 28.  
Phase I component: Estimating the MTD of hCG for Arm 1 and Arm 2A 
The first phase is a dose finding study. The dose escalation will be two 
independent processes with CRM for Arm 1 (high-risk aGVHD) and Arm 2A 
(steroid-dependent aGVHD). The target toxicity rate was determined based 
on clinical input and acceptability. Continual reassessment method (CRM) 

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is originally proposed by O’Quigley et al [31]. The goal for CRM is to identify 
the dose level which corresponds to the desired toxicity rate of 25% or less. 
We are choosing a lower target toxicity than would be chosen under the 
standard design. A standard “3+3” design will often choose a dose with 
corresponding to a toxicity rate of 33% or lower. The trial will stop if the 
posterior probability (that the lowest dose is more toxic than the target dose) 
is 0.90. 
Dose Level 
hCG Dose 
-2** 
125 USP hCG/875 pg EGF/m2 
-1* 
250 USP hCG/1,750 pg EGF/m2 
1 
500 USP hCG/3,500 pg EGF/m2 
2 
1,000 USP hCG/7,000 pg EGF/m2 
3 
2,000 USP hCG/14,000 pg EGF/m2 
 
*Dose level -1 (250 USP hCG/1,750 pg EGF/m2) will be used only if Dose Level 1 (500 USP 
hCG/3,500 pg EGF/m2) proves too toxic. 
**Dose level -2 (125 USP hCG/875 pg EGF/m2) will be used only if Dose Level -1 (250 USP 
hCG/1,750 pg EGF/m2) proves too toxic. 
 
 
Initial estimates of toxicity or the “skeleton” estimates are given by the 
clinicians. The estimates of toxicity for Arm 1 and Arm2 are listed as 1%, 
2%, 5%, 10% and 20% for the 5 dose levels, respectively. The 1st 2 patients 
will be enrolled in dose level 1. 
The next cohort of 2 patients will not begin treatment until the last patient in 
the current cohort has reached day 21. Each new cohort of 2 patients will 
be assigned by the study statistician (Qing Cao email: caox075@umn.edu).  
The CRM assumes the working dose-toxicity model or power model. The 
probability (toxicity at di) = piexp(α) for each dose level i. The parameter α is 
distributed a priori as a normal random variable with mean 0 and variance 
2. After simulation, a variance of 2 was predicted to balance both patient 
safety and accuracy.  Pre-specified constants pi for each dose are 1st 
determined by using the skeleton estimates at the beginning of the trial. 
After initiation of the trial and collection of events, the unknown parameter 
α is continuously updated. Calculation of posterior means of probability of 
toxicity from dose level i are calculated from Bayes theorem using the 
likelihood function based on a binomial distribution for the binary outcome 
of toxicity. After calculation of these means, each new cohort is assigned to 
the dose with an estimated toxicity probability closest to the pre-specified 
target toxicity of 25%. 
Eventually the MTD will be identified when the total sample size of 20 
patients is exhausted.  

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The dose limiting toxicity (DLT) is defined as any of the following occurring 
by day 21 after the 1st dose of hCG.  
• In males and females:  Grade 2-4 thromboemcolic event (CTCAE v4)  
• In males and females:  Grade 3-4 ascites (CTCAE v4)  
• Specific to females:  Ovarian hyperstimulation syndrome, defined as the 
rapid development of enlarged, cystic, painful ovaries 
 
Software within the BMA-CRMSimulator Version 2.1.0, downloaded from 
the MD Anderson biostatistics department website, will be used to calculate 
the posterior means of the toxicity probabilities for all of the doses under 
consideration:  
http://biostatistics.mdanderson.org/SoftwareDownload. Dose escalation of 
more than one level is not permitted with this design. 
Phase I component: Estimating the MTD of hCG for Arm 2B 
The phase I design for Arm2B (steroid-refractory aGVHD) is similar to Arm 
1 and Arm 2B. The goal for CRM is to identify the dose level which 
corresponds to the desired toxicity rate of 30% or less. We are choosing a 
lower target toxicity than would be chosen under the standard design. A 
standard “3+3” design will often choose a dose with corresponding to a 
toxicity rate of 33% or lower. The trial will stop if the posterior probability 
(that the lowest dose is more toxic than the target dose) is 0.90. 
Dose Level 
hCG Dose 
-2** 
125 USP hCG/875 pg EGF/m2 
-1* 
250 USP hCG/1,750 pg EGF/m2 
1 
500 USP hCG/3,500 pg EGF/m2 
2 
1,000 USP hCG/7,000 pg EGF/m2 
3 
2,000 USP hCG/14,000 pg EGF/m2 
4 
3,500 USP hCG/24,500 pg EGF/m2 
5 
5,000 USP hCG/35,000 pg EGF/m2 
 
*Dose level -1 (250 USP hCG/1,750 pg EGF/m2) will be used only if Dose Level 1 (500 USP 
hCG/3,500 pg EGF/m2) proves too toxic. 
**Dose level -2 (125 USP hCG/875 pg EGF/m2) will be used only if Dose Level -1 (250 USP 
hCG/1,750 pg EGF/m2) proves too toxic. 
 
 
Initial estimates of toxicity or the “skeleton” estimates are given by the 
clinicians. The estimates of toxicity for Arm 1 and Arm2 are listed as 1%, 
2%, 5%, 10%, 15%, 20% and 25% for the 7 dose levels, respectively. The 
1st 2 patients will be enrolled in dose level 1. The next cohort of 2 patients 
will not begin treatment until the last patient in the current cohort has 
reached day 21. Each new cohort of 2 patients will be assigned by the study 

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statistician (Qing Cao email: caox075@umn.edu).  With same calculation 
described in Arm 1 and Arm 2A, each new cohort is assigned to the dose 
with an estimated toxicity probability closest to the pre-specified target 
toxicity of 30%. DLT has the same definition as the other two arms.  
Operating Characteristics for Phase I (Arm 1 and Arm 2A) 
Operating characteristics are calculated by BMA-CRMSimulator for two 
different scenarios, one in which the “true” probabilities of DLTs are the 
same as those specified by the clinicians and one in which the “true” 
probabilities of DLTs are much higher than expected. Table 2 shows that 
there is a 88% chance of selecting the maximum dose of 2,000 USP 
hCG/14,000 pg EGF/m2, which is closest to our target toxicity of 25%. Table 
3 shows that there is a 11% chance of stopping the study or a 28% chance 
of selecting the dose associated with a 25% toxicity rate. 
Table 2. Simulations based on “true” probabilities equal to initial skeleton 
estimates. 
Dose 
Actual probability in 
theory of having a 
DLT at each dose  
Probability of Selecting 
the specified dose given 
the probabilities of having 
a DLT listed in the 2nd 
column  
Estimated 
Number 
of 
subjects treated at each 
dose level during the study 
125 USP hCG/875 pg EGF/m2 
0.01 
0.00 
0.2 
250 USP hCG/1,750 pg EGF/m2 
0.02 
0.00 
0.3 
500 USP hCG/3,500 pg EGF/m2 
0.05 
0.01 
2.9 
1,000 USP hCG/7,000 pg EGF/m2 
0.10 
0.11 
3.9 
2,000 USP hCG/14,000 pg EGF/m2 
0.20 
0.88 
12.7 
 
Table 3. Simulations based on “true” probabilities equal to excessively high 
toxicity. 
                     Probability of stopping: 11% 
Dose 
Actual probability 
in theory of 
having a DLT at 
each dose  
Probability of Selecting 
the specified dose given 
the probabilities of 
having a DLT listed in 
the 2nd column  
Estimated Number of 
subjects treated at 
each dose level 
during the study 
125 USP hCG/875 pg EGF/m2 
0.20 
0.29 
6.2 
250 USP hCG/1,750 pg EGF/m2 
0.25 
0.28 
3.7 
500 USP hCG/3,500 pg EGF/m2 
0.35 
0.22 
5.2 
1,000 USP hCG/7,000 pg EGF/m2 
0.45 
0.09 
2.3 
2,000 USP hCG/14,000 pg EGF/m2 
0.55 
0.01 
1.0 
 
Operating Characteristics for Phase I (Arm 2B)  
Operating characteristics are calculated by BMA-CRMSimulator for two 
different scenarios, one in which the “true” probabilities of DLTs are the 

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same as those specified by the clinicians and one in which the “true” 
probabilities of DLTs are much higher than expected. Table 4 shows that 
there is a 78% chance of selecting the maximum dose of 5,000 USP 
hCG/35,000 pg EGF/m2, which is closest to our target toxicity of 30%. Table 
5 shows that there is a 11% chance of stopping the study or a 29% chance 
of selecting the dose associated with a 30% toxicity rate.  
Table 4. Simulations based on “true” probabilities equal to initial 
skeleton estimates. 
Dose 
Actual probability in 
theory of having a 
DLT at each dose  
Probability of Selecting 
the specified dose given 
the probabilities of having 
a DLT listed in the 2nd 
column  
Estimated 
Number 
of 
subjects treated at each 
dose level during the study 
125 USP hCG/875 pg EGF/m2 
0.01 
0.00 
0.2 
250 USP hCG/1,750 pg EGF/m2 
0.02 
0.00 
0.0 
500 USP hCG/3,500 pg EGF/m2 
0.05 
0.00 
2.3 
1,000 USP hCG/7,000 pg EGF/m2 
0.10 
0.02 
2.9 
2,000 USP hCG/14,000 pg EGF/m2 
0.15 
0.07 
3.1 
3,500 USP hCG/24,500 pg EGF/m2 
0.20 
0.14 
2.9 
5,000 USP hCG/35,000 pg EGF/m2 
0.25 
0.78 
8.6 
 
Table 5. Simulations based on “true” probabilities equal to excessively high 
toxicity. 
                     Probability of stopping: 11% 
Dose 
Actual probability 
in theory of 
having a DLT at 
each dose  
Probability of Selecting 
the specified dose given 
the probabilities of 
having a DLT listed in 
the 2nd column  
Estimated Number of 
subjects treated at 
each dose level 
during the study 
125 USP hCG/875 pg EGF/m2 
0.25 
0.28 
6.7 
250 USP hCG/1,750 pg EGF/m2 
0.30 
0.29 
3.1 
500 USP hCG/3,500 pg EGF/m2 
0.40 
0.23 
5.3 
1,000 USP hCG/7,000 pg EGF/m2 
0.50 
0.07 
2.6 
2,000 USP hCG/14,000 pg EGF/m2 
0.60 
0.02 
0.7 
3,500 USP hCG/24,500 pg EGF/m2 
0.70 
0.00 
0.2 
5,000 USP hCG/35,000 pg EGF/m2 
0.75 
0.00 
0.1 
Phase II extension: Studying the efficacy of hCG (Arm 1, Arm 2A and 
Arm 2B) 
After completion of the dose finding trial for each arm, the final doses will 
be carried forward into a two-stage phase II extension trial to confirm safety 
and make a preliminary determination of the activity level for Arm 1 and Arm 
2. If the phase I trial enrolls fewer than 13 patients at the MTD, we will 
employ Simon’s Minmax two-stage design (Simon, 1989) with the possibility 
to discontinue after the 1st stage if the response rate is low.  
• Stage 1: Enroll a total of 12 patients (including all treated at the MTD in 
phase I). If 5 or more of these respond, the trial will continue to stage 2. 

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• Stage 2: Enroll an additional 14 patients. If 14 or more out of 26 patients 
respond, hCG will be considered promising for further investigation.  
If phase I trial enrolls more than 12 patients at the MTD, we will adjust the 
futility rule above for the two stage design with the giving number of patients 
enrolled at the MTD.  
13.2 Sample Size Consideration 
Resulting from laboratory studies conducted on the first several patients on 
this study, the investigative team plans to test higher doses in steroid-
refractory patients with aGVHD (separating of Arms into 2A and 2B).  This 
necessitates expanding the number of participants treated on the arms as 
described below: 
 
Arm 1 Treated Patients 
Arm 2A Treated Patients 
Arm 2B Treated Patients 
Level 1: N = 2, DLT = 0 
Level 1: N = 1, DLT = 0 
Level 1: N = 1, DLT = 0  
Level 2: N = 2, DLT = 0 
Level 2: N = 0, DLT = 0 
Level 2: N = 2, DLT = 0 
Level 3: N = 6, DLT = 0 
Level 3: N = 3, DLT = 0 
Level 3: N = 4, DLT = 0 
Enroll 10 more patients 
Next dose level 3: N = 2 
Enroll 16 more patients  
Next dose level 3: N = 2 
Enroll 13 more patients  
Next dose level 4: N = 2 
• 
The next dose was calculated with R package “dfcrm”.  
• 
The next dose for Arm 2B was dose level 5, but restrict the dose jump, so the next 
dose will be dose level 4. 
 
We now anticipate recruitment of 20 participants in each of the 3 arms to 
complete phase I. 
In phase I, based on resources, the maximum number of allowable 
evaluable patients is 20 for each arm (total of 60 for all three arms). The 
simulations show it should be sufficient and safe to define the MTD for future 
efficacy trials.  
For Arm 1 or 2A, if fewer than 13 patients are enrolled at the MTD, the  
phase II extension trial using Simon’s two-stage Minmax design will require 
26 patients to test the null hypothesis that the response rate at day 28 is 
<40% versus the alternative that the response rate is ≥ 65% after assuming 
80% power and a significance level of 0.05 and using a maximum of 26 
patients. We will choose the rule that minimizes the expected sample size 
giving a true response rate equivalent to the null hypothesis. We expect that 
for each arm, phase I and II will require approximately 33 patients due to 
use of patients from the phase I trial in the phase II extension (table 2 shows 
about 13 patients will be treated under MTD). But the sample size for each 

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arm could be as high as 44 when only two patients were treated at MTD in 
phase I. Therefore we expect the sample size of the phase I/II study for Arm 
1 and Arm 2A will be 66 to 88 patients.  
For Arm 2B, same as the other two arms, if fewer than 13 patients are 
enrolled at the MTD, the phase II extension trial using Simon’s two-stage 
Minmax design will require 26 patients to test the null hypothesis that the 
response rate at day 28 is <40% versus the alternative that the response 
rate is ≥ 65% after assuming 80% power and a significance level of 0.05 
and using a maximum of 26 patients. We expect that for each arm, phase I 
and II will require approximately 37 patients due to use of patients from the 
phase I trial in the phase II extension (table 4 shows about 9 patients will be 
treated under MTD). But the sample size could be as high as 44 when only 
two patients were treated at MTD in phase I. We expected the sample size 
of phase I/II study for Arm 2B will be 37 to 44 patients. 
The number of evaluable patients per year will be approximately 20 for each 
arm. We expect accrual to be completed within 7 years.  
13.3 Statistical Analysis 
SAS 9.3 (Cary, NC, USA) will be used for the statistical analyses.  For binary 
endpoints such as toxicity, summary statistics including proportions and 
their 95% confidence intervals will be calculated for each arm. All toxicity 
and safety assessments will be presented in the data listings and 
summarized with descriptive statistics.  Response at day 28 will be estimate 
with simple proportions and descriptive plots.  
 
The secondary endpoint, proportions of response among the surviving 
patients at day 7, 14, 56 and 180, treatment failure proportion for acute 
GVHD at day 28, 56 and 180 after initiation of protocol therapy, the 
incidence of acute GVHD flare after CR/PR requiring increase of steroids or 
other systemic treatment at days 28, 56, and 180 will be estimated. Overall 
survival and disease free survival probabilities after the initial treatment will 
be estimated using Kaplan-Meier’s method. Treatment-related mortality and 
the incidence of chronic GVHD after the initial treatment will be estimated 
by competing risk analysis. For other correlative endpoints, continuous 
measures will be summarized with mean and SD for each arm and time 
point (if skewed, we will report median and interquatile range) and 
categorical measures will be summarized with frequency and percentage.  
 
 

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13.4 Early Stopping Rule 
During Phase I CRM, the trial will stop if the posterior probability that the 
lowest dose is more toxic than the target is greater than 90%. Stopping rules 
will also be employed to monitor excess toxicity outside of the phase I trial. 
Stopping rule was developed using Pocock stopping boundaries [32]. The 
stopping rule will be monitored for Arm 1, 2A and Arm 2B separately.   
 
Early Stopping Rule for Toxicity 
Stopping rules were developed for excessive Grade 4/5 non-hematologic, 
non-infectious toxicity attributed to the study drug occurring by day 28 after 
the last dose of hCG. The goal is to construct a boundary based on toxicity 
such that the probability of early stopping is at most 10% if the toxicity rate 
is equal to 20% and assuming 13 more patients of each arm are enrolled 
after phase I. Given these parameters, the upper stopping boundary for 
toxicity is 2 events out of 2 patients, 3 events out of 3 patients, 5 out of 8, 
or 6 at any time. If the true toxicity rate is as high as 50% then chance of 
early stopping is 80% and the expected sample size is 7.3.  
14 
Conduct of the Study 
14.1 Good Clinical Practice 
The study will be conducted in accordance with the appropriate regulatory 
requirement(s).  Essential clinical documents will be maintained to 
demonstrate the validity of the study and the integrity of the data collected.  
Master files should be established at the beginning of the study, maintained 
for the duration of the study and retained according to the appropriate 
regulations.   
14.2 
Ethical Considerations 
The study will be conducted in accordance with ethical principles founded 
in the Declaration of Helsinki.  The IRB will review all appropriate study 
documentation in order to safeguard the rights, safety and well-being of the 
patients.  The study will only be conducted at sites where IRB approval has 
been obtained.  The protocol, consent, written information given to the 
patients, safety updates, annual progress reports, and any revisions to 
these documents will be provided to the IRB by the investigator.   
14.3 
Informed Consent 
All potential study participants will be given a copy of the IRB-approved 
consent to review. The investigator or designee will explain all aspects of 

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the study in lay language and answer all questions regarding the study. If 
the participant decides to participate in the study, he/she will be asked to 
sign and date the consent document. Patients who refuse to participate or 
who withdraw from the study will be treated without prejudice. 
15 
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