Conditions: Graft vs Host Disease Immune System Disorders
Keywords: Acute Graft vs Host Disease GVHD
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 180
Healthy volunteers: no

Brief summary:
The study is a randomized Phase II, four arm treatment trial. The primary purpose of the study is to define new agents with promising activity against acute graft-versus-host disease (GVHD) suitable for testing against corticosteroids alone in a subsequent Phase III trial.

Detailed description:
BACKGROUND:

Acute graft-versus-host disease (GVHD) is the major complication of allogeneic hematopoietic stem cell (HSC) transplantation. Acute GVHD produces significant morbidity and complicates patient management resulting in organ toxicity, frequent infections, malnutrition, and substantial delay in recovery from transplantation. Corticosteroids have been the primary therapy for acute GVHD for over three decades. Various additional immunosuppressive strategies have been tested as GVHD therapy but neither anti-thymocyte globulin (ATG), CD5-immunotoxins, IL-1 antagonists nor other agents have been demonstrably helpful in either control of GVHD symptoms or improvement in survival. Published response rates of complete response (CR) to acute GVHD therapy with corticosteroids range from 25-41%. These rates will be used as benchmarks for assessing efficacy of promising new agents. New immunosuppressive agents and strategies are required to improve the management of GVHD and decrease the toxicities of the immunosuppressive regimens.

DESIGN NARRATIVE:

In this trial, patients with newly diagnosed acute GVHD will be randomly assigned to receive corticosteroids plus one of four new agents (etanercept, MMF, denileukin diftitox \[Ontak\], and pentostatin). A control arm of only corticosteroids will not be employed. Each agent will be assessed for safety and efficacy (at least 35% complete remission \[CR\] rate at Day 28 of therapy can be expected from previously untreated patients).

Arms:
arm 1: Enroll within 48 hours of new onset acute GVHD and randomize to Etanercept arm 2: Enroll within 48 hours of new onset acute GVHD and randomize to Mycophenolate Mofetil arm 3: Enroll within 48 hours of new onset acute GVHD and randomize to Denileukin Diftitox arm 4: Enroll within 48 hours of new onset acute GVHD and randomize to Pentostatin

Interventions: intervention 1: Etanercept intervention 2: Mycophenolate Mofetil intervention 3: Denileukin Diftitox intervention 4: Pentostatin
Intervention descriptions: intervention 1: Etanercept \[25 mg subcutaneously twice weekly for up to 4 weeks; discontinue if in complete response by 4 weeks\]. intervention 2: Mycophenolate mofetil (MMF) \[20 mg/kg (maximum 1 gm) orally or intravenously twice daily; continue through prednisone taper, then taper MMF over 4 weeks\]. intervention 3: Denileukin Diftitox (ONTAK®) \[9 mcg/kg intravenously Days 1, 3, 5, 15, 17, 19\]. intervention 4: Pentostatin \[1.5 mg/m2 daily for 3 days; Days 1-3 and repeat Days 15-17

Protocol document:
Prot_SAP_ICF_000.pdf: 
 
 
Initial Systemic Treatment of Acute GVHD: A Phase II Randomized 
Trial Evaluating Etanercept, Mycophenolate Mofetil (MMF), 
Denileukin Diftitox (ONTAK), and Pentostatin  
in Combination with Corticosteroids 
 
BMT CTN PROTOCOL 0302 
VERSION 7.0 
 
 
Study Chairperson 
Daniel Weisdorf, M.D. 1 
 
 
Protocol Team 
Joseph Antin, M.D. 2 
Nelson Chao, M.D. 3 
Nancy DiFronzo, Ph.D. 4 
James Ferrara, M.D. 5 
Mary Horowitz, M.D., M.S. 6 
Eric Leifer, Ph.D. 4 
Brent Logan, Ph.D. 6 
Pam Lucarelli 7 
Richard Nash, M.D. 8 
Robert Soiffer, M.D. 2 
Georgia Vogelsang, M.D. 9 
 
 
 
Sponsored by the National Institutes of Health 
National Heart, Lung and Blood Institute 
National Cancer Institute 
 
 
 
1 University of Minnesota  
2 Dana Farber Cancer Institute  
3 Duke University Medical Center 
4 National Heart, Lung, and Blood Institute 
5 University of Michigan 
6 Center for International Blood and Marrow 
Transplant Research, Medical College of 
Wisconsin 
7 The EMMES Corporation  
8 Fred Hutchinson Cancer Research Center 
9 Johns Hopkins University 
 
 


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Acute GVHD Protocol – 0302 
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Core Study Participants: 
Non-Core Study Participants: 
Case Western Reserve University Consortium 
Oregon Health & Science University - Adult 
University Hospitals of Cleveland 
Washington University/Barnes Jewish Hospital 
City of Hope National Medical Center 
Dana Farber/Partners Cancer Center 
Duke University Medical Center 
Fred Hutchinson Cancer Research Center 
Johns Hopkins University 
Memorial Sloan-Kettering Cancer Center 
Pediatric Blood and Marrow Transplant Consortium 
Children’s Hospital of Pittsburgh 
Children’s Mercy Hospital, University of Missouri 
Children’s National Medical Center, Washington, DC 
Columbus Children’s Hospital 
Hackensack University Medical Center 
Oregon Health and Science University 
Texas Transplant Institute 
University of Oklahoma Health Science Center 
Washington University/St. Louis Children’s 
Hospital 
Stanford Hospital and Clinics 
UCSD/SCRIPPS School of Medicine 
University of Florida College of Medicine 
University of Michigan Medical Center 
University of Minnesota 
University of Nebraska Medical Center 
University of Pennsylvania Hospital 
University of Texas, MD Anderson Cancer Research Center 
 
 
Roswell Park Cancer Institute 
 


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PROTOCOL SYNOPSIS – BMT CTN PROTOCOL 0302 
 
Initial Systemic Treatment of Acute GVHD: A Phase II Randomized Trial Evaluating 
Etanercept, Mycophenolate Mofetil (MMF), Denileukin Diftitox (ONTAK) and Pentostatin 
in Addition to Corticosteroids 
 
Principal Investigator: 
Daniel Weisdorf, M.D. 
 
Study Design: 
The study is a randomized Phase II, four arm treatment trial.  The 
primary purpose of the study is to define new agents with promising 
activity against acute GVHD suitable for testing against corticosteroids 
alone in a subsequent Phase III trial. 
 
 
Corticosteroids have been used as primary therapy for acute GVHD for 
many years.  Historical published and unpublished data from the 
University of Minnesota, Dana Farber Cancer Institute and the Fred 
Hutchinson Cancer Center define an expected 35% complete response 
(CR) at Day +28 of corticosteroid therapy for previously untreated 
patients with acute GVHD. 
 
 
In this trial, patients with newly diagnosed acute GVHD will receive 
corticosteroids plus one of four new agents.  A control arm of only 
corticosteroids will not be employed.  Each agent will be assessed for 
safety (stopping rules defined) and efficacy (≥ 35% CR rate at Day 28 of 
therapy). 
 
Primary Objective: 
The primary objective is to estimate the proportion of CR at Day 28 of 
therapy for newly diagnosed acute GVHD for each of these agents given 
in combination with corticosteroids. 
 
Secondary Objectives: 
Secondary objectives are to determine: proportion of partial response 
(PR), mixed response and progression at Day 28; proportion of treatment 
failure (no response, progression, administration of additional therapy for 
GVHD, or mortality) by Day 14; the incidence of GVHD flares requiring 
increasing therapy before Day 90.  In addition, the following endpoints 
will be examined: incidence of discontinuation of immune suppression 
without flare by Days 90, 180 and 270 post-therapy, incidence of chronic 
GVHD by 9 months, overall survival at 6 and 9 months post initiation of 
therapy, incidence of systemic infections within 3 months of initiation of 
therapy, incidence of EBV-associated lymphoma. 
 
Eligibility: 
Patients must be greater than or equal to 6 years of age and have acute 
GVHD requiring systemic therapy.  No previous systemic immune 
suppressive therapy for acute GVHD is allowed except for a maximum 
48 
hours 
of 
prior 
corticosteroid 
therapy 
(≥ 
1 
mg/kg/day 
methylprednisolone).  Patients receiving ONTAK, pentostatin, or 
etanercept within 7 days of screening will be excluded.  Patients 
receiving MMF for GVHD prophylaxis will be randomized into one of 
the other three treatment arms.  Patients must have an absolute neutrophil 


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ii 
count (ANC) greater than 500/µL and an estimated creatinine clearance 
greater than 30 mL/minute. 
 
Treatment Description: 
The treatment schedules are as follows: Etanercept [25 mg 
subcutaneously twice weekly for up to 4 weeks; discontinue if in CR by 
4 weeks].  Mycophenolate mofetil (MMF) [20 mg/kg (maximum 1 gm) 
PO or IV BID; continue through prednisone taper, then taper MMF over 
4 weeks].  Denileukin Diftitox (ONTAK®) [9 mcg/kg IV Days 1, 3, 5, 
15, 17, 19].  Pentostatin [1.5 mg/m2 daily for 3 days; Days 1-3 and 
repeat Days 15-17]. 
 
 
All patients will receive methylprednisolone 2 mg/kg/day IV (or 
prednisone 2.5 mg/kg/day PO) divided in 2-3 daily doses for at least 7 
days.  Prednisone may be tapered as tolerated to no less than  
0.75 mg/kg/day (methylprednisolone 0.6 mg/kg/day) at Day 28 of 
therapy.  If not in CR at Day 28, patients will be followed for study 
endpoints, but assigned therapy can be continued or changed per 
institutional guidelines. 
 
 
In addition to prescribed study drug plus corticosteroids, all patients 
should receive transfusion support per institutional practice; anti-
infective 
prophylaxis 
directed 
towards 
CMV, 
gram 
positive 
(encapsulated) bacteria, pneumocystis carinii and fungal infections  
 
 
Weekly GVHD organ stage scores, overall clinical grade, biopsy 
information for GVHD and relevant differential diagnosis will be 
recorded and reported to the BMT CTN Data Coordinating Center 
(DCC). 
 
Accrual Objective: 
A maximum of 180 patients, but at most 65 per arm, will be randomized 
to one of the four treatment arms.  After 80 patients are evaluable for the 
primary endpoint, if in any experimental arm, observed responses 
suggest a posterior probability > 85% that an arm will have a true CR  
< 35%, enrollment to the arm will cease and the arm will be excluded 
from further consideration due to lack of evidence for efficacy. 
 
Accrual Period: 
The estimated accrual period is 12 months. 
 
Study Duration: 
Patients will be followed for 9 months following initiation of therapy. 
 


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STUDY DESIGN SCHEMATIC 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
* Steroids = 2.5 mg/kg prednisone/day PO or IV equivalent in methylprednisolone (2 mg/kg) 
divided 2-3 times daily 
 
Primary Endpoint: 
Proportion of CR at Day 28 of therapy. 
Enroll within 48 hours  
of new onset acute GVHD meeting other eligibility 
criteria and randomize to 1 of 4 study drugs 
Patient taking MMF for GVHD prophylaxis 
within 7 days of GVHD onset? 
Yes 
No 
Randomize to ONTAK, pentostatin, 
or etanercept and steroids* 
Randomize to MMF, ONTAK, 
pentostatin, or etanercept and steroids* 
Continue to randomize until 180 patients 
enrolled or max 65 patients per arm 
Continue to randomize until 80 patients 
evaluable for primary endpoint, assess 
MMF use and efficacy of 4 treatment 
arms, adjust randomization if necessary 


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STUDY SCHEMA 
 
Aim:  To determine if any of four new agents, given in addition to corticosteroids as initial therapy for 
acute GVHD improves response rate and overall clinical outcome. 
 
Inclusion Criteria 
1. Prior allogeneic hematopoietic stem cell transplant using 
either bone marrow, PBSC or cord blood. 
2. De novo acute GVHD diagnosed within 48 hours prior 
to enrollment.  Biopsy confirmation of GVHD is strongly 
recommended but not required.  Enrollment should not be 
delayed awaiting biopsy or pathology results.  The patient 
must have had no previous systemic immune suppressive 
therapy given for treatment of acute GVHD except for a 
maximum 48 hours of prior corticosteroid therapy 
(≥ 1 mg/kg/day methylprednisolone). 
3. GVHD developing following DLI for prophylaxis or 
planned DLI. 
4. ≥ 6 years of age at enrollment. 
5. Clinical status at enrollment to allow tapering of steroids 
to not less than 0.6 mg/kg/day methylprednisolone  
(0.75 mg/kg/day prednisone) at Day 28 of therapy (e.g. 
persisting malignant disease suggesting the need for 
accelerated taper of immunosuppression). 
6. ANC greater than 500/µL. 
7. Estimated creatinine clearance greater than 30 mL/minute 
8. Signed informed consent and/or assent. 
9. Assent and educational materials provided to, and 
reviewed with, patients under the age of 18. 
Exclusion Criteria 
1. ONTAK, pentostatin or etanercept given within 7 
days of enrollment. 
2. Isolated limited skin GVHD as the sole manifestation 
of acute GVHD. 
3. Active uncontrolled infection. 
4. Patients that have undergone an unscheduled (or not 
part of original transplant therapy plan) DLI. 
5. Patients unlikely to be available at the transplant 
center on Day 28 and 56 of therapy. 
6. A clinical syndrome resembling de novo chronic 
GVHD developing at any time after 
allotransplantation. 
7. Other investigational therapeutics for GVHD within 
30 days, including agents used for GVHD 
prophylaxis. 
8. If any prior steroid therapy (for indication other than 
GVHD), treatment at doses > 0.5 mg/kg/d methyl-
prednisolone within 7 days prior to onset of GVHD. 
9. Patients who are pregnant, breast feeding, or if 
sexually active, unwilling to use effective birth 
control for the duration of the study. 
10.Adults unable to provide informed consent 
11.Patients with a history of intolerance to any of the 
study drugs. 
 
If any improvement after 7 days of full dose corticosteroids + study drug: Taper steroids as tolerated 
to no less than 0.75 mg/kg/day prednisone (or 0.6 mg/kg/day methylprednisolone) on Day 28.  
Improvement is defined as any clinically recognizable lessening of skin rash, redness, or extent; lessening 
of diarrhea or lowered bilirubin (though it does not have to be greater than or equal to one stage 
improvement in any involved organ), without worsening in any organ. 
 
Suggested prednisone taper for responders 
(round to nearest 5 mg of prednisone): 
2.5 mg/kg/day divided in 2-3 doses Day 0-6 
2.5 mg/kg/day once daily Day 7-13 
2 mg/kg/day Day 14-21 
1.4 mg/kg/day Day 21-28 
0.75 mg/kg/day Day 29-35 
0.6 mg/kg/day Day 36-42 
0.4 mg/kg/day Day 43-49 
0.25 mg/kg/day Day 50-56 
0.1 mg/kg/day Day 57-63 
0.1 mg/kg every other day, Day 63-69;  
Discontinue on Day 70 
 
Primary endpoint: 
Proportion of CR at Day 28 of therapy 
Secondary endpoints: 
- Proportion of PR, mixed response and progression at Day 28 
- Proportion of NR, progression, administration of additional 
therapy for GVHD, or mortality by Day 14 
- Incidence of GVHD flares requiring increasing therapy 
before Day 90 
- Incidence of discontinuing immune suppression without flare 
by Day 90, 180 and 270 post therapy 
- Incidence of Chronic GVHD by 9 months 
- Survival at 6 and 9 months after initiation of treatment 
- Incidence of systemic infections within 3 months of therapy 
- Incidence of EBV lymphoma within 9 months of therapy 
 
If acute GVHD progresses within 7 days or no response within 14 days, then treat with alternative 
secondary GVHD therapy off study, but follow for study endpoints. 


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TABLE OF CONTENTS 
 
1 
BACKGROUND AND RATIONALE .............................................................................. 1-1 
1.1 
Study Goal ........................................................................................................................... 1-1 
1.2 
Background Data: Therapy of Acute GVHD ..................................................................... 1-2 
1.2.1 
Daclizumab ........................................................................................................................... 1-2 
1.2.2 
Infliximab ............................................................................................................................. 1-2 
1.2.3 
Etanercept ............................................................................................................................. 1-2 
1.2.4 
Sirolimus ............................................................................................................................... 1-3 
1.2.5 
Mycophenolate Mofetil (MMF) ............................................................................................. 1-3 
1.2.6 
Pentostatin............................................................................................................................. 1-3 
1.2.7 
Denileukin Diftitox (ONTAK) ............................................................................................... 1-3 
1.2.8 
Study Agents ......................................................................................................................... 1-4 
1.2.9 
Staging of Acute GVHD ........................................................................................................ 1-4 
2 
STUDY DESIGN............................................................................................................... 2-1 
2.1 
Study Design ........................................................................................................................ 2-1 
2.2 
Primary Objective and Rationale for Study Design ........................................................... 2-1 
2.3 
Eligibility ............................................................................................................................. 2-2 
2.3.1 
Inclusion Criteria ................................................................................................................... 2-2 
2.3.2 
Exclusion Criteria .................................................................................................................. 2-4 
2.3.3 
Informed Consent .................................................................................................................. 2-5 
2.4 
Treatment Plan .................................................................................................................... 2-5 
2.4.1 
Risks of All Study Drugs ....................................................................................................... 2-5 
2.4.1.1 
Risks of infection ........................................................................................................ 2-5 
2.4.1.2 
Drug associated toxicities ............................................................................................ 2-5 
2.4.2 
Etanercept (ENBREL®, Amgen, Inc.) .................................................................................... 2-5 
2.4.2.1 
Mechanism of action ................................................................................................... 2-5 
2.4.2.2 
Etanercept drug supply ................................................................................................ 2-6 
2.4.2.3 
Dose and administration .............................................................................................. 2-6 
2.4.2.4 
Drug interactions ......................................................................................................... 2-6 
2.4.2.5 
Risks and toxicities ...................................................................................................... 2-6 
2.4.2.6 
Discontinuation ........................................................................................................... 2-7 
2.4.3 
Mycophenolate Mofetil (CellCept®, Roche) ........................................................................... 2-7 
2.4.3.1 
Mechanism of action ................................................................................................... 2-7 
2.4.3.2 
MMF drug supply........................................................................................................ 2-7 
2.4.3.3 
Dose and administration .............................................................................................. 2-8 
2.4.3.4 
Risks and toxicities ...................................................................................................... 2-8 
2.4.3.5 
Guidelines for MMF dose adjustment .......................................................................... 2-8 
2.4.3.6 
Drug interactions ......................................................................................................... 2-9 
2.4.3.7 
Discontinuation ........................................................................................................... 2-9 
2.4.4 
Denileukin Diftitox (ONTAK®, Ligand Pharmaceuticals) ...................................................... 2-9 
2.4.4.1 
Mechanism of action ................................................................................................... 2-9 
2.4.4.2 
ONTAK drug supply ................................................................................................... 2-9 
2.4.4.3 
Dose and administration ............................................................................................ 2-10 
2.4.4.4 
Drug interactions ....................................................................................................... 2-10 
2.4.4.5 
Risks and toxicities .................................................................................................... 2-10 
2.4.4.6 
Guidelines for dose adjustment .................................................................................. 2-10 
2.4.5 
Pentostatin (Nipent®, Supergen Pharmaceuticals)................................................................. 2-10 


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2.4.5.1 
Mechanism of action ................................................................................................. 2-10 
2.4.5.2 
Pentostatin drug supply ............................................................................................. 2-11 
2.4.5.3 
Dose and administration guidelines............................................................................ 2-11 
2.4.5.4 
Drug interactions ....................................................................................................... 2-11 
2.4.5.5 
Risks and toxicities .................................................................................................... 2-11 
2.4.5.6 
Guidelines for dose adjustment .................................................................................. 2-11 
2.4.6 
Corticosteroids (prednisone or methylprednisolone)............................................................. 2-12 
2.4.6.1 
Steroid taper .............................................................................................................. 2-12 
2.4.7 
Cyclosporine or Tacrolimus ................................................................................................. 2-13 
2.4.8 
GVHD Progression or Non-response ................................................................................... 2-13 
2.4.9 
GVHD Complete Response or Partial Response .................................................................. 2-13 
2.4.10 
Acute GVHD Flare .............................................................................................................. 2-13 
2.4.11 
Supportive Care ................................................................................................................... 2-14 
2.5 
GVHD Scoring ................................................................................................................... 2-14 
2.5.1 
GVHD Scoring .................................................................................................................... 2-15 
2.5.2 
Chronic GVHD ................................................................................................................... 2-15 
2.5.3 
Studies of GVHD Biology ................................................................................................... 2-15 
3 
STUDY ENDPOINTS ....................................................................................................... 3-1 
3.1 
Primary Endpoint................................................................................................................ 3-1 
3.1.1 
Staging and Grading of Acute GVHD .................................................................................... 3-1 
3.2 
Secondary Endpoints ........................................................................................................... 3-2 
3.2.1 
Proportion of Partial Response (PR), Mixed Response (MR), No Response (NR) and 
Progression ........................................................................................................................... 3-2 
3.2.2 
Proportion of Primary Treatment Failures .............................................................................. 3-2 
3.2.3 
GVHD Flares ........................................................................................................................ 3-2 
3.2.4 
Immunosuppression Discontinuation ..................................................................................... 3-2 
3.2.5 
Chronic GVHD ..................................................................................................................... 3-2 
3.2.6 
Survival................................................................................................................................. 3-2 
3.2.7 
Systemic Infections ............................................................................................................... 3-3 
4 
PATIENT ENROLLMENT AND EVALUATION ......................................................... 4-1 
4.1 
Enrollment and Randomization .......................................................................................... 4-1 
4.1.1 
Randomization ...................................................................................................................... 4-1 
4.2 
Study Monitoring ................................................................................................................ 4-1 
4.2.1 
Follow-up Schedule ............................................................................................................... 4-1 
4.2.2 
Assessments .......................................................................................................................... 4-3 
4.2.3 
Weekly GVHD Monitoring ................................................................................................... 4-4 
4.2.4 
Serious Adverse Event (SAE) Reporting................................................................................ 4-4 
5 
STATISTICAL CONSIDERATIONS.............................................................................. 5-1 
5.1 
Study Design Synopsis ......................................................................................................... 5-1 
5.2 
Accrual ................................................................................................................................. 5-1 
5.3 
Randomization..................................................................................................................... 5-1 
5.4 
Primary Endpoint................................................................................................................ 5-2 
5.5 
Primary Hypothesis ............................................................................................................. 5-2 
5.6 
Study Design ........................................................................................................................ 5-2 
5.7 
Power and Operating Characteristics of Study Design ...................................................... 5-3 
5.8 
Stopping Guidelines............................................................................................................. 5-4 
5.8.1 
Stopping Guidelines for Mortality ......................................................................................... 5-5 
5.9 
Demographic and Baseline Characteristics ........................................................................ 5-6 


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5.10 
Endpoints and Analysis Plan............................................................................................... 5-6 
 
 
LIST OF APPENDICES 
 
APPENDIX A 
REFERENCES 
APPENDIX B 
INFORMED CONSENTS (PARTICIPANT AND ASSENT) AND 
EDUCATIONAL MATERIAL 
APPENDIX C 
LABORATORY PROCEDURES 
APPENDIX D 
HUMAN SUBJECTS 
 
 
 


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1-1 
 
CHAPTER 1 
 
 
1 
BACKGROUND AND RATIONALE 
 
Acute graft-versus-host disease (GVHD) is the major complication of allogeneic hematopoietic 
stem cell (HSC) transplantation.  Acute GVHD produces significant morbidity and complicates 
patient management resulting in organ toxicity, frequent infections, malnutrition and substantial 
delay in recovery from transplantation.  
 
Acute GVHD usually occurs within the first 30-60 days of allogeneic HSCT and may involve the 
skin, liver and intestinal tract.  T lymphocytes contained in the donor graft respond in vivo to 
disparate major (HLA) or minor (non-HLA) histocompatibility antigens on host tissues and a 
cascade of events leads to the signs and symptoms of acute GVHD.  Greater HLA disparity 
between the donor and the recipient results in a higher incidence of acute GVHD. 
 
The syndrome of acute GVHD includes the following signs and symptoms.  Skin involvement 
(maculopapular exanthem) is usually the first sign of acute GVHD.  Lesions may be pruritic or 
painful, red to violaceous in color, and often involve the palms and soles. Acute GVHD of the 
intestines may involve the stomach, small bowel and colon producing persistent nausea and 
vomiting or profuse diarrhea, intestinal bleeding, crampy abdominal pain and ileus.  Liver 
GVHD produces cholestatic liver injury with hyperbilirubinemia and some hepatocellular 
enzyme elevations.   
 
The use of cyclosporine (CSA) or tacrolimus (FK) plus short course methotrexate (MTX) has 
lowered the risk for acute GVHD compared to single agent or other combination therapy, but 
still 35-50% of patients develop Grade 2-4 acute GVHD [2-4, 5].  Older recipients or those 
transplanted from alternative (unrelated or partially matched) donors may develop more frequent 
or possibly more therapy-resistant acute GVHD. 
 
GVHD Therapy 
Corticosteroids have been the primary therapy for acute GVHD for over three decades.  Various 
additional immunosuppressive strategies have been tested as GVHD therapy but neither anti-
thymocyte globulin (ATG), CD5-immunotoxins, IL-1 antagonists nor other agents have been 
demonstrably helpful in either control of GVHD symptoms or improvement in survival [6-11].  
Published response rates of complete response (CR) to acute GVHD therapy with corticosteroids 
range from 25-41% [12-14].  These rates will be used as benchmarks for assessing efficacy of 
promising new agents.  New immunosuppressive agents and strategies are required to improve 
the management of GVHD and decrease the toxicities of the immunosuppressive regimens. 
 
1.1 
Study Goal 
 
This study will test four new agents (etanercept, MMF, denileukin diftitox (ONTAK) and 
pentostatin), each in combination with corticosteroids as initial therapy of acute GVHD in a 
prospective, randomized Phase II trial.  The primary purpose of the study is to identify agents 


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1-2 
with sufficient promise for definitive testing in a subsequent Phase III trial in comparison to 
corticosteroids alone. 
 
1.2 
Background Data: Therapy of Acute GVHD 
 
Recently, several new agents have been tested for GVHD therapy.  Relevant data (peer-reviewed 
manuscripts, abstracts and/or personal communication) about the most promising new agents are 
briefly summarized below. 
 
1.2.1 Daclizumab 
 
A report from Germany [15] summarized GVHD treatment in 16 patients; 12 had steroid 
resistant acute GVHD.  Patients (n=7) received daclizumab in a dosage of 1 mg/kg body weight 
(BW) on Day 1-5 and once every week thereafter until Day 28.  Eight patients received 1 mg/kg 
on Day 1 and 2, followed by one dose per week through Day 28.  Of the 12 evaluable patients, 6 
responded to therapy.  However, the rate of infections was high with 14 of 16 patients 
experiencing infections, 2 of them fatal.  Four of 16 (25%) patients survived. 
 
Przepiorka et al., [16] reported on 43 patients with steroid refractory acute GVHD.  Daclizumab 
was administered at 1 mg/kg IV on study Day 1, 8, 15, 22 and 29 (n=24) or at 1 mg/kg IV on 
study Day 1, 4, 8, 15 and 22 (n=19) given either weekly (n=29) or 3 times per week (n=19).  CR 
at Day 42 was 29% using the weekly regimen and 47% using the 3 times per week regimen.  
Improved 120 day survival (29% versus 53%) was also seen with the second regimen. 
 
Wolff [17] reported a study of 12 patients treated with daclizumab and steroids.  Six patients 
achieved CR, 4 partial responses, 1 non-response and 1 early death.  A randomized trial of 
daclizumab plus corticosteroids versus corticosteroids alone (Dana Farber/ Minnesota/other 
centers) showed no benefit and some added toxicity with daclizumab [42]. 
 
1.2.2 Infliximab 
 
Couriel et al., [18] reported a preliminary analysis of a randomized trial (n=60) showing no 
benefit of infliximab over steroids alone. The trial was stopped early.  However, Sleight et al. 
[19] reported a series of 24 pediatric patients with 16 of the patients refractory to steroids.  In this 
series, the patients received infliximab plus various other therapies.  Improvement was seen in 
15/16 evaluable patients with skin GVHD, 11/14 with gut GVHD, and 1/3 with liver GVHD; 
only 2 of 16 are surviving.  Kobbe et al. [20] reported PR in 3 of 4 patients receiving  
10 mg/kg/week. 
 
1.2.3 Etanercept  
 
In a report of 10 patients with chronic GVHD, Chiang et al. [21] found improvement of GVHD 
in 7 of the 8 patients completing an 8-week course.  In another series, 13 patients received 25 mg 
subcutaneously twice weekly for up to 8 weeks [22].  Ten of 13 had a CR, most within 2 weeks.  
CR was more frequent for Grade 2 than Grade 3 acute GVHD. 
 


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1.2.4 Sirolimus 
 
Benito et al. [23] reported 21 cases of steroid refractory disease treated with sirolimus at a 
median of 37 days post-transplant.  Ten patients stopped treatment early (5 NR, 2 
myelosuppression, 2 seizures).  Ten patients had thrombotic microangiopathy (TMA).  However, 
in 5 of 10, TMA did not appear correlated with sirolimus therapy.  Of the 18 patients who 
received at least 6 doses, 12 responded (5 CR and 7 PR).  Six of the 21 patients are surviving. [J. 
Antin, personal communication].  It is recognized that sirolimus has complex pharmacology and 
drug interactions.  The formulation is currently not suitable for concurrent use with voriconazole.  
Information on dose modifications required for combined use of sirolimus and voriconazole may 
be available during 2004.  It is promising but additional clinical pharmacology is needed. 
 
1.2.5 Mycophenolate Mofetil (MMF) 
 
Basara et al. [24] reported that treatment with MMF improved GVHD in 65% of 17 patients with 
acute GVHD (N=24; 17 acute GVHD; 65% improved).  A report from Seattle (R Nash personal 
communication) showed similar results with 6 CR and 2 PR in 19 patients. 
 
1.2.6 Pentostatin 
 
Bolanos-Meade et al. [25] reported a dose escalation trial for treatment of steroid resistant acute 
GVHD using 1-4 mg/m2 daily x 3 days.  The best dose was 1.5mg/m2 x 3 days, then repeated q 2 
weeks.  Of 20 treated patients, 11 achieved CR, 3 achieved PR, 3 had mixed responses and 3 
progressed while on therapy.  A recent update (personal communication, G. Vogelsang) totaling 
23 treated patients showed 14 CR and 3 PR among 22 evaluable patients.  At higher doses, late 
infections were considered dose limiting.  The drug was well tolerated with only modest 
elevations of liver function tests and thrombocytopenia, each observed in only a single patient. 
 
1.2.7 Denileukin Diftitox (ONTAK) 
 
Shaughnessy et al. [26] described 11 patients treated with ONTAK 4.5 g/kg/d x 5 days 
followed by the same dose weekly x 4.  Of the 11, there were 5 responders, including 2 CR and 1 
PR with 2 lesser responses. 
 
Ho et al. reported 10 patients receiving 9 mcg/kg IV Days 1, 15 or Days 1, 3, 5, 15, 17 and 19.  
CR was seen in 3 patients, PR in 4 and mixed response in 2 (78% CR+PR).  A subsequent update 
[27] included 28 patients with steroid refractory GVHD [grade II (n = 10), grade III (n = 13), 
grade IV (n = 5)].  Seven received ONTAK at dose level I (9 g/kg IV on Days 1 and 15), 16 at 
dose level II (9 g/kg IV Days 1, 3, 5, 15, 17 and 19), and 5 at dose level III (9g/kg IV Days 1-
5, 15-19), which yielded dose-limiting toxicity (1 acute renal failure, 3 elevated hepatic 
transaminases).  At Day 28, 7 of 22 evaluable (32%) had CR and 9 (41%) PR, for an overall 
response rate of 73%.  Among the 9 with PR, 3 additional patients subsequently entered CR after 
Day 29 without additional GVHD therapy, resulting in an overall CR rate of 45%.  Response rate 
was highest at dose level II (CR = 45% and PR = 27%). Of the 22 evaluable for GVHD response, 
9 (41%) are alive.  
 


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1.2.8 Study Agents 
 
Based on the available data presented above, the four agents considered most promising for 
Phase II multi-center testing were etanercept, MMF, ONTAK and pentostatin.  Each study agent 
has promising preliminary data reported from single or several center studies, suitable safety 
experience and tolerable applicability for multi-center Phase II testing as initial systemic therapy 
for acute GVHD. 
 
1.2.9 Staging of Acute GVHD 
 
Several schemas for staging and grading of acute GVHD have been published [28, 41].  In this 
study we will classify patients by the CIBMTR grading schema (Grades A-D), which has been 
shown to be more predictive of survival than earlier versions [7, 28].   
 


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CHAPTER 2 
 
 
2 
STUDY DESIGN 
 
2.1 
Study Design 
 
This trial is designed to evaluate the safety and efficacy of four new agents for acute GVHD in a 
prospective, multi-center, two-stage, randomized, four-arm Phase II trial.  Each study arm will 
include corticosteroids plus a new agent.  After 80 patients are evaluable for the primary 
endpoint, each arm will be assessed for safety using Bayesian posterior probability rules. 
 
Study Design without Steroid Only Control 
In this Phase II trial design, a corticosteroid only control arm will not be used since such an arm 
would not aid in judging which experimental arm was suitable for consideration in a subsequent 
Phase III randomized trial given the limited statistical power for comparison between arms.  
Each agent will be tested for potential efficacy against well established historical data published 
from several centers (≥ 35% CR rate at Day 28 of therapy).  A control cohort would not assist 
this comparison and would delay completion of this prospective Phase II test of individual agents 
given as an adjunct to corticosteroids. 
 
2.2 
Primary Objective and Rationale for Study Design 
 
The primary purpose of the study is to define new agents with promising activity against acute 
GVHD suitable for testing against corticosteroids alone in a subsequent comparative, Phase III 
trial.  Careful review of published and unpublished data on therapy of acute GVHD documents 
an expected complete response (CR) rate for steroids alone of 35%.  Each of the four study 
agents selected are reported to have promising activity as therapy of acute GVHD in preliminary 
testing but have limited Phase II data available.  This trial will more formally test their safety and 
efficacy as supplemental to corticosteroids alone.  
 
The choice of agents for study in the subsequent Phase III trial against steroids alone will include 
consideration of the initial CR rate (the primary endpoint) as well as several important 
assessments of other safety and efficacy parameters (the secondary endpoints).  Those 
considered suitable for subsequent testing will have demonstrable primary efficacy as well as 
satisfactory estimates (developed within this randomized Phase II trial) of GVHD control 
without excessive rates of the anticipated complications of infection, GVHD flare, chronic 
GVHD or early mortality.  Thus, in this Phase II trial, a control arm will be omitted and deferred 
until the formal, prospective Phase III trial, which will test the best agent(s) against the control of 
steroids alone. 
 


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2.3 
Eligibility 
 
2.3.1 Inclusion Criteria 
 
Biopsy of involved tissue is strongly encouraged, but not required for study entry.  The clinical 
diagnosis of acute GVHD requiring systemic therapy with corticosteroids is necessary for 
enrollment on the trial.  Enrollment and randomization includes commitment to continue both 
steroids and study drug as indicated by the protocol as well as the required follow-up 
observations.  If the intention to treat a patient is dependent on histologic results, the patient 
should not be enrolled on the BMT 0302 study until the results are known.  Patients should only 
be enrolled with a clinically established diagnosis.  Histologic confirmation of acute GVHD is 
not required, but can be used as supportive evidence for the diagnosis.  If an unexpected biopsy 
result occurs, the center should contact the protocol chair. 
 
1. Prior allogeneic hematopoietic stem cell transplant using either bone marrow, peripheral 
blood stem cells or cord blood.  Recipients of non-myeloablative transplants are also 
eligible. 
 
2. De novo acute GVHD requiring systemic therapy within 48 hours of diagnosis [28].  
GVHD is defined as the presence of skin rash and/or persistent nausea, vomiting, and/or 
diarrhea and/or cholestasis as the clinical criteria compatible with the diagnosis of acute 
GVHD when present in the right clinical setting and not directly attributable to other 
etiologies such as drug rash, enteric infection, or hepatotoxic syndromes (e.g., veno 
occlusive disease of the liver).  The patient must have had no previous systemic immune 
suppressive therapy given for treatment of acute GVHD except for a maximum 48 hours 
of prior corticosteroid therapy ≥ 1 mg/kg/day methylprednisolone. 
 
Note that patients with stage II skin or isolated upper gastrointestinal (GI) only are 
eligible.  However, the protocol team does not recommend initiating systemic therapy 
with steroids for limited skin or upper GI only GVHD, but if the institutional treatment 
plan and treating physician initiates corticosteroid therapy at  2.0 mg/kg/day 
methylprednisolone (2.5 mg/kg/day prednisone), the patients are eligible for enrollment.   
 
3. Patients that have undergone a donor lymphocyte infusion (DLI) as part of their original 
transplant therapy plan. 
 
4. Greater than or equal to 6 years of age at the time of enrollment. 
 
5. Clinical status at enrollment to allow tapering of steroids to not less than 0.6 mg/kg/day 
methylprednisolone (0.75 mg/kg/day prednisone) at Day 28 of therapy. 
 
6. Absolute neutrophil count (ANC) greater than 500/µL. 
 
7. Estimated creatinine clearance greater than 30 mL/minute (see Table 2.3.1). 
 
8. Written informed consent and/or assent from patient, parent or guardian. 


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9. Documentation that the assent document and education materials have been provided to, 
and reviewed with, patients under the age of 18. 
 
 
Table 2.3.1 
ESTIMATED CREATININE CLEARANCE (mL/min) 
 
Serum 
Creatinine 
Ideal Body 
Weight   20 kg 
10 years/15 
40 kg 
20years/ 
30/40 
60 kg 
20years/ 
30/40 
80 kg 
20years/ 
30/40 
100 kg 
20years/ 
30/40 
0.8 
Male 45/43 
Female 38/37 
Male 
83/76/69 
Female 
71/65/59 
Male 
125/115/104 
Female 
106/97/89 
 
 
1.0 
Male 36/35 
Female 31/30 
Male 
67/61/56 
Female 
57/52/47 
Male 
100/92/83 
Female 
85/78/71 
 
 
1.2 
Male 30/29 
Female 26/25 
Male 
56/51/46 
Female 
47/43/39 
Male 
83/76/83 
Female 
59/65/71 
Male 
111/102/93 
Female 
94/87/79 
 
1.4 
Male 26/25 
Female 22/21 
Male 
48/44/40 
Female 
41/37/34 
Male 
71/66/60 
Female 
51/56/51 
Male 
95/87/79 
Female 
81/74/68 
Male 
119/109/99 
Female 
101/93/84 
1.6 
Male 23/22 
Female 19/18 
Male 
42/38/35 
Female 
35/33/30 
Male 
63/57/52 
Female 
53/49/44 
Male 
83/76/69 
Female 
71/65/59 
Male 
104/96/87 
Female 
89/81/74 
1.8 
Male 20/19 
Female 17/16 
Male 
37/34/31 
Female 
32/29/26 
Male 
56/51/46 
Female 
47/43/39 
Male 
74/68/62 
Female 
63/58/53 
Male 
93/85/77 
Female 
79/72/66 
2.0 
Male 18/17 
Female 15/15 
Male 
33/31/28 
Female 
28/26/24 
Male 
50/46/42 
Female 
43/39/35 
Male 
67/61/56 
Female 
57/52/47 
Male 
83/76/69 
Female 
71/65/59 
2.2 
 
Male 
30/28/25 
Female 
26/24/22 
Male 
46/42/38 
Female 
39/35/32 
Male 
61/56/51 
Female 
52/47/43 
Male 
76/69/63 
Female 
64/59/54 
Note: Modified from Cockcroft Gault equation; (140 – age)*wt/([Cr]*72= clearance  Females: 0.85 x the 
calculated result. 


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Calculated or measured creatinine clearance for patients can also be used. 
 
 
2.3.2 Exclusion Criteria 
 
1. Patients receiving ONTAK, pentostatin or etanercept within seven days of screening for 
enrollment. 
 
2. Patients with isolated limited skin GVHD (stage I) as the sole manifestation of acute 
GVHD. 
 
3. Patients with uncontrolled infections will be excluded.  For bacterial or viral infections, 
patients must be receiving definitive therapy and have no signs of progressing infection 
for 72 hours prior to enrollment.  For fungal infections patients must be receiving 
definitive systemic anti-fungal therapy and have no signs of progressing infection for 1 
week prior to enrollment. 
 
Progressing infection is defined as hemodynamic instability attributable to sepsis or new 
symptoms, worsening physical signs or radiographic findings attributable to infection.  
Persisting fever without other signs or symptoms will not be interpreted as progressing 
infection. 
 
4. Patients that have undergone an unscheduled DLI or a DLI that was not part of their 
original transplant therapy plan or GVHD developing following DLI given for treatment 
of persistent or recurrent malignancy after transplantation would be excluded. 
 
5. Patients unlikely to be available at the transplantation center on Day 28 and 56 of 
therapy. 
 
6. A clinical syndrome resembling de novo chronic GVHD developing at any time after 
allotransplantation.  See Chapter 2 of the BMT CTN Manual of Procedures for details of 
de novo chronic GVHD. 
 
7. Patients receiving other investigational agents for GVHD, including GVHD prophylaxis 
agents within 30 days of screening for eligibility.  The following agents are not 
considered experimental and therefore are not excluded: cyclosporine, tacrolimus, 
sirolimus, 
corticosteroids 
(prednisone, 
methylprednisolone, 
prednisolone, 
dexamethasone, or other), antithymocyte globulin (atgam, thymoglobulin or other), and 
methotrexate. 
 
8. Patients receiving methyprednisolone > 0.5 mg/kg/day (or 0.6 mg/kg/day predisone) 
within 7 days of onset of acute GVHD.  If steroid therapy has been administered for a 
non-GVHD related condition and tapered to ≤ 0.5 mg/kg/day methylprednisolone (0.6 
mg/kg/day prednisone) for seven or more days prior to the onset of acute GVHD, the 
patient is eligible. 
 


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9. Patients who are pregnant, breast feeding, or, if sexually active, unwilling to use effective 
birth control for the duration of the study. 
 
10. Adults unable to provide informed consent. 
 
11. Patients with a history of intolerance to any of the study drugs. 
 
2.3.3 Informed Consent 
 
The informed consent process will begin at recognition of subject eligibility and consent will be 
obtained per institutional practices before study therapy is initiated. 
 
2.4 
Treatment Plan 
 
Treatment should be initiated as soon as possible after randomization.  A maximum of 48 hours 
is allowable. 
 
2.4.1 Risks of All Study Drugs   
 
2.4.1.1 Risks of infection  
 
All four study drugs are potent immunosuppressives.  All have been associated with increased 
risks of opportunistic infection and in this setting, where corticosteroids and the intrinsic 
immunosuppression of GVHD are apparent, particular caution must be paid to the risks of 
additional infection.  Anti-infective prophylaxis (see Section 2.4.11) is required for all patients 
enrolled in this trial. 
 
Each of these study drugs can add to risks of infection due to their immunosuppressive potency.  
Patients with sustained bacteremia (≥ 2 days of bacteremia), persistent (≥ 5 days) culture 
negative fever or hemodynamic instability (sepsis syndrome with hypotension requiring  
> 30 ml/kg fluid resuscitation or pressor support) should have their study drug held at the 
attending physician's discretion.  Study drug can be reinstituted when control of the infection 
episode and hemodynamic stability is achieved.  Replacement doses or extension of study drug 
therapy is not recommended. 
 
2.4.1.2 Drug associated toxicities  
 
Study drug should be held for any Grade 3-4 toxicities that are considered probably related to 
study drug. 
 
2.4.2 Etanercept (ENBREL®, Amgen, Inc.) 
 
2.4.2.1 Mechanism of action 
 
ENBREL is a recombinant DNA derived protein composed of tumor necrosis factor (TNF) 
receptor linked to the FC portion of human IgGl.  Etanercept binds TNF and blocks its 


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interaction with target cell surface receptors [29-31].  It has anti-inflammatory activity that has 
been demonstrated in rheumatoid arthritis and tested in other inflammatory states including 
Wegener’s granulomatosis, inflammatory bowel disease, and other autoimmune and 
inflammatory states.   
 
2.4.2.2 Etanercept drug supply 
 
Etanercept will be provided by the BMT CTN for 4 weeks or approximately 8 doses.  The 
unreconstituted drug is supplied in vials containing 25 mg etanercept and must be stored between 
2ºC and 8ºC (without freezing) in the refrigerator.  The reconstituted vial (using 1 mL of 
bacteriostatic water, added slowly without shaking to minimize foaming) creates a solution of  
25 mg/mL after approximately 10 minutes of dissolution.  Reconstituted solutions should be 
administered as soon as possible after reconstitution.  If not administered immediately following 
reconstitution, reconstituted etanercept can be stored under refrigeration (2ºC - 8ºC) for up to 28 
days.  No other diluents except for bacteriostatic water for injection can be used and the drug 
should not be filtered when infused. 
 
2.4.2.3 Dose and administration 
 
Etanercept is to be administered subcutaneously within 28 days of reconstitution and stored in 
the refrigerator before use. 
 
It is to be administered no more often than once every 72 hours.  It is administered 
subcutaneously in rotating sites and should not be reinjected into a site with residual injection 
site reaction (new sites must be at least 1 inch from previous injection site).  The thigh, abdomen, 
and upper arm are preferred sites of injection.   
 
Patients will receive 25 mg subcutaneously twice weekly for up to 4 weeks.  Dosing for patients 
less than 17 years of age or with body surface area (BSA) < 0.6 m2 should be 0.4 mg/kg 
subcutaneously twice weekly with a maximum of 25 mg within 24 hours (the adult dose).  No 
premedications are required.   
 
2.4.2.4 Drug interactions 
 
No specific drug interactions have been recognized and no dose modifications will be required 
for metabolic or excretory dysfunction (liver or renal impairment). 
 
2.4.2.5 Risks and toxicities 
 
Administration of etanercept may cause injection site reactions with redness, tenderness, or 
warmth in up to one-third of patients.  Headache, dizziness, and rash may occur in a fraction of 
patients and upper respiratory tract symptoms with cough, sinusitis, rhinitis, and pharyngitis can 
occur.  It is uncertain whether there are infectious or inflammatory symptoms accompanying 
long-term use of etanercept.  
 


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Serious opportunistic infections, including tuberculosis, have been identified.  Rarely, 
pancytopenia has been observed. 
 
2.4.2.6 Discontinuation 
 
If patient is in CR (no signs of acute GVHD symptoms) by 4 weeks after therapy was initiated, 
etanercept is discontinued. 
 
For additional information on this study drug please see "Prescribing Information", at 
http://www.enbrel.com/ 
 
2.4.3 Mycophenolate Mofetil (CellCept®, Roche) 
 
2.4.3.1 Mechanism of action 
 
Mycophenolate mofetil (MMF) is a morpholinoethyl ester of mycophenolic acid (MPA). A 
product of several penicillium species, MPA possesses antibacterial, antifungal, antiviral, 
antitumor and immunosuppressive properties.  MMF is a pro-drug since the immunosuppressive 
activity is evident only after hydrolysis to MPA [32-34].  MMF was developed to enhance the 
bioavailability of MPA.  MPA mediates its effect by inhibiting inosine monophosphate 
dehydrogenase (IMPDH), an enzyme that catalyzes the oxidation of inosine monophosphate to 
xanthine monophosphate, an intermediate metabolite in the synthesis of guanosine triphosphate 
(GTP).  Lymphocytes rely on the de novo purine synthesis pathway for the nucleotides necessary 
for DNA synthesis; other cells can also rely on the salvage pathway.  The action of MPA results 
in the depletion of the nucleotide pool in cell synthesis.  An additional mode of action of MPA 
may be that, by depletion of GTP, it inhibits recruitment of leukocytes to sites of inflammation 
by inhibiting the glycosylation of lymphocyte glycoproteins involved in intercellular adhesion.  
Pharmacokinetics will be studied twice in each subject receiving MMF (Appendix C-5). 
 
2.4.3.2 MMF drug supply 
 
MMF will be provided by the BMT CTN for up to 8 weeks.  Oral MMF is available as  
500 mg tablets and 250 mg hard gelatin capsules in bottles of 100 each.  Capsules should be 
stored at room temperature.  A liquid suspension is also available (200 mg/mL, 225 mL bottle) 
and should be stored at 25ºC.  Once reconstituted the solution may be stored at room temperature 
or under refrigeration (do not freeze) for 60 days.  Enteric coated MMF (Myfortic) should not be 
used. 
 
Intravenous: MMF for intravenous use will be supplied as a lyophilized powder.  It will be 
reconstituted by injecting 5% Dextrose in Water (D5W) into each vial.  
 
Intravenous MMF does not contain an antibacterial preservative; therefore, it should be prepared 
under aseptic conditions.  Reconstitute each vial with 14 mL D5W.  Gently shake vial to dissolve 
the drug.  Further dilute into D5W for a final concentration of 6 mg/mL.  Dilute 1-g doses in  
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with normal saline and other saline containing diluents.  Only D5W solution should be used for 
reconstitution and infusion solution.  Infusion lines should be flushed with D5W only. 
 
The Pharmacist at each center should prepare each dose of drug to avoid errors.  Because of the 
potential teratogenic effects of MMF, pharmacists should follow the guidelines established at 
their centers for the preparation of similar types of solutions. 
 
2.4.3.3 Dose and administration 
 
Patients with body surface area (BSA) > 1.5 m2 will receive 20 mg/kg (actual body weight) PO 
or IV twice daily (maximum 1 g per dose).  Intravenous doses are infused over a two-hour 
period.   
 
For patients from 1.25 to 1.5 m2, administer 750 mg IV or as capsules PO twice daily.   
 
For patients < 1.5 m2 receiving suspension (not capsules), MMF as oral suspension can be given 
at a dose of 600 mg/m2 PO twice daily (maximum 1 g twice daily).  
 
If patients with GI GVHD are assigned to MMF, and they cannot tolerate oral fluids greater than 
500 mL per day (200 mL/m2), then MMF should be administered intravenously for the first 3-7 
days of therapy and converted to oral MMF as soon as possible after the initial 3 days of therapy 
if oral medications and this limited fluid intake are tolerated. 
 
A study of MMF absorption and pharmacokinetics is part of the trial (Appendix C-5) but levels 
will not be used for dose modification. 
 
2.4.3.4 Risks and toxicities 
 
Administration of MMF may produce diarrhea, leukopenia, sepsis, vomiting and possibly a 
higher incidence of certain viral infections (CMV, VZV, Herpes Simplex).  Gastrointestinal 
bleeding and perforation have also been described infrequently.  
 
2.4.3.5 Guidelines for MMF dose adjustment 
 
If any toxicity is judged on clinical grounds related to MMF administration, dose adjustment can 
occur as follows:   
 
Gastrointestinal (GI) Toxicity: If GI toxicity requires medication for control of persistent 
vomiting or diarrhea (not related to GVHD), a dose reduction to 50% can be instituted or, if 
symptoms persist and are severe (Grade 3 or 4), the drug can be stopped.  If GI toxicity does not 
resolve within 48-72 hours of stopping MMF, then this toxicity is not likely related to MMF and 
the drug should be restarted.  The study site principal investigator should be consulted before 
discontinuing MMF for GI toxicity.   
 


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Myelosuppression:  If the ANC is less than 1000/L, MMF dosing should be reduced by 50%.  
If the ANC is less than 500/L, the drug should be discontinued until neutrophil recovery occurs.  
Use of filgrastim and/or discontinuation of other myelosuppressive agents is allowed. 
 
Guidelines for Renal Insufficiency: As free MPA exposure has not been directly correlated 
with renal dysfunction, MMF dosing should not be modified unless renal failure requiring 
dialysis is required.  If dialysis is needed, MMF can be reduced to 25% - 50% of the starting 
dose. 
 
Hepatic Dysfunction: No adjustments of MMF are required for liver dysfunction. 
 
2.4.3.6 Drug interactions 
 
MMF activity is decreased with oral administration of antacids and cholestyramine.  Acyclovir 
or ganciclovir blood levels may increase during co-administration with MMF due to competition 
for tubular secretion of these drugs, especially if renal impairment is present.  Probenecid may 
also augment MPA levels due to inhibition of tubular secretion.  High doses of salicylates (or 
other highly protein bound drugs) may increase the free fraction of MPA and exaggerate the 
potential for myelosuppression. 
 
2.4.3.7 Discontinuation 
 
If in CR at Day 28, continue MMF at the same dose through the completion of the prednisone 
taper, then taper MMF and discontinue over four more weeks. 
 
2.4.4 Denileukin Diftitox (ONTAK®, Ligand Pharmaceuticals) 
 
2.4.4.1 Mechanism of action 
 
ONTAK is a fusion protein combining sequences from IL-2 with sequences from diphtheria, 
DAB-389.  It binds with the CD25 and delivers the toxin that inhibits intracellular protein 
synthesis, rapidly leading to death in any cells that are bound.  It has activity against cutaneous T 
cell lymphoma, but its anti T-lymphocyte lytic effects have been used for immunosuppressive 
therapy in treatment of GVHD and other conditions [35, 36]. 
 
ONTAK is administered intravenously and is distributed within 2-5 minutes and is metabolized 
in the liver by proteolytic degradation with a terminal half-life 70-80 minutes before inactivation. 
 
2.4.4.2 ONTAK drug supply 
 
ONTAK will be provided by the BMT CTN for 4 weeks or 6 doses.  It is supplied as a frozen 
solution ( -10C) containing 150 micrograms/mL (2 mL).  It is diluted for administration using 
preservative free normal saline.  It is recommended that 9 mL of saline is added for each mL of 
ONTAK maintaining a concentration of at least 15 micrograms/mL during preparation.  It is 
swirled gently with vigorous shaking avoided and should be administered within 6 hours of 
thawing and dilution.  It should not be put into glass containers or syringes.  


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2.4.4.3 Dose and administration 
 
ONTAK will be administered at 9 micrograms/kg (actual body weight) intravenously on Days 1, 
3, 5, 15, 17, and 19 of study.  If ONTAK is unavailable on Day 1, it should be administered on 
Days 2, 4, 6, 15, 17 and 19 of the study.  The rate of infusion for initial ONTAK administration 
should be over approximately 1 hour, with subsequent infusions shortened to no less than 15 
minutes as tolerated by the patient.  No dose modifications are permitted. 
 
2.4.4.4 Drug interactions 
 
Specific drug interactions have not been reported. 
 
2.4.4.5 Risks and toxicities 
 
Administration of ONTAK may cause infusion reactions with fever, chills, or hypotension 
sometimes resembling acute hypersensitivity events during or within 24 hours of infusion.  
Dyspnea and chest tightness, flushing, and pruritis may occur.   
 
Additionally, vascular leak with hypoalbuminemia, edema and, if severe, hypotension may occur 
in patients receiving ONTAK.  Lymphopenia with risk of infection and anemia may develop.  
Gastrointestinal side effects of diarrhea, anorexia, nausea, and vomiting are possible but are seen 
more frequently in doses considerably higher than used in this trial.  Reversible hepatic 
transaminase elevations can also be seen.  Myelosuppression with leukopenia or 
thrombocytopenia can occur, but is uncommon. 
 
2.4.4.6 Guidelines for dose adjustment  
 
ONTAK should be stopped for Grade 4 drug associated toxicity and drug held for 4 days for 
Grade 3 toxicity that improves to less than Grade 3 within the 4 days. 
 
Flulike symptoms with headache, fever, chills, arthralgia and myalgias may occur, but tend to 
decrease in incidence and severity with repeat treatments.  Slowing the infusion to 30 minutes 
has reduced the severity of these symptoms. 
 
Infusion-associated reactions can be managed by temporarily discontinuing or interrupting the 
infusion and then restarting at a slower rate.  Administering the dose of corticosteroids due for 
GVHD therapy as a pre-medication along with acetaminophen and/or diphenhydramine may 
minimize infusion-associated reactions. 
 
2.4.5 Pentostatin (Nipent®, Supergen Pharmaceuticals) 
 
2.4.5.1 Mechanism of action 
 
Pentostatin is a purine anti-metabolite that inhibits adenosine deaminase preventing the 
deamination of adenosine to inosine.  Accumulation of deoxyadenosine and dATP leads to 


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reduction in DNA synthesis and subsequent cell death.  The drug distributes rapidly to body 
tissues and has a terminal half-life of 5-15 hours with urinary excretion, mostly as unchanged 
drug [37, 38, 39]. 
 
2.4.5.2 Pentostatin drug supply 
 
Pentostatin will be provided by the BMT CTN for 4 weeks or 6 doses.  Pentostatin is supplied as 
a powder for reconstitution (10 mg/vial) in preparation for injection.  Vials are stored in the 
refrigerator between 2C and 8C.  It is diluted for infusion with 5% dextrose injection USP or 
0.9% saline.  Because it contains no preservatives, the diluted prepared solution must be used 
within 24 hours.   
 
2.4.5.3 Dose and administration guidelines 
 
Patients will receive 1.5 mg/m2 daily (BSA calculated using actual body weight) for 3 days on 
Days 1-3 and Days 15-17 of study for a total of 6 doses.  If pentostatin is unavailable on Day 1, it 
should be administered on Days 2-4 and 15-17 of the study.  It will be infused intravenously over 
15-30 minutes. 
 
2.4.5.4 Drug interactions 
 
Fludarabine and cyclophosphamide should not be co-administered as pentostatin toxicity is 
exaggerated. 
 
2.4.5.5 Risks and toxicities 
 
Mild to moderate nausea, vomiting, anorexia, diarrhea and skin rash are the most common 
adverse reactions, occurring in 30-50% of patients.  Thrombocytopenia, anemia, and to a lesser 
extent, leukopenia may occur, though of modest severity, even with higher doses than used in 
this trial.  Modest elevation of hepatic enzymes and increased risk of infection may occur. 
 
2.4.5.6 Guidelines for dose adjustment  
 
Myelosuppression:  If the ANC is less than 1000/L, pentostatin dosing should be reduced by 
50%.  If the ANC is less than 500/L, the drug should be discontinued until neutrophil recovery 
occurs.  Use of filgrastim and/or discontinuation of other myelosuppressive agents is allowed. 
 
Renal Insufficiency:  Pentostatin dose reductions for renal insufficiency are made based on 
estimated creatinine clearance (Table 2.3.1).  For clearance of 30-50 mL per minute, the dose 
should be reduced to 50% or 0.75 mg/m2 IV in the same schedule.  The drug should not be used 
if clearance is less than 30 mL per minute. 
 
Hepatic Insufficiency:  Dose reduction is not required for liver dysfunction.   
 


BMT CLINICAL TRIALS NETWORK 
 
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2-12 
2.4.6 Corticosteroids (prednisone or methylprednisolone) 
 
 All patients will receive methylprednisolone 2 mg/kg/day IV (or prednisone  
2.5 mg/kg/day PO) divided in 2-3 daily doses for 7 days.  
 
 Patients unable to tolerate oral fluids of at least 500 mL/day (200 mL/m2) must be treated 
with IV steroids for at least the first 3 days to ensure bioavailability. 
 
 For responding patients between Day 8 and Day 28, the dose of methylprednisolone must 
be at least 0.6 mg/kg/day (0.75 mg/kg/day prednisone).  
 
 Clinical judgment should guide the pace of the steroid taper between Day 8 and Day 28 
and a suggested taper schedule for responding patients (whose GVHD improves in at 
least one organ by at least one stage and without worsening in any other organ) is shown 
 
 For patients that have not showed improvement in GVHD within the first week, the taper 
can be slower than the recommended rate, but should not be faster. 
 
 After Day 28, the steroid dose is determined by the clinical judgment of the treating 
physician. 
 
2.4.6.1 Steroid taper 
 
Suggested taper for responders 
(methylprednisolone IV)  
 
2 mg/kg/day divided in 2-3 doses Days 0-6 
2 mg/kg/day once daily Days 7-13 
1.5 mg/kg/day Days 14-21 
1.0 mg/kg/day Days 22-28 
0.5 mg/kg/day Days 29-35 
0.4 mg/kg/day Days 36-42 
0.3 mg/kg/day Days 43-49 
0.2 mg/kg/day Days 50-56 
0.1 mg/kg/day Days 57-63 
0.1 mg/kg every other day Days 64-69 
Discontinue on Day 70 
 
Suggested taper for responders 
(prednisone orally) 
 
2.5 mg/kg/day divided 2-3 doses Days 0-6 
2.5 mg/kg/day once daily Days 7-13 
2 mg/kg/day Days 14-21 
1.4 mg/kg/day Days 22-28 
0.75 mg/kg/day Days 29-35 
0.6 mg/kg/day Days 36-42 
0.4 mg/kg/day Days 43-49 
0.25 mg/kg/day Days 50-56 
0.1 mg/kg/day Days 57-63 
0.1 mg/kg every other day Days 64-69  
Discontinue on Day 70 
 
 


BMT CLINICAL TRIALS NETWORK 
 
Acute GVHD Protocol – 0302 
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2-13 
2.4.7 Cyclosporine or Tacrolimus 
 
Cyclosporine or tacrolimus should be continued at therapeutic doses adjusted as necessary for 
renal, central nervous system (CNS) or other toxicity using conventional management 
guidelines.  The recommended therapeutic trough level of cyclosporine is 200-400 ng/mL 
measured by whole blood HPLC; institutional levels for other assays.  The recommended 
therapeutic trough level for tacrolimus is 5-15 ng/mL.   
 
2.4.8 GVHD Progression or Non-response 
 
If acute GVHD progresses (new organ involvement or increased organ specific symptoms 
sufficient to increase the organ stage by one or more) within 7 days or there is no response (no 
reduction in any GVHD organ staging) within 14 days, the patient may be treated with 
alternative secondary GVHD therapy and/or taken off assigned study therapy.  Patient must still 
be followed for all study endpoints. 
 
2.4.9 GVHD Complete Response or Partial Response 
 
Complete response at Day 28 is the primary endpoint evaluation for the trial.  Patients with 
complete response will continue corticosteroids according to the taper above.  Etanercept, 
ONTAK, or pentostatin will not be further administered.  MMF (as per Section 2.4.3 above) will 
be continued through the duration of prednisone taper and then tapered over 4 weeks and 
stopped. 
 
Patients not in CR at Day 28 will not have further study drug supplied (with the exception of 
MMF), but may continue assigned study drug or alternative therapy at the investigator or 
institutional discretion.  Patients must still be followed for all study endpoints. 
 
2.4.10 Acute GVHD Flare 
 
Patients who have achieved CR at Day 28 and who have subsequent flare of acute GVHD 
(recurrence of GVHD signs or symptoms) can be treated with the same or alternative agents at 
the investigator/institutional discretion.  Alternatively they can have a modest increment of 
corticosteroid dosing (recommended increase is prednisone 0.25 mg/kg/day x 7 days) and 
resume a taper if a response is observed. 
 


BMT CLINICAL TRIALS NETWORK 
 
Acute GVHD Protocol – 0302 
Version 7.0 dated August 20, 2007 
 
2-14 
2.4.11 Supportive Care 
 
In addition to prescribed study drug plus corticosteroids, all patients should receive the 
following: 
 Transfusion support per institutional practice 
 Anti-infective prophylaxis or preemptive therapy directed towards: CMV, gram positive 
(encapsulated) bacteria, pneumocystis carinii and fungal infections.  Patients enrolled on 
BMT CTN Protocol #0101 (Fungal Prophylaxis) should continue on their assigned study 
drug. 
 
2.5 
GVHD Scoring 
 
Weekly GVHD organ stage scores, overall clinical grade, biopsy information for GVHD and 
relevant differential diagnosis will be recorded and reported to the DCC.  Organ involvement, 
biopsy information, staging, differential diagnosis, and GVHD therapy will be documented in the 
medical record using the BMT CTN GVHD scoring stamp or equivalent. 
 
Symptoms of chronic GVHD, if present, will be reported according to the CTN MOP (Chapter 
2) and reported on the GVHD symptom record. 
 
Patients receiving alternative GVHD therapy (progression, PR or no CR at Day 28) should still 
have weekly scoring performed through week 8 and follow-up for all study endpoints. 
 


BMT CLINICAL TRIALS NETWORK 
 
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Version 7.0 dated August 20, 2007 
 
2-15 
2.5.1 GVHD Scoring 
 
An example acute GVHD weekly data record (stamp) is shown below. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2.5.2 Chronic GVHD  
 
Patients developing sign/symptoms of chronic GVHD (CGVHD) will have symptoms recorded 
on the CGVHD scoring form at the scheduled follow-up visits. 
 
2.5.3 Studies of GVHD Biology  
 
Laboratory studies to investigate biologic parameters of response and differential response to the 
four study drugs will be included in this clinical trial. 
 Skin biopsies (pre and on Day 28 of study therapy) for clinical diagnostic pathology, 
immunopathology, and other investigations (Appendix C-4) are to be performed. 
 Immunophenotyping: Cytokine assays to correlate with GVHD response and cytokine 
gene polymorphisms will be examined (Appendix C-1). 
 Proteomics (Appendix C-6) of sera collected before and after therapy will be performed. 
 
Clinical Acute GVHD Assessment 
 
Date  ___________ 
Patient ID  ____________________________________         Karnofsky/Lansky _______ 
 
    
CODES 
 
 
 
 
 DIFFERENTIAL DIAGNOSIS 
 
0 
1 
2 
3 
4 
5 
 
 GVHD 
Drug 
 Rxn 
 Cond 
  Reg 
TPN 
Infect 
VOD 
Other 
Skin 
     
 
% body rash:____ 
      
   
 
 
Lower GI 
      Vol: ___________ 
          
 
 
Upper GI 
  
 
 
 
 
 
          
 
 
Liver 
     
 
Max bili: ________ 
            
 
 
Treatment: 
 
CSA 
 Tacrolimus 
 Pred 
 Methylpred 
 Ontak 
 
 
Pentostatin 
 MMF 
 Etanercept 
 Other _______________________ 
 
Code Definitions: 
Skin: 
0  No rash 
1  Maculopapular rash,  
    < 25% of body surface 
2  Maculopapular rash,  
    25-50% of body surface 
3  Generalized erythroderma 
4  Generalized erythroderma with bullous 
  formation and desquamation 
Lower GI (Diarrhea): 
0  None 
1  ≤ 500 mL/day or < 280 mL/m2 
2  501-1000 mL/day or 280- 555 mL/m2 
3  1001-1500 mL/day or 556- 833 mL/m2 
4  > 1500 mL/day or > 833 mL/m2 
5   Severe abdominal pain with or without 
ileus, or stool with frank blood or melena 
Upper GI: 
  0 No protracted 
nausea and vomiting 
  1 Persistent nausea, 
vomiting or anorexia 
Liver (Bilirubin): 
0  < 2.0 mg/dl 
1  2.1-3.0 mg/dl 
2  3.1-6.0 mg/dl  
3  6.1-15.0 mg/dl 
4  > 15.1 mg/dl 
 
Signature __________________________________________ 


BMT CLINICAL TRIALS NETWORK 
 
Acute GVHD Protocol – 0302 
Version 7.0 dated August 20, 2007 
 
2-16 
Definite and Possible Manifestations of Chronic GVHD 
Organ System 
Definite manifestations of chronic GVHD 
Possible manifestations of chronic GVHD 
Skin 
Scleroderma (superficial or fasciitis), lichen 
planus, vitiligo, scarring alopecia, 
hyperkeratosis pilaris, contractures from skin 
immobility, nail bed dysplasia 
Eczematoid rash, dry skin, maculopapular 
rash, hyperpigmentation, hair loss 
Mucous 
membranes 
Lichen planus, non-infectious ulcers, corneal 
erosions/non-infectious conjunctivitis 
Xerostomia, keratoconjunctivitis sicca  
GI tract 
Esophageal strictures, steatorrhea 
Anorexia, malabsorption, weight loss, 
diarrhea, abdominal pain 
Liver 
None 
Elevation of alkaline phosphatase, 
transaminitis, cholangitis, 
hyperbilirubinemia 
GU 
Vaginal stricture, lichen planus 
Non-infectious vaginitis, vaginal atrophy 
Musculoskeletal/ 
Serosa 
Non-septic arthritis, myositis, myasthenia, 
polyserositis, contractures from joint 
immobilization 
Arthralgia 
Hematologic 
None 
Thrombocytopenia, eosinophilia, 
autoimmune cytopenias 
Lung 
Bronchiolitis obliterans  
Bronchiolitis obliterans with organizing 
pneumonia, interstitial pneumonitis 
From BMT CTN MOP Chapter 2 
 
 


BMT CLINICAL TRIALS NETWORK 
 
Acute GVHD Protocol – 0302 
Version 7.0 dated August 20, 2007 
 
3-1 
 
CHAPTER 3 
 
 
3 
STUDY ENDPOINTS 
 
3.1 
Primary Endpoint 
 
The primary endpoint is proportion of CR at Day 28 of therapy.  CR is defined as resolution of 
all signs and symptoms of GVHD in all evaluable organs in comparison to Day 1 scoring.  
For a response to be scored as CR at Day 28 or later, the participant must still be in CR on that 
day and have had no intervening salvage therapy for an earlier progression, PR or NR. 
 
3.1.1 Staging and Grading of Acute GVHD 
 
Staging* 
 
Stage 0 
Stage 1 
Stage 2 
Stage 3 
Stage 4 
Skin 
No rash 
Rash < 25% 
BSA 
25-50% 
> 50% 
Generalized 
erythroderma 
Plus bullae and 
desquamation 
Gut 
< 500 mL 
diarrhea/day 
501-1000 
mL/day 
1001–1500 
mL/day 
> 1500 
mL/day 
Severe 
abdominal pain 
& ileus 
UGI 
 
Severe 
nausea/vomiting 
 
 
 
Liver 
Bilirubin 
≤  2 mg/dl 
2.1-3 mg/dl 
3.1-6mg/dl 
6.1-15mg/dl 
> 15 mg/dl 
 
 
Grading Index of Acute GVHD*  
 
Grade A 
Grade B 
Grade C 
Grade D 
Skin 
1 
2 
3 
4 
Gut 
0 
1-2 
3 
4 
Upper GI 
0 
1 
 
 
Liver 
0 
1-2 
3 
4 
 
*[See Appendix A, Endnote 28] 
 


BMT CLINICAL TRIALS NETWORK 
 
Acute GVHD Protocol – 0302 
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3-2 
3.2 
Secondary Endpoints 
 
3.2.1 Proportion of Partial Response (PR), Mixed Response (MR), No Response (NR) and 
Progression 
 
Partial response (PR) is defined as improvement in one or more organs involved with 
GVHD symptoms without progression in others.  For a response to be scored as PR at Day 28 
or later, the participant must still be in PR on that day and have had no intervening salvage 
therapy for an earlier progression, PR or no response (NR).  Mixed response (MR) is defined as 
improvement in one or more organs with deterioration in another organ manifesting 
symptoms of GVHD or development of symptoms of GVHD in a new organ.  Progression is 
defined as deterioration in at least one organ without any improvement in others.  No 
response (NR) is defined as absence of any improvement or progression as defined.  Scoring 
of PR, MR, NR and progression are in comparison to the participant’s GVHD status (score) on 
Day 0 of the study. 
 
3.2.2 Proportion of Primary Treatment Failures 
 
No response, progression, administration of additional therapy for GVHD, or mortality by Day 
14 post-initiation of treatment will be considered a primary treatment failure. 
 
3.2.3 GVHD Flares  
 
Flares are defined as any increase in symptoms of or therapy for acute GVHD after an initial 
response (i.e., progression from an earlier CR or PR).  The rate of flares prior to Day 90 will be 
examined. 
 
3.2.4 Immunosuppression Discontinuation 
 
Immunosuppression discontinuation will be assessed by Day 90, Day 180 and Day 270.  It is 
defined as the discontinuation of corticosteroids and all additional immunosuppressives, except 
cyclosporine or tacrolimus, for treatment of acute GVHD without subsequent flare by Day 90 
post-initiation of therapy and later by discontinuation of all immunosuppressive medications, 
including cyclosporine or tacrolimus.  Note that discontinuation of MMF will be 4 weeks later 
than other study drugs even in patients with CR. 
 
3.2.5 Chronic GVHD 
 
Chronic GVHD is defined per the BMT CTN Manual of Procedures (MOP) Chapter 2.  The 
incidence of chronic GVHD at 9 months will be computed for each treatment arm. 
 
3.2.6 Survival 
 
Overall survival at Days 180 and 270 will be computed for each treatment arm. 
 


BMT CLINICAL TRIALS NETWORK 
 
Acute GVHD Protocol – 0302 
Version 7.0 dated August 20, 2007 
 
3-3 
3.2.7 Systemic Infections  
 
All microbiologically documented infections occurring within three months of initiation of 
therapy will be reported by site of disease, date of onset, and severity.  For definitions see the 
BMT CTN MOP.  Post-transplant lymphoproliferative disorder will be reported through 9 
months post-initiation of therapy. 
 
 


BMT CLINICAL TRIALS NETWORK 
 
Acute GVHD Protocol – 0302 
Version 7.0 dated August 20, 2007 
 
4-1 
 
CHAPTER 4 
 
 
4 
PATIENT ENROLLMENT AND EVALUATION 
 
4.1 
Enrollment and Randomization 
 
Patients will be registered using the BMT CTN Advantage Electronic Data Capture (EDC).  The 
following procedures shall be followed: 
1. An authorized user at the clinical center completes the initial screening by entering 
patient demographics and Segment A information (inclusion/exclusion criteria) of the 
Eligibility Form within 48 hours of diagnosis of acute GVHD. 
2. If the patient is eligible, a study number and random treatment assignment is generated. 
3. A visit schedule based on treatment start date is displayed for printing. 
 
If a connection is interrupted during a randomization session, the process is completely canceled 
and logged.  A backup manual registration and randomization system will also be available to 
provide for short-term system failure or unavailability. 
 
4.1.1 Randomization  
 
Patients will be randomized within 48 hours of diagnosis of acute GVHD.  Patients who have 
received MMF for GVHD prophylaxis, within 7 days of GVHD onset, will be randomized in a 
1:1:1 ratio to receive ONTAK, pentostatin or etanercept.  Patients who have not received MMF 
for GVHD prophylaxis will be randomized in a 2:1:1:1 ratio (MMF and the other three arms 
respectively) to one of the four treatment arms.  Treatment should be initiated as soon as possible 
after randomization.  A maximum of 48 hours is allowable. 
 
No stratification factors will be used.  Strata that were considered included: Grade B versus 
Grades C/D, age, unrelated donor, single organ versus multi-organ GVHD, full versus non-
ablative conditioning, GVHD prophylaxis with a calcineurin inhibitor.  However, none of these 
consistently identify GVHD patient cohorts with different CR rates.  Therefore, it was decided 
that no stratification factors would be used in this trial. 
 
4.2 
Study Monitoring 
 
4.2.1 Follow-up Schedule 
 
The Follow-up Schedule for scheduled study visits is outlined in Table 4.2.1.  A detailed 
description of each of the forms and the procedures required for forms completion and 
submission can be found in the Data Management Handbook and User’s Guide. 
 
Follow-up Assessments: The timing of follow-up visits is based on the date of randomization.  
Following randomization, the Transplant Center can print a Patient Visit Schedule listing target 


BMT CLINICAL TRIALS NETWORK 
 
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4-2 
dates for assessments.  Weeks 1-8 visits are primarily for acute GVHD scoring.  The subsequent 
visits are for follow-up reports. 
 
Criteria for Forms Submission: Criteria for timeliness of submission for all study forms are 
detailed in the Data Management Handbook and User’s Guide.  Forms that are not received at 
the DCC within the specified time will be considered delinquent.  Transplant Centers can view 
past due forms via the Web-based data entry system.  A missing form will continue to appear 
until the form is entered into the DCC’s master database, or until an exception is granted and 
entered into the Missing Form Exception File, as detailed in the Data Management Handbook 
and User’s Guide. 
 
Reporting Patient Deaths: The Recipient Death Information must be entered into the web-
based data entry system within 24 hours of knowledge of a patient’s death.  If the cause of death 
is unknown at that time, it need not be recorded at that time.  However, once the cause of death is 
determined, the form must be updated. 
 
CIBMTR Data Reporting: All transplant centers participating in BMT CTN protocols are 
required to register all of their transplant patients, not just those enrolled on BMT CTN 
protocols, with the CIBMTR, using standard CIBMTR Preregistration and Transplant Essential 
Data Forms (instructions available at www.ibmtr.org).  CIBMTR Day 100 Report Forms 
(including the Core, Graft and Disease Inserts) and CIBMTR Follow-up Forms (including the 
Core and Disease Inserts) are required for patients enrolled on BMT CTN protocols and will be 
submitted directly to the CIBMTR at the times specified in the Data Management Handbook and 
User’s Guide.   
 


BMT CLINICAL TRIALS NETWORK 
 
Acute GVHD Protocol – 0302 
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4-3 
Table 4.2.1 
FOLLOW-UP SCHEDULE 
 
Assessment Time 
Target Day1 
(Days Post-Enrollment) 
1 week 
7 days 
2 weeks 
14 days 
3 weeks 
21 days 
4 weeks 
28 days 
5 weeks 
35 days 
6 weeks 
42 days 
7 weeks 
49 days 
8 weeks 
56 days 
90 days 
90 days 
120 days 
120 days 
6 months 
180 days 
9 months 
270 days 
 
1.Target day range = 2 days up to Week 8, and 14 days for 
Days 90, 120, and 28 days for 6 and 9 months, post-
randomization. 
 
 
4.2.2 Assessments  
 
All assessments are considered standard-of-care unless identified below by “*.” 
 
Pre-Randomization    
1. Recommended biopsy of involved tissue* 
2. History and physical exam including height and weight 
3. Pregnancy test (if applicable) 
4. Complete acute GVHD staging and grading information including assessments of rash, 
diarrhea, nausea/vomiting, weight and liver function tests 
5. CBC with differential, platelet count 
6. Liver function tests (bilirubin, alkaline phosphatase, AST, ALT) plus creatinine 
7. Tacrolimus/cyclosporine level 
8. Samples for laboratory studies (see Appendix C)* 
Post-Randomization  
1. Recommended skin biopsy if skin involved on Day 28 (see Appendix C-4)* 


BMT CLINICAL TRIALS NETWORK 
 
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4-4 
2. Karnofsky or Lansky performance status on Days 28, 90, 180 and 270 
3. Complete acute GVHD staging and grading information including assessments of rash, 
diarrhea, nausea/vomiting, weight and liver function tests weekly until Day 56, and 3, 4, 
6 and 9 months, creatinine weekly until Day 28, and recommended creatinine on Days 
35, 42, 49, 56, and 3, 4, 6, and 9 months. 
4. Chronic GVHD evaluation (if present) 3, 4, 6 and 9 months 
5. CBC with differential, platelet count weekly until Day 56 
6. Toxicity evaluation weekly until Day 56 
7. Steroid dose weekly until Day 56, and 3, 4, 6 and 9 months 
8. Tacrolimus/cyclosporine levels weekly until Day 28 
9. MMF levels in weeks 1 and 2 (4 levels at time 0, 1, 2, 6 hours on oral MMF; 0, 2, 4, 6 
hours if on IV MMF) (see Appendix C-5)* 
10. Samples for laboratory studies on Days 0, 14, 28 and 90 (see Appendix C)* 
11. Infections through Day 90 and occurrence of Post-transplant Lymphoproliferative 
Disorder (PTLD) through Day 270. 
 
4.2.3 Weekly GVHD Monitoring  
 
GVHD scoring will be performed weekly for 8 weeks from study entry.  Days 1, 28 and 56 (+7 
days) scoring must be performed by direct observation at the Transplant Center. 
 
4.2.4 Serious Adverse Event (SAE) Reporting 
 
Serious Adverse Events will be consistent with BMT CTN procedures (BMT CTN 
Administrative Manual of Procedures, Chapter 6) and reported through an expedited Adverse 
Event (AE) reporting system.  Serious and unexpected Grades 3-5 adverse events should be 
reported within three working days.  Deaths are to be reported within 24 hours.  Other SAEs will 
be tracked periodically as defined in the Form Submission Schedule, and staged according to 
NCI Common Terminology Criteria for Adverse Events (CTCAE), Version 3.0.   
 
The Data and Safety Monitoring Board will receive summary reports of all Grade 3-5 
unexpected adverse experiences on an annual basis. 
 
 


BMT CLINICAL TRIALS NETWORK 
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Version 7.0 dated August 20, 2007 
 
4-5 
Table 4.2.2  REQUIRED ASSESSMENTS 
 
 
 
(Day 0) 
Days Post Randomization 
 
Baseline  
7 
14 
21 
28 
35 
42 
49 
56 
90 
120 
180 
270 
Suggested biopsy of involved tissue (+ extra skin biopsy 
Days 1 & 281) 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
History and physical exam 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy test (if applicable) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Karnofsky/Lansky performance status 
 
 
 
 
X 
 
 
 
 
X 
 
X 
X 
Acute GVHD evaluation 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Chronic GVHD evaluation 
 
 
 
 
 
 
 
 
X 
X 
X 
X 
X 
CBC with differential, platelet count 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
Basic chemistry (creatinine) 
X 
X 
X 
X 
X 
X2 
X2 
X2 
X2 
X2 
X2 
X2 
X2 
Liver function tests (alkaline phosphatase, bilirubin, AST, 
ALT) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Toxicity evaluation 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
Steroid dose 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Tacrolimus/cyclosporine levels 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
MMF levels (see Appendix C-51) 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
Blood and serum for ancillary laboratory studies (see 
Appendix C1) 
X 
 
X 
 
X 
 
 
 
 
X 
 
 
 
1 Research procedures beyond that required for usual care. 
2 Recommended 
 
 


BMT CLINICAL TRIALS NETWORK 
Acute GVHD Protocol – 0302 
Version 7.0 dated August 20, 2007 
5-1 
 
CHAPTER 5 
 
 
5 
STATISTICAL CONSIDERATIONS 
 
5.1 
Study Design Synopsis 
 
This study is a multi-center, randomized two-stage Phase II trial designed to evaluate several 
frontline treatments for acute GVHD.  After 20 patients per treatment are evaluable (stage I), a 
stopping rule for lack of efficacy will be applied, so that future patients may be randomized to 
the most promising treatments.  The remaining patients will be randomized in stage II to the 
treatments passing through stage I, to a maximum of 65 patients per arm or 180 patients total. 
 
5.2 
Accrual 
 
It is estimated that nine months of accrual, or approximately 20 patients per month, will be 
necessary to enroll the targeted sample size.  Only Core Centers will enroll patients on this study.  
 
5.3 
Randomization 
 
Randomization will occur separately in two strata.  Stratum 1 consists of patients who had 
received MMF previously as GVHD prophylaxis and are not eligible to receive MMF again as 
GVHD treatment, while stratum 2 consists of patients who did not receive MMF previously as 
GVHD prophylaxis.  Patients in stratum 1 will be randomized in equal proportions to the other 
three treatment arms.  Patients in stratum 2 will be randomized in unequal proportions to all four 
arms (initially 40% to MMF and 20% to each of the other three arms).  This unequal allocation 
was chosen to target approximately equal sample sizes per arm (45) by the end of the study, 
assuming 33% of patients will belong to stratum 1.  Randomization in each stratum will occur 
using two sets of permuted blocks of varying sizes.  One set of permuted blocks will determine 
MMF vs. not MMF for patients in stratum 2, while the other set will determine which of the 
other three arms the patient is assigned to.  After 80 patients have been enrolled and followed up 
for the primary endpoint, two things will be assessed.  First a stopping rule will be applied as 
described below to determine whether any of the arms should be closed for lack of efficacy.  
Second, the proportion of patients entering stratum 1 will be assessed.  If the MMF treatment 
arm is closed for lack of efficacy, then we will revert to equal randomization for any remaining 
treatment arms.  Otherwise, based on the number of non-MMF arms remaining (i.e. not closed 
for lack of efficacy) and the proportion of patients entering stratum 1, we will adjust the 
allocation probability P to MMF treatment in stratum 2 according to the following formula: 
 
P=(k*i+3*(180-i)-4*k*y)/(3*(k+1)*(180-i)*(1-r)), 
 
where r=observed percent of MMF prophylaxis use so far, i=80 is the number accrued, k is the 
number of non-MMF treatment arms remaining, and y is the number assigned to MMF 
treatment.  This formula is obtained by setting the expected final number of patients assuming 
allocation of P in stratum 2 to MMF treatment equal to the expected final number of patients in 


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each of the other treatment arms.  Some examples of the new allocation rates are given in the 
table below, as a function of the observed MMF prophylaxis rate and the number of remaining 
arms.  Also given are the expected sample sizes after 80 patients and after 180 patients in both 
the MMF arm and a representative non-MMF arm. 
 
Evaluated at 
After 80 Patients 
New  
P 
After 180 Patients 
MMF  
Rate, r 
# of non-MMF 
arms remaining, 
k 
#  
MMF 
#  
other trt 
#  
MMF 
#  
other trt 
0.2 
3 
26 
18 
0.243 
45 
45 
0.2 
2 
26 
18 
0.354 
54 
54 
0.2 
1 
26 
18 
0.578 
65 
65 
0.33 
3 
21 
20 
0.352 
45 
45 
0.33 
2 
21 
20 
0.478 
53 
53 
0.33 
1 
21 
20 
0.732 
65 
65 
0.45 
3 
18 
21 
0.498 
45 
45 
0.45 
2 
18 
21 
0.645 
53 
53 
0.45 
1 
18 
21 
0.938 
65 
65 
0.5 
3 
16 
21 
0.580 
45 
45 
0.5 
2 
16 
21 
0.738 
53 
53 
0.5 
1 
16 
21 
1.000 
60 
65 
 
 
5.4 
Primary Endpoint 
 
The primary endpoint for the trial is the proportion of complete response to acute GVHD therapy 
at Day 28. 
 
5.5 
Primary Hypothesis 
 
Each arm will be compared to the historical rate of 35% CR at Day 28 of therapy.  This will be 
done by computing a “Bayesian posterior probability of efficacy,” i.e., the Bayesian posterior 
probability that the response rate is > 35%, using a Beta (3.5, 6.5) prior distribution.  This prior 
has a mean of 35% and a “weight” of 10 patients.  If this posterior probability of efficacy is  
> 90%, the treatment is considered a candidate for further investigation based on the primary 
endpoint.  No statistical comparisons between the arms will be performed: the purpose of the 
trial is only to identify treatments for further investigation through comparisons to a historical 
control of corticosteroids alone and is underpowered for comparisons among the arms. 
 
5.6 
Study Design 
 
Stage I: Randomize patients in two strata as described above until 80 patients are evaluable for 
the primary endpoint.  After 80 patients are evaluable, apply the following stopping rule for lack 
of efficacy: if the Bayesian posterior probability of efficacy for a particular arm is < 20%, close 
the arm to further accrual.  This Bayesian stopping rule is equivalent to stopping if the number of 
patients alive in CR at Day 28 is less than or equal to 4 out of 20. 


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Stage II: Continue randomizing patients after modifying the randomization scheme as described 
above.  If the MMF treatment arm is dropped for lack of efficacy, then the randomization will be 
done equally among all remaining treatment arms.  If not, then the randomization probabilities 
will be recomputed according to the rate of MMF prophylaxis use in the first 80 patients as well 
as the number of remaining non-MMF treatment arms that passed successfully through stage I.  
This randomization will be done up until a maximum of 65 per group or 180 patients have been 
accrued.  At the end of the study, a treatment identifying a good candidate will be based on the 
primary endpoint if the Bayesian posterior probability of efficacy is > 90%.   
 
This Bayesian rule corresponds to a cutoff in terms of numbers of patients alive in CR at Day 28, 
which depends on the final enrolled sample size and therefore the number of treatment arms 
passing through stage I.  Sample cutoffs are given in Table 5.6 below.   
 
Table 5.6 
CUTOFFS FOR PATIENTS ALIVE IN CR AT DAY 28 
 
Number of Treatments 
Passing through Stage I 
Final n per 
Treatment Group 
Declare Treatment a Good 
Candidate if # of CRs is ≥ x 
1 
65 
29 
2 
65 
29 
3 
52 
24 
4 
45 
21 
 
 
5.7 
Power and Operating Characteristics of Study Design 
 
This design, including the stopping rules for lack of efficacy as well as stopping for mortality 
discussed in Section 5.8, was simulated for several configurations of true success rates, which 
represent improvements over the historical rate of 0%, 10%, 15%, and 20%.  Table 5.7 below 
gives the configurations of true CR rates considered, with bold entries denoting effective 
treatments relative to the historical control rate of 35%.  Note that because there are multiple 
decisions being performed on the various treatments, there is no simple definition of power or 
type I error.  Therefore, for each configuration of success rates, several measures of power and 
type I error are presented which are appropriate for a design with multiple arms.  Also note that 
these operating characteristics apply for the situation when the true proportion r in stratum 2 is 
33%.  Deviations from this will have minimal impact on the operating characteristics because the 
final sample sizes are similar regardless of r. 
 
First we report the marginal probability of identifying treatment arm 1 as a good candidate.  This 
can be interpreted as the power for arm 1 individually, if arm 1 is effective, and the type I error 
rate for arm 1 individually if arm 1 is not effective.  Next we report the probability that all 
effective treatments are identified as good candidates and no ineffective treatments are identified, 
which can be interpreted as a “perfect” decision on all treatments.  Third, we present the 
probability that at least one effective treatment is identified as a good candidate and no 
ineffective treatments are identified.  This can be interpreted as a “perfect” decision on the 
ineffective treatments and a “good” decision on the effective treatments (i.e. we correctly 


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identified at least one good candidate).  Finally, we present the probability that at least one 
ineffective treatment is identified as a good candidate, while none of the effective treatments are 
identified.  This can be interpreted as a type I error and is an undesirable result.  Results are 
based on 10000 simulated datasets for each scenario.  Note that this two-stage design has 
approximately 75% marginal probability or power to detect an improvement of 15% in the CR 
rate over the historical control rate, and more than 90% marginal power to detect a 20% 
improvement.  Also, if there are two or more effective treatments with 50% CR rates, the design 
has at least 80% power to identify at least one of them as a good candidate while not identifying 
any ineffective treatments. 
 
Table 5.7 
POWER AND OPERATING CHARACTERISTICS 
 
True CR Rates  
(Effective arms denoted in bold) 
Probability of Identifying as Good Candidates: 
Arm 1 
Arm 2 
Arm 3 
Arm 4 
Arm 1 
All effective 
treatments and 
no ineffective 
treatments 
At least one 
effective 
treatment and 
no ineffective 
treatments 
No effective 
treatments and 
at least one 
ineffective 
treatment  
0.35 
0.35 
0.35 
0.35 
7% 
- 
- 
27% 
0.45 
0.35 
0.35 
0.35 
49% 
40% 
40% 
10% 
0.50 
0.35 
0.35 
0.35 
75% 
59% 
59% 
5% 
0.55 
0.35 
0.35 
0.35 
91% 
72% 
72% 
2% 
0.50 
0.50 
0.35 
0.35 
74% 
47% 
80% 
1% 
0.50 
0.50 
0.50 
0.35 
72% 
35% 
91% 
0% 
0.50 
0.50 
0.50 
0.50 
72% 
27% 
99% 
- 
 
Frequentist confidence intervals (CI) will also be constructed for the CR rate at Day 28.  For a 
treatment which is declared a good candidate (≥ 29/65 or 24/52 or 21/45 CRs), a 90% CI will 
have a lower bound of at least 34% and a 95% CI will have a lower bound of at least 32%. 
 
5.8 
Stopping Guidelines 
 
At the end of stage I, lack of efficacy stopping guidelines will be implemented separately for 
each arm.  If the Bayesian posterior probability of efficacy for a particular arm is < 20%, it will 
be recommended that the arm be closed to further accrual.  For lack of efficacy, an arm will be 
closed if the posterior probability is ≤ 20% that the CR rate at Day 28 is greater than 35%.  This 
is equivalent to stopping for lack of efficacy if 4 or fewer of the first 20 patients are CRs.  Once 
the stopping criteria have been met for a particular arm, no more patients will be randomized to 
that arm.   
 
After 20 patients have been accrued, this stopping rule for lack of efficacy results in the 
following stopping probabilities as a function of the true CR rate. 
 


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Table 5.8 
STOPPING GUIDELINES 
 
N  
Evaluable 
Stop if # of 
CRs are 
Probability of Stopping for Lack of Efficacy 
CR=15% 
CR=25% 
CR=35% 
CR=45% 
20 
≤ 4 
83% 
42% 
12% 
2% 
 
5.8.1 Stopping Guidelines for Mortality 
 
Mortality of 25% at Day 28 of therapy would be considered unexpected and an unacceptable 
number of excess deaths compared to the anticipated and historical rate of approximately 10%.   
 
Therefore, a stopping rule for Day 28 mortality will be applied after every 10 patients are 
accrued per group.  A particular arm will close at interim look t if the number of deaths on that 
arm is >= xt., e.g., if four or more deaths in the first 10 patients or six or more deaths in the first 
20 patients, etc. as detailed in Table 5.8.1 below.  Looking after every 10 patients per group 
corresponds approximately to looking at mortality every 2 months starting with the third month 
(i.e. after months 3, 5, 7, 9), assuming an accrual rate of 20 patients/month.  If one or more arms 
should close after stage I, we will review the mortality data slightly more frequently. 
 
The stopping rules and probabilities of stopping at each interim look are given in the table below.  
Note that the final sample size per arm is variable due to the stopping rule for lack of efficacy; 
therefore, the cumulative stopping probabilities are also given for cumulative sample sizes of 40 
and 60.  In summary, a treatment with a 10% mortality rate, which is what is expected based on 
historical data, will have a 2.6% chance of triggering the stopping rule for mortality.  If that 
mortality rate for a treatment under investigation should increase to 25%, a 15% increase in 
mortality over the historical rate, we will have a 65.2% chance of triggering the stopping rule 
within 40 patients and a 76.8% chance of triggering the stopping rule within 65 patients. 
 


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Table 5.8.1 
STOPPING PROBABILITIES FOR VARIOUS MORTALITY RATES AT DAY 28  
(Stopping rule is defined as stop if the number of deaths at look t is ≥ xt) 
 
 
Look 
(t) 
 
Total 
N 
 
xt 
Probabilities of Stopping at Look t for True Mortality Rates of  
p = 0.1 
0.15 
0.2 
0.25 
0.3 
1 
10 
4 
1.4% 
5.3% 
12.7% 
22.8% 
35.2% 
2 
20 
6 
0.7% 
4.2% 
11.1% 
20.4% 
26.4% 
3 
30 
8 
0.3% 
2.7% 
7.5% 
13.2% 
15.0% 
4 
40 
10 
0.2% 
1.8% 
5.8% 
8.8% 
9.0% 
5 
50 
12 
0.0% 
1.6% 
4.7% 
6.5% 
5.4% 
6 
60 
14 
0.0% 
1.0% 
3.8% 
5.1% 
3.2% 
Cumulative probability of 
stopping after 40 patients 
2.6% 
14.0% 
37.1% 
65.2% 
85.6% 
Cumulative probability of 
stopping after 60 patients 
2.6% 
16.6% 
45.6% 
76.8% 
94.2% 
 
If lack of efficacy or mortality rates significantly exceed pre-set thresholds, the NHLBI will be 
notified in order that the DSMB can be advised.  Policies and composition of the DSMB are 
described in the BMT CTN’s Manual of Procedures. 
 
5.9 
Demographic and Baseline Characteristics 
 
Demographics and baseline characteristics will be summarized for all patients.  Between group 
comparisons will be performed for continuous variables via a t-test and for categorical variables, 
via the chi-square test.  Covariates considered will include age (recipient and donor), sex, type of 
donor (related versus unrelated), graft source (marrow versus peripheral blood versus umbilical 
cord blood), HLA match/mismatch, GVHD Grades B versus C/D at enrollment, single versus 
multiorgan GVHD, skin GI, liver involvement, CMV seropositivity in recipient or donor, use of 
CSA versus tacrolimus prophylaxis, and day of onset of GVHD. 
 
5.10 Endpoints and Analysis Plan 
 
The primary endpoint is the proportion of CR at Day 28.   
 
The secondary endpoints are: 
 Proportion of PR, mixed response, no response, and progression at Day 28 
 Proportion of treatment failures by Day 14 
 Incidence of GVHD flares before Day 90 
 Incidence of discontinuing immune suppression without flare by Days 90, 180 and 270 of 
therapy 
 Incidence of chronic GVHD within 9 months 
 Survival at 6 and 9 months after initiation of therapy 


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 Incidence of systemic infections within 3 months of therapy 
 Incidence of EBV lymphoma within 9 months of therapy 
 
In addition to the estimation of the Bayesian posterior probabilities as discussed above, estimates 
and 95% confidence intervals will be constructed for all endpoints, separately for each study 
group.  These may include point estimates, Kaplan-Meier survival curves, or cumulative 
incidence curves depending on the secondary endpoint of interest.  Acceptance of a particular 
study agent as sufficiently promising for future Phase III testing will include efficacy in the 
primary study endpoint as well as the secondary endpoints.  Toxicities, secondary infections and 
9-month survival may be of particular importance in assessing the therapeutic index for each 
study agent. 


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APPENDIX A 
 
REFERENCES 


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APPENDIX A 
 
REFERENCES 
 
 
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21. Chiang KY, Abhyankar S, Bridges K, Godder K, Henslee-Downey JP. Recombinant human 
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33. Eugui EM, Mirkovich A, Allison AC.  Lymphocyte-selective antiproliferative and 
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B-1 
 
 
 
 
APPENDIX B 
 
INFORMED CONSENTS 
AND 
EDUCATIONAL MATERIAL 
 
 
 
INFORMED CONSENT TO PARTICIPATE IN RESEARCH 
 
ASSENT TO PARTICIPATE IN RESEARCH 
 
CLINICAL INFORMATION FOR RESEARCH PROTOCOL 
 
 
 


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Participant Consent 
Page 1 of 10 
 
 
Informed Consent to Participate in Research 
 
 
 
 
 
 
 
 
 
We invite you to participate in this research study.  About 180 patients will participate at up to 
27 centers around the country.  Your study participation will last about 9 months.  This is a study 
for patients who have a new diagnosis of graft-versus-host disease (GVHD) after receiving an 
allogeneic (donor) stem cell transplant.   
 
GVHD is a medical condition where the donor cells attack and damage your tissues after you 
have a transplant.  GVHD can cause:  
 Skin rashes,  
 Intestinal problems like feeling sick to your stomach (nausea), throwing up (vomiting), 
diarrhea, or,  
 Liver damage like hepatitis or jaundice.   
 
GVHD can also increase your risk of infection.  Sometimes, GVHD can be controlled with 
treatments that use corticosteroids (like prednisone).  More often, patients need long-term drug 
therapy to control their GVHD symptoms and to suppress the immune system.  Long-term drug 
therapy has risks and side effects.  Because of the risks of continued GVHD, new drugs to 
control GVHD will be tested in this study.   
 
All four drugs being tested in this study are approved by the US Food and Drug Administration 
(FDA) for treating diseases other than GVHD.  This study will test whether these new drugs can 
help treat GVHD better than the standard therapy of steroids alone.  One of the study drugs is 
given either orally, or if you cannot take pills, then it will be given by IV infusion.  The other 3 
study drugs are given either by IV or by injection under the skin. 
 
This consent form tells you about the study.  The study investigators have found that you are 
eligible to participate in this study, and this form must be signed before any treatment related to 
the study is given to you.  The doctors in charge of this study (the investigators) or other staff 
will also discuss this study with you and answer any questions you might have.  Before you 
decide to join this study, please read this information and ask any questions about things you do 
not understand.  Some patients find it helpful to have a family member or friend with them to 
help ask questions and listen to information.   
 


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This study will give more information to doctors about future treatment choices for GVHD.  
Importantly, 
 You will not be paid to be in this study.   
 You or your insurance company will pay for all medical bills for your treatment. 
 You will not be charged for research tests. 
 You will also face the same risks and benefits as any other transplant patient. 
 
Before you decide to join the study, please read the information below.  Feel free to ask 
questions to understand your rights and protections.  It is your choice to take part in this study.  
You and your doctor will discuss other treatment options if you decide not to be in this 
study. 
 
Your Name:  
 
 
 
 
 
 
 
 
 
 
 
 
Title of Research Study: Initial Systemic Treatment of Acute GVHD: A Phase II Randomized 
Trial Evaluating Etanercept, Mycophenolate Mofetil (MMF), ONTAK and Pentostatin in 
addition to Corticosteroids 
 
Principal Investigator:  Daniel Weisdorf, M.D., University of Minnesota, MMC 480, 
Minneapolis, MN  55455, 612-624-3101 
 
Transplant Center Principal Investigator: _______________ 
 
Study Sponsor:  This study is sponsored by the National Institutes of Health (NIH) by providing 
financial support for this study through the Blood and Marrow Transplant Clinical Trials 
Network (BMT CTN).  
 
The drugs in this study were donated by the companies that made them, and these companies 
also gave some financial support to help pay the costs of this study.  These companies did not 
plan or design this study.  In addition, they will not have a part in analyzing the results of this 
study. 
 
The Purpose of the Study 
 
The study is to test new treatments for acute Graft-versus-Host Disease (GVHD).  GVHD is a 
medical condition where the donor graft attacks and damages your tissues after you have a 
transplant.    If you have limited GVHD of the skin or only GVHD in your upper gastrointestinal 
(GI) tract, your doctor may decide that you need systemic treatment, though not all patients 
receive systemic treatment for this type of GVHD.  By offering you this study, your doctor 
recommends that you get additional treatment. 
 
Four new drugs are being tested: 
 Etanercept (ENBREL®) 
 Mycophenolate Mofetil (MMF) (CellCept®) 
 Denileukin Diftitox (ONTAK®) 
 Pentostatin (Nipent®) 


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In this study, use of one of these four drugs, along with prednisone or methylprednisolone, may 
increase the control of your GVHD.  It is uncertain whether use of any of these new drugs will 
improve control of GVHD. 
 
Each drug will be given along with the standard therapy of prednisone or methylprednisolone 
which is the usual therapy for acute GVHD.  There is no placebo or sugar pill treatment in this 
trial.  Everyone in the study will receive prednisone or methylprednisolone plus one of the four 
new drugs, unless you are already receiving MMF; then only one of the other 3 drugs will be 
assigned to you.  All patients in the study will be randomly assigned (like flipping a coin) to 
receive one of the four new study drugs with some statistical adjustment to ensure that about the 
same number of patients will receive each of the new study drugs. 
 
What will be done if you take part in this study? 
In addition to receiving prednisone plus the assigned study drug, for your routine care (outside 
this study), you will be watched closely for signs and symptoms of:  
 GVHD,   
 Changes in your blood counts,  
 Changes in liver and kidney function, and  
 Any signs of infection.   
 
For the study, the GVHD signs and symptoms will be recorded at least once a week for the first 8 
weeks.  The exam at week 4 and week 8 must be done at the transplant center.  Other weekly 
exams and tests may be done at the transplant center, or at a local doctor’s office if your 
condition improves and it is closer to where you live.  
 
If you are randomly chosen to receive MMF, you will have additional blood samples drawn to 
study the actions of this drug in your body.  You will have 5 mL (about 1 teaspoon) of blood 
drawn at 4 different times (total of 20 mL) on one day between Days 3 and 7 and again on 
another day between Days 10 and 14 (for a total of 40 mL).  This blood will be drawn from an 
existing central venous catheter or a temporary peripheral venous catheter. 
 
 
STUDY DRUGS 
 
What are the possible discomforts and risks? 
 
Infections 
 
Because GVHD is caused by an immune attack on your tissues from the transplanted donor cells, 
all treatments for GVHD include drugs to suppress (turn off) this immune attack. 
 
The risk of infection is increased in patients with GVHD and those taking immune suppressing 
corticosteroids like prednisone or methylprednisolone, the standard therapy for GVHD.  All four 
study drugs can also increase your chance of infection.  Therefore, you will take several 
protective antibiotics and be watched carefully for any infections while you are being treated for 


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GVHD.  Tell your doctors promptly if you get a fever, chills, a cough or any other symptoms 
that might be part of an infection. 
 
Side Effects of Study Drugs 
 
All drugs can have side effects, both the standard therapy (steroids) and the new drugs being 
tested in this study.  Your doctors will watch you carefully for any side effects and will modify 
your treatment if they develop.  Experience with pediatric patients treated with these agents for 
GVHD is limited. 
 
Etanercept (ENBREL):  Etanercept is an anti-inflammatory and immune suppressive drug.   
 
Etanercept can cause certain side effects.  Some patients develop redness or soreness at the 
injection sites.  Sometimes headache, dizziness, or rash can occur.  A few people develop fever, 
upper respiratory tract symptoms (like a cold with cough, stuffy nose or sinusitis) and rarely 
some serious infections have developed in people taking etanercept.   Rarely, blood counts can 
be lowered in people taking etanercept. 
 
In this study, the drug will be given by injection under the skin (subcutaneously) twice weekly 
for up to four weeks. 
 
Mycophenolate Mofetil (MMF) (CellCept):  MMF is a potent immunosuppressive drug that 
blocks the growth of lymphocytes (immune cells), which can cause GVHD.  
 
MMF can cause certain side effects.  Occasionally it can lower the blood counts.  More 
frequently, it can cause nausea, vomiting or diarrhea.  Rarely, serious gut injury can occur. 
 
In this study, it is given twice daily as capsules or a liquid.  It may be continued for 8-10 weeks 
or as long as GVHD is active. 
 
Denileukin Diftitox (ONTAK):  ONTAK is an immune suppressive drug, but has also been 
used to treat certain kinds of lymphocyte cancers called T cell lymphomas. 
 
ONTAK can cause certain side effects.  Most patients develop a headache, skin rash, fever, 
chills, muscle aches, and joint aches while the infusion is given or within a day of each infusion.  
Dyspnea (shortness of breath), chest tightness, and flushing of the skin may occur.  Slowing the 
infusion may minimize these side effects.  Occasionally, acute allergic-like reactions with 
itching, temperature, chills, or a rash can develop.  These side effects may be relieved by 
temporarily stopping and then restarting the infusion and giving medications before the dose of 
ONTAK like acetaminophen (Tylenol) or diphenhydramine (Benadryl) to try to prevent these 
reactions. 
 
In a few patients, ONTAK can cause fluid accumulation (edema).  This can lead to swelling in 
the tissues, legs, or rarely fluid in the lungs or low blood pressure.  If given in higher doses than 
in this study, anorexia (loss of appetite), upset stomach with nausea, vomiting or diarrhea can 
occur.  Rarely, liver problems or low blood counts can develop. 


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In addition, new information has suggested that rarely, vision changes or vision loss can occur in 
patients treated with ONTAK. 
 
In this study, ONTAK will be given intravenously (over approximately 60 minutes) Days 1, 3, 5 
of the first week and repeated in the third week (Days 15, 17, 19) of your treatment. 
 
Pentostatin (Nipent):  Pentostatin is an immune suppressive drug that is sometimes used as an 
anticancer chemotherapy drug as well.   
 
Pentostatin can cause certain side effects.  Up to half of the patients develop skin rash, nausea, 
vomiting, anorexia (loss of appetite), mouth sores, or diarrhea, although these side effects usually 
occur when patients are given higher dosages of the drug than you will receive on this study.  
Rarely, patients have kidney trouble.  In a few patients treated with higher dosages of 
pentostatin, low platelet counts, anemia, or increased need for transfusions have developed.  
Rarely, low white blood cell counts can occur.  A small number of patients also develop 
temporary abnormalities in their liver function blood tests. 
 
In this study, pentostatin will be given intravenously (over 15-30 minutes) on Days 1, 2, 3, of the 
first week and repeated in the third week on Days 15, 16 and 17 of your treatment. 
 
Voluntary Participation 
 
You are free to choose to participate or decline participation now or at any time in your treatment 
course.  If you decline to participate now or withdraw your consent after treatment is started, 
standard GVHD therapy will be available to you.  None of your rights are waived by enrolling in 
this research study and therapy for any medical conditions will continue even if you withdraw 
from the study.  Your doctors have made themselves available to answer any of your questions 
about participation in this study and will continue to do so throughout your treatment. 
Throughout the study, the researchers will tell you of new information that might affect your 
decision to remain in the study.  If you have additional questions about the study or about your 
rights as a participant in this study, you may contact [the Principal Investigator at your transplant 
center].  If you have any questions or concerns, please ask your doctors or the study 
coordinators. 
 
You can be taken off the study (with or without your consent) for any of the following reasons: 
 You do not qualify to be in the study because you do not meet the study requirements.  Ask 
your doctor if you would like more information about this. 
 You need a medical treatment not allowed in this study. 
 The investigator decides that continuing in the study would be harmful to you. 
 The study treatments have a bad effect on you. 
 You become pregnant and the study treatment could be harmful to the fetus. 
 You are unable to keep appointments or take study drugs as directed. 
 Other study-specific reasons; for example, if the dose of study drug you are taking has been 
found to be unsafe. 


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 The study is cancelled by the Food and Drug Administration (FDA) or the National 
Institutes of Health (NIH). 
 
Benefits 
 
Although this study cannot be guaranteed to be of benefit to you, it is hoped that your taking part 
may help in the treatment of your GVHD.  A possible advantage of this study is that one of the 
study drugs may treat GVHD better than the others.  However, you may not benefit from this 
treatment.  It is also hoped that the information learned from this study will help your doctors 
treat patients in the future who get GVHD. 
 
Compensation 
 
In the event that this research activity results in an injury, treatment will be available including 
first aid, emergency treatment and follow-up care as needed.  Care for such injuries will be billed 
in the ordinary manner, to your insurance company.  If you think you have suffered a research 
related injury, let the study physicians know right away.  Unexpected side effects or accidents 
might result in your getting sicker than anticipated in the course of this treatment.  All available 
medical care will be provided to you, but you and your insurance company (3rd party payer) are 
responsible for the costs of all such care. 
 
Confidentiality 
 
All necessary steps will be undertaken to avoid your being identified in any public presentations.  
However, the results of this study treatment may be published in scientific journals in the future, 
but no one patient (including you) will be identified.  Information concerning your transplant 
course may be reviewed or transmitted to national and international transplant registries, 
including the Center for International Blood and Marrow Transplant Research (CIBMTR), 
Autologous Blood and Marrow Transplant Registry (ABMTR), the National Marrow Donor 
Program (NMDP), to the Food and Drug and Administration (FDA), Data Coordinating Center 
of the National Institutes of Health (NIH), Blood and Marrow Transplant Clinical Trials Network 
(BMT CTN), and to other authorized study organizations.  However, you will not be identified 
by name in publications or reports coming from such groups or review. 
 
Information related to or resulting from your stem cell transplant will be reported to the 
CIBMTR.  The CIBMTR is a voluntary organization of basic and clinical scientists working 
together in an effort to gather information on results of stem cell and marrow transplants.  This 
information is used to guide clinical decisions and identify ways to improve transplant outcomes.  
Scientific data or medical information (not identifiable with you) that could be useful to others 
may be presented at meetings and/or published in medical journals. 
 
You will be given a copy of this form. 
 


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HIPAA1 authorization to use and disclose individual health information for research 
purposes 
 
a. Purpose:  As a research participant, I authorize the Principal Investigator and the 
researcher’s staff to use and disclose my individual health information for the purpose of 
conducting the research study entitled Initial Systemic Treatment of Acute GVHD: A 
Phase II Randomized Trial Evaluating Etanercept, Mycophenolate Mofetil (MMF), 
Denileukin Diftitox (ONTAK), and Pentostatin in Combination with Corticosteroids 
 
b. Individual Health Information to be Used or Disclosed: My individual health information 
that may be used or disclosed to conduct this research includes: demographic information 
(e.g., age, date of birth, sex, weight), medical history (e.g., diagnosis, complications with 
prior treatment), physical examination findings, and laboratory test results obtained at the 
time of work up and after treatment (e.g., blood tests, biopsy results).  
 
c. Parties Who May Disclose My Individual Health Information: The researcher and the 
researcher’s staff may obtain my individual health information from: 
(list hospitals, clinics or providers from which health care information can be requested) 
 
______________________________________________________________________ 
 
______________________________________________________________________ 
 
______________________________________________________________________ 
 
d. Parties Who May Receive or Use My Individual Health Information: The individual 
health information disclosed by parties listed in item c and information disclosed by me 
during the course of the research may be received and used by the following parties: 
 
Principal Investigator and the researcher’s staff 
 
Dr. Dan Weisdorf, Study Chairperson and staff/laboratories at University of 
Minnesota  
 
Staff/laboratories identified in the protocol for the evaluation of other laboratory 
samples; e.g., Dr. Pamela Jacobson/University of Minnesota and Dr. Brian 
Nickoloff/Loyola University of Chicago 
 
National Heart, Lung, and Blood Institute (NHLBI) and the National Cancer 
Institute (NCI), both of the National Institutes of Health (NIH), study sponsors  
 
Blood and Marrow Transplant Clinical Trials Network (BMT CTN), data 
coordinating center 
 
U.S. government agencies that are responsible for overseeing research such as the 
Food and Drug Administration (FDA) and the Office of Human Research 
Protections (OHRP) 
                                                   
1 HIPAA is the Health Insurance Portability and Accountability Act of 1996, a federal law related to privacy of health 
information 


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 
U.S. government agencies that are responsible for overseeing public health 
concerns such as the Centers for Disease Control (CDC) and federal, state and local 
health departments. 
 
e. Right to Refuse to Sign this Authorization: I do not have to sign this Authorization.  If I 
decide not to sign the Authorization, I will not be allowed to participate in this study or 
receive any research-related treatment that is provided through the study.  However, my 
decision not to sign this authorization will not affect any other treatment, payment, or 
enrollment in health plans or eligibility for benefits.   
 
f. Right to Revoke:  I can change my mind and withdraw this authorization at any time by 
sending a written notice to the Principal Investigator to inform the researcher of my 
decision.  If I withdraw this authorization, the researcher may only use and disclose the 
protected health information already collected for this research study.  No further health 
information about me will be collected by or disclosed to the researcher for this study. 
 
g. Potential for Re-disclosure: My individual health information disclosed under this 
authorization may be subject to re-disclosure outside the research study and no longer 
protected.  Examples include potential disclosures for law enforcement purposes, 
mandated reporting or abuse or neglect, judicial proceedings, health oversight activities 
and public health measures. 
 
h. This authorization does not have an expiration date. 
 
Blood Samples for Research 
You will have tests to measure the effect of GVHD and the drugs on your immune system. 
 
For these tests you will need to give a blood sample (3-5 teaspoonsful) at Days 0, 14 and 28 of 
the study, and 1 teaspoonful on Day 90.  If your GVHD comes back, you will also need to give 1 
teaspoonful of blood. 
 
Additionally, to understand how certain genes may affect the development of GVHD in you, 
some genes that affect inflammation hormones (cytokines) will be studied in your blood or in a 
swab of the tissues in your mouth.   
 
Skin Samples for Research 
GVHD can cause a skin rash.  To study how GVHD may affect your skin and the genes in your 
skin cells, a small skin sample (skin biopsy) will be taken before you start treatment and another 
28 days after you start treatment for your GVHD.  The skin samples are taken from an area 
where you currently have a rash.  Each sample is a 1/8” (one-eighth of an inch) circle of skin. 
 
These skin samples are not required for your treatment, but will be used to study what happens to 
your skin during treatment.  This may better explain how acute GVHD injures the skin cells and 
the effect of treatment by the different study drugs. 
 


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The samples collected for research purposes will be sent to laboratories that have contracts with 
the National Marrow Donor Program (NMDP) to conduct these research tests.  They will be 
labeled with unique codes that do not contain information that could identify you.  A link to this 
code does exist.  The link is stored at the Data Coordinating Center for the Blood and Marrow 
Transplant Clinical Trials Network (BMT CTN).  The staff at the laboratories where your 
samples are being tested do not have a link to this code.  Your samples will be stored at these 
laboratories until the entire sample has been used for the research tests or until the end of the 
study. 
 
If any of your samples are leftover after the research studies are completed, these samples will 
either be destroyed or be sent to the National Heart Lung and Blood Institute (NHLBI) sample 
repository in Maryland.  If your leftover samples are sent to the repository, they will be given an 
anonymous code.  These leftover samples stored at the repository can never be linked to you.  
Any research performed on these leftover samples must first be approved by an advisory panel at 
the NHLBI. 
 
Research Samples are Optional 
It is your choice to volunteer to give these extra blood and skin samples.  Your blood samples 
and skin biopsy will be collected confidentially (your name will not be attached to them).  Only 
the study doctors or personnel working with them will study your skin and blood samples. 
 
If you agree to allow your blood and skin samples to be used for research, you can change your 
mind later.  If you do, please contact [the Principal Investigator at your transplant center] in 
writing to state that you are withdrawing permission for your blood or your skin biopsy to be 
used for research.  His mailing address is on the first page of this consent form.  Any unused 
blood or biopsy tissue will be destroyed if you withdraw your permission.    
 
If you choose not to participate in this additional research there will be no change in your care.   
 
Please indicate your choice(s) below: 
 
Blood Samples 
 
_____ I give permission to use additional samples of my blood for GVHD-related research. 
 
_____  No, I do not wish samples of my blood to be used for additional research. 
 
Skin Samples 
 
_____  I give permission to use additional sample of my skin for GVHD-related research. 
 
_____  No, I do not wish samples of my skin to be used for additional research. 
 
Patient's Signature:  
 
 
 
 
 
 
 
 
 
 
Date:  
 
 
 
 
 
 
 
 
 
 
 


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Statement of Consent 
 
I consent to participate in this study treatment plan for GVHD including the use of prednisone 
plus one of four study drugs that will be assigned to me in random fashion.  I have asked 
questions that I have now and have received answers.  I know that I can ask additional questions 
in the future. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Signature 
 
 
 
 
 
 
     Date 
 
 
 
I have explained in full the details of this study treatment plan for acute GVHD and answered as 
best as possible all questions that were asked.   
 
 
 
 
 
 
 
 
                                                                              
    
Signature of Counseling Physician 
 
 
 
      Date 


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Assent Consent 
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Assent to Participate in Research (Ages 6 to 17 years old) 
 
 
 
 
 
 
 
1. Title of Research Study 
Initial Systemic Treatment of Acute GVHD: A Phase II Randomized Trial Evaluating Etanercept, 
Mycophenolate Mofetil (MMF), ONTAK and Pentostatin in addition to Corticosteroids 
 
2. Purpose of the Study 
After your donor transplant, you have developed a new problem called graft-vs-host disease (GVHD).  
The donor cells are attacking your tissues and can cause skin, stomach, or liver trouble.  Your doctors 
will give you standard drugs to treat GVHD (like prednisone), but want to test whether one of four 
new drugs can treat GVHD even better.  The four drugs (etanercept, ONTAK, MMF, or pentostatin) 
all can treat some diseases.  Some patients with GVHD have gotten better using these drugs. 
 
Your doctors want you to join a study where you will get only one of these drugs in addition to the 
standard one, prednisone.  Your doctors will watch you carefully and check to see if your GVHD is 
improving or if any of the drugs are causing new problems. 
 
These treatments may cause infections, so your doctors will watch you closely for fever or chills.  If 
you feel sicker, tell your doctors. 
 
If you have any questions don’t hesitate to ask your doctors and tell them whether you understand 
their answers. 
 
Your parents (or a guardian) are also asked for their permission for you to join this treatment study. 
 
 
I agree to be in the GVHD study. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Patient’s Signature 
 
 
 
 
 
Date 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Doctor’s Signature 
 
 
 
 
 
Date 
 


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Educational Materials 
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CLINICAL INFORMATION FOR RESEARCH PROTOCOL  
(Appropriate for Ages 6 – 17) 
 
After your transplant, you have developed a new problem called graft vs. host disease (GVHD).  
The donor cells are attacking your tissues and can cause skin, stomach, or liver trouble.  Your 
doctors will give you standard drugs to treat GVHD (prednisone), but want to test whether 
adding one of four new drugs can treat GVHD even better.  The four drugs (etanercept, ONTAK, 
MMF or pentostatin) can treat some diseases, but have not been well studied for patients with 
GVHD.  Some patients with GVHD have gotten better using these drugs.  If you join the study, 
you will receive prednisone, a medicine given to all patients with GVHD.  In addition, the 
doctors would randomly choose (like flipping a four-sided coin) which of the four study drugs 
you will receive, unless you are already receiving MMF; then it would be like a 3-sided coin 
assigning you one of the other three new drugs. 
 
The first drug, etanercept, is given in a shot under the skin twice a week for four weeks.  
Sometimes the skin spot where the shot was given can get red or sore and sometimes people get 
headaches or dizzy. 
 
The second drug, MMF, is given twice daily as pills or a liquid.  If you can’t take any pills it can 
be given through a vein (IV) or your Hickman catheter.  It sometimes causes stomach upset or 
diarrhea and sometimes lowers blood counts. 
 
The third drug, ONTAK, is given through your IV or through your Hickman catheter three days 
a week the 1st and 3rd week of treatment.  Sometimes people get a headache, skin rash, fever, or 
chills.  Sometimes people have some breathing trouble or swelling of their feet. 
 
The fourth drug, pentostatin, is given through you IV on Days 1, 2, 3, and 15, 16, 17 of your 
GVHD treatment.  It can cause a rash, stomach trouble, or diarrhea.  It can lower blood counts in 
some people. 
 
You will only receive one of the study drugs along with prednisone.  Your doctors will watch 
you carefully for any side effects and to see how well your GVHD is responding to the treatment.  
Any of these drugs can increase your chance of getting an infection, so your doctors will watch 
you carefully for fever or other problems as well. 
 
Any time you want, you can ask questions of your doctors or your parents.  Make sure you 
understand their answers and ask more questions if you don’t. 
 
While you are treated for GVHD, you will have exams and blood tests to check how well you are 
doing.  If you have a rash, the doctors may want a small skin biopsy (numbing the skin and 
taking a 1/8” circle of skin) to look at under the microscope.  This will be used to study how the 
skin GVHD is changing. 
 
The doctors want to improve treatments for GVHD and hope that this study will identify new 
drugs that are a better treatment than prednisone alone.  
 


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C-1 
 
 
 
 
APPENDIX C 
 
LABORATORY PROCEDURES 
 


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C-2 
 
APPENDIX C 
 
LABORATORY PROCEDURES 
 
 
ANCILLARY LABORATORY STUDIES 
Additional laboratory testing will be performed to characterize the immunologic responses to the 
various study agents and these are listed below. 
 
 
C-1. 
IMMUNOPHENOTYPING 
 
Lymphocyte phenotyping: at baseline (before therapy), Day 14 and Day 28 of study treatment.  
Peripheral blood lymphocyte phenotyping will be performed including, but not limited to, 
enumeration of CD3, 4, 8, 25, 69, 20, CD4+25+. 
 
The lymphocyte phenotyping will be performed on freshly drawn cells at the transplant center 
and recorded as cells of each subset phenotype per mL of blood. 
 
 
C-2. 
CYTOKINE ANALYSIS 
 
Serum for cytokines IL-2, TNF, IFN-gamma will be collected on Days 0, 14 and 28 of therapy.  
Additionally, serum samples will be analyzed by mass spectroscopy for proteomics analysis.  A 
7mL peripheral blood sample will be collected in a red top tube, spun, aliquoted into three  
1.8 mL cryovials and frozen at -20° C at the transplant center and then batch shipped quarterly 
on dry ice to the BMT CTN NIH sample repository for distribution and assay. 
 
 
C-3. 
CYTOKINE GENE POLYMORPHISM 
 
A 3 mL peripheral blood sample (for DNA) will be collected in an ACD-A anticoagulant- 
containing tube on Day 0 to analyze cytokine gene polymorphisms.  This sample will represent 
donor DNA.  To assess recipient cytokine genes, if available, pre-transplant recipient blood 
collected in an ACD-A anticoagulant-containing tube or an EDTA tube for DNA will be used or 
alternatively, a buccal swab will be collected for recipient DNA. 
 
Blood will be aliquoted into two 1.8 ml cryovials and frozen at -20° C.  The buccal swab should 
be allowed to dry at room temperature.  Remove the tip of the swab and store in a 1.8 mL 
cryovial.  Freeze cryovial at 20° C.  Samples will be batch shipped quarterly on dry ice to the 
BMT CTN NIH sample repository. 
 


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C-3 
 
C-4. 
SKIN BIOPSY -- IMMUNOPATHOLOGY AND GENE EXPRESSION 
 
It is recommended that a skin biopsy be collected on Day 0 and Day 28 for pathology, 
immunopathology for lymphoid cell infiltration, immunochemical analysis and gene expression 
profiling.   
 
There are four aims in analyzing the skin biopsies obtained through this protocol: 
(1) To identify the nature of the dermal infiltrate and examine the effects of the different 
treatments on the composition of the infiltrate. 
(2) To analyze the endothelial adhesion molecules and chemokines expressed in the skin and 
determine how the different treatments impact these molecular mediators of lymphocyte 
trafficking. 
(3) To examine the deposition and molecular structure of hyaluronic acid in GVHD in the 
skin and how different treatments modify the hyaluronic acid deposits. 
(4) To compare the level of gene expression of immune response-associated gene products 
(e.g., cytokines) and characterize the changes in expression among different treatments 
within different cells present in skin (e.g., endothelial cells, infiltrating cells, 
keratinocytes, etc.). 
 
Aims (1) and (2) will require immunohistochemistry of fixed sections using antibodies to subset-
specific markers to define the infiltrating cells and to adhesion molecules and chemokines.  Aims 
(3) and (4) will require frozen tissue, from which sections will be cut for hyaluronic acid studies 
(Aim 3) or for laser capture dissection of discrete cells within the skin followed by gene 
expression profiling by arrays (Aim 4). 
 
Sample Handling Requirements: 
At the time of clinical diagnosis of acute cutaneous GVHD (prior to initiating any therapy), two 
4 mm punch biopsies will be obtained.  One biopsy will be placed in buffered formalin for 
routine histopathology per local institutional practice.  The second biopsy will be snap frozen in 
liquid nitrogen and sent for research studies.  The freezing step will be performed as follows: 
Note: The skin biopsy should be snap frozen immediately.  If this is not possible, the skin 
sample must be transported from the clinic on a saline soaked gauze pad and snap frozen 
within 60 minutes of the procedure. 
(1) The skin sample is placed in a 1.8 mL cryovial and the cryovial closed 
(2) The cryovial is then immersed vertically into liquid nitrogen 
(3) Once frozen in liquid nitrogen, the sample must be immediately transferred to a -80ºC 
freezer for long-term storage.  For handling of the cryovial, it is imperative that only the 
very bottom of the tube or the very top of the cap is handled (i.e, do not touch the sides of 
the cylinder), so as to never transfer heat to the skin sample.  All transfers between 
freezers must be performed quickly, to prevent thermal warming of the skin sample.   
 
Skin biopsies are obtained again on Day 28 and handled precisely as described above. 
 


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The frozen skin samples will be batch-shipped quarterly on dry ice by overnight delivery to the 
laboratory of Dr. Brian Nickoloff at Loyola University of Chicago. 
 
 
C-5. 
MYCOPHENOLATE PHARMACOKINETICS 
 
Added sampling for MMF and metabolite concentration and clearance calculations will be 
conducted during weeks 1 (between Days 3-7) and week 2 (Days 10-14) for patients assigned to 
receive MMF.  Sampling will be done at hour 0 (pre-dose), 1, 2 and 6 hours for patients 
receiving oral MMF and hour 0, 2, 4 and 6 hours for patients receiving IV (infusion over 2 
hours) MMF.   
 
Pharmacokinetic Methods: 
Pharmacokinetics will be studied twice in each subject following the initiation of MMF and 
corticosteroids for the treatment of acute GVHD.  Patients will be sampled at steady-state once in 
week 1 (between Days 3-7) and again in week 2 (between Days 10-14) of MMF treatment.  
Steady-state is defined as a minimum of 4 identical mycophenolate mofetil doses given 12 hours 
apart. 
 
Blood samples will be obtained at times 0, 1, 2, 6 hours following the administration of the oral 
dose and at 0, 2, 4 and 6 hours after the start of the IV infusion if receiving IV MMF.  
Intravenous MMF must be administered at a constant rate over 2 hours.  Five mL of blood will 
be drawn at each time point from an existing central venous catheter or a temporary peripheral 
venous catheter.   
 
Serum albumin, serum creatinine and total bilirubin will be measured, height and weight, 
medical history, and a detailed list of concomitant medications will be obtained on the day of 
each pharmacokinetic sampling.  The pharmacokinetic form (included in the shipping box) will 
be completed for each pharmacokinetic sampling period. 
 
MPA and MPAG Plasma Sample Processing and Storage: 
 
Blood samples (5 mL) will be collected in a 10 mL purple top tube containing EDTA 
anticoagulant and placed on wet ice.  Optimally, samples will be processed within 30 minutes of 
withdrawal, however samples are stable for up to 4 hours at room temperature or 8 hours if 
refrigerated at 1-4 degrees C.  Samples should be prepared by centrifugation at 3400 rpm x 10 
minutes, plasma removed and divided equally between two 5 mL screw top plastic tubes and 
then frozen at -20 degrees C.  Samples should be stored frozen at -20 degrees C and then batch 
shipped after the completion of week 1 and 2 for each patient, frozen on dry ice in the provided 
shipping container to the University of Minnesota. 
 
Total MPA and MPAG, and unbound MPA will be measured with high-pressure liquid 
chromatography methods.  Total MPA and MPAG concentrations are simultaneously measured.  
Unbound MPA concentrations are measured separately.  The total MPA standard curve is 
prepared over the concentrations of 0.01 to 10 mcg/mL.  MPAG standard curve is 1-100 


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mcg/mL.  The unbound MPA standard curve is prepared over the concentrations of 0.0025 to  
0.1 mcg/mL.  Inter and intraday coefficients of variation are < 10% for both assays. 
 
Data Analysis: 
Exposure measures will be determined from four blood samples from which a 12-hour AUC will 
be calculated using the limited sampling model developed from data in a previous 
pharmacokinetic trial [40].  The primary exposure measures of interest are total and unbound 
MPA area under the curve (AUC), total and unbound MPA Cmax and Cmin, and free fraction.  
For each patient, an AUC will be calculated for each sampling period and will be used to 
investigate the interpatient variability, as measured by standard statistics such as standard 
deviation and coefficient of variation (CV).  Graphs and correlations will be used to examine the 
distribution of values and bivariate relationships.  The relationships between exposure measures 
and individual characteristics (age, gender, diagnosis, serum albumin, renal and hepatic function, 
site and grade of GVHD, height, weight, BSA) will be examined in order to identify potentially 
important variables that determine exposure.  Intrapatient variability will be expressed by 
comparing the change in total exposure measures between the two sampling times.  Clinical 
covariates (serum creatinine and albumin, total bilirubin, grade and site of GVHD at time of 
sampling) will be assessed for their relationship to changes in pharmacokinetic parameters.  
Exploratory analyses will be conducted to evaluate the relationship between acute GVHD 
resolution and MPA exposure.  
 
If MMF is given by IV, it will be infused over two hours.  Since the infusion pump must be shut 
off during pharmacokinetic sampling, the pump infusion rate should be changed accordingly to 
ensure complete delivery of dose over two hours.  The line should be completely flushed at the 
end of the infusion to ensure administration of the entire dose.  Mycophenolate samples may be 
drawn from either a peripheral or central line.  If samples are drawn from the central line, 
infusion pumps on all lumens must be shut off during the draw and a waste drawn that is 
equivalent to the volume of catheter.  For intravenous pharmacokinetics, blood will be drawn 
through a lumen is not being used for mycophenolate infusion.  This will avoid contamination of 
sample.  If possible, no medications or IV fluids should be co-infused with mycophenolate.   
 
 
C-6. 
PROTEIN PROFILING 
 
Serum specimens will be obtained at the time of study entry (Day 0) and then again at Day 14, 
Day 28 and Day 90 or at the time of GVHD recurrence.  These samples will be analyzed by mass 
spectrometry and time of flight analysis to determine if there are protein signatures associated 
with the onset GVHD and response to therapy. 
 
The goals for the proteonomic analysis are: 
1. To determine whether specific, predictable protein signatures in the serum or urine of 
patients experiencing acute GVHD 
2. Identify whether response to treatment correlates with changes in serum protein 
signatures 


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3. To investigate whether proteomonic signatures at the time of recurrent GVHD are similar 
to that of the initial episode of GVHD 
4. To determine whether protein signatures present after the start of therapy can predict 
treatment failure 
5. To use proteonomic analysis to identify changes in novel proteins that may be involved in 
the pathogenesis of GVHD and are predictive for the onset of acute GVHD 
 
Sample Acquisition Times: 
Samples should be obtained at the below stated times: 
 Study enrollment (Day 0) 
 Day 14 
 Day 28 
 At the time of disease flare 
 After steroid taper (Day 90) 
 
Sample Handling Requirements: 
 
On Days 0, 14, and 28, the collection and processing of the samples is the same as that for the 
cytokine analysis sample.  There is no need to collect this sample separately on Days 0, 14, 
and 28. 
 
On Day 90 or at time of GVHD recurrence, collect 5 mL peripheral blood in a red top tube, 
allow sample to clot for 30 minutes, then centrifuge at 900 x g or 2100 RPM.  Extract serum 
carefully without disturbing the clot.  Transfer and divide serum equally into two 1.8 mL 
cryovials and freeze at -20° C.  Batch ship quarterly to the BMT CTN NIH sample repository, on 
dry ice. 


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SCHEDULE OF LABORATORY EVALUATIONS 
 
Type of Sample 
Method 
Type of Storage 
Dates Samples 
Obtained 
Shipping 
Specifications 
Test 
Location 
Immunophenotyping 
Peripheral blood lymphocyte 
phenotyping, including CD3, 4, 
8, 25, 69, 20, CD4+ 25+ on 
freshly drawn cells. 
According to 
institutional 
practice 
Day 0, Day 14 and 
Day 28 
N/A 
Transplant 
Center 
Cytokine Analysis and 
Protein Profiling 
7 mL peripheral blood sample 
(10 mL red top tube) placed 
upright and allowed to clot for 
approximately 30 minutes at 
room temperature, centrifuge at 
2100 RPM1.  Extract serum 
carefully without disturbing clot.  
Transfer and divide serum into 
three 1.8 mL cryovials. 
Frozen at -20C 
Day 0, Day 14 and 
Day 28  
Shipped quarterly 
frozen on dry ice in a 
provided shipping 
container, FedEx 
priority overnight to 
the BMT CTN NIH 
sample repository 
Dr. Susan 
Hsu’s 
Laboratory, 
ARC Penn 
Jersey 
Protein Profiling  
5 mL peripheral blood sample 
(10 mL red top tube) placed 
upright and allowed to clot for 
approximately 30 minutes at 
room temperature, centrifuge at 
2100 RPM1.  Extract serum 
carefully without disturbing clot.  
Transfer and divide serum into 
two 1.8 mL cryovials. 
Frozen at -20C 
Day 90 and at the time 
of GVHD recurrence 
 
 
Shipped quarterly 
frozen on dry ice in a 
provided shipping 
container, FedEx 
priority overnight to 
the BMT CTN NIH 
sample repository 
Dr. Gary 
Nelsestuen’s 
Laboratory, 
University of 
Minnesota 
Note: 
1If necessary, samples can be stored at 2-8ºC overnight and centrifuged the next day.  However, same day processing is preferred. 


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Type of Sample 
Method 
Type of Storage 
Dates Samples 
Obtained 
Shipping 
Specifications 
Test 
Location 
Cytokine Gene 
Polymorphism 
3 mL peripheral blood sample 
(ACD-A anticoagulant yellow 
top tube or EDTA purple top 
tube) aliquoted into two 1.8 mL 
cryovials.  
Buccal Swab allowed to dry at 
room temperature and transferred 
into a 1.8 mL cryovial. 
Frozen at -20C 
Pre-transplant (if blood 
available) and Day 0.  
Buccal Swab if pre-
transplant blood not 
available 
Shipped quarterly 
frozen on dry ice in a 
provided shipping 
container, FedEx 
priority overnight to 
the BMT CTN NIH 
sample repository 
Dr. John 
Hansen’s 
Laboratory, 
Fred 
Hutchinson 
Cancer 
Research 
Center 
Skin Biopsy 
Two 4 mm punch biopsies, one 
biopsy in buffered formalin for 
routine histopathology.  The 
second snap frozen in liquid 
nitrogen for research studies. 
Second biopsy 
frozen at -80C  
Day 0 and Day 28 
Shipped quarterly on 
dry ice to Loyola 
University 
Dr. 
Nickloff’s 
Laboratory, 
Loyola 
University 
Pharmacokinetics 
(Patients assigned to 
MMF arm only) 
5 mL of peripheral blood sample 
(10 mL purple top tube 
containing EDTA anticoagulant) 
will be drawn at each timepoint, 
and medical information 
recorded.  Sample placed on wet 
ice, then centrifuge for 10 
minutes at 3400 RPM within 30 
minutes of collection2.  Remove 
plasma and divide equally 
between two 5 mL plastic tubes. 
Frozen at -20C 
Week 1 and Week 2 
(Sampling will be done 
at 0, 1, 2, and 6 hours) 
Shipped after 
completion of week 1 
and week 2 on dry ice 
to University of 
Minnesota 
Dr. Pamala 
Jacobson, 
University of 
Minnesota 
Note: 
2Optimally, samples will be processed within 30 minutes of collection; however, samples are stable for up to 4 hours at room temperature or 8 hours if 
refrigerated at 1-4ºC. 


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APPENDIX D 
 
HUMAN SUBJECTS 
 
 
 
 


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APPENDIX D 
 
HUMAN SUBJECTS 
 
 
Subject consent: Candidates for the study will be identified as described in Chapter 4 of the 
protocol.  The Principal Investigator or his/her designee at each transplant center will contact the 
candidates and enroll them onto the study.  The study coordinator at each center will provide the 
patient with information about the purpose of the study and obtain consent.  The Network will 
provide a template of the consent form to each center.  Each center will customize the template 
according to their local requirements and submit it for review by the local Internal Review Board 
(IRB).  The DCC will verify the adequacy of the consent forms.  Each center must provide 
evidence of IRB approval.  
 
Confidentiality: Confidentiality will be maintained by individual names being masked and 
assigned a patient identifier code.  The code relaying the patient’s identity with the ID code will 
be kept separately at the center.  The ID code will be transmitted to the network.   
 
Participation of women, children, minorities and other populations: Women, children and 
ethnic minorities will be included in this study. 
 
Accrual will be monitored within each center with the expectation that the enrolled patient 
population is representative of the transplanted patient population at each center.  Representation 
will be examined by comparing gender, race, ethnicity and age distributions.  Accrual of 
minority patients will be expected to be in proportion to the number of minority patients 
transplanted at each center.  The DCC and NHLBI will discuss enrollment anomalies with the 
centers.