A Phase III Randomized, Multicenter Trial Comparing Sirolimus/Tacrolimus With Tacrolimus/Methotrexate as Graft-versus-Host Disease (GVHD) Prophylaxis After HLA-Matched, Related Peripheral Blood Stem Cell Transplantation (BMT CTN #0402)
Phase: PHASE3
Conditions: Leukemia, Myelocytic, Acute, Leukemia, Lymphocytic, Acute, Leukemia, Myeloid, Chronic, Myelodysplastic Syndromes
Sponsor: Medical College of Wisconsin

BMT CTN 0402 
A Phase III Randomized, Multicenter Trial 
Comparing Sirolimus/Tacrolimus with 
Tacrolimus/Methotrexate as Graft-Versus-Host 
Disease (GVHD) Prophylaxis After HLA-Matched, 
Related Peripheral Blood Stem Cell 
Transplantation 
NCT00406393 

 
 
 
A Phase III Randomized, Multicenter Trial Comparing  
Sirolimus/Tacrolimus with Tacrolimus/Methotrexate as Graft-
Versus-Host Disease (GVHD) Prophylaxis After HLA-Matched, 
Related Peripheral Blood Stem Cell Transplantation 
 
 
BMT CTN PROTOCOL 0402 
Version 4.0 
 
Study Chairpersons 
Corey Cutler, M.D., M.P.H., F.R.C.P.C.1 
Joseph H. Antin, M.D.1 
 
Protocol Team  
 
James Ferrara, M.D.2 
Stephan Grupp, M.D., Ph.D.3 
Mary Horowitz, M.D., M.S.4 
Laura Johnston, M.D.5 
Brent Logan, Ph.D.4 
Margaret MacMillan, M.D.6 
Philip McCarthy, M.D.7 
Ryotaro Nakamura, M.D.8 
David Porter, M.D.9 
Jennierose D’Elia10 
 
 
Sponsored by the National Institutes of Health 
National Heart, Lung, and Blood Institute 
National Cancer Institute 
 
 
1 
Dana Farber Cancer Institute 
2 
University of Michigan 
3 
Children’s Hospital of Philadelphia 
4 
Center for International Blood and 
Marrow Transplant Research, Medical 
College of Wisconsin 
5 
Stanford University  
6 
University of Minnesota 
7 
Roswell Park Cancer Institute 
8 
City of Hope National Medical Center 
9 
University of Pennsylvania 
10 The EMMES Corporation 
 

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Core Study Participants: 
 Affiliate Study Participants: 
City of Hope National Medical Center 
Dana Farber Cancer Institute 
Duke University Medical Center 
Ohio State University 
Stanford University Medical Center 
University Hospitals of Cleveland 
University of California, San Diego 
University of Florida College of Medicine 
University of Michigan Medical Center 
University of Minnesota 
University of Pennsylvania Cancer Center 
University of Washington, Barnes Jewish Hospital 
 Baylor University Medical Center 
Emory University Medical Center 
Henry Ford Health System 
Hospital Saint-Louis 
Indiana University 
Mayo Clinic 
Roswell Park Cancer Institute 
Texas Transplant Institute 
Virginia Commonwealth University MCV Hospitals 
University of Iowa Hospitals and Clinics 
University of Oklahoma Medical Center 
University of Pittsburgh Cancer Institute 
University of Wisconsin Hospital & Clinics 
 

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GVHD Prophylaxis – Protocol 0402 
 
 
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PROTOCOL SYNOPSIS – BMT CTN PROTOCOL 0402 
 
A Phase III Randomized, Multicenter Trial Comparing  
Sirolimus/Tacrolimus with Tacrolimus/Methotrexate as Graft vs. Host Disease (GVHD) 
Prophylaxis After HLA-Matched, Related Peripheral Blood Stem Cell Transplantation 
 
 
Principal Investigators: 
Corey Cutler, M.D., M.P.H., F.R.C.P.C. 
 
Joseph H. Antin, M.D.  
 
Study Design: 
The study is designed as a Phase III, randomized, open label, 
multicenter, prospective, comparative trial of sirolimus and 
tacrolimus 
versus 
tacrolimus 
and 
methotrexate 
as 
GVHD 
prophylaxis after HLA-matched, related peripheral blood stem cell 
transplantation 
in 
patients 
with 
hematologic 
malignancies.  
Recipients will be stratified by transplant center and will be 
randomized to the sirolimus/tacrolimus or tacrolimus/methotrexate 
arms in a 1:1 ratio. 
 
Primary Objective: 
The primary objective is to compare the rates of 114-day Grades II-
IV acute GVHD free survival between patients in the two study arms 
from the time of randomization using an intent-to-treat analysis. 
 
Secondary Objectives: 
Patients randomized to the two study arms will be compared for the 
following endpoints:  time to neutrophil and platelet engraftment, the 
incidence of Grades III-IV acute GVHD, mucositis severity, time to 
first hospital discharge after transplantation, all infections, CMV 
reactivation and thrombotic microangiopathy incidence and VOD 
during the 100 days post-transplantation, malignant disease relapse 
and 1-year relapse-free and overall survival. 
 
Eligibility Criteria: 
Eligible patients are between 2 and 60 years of age, have acute 
leukemia, myelodysplasia, chronic myeloid leukemia, adequate 
organ function, a serologic (or higher resolution) 6/6 Class I HLA-A 
and B and molecular Class II DRB1 matched sibling donor, and are 
able to give signed informed consent prior to enrollment.   
 
Treatment Description: 
Patients will receive one of two conditioning regimens described in 
the protocol, at the discretion of the transplant physician.  The 
transplant physician must choose among these regimens prior to 
GVHD prophylaxis assignment by randomization.  Conditioning 
regimens will vary by center but at each center will be the same for 
patients randomized to either GVHD prophylaxis regimen.  Stem 
cell donors will donate peripheral blood stem cells according to local 
institutional practices.  Peripheral blood stem cells will not be 
manipulated or T-depleted prior to administration.  Standard post-

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transplant care will be administered.  Patients will be randomized to 
one of two GVHD prophylaxis regimens and will be followed for the 
endpoints of interest. 
 
Accrual Objective: 
Patients who are candidates for transplantation of G-CSF-mobilized 
peripheral blood stem cells from HLA-matched, related donors will 
be targeted for accrual.  Approximately 156 patients will be accrued 
per study arm (total of 312 patients). 
 
Accrual Period: 
The estimated accrual period is three years. 
 
Study Duration: 
Patients will be followed for 114 days post randomization for 
evaluation of the primary endpoint, with additional follow-up to two 
years after transplantation for evaluation of secondary endpoints. 

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TREATMENT SCHEMA 
 
 
 
 
 
Infusion of peripheral blood stem cells 
from 6/6 HLA-matched sibling donor 
Sirolimus + Tacrolimus 
Tacrolimus + Methotrexate 
Randomization continues to 312 total subjects,  
156 per arm. 
Randomization 
Declare Conditioning Regimen 

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TABLE OF CONTENTS 
 
 
1. 
BACKGROUND AND RATIONALE .............................................................................. 1-1 
1.1. 
Allogeneic Stem Cell Transplantation .............................................................................. 1-1 
1.2. 
GVHD Prophylaxis ........................................................................................................... 1-1 
1.3. 
Sirolimus ........................................................................................................................... 1-3 
1.3.1. 
Preliminary Experience with Sirolimus in Stem Cell Transplantation ................................... 1-4 
1.3.1.1. 
Therapy of established GVHD ....................................................................................... 1-4 
1.3.1.2. 
Prophylaxis of GVHD .................................................................................................... 1-4 
2. 
STUDY DESIGN ............................................................................................................... 2-1 
2.1. 
Study Overview ................................................................................................................. 2-1 
2.2. 
Hypothesis and Study Objectives ..................................................................................... 2-1 
2.2.1. 
Primary Hypothesis ............................................................................................................. 2-1 
2.2.2. 
Secondary Hypotheses ........................................................................................................ 2-1 
2.2.3. 
Primary Objective ............................................................................................................... 2-2 
2.2.4. 
Secondary Objectives .......................................................................................................... 2-2 
2.3. 
Patient Eligibility for Randomization .............................................................................. 2-2 
2.3.1. 
Patient Eligibility for Randomization................................................................................... 2-2 
2.3.2. 
Patient Exclusion Criteria .................................................................................................... 2-4 
2.4. 
Conditioning ...................................................................................................................... 2-5 
2.4.1. 
Additional Drugs ................................................................................................................. 2-5 
2.4.2. 
Choice of Conditioning Regimen......................................................................................... 2-5 
2.4.3. 
Order of Administration of TBI and Cyclophosphamide ...................................................... 2-5 
2.4.3.1. 
TBI administration ......................................................................................................... 2-5 
2.4.3.2. 
Cyclophosphamide administration.................................................................................. 2-5 
2.5. 
Stem Cell Transplantation ................................................................................................ 2-6 
2.5.1. 
Stem Cell Collection ........................................................................................................... 2-6 
2.5.2. 
Stem Cell Administration .................................................................................................... 2-6 
2.6. 
GVHD Prophylaxis ........................................................................................................... 2-6 
2.6.1. 
Sirolimus/Tacrolimus .......................................................................................................... 2-7 
2.6.1.1. 
Dosing ........................................................................................................................... 2-7 
2.6.1.2. 
Monitoring ..................................................................................................................... 2-7 
2.6.1.3. 
Target serum levels and dose modifications.................................................................... 2-7 
2.6.1.4. 
Tapering ........................................................................................................................ 2-8 
2.6.2. 
Tacrolimus/Methotrexate..................................................................................................... 2-8 
2.6.2.1. 
Dosing ........................................................................................................................... 2-8 
2.6.2.2. 
Monitoring ..................................................................................................................... 2-8 
2.6.2.3. 
Target serum levels and dose modification ..................................................................... 2-8 
2.6.2.4. 
Tapering ........................................................................................................................ 2-9 
2.7. 
Supportive Care ................................................................................................................ 2-9 
2.7.1. 
Growth Factors ................................................................................................................... 2-9 
2.7.2. 
Blood Products .................................................................................................................... 2-9 
2.7.3. 
Prophylaxis Against Infections ............................................................................................ 2-9 
2.7.4. 
Intravenous Immune Globulin (IVIG)................................................................................ 2-10 
2.7.5. 
Kepivance ......................................................................................................................... 2-10 
2.8. 
Description of Study Drugs............................................................................................. 2-10 

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2.8.1. 
Administration and Storage ............................................................................................... 2-10 
2.8.1.1. 
Sirolimus (Rapamune) ............................................................................................... 2-10 
2.8.1.2. 
Tacrolimus (Prograf) .................................................................................................. 2-11 
2.8.1.3. 
Methotrexate ................................................................................................................ 2-13 
2.8.2. 
Adverse Reactions............................................................................................................. 2-13 
2.8.2.1. 
Sirolimus ..................................................................................................................... 2-13 
2.8.2.2. 
Tacrolimus ................................................................................................................... 2-14 
2.8.2.3. 
Methotrexate ................................................................................................................ 2-14 
2.8.3. 
Drug Interactions .............................................................................................................. 2-14 
2.8.3.1. 
Sirolimus ..................................................................................................................... 2-14 
2.8.3.2. 
Tacrolimus ................................................................................................................... 2-15 
2.8.3.3. 
Methotrexate ................................................................................................................ 2-15 
2.9. 
Management of Toxicities ............................................................................................... 2-16 
2.9.1. 
GVHD .............................................................................................................................. 2-16 
2.9.2. 
VOD ................................................................................................................................. 2-16 
2.9.3. 
Idiopathic Pneumonia Syndrome/Diffuse Alveolar Hemorrhage ........................................ 2-16 
2.9.4. 
Thrombotic Microangiopathy ............................................................................................ 2-17 
2.9.5. 
Hyperlipidemia ................................................................................................................. 2-17 
2.9.6. 
Non-Engraftment .............................................................................................................. 2-17 
2.9.7. 
Other Toxicities ................................................................................................................ 2-18 
3. 
STUDY ENDPOINTS ....................................................................................................... 3-1 
3.1. 
Primary Endpoint ............................................................................................................. 3-1 
3.2. 
Secondary Endpoints ........................................................................................................ 3-1 
3.2.1. 
Neutrophil and Platelet Engraftment .................................................................................... 3-1 
3.2.2. 
Acute GVHD of Grades III-IV ............................................................................................ 3-1 
3.2.3. 
Mucositis Severity............................................................................................................... 3-1 
3.2.4. 
Time to First Hospital Discharge ......................................................................................... 3-2 
3.2.5. 
Infections ............................................................................................................................ 3-2 
3.2.6. 
CMV Reactivation .............................................................................................................. 3-2 
3.2.7. 
Thrombotic Microangiopathy .............................................................................................. 3-2 
3.2.8. 
VOD ................................................................................................................................... 3-2 
3.2.9. 
Chronic GVHD ................................................................................................................... 3-3 
3.2.10. 
Relapse of the Original Malignancy..................................................................................... 3-3 
3.2.11. 
Survival .............................................................................................................................. 3-4 
4. 
PATIENT REGISTRATION, RANDOMIZATION AND ENROLLMENT .................. 4-1 
4.1. 
Approaching Patients, Eligibility Screening and Obtaining Consent ............................. 4-1 
4.2. 
Transplant Protocol Registration ..................................................................................... 4-1 
4.3. 
Randomization .................................................................................................................. 4-1 
4.4. 
Treatment Scheduling ....................................................................................................... 4-1 
4.5. 
Patient Evaluation............................................................................................................. 4-1 
4.5.1. 
Recipient Evaluation ........................................................................................................... 4-1 
4.6. 
Study Monitoring .............................................................................................................. 4-2 
4.6.1. 
Patient Assessments ............................................................................................................ 4-2 
4.6.1.1. 
Pre-transplant evaluations .............................................................................................. 4-4 
4.6.1.2. 
Post-transplant evaluations ............................................................................................. 4-4 
4.6.2. 
Case Report Forms .............................................................................................................. 4-5 
4.6.3. 
Reporting Patient Deaths ..................................................................................................... 4-5 
4.6.4. 
Reporting Serious Adverse Events....................................................................................... 4-5 

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4.6.4.1. 
Patient SAEs .................................................................................................................. 4-5 
4.6.5. 
CIBMTR Data Reporting .................................................................................................... 4-6 
5. 
STATISTICAL CONSIDERATIONS .............................................................................. 5-1 
5.1. 
Study Design...................................................................................................................... 5-1 
5.1.1. 
Accrual ............................................................................................................................... 5-1 
5.1.2. 
Randomization .................................................................................................................... 5-1 
5.1.3. 
Primary Endpoint ................................................................................................................ 5-1 
5.1.4. 
Primary Hypothesis ............................................................................................................. 5-1 
5.2. 
Sample Size and Power Considerations ........................................................................... 5-1 
5.3. 
Interim Analysis and Stopping Guidelines ....................................................................... 5-2 
5.3.1. 
Interim Analysis for Efficacy .............................................................................................. 5-2 
5.3.2. 
Operating Characteristics of the Design ............................................................................... 5-3 
5.3.3. 
Guidelines for Safety Monitoring ........................................................................................ 5-3 
5.4. 
Demographic and Baseline Characteristics...................................................................... 5-6 
5.5. 
Analysis of Secondary Endpoints ..................................................................................... 5-6 
5.6. 
Safety Analysis .................................................................................................................. 5-7 
 
 
LIST OF APPENDICES 
 
APPENDIX A 
SAMPLE TAPERING SCHEDULES FOR SIROLIMUS AND 
TACROLIMUS 
APPENDIX B 
INFORMED CONSENTS 
APPENDIX C 
LABORATORY SAMPLES 
APPENDIX D 
RECOMMENDED LEUCOVORIN RESCUE SCHEMA 
APPENDIX E 
CONSENSUS GVHD GRADING 
APPENDIX F 
MODIFIED OMAS SCORING SYSTEM SAMPLE CASE REPORT 
FORM 
APPENDIX G 
HUMAN SUBJECTS 
APPENDIX H 
GUIDELINES FOR SIMULTANEOUS USE OF SIROLIMUS AND 
ORAL VORICONAZOLE 
APPENDIX I 
REFERENCES 
 
 
 

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CHAPTER 1 
 
 
1. BACKGROUND AND RATIONALE 
 
1.1. 
Allogeneic Stem Cell Transplantation 
 
Hematopoietic stem cell transplantation (HSCT) is an accepted therapy for hematologic 
malignancies, marrow failure, and some congenital disorders.  However, both transplant-related 
toxicity and graft-versus-host disease (GVHD) continue to be major problems that limit the 
general value of HSCT.   
 
Transplant-related toxicity results from the conditioning regimen and agents used to control 
GVHD.  Patient and donor age, disease remission status, number of prior chemotherapy 
regimens, comorbid diseases and many other factors influence short and long-term risks of 
transplant-related morbidity and mortality.  Modifications to the chemotherapeutic and GVHD 
prophylaxis agents that reduce transplant-related toxicity would be a welcome improvement. 
 
Acute GVHD results from a complex interaction of donor T cells and recipient tissues that 
involves recognition of major and/or minor histocompatibility antigens in an inflammatory 
milieu.  Clinical injury is thought to derive from direct T cell injury through perforin/granzyme, 
Fas/FasL interactions, and the effects of inflammatory cytokines.  The context of this recognition 
is a critical factor.  HSCT recipients recently subjected to conditioning regimen toxicity and 
often having active infection present a pro-inflammatory milieu for T cell recognition and 
activation.  This may in part be mediated through direct and indirect cytokine effects.  It is 
reasonable to propose that drugs that result in additional tissue injury may contribute to GVHD. 
They also clearly increase transplantation-related morbidity.   
 
Grade II-IV acute GVHD occurs in 35-40% of patients undergoing HLA-matched related donor 
stem cell transplantation1.  Fifteen percent develop severe, Grade III-IV disease.  Approximately 
40% of patients with acute GVHD will have durable responses to corticosteroid therapy; there 
has been little change in this response rate over the past 20 years despite addition or substitution 
of other immunosuppressive drugs to GVHD treatment regimens2.  The prognosis for the 60% of 
patients without long-lasting responses is poor3.  A strategy that minimizes the incidence of 
GVHD, without other adverse effects, rather than improves treatment of established GVHD, 
seems more likely to improve overall survival after allogeneic transplantation. 
 
1.2. 
GVHD Prophylaxis 
GVHD can be prevented or decreased with a variety of pharmacologic agents4 and non-
pharmacologic techniques5.  Early transplants were done using post-transplant methotrexate to 
prevent GVHD; in the 1980s cyclosporine was demonstrated to be superior to methotrexate and 
in 1986 the combined use of cyclosporine and methotrexate was shown to be superior to single 
agent prophylaxis6.  Recently, other immunophilin inhibitors, such as tacrolimus, first used 
successfully to prevent rejection after solid organ transplantation, have been developed as 

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GVHD prophylactic agents because of favorable toxicity profiles in comparison with 
cyclosporine7.  As monotherapy for prevention of GVHD after allogeneic transplantation, 
tacrolimus has demonstrated safety and efficacy in rodent studies8 as well as in trials of human 
subjects9.    
 
Trials evaluating combination therapy as prophylaxis for GVHD in comparison with single agent 
therapy focused mainly on cyclosporine rather than tacrolimus.  The Seattle group initially 
demonstrated the superiority of cyclosporine/methotrexate over cyclosporine alone for GVHD 
prophylaxis6.  Since then, several trials have demonstrated the efficacy of adding a second agent 
to cyclosporine10, 11, 12.  There are few studies evaluating single agent tacrolimus with tacrolimus-
based combination therapy.  However, in one small trial evaluating GVHD after peripheral blood 
stem cell transplantation, where GVHD rates may be higher than after bone marrow 
transplantation13, no statistically significant increase in GVHD was noted in patients who 
received tacrolimus monotherapy in comparison with patients who received combination 
tacrolimus/methotrexate14.  The addition of a third agent to established 2-drug GVHD 
prophylaxis regimens has not shown a benefit15, 16. 
 
Large Phase III studies comparing tacrolimus/methotrexate versus cyclosporine/methotrexate for 
both matched, related and unrelated donors have been performed.  In the matched, related donor 
setting, 329 patients were randomized to receive either tacrolimus/methotrexate or 
cyclosporine/methotrexate.  The incidence of Grade II-IV acute GVHD was 31.9% in the 
tacrolimus arm and 44.4% in the cyclosporine arm17.  Similarly, in the unrelated donor study, the 
incidence of Grade II-IV acute GVHD was 56% among the 46 patients randomized to tacrolimus 
and was 74% among the 63 patients randomized to receive cyclosporine18.   
 
Despite data from retrospective database studies19, prospective Phase II studies20 and the data 
from two randomized controlled trials17, 18, cyclosporine-based regimens remain the most 
common form of GVHD prophylaxis in the HLA-identical sibling and unrelated donor settings, 
however, the use of tacrolimus and tacrolimus-based regimens is increasing.  Table 1.2 
demonstrates current GVHD prophylaxis agent use, according to a recent Center for 
International Blood and Marrow Transplant Research (CIBMTR) analysis.   
 
 
Table 1.2 – Current Use of GVHD Regimens, as Reported to the IBMTR 
 
Regimen 
Matched, Related Donor 
Unrelated Donor 
Methotrexate alone 
4% 
1% 
Cyclosporine alone 
6% 
2% 
Tacrolimus ± other 
5% 
6% 
Cyclosporine ± other 
6% 
27% 
Cyclosporine, Methotrexate ± other 
42% 
32% 
Tacrolimus, Methotrexate ± other 
24% 
24% 
T cell depletion ± other 
10% 
5% 
Other 
2% 
4% 

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Methotrexate has a long history of use as a GVHD prophylaxis agent in HSCT.  It has been 
shown to complement the effects of cyclosporine in preventing severe GVHD6.  However, it also 
may increase the risk of other complications such as mucositis and hemorrhagic 
pneumonia/interstitial pneumonitis21 and as a consequence, may increase the incidence of GVHD 
through tissue injury22, 23.  Methotrexate is an antiproliferative agent and is associated with 
slower recovery of neutrophils after allogeneic transplantation.  In a retrospective study of 
peripheral blood stem cell transplantation trials, the use of full-dose methotrexate was associated 
with a 2.5 day median delay in the time to neutrophil engraftment24.   
 
1.3. 
Sirolimus 
 
Sirolimus (Rapamune®, Wyeth) is a naturally occurring compound originally isolated from a soil 
saprophyte (Streptomyces hygroscopicus) found uniquely on Easter Island (Rapa Nui).   In 
addition to its immunosuppressive properties, sirolimus has antifungal, antiviral and 
antineoplastic properties. 
 
Although structurally similar to calcineurin inhibitors, sirolimus binds uniquely to FK binding 
protein 12 (FKBP12) and then complexes with mTOR (mammalian Target of Rapamycin).  
Sirolimus does not interact with calcineurin or its downstream effectors.  The sirolimus-
FKBP12-mTOR complex inhibits several distinct biochemical pathways, resulting in a reduction 
in DNA transcription, DNA translation, protein synthesis and cell cycling, ultimately leading to 
T cell immunosuppression.  Upstream pathways that interact with mTOR include the PTEN/pI3 
kinase/Akt pathway and the Janus kinase pathway, which is important in mediating IL-2 driven 
signaling from the T cell receptor, among others25.  Although the mechanisms are less clear, this 
compound appears to exert some of its immunosuppressive properties via inhibition of dendritic 
cell activity through a reduction in antigen uptake26, 27, cellular maturation28, intracellular 
signaling29 and apoptosis induction30, 31. 
 
Sirolimus is widely used in solid organ transplantation.  In renal transplantation, several 
randomized trials demonstrate that addition of sirolimus to an established immunosuppressive 
regimen is associated with improved allograft survival32, 33 and long-term renal function34.  
Although not tested in a randomized fashion, there is ample evidence to suggest that the 
introduction of sirolimus to facilitate early calcineurin minimization after liver transplantation 
has lead to a reduction in adverse outcomes without increasing the graft rejection rate35, 36, 37, 38. 
 
Sirolimus has been used in combination with a variety of other immunosuppressive agents in the 
solid organ transplant setting.  In vitro studies suggest that the combination of sirolimus and 
tacrolimus is more effective than the combination of sirolimus and cyclosporine in reducing 
memory T cell production, apoptosis induction and cytokine production39.  When tested in a 
comparative fashion after renal transplantation, early results suggested fewer episodes of acute 
rejection and better preservation of renal function with sirolimus and tacrolimus versus sirolimus 
and cyclosporine40, 41.  The use of combination immunosuppression with sirolimus in stem cell 
transplantation is attractive, since the drug has non-overlapping toxicities with calcineurin 
inhibitors and a different mechanism of action. 

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1.3.1. Preliminary Experience with Sirolimus in Stem Cell Transplantation 
 
1.3.1.1. 
Therapy of established GVHD 
 
In the only published report on therapy of established steroid-refractory acute GVHD, Benito et 
al described their experience treating 21 patients with sirolimus.  Most patients were treated with 
4-5 mg/m2/day after an oral loading dose of 15 mg/m2.  Although the drug was active in this 
patient population (overall response rate 57%), the drug proved to be too toxic at the doses used.  
Five patients developed thrombotic microangiopathy and many others had reversible 
cytopenias42. 
 
The efficacy of sirolimus as therapy for chronic GVHD in combination with calcineurin 
inhibitors has been published in abstract only.   Johnston et al described a 56% overall response 
rate when sirolimus was given with either tacrolimus or cyclosporine in patients with established 
chronic GVHD43, while Couriel et at described a 68% response rate to the combination of 
sirolimus and tacrolimus in patients with steroid-refractory chronic GVHD44. 
 
1.3.1.2. 
Prophylaxis of GVHD 
 
Unrelated Donor Transplantation 
At the Dana-Farber Cancer Institute, 65 patients (median age 39.5 years, range 19-62) have 
undergone HLA-matched unrelated and single HLA-antigen mismatched related and unrelated 
bone marrow transplantation using the combination of sirolimus, tacrolimus and abbreviated 
methotrexate as GVHD prophylaxis.  Presumably due to a reduction in methotrexate dose, rapid 
neutrophil and platelet engraftment was noted.  The oral formulation of sirolimus was tolerated 
and patients maintained adequate serum levels during the trial (sirolimus 3-12 ng/mL, tacrolimus 
5-10 ng/mL).  Among the first 41 patients analyzed as part of a Phase II clinical trial, the 
incidence of Grade II-IV acute GVHD was 26% and the incidence of Grade III-IV acute GVHD 
was 13%.  Overall, the combination was safe with only moderate transplant-related toxicity.  
Overall survival for the entire group of patients (n=65) at one year is 56% and at 2 years is 50% 
in this high-risk population45.   
 
Related Donor Transplantation 
At the Dana-Farber Cancer Institute, over 50 patients (median age 41 years, range 18-59) have 
undergone HLA-matched, related peripheral blood stem cell transplantation using the 
combination of sirolimus and tacrolimus without methotrexate.  The hypothesis tested in this trial 
was that the omission of methotrexate would not increase the rate of GVHD and would reduce 
transplant-related toxicity.  To date, sirolimus has been well tolerated and oral administration has 
been effective in attaining and maintaining adequate serum drug levels.  Among the first 38 
patients followed for greater than 100 days, the median times to neutrophil and platelet 
engraftment were 14 days (range 11 to 17 days) and 13 days (range 10 to 47 days), respectively.  
Platelet engraftment occurred sooner in comparison with recent IBMTR analyses (13 vs. 18 
days, unpublished data).  The rates of Grade II-IV and III-IV acute GVHD were 16% and 5%, 
respectively, at 100 days.  Transplant-related toxicity has been limited: 4 patients developed 

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severe veno-occlusive disease of the liver (11%) and no patient developed idiopathic pneumonia 
syndrome.  CMV reactivation rates were low (<10%) and no patient developed an invasive 
fungal infection during the first 100 days after transplantation when prophylactic antifungal 
agents were not used.  Thrombotic microangiopathy occurred in 4 patients (11%); it resolved in 
all cases when tacrolimus was discontinued.  Transplant-related mortality at 100 days was 5%. 
Overall survival at 1 year is 72%46, now with a median follow-up of 13 months.  A summary of 
the major clinical results along with results from the randomized trial that compared 
tacrolimus/methotrexate with cyclosporine/methotrexate is found in Table 1.3.     
 
 
Table 1.3 – Clinical Outcomes Associated With Alternate GVHD Prophylaxis Regimens 
After Matched, Related Allogeneic Stem Cell Transplantation 
 
 
Tacrolimus/ 
Sirolimus 
Tacrolimus/ 
Methotrexate 
Cyclosporine/ 
Methotrexate 
Sample Size 
38 
165 
164 
Median Age (range) 
41 (19-59) 
40 (17-61) 
40 (16-63) 
% Male 
50 
56 
61 
Stem Cell Source 
PBSC 
BM 
BM 
Neutrophil Recovery (median days) 
14 
19  
20  
GVHD (%) 
 
 
 
   Acute Grade II-IV 
16 
31.9 
44.4 
   Acute Grade III-IV 
5 
13.3 
17.1 
   Chronic 
31 
55.9 
49.4 
Survival (%) 
 
 
 
   100-Day Treatment-Related Mortality  
5 
32 
21 
   1 Year Relapse-Free Survival 
69 
47 
58 
   1 Year Overall Survival 
72 
52 
64 
 
 

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CHAPTER 2 
 
 
2. STUDY DESIGN 
 
2.1. 
Study Overview 
 
This is a Phase III randomized, open label, multicenter clinical trial sponsored by the Blood and 
Marrow Transplant Clinical Trials Network (BMT CTN).  The objective of the trial is to test the 
null hypothesis that there is no difference in acute GVHD-free survival at 100 days after 
peripheral blood stem cell transplantation using sirolimus and tacrolimus versus tacrolimus and 
methotrexate for GVHD prophylaxis.  Conditioning regimens will vary by center but at each 
center will be the same for patients randomized to either GVHD prophylaxis regimen.  Because 
randomization is expected to occur approximately 14 days prior to transplant, the primary 
endpoint of this trial is Grades II-IV acute GVHD-free survival at 114 days post-randomization.  
Secondary analyses will consider neutrophil and platelet recovery, time to hospital discharge, 
acute and chronic GVHD, relapse, infections, adverse events (mucositis, thrombotic 
microangiopathy and CMV reactivation) and overall survival.  Accrual is anticipated for three 
years with a follow-up period of two years. 
 
2.2. 
Hypothesis and Study Objectives 
 
2.2.1. Primary Hypothesis 
 
The primary hypothesis to be tested in this randomized, controlled trial is that the combination of 
sirolimus and tacrolimus (study group) will result in a 15% improvement in 114-day Grades II-
IV acute GVHD-free survival from the time of randomization in comparison to the combination 
of tacrolimus and methotrexate (control group) after myeloablative HLA-matched, related donor 
allogeneic peripheral blood stem cell transplantation for acute myelogenous leukemia (AML), 
acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML) or myelodysplastic 
syndrome (MDS).  
 
2.2.2. Secondary Hypotheses 
 
Secondary hypotheses to be tested in this clinical trial include:  
 Stable neutrophil and platelet engraftment will occur sooner with study therapy;  
 Hospital discharge will occur sooner in the study group;  
 Incidence of Grades II-IV and III-IV of acute GVHD will be lower in the study group; 
 Mucositis incidence and severity will be reduced with study therapy;  
 Infectious events will be less frequent in the study group; 
 CMV reactivation will be less frequent in the study group;  
 Chronic GVHD incidence will be lower in the study group; and, 

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 Relapse-free and overall survival will be higher in the study group. 
 
2.2.3. Primary Objective 
 
The primary objective is to compare rates of 114-day Grades II-IV acute GVHD-free survival 
post randomization for HLA-matched, related donor allogeneic peripheral blood stem cell 
transplantation using two different GVHD prophylaxis regimens. 
 
2.2.4. Secondary Objectives 
 
Secondary objectives are to compare patients receiving sirolimus and tacrolimus with those 
receiving methotrexate and tacrolimus for: 
1. Time to stable neutrophil and platelet engraftment after transplantation; 
2. Incidences of Grades III-IV acute GVHD; 
3. Mucositis severity; 
4. Time to first hospital discharge; 
5. Incidence of infections in the first 100 days post-transplantation; 
6. CMV reactivation in the first 100 days post-transplantation; 
7. Thrombotic microangiopathy incidence in the first 100 days post-transplantation; 
8. VOD incidence in the first 100 days post-transplant; 
9. Incidence of chronic GVHD; 
10. Relapse of primary malignant disease in distinct disease subgroups; and, 
11. 1-year relapse-free and overall survival. 
 
2.3. 
Patient Eligibility for Randomization 
 
2.3.1. Patient Eligibility for Randomization 
 
Diagnoses to be included: 
1. Acute Myelogenous Leukemia at the following stages: 
 First Remission 
 Second or subsequent remission 
Complete remission is defined as the absence of blasts in the peripheral circulation at 
the time of enrollment and < 5% blasts in the marrow within 28 days of enrollment.  
2. Acute Lymphoblastic Leukemia at the following stages: 
 First Remission 
 Second or subsequent remission 
Complete remission is defined as the absence of blasts in the peripheral circulation at 
the time of enrollment and < 5% blasts in the marrow within 28 days of enrollment. 

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3. Acute Biphenotypic Leukemia at the following stages: 
 
First remission 
 
Second or subsequent remission 
Complete remission is defined as the absence of blasts in the peripheral circulation at 
the time of enrollment and < 5% blasts in the marrow within 28 days of enrollment. 
4. Chronic Myelogenous Leukemia at the following stages: 
 First or Subsequent Chronic Phase: 
o Stable, not hematologic remission: blasts present in marrow and/or peripheral 
blood, but disease does not qualify as accelerated or blast phase 
o Hematological remission: no blast cells or precursor cells in the blood or marrow 
o Partial cytogenetic remission: Ph+ metaphases > 0% but < 35% 
o Complete cytogenetic remission: absence of Ph+ metaphases 
 Accelerated Phase - any one of the following symptoms: 
o WBC difficult to control (> 50 x 109/L despite therapy) 
o Rapid doubling of WBC (< 5 days) 
o 10% blasts in blood or marrow 
o 20% blasts and/or promyelocytes in blood or marrow 
o 20% basophils and/or eosinophils in blood 
o Anemia or thrombocytopenia unresponsive to standard treatment 
o Persistent thrombocytosis (> 1000 x 109/L) 
o Cytogenetic abnormalities in addition to Ph+ 
o Increasing splenomegaly 
o Marrow fibrosis 
5. Myelodysplastic syndromes at any of the following stages: 
 Refractory anemia 
 Refractory anemia with ringed sideroblasts 
 Refractory cytopenia with multilineage dysplasia 
 Refractory cytopenia with multilineage dysplasia and ringed sideroblasts 
 Refractory anemia with excess blasts-1 (5-10% blasts) 
 Refractory anemia with excess blasts-2 (10-20% blasts) 
 Myelodysplastic syndrome, unclassified 
 MDS associated with isolated del (5q)  
 Chronic Myelomonocytic Leukemia 
 
Other eligibility criteria include: 
1. 6/6 HLA-matched sibling, defined by Class I (HLA-A and B) serologic typing (or higher 
resolution) and Class II (HLA-DRBI) molecular typing willing to donate peripheral blood 
stem cells and meeting institutional criteria for stem cell donation.  The donor must be 
medically eligible to donate stem cells according to individual transplant center criteria.  
Pediatric patients in whom a pediatric sibling donor is not anticipated to be a suitable 
leukapheresis candidate are not eligible. 
2. Karnofsky ≥ 70% or Lansky performance status ≥ 70% for patients < 16 years old at time 
of registration. 

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3. Age range: 2.0 – 60.0 years at time of registration. 
4. For children less than age 18, willingness and ability to take oral medications per the 
treating physician.  
5. Signed informed consent. 
 
2.3.2. Patient Exclusion Criteria 
 
Patients will be excluded from this trial if they meet the following criteria: 
1. Prior allogeneic or autologous transplants using any hematopoietic stem cell source. 
2. Seropositive for the human immunodeficiency virus (HIV). 
3. Uncontrolled bacterial, viral or fungal infections (currently taking medication and 
progression of clinical symptoms). 
4. Pregnancy (positive serum β-HCG) or breastfeeding.  
5. Renal function: Serum creatinine outside the normal range for age; or calculated creatinine 
clearance < 50 mL/min/1.72m2. 
6. Hepatic function: Most recent direct bilirubin, ALT or AST greater than two times the 
upper limit of normal for the laboratory. 
7. Pulmonary disease: In adults, FVC or FEV1 < 60% predicted (corrected for hemoglobin).  
In children, overt hypoxemia as measured by an oxygen saturation < 92%. 
8. Cardiac ejection fraction < 45% in adults and children, or < 26% shortening fraction in 
children. 
9. Cholesterol > 500 mg/dL or Triglycerides > 500 mg/dL while being treated or not on 
appropriate lipid-lowering therapy.  
10. Patient with prior history of allergy to sirolimus. 
11. Patient requiring voriconazole at time of study registration. 
12. Patients receiving another investigational drug unless cleared by the Protocol Officer or 
Protocol Chair. 
13. Patients with prior malignancies except resected basal cell carcinoma or treated 
carcinoma in-situ.  Cancer treated with curative intent > 5 years previously will be 
allowed.  Cancer treated with curative intent < 5 years previously will not be allowed 
unless approved by the Protocol Officer or Protocol Chair. 
 

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2.4. 
Conditioning 
 
Two groups of myeloablative conditioning regimens will be allowed: 
1. Cyclophosphamide and Total Body Irradiation (CY-TBI) with a TBI dose of at least  
1200 cGy of fractionated TBI*.  
2. Etoposide and Total Body Irradiation (VP16-TBI) with a TBI dose of at least 1200 cGy 
of fractionated TBI.  
 
2.4.1. Additional Drugs 
 
Additional drugs, including anti-T cell antibodies, may not be used in the conditioning regimen. 
 
2.4.2. Choice of Conditioning Regimen  
 
Transplant centers may use different regimens for patients with different diseases as required by 
institutional protocol.  However, the transplant center must declare before randomization what 
conditioning regimen will be used for each patient.  The specified regimens will be used for the 
patient whether randomized to receive sirolimus/tacrolimus or tacrolimus/methotrexate. 
 
2.4.3. Order of Administration of TBI and Cyclophosphamide 
 
The order of administration of cyclophosphamide and TBI is at the discretion of the transplant 
center.  Within each institution, all patients should receive the cyclophosphamide and TBI in the 
same order.  If cyclophosphamide or VP16 is given last, there should be at least a one-day rest 
period before the peripheral blood stem cell infusion. 
 
2.4.3.1. 
TBI administration 
 
Fractionated TBI will be administered according to the institutional protocol.  The Institution 
will also define radiation sources, dose rates, details of lung shielding and sites receiving boost 
radiation.  TBI may be delivered from either linear accelerator or Cobalt sources.  Lung shielding 
is preferred but not required during TBI.   
 
2.4.3.2. 
Cyclophosphamide administration 
 
Cyclophosphamide will be administered intravenously.  Mesna is allowed, but not required. 
 
                                                   
* The conditioning regimen, from which the Phase II data is derived, is comprised of Cytoxan 1800 mg/m2 x 2 doses 
and TBI 14 Gy in 7 fractions with lung shielding. 

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2.5. 
Stem Cell Transplantation 
 
2.5.1. Stem Cell Collection 
 
HLA-matched sibling donors will undergo G-CSF mobilization according to local institutional 
practices.  Peripheral blood stem cells will be collected by large volume apheresis according to 
local institutional guidelines.  Plasma and red cell depletion are allowed for volume reduction or 
ABO incompatibility but any other form of graft manipulation (including ex-vivo T cell 
depletion) is not permitted. 
 
The target stem cell dose is between 2 x 106/kg and 10 x 106/kg (actual body weight) CD34+ 
stem cells.    
 
Up to two leukapheresis procedures may be performed to obtain the minimum CD34+ stem cell 
target.  If, after two leukapheresis procedures, fewer than 2 x 106/kg CD34+ stem cells have been 
collected, transplant centers will have the discretion to continue peripheral blood stem cell 
harvesting or to proceed to bone marrow harvesting to obtain sufficient stem cells.  Regardless of 
the decision, subjects will be followed and analyzed with their treatment assignment group in an 
intent-to-treat manner. 
 
If more than 10 x 106/kg CD34+ stem cells are collected, the excess should either be discarded or 
cryopreserved for future use, but should not be administered to the patient. 
 
2.5.2. Stem Cell Administration 
 
Peripheral blood stem cells will be administered on Day 0 to all patients according to individual 
institutional guidelines after appropriate processing and quantification has been performed by the 
local laboratory.  Stem cells are administered through an indwelling central venous catheter.  If 
infusion occurs over two days, Day 0 is the day infusion is completed. 
 
2.6. 
GVHD Prophylaxis  
 
This is a randomized, controlled clinical trial comparing two GVHD prophylaxis regimens:  
sirolimus/tacrolimus and tacrolimus/methotrexate.  All patients will be randomly assigned to one 
of the two GVHD prophylaxis regimens.  GVHD prophylaxis begins on Day –3.  Blinding will 
not be performed.  All patients will be analyzed with their treatment assignment group (intent-to-
treat analysis). 
 

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2.6.1. Sirolimus/Tacrolimus 
 
2.6.1.1. 
Dosing 
 
Adults: Sirolimus will be given in a loading dose of 12 mg on Day –3 followed by a daily oral 
dose of 4 mg per day.  Doses may be repeated if the subject vomits within 15 minutes of an oral 
dose.   
 
Children: Children aged < 12.0 years OR weighing < 40.0 kg will be given an oral loading dose 
of sirolimus of 3 mg/m2 followed by a daily oral dose of 1 mg/m2, rounded to the nearest full 
milligram. 
 
Adults and Children:  Tacrolimus will be given at a dose of 0.02 mg/kg every 24 hours as a 
continuous intravenous infusion beginning on Day –3.  An effort will be made to convert the 
tacrolimus to oral dosing at 2-3 times the total 24-hour intravenous dose, split into 2 doses given 
every 12 hours as soon as clinically feasible.   
 
2.6.1.2. 
Monitoring 
 
Levels of sirolimus and tacrolimus will be drawn at least twice per week while hospitalized, then 
weekly or monthly thereafter unless a change in medication (e.g. use of itraconazole) or renal 
function might result in an acute change in level.  At that point levels will be measured as 
clinically indicated.  Levels of sirolimus must be assayed by HPLC or HPLC-MS.  ELISA or 
other immunoassays are not permitted.  Tacrolimus level is measured according to local 
institutional practices. 
 
2.6.1.3. 
Target serum levels and dose modifications 
 
The target serum level for sirolimus is 3-12 ng/mL. 
 
Dose adjustments are based on clinical judgment of the treating physician after considering 
clinical toxicity, serum levels, GVHD, concomitant drug use and the rate of rise or decline of the 
serum level.  For levels < 3 ng/mL, it is suggested, but not required, that the dose of sirolimus be 
increased by approximately 25% increments no more frequently than every 2 days, rounded to 
the nearest full milligram until the target range is achieved.  Conversely, for levels > 12 ng/mL, it 
is suggested, but not required, that the dose be decreased by approximately 25% no more 
frequently than every 2 days until the target level is achieved.  Alternatively, sirolimus can be 
held entirely as long as serum levels are monitored and the drug is restarted when the level 
returns to the therapeutic range and the treating physician feels it is appropriate to restart the 
agent. 
 
The target serum level for tacrolimus is 5-10 ng/mL. 
 
Dose adjustments are based on clinical judgment of the treating physician after considering 
clinical toxicity, serum levels, GVHD, concomitant drug use and the rate of rise or decline of the 

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serum level.  For levels < 5 ng/mL, it is suggested, but not required, that the dose of tacrolimus 
be increased by approximately 25% increments every 1-2 days, rounded to the nearest  
0.5 milligram (when dosing is oral) until the target range is achieved.  Conversely, for levels  
> 10 ng/mL, it is suggested, but not required, that the dose of tacrolimus be decreased by 
approximately 25% every 1-2 days until the target level is achieved.  Alternatively, tacrolimus 
can be held entirely as long as serum levels are monitored and the drug is restarted when the 
level returns to the therapeutic range and the treating physician feels it is appropriate to restart 
the agent. 
 
2.6.1.4. 
Tapering 
 
Tapering of sirolimus and tacrolimus will begin at Day 100 in the absence of GVHD and disease 
relapse.  The goal of tapering is the complete discontinuation of immunosuppressant medications 
by 6 months.  Sirolimus and tacrolimus should be tapered in an alternating fashion every other 
week when clinically feasible.  Similar reductions in dosages should be employed for each drug 
at each tapering event, with modifications at the end of the tapering schedule to allow near 
simultaneous discontinuation of sirolimus and tacrolimus.  Sample tapering schedules for adults 
and children are found in Appendix A. 
 
2.6.2. Tacrolimus/Methotrexate 
 
2.6.2.1. 
Dosing 
 
Dosing of tacrolimus is as described in Section 2.6.1.1 above. 
 
Methotrexate will be given at a dose of 15 mg/m2 on Day 1 after transplantation, and at a dose of 
10 mg/m2 on Days 3, 6 and 11 after transplantation.  Attempts should be made to administer all 
four doses of methotrexate.  Omission of any of the doses of methotrexate is at the treating 
physician’s discretion, will not be considered a protocol violation, but will be recorded as a 
deviation from the planned GVHD prophylaxis regimen.   
 
Leucovorin rescue may be given at the treating physician’s discretion.  Patients at risk for 
methotrexate toxicity include those with large fluid collections (ascites, pleural effusions) or with 
decreased renal function.  A recommended leucovorin rescue schema can be found in Appendix 
D. 
 
2.6.2.2. 
Monitoring 
Levels of tacrolimus will be drawn at least twice per week while hospitalized, then weekly or 
monthly thereafter unless a change in medication (e.g. use of itraconazole) or renal function 
might result in an acute change in level.  At that point levels will be measured as clinically 
indicated.   
 
2.6.2.3. 
Target serum levels and dose modification 
 
Target dosing and dose modifications for tacrolimus are as described in Section 2.6.1.3 above. 

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2.6.2.4. 
Tapering 
 
Tapering of tacrolimus is as described in Section 2.6.1.4 above.  Tapering should commence at 
Day 100 in the absence of GVHD and disease relapse.  The goal of tapering is the complete 
discontinuation of immunosuppressant medications by six months.  Tacrolimus should be 
tapered every other week when clinically feasible.  Similar reductions in dosages should be 
employed at each tapering event, to allow complete discontinuation by six months.  
 
2.7. 
Supportive Care 
 
All supportive care will be given in keeping with BMT CTN Manual of Procedures (MOP) and 
local institutional practice.  Supportive care should be administered in a similar fashion to 
subjects randomized to both arms of the study. 
 
2.7.1. Growth Factors 
 
Growth factors will not be used following transplant unless absolute neutrophil cell count (ANC) 
recovery to 0.5x109/L has not occurred by Day +14.  If by Day +14, the ANC is still < 0.5x109/L 
or if, after initial recovery, the ANC drops below 1 x 109/L, G-CSF may be given per 
institutional guidelines. 
 
2.7.2. Blood Products 
 
Transfusion thresholds for blood product support will be consistent with BMT CTN MOP and 
standard institutional guidelines.  All blood products will be irradiated.  Patients who are CMV 
negative will receive CMV negative or filtered blood products from study entry. 
 
2.7.3. Prophylaxis Against Infections 
 
All patients will receive prophylaxis against bacterial, fungal and viral infections during the peri-
transplant period according to the BMT CTN MOP.  Prophylaxis must be the same for patients 
on both arms of the protocol.  This will include: 
1. Anti-bacterial prophylaxis: In keeping with the BMT CTN MOP and local institutional 
standards for allogeneic transplants.  Prophylactic antibacterial antibiotics should be used 
for patients during the neutropenic (ANC < 500/mcL) period. 
2. Pneumocystis carinii: Prophylaxis will start at the time of hospital discharge or on Day 30 
post-transplant according to institutional practice.  Prophylaxis should be continued 
according to institutional practices. 
3. Antifungal therapy: Prophylaxis with fluconazole or other antifungal agents can be given 
as per local institutional guidelines.  Fluconazole and other azoles are expected to 
increase serum tacrolimus and sirolimus levels, therefore, dosages of sirolimus and 
tacrolimus should be adjusted accordingly.  Due to extreme interactions with 
sirolimus, voriconazole is contraindicated during sirolimus therapy and should not 

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be used by patients randomized to receive sirolimus.  At this time, voriconazole is 
not considered standard antifungal prophylaxis and should not be used as 
prophylaxis in the tacrolimus/methotrexate arm; however, in the event of suspected 
or documented fungal infection, voriconazole may be used in this study arm if 
deemed clinically necessary.  For guidelines on the simultaneous use of sirolimus 
and oral voriconazole, see Appendix H. 
4. HSV/VZV: Prophylaxis will begin with conditioning therapy and continue up to the time 
of discharge.  At that time, prophylaxis should continue as per institutional guidelines.   
5. CMV: Monitoring and preemptive or prophylactic treatment strategy will be in 
accordance with the BMT CTN MOP and local institutional practice. 
 
2.7.4. Intravenous Immune Globulin (IVIG) 
 
IVIG administration will be left to local institutional standard practice. 
 
2.7.5. Kepivance 
 
Kepivance is allowed, but not recommended because mucositis assessment is a secondary 
endpoint to the protocol. 
 
2.8. 
Description of Study Drugs 
 
2.8.1. Administration and Storage 
 
2.8.1.1. 
Sirolimus (Rapamune) 
 
Sirolimus will be provided by the BMT CTN for up to six months. 
 
Similar to the calcineurin inhibitors, sirolimus has low oral bioavailability due to first pass 
metabolism by the liver and intestinal wall as well as poor absorption because it is 
countertransported in the gut lumen by the multidrug efflux pump, P-glycoprotein (P-gp).  The 
median tmax value for the oral suspension is < 1 hour indicating that there is rapid absorption. 
However, systemic availability is ~14% in stable renal transplant patients.  Total body clearance 
is 127 to 240 mL/hr/kg and is not related to dose.  Half-life is 57 to 63 hours.  It is extensively 
bound by blood cells and plasma proteins.  It is excreted by hepatic and gut metabolism.  In 
Phase III studies, whole blood trough levels for a 2 mg/day dose were 8.59  4.01 ng/mL and 
correlate with the AUC (r2=0.95). Only ~2% is excreted in the urine.   
 
The mean bioavailability of sirolimus after administration of the tablet is about 27% higher 
relative to the oral solution.  Sirolimus oral tablets are not bioequivalent to the oral solution; 
however, clinical equivalence has been demonstrated at the 2-mg dose level.  Differences in 
absorption kinetics and bioavailability in renal transplant patients are shown in Table 2.8a 
 
 

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Table 2.8a – Sirolimus Pharmacokinetics 
 
Formulation, 
2 mg 
Cmax,ss 
(ng/mL) 
tmax 
(h) 
AUC 0 
(ngh/mL) 
CL/F/WT 
(mL/h/kg) 
Oral Solution  
14.4 ± 5.3 
2.12 ± 0.84 
194 ± 78 
173 ± 50 
Tablet 
15.0 ± 4.9 
3.46 ± 2.40 
230 ± 67 
139 ± 63 
 
 
Renal failure does not affect the excretion of sirolimus, but excretion is reduced in liver failure as 
shown in Table 2.8b. 
 
Table 2.8b – Sirolimus Pharmacokinetics in Hepatic Impairment 
 
Population 
Cmax,ss 
(ng/mL) 
tmax 
(h) 
AUC 0 
(ngh/mL) 
CL/F/WT 
(mL/h/kg) 
Healthy Subjects 
78.2  18.3 
0.83  0.17 
970  272 
215  76 
Hepatic Impairment 
77.9  23.1 
0.84  0.17 
1567  616 
4  62 
 
 
Sirolimus oral solution: Sirolimus oral solution (bottles and foil pouches) should be stored 
protected from light and refrigerated at 2C to 8C (36F to 46F).  For dilution, the appropriate 
dose of sirolimus oral solution should be measured using an amber oral syringe.  The dose 
should be added to a glass or plastic container that holds at least 60 milliliters (mL).  Before 
taking the dose, it should be diluted with water or orange juice; IT SHOULD NOT BE 
DILUTED WITH GRAPEFRUIT JUICE.  After mixing with the diluent, the dose should be 
taken immediately.  The syringe should be discarded after one use.  Sirolimus oral solution 
provided in bottles may develop a slight haze when refrigerated.  If such a haze occurs allow the 
product to stand at room temperature and shake gently until the haze disappears.  The presence 
of this haze does not affect the quality of the product. 
 
Sirolimus tablets:  Sirolimus tablets are available as white triangular shaped tablets marked 
“RAPAMUNE 1 mg” in bottles of 100 tablets or as cartons containing ten blister cards of ten 
tablets each.  Sirolimus tablets should be stored at 20° to 25°C (68°-77°F).  Cartons should be 
used to protect the blister cards and strips from light.  Sirolimus tablets should be dispensed in a 
tight, light-resistant container.  
 
2.8.1.2. 
Tacrolimus (Prograf) 
 
The pharmacokinetic parameters of tacrolimus have been determined following intravenous and 
oral administration in healthy volunteers, kidney transplant and liver transplant patients. 
Pharmacokinetic parameters for healthy volunteers are found in the Table 2.8c. 
 

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Table 2.8c – Tacrolimus Pharmacokinetics 
 
 
Parameters 
Population 
N 
Route, Dose 
Cmax 
(ng/mL) 
tmax 
(hr) 
AUC 
(ng*h/mL) 
t½       (hr) 
Cl   (L/hr/kg) 
V     (L/kg) 
Healthy 
Volunteers 
8 
Iv 
(0.025 mg/kg/4hr) 
--- 
--- 
598±125* 
34.2±7.7 
0.04±0.009 
1.91±0.31 
16 
po (5 mg) 
29.7±7.2 
1.6±0.7 
243±73** 
34.8±11.4 
0.041±0.008 
*** 
1.94±0.53 
*** 
* AUC0-120  ** AUC0-72   *** Corrected for individual bioavailability 
 
Absorption of tacrolimus from the gastrointestinal tract after oral administration is incomplete 
and variable.  The absolute bioavailability of tacrolimus was 17 ± 10% in adult kidney transplant 
patients (N=26), 22 ± 6% in adult liver transplant patients (N=17), and 18 ± 5% in healthy 
volunteers (N=16).  
 
A single dose study conducted in 32 healthy volunteers established the bioequivalence of the 1 
mg and 5 mg capsules.  Another single dose study in 32 healthy volunteers established the 
bioequivalence of the 0.5 mg and 1 mg capsules.  Tacrolimus maximum blood concentrations  
(Cmax) and area under the curve (AUC) appeared to increase in a dose-proportional fashion in 18 
fasted healthy volunteers receiving a single oral dose of 3, 7 and 10 mg.  In 18 kidney transplant 
patients, tacrolimus trough concentrations from 3 to 30 ng/mL measured at 10-12 hours post-
dose (Cmin) correlated well with the AUC (correlation coefficient 0.93). In 24 liver transplant 
patients over a concentration range of 10 to 60 ng/mL, the correlation coefficient was 0.94.  The 
rate and extent of tacrolimus absorption were greatest under fasted conditions.  The presence and 
composition of food decreased both the rate and extent of tacrolimus absorption when 
administered to 15 healthy volunteers.  The effect was most pronounced with a high-fat meal 
(848 kcal, 46% fat): mean AUC and Cmax were decreased 37% and 77%, respectively; Tmax was 
lengthened 5-fold.  A high-carbohydrate meal (668 kcal, 85% carbohydrate) decreased mean 
AUC and mean Cmax by 28% and 65%, respectively.  In healthy volunteers (N=16), the time of 
the meal also affected tacrolimus bioavailability.  When given immediately following the meal, 
mean Cmax was reduced 71%, and mean AUC was reduced 39%, relative to the fasted condition.  
When administered 1.5 hours following the meal, mean Cmax was reduced 63%, and mean AUC 
was reduced 39%, relative to the fasted condition.  In 11 liver transplant patients, tacrolimus 
administered 15 minutes after a high fat (400 kcal, 34% fat) breakfast, resulted in decreased 
AUC (27 ± 18%) and Cmax (50 ± 19%), as compared to a fasted state.  
 
The plasma protein binding of tacrolimus is approximately 99% and is independent of 
concentration over a range of 5-50 ng/mL.  Tacrolimus is bound mainly to albumin and alpha-1-
acid glycoprotein, and has a high level of association with erythrocytes.  The distribution of 
tacrolimus between whole blood and plasma depends on several factors, such as hematocrit, 
temperature at the time of plasma separation, drug concentration, and plasma protein 

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concentration.  In a U.S. study, the ratio of whole blood concentration to plasma concentration 
averaged 35 ng/mL (range 12 to 67).  
 
Tacrolimus injection must be diluted with 0.9% Sodium Chloride Injection or 5% Dextrose 
Injection to a concentration between 0.004 mg/mL and 0.02 mg/mL prior to use.  Diluted 
infusion solution should be stored in glass or polyethylene containers and should be discarded 
after 24 hours.  The diluted infusion solution should not be stored in a PVC container due to 
decreased stability and the potential for extraction of phthalates. In situations where more dilute 
solutions are utilized (e.g., pediatric dosing, etc.), PVC-free tubing should likewise be used to 
minimize the potential for significant drug adsorption onto the tubing.  Parenteral drug products 
should be inspected visually for particulate matter and discoloration prior to administration, 
whenever solution and container permit.  Due to the chemical instability of tacrolimus in alkaline 
media, tacrolimus injection should not be mixed or co-infused with solutions of pH 9 or greater 
(e.g., ganciclovir or acyclovir).  Supplied as a sterile solution in 1-mL ampoules containing the 
equivalent of 5 mg of anhydrous tacrolimus per mL, in boxes of 10 ampoules.  Store between 
5°C and 25°C (41°F and 77°F).  Tacrolimus capsules (1 and 5 mg) are stored at controlled room 
temperature, 15°C-30°C (59°F-86°F).  
 
2.8.1.3. 
Methotrexate 
 
Methotrexate sodium for injection should be reconstituted with an appropriate sterile, 
preservative free medium such as 5% dextrose solution, USP, or sodium chloride injection, USP.  
Reconstitute the 20 mg vial to a concentration no greater than 25 mg/mL.  The 1 gram vial 
should be reconstituted with 19.4 mL to a concentration of 50 mg/mL.  Reconstitute immediately 
prior to use.  When high doses of methotrexate are administered by IV infusion, the total dose is 
diluted in 5% dextrose solution. 
 
Each 20 mg and 1 g vial of lyophilized powder contains methotrexate sodium equivalent to 20 
mg and 1 g methotrexate respectively.  Store at controlled room temperature, 20°-25° C  
(68°-77° F); excursions permitted to 15°-30° C (59°-86° F).  Protect from light. 
 
2.8.2. Adverse Reactions 
 
2.8.2.1. 
Sirolimus 
 
Despite the similarity between sirolimus and tacrolimus, sirolimus is not associated with 
neurotoxicity or nephrotoxicity because of its inability to inhibit calcineurin.  Phase III clinical 
trials have indicated that the primary toxicities are hypertriglyceridemia, hypercholesterolemia, 
mild thrombocytopenia, anemia, leukopenia, hypokalemia, elevated LDH, arthralgia, epistaxis, 
edema, and infections.  Clinically significant elevations in hepatic transaminases without 
sequelae were noted in the prior Phase II study with this drug.  However, the incidence of veno-
occlusive disease of the liver was not noted to be higher than expected in trials of unrelated 
transplantation.  A syndrome of thrombotic microangiopathy, comprised of microangiopathic 
hemolytic anemia, thrombocytopenia and renal dysfunction has been described in association 

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with sirolimus and tacrolimus use.  In common with all other immunosuppressants, it may 
increase the risk of opportunistic infections and post-transplant lymphoproliferative disorders. 
 
2.8.2.2. 
Tacrolimus 
 
There is a spectrum of well-described toxic effects of tacrolimus.  The primary toxicities in large, 
controlled trials were reversible renal insufficiency (doubling of creatinine in 82%), hypertension 
requiring 
the 
use 
of 
antihypertensive 
medications 
(21%), 
hyperglycemia 
(12%), 
hypomagnesemia, hypokalemia, and neurologic toxicity.  In addition there is an infusional 
toxicity from the cremaphor diluent.  There is an increased risk of opportunistic infections and 
secondary malignancies.  Some of these toxicities are recognized complications of 
transplantation.  
 
2.8.2.3. 
Methotrexate 
 
The most frequently reported adverse reactions associated with methotrexate use as GVHD 
prophylaxis include ulcerative stomatitis, leucopenia and suppressed hematopoiesis, nausea, and 
abdominal distress.  Other frequently reported adverse effects are malaise, undue fatigue, chills 
and fever, dizziness and decreased resistance to infection.  Methotrexate may be associated with 
increased rates of pulmonary complications after transplantation.  The risk of infections is due to 
the suppression of hematopoiesis after transplantation.  
 
2.8.3. Drug Interactions 
 
2.8.3.1. 
Sirolimus 
 
Sirolimus is known to be a substrate for both cytochrome CYP3A4 and P-glycoprotein. 
 
Drugs that may increase sirolimus blood concentrations include:  
 
Calcium channel blockers: diltiazem, nicardipine, verapamil.  
 
Calcineurin inhibitors: cyclosporine. 
 
Antifungal agents: ketoconazole, clotrimazole, fluconazole, itraconazole.  
 
Macrolide antibiotics: clarithromycin, erythromycin, troleandomycin.  
 
Gastrointestinal prokinetic agents: cisapride, metoclopramide.  
 
Other drugs: rifampin, bromocriptine, cimetidine, danazol, HIV-protease inhibitors (e.g., 
ritonavir, indinavir).  
 
Due to extreme interactions with voriconazole, this drug is contraindicated during 
sirolimus therapy.  It is recommended that drugs with known interactions with sirolimus 
be avoided when clinically feasible.  When these medications are required, dose 
modifications of sirolimus may be required.  For guidelines on the simultaneous use of 
sirolimus and oral voriconazole, see Appendix H. 
 

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Drugs that may decrease sirolimus concentrations include: 
 
Anticonvulsants: carbamazepine, phenobarbital, phenytoin. 
 
Antibiotics: rifabutin, rifapentine.  
 
Herbal preparations: St. John's Wort (Hypericum perforatum) could result in reduced 
sirolimus concentrations.  
 
Care should be exercised when drugs or other substances that are metabolized by CYP3A4 are 
administered concomitantly with sirolimus.  Grapefruit juice reduces CYP3A4-mediated 
metabolism of sirolimus and must not be used for dilution. 
 
2.8.3.2. 
Tacrolimus 
 
Tacrolimus is extensively metabolized by the cytochrome P-450 (CYP3A4) system.  
  
Drugs that may increase tacrolimus blood concentrations include:  
 
Calcium channel blockers: diltiazem, nicardipine, nifedipine, verapamil 
 
Antifungal agents: ketoconazole, clotrimazole, fluconazole, itraconazole 
 
Macrolide antibiotics: clarithromycin, erythromycin, troleandomycin.  
 
Gastrointestinal prokinetic agents: cisapride, metoclopramide.  
 
Other drugs: bromocriptine, cimetidine, cyclosporine, danazol, ethinyl estradiol, 
omeprazole, nefazodone, HIV-protease inhibitors (e.g., ritonavir, indinavir).  
 
Drugs that may decrease tacrolimus concentrations include: 
 
Anticonvulsants: carbamazepine, phenobarbital, phenytoin. 
 
Antibiotics: rifabutin, rifapentine.  
 
Herbal preparations: St. John's Wort (hypericum perforatum) could result in reduced 
tacrolimus concentrations.  
 
Due to extreme interactions with voriconazole, tacrolimus doses should be empirically 
lowered by 50% when concomitant therapy with voriconazole is initiated.  It is 
recommended that drugs with known interactions with tacrolimus be avoided when 
clinically feasible.  When these medications are required, dose modifications of tacrolimus 
may be required. 
 
2.8.3.3. 
Methotrexate 
 
Methotrexate is partially bound to serum albumin, and toxicity may be increased because of 
displacement by certain drugs, such as salicylates, phenylbutazone, phenytoin, and sulfonamides.  
Renal tubular transport is also diminished by probenecid; use of methotrexate with this drug 
should be carefully monitored. 
 

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Oral antibiotics such as tetracycline, chloramphenicol, and nonabsorbable broad-spectrum 
antibiotics, may decrease intestinal absorption of methotrexate or interfere with the enterohepatic 
circulation by inhibiting bowel flora and suppressing metabolism of the drug by bacteria. 
Penicillins may reduce the renal clearance of methotrexate; increased serum concentrations of 
methotrexate with concomitant hematologic and gastrointestinal toxicity have been observed 
with high and low dose methotrexate. Use of methotrexate with penicillins should be carefully 
monitored.  
 
2.9. 
Management of Toxicities 
 
Toxicities will be scored as per the NCI’s Common Terminology Criteria for Adverse Events 
(CTCAE) version 3.0 (see http://ctep.cancer.gov/forms/CTCAEv3.pdf).  Toxicities of the 
blood/bone marrow, gut, liver, and skin will be excluded from the common toxicity criteria and 
evaluated separately since they are likely to be affected by GVHD.  GVHD severity will be 
determined clinically, however, biopsies of affected organs are strongly encouraged whenever 
possible.  
 
2.9.1. GVHD 
 
Specific therapy of GVHD will not be mandated by protocol, although it is recommended that 
only Grade II-IV GVHD be treated with systemic therapy.  A recommendation for initial therapy 
of GVHD is methylprednisolone, 2 mg/kg/d IV (or an equivalent corticosteroid).  If the patient 
responds to steroids, the steroid will be tapered as tolerated clinically, but with the intent of 
decreasing the dose by 10%/week.  For steroid non-responders, treatment for GVHD will be per 
local institutional standards.  Tacrolimus and sirolimus (in the appropriate treatment group) 
should be continued during treatment of acute GVHD. 
 
Whenever possible, the diagnosis of GVHD should be confirmed with a biopsy of the involved 
organ and histological examination of a biopsy specimen by a pathologist experienced in the 
diagnosis of GVHD.  Due to the early engraftment anticipated with the omission of methotrexate 
in the sirolimus arm, conditioning-related diarrhea may coincide with engraftment, therefore, 
sigmoidoscopy and biopsy are strongly encouraged to diagnose GVHD of the intestine.  
Diagnosis of isolated hepatic GVHD must be made by liver biopsy. 
 
2.9.2. VOD 
 
Treatment of veno-occlusive disease of the liver will be treated per local institutional standards.  
Participation in clinical trials is permissible.  Tacrolimus and sirolimus (in the appropriate 
treatment group) should be continued during VOD therapy.  Levels should be measured 
frequently in the context of hepatic and/or renal failure. 
 
2.9.3. Idiopathic Pneumonia Syndrome/Diffuse Alveolar Hemorrhage 
 
Treatment of idiopathic pneumonia syndrome/diffuse alveolar hemorrhage will be treated per 
local institutional standards.  Participation in clinical trials is permissible.  Tacrolimus and 

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sirolimus (in the appropriate treatment group) should be continued during idiopathic pneumonia 
syndrome/diffuse alveolar hemorrhage therapy.   
 
2.9.4. Thrombotic Microangiopathy 
 
Thrombotic microangiopathy will be defined according to BMT CTN guidelines.  The diagnostic 
criteria include the concurrent occurrence of: 
1- Red cell fragmentation on a manual differential (2 + schistocytes) with a negative 
Coombs test 
and 
2- LDH > normal 
and either 
3- Renal dysfunction (doubling of serum creatinine or a decrease > 50% in the measured 
creatinine clearance) 
or 
Neurological dysfunction unexplained by another etiology 
 
The development of thrombotic microangiopathy should be managed conservatively.  
Tacrolimus should be discontinued from the GVHD regimen.  At the treating physician’s 
discretion, Mycophenolate Mofetil can be substituted for GVHD prophylaxis.  Sirolimus should 
be held if serum levels are supratherapeutic and restarted once in the target range at an 
appropriate dose.  The use of plasmapheresis and plasma exchange does not appear to be of 
significant clinical utility in the management of transplant-associated microangiopathy, but may 
be used at the discretion of the treating physician.  
 
2.9.5. Hyperlipidemia 
 
Increases in triglyceride and cholesterol levels as a result of prolonged exposure to sirolimus will 
be managed with dietary manipulations and HMG CoA inhibitors or other standard therapy as 
necessary to reduce levels to the normal range.  Study drugs should be continued during therapy 
for hypercholesterolemia and hypertriglyceridemia unless the lipid levels are uncontrollable with 
standard therapy and are deemed to be a risk to the subject, per the treating physician. 
 
2.9.6. Non-Engraftment 
 
Engraftment will be considered to be three consecutive measurements ANC > 500mcL over 
three or more days. If the ANC < 100 on Day 28, evaluation for graft failure will be initiated, 
including bone marrow aspiration/biopsy, cytogenetics and/or fluorescence in situ hybridization 
(FISH) if appropriate, and RFLP analysis if appropriate.  Plans for a second stem cell infusion 
will be made depending on the results of the work-up. 
 

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2.9.7. Other Toxicities 
 
All other associated toxicities that may be attributable to sirolimus will be managed 
symptomatically.  If that is unsuccessful and the toxicity can be attributed to sirolimus, this drug 
will be discontinued. 
 

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CHAPTER 3 
 
 
3. STUDY ENDPOINTS 
 
3.1. 
Primary Endpoint 
 
The primary endpoint is 114-day Grades II-IV acute GVHD-free survival from the time of 
randomization.  All randomized patients will be analyzed for this endpoint.  The event occurs at 
the first of either of two events:  the development of Grade II-IV GVHD or death from any cause 
prior to 114 days from randomization.  
 
3.2. 
Secondary Endpoints 
 
3.2.1. Neutrophil and Platelet Engraftment 
 
Neutrophil engraftment is defined as achieving an Absolute Neutrophil Count (ANC) > 500/mcL 
for three consecutive measurements on different days.  The first of the three days will be 
designated the day of neutrophil engraftment.  The ANC is calculated by the following formula: 
 
ANC = Total WBC x (%Neutrophils + %Bands) 
 
Platelet engraftment is defined as a platelet count > 20,000/mcL for three consecutive 
measurements over three or more days.  The first of the three days will be designated the day of 
platelet engraftment.  Subjects must not have had platelet transfusions during the preceding 3 
days or in the following 7 days after the day of engraftment, unless the platelet transfusion is 
being given specifically to achieve a platelet threshold to allow an elective invasive procedure, 
such as a central catheter removal.  The time to a platelet count > 100,000/mcL will be collected 
as well. 
 
This endpoint will be evaluated through 100 days.  All patients failing to meet the neutrophil 
engraftment target at this time will be considered to have non-engraftment.  
 
3.2.2. Acute GVHD of Grades III-IV 
 
Acute GVHD is graded according to the BMT CTN MOP (see Appendix E).  The first day of 
acute GVHD onset at a certain grade will be used to calculate cumulative incidence curves for 
that GVHD grade (e.g., if the onset of Grade I acute GVHD is on Day 19 post-transplant and 
onset of Grade III is on Day 70 post-transplant, time to Grade III is Day 70).  This endpoint will 
be evaluated through 114 days post-randomization. 
 
3.2.3. Mucositis Severity 
 
Mucositis severity will be scored per the modified OMAS scoring system (see Appendix F).  
Mucositis scores will be recorded three times per week (Monday, Wednesday and Friday) from 

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Day 0 to the shorter of Day 21 after transplantation or the time of discharge.  Both peak and 
mean mucositis scores will be analyzed. 
 
3.2.4. Time to First Hospital Discharge 
 
The time from stem cell transplantation (Day 0) to the date of first hospital discharge will be 
recorded as the length of the initial hospital stay.  Patients remaining in the hospital on Day 100 
will be censored for this outcome. 
 
3.2.5. Infections 
 
Microbiologically documented infections will be reported by site of disease, date of onset, 
severity and resolution, if any.  For definitions, see the BMT CTN MOP. 
 
3.2.6. CMV Reactivation  
 
CMV reactivation is defined as the presence of viral DNA level  2 pg/mL on two consecutive 
measurements using a viral hybrid capture or other suitable measurement technique.  
 
3.2.7. Thrombotic Microangiopathy 
 
The occurrence of thrombotic microangiopathy (according to the definition in Section 2.9.4) 
within the first 100 days after stem cell transplantation will be recorded.  The first day of onset 
will be used for reporting purposes.  The incidence of TMA will be compared between study 
groups using cumulative incidence curves.  
 
3.2.8. VOD 
 
VOD will be defined as the occurrence of VOD (based on the Baltimore Criteria for the 
diagnosis of VOD) in conjunction with other end-organ dysfunction. 
 
Baltimore Criteria for the Diagnosis of VOD: 
Jaundice (bilirubin ≥ 2 mg/dL) 
and at least 2 of the following 3 clinical findings: 
• 
Ascites 
• 
Weight gain ≥ 5% above baseline weight (defined as weight on the first day of 
conditioning or the weight on the date of admission) 
• 
Hepatomegaly (with pre-existing hepatomegaly there must be documentation by 
physical exam or imaging that liver size is increased over baseline) 
 
Severe VOD is defined as meeting the above criteria and also demonstrating multi-organ failure, 
i.e., presence of one or both of the following: 
• 
Renal dysfunction: 
• 
Serum creatinine ≥ 3 x value on the date of admission or ≥ 3 x lowest value 
during conditioning prior to SCT (whichever is lowest) 

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• 
Creatinine clearance or GFR ≤ 40% of admission value 
• 
Dialysis dependence 
• 
Pulmonary dysfunction:  
• 
Documentation of oxygen saturation ≤ 90% on room air or requirement for 
oxygen supplementation/ventilator dependence. Dysfunction must be attributable 
to fluid overload or mechanical impingement from abdominal distention or 
hepatic enlargement and not to an infection 
 
3.2.9. Chronic GVHD 
 
Chronic GVHD is scored according to the BMT CTN MOP.  The first day of chronic GVHD 
onset will be used to calculate cumulative incidence curves. 
 
3.2.10. Relapse of the Original Malignancy 
 
Relapse of Malignancy - Testing for recurrent malignancy in the blood, marrow or other sites 
will be used to assess relapse after transplantation.  For the purpose of this study, relapse is 
defined by either morphological or cytogenetic evidence of AML, ALL, CML, MDS or CMML 
consistent with pre-transplant features.   
 
Minimal Residual Disease - Minimal residual disease is defined by the sole evidence of 
malignant cells by flow cytometry, or fluorescent in situ hybridization (FISH), or Southern blot, 
or Western blot, or polymerase chain reaction (PCR), or other techniques, in absence of 
morphological or cytogenetic evidence of disease in blood or marrow.  Since the frequency of 
testing for minimal residual disease is highly variable among centers, and the sensitivity is highly 
variable among laboratory techniques, evidence of minimal residual disease will not be sufficient 
to meet the definition of relapse in the context of this study, even if transplant physicians will 
utilize the information to alter therapy.  Data on tapering immunosuppression, infusing donor T 
cells, administering chemotherapy or biological agents to attempt reducing the tumor load will be 
captured in the case report forms. 
 
Acute Leukemia - Relapse will be diagnosed when: 
1. Leukemic blasts (> 25%) are documented in the blood or bone marrow after 
transplantation, or  
2. Leukemic blasts > 5% and < 25% are documented in the blood or bone marrow and 
supported by reappearance of cytogenetic abnormality, or  
3. Leukemic blasts > 5% and < 25% are documented in the blood or bone marrow on at 
least 2 occasions, or  
4. There is leukemia detected at an extramedullary site.  
 
Chronic Myelogenous Leukemia - Relapse will be diagnosed when: 
1. Immature hematopoietic cells are persistently documented in the peripheral blood, or  

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2. There is myeloid hyperplasia in the bone marrow in the presence of a cytogenetic relapse, 
as defined by: 
a) 50% of the metaphases exhibit the characteristic 9;22 translocation with at least ten 
metaphases analyzed, or 
b) At least one metaphase exhibits the 9;22 translocation on each of two separate 
consecutive examinations at least one month apart, regardless of number of 
metaphases analyzed. 
Myelodysplastic Syndrome - Relapse will be diagnosed when: 
1. Reappearance of pre-transplant morphologic abnormalities, detected in two consecutive 
bone marrow specimens taken at least one month apart, or 
2. Satisfying above criteria for evolution into acute leukemia, or   
3. Reappearance of pre-transplant cytogenetic abnormalities in at least 50% of metaphases 
with at least ten metaphases examined, or 
4. Reappearance of pre-transplant cytogenetic abnormality in at least two metaphases on 
each of two separate consecutive examinations at least one month apart, regardless of the 
number of metaphases analyzed. 
 
3.2.11. Survival  
 
Both overall survival and relapse-free survival will be assessed one and two years from the time 
of randomization.  All patients will be assessed in an intention-to-treat fashion.  Patients alive at 
the time of last observation will be censored. 
 

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CHAPTER 4 
 
 
4. PATIENT REGISTRATION, RANDOMIZATION AND ENROLLMENT 
 
4.1. 
Approaching Patients, Eligibility Screening and Obtaining Consent 
 
Subjects will be approached for this study after the decision to proceed with transplantation is 
made and a suitable HLA-matched sibling identified.  Transplant physicians will evaluate the 
patient eligibility for randomization onto this study (see Section 2.3).  Eligibility criteria will be 
verified and ineligible patients will proceed off study and no further follow-up will be obtained 
Eligible patients willing to participate in the trial will sign an Institutional Review Board (IRB) 
approved consent form.  Transplant center personnel will record the documentation of patient 
consent in EMMES AdvantageEDCSM (Electronic Data Capture, an Internet-based data entry 
system) and patients will be registered through AdvantageEDC. 
 
4.2. 
Transplant Protocol Registration 
 
Before randomization occurs, the transplant center must state through AdvantageEDC which 
conditioning regimen will be used for the enrolled subject.  Such a registration step will avoid 
potential biases that preferential use of a certain regimen on one treatment arm could confer to 
the study.  At this stage, the transplant center will also verify that the patient is still a candidate 
for transplantation, and eligible for the trial. 
 
4.3. 
Randomization 
 
Once the subject is deemed eligible and has given written informed consent, and the transplant 
center has confirmed patient eligibility and registered the patient’s conditioning regimen, 
randomization occurs.  Randomization should occur within 7 days of the planned initiation 
of conditioning therapy.   See Section 5.1.2 for further details on the randomization process. 
 
4.4. 
Treatment Scheduling 
 
Treatment should be initiated as soon as possible after randomization.  This will prevent subject 
attrition prior to stem cell transplantation for reasons such as disease progression.  Consequently, 
all treatments related to the transplant should be scheduled PRIOR to randomization.  This 
includes planning an admission date and ensuring that the stem cell donor can be mobilized and 
undergo apheresis in a coordinated fashion with the planned transplant.   
 
4.5. 
Patient Evaluation 
 
4.5.1. Recipient Evaluation 
 
The patient pre-transplant evaluation must be completed within six weeks of conditioning for 
transplantation.  This step is necessary because patient organ function, infection status and status 

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of malignancy may vary over time.  This evaluation will protect patients with a new 
contraindication to transplant from initiating transplant therapy at an unsafe time. 
 
4.6. 
Study Monitoring 
 
A visit schedule based on transplant date is displayed for printing in AdvantageEDC and is 
shown in Table 4.6a. 
 
Table 4.6a – Follow-Up Schedule 
 
Study Visit 
Target Day Post-Transplant 
Baseline 
 28 days from conditioning 
1 week 
7  2 days 
2 week 
14  2 days 
3 week 
21  2 days 
4 week 
28  2 days 
5 week 
35  2 days 
6 week 
42  2 days 
7 week 
49  2 days 
8 week 
56  2 days 
9 week 
63  2 days 
10 week 
70  2 days 
12 week 
84  2 days 
100 day 
100  5 days 
6 month 
180  14 days 
9 month 
270  days 
12 month 
365  14 days 
24 month 
730  28 days 
 
4.6.1. Patient Assessments 
 
Table 4.6b summarizes patient clinical assessments over the course of the study. 

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Table 4.6b – Patient Clinical Assessments 
 
Study Assessments/  
Testing 
Base
line 
Days Post-Transplant 
7 
14 
21 
28 
35 
42 
49 
56 
63 
70 
77 
84 
91 
98 
100 
180 
270 
365 
730 
History, Physical Exam, 
Weight, Height, Karnofsky/ 
Lansky Performance Status 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
 
 
X 
X 
X 
X 
X 
CBC1, Differential, Platelet 
Count, Blood Chemistries2 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
 
 
X 
X 
X 
X 
X 
Infectious Disease Titers3 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Serum Cholesterol and 
Triglyceride Levels 
X 
 
 
 
X 
 
 
 
X 
 
 
 
 
 
 
X 
X 
X 
X 
X 
EKG, PFT, Chest X-ray 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Bone Marrow Aspirate and 
Biopsy for Pathology and 
Cytogenetics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
X 
X 
ß-HCG Serum Pregnancy 
Test (females only) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Sirolimus/Tacrolimus Level4 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
 
 
X 
X 
X 
X 
X 
Mucositis Score5 
 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
GVHD and Toxicity 
Assessments6 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Immune Reconstitution 
Assays 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
X 
X 
 
X 
X 
Whole Blood Sample7  
X 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
X 
X 
 
X 
XX 
1 CBC performed three times weekly from Day 0 until ANC > 500/mcL for three days and platelet count > 20,000/mcL after nadir, while hospitalized.  CBC then performed weekly 
through 8 weeks and every other week through Day 100 post-transplant. 
2 Blood chemistries include: creatinine, bilirubin, alkaline phosphatase, AST, and ALT.  Blood chemistries performed twice weekly until Day 28. Blood chemistries performed weekly 
after Day 28 until 12 weeks post-transplant. 
3 Infectious disease titers include: CMV, hepatitis panel (HepB SAb, HepB SAg, HepB Core Ab, HepC Ab), herpes simplex virus, varicella zoster virus, syphilis, HIV and HTLV ½ 
antibody. 
4 Sirolimus and Tacrolimus levels are drawn twice weekly while hospitalized, then weekly through week 10, every other week through 100 days post-transplant.  Applicable only for 
patients receiving these drugs. 
5 Every Monday, Wednesday and Friday through Day 21 or hospital discharge.  See Appendix F 
6 Morbidity assessments performed weekly until Day 100 post-transplant.  GVHD assessments performed weekly until Day 98. 
7 Sample amount of 16 mL, or 8mL for patients < 12 years old.  To be cryopreserved at the transplant center for future testing.  The center may make further adjustments in 
blood volumes collected from patients less than 12 years old to meet the institution’s Human Investigational Committee’s Guidelines. 

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4.6.1.1. 
Pre-transplant evaluations 
 
The following observations should be made < six weeks before initiation of conditioning 
therapy: 
1. History, physical examination, height and weight. 
2. Karnofsky/Lansky performance status. 
3. CBC with differential and platelet count, creatinine, bilirubin, alkaline phosphatase, AST, 
ALT. 
4. CMV antibody test, hepatitis panel (HepB SAb, HepB SAg, HepB Core Ab, HepC Ab), 
herpes simplex virus, varicella zoster virus, syphilis, HIV and HTLV1/2 antibody. 
5. Serum cholesterol and triglyceride levels. 
6. EKG. 
7. Pulmonary function tests, including DLCO, FEV1, and FVC. 
8. Bone marrow aspirates for pathology and cytogenetics. 
9. Chest X-ray. 
10. -HCG for serum pregnancy test (females only). 
11. Blood (16 mL, or 8 mL for patients < 12 years old) from recipient to be cryopreserved at 
the transplant center for future testing if consent obtained.  This should be collected and 
processed per Appendix C.  The center may make further adjustments in blood volumes 
collected from patients less than 12 years old to meet the institution’s Human 
Investigational Committee’s Guidelines. 
12. Blood (16 mL, or 8 mL for patients < 12 years old) from donor to be cryopreserved at the 
transplant center for genetic testing if consent obtained.  This should be collected and 
processed per Appendix C.  The center may make further adjustments in blood volumes 
collected from patients less than 12 years old to meet the institution’s Human 
Investigational Committee’s Guidelines. 
 
4.6.1.2. 
Post-transplant evaluations 
 
The following observations should be made according to Table 4.6b: 
1. History and physical exam to assess GVHD and other morbidity weekly through Day 100 
post-transplant, then at 6, 9, 12 and 24 months.  GVHD evaluation and grading to be in 
keeping with BMT CTN MOP. 
2. Data on occurrence of infections and recorded as per the CTN MOP. 
3. CBC at least three times a week from Day 0 until ANC > 500/mcL for 3 days and platelet 
count > 20,000/mcL for 3 days (while hospitalized only) after nadir reached.  A manual 
differential must be performed.  Thereafter CBC weekly until 10 weeks, every other 
week through 100 days, then at 6, 9, 12 and 24 months post-transplant. 

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4. Creatinine, bilirubin, alkaline phosphatase, ALT, AST, twice a week until hospital 
discharge and then weekly until 10 weeks, every other week through 100 days, then at 6, 
9, 12 and 24 months post-transplant. 
5. Tacrolimus and sirolimus levels twice a week until hospital discharge and then weekly 
until 10 weeks, every other week through 100 days, then at 6, 9, 12 and 24 months post-
transplant (only when subject is using the medication). 
6. Mucositis assessment three times per week (Monday, Wednesday and Friday) while 
hospitalized or until Day 21 (see Appendix F for modified OMAS scoring system). 
7. Serum cholesterol and triglyceride level at months 1 and 2, 100 days, months 6, 9, 12 and 
24 post-transplant. 
8. Bone marrow aspirate and biopsy to pathology, aspirate and cytogenetics at 100 days, 12 
and 24 months post-transplant. 
9. Immune reconstitution blood samples at Days 28, 100, 180, 365 and 730 (see Appendix 
C). 
10. Investigational future testing blood samples (16 mL, or 8 mL for patients < 12 years old) 
at Days 28, 100, 180, 365 and 730 if consent obtained (see Appendix C).  The center may 
make further adjustments in blood volumes collected from patients less than 12 years old 
to meet the institution’s Human Investigational Committee’s Guidelines. 
 
4.6.2. Case Report Forms 
 
A description of the forms, the procedures required for forms completion and timeliness of 
submission can be found in the Data Management Handbook and User’s Guide.  Forms that are 
not received within the specified time are considered delinquent.  Transplant centers can view 
submitted past due, and expected forms via AdvantageEDC.  A missing form will continue to be 
requested either until the form is reported, or until an exception is granted. 
 
4.6.3. Reporting Patient Deaths 
 
Recipient death information must be reported to the BMT CTN Data Coordinating Center (DCC) 
within one business day of the event.  Death after the patient has left the transplant center must 
be reported within one business day of the event notification to the transplant center. If the cause 
of death is unknown, it need not be recorded at the time of initial reporting.  However, once the 
cause of death is determined, the form must be updated. 
 
4.6.4. Reporting Serious Adverse Events 
 
4.6.4.1. 
Patient SAEs 
 
Reporting of patient serious adverse events (SAE) will be consistent with standard BMT CTN 
procedures.  Unexpected, grades 3-5 adverse events (AEs) will be reported through an expedited 
AE reporting system via the web-based electronic data capture system, AdvantageEDC.  
Unexpected, grades 4-5AEs must be reported within 24 hours of knowledge of the event.  

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Unexpected, grade 3 AEs must be reported within three business days of knowledge of the event.  
Other SAEs will be tracked periodically as defined in the Form Submission Schedule, 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 adverse experiences 
on at least an annual basis. 
 
4.6.5. CIBMTR Data Reporting 
 
All transplant centers will be required to pre-register patients with the Center for International 
Blood and Marrow Transplant Research (CIBMTR) for all transplant patients whether or not 
they enroll in a BMT CTN Protocol.  In addition, the transplant center must complete the 
CIBMTR Day 100 Report Form (including the Core, Graft and Disease Inserts) and CIBMTR 
Follow-up Form (including the Core and Disease Inserts) yearly for all patients enrolled in BMT 
CTN protocols.  CIBMTR forms will be submitted directly to the CIBMTR at the times specified 
on the Form Submission Schedule. 
 
 

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CHAPTER 5 
 
 
5. STATISTICAL CONSIDERATIONS 
 
5.1. 
Study Design 
 
The study is designed as a Phase III, randomized, open label, multicenter, prospective 
comparative study of sirolimus and tacrolimus versus tacrolimus and methotrexate as GVHD 
prophylaxis after HLA-matched, related peripheral blood stem cell transplantation in patients 
with hematologic malignancies.  The target enrollment is 312 patients. 
 
5.1.1. Accrual 
 
It is estimated that three years of accrual will be necessary to enroll the targeted sample size.  
Both Core and non-Core Centers will enroll patients on this study.  Accrual will be reported by 
race, ethnicity, gender, and age (pediatric patients will be defined as < 18 years). 
 
5.1.2. Randomization 
 
All patients will be randomized within 7 days prior to the initiation of conditioning therapy.  
Randomization will be performed in a 1:1 ratio using random block sizes for the two arms.  
Randomization will be stratified by transplant center. 
 
5.1.3. Primary Endpoint 
 
The primary endpoint is the Grades II-IV acute GVHD-free survival proportion at 114 days post-
randomization.  The primary analysis will be performed using the intent-to-treat principle so that 
all randomized patients will be included in the analysis.  Death or the occurrence of Grade II-IV 
acute GVHD by Day 114 post-randomization will be considered events for this endpoint. 
 
5.1.4. Primary Hypothesis 
 
The primary null hypothesis of the study is that there is no difference between the GVHD-free 
survival rates for sirolimus and tacrolimus compared to tacrolimus and methotrexate. 
 
Ho: 
pst = ptm 
Ha: 
pst  ptm 
 
5.2. 
Sample Size and Power Considerations 
 
GVHD-free survival at 114 days post-randomization will be compared between the standard and 
experimental therapy arms using the standardized difference in the Kaplan Meier estimates of 
survival.  The final analysis will be performed after all patients have been followed for a 
minimum of 114 days post-randomization.  At this time point, all individuals will have been 

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completely observed for the primary outcome.  Note that if there is no loss to follow up, the 
comparison of the Kaplan-Meier estimates is equivalent to the usual two-sample Z test of 
binomial proportions.  However, the Kaplan Meier test will be used in the event of loss to follow 
up as well as in the interim analyses conducted with incomplete follow-up.  The sample size of 
156 patients per group is sufficient to maintain type I error of 5% across all planned interim 
analyses (see below) while providing 80% statistical power for a two-sided test to detect an 
increase in the proportion surviving without Grade II-IV GVHD at 114 days from 0.60 in the 
tacrolimus/methotrexate arm to 0.75 in the sirolimus/tacrolimus arm.   
 
5.3. 
Interim Analysis and Stopping Guidelines 
 
Interim analyses for efficacy will be conducted at times coincident with regularly scheduled 
meetings of the NHLBI-appointed Data and Safety Monitoring Board (DSMB) at approximately 
six month intervals.  Monitoring of key safety endpoints will be conducted monthly, and if 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. 
 
5.3.1. Interim Analysis for Efficacy 
 
Analyses will be performed as described below for the primary endpoint.  Toxicity, adverse 
events, and other safety endpoints will be monitored regularly and reported to the DSMB at each 
interim analysis.  
 
At the time of each interim analysis, the test statistic based on the Kaplan-Meier proportion will 
be compared to the critical value shown below.  All patients randomized prior to the time of the 
interim analyses will be used to compute the Kaplan-Meier estimate.  If the test statistic exceeds 
the critical value, the DSMB will discuss the continuation of the trial. 
 
At each interim analysis time point, a two-sided test to detect either an increase or decrease in the 
proportion of patients surviving without Grade II-IV GVHD at 114 days will be performed, as 
described above for the final analysis.  In order to preserve the over-all type I error rate at 5%, 
the critical value for the test statistic will be inflated above 1.96, the value that would be used if 
no repeated testing were used.  Equivalently, the nominal p-value at which an observed 
difference is declared significant will be reduced below 0.05.  The actual critical values and 
nominal p-values will be computed using statistical methods for group sequential testing with 
O’Brien Fleming boundaries.   
 
As an example, Table 5.3a shows the critical values and nominal p-values for tests conducted 
every six months starting with the 9th month after the study opens to enrollment.  The column 
labeled “followed to 114 days” shows the expected number of individuals who have reached the 
114-day post-randomization follow-up time point, assuming uniform accrual over a three-year 
period.  The fraction of patients followed to 114 days, as compared to a denominator comprised 
of the total sample of 312, quantifies the “statistical information” from which the critical values, 
nominal type I errors and cumulative type I error are computed.   

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Table 5.3a – Critical Values and Cumulative Type I Error 
 
Calendar 
Time 
(Months) 
Followed to 114 
Days 
Critical 
Value 
Nominal Type I 
Error 
Cumulative Type I 
Error 
9 
46 
5.029 
.0000 
.0000 
15 
98 
3.556 
.0004 
.0004 
21 
150 
2.903 
.0034 
.0038 
27 
202 
2.514 
.0094 
.0132 
33 
254 
2.249 
.0158 
.0290 
39.3 (Final) 
312 
2.053 
.0210 
.0500 
 
In practice, the rate of accrual or timing of DSMB meetings may not be as anticipated.  To 
permit necessary flexibility in scheduling interim analyses, the critical values will be recomputed 
to correspond to the actual available statistical information using the “use-function” approach of 
Lan and DeMets. 
 
5.3.2. Operating Characteristics of the Design  
 
Under the assumption that time to GVHD or death is exponentially distributed, the statistical 
power to reject the null hypothesis of equal 100-day GVHD-free survival is shown in Table 5.3b 
under a variety of scenarios, using the comparison of the Kaplan-Meier estimates.  These 
simulation results assume uniform accrual of patients over three years.  This table shows that the 
target sample size of 312 patients has 80% power to detect a 15% improvement in GVHD-free 
survival. 
 
Table 5.3b – Power to Reject the Null Hypothesis under Various Scenarios 
 
 
Proportion Surviving 
Power at Interim and Final Analyses 
Overall 
 
GVHD-Free at Day 114 
By Month of Scheduled Analysis 
N 
Standard 
Experiment 
9 
15 
21 
27 
33 
39.3 
Power 
312 
.60 
.60 
.000 
.001 
.004 
.011 
.018 
.021 
.055 
312 
.60 
.70 
.000 
.011 
.057 
.116 
.144 
.123 
.451 
312 
.60 
.75 
.001 
.044 
.177 
.248 
.207 
.125 
.802 
312 
.60 
.80 
.003 
.136 
.358 
.293 
.133 
.049 
.972 
* from simulation with 10,000 replications, assuming exponential time to failure 
 
 
5.3.3. Guidelines for Safety Monitoring 
 
The cumulative incidences of thrombotic microangiopathy and severe veno-occlusive disease 
(VOD) at 100 days post-transplant will be monitored separately for each arm.  Death without 
each type of toxicity will be considered as a competing risk for that event.  Refer to Section 3.2 
for definitions of these safety monitoring endpoints.  Monitoring will be performed monthly until 

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enrollment to that treatment arm is closed.  Each month, the null hypothesis that the cumulative 
incidence of thrombotic microangiopathy is less than or equal to 10% will be tested against the 
alternative that it is greater than 10%.  Similarly, the cumulative incidence of severe VOD will 
be tested against a null value of 10%.  Since the busulfan/cyclophosphamide conditioning 
regimen was removed early in the study due to toxicity, only patients who receive TBI-based 
conditioning regimens will be included in the safety monitoring analysis for severe VOD. 
 
A truncated Sequential Probability Ratio Test (SPRT) for a binomial outcome will be used for 
each endpoint, as described in greater detail below.  Note that in the absence of censoring, the 
binomial proportion of patients experiencing the toxicity prior to death is equal to the cumulative 
incidence. This sequential testing procedure conserves type I error across all of the monthly 
examinations for a single toxicity endpoint, but not across the multiple safety endpoints or two 
treatment arms.  Thus for a single endpoint and study arm, the type I error is approximately 5%, 
and across both safety endpoints and both treatment arms, the study-wise type I error is < 20%. 
 
The rationale for not conserving type I error across multiple safety endpoints and treatments is 
twofold.  First, adjusting the size of the test for multiple comparisons would reduce statistical 
power to detect adverse outcomes, which seems imprudent.  Secondly, the procedure is a 
guideline for requiring additional review by the Data and Safety Monitoring Board, and is not a 
formal “stopping rule” that would mandate automatic closure of study enrollment.   
 
The SPRT can be represented graphically.  At each monthly interim analysis, the total number of 
patients enrolled is plotted against the total number of endpoints observed in those patients, (e.g., 
patients with thrombotic microangiopathy).  The continuation region of the SPRT is defined by 
two parallel lines.  Only the upper boundary will be used for monitoring each treatment arm to 
protect against poor 100-day incidences of rates of thrombotic microangiopathy.  If the graph 
falls above the upper boundary, the SPRT rejects the null hypothesis, and concludes that there 
are more events of thrombotic microangiopathy than predicted by the observed number of 
patients enrolled on study.  Otherwise, the SPRT continues until enrollment to the treatment arm 
reaches the target goal.   
 
The usual measures of performance of an SPRT are the error probabilities  and  of rejecting 
H0 when  = 0 and of accepting H1 when  = 1, respectively, and the expected sample size 
E(N|i).  The tests to be used in this protocol were developed from the following SPRTs:   
 
 A SPRT contrasting 10% versus 20% 100-day cumulative incidence of thrombotic 
microangiopathy, with nominal type I and II errors of 5.5% and 20%, respectively.  The 
common slope of the parallel lines is 0.145 and the intercept for the upper boundary is 
3.301. 
 A SPRT contrasting 10% versus 20% 100-day cumulative incidence of severe VOD, with 
nominal type I and II errors of 5.5% and 20%, respectively.  The common slope of the 
parallel lines is 0.145 and the intercept for the upper boundary is 3.301. 
 

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Note that since the test uses only the upper boundary, and is truncated by a finite sample size, the 
size of the test will be slightly lower than the nominal level of 5.5% and closer to 5%.  
 
Table 5.3c demonstrates the stopping points for various numbers of enrolled patients for 
illustration. 
 
Table 5.3c – Sample Stopping Boundaries for SPRT 
 
Number of Patients 
Enrolled, n 
Stop if Number of 
Events in First n 
Patients is >= x 
Number of Patients 
Enrolled, n 
Stop if Number of 
Events in First n 
Patients is >=x 
15 
6 
90 
17 
30 
8 
105 
19 
45 
10 
120 
21 
60 
13 
135 
23 
75 
15 
150 
26 
 
Note that because there is a lag from transplant to complete follow up (100 days), the stopping 
rule may be triggered before complete follow up is available.  For example, there may be 6 
events in the first 15 patients even though the first 15 patients do not all have 100 days of follow 
up.  This implies that the plot of the number of patients transplanted against events observed in 
those patients may change for earlier accrual points as follow up is completed, and will therefore 
be updated each month.   
 
The actual operating characteristics of the truncated test, shown in Table 5.3d, were determined 
in a simulation study that assumed uniform accrual of 156 individuals over a three-year time 
period.  No assumptions are made about the time to event distribution for either the toxicity 
event of interest or the competing event of death prior to observing that toxicity.  The monitoring 
rule is based only on the binomial outcome of whether a patient experienced the toxicity by 100 
days and prior to death.  Because the simulations only use a binomial outcome, the mean month 
stopped is calculated based on the accrual time of the patient triggering the stopping rule + 100 
days for complete follow up.  In practice, because of the potential for the stopping rule to be 
triggered prior to complete follow up of a particular patient, the actual mean month stopped may 
be earlier than shown in the simulation results.  However, this is difficult to estimate because it is 
dependent on assumptions about the time to event distribution for both the toxicity event and the 
competing risk, as well as the dependence between them.   
 

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Table 5.3d – Operating Characteristics of Sequential Testing Procedure 
 from a Simulation Study with 10,000 Replications 
 
Thrombotic Microangiopathy 
True 100-Day Rate 
10% 
15% 
20% 
Probability Reject Null 
0.052 
0.484 
0.934 
Mean Month Stopped 
38.1 
30.3 
18.6 
Mean # Endpoints in 100 Days 
15.2 
18.1 
14.9 
Mean # Patients Enrolled 
151.4 
120.3 
73.9 
 
 
Severe VOD 
True 100-Day Rate 
10% 
15% 
20% 
Probability Reject Null 
0.052 
0.484 
0.934 
Mean Month Stopped 
38.1 
30.3 
18.6 
Mean # Endpoints in 100 Days 
15.2 
18.1 
14.9 
Mean # Patients Enrolled 
151.4 
120.3 
73.9 
 
For example, the testing procedure for thrombotic microangiopathy rejects the null hypothesis in 
favor of the alternative 5% of the time when the true 100-day incidence is 10%, and 93% of the 
time when the rate is 20%.  When the true 100-day toxicity incidence is 20%, on average, the 
Data and Safety Monitoring Board will be consulted 18.6 months after opening, when 14.9 
events have been observed in 73.9 patients.   
 
5.4. 
Demographic and Baseline Characteristics 
 
Demographics and baseline characteristics will be summarized for all patients.  Characteristics to 
be examined are: age, gender, race/ethnicity, performance status, primary disease, risk status, 
donor age, donor gender, and donor ethnicity.  Between group comparisons will be performed 
for continuous variables via a t-test and for categorical variables, via the chi-square test. 
 
5.5. 
Analysis of Secondary Endpoints 
 
1. Time to stable neutrophil and platelet engraftment after transplantation; cumulative 
incidence curves will be computed by treatment.  A log-rank test will be used to compare 
the two arms.  A Cox proportional hazards model will be fit to control for important 
prognostic variables. 
2. Time to Grades II-IV or Grades III-IV acute GVHD; cumulative incidence curves 
will be computed by treatment.  A log-rank test will be used to compare the two arms.  A 
Cox proportional hazards model will be fit to control for important prognostic variables. 
3. Mucositis severity; median (range) and maximal mucositis scores will be summarized by 
treatment arm and compared using a Mann-Whitney Wilcoxon test. 

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4. Time to first hospital discharge; cumulative incidence curves will be computed by 
treatment.  A log-rank test will be used to compare the two arms.  A Cox proportional 
hazards model will be fit to control for important prognostic variables. 
5. Incidence of infection; cumulative incidence curves will be computed by treatment.  A 
log-rank test will be used to compare the two arms.  A Cox proportional hazards model 
will be fit to control for important prognostic variables. 
6. Incidence of CMV reactivation; cumulative incidence curves will be computed by 
treatment.  A log-rank test will be used to compare the two arms.  A Cox proportional 
hazards model will be fit to control for important prognostic variables. 
7. Incidence of thrombotic microangiopathy; cumulative incidence curves will be 
computed by treatment.  A log-rank test will be used to compare the two arms.  A Cox 
proportional hazards model will be fit to control for important prognostic variables. 
8. Time to chronic GVHD; cumulative incidence curves will be computed by treatment.  A 
log-rank test will be used to compare the two arms.  A Cox proportional hazards model 
will be fit to control for important prognostic variables. 
9. Relapse of primary malignant disease in distinct disease subgroups; cumulative 
incidence curves will be computed by treatment.  A log-rank test will be used to compare 
the two arms.  Also, a Cox proportional hazards model will be fit to control for important 
prognostic variables.  All patients will be followed for a minimum of one year post-
transplant for relapse. 
10. Relapse-free and overall survival; Kaplan-Meier curves will be computed by treatment.  
A log-rank test will be used to compare the two arms.  Also, a Cox proportional hazards 
model will be fit to control for important prognostic variables.  All patients will be 
followed for a minimum of one year post-transplant for mortality and relapse. 
Two-sided tests will be used throughout.  A p-value of 0.05 or less will be considered 
statistically significant. 
 
5.6. 
Safety Analysis 
 
All entered patients will be included in the safety analysis: 
1. Serious Adverse Events: All reported serious adverse events potentially associated with 
study drug will be carefully examined with respect to the severity and relationship to 
study drug.  Adverse events will be graded according to the NCI Common Terminology 
Criteria for Adverse Events Version 3.0.  The incidence for each reported study drug 
associated adverse event delineated in Section 2.8.2 will be presented for each group. 
2. Clinical Laboratory Tests: Laboratory tests will be performed during the study. 
Descriptive statistics will be calculated for each laboratory test.  Abnormal laboratory test 
results will be summarized in the report.  Laboratory data will be cross-classified into 
below, within, and above the reference range according to their values at screening and 
during the study.  
  

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APPENDIX A 
 
SAMPLE TAPERING SCHEDULES 
FOR SIROLIMUS AND TACROLIMUS 
 
 

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APPENDIX A 
 
SAMPLE TAPERING SCHEDULES FOR SIROLIMUS AND TACROLIMUS 
 
 
EXAMPLE 1:  
 
 
Starting doses: Sirolimus 4 mg qd 
 
 
 
 
Tacrolimus 2 mg bid 
 
 
 
Day 100 
 
Sirolimus 
3 mg  
Tacrolimus  
2 mg bid 
 
 
Day 100 + 2 Weeks 
Sirolimus  
3 mg  
Tacrolimus  
1.5 mg bid 
 
 
Day 100 + 4 Weeks 
Sirolimus  
2 mg  
Tacrolimus  
1.5 mg bid 
Day 100 + 6 Weeks 
Sirolimus  
2 mg  
Tacrolimus  
1.0 mg bid  
Day 100 + 8 Weeks 
Sirolimus  
1 mg  
Tacrolimus  
1.0 mg bid  
Day 100 + 10 Weeks Sirolimus  
1 mg  
Tacrolimus  
0.5 mg bid  
Day 100 + 12 Weeks Sirolimus  
0 mg  
Tacrolimus  
0.5 mg bid  
Day 100 + 14 Weeks Sirolimus  
0 mg  
Tacrolimus  
0 mg bid  
 
EXAMPLE 2: 
 
 
Starting doses: Sirolimus 2 mg qd 
 
 
 
 
Tacrolimus 3 mg bid 
 
Day 100 
 
Sirolimus 
2 mg  
Tacrolimus  
2.0 mg bid 
 
 
Day 100 + 2 Weeks 
Sirolimus  
1.5 mg  
Tacrolimus  
2.0 mg bid 
 
 
Day 100 + 4 Weeks 
Sirolimus  
1.5 mg  
Tacrolimus  
1.0 mg bid 
Day 100 + 6 Weeks 
Sirolimus  
1 mg  
Tacrolimus  
1.0 mg bid  
Day 100 + 8 Weeks 
Sirolimus  
1 mg  
Tacrolimus  
0.5 mg bid  
Day 100 + 10 Weeks Sirolimus  
1 mg qod* 
Tacrolimus  
0.5 mg bid  
Day 100 + 12 Weeks Sirolimus  
1 mg qod 
Tacrolimus  
0 mg bid 
Day 100 + 14 Weeks Sirolimus 
0 mg  
Tacrolimus  
0 mg  
 
* 1 mg qod may be replaced by 0.5 mg qd for sirolimus, however, this formulation does not exist 
in capsule format.  Caplets may be manually split. 
 

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APPENDIX B 
 
INFORMED CONSENTS 
 
 
 Participant Consent 
 
 Donor Consent 

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IRB # 
 
 
Participant Consent 
Page 1 of 19 
Participant initials ______ 
 
Informed Consent to Participate in Research 
 
 
 
 
 
 
 
 
 
 
Principal Investigator Contact Information 
(Insert contact information for PI at your site) 
 
 
Study Sponsor: This study is sponsored by the National Institutes of Health (NIH) by providing 
financial support through the Blood and Marrow Transplant Clinical Trials Network (BMT 
CTN). 
 
 
Introduction 
This is a clinical trial, which is a research study to answer specific medical questions.  The 
information from this study will help future patients.  The Study doctor (the person in charge of 
the research) will explain the clinical trial to you.  Clinical trials include only people who choose 
to join the study.  
 
Please take your time to decide if you want to join this study.  Some people find it helpful to talk 
about the study with their family and friends before they make a decision.  It may also be useful 
to talk with your doctor and other people on your health care team about the study.  If you have 
questions or want to know more about the study, you can ask them for more information. 
 
You are asked to join this study because: 
1. You have a disease that can be treated by a peripheral blood stem cell transplant; and,  
2. You have a brother or sister who has agreed to be your donor.  
 
Before you decide whether or not to join the study, please read the information below.  Feel free 
to ask questions to understand your rights and protections.  Participating in this study is your 
choice.  If you decide not to be in this study, you and your doctor will discuss other treatment 
options. 
 

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Participant initials ______ 
Why is this study being done? 
Stem cell transplantation is a standard therapy for acute and chronic leukemias and 
myelodysplastic disorders.  A common problem that may occur after a stem cell transplant is a 
condition known as graft-versus-host disease (GVHD).  The word “graft” refers to the donor 
blood cells that you will receive during your transplant.  The word “host” refers to the person (in 
this case, you) receiving the cells.  GVHD is a complication where the donor graft attacks and 
damages some of your (the transplant recipient's) tissues.   
 GVHD can cause skin rash, intestinal problems such as nausea, vomiting, or diarrhea,   
 It may also damage your liver and cause hepatitis or jaundice.   
 GVHD may also increase your risks of infection. 
 
The purpose of this study is to compare two combinations of medications to see which is better 
at preventing GVHD.  These combinations of medications in this study are: 
 
A- Sirolimus and tacrolimus  
B- Methotrexate and tacrolimus. 
 
Doctors want to know if combination A is better than combination B or if they give the same 
results.  The study will help doctors make GVHD treatment choices for future transplant patients. 
 
 
How many people will take part in the study? 
About 312 people throughout the country will take part in this study.  Each study group will have 
156 patients.   
 
 
What will happen if I take part in this research study? 
Before you begin the study -- You will need to have several exams, tests or procedures to find 
out if you can be in the study.  These exams, tests or procedures are part of regular cancer care 
and may be done even if you do not join the study.  If you have had some of them recently, they 
may not need to be repeated.  This will be up to your doctor with the study. 
 Medical history and physical exam, including height and weight 
 Blood tests  
 Urine tests 
 Heart function tests 
 Lung tests, including a Pulmonary Function Test (PFT) 
 Bone marrow biopsies and aspirates 

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 If you are a woman able to have children, a pregnancy test will also be performed, using 
a blood sample.  If you are pregnant, you will not be able to take part in this study. 
 
Before your transplant, your doctor will choose from one of two conditioning regimens.  The 
conditioning regimen prepares your body for transplant.  It uses treatments such as chemotherapy 
and radiation to destroy the cancer cells and the cells that make up your immune system.  Your 
doctor will decide which conditioning regimen you will receive before you join one of the study 
groups.  
 
During the study -- If the exams, tests and procedures show that you can be in the study, the next 
step is for you to be randomized into one of the study groups described below.  Randomization 
means that you are put into a group by chance, similar to the flip of a coin.  A computer program 
will place you in one of the study groups.  Neither you nor your doctor can choose the group you 
will be in. 
 
You will begin your conditioning chemotherapy and/or radiotherapy several days prior to the 
transplant.  The treatments to prevent GVHD will begin three days before transplantation for 
both study groups.   
 
A. Sirolimus/tacrolimus group:  
 You will take a sirolimus pill by mouth once per day.    
 Tacrolimus will be given by intravenous infusion (through your vein) every day 
while in the hospital.   
 Before you leave the hospital the tacrolimus will be changed to a pill that you will 
take by mouth.   
 These medications will be continued for at least 100 days from the time of 
transplantation.  
 The amount of drug given will slowly be decreased and eventually stopped.  This 
process occurs over several months. 
 
B. Methotrexate/tacrolimus group:  
 Tacrolimus will be given by intravenous infusion (through your vein) every day 
while in the hospital.   
 Before you leave the hospital the tacrolimus will be changed to a pill that you will 
take by mouth.  
 You will take methotrexate on 4 separate days.  The methotrexate will be given by 
intravenous infusion on the 1st, 3rd, 6th and 11th days after your transplant. 
 The tacrolimus will be continued for at least 100 days from the time of 
transplantation.   

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 The amount of drug given will slowly be decreased and eventually stopped.  This 
process occurs over several months. 
 
Peripheral blood stem cells from your donor will be given to you 3 days after starting the GVHD 
treatments.  
 
Following the transplant, you will have the following standard tests and evaluations: 
 Medical history  
 Physical examination, including height and weight 
 Blood tests  
 Urine tests 
 
You will have the following study tests performed: 
 Blood tests to measure the levels of sirolimus and tacrolimus  
 Blood tests to measure your cholesterol and triglycerides (fat) levels 
 Oral exams to measure any side effects that occur in your mouth and throat 
(mucositis) 
 
You will be asked to return to the transplant center for regular follow up care.  The standard tests 
and study tests will be done at that time. 
 
 
How long will I be in the study? 
You will be in the study for up to two years.  You will need to take medication for at least 3 
months and possibly longer depending on how long it takes to gradually take you off of the 
medication.  You will also need to answer questions about your medical health at regular times 
for up to two years.  
 
Follow-up for your transplant will last as long as you require care.  However, we would like to 
keep track of your medical condition for the rest of your life by contacting you and the doctor 
providing your regular medical care by phone or mail once a year.  Keeping in touch with you 
and checking on your condition every year helps us look at the long-term effects of the study and 
transplantation in general.  Many transplant centers include this type of long-term follow-up as 
part of their regular medical care.  It is not necessary for you to agree to follow-up for longer 
than two years to participate in this study. 
 
 
Can I stop being in the study? 
Yes. You can decide to stop at any time.  Tell your doctor if you are thinking about stopping or 
decide to stop.  He or she will tell you how to stop safely.  

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It is important to tell your doctor if you are thinking about stopping so any risks from the 
medications can be evaluated.  Another reason to tell your doctor that you are thinking about 
stopping is to discuss what follow-up care and testing could be most helpful for you. 
 
 
Can the Principal Investigator withdraw me from the study? 
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. 
 The study is cancelled by the Food and Drug Administration (FDA) or the National 
Institutes of Health (NIH). 
 
 
What side effects or risks can I expect from being in the study? 
Everyone taking part in the study will be watched carefully for any side effects.  However, 
doctors don't know all the side effects that may happen.  Side effects may be mild or very 
serious.  Your health care team may give you medicines to help lessen side effects.  In some 
cases, side effects can be serious, long lasting, or may never go away. 
 
You should talk to your study doctor about any side effects that you have while taking part in the 
study.  
 

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Potential Side Effects and Risks from Study Drugs 
Sirolimus and Tacrolimus 
Likely (10-25%) 
Less Likely but Serious (< 10%) 
 Elevation of blood lipids (fats, including 
cholesterol and triglycerides) 
 Mild reduction in blood counts, including 
platelets, red cells (anemia) and white 
blood cells 
 Low blood potassium 
 Muscle aches and pains 
 Fluid retention  
 High blood pressure 
 Tremor  
 Swollen gums 
 Increased facial hair 
 
 Lung inflammation 
 Reversible kidney damage, which may 
lead to acute kidney failure and require 
hospitalization, possibly dialysis and 
may lead to permanent kidney failure 
 Thrombotic Microangiopathy, a syndrome 
comprised of abnormal kidney function 
and destruction of red blood cells and 
platelets.  Temporary dialysis may be 
required for the kidney failure 
 Neurological dysfunction, including a 
decreased level of consciousness, coma, 
blindness and seizures 
 Veno-occlusive disease, which causes 
severe damage to the liver 
 
Methotrexate and Tacrolimus 
Likely 
Less Likely but Serious 
 
Delay in the time it takes for your blood 
counts to recover.  This may increase 
the risk of infection and bleeding after 
transplantation, may increase the 
number of transfusions required during 
the transplantation and may prolong 
your hospital stay. 
 
Worsening of mucositis (inflammation 
of the lining of the mouth, throat and 
stomach) 
 
Low blood potassium 
 
Muscle aches and pains 
 
Fluid retention  
 
High blood pressure 
 
Tremor  
 
Swollen gums 
 
Increased facial hair 
 Lung inflammation 
 Reversible kidney damage, which may 
lead to acute kidney failure and require 
hospitalization, possibly dialysis and 
may lead to permanent kidney failure 
 Thrombotic Microangiopathy, a syndrome 
comprised of kidney dysfunction and 
destruction of red blood cells and 
platelets.  Temporary dialysis may be 
required for the kidney failure 
 Neurological dysfunction, including a 
decreased level of consciousness, coma, 
blindness and seizures 
 
 

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These side effects usually get better completely after you stop taking the drugs, but some long-
term problems, such as kidney damage, may occur. 
 
A standard combination of medications to prevent GVHD is the combination of tacrolimus and 
methotrexate.  We are trying to determine if the combination of sirolimus and tacrolimus is as 
effective as tacrolimus and methotrexate.  Because this is a comparative trial to study how to 
prevent GVHD, there is a risk that you will receive a less effective GVHD prevention program 
than the standard combination.  Even though this study is trying to prevent GVHD from 
developing, there is a chance that you will still develop GVHD, no matter which combination 
you receive.  All study participants who develop GVHD will be treated for GVHD.  The 
treatment you receive will be up to your doctor. 
 
Refer to Attachment A for additional risks and toxicities for all transplant patients.  
 
 
Are there benefits to taking part in the study? 
There may or may not be direct benefits to you from participating in this study.  We hope that 
information gathered in this trial will benefit future transplant patients. 
 
 
What other choices do I have if I do not take part in the study? 
If you do not want to join this study, you should know your other options.  These options 
include: 
 
Bone marrow transplantation 
 
Peripheral Blood Stem Cell Transplantation using standard treatment (but not included in 
study) 
 
No transplant 
 
Other chemotherapy 
 
No therapy for your cancer with care to help you feel comfortable 
 
You should know about your treatment choices before you decide if you will take part in this 
study. 
 
 
What are the costs of taking part in this study? 
You and/or your insurance company will pay all standard care relating to your stem cell 
transplant.   
 
You will not be billed for any tests or procedures that are only for research.  
 
You will not be paid to be in this study.  

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Sirolimus will be provided free of charge by Wyeth, Inc for at least 6 months.  At that time, you 
or your insurance carrier will responsible for the costs of this medication.  Methotrexate and 
tacrolimus will be the responsibility of you or your insurance carrier.   
 
The companies that make the drugs used in this study did not plan or design this clinical trial.  
They will also not have a part in analyzing the results of this study. 
 
For more information on clinical trials and insurance coverage, you can visit the National Cancer 
Institute’s Web site at http://cancer.gov/clinicaltrials/understanding/insurance-coverage.  You 
can print a copy of the “Clinical Trials and Insurance Coverage” information from this Web site. 
 
Another way to get the information is to call 1-800-4-CANCER (1-800-422-6237) and ask them 
to send you a free copy. 
 
 
What if I am injured as a result of being in this study? 
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. If you have any questions about study-related injuries, 
you may contact [insert name of person at institution] at [insert phone number]. 
 
 
What are my rights if I take part in this study? 
Taking part in this study is your choice.  You may choose either to take part or not to take part in 
the study.  If you decide to take part in this study, you may leave the study at any time.  No 
matter what decision you make, there will be no penalty to you and you will not lose any of your 
regular benefits.  Leaving the study will not affect your medical care.  You can still get your 
medical care from our institution.  
 
We will tell you about new information or changes in the study that may affect your health or 
your willingness to continue in the study. 
 
In the case of injury resulting from this study, you do not lose any of your legal rights to seek 
payment by signing this form.  
 
If you have questions about your rights as a study subject, you may call the Institutional Review 
Board (IRB) office at [insert phone number]. 
 

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Will my medical information be kept private? 
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) and 
the National Marrow Donor Program (NMDP), to the Food and Drug and Administration (FDA), 
Data Coordinating Center of the Blood and Marrow Transplant Clinical Trials Network (BMT 
CTN), the EMMES Corporation (which is helping to coordinate this study) 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. 
 
 
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 A Phase III Randomized, Multicenter Trial 
Comparing  Sirolimus/Tacrolimus with Tacrolimus/Methotrexate as GVHD Prophylaxis 
After HLA-Matched, Related Peripheral Blood Stem Cell Transplantation. 
 
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 transplantation (e.g., blood tests, biopsy results).  
 
                                                   
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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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 Investigators and the researcher’s staff, including Dr. Corey Cutler and 
Dr. Joseph Antin, Study Chairpersons and staff/laboratories at Dana Farber Cancer 
Institute 
 Staff/laboratories identified in the protocol for the evaluation of other laboratory 
samples 
 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 and 
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) 
 U.S. government agencies responsible for overseeing public health concerns such as 
the Centers for Disease Control (CDC) and federal, state and local health 
departments. 
 Others: 
 
 
 
 
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 

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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. DNA from your stored blood sample might be used in genome-wide association (GWA) 
or pharmacogenomics studies for a future project either done or supported by the 
National Institutes of Health (NIH).  Genome-wide association studies are a way for 
scientists to identify genes involved in human disease.  This method searches the genome 
for small genetic changes that are more common in people with a particular disease than 
in people without the disease.  Each study can look at hundreds of thousands of genetic 
changes at the same time.  Researchers use data from this type of study to find genes that 
may add to a person’s risk of developing a certain disease.  Pharmacogenomics studies 
are similar genetic tests but look specifically at genes related to how the body breaks 
down medications.  
i. If your coded samples are used in such a study, the researcher is required to add your test 
results and sample information into a shared, public research database.  This public 
database is called the NIH Genotype and Phenotype Database and it is managed by the 
National Center for Biotechnology Information (NCBI).  The NCBI will never have any 
information that would identify you, or link you to your information or research samples.  
A new federal law (2009), called the Genetic Information Nondiscrimination Act (GINA) 
generally makes it illegal for health insurance companies, group health plans, and 
employers of 15 or more persons to discriminate against you based on your genetic 
information.  Health insurance companies and group health plans may not request your 
genetic information that we get from this research.  This means that they must not use 
your genetic information when making decisions regarding insurability.  Be aware that 
this new federal law will not protect you against genetic discrimination by companies that 
sell life insurance, disability insurance, or long-term care insurance. 
j. This authorization does not have an expiration date. 
 
************************************************ 
About Using Blood for Research 
Please note: This section of the informed consent form is about future research studies that 
will be done using blood samples from people who are taking part in the main study described 
above.  You may give small blood samples for these future research studies if you want to.  
You can still be a part of the main study even if you say 'no' to giving blood samples for future 

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research studies.  You can say "yes" or "no" to giving blood samples for future research 
studies.  Please mark your choice at the end of this section. 
 
We would like to have six small (3 teaspoons or 16 mL) blood samples for future research.  
Patients less than 12 years old will provide 1½ teaspoons or 8 mL).  If you agree, these samples 
will be obtained at the time other blood samples are drawn on 6 occasions pre-transplant and on 
Day 28, 100, 180, 365 and 730.  They will be kept and may be used in research to learn more 
about GVHD, cancer and other diseases.  Usually the blood can be drawn from your central 
venous catheter at the time of the other blood collections.  If this is not possible, it will be taken 
from a vein.  When the sample is given to investigators for research, no information about your 
name, address, phone number or other information that will let the researcher know who you are 
will be provided. 
 
The samples collected for research purposes will be sent to the National Heart, Lung, and Blood 
Institute (NHLBI) sample repository in Maryland.  The samples 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 and Coordinating Center for the Blood and Marrow Transplant Clinical 
Trials Network (BMT CTN).  The staff at the repository where your sample is being stored does 
not have a link to this code.  Your samples will be stored at this repository until the samples have 
been used for the research tests or until the end of the study.  Any research performed on the 
samples must first be approved by an advisory panel at the NHLBI. 
 
The research that may be done with your blood is not designed specifically to help you.  It might 
help people who have GVHD, cancer and other diseases in the future.  
 
Reports about research done with your blood will not be given to you or your doctor.  These 
reports will not be put in your health record.  The research will not have an effect on your care.  
 
Things to Think About: The choice to let us have blood samples for future research is up to 
you.  No matter what you decide to do, it will not affect your care.  
 
If you decide now that your blood can be kept for research, you can change your mind at any 
time.  Just contact your study doctor and let him or her know that you do not want us to use your 
blood sample.  Then any blood that remains will no longer be used for research.  
 
In the future, people who do research on these blood samples may need to know more about your 
health.  While the study doctor or others involved in running this study may give the researchers 
reports about your health, it will not give them your name, address, phone number, or any other 
information that will let the researchers know who you are.  
 
Sometimes blood is used for genetic research (about diseases that are passed on in families).  
Even if your blood is used for this kind of research, the results will not be put in your health 
records.  

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Participant initials ______ 
 
Your blood will be used only for research and will not be sold.  The research done with your 
blood may help to develop new products in the future.  
 
Benefits: The benefits of research using blood include learning more about what causes cancer 
and other diseases, how to prevent them, and how to treat them.  
 
Risks: The greatest risk to you is the release of information from your health records.  We will 
do our best to make sure that your personal information will be kept private.  The chance that 
this information will be given to someone else is very small.  
 
Making Your Choice: Please read each sentence below and think about your choice.  After 
reading each sentence, please indicate your choice below.  If you have any questions, please talk 
to your doctor or nurse, or call our research review board at ___________________.  
 
No matter what you decide to do, it will not affect your care. 
 
 Yes, I agree to have small blood samples drawn for future research. 
 
 No, I do not agree to have small blood samples drawn for future research. 
 
 ____________________________________   _____________________________  
Signature 
Date 
************************************************ 
 
If you have any questions about this study, you may contact the study Principal Investigator 
listed on the first page of this form. 
 
If you have questions about your rights as a research participant, you may contact (INSERT 
CONTACT INFORMATION) 
 

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CONSENT AND ASSENT INSTRUCTIONS 
 
 
CONSENT: Subjects 18 years and older must sign on the subject line below.  For subjects 
under 18, consent is provided by the Legally Authorized Representative. 
 
ASSENT: 
Is required for subjects under the age of 18, using the Assent Section on the 
following page. 
 
I have been informed about this study’s purpose, procedures, possible benefits and risks.  I 
have been given the chance to ask questions.  My questions have all been answered 
satisfactorily.  I understand that I can ask other questions at any time. 
 
I voluntarily agree to take part, or to allow my child to take part, in this study. 
 
By signing this consent form, I have not given up any of the legal rights that I (my child) 
otherwise would have as a subject in a research study. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Subject‘s Signature 
 
 
 
 
 
Date 
 
 
If you are not the subject, please print your name 
 
 
 
 
 
and indicate one of the following: 
 
 
 
The subject’s parent  
 
     The subject’s guardian 
 
 
A surrogate 
 
 
 
     A durable power of attorney 
 
 
A proxy 
 
 
 
     Other, please explain: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Legally Authorized Representative Signature 
 
Date 
 
 
As a representative of this study, I have explained the purpose, the procedures, the benefits, 
and the risks that are involved in this research study: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Signature of person conducting informed consent  
Date 
 

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ASSENT SIGNATURES:  For subjects under the age of 18 years. 
 
Assent of Minor 
 
I have been told what I will be asked to do if I am in this study.  I have been told that I don’t 
have to be in this study.  I may quit the study at any time, and no one will be mad at me.  I 
have had a chance to discuss the study and ask questions.  My questions have been answered.  
I agree to be in the study and do what I am asked to do so long as I continue in the study. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Signature of Minor 
 
 
 
Date 
 
 
Age (years) 
Study Personnel 
 
 
I have explained the purposes, procedures, and risks involved in this research study in detail 
to: 
 
 
 
 
 
 
 
 
 
 
 
 
 
          
Print name(s) of Parents/Authorized Consenting Party, and  
 
who in my opinion   
IS/ 
IS NOT capable of assenting to participate in this study. 
 
 
Print child’s name 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Signature of Person Conducting Assent   
 
Date 
 
 

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ATTACHMENT A 
 
Additional Risks and Toxicities Related to the Standard Transplant Procedure 
 
 
There are certain risks related to a peripheral blood stem cell (PBSC) transplant.  There are risks 
from the medications and/or irradiation therapy you will receive as part of the conditioning for 
the transplant, and risks related to the transplant itself.  Most of these risks and side effects are 
listed below, but they will vary from person to person.  There may be more risks involved with 
transplantation, and there may be more side effects.  Your doctor may give you medications to 
lessen some of the side-effects.  
 
Risks Related to the Transplant Conditioning Regimen 
 
You and your doctor will choose a conditioning regimen that may include some of the 
following therapies. 
 
Cyclophosphamide (Cytoxan): This is a common medication used to treat cancer.  This 
medication kills cancer cells by stopping them from growing.  Cyclophosphamide may cause you 
to have diarrhea (loose stools), nausea (feeling sick to your stomach), vomiting (throwing up), 
short-term hair loss, short-term bladder problems, and, at times, bleeding from the bladder.  A 
few patients may have bladder damage and bleeding for a longer time.  You will be given large 
amounts of a sterile solution through your central line to protect your bladder.  A bladder 
catheter (thin plastic tube) may be inserted into your bladder, if your physician thinks that it can 
help you.  Cyclophosphamide slows the making of new red blood cells, white blood cells, and 
platelets.  This causes a risk of infection and/or severe bleeding until the transplanted donor cells 
begin to work in you.  You will get blood transfusions as needed.  Cyclophosphamide also 
lowers your defense system.  As a result, you may have more infections for several months after 
transplant.  In a small number of patients, cyclophosphamide can damage the heart muscle 
causing heart failure.  Sometimes cyclophosphamide causes abnormal heart function.  If this 
occurs you may have shortness of breath and have fluids build-up in your body.  This medication 
can also cause the lungs to become scarred.  If scarring of the lungs occurs it will usually happen 
three to six months after you receive the medication.  Scarring of the lungs can cause death.  
Cyclophosphamide can damage the male (testes) or female (ovaries) sex glands.  In men, the 
number of sperm may be reduced but you would still be able to have intercourse.  Women who 
are still menstruating may have irregular periods or may no longer have any periods.  Whether 
you are a man or woman, this medication will likely greatly decrease your chances of being able 
to have a child.  It is not known whether the use of cyclophosphamide will cause more side 
effects or problems with your health in the future. 
 
Etoposide (VP-16): This medication disrupts the growth of cancer cells and destroys them.  
While taking etoposide you most likely will have diarrhea (loose stools), nausea (feeling sick to 

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your stomach), vomiting (throwing up), lower white blood cell count that increases your risk of 
infection, lower platelet count that increases your risk of bleeding, hair loss, stopping of 
menstrual periods in women, temporary reduced or no sperm production in men.  Less likely side 
effects that you may experience are fatigue, sores in the mouth or on the lips, fever, rash, and 
loss of appetite. Rare side effects that you may experience are damage to your heart, which may 
result in a heart attack or heart failure.  Your blood pressure may fall during the infusion of 
etoposide, and etoposide rarely causes numbness and tingling in the fingers and toes, in a 
condition termed, neuropathy.  This may be reversible.  Etoposide can rarely cause changes in 
your liver function. 
 
Total Body Irradiation (TBI):  TBI may cause you to have diarrhea (loose stools), nausea 
(feeling sick to your stomach), vomiting (throwing up), and painful swelling of the saliva gland 
for a few days.  You may also experience short-term hair loss.  TBI kills both sick and normal 
marrow, leading to a lack of red blood cells, white blood cells, and platelets.  The short-term loss 
of these blood cells could cause you to become anemic, develop an infection, and/or bleeding.  
This will continue until the transplanted donor cells begin to work in you.  You will get blood 
transfusions as needed.  There is a risk that cataracts (cloudiness) may develop in your eyes.  
This may mean partial loss of vision, and you may need contact lenses or surgery to remove the 
cataracts.  The TBI dose used will probably result in sterility (not being able to have children.)  It 
is not known whether the use of TBI will cause more side effects or problems with your health in 
the future. 
 
The conditioning regimens allowed by this protocol are likely to cause women to enter pre-
mature menopause.  Men who receive these conditioning regimens are unlikely to be able to 
father children.  However, there is no guarantee that this will happen to you, so you should 
discuss the need for birth control with your doctor.  In any case, you are not protected from 
sexually transmitted diseases as a result of having these treatments. 
 
Risks Related to the Infusion Peripheral Blood Stem Cells (PBSC) 
 
The infusion of bone marrow or PBSC usually has few side effects.  Rarely the infusion may 
cause a headache, chest pain or trouble breathing, a slight fever, or blood in the urine.  Very 
rarely, it may cause an allergic reaction (which shows up as a severe drop in blood pressure and 
may cause loss of consciousness).   
 
Risks Related to the Transplant Procedure 
 
The following risks are not specifically related to any one medication or the transplanted donor 
cells, but they are risks that are a part of the transplant procedure. 
 
Bleeding: Platelets help your blood to clot.  Your platelets will be low until the new bone 
marrow grows and, as a result, bleeding may occur.  This can be minor bleeding, such as 
nosebleeds or bruising, but more serious, life-threatening bleeding in the lungs and brain can 

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occur if the platelet count remains low.  Usually, there is success in preventing major bleeding 
problems with transfusions of platelets.  However, some patients may not respond well to 
transfused platelets and may be at serious risk for bleeding.  
 
Veno-Occlusive Disease (VOD): This can occur as a result of high dose chemotherapy, 
irradiation therapy, or both.  VOD causes severe damage to the liver.  Symptoms include 
jaundice (yellowing of the skin and eyes), weight gain, and extra fluid build-up in the abdominal 
cavity and other parts of the body.  It can often be managed successfully, and completely resolve.  
However, complications can arise that may be fatal. 
 
Mouth Sores and Diarrhea: The large doses of medicines and irradiation cause irritation in the 
lining of the mouth and intestines.  This can result in painful mouth sores and diarrhea.  If you 
have severe mouth sores you will be given medicine to help control the pain.  If your mouth 
sores are severe you may not be able to eat normally until the sores are healed.  Mouth sores get 
better when the white blood count starts to rise, and engraftment occurs. 
 
Capillary Leak Syndrome: This may occur as a result of chemotherapy and irradiation therapy.  
The blood vessels may become ‘leaky’ and fluid enters the abdominal cavity, lungs, and other 
tissues.  You may gain water weight and not go to the bathroom as often as you normally do.  
Capillary leak syndrome can be difficult to manage if extra fluid enters the lungs and causes 
difficulty breathing.  You may die if there is continued fluid collection in the lungs.  
 
Unexpected Organ Damage and Other Side Effects: It is possible you may experience 
unexpected, life-threatening heart, lung, kidney, or liver damage as a result of the transplant.  
Occasionally, the high doses of chemotherapy and radiation cause severe lung damage that 
cannot always be treated.  If this happens, you may need to use oxygen or even a respirator.  The 
lung damage may get worse and be life-threatening.  Rarely, multi-organ failure (such as lung 
and kidney failure) may occur, which is often fatal.  A form of kidney failure, called thrombotic 
microangiopathy may result in reversible or irreversible kidney damage, and may require 
temporary or permanent dialysis therapy. 
 
Late Effects: You may experience side effects that occur several months to many years after 
your transplant.  You may experience poor function of the thyroid gland, requiring you to take 
thyroid medication.  Poor thyroid function can result in fatigue, weight gain, hair loss, 
depression, dry skin and dry hair; however, this is easily corrected by the medication.  As a result 
of irradiation, cataracts may occur earlier in life compared to a person who had not had a 
transplant.  If you develop cataracts (cloudiness in the eyes) they may require treatment.  It is 
rare, but your kidneys could be affected, causing anemia (low red blood cell count) or high blood 
pressure.  There is also a risk you may develop a second cancer as a result of the chemotherapy, 
irradiation and/or underlying disease.  If secondary cancers occur they generally do not occur 
until 10 to 15 years after the transplant.  The long-term effects upon heart, lung, and brain are 
unknown. 
 

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Fluid Build-up: You will receive intravenous fluids during the transplant process and you may 
have difficulty eliminating this fluid.  Furosemide is a medication that is often given to help 
eliminate this excess fluid.  This medication may cause hearing loss and loss of body chemicals 
such as potassium and sodium. 
 

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Informed Consent to Participate in Research  
 
Study Title: A Phase III Randomized, Multicenter Trial Comparing Sirolimus/Tacrolimus with 
Tacrolimus/Methotrexate as GVHD Prophylaxis After HLA-Matched, Related Peripheral Blood 
Stem Cell Transplantation 
 
Principal Investigator Contact Information: (Insert contact information for PI at your site.) 
 
Introduction 
We invite you to take part in a research study sponsored by the National Institutes of Health 
(NIH) and the Blood and Marrow Transplant Clinical Trials Network (BMT CTN). 
 
First, we want you to know that: 
 Taking part in NIH research is entirely voluntary. 
 You may choose not to take part, or you may withdraw from the study at any time.  In 
either case, you will not lose any benefits to which you are otherwise entitled.  
 You may receive no benefit from taking part.  The research may give us knowledge 
that may help people in the future. 
 
Second, some people have personal, religious or ethical beliefs that may limit the kinds of 
medical or research treatments they would want to receive (such as blood transfusions).  If you 
have such beliefs, please discuss them with your doctors or research team before you agree to the 
study. 
 
Now we will describe this research study.  Before you decide to take part, please take as much 
time as you need to ask any questions and discuss this study with your family, friends, or your 
personal physician or other health professional. 
 
 
Purpose 
During the course of this study, we will attempt to learn about genetic factors that may have an 
influence on the risks of Graft-vs-Host Disease (GVHD) and long-term outcome after allogeneic 
stem cell transplantation.  GVHD is an immune reaction that occurs after transplantation of stem 
cells from one individual (the donor) to another (the recipient).  GVHD is a form of rejection of 
the recipient tissues (such as the skin, the intestinal tract and the liver) by the donor immune 
system.  We are interested in studying the small variations or differences in genes, called 
polymorphisms or variants that could influence the immune system to cause GVHD.  At this 
time, there are several candidate genes that may influence the incidence of GVHD.  We would 
like to store your DNA (which will be isolated from your blood) to eventually study some of 
these genes.  We invite you to participate in this study so that we can learn more about these 
genetic factors. 

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Procedures 
Sixteen (16) milliliters of blood will be used for the genetic analysis.  We ask that you submit a 
sample of no more than 16 mL, equivalent to three teaspoons.  For donors less than 12 years old, 
a sample of no more than 8 mL, equivalent to 1½ teaspoons, will be requested.  If you withdraw 
from the study, your samples will not be used for other research studies or tested further.   
 
Clinical information (e.g., HLA typing) about you will be collected.  The NIH will not have 
access to the names of the patients enrolled in this study.  The clinical information will be coded 
and compared to the genetic analysis at a later date.   
 
The samples collected for research purposes will be sent to laboratories that have contracts with 
the BMT CTN to conduct these research tests.  They will be labeled with unique codes that do 
not contain information that could identify you.  The link is stored at the Data Coordinating 
Center for the BMT CTN.  The staff at the laboratories where your samples are being tested do 
not have a link to this code.   
 
In the laboratory, we will isolate DNA from your blood.  At a later date, we will test the DNA 
for the polymorphisms in your genes and in the genes in your stem cell recipient.  The 
polymorphisms will be correlated with clinical outcomes after transplantation. 
 
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. 
 
If you agree to allow your blood to be kept for research, you are free to change your mind at any 
time.  We ask that you tell [the Principal Investigator] at [address] in writing and let him/her 
know you are withdrawing your permission for your sample to be used for research.  Any unused 
sample will be destroyed. 
 
 
Alternatives 
You may choose not to participate in this part of the study.  The decision to participate in this 
study will not affect the care given to you by your physicians.  
 
 

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Risk and Discomfort 
There is a small risk of an infection or fainting from the blood draw. 
 
At no time will this information be made available to those not directly involved in the study 
without your written consent.  Only the results of the proposed analysis will be collected, 
presented and published.  At no time, will the name or an identifier of patients or stem cell 
donors be available to anyone except those conducting the study.  The investigators who will 
conduct the genetic analysis will not have access to the names of the patients or donors enrolled 
in this study.  The clinical information will be coded and compared to the genetic analyses to be 
performed at a later date.   
 
 
Benefits 
It is possible that the information from this study could be important for future transplant donors 
and recipients.  However individual results will not be reported directly to you.   
 
This study will increase our understanding of the factors that influence the risk for developing 
GVHD after stem cell transplantation.  We hope that it will eventually contribute to 
improvements in prevention and treatment of GVHD.  There may be no direct benefit to you or 
your stem cell recipient from this study.  If there are any questions, we will attempt to answer 
them with the most recent information.   
 
 
HIPAA2 AUTHORIZATION TO USE AND DISCLOSE INDIVIDUAL HEALTH 
INFORMATION FOR RESEARCH PURPOSES 
 
1. 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 A Phase III Randomized, Multicenter Trial Comparing 
Sirolimus/Tacrolimus with Tacrolimus/Methotrexate as GVHD Prophylaxis After HLA-
Matched, Related Peripheral Blood Stem Cell Transplantation. 
 
2. 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, physical examination findings, and genetic 
test results.  
 
 
                                                   
2 HIPAA is the Health Insurance Portability and Accountability Act of 1996, a federal law related to privacy of health 
information. 

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3. 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): 
 
___________________________________________________________________ 
 
___________________________________________________________________ 
 
___________________________________________________________________ 
 
 
4. Parties Who May Receive or Use My Individual Health Information: The individual health 
information disclosed by parties listed in item 3 and information disclosed by me during the 
course of the research may be received and used by the following parties: 
 Principal Investigators and the researcher’s staff, including Dr. Corey Cutler and Dr. 
Joseph Antin, Study Chairpersons and staff/laboratories at Dana Farber Cancer 
Institute 
 National Heart, Lung and Blood Institute (NHLBI) and 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) 
 U.S. government agencies responsible for overseeing public health concerns such as 
the Centers for Disease Control (CDC) and federal, state and local health 
departments. 
 
5. 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.   
 
6. 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. 
 

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7. 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. 
 
8. DNA from your stored blood sample might be used in genome-wide association (GWA) or 
pharmacogenomics studies for a future project either done or supported by the National 
Institutes of Health (NIH).  Genome-wide association studies are a way for scientists to 
identify genes involved in human disease.  This method searches the genome for small 
genetic changes that are more common in people with a particular disease than in people 
without the disease.  Each study can look at hundreds of thousands of genetic changes at the 
same time.  Researchers use data from this type of study to find genes that may add to a 
person’s risk of developing a certain disease.  Pharmacogenomics studies are similar genetic 
tests but look specifically at genes related to how the body breaks down medications.  
 
If your coded samples are used in such a study, the researcher is required to add your test 
results and sample information into a shared, public research database.  This public database 
is called the NIH Genotype and Phenotype Database and it is managed by the National 
Center for Biotechnology Information (NCBI).  The NCBI will never have any information 
that would identify you, or link you to your information or research samples.  
 
9. A new federal law (2009), called the Genetic Information Nondiscrimination Act (GINA) 
generally makes it illegal for health insurance companies, group health plans, and employers 
of 15 or more persons to discriminate against you based on your genetic information.  Health 
insurance companies and group health plans may not request your genetic information that 
we get from this research.  This means that they must not use your genetic information when 
making decisions regarding insurability.  Be aware that this new federal law will not protect 
you against genetic discrimination by companies that sell life insurance, disability insurance, 
or long-term care insurance. 
 
10. This authorization does not have an expiration date. 
 
 

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OTHER PERTINENT INFORMATION 
 
1.  Confidentiality. When results of an NIH research study are reported in medical journals or at scientific meetings, the people 
who take part are not named and identified.  In most cases, the NIH will not release any information about your research 
involvement without your written permission.  However, if you sign a release of information form, for example, for an insurance 
company, the NIH will give the insurance company information from your medical record.  This information might affect (either 
favorably or unfavorably) the willingness of the insurance company to sell you insurance. 
 
The Federal Privacy Act protects the confidentiality of your NIH medical records.  However, you should know that the Act 
allows release of some information from your medical record without your permission, for example, if it is required by the Food 
and Drug Administration (FDA), members of Congress, law enforcement officials, or other authorized people. 
 
2.  Policy Regarding Research-Related Injuries.  The Clinical Center will provide short-term medical care for any injury 
resulting from your participation in research here.  In general, no long-term medical care or financial compensation for research-
related injuries will be provided by the National Institutes of Health, the Clinical Center, or the Federal Government.  However, 
you have the right to pursue legal remedy if you believe that your injury justifies such action. 
 
3.  Payments.  The amount of payment to research volunteers is guided by the National Institutes of Health policies.  In general, 
patients are not paid for taking part in research studies at the National Institutes of Health. 
 
4.  Problems or Questions.  If you have any problems or questions about this study, or about your rights as a research 
participant, or about any research-related injury, contact the Principal Investigator at (xxx) xxx-xxxx. 
 
5.  Consent Document.  Please keep a copy of this document in case you want to read it again. 
 
COMPLETE APPROPRIATE ITEM BELOW, A or B 
A. Adult Patient's Consent. 
I have read the explanation about this study and have been 
given the opportunity to discuss it and to ask questions. I 
hereby consent to take part in this study. 
 
 
Signature of Adult Patient & Date Signed 
 
 
 
THIS CONSENT DOCUMENT HAS BEEN APPROVED 
FOR USE FROM _________ THROUGH __________. 
B. Parent's Permission for Minor Patient. 
I have read the explanation about this study and have been 
given the opportunity to discuss it and to ask questions. I 
hereby give permission for my child to take part in this study. 
(Attach NIH 2514-2, Minor’s Assent, if applicable) 
 
Signature of Parent(s)/Guardian & Date Signed 
If other than parent, specify relationship:  
 
 
 
Child's Verbal Assent (if applicable). 
The information in the above consent form has been 
adequately described to my child in language that my child 
can understand, and my child willingly agrees to participate 
in the study. 
 
Signature of Parent(s)/Guardian & Date Signed 
 
Signature of Investigator & Date Signed 
 
Signature of Witness & Date Signed 
 
 
 

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APPENDIX C 
 
LABORATORY SAMPLES 

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APPENDIX C 
 
LABORATORY SAMPLES 
 
 
1. HLA TYPING 
 
Before Transplantation:  HLA typing will be performed for all patients and donors in 
American Society of Histocompatibility and Immunogenetics (ASHI)-approved laboratories 
designated by the transplant centers.  HLA typing must be performed by serologic (or higher 
resolution) methods for HLA-A and B, and molecular typing for DRB1 consistent with NMDP 
standard procedures. 
 
 
2. PATHOLOGY/CYTOGENETICS STUDIES 
 
A bone marrow biopsy/aspirate is required from the patient within four weeks prior to the 
initiation of conditioning therapy.  A bone marrow aspirate and biopsy with cytogenetics is 
required at Day 100 and 12 months and 24 months post transplant. 
 
Pathology and cytogenetic studies will be conducted per institutional guidelines. 
 
 
3. FLOW CYTOMETRY/IMMUNE RECONSTITUTION ASSAY 
 
Flow cytometry on peripheral blood specimens from the patient will be performed at the 
following timepoints: Day 28, Day 100, Day 180, Day 365 and Day 730. 
Tubes will be collected and shipped to: 
 
Esoterix Clinical Trials Services 
Attn: Shiela Rines 
750 Walnut Avenue 
Cranford, NJ  07016 
 
Assays will be performed to determine the number of CD2, CD3, CD4, CD8, CD19, CD25, 
CD45Ro/Ra and CD56 cells. 
 
Immunophenotype assays of lymphoid subsets and dendritic cells:  Samples of peripheral 
blood will be obtained from patients at 28, 100, 180, 365 and 730 days post-transplant.  Sample 
processing and testing should be performed as quickly after sample receipt as possible.  
Laboratory will perform and report to EMMES a complete blood count (CBC) and manual cell 
differential & morphology assessment on the EDTA containing samples submitted at each time 
point. Laboratory will calculate the absolute numbers of subsets of immune cells in the blood 
based upon the absolute number of leukocytes in the blood and the percentage of leukocytes that 
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The flow cytometric analysis will be performed using a panel of flow cytometry assays, each 
containing multiple fluorescently labeled monoclonal antibodies.  The FACS panel that will be 
used is shown below.  Please note that all necessary controls are not shown in this table. 
 
 
Flow Cytometry for Immune Reconstitution 
 
Tube  
# 
Class of  
Cells 
Cell Types 
Measured Blood Cell 
Subsets 
Antibodies 
1 
Lymphoid 
Subsets 
T cells 
CD4 
CD8 
CD25+  
anti-CD4(PerCP) 
anti-CD8 (FITC) 
anti-CD25 (PE) 
anti-CD3(APC) 
2 
Lymphoid 
Subsets 
NK and  T cells 
CD56+  
 T cells 
anti-CD56 (PE) 
anti- TCR (FITC) 
anti-CD16 (PerCP) 
anti-CD3 (APC) 
3 
Lymphoid 
Subsets 
CD8 T cell 
Subsets 
Naïve and activated CD8 
T cells 
anti-CD8 (APC) 
anti-Ki67(FITC) 
anti-CD62L (PE) 
anti-CD45RA (TC) 
4 
Lymphoid 
Subsets 
CD4 T cell 
Subsets 
Naïve and activated CD4 
T cells 
anti-CD4 (APC) 
anti-Ki67(FITC) 
anti-CD62L(PE) 
anti-CD45RA (TC) 
5 
Lymphoid 
Subsets 
B-cells & 
monocytes 
CD5+ 
CD27 
B cells 
CD14+ monocytes 
anti-CD5 (PerCP) 
anti-CD27 (FITC) 
anti-CD14(PE) 
anti-CD19 (APC) 
6 
Dendritic Cell 
Subsets 
DC subsets 
DC1 and DC2 subsets 
anti-lineage (FITC) 
anti-CD123(PE) 
anti-HLADR (PerCP) 
anti-CD11c(APC) 
7 
Isotype 
Controls 
Isotype Ig 
Non-specific staining 
irrelevant (FITC) (PE) (PerCP) 
(APC) conjugates to murine 
monoclonal Ab 
 
 

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5. DONOR GENETIC STUDIES 
 
Background 
 
The science of allogeneic stem cell transplantation continues to evolve.  Until recently, only a 
few donor characteristics, such as donor age, sex and ABO blood type, were known to influence 
transplant outcome.  However, recently, a number of donor molecular characteristics have been 
identified that potentially influence recipient outcome.  These donor characteristics are 
predominantly polymorphisms in previously described genes.  
 
The gene polymorphisms fall into several distinct categories.  Polymorphisms in cytokine genes 
(such as IL-6, IL-10 and others) have been linked to differences in acute1-13 and chronic14-16 
GVHD outcome, and also to differences in host response to infectious agents after 
transplantation.17,18 A second group of polymorphisms include genes not directly involved in the 
immune responses or regulation the immune response, but which are presented in the context of 
MHC I or II and are termed minor histocompatibility antigens.19-23 Yet a third group of distinct 
polymorphisms are in the family of the Natural Killer (NK) cell receptor and ligand group, 
collectively termed the Killer Immunoglobulin-like Receptor (KIR) family, which have been 
linked to differences to both GVHD and relapse rates after transplantation.24-28 There is a fourth 
group of genes only loosely related to the immune system within which polymorphisms can 
influence rates of GVHD.29-33 Finally, genes involved in drug metabolism have been linked to 
toxicity and GVHD after allogeneic transplantation.34, 35 
 
Current donor search strategies focus primarily on HLA typing, and then use ancillary factors 
such as age, sex, parity and CMV serostatus to select an appropriate donor.  Recently, several 
investigators have suggested that search strategies that include donor genotype of cytokines and 
other relevant genes, be incorporated into search strategies.36-38  
 
The purpose of this study is to collect donor samples for DNA and serum banking, from which 
studies of these polymorphisms can be performed.  Since the results of the polymorphism 
analysis will be correlated with clinical results after transplantation, the samples will be stored at 
the time of stem cell donation, and will be analyzed at a later date, when transplant outcomes can 
be measured. 
 
Research Plan 
 
Prior to administration of G-CSF, donors will provide 16 mL (or 8 mL for donors less than 12 
years old) of peripheral blood.  The center may make further adjustments in blood volumes 
collected from patients less than 12 years old to meet the institution’s Human Investigational 
Committee’s Guidelines.  The samples will be processed, stored under appropriate conditions, 
and shipped quarterly to the NHLBI repository.  No analysis will occur until all samples have 
been received and pertinent research questions have been developed.   
 

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6. REPOSITORY INFORMATION 
 
Standard procedures for collection, storage, and shipping of specimens will be followed 
according to the BMT CTN guidelines.  Samples will be given a unique alphanumeric code that 
contains no personal identifiers.  Transplant Center Coordinators will hold the link to the code.  
Laboratory staff will not have access to the link.  All laboratory studies will be performed at 
laboratories under contract with the NMDP on behalf of the BMT CTN. 
 
Frozen samples should be batched and shipped quarterly to the Repository in compliance with 
the shipping procedures specified in the BMT CTN MOP and the BMT CTN 0402 Laboratory 
Sample Information Guide:  
 
Christina Demasco 
NHLBI Repository 
SeraCare BioServices 
217 Perry Parkway 
Gaithersburg, Maryland  20877 
Phone: (301) 208-8100 
Fax: 
(301) 208-8829 
Email: 
nhlbi@bbii.com 

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C-7 
SCHEDULE OF LABORATORY EVALUATIONS 
 
Type of Sample 
Method 
Type of Storage 
Dates Samples Obtained 
Shipping  
Specifications 
Test  
Location 
HLA Typing 
According to 
institutional practice  
According to institutional 
practice 
Prior to conditioning 
regimen. 
N/A 
Transplant 
Center 
Pathology/ 
Cytogenetic Studies 
According to 
institutional practice 
According to institutional 
practice 
Within four weeks prior to 
the initiation of 
conditioning therapy and on 
Day 100, 365 and 730 post-
transplant. 
N/A 
Transplant 
Center 
Flow Cytometry/ 
Immune 
Reconstitution 
8.5 mL peripheral 
blood  
8.5 mL Yellow Top 
ACD-A 
Anticoagulation 
Vacutainer  
(003 IR-Blood V) 
No additional processing –  
do not centifuge. 
Day 28, 100, 180, 365 and 
730 post-transplant. 
Shipped 
overnight on cold 
ice packs (4°C) 
Esoterix 
Clinical 
Trials 
Services 
3 mL peripheral 
blood  
3 mL Lavendar Top 
EDTA 
Anticoagulation 
Vacutainer  
(004 IR-Blood V) 
No additional processing –  
do not centifuge. 
Day 28, 100, 180, 365 and 
730 post-transplant. 
Shipped 
overnight on cold 
ice packs (4°C) 
Esoterix 
Clinical 
Trials 
Services 

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Type of Sample 
Method 
Type of Storage 
Dates Samples Obtained 
Shipping  
Specifications 
Test  
Location 
Investigational 
Future Testing 
10 mL peripheral 
blood (10 mL red 
top tube) 
OR 
5 mL peripheral 
blood (6 mL red top 
tube) for patients  
< 12 years old1 
Place upright in rack for 30 
minutes.  Centrifuge at 900 x g 
or 2100 rpm for 10 minutes.  
Extract serum, transfer 500 ul 
aliquots to 10 cryovials2. 
Store at -70° C. 
Baseline (prior to the 
initiation of conditioning 
therapy) and on Day 28, 
100, 180, 365 and 730 post-
transplant. 
Frozen shipment 
quarterly to 
Repository in 
compliance with 
shipping 
procedures 
specified in the 
BMT CTN MOP 
TBD 
6 mL peripheral 
blood (7 mL 
lavender top tube)  
OR 
3 mL peripheral 
blood (4 mL 
lavender top tube) 
for patients  
< 12 years old1 
Centrifuge at 900 x g or 2100 
rpm for 10 minutes within 30 
minutes of collection.  Extract 
plasma, transfer 500 ul aliquots 
to 6 cryovials2. 
Store at -70° C. 
Baseline (prior to the 
initiation of conditioning 
therapy) and on Day 28, 
100, 180, 365 and 730 post-
transplant. 
Frozen shipment 
quarterly to 
Repository in 
compliance with 
shipping 
procedures 
specified in the 
BMT CTN MOP 
TBD 
 
 
 
 
 
 

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Type of Sample 
Method 
Type of Storage 
Dates Samples Obtained 
Shipping  
Specifications 
Test  
Location 
Donor Genetic 
Studies 
10 mL peripheral 
blood (10 mL 
lavendar top tube) 
OR 
5 mL peripheral 
blood (6 mL 
lavendar top tube) 
for donors  
< 12 years old1 
Place upright in rack for 30 
minutes.  Centrifuge at 900 x g 
or 2100 rpm for 10 minutes.  
Extract pasma, transfer 500 ul 
aliquots to 10 cryovials. 
Store at -70° C. 
Resuspend cellular component 
in RBC lysis buffer, divide and 
transfer into 6 cryovials.  Respin 
at 900 x g or 2100 rpm for 10 
minutes.  Discard buffer and 
freeze pellet at -70° C 
immediately. 
Prior to administration of 
G-CSF 
Frozen shipment 
quarterly to 
Repository in 
compliance with 
shipping 
procedures 
specified in the 
BMT CTN MOP 
TBD 
Notes: 
1 The center may make further adjustments in blood volumes collected from patients less than 12 years old to meet the institution’s Human 
Investigational Committee’s Guidelines. 
2 Use half of the amount of cryovials for patients less than 12 years old. 
 
 

BMT CLINICAL TRIALS NETWORK 
 
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Version 4.0 dated December 3, 2010 
 
 
C-10 
Appendix C Reference List 
 
 
(1)  Cavet J, Middleton PG, Segall M et al. Recipient tumor necrosis factor-alpha and 
interleukin-10 gene polymorphisms associate with early mortality and acute graft-versus-
host disease severity in HLA-matched sibling bone marrow transplants. Blood. 
1999;94:3941-3946. 
 
(2)  Cavet J, Dickinson AM, Norden J et al. Interferon-gamma and interleukin-6 gene 
polymorphisms associate with graft-versus-host disease in HLA-matched sibling bone 
marrow transplantation. Blood. 2001;98:1594-1600. 
 
(3)  Cullup H, Dickinson AM, Jackson GH et al. Donor interleukin 1 receptor antagonist 
genotype associated with acute graft-versus-host disease in human leucocyte antigen-
matched sibling allogeneic transplants. Br J Haematol. 2001;113:807-813. 
 
(4)  Hattori H, Matsuzaki A, Suminoe A et al. Polymorphisms of transforming growth factor-
beta1 and transforming growth factor-beta1 type II receptor genes are associated with 
acute graft-versus-host disease in children with HLA-matched sibling bone marrow 
transplantation. Bone Marrow Transplant. 2002;30:665-671. 
 
(5)  Karabon L, Wysoczanska B, Bogunia-Kubik K, Suchnicki K, Lange A. IL-6 and IL-10 
promoter gene polymorphisms of patients and donors of allogeneic sibling hematopoietic 
stem cell transplants associate with the risk of acute graft-versus-host disease. Hum 
Immunol. 2005;66:700-710. 
 
(6)  Lin MT, Storer B, Martin PJ et al. Relation of an interleukin-10 promoter polymorphism 
to graft-versus-host disease and survival after hematopoietic-cell transplantation. N Engl 
J Med. 2003;349:2201-2210. 
 
(7)  Lin MT, Storer B, Martin PJ et al. Genetic variation in the IL-10 pathway modulates 
severity of acute graft-versus-host disease following hematopoietic cell transplantation: 
synergism between IL-10 genotype of patient and IL-10 receptor beta genotype of donor. 
Blood. 2005;106:3995-4001. 
 
(8)  MacMillan ML, Radloff GA, Kiffmeyer WR et al. High-producer interleukin-2 genotype 
increases risk for acute graft-versus-host disease after unrelated donor bone marrow 
transplantation. Transplantation. 2003;76:1758-1762. 
 
(9)  Middleton PG, Taylor PR, Jackson G, Proctor SJ, Dickinson AM. Cytokine gene 
polymorphisms associating with severe acute graft-versus-host disease in HLA-identical 
sibling transplants. Blood. 1998;92:3943-3948. 
 (10)  Mlynarczewska A, Wysoczanska B, Karabon L, Bogunia-Kubik K, Lange A. Lack of 
IFN-gamma 2/2 homozygous genotype independently of recipient age and intensity of 
conditioning regimen influences the risk of aGVHD manifestation after HLA-matched 
sibling haematopoietic stem cell transplantation. Bone Marrow Transplant. 2004;34:339-
344. 

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C-11 
 (11)  Nordlander A, Uzunel M, Mattsson J, Remberger M. The TNFd4 allele is correlated to 
moderate-to-severe acute 
graft-versus-host disease after allogeneic stem 
cell 
transplantation. Br J Haematol. 2002;119:1133-1136. 
 (12)  Stark GL, Dickinson AM, Jackson GH et al. Tumour necrosis factor receptor type II 
196M/R genotype correlates with circulating soluble receptor levels in normal subjects 
and with graft-versus-host disease after sibling allogeneic bone marrow transplantation. 
Transplantation. 2003;76:1742-1749. 
 (13)  Takahashi H, Furukawa T, Hashimoto S et al. Contribution of TNF-alpha and IL-10 gene 
polymorphisms to graft-versus-host disease following allo-hematopoietic stem cell 
transplantation. Bone Marrow Transplant. 2000;26:1317-1323. 
 (14)  Bogunia-Kubik K, Mlynarczewska A, Wysoczanska B, Lange A. Recipient interferon-
gamma 3/3 genotype contributes to the development of chronic graft-versus-host disease 
after allogeneic hematopoietic stem cell transplantation. Haematologica. 2005;90:425-
426. 
 (15)  Cullup H, Dickinson AM, Cavet J, Jackson GH, Middleton PG. Polymorphisms of 
interleukin-1alpha constitute independent risk factors for chronic graft-versus-host 
disease after allogeneic bone marrow transplantation. Br J Haematol. 2003;122:778-787. 
 (16)  Kim DH, Lee NY, Sohn SK et al. IL-10 promoter gene polymorphism associated with the 
occurrence of chronic GVHD and its clinical course during systemic immunosuppressive 
treatment for chronic GVHD after allogeneic peripheral blood stem cell transplantation. 
Transplantation. 2005;79:1615-1622. 
 (17)  Bogunia-Kubik K, Mlynarczewska A, Jaskula E, Lange A. The presence of IFNG 3/3 
genotype in the recipient associates with increased risk for Epstein-Barr virus reactivation 
after allogeneic haematopoietic stem cell transplantation. Br J Haematol. 2006;132:326-
332. 
 (18)  Seo KW, Kim DH, Sohn SK et al. Protective role of interleukin-10 promoter gene 
polymorphism in the pathogenesis of invasive pulmonary aspergillosis after allogeneic 
stem cell transplantation. Bone Marrow Transplant. 2005;36:1089-1095. 
 (19)  Akatsuka Y, Warren EH, Gooley TA et al. Disparity for a newly identified minor 
histocompatibility antigen, HA-8, correlates with acute graft-versus-host disease after 
haematopoietic stem cell transplantation from an HLA-identical sibling. Br J Haematol. 
2003;123:671-675. 
 (20)  Goulmy E. Human minor histocompatibility antigens. Curr Opin Immunol. 1996;8:75-81. 
 (21)  Kernan NA, Dupont B. Minor histocompatibility antigens and marrow transplantation. N 
Engl J Med. 1996;334:323-324. 

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C-12 
 (22)  Kotzampasaki 
EM, 
Spyropoulou-Vlachou 
MS, 
Kalofoutis 
C 
et 
al. 
Minor 
histocompatibility antigen HA-1 and HPA-5 polymorphisms in HLA-identical related 
bone marrow transplantation. Transplant Proc. 2004;36:1735-1738. 
 (23)  Miklos DB, Kim HT, Miller KH et al. Antibody responses to H-Y minor 
histocompatibility antigens correlate with chronic graft-versus-host disease and disease 
remission. Blood. 2005;105:2973-2978. 
 (24)  Chen C, Huang SL. Killer cell inhibitory receptors involved in graft-versus-host disease 
and graft-versus-leukemia effect in allogeneic hematopoietic stem cell transplantation - 
review. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2004;12:236-243. 
 (25)  Gagne K, Brizard G, Gueglio B et al. Relevance of KIR gene polymorphisms in bone 
marrow transplantation outcome. Hum Immunol. 2002;63:271-280. 
 (26)  Beelen DW, Ottinger HD, Ferencik S et al. Genotypic inhibitory killer immunoglobulin-
like receptor ligand incompatibility enhances the long-term antileukemic effect of 
unmodified allogeneic hematopoietic stem cell transplantation in patients with myeloid 
leukemias. Blood. 2005;105:2594-2600. 
 (27)  Igarashi T, Wynberg J, Srinivasan R et al. Enhanced cytotoxicity of allogeneic NK cells 
with killer immunoglobulin-like receptor ligand incompatibility against melanoma and 
renal cell carcinoma cells. Blood. 2004;104:170-177. 
 (28)  Schaffer M, Malmberg KJ, Ringden O, Ljunggren HG, Remberger M. Increased 
infection-related mortality in KIR-ligand-mismatched unrelated allogeneic hematopoietic 
stem-cell transplantation. Transplantation. 2004;78:1081-1085. 
 (29)  Bogunia-Kubik K, Lange A. HSP70-hom gene polymorphism in allogeneic 
hematopoietic stem-cell transplant recipients correlates with the development of acute 
graft-versus-host disease. Transplantation. 2005;79:815-820. 
 (30)  Holler E, Rogler G, Herfarth H et al. Both donor and recipient NOD2/CARD15 
mutations associate with transplant-related mortality and GvHD following allogeneic 
stem cell transplantation. Blood. 2004;104:889-894. 
 (31)  Middleton PG, Cullup H, Dickinson AM et al. Vitamin D receptor gene polymorphism 
associates with graft-versus-host disease and survival in HLA-matched sibling allogeneic 
bone marrow transplantation. Bone Marrow Transplant. 2002;30:223-228. 
 (32)  Middleton PG, Norden J, Cullup H et al. Oestrogen receptor alpha gene polymorphism 
associates with occurrence of graft-versus-host disease and reduced survival in HLA-
matched sib-allo BMT. Bone Marrow Transplant. 2003;32:41-47. 
 (33)  Rozman P, Karas M, Kosir A et al. Are human platelet alloantigens (HPA) minor 
transplantation antigens in clinical bone marrow transplantation? Bone Marrow 
Transplant. 2003;31:497-506. 

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C-13 
 (34)  Kalayoglu-Besisik 
S, 
Caliskan 
Y, 
Sargin 
D, 
Gurses 
N, 
Ozbek 
U. 
Methylenetetrahydrofolate reductase C677T polymorphism and toxicity in allogeneic 
hematopoietic cell transplantation. Transplantation. 2003;76:1775-1777. 
 (35)  Robien K, Schubert MM, Bruemmer B et al. Predictors of oral mucositis in patients 
receiving hematopoietic cell transplants for chronic myelogenous leukemia. J Clin Oncol. 
2004;22:1268-1275. 
 (36)  Dickinson AM, Middleton PG, Rocha V, Gluckman E, Holler E. Genetic polymorphisms 
predicting the outcome of bone marrow transplants. Br J Haematol. 2004;127:479-490. 
 (37)  Kallianpur AR. Genomic screening and complications of hematopoietic stem cell 
transplantation: has the time come? Bone Marrow Transplant. 2005;35:1-16. 
 (38)  Socie G, Loiseau P, Tamouza R et al. Both genetic and clinical factors predict the 
development of graft-versus-host disease after allogeneic hematopoietic stem cell 
transplantation. Transplantation. 2001;72:699-706. 
 

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D-1 
 
 
 
 
APPENDIX D 
 
RECOMMENDED LEUCOVORIN RESCUE SCHEMA 
 
 
 

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D-2 
APPENDIX D 
 
RECOMMENDED LEUCOVORIN RESCUE SCHEMA 
 
 
12 hours after d+1 methotrexate 
Leucovorin 15 mg/m2 iv q6h x 3 doses 
 
Maximum single dose 30 mg 
 
12 hours after d+3 methotrexate 
Leucovorin 10 mg/m2 iv q6h x 6 doses 
 
Maximum single dose 25 mg 
 
24 hours after d+6, 11 methotrexate 
Leucovorin 10 mg/m2 iv q6h x 8 doses 
 
Maximum single dose 25 mg 
 
 
Methotrexate levels are not reliable during leucovorin therapy. 
 
 

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E-1 
 
 
 
 
APPENDIX E 
 
CONSENSUS GVHD GRADING 
 

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E-2 
APPENDIX E 
 
CONSENSUS GVHD GRADING 
 
 
Organ Staging of GVHD 
 
Stage 
Skin 
Liver 
Gut 
0 
No rash due to GVHD 
Bilirubin 
< 2 mg/dL 
< 500 mL (5 mL/kg for patients under 12 
years) diarrhea per day 
1 
Maculopapular rash < 25% of 
body surface a 
Bilirubin 
2-3 mg/dLb 
500 to 999 mL diarrhea per day c 
(5.1-10 mL/kg for patients under 12 
years), or persistent nausea with 
histologic evidence of GVHD in stomach 
or duodenum 
2 
Maculopapular rash 25-50% of 
body surface a 
Bilirubin 
3.1-6 mg/dLb 
1,000 to 1,499 mL diarrhea per day 
(10.1-15.0 mL/ kg for patients under 12 
years) 
3 
Maculopapular rash > 50% of 
body surface a 
Bilirubin 
6.1-15 mg/dL b 
1,500 or more mL diarrhea per day c 
(more than 15.0 mL/kg for patients under 
12 years of age) 
4 
Generalized erythroderma with 
bullous formation 
Bilirubin 
> 15 mg/dLb 
Severe abdominal pain with or without 
ileus 
a Use “Rule of Nines” to determine extent of rash. 
b Range given as total bilirubin.  Downgrade one stage if an additional cause of elevated bilirubin has 
been documented. 
c Downgrade one stage if an additional cause of diarrhea has been documented. 
 
 
Overall Clinical Grading of Severity of Acute GVHD 
 
Grade 
 
Degree of Organ Involvement 
0 
 
No Stage 1-4 of any organ 
I 
 
Stage 1-2 rash and no liver or gut involvement  
II 
 
Stage 3 rash, or Stage 1 liver involvement, or Stage 1 gut involvement  
III 
 
None to Stage 3 skin rash with Stage 2-3 liver involvement, or Stage 2-4 gut 
involvement  
IV 
 
Stage 4 skin rash, or Stage 4 liver involvement 
  Adapted from Thomas et al., NEJM, 1975, pp. 895-90 

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F-1 
 
 
 
 
APPENDIX F 
 
MODIFIED OMAS 
 
MUCOSITIS SCORING SYSTEM 
 
SAMPLE CASE REPORT FORM 
 

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F-2 
APPENDIX F 
 
MODIFIED OMAS MUCOSITIS SCORING SYSTEM SAMPLE  
CASE REPORT FORM 
 
 
Site: 
 
 
 
 
Protocol: 
Subject ID: 
 
 
 
Date:  
 
Assessor’s initials: 
 
 
Site 
Ulceration 
Erythema 
          
Maxillary labial mucosa 
Y     N 
Y     N 
NE* 
Mandibular labial 
mucosa 
Y     N 
Y     N 
NE 
Right cheek 
Y     N 
Y     N 
NE 
Left cheek 
Y     N 
Y     N 
NE 
Right lateral  
& ventral tongue 
Y     N 
Y     N 
NE 
Left lateral  
& ventral tongue 
Y     N 
Y     N 
NE 
Floor of mouth 
Y     N 
Y     N 
NE 
Soft palate 
Y     N 
Y     N 
NE 
 
 
 
 
 
 
 
 
 
 
*not evaluable 
 
 
Ability to eat:    solids   liquids     NPO      
 
 
Mouth Pain       Y     N 
 
 
 
WHO score: 
0 
Normal mucosa 
1 
Erythema & mouth pain 
2 
Ulceration & ability to eat solids 
3 
Ulceration & liquids only 
4 
Ulceration & NPO 
 

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G-1 
 
 
 
 
APPENDIX G 
 
HUMAN SUBJECTS 
 

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G-2 
APPENDIX G 
 
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. 
 

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H-1 
 
 
 
 
APPENDIX H 
 
GUIDELINES FOR SIMULTANEOUS USE OF SIROLIMUS AND 
ORAL VORICONAZOLE 
 

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H-2 
APPENDIX H 
 
GUIDELINES FOR SIMULTANEOUS USE OF SIROLIMUS AND  
ORAL VORICONAZOLE 
 
 
Background 
Voriconazole is currently considered the best initial treatment of invasive aspergillus1 and other 
invasive fungal infections such as those caused by Fusarium sp. and Scedosporium 
apiospermum2.  However, the use of voriconazole in combination with sirolimus was 
contraindicated at the time of voriconazole approval due to a substantial increase in sirolimus 
drug exposure in healthy subjects because voriconazole inhibits several CYP isoenzymes3.  This 
inhibition results in a sharp increase in sirolimus bioavailability; voriconazole increased 
sirolimus peak plasma concentration (Cmax) and area under the plasma-concentration-time curve 
(AUC) by 6.6 fold and 11.1 fold, respectively 3, 4.  Recently the group at Dana-Farber Cancer 
Institute has demonstrated that co-administration of voriconazole and sirolimus is safe if a 90% 
reduction in sirolimus dosing is undertaken5.  Moreover, Pfizer Inc. has now undertaken a series 
of pharmacokinetic studies in normal volunteers that confirms that a 90% dose reduction of 
sirolimus results in reliable levels of sirolimus in the target range6.  The increase in 
bioavailability is due to transient inhibition of CYP enzymes in the intestinal mucosa.  Inhibition 
of liver CYP enzymes will prolong the half-life but not increase the bioavailability of the drug.  
The effects of intravenous voriconazole are less pronounced and dose modifications are not 
required when the intravenous formulation is used.  The effect of voriconazole on CYP is 
transient; therefore, if sirolimus and voriconazole are staggered there is a progressively less 
potent effect on sirolimus level.  If the drugs are staggered by as much as 5 hours, there is no 
effect on bioavailability.  If voriconazole and sirolimus are separated by 1, 2, 3, or 4 hours, the 
effect on bioavailability is less predictable.  
 
Suggested Dose Adjustments 
A 90% dose reduction of sirolimus is an effective strategy for the co-administration of sirolimus 
and oral voriconazole.  Most adults receive sirolimus tablets, which have a minimum strength of 
1 mg and which cannot be crushed or broken.  However, there is a liquid formulation of 
sirolimus at a concentration of 1 mg/mL.  An appropriate fractional dose can be drawn into a  
1 mL syringe and swallowed directly.  The liquid has an unpleasant taste that can be masked by 
mixing with juice or milk.  Alternatively, empty gelatin capsules can be dispensed along with the 
liquid sirolimus.  Because the drug is not in aqueous solution, the liquid can be placed in the 
capsule by the patient, family member or health care provider without causing dissolution of the 
gelatin.  The capsule can then be taken orally.  Blood levels of sirolimus should be measured 
after the dosage change.  Changes in levels due to altered bioavailability should be apparent 
within 24-48 hours.  There will also be a prolongation of sirolimus half-life that may require 
dose adjustments in the weeks following the initiation of voriconazole therapy.  If voriconazole 
is stopped or changed to intravenous formulation, the dose of sirolimus should be increased by 
90% and blood levels should be monitored.  
 

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H-3 
Example: Patient is on 2 mg sirolimus daily. Voriconazole is taken at the same time as the 
sirolimus and the new sirolimus dose is 0.2 mg dispensed as 0.2 mL taken directly or placed in 
gelatin capsules. 
 
 
 
Endnotes for Appendix H 
 
1 
Herbrecht R, Denning DW, Patterson TF, Bennett JE, Greene RE, Oestmann JW, et al. 
Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis. N Engl J 
Med 2002; 347:408-415. 
 
2 
Center for Drug Evaluation and Research, FDA. NDA 21-266 and 21-267, VfendTM 
(Voriconazole). 24 May 2002  [Accessed 21 August 2005]; Available from: 
http://www.fda.gov/cder/foi/appletter/2002/21266ltr.pdf; Perfect JR, Marr KA, Walsh TJ, 
Greenberg RN, DuPont B, de la Torre-Cisneros J, et al. Voriconazole treatment for less-
common, emerging, or refractory fungal infections. Clin Infect Dis 2003; 36(9):1122-1131. 
 
3 
Product Information: Vfend(R), voriconazole. Pfizer Inc, New York, NY, 2005. 
 
4 
Abel S, Allan R, Fielding A. Effect of voriconazole on the pharmacokinetics of sirolimus 
(poster). In: 12th European Congress of Clinical Microbiology and Infectious Diseases; April 
24-27, 2002; Milan, Italy; April 24-27, 2002.12. 
 
5 
Marty FM, Lowry CM, Cutler CS, Campbell BJ, Fiumara K, Baden LR, Antin JH. 
Voriconazole and sirolimus co-administration following allogeneic hematopoietic stem cell 
transplantation. Biol Blood Marrow Transplant, 2006, in press. 
 
6 
Bharat Damle, Clinical Pharmacology Pfizer Global Clinical Research and Development, 
personal communication. 

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I-1 
 
 
 
 
APPENDIX I 
 
REFERENCES 
 

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I-2 
APPENDIX I 
 
REFERENCES 
 
 
                                                   
1 Champlin RE, Schmitz N, Horowitz MM et al. Blood stem cells compared with bone marrow 
as a source of hematopoietic cells for allogeneic transplantation. Blood. 2000;95:3702-3709. 
2 Weisdorf D, Haake R, Blazar B et al. Treatment of moderate/severe acute graft-versus-host 
disease after allogeneic bone marrow transplantation: an analysis of clinical risk features and 
outcome. Blood. 1990;75:1024-1030. 
3 Martin PJ, Schoch G, Fisher L et al. A retrospective analysis of therapy for acute graft-versus-
host disease: secondary treatment. Blood. 1991;77:1821-1828. 
4 Simpson D. New developments in the prophylaxis and treatment of graft versus host disease. 
Expert Opin Pharmacother. 2001;2:1109-1117. 
5 Ho VT, Soiffer RJ. The history and future of T-cell depletion as graft-versus-host disease 
prophylaxis for allogeneic hematopoietic stem cell transplantation. Blood. 2001;98:3192-
3204. 
6 Storb R, Deeg HJ, Whitehead J et al. Methotrexate and cyclosporine compared with 
cyclosporine alone for prophylaxis of acute graft versus host disease after marrow 
transplantation for leukemia. N Engl J Med. 1986;314:729-735. 
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