Conditions: Leukemia, Myeloid, Acute Myelodysplastic Syndromes Precursor Cell Lymphoblastic Leukemia-Lymphoma
Allocation: NON_RANDOMIZED | Intervention model: PARALLEL
Enrollment: 54
Healthy volunteers: no

Brief summary:
The purpose of this phase I/II study is to define the maximum tolerated dose of 5-AzaC and the effect on grade II-IV GvHD when given after matched unrelated donor transplant (MUD).

Detailed description:

Arms:
arm 1: Conditioning treatment

Transplant on Day 0

15 mg/m\^2 azacitidine Days 7-11

15 mg/m\^2 azacitidine Days 35-39

15 mg/m\^2 azacitidine Days 63-67

15 mg/m\^2 azacitidine Days 91-95 arm 2: Conditioning treatment

Transplant on Day 0

30 mg/m\^2 azacitidine Days 7-11

30 mg/m\^2 azacitidine Days 35-39

30 mg/m\^2 azacitidine Days 63-67

30 mg/m\^2 azacitidine Days 91-95 arm 3: Conditioning treatment

Transplant on Day 0

37.5 mg/m\^2 azacitidine Days 7-11

37.5 mg/m\^2 azacitidine Days 35-39

37.5 mg/m\^2 azacitidine Days 63-67

37.5 mg/m\^2 azacitidine Days 91-95 arm 4: Conditioning treatment

Transplant on Day 0

45 mg/m\^2 azacitidine Days 7-11

45 mg/m\^2 azacitidine Days 35-39

45 mg/m\^2 azacitidine Days 63-67

45 mg/m\^2 azacitidine Days 91-95 arm 5: Conditioning treatment

Transplant on Day 0

Dose determined in Phase I - 45 mg/m\^2 azacitidine Days 7-11

Dose determined in Phase I - 45 mg/m\^2 azacitidine Days 35-39

Dose determined in Phase I - 45 mg/m\^2 azacitidine Days 63-67

Dose determined in Phase I - 45 mg/m\^2 attitudinize Days 91-95

Interventions: intervention 1: Azacitidine
Intervention descriptions: intervention 1: None

Protocol document:
Prot_SAP_000.pdf: 
Amendment 11: Version 09/06/18 
 
Page 1 of 96 
 
Phase I/II Trial of Intravenous Azacitidine in Patients Undergoing 
Matched Unrelated Stem Cell Transplantation 
 
Washington University School of Medicine 
Division of Oncology 
660 South Euclid Avenue, Campus Box 8056 
St. Louis, MO  63110 
 
Protocol#:  201303012 
IND #: 117649 
ClinicalTrials.gov#: NCT01747499 
Protocol Version Date:  09/06/18 
Study Drug: Azacitidine 
 
 
 
 
 
Principal Investigator: 
Mark A. Schroeder, M.D. 
Telephone: 314-454-8304 // Fax: 314-454-7551 
Email: markschroeder@wustl.edu 
 
Sub-Investigators: 
 
John F. DiPersio, M.D., Ph.D. 
Peter Westervelt, M.D., Ph.D. 
Linda Eissenberg, Ph.D. 
Amanda Cashen, M.D. 
Keith Stockerl-Goldstein, M.D. 
Camille Abboud, M.D. 
Ravi Vij, M.D. 
Geoffrey Uy, M.D. 
Mathew Walter, M.D.  
Todd Fehniger, MD., Ph.D. 
Armin Ghobadi, M.D. 
Meagan Jacoby, M.D., Ph.D. 
Iskra Pusic, M.D. 
Lukas Wartman,  M.D. 
John Welch, M.D., Ph.D. 
Tanya Wildes, M.D. 
Terrence Wong, M.D., Ph.D. 
 
Statistician: 
 
Esther Lu (Jingxia Liu), Ph.D. 
 
CONFIDENTIAL 
The information contained in this document is regarded as confidential and, except to the extent necessary to 
obtain informed consent, may not be disclosed to another party unless law or regulations require such 
disclosure.  Persons to whom the information is disclosed must be informed that the information is confidential 
and may not be further disclosed by them 
 


Amendment 11: Version 09/06/18 
 
Page 2 of 96 
 
Phase I/II Trial of Intravenous Azacitidine in Patients Undergoing Matched Unrelated 
Stem Cell Transplantation 
 
Protocol Revision History 
 
Initial Approval Version 
 
 
 
 
 
 
03/04/2013 
Amendment #1 Version 
 
 
 
 
 
 
11/15/2013 
Amendment #2 Version 
 
 
 
 
 
 
01/17/2014 
Amendment #3 Version 
 
 
 
 
 
 
03/18/2014 
Amendment #4 Verison  
 
 
 
 
 
 
05/02/2014 
Amendment #5 Version 
 
 
 
 
 
 
09/24/2014 
Amendment #6 Version 
 
 
 
 
 
 
10/08/2015 
Amendment #7 Version 
 
 
 
 
 
 
05/11/2016 
Amendment #8 Version 
 
 
 
 
 
 
08/02/2016 
Amendment #9 Version 
 
 
 
 
 
 
05/11/2017 
Amendment #10 Version 
 
 
 
 
 
 
05/03/2018 
Amendment #11 Version 
 
 
 
 
 
 
09/06/2018 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Amendment 11: Version 09/06/18 
 
Page 3 of 96 
SCHEMA 
 
Azacitidine dosing cohorts 
 
Cohort  1 
Cohort 2 
Cohort 3 
Cohort 4 
15 mg/m2 
30 mg/m2 
37.5 mg/m2 
45 mg/m2 
 
 
 
 
 
 
 
 
 
 
 
 


Amendment 11: Version 09/06/18 
 
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Glossary of Abbreviations 
5-AzaC 
5’-azacytidine (azacytidine, azacitidine) 
AE 
Adverse event 
aGvHD 
Acute Graft versus Host Disease 
ALL 
Acute Lymphoid Leukemia 
ALLO-HSCT 
Allogeneic hematopoietic stem cell transplant 
ALT  
Alanine transaminase  
AML 
Acute myeloid leukemia 
ANC 
Absolute neutrophil count 
ASP 
Aspirate 
AST  
Aspartate transaminase  
AUC 
Area under the curve 
BJH 
Barnes-Jewish Hospital 
BM 
Bone marrow 
BMT 
Bone marrow transplant 
BUN 
Blood urea nitrogen 
CALGB 
Cancer and Leukemia Group B 
CBC 
Complete blood count 
CCR 
Conventional care 
CFR 
Code of Federal Regulations 
cGvHD 
Chronic Graft vs. Host Disease 
CI 
Confidence interval 
CIBMTR 
Center for International Blood and Marrow Transplant Research 
CML 
Chronic Myelogenous Leukemia 
CMML 
Chronic myelomonocytic leukemia 
CMP 
Complete metabolic panel  
CR 
Complete remission 
CRc 
Cytogenetic complete remission  
CRi 
Complete remission incomplete 
CRm 
Morphologic complete remission 
CRF 
Case report form 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTEP 
Cancer Therapy Evaluation Program 
DC 
Dendritic cell 
DLCO 
Diffusing capacity of the lung for carbon monoxide 
DLI 
Donor lymphocyte infusion 
DLTs 
Dose limiting toxicities 
DMSO 
Dimethyl sulfoxide 
DNA 
deoxyribonucleic acid 
DOB 
Date of birth 
DSM 
Data and safety monitoring  
DSMC 
Data and Safety Monitoring Committee  
ECG  
Electrocardiogram 
ECOG 
Eastern Cooperative Oncology Group 
EDTA 
ethylenediaminetetraacetic acid   
FAB 
French American British  
FCBP 
Females of child bearing potential 
FDA 
Food and Drug Administration 
G-CSF 
Granulocyte colony stimulating factor, filgrastim (NEUPOGEN) 
GvHD 
Graft vs. Host Disease 
GvL 
Graft vs. Leukemia 
HCT 
Hematopoietic cell transplant 
HIV 
Human Immunodeficiency Virus 
HRPO 
Human Research Protection Office (IRB) 
HSCT 
Hematopoietic stem cell transplant 
HSV 
Herpes simplex virus 


Amendment 11: Version 09/06/18 
 
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HTLV 
Human T-lymphotropic virus 
IBMTR 
International Bone Marrow Transplant Registry 
IBW 
Ideal body weight 
ICH 
International Conference on Harmonization 
IND 
Investigational New Drug 
IPSS 
International prognostic scoring system 
IRB 
Institutional Review Board   
IV 
Intravenous (i.v.) 
IWG 
International Working Group 
LDH 
Lactate dehydrogenase 
LLN 
Lower limit of normal 
MA 
Myeloablative 
MDS 
Myelodysplastic syndrome 
MED 
Minimum effective dose 
MM 
Multiple myeloma 
MRI 
Magnetic resonance imaging 
MTD 
Maximum tolerated dose 
MUD 
Matched unrelated donor 
MUGA 
Multiple Gated Acquistion scan 
NCI 
National Cancer Institute 
NIH 
NMDP 
National Institutes of Health 
National Marrow Donor Program 
OHRP 
Office of Human Research Protections  
OS 
Overall survival 
PB 
Peripheral blood 
PBSC 
Peripheral blood stem cell 
PD 
Progressive disease  
PI 
Principal investigator 
PK 
Pharmacokinetic 
PR 
Partial response (Partial remission)  
QASMC 
Quality Assurance and Safety Monitoring Committee 
RAEB 
Refractory anemia with excess blasts 
RAEB-T 
Refractory anemia with excess blasts in transformation 
RFS 
Relapse-free survival 
RIC 
Reduced intensity conditioning regimen 
RNA 
Ribonucleic acid 
ROS 
Review of Systems 
RR 
Response rate 
SAE 
Serious adverse event 
SC 
Subcutaneous 
SCC 
Siteman Cancer Center 
SCT 
Stem cell transplant 
SD 
Stable disease 
SLCH 
St. Louis Children’s Hospital 
TBI 
Total body irradiation 
TCD 
T cell depleted 
Tconv 
Conventional T cell 
TF 
Treatment failure 
TREC 
T cell receptor excision circle 
Treg 
Regulatory T cell 
TRM 
Treatment-related mortality 
ULN 
Upper limit of normal 
UPN 
Unique patient number 
WHO 
World Health Organization 
WU 
Washington University 


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TABLE OF CONTENTS 
Phase I/II Trial of Intravenous Azacitidine in Patients Undergoing Matched Unrelated Stem Cell 
Transplantation ............................................................................................................................... 2 
Amendment #8 Version 
      08/02/2016 ..................................................................................... 2 
SCHEMA ........................................................................................................................................ 3 
Glossary of Abbreviations .............................................................................................................. 4 
1.0 
BACKGROUND AND RATIONALE ............................................................................... 9 
1.1 
Graft-versus-Host Disease after Allogeneic Stem Cell Transplantation for Acute 
Myeloid Leukemia and Myelodysplastic Syndrome .................................................................. 9 
1.2 
Investigational Agent:  Azacitidine (5’-Azacytidine, Vidaza, Celgene) ...................... 20 
1.3 
Study Rationale ............................................................................................................. 26 
1.4 
Correlative Studies Background ................................................................................... 26 
2.0 
OBJECTIVES ................................................................................................................... 28 
2.1 
Primary Objective ......................................................................................................... 28 
2.2 
Secondary Objectives.................................................................................................... 29 
3.0 
PATIENT SELECTION ................................................................................................... 29 
3.1 
Inclusion Criteria .......................................................................................................... 29 
3.2 
Exclusion Criteria ......................................................................................................... 31 
3.3 
Donor Selection ............................................................................................................ 32 
3.4 
Eligibility of Women and Minorities ............................................................................ 32 
4.0 
REGISTRATION PROCEDURES .................................................................................. 32 
4.1 
Confirmation of Patient Eligibility ............................................................................... 32 
4.2 
Patient Registration in the Siteman Cancer Center OnCore Database ......................... 32 
4.3 
Assignment of UPN ...................................................................................................... 33 
5.0 
TREATMENT PLAN ....................................................................................................... 33 
5.1 
Overall Treatment Plan ................................................................................................. 33 
5.2 
Treatment Schema ........................................................................................................ 34 
5.3 
Definition of MTD, DLT, Dose Escalation Criteria, and Toxicity, Response, and DLT 
Evaluations ................................................................................................................................ 34 
5.4 
Replacement of Ineligible Patients ............................................................................... 36 
6.0 
ADMINISTRATION OF PROTOCOL TREATMENT .................................................. 36 
6.1 
Conditioning Regimens for Transplant ......................................................................... 36 
6.2 
GvHD Prophylaxis ........................................................................................................ 36 
6.3 
Stem Cell Transplantation (Day 0) ............................................................................... 37 
6.4 
Azacitidine Administration ........................................................................................... 37 
6.5 
Growth Factor Administration ...................................................................................... 39 
6.6 
General Concomitant Medication and Supportive Care Guidelines ............................. 39 
6.7 
Prohibited Medications ................................................................................................. 39 
6.8 
Women of Childbearing Potential ................................................................................ 39 
6.9 
Treatment of Acute GvHD ............................................................................................ 40 
6.10 
Failure to Engraft .......................................................................................................... 40 
6.11 
Duration of Therapy ...................................................................................................... 40 


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6.12 
Post-Treatment Follow-Up ........................................................................................... 41 
6.13 
End of Study Definition ................................................................................................ 41 
6.14 
Definition of Completed Patients.................................................................................. 41 
7.0 
PHARMACEUTICAL INFORMATION ......................................................................... 41 
7.1 
Azacitidine .................................................................................................................... 41 
7.2 
Methotrexate ................................................................................................................. 43 
7.3 
Tacrolimus .................................................................................................................... 43 
8.0 
SCHEDULE OF ASSESSMENTS AND STUDY CALENDERS .................................. 45 
8.1 
Study Calendar – Screening and Treatment .................................................................. 45 
8.2 
Schedule of Assessments – Screening and Treatment .................................................. 46 
8.3 
Study Calendar – Post-Treatment Follow-Up ............................................................... 49 
8.4 
Schedule of Assessments – Post-Treatment Follow-Up ............................................... 49 
9.0 
CORRELATIVE STUDIES ............................................................................................. 51 
9.1 
Treg Analysis ................................................................................................................ 51 
9.2 
Recipient Immune-Reconstitution Analysis ................................................................. 51 
9.3 
Pharmacokinetic (PK) Studies ...................................................................................... 52 
9.4 
Questionnaires............................................................................................................... 54 
10.0 
DATA SUBMISSION SCHEDULE ................................................................................ 54 
11.0 
REGULATORY AND REPORTING REQUIREMENTS .............................................. 56 
11.1 
Definitions..................................................................................................................... 56 
11.2 
Reportable Events ......................................................................................................... 57 
11.3 
Unanticipated Problems ................................................................................................ 57 
11.4 
Noncompliance ............................................................................................................. 58 
11.5 
Serious Noncompliance ................................................................................................ 58 
11.6 
Protocol Exceptions ...................................................................................................... 58 
11.7 
Reporting to the Human Research Protection Office (HRPO) at Washington University
 
58 
11.8 
Reporting to the Quality Assurance and Safety Monitoring Committee (QASMC) at 
Washington University ............................................................................................................. 58 
11.9 
Reporting to the FDA.................................................................................................... 59 
11.10 
Reports to Celgene .................................................................................................... 59 
11.11 
Pregnancies ............................................................................................................... 60 
11.12 
Timeframe for Reporting Required Events............................................................... 61 
12.0 
DATA AND SAFETY MONITORING ........................................................................... 61 
13.0 
STUDY EFFICACY AND DISCONTINUATION ......................................................... 62 
13.1 
Criteria to Measure Efficacy in Mitigation of GvHD ................................................... 62 
13.2 
Definitions for Safety and Efficacy Assessments ......................................................... 62 
13.3 
Response Review .......................................................................................................... 64 
14.0 
STATISTICAL CONSIDERATIONS.............................................................................. 65 
14.1 
Study Design ................................................................................................................. 65 
14.2 
Sample Size Calculation ............................................................................................... 66 
14.3 
Accrual .......................................................................................................................... 66 
14.4 
Statistical Analyses ....................................................................................................... 66 


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15.0 
REFERENCES ................................................................................................................. 70 
APPENDIX 1: ECOG Performance Status Scale ......................................................................... 75 
APPENDIX 2: Registration Form ................................................................................................ 76 
APPENDIX 3: Acute GVHD Assessment .................................................................................... 77 
APPENDIX 4: Chronic GVHD Assessment ................................................................................ 78 
APPENDIX 5: FACT-BMT (Version 4) ...................................................................................... 90 
APPENDIX 6: Human Activity Profile ........................................................................................ 94 
APPENDIX 7: Immunosuppressive Medication Questionnaire ................................................... 96 
 


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1.0 
BACKGROUND AND RATIONALE 
 
1.1 
Graft-versus-Host Disease after Allogeneic Stem Cell Transplantation for 
Acute Myeloid Leukemia and Myelodysplastic Syndrome 
 
1.1.1 Myelodysplastic Syndrome and Acute Myeloid Leukemia  
 
1.1.1.1 General Background 
 
Myelodysplastic syndromes (MDS) are clonal hematologic disorders 
characterized by ineffective hematopoiesis in one or more hematopoietic 
lineages.  MDS is estimated to affect 3/100,000 persons per year in the 
United States with peak incidence occurring at age >601,2  The three-year 
overall survival estimate based on registry data is approximately 35-45%1,2 
but median survival is less than six months in high-risk MDS.3 
Approximately half of all MDS cases will transform to acute myeloid 
leukemia (AML).  The only available curative therapy for MDS remains 
allogeneic hematopoietic stem cell transplantation (allo-HSCT).  However, 
few patients are good candidates for this intensive therapy and the incidence 
of relapse and mortality is increased in high-risk and older patients.4-6  The 
hallmarks of MDS include peripheral blood cytopenias and bone marrow 
hyperplasia with dysplastic hematopoietic precursor morphology.  MDS 
and AML are similar diseases in the elderly and likely represent a spectrum 
of neoplastic evolution in this patient population.  Front-line therapy for 
MDS includes supportive care with transfusions and growth factors in low-
risk disease. 
 
In AML, the most frequently diagnosed form of adult leukemia, myeloid 
precursor cell differentiation is inhibited leading to an accumulation of 
immature and blast cells in the bone marrow, peripheral blood and/or other 
organs.  Diagnosis of AML is based on cellular morphology, immunologic, 
cytogenetic, and molecular features and prompts rapid treatment since the 
median survival of untreated AML patients is less than 3 months.7,8 The 
Leukemia and Lymphoma Society expected ~12,000 cases in the United 
States in 2010 and the incidence is anticipated to rise as the population ages 
and the number of cases swells exponentially from 0.8 per 100,000 people 
at age 45 to >15 per 100,000 people at age 75.9 
 
When given standard induction chemotherapy for AML (cytosine 
arabinoside and an anthracycline), a complete remission is possible in 60-
80% of patients younger than 60 years7,8 and in 45-60% of patients older 
than 60 years.10-13 Unfortunately nearly all of these patients relapse (median 
4-8 months) due to minimal residual disease unless given additional 
consolidation therapy.   Such treatment usually consists of additional 
cytotoxic chemotherapy with or without allo-HSCT.  Those patients most 
apt to be helped by allo-HSCT can be identified by pre-treatment variables 
such as AML subtype, unfavorable cytogenetics, FLT-ITD mutational 


Amendment 11: Version 09/06/18 
 
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status, and pre-existing hematological disorders.  After a nonmyeloablative 
allo-HSCT, relapse remains the most common reason for treatment failure 
with a median relapse time of 3-6 months.  If a myeloablative preparative 
regimen is used, median relapse time is somewhat extended. 
 
The Center for International Blood and Marrow Transplant Research 
(CIBMTR) maintains a registry of hematopoietic cell transplantation (HCT) 
outcomes from more than 500 centers worldwide. Recently, the CIBMTR 
reported the registry outcomes of adults with AML undergoing a matched 
unrelated donor HCT.14 Two-thirds of patients lack a suitable related donor.  
Of 2223 adult AML patients undergoing allogeneic HCT between 2002 and 
2006 who were evaluated, 1193 had 8/8 HLA matched unrelated donor 
transplants.  The Day 100 incidence of clinically significant acute graft-
versus-host disease (GvHD) in this group was 51% (95% CI 48-54), with 
life threatening acute GvHD occurring in 25% (95% CI 23–28).  The overall 
survival and relapse free survival at 1 year was 52% (95% CI 50–55) and 
35% (95% CI 32–37) respectively. 
 
Relapse is a major problem after allo-HSCT. The National Cancer Institute 
international workshop on the biology, prevention, and treatment of relapse 
after allo-HSCT summarized current data on the outcomes of patients with 
AML and MDS who relapse after transplant.15  The incidence of relapse for 
AML in first remission is 10–40%.  This incidence increases to >40–50% 
in advanced disease.  The outcomes after relapse are poor and depend on 
patient age, remission duration, cytogenetics, disease stage at relapse, and 
other comorbidities.  Standard treatment for relapse after allo-HSCT 
includes chemotherapy and donor leukocyte infusion(s), but this is rarely 
curative and most patients relapsing will end up dying from their disease.  
For patients with MDS, the risk of relapse post allo-HSCT is 20-60%. Those 
with low risk disease have a recurrence rate of 5–10%, and the rate for those 
with high-risk disease is 10–40% (similar to AML).  Those relapsing with 
MDS after allo-HSCT also have poor long-term survival, which depends on 
factors similar to those for AML patients (disease activity at transplant, 
cytogenetics, age, therapy related MDS). Long-term survival after relapse 
of MDS post allo-HSCT is 0%-40%.  In conclusion, the outcomes of relapse 
of MDS and AML after allo-HSCT are dismal and strategies to reduce this 
major cause of morbidity and mortality after transplant are needed. 
 
We evaluated our own institutional data on relapse and GvHD after allo-
HSCT at Washington University School of Medicine in Saint Louis. Our 
data collected over a recent two year period (2010 – 2011) suggests similar 
outcomes to that reported by the CIBMTR.  Clinically significant GvHD 
occurred in 52–66% of recipients of unrelated donor transplants, similar to 
the 51% observed by the CIBMTR.  Life-threatening GvHD occurred in 
17–21% of recipients. The overall survival and relapse free survival at 1 
year was 46–49% and 30–36%, respectively, dependent on the type of 


Amendment 11: Version 09/06/18 
 
Page 11 of 96 
conditioning regimen used.  Table 1 summarizes these findings. 
 
Table 1.  Washington University Transplant Outcomes (2010 – 2011) 
Donor 
Preparative 
Regimen 
n 
aGvHD 
Gr II-IV 
aGvHD 
Gr III-
IV 
1 yr 
NRM 
1 yr 
OS 
1 yr 
Relapse 
MRD 
 
 
 
 
 
 
 
 
Myeloablative 
79 
43% 
9% 
15% 
57% 
32% 
 
Reduced Intensity 
33 
56% 
12% 
18% 
52% 
30% 
 
 
 
 
 
 
 
 
URD 
 
 
 
 
 
 
 
 
Myeloablative 
116 
66% 
17% 
25% 
49% 
36% 
 
Reduced Intensity 
47 
52% 
21% 
13% 
46% 
30% 
Incidence of acute GvHD, non-relapse mortality (NRM), overall survival (OS), and relapse after 8/8 
HLA matched related (MRD) and unrelated donor (URD) allogeneic transplant in patients with AML or 
MDS. 
 
In the event of a relapse, a remission may be re-established by salvage 
chemotherapy followed by a donor lymphocyte infusion (DLI) in which the 
patient receives additional lymphocytes from the same donor without 
prophylactic immunosuppression.  A retrospective analysis16 of ~400 AML 
patients in 41 centers indicates that such therapy doubled the estimated 
survival at 2 years from ~9% to ~21%.  The drawback of providing a new 
infusion of allogeneic cells is that many patients suffered from acute GvHD 
(43%) and 80% of these had grade II to IV disease.  In patients surviving at 
Day 100 post-DLI, 46% had chronic GvHD.  
 
DLI recipients also experience pancytopenia in 18-50% of cases.  Infusing 
more donor stem cells can often correct the marrow aplasia, especially if it 
was due to bone marrow failure.  While it usually resolves without therapy 
and only 2-5% have sustained marrow aplasia, the cause of the 
pancytopenia is unclear. We retrospectively examined data from 20 
relapsed AML patients at Barnes Jewish Hospital in St. Louis who had 
received a DLI after allo-HSCT (Table 2).   
 
 


Amendment 11: Version 09/06/18 
 
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Table 2.  Effect of time between chemotherapy and DLI on recovery from pancytopenia 
Patient 
Time interval (days) 
GVHD 100 
days after 
DLI 
(Y: 1, N: 2) 
Mobilized: 1 
Non-mobilized: 2 
#CD3+ cells 
infused  
(x 106) 
#CD34+ cells 
infused 
 (x 106) 
Chemotherapy 
to DLI 
Chemotherapy 
to neutrophil 
count recovery1 
DLI to 
neutrophil count  
recovery 
 
1 
15 
25 
10 
2 
1 
10 
1 
2 
13 
28 
15 
1 
1 
10 
0.8 
3 
37 
28 
0 (-9) 
1 
2 
10 
0.03 
4 
14 
21 
17 
n/a2 
1 
10 
0.39 
5 
55 
28 
0(-27) 
1 
2 
10 
0.01 
6 
17 
28 
11 
2 
1 
10 
0.44 
7 
n/a3 
n/a 
0 
1 
2 
10 
0.04 
8 
27 
32 
5 
2 
2 
1 
0 
9 
38 
43 
5 
n/a 
1 
100 
1.76 
10 
18 
35 
17 
2 
2 
89 
0.15 
11 
70 
24 
0(-46) 
2 
2 
10 
0.03 
12 
28 
29 
1 
1 
1 
10 
0.53 
13 
15 
39 
24 
2 
1 
10 
2 
14 
46 
26 
0(-20) 
2 
2 
10 
0.01 
15 
15 
31 
16 
2 
1 
10 
0.13 
16 
14 
34 
20 
n/a 
1 
10 
0.33 
17 
16 
21 
5 
1 
1 
30 
1.1 
18 
92 
27 
0(-65) 
1 
2 
10 
0.01 
19 
11 
25 
14 
2 
1 
10 
0.12 
20 
15 
49 
34 
n/a 
1 
10 
0.51 
Mean 
(Range) 
27.8 (11-92) 
 
28.6 (21-49) 
 
13.8 (1-34) 
 
Yes: 7 (35%) 
No:  9 (45%) 
N/A: 4 (20%) 
Mob: 12 (60%) 
Non-Mob: 8 (40%) 
19 (1-100) 
0.47 (0-2) 
SD 
22.2 
9.5 
9.5 
 
 
 
 
Median 
16.5 
28 
(14.5) 
 
 
10 
0.24 
1Defined as absolute neutrophil count >500/mm3 
2Not applicable.   
3Patient 7 received no chemotherapy. 
 
Nineteen patients had chemotherapy prior to DLI.  Among these patients, 
there was a striking drop in the time required to recover absolute neutrophil 
counts (ANC) post-DLI in those who received the infusion ≥27 days after 
pre-DLI chemotherapy (median = 0 days, range = 0-5 days) compared to 
those who received an earlier infusion (median = 16 days, range 5-34 days). 
The DLI caused no new cytopenia in these patients.  
 
In conclusion, the current treatments for relapse after allo-HSCT are 
unsatisfactory and novel approaches are needed to both prevent 
relapse and decrease the incidence of GvHD and its associated 
morbidity and mortality. 
 
1.1.1.2 Role of DNA Methylation in MDS and AML 
 
Epigenetic mechanisms, such as DNA methylation of CpG dinucleotides 
and deacetylation of histones, have evolved to allow genetically identical 
cells to express different phenotypes, especially during differentiation into 
the tissues and organs of multicellular organisms.  Genes with heavily 
methylated promoter regions are generally silent, while those with 
hypomethylation tend to be active.  Alteration of the methylation state may 


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Page 13 of 96 
lead to unintended expression or shut down of genes resulting in aberrant or 
pathological phenotypes, such as uncontrolled proliferation, angiogenesis, 
and altered cell adhesion.  In addition altered methylation can lead to 
chromosome instability, a hallmark of many tumor cells.  Virtually all 
cancers are characterized by changes in the methylation state, suggesting 
that this is a predisposing “hit” in Knudson’s two-hit model of 
carcinogenesis (for a review see Lopez17).  Because hypermethylation is 
associated with only rare genes in healthy individuals, the majority of 
methylation changes in cancer cells are specific to the disease.   Loss of 
heterozygosity resulting from progressive DNA methylation has been 
reported as a critical event in MDS patients that progress to AML.18  
Recently, the hypomethylating agent azacitidine has been shown to reverse 
hypermethylation in patients with MDS and secondary AML.19  Blocking 
the hypermethylation of tumor suppressor genes may halt or delay 
progression to AML. 
  
Aberrant methylation has long been recognized as being associated with 
AML, prompting Figueroa, et al.20 to perform a retrospective analysis of the 
methylation patterns of ~14,000 genes in 344 patients.  Their work revealed 
16 different AML groups with internally common clinical outcomes.  Five 
of these established new AML subtypes.  The majority of these groups 
featured distinct strong hypermethylation patterns, although a few were also 
characterized by hypomethylation.  Some of the groups had normal 
genotypes, which emphasizes the importance that epigenetics has in the 
establishment of disease.  A set of 15 genes was identified whose 
methylation status could be used to predict the clinical prognosis for the 
patients, and for some of the groups this status represents the only currently 
available diagnostic method. 
 
Sudan et al.21 retrospectively explored the use of azacitidine (5-AzaC), a 
drug that decreases DNA methylation, in AML outpatients (median age=66) 
using multiple cycles of 75mg aza/m2/day for 7 days every 4 weeks.  Those 
who responded to therapy (60%) survived with a median of over 15 months 
while those who did not had a median of only 2.5 months.  The treatment 
was deemed well tolerated with only 8 out of 20 needing hospitalization for 
complications during any of the treatment cycles.  In comparison the 
patients receiving traditional chemotherapy were hospitalized during 
infusion and during pancytopenia, if it occurred, for 2-4 more weeks.  The 
FDA has approved a dosing of 525 mg aza/m2 per treatment cycle.  
 
Histocompatibility antigens22 as well as some tumor specific antigens23 
become upregulated upon treatment of cells with hypomethylating agents.  
Thus Lübbert et al.24 reasoned that pre-treating AML or chronic myeloid 
leukemia patients with a low dose of azacitidine (less than a third of the 
FDA approved dose per treatment cycle) prior to DLI might increase the 
anti-leukemia effect of the infused cells.  Care was taken to allow a 7-day 


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wash-out period between the last injection of azacitidine and the DLI so as 
not to affect the function of the donated lymphocytes.  Many of the patients 
were given one or more additional cycles of azacitidine approximately 
every 3 weeks after the DLI.  Overall survival at 2 years was 16%.  Notably, 
only 2 out of 19 patients had de novo cases of GvHD despite initial concerns 
that even low doses of a non-tissue specific demethylating agent might 
increase GvHD by upregulating target genes on normal tissues in addition 
to those on cancer cells. 
 
1.1.2 Graft versus Host Disease 
  
1.1.2.1 GvHD correlates with GvL  
 
Allogeneic bone marrow transplant (BMT) cures leukemia by means of 
cytoreduction induced by the preparative regimen and by transfer of 
immunocompetent alloreactive donor T cells from the bone marrow 
allograft that exert an anti-leukemic effect called the Graft-versus-
Leukemia (GVL) effect.25,26  For many years, the evidence for this GvL 
effect was derived from experimental animal models and retrospective 
comparative analyses of leukemia relapse rates after allogeneic and 
syngeneic BMT in studies with patients experiencing the presence or 
absence of acute or chronic GvHD, and between patients receiving 
unmodified or T cell depleted BMT.  More recently, direct evidence for this 
anti-leukemic effect was demonstrated by the infusion of unaltered donor 
peripheral blood leukocytes into patients who had relapsed after allogeneic 
BMT (for a review see Schleunig27).   
 
The International Bone Marrow Transplant Registry (IBMTR) analyzed 
data from 2,254 patients who underwent HLA-identical sibling BMT for 
leukemia (Chronic Myeloid Leukemia [CML] in chronic phase, AML and 
Acute Lymphoblastic Leukemia [ALL] in first remission).  After adjusting 
for other variables that affect relapse, they showed that there was a 
statistically significant reduction in the relapse risk for patients who 
developed GvHD.25,26 For patients that received unaltered BMT and did not 
develop GvHD, the relative risk for relapse was defined as 1.0.  Patients 
who developed only acute GvHD had a relative risk of relapse of 0.68 
(p=0.03), and patients who developed only chronic GvHD had a relative 
risk of 0.43 (p=0.01), while patients who developed both acute and chronic 
GvHD had a relative risk of 0.33 (p=0.0001).  The relative risk for relapse 
for patients who developed both acute and chronic GvHD was 0.34 for 
AML.  Thus, there is a strong association between anti-leukemic effect and 
GvHD, especially chronic GvHD. 
 
It was also shown that the anti-leukemic effect parallels the severity of 
GvHD.  The removal of mature T cells from the bone marrow graft 
represents the most effective way to prevent the development of acute and 


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chronic GvHD.  This benefit of T cell depletion is offset by increased graft 
failure and leukemia relapse, so that overall survival is not improved.  The 
IBMTR compared HLA-identical sibling transplants in 731 recipients of T 
cell depleted transplants with 2,480 recipients of non-T cell depleted 
transplants.  The relative risk of relapse after T cell depleted transplant was 
significantly increased compared to non-T cell depleted transplant for all 
types of leukemia (ALL, AML, CML) and all stages (early, intermediate, 
advanced) of disease with the exception of AML in or beyond second 
remission and CML in blast crisis.28   Because T cell depletion decreases 
GvHD, which affects leukemia relapse, the analysis was repeated after 
adjusting for incidence and severity of acute and chronic GvHD to examine 
if T cell depletion independently affects leukemia relapse.  After adjusting 
for GvHD, the relative relapse risks with T cell depletion for AML and ALL 
in first remission and CML in chronic phase were 1.66 (p=N5), 1.55 (p=N5) 
and 4.87 (p < 0.0001) respectively compared to non-T cell depleted 
transplants.  Therefore it appears that T cell depletion increases leukemia 
relapse in AML and ALL via loss of GvHD.  Thus, there is need for 
transplant strategies that effectively eliminate GvHD while maintaining the 
graft-versus-tumor effect. 
 
Demonstration that the GVL effect exists in the absence of GvHD is best 
illustrated by transplant studies using identical-twin donors that are 
associated with no or minimal GvHD and a relatively high rate of leukemia 
relapse. In a matched-pair analysis of IBMTR data, leukemia relapse rates 
were compared between 103 patients transplanted from identical-twin 
donors and 1,030 patients transplanted from HLA-identical siblings.28,29  
The 3-year probability of relapse for AML was 52% after an identical twin 
transplant compared with 16% after an HLA-identical sibling transplant.  
After adjustment for acute and chronic GvHD the relative risks for relapse 
of syngeneic compared to allogeneic transplants was 3.1 (95% confidence 
interval, 1.9 to 5.1) for AML.  Therefore, it appears that there is an 
allogeneic GvL effect for AML that is independent of GvHD, yet still 
dependent on T cells. 
 
Despite the potential positive impact on reducing disease recurrence 
after transplant, the negative impact of acute GvHD on morbidity and 
mortality after allogeneic transplant is significant. According to a recent 
study by the CIBMTR30, including 4224 patients with AML and 1517 
patients with MDS, those patients that develop acute GvHD after transplant 
have an increased risk of treatment related mortality (HR 2.51 (95% CI 2.18 
– 2.89)), and lower overall survival (HR 1.71 (95% CI 1.55 – 1.89)) based 
on a time dependent multivariate analysis. Our own institutional incidence 
of acute GvHD after allo-HSCT is similar to that reported by the CIBMTR 
registry (Table 1). Clinically significant acute GvHD occurred in 52–66% 
of recipients of unrelated donor transplants and life threatening acute GvHD 
occured in 17–21% of recipients. Acute GvHD remains a significant 


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problem in our study population; thus, finding a means to harness the 
GvL effect while reducing or eliminating GvHD is a major goal in 
designing novel transplant trials. 
 
1.1.2.2 Regulatory T cells and GvHD  
 
Since GvHD is a result of donor cells recognizing recipient cells as foreign 
entities, GvHD should be controlled by those cells that mediate self-
tolerance and homeostasis: regulatory T cells (Tregs).  These are the only 
cells known to express FOXP3, the forkhead transcription factor, which has 
a prominent role in maintaining self-tolerance.  Defects in FOXP3 
expression lead to severe immunosuppressive diseases both in humans and 
mice,31-33 and depleting rodents of Tregs mimics these diseases.  Ectopic 
expression of FOXP3 in conventional mouse CD4+ T cells leads to 
acquisition of suppressive activity and expression of molecules 
characteristic of Tregs, including CD25.  These results suggest that FOXP3 
is a master regulator that differentiates conventional CD4+ cells into Tregs.  
 
In a recent prospective clinical trial Magenau et al34 found that the ultimate 
severity of GvHD could be predicted by the relative frequency of Tregs at 
its onset.  Patients who received autologous BMT had average ratios of 
Tregs to conventional T cells similar to those of healthy individuals (1.09 
and 1.17 respectively), as did patients who received an allogeneic BMT, but 
did not acquire GvHD (~1.06).  In contrast when patients first began to 
suffer from GvHD, their average ratio was ~0.75.   The lower the frequency 
of CD4+CD25hiFOXP3+ cells, the more severe the ultimate GvHD.  
Frequencies below 0.5% were associated with poor outcomes in terms of 
overall survival, non-relapse mortality, maximum GvHD grade, and 
treatment response at 4 weeks. 
 
In an animal model Tregs controlled GvHD while maintaining effective 
GvL.  Edinger et al.35 showed that Treg cells (CD4+CD25+FOXP3+) 
coinfused into mice along with conventional T cells (CD4+CD25- and 
CD8+CD25-), inhibited growth of a luciferase-tagged leukemia cell line as 
determined by biolumenscence measurements.  In addition the Tregs 
suppressed GvHD by limiting the normal increase in number of 
conventional T cells even though these cells still migrated to the skin, lymph 
nodes, and gut.  Most animals (~67%) survived the entire 60d observation 
period.  
 
Similar administration of Tregs to patients might thus be expected to have 
a therapeutic benefit.  However the difficulty in developing a rapid 
antibody-based purification method has thwarted attempts to do this.  
FOXP3, the most reliable marker for Tregs, is an intracellular protein, thus 
precluding its use as a selectable marker.  Although cell surface expression 
of CD25 closely correlates with FOXP3 expression, the suppressive Treg 


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effect is found only in those few cells with the highest CD25 expression.  In 
addition there is a threshold level of FOXP3 and CD25 expression needed 
for fully developed Treg functions:  CD4+CD25midFOXP3low cells still 
retain a naïve T cell phenotype (CD45RAhiCD45RO-).  Selection for cells 
lacking CD127 has also been explored as a means to enrich for Tregs, but 
when used along with CD25+ selection, it results in co-purification of a 
subset of FOXP3low cells. 
 
In lieu of infusing purified Tregs into patients to achieve an antileukemic 
effect with decreased GvHD, post-transplant treatment strategies that 
increase the proportion or absolute numbers of Tregs could prove beneficial.  
In a kidney transplant setting it has been shown that switching patients from 
immunotherapy with multiple agents to one using only rapamycin 
(sirolimus) 
resulted 
in 
an 
increase 
in 
the 
number 
of 
CD4+CD25bright+FoxP3+ Treg cells.36  This increase did not occur when a 
switch was made to tacrolimus.  Even haploidentical patients had a 
decreased incidence of GvHD when rapamycin was used post-bone marrow 
transplant.37  Likewise a brief high dose of cyclophosphamide post-BMT 
tended to increase CD4+CD25+FoxP3+ numbers, and patients with the 
highest numbers had the least severe GvHD.38-40  However relapse rates 
remained high with either rapamycin or hi dose cyclophosphamide therapy. 
When mice were given azacitidine post-transplant, we found an increase in 
the number of Tregs, decreased GvHD, and very effective tumor control 
(see section 1.1.2.3). In the trial described here we will begin to examine 
the safety and effectiveness of azacitidine treatment administered following 
allogeneic stem cell transplant as GvHD prophylaxis.  
 
1.1.2.3 DNA methylation, Tregs, and GvHD 
 
Just as DNA methylation plays a role in the phenotype of cancer cells, DNA 
methylation also affects the differentiation of immune cells with important 
consequences for controlling both tumor growth and GvHD.  In both mice 
and humans a demethylated FOXP3 locus represents the most reliable 
criterion for natural Tregs.33,41-43   The Foxp3 locus is heavily methylated 
and silenced in all cell types examined with the sole exception of Tregs, the 
only cell type in which it is known to be expressed.  
 
Reasoning that hypomethylating agents such as azacitidine or decitabine 
might affect the expression of FOXP3, Sanchez-Arbaca et al.44 treated 
CD4+CD25-FOXP3- T cells with 100 nM of the hypomethylating agent, 
azacitidine for 14 days.  They found that this treatment decreased the 
fraction of the FOXP3 promoters existing in a methylated state.  
Simultaneously the percentage of cells with a CD4+CD25+FOXP3+ 
phenotype had increased from 6% before drug treatment to 27% afterwards.  
Unfortunately these cells neither proliferated nor became activated in vitro.  
Nonetheless with the hope of limiting GvHD, these investigators then 


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explored whether azacitidine could induce Tregs in vivo when provided 
after an allogeneic T cell infusion in mice.  They saw the best overall 
survival and least GvHD when the drug was provided at 1 mg/kg in 2 cycles, 
the first at 60 and 80 hours post-transplant, and the second at 19, 21, and 23 
days post-transplant.  However they saw a significant increase in the 
number of Tregs only when the second cycle of azacitidine was given.   
 
Recently in the laboratory of one of our sub-investigators (J.F. DiPersio), 
Choi, et al.45 also demonstrated a dramatic increase in the percentage of 
human or mouse cells expressing FOXP3 in vitro increases after treatment 
with azacitidine or decitabine, another hypomethylating agent.  We 
extended these observations to our allogeneic mouse model in which mice 
were irradiated and then infused the following day with T cell depleted 
(TCD) bone marrow  (BM) to promote recovery from the irradiation-
induced myelosuppression.  Eleven days later the mice were given an 
infusion of mismatched conventional T cells.  Only 3 out of 23 of these 
animals survived.  In contrast every mouse (23/23) survived, became 
engrafted with donor cells, and had no GvHD-related symptoms (weight 
loss, ruffled fur, or diarrhea) when given a single course of four azacitidine 
injections at 2mg/kg, every other day beginning 4 days post-infusion of  
conventional T cells (Tconv, figure 1). When the hypomethylating agent 
decitabine was used instead, the majority of mice died from pancytopenia.  
Azacitidine-treated mice also had high levels of donor CD3+ T cells and 
B220+ B cells as has been seen clinically in patients who experience less 
severe GvHD. 
 
While Sanchez-Abarca44 had found that azacitidine inhibits the proliferation 
and activation of T cells in vitro, our dosing method permitted 
carboxyfluorescein succinimidyl ester-labeled allogeneic donor cells to 
proliferate vigorously in vivo.  The proportion of FOXP3+ cells also 
increased (figure 1), suggesting that Tregs were at least partly responsible 
for mediating these effects.  Notably this increase in Tregs after only 1 cycle 
of azacitidine treatment occurred even when the transplant was depleted of 
Tregs prior to infusion.  This result suggests that azacitidine was not just 
inducing an expansion of Tregs already present in the infused population.  
Instead, Tconv cells were actually being converted into Tregs post-infusion 
as a result of treatment with azacitidine.  
  


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Figure 1. AzaC treatment of mice that underwent a transplantation with delayed allogeneic T 
cells mitigates GVHD. (A) Schema of the experiments. B6 mice (CD45.2) TCD BM (5 x 106 cells) 
were used as a stem cell source. To induce GVHD, 2 x 106 Tconv (B6, CD45.1) were given on day 
11 after SCT followed by the treatment with AzaC/Dec/PBS (every other day; 4 doses) starting on 
day 15 after SCT. (B-F) Mice treated with AzaC (2 mg/kg) show significantly higher survival rate 
(B), less weight loss (C), and more B cells (E) and T cells (F) with donor engraftment (D) than mice 
infused with pbsTs. (G) AzaC group also show increased Treg population in peripheral blood. T rec: 
T cells from recipient; T BM: T cells from donor BM; T donT: T cells from donor T cells; B BM: B 
cells from donor BM. (D-G) Analyzed 1 month after transplantation. A pool of 4 independent 
experiments (Excerpted from Choi et al.,45, figure 4).  
 
As emphasized earlier (Section 1.1.2.1), finding a means to decrease GvHD 
while retaining a GvL effect is a major goal in this field.  Consequently we 
next examined the effect of azacitidine in our well-established GvL model 
using luciferase-tagged tumor cells and bioluminescence imaging of the 
tumor-injected mice.  Irradiated mice were infused as above with T cell 
depleted bone marrow, but this time along with an A20 leukemia cell line, 
and the tumor became well established during the 11 days allowed for bone 
marrow recovery.  At this point conventional T cells were provided, 
followed again by the single low dose course of azacitidine described above.  
In the absence of T cells, this low dose of azacitidine did nothing to control 
tumor growth.  Remarkably, mice treated with azacitidine after the T cell 
infusion had both a much lower leukemic burden and significantly better 
survival than tumor-injected mice given the T cells but no azacitidine 
(figure 2).  These data suggest that our method not only preferentially 
suppresses GvHD, but that it also leaves the strong GvL effect intact.  Such 
an effect is a major goal in the treatment of AML patients. 


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Figure 2. AzaC treatment of mice that underwent a transplantation with delayed 
allogeneic T cells mitigates GVHD while preserving GVL (A) Schema of the experiments. 
B6 mice (CD45.2) TCD BM (5 x 106 cells) were used as a stem cell source. To induce GVHD, 10 
x 106 Tconv (B6, CD45.1) were given on day 11 after SCT followed by the treatment with AzaC 
or PBS (every other day; 4 doses) starting on day 15 after SCT. For examination of GVL effect, 
1 x 104 A20-luc/egfp leukemic cells were given along with TCD BM. (B-C) Mice treated with 
AzaC show significantly higher survival rate (B) and lower leukemic burden (C). Y axis in top 
panels indicates photon flux (photons/sec) in log scale measured from the dorsal and the 
ventral view with a region of interest drawn over the entire body of each mouse. Actual 
images of 1 representative mouse from each group are shown in bottom panels (scale: 
photons/sec/cm2/sr). A pool of 3 independent experiments (Excerpted from Choi et al.45 
Figure 6).   
 
1.2 
Investigational Agent:  Azacitidine (5’-Azacytidine, Vidaza, Celgene)  
 
In this trial we hope to extend to patients the observation from our mouse model that 
azacitidine treatment post-DLI mitigates GvHD while retaining GvL.  This effect was not 
seen with another hypomethylating agent, decitabine, as mouse survival was compromised 
by pancytopenia.  
 
 


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1.2.1 Mechanism of Action 
 
Azacitidine is a pro-drug that is metabolized in vivo to its more active substrate, 5-
azacitidine, a pyrimidine nucleoside analog of cytidine.  The FDA has approved its 
use in treating MDS and other leukemias, e.g. AML.  Its cytotoxic effects are 
directly related to dose, and this is greatest in cells that are proliferating and 
metabolically active.46-48   
 
Azacitidine incorporates primarily into RNA and is incorporated into DNA only 
after 5-aza-ribonucleotides are converted into 5-aza-deoxyribonucleotides by 
ribonucleotide reductase.  Normally the mammalian enzymes DNMT1 and 
DNMT3b catalyze the covalent attachment of methyl groups to the C5 position of 
cytosine in CpG dinucleotides.  DNMT3b is believed to establish de novo 
methylation patterns during mammalian development, while DNMT1 transfers and 
maintains the pattern in daughter strands of DNA.  In treated cells 5-aza-
deoxyribonucleotides can covalently trap DNMTl, thus depleting it from the 
cellular pool and resulting in global demethylation.49,50 A third highly conserved 
enzyme, DNMT2, has limited DNA methytransferase activity, but is noteworthy 
for its recently discovered ability to specifically methylate cytosine 38 in 
tRNAAsp.51 DNMT2 is expressed predominantly in testis, but Schaefer et al.52 
discovered that it is also expressed in many human cancer cell lines, including three 
representing myeloid leukemias: HL-60, K562, and ML-1.  In these cells 5-AzaC, 
but not decitabine, caused hypomethylation of tRNAAsp, thus altering incorporation 
of this amino acid into proteins and consequently affecting cellular metabolism.  
Hypomethylation of tRNAAsp was greatest after one 3-day azacitidine treatment 
period.  
 
1.2.2 Toxicology and Safety 
 
1.2.2.1 Preclinical studies   
 
Cytotoxic effects from azacitidine are directly related to dose and are 
greatest in cells that are proliferating and metabolically active.46-48  Studies 
in mice, rats, dogs, and Rhesus monkeys identified the bone marrow, liver, 
kidneys, and lymphoid tissues (spleen, lymph nodes, and thymus) as the 
main target organs of toxicity for azacitidine.53 The lethal single dose of 
intravenous azacitidine in mice, rats, and dogs was approximately 250 
mg/m2.  Repeated daily dosing increases the toxicity.53 
 
In studies on carcinogenicity, mice had an increase in hematopoietic tumors 
when given 3 injections/week for 52 weeks of 2.2mg/kg azacitidine (~6.6 
mg/m2 which corresponds to ~8% of the recommended human daily dose 
on a mg/m2 basis).  There was also an increase in the number of 
lymphoreticular system, lung, mammary gland, and skin tumors in those 
given 2.0 mg/kg once a week for 50 weeks.  An increase in testicular tumors 
was seen in rats given twice weekly doses of 15-60 mg aza/m2 (20-80% of 
the recommended human dose on a mg/m2 basis).  These tumors may in part 


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be due to the mutagenic potential demonstrated in mouse lymphoma cells 
and human lymphoblast cells and the clastogenic potential seen in L5178Y 
mouse cells and Syrian hamster embryo cells. 
 
In studies on reproductive effects, male mice given 9.9 mg aza/m2 daily 
(corresponding to ~9% of the recommended human daily dose) for 3 days 
prior to mating with untreated females had both decreased fertility and loss 
of offspring.  Male rats had decreased testicular and epididymal weight, 
lower sperm counts, as well as decreased pregnancy weight and a loss of 
embryos in their untreated female mates when treated 3 times per week for 
11 or 16 weeks at 15-30 mg/m2 (~20-40%, the recommended human daily 
dose).  A similar study showed an increase in abnormal embryos in mated 
females after 2 days gestation.54  
 
1.2.2.2 Clinical Toxicities  
 
The FDA approved starting dose of azacitidine for MDS is 75 mg/m2/day 
for 7 days every 28 days. Azacitidine at a dose of 75 mg/m2 single SC 
injection is well tolerated.55 Three trials of azacitidine in MDS were updated 
using the IWG response criteria and showed overall response rates between 
40% and 70% (10-17% CR, 23-36% hematologic improvement).56   A large 
randomized Phase III trial in higher-risk MDS patients, classified by FAB 
as RAEB, RAEB-T, or CMML with 0-29% marrow blasts, and an IPSS of 
Intermediate -2 or High was performed using azacitidine (75 mg/m2/day x 
7days in 28 day cycles) versus conventional care regimens.57 Three hundred 
fifty-eight patients were randomized to azacitidine (N=179) or conventional 
care (CCR) (N=179): best supportive care only (N=105, 59%), low-dose 
cytarabine (N=49, 27%), or standard chemotherapy (N=25, 14%). At 
baseline, 95% of patients were higher risk: RAEB (58%), RAEB-T/WHO 
AML (34%), CMML (3%), and other (5%). By IPSS, 87% were higher risk: 
Intermediate -2 (40%), High (47%), and 13% Indeterminate/other. 
Azacitidine was administered for a median of 9 cycles; low-dose cytarabine 
for 4 cycles. Median follow-up for the survival analysis was 21.1 months. 
Azacitidine demonstrated statistically superior overall survival compared to 
CCR, with a median overall survival of 24.4 months vs. 15 months for CCR 
(stratified log-rank p=0.0001, hazard ration 0.58). Two-year survival 
approximately doubled in the azacitidine arm compared to CCR: 51% vs. 
26% (p<0.0001). Azacitidine was well tolerated with safety data consistent 
with previous reports.  The efficacy and safety of azacitidine has been 
established by the subcutaneous and intravenous routes and is described 
further in the package insert and the investigator’s brochure, which contains 
comprehensive 
pharmacology, 
toxicology, 
pharmacokinetics, 
pharmacodynamics, metabolism, preclinical, and clinical efficacy and 
safety data information. 
 
In AML patients, Sudan et al.21 retrospectively explored the use of 
azacitidine in outpatients (median age=66) using 1 or more cycles of 75 


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mg/m2/day for 7 days every 4 weeks.  Those who responded to therapy 
(60%) survived with a median of over 15 months while nonresponders had 
a median of only 2.5 months.  Only 8 out of 20 needed hospitalization for 
complications, while all patients receiving traditional chemotherapy were 
hospitialized during infusion and for 2-4 more weeks during pancytopenia.  
Infection was the most common toxic event.  In an analysis of 3 sequential 
Cancer and Leukemia Group B (CALGB) trials, AML patients receiving an 
azacitidine dosing schedule similar to the one above had a median survival 
of 19.3 months compared to 12.9 months in those with conventional 
treatment.  There was no increase in infection or bleeding.  Similar 
observations on the drug’s safety come from a phase III trial reported by 
Fenaux, et al.57 in which patients receiving azaciditine had significantly 
fewer morbidities and an overall survival of 24.5 months compared to 16.0 
months for conventionally treated patients.  More azacitidine-treated 
patients achieved transfusion independence, and although infections were a 
problem, fewer required intravenous antibiotics.  The most frequent 
hematological 
problems 
were 
leukopenia, 
granulocytopenia, 
thrombocytopenia, neutropenia, and anemia.  Such toxicity was usually 
transient, and patients recovered prior to their next cycle of azacitidine 
treatment.   
 
Azacitidine has been used along with other drugs in AML patients.  When 
combined with hydroxyurea and gemotuzumab treatment, there was a low 
overall toxicity (5% early death due to disease progression). A study 
combining azacitidine with all-trans retinoic acid and escalating doses of 
valproic acid eventually resulted in dose-limiting neurotoxicity.  In a study 
of patients with a variety of advanced malignancies, Braiteh et al.58 
examined the safety of azacitidine in combination with valproic acid, a 
histone deacetylating agent that also causes epigenetic modifications.  The 
initial dosing used 20 mg/m2 and was escalated to a maximum tolerated 
dose of 75 mg/m2.  Grade 3 or 4 toxicities were neutropenic fever and 
thrombocytopenia.  At the grade 1 or 2 level, the most common problems 
were somnolence and tremor.   
 
1.2.3 Rationale for Dosing Regimen  
 
The FDA determined that there is an acceptable safety profile using the approved 
dosing of azacitidine in multiple cycles of 75 mg/m2 every 28 days for MDS. The 
study proposed here is designed as a traditional 3+3 phase I trial with the intention 
of defining the Maximum Tolerated Dose (MTD) of azacitidine when given 
intravenously daily on Days 7, 8, 9, 10, and 11 in conjunction with a matched (8/8) 
unrelated peripheral blood transplant. In order to optimally suppress relapse and 
GvHD, the same dose of azacitidine will be given daily for 5 days beginning on 
approximately Days 35, 63, and 91 post-transplant.  Appropriate dose reductions of 
azacitidine in the last 3 cycles will be performed based on the guidelines in Section 
5.4. Allowed conditioning regimens for transplant are shown in Section 5.1.1. 


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Standard mini-dose methotrexate and tacrolimus will be given for GvHD 
prophylaxis (Section 5.1.2).  For the Phase I portion of this trial, patients with other 
hematological malignancies will be eligible.  After determination of the MTD from 
the first cycle of azacitidine (days 7-11), we will use this dose to perform a phase 
II study in patients with AML in remission 1 or 2 or MDS (≤70 years old).  The 
primary endpoint to this study will be grade II–IV acute GvHD.  The study is 
powered based on the mean incidence of acute GvHD by Day +180 from registry 
data of unrelated allo-HSCT for AML CR1 or 2 (n = 404) provided by the CIBMTR 
which is 44% (95% CI 39% – 49%). (The data presented here are preliminary and 
were obtained from the Statistical Center of the Center for International Blood and 
Marrow Transplant Research. The analysis has not been reviewed or approved by 
the Advisory or Scientific Committee of the CIBMTR.).  Of note is that the actuarial 
risk of aGvHD provided by the CIBMTR is slightly less than our own institutional 
data (see Table 1) perhaps because we are evaluating all diseases undergoing allo-
HSCT.  We feel a reduction in the incidence of acute GvHD at Day +180 from 
44% to ≤ 20% would be clinically significant. The only change to the standard 
of care is the addition of four 5-day cycles of intravenous azacitidine beginning on 
Days 7, 35, 63, and 91 post-transplant (total of 20 doses per each subject). In our 
mouse studies (see Section 1.1.2.3), early dosing with azacitidine eliminated GvHD 
while retaining GvL. 
 
To date no studies have administered azacitidine early after allogeneic stem cell 
transplant prior to neutrophil engraftment.  Thus the dose selected for the phase I 
portion of this trial is based on our own limited institutional data obtained from an 
ongoing study of azacitidine given during marrow aplasia after chemotherapy and 
DLI for relapsed AML or MDS after allogeneic stem cell transplant.  Data from the 
first 3 patients dosed at 45 mg/m2 on Days 4, 6, 8, and 10 after DLI is summarized 
in Table 3.   Only 1 of the 3 treated patients had delayed count recovery, defined as 
absence of ANC>500 cells/uL greater than 42 days post chemotherapy.  This cohort 
is currently being expanded and if a second DLT (delayed count recovery) is 
observed, we will proceed to the dose minus 1 level of 30 mg/m2.  Additional data 
on azacitidine use after allo-HSCT is available from 2 other studies.59,60 In the de 
Lima study, an adaptive Bayesian approach was used to determine the optimal dose 
based on time to toxicity. Azacitidine was given daily for 5 days starting around 
Day +40 after transplant at the following doses: 8, 16, 24, 32, and 40 mg/m2 and 4 
schedules: 1, 2, 3, or 4 cycles, each with 5 days of drug and 25 days of rest. They 
found that reversible thrombocytopenia was the dose limiting toxicity (DLT) and 
the best dose was 32 mg/m2 given for 4 cycles. At a median follow up of 20.5 
months, the 1 year overall survival was 77%.  In another trial by Goodyear et al., a 
dose of 36 mg/m2 starting on Day +42 in patients with stable engraftment was given 
after reduced intensity allogeneic transplant.  Azacitidine was given daily for 5 days 
on a 28 day cycle for up to 10 cycles and a dose reduction was planned to 24 mg/m2 
if grade 3–4 hematologic toxicity occurred for >2 weeks. Of note, no grade 3–4 
hematologic toxicities were observed. This dosing schedule resulted in limited 
acute GvHD (3/27 (11.1%) in the treatment group and 7/19 (37%) in the control 
group) and no significant reduction in blood counts or donor engraftment at Day 


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+90 post allo-HSCT (full donor peripheral blood chimerism: 19/24 treatment group, 
19/19 control group).  This was also associated with an increase in circulating Tregs 
up to 3 fold compared to time matched controls. This increase in Treg numbers was 
only observed early after transplant at cycle 3 of treatment and not when measured 
after 6 or 9 cycles of azacitidine treatment. Based on this data, we believe that a 
dose between 30 and 45 mg/m2 for 4 cycles of treatment will likely be the 
optimal dose to establish tolerance and efficacy but because aza has never been 
used after transplant we will start dosing at 50% of this dose (15 mg/m2).  
 
Table 3: Azacitidine post DLI preliminary study outcomes 
Subject Azacitidine 
Dose 
Adverse Events 
DLT for count 
recovery? (Y/N) 
Outcome 
001 
None 
- 
 
002 
None 
- 
 
003 
None 
- 
 
004 
45 mg/m2 
N 
 
005 
45 mg/m2 
Y 
Delayed count recovery to day 
+56 
006 
45 mg/m2 
N 
Grade III GI Acute GvHD 
(patient had grade IV GI GvHD at 
the time of original transplant) 
 
In addition to these studies in humans our preliminary data in mice also supports 
the starting dose of 30 mg/m2. We chose to use 2 mg/kg dose in our mouse studies 
because it approximates using 75 mg/m2 in a 70 kg, 1.7 m person.  Unpublished 
data from our mouse model indicate that using 1 mg/kg for each of the 4 injections 
prevented GvHD as effectively as using 2 mg/kg.  Since 1 mg/kg is ~30 mg/m2, we 
have chosen this as our starting dose in this trial. 
 
1.2.4 Pharmacokinetics   
 
Azacitidine is rapidly absorbed when given subcutaneously to humans, with 
maximum plasma concentrations found 0.5 to 2 hours after dosing.  In patients with 
MDS, the peak plasma concentration was reached 11 min after intravenous 
administration (Cmax=12 µM [2750 ng/ml]).55  Azacitidine and its metabolites are 
renally cleared.  The effects of renal or hepatic impairment, gender, age, or race on 
the pharmacokinetics of azacitidine have not been studied.  The bioavailability after 
subcutaneous administration is 89% (range 52% to 128%) with a mean half-life of 
0.69 hours.53 A much lower peak serum concentration of 3 µM azacitidine is 
reached after subcutaneous administration of a therapeutic dose of 75 mg/m2.55 
 
1.2.5 Route of elimination 
 
Azacitidine and its metabolites are renally cleared.  
 


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1.2.6 Metabolism of drug in humans 
 
An in vitro study suggests that azacitidine may be metabolized by the liver, 
however the effect on azacitidine metabolism by microsomal enzyme inhibitors or 
inducers has not been studied.54  
 
1.2.7 Drug interactions  
 
There have not been extensive drug interaction studies with azacitidine.  
Azacitidine neither inhibited nor induced cytochrome P450 (CYP) enzymes in 
cultured human hepatocytes at 70uM or lower.61  A much lower peak serum 
concentration of 3 uM azacitidine is reached after subcutaneous administration of 
a therapeutic dose of 75 mg/m2.55   
 
1.3 
Study Rationale  
 
In this trial, we will begin to explore the possibility that, as in mice, azacitidine given after 
allo-HSCT may mitigate GvHD while retaining GvL (see Section 1.1.2.3).  Lübbert et al2424 
(Section 1.1.1.2) observed a low rate of GvHD when they treated patients with azacitidine 
both prior to DLI and for multiple rounds after DLI.  In our mouse study (Section 1.1.2.3), 
a single cycle of azacitidine post-DLI resulted in effective tumor control and a profound 
decrease in GvHD.  This effect was hypothesized to be due to azacitidine-induced 
demethylation of the FOXP3 locus with subsequent conversion of conventional T cells into 
Tregs.  It is noteworthy that in patients a higher relative frequency of Tregs after allogenic 
HSCT is associated with a lower rate and severity of GVHD, a lower rate of non-relapse 
mortality, and equivalent relapse mortality.34 We hypothesize that providing a single cycle 
of azacitidine early after allogeneic stem cell infusion and continuing cycles after count 
recovery on ~Days 35, 63, and 91 will be safe and well tolerated and associated with 
reduced rates of GVHD without affecting the rate of complete remission (GVL).  The 
purpose of this phase I/II study is to define the maximum tolerated dose of 5-AzaC 
and the effect on grade II-IV GvHD when given after matched unrelated donor 
transplant (MUD).  
 
1.4 
Correlative Studies Background 
  
1.4.1 Pharmacokinetics 
 
This will be the first time azacitidine has been used upfront after transplant and its 
pharmacokinetics (PK) in recipients remains unknown.  In the phase I portion of 
the trial only, we will obtain serial blood samples prior to and after the first dose of 
azacitidine administered on Day +7.  Plasma will be isolated and cryopreserved for 
later PK analyses to determine AUC, Cmax, and half-life of azacitidine in this 
setting.  The PK data will be used to correlate and interpret clinical results and will 
help to guide future studies. 
 
1.4.2 Immune Reconstitution after Transplantation 
 


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Immune reconstitution after transplantation is an important determinate for risk of 
infection after transplantation. To understand the kinetics of immune reconstitution 
after transplantation with a mobilized peripheral blood stem cell product and early 
administration of azacitidine after transplant, we will collect peripheral blood 
samples at pre-determined time points from the recipient.  These samples will be 
analyzed by multiparameter flow cytometry for lymphocyte subsets (including T-
regs, naïve/central memory/effector memory, Th1/Th2/Th17 and NKT cells) and 
circulating dendritic cells.  To determine the number of bone marrow derived T-
cells that are recent thymic emigrants, PCR TREC analysis will be used. 
 
1.4.3 Regulatory T Cell Frequency and Number 
 
As indicated in Section 1.1.2.3, we have seen that azacitidine treatment post-DLI 
in mice increases the frequency of Tregs resulting in a suppression of GvHD while 
the GvL effect is maintained.  Our hypothesis is that a similar increase in Tregs will 
occur in AML/MDS patients who are given 4 cycles of azacitidine beginning on 
Days 7, ~35, ~63, and ~91 post allo-HSCT resulting in a corresponding decrease in 
GvHD, while the GvL affect from the MUD will be retained. 
 
We will assess relative Treg numbers and percentages within the peripheral blood 
before (from the donor PBSC product) and after transplant (recipient peripheral 
blood).  In addition, we will examine several subsets that may have functional 
significance. Miyara et al62. determined that Tregs can be separated into 3 subsets 
based on their expression of cell surface markers.   These subsets differ in their 
proliferative capacity, effector functions, and ability to secrete specific cytokines.  
Upon stimulation resting Tregs (rTreg, CD4+CD25++CD45RA+) respond with 
more active and sustained proliferation than do effector or “activated” Tregs (aTreg, 
CD4+CD25+++CD45RA-), with the latter dying after only a few days.  rTregs are 
able to differentiate into activated T cells in response to stimulus.  These aTregs not 
only more effectively suppress proliferation of naïve T cells (CD4+CD25-
CD45RA+), but also suppress rTregs as a form of negative feedback.  A third 
subpopulation of Tregs (CD4+25++CD45-) is nonsuppressive, but they secrete 
abundant amounts of proinflammatory cytokines such as IL-17.  The proportion of 
each subset varies with age and disease state. We will use flow cytometry to 
measure the proportion of rTregs and aTregs in the donor PBSC product pre-Allo-
HSCT, and in the recipient peripheral blood at several times after subsequent 5-
AzaC treatment to explore the effect of 5-AzaC on frequency of both rTregs and 
aTregs and whether there is a connection between these frequencies and GvHD.  
Importantly, as a control for these correlative studies we will utilize a biospecimen 
bank of cryopreserved peripheral blood mononuclear cells from patients with AML 
or MDS in CR1 or 2 undergoing a 8/8 MUD over the last 2 years, collected at serial 
time points after transplant (pre-transplant, Day +30, Day+100, and Day +365).  
 
In addition to the Treg subsets, the differentiation state of Tregs within the 
peripheral blood will be assessed by flow cytometry (see review by Sakaguchi et 
al63). Naïve Tregs are phenotypically CD45RA+CD45RO-FOXP3low, indicating 


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that they have not yet undergone T cell receptor-stimulated maturation in the 
thymus.  As recent emigrants from the thymus, most naïve Tregs also express CD31.  
However because they are believed to require constant stimulation by their cognate 
antigen to remain in the periphery, they are not strictly “naïve” T cells.  In humans, 
but not mice, these cells have potent suppressive activity and can also be found in 
cord blood.  Cells that are actively proliferating express Ki67, thus providing a 
means to distinguish resting (naïve) Tregs from activated Tregs.  Once naïve Tregs 
have undergone T cell receptor-stimulated maturation, they not only proliferate, but 
are also very resistant to apoptosis.  When activated these cells convert to highly 
suppressive and proliferative CD45RO+CD25hiFOXP3hi “effector” Treg cells, with 
no memory functions despite the association of CD45RO with conventional 
memory T cells.  Effector Tregs are more prevalent in adults and the elderly than 
in utero or in cord blood.  Using flow cytometry for a variety of these markers, the 
proportion of Tregs and other T cell subsets (naïve/central memory/effector 
memory and Th1/Th2/Th17) within the blood will be determined.  
 
Finally as indicated in section 1.1.2.2, Magenau et al34  have found that the 
frequency of Tregs within the blood at onset of GvHD has predictive value for the 
ultimate severity of GvHD symptoms and patient outcome. In our trial we will use 
flow cytometry to measure the proportion of naïve (resting) Tregs (rTregs) and 
activated (effector) Tregs (aTregs) in patients pre-Allo-HSCT, immediately prior 
to each cycle, and at ~14 days and ~28 days after each cycle of 5-AzaC to explore 
the effect of 5-AzaC on frequency of both rTregs and aTregs and whether there is 
an association between these frequencies and GvHD. Based on the observations 
above, we will define Tregs subtypes as follows: 
 
Naive (resting) Tregs (CD4+CD25++FoxP3lowCD45RA+CD31+) 
Activated (effector) Tregs (CD4+CD25+++FoxP3highCD45RA-CD31-) 
 
Based on these correlative studies we hope to have a better understanding of 
the kinetics of Treg and other T cell subsets (naïve/central memory/effector 
memory and Th1/Th2/Th17) recovery after Allo-HSCT and determine the 
relative frequency of peripheral blood Tregs from patients treated with 5-
AzaC after allo-HSCT compared with matched controls and correlate their 
levels with the development of aGvHD. 
 
 
2.0 
OBJECTIVES 
 
2.1 
Primary Objective  
 
Phase I: to determine the maximum tolerated dose (MTD) of azacitidine in patients 
undergoing matched (8 out of 8 HLA matched) unrelated donor transplant for any 
hematological malignancy (excluding myelofibrosis) in remission or with stable minimal 
residual disease. 
 


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Phase II: to determine the effect of azacitidine on grade II-IV GvHD in patients undergoing 
matched (8 out of 8 HLA matched) unrelated donor transplant for AML in remission 1 or 
2 or MDS. 
 
2.2 
Secondary Objectives 
 
1. 
To determine the rate of Grades III-IV aGVHD at Day +180. 
 
2. 
To determine the rate of cGVHD at one year after transplant. 
 
3. 
To determine overall survival at one year after transplant. 
 
4. 
To determine the effects of azacitidine on frequency and absolute number of Tregs 
by comparing to a standard care group of matched historical controls. 
 
5. 
To determine treatment-related mortality at Day 100. 
 
6. 
To determine the rate of relapse-free survival at one year after transplant. 
 
 
3.0 
PATIENT SELECTION 
 
3.1 
Inclusion Criteria  
 
Patients must meet the following criteria within 30 days prior to Day 0 unless otherwise 
noted. 
 
1. Phase I: Diagnosis of a hematological malignancy listed below (excluding 
myelofibrosis) in remission or with stable minimal residual disease 
a. Acute myelogenous leukemia (AML) in 1st or subsequent remission or in relapse 
after any remission. 
b. Acute lymphoblastic leukemia (ALL) in 1st or subsequent remission or in relapse 
after any remission 
c. Myelodysplastic syndrome either intermediate 1 or 2, or high risk by the 
International Prognostic Scoring System, 
d. Chronic myelogenous leukemia (CML) in accelerated or second chronic phase, 
e. Non-Hodgkin’s lymphoma (NHL) or Hodgkin’s disease (HD) in 2nd or greater 
complete remission, partial remission, or refractory relapse,  
f. Chronic lymphocytic leukemia (CLL), Rai Stage 2-4, failing at least 2 prior 
regimens,  
g. Multiple myeloma (MM), Stage 2-3, 
h. Myeloproliferative disorder or neoplasm 
 
Phase II: Diagnosis of AML in remission 1 or 2 or a diagnosis of Myelodysplastic 
syndrome either intermediate 1 or 2, or high risk by the International Prognostic 
Scoring System. 


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2. Patients with MDS must be transplant candidates by current clinical standards. 
 
3. Patients who have been treated with hypomethylating agents prior to entering the study 
are eligible. 
 
4. Must have matched unrelated donor (8 of 8 HLA match at A, B, C, and DR loci) by 
high resolution DNA typing. 
 
5. Must have donor peripheral blood stem cells mobilized by NMDP standards.  No bone 
marrow donors. 
  
6. Must have 4-8 x 106 CD34+ cells/kg (recipient weight) infused on Day 0. 
 
7. Must have at least one additional aliquot of ≥1 x 106 CD34/kg cryopreserved cells 
stored at the time of transplant. 
 
8. Must receive a myeloablative or reduced intensity conditioning regimen for SCT as 
defined by the CIBMTR64 
a. Cyclophosphamide and single dose total body irradiation 
b. Fludarabine and busulfan 
c. Fractionated TBI and cyclophosphamide 
d. Busulfan and cyclophosphamide 
 
9. Must be able to receive GVHD prophylaxis with tacrolimus and methotrexate as 
outlined in Section 6.2. 
 
10. Must be > 18 years old and ≤ 70 years old.  Azacitidine is not approved by the FDA 
for use in children. 
 
11. Must have an Eastern Cooperative Oncology Group (ECOG) performance status < 2 
(see Appendix 1). 
 
12. Must have laboratory results indicating: 
a. Total bilirubin < 2.0 mg/dl, unless a diagnosis of Gilbert’s disease 
b. AST/ALT < 3 X the upper limit of institutional normal  
c. Serum creatinine < 2.0 mg/dl 
 
13. Patient must have ability to understand and willingness to provide written informed 
consent prior to participation in the study and any related procedures being performed.  
 
14. The effects of azacitidine on the developing human fetus at the recommended 
therapeutic dose are unknown.  For this reason and because category D agents as well 
as other therapeutic agents used in this trial are known to be teratogenic, women of 
childbearing age must have a negative serum pregnancy test (ß-human chorionic 
gonadotropin) within 72 hours prior to initiating the conditioning regimen and be 


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willing to not become pregnant by using effective contraception while undergoing 
treatment and for at least 3 months after the last dose of azacitidine. 
 
15. Men must be willing not to father a new child while receiving therapy. They must use 
an effective barrier method of contraception during the study and for 3 months 
following the last dose.  
 
3.2 
Exclusion Criteria 
 
1. Must not have myelofibrosis or other disease known to prolong neutrophil engraftment 
to > 28 days after transplant. 
 
2. Must not be receiving any other investigational agents within 14 days of first dose of 
azacitidine (Day 7).  
 
3. Must not have nonmyeloablative conditioning as defined below64: 
a. TBI </= 2 Gy ± purine analog 
b. Flu + Cy ± ATG 
c. Flu +AraC + Ida 
d. Cladribine + AraC 
e. Total Lymphoid Irradiation + ATG 
 
4. Must not recieve antithymocyte globulin as part of pre-transplant conditioning 
regimens. Antithymocyte globulin is excluded due to its potential impact on 
modulating the incidence of GvHD or GvL. 
 
5. Must not have uncontrolled intercurrent illness including ongoing or active infection, 
symptomatic congestive heart failure, unstable angina pectoris, or psychiatric 
illness/social situations that would limit compliance with study requirements.  
 
6. Must not be pregnant or breastfeeding.  Pregnant women are excluded from this study 
because azacitidine is a Category D agent with the potential for teratogenic or 
abortifacient effects.  Because there is an unknown but potential risk for adverse events 
in nursing infants secondary to treatment of the mother with azacitidine, breastfeeding 
should be discontinued if the mother is treated azacitidine.  These potential risks may 
also apply to other agents used in this study. 
 
7. Must not have a known or suspected hypersensitivity to azacitidine, mannitol, or 
compounds of similar composition to azacitidine. 
 
8. Must not have an advanced malignant hepatic tumor. 
 
9. Must not be HIV, HBV or HCV positive. 
 
10. Must not have undergone a prior allogeneic donor (related, unrelated, or cord) 
transplant. Prior autologous transplant is not exclusionary.  


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3.3 
Donor Selection 
 
Donors will not receive any study procedures on this protocol. Donors will sign the 
standard NMDP donor consent and undergo standard mobilization prior to leukopheresis 
according to NMDP guidelines.  
 
Donors will be tested for relevant communicable diseases and only donors free from risk 
factors for, and no clinical evidence of, infection due to relevant communicable disease 
agents and diseases including: Human immunodeficiency virus; Hepatitis B virus; 
Hepatitis C virus; Human transmissible spongiform encephalopathy, including 
Creutzfeldt-Jakob disease; Treponema pallidum and HTLV 1 and 2 will be eligible to 
donate.  
 
Donor must be HIV-1&2 antibody, HTLV-I&II antibody, HBV and HCV sero-negative, 
by FDA licensed test. 
 
3.4 
Eligibility of Women and Minorities 
 
Both men and women and members of all races and ethnic groups are eligible for this trial.  
 
 
4.0 
REGISTRATION PROCEDURES  
 
Patients must not start any protocol intervention prior to registration through the Siteman 
Cancer Center. 
 
The following steps must be taken before registering patients to this study: 
 
1. Confirmation of patient eligibility 
2. Registration of patient in the Siteman Cancer Center OnCore database 
3. Assignment of unique patient number (UPN) 
 
 
4.1 
Confirmation of Patient Eligibility 
 
Confirm patient eligibility by collecting the information listed below and completeing 
registration form (Appendix 2): 
 
1. Registering MD’s name 
2. Patient’s race, sex, and DOB 
3. Three letters (or two letters and a dash) for the patient’s initials 
4. Copy of signed consent form  
5. Completed eligibility checklist, signed and dated by a member of the study team 
6. Copy of appropriate source documentation confirming patient eligibility 
 
4.2 
Patient Registration in the Siteman Cancer Center OnCore Database 
 


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All patients must be registered through the Siteman Cancer Center OnCore database. 
 
4.3 
Assignment of UPN 
 
Each patient will be identified with a unique patient number (UPN) for this study.  All data 
will be recorded with this identification number on the appropriate CRFs. 
 
 
5.0 
TREATMENT PLAN 
 
5.1 
Overall Treatment Plan 
 
Patients with advanced hematological malignancies (except myelofibrosis) who are 
eligible for hematopoietic cell transplantation and who have a matched (8/8 HLA antigen 
matched) unrelated donor will be eligible for phase I of this study. Cohorts of patients in 
the phase I portion of the trial will receive escalating doses of azacitidine (as described in 
Section 5.3.3) to determine the maximum tolerated dose (MTD).  To ensure engraftment, 
the treatment of the first 3 subjects will be staggered by an interval of 4 weeks. After the 
first 3 subjects are treated and engraftment confirmed, enrollment and treatment can occur 
weekly. 
 
Phase I Azacitidine dosing cohorts 
 
Cohort 1 
(Starting Dose) 
Cohort 2 
Cohort 3 
Cohort 4 
15 mg/m2 
30 mg/m2 
37.5 mg/m2 
45 mg/m2 
 
Forty-six additional patients with AML in 1st or 2nd remission or MDS who are eligible for 
hematopoietic cell transplantation and who have a matched (8/8 HLA antigen matched) 
unrelated donor will be enrolled in the phase II portion of this study to be treated with the 
dose of azacitidine determined to be the MTD during phase I.   


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5.2 
Treatment Schema 
 
                                  
 
 
5.3 
Definition of MTD, DLT, Dose Escalation Criteria, and Toxicity, Response, 
and DLT Evaluations 
 
5.3.1 Definition of MTD (Maximum Tolerated Dose) 
 
The maximum tolerated dose (MTD) is defined as the dose level immediately 
below the dose level at which 2 patients of a cohort (of 2 to 6 patients) experience 
dose-limiting toxicity as descrbed in Section 5.3.2.  To ensure engraftment, the 
treatment of the first 3 subjects will be staggered by an interval of 4 weeks. After 
the first 3 subjects are treated and engrafted enrollment and treatment can occur 
weekly. Dose escalations will proceed until the MTD has been reached. 
 
5.3.2 DLTs (Dose Limiting Toxicities) Cycle 1 
 
Dose limiting toxicities (DLTs) are: 
• failure to have neutrophil engraftment (ANC >500 x 3 days) by Day 28 
post-stem cell infusion 
• grade 4 or higher organ toxicities (neurologic, pulmonary, cardiac, 
gastrointestinal, genitourinary, renal, hepatic, cutaneous) not attributable to 
any other causes that occur through Day 28 (not due to primary malignancy, 
or infection) 
 
5.3.3 DLTs (Dose Limiting Toxicities) Cycles 2-4 
 
• Grade 3 or higher organ toxicity at least possibly related to azacitidine 
• Failure to administer at least 75% of the azacitidine dose due to 
hematologic toxicity. 
 
5.3.4 Dose Escalation Criteria 
 
All subjects receiving a single dose of azacitidine are evaluable for toxicity. Dose 
escalation is determined by DLTs in the first cycle regardless of future ability to 
receive azacitidine or the development of non-hematologic DLTs in Cycles 2-4.  If 
a subject is only able to receive the first cycle of azacitidine, this is adequate to 
escalate dosing to a subsequent cohort.  


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Dose escalations will proceed as follows after the occurrence of dose-limiting 
toxicity (DLT): 
 
Number of Patients 
with DLT at a Given 
Dose Level 
Escalation Decision Rule 
0 out of 3 
Enter 3 patients at the next dose level. 
>2 
Dose escalation will be stopped.  This dose level will be 
declared the maximally administered dose (highest dose 
administered).  Three (3) additional patients will be 
entered at the next lowest dose level if only 3 patients 
were treated previously at that dose. 
1 out of 3 
Enter at least 3 more patients at this dose level. 
 
If 0 of these 3 patients experience DLT, proceed to the 
next dose level. 
 
If 1 or more of this group suffer DLT, then dose 
escalation is stopped, and this dose is declared the 
maximally administered dose.  Three (3) additional 
patients will be entered at the next lowest dose level if 
only 3 patients were treated previously at that dose. 
<1 out of 6 at highest 
dose level below the 
maximally 
administered dose 
This is generally the recommended phase 2 dose. At 
least 6 patients must be entered at the recommended 
phase 2 dose. 
 
If >1 patient in the de-escalation cohort (Cohort -1) has a DLT, then the trial will 
be stopped and no phase II portion of the trial will be performed.  
 
If 0 of 3 in Cohort 3 experience a DLT then 3 additional subjects will be enrolled. 
If <1 out of 6 at this highest dose level experience a DLT then 45 mg/m2 will be 
confirmed as MTD. 
 
5.3.5 Toxicity, Response, and DLT Evaluations 
 
All patients who receive any dose of azacitidine are evaluable for toxicity.  Patients 
are evaluated for toxicity from the first dose of azacitidine (Day 7) until Day +140 
(or 30 days after the last dose of azacitidine for patients who fail to complete all 4 
cycles). 
 
All patients are evaluable for effects of azacitidine on acute GvHD after receiving 
at least 1 dose of azacitidine unless they die or are removed from the study prior to 
Day 100 without acute GvHD. 
 


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All patients are evaluable for response of the hematologic malignancy to the stem 
cell transplant unless they are discontinued from the study prior to Day 100 and 
have not had any disease assessment. 
 
A patient in phase I is evaluable for DLT assessment only through Day 28.  Whether 
phase I subjects have toxicity from study drug in subsequent cycles or are unable 
to receive subsequent cycles of azacitidine will not have a bearing on the 
determination of the MTD and phase II dosing. 
 
5.4 
Replacement of Ineligible Patients 
 
Should a patient be enrolled and sunsequently be unable to start treatment with azacitidine 
on Day +7 as per protocol (due to declining performance status, unacceptable lab results, 
inadequate stem cell product infusion, or other reason), the patient will be removed from 
the study and will be replaced. 
 
 
6.0 
ADMINISTRATION OF PROTOCOL TREATMENT 
 
6.1 
Conditioning Regimens for Transplant 
 
Although including myelo-ablative (MA) and reduced-intensity conditioning (RIC) 
regimens may result in added variability, retrospective studies comparing MA and RIC in 
AML/MDS show similar outcomes of survival, treatment related mortality and GvHD.65,66 
Both types of regimens are being allowed in this protocol to tailor conditioning at the 
discretion of the treating physician and to enhance patient enrollment.  Historically about 
50% of our transplants for AML and MDS are performed using RIC. 
 
Recipients will undergo institutionally standard myeloablative or reduced intensity 
chemotherapy or chemoradiotherapy using one of the following conditioning regimens, 
which, will be administered at the discretion of the treating physician: 
• cyclophosphamide and single dose total body irradiation (TBI) 
• fludarabine and busulfan 
• fractionated TBI and cyclophosphamide 
• busulfan and cyclophosphamide 
 
Antithymocyte globulin will not be permitted as part of pre-transplant conditioning 
regimens in this protocol due to its potential impact on modulating the incidence of GvHD 
or GvL. Non-myeloablative conditioning as defined by the CIBMTR working group will 
not be permitted on this trial. 
 
6.2 
GvHD Prophylaxis 
 
Tacrolimus + mini-methotrexate is the only prophylactic GvHD regimen allowed on this 
trial. 
 


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Tacrolimus will be administered beginning on Day -2 and should continue for a minimum 
of 100 days prior to tapering.  Dosing and adjustments are at the discretion of the treating 
physician with goal trough between 5 and 10. 
 
Methotrexate will be administered as an intravenous dose of 10mg/m2 on Day +1 post-
transplant and 7.5mg/m2 on Days +3 and +6 (mini-methotrexate).  Dose adjustments per 
institutional guidelines are allowed for renal and liver dysfunction and mucositis. 
 
6.3 
Stem Cell Transplantation (Day 0) 
 
On Day 0 the allograft will be infused per standard institutional practice. The maximum 
CD34+ cell dose to be given is 8.0x106.  The reason for this is because of concern for a 
higher risk of both acute and chronic GVHD that has been seen with G-CSF mobilized 
peripheral blood transplants at higher CD34 cell doses.67,68 
 
In the event that < 4.0 x 106 CD34+ cells/kg are infused, the recipient will be removed 
from the trial and no study drug will be administered at Day +7.  
 
In addition, at least one aliquot of ≥1 x 106 CD34+ cells/kg must be stored per institutional 
guidelines at the time of transplant and reserved to treat potential graft failure. If this is 
not possible, the subject will be excluded from the trial and not receive study drug. At 
the discretion of the transplant physician, additional cells may be cryopreserved for use at 
a later time. 
 
6.4 
Azacitidine Administration 
 
Azacitidine will be administered for 5 consecutive days each cycle. Cycle 1 begins on Day 
+7.  Patients will receive up to 3 more cycles of treatment starting on Days +35, +63, and 
+91 (+/- 4 days). It is recommended that cycles 2 through 4 be started on a Monday.   
 
The starting dose will be 15 mg/m2 during phase I; the phase II dose will be the MTD as 
determined in phase I.  The dose escalation schedule for Phase I may be found in the table 
below: 
 
Phase I Azacitidine dosing cohorts 
 
Cohort 1 
Cohort 2 
Cohort 3 
Cohort 4 
15 mg/m2 
30 mg/m2 
37.5 mg/m2 
45 mg/m2 
 
Azacitidine dose should be calculated based on adjusted ideal body weight.  Adjusted 
body weight = [0.4 (Actual weight – IBW)] + IBW 
 
Ondansetron 4 mg IV will be administered prior to all azacitidine doses.  
 
Azacitidine solution will be administered intravenously. Azacitidine reconstituted for 
intravenous administration may be stored at 25°C (77°F), but administration must be 


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completed within 1 hour of reconstitution.54 Administer the total dose over a period of 15 
minutes.  
 
Subjects should have a dedicated line for azacitidine administration.  Before and after 
administration, the line must be flushed with a minimum of 20 mL of normal saline.  If a 
dedicated line is not possible, the existing line must be flushed before and after drug 
administration. 
 
 
Azacitidine is incompatible with 5% Dextrose solutions, Hespan, or solutions that contain 
bicarbonate.  These solutions have the potential to increase the rate of degradation of 
azacitidine and should therefore be avoided.54  
 
6.4.1 Azacitidine Dose Modifications 
 
Azacitidine should not be administered at any time if creatinine > 2.0mg/dl, or if total 
bilirubin > 2.0mg/dl, or AST or ALT > 3.0 x ULN with an albumin <3.0 g/dl. 
 
Any patient that develops grade 3 or 4 non-hematologic toxicity (excluding 
nausea/vomiting, electrolyte abnormalities, or infection) related to azacitidine (definitely, 
probably, or possibly) will have study treatment discontinued.  
 
Cycle 
 
1 
No dose modification. 
2 
No dose modification, but cycle may be held for up to 2 weeks if ANC 
<500/mm3 and/or Platelets <20,000/mm3.  If hold exceeds 2 weeks, the 
cycle is to be skipped. 
If platelet count recovers to >20,000/mm3 and ANC>500/mm3 at 
subsequent cycles, azacitidine treatment may be resumed.  
3 & 4 
Depending on counts from preceding cycle and current counts: 
ANC 
Platelets 
Dose Modification 
≥1500/mm3 
≥100,000/mm3 
Full dose 
<1500/mm3 - ≥500/mm3 
<100,000/mm3 - 
≥20,000/mm3 
If 
ANC 
or 
platelets do not 
recover within 
75% of start of 
prior 
cycles 
then 
reduce 
dose 
of 
azacitidine 
by 
25%. * 
<500/mm3 
<20,000/mm3 
Hold dose 
If platelet count recovers to ≥20,000/mm3 and ANC to ≥500/mm3 at 
subsequent cycles, azacitidine treatment may be resumed.  
 


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*Previous dose level may be resumed at subsequent cycles if the platelet and ANC have 
recovered to 75% of peak levels after transplant. 
 
Treatment may be delayed by up to 2 weeks to allow resolution of grade 3 toxicity at least 
possibly related to azacitidine. If hold exceeds 2 weeks, the cycle is to be skipped. 
 
Azacitidine should not be administered during Cycles 2 – 4 if patient is receiving G-CSF 
to support ANC>500 
 
 
Additional dose modifications may be allowed for other hematologic or non-hematologic 
toxicities in Cycles 2-4 with consultation of the PI.  
 
 
6.5 
Growth Factor Administration  
 
G-CSF will be administered starting on Day + 12 at a dose of 5 mcg/kg/day(actual recipient 
weight) by subcutaneous injection until the absolute neutrophil count is > 1500/ul for 2 
consecutive days or > 5000/ul for one day. Dose will be rounded to the nearest vial size. 
 
6.6 
General Concomitant Medication and Supportive Care Guidelines 
 
Concomitant medications and supportive care measures will be given per institutional 
guidelines and at the discretion of the treating physician whenever medically necessary 
with the exceptions of the prohibited medications listed in Section 6.7. 
 
6.7 
Prohibited Medications 
 
The following medications are prohibited for the duration of the patient’s study 
participation. Patients who require treatment with any prohibited medication will be 
removed from the protocol.  The PI should be notified immediately, prior to their 
administration when possible. 
 
• Agents or therapies with the intent to treat the patient’s malignancy 
• Experimental/investigational medications 
• Conditioning regimen agents other than those specified in this protocol.  
• All prophylactic medications for GVHD other than those specified in this protocol.  
The use of corticosteroids for reasons other than GVHD prophylaxis will be 
permitted with documentation of the rationale for usage. 
• Any medication to accelerate platelet engraftment. 
 
6.8 
Women of Childbearing Potential 
 
Women of childbearing potential (defined as women with regular menses, women with 
amenorrhea, women with irregular cycles, women using a contraceptive method that 


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precludes withdrawal bleeding, and women who have had a tubal ligation) are required to 
have a negative pregnancy test prior to initiating the conditioning regimen.   
 
Female and male patients (along with their female partners) are required to use two forms 
of acceptable contraception, including one barrier method, during participation in the study 
and for 3 months following the last dose of azacitidine.  
 
If a patient is suspected to be pregnant, azacitidine should be immediately discontinued.  In 
addition a positive urine test must be confirmed by a serum pregnancy test.  If it is 
confirmed that the patient is not pregnant, the patient may resume dosing.  If a female 
patient or female partner of a male patient becomes pregnant during therapy or within 4 
weeks after the last dose of azacitidine, the investigator must be notified in order to 
facilitate outcome follow-up. 
 
6.9 
Treatment of Acute GvHD 
 
In the event that a subject develops acute GvHD the following steps will be taken: 
 
1. Acute GvHD will be treated per the standard of care with high dose steroids. 
2. Subsequent cycles of azacitidine will be continued unless treatment is contra-
indicated by cytopenias (see Section 6.4.7.1) or if the subject develops steroid 
refractory GvHD. Steroid refractory acute GvHD will be defined as no 
improvement in GvHD within 7 days of starting high dose system steroids (2mg/kg) 
or progression of GvHD while on 2mg/kg steroids. Subjects who develop steroid 
refractory acute GvHD will still be followed for chronic GvHD and survival. 
3. If steroid refractory GvHD develops, the subject will be taken off study and further 
treatment is at the discretion of the treating physician. 
 
6.10 
Failure to Engraft 
 
If any patient transplanted with an allograft on study does not have evidence of an absolute 
neutrophil count (ANC) of at least 500/ul by Day 28, this is considered failure to engraft.  
At this time, the cryopreserved back-up product will be infused per institutional guidelines 
and the donor will be contacted to plan re-collection immediately. 
 
6.11 
Duration of Therapy 
 
Study treatment may continue for up to 4 cycles or until one of the following criteria 
applies: 
 
• Death 
• Failure to engraft at Day 28 
• Relapse of the patient’s primary diagnosis  
• Development of steroid refractory acute GvHD 
• Adverse event(s) that, in the judgment of the Investigator, may cause severe or 
permanent harm or which rule out continuation of study drug. 


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• Any grade 4 organ toxicity that is at least possibly related to azacitidine 
• General or specific changes in the recipient’s condition rendering him/her 
unacceptable for further treatment in the judgment of the investigator 
• Suspected pregnancy  
• Serious noncompliance with the study protocol 
• Lost to follow-up 
• Patient withdraws consent 
• The PI decides to remove the recipient from study 
• The Siteman Cancer Center decides to close the study 
 
 
 
6.12 
Post-Treatment Follow-Up 
 
Patients will be followed for 2 years after first dose of study drug. Patients who prematurely 
discontinue treatment should still complete the remaining study visits (if willing and able).  
 
6.13 
End of Study Definition 
 
The end of study is defined as any one of the following (whichever occurs first): 
•  The date of the two-year follow-up 
•   The date of death 
• Lost to follow-up 
• Patient withdraws consent 
 
6.14 
Definition of Completed Patients 
 
A completed patient is one who receives treatment and completes 2-year follow-up visit. 
 
7.0 
PHARMACEUTICAL INFORMATION  
 
7.1 
Azacitidine 
 
[Information in this section was directly excerpted or adapted from the investigator 
brochure.54] 
 
7.1.1 Mechanism of Action/Classification  
 
Azacitidine is a pyrimidine nucleoside analog of cytidine.  Azacitidine causes 
hypomethylation of DNA and direct cytotoxicity.  Hypomethylation may restore 
normal function to genes that are critical for differentiation and proliferation.  The 
cytotoxic effects are cell cycle dependent and cause the death of rapidly dividing 
cells.  For additional information, please see Section 1.2.1. 
 
7.1.2 Pharmacodynamics/kinetics 
 


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Azacitidine is rapidly absorbed when given subcutaneously to humans, with 
maximum plasma concentrations found 0.5 to 2 hours after dosing.  In patients with 
MDS, the peak plasma concentration was reached 11 min after intravenous 
administration (Cmax=12 µM [2750 ng/ml]).55  Azacitidine and its metabolites are 
renally cleared.  The effects of renal or hepatic impairment, gender, age, or race on 
the pharmacokinetics of azacitidine have not been studied.  The bioavailability after 
subcutaneous administration is 89% (range 52% to 128%) with a mean half-life of 
0.69 hours.53 A much lower peak serum concentration of 3 µM azacitidine is 
reached after subcutaneous administration of a therapeutic dose of 75 mg/m2.55 
 
The pharmacokinetics of azacitidine have been studied in 6 MDS patients following 
a single 75 mg/m2 subcutaneous (SC) dose and a single 75 mg/m2 intravenous (IV) 
dose.  Azacitidine is rapidly absorbed after SC administration; the peak plasma 
azacitidine concentration of 750 ± 403 ng/ml occurred in 0.5 hour.  The 
bioavailability of SC azacitidine relative to IV azacitidine is approximately 89%, 
based on area under the curve.  Mean volume of distribution following IV dosing 
is 76 ± 26 L.  Mean apparent SC clearance is 167 ± 49 L/hour and mean half-life 
after SC administration is 41 ± 8 minutes.  Urinary excretion is the primary route 
of elimination of azacitidine and its metabolites (50-85%).  The mean elimination 
half-lives of azacitidine and its metabolites were similar after IV and SC 
administrations, about 4 hours.  
 
7.1.3 Formulation 
 
VIDAZA (azacitidine for injection) is supplied as a lyophilized powder in 100 mg 
single-use vials packaged in cartons of 1 vial. 
 
7.1.4 Availability 
 
Azacitidine (VIDAZA) is commercially available through Celgene Corporation 
who will be providing azacitidine for this study.  
 
7.1.5 Preparation 
 
Reconstitute the appropriate number of VIDAZA vials to achieve the desired dose. 
Reconstitute each vial with 10 mL sterile water for injection. Vigorously shake or 
roll the vial until all solids are dissolved. The resulting solution will contain 
azacitidine 10 mg/mL. The solution should be clear. Parenteral drug product should 
be inspected visually for particulate matter and discoloration prior to administration, 
whenever solution and container permit. Withdraw the required amount of 
VIDAZA solution to deliver the desired dose and inject into a 100 mL infusion bag 
of 0.9% Sodium Chloride.54   
 
VIDAZA is incompatible with 5% Dextrose solutions, Hespan, or solutions that 
contain bicarbonate.  These solutions have the potential to increase the rate of 
degradation of VIDAZA and should therefore be avoided.54  


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7.1.6 Storage and Stability  
 
VIDAZA is a cytotoxic drug and, as with other potentially toxic compounds, 
caution should be exercised when handling and preparing VIDAZA suspensions. 
The VIDAZA vial is single-use and does not contain any preservatives. Unused 
portions of each vial should be discarded properly. Store unreconstituted vials at 
25º C (77º F); excursions permitted to 15º-30º C (59º-86º F).  Procedures for proper 
handling and disposal of anticancer drugs should be applied. If reconstituted 
VIDAZA comes into contact with the skin, immediately and thoroughly wash with 
soap and water. If it comes into contact with mucous membranes, flush thoroughly 
with water 54.  
 
7.1.7 Administration  
 
See Section 6.4 for Azacitidine administration instructions. 
 
7.1.8 Nursing Implications 
 
Azacitidine will be administered at a facility capable of managing hypersensitivity 
reactions.   
 
 
7.2 
Methotrexate 
 
7.2.1 Supply, Storage, and Preparation 
 
Commercially available in 2 mL, 4 mL, 8 mL, 10 mL vials for injection at a 
concentration of 25 mg/mL.  In addition, methotrexate is available in 1000 mg vials 
for injection. Please refer to the package insert for additional information. Stability 
and compatibility of methotrexate sodium solutions depend on several factors 
including the formulation of methotrexate sodium used, presence of preservatives, 
concentration of drug, specific diluents used, resulting pH, and temperature; the 
manufacturer’s labeling and specialized references should be consulted for specific 
information. Methotrexate sodium solutions should be inspected visually for 
particulate matter and discoloration whenever solution or container permits.  
Administer via slow IV push.  
 
 
7.3 
Tacrolimus  
 
7.3.1 Supply, Preparation, and Administration 
 
Tacrolimus is a commercially available macrolide compound with potent 
immunosuppressant properties.  Tacrolimus is available for oral administration as 
capsules containing the equivalent of 0.5 mg, 1 mg, or 5 mg of anhydrous 
tacrolimus. For IV use, tacrolimus is available as a sterile solution in 1mL ampuls 


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Page 44 of 96 
containing the equivalent of 5 mg of anhydrous tacrolimus per mL. Store tacrolimus 
at controlled room temperature (15-30˚C). Diluted solutions should be stored in 
glass or polyethylene containers and should be discarded after 24 hours. Tacrolimus 
for injection must be diluted prior to IV infusion.  For IV infusion, the concentrate 
is diluted with 0.9% sodium chloride or 5% dextrose injection to a concentration of 
4-20 mcg/mL.  Diluted solutions should be stored in glass or polyethylene 
containers and should be discarded after 24 hours. A plasticized polyvinyl chloride 
(PVC) container should not be used because stability of the solution is decreased 
and polyoxyl 60 hydrogenated castor oil contained in the formulation may leach 
phthalates from PVC containers.  In addition, PVC-free tubing should be utilized 
to minimize the potential for significant drug absorption onto the tubing.   
Tacrolimus concentrate for injection and diluted solutions of the drug should be 
inspected visually for particulate matter and discoloration prior to administration 
whenever solution and container permit.  Tacrolimus is to be initiated on Day –2.  
Begin tapering between Day +90 to +120 in the absence of GVHD as tolerated with 
a goal of stopping by Day +150 to +180. Serum levels should be frequently checked 
according to institutional guidelines. 
 
 
 


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8.0 
SCHEDULE OF ASSESSMENTS AND STUDY CALENDERS 
 
8.1 
Study Calendar – Screening and Treatment 
 
  
  
Screening1 
Cycle 12 
Cycle 22 
Cycle 32 
Cycle 42 
D100 
(-3/+7) 
D7 
Daily D8-11 
D358 
Daily D36-398 
D638 
Daily D64-678 
D918 
Daily D92-958 
Treatment 
  
  
  
  
  
  
  
  
  
  
Azacitidine infusion3 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
Labs/Procedures 
 
 
 
 
 
 
 
 
 
 
CBC & CMP 
X 
X4 
X9 
X4 
 
X4 
 
X4 
 
X 
Pregnancy test 
X 
 
 
 
 
 
 
 
 
 
Virology screen 
X 
 
 
 
 
 
 
 
 
 
Bone marrow biopsy 
X 
 
 
X 
 
X 
 
X 
 
 
Disease staging5 
X 
 
 
X 
 
X 
 
X 
 
X 
Treg analysis 
X6 
 
 
X 
 
X 
 
X 
 
X 
TREC analysis 
 
X 
 
X 
 
X 
 
X 
 
X 
Lymph 13 / 
DC subset analysis 
X6 
X 
 
X 
 
X 
 
X 
 
X 
Chimerism analysis 
X 
 
 
X 
 
X 
 
X 
 
X 
Pharmacokinetics7 
 
X 
 
 
 
 
 
 
 
 
Assessments 
 
 
 
 
 
 
 
 
 
 
Physical exam and ROS 
X 
X 
X9 
X 
 
X 
 
X 
 
X 
ECOG PS  
X 
X 
 
X 
 
X 
 
X 
 
X 
Acute GVHD assessment10 
 
X 
 
X 
 
X 
 
X 
 
X 
Immunosupressive 
Medication Questionnaire 
 
X 
 
X 
 
X 
 
X 
 
X 
Quality of Life assessments 
X 
 
 
 
 
 
 
 
 
 
1: Screening assessments should occur within 30 days prior to registration, except as noted in Section 8.2.1 
2: It is recommended that the first day of the cycle take place on a Monday 
3: All required labs/procedures and assessments should be completed prior to azacitidine dose 
4: Results must be reviewed prior to azacitidine dosing 
5: Clinical assessment only unless standard practice calls for a diagnostic procedure at these time points post-transplant 
6: From recipient and infused product 
7: Only for phase I patients; required timepoints: pre-dose, 2 hours, 4 hours, 6 hours, 12 hours and 24 hours post-dose 
8: +/- 4 days 
9: At least weekly review of systems, exam and laboratory testing until resolution of regimen-related toxicity and neutrophil recovery 
10: Assessment every 1-2 weeks through Day 100. After Day 100, every 4 - 6 weeks (+/- 3 days) through Day 180. Patients will continue to be followed after relapse and/or after 
starting alternate therapy through Day 180. 


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8.2 
Schedule of Assessments – Screening and Treatment 
 
8.2.1 Screening 
 
Within 30 days prior to registration (except where noted) the patient will undergo 
screening assessments as follows: 
• Physical exam and review of systems (ROS) 
• ECOG performance status 
• Quality of Life assessments 
• Complete blood count (CBC) with differential and platelet count 
• Complete metabolic panel (CMP): sodium, potassium, chloride, bicarbonate, 
blood urea nitrogen (BUN), creatinine, glucose, calcium, total protein, albumin, 
total bilirubin, AST, ALT, alkaline phosphatase 
• Pregnancy test for female patients of childbearing potential (serum or urine) 
• Virology screen (HIV 1 and 2 and hepatitis B and C) (within 60 days) 
• Bone marrow aspirate and biopsy (within 60 days) 
• Disease-specific staging (within 60 days) 
• Correlative Studies 
o Treg analysis (from recipient and infused product) 
o Lymph 13/DC subset analysis (from recipient and infused product) 
o Chimerism analysis 
 
8.2.2 Treatment Schedule and Assessments 
 
Up to four cycles of azacitidine treatment will be administered. If a patient does not 
receive treatment during a cycle, all other labs/procedures and assessments should 
still be completed. 
 
Cycle 1 (Day +7 to Day +35) 
Azacitidine will be administered on Days +7 to +11.  On Day +7, the following 
assessments must take place prior to azacitidine infusion: 
• Physical exam and ROS 
• ECOG performance status 
• Acute GVHD assessment every 1 – 2 weeks through day +100. After Day 100, 
Acute GvHD assessment every 2 weeks (+/- 3 days) through Day 180. 
• Immunosupressive Medication Questionnaire 
• CBC with differential and platelet count (results must be reviewed prior to 
azacitidine dosing) 
• CMP (results must be reviewed prior to azacitidine dosing) 
• Correlative Studies 
o TREC analysis 
o Lymph 13/DC subset analysis  
o Pharmacokinetics (phase I patients only): 
 Pre-dose 
 2 hours 
 4 hours 


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 6 hours 
 12 hours 
 24 hours 
 
Day +11 to Day +35 
At least weekly review of systems, exam and laboratory testing (CBC and CMP) 
until resolution of regimen-related toxicity and neutrophil recovery  
 
 
Cycle 2 (Day +35 to Day +39) [+/- 4 days] 
Azacitidine will be administered on Days +35 to +39 (+/- 4 days).  On Day +35 
(+/- 4 days), the following assessments must take place prior to azacitidine infusion: 
• Physical exam and ROS 
• ECOG performance status 
• Acute GVHD assessment every 1 – 2 weeks through day +100. After Day 
100, Acute GvHD assessment every 2 weeks (+/- 3 days) through Day 180. 
• Immunosupressive Medication Questionnaire 
• CBC with differential and platelet count (results must be reviewed prior to 
azacitidine dosing) 
• CMP (results must be reviewed prior to azacitidine dosing) 
• Bone marrow biopsy and aspirate 
• Disease-specfic staging 
• Correlative Studies 
o Treg analysis 
o TREC analysis 
o Lymph 13/DC subset analysis  
o Chimerism analysis 
 
Cycle 3 (Day +63 to Day +67) [+/- 4 days] 
Azacitidine will be administered on Days +63 to +67 (+/- 4 days).  On Day +63 
(+/- 4 days), the following assessments must take place prior to azacitidine infusion: 
• Physical exam and ROS 
• ECOG performance status 
• Acute GVHD assessment every 1 – 2 weeks through day +100.  After Day 
100, Acute GvHD assessment every 2 weeks (+/- 3 days) through Day 180. 
• Immunosupressive Medication Questionnaire 
• CBC with differential and platelet count (results must be reviewed prior to 
azacitidine dosing) 
• CMP (results must be reviewed prior to azacitidine dosing) 
• Bone marrow biopsy and aspirate 
• Disease-specfic staging 
• Correlative Studies 
o Treg analysis 
o TREC analysis 
o Lymph 13/DC subset analysis  
o Chimerism analysis 


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Cycle 4 (Day +91 to Day +95) [+/- 4 days] 
Azacitidine will be administered on Days +91 to +95 (+/- 4 days).  On Day +91 
(+/- 4 days), the following assessments must take place prior to azacitidine infusion: 
 
• Physical exam and ROS 
• ECOG performance status 
• Acute GVHD assessment every 1 – 2 weeks through Day +100. After Day 
100, Acute GvHD assessment every 2 weeks (+/- 3 days) through Day 180. 
• Immunosupressive Medication Questionnaire 
• CBC with differential and platelet count (results must be reviewed prior to 
azacitidine dosing) 
• CMP (results must be reviewed prior to azacitidine dosing) 
• Bone marrow biopsy and aspirate 
• Disease-specfic staging 
• Correlative Studies 
o Treg analysis 
o TREC analysis 
o Lymph 13/DC subset analysis  
o Chimerism analysis 
 
End of Treatment Day +100 [- 3/+7 days] 
• Physical exam and ROS 
• ECOG performance status 
• Acute GvHD assessment every 1 – 2 weeks through Day 100. After Day 100, 
Acute GvHD assessment every 4 - 6 weeks (+/- 3 days) through Day 180. 
• Immunosupressive Medication Questionnaire 
• CBC with differential and platelet count 
• CMP 
• Disease-specfic staging 
• Correlative Studies 
o Treg analysis 
o TREC analysis 
o Lymph 13/DC subset analysis  
o Chimerism analysis 
 
 
 


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8.3 
Study Calendar – Post-Treatment Follow-Up 
 
  
  
D140   
(±7) 
D180  
(±14) 
D270  
(±14) 
D365  
(±14) 
Mos. 15, 
18, 21, 24 
(±28) 
Labs/Procedures 
  
  
  
  
  
CBC & CMP 
X 
X 
X 
X 
  
Bone marrow biopsy 
  
X 
  
X 
  
Disease staging1 
X 
X 
X 
X 
  
Treg analysis 
X 
X 
X 
X 
  
TREC analysis 
X 
X 
X 
X 
  
Lymph 13/DC subset analysis 
X 
X 
X 
X 
  
Chimerism analysis 
X 
X 
X 
X 
  
Assessments 
  
  
  
  
  
Physical exam and ROS 
X 
X 
X 
X 
X 
ECOG PS  
X 
X 
X 
X 
  
Acute GVHD assessment2 
X 
X 
  
  
  
Chronic GVHD assessment4 
X 
X 
X 
X 
X3 
Immunosupressive Medication 
Questionnaire 
X 
X 
 
 
 
Quality of Life assessments 
  
X 
  
X 
  
1: Clinical assessment only unless standard practice calls for a diagnostic procedure at these time points 
post transplant 
2: Assessment every 4 - 6 weeks through day +180. Patients will continue to be followed after relapse 
and/or after starting alternate therapy through Day 180. 
3: Only for patients who are in remission. 
4: Patients will continue to be followed after relapse and/or starting alternate therapy through Day 365. 
 
8.4 
Schedule of Assessments – Post-Treatment Follow-Up 
 
Patients will be followed for 2 years after first dose of study drug. Patients who prematurely 
discontinue treatment should still complete post-treatment follow-up visits (if willing and 
able).  
 
Day +140 [+/- 7 days] 
• Physical exam and ROS 
• ECOG performance status 
• Acute GVHD assessment every 4 – 6 weeks (+/- 3 days) through day +180.  
• Chronic GVHD assessment 
• Immunosupressive Medication Questionnaire 
• CBC with differential and platelet count  
• CMP 
• Disease-specfic staging 
• Correlative Studies 
o Treg analysis 
o TREC analysis 
o Lymph 13/DC subset analysis  


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o Chimerism analysis 
 
 
Day +180 [+/- 14 days] 
• Physical exam and ROS 
• ECOG performance status 
• Acute GVHD assessment (+/-3 days) 
• Chronic GVHD assessment 
• Immunosupressive Medication Questionnaire 
• QOL assessments 
• CBC with differential and platelet count  
• CMP 
• Bone marrow biopsy and aspirate 
• Disease-specfic staging 
• Correlative Studies 
o Treg analysis 
o TREC analysis 
o Lymph 13/DC subset analysis  
o Chimerism analysis 
o TRM analysis 
 
Day +270 [+/- 14 days] 
• Physical exam and ROS 
• ECOG performance status 
• Chronic GVHD assessment 
• CBC with differential and platelet count 
• CMP 
• Disease-specfic staging 
• Correlative Studies 
o Treg analysis 
o TREC analysis 
o Lymph 13/DC subset analysis  
o Chimerism analysis 
 
Day +365 [+/- 14 days] 
• Physical exam and ROS 
• ECOG performance status 
• Chronic GVHD assessment 
• QOL assessments 
• (CBC with differential and platelet count 
• CMP 
• Bone marrow biopsy and aspirate 
• Disease-specfic staging 
• Correlative Studies 
o Treg analysis 


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o TREC analysis 
o Lymph 13/DC subset analysis  
o Chimerism analysis 
 
Months 15, 18, 21, and 24 [+/- 28 days] 
• Physical exam 
• Chronic GVHD assessment (only for patients who are in remission) 
 
 
9.0 
CORRELATIVE STUDIES 
 
9.1 
Treg Analysis 
 
As discussed in Section 1.1.2.3, we have evidence that providing azacitidine to mice post-
DLI increases Treg numbers and frequencies.  A higher Treg number is associated with a 
lower rate of GVHD in both animal and human studies (see Section 1.4).  We will explore 
the correlation between azacitidine administration to patients and the frequency and 
absolute number of resting and activated Tregs in peripheral blood.  Ten mL of blood in 
sodium heparin tube(s) will be collected at the following time points: 
o Baseline sample from the recipient   
o Peripheral blood stem cell product (not to exceed 1% of the total volume, 0.5 – 1 
mL typically) 
o Prior to beginning Cycle 2 (~Day 35 (pre-dose)) 
o Prior to beginning Cycle 3 (~Day 63 (pre-dose)) 
o Prior to beginning Cycle 4 (~Day 91 (pre-dose)) 
o Day 100 
o Day 140 
o Day 180 
o Day 270 
o Day 365 
 
Note that no Day 7 sample will be obtained because of significant cytopenias at this time 
after transplant. 
 
9.2 
Recipient Immune-Reconstitution Analysis 
 
9.2.1 TREC Analysis 
 
The purpose of the TREC (T-cell receptor excision circle) assay is to evaluate de 
novo synthesis of T cells using a real-time quantitative polymerase chain reaction. 
Ten mL of peripheral blood in EDTA tube(s) will be collected at the following time 
points: 
• Prior to beginning cycle 1 (Day 7(pre-dose))* 
• Prior to beginning Cycle 2 (~Day 35 (pre-dose)) 
• Prior to beginning Cycle 3 (~Day 63 (pre-dose)) 
• Prior to beginning Cycle 4 (~Day 91 (pre-dose)) 
• Day 100 


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• Day 140 
• Day 180 
• Day 270 
• Day 365 
*Forty mL of peripheral blood in EDTA tube(s) will be collected on Day 7 
 
9.2.2 Lymphocyte Subset-13 (“Lymph 13”) and DC Subset Analysis 
 
Lymphocyte subset analysis requires a 10mL blood sample collected in EDTA 
tube(s). DC subset analysis will be performed on a separate 10mL blood sample 
collected in sodium heparin tube(s). Blood for both of these analyses will be 
collected at the following time points: 
• Baseline sample from the recipient  
• Peripheral blood stem cell product (not to exceed 1% of the total volume, 
0.5 – 1 mL typically) 
• Prior to beginning cycle 1 (Day 7(pre-dose)) 
• Prior to beginning Cycle 2 (~Day 35 (pre-dose)) 
• Prior to beginning Cycle 3 (~Day 63 (pre-dose)) 
• Prior to beginning Cycle 4 (~Day 91 (pre-dose)) 
• Day 100 
• Day 140 
• Day 180 
• Day 270 
• Day 365 
 
9.2.3 Chimerism Analysis 
 
Chimerism analysis requires 30 mL EDTA tube(s) of peripheral blood will be 
collected at each the following time points: 
• Baseline 
• Prior to beginning Cycle 2 (~Day 35 (pre-dose)) 
• Prior to beginning Cycle 3 (~Day 63 (pre-dose)) 
• Prior to beginning Cycle 4 (~Day 91 (pre-dose)) 
• Day 100 
• Day 180 
• Day 270 
• Day 365 
 
9.2.4 TRM Analysis 
 
TRM analysis on day 180 of the study will be planned to access for TRM.   
 
9.3 
Pharmacokinetic (PK) Studies 
 


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Pharmacokinetic studies of azacitidine will be performed during the first cycle on the first 
day of azacitidine treatment (Day 7).  Four ml of blood will be drawn into EDTA tube(s) 
at the following time points: 
• Pre-dose 
• 2 hours post-dose 
• 4 hours post-dose 
• 6 hours post-dose 
• 12 hours post-dose 
• 24 hours post-dose 
 
 
9.3.1 Sample Delivery 
 
Samples must be maintained at room temperature and delivered as soon as possible 
to the DiPersio laboratory, room 626 P Southwest Tower, Washington University. 
Specimens will be identified by initials and study number.  
 
9.3.2 Sample Processing 
 
Peripheral blood samples will be processed for plasma if needed and then red cell 
lysed or purified by ficoll gradient.  Cells may be analyzed by FACS or used in 
functional studies, or will be cryopreserved viably at 107/vial in 90% fetal bovine 
serum with 10% DMSO. Fresh peripheral blood may also be used for correlative 
studies at the discretion of the Principal Investigator.  Plasma will be divided into 
1-2 mL aliquots and frozen at -80oC. 
 
Samples delivered to the DiPersio lab will be analyzed by multiparameter flow 
cytometry to delineate the major classes of B, T (including Treg subsets, 
naïve/central memory/effector memory and Th1/Th2/Th17), and NK cells, 
monocytes and DCs using the flow cytometry panels recently described by the 
Human Immunophenotyping Consortium.69 Intracellular staining of FOXP3 will be 
performed after fixation and permeabilization according to the manufacturer’s 
recommendation.  
 
The percentage and absolute number of the following cell types will be determined 
by FACS analysis: 
 
• Resting (naïve) Tregs:  CD4+CD25++FoxP3lowCD45RA+CD31+  
• Activated (effector) Tregs:   CD4+CD25+++FoxP3highCD45RA- CD31-  
• Total T cells (CD3+ cells)  
• Total CD4+ and CD8+ cells  
• Naïve (CCR7+CD45RA+), central memory (CCR7+CD45RA-), effector 
memory (CCR7-CD45RA-) and effector (CCR7-CD45RA+) CD3+CD4+ 
and CD3+CD8+ T cells 
• Th1 (CXCR3+CCR6-), Th2 (CXCR3-CCR6-) and Th17 (CXCR3-CCR6+) 
CD3+CD4+ T cells 


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• naïve (CD19+CD27-) and memory (CD19+CD27+) B cells 
• classical (CD14+CD16-) and non-classical (CD14+CD16+) monocytes 
• myeloid (CD14-HLA-DR+CD11c+) and plasmacytoid (CD14-HLA-
DR+CD123+) dendritic cells 
• NKT cells (CD3+CD56+) 
• NK cells (CD56+CD16- and CD56+CD16+). 
 
Absolute Treg numbers will be calculated by multiplying Treg frequency by the 
absolute lymphocyte count (ALC) obtained by an automated differential 
assessment. 
 
Lymph 13 processing to be done locally by the BJH flow lab and DC subset 
analysis will be performed by the DiPersio Lab. 
 
9.4 
Questionnaires 
 
9.4.1 GVHD Assessments 
 
Acute GVHD will be accessed using modified Glucksberg criteria (Appendix 3).  
Chronic GVHD will be accessed using the NIH concensus criteria (Appendix 4). 
 
9.4.2 Quality of Life Assessments 
 
Quality of Life (QOL) will be assessed by a patient self report questionnaire FACT-
BMT and Human Activity Profile (HAP) (Appendices 5 and 6).  
 
9.4.3 Immunosupressive Medication Questionnaire 
 
The use of immunsupressive medications will be captured using the 
Immunosuppressive Medication Questionnaire (Appendix 7). 
 
 
10.0 
DATA SUBMISSION SCHEDULE 
 
Case report forms will be completed according to the schedule listed in this section.  There is a 
window of up to 14 days allowed between the time point listed in the submission schedule and the 
date when the form must be completed. 
 
 
 
 
 
 
 
 
 


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Case Report Form 
Submission Schedule 
Consent form 
Prior to starting treatment 
On Study 
Medical History 
Treatment History 
Baseline 
Safety Labs 
Correlative Studies  
Baseline 
Cycle 1 
Cycle 2 
Cycle 3 
Cycle 4 
Day +100 
Day +140 
Day +180 
Day +270 
Day +365 
Bone Marrow 
Response Assessment  
Baseline (Bone Marrow only) 
Cycle 2 
Cycle 3 
Cycle 4 
Day +180 
Day +365 
FACT-BMT 
Human Activity Profile 1 of 2 
Human Activity Profile 2 of 2 
Baseline 
Day +180 
Day +365 
Azacitidine Dosing 
Cycle 1 
Cycle 2 
Cycle 3 
Cycle 4 
DLT Evaluation 
ANC Engraftment 
Cycle 1 
Platelet Engraftment 
Day +100 
Follow-Up 
Month 15 
Month 18 
Month 21 
Month 24 
End of Study 
Survival 
End of study 
Acute GVHD Assessment 
Every 1-2 weeks through Day +100 
Every 2 weeks through Day +180 
ChronicGVHD-Skin-Nails-Scalp-
Body Hair 
Chronic GVHD-Mouth-Eyes 
Day +140 
Day +180 
Day +270 


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Chronic GVHD-GI-Liver-Lungs 
Chronic GVHD-Muscle-Fascia-
Joints -Genitalia 
Chronic GVHD-General 
Day +365 
Month 15 
Month 18 
Month 21 
Month 24 
Immunosuppressive Medications 
Continuous 
Adverse Events 
Continuous 
 
11.0 
REGULATORY AND REPORTING REQUIREMENTS 
 
The entities providing oversight of safety and compliance with the protocol require reporting as 
outline below. 
 
The Washington University Human Research Protection Office (HRPO) requires that all events 
meeting the definition of unanticipated problem or serious noncompliance be reported as outlined 
in Section 11.7. 
 
The FDA requires that all serious and unexpected adverse events be reported as outlined in Section 
11.8.  In addition, any fatal or life-threatening adverse experiences where there is a reasonable 
possibility of relationship to study intervention must be reported. 
 
Celegene requires that all AEs be reported as outlined in Section 11.9. 
 
11.1 
Definitions 
 
11.1.1 Adverse Events (AEs) 
 
Definition:  any unfavorable medical occurrence in a human subject including any 
abnormal sign, symptom, or disease. 
 
Grading:  the descriptions and grading scales found in the revised NCI Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for 
all toxicity reporting.  A copy of the CTCAE version 4.0 can be downloaded from 
the CTEP website. 
 
Attribution (relatedness), Expectedness, and Seriousness: the definitions for the 
terms listed that should be used are those provided by the Department of Health 
and Human Services’ Office for Human Research Protections (OHRP).  A copy of 
this guidance can be found on OHRP’s website: 
(http://www.hhs.gov/ohrp/policy/advevntguid.html).   
 
11.1.2 Serious Adverse Event (SAE) 
 
Definition:  any adverse drug experience occurring at any dose that results in any 
of the following outcomes: 
• Death 


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• A life-threatening adverse drug experience 
• Inpatient hospitalization or prolongation of existing hospitalization 
• A persistent or significant disability/incapacity (i.e., a substantial disruption 
of a person’s ability to conduct normal life functions) 
• A congenital anomaly/birth defect 
• Any other experience which, based upon appropriate medical judgment, 
may jeopardize the subject and may require medical or surgical intervention 
to prevent one of the outcomes listed above 
 
All unexpected SAEs must be reported to the FDA. 
 
11.1.3 Unexpected Adverse Experience 
 
Definition: any adverse drug experience, the specificity or severity of which is not 
consistent with the current investigator brochure (or risk information, if an IB is not 
required or available). 
Events that are both serious AND unexpected must be reported to the FDA. 
 
11.1.4 Life-Threatening Adverse Experience 
 
Definition: any adverse drug experience that places the subject (in the view of the 
investigator) at immediate risk of death from the reaction as it occurred, i.e., it does 
not include a reaction that, had it occurred in a more severe form, might have caused 
death. 
Life-threatening adverse experiences must be reported to the FDA. 
 
11.2 
Reportable Events 
 
All AEs will be collected and reviewed for phase I patients regardless of grade. Only 
selected AEs as described below will be collected and reviewed for phase II patients: 
• All grade 3, 4, or 5 AEs 
• Any AE that requires azacitidine treatment to be delayed, held, or discontinued 
• Any AE that requires modification of GVHD prophylaxis medications 
• Other AEs of interest as determined following completion of the phase I portion of 
this study 
 
11.3 
Unanticipated Problems 
 
Definition: 
• unexpected (in terms of nature, severity, or frequency) given (a) the research 
procedures that are described in the protocol-related documents, such as the IRB-
approved research protocol and informed consent document; and (b) the 
characteristics of the subject population being studied; 


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• related or possibly related to participation in the research (in this guidance 
document, possibly related means there is a reasonable possibility that the incident, 
experience, or outcome may have been caused by the procedures involved in the 
research); and 
• suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was previously 
known or recognized. 
 
11.4 
Noncompliance 
 
Definition: failure to follow any applicable regulation or institutional policies that govern 
human 
subjects 
research 
or 
failure 
to 
follow 
the 
determinations 
of 
the 
IRB.  Noncompliance may occur due to lack of knowledge or due to deliberate choice to 
ignore regulations, institutional policies, or determinations of the IRB. 
 
11.5 
Serious Noncompliance 
 
Definition: noncompliance that materially increases risks, that results in substantial harm 
to subjects or others, or that materially compromises the rights or welfare of participants. 
 
11.6 
Protocol Exceptions 
 
Protocol exceptions are not allowed. 
 
11.7 
Reporting to the Human Research Protection Office (HRPO) at Washington 
University 
 
The PI is required to promptly notify the IRB of the following events: 
 
• Any unanticipated problems involving risks to participants or others which occur 
at WU, any BJH or SLCH institution, or that impacts participants or the conduct of 
the study. 
• Noncompliance with federal regulations or the requirements or determinations of 
the IRB. 
• Receipt of new information that may impact the willingness of participants to 
participate or continue participation in the research study. 
 
These events must be reported to the IRB within 10 working days of the occurrence of the 
event or notification to the PI of the event.  The death of a research participant that qualifies 
as a reportable event should be reported within 1 working day of the occurrence of the 
event or notification to the PI of the event. 
 
 
11.8 
Reporting to the Quality Assurance and Safety Monitoring Committee 
(QASMC) at Washington University 
 


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The PI is required to notify the QASMC of any unanticipated problem occurring at WU or 
any BJH or SLCH institution that has been reported to and acknowledged by HRPO as 
reportable.  (Unanticipated problems reported to HRPO and withdrawn during the review 
process need not be reported to QASMC.) 
 
QASMC must be notified within 10 days of receipt of IRB acknowledgment via email to 
a QASMC auditor. 
 
 
11.9 
Reporting to the FDA 
 
The conduct of the study will comply with all FDA safety reporting requirements.  
PLEASE NOTE THAT REPORTIING REQUIREMENTS FOR THE FDA DIFFER 
FROM REPORTING REQUIREMENTS FOR HRPO/QASMC.  It is the 
responsibility of the investigator to report any unanticipated problem to the FDA as 
follows: 
 
Report any unexpected fatal or life-threatening adverse experiences (Section 11.1.4) 
associated with use of the drug  by telephone or fax no later than 7 calendar days 
after initial receipt of the information.  Report any serious, unexpected adverse 
experiences (Section 11.1.3), as well as results from animal studies that suggest 
significant clinical risk within 15 calendar days after initial receipt of this 
information.   
 
All MedWatch forms will be sent by the investigator or investigator’s team to the FDA at 
the following address or by fax: 
 
Food and Drug Administration 
Center for Drug Evaluation and Research 
Division of Oncology Drug Products 
5901-B Ammendale Rd. 
Beltsville, MD 20705-1266 
FAX: 1-800-FDA-0178 
 
 
11.10 Reports to Celgene 
 
The investigator will also provide Celgene Corporation (as a supplier of azacitidine 
(VIDAZA) for this study) a copy of the Investigator IND annual report at the time of the 
investigators submission to the FDA.  Submit reports to Celgene at: 
 
Celgene Corporation / Attn: Medical Affairs Operations 
Connell Corporate Park 
400 Connell Drive, Suite 700 
Berkeley Heights, NJ 07922 
Tel: (908) 673-9000 


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All AE reports must include the patient number, age, sex, weight, severity of reaction (mild, 
moderate, severe), relationship to drug, date and time of administration of test medications 
and all concomitant medications, and medical treatment provided. The investigator is 
responsible for evaluating all adverse events to determine whether criteria for “serious” 
and as defined above are present.  The investigator is responsible for reporting adverse 
events to Celgene as described below. 
 
11.10.1  Expedited Reporting by Investigator to Celgene 
 
Serious adverse events/unanticipated problems (SAE) are defined above.  The 
investigator must inform Celgene of all SAEs within 24 hours of being aware of 
the event (regardless of of whether the SAE is expected or not). This must be 
documented on Celgene SAE form or a FDA 3500 or MEDWATCH form.  This 
form must be completed and supplied to Celgene within 24 hours/1 business day.  
The initial report must be as complete as possible, including details of the current 
illness and (serious) adverse event, and an assessment of the causal relationship 
between the event and the investigational product(s).  Information not available at 
the time of the initial report (e.g., an end date for the adverse event or laboratory 
values received after the report) must be documented on a follow-up MEDWATCH.  
A final report to document resolution of the SAE is required.  The Celgene tracking 
number (VZ-CL-AML-PI-002423) and the institutional protocol number should be 
included on SAE reports (or on the fax cover letter) sent to Celgene.  A copy of the 
fax transmission confirmation of the SAE report to Celgene should be attached 
retained with the patient records. Celgene drug safety contact information is as 
follows: 
 
Celgene Corporation 
Global Drug Safety and Risk Management 
Connell Corporate Park 
300 Connell Dr., Suite 6000 
Berkley Heights, NJ 07922 
Toll Free: (800) 640-7854 / Phone: (908) 673-9667 / Fax: (908) 673-9115 
E-mail: drugsafety@celgene.com 
 
 
11.11 Pregnancies 
 
Pregnancy of a female subject or the female partner of a male subject occurring while the 
subject is on azacitidine or within 4 weeks after the subject’s last dose of azacitidine are 
considered expedited reportable events.  If the subject is on azacitidine, it is to be 
discontinued immediately.  The pregnancy must be reported to Celgene Drug Safety within 
24 hours of the Investigator’s knowledge of the pregnancy by phone and facsimile using 
the SAE Form. 
 


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The Investigator will follow the pregnant female until completion of the pregnancy, and 
must notify Celgene Drug Safety of the outcome as specified below.  The Investigator will 
provide this information as a follow-up to the initial SAE. 
 
If the outcome of the pregnancy meets the criteria for immediate classification as a SAE 
(i.e., spontaneous abortion [any congenital anomaly detected in an aborted fetus is to be 
documented], stillbirth, neonatal death, or congenital anomaly), the Investigator should 
follow the procedures for reporting SAEs (i.e., report the event to Celgene Drug Safety by 
facsimile within 24 hours of the Investigator’s knowledge of the event). 
 
Any suspected fetal exposure to azacitidine must be reported to Celgene within 24 hours 
of being made aware of the event.  The pregnant female should be referred to an 
obstetrician/gynecologist experienced in reproductive toxicity for further evaluation and 
counseling.  All neonatal deaths that occur within 30 days of birth should be reported, 
without regard to causality, as SAEs.  In addition, any infant death after 30 days that the 
Investigator suspects is related to the in utero exposure to the study drug should also be 
reported.  In the case of a live “normal” birth, Celgene Drug Safety should be advised as 
soon as the information is available. 
 
11.12 Timeframe for Reporting Required Events 
 
AEs will be collected from the time of the first dose of azacitidine (Day +7) through the 
Day 140 visit or 30 days after last dose of azacitidine (for patients that do no complete all 
four cycles of treatment).  
 
 
12.0 
DATA AND SAFETY MONITORING   
 
In compliance with the Washington University Institutional Data and Safety Monitoring Plan, the 
Data and Safety Monitoring Committee (DSMC) will meet to review toxicity data at least every 6 
months.  The report will be prepared by the statistician with assistance from the study team and 
will be submitted to the Washington University Quality Assurance and Safety Monitoring 
Committee (QASMC). 
 
Additionally, during the phase I dose escalation, the Principal Investigator will review all patient 
data at least monthly (or before each dose-escalation if occurring sooner than monthly), and 
provide a semi-annual report to the Quality Assurance and Safety Monitoring Committee 
(QASMC).  In both instances, this report will include: 
 
• HRPO protocol number, protocol title, Principal Investigator name, data coordinator 
name, regulatory coordinator name, and statistician 
• Date of initial HRPO approval, date of most recent consent HRPO approval/revision, 
date of HRPO expiration, date of most recent QA audit, study status, and phase of study 
• History of study including summary of substantive amendments; summary of accrual 
suspensions including start/stop dates and reason; and summary of protocol error(s), or 
breach of confidentiality including start/stop dates and reason 


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• Study-wide target accrual and study-wide actual accrual  
• Protocol activation date  
• Average rate of accrual observed in year 1, year 2, and subsequent years  
• Expected accrual end date and accrual by cohort 
• Objectives of protocol with supporting data and list the number of participants who 
have met each objective 
• Measures of efficacy  
• Early stopping rules with supporting data and list the number of participants who have 
met the early stopping rules 
• Summary of toxicities separated by cohorts with the number of dose-limiting toxicities 
indicated 
• Abstract submissions/publications 
• Summary of any recent literature that may affect the safety or ethics of the study  
 
The study principal investigator and Research Patient Coordinator will monitor for serious 
toxicities on an ongoing basis during both the phase I (as outlined above) and every 6 months 
during the phase II portion of the trial. Once the principal investigator or Research Patient 
Coordinator becomes aware of an adverse event, the AE will be reported to the HRPO and 
QASMC according to institutional guidelines. 
 
A DSMC will consist of no fewer than 3 members including 2 clinical investigators and a 
biostatistician.  Like investigators, DSMC members are subject to the Washington University 
School of Medicine policies regarding standards of conduct. Individuals invited to serve on the 
DSMC will disclose any potential conflicts of interest to the trial principal investigator and/or 
appropriate university officials, in accordance with institution policies. Potential conflicts that 
develop during a trial or a member’s tenure on a DSMC must also be disclosed.  
 
Refer to the Washington University Quality Assurance and Data Safety Monitoring Committee 
Policies and Procedures for full details on the responsibilities of the DSMC at 
http://www.siteman.wustl.edu/uploadedFiles/Research_Programs/Clinical_Research_Resources/
Protocol_Review_and_Monitoring_Committee/QASMCQualityAssurcance.pdf 
 
 
13.0 
STUDY EFFICACY AND DISCONTINUATION 
 
13.1 
Criteria to Measure Efficacy in Mitigation of GvHD 
 
GVHD rate and severity will be assessed based on modified Glucksberg criteria.70  Grade 
II-IV and III-IV aGVHD in first100 days after transplant will be assessed.  
 
13.2 
Definitions for Safety and Efficacy Assessments  
 
13.2.1 Neutrophil Engraftment 
 


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Time to neutrophil engraftment is measured by determining the first of 3 
consecutive measurements of neutrophil count ≥ 500/ul following conditioning 
regimen-induced nadir. 
 
13.2.2 Platelet Engraftment 
 
Time to platelet engraftment is measured by determining the first of 3 consecutive 
measurements of platelet count ≥ 20,000/ul without platelet transfusion support for 
7 days. 
 
13.2.3 Primary Graft Failure 
 
Failure of neutrophil engraftment by Day 28. 
 
13.2.4 Secondary Graft Failure 
 
Primary engraftment followed by a drop in the neutrophil count to less than 500/ml 
for more than 3 consecutive days without any apparent cause such as drugs or 
opportunistic infection. 
 
13.2.5 Full Donor Chimerism 
 
Greater than or equal to 95% donor cells within the bone marrow. 
 
13.2.6 Acute GVHD 
 
Incidence and severity of acute GVHD will be assessed based on the modified 
Glucksberg criteria70 and Seattle criteria.71  Attempts should be made to confirm 
the diagnosis pathologically by biopsy of target organ(s). 
 
13.2.7 Steroid Refractory Acute GVHD 
 
Steroid refractory acute GvHD will be defined as no improvement in GvHD within 
7 days of starting high dose system steroids (2mg/kg) or progression of GvHD 
while on 2mg/kg steroids. 
13.2.8 Chronic GVHD 
 
Incidence and severity of chronic GVHD will be assessed based on the NIH 
consensus criteria and global severity scoring system.72 Attempts should be made 
to confirm the diagnosis pathologically by biopsy of target organ(s). 
 
 
13.2.9 Treatment-Related Mortality 
 


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Death that results from a transplant procedure-related complication (e.g. infection, 
organ failure, hemorrhage, GVHD) rather than from relapse of the underlying 
disease or an unrelated cause. 
 
 
13.2.10 Adverse Events 
 
Adverse events will be assessed and graded according to NCI Common Toxicity 
Criteria version 4 as outlined in Section 10.2. 
 
13.2.11 Determination of Relapse 
 
A patient will be considered relapsed when there is a recurrence of the original 
malignant disease after transplantation. The time to relapse is the time from the date 
of transplant to date of the first observation of hematologic, radiographic, or 
cytogenetic changes, which result in characterization as relapse. 
 
13.2.12 Determination of Survival 
 
Survival will be measured by assessing if the patient remains alive by visual 
observation, telephone call or notification of death medical record or death 
certificate. 
 
13.2.13 Evaluable for Toxicity 
 
All subjects will be evaluable for toxicity from the time of their first dose of 
azacitidine. 
 
13.2.14 Overall Survival 
 
Defined as the date of transplant to the date of death from any cause. 
 
13.2.15 Duration of Remission 
 
Defined as the interval from the date complete remission is documented to the date 
of recurrence. 
 
 
13.2.16 Disease-Free Survival 
 
Defined as the interval from the date of first documentation of a CR to date of 
relapse. 
 
 
13.3 
Response Review 
 


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At the end of the study all responses will be reviewed by an expert independent of the 
study. 
 
 
 
14.0 
STATISTICAL CONSIDERATIONS   
 
14.1 
Study Design 
 
The primary objectives of this study are: (1) to determine the maximum tolerated dose 
of azacitidine in patients undergoing matched (8 out of 8) unrelated donor transplant for 
any hematological malignancy in remission or with stable disease (phase I) and (2) to 
determine the effect of azacitidine on grade II-IV GvHD in patients undergoing matched 
(8 out of 8) unrelated donor transplant for AML in remission 1 or 2 or MDS (phase II). 
 
14.1.1 Phase I 
 
A 3+3 phase I clinical trial will be conducted to assess the toxicity profile and 
establish the MTD of azacitidine after allo-HSCT in patients with any 
hematological malignancy in remission or with stable disease. This is a dose 
escalating phase I, single arm, single institute, open label, non-randomized study. 
Three cohorts are planned; in addition, there exists one dose de-escalation cohort. 
A minimum of 12 and maximum of 24 patients will be treated in the phase I portion. 
 
14.1.2 Stopping Rules for Phase I 
 
If >1 patient in cohort 1 has a DLT, then the trial will be stopped and no phase II 
portion of the trial will be performed. 
 
If graft failure with donor engraftment or loss of donor engraftment by Day 100 
occurs in ≥20% of subjects, then trial will be stopped. 
 
If grade III – IV acute GvHD occurs in ≥50% of subjects, then the trial will be 
stopped. 
 
14.1.3 Phase II 
 
Provided there are no reasons for stopping the trial in phase I, an open label single 
arm phase II trial will be conducted based on the MTD determined from the phase 
I portion.  A total of 46 patients will be treated in the phase II portion of the trial.  
Efficacy of acute GvHD prevention will be compared to matched historical controls 
(matched for age, sex and disease) from the CIBMTR registry. 
 
Toxicity will be assessed on an ongoing basis every 6 months during the phase II 
portion. The interim analyses will be performed as per Section 14.4.4.  
 


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14.2 
Sample Size Calculation 
 
14.2.1 Phase I 
 
Given the 3+3 design of the phase I portion of this trial, a minimum of 12 patients 
and a maximum of 24 will be enrolled in the phase I portion of the trial. 
 
14.2.2 Phase II 
 
Given the exploratory nature of this study we are willing to accept an alpha of 0.1. 
Using a two-sided alpha of 0.1, 80% power and a Fisher’s exact test, we will need 
a sample size of 46 per group to detect a statistical difference if the incidence of 
grade II-IV acute GvHD in the azacitidine group is 20%, assuming the incidence of 
44% in the standard care group (matched historical controls from the CIBMTR 
registry).  
 
14.2.3 Correlative Studies 
 
Our pilot data shows that the mean change in Tregs in patients undergoing 
peripheral blood stem cell transplant is 4%, and if we assume that the mean change 
in the azacitidine group will be at least 2 fold (8%) (based on Goodyear et al.59), 
and assuming the standard deviation is 2.5%, the effect size is calculated as (8%-
4%)/2.5%=1.6. Therefore we need 8 subjects in each group. (See effect size table 
below). 
 
Effect size 
Sample size per group 
0.6 
45 
0.7 
34 
0.8 
26 
0.9 
21 
1 
17 
1.2 
12 
1.6 
8 
 
 
14.3 
Accrual 
 
We perform over 100 unrelated allogeneic HSCT per year at Washington University, 
approximately 50% of which are for AML.  We estimate around 30 patients/year may be 
eligible for this trial.  We anticipate enrollment of approximately 2-3 patients per month.  
Historically, around 50% of transplants are performed using reduced intensity conditioning. 
 
 
14.4 
Statistical Analyses 
 
14.4.1 Descriptive Analysis 


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14.4.1.1 
Patient Disposition 
 
The number of patients discontinued, the reasons for discontinuation, and 
the amount of therapy administered will be summarized by patient and by 
reason for discontinuation. 
 
14.4.1.2 
Protocol Deviations 
 
All significant deviations will be summarized by patient and by type of 
deviation. 
 
14.4.1.3 
Demographics and Baseline Characteristics 
 
Descriptive summary statistics will be provided for demographic and 
important baseline characteristics including gender, age, performance status, 
FAB/WHO AML subtype, and cytogenetic abnormalities.  For continuous 
variables, the number of patients, mean, standard deviation, median, 
minimum and maximum will be provided.  For categorical variables the 
number and percentage of patients in each category will be summarized. 
 
14.4.2 Primary and Secondary Endpoint Analysis 
 
MTD will be determined as described in Section 5.3.1.   
 
Type and grade of GVHD will be documented based on modified Glucksberg 
criteria.70 
GVHD rate, and survival at 100, 180, and 365 days after transplant will be 
summarized using descriptive statistics. An exploratory descriptive stratified 
analysis by transplant conditioning regimen, and acute GvHD at day 180 and at one 
year after transplant will be performed, but the study is not powered to detect a 
difference between conditioning groups given its exploratory nature and large 
retrospective studies showing similar outcomes.65,66 
                        
Anyone dying prior to Day 100 without acute GvHD will be censored in assessing 
the incidence of Grade II-IV acute GvHD in the first 100 days after transplant in 
patients with MDS and AML.  Subjects dying prior to study drug will be excluded 
and replaced. 
 
Overall survival (OS) is defined as the date from transplant to death or last follow-
up. Relapse-free survival (RFS) is defined as the time from the date of transplant to 
the date of relapse or last follow-up.  Kaplan-Meier (KM) curves for OS and RFS 
will be generated. 
 
Treg frequency and number will be assessed by paired analysis with control banked 
samples at pre-transplant (donor PBSC product), Day 30, and Day 100. Paired t-


Amendment 11: Version 09/06/18 
 
Page 68 of 96 
test or Wilcoxon signed rank test will be used to compare Day 30 (or 100) change 
from the pre-transplant between azacitidine and standard care group. Repeated 
analysis which accounts for the correlation across the three time points (pre-
transplant, Day 30, and Day 100) per subject will be considered as appropriate.  
 
14.4.3 Safety Analysis  
 
Subjects who receive at least one dose of azacitidine will be monitored for safety.  
AEs will be coded according to CTCAE v 4.0.  The results will be tabulated to 
examine their frequency, organ systems affected, and relationship to study 
treatment.  
 
The results of laboratory assessments will be evaluated similarly.  Interim safety 
data will be examined on an ongoing basis to ensure subject safety. In addition the 
Quality Assurance and Safety Monitoring Committee (QASMC) will review the 
DSM semi-annually. 
 
Applicable laboratory parameters will be graded according to NCI CTCAE v4.0.  
The incidence of maximum grade (number and percent of patients experiencing the 
maximum grade during the study) will be summarized for each laboratory 
parameter. Graphical display of selected hematological parameters will also be 
provided. 
 
Vital signs, physical examination findings, ECOG performance status scores, and 
weights will be presented in listing format. 
 
14.4.4 Interim Analysis  
 
During the phase II portion of the study three interim analyses will be planned for 
TRM and hematologic toxicity as defined in sections 13.2.1-13.2.3.  A four-stage 
group sequential design with an O’Brien-Fleming method will be used to stop the 
trial early.  
 
Assuming TRM in the azacitidine group is 30% and TRM in the standard care 
group is 10%, the design uses a one-sided alternative hypothesis with early stopping 
to reject the null hypothesis - TRM in the azacitidine group is same as TRM in the 
standard care group. The following table displays the required sample size and 
boundary values at each interim analysis. At each interim stage, if the standardized 
test statistics is larger than the corresponding boundary, then trial is stopped and we 
reject the null hypothesis. Otherwise, the process continues to next interim. At the 
final analysis, 4th interim, the trial stops and TRM in the azacitidine group is 
statistically larger than TRM in the standard care group if the standardized statistics 
>=0.13705. Otherwise, TRM in the azacitidine group is statistically same from 
TRM in the standard care group. 
 
 


Amendment 11: Version 09/06/18 
 
Page 69 of 96 
Interim 
N 
(Azacitidine) 
N 
(Standard care) 
Boundary 
1 
12 
12 
0.54819 
2 
24 
24 
0.27409 
3 
36 
36 
0.18273 
4 
48 
48 
0.13705 
 
 
Assuming hematologic toxicity in the azacitidine group is 20% and hematologic 
toxicity in the standard care group is 5%, the design uses a one-sided alternative 
hypothesis with early stopping to reject the null hypothesis - hematologic toxicity 
in the azacitidine group is same as in the standard care group. 
 
Interim 
N 
(Azacitidine) 
N 
(Standard care) 
Boundary 
1 
15 
15 
0.41114 
2 
30 
30 
0.20557 
3 
45 
45 
0.13705 
4 
59 
59 
0.10278 
 


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15.0 
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34. 
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von Reyn Cream L, Ehmann WC, Rybka WB, et al: Sirolimus in unmanipulated 
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Silverman LR, McKenzie DR, Peterson BL, et al: Further analysis of trials with 
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Fenaux P, Mufti GJ, Hellstrom-Lindberg E, et al: Azacitidine prolongs overall 
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Braiteh F, Soriano AO, Garcia-Manero G, et al: Phase I study of epigenetic 
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Zaucha JM, Gooley T, Bensinger WI, et al: CD34 cell dose in granulocyte 
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68. 
Przepiorka D, Anderlini P, Saliba R, et al: Chronic graft-versus-host disease after 
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71. 
Glucksberg H, Storb R, Fefer A, et al: Clinical manifestations of graft-versus-host 
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Filipovich AH, Weisdorf D, Pavletic S, et al: National Institutes of Health 
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APPENDIX 1: ECOG Performance Status Scale 
 
 
Grade 
 
 
Description 
0 
Normal activity.  Fully active, able to carry on all pre-disease 
performance without restriction. 
1 
Symptoms, but ambulatory.  Restricted in physically strenuous 
activity, but ambulatory and able to carry out work of a light or 
sedentary nature (e.g., light housework, office work). 
2 
In bed <50% of the time.  Ambulatory and capable of all self-care, but 
unable to carry out any work activities.  Up and about more than 50% 
of waking hours. 
3 
 
In bed >50% of the time.  Capable of only limited self-care, confined 
to bed or chair more than 50% of waking hours. 
 
4 
100% bedridden.  Completely disabled.  Cannot carry on any self-care.  
Totally confined to bed or chair. 
5 
 
Dead. 
 
 
 
 


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APPENDIX 2: Registration Form 
 
Patient Initials  ____-____-____ 
Date of Birth: ____/____/____           Age: _____yr   
Sex: 
 Male 
 Female 
 
Race:             
 White 
                      
 Black or African American 
 
 Native Hawaiian or Other Pacific          
Islander 
                      
 Asian 
                      
 American Indian or Alaska Native 
                      
 Unknown 
 
Ethnicity:   
 Hispanic or Latino 
 
 
 Non-Hispanic 
 
 
 Unknown 
Diagnosis:   
 AML     
 
 MDS 
 
 Other 
 
Specify Other: ______________________ 
Informed Consent Date: ____/____/____   
Proposed Registration Date: ____/____/____   
Current Zip Code:_______________   
ECOG at Baseline: 
 0       
 1      
 2      
Does the participant satisfy all of the eligibility criteria?       
 Yes         
 No 
Were any waivers granted?       
 Yes         
 No  
 
Person completing this form 
 
______________________________________________________________________________ 
Printed name 
 
 
 
 
Signature  
 
 
 
Date 
 
Registering MD 
 
______________________________________________________________________________ 
Printed name 
 
 
 
 
Signature  
 
 
 
Date 
 
Please include: 
1. Copy of signed consent form  
2. Completed eligibility checklist, signed and dated by a member of the study team 
3. Copy of appropriate source documentation confirming patient eligibility 
 
--------------------------------------------------------------------------------------------------------------------- 
To be completed by PI or designee 
 
Assigned UPN ___________________________ 
Dose Level:       
 1     
 2     
 3     
 4 
 
 
Signature _________________________________  Date___________________ 
 
 


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APPENDIX 3: Acute GVHD Assessment 
 
Date of Assessment: _________________________     
 
Date of GvHD Onset: ________________________         
 
Acute GVHD Stage 
Skin1 
Stage 1  Rash on < 25% of skin 
 
Stage 2  Rash on 25-50% of skin 
 
Stage 3  Rash on > 50% of skin 
 
Stage 4  Generalized erythoderma with bollous formation 
 
Liver2 
Stage 1 
Bilirubin 2-3 mg/dl 
 
Stage 2 
Bilirubin 3-6 mg/dl 
 
Stage 3 
Bilirubin 6-15 mg/dl 
 
Stage 4 
Bilirubin >15 mg/dl 
 
Gut 
Stage 1  Diarrhea >500 ml/day or persistent nausea3 
 
Stage 2  Diarrhea >1000 ml/day 
 
Stage 3  Diarrhea >1500 ml/day 
 
Stage 4  Severe abdominal pain with or without ileus 
 
Acute GVHD Grade 
 
Skin 
Liver 
Gut 
[  ] Grade 1 
Stage 1-2 
None 
None 
[  ] Grade 2 
Stage 3 or 
Stage 1 or 
Stage 1 
[  ] Grade 3 
-- 
Stage 2-3 or 
Stage 2-4 
[  ] Grade 4 
Stage 4 
Stage 4 
-- 
 
1. 
Use ‘Rule of Nines’ to determine extent of rash 
 
2. 
Based on peak total blirubin. Decrease by one stage if another cause of hyperbilrubinemia has been documented 
3. 
Persistent nausea with histologic confirmation of GVHD in the stomach or duodenum 
 
Reference: D Przepiorka, D Weisdorf, P Martin et al. Consensus conference on acute GVHD grading. Bone marrow 
transplantation. 1995;15:825-828                                                              


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APPENDIX 4: Chronic GVHD Assessment 
 
Date of Assessment: _________________________              
 
SKIN (Patient History and Exam) 
Score 0 
 
Score 1 
 
Score 2 
 
Score 3 
 
 
Skin 
Score 
0 
1 
2 
3 
No Symptoms 
< 18% BSA with 
disease signs but 
NO sclerotic 
features 
19-50% BSA OR 
involvement with 
superficial sclerotic 
features “not 
hidebound” (able to 
pinch 
>50% BSA OR deep 
sclerotic features 
“hidebound” (unable 
to pinch) OR 
impaired mobility, 
ulceration or severe 
pruritus 
NIH item that scores maximum severity based on: 
→ Percent body surface area involved by erythema OR 
→ Degree of sclerotic features OR 
→ Impaired mobility OR 
→ Ulceration OR 
→ Severe pruritus  
 
Maculopapular rash  
 
 
  
 
Erythroderma  
 
 
Lichen planus-like features 
 
 
  
 
Poikiloderma  
 
 
Papulosquamous lesions or ichthyosis 
  
 
Sclerotic features 
 
 
Hyperpigmentation 
 
 
 
  
 
Pruritus 
 
 
 
Hypopigmentation 
 
 
 
  
 
Hair Involvement 
 
 
Keratosis pilaris 
 
 
 
  
 
Nail Involvement 
 
 
Erythema 
 
 
 
 
 
 
% BSA involved: ________ 
 
 
 
 
 
 
 
 
 
 
 


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*% BSA Reference 
 
 


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cGVHD signs and symptoms seen with NAILS, SCALP, BODY HAIR 
Score 0 
 
Score 1 
 
Score 2 
 
Score 3 
 
 
Check all that apply: 
 
Distinctive (seen in Chronic GVHD, but insufficient alone to establish diagnosis of Chronic 
GVHD) 
Dystrophy 
 
 
 
 
 
 
Longitudinal ridging, splitting, or brittle features 
 
Onycholysis 
 
 
Pterygium unguis 
 
 
 
 
 
Nail loss (usually symmetic; affects most nails) 
 
New onset of scarring or nonscarring scalp   
 
alopecia (after recovery from chemoradiotherapy)  
Scaling, papulosquamous lesions 
 
 
 
 
Other features/common (seen with both Acute and Chronic GVHD) 
Thinning scalp hair, typically patchy, course or dull  
 
(not explained by endocrine or other causes)  
 
Premature gray hair 
 
 
 
 
 
 
Please describe: 
Patient timeline, extent of finding, duration/rapidity of onset, relationship to immunosuppression 
and likely attributability to GVHD 
______________________________________________________________________________
______________________________________________________________________________
________________________________________ 
 
 
 
 
 
 
 


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cGVHD signs and symptoms seen with MOUTH 
Score 0 
 
Score 1 
 
Score 2 
 
Score 3 
 
 
Oral 
Score 
0 
1 
2 
3 
No Symptoms 
Mild symptoms with 
disease signs but not 
limiting oral intake 
significantly 
Moderate symptoms 
with disease signs 
with 
partial 
limitation 
of 
oral 
intake 
Severe 
symptoms 
with disease signs on 
examination 
with 
major limitation of 
oral intake 
 
Oral Exam 
Check all that apply: 
 
Diagnostic (sufficient to establish Chronic GVHD) 
Lichen-type features  
 
 
 
 
Hyperkeratotic plaques 
 
 
 
 
Restriction of mouth opening from sclerosis  
 
 
Distinctive (seen in Chronic GVHD, but insufficient alone to establish diagnosis of Chronic 
GVHD) 
Xerostomia 
 
 
 
 
 
 
Mucocele 
 
 
 
 
 
 
Muscosal atrophy 
 
 
 
 
 
Pseudomembranes 
 
 
 
 
 
Ulcers  
 
 
 
 
 
 
 
Other features/common (seen with both Acute and Chronic GVHD) 
Gingivitis 
 
 
 
 
 
 
Mucositis 
 
 
 
 
 
 
Erythema 
 
 
 
 
 
 
Pain 
 
 
 
 
 
 
 
 
Please describe: 
Patient timeline, extent of finding, duration/rapidity of onset, relationship to immunosuppression 
and likely attributability to GVHD 
______________________________________________________________________________
______________________________________________________________________________
________________________________________ 
 
 
 


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cGVHD signs and symptoms seen with EYES 
Score 0 
 
Score 1 
 
Score 2 
 
Score 3 
 
 
Eye 
Score 
0 
1 
2 
3 
No Symptoms 
Mild dry eye symptoms 
not affecting ADL 
(requiring eye drops 
<3x per day) OR 
asymptomatic signs of 
keratoconjunctivitis 
sicca 
Moderate dry eye 
symptoms partially 
affecting ADL 
(requiring eye drops 
>3x per day or 
punctal plugs) 
without vision 
impairment 
Severe dry eye 
symptoms 
significantly affecting 
ADL (special 
eyewear to relieve 
pain) OR unable to 
work because of 
ocular symptoms OR 
loss of vision cause 
by 
keratoconjunctivitis 
sicca 
 
Nuances for a patient using drops < 3 x per day: 
  But wearing a Boston Scleral lens → Score 3 
  But had punctal plugs placed  7 days → Score 2 
  But had plugs placed a month ago →Score 1 
 
Check all that apply: 
 
Distinctive (seen in Chronic GVHD, but insufficient alone to establish diagnosis of Chronic 
GVHD) 
New onset dry, gritty, or painful eyes 
 
 
Cicatricial conjunctivitis 
 
 
 
 
Keratoconjunctivitis sicca 
 
 
 
 
Confluent areas of punctate keratopathy 
 
 
 
 
Other features/common (seen with both Acute and Chronic GVHD) 
Photophobia  
 
 
 
 
 
Periorbital hyperpigmentation 
 
 
 
Blepharitis (erythema of the eyelids with edema) 
 
 
Please describe: 
Patient timeline, extent of finding, duration/rapidity of onset, relationship to immunosuppression 
and likely attributability to GVHD 
______________________________________________________________________________
______________________________________________________________________________
________________________________________ 
 
 
 


Amendment 11: Version 09/06/18 
 
Page 83 of 96 
cGVHD signs and symptoms seen with GI TRACT 
Score 0 
 
Score 1 
 
Score 2 
 
Score 3 
 
 
GI Tract 
Score 
0 
1 
2 
3 
No Symptoms 
Symptoms such as 
dysphagia, anorexia, 
nausea, 
abdominal 
pain 
or 
diarrhea 
without 
significant 
weight loss (<5%) 
Symptoms with mild 
to moderate weight 
loss (5-15%) 
Symptoms associated 
with 
significant 
weight loss >15%, 
requires 
nutritional 
supplement for most 
calorie 
needs 
OR 
esophageal dilation  
 
Check all that apply: 
 
Diagnostic (sufficient to establish Chronic GVHD) 
Esophageal web 
 
 
 
 
 
Strictures or stenosis in the upper to mid third 
 
of the esophagus 
 
 
 
 
 
 
Other features/common (seen with both Acute and Chronic GVHD) 
Exocrine – pancreatic insufficiency  
 
 
Anorexia 
 
 
 
 
 
 
Nausea 
 
 
 
 
 
 
Vomiting 
 
 
 
 
 
 
Diarrhea 
 
 
 
 
 
 
Weight loss 
 
 
 
 
 
 
Failure to thrive (infants and children) 
 
 
 
Please describe: 
Patient timeline, extent of finding, duration/rapidity of onset, relationship to immunosuppression 
and likely attributability to GVHD 
______________________________________________________________________________
______________________________________________________________________________
________________________________________ 
 
 
 


Amendment 11: Version 09/06/18 
 
Page 84 of 96 
cGVHD signs and symptoms seen with LIVER 
Score 0 
 
Score 1 
 
Score 2 
 
Score 3 
 
 
Liver 
Score 
0 
1 
2 
3 
Normal LFT 
Elevated Bilirubin, 
AP, AST or ALT 
<2x ULN 
Bilirubin >3 mg/dl or 
Bilirubin, 
enzymes 
2-5 x ULN 
Bilirubin or enzymes 
>5 x ULN 
 
Check all that apply: 
 
Other features/common (seen with both Acute and Chronic GVHD) 
Total Bilirubin, alkaline phosphatase  
 >2x upper limit of normal 
 
 
 
 
ALT or AST > 2 x upper limit of normal 
 
 
 
Please describe: 
Patient timeline, extent of finding, duration/rapidity of onset, relationship to immunosuppression 
and likely attributability to GVHD 
______________________________________________________________________________
______________________________________________________________________________
________________________________________ 
 
 


Amendment 11: Version 09/06/18 
 
Page 85 of 96 
 
cGVHD signs and symptoms seen with LUNGS 
Score 0 
 
Score 1 
 
Score 2 
 
Score 3 
 
 
Lung 
Score 
0 
1 
2 
3 
No Symptoms 
 
FEV1 > 80%   OR 
LFS = 2 
Mild 
symptoms 
(shortness of breath 
after 
climbing 
1 
flight of steps) 
FEV1 60-79%   OR 
LFS 3-5 
Moderate symptoms 
(shortness of breath 
after walking on flat 
ground) 
FEV1 40-59%   OR 
LFS 6-9 
Severe 
symptoms 
(shortness 
at 
rest 
requiring O2) 
FEV1 >39%   OR 
LFS 10-12 
 
FEV1:______  
 
Not done
 
 
NA
 
DLCO: ______ 
 
Not done
 
 
NA
 
LFS: ________ 
 
Not done
 
 
NA
 
 
Check all that apply: 
 
Diagnostic (sufficient to establish Chronic GVHD) 
Bronchiolitis obliterans diagnosed with lung biopsy 
 
 
Distinctive (seen in Chronic GVHD, but insufficient alone to establish diagnosis of Chronic 
GVHD) 
Bronchiolitis obliterans diagnosed with PFTs 
and radiology  
 
 
 
 
 
 
Other features/common (seen with both Acute and Chronic GVHD) 
Bronchiolitis obliterans with organizing pneumonia (BOOP) 
 
 
Please describe: 
Patient timeline, extent of finding, duration/rapidity of onset, relationship to immunosuppression 
and likely attributability to GVHD 
______________________________________________________________________________
______________________________________________________________________________
________________________________________ 
 
 


Amendment 11: Version 09/06/18 
 
Page 86 of 96 
 
cGVHD signs and symptoms seen with MUSCLE, FASCIA, JOINTS 
Score 0 
 
Score 1 
 
Score 2 
 
Score 3 
 
 
Muscles, 
Fascia 
& 
Joints 
Score 
0 
1 
2 
3 
No Symptoms 
 
 
Mild tightness of 
arms or legs, normal 
or mild decreased 
range 
of 
motion 
(ROM) AND not 
affecting ADL  
Tightness of arms or 
legs 
OR 
joint 
contractures, 
erythema 
thought 
due 
to 
fasciitis, 
moderate 
decrease 
ROM AND mild to 
moderate limitation 
to ADL 
Contractures WITH 
significant decrease of 
ROM 
AND 
significant limitation 
of ADL (unable to tie 
shoes, button shirt, 
dress self etc.) 
 
Check all that apply: 
 
Diagnostic (sufficient to establish Chronic GVHD) 
 
Fasciitis 
 
 
 
 
 
 
Joint stiffness or contracture secondary to sclerosis 
 
 
Distinctive (seen in Chronic GVHD, but insufficient alone to establish diagnosis of Chronic 
GVHD) 
Myositis or polymyositis 
 
 
 
 
 
Other features/common (seen with both Acute and Chronic GVHD) 
Edema  
 
 
 
 
 
 
Muscle cramps 
 
 
 
 
 
Arthralgia or arthritis  
 
 
 
 
 
Please describe: 
Patient timeline, extent of finding, duration/rapidity of onset, relationship to immunosuppression 
and likely attributability to GVHD 
______________________________________________________________________________
______________________________________________________________________________
________________________________________ 
 
 
 


Amendment 11: Version 09/06/18 
 
Page 87 of 96 
cGVHD signs and symptoms seen with GENITALIA 
Score 0 
 
Score 1 
 
Score 2 
 
Score 3 
 
 
Genitalia 
Score 
0 
1 
2 
3 
No Symptoms 
 
 
Symptomatic with 
mild signs on exam 
AND no effect on 
coitus and minimal 
discomfort with 
gynecologic exam 
Symptomatic with 
moderate signs on 
exam AND with 
mild dyspareunia 
OR with discomfort 
with gynecologic 
exam 
Symptomatic WITH 
advanced sign 
(stricture, labial, 
agglutination, or 
severe ulceration) 
AND severe pain 
with coitus or 
inability to insert 
vaginal speculum  
 
Check all that apply: 
 
Diagnostic (sufficient to establish Chronic GVHD) 
Lichen planus-like features 
 
 
 
 
Vaginal scarring or stenosis  
 
 
 
 
Distinctive (seen in Chronic GVHD, but insufficient alone to establish diagnosis of Chronic 
GVHD) 
Erosion 
 
 
 
 
 
 
Fissures 
 
 
 
 
 
 
Ulcers  
 
 
 
 
 
 
 
 


Amendment 11: Version 09/06/18 
 
Page 88 of 96 
 
Other Indicators 
None 
Mild 
Moderate 
Severe 
Not Assessed 
Ascites (serositis) 
 
 
 
 
 
Myasthenia Gravis 
 
 
 
 
 
Polymyositis 
 
 
 
 
 
Platelets less than 
100,000/ul 
 
 
 
 
 
Pericardial effusion 
 
 
 
 
 
Nephrotic syndrome 
 
 
 
 
 
Cardiomyopathy 
 
 
 
 
 
Cardiac conduction defects 
 
 
 
 
 
Progressive onset 
 
 
 
 
 
Pleural Effusion(s) 
 
 
 
 
 
Peripheral Neuropathy 
 
 
 
 
 
Eosinophilia >500ul 
 
 
 
 
 
Coronary artery 
i
l
t
 
 
 
 
 
 
Other, specify:_________________________________________________ 
 
 


Amendment 11: Version 09/06/18 
 
Page 89 of 96 
 
Does the patient have chronic GVHD?  
Yes 
    No 
    
If yes, specify Severity: 
Mild 
 
Moderate 
  
Severe 
    
 
 
 NIH CGVHD Global Severity Category reflects overall disability 
SEVERITY 
ORGAN SCORE 
NO. OF ORGANS 
Mild 
All 1 (0 for Lung) 
1-2 
Moderate 
All 1 (0 for Lung) 
3 or more 
At least one 2 (1 for 
Lung) 
1-2 
Severe 
At least one 3 (2 for 
Lung) 
1 or more 
 
Were there additional exams performed?   Yes 
 No 
    
If yes, check all that apply: 
Procedure 
Date 
Result 
Biopsy               
 
 
CT Scan             
 
 
 
 
 
MRI                   
 
 
 
 
 
Photo                
 
 
Schirmer Eye Test    
 
 
 PFT 
 
 
 LFT 
 
 
Ultrasound/Echocardiogram    
 
X-Ray                 
 
 
Other:__________________   
 
 
Signature: ________________________________   Date: __________________ 
 
 


Amendment 11: Version 09/06/18 
 
Page 90 of 96 
 
APPENDIX 5: FACT-BMT (Version 4) 
Below is a list of statements that other people with your illness have said are important. Please 
circle or mark one number per line to indicate your response as it applies to the past 7 
days. 
 
 
PHYSICAL WELL-BEING 
 
Not 
at all 
A little 
bit 
Some-
what 
Quite
a bit 
Very 
much 
GP1 
I have a lack of 
energy
 .......................................................................................... 
0 
1 
2 
3 
4 
GP2 
I have 
nausea
 .......................................................................................... 
0 
1 
2 
3 
4 
GP3 
Because of my physical condition, I have trouble 
meeting the needs of my 
family
 .......................................................................................... 
 
0 
 
1 
 
2 
 
3 
 
4 
GP4 
I have 
pain
 .......................................................................................... 
0 
1 
2 
3 
4 
GP5 
I am bothered by side effects of 
treatment
 .......................................................................................... 
0 
1 
2 
3 
4 
GP6 
I feel 
ill
 .......................................................................................... 
0 
1 
2 
3 
4 
GP7 
I am forced to spend time in 
bed
0 
1 
2 
3 
4 
 
 
SOCIAL/FAMILY WELL-BEING 
 
Not 
at all 
A little 
bit 
Some-
what 
Quite
a bit 
Very 
much 
GS1 
I feel close to my 
friends
 .......................................................................................... 
0 
1 
2 
3 
4 


Amendment 11: Version 09/06/18 
 
Page 91 of 96 
 
 
Please circle or mark one number per line to indicate your response as it applies to the past 
7 days. 
 
 
EMOTIONAL WELL-BEING 
Not 
at all 
A little 
bit 
Some-
what 
Quite
a bit 
Very 
much 
GE1 
I feel 
sad
 .......................................................................................... 
0 
1 
2 
3 
4 
GE2 
I am satisfied with how I am coping with my 
illness
 .......................................................................................... 
0 
1 
2 
3 
4 
GE3 
I am losing hope in the fight against my 
illness
 .......................................................................................... 
0 
1 
2 
3 
4 
GS2 
I get emotional support from my 
family
 .......................................................................................... 
0 
1 
2 
3 
4 
GS3 
I get support from my 
friends
 .......................................................................................... 
0 
1 
2 
3 
4 
GS4 
My family has accepted my 
illness
 .......................................................................................... 
0 
1 
2 
3 
4 
GS5 
I am satisfied with family communication about my 
illness
 .......................................................................................... 
 
0 
 
1 
 
2 
 
3 
 
4 
GS6 
I feel close to my partner (or the person who is my main 
support)
 .......................................................................................... 
 
0 
 
1 
 
2 
 
3 
 
4 
Q1 
Regardless of your current level of sexual activity, please 
answer the following question. If you prefer not to answer it, 
please mark this box           and go to the next section. 
 
 
 
 
 
GS7 
I am satisfied with my sex 
life
0 
1 
2 
3 
4 


Amendment 11: Version 09/06/18 
 
Page 92 of 96 
GE4 
I feel 
nervous
 .......................................................................................... 
0 
1 
2 
3 
4 
GE5 
I worry about 
dying
 .......................................................................................... 
0 
1 
2 
3 
4 
GE6 
I worry that my condition will get 
0 
1 
2 
3 
4 
 
 
 
FUNCTIONAL WELL-BEING 
 
Not 
at all 
A little 
bit 
Some-
what 
Quite
a bit 
Very 
much 
 
GF1 
I am able to work (include work at 
home)
 .......................................................................................... 
0 
1 
2 
3 
4 
GF2 
My work (include work at home) is 
fulfilling
 .......................................................................................... 
0 
1 
2 
3 
4 
GF3 
I am able to enjoy 
life
 .......................................................................................... 
0 
1 
2 
3 
4 
GF4 
I have accepted my 
illness
 .......................................................................................... 
0 
1 
2 
3 
4 
GF5 
I am sleeping 
well
 .......................................................................................... 
0 
1 
2 
3 
4 
GF6 
I am enjoying the things I usually do for 
fun
 .......................................................................................... 
0 
1 
2 
3 
4 
GF7 
I am content with the quality of my life right 
0 
1 
2 
3 
4 
 
 
 


Amendment 11: Version 09/06/18 
 
Page 93 of 96 
Please circle or mark one number per line to indicate your response as it applies to the past 
7 days. 
 
 
ADDITIONAL CONCERNS 
 
Not 
at all 
A little 
bit 
Some-
what 
Quite 
a bit 
Very 
much 
BMT1 
I am concerned about keeping my job (include work at 
home)
 
0 
 
1 
 
2 
 
3 
 
4 
BMT2 
I feel distant from other 
people
0 
1 
2 
3 
4 
BMT3 
I worry that the transplant will not 
work
0 
1 
2 
3 
4 
BMT4 
The effects of treatment are worse than I had 
imagined
0 
1 
2 
3 
4 
C6 
I have a good 
appetite
0 
1 
2 
3 
4 
C7 
I like the appearance of my 
body
0 
1 
2 
3 
4 
BMT5 
I am able to get around by 
myself
0 
1 
2 
3 
4 
BMT6 
I get tired 
easily
0 
1 
2 
3 
4 
BL4 
I am interested in 
sex
0 
1 
2 
3 
4 
BMT7 
I have concerns about my ability to have 
children
0 
1 
2 
3 
4 
BMT8 
I have confidence in my 
nurse(s)
0 
1 
2 
3 
4 
BMT9 
I regret having the bone marrow 
transplant
0 
1 
2 
3 
4 
BMT10 
I can remember 
things
0 
1 
2 
3 
4 
Br1 
I am able to 
concentrate
0 
1 
2 
3 
4 
BMT11 
I have frequent 
colds/infections
0 
1 
2 
3 
4 
BMT12 
My eyesight is 
blurry
0 
1 
2 
3 
4 
BMT13 
I am bothered by a change in the way food 
tastes
0 
1 
2 
3 
4 
BMT14 
I have 
tremors
0 
1 
2 
3 
4 
B1 
I have been short of 
breath
0 
1 
2 
3 
4 
BMT15 
I am bothered by skin problems (e.g., rash, 
itching)
0 
1 
2 
3 
4 
BMT16 
I have trouble with my 
bowels
0 
1 
2 
3 
4 
BMT17 
My illness is a personal hardship for my close family 
members
 
0 
 
1 
 
2 
 
3 
 
4 
BMT18 
The cost of my treatment is a burden on me or my 
family
..........................................................................................
 
0 
 
1 
 
2 
 
3 
 
4 


Amendment 11: Version 09/06/18 
 
Page 94 of 96 
APPENDIX 6: Human Activity Profile 
This is a list of common physical activities.  For each activity, check whether you currently engage in it, no 
longer engage in it, or have never engaged in it.  The best way to decide is to ask yourself whether you 
would engage in the activity today if you had the opportunity.  Please read the instructions and then 
complete the items as accurately as you can. 
 
Patient Initials:   
 
 
 
 
 
 
Study ID#:   
 
 
 
 
 
Still 
Doing 
Stopped 
Doing 
Never 
Did 
1. Getting in or out of chairs or bed (without assistance) 
 
 
 
2. Listening to the radio 
 
 
 
3. Reading books, magazines, or newspapers 
 
 
 
4. Writing (letters, notes) 
 
 
 
5. Working at a desk or table 
 
 
 
6. Standing (for more than 1 minute) 
 
 
 
7. Standing (for more than 5 minutes) 
 
 
 
8. Dressing or undressing (without assistance) 
 
 
 
9. Getting clothes from drawers or closets 
 
 
 
10. Getting in or out of a car (without assistance) 
 
 
 
11. Dining at a restaurant 
 
 
 
12. Playing cards/table games 
 
 
 
13. Taking a bath (no assistance needed) 
 
 
 
14. Putting on shoes, stockings, or socks (no rest or break needed) 
 
 
 
15. Attending a movie, play, church event, or sports activity 
 
 
 
16. Walking 30 yards (27 meters) 
 
 
 
17. Walking 30 yards (nonstop) 
 
 
 
18. Dressing/undressing (no rest or break needed) 
 
 
 
19. Using public transportation or driving a car (99 miles or less) 
 
 
 
20. Using public transportation or driving a car (100 miles or more) 
 
 
 
21. Cooking your own meals 
 
 
 
22. Washing or drying dishes 
 
 
 
23. Putting groceries on shelves 
 
 
 
24. Ironing or folding clothes 
 
 
 
25. Dusting/polishing furniture or polishing a car 
 
 
 
26. Showering 
 
 
 
27. Climbing 6 steps 
 
 
 
28. Climbing 6 steps (nonstop) 
 
 
 
29. Climbing 9 steps 
 
 
 
30. Climbing 12 steps 
 
 
 
31. Walking ½ block on level ground 
 
 
 
32. Walking ½ block on level ground (nonstop) 
 
 
 
33. Making a bed (not changing sheets) 
 
 
 
34. Cleaning windows 
 
 
 
35. Kneeling, squatting to do light work 
 
 
 
36. Carrying a light load of groceries 
 
 
 
37. Climbing 9 steps (nonstop) 
 
 
 
38. Climbing 12 steps (nonstop) 
 
 
 
39. Walking ½ block uphill 
 
 
 
40. Walking ½ block uphill (nonstop) 
 
 
 
41. Shopping (by yourself) 
 
 
 
42. Washing clothes (by yourself) 
 
 
 
43. Walking 1 block on level ground 
 
 
 
44. Walking 2 blocks on level ground 
 
 
 


Amendment 11: Version 09/06/18 
 
Page 95 of 96 
 
Still 
Doing 
Stopped 
Doing 
Never 
Did 
45. Walking 1 block on level ground (nonstop) 
 
 
 
46. Walking 2 blocks on level ground (nonstop) 
 
 
 
47. Scrubbing (floors, walls, or cars) 
 
 
 
48. Making a bed (changing sheets) 
 
 
 
49. Sweeping 
 
 
 
50. Sweeping (5 minutes nonstop) 
 
 
 
51. Carrying a large suitcase or bowling (one game) 
 
 
 
52. Vacuuming carpets 
 
 
 
53. Vacuuming carpets (5 minutes nonstop) 
 
 
 
54. Painting (interior/exterior) 
 
 
 
55. Walking 6 blocks on level ground 
 
 
 
56. Walking 6 blocks on level ground (nonstop) 
 
 
 
57. Carrying out the garbage 
 
 
 
58. Carrying a heavy load of groceries 
 
 
 
59. Climbing 24 steps 
 
 
 
60. Climbing 36 steps 
 
 
 
61. Climbing 24 steps (nonstop) 
 
 
 
62. Climbing 36 steps (nonstop) 
 
 
 
63. Walking 1 mile 
 
 
 
64. Walking 1 mile (nonstop) 
 
 
 
65. Running 110 yards (100 meters) or playing softball/baseball 
 
 
 
66. Dancing (social) 
 
 
 
67. Doing calisthenics or aerobic dancing (5 minutes nonstop) 
 
 
 
68. Mowing the lawn (power mower but not a riding mower) 
 
 
 
69. Walking 2 miles 
 
 
 
70. Walking 2 miles (nonstop) 
 
 
 
71. Climbing 50 steps (2 ½ floors) 
 
 
 
72. Shoveling, digging, or spading 
 
 
 
73. Shoveling, digging, or spading (5 minutes nonstop) 
 
 
 
74. Climbing 50 steps (nonstop) 
 
 
 
75. Walking 3 miles or golfing 18 holes without a riding cart 
 
 
 
76. Walking 3 miles (nonstop) 
 
 
 
77. Swimming 25 yards 
 
 
 
78. Swimming 25 yards (nonstop) 
 
 
 
79. Bicycling 1 mile 
 
 
 
80. Bicycling 2 miles 
 
 
 
81. Bicycling 1 mile (nonstop) 
 
 
 
82. Bicycling 2 miles (nonstop) 
 
 
 
83. Running or jogging ¼ mile 
 
 
 
84. Running or jogging ½ mile 
 
 
 
85. Playing tennis or racquetball 
 
 
 
86. Playing basketball/soccer (game play) 
 
 
 
87. Running or jogging ¼ mile (nonstop) 
 
 
 
88. Running or jogging ½ mile (nonstop) 
 
 
 
89. Running or jogging 1 mile 
 
 
 
90. Running or jogging 2 miles 
 
 
 
91. Running or jogging 3 miles 
 
 
 
92. Running or jogging 1 mile in 12 minutes or less 
 
 
 
93. Running or jogging 2 miles in 20 minutes or less 
 
 
 
94. Running or jogging 3 miles in 30 minutes or less 
 
 
 
Fix, A.J., and D.M. Daughton, Human Activity Profile Professional Manual. Odessa, Fla: Pyschololgical 
Assessment Resources, Inc; 1988. 


Amendment 11: Version 09/06/18 
 
Page 96 of 96 
 
APPENDIX 7: Immunosuppressive Medication Questionnaire 
 
This form should be completed by the patients RN, NP, or MD 
 
Date of Assessment: _________________________              
 
Current Daily Tacrolimus Dose____________________________mg 
 
 
List any other medication administered for immunosupression within the past 30 days 
 
Drug__________________________ Daily Dose___________________________ 
 
Start Date______________________ Stop Date__________________________ 
 
 
Drug__________________________ Daily Dose___________________________ 
 
Start Date______________________ Stop Date__________________________ 
 
 
Drug__________________________ Daily Dose___________________________ 
 
Start Date______________________ Stop Date__________________________ 
 
 
Add additional lines below as needed 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Printed Name_____________________________ Signature_____________________________