Conditions: Myelodysplastic Syndromes (MDS) Acute Myelogenous Leukemia (AML)
Keywords: 5'-Azacitidine Stem cell transplant 13-192
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 32
Healthy volunteers: no

Brief summary:
The purpose of this study is to learn if 5'-Azacitidine will help to lower the risk of the disease coming back after a stem cell transplant in patients with MDS and AML. This study will also be looking at the side effects of this medicine.

5'-Azacitidine is an FDA approved drug for treatment of MDS and AML, as well as patients whose disease came back after transplant, where it helped going into remission. It is unclear if 5'-Azacitidine can prevent the disease from coming back after transplant. This study will help show if getting 5'-Azacitidine soon after transplant can lower the risk of your disease coming back.

Detailed description:

Arms:
arm 1: This is a single arm phase II trial to assess the efficacy and confirm the safety of maintenance therapy with 5'-azacitadine compared to historical control after TCD allogeneic hematopoietic stem cell transplant for patients with MDS and AML who are at high risk of relapse.

Interventions: intervention 1: low dose 5'-azacitidine
Intervention descriptions: intervention 1: 5'-azacitadine will be given at a low dose of 32mg/m2 S.C for 5 days every 28 days (a cycle). Dose de-escalation will be permitted for hematologic and non- hematologic toxicities. Patients will start taking the study drug between days 60-120 post TCD allogeneic hematopoietic stem cell transplant and up to a year post-transplant or until there is a toxicity that requires cessation of therapy. Therefore patients will get between 8-10 cycles. Patients who come off-study for reasons unrelated to toxicities before completing 4 cycles will be replaced Since most cases of relapse occur early post transplant, in the first year, this is the most appropriate time to intervene. Treatment will start as soon as possible.

Protocol document:
Prot_SAP_000.pdf: 
Memorial Sloan Kettering Cancer Center 
IRB Number: 13-192 A(11) 
Approval date: 13-May-2019 
Page 1 of 37 
 
 
 
 
 
 
 
 
A single arm phase II trial of maintenance  low dose 5’-azacitidine post T cell 
depleted allogeneic stem cell transplantation for patients with Myelodysplastic 
Syndrome and Acute Myelogenous Leukemia with high risk for post-transplant 
relapse 
 
PROTOCOL FACE PAGE FOR 
MSKCC THERAPEUTIC/DIAGNOSTIC   PROTOCOL 
 
 
Principal 
Investigator/Department: 
Roni Tamari, M.D. 
Medicine 
Co-Principal 
Investigator(s)/Department: 
Maria Cancio, M.D. 
Medicine 
Investigator(s)/Department: 
Juliet Barker, M.B.B.S 
Christina Cho, M.D. 
David Chung, M.D., Ph.D. 
Parastoo Dahi, M.D. Sergio 
Giralt, M.D. Boglarka 
Gyurkocza, M.D.           
Alan Hanash, M.D., Ph.D. 
Katherine Hsu, M.D., Ph.D. 
Ann Jakubowski, M.D., Ph.D. 
Scott James, M.D., Ph.D. 
Oscar Lahoud, M.D. 
Heather Landau, M.D. 
Esperanza Papadopoulos, M.D. 
Jonathan Peled, M.D. 
Miguel-Angel  Perales, M.D. 
Ioannis Politikos, M.D. 
Doris Ponce, M.D. 
Craig Sauter, M.D. 
Michael Scordo, M.D. 
Brian Shaffer, M.D. 
Gunjan Shah, M.D. 
Melody Smith, M.D. 
Marcel van den Brink, M.D., Ph.D. 
James Young, M.D. 
Virginia Klimek, M.D. 
Martin Tallman, M.D. 
Farid Boulad, M.D. 
Kevin Curran, M.D 
Nancy Kernan, M.D. 
Medicine 
Medicine 
Medicine 
Medicine 
Medicine 
Medicine 
Medicine 
Medicine 
Medicine 
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Medicine 
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Medicine 
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Medicine 
Medicine 
Medicine 
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Medicine 
Medicine 
Medicine 
Medicine 
Medicine 
Medicine 
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Pediatrics 
Pediatrics 
Pediatrics 


Memorial Sloan Kettering Cancer Center 
IRB Number: 13-192 A(11) 
Approval date: 13-May-2019 
Page 2 of 37 
 
 
 
 
 
 
Richard O’Reilly, M.D. 
Susan Prockop, M.D. 
Andromachi Scaradavou, M.D 
Barbara Spitzer, M.D. 
Sean Devlin, Ph.D. 
Audrey Hamilton, M.D. 
Marina Shcherba, D.O. 
Paul Hamlin, MD 
Matthew Matasar, M.D. 
Arlyn Apollo, M.D. 
Pamela Drullinsky,  M.D. 
Zoe Goldberg, M.D. 
Kenneth Ng, M.D. 
Tiffany Troso-Sandoval, M.D. 
Philip Caron, M.D., Ph.D. 
Hani Hassoun, M.D. 
Andrew Zelenetz, M.D. 
Michael Mauro, M.D. 
Shani Irby, NP 
Megan Heavey Scott, NP 
Abigail Cohen, NP 
Nicole Lestrange, NP 
Chelsea Mintz, NP 
Carter Hibbs, NP 
Allison Tucker, NP 
Tara Wolff, NP 
Stefanie Kelly, NP 
Shelia Kenny, NP 
Elaina Preston, PA 
Naomi Cazeau, NP 
Christina Kiss 
Courtney McElrath, NP 
Meighan Palazzo, NP 
Melissa Cortes, NP 
Marci Andrejko, NP 
Jason Batalha, NP 
Azadeh Namakydoust, MD 
Colette Owens , MD 
Neha Korde , MD 
Regina Byrne, NP 
Ildefonso Rodriguez-Rivera, MD 
Anita Kumar, MD 
Janet Cogswell, RN 
Lisa Sarmasti, RN 
Pediatrics 
Pediatrics 
Pediatrics 
Pediatrics 
Epidemiology and Biostatistics 
Medicine 
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Memorial Sloan Kettering Cancer Center 
IRB Number: 13-192 A(11) 
Approval date: 13-May-2019 
Page 3 of 37 
 
 
 
 
 
 
Jennifer Howgate-Klingaman,  RN 
Geri Arnell, RN 
Megan Stasi, RN 
Sherie Mar-Chaim, RN 
Nursing 
Nursing 
Nursing 
Nursing 
Maureen Kennedy, RN 
Nursing 
Nicole Heinz, RN 
Nursing 
Raylene Langish, RN 
Nursing 
Jessica Marchisotto, RN 
Nursing 
Irene Kelly, RN 
Nursing 
Gloria Wasilewski, RN 
Nursing 
Natasia Rodriguez, RN 
Nursing 
Consenting 
Juliet Barker, M.B.B.S 
Medicine 
Professional(s)/Department: 
Christina Cho, M.D. 
Medicine 
David Chung, M.D., Ph.D. 
Medicine 
Parastoo Dahi, M.D. 
Medicine 
Sergio Giralt, M.D. 
Medicine 
Boglarka Gyurkocza, M.D. 
Medicine 
Alan Hanash, M.D., Ph.D 
Medicine 
Katherine Hsu, M.D., Ph.D. 
Medicine 
Ann Jakubowski, M.D., Ph.D. 
Medicine 
Scott James, M.D., Ph.D. 
Medicine 
Oscar Lahoud, M.D. 
Medicine 
Heather Landau, M.D. 
Medicine 
Esperanza Papadopoulos, M.D. 
Medicine 
Jonathan Peled, M.D. 
Medicine 
Miguel-Angel  Perales, M.D. 
Medicine 
Ioannis Politikos, M.D. 
Medicine 
Doris Ponce, M.D. 
Medicine 
Craig Sauter, M.D. 
Medicine 
Michael Scordo, M.D. 
Medicine 
Brian Shaffer, M.D. 
Medicine 
Gunjan Shah, M.D. 
Medicine 
Melody Smith, M.D. 
Roni Tamari, M.D. 
Medicine 
Medicine 
Marcel van den Brink, M.D., Ph.D. 
Medicine 
James Young, M.D 
Medicine 
Virginia Klimek, M.D. 
Medicine 
Martin Tallman, M.D. 
Medicine 
Farid Boulad, M.D. 
Pediatrics 
Kevin Curran, M.D 
Pediatrics 
Nancy Kernan, M.D. 
Richard O’Reilly, M.D. 
Pediatrics 
Pediatrics 
Susan Prockop, M.D. 
Pediatrics 
Andromachi Scaradavou, M.D. 
Pediatrics 


Memorial Sloan Kettering Cancer Center 
IRB Number: 13-192 A(11) 
Approval date: 13-May-2019 
Page 4 of 37 
 
 
 
 
 
 
Maria Cancio, M.D. 
Pediatrics 
Barbara Spitzer, M.D. 
Pediatrics 
Audrey Hamilton, M.D. 
Medicine 
Paul Hamlin, M.D. 
Medicine 
Marina Shcherba, DO 
Medicine 
Matthew Matasar, M.D. 
Medicine 
Arlyn Apollo, M.D. 
Medicine 
Pamela Drullinsky, M.D. 
Medicine 
Zoe Goldberg, M.D. 
Medicine 
Kenneth Ng, M.D. 
Medicine 
Tiffany Troso-Sandoval, M.D. 
Medicine 
Philip Caron, M.D., Ph.D. 
Medicine 
Hani Hassoun, M.D. 
Medicine 
Andrew Zelenetz, M.D. 
Medicine 
Michael Mauro, M.D. 
Medicine 
Azadeh Namakydoust, MD 
Medicine 
Colette Owens, MD 
Medicine 
Neha Korde, MD 
Medicine 
Ildefonso Rodriguez-Rivera, MD 
Medicine 
Anita Kumar, MD 
Medicine 
 
 
Please Note: A Consenting Professional must have completed the mandatory Human 
Subjects Education and Certification Program. 
 
OneMSK  Sites 
Manhattan 
All Protocol Activities 
Basking Ridge 
Consent and Followup 
Westchester 
Consent and Followup 
Nassau 
Consent and Followup 
Commack 
Consent and Followup 
Monmouth 
Consent and Followup 


Memorial Sloan Kettering Cancer Center 
IRB Number: 13-192 A(11) 
Approval date: 13-May-2019 
Page 5 of 37 
 
 
 
 
 
 
 
 
 
 
Memorial Sloan-Kettering Cancer Center 
1275 York Avenue 
New York, New York 10065 
Bergen 
Consent and Followup 


Memorial Sloan Kettering Cancer Center 
IRB Number: 13-192 A(11) 
Approval date: 13-May-2019 
Page 6 of 37 
 
 
 
 
Table of Contents 
1.0 
PROTOCOL SUMMARY AND/OR SCHEMA ........................................................................ 7 
2.0 
OBJECTIVES AND SCIENTIFIC AIMS ................................................................................. 8 
3.0 
BACKGROUND AND RATIONALE ....................................................................................... 9 
4.0 
OVERVIEW OF STUDY DESIGN/INTERVENTION ................................................................. 16 
4.1 
Design .................................................................................................................................. 16 
4.2 
Intervention .............................................................................................................................. 16 
5.0 
THERAPEUTIC/DIAGNOSTIC AGENTS ............................................................................. 17 
6.0 
CRITERIA FOR SUBJECT ELIGIBILITY ................................................................................. 18 
6.1 
Subject Inclusion Criteria ........................................................................................................ 18 
6.2 
Subject Exclusion Criteria ....................................................................................................... 18 
7.0 
RECRUITMENT PLAN ......................................................................................................................... 19 
8.0 
PRETREATMENT EVALUATION ....................................................................................... 19 
9.0 
TREATMENT/INTERVENTION PLAN ................................................................................. 20 
10.0 
EVALUATION DURING TREATMENT/INTERVENTION ..................................................... 17 
11.0 
TOXICITIES/SIDE EFFECTS .................................................................................................... 23 
12.0 
CRITERIA FOR THERAPEUTIC RESPONSE/OUTCOME ASSESSMENT ......................... 25 
13.0 
CRITERIA FOR REMOVAL FROM STUDY ........................................................................ 26 
14.0 
BIOSTATISTICS ................................................................................................................. 26 
15.0 
RESEARCH PARTICIPANT REGISTRATION AND RANDOMIZATION PROCEDURES 28 
15.1  Research Participant Registration .......................................................................................... 28 
16.0 
DAT A M ANAGEMENT ISSUES .......................................................................................... 29 
16.1 
Quality Assurance ................................................................................................................ 29 
16.2 
Data and Safety Monitoring .................................................................................................... 29 
17.0 
PROTECTION OF HUMAN SUBJECTS ............................................................................. 30 
17.1 
Privacy .................................................................................................................................. 30 
17.2 
Serious Adverse Event (SAE) Reporting ................................................................................ 30 
18.0 
INFORMED CONSENT PROCEDURES................................................................................... 32 
19.0 
REFERENCES ........................................................................................................................... 33 


Memorial Sloan Kettering Cancer Center 
IRB Number: 13-192 A(11) 
Approval date: 13-May-2019 
Page 7 of 37 
 
 
 
 
 
 
 
 
1.0 
PROTOCOL SUMMARY AND/OR SCHEMA 
 
This is a single arm phase II trial to assess the efficacy of 5’-azacitidine to reduce the high 
relapse rate after T cell depleted (TCD) hematopoietic stem cell transplant (HSCT) from 
histocompatible matched or mismatched related or unrelated donor for patients with 
Myelodysplastic syndrome (MDS) and Acute Myelogenous Leukemia (AML) who are at 
high risk for relapse. Candidates for this trial will include MDS patients who have an 
International  Prognostic Scoring System (IPSS) Intermediate-1 with poor cytogenetics and 
higher IPSS as well as AML patients with high risk cytogenetics and molecular mutations 
who on day +28-30 post T cell depleted transplant and prior to enrollment are in remission 
with no evidence of excess blasts or cytogenetics and/or molecular abnormalities. 
 
Hematopoietic stem cell donors for this trial will include individuals who are 10/10 HLA 
matched and up to two-antigen mismatched at the HLA A, B, C DRB1 and DQB1 loci, as 
defined by high-resolution methods. The stem cell source can be either PBSC or bone 
marrow. 
 
Patients will be prepared for transplantation with a chemotherapy only myeloablative 
regimen or a total body irradiation (TBI)-containing myeloablative regimen. 
 
Maintenance therapy with 5’-azacitidine will be given at a dose of 32 mg/m2/day for 5 
consecutive days subcutaneously, every 28 days (a cycle). This treatment can start 
between 60-120 days  posttransplant depending on patient’s clinical and hematological 
status and will be given for up to a year post transplant. 
 
The primary endpoint of the study is the relapse rate after treatment initiation. Patients will 
also be monitored for regimen related toxicities, incidence of infections, incidence and 
severity of acute and chronic graft versus host disease (GvHD), marrow and T cell 
chimerism and characteristics of immune reconstitution. Correlative studies will look at the 
effects of DNA methylation and relapse risk in response to treatment with 5’-Azacitidine, 
the effect of maintenance 5’-azacitidine on expression of cell markers that are highly 
specific and sensitive for the detection of residual MDS/leukemic cells as well as the effect 
of treatment with 5’-Azacitidine on augmenting GVL by increasing regulatory T cells. 


Memorial Sloan Kettering Cancer Center 
IRB Number: 13-192 A(11) 
Approval date: 13-May-2019 
Page 8 of 37 
 
 
 
 
 
 
 
 
 
 
 
2.1 
OBJECTIVES AND SCIENTIFIC AIMS 
 
The aim of this prospective phase II trial is to evaluate the efficacy and confirm the safety of 5’- 
azacitidine after allogeneic T cell depleted (TCD) hematopoietic stem cell transplant (HSCT) from 
histocompatible matched or mismatched related or unrelated donor in patients with 
Myelodysplastic syndrome (MDS) and Acute Myelogenous Leukemia (AML) who are at high risk 
for relapse. 
 
Primary objective: 
 
To evaluate whether maintenance treatment post transplant with 5-azacitidine can reduce the 
relapse rate over the current standard of care for high-risk MDS and AML patients. 
 
Secondary objectives: 
 
1. To assess overall survival from the time of 5-azacitadine initiation. 
 
2. To confirm the safety and tolerability of low dose 5’-azacitadine by measuring the 
frequency of transfusions, frequency of bleeding, frequency of serious infections, and use 
of G-CSF support. 
 
3. To evaluate graft function and myeloid and T cell chimerism after administration of low 
dose 5’-azacitdine 
 
4. To evaluate the incidence of acute and chronic GvHD following the initiation of 5- 
azacitadine. 
T cell depleted 
transplant 
If marrow prior to 
enrollement  is in 
morphologic  remission+ 
stable counts+ no 
toxicities 
Eligiblility  met for 
enrollment 
5'-azacitidine 
32mg/m2/day,  1-5 
days every 28 days 
BM aspirate +/- 
biopsy for disease 
status and 
correlative  studies 
every 3 months. 


Memorial Sloan Kettering Cancer Center 
IRB Number: 13-192 A(11) 
Approval date: 13-May-2019 
Page 9 of 37 
 
 
 
 
 
 
 
Correlative Objectives: 
 
Correlate treatment with low dose 5’-Azacitidine with: 
 
1. Gene specific methylation in the hematopoietic stem cell (HSC) compartment in CD 34 
cells subpopulations. 
 
2. Recurrence of cytogenetic abnormalities in the hematopoietic stem cell (HSC) 
compartment in CD34 cells subpopulations. 
 
3. Normalization of myeloid progenitor frequencies known to be altered in MDS. 
 
4. Level of regulatory T cells (CD4+CD25-FoxP30). 
 
 
 
 
 
 
 
 
3.0 
BACKGROUND AND RATIONALE 
 
3.1 
Advanced Myelodysplastic  Syndrome and high risk Acute Myelogenous  Leukemia 
 
Myelodysplastic  syndromes (MDS) are hematopoietic stem cell disorders characterized by 
cytopenias with a variable risk of transformation to acute leukemia. The median survival of MDS 
patients with refractory cytopenias without an excess of blasts is about 5 years. The more 
advanced forms of MDS, defined by an increase in the blast count and presence of several 
cytogenetics changes, have much worse prognosis and shorter survival. The median survival for 
patients with refractory anemia with excess of blasts (RAEB-1, blasts 5-9%) is 1.5 years and for 
patients with RAEB-2 (blasts 10-19%) is one year1.  These patients are more likely to develop 
and die from acute myelogenous leukemia (AML). Although a variety of non-transplant options 
are currently being developed and survival has improved, none of these treatments is curative in 
patients with advanced MDS and AML evolved from MDS.2,3,4 
 
AML is also a group of heterogeneous disorders with regard to morphology and chromosome 
aberrations detected in the leukemic cells. Slovak et al in a SWOG/ECOG study looked at the 
correlation between cytogenetic changes at presentation and outcome after therapy for de-novo 
AML and identified 3 prognostic groups based on cytogenetics; low, intermediate and high risk 
with 5 years overall survival of 55%, 38% and 11%, respectively.5  These prognostic differences 
among the three groups remained also after achieving remission, with worse outcome seen in the 


Memorial Sloan Kettering Cancer Center 
IRB Number: 13-192 A(11) 
Approval date: 13-May-2019 
Page 10 of 37 
 
 
 
 
unfavorable group, which is the group that benefited the most from allogeneic hematopoietic stem 
cell transplantation. Although progress has been made in defining prognostic markers for AML, a 
substantial percentage of patients lack a specific cytogenetic abnormality of prognostic 
significance. Recent studies have identified several recurrent somatic mutation in patients with 
AML including mutations in FLT3, NPM1, CEBPA, TETA, ASXL1, IDH1/IDH2,  DNMT3A,  PHF6 
and many more that have prognostic implications for patients with AML, and in specifics to the 
intermediate cytogenetic risk group 6,7,8,9,10,11,12,13. More recently Patel et al reported the prognostic 
relevance of integrated genetic profiling in AML which has a very important impact on 
understanding the biology of the disease, improving prognostic models and affecting therapeutic 
decision making.14 
3.2 
Allogeneic Hematopoietic Stem cell Transplantation for MDS and AML 
 
Allogeneic hematopoietic stem cell transplantation (HSCT) is currently the only curative treatment 
available for patients with myelodysplastic syndrome and high risk acute 
leukemia15,16,17,18,19,20,21,22,23,24,25. However, the success rate in these patients has been hindered   
by two major problems: (i) a high transplant-related mortality secondary to graft-versus-host 
disease (GvHD) and infectious complications and (ii) a high post transplant disease relapse, 
particularly in patients with advanced MDS and AML. 
 
The two main strategies that have been used to reduce GvHD are: (i) Pharmacologic  
manipulation of donor T cells in the patient after administration of the allograft with 
immunosuppressants: calcineurin inhibitors, methotrexate, mycophenolate, monoclonal  
antibodies such as alemtuzumab (anti CD52 antibody –Campath), and polyclonal anti T cell 
antibodies such as antithymocyte globulin. Although the incidence of GvHD has improved by 
combining these agents, this complication still remains a significant problem in HLA matched 
transplant and more notable in non-HLA matched transplants.  (ii) Removal of donor T cells of the 
allograft before infusion to the recipient. This approach also removes the cells that in part mediate 
the graft versus-leukemia-effect and potentially could increase the risk of relapse secondary to 
possible loss of graft versus leukemia effect (GVL). 
 
The MSKCC HSCT program has focused its research on T cell depletion and methods of T cell 
depletion that have been investigated includes: agglutination of T cells with first soybean lectin 
and then sheep red blood cells to deplete the T cells from marrow grafts and CD34 positive 
selection in peripheral blood stem cell grafts. Our initial experience with T cell depletion 
transplants demonstrated significant decrease in the incidence of GvHD even in mismatched 
transplants without increase in the incidence of relapse in patients transplanted in remission or 
with minimal residual disease. In our series of 232 consecutive adult leukemic patients (median 
age 41) transplanted with marrow grafts from HLA-matched siblings depleted of T-cells with 
soybean agglutinin and E rosette depletion, the incidence of grade II-IV GvHD was 3% and the 
incidence of chronic graft vs. host disease was 5% 24.  Similarly, in over 100 recipients of T cell 
depleted unrelated marrow grafts, the incidence of grade II-IV  aGvHD and of chronic GvHD was 
8%. In two recent studies26,27 using ex-vivo TCD with the Miltenyi ClinMACS device in patients 


Memorial Sloan Kettering Cancer Center 
IRB Number: 13-192 A(11) 
Approval date: 13-May-2019 
Page 11 of 37 
 
 
 
 
with hematologic malignancies with favorable and unfavorable risk disease who received a 
matched28 related donor (MRD) or matched unrelated donor (MUD) graft the incidence of GvHD 
was also low (aGvHD-23% and cGvHD- 7% in the MRD and aGvHD- 9% and cGvHD- 29% in the 
MUD). 
 
The second main obstacle to successful allogeneic HSCT is the high relapse rate, particularly in 
patients with advanced MDS and AML, in whom the relapse rate can be as high as 40% in the 
first 2 years post transplant 29,30 and even higher-60%, for patients who are not at remission at 
time of transplant. 31,32 
 
Strategies to reduce relapse rates after allogeneic stem cell transplantation include interventions 
(i) before transplantation, for instance therapy with induction chemotherapy or hypomethylating 
agents 32, to achieve remission prior to transplant, and (ii) post transplant interventions usually 
involving some form of immune manipulation such as withdrawal of immunosuppression, use of 
cytokines in order to enhance the immune response, or infusion of donor lymphocytes (DLI). 
These strategies have been successful at decreasing relapse rates, but on the expense of higher 
rates of graft-versus-host disease. 33,34,35 
 
Our early experience with TCD in MDS established that optimization of the patient’s disease 
status, that is induction of remission prior to transplant, can result in a lower incidence of 
posttransplant relapse.32  The relapse rate at 1-year posttransplant for patients transplanted with 
active disease was 60% whereas patients transplanted in remission was 20%.  Further analysis of 
our early experience with our most recent experience demonstrated that the relapse rate was 
similar whether the patient was conditioned for transplant with a TBI containing preparative 
regimen or a chemotherapy containing preparative regimen combining Busulphan, Melphalan, 
and Fludarabine.  Moreover, recent analysis (unpublished data) of transplant outcomes in children 
and adults who underwent T cell depleted transplant at MSKCC between 1985-2011 for MDS and 
AML demonstrated that poor risk cytogenetics at diagnosis  is a very strong predictor of  relapse 
(figure 1). 
 
Since the focus of the MSKCC transplant program has been T cell depletion, patients with MDS 
and AML have been transplanted with this type of allograft. A few studies have looked at 
preemptive/maintenance intervention post transplant to reduce the risk of relapse, though, to the 
best of our knowledge not in the unique setting of T cell depleted transplant. Despite the common 
perception that TCD transplant is associated with higher risk of relapse, due to loss of GVL effect, 
the relapse rate after T cell depleted myeloablative transplants in our MDS/AML patients in 
remission or second refractory cytopenia phase is lower or at least similar to that seen in 
recipients of T cell replete myeloablative transplants reported by other centers. We therefore 
believe that with our long term experience with TCD transplant for MDS and AML this is an 
optimal setting to evaluate the potential benefit of post transplant intervention to reduce the risk of 
relapse. 


Memorial Sloan Kettering Cancer Center 
IRB Number: 13-192 A(11) 
Approval date: 13-May-2019 
Page 12 of 37 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
P=0.02 
1.0 
0.8 
Good N=88 
Intermediate N=35 
High N=49 
0.6 
CI 
0.4 
0.2 
0.0 
0 
1 
2 
3 
4 
5 
6 
7 
Years following SCT 


Memorial Sloan Kettering Cancer Center 
IRB Number: 13-192 A(11) 
Approval date: 13-May-2019 
Page 13 of 37 
 
 
 
 
 
 
 
3.3 5-Azacitidine and summary of results of trials in patients with MDS and AML 
 
Azacitidine (5-azacytidine) is a chemical analogue of the cytosine nucleoside used in DNA and 
RNA. Azacitidine is thought to induce antineoplastic activity via two mechanisms; inhibition of DNA 
methyltransferase at low doses, causing hypomethylation of DNA, and direct cytotoxicity in 
abnormal hematopoietic cells in the bone marrow through its incorporation into DNA and RNA at 
high doses, resulting in cell death. 
 
Epigenetics changes consist of biochemical modifications to chromatin that do not alter the 
sequence of the DNA itself. The primary epigenetic modifications are DNA methylation and histone 
modification, both of which are potentially reversible. 36,37  DNA methylation plays an important role 
in gene regulation, and aberrant methylation of promoter regions can occur in malignancies, and is 
correlated with gene silencing, particularly of tumor suppressor genes 38. MDS and AML appear to 
be more amenable to hypomethylating agents, compared to solid tumors, though the exact 
mechanism of response in unknown. Currently, two DNA hypomethylating agents have been 
approved for patients with MDS: 5’-azacitadine and Decitabine (5-aza-2’-deoxycitidine). Both drugs 
are DNA methyltransferase (DNMT) inhibitors that have demethylating effects. 
 
Two randomized trials comparing 5’-azacitadine to best supportive and conventional care 
regimens in patients with MDS, demonstrated that 5’-azacitadine was superior in terms of time to 
progression to AML and in overall survival. 2,3 
Only small phase II studies have investigated the use of 5’-azacitadine or decitabine to induce 
remission prior to allogeneic SCT. Lubbert, et al treated 15 patients with MDS or AML at a median 
age of 65 with low dose decitabine before allogeneic SCT 39 and showed that this can be effective 
treatment, which can induce response and even remission prior to allogeneic HSCT. With a 
median of 5 cycles, 33% of the patients achieved a CR and 6% achieved a PR prior to 
transplantation. 
 
Data on use of 5’-Azacitadine in pediatric patients is much more limited; however several studies 
have incorporated 5’-azacitadine in combination with other chemotherapies for pediatric myeloid 
leukemia patients in either the up-front setting at a dose of 100mg/m2 X 4 days  or in relapsed or 
refractory patients at doses of 250-300 mg/m2/day for 2 days with improvement in response rates 
and overall well tolerated, though with high incidence of myelosuppression. 40,4142 
3.4 
Summary of results of phase I-II trials of 5’-azacitadine post allogeneic HSCT in patients 
with MDS/AML 
Risk for relapse based on cytogenetic changes (as per IPSS criteria) at diagnosis in 
172 patients who underwent T cell depleted transplant at MSKCC between 1985- 
2011. 


Memorial Sloan Kettering Cancer Center 
IRB Number: 13-192 A(11) 
Approval date: 13-May-2019 
Page 14 of 37 
 
 
 
 
Several studies have assessed the safety and efficacy of 5’-azacitadine after allogeneic HSCT in 
patients with MDS and AML as salvage therapy at disease recurrence or as a preventive measure 
to reduce the risk of relapse. A retrospective study by Jabbour et al in 17 AML patients who 
received an allogeneic SCT and then 5’-azacitidine at the time of relapse (n=9) or as maintenance 
(n=8) 43 showed a significant response.   5’-azacitadine was given at different doses: 16, 24 and 
40 mg/m2/day for 5 consecutive days every 4 weeks, with median of 8 cycles. Among the patients 
in the relapsed disease group, the response rate was 55% (5/9). The 8 patients who received 5’- 
azacitadine as maintenance treatment remained in CR for median of 17 months (range 14-26 
months), however 37.5% (3/8) relapsed eventually.  The one-year EFS and OS were 55% and 
90%. The majority of patients experienced grade 1-2 hematologic toxicity but no dose reductions 
were required. 
 
In a phase I study by De Lima et al, 45 patients with AML (n=37) and MDS (n=8) received 
different dosages of 5’-azacitadine (8, 16, 24, 32 and 40mg/m2/day for 5 days) for 1-4 cycles. 
44This study showed that 5’-azacitadine is safe and well tolerated after allogeneic SCT, with a 
recommended dose of 32mg/m2/day for 5 days. 
 
The Relaza study in Europe looked at patients with MDS (n=3) and AML (n=17) with evidence of 
MRD by CD34+ donor chimerism <80% without evidence of hematologic relapse who were 
treated with 5’-azacitadine at a dose of 75 mg/m2 /day for 7 days for 4 cycles followed by more 
cycles depending on response. After the initial 4 cycles, 50% of patients had a major response. 
Grade 3-4 hematologic toxicity, mostly neutropenia and thrombocytopenia, resulted in dose 
reduction in 45% of patients.45 
Two main conclusions can be drawn from the above studies: (i) Low dose of 5’-Azacitidine - 32 
mg/m2/day was found to be tolerable in the post transplant setting with relatively safe toxicity 
profile.  (ii) 5’-Azacitidine was effective in inducing remission in patients who relapsed after 
allogeneic HSCT as well as maintaining remission when given for maintenance. Of note is that  
the patients enrolled in these studies were patients at very high risk, as majority were not in CR at 
time of transplantation, which is different from patients undergoing T cell depleted HSCT. 
 
3.5 Mechanism of activity of 5’-azacitidine post allogeneic hematopoietic stem cell transplant 
 
The administration of 5’-azacitidine to recipients of allogeneic HSCT can affect both stem cells 
and T cells, irrespective of origin, donor derived or residual host cells and therefore consolidate 
the effect of the transplant by two different pathways; 
 
(i) 
Eliminating residual leukemic stem cells-  Following myeloablative conditioning, the 
stem cell population will consist of largely donor derived normal hematopoietic stem 
cells and very minimal residual host MDS and AML stem cells. This latter cells are 
the ones shown to be the disease initiating cells and are likely responsible for 
relapse. 46,47,48, 49 Since 5’-azacitidine and other hypomethylating agents have their 
preferential effect on leukemic stem cells as it has been well documented in MDS 


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patients receiving these agents 50,51 we hypothesize that early administration of this 
agent post transplant can act upon the residual MDS and AML HSC and prevent its 
expansion, while awaiting donor derived GVL effect which is a later event. 
 
 
(ii) 
Augmenting GVL effect- Regulatory T cells (Tregs) are known to contribute to the 
maintenance of self-tolerance by regulating inflammatory responses and 
suppression of autoimmunity and GvHD in mouse models. The major population of 
Tregs is naturally occurring Tregs (nTreg). These cells are generated in the thymus 
and defined by CD4+CD25+FOXP3+. Small numbers of Treg can also be generated 
in the periphery from naïve CD4+CD25- T cells by T cell receptor stimulation. 
FOXP3 is a transcription factor exclusively expressed in nTregs. Mutation in FOXP3 
lead to autoimmune disease due to loss of functional nTregs and forced expression 
of FOXP3 in CD4+CD25- T cells induce regulatory properties. Recent reports 
demonstrated that the FoxP3 locus in both humans and mice is unmethylated in 
Tregs while heavily methylated and silenced in CD4+CD25-T cells. Choi et al 
demonstrated in a mouse model that hypomethylating agents (both 5’-azacitidine 
and decitabine) are able to induce FOXP3 expression in CD4+CD25- T cells. 
Interestingly, 5’-azacitidine given to mice after HLA mismatched transplant resulted 
in less GVHD while preserving GVL by peripheral conversion of alloreactive T cells 
into FOXP3+ Tregs and epigenetic modulation of genes downstream of Foxp3 
required for the suppressor function of Tregs. 52 
 
Goodyear et al, have recently shown that the administration of 5’-azacitidine post- 
transplant increased peripheral Tregs and suggested another mechanism of anti- 
leukemic effect, through up regulation of expression of epigenetically silenced tumor 
antigens such as MAGE-A1  (melanoma-associated Ag1) and WT-1 (Wilm tumor 
Ag1).  5’-azacitidine has the capacity to induce a CD8+ T cell response to tumor 
antigens in patients with hematologic malignancies. This effect of 5’-azacitidine on 
Tregs expression was confined to the immediate period after transplant.53 
 
These effects on T cells were observed in recipients of unmodified transplant and 
are unknown in the setting of TCD transplant. When Tregs recovery was studied 
after T cell depleted transplant, in adult patients who received myeloablative 
conditioning regimens in combination of ATG, slow recovery of Tregs was 
associated with higher incidence of opportunistic infections and extensive chronic 
GVHD. This study demonstrated Treg recovery, at least age appropriate, by the 
second year post transplant.54 
 
3.6 Correlative studies 


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Methylation- Although, in their study, De Lima et al did not demonstrate an effect on DNA 
methylation using unfractionated marrow cells it is possible that the shorter period of treatment (4 
cycles) as well as lower average doses (16-40mg/m2) were responsible for that. 
In this proposed study, the gene specific methylation in sorted hematopoietic and leukemic stem 
cells from marrow samples will be analyzed at several points: at diagnosis (if available), prior to 
transplant, posttransplant prior to initiation of 5’-azacitadine and every 3 months. The purpose to 
this part of the study is to determine if maintenance 5’-Azacitadine post transplant has an effect 
on methylation. 
Minimal Residual Disease (MRD)- Currently there is still no one acceptable test with high 
specificity and sensitivity for detection of minimal residual myeloid disease. We plan to assess 
disease status by morphologic evaluation and immunophenotyping. 
Moreover, In collaboration with Stephen Chung, MD and Christopher Y Park, MD PhD, a panel of 
aberrantly expressed cell surface proteins that are highly specific and sensitive for the detection 
of cells bearing these antigens will be used to assess for residual MDS/AML cells. This will allow 
us to determine whether persistence of cells bearing these antigens can predict for relapse after 
transplant. This analysis will be performed by multi-parameter flow cytometry. 
Cytogenetic changes in the stem cells- In collaboration with Christopher Y Park, MD PhD, and 
Steve Chung, MD fluorescence activated cell sorting (FACS) and FISH technique will be used to 
assess for residual involvement of the HSC compartment following transplantation, assessing for 
the effect of 5’-azacitadine therapy on residual MDS HSC over serial time points (e.g. every three 
months). 
Graft-versus-Leukemia- Last, we’ll assess the effect of 5’-Azacitadine on T cell differentiation 
with specific interest on Tregs. This will be done by the flow cytometry lab and will be part of 
routine post transplant “FLOW7 BMT” which assess for CD4, CD8 and cD19. In addition, the 
effect of treatment with post transplant 5’-Azacitadine on T regs will be studied. 
 
 
4.1 
OVERVIEW OF STUDY DESIGN/INTERVENTION 
 
4.2 
Design 
 
This is a single arm phase II trial to assess the efficacy and confirm the safety of maintenance 
therapy with 5’-azacitadine compared to historical control after TCD allogeneic hematopoietic 
stem cell transplant for patients with MDS and AML who are at high risk of relapse. 
 
4.3 
Intervention 
 
5’-azacitadine will be given at a low dose of 32mg/m2 S.C for 5 days every 28 days (a cycle). 
Dose de-escalation will be permitted for hematologic and non- hematologic toxicities. 


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Patients will start taking the study drug between days 60-120 post TCD allogeneic hematopoietic 
stem cell transplant and up to a year post-transplant or until there is a toxicity that requires 
cessation of therapy. Therefore patients will get between 8-10 cycles. Patients who come off- 
study for reasons unrelated to toxicities before completing 4 cycles will be replaced. Since most 
cases of relapse occur early post transplant, in the first year, this is the most appropriate time to 
intervene. Treatment will start as soon as possible. 
 
5.0 
THERAPEUTIC/DIAGNOSTIC AGENTS 
 
5’-azacitadine- Vidaza TM 
 
 
Source and pharmacology: 5’-azacitadine contains AZA, which is a pyrimidine nucleoside 
analog of cytidine. AZA is a 4-amino-1-a-D-ribofuranosyl-s-triazin-2(1H)-one. The empirical 
formula is C8H12N4O5. The molecular weight is 244. The finished product is supplied in a sterile 
form for reconstitution and subcutaneous injection only. Vials of 5’-azacitadine contain 100 mg of 
5-azacitadine and 100 mg mannitol as a sterilized lyophilized powder. 
Solution preparation: 5’-azacitadine should be reconstituted aseptically with 4ml sterile water for 
injection. The diluent should be injected slowly into the vial. The vial should be inverted 2-3 times 
and gently rotated until a uniform suspension is achieved. The suspension will be cloudy. The 
resulting suspension will contain 5’-azacitadine 25mg/ml. 
Preparation for immediate release: doses greater than 4 mL should be divided equally into the 
syringes. The product may be held at room temperature for up to 1 hour, but must be 
administrated within 1 hour after reconstitution. 
Preparation for delayed release: the reconstituted product may be kept in the vial or drawn into 
a syringe. Doses greater than 4 mL should be divided equally into two syringes. The product must 
be refrigerated immediately, and may be held under refrigerated conditions (20C-80C, 360F-460F) 
for up to 8 hours. After removal from refrigerated conditions, the suspension may be allowed to 
equilibrate to room temperature for up to 30 minutes prior to administration. 
Storage: Store un-reconstituted vials at 250C (770F); excursions permitted to 150-300C (590F- 
860F) 
Stability: reconstituted 5-azacitadine may be stored for up to 1 hour at 250C (770F) or for up to 8 
hours between 2-80C (360F-460F). The 5-azacitadinevial is single-use and does not contain any 
preservatives. Unused portions of each vial should be discarded properly. Do not save any 
unused portions for later administration. 
Administration: to provide a homogeneous suspension, the contents of the syringe must be re- 
suspended by inverting the syringe 2-3 times and gentle rolling the syringe between the palms for 
30 seconds immediately prior to administration. 5-azacitadine is administrated subcutaneously. 
Doses greater than 4 mL should be divided equally into 2 syringes and injected into 2 separate 


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sites. Rotate sites for each injection (thigh, abdomen, or upper arm). New injections should be 
given at least one inch from an old site and never into areas were the site is tender, bruised, red, 
or hard. 
 
 
 
6.1 
CRITERIA FOR SUBJECT ELIGIBILITY 
 
Patients who have undergone T cell depleted allogeneic hematopoietic stem cell transplantation 
at MSKCC for: 
 
1. De novo myelodysplastic syndromes (MDS): IPSS-1 with poor risk cytogenetics or 
higher IPSS. 
2. Acute myelogenous leukemia (AML) in first remission that required more than 1 cyc le 
of treatment to achieve remission or with the following cytogenetic abnormalities: FLT3 
mutation, deletion/monosomy of chromosome 5 or 7, MLL gene rearrangement, or 
more than or equal to 3 cytogenetics abnormalities. Also patients in second or greater 
remission. 
3. Patients with Secondary MDS/AML. 
4. Patients will be considered eligible for the study if after transplant they achieved 
hematologic (<5% blasts) and cytogenetic remission. 
5. Patients will be eligible to enter the study between 60-120 days post transplant. 
 
 
6.2 
Subject Inclusion Criteria 
 
1. Age: pediatrics and adults patients – 1 year old-75 years old. 
2. Karnofsky performance status >=60% for patients >16yo and Lansky performance 
status >=60% for patients ≤16yo 
3. Stable blood counts (ANC>1000/uL, Hb>8gr/dL, Plt>50,000/ uL) not supported by 
transfusions. 
4. Renal: Serum creatinine <1.5 ULN 
5. Hepatic: <3xULN ALT and <1.5 total serum bilirubin, unless there is congenital benign 
hyperbilirubinemia. 
6. Cardiac: Adequate cardiac function measured by LVEF>50%. If asymptomatic, pre- 
transplant echocardiogram is adequate. If symptomatic, echocardiogram needs to be 
repeated. 
7. Each patient must be willing to participate as a research subject and must sign an 
informed consent form. 
 
Patients who were treated with 5’-azacitadine without response prior to transplant would 
be eligible to participate on this protocol. 
 
6.3 
Subject Exclusion Criteria 


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Patients will be excluded from the trial if at time of enrollment: 
 
1. Active uncontrolled bacterial, fungal or viral infection. 
2. Evidence of uncontrolled graft-versus-host disease. 
3. Pulmonary: new onset hypoxia 
4. Known or suspected hypersensitivity to 5’-azacitadine or mannitol. 
5. Evidence of residual disease either by increased blasts count (>5%) or persistence of 
previous known cytogenetics abnormalities. 
6. Peripheral blood neutrophil chimerism: less than 95% donor. 
 
 
 
7.0 
RECRUITMENT PLAN 
 
Patients who fulfill the eligibility criteria as listed in section 6.0 will be recruited for this 
study by an attending physician of the allogeneic BMT services in Medicine and 
Pediatrics. In the period of time between 60-120 days post-transplant, patients who are 
eligible for the study protocol will be offered to participate. A discussion will take place 
about the need for treatment with 5’- azacitidine which involves 5 days of subcutaneous 
injection every month for a year post transplant, the rational of decreasing risk of relapse, 
and the potential side effects, which at this part are mostly known to be related to 
myelosuppression and decreased blood counts.  This protocol will take due notice of 
NIH/ADAMHA  policies concerning inclusion of women and minorities in clinical research 
population. 
 
 
8.1 
PRETREATMENT EVALUATION 
 
The patient will undergo comprehensive medical evaluation after stable engraftment has 
been documented. This evaluation includes: 
1. 
Complete physical exam and medical history 
2. 
Bone marrow tests post HSCT will be performed to determine remission status by 
morphology, cytogenetics and molecular studies (if indicated) and chimerism as part of 
routine post transplant assessments. At the same time, bone marrow sample will also be 
taken for baseline correlative studies under the biospecimen protocol. Any bone marrow 
sample collected prior to starting treatment with Azacitidine can be used as baseline for 
correlative studies. 
3. 
Complete blood counts (CBC) 
4. 
Complete metabolic panel 
5. Pregnancy test will be checked for women at child bearing age prior to initiation of 
treatment 


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6. 
Cardiac: Adequate cardiac function measured by LVEF>50%. If asymptomatic, pre- 
transplant echocardiogram is adequate. If symptomatic, echocardiogram needs to be 
repeated. 
 
 
9.1 
TREATMENT/INTERVENTION PLAN 
 
1. 
5’-azacitadine will be given at a dose of 32mg/m2 for 5 consecutive days subcutaneously. 
Treatment will be given every 28 days (cycle). Treatment will be given up to a year post 
transplant. 
2. 
CBC will be checked weekly during the first 2 cycles, and if stable every 2 weeks with 
following cycles. CBC results from tests performed externally will be accepted for Days 8, 
15, and 22 of C1 and C2 and Day 15 of subsequent cycles. Comprehensive metabolic 
panel will be checked at the beginning of every cycle. 
3. 
Pregnancy test will be checked for women at child bearing age prior to start of each 
cycle. 
4. 
Bone marrow studies (day 100, and 6, 12, 18 and 24 months post transplant) for 
assessment of disease status and chimerism. Peripheral blood chimerism status at day 
100, and 6, 12, 18 and 24 months post transplant will be tested as part of routine post 
transplant evaluation 
5. 
Peripheral blood lymphocytes phenotyping and in-vitro response to standard panel of 
mitogen will be assessed as part of routine post transplant assessment 
6. 
Bone marrow samples for correlative studies will be tested at the same time that patients 
are undergoing routine BM studies. These tests are considered experimental. 
7. 
Peripheral blood samples for correlative studies will be collected prior to start of 
Azacitidine treatment on C1D1 and at 12 and 18 months post transplant. These tests are 
considered experimental. 
 
 
Dose modifications 
 
Eve nt 
Modifica tion 
ANC <1000/uL for > 4 days, 
Drug held until resolved  (ANC 1500) and up to 60 
days. If after 60 days there is no neutrophils 
recovery  the treatment will be permanently 
discontinued. 
• 
If by next cycle ANC is 1000-1500,  not G- 
CSF dependent 
Resume therapy   with 50% dose reduction 
• 
If by next cycle ANC fully recovered, 
>1500 not G-CSF  dependent 
Resume full therapy dose 
Pla te le ts <10,000/uL for > 4 days , or if transfusion 
dependent  to keep count>10,000/  uL 
Drug held until resolved  (>50,000) and up to 60 
days. 


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• 
If by next cycle Platelets are 10,000- 
50,000, not transfusion  dependent 
Resume therapy  but with 50% dose reduction 
• 
If by next cycle Platelets >50,000 
Resume full therapy dose 
He moglobin < 8g/dL for > 4 days or if transfusion 
dependent 
Drug held until resolved  (> 8g/dL) and up to 60 
days. 
• 
If by next cycle hemoglobin  >8g/dL 
Resume full therapy dose 
He pa tic tox icity – 
 Drug related grade 3-4 
 Drug related grade 1-2 
Drug  stopped 
Next cycle delayed up to 60 days for resolution or 
reduced by 25% on subsequent cycles (resume full 
dose on next cycle once resolved) 
Cre a tinine > 50% above  age-adjusted  ULN 
Drug held 
Cre a tinine ≤ 50% above  age-adjusted  ULN after a 
maximum delay of 45 days 
Dose reduced 50% on next cycle (resume full dose 
on next cycle once resolved) 
Se rum bica rbona te le ve l (CO2)  - Unexplained 
reduction; < 20mEq/L 
Dose reduced by 50% on next cycle 
Ca rdia c, pulmona ry, de rma tologic, or 
ne urologic tox icity  - Drug related grade 3-4 
Drug  stopped 
Acute  infe ction 
Drug held (resume full dose once resolved) 


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10.0 
EVALUATION DURING TREATMENT/INTERVENTION 
 
During the treatment patients will be seen in clinic every 4 weeks (prior to every cycle) and more 
frequently if required by primary BMT attending. All patients will be closely monitored and 
evaluated as per MSKCC BMT standard of care guidelines. Evaluations may be withheld if the 
treating physician feels that there is a strong contra-indication to perform the study (e.g. patient 
has relapsed or is terminally ill). Also, additional tests will be performed as clinically indicated. 
Study specific assessment schedule listed in table below. 
 
Schedule of Study Assessments 
 
 
Procedures 
Pre- 
Treatment 
Post  Tre atme nt Sta rt 
C1 & C2 
C3+ 
D1 
D8 
D15 
D22 
D1 
D15 
Window 
45 
(+/-) 
(+/-) 
(+/-) 
(+/-) 
(+/-) 
(+/-) 
7 
3 
3 
3 
7 
3 
Eligibility 
X 
Informed  consent 
X 
History/Physical 
X 
Pregnancy Test (1) 
X 
X 
X 
Blood Chemistry/CMP (2) 
X 
X 
X 
CBC (2) 
X 
X 
X 
X 
X 
X 
X 
Echo-  if no new symptoms,  pretransplant is 
adequate 
X 
Toxicity assessment (3) 
X 
X 
Correlative  studies – Peripheral  blood for Tregs  expression 
studies (4) 
X* 
 
Procedures 
Post Tra nsp la nt 
100 
days 
6 
months 
12 
months 
18 
months 
24 
months 
Window 
(+/-) 21 (+/-) 14 (+/-) 14 
(+/-) 14 
(+/-) 14 
Chimerism (Peripheral  Blood) (5) 
X 
X 
X 
X 
X 
Bone marrow aspirate and/or biopsy (6) 
X 
X 
X 
X 
X 
Peripheral  blood  lymphocytes phenotyping  and in-vitro 
response (7) 
X 
X 
X 
X 
X 
Correlative  studies - BM sample for methylation assays, 
flow studies for MRD (8) 
X 
X 
X 
X 
X 
Correlative  studies – Peripheral  blood for Tregs  expression 
studies 
X 
X 
 
*Peripheral blood samples will be collected prior to start of Azacitidine treatment on C1D1 
only 


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(1) Pregnancy Test – Not required for Cycle 1 Day 1 if done at screening, but required for Day 1 of 
each subsequent cycle for FCBP. 
(2) CBC – checked weekly during the first 2 cycles, and if stable, every 2 weeks with subsequent 
cycles. Outside CBC results will be accepted for D8, 15 and 22 of C1 and C2 and for D15 of 
subsequent cycles. CBC/Chemistry must result prior to IP administration on or within 7 days of 
Day 1 of each cycle. If a CBC is performed within overlapping windows for two time-points, it is 
not necessary to repeat unless clinically indicated. 
(3) Toxicity Assessment - Not required for Cycle 1 Day 1, but required for Day 1 of each subsequent 
cycle or within 7days of Day 1 of each cycle. Patients who pass away, complete treatment or end 
treatment before the start of the next cycle will have toxicities captured at the end of the last  
cycle started. 
(4) Peripheral blood samples (2 GTT) will be collected and stored at HOTB prior to start of 
Azacitidine treatment on C1D1 and at 12 and 18 months post-transplant 
(5) Peripheral blood will be checked for neutrophil, B cells, and T cell chimerism 
(6) In the first year bone marrow tests will be performed to assess disease status by morphology, 
cytogenetics, and molecular as per BMT standard of care time-points. Following the first year 
these studies will be performed at 18 and 24 months post transplant. 
(7) Peripheral blood will be checked for lymphocytes phenotyping as well as in-vitro response to 
standard panel of mitogens as per BMT standard of care. 
(8) Correlative studies include global and specific genes methylation status, Fluorescence activated 
cell sorting (FACS) and FISH assays will be used to assess for residual involvement for the HSC 
compartment 
 
 
10.1 Correlative Studies 
 
Bone marrow samples will be obtained on Day+100 and at 6, 12,18 and 24 months post transplant 
as part as our routine post transplant follow up. In addition, another BM aspirate sample, 3 cc, will be 
sent to HOTB. 
 
Using a panel of aberrantly expressed cell surface proteins that are highly specific and sensitive for 
the detection of cells bearing these antigens, the samples will be assessed for residual MDS/AML 
cells. This analysis will be performed by multiparameter flow cytometry. 
 
By using fluorescence activated cell sorting (FACS) and FISH technique, the samples will be 
assessed for residual involvement of the HSC compartment following transplantation. 
 
Global methylation assay will also be assessed on the sorted cells by using anti- 5'-methylcytosine 
monoclonal antibody which will be provided by Dr. Ari Melnick. 
Peripheral blood sample will be obtained prior to start of Azacitidine treatment on C1D1 and at 12 
and 18 months post transplant and will be stored at the Hematology-Oncology Tissue Bank (HOTB). 


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These samples will be sent to the clinical lab to assess azacitidine‘s effect on Tregs expression- 
peripheral blood will be examined by flow using the markers CD3+CD4+CD25HICD127NEG. 
 
 
11.1 TOXICITIES/SIDE EFFECTS 
 
Toxicities will be graded according to NCI CTAE version 4.0 grades 3-4 only, except grades 
1-4 hepatic toxicity (increased AST, ALT, alkaline phosphatase and bilirubin) and grades 1-4 
GI toxicity (nausea, vomiting, diarrhea). The side effects and toxicities related to 5- 
azacitadine are listed below. 
 
5’-azacitadine 
 
Likely 
 
• 
Nausea 
• 
Vomiting 
• 
Diarrhea 
• 
Constipation 
• 
Lowered white blood cell count that may lead to infection 
• 
Lowered platelet count that may lead to an increase in bruising or bleeding 
• 
Lowered red blood cells that may cause anemia, tiredness, or shortness of breath 
• 
Chest pain 
• 
Fever 
• 
Fatigue and weakness 
• 
Headache 
• 
Dizziness 
• 
Bruising 
• 
Rash 
• 
Dry skin 
• 
Skin nodules 
• 
Erythema 
• 
Weight loss 
• 
Abdominal pain 
• 
Loss of appetite 
• 
Joint pain 
• 
Muscle Pain 
• 
Breathlessness 
• 
Upper respiratory tract infections as well as infections of the throat and sinuses 
• 
Pneumonia 


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• 
Injection site bruising, pain or reaction 
• 
Anxiety 
• 
Insomnia 
 
 
Less Likely: 
• 
Abnormal heart beat 
• 
Fainting spells 
• 
Skin infections 
• 
Upper abdominal pain 
• 
Bleeding from the gums 
• 
Difficulty eating 
• 
Hemorrhoids 
• 
Pain when urinating 
• 
Urinary tract infection 
• 
Muscle cramps 
• 
Wheezing 
• 
Fluid on the lungs 
• 
Increased sweating 
• 
Night sweats 
 
 
Rare But Serious: 
• 
Convulsions 
• 
Anaphylaxis 
• 
Bone marrow depression 
• 
Congestive Heart Failure 
• 
Stupor 
• 
Coma 
 
 
12.1 CRITERIA FOR THERAPEUTIC RESPONSE/OUTCOME ASSESSMENT 
 
Definitions of events in the study period that are important for analysis and treatment 
 
1. Disease relapse 
 
Relapse of MDS or AML will be analyzed as to type and genetic origin of the leukemic cells. 
These will be defined by morphologic and/or cytogenetic criteria: an increasing number of 
blasts in the marrow over 5%, by presence of circulating blasts, or by presence of blasts in 
any extramedullary site as well as presence of previous cytogenetic abnormalities. Other 


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studies assessing for MRD, FACS and FISH assays will be evaluated but would not be 
considered disease relapse if positive since they are experimental.. 
2. Transplant related mortality 
 
Includes fatal complications resulting from the therapy such as severe infection, 
hemorrhages and graft failure. 
 
3. Immunologic  reconstitution 
 
Our previous studies have identified time points at which the various immunologic functions 
can be expected to return. Immunophenotyping and NK cell function will be performed on 
circulating lymphocytes of all patients. 
 
T cell proliferation in response to PHA, candida, viral antigens and tetanus following 
immunization will be performed at approximately 6 months post transplant and every 3-6 
months thereafter until normal values are reached. 
 
4. Engraftment and chimerism 
 
Engraftment will be documented by analysis of bone marrow cells as well peripheral blood 
cells for chimerism by standard karyotype or short tandem repeat analysis every 3 up to one 
year posttransplant. 
 
 
13.0 
CRITERIAFOR REMOVAL FROM STUDY 
 
If at any time the patient is found to be ineligible for the protocol as designated in the section on 
Criteria for Patient/Subject Eligibility, the patient will be removed from the study. Intolerable toxicity 
would take a subject off protocol therapy, but not off study to continue to follow them since overall 
survival is a secondary objective. Also patients may be removed from the study if requested by the 
patient. Patients will need to receive a minimum of 4 cycles to be considered evaluable for protocol 
objectives. Patients who receive less than 4 cycles due to treatment-related toxicities will be 
considered evaluable for toxicity only and will not be replaced on study. Patients who relapse prior to 
receiving 4 cycles are considered evaluable per protocol. Patients who come off-study for reasons 
unrelated to toxicities before completing 4 cycles are considered inevaluable per protocol and will be 
replaced. Management will continue as per standard allogeneic HSCT guidelines. 
14.1 
BIOSTATISTICS 
 
This phase II trial will explore the efficacy of treating high-risk MDS and AML patients with 5’- 
azacitidine following TCD-HSCT. Based on historical MSKCC data for patients with advanced MDS 
and AML (adults and pediatrics) with high relapse risk, who meet the entry criteria, the two-year 


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cumulative incidence of relapse is 35%. Therefore, this intervention would be considered promising 
if the two-year relapse proportion is 15% or less. If the relapse proportion is 35% or greater, the 5- 
azacitibine intervention would not be considered promising. Therefore, using a single stage exact 
design, a total of 32 patients will accrue on study. At the end of the trial, if seven or fewer patients 
relapse by two years post treatment initiation, the trial will be considered promising. If, however, 
more than 8 patients relapse at any point the study will be considered unpromising and will be 
closed for further accrual. The type I and type II errors for this trial are 0.08 and 0.10, respectively. It 
is anticipated that enrollment will be completed in 5 years with an accrual of approximately 6-7 
patients/year. 
 
If a patient is found to be ineligible for reasons unrelated to disease relapse or progression, or is lost 
to follow-up, s/he will be replaced for the primary endpoint. If a patient dies for reasons unrelated to 
disease relapse or progression (i.e., treatment related mortality), s/he will remain as part of the 32 
patient cohort. Treatment-related mortality will be monitored as part of the stopping rules discussed 
below. 
 
In order to reduce patient risk and confirm the safety of 5’-azacitidine in this setting, the study design 
includes early termination in event of severe neutropenia, severe thrombocytopenia, and treatment- 
related mortality. The boundaries are based on accrual of 32 patients. The calculations in the table 
below are based on marginal probabilities. 
 
 
 
Failure Type 
# of failures needed 
to stop the study 
Failure rate 
in the 
population 
Probability 
boundary 
is crossed 
Neutropenia 
(Grade III-IV) 
3 in the first 8 patients 
4 in the first 14 patients 
5 in the first 20 patients 
6 in the first 27 patients 
7 at any point 
0.1 
0.1 
0.3 
0.92 
Thrombocytopenia 
(Grade III- 
IV) 
3 in the first 8 patients 
4 in the first 14 patients 
5 in the first 20 patients 
6 in the first 27 patients 
7 at any point 
0.1 
0.1 
0.3 
0.92 
3 in the first 5 patients 
4 in the first 8 patients 
5 in the first 12 patients 
0.15 
0.07 


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Treatment-related mortality 
6 in the first 16 patients 
7 in the first 20 patients 
8 in the first 25 patients 
9 in the first 29 patients 
10 at any point 
0.4 
0.93 
 
 
In addition to the primary endpoint, there are a number of secondary and correlative objectives. 
Secondary Objectives: 
1. Kaplan-Meier methodology will be used to compare overall survival. 
2. The safety will be described by tabulating the number of transfusions, frequencies of 
bleeding and serious infections, and the use of G-CSF support. 
3. We will also evaluate the following metrics: graft function; myeloid and T-cell chimerism; and 
acute and chronic GVHD incidence. Graft function will be examined after enrollment by 
summary statistics of hemoglobin levels, neutrophils and platelets. Myeloid  and T-cell 
chimerism will be summarized as a continuous measurement at enrollment, 6, 9,12,18 
months post-transplant and 24 months post-transplant. Cumulative incidence function will be 
used to estimate grade II-IV  acute GVHD and chronic GVHD incidence. 
 
 
 
 
Correlative Studies: 
 
1. The global DNA methylation status will be evaluated at baseline, and the change from 
baseline will be explored among individuals who do and do not relapse by the 24 month BM 
evaluation. . This continuous measurement will be compared using a rank-sum test. 
2. The reoccurrence of cytogenetic abnormalities will be evaluated in the CD34 subpopulation 
of cells at the time of trial enrollment. This exploratory measure is calculated as the 
proportion of involved cytogenetic cells out of the total number of stem cells and may be a 
marker of disease burden following transplant. This proportion will be categorized and 
relapse will be contrasted across these categories using a permutation-based logrank test. 
3. The composition of myeloid progenitor cells will be compared pre- and post- 5’-azacitidine 
treatment to explore whether normalization occurs following treatment. These paired 
measurements will be compared using McNemar’s test. 
4. The levels of regulatory T-cells and CD8+ will be graphically displayed across baseline 
and post-treatment time points. 
15.1 
RESEARCH PARTICIPANT REGISTRATION AND RANDOMIZATION PROCEDURES 
 
15.2 
Research Participant Registration 


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Confirm eligibility  as defined in the section entitled  Inclusion/Exclusion  Criteria. Obtain informed 
consent, by following  procedures  defined in section entitled  Informed  Consent Procedures. 
During the registration  process registering  individuals  will be required to complete a protocol 
specific Eligibility  Checklist. The individual  signing the Eligibility  Checklist is confirming whether 
or not the participant  is eligible  to enroll in the study. Study staff are responsible  for ensuring 
that all institutional  requirements  necessary to enroll a participant  to the study have been 
completed. See related Clinical Research Policy and Procedure #401 (Protocol Participant 
Registration). 
16.1 
DATA MANAGEMENT ISSUES 
 
A Research Study Assistant (RSA) will be assigned to the study and will be responsible for both 
pediatric and adult accruals. The responsibilities of the RSA and principal investigator include project 
compliance, data collection, abstraction and entry, data reporting, regulatory monitoring, problem 
resolution and prioritization, and coordinate the activities of the protocol study team. The data 
collected for this study will be entered into The Clinical Research Data Base (CRDB), a secure 
database. Source documentation will be available to support the computerized patient record. 
16.2 
Quality Assurance 
 
Weekly registration reports will be generated to monitor patient accruals and completeness of 
registration data. Routine data quality reports will be generated to assess missing data and 
inconsistencies. Accrual rates and extent and accuracy of evaluations and follow-up will be 
monitored periodically throughout the study period and potential problems will be brought to the 
attention of the study team for discussion and action. Random-sample data quality and protocol 
compliance audits will be conducted by the study team, at a minimum of two times per year, more 
frequently if indicated. 
16.3 
Data and Safety Monitoring 
 
The Data and Safety Monitoring (DSM) Plans at Memorial Sloan-Kettering Cancer Center were 
approved by the National Cancer Institute in September 2001. The plans address the new policies 
set forth by the NCI in the document entitled “Policy of the National Cancer Institute for Data and 
Safety Monitoring of Clinical Trials” which can be found at:  
http://cancertrials.nci.nih.gov/researchers/dsm/index.html. The DSM Plans at MSKCC were 
established and are monitored by the Clinical Research Administration. The MSKCC Data and 
Safety Monitoring Plans can be found on the MSKCC Intranet at:  
http://mskweb2.mskcc.org/irb/index.htm 
There are several different mechanisms by which clinical trials are monitored for data, safety and 
quality. There are institutional processes in place for quality assurance (e.g., protocol monitoring, 
compliance and data verification audits, therapeutic response, and staff education on clinical 
research QA) and departmental procedures for quality control, plus there are two institutional 
committees that are responsible for monitoring the activities of our clinical trials programs. The 


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Page 30 of 37 
 
 
 
 
committees: Data and Safety Monitoring Committee (DSMC) for Phase I and II clinical trials, and the 
Data and Safety Monitoring Board (DSMB) for Phase III clinical trials, report to the Center’s 
Research Council and Institutional Review Board.  During the protocol development and review 
process, each protocol will be assessed for its level of risk and degree of monitoring required. Every 
type of protocol (e.g., NIH sponsored, in-house sponsored, industrial sponsored, NCI cooperative 
group, etc.) Will be addressed and the monitoring procedures will be established at the time of 
protocol activation. 
17.1 
PROTECTION OF HUMAN SUBJECTS 
 
Risks: From the studies that have been done so far it was demonstrated that post transplant 5’- 
Azacitadine can be safely given in patients with myelodysplastic syndrome and acute myelogenous 
leukemia and that these patients may benefit from the treatment.  However, given this is a new 
treatment approach in T cell depleted transplant, it is possible that there are side effects that have 
not yet been seen. 
Benefits: The information from this study will help future cancer patients. 
Possible toxicities/side effects: Toxicities and side effects of the agents used are listed in section 
11 and reporting of serious adverse events is found in section 17.2. 
Consent Process: Participation in this study is voluntary. All patients will be required to sign a 
statement of informed consent which must conform to MSKCC IRB guidelines. For minors consent 
will be obtained from parents or guardians of unemancipated minors. Minors between 7-17 years old 
will be assented. This protocol will take due notice of NIH/ADAMHA  policies concerning inclusion of 
women and minorities in clinical research population. 
Alternatives:  Alternative treatment options will be presented to the patient prior to taking part in this 
study. Alternative treatment options may include not participating in this study and follow up as per 
regular standard of care post transplant, or taking part in another study of post transplant 
maintenance therapy, should that becomes available. 
Costs: The patient’s health plan/insurance company will need to pay for all of the costs of standard 
medical care. Patients will not be paid for taking part in this study. Research tests will be done at no 
cost to the patient. 
Confidentiality: Every effort will be made to maintain patient confidentiality. Research and hospital 
records are confidential. 
17.2 
Privacy 
 
MSKCC’s Privacy Office may allow the use and disclosure of protected health information pursuant 
to a completed and signed Research Authorization form. The use and disclosure of protected health 
information will be limited to the individuals described in the Research Authorization form. A 
Research Authorization form must be completed by the Principal Investigator and approved by the 
IRB and Privacy Board (IRB/PB). 
17.3 
Serious Adverse Event (SAE) Reporting 


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An adverse event is considered serious if it results in ANY of the following outcomes: 
• 
Death 
• 
A life-threatening adverse event 
• 
An adverse event that results in inpatient hospitalization or prolongation of existing 
hospitalization 
• 
A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
• 
A congenital anomaly/birth defect 
• 
Important Medical Events (IME) that may not result in death, be life threatening, or 
require hospitalization may be considered serious when, based upon medical judgment, 
they may jeopardize the patient or subject and may require medical or surgical 
intervention to prevent one of the outcomes listed in this definition 
Note: Hospital admission for a planned procedure/disease treatment is not considered an 
SAE. 
 
SAE reporting is required as soon as the participant signs consent. SAE reporting is 
required for 30-days after the participant’s last investigational treatment or intervention. Any 
events that occur after the 30-day period and that are at least possibly related to protocol 
treatment must be reported. 
 
If an SAE requires submission to the IRB office per IRB SOP RR-408 ‘Reporting of Serious 
Adverse Events’, the SAE report must be sent to the IRB within 5 calendar days of the event. 
The IRB requires a Clinical Research Database (CRDB) SAE report be submitted 
electronically to the SAE Office as follows: 
 
For IND/IDE trials: Reports that include a Grade 5 SAE should be sent to  
saegrade5@mskcc.org.  All other reports should be sent to saemskind@mskcc.org. 
 
For all other trials: Reports that include a Grade 5 SAE should be sent to  
saegrade5@mskcc.org.  All other reports should be sent to sae@mskcc.org. 
 
The report should contain the following information: 
Fields populated from CRDB: 
• 
Subject’s initials 
• 
Medical record number 
• 
Disease/histology (if applicable) 
• 
Protocol number and title 
Data needing to be entered: 
• 
The date the adverse event occurred 


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Page 32 of 37 
 
 
 
 
• 
The adverse event 
• 
The grade of the event 
• 
Relationship of the adverse event to the treatment (drug, device, or intervention) 
• 
If the AE was expected 
• 
The severity of the AE 
• 
The intervention 
• 
Detailed text that includes the following 
o A explanation of how the AE was handled 
o A description of the subject’s condition 
o Indication if the subject remains on the study 
• 
If an amendment will need to be made to the protocol and/or consent form 
• 
If the SAE is an Unanticipated Problem 
 
The PI’s signature and the date it was signed are required on the completed report. 
 
 
 
All patients will be followed for safety and toxicity related to the study. The reportable serious 
adverse events (SAEs) will be defined according to the current MSKCC Adult and Pediatric 
BMT Adverse Event Reporting Guide.. 
18.1 
INFORMED CONSENT PROCEDURES 
 
Before protocol-specified procedures are carried out, consenting professionals will explain full details 
of the protocol and study procedures as well as the risks involved to participants prior to their 
inclusion in the study. Participants will also be informed that they are free to withdraw from the study 
at any time. All participants must sign an IRB/PB-approved consent form indicating their consent to 
participate. This consent form meets the requirements of the Code of Federal Regulations and the 
Institutional Review Board/Privacy Board of this Center. The consent form will include the following: 
1. The nature and objectives, potential risks and benefits of the intended study. 
2. The length of study and the likely follow-up required. 
3. Alternatives to the proposed study. (This will include available standard and 
investigational therapies. In addition, patients will be offered an option of supportive 
care for therapeutic studies.) 
4. The name of the investigator(s) responsible for the protocol. 
5. The right of the participant to accept or refuse study interventions/interactions and to 
withdraw from participation at any time. 
 
Before any protocol-specific procedures can be carried out, the consenting professional will fully 
explain the aspects of patient privacy concerning research specific information.  In addition to  
signing the IRB Informed Consent, all patients must agree to the Research Authorization component 
of the informed consent form. 


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IRB Number: 13-192 A(11) 
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Page 33 of 37 
 
 
 
 
Each participant and consenting professional will sign the consent form. The participant must 
receive a copy of the signed informed consent form. 
 
 
19.0 
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IRB Number: 13-192 A(11) 
Approval date: 13-May-2019 
Page 34 of 37 
 
 
 
 
 
 
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Memorial Sloan Kettering Cancer Center 
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Approval date: 13-May-2019 
 
 
26   Devine SM, Carter S, Soiffer RJ, Low risk of chronic graft-versus-host disease and relapse 
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Approval date: 13-May-2019 
 
 
38   Kroger N Epigenetic modulation and other options to improve outcomes of stem cell 
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IRB Number: 13-192 A(11) 
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45. 
 
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52   Choi J, Ritchey J, Prior JL et al In vivo administration of hypomethylating agents mitigate graft- 
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53   Goodyear OC, Dennis M, Jilani NY et al Azacitidine augments expansion of regulatory T cells 
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54 Lewin SR, Heller G, Zhang L et al Direct evidence for new T-cell generation by patients after 
either T-cell-depleted or unmodified allogeneic hematopoietic stem cell transplantations. Blood 
2002;100:2235-42.