Conditions: Leukemia, Erythroblastic, Acute Myelodysplastic Syndromes
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 40
Healthy volunteers: no

Brief summary:
The purpose of this study is to determine whether 5-Azacytidine priming before the conditioning regimen for subjects receiving a hematopoietic stem cell transplant is an effective treatment for high risk myeloid malignancies in complete remission (CR).

Detailed description:
This open label two-step phase II study is designed to determine the safety and efficacy of epigenetic priming with 5-Azacytidine immediately prior to reduced intensity conditioning for an in vivo T-cell depleted hematopoietic stem cell transplantation for high risk myeloid malignancies in complete remission (CR).

Subjects will be given a five day course of subcutaneous 5-azacytidine, followed by a reduced intensity conditioning regimen of fludarabine, melphalan and total body irradiation (TBI) prior to an allogeneic hematopoietic stem cell transplantation from a related or unrelated Human Leukocyte Antigen (HLA) matched donor.

The effect of 5-azacytidine on global gene methylation will be assessed. Evaluations for safety, in particular for graft failure, transplant related mortality and acute graft versus host disease will be made on a weekly basis. Efficacy, as defined by disease free survival, will be evaluated with a bone marrow biopsy at the standard time points, which are one-, three-, six-, and twelve-months after transplant and upon clinical suspicion within regular follow-up visits - weekly for the first 3 months, then biweekly for 3 months, then monthly until one-year post-stem cell transplant. Thereafter, unless otherwise dictated by the clinical scenario, the follow up visits will be every 3 months.

Arms:
arm 1: Patients will be given a five day course of subcutaneous 5-azacytidine, followed by a reduced intensity conditioning regimen of fludarabine and melphalan with or without total body irradiation prior to an allogeneic hematopoietic stem cell transplantation from a related or unrelated HLA matched donor.

Interventions: intervention 1: 5-Azacytidine intervention 2: Fludarabine intervention 3: Melphalan intervention 4: Alemtuzumab intervention 5: Total Body Irradiation
Intervention descriptions: intervention 1: Patients will be given a five day course of subcutaneous 5-azacytidine followed by a reduced intensity conditioning regimen of fludarabine and melphalan with or without total body irradiation prior to an allogeneic hematopoietic stem cell transplantation from a related or unrelated HLA matched donor. 5-Azacytidine 75 mg/m2 subcutaneously daily at the same time on days -11, -10, -9,

-8 and -7. This will be administered on an outpatient basis if possible. intervention 2: Conditioning regimen: Hospital admission will usually take place on the first day of the conditioning regimen. Fludarabine will be given at 40 mg/m2 intravenously daily at the same time over 30 minutes on days -6,-5,-4,-3. intervention 3: Conditioning regimen: Hospital admission will usually take place on the first day of the conditioning regimen. Melphalan will be given at 140 mg/m2 IV on day -3. intervention 4: Conditioning regimen: Hospital admission will usually take place on the first day of the conditioning regimen. Alemtuzumab will be given at 30 mg subcutaneously on Days -4 and -2 for unrelated donors, and Day -2 for related donors. intervention 5: Conditioning regimen: Hospital admission will usually take place on the first day of the conditioning regimen. Total Body Irradiation (TBI) will be given at 2 doses of 200 cGy each on one day of the conditioning regimen (between days -6 and -3).

Protocol document:
Prot_SAP_000.pdf: 
Title: Epigenetic priming with 5-Azacytidine prior to in vivo T-cell depleted allogeneic stem cell 
transplantation for patients with high-risk myeloid malignancies in morphologic remission 
NCT02497404 
Initial IRB Approval Date: July 7, 2014 
Amendment History 
• 
Original Protocol Date: January 6, 2014 
• 
Amendment #1: April 9, 2015 
• 
Amendment #2: November 2, 2015 
• 
Amendment #3: April 16, 2018 


                                                                                                 IRB Protocol Number: 1306014009 
                                                                                                Version Date: 16APR2018 
 
 
Tomer 
 
 
 
Epigenetic priming with 5-Azacytidine prior to in vivo T-cell depleted allogeneic stem cell 
transplantation for patients with high risk myeloid malignancies in morphologic remission 
 
 
Principal Investigator: 
 
Sebastian Mayer, MD 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 


 
 
Statistician: 
 
Regulatory Coordinator: 
 
 
 
                                           
 
 
                   
      
                                             
 
                                            
 
 
      
 
 
 
 
 
 
 
      
 
                                 
 
 
  
 
Responsible Research Nurse: 
Responsible Data Manager: 
 
                                             
 
 
 
                             
        
 
                                            
 
                                          
    
       
                                   
 


 
 
i
 
SCHEMA 
 
 
PROTOCOL TITLE:  
Epigenetic priming with 5-Azacytidine prior to in vivo T-cell depleted allogeneic stem cell 
transplantation for patients with high risk myeloid malignancies in morphologic remission 
 
PROTOCOL NUMBER 
1306014009 
FINAL PROTOCOL DATE 
 
STUDY DRUG 
5-Azacytidine 
INDICATION 
Poor risk AML in CR1, High Risk MDS in 
CR, AML in CR>1 
STUDY PHASE 
Phase II 
STUDY OBJECTIVES: 
 
Primary Objective: 
 
To evaluate  6 month, 1-year and 2-year disease-free survival and overall survival after 
allogeneic stem cell transplantation with 5-Azacytidine priming immediately prior to 
conditioning   and Alemtuzumab in vivo T- cell depletion in patients with poor risk myeloid 
malignancies in complete morphologic remission. 
 
Secondary Objectives: 
 
To evaluate the incidence and severity of acute and chronic GVHD after 5-azacytidine 
priming prior to conditioning for allogeneic stem cell transplantation with 
Alemtuzumab-based in vivo T-cell depletion in patients with poor risk myeloid 
malignancies in complete morphologic remission.    
 
To evaluate engraftment and chimerism patterns after 5-azacytdine primed conditioning for 
stem cell transplantation with Alemtuzumab-based in vivo T-cell depletion in patients with 
poor risk myeloid malignancies in complete morphologic remission.    
 
To evaluate changes in global gene methylation by 5-azacytidine prior to conditioning for 
stem cell transplantation with Alemtuzumab-based in vivo T-cell depletion in patients with 
poor risk myeloid malignancies in complete morphologic remission.    
 
STUDY DESIGN: 
This open label two-step phase II study is designed to determine the safety and efficacy of 
epigenetic priming with the hypomethylating agent 5-Azacytidine immediately prior to 
reduced intensity conditioning for an in vivo T-cell depleted hematopoietic stem cell 
transplantation for high risk myeloid malignancies in complete morphologic remission (CR). 


 
 
ii
 
The inclusion criteria are AML with poor risk cytogenetics or other poor risk characteristics in 
CR 1 or beyond, high risk MDS in CR, other AML in CR 2 or beyond (excluding 
promyelocytic leukemia) and secondary AML after MDS/MPN in CR. 
These patients will be given a five day course of subcutaneous 5-azacytidine, followed by a 
reduced intensity conditioning regimen of fludarabine and melphalan with total body 
irradiation prior to an allogeneic hematopoietic stem cell transplantation from a related or 
unrelated HLA matched donor. 
The effect of 5-azacytidine on global gene methylation will be assessed. Evaluations for 
safety, in particular for graft failure, transplant related mortality and acute graft versus host 
disease will be made on a weekly basis. Efficacy, as defined by disease free survival, will be 
evaluated with a bone marrow biopsy at the standard time points, which are one-, three-, six-, 
and twelve-months after transplant and upon clinical suspicion within regular follow-up visits 
- weekly for the first 3 months, then biweekly for 3 months, then monthly until one-year post-
SCT. Thereafter, unless otherwise dictated by the clinical scenario, the follow up visits will be 
every 3 months. 
Patients will be followed for the primary endpoints disease relapse or death and engraftment 
kinetics, incidence of acute and chronic graft versus host disease will be analyzed. 
 
 


 
 
iii
 
Contents 
SCHEMA ......................................................................................................................................... I 
1. 
OBJECTIVES ...................................................................................................................... 1 
1.1 Primary Objectives................................................................................................................ 1 
1.2 Secondary Objectives............................................................................................................ 1 
2. 
BACKGROUND ................................................................................................................. 1 
2.1      Disease ............................................................................................................................. 1 
2.2       Investigational Agent ....................................................................................................... 3 
2.3     Rationale ........................................................................................................................... 3 
2.4      Correlative Studies Background ....................................................................................... 4 
3.PATIENT SELECTION .............................................................................................................. 5 
3.1  Inclusion Criteria .................................................................................................................. 5 
3.2  Exclusion Criteria ................................................................................................................. 6 
4. 
REGISTRATION PROCEDURES ..................................................................................... 6 
5. 
TREATMENT PLAN, DONOR SELECTION AND EVALUATIONS ............................ 7 
5.1  Agent Administration ........................................................................................................... 7 
5.2  General Concomitant Medication and Supportive Care Guidelines .................................... 8 
5.3  Evaluations ........................................................................................................................... 8 
5.4  Duration of Therapy and Criteria for Removal From Study .............................................. 11 
5.5  Duration of Follow Up ....................................................................................................... 11 
6. DOSING DELAYS/DOSE MODIFICATIONS....................................................................... 12 
7. ADVERSE EVENT REPORTING REQUIREMENTS ........................................................... 12 
7.1  Investigational Agent Risks: .............................................................................................. 12 
7.2 Adverse Event Characteristics ............................................................................................ 12 
7.3 Recording and reporting  of Adverse Events and SAE ....................................................... 13 
7.4 Reporting and Exclusions ................................................................................................... 13 
    7.5 Reporting of SAE to FDA................................................................................................... 14 
8. 
PHARMACEUTICAL INFORMATION .......................................................................... 14 
8.1 Investigational Agent .......................................................................................................... 14 
8.2 Availability .......................................................................................................................... 14 
8.3 Agent Ordering ................................................................................................................... 15 
8.4 Agent Accountability .......................................................................................................... 15 
8.5 Other agents: ....................................................................................................................... 15 


 
 
iv 
 
9. 
CORRELATIVE/SPECIAL STUDIES ............................................................................. 18 
9.1 Laboratory Correlative Studies ........................................................................................... 18 
10. 
MEASUREMENT OF EFFECT........................................................................................ 19 
11. 
DATA REPORTING / REGULATORY CONSIDERATIONS ....................................... 20 
11.1 Data Collection ................................................................................................................. 20 
    11.1.1 REDCap ......................................................................................................................... 20 
11.1.2 Regulatory Considerations ............................................................................................. 20 
12. 
STATISTICAL CONSIDERATIONS............................................................................... 20 
12.1 Study Design/Endpoints and stopping rules for lack of efficacy ...................................... 20 
12.2 Sample Size/Accrual Rate ................................................................................................. 21 
12.3 Analysis of  Endpoints ...................................................................................................... 21 
12.4 Stopping rules for graft failure and acute graft versus host disease ................................. 21 
REFERENCES ............................................................................................................................. 22 
APPENDIX A ............................................................................................................................... 27 
APPENDIX B ............................................................................................................................... 28 
 


 
 
  
 
1. 
OBJECTIVES 
1.1. 
Primary Objectives  
To evaluate disease free survival and overall survival after allogeneic stem cell 
transplantation with 5-azacytidine priming immediately prior to conditioning  
and Alemtuzumab-based in vivo T- cell depletion in patients with poor risk 
myeloid malignancies in complete morphologic remission. 
1.2. 
Secondary Objectives 
To evaluate the incidence and severity of acute and chronic GVHD after 5-
azacytidine priming prior to conditioning for allogeneic stem cell transplantation 
with Alemtuzumab-based in vivo T-cell depletion in patients with poor risk 
myeloid malignancies in complete morphologic remission.    
 
To evaluate engraftment and chimerism patterns after 5-azacytidine primed 
conditioning for stem cell transplantation with Alemtuzumab-based in vivo T-cell 
depletion in patients with poor risk myeloid malignancies in complete morphologic 
remission.    
 
 To evaluate changes in global gene methylation induced by 5-azacytidine prior to 
conditioning for stem cell transplantation with Alemtuzumab-based in vivo T-cell 
depletion in patients with poor risk myeloid malignancies in complete morphologic 
remission.    
 
 
2. 
BACKGROUND 
 
      2.1 Disease 
 
 
 
Poor risk AML and MDS and allogeneic stem cell transplantation 
 
 
The prognosis of AML with poor risk cytogenetics treated with conventional 
chemotherapy is dismal with 5 year overall survival rates ranging from 4-14%1. 
Similarly, the outlook for patients with intermediate-2 or high grade MDS is grim, 
with median survivals of 1.2 and 0.4 years, respectively2. 
 
 
Allogeneic stem cell transplantation remains the most promising therapeutic modality 
and a multitude of trials have confirmed its potentially curative role for these 
diseases. But even after transplant outcomes are worse for patients with adverse risk 
factors, particularly patients with monosomal karyotypes3.  A study by van Straaten 
of allo transplant for patients with AML or MDS with chromosome 5 and 7 
abnormalities showed a 25% 3-year overall survival rate.  Benefits were confined to 


 
 
2
 
patients younger than 40 years of age with overall survival of 38% versus 8% for 
those older than 404. 
  
 
 
More recent studies for both high risk AML and MDS as well as secondary AML still 
show a long term overall survival rate between 20% and 45%  after allogeneic 
transplants5,6,7.  Recurrence of leukemia constitutes the most important cause of 
treatment failure for patients transplanted in remission. The ultimate objective of our 
proposed study is to develop strategies to further improve the outcomes of allogeneic 
transplantation in high risk patients. 
 
 
 
Evolution of transplant related mortality and relapse rate 
 
In the past decade, the outcome of stem cell transplantation has significantly 
improved, in part due to the advances of conditioning regimens as well as improved 
supportive care. While the risk of non-relapse mortality has been reduced by 52% 
since the years 1993 through 1997, the rates of relapse have undergone a more 
modest reduction of 21%8.Reductions in the intensity of the pretransplant 
conditioning have opened the option of an allogeneic stem cell transplant to patients 
previously thought not fit for the procedure but the success of non-myeloablative 
transplants is compromised by relapse rates ranging from 40-50% (9,10). 
A recent retrospective analysis of over 5900 patients transplanted for AML in CR 
over the last 25 years showed that the improvement in overall survival is mainly due 
to reductions in NRM while relapse rates of the underlying malignancy have 
remained unchanged (11).  The development of conditioning regimens with increased 
antileukemic efficacy but limited toxicity represents a major challenge.  
It is with this purpose that we propose a novel strategy to enhance the susceptibility 
of leukemic cells to the conditioning regimen. 
 
DNA methylation and sensitivity to chemotherapy 
 
Epigenetic silencing through hypermethylation of genes that govern apoptosis can 
confer refractoriness to chemotherapy, a process that may be reversed through 
hypomethylating agents (12, 13). There is now burgeoning preclinical evidence for a 
chemosensitizing effect of the hypomethylating agents 5-Azacytidine and Decitabine 
for a range of hematologic malignancies and solid tumors as well as their ability to 
restore disrupted proapoptotic pathways (13, 14, 15, 16, 17 ). 
Furthermore, a prospective study conducted at our center by Scandura et al 
demonstrated the feasibility of priming with decitabine prior to induction 
chemotherapy for AML (18).  Although it was a phase 1 study not designed to assess 
efficacy there was an encouraging CR rate of 83% for intermediate and poor risk 
AML patients. 
In the current study we plan to use azacytidine based chemosensitization prior to 
transplant with a well-established conditioning regimen,  
 
 


 
 
3
 
2.2 Investigational Agent 
 
 
 
5-Azacytidine 
 
5-Azacitidine (4-amino-1-β-D-ribofuranosyl-s-triazin-2(1H)-one) is an analog of 
the naturally occurring pyrimidine nucleoside, cytidine. It differs from cytidine 
in having nitrogen in the 5-position of the heterocyclic ring 
Therapeutic classification: At lower doses acts as DNA-hypomethylating agent. 
At higher doses acts as a cytidine analog. 
 
Pharmacological data:  5-azacytidine is supplied as a lyophilized powder in 
single-use vials containing 100 mg of each 5-azacytidine and mannitol as a 
freeze dried cake or powder. It is reconstituted with 4ml of sterile water per 100 
mg of drug. 
Dosing information: The usual dose is 75 to 100 mg per m2 by subcutaneous 
injection daily for 7 days, repeated every 4 weeks. 
Drug interactions: none known 
Side effects: Myelosuppression, with neutropenia, thrombocytopenia, anemia, 
and febrile neutropenia is the major dose limiting toxicity of 5-azacytidine. 
Nausea, vomiting, diarrhea, dyspepsia and abdominal pain are common. 
Signs of CNS depression, including lethargy, confusion and coma have been 
reported. 
Laboratory abnormalities such as hyperglycemia, hypomagnesemia, 
hypokalemia, and elevations of liver function tests are common. 
Mechanism of action: At lower doses, 5-azacytidine causes hypomethylation of 
DNA. In neoplastic cells, this hypomethylation can either induce apoptosis or 
restore normal function to genes that are critical for the control of cellular 
differentiation, proliferation and apoptosis. At higher doses, it acts as a cytidine 
analog, becoming incorporated into nucleic acids, and causing DNA transcription 
and replication errors. Nonproliferating cells are relatively insensitive to 5-
azacytidine. 
Pharmacokinetics/metabolism: The currently available form of the drug is not 
orally bioavailable. Following administration, the drug is activated inside cells to 
its triphosphate form. It is metabolized in the liver and excreted primarily in the 
urine. The elimination half time is about 4 hours. 
 
2.3Rationale 
 
We are basing the conditioning regimen and graft versus host prophylaxis on a study 
previously published by van Besien et al. combining a Fludarabine/Melphalan based 
reduced intensity conditioning regimen with Alemtuzumab as an in vivo T-cell 
depleting agent with standard risk AML and MDS as indication.  While retaining the 
efficacy of the prior regimen of Fludarabine/Melphalan without T-cell depletion, this 
regimen has shown remarkably low rates of grade 3-4 acute GVHD (10%) and 
extensive chronic GVHD (18%). Overall survival at 18 months was 48% (19,20,21). 


 
 
4
 
 
The disappointing lack of improvement in relapse rates for AML post allogeneic stem 
cell transplantation in the last 20 years despite multiple variations of conditioning 
regimens has called our attention to new ways of increasing the therapeutic efficacy of 
already existing conditioning regimens (8,9). 
 
Given the wealth of preclinical data on the chemotherapy sensitizing effect of 
hypomethylating agents on a wide range of tumors (12-17) and the proof of principle 
of clinical feasibility of this concept in AML (18) there is rationale to employ 
epigenetic priming before conditioning for an allogeneic stem cell transplant. In the 
study conducted by Scandura et al, increased hematologic toxicity had been a concern 
initially, but in fact there was a suggestion of more rapid platelet recovery and no 
delay in neutrophil recovery in the patients undergoing pre induction Decitabine, 
dispelling this fear. 
Furthermore, in the case of allogeneic transplantation, hematologic toxicity is not a 
major concern as recovery is based on engraftment of allogeneic cells and given the 
short half life of 5-Azacytidine (4 hours), there should no longer be any clinically 
relevant serum drug levels at the time of transplantation. 
 
The dosing of 5-azacytidine is derived from both clinical experience and preclinical 
efficacy studies comparing the hypomethylating activity of 5-azacyidine and 
decitabine which showed a 2 to 10 fold stronger hypomethylating activity on AML 
cells for decitabine compared to 5-azacytidine.(22). 
The routine dose for 5-azacytidine to treat MDS is 75 mg/m2 for 7 days. (23). Our 
epigenetic priming study employed decitabine at 20 mg/m2 /d either as 1h infusion or 
24h CVI for 3, 5 or 7 days and no significant difference in the global hypomethylating 
activity was found. 
Given the possibility of administering 5-azacytidine subcutaneously without the need 
for hospital admission we chose this drug over decitabine. A dosing schedule of 50 
mg/m2/d for 5 days appears sufficient to induce global hypomethylation in the genome 
of hematopoietic cells. This hypothesis will be verified by pre- and post-priming 
measurements of the methylation status of the HIST1H2AA gene in bone marrow 
samples (see below).  
 
2.4 Correlative Studies Background 
 
 We will use the pharmacodynamic measurement of DNA hypomethylation to assess 
and efficacy of our 5-azacytidine dosing schedule. At the dose level used in this 
protocol, 5-azacytidine primarily acts to induce DNA hypomethylation and the site and 
extent of demethylation is thought to underlie its clinical activity. Previous studies 
have established that 5-azacytidine is a potent hypomethylating agent capable of 
decreasing both universal cytosine methylation (at repetitive elements, or in total 
hydrolyzed genomic DNA) and select CpG Island methylation.  


 
 
5
 
As a measure of 5-azacytidine’s ability to induce genome wide hypomethylation we 
will measure the methylation status of the HIST1H2AA gene in in CD 34+ bone 
marrow cells, which can serve as a surrogate marker for global gene methylation status 
(18). 
 
We will then compare patient samples obtained before, and immediately following 
priming treatment to establish the pharmacodynamic activity of 5-azacytidine.  
3. PATIENT SELECTION 
 
3.1 
Inclusion Criteria 
 
3.1.1 Patients must have histologically or cytologically confirmed AML or MDS as 
specified below:  
 
 
Acute myeloid leukemia with poor risk cytogenetics (1) in complete 
morphologic remission.  These include: del (5q)/-5, del (7q)/-7, abn 3q, 9q, 
11q, 20q, 21q, 17p, t(6;9), t(9;22) or complex karyotypes (≥ 3 unrelated 
abnormalities) 
 
 
 
Acute myeloid leukemia with either Flt-3-, TET 2-, p53-, DNMT3A- or ASXL 
1-mutation, mutations of genes involved in the chromatin/spliceosome 
category (EZH2, SRSF2, U2AF1, ZRSR2), BCOR and RUNX1, as well as MLL 
rearrangement EVI1 overexpression in complete morphologic remission 
 
Acute myeloid leukemia with a white blood cell count of ≥50000/mcL at 
presentation in first complete morphologic remission 
 
Acute myeloid leukemia in first complete morphologic remission, having 
required more than one course of induction chemotherapy to attain 
remission status 
 
 
Acute myeloid leukemia, all types, excluding M3 (Promyelocytic leukemia) 
in second or higher complete morphologic remission  
 
 
Myelodysplastic syndromes (MDS) (intermediate-2, high risk and CMML 
with bone marrow blasts <5%) 
 
Secondary acute myeloid leukemia on the basis of prior MDS or prior 
myeloproliferative neoplasm (MPN) in complete morphologic remission  
 


 
 
6
 
3.1.2 Age >18 years. 
 
3.1.3 Life expectancy not severely limited by concomitant disease. 
 
3.1.4 KPS at least 70. 
 
3.1.5 Adequate organ function as defined below: 
Serum Bilirubin: <2.0 mg/dL 
 
 
      ALT (SGPT): <3X upper limit of normal 
Creatinine Clearance: >60 mL/min (eGFR as estimated by the modified 
MDRD equation) 
 
Patients with decreased LVEF or abnormal PFTs will be evaluated by 
cardiology or pulmonary prior to enrollment on this protocol and their 
eligibility will be determined by the compound of their clinical 
cardiopulmonary status and testing results as patient with abnormal echo 
and /or PFTs can have adequate cardiopulmonary functioning 
 
3.1.6 Ability to understand and the willingness to sign a written informed consent        
document. 
 
3.2 
Exclusion Criteria  
 
 
 
 
 
3.2.1 Evidence of chronic active hepatitis or cirrhosis. 
 
3.2.2 HIV infection. 
 
3.2.3 Uncontrolled illness including, but not limited to, ongoing or active infection,   
symptomatic congestive heart failure, unstable angina pectoris, cardiac 
arrhythmia, or psychiatric illness/social situations that would limit 
compliance with study requirements.  
 
3.2.4 Pregnant and lactating women are excluded from the study because the risks 
to an unborn fetus or potential risks in nursing infants are unknown. 
 
3.2.5 There are no prior therapies or concomitant medications that would render 
the patients ineligible. 
 
4. REGISTRATION PROCEDURES 
 
Central Patient Registration 
 
Patients will be centrally registered with the Weill Cornell Medical College 
(WCMC), Division of Hematology and Medical Oncology Clinical Research Office.  


 
 
7
 
To register a patient, fax the following documents to the Clinical Research Office at 
(646) 962-1610: 
 
 WCMC Patient registration form 
 
First and last page of the fully executed informed consent form, plus 
additional pages if checkboxes for correlative studies are required. 
 Fully executed HIPAA research authorization form 
 Eligibility checklist signed and dated by investigator and research nurse 
 Documentation of any eligibility waivers granted  
 For inpatients, signed consent documentation template 
 
Central registration information is reviewed and entered into the HemOnc centralized 
research database.  Documentation of patient registration will be faxed to the 
Investigational Pharmacy to allow for release of study agent. 
 
5. 
TREATMENT PLAN, DONOR SELECTION AND EVALUATIONS 
 
5.1 
Agent Administration 
 
Treatment will be administered on an outpatient basis.   
 
5-Azacytidine priming 
 
5-Azacytidine 50 mg/m2 subcutaneously daily at the same time on days -11, -10, -9,  
-8 and -7. This will be administered on an outpatient basis.  
 
Hospital admission will usually take place on the first day of the conditioning 
regimen. 
 
Conditioning with Fludarabine-Melphalan-Alemtuzumab 
 
Fludarabine 40 mg/m2 intravenously daily at the same time over 30 minutes on days -
6,-5,-4,-3.  
Melphalan 140 mg/m2 IV on day -3.   
Alemtuzumab, 30 mg subcutaneously on days -4 and -2 for unrelated donors, and on 
day -2 for related donors. 
 
Total Body Irradiation (TBI) 
 
Given a recent publication indicating better disease control for this condition regimen 
with the inclusion of low dose TBI (24), patients will receive 2 doses of TBI of 200 
cGy each on one day of the conditioning regimen (between days -6 and -3) 
 


 
 
8
 
Stem cell infusion 
 
On day 0 the stem cell product will be infused according to BMT unit policy. 
 
Allogeneic hematopoietic stem cells (HSC) may be infused fresh or after 
cryopreservation depending on donor availability. 
 
All HSC procurement through the NMDP will done in strict compliance with the 
protocols, policies, and procedures established by the NMDP. 
 
 
 
 
 
Graft versus Host Disease prophylaxis 
 
Tacrolimus 0.03 mg/kg/day IV CI over 24 hr from 4 PM day -2 until engraftment or 
when patient is able to take PO, then tacrolimus 0.09 mg/kg PO in 2 divided doses.  
Tacrolimus should be given at full dose to maintain levels of 5-15 ng/mL through day 
100. Thereafter, tacrolimus will be tapered by 20% every week. 
 
In recipients of unrelated donor transplants, tacrolimus will be continued until day 
180. Thereafter it will be tapered by 20% per week. 
 
Infection, toxicity or other clinical circumstances may prompt earlier discontinuation 
or adjustment of doses. In the presence of GVHD, a clinical decision by the attending 
physician will determine if tacrolimus can be tapered or should be continued. PO 
tacrolimus can be used when IV access for CI tacrolimus is unavailable.  
 
       5.2        General Concomitant Medication and Supportive Care Guidelines 
Infection prophylaxis, growth factor administration and supportive care will be as 
per BMT unit policy. CMV prophylaxis with high dose ganciclovir-acyclovir-
valacyclovir is strongly recommended. 
 
       5.3        Evaluations 
 
 
 
Pre-Conditioning Evaluation 
 
Pre-Transplant Evaluation will follow transplant program policies. 
 
Prior to of Azacytidine priming, a bone marrow biopsy and aspiration will be 
performed to ascertain the patient’s status in complete morphologic remission. This is 
part of the standard of care evaluation for every patient prior to an allogeneic stem 
cell transplantation. 
 
An additional 3-5 cc of bone marrow aspirate will be set aside for measurement of 
HIST1H2AA methylation level. 
 


 
 
9
 
On the first day after completion of the course of 5-Azacytidine, another bone 
marrow aspiration will be obtained and again there will be sample of 3-5 cc set aside 
for HIST1H2AA methylation level evaluation. This bone marrow aspiration is solely 
for research purposes and will serve to evaluate the changes in global gene 
methylation in response to 5-Azacytidine as well as the impact of 5-Azacytidine on 
the morphology and cellularity of the bone marrow. 
 
Post-transplant Evaluation  
 
Post-transplant Evaluation will follow the routine for the allogeneic transplant 
patient.  The tests outlined in Table 1 are strongly recommended, but can be adjusted 
as dictated by clinical circumstances.  
 
Table 1: The follow-up schedule for scheduled study visits is outlined below. These tests may be 
adjusted as warranted by clinical circumstances and evolving transplant policy. Please also refer 
to institutional transplant work up guidelines.  
 
 
 
Prior 
to 
aza 
(between 
day-53 
and -11) 
After 
aza 
Day -
6 
14 
21 
28 
60 
70 
100 
180 
365 
q365 
Physical exam, height, weight, and KPS 
performance status 
X 
 
 
 
X 
 
 
X 
X 
X 
 
GVHD and other morbidity assessments5 
 
 
 
X 
 
 
X 
X 
X 
 
Toxicity assessments 
X 
 
 
 
X 
 
 
X 
X 
X 
 
Electrocardiogram 
X 
 
 
 
 
 
 
 
 
 
 
Infectious disease titers3 
X 
 
 
 
 
 
 
 
 
 
 
Chest CT or x-ray4 
X 
 
 
 
 
 
 
 
 
 
 
LVEF, or shortening fraction4 
X 
 
 
 
 
 
 
 
 
 
 
DLCO, FEV I and FVC and 02-saturation4 
X 
 
 
 
 
 
 
 
 
 
 
HLA typing6 
X 
 
 
 
 
 
 
 
 
 
 
B-HCG serum pregnancy test (pre-
menopausal females only) within 4 
weeks of conditioning 
X 
 
 
 
 
 
 
 
 
 
 


 
 
10
 
 
Prior 
to 
aza 
(between 
day-53 
and -11) 
After 
aza 
Day -
6 
14 
21 
28 
60 
70 
100 
180 
365 
q365 
CBC1 , differential, platelet count, and 
blood chemistries2 
X 
X 
X 
X 
X 
 
 
X 
X 
X 
 
Bone marrow biopsy and aspirate for 
pathology 
X 
X 
 
X 
 
 
X 
X 
X 
X 
Chimerism 
X 
X 
 
X 
X 
X 
X 
X 
X 
 
Lymphocyte Subsets and Ig Levels 
X 
 
 
 
X 
 
 
X 
X 
X 
 
Pneumococcal antibody levels5 
X 
 
 
 
 
 
 
 
X 
X 
 
HLA Antibodies7 
X 
 
 
 
 
 
 
 
Research Blood and Marrow Samples 
 
X 
(this 
will 
occur 
within 10 
days prior 
to 
Aza 
priming) 
X 
 
 
X 
X 
 
X 
X 
X 
X 
 
Note: 
*The exact day of the tests is approximate. Tests can be scheduled several days before or after. 
The window is up to five days before and after in the first three weeks. Up to seven days before 
and after in the first 100 days and up to one month before and after at subsequent time points. 
**Baseline tests: see also section 7.0 
1. CBC performed at least three times a week from Day 0 until ANC >500 mcL for three days 
after nadir. CBC performed twice weekly until Day 28. CBC performed approximately 
weekly after Day 28 until 12 weeks post-transplant. 
2. Blood chemistries to include a standard complete metabolic panel, LDH, and magnesium. 
3. Infectious disease titers include: CMV, Hepatitis panel (HepBSAb, HepBSAg, HepB Core 
Ab, HepCAb), herpes simplex virus, syphilis, HIV and HTLV I/II antibody. 
4. CT scan and PFTs can be obtained up to 2 months prior to conditioning, unless there have 
been intercurrent infections affecting the respiratory tract. 
5. Echocardiogram and ECG can be obtained up to 2 months prior to conditioning, unless the 
patient received cardiotoxic chemotherapy or had any cardiac event (ie MI) in the interim. 
6. HLA Typing can occur at any time prior to the administration of 5-azacytidine as HLA 
Typing does not change. 


 
 
11
 
7.  Testing for HLA antibodies can be performed at any time prior to the administration of 5-
azacytidine as the frequency of testing will vary depending on the donor being used for 
transplant. 
 
Blood and bone marrow samples 
 
 Samples of bone marrow will be obtained at the following time points: 
 
 
 
Prior to priming with 5-Azacytidine (bone marrow) within 10 days before therapy – 
this is research-specific 
 
After priming with 5-Azacytidine on day -6 (bone marrow) – this is research-specific 
 
Day +28 +/-  4 days (BM and blood) 
Day +60 +/- 7 days (Blood) 
 
Day +100 +/-  7 days (BM and blood) 
 
Day +180 +/- 7 days (BM and blood) 
 
At relapse (BM and blood) 
 
One year and yearly thereafter (BM and blood) 
 
The 2 bone marrow samples will be examined for global gene methylation status by 
measuring the methylation status of HIST1H2AA by Dr. Joseph Scandura (see section 9). 
 
 
    5.4        Duration of Therapy and Criteria for Removal from Study 
 
As the stem cell transplantation is a non-repetitive intervention there will be no cycles 
of treatment. Patients will be followed for disease free and overall survival as well as 
for secondary endpoints.  They will remain on study until one of the following occurs 
 
o Disease recurrence 
 
o Intercurrent illness that prevents further administration of treatment 
 
o Unacceptable adverse event(s) 
 
o Patient decides to withdraw from the study, or 
 
o General or specific changes in the patient’s condition render the patient 
unacceptable for further treatment in the judgment of the investigator. 
 
 
5.5 
Duration of Follow Up 
 


 
 
12
 
Patients will be followed for 5 years after removal from study or until death, 
whichever occurs first.  Patients removed from study for unacceptable adverse events 
will be followed until resolution or stabilization of the adverse event. 
6. DOSING DELAYS/DOSE MODIFICATIONS 
  
Given the higher than expected rate of grade 3 or 4 renal toxicity after the first 12 patients were 
treated with azacytidine 75m/m2 daily x 5 days (2 with grade 3, 2 with grade 4) we will reduce 
the dose of Azacytidine to 50 mg/m2 daily x 5 days. Should renal toxicity grade 3 or 4 occur by 
day 15 in 2 or more of the first six patients, then the dose of azacytidine will be further reduced 
to 32 mg/m2 / day x 5 days. If 2 or more grade 3 or 4 toxicities occur among the first six patients 
at the 32 mg/m2/d level, the trial will be considered too toxic and closed for accrual. If one or 
fewer grade 3 or 4 renal toxicities occur, accrual will continue at this dose level with close 
continuous observation for renal toxicity. 
7. ADVERSE EVENT REPORTING REQUIREMENTS  
 
Adverse event (AE) monitoring and reporting is a routine part of every clinical trial.  The 
investigator will be required to provide appropriate information concerning any findings that 
suggest significant hazards, contraindications, side effects, or precautions pertinent to the 
safe use of the drug under investigation.  Safety will be monitored by evaluation of adverse 
events reported by patients or observed by investigators or research staff, as well as by other 
investigations such as clinical laboratory tests, x-rays, electrocardiographs, etc. 
For the purposes of this study, the pre-conditioning 5-azacytidine administration is the only 
investigational component of the treatment. All other components (conditioning therapy, 
gvhd prophylaxis, transplant etc.) are considered standard interventions.  
 
7.1 Investigational Agent Risks:  
 
5-Azacytidine: 
 
Myelosuppression, with neutropenia, thrombocytopenia, anemia, and febrile 
neutropenia is the major dose limiting toxicity of 5-azacytidine. 
Nausea, vomiting, diarrhea, dyspepsia and abdominal pain are common. 
Signs of CNS depression, including lethargy, confusion and coma have been reported. 
Laboratory abnormalities such as hyperglycemia, hypomagnesemia, hypokalemia, and 
elevations of liver function tests are common. 
 
7.2 Adverse Event Characteristics  
 
 
CTCAE term (AE description) and grade:  The descriptions and grading scales 
found in the revised NCI Common Terminology Criteria for Adverse Events 


 
 
13
 
(CTCAE) version 4.0 will be utilized for AE reporting.  A copy of the CTCAE 
version 4.0 can be downloaded from the CTEP web site (http://ctep.cancer.gov). 
 
 
Attribution of the AE: 
- 
Definite – The AE is clearly related to the study treatment. 
- 
Probable – The AE is likely related to the study treatment. 
- 
Possible – The AE may be related to the study treatment. 
- 
Unlikely – The AE is doubtfully related to the study treatment. 
- 
Unrelated – The AE is clearly NOT related to the study treatment. 
 
7.3 Recording and Reporting of Adverse Events and SAE 
 
 All infections, acute GVHD and any other grade 3 or higher adverse events will be 
recorded on a patient specific adverse event log.  The AE log will be maintained by the 
research staff and kept in the patient’s research chart. 
 Stem Cell transplant is a complex procedure with prolonged initial admission and 
numerous immediate and delayed complications as well as frequent readmissions. 
 
 Expected adverse events are those listed in the consent form and include regimen-related 
toxicities, myelosuppression, opportunistic infections and GVHD.   Expected adverse 
events of grade III and higher CTC severity will be captured in the transplant database 
and reported to the IRB upon request.  
 
 
 Grade three or higher adverse events that are judged to be unexpected and possibly 
related to the investigational procedure, will be reported to the study chairman within 48 
hours of investigator notification. Such events will be reported to the local IRB within 
the institution’s prescribed time period. 
 
 All fatal Adverse Events will also be reported to the study chairman within 48 hours and 
reported to the local IRB within the institution’s prescribed time period. 
 
 
 
 
 
Immediate Reporting 
Report quarterly 
Hematopoietic Toxicity 
Graft failure and death 
Time to Hematopoietic 
recovery 
Extramedullary Toxicity 
Fatal toxicity.  
Grade III-IV Toxicity 
deemed unexpected and 
possibly related to the 
investigational procedure 
Grade III-IV toxicities not 
deemed 
expected/unrelated 
Infections 
Fatal  
Grade II-IV 


 
 
14
 
Acute GVHD 
Fatal or Grade IV 
Grade II-III 
Chronic GVHD 
Fatal  
Limited and extensive 
 
 
 
 
7.4     Reporting and Exclusions 
 
 
 
 
7. 4.1  Evaluation of toxicity.  All patients will be evaluable for toxicity 
from the time of their first treatment with 5-Azacytidine. 
 
 
 
 
7. 4.2  Evaluation of efficacy.  All patients included in the study will be 
assessed for the specified endpoints if they have proceeded through stem 
cell transplantation   
 
7.5 Reporting of SAE to FDA   
 
If a reportable AE occurs on this study, the event will be filed on a 
MedWatch form with the FDA. 
 
CDER INDs:  
Food and Drug Administration 
Center for Drug Evaluation and Research 
Division of Oncology Products  
5901-B Ammendale Road  
Beltsville, MD 20705-1266  
 
8. 
PHARMACEUTICAL INFORMATION 
 
A list of the adverse events and potential risks associated with Investigational Agent can be 
found in Section 7.1. 
 
8.1 
Investigational Agent  
 
5-azacytidine is supplied as a lyophilized powder in single-use vials containing 100 
mg of each azacytidine and mannitol as a freeze dried cake or powder. It is 
reconstituted with 4ml of sterile water per 100 mg of drug. 
 
 
8.2 
Availability 
 
5-azacytidine is an approved drug and will be commercially available. 


 
 
15
 
 
 
 
8.3 
 Agent Ordering 
 
 
 
See above 
 
8.4 
Agent Accountability 
 
The investigator, or a responsible party designated by the investigator, will maintain a 
careful record of the inventory and disposition of all agents on a Drug Accountability 
Record Form (DARF).  
 
8.5 
Other agents and procedures: 
 
 
Melphalan: 
 
Therapeutic Classification: Alkylating agent. 
Pharmaceutical data: Supplied as 50 mg vials reconstitute in 10 ml of diluent. 
Stability and storage: Reconstituted solution retains 90% potency for about 3 hours at 30o 
C. Storage at 5o C results in precipitation. Intact packages can be stored at room 
temperature. 
Route of Administration: intravenous, oral, intraperitoneal. 
Side effects: Dose limiting toxicity is hematological. Possible delayed toxicity in patients 
with irradiated bone marrow. Gastrointestinal toxicity is infrequent and usually mild; 
occasional azotemia has been reported in certain cases. Tissue necrosis may result if 
infiltration occurs.  
Mechanism of action: Interferes with DNA replication and transcription of RNA and 
ultimately results in the disruption of nucleic acid function. 
Human Pharmacology: Melphalan is incompletely and erratically absorbed, has a 
prolonged plasma half-life, and is excreted largely unchanged in the urine. Intravenous 
melphalan result in very high peak concentration, plasma levels followed by an initial 
rapid decay curve (half-life 30-60 minutes). Most of the administered dose is recovered in 
the urine, almost none in the feces, and no significant quantity of any metabolite has been 
identified.   
Human Toxicology: Myelosuppresion is the major toxicity and is dose related. Nausea and 
vomiting is almost uniformly observed, mucositis and diarrhea have also been observed. 
Transient rises in BUN and creatinine have occurred with high dose melphalan as also 
acute renal failure. Hypersensitivity reactions to high dose melphalan have been reported. 
 
Fludarabine: 
 
Fludarabine is the 2-fluoro, 5-phosphate derivative of vidarabine.     


 
 
16
 
 
DOSING INFORMATION:  Doses of 25 mg/mL(2)/day (30-minute infusion) for 5 days 
every 4 weeks has been effective previously treated patients with chronic lymphocytic 
leukemia; in non-Hodgkin's lymphoma, a loading dose of 20 mg/m(2) intravenously 
followed by a continuous intravenous infusion of 30 mg/m(2)/24 hours for 48 hours, has 
been effective; dose reductions are suggested in renal insufficiency. 
 
PHARMACOKINETICS:  Following intravenous administration, fludarabine phosphate is 
rapidly dephosphorylated to 2-fluoro-vidarabine, which subsequently enters tumor cells 
and is phosphorylated to the active triphosphate derivative; peak plasma levels of 
2-fluoro-vidarabine have ranged from 0.3 to 0.9 mcg/mL following a short infusion of 25 
mg/m(2) fludarabine; 24% of a dose of fludarabine is recovered in the urine as 
2-fluoro-vidarabine; the elimination half-life of 2-fluoro-vidarabine is 9 hours. 
     
CAUTIONS:  Myelosuppression, particularly neutropenia, is the predominant adverse 
effect; a severe neurotoxicity has been observed, mainly with higher doses; other adverse 
effects include nausea, vomiting, diarrhea, stomatitis, skin rash, and somnolence; 
pneumonitis has been reported in 1 patient.        
                                       
CLINICAL APPLICATIONS:  Intravenous fludarabine has been highly effective in 
heavily pretreated patients with chronic lymphocytic leukemia; the drug has also produced 
responses in patients with non-Hodgkin's lymphoma and acute leukemia; however, 
neurotoxicity has been a major concern, even with low doses, and more studies are needed 
to clarify its ultimate place in therapy. 
 
Alemtuzumab: 
 
Therapeutic Classification: Humanized monoclonal antibody 
 
Side effects: The infusion of Alemtuzumab has been associated with fever, nausea, 
headache, vomiting, rash, chills and rigor. Occasionally hypotension and bronchospasm 
have been reported. This can be managed by adequate premedication. 
 
Pharmaceutical Data: Alemtuzumab is supplied as a purified preparation diluted in PBS 
with 0.05 mm EDTA. Each Ampoule contains 30 mg of Alemtuzumab. 
 
Administration: 20 mg of Alemtuzumab is given intravenously or subcutaneously for 3 to 
5 days. 
 
Mechanism of Action: Alemtuzumab is a monoclonal antibody directed against CD52, an 
epitope that is abundantly expressed on T- and B- lymphocytes, but not on NK cells. It has 


 
 
17
 
been extensively used for the prevention of GVHD both in vitro and in vivo. 
 
 
Tacrolimus: 
 
  
TACROLIMUS is a macrolide compound with potent immunosuppressant properties.                              
    
DOSING INFORMATION:  TACROLIMUS is usually given intravenously initially in 
doses of 0.03 mg/kg/day for 3 days, followed by conversion to oral therapy (0.09 mg/kg 
twice daily); dose adjustments are required in patients with hepatic dysfunction.                      
   
PHARMACOKINETICS:  The oral absorption of TACROLIMUS is erratic and 
incomplete; absolute bioavailability is approximately 25%; peak serum levels are seen 1 to 
4 h after an oral dose, and therapeutic serum concentrations have ranged from 0.2 to 6 
ng/mL; TACROLIMUS is extensively metabolized in the liver, with only small amounts of 
unchanged drug (2% or less) being recovered in the urine; the elimination half-life of 
TACROLIMUS is approximately 10 h.     
                                    
   
CAUTIONS:  Common adverse effects of TACROLIMUS are headache, hyperesthesia, 
tremors, circumoral numbness, insomnia, nausea, abdominal discomfort, and appetite       
insomnia, nausea, abdominal discomfort, and appetite changes; all of these effects occur 
primarily with IV TACROLIMUS and are more frequent during combined use of 
TACROLIMUS and CYCLOSPORINE; other adverse effects include nephrotoxicity, 
hyperkalemia, hyperuricemia, hyperglycemia, dysphasia, photophobia, flushing, and 
lymphoproliferative disorder; unlike CYCLOSPORINE, hirsutism, gingival hyperplasia, 
and hypertension are generally not seen with TACROLIMUS; combined therapy with 
CYCLOSPORINE has resulted in increases in cyclosporine serum levels and more severe 
nephrotoxicity.               
 
CLINICAL APPLICATIONS:  TACROLIMUS is primarily indicated for prophylaxis of 
organ rejection in patients receiving an allogeneic liver transplant.                           
 
 
Bone marrow biopsy and aspiration: 
 
-Bleeding 
 
-Pain 
 
-Infection 
 
All these risks are minimized by application of sterile technique, local anesthesia and 
pressure dressing. 
 
 
Stem Cell infusion: 
 
Chest pain 
Chills 


 
 
18
 
Drop in blood pressure 
Fever 
Flushing 
Headache 
Hives 
Nausea 
Pain 
Shortness of breath 
 
The risks are minimized by premedication with Acetaminophen and Diphenhydramine 
and close monitoring during the stem cell infusion enabling the team to intervene 
immediately should an adverse event occur. 
 
Stem Cell Transplantation- generic risks and complications: 
 
Graft failure 
Acute and chronic graft versus host disease 
Hemorrhage to vital organs 
Venoocclusive disease of the liver (Sinusoidal obstruction syndrome – SOS) 
Anemia, leukopenia, thrombocytopenia 
Infections (bacterial, viral, fungal, protozoal) 
Mucositis of oral cavity, esophagus, stomach and bowels 
Damage to kidneys, liver, lungs and heart 
Nausea, vomiting and resulting malnutrition 
Pain 
Cataracts 
Premature menopause and infertility 
Osteoporosis 
Avascular necrosis of bones due to steroid therapy 
Thyroid dysfunction 
Death 
 
The patients are monitored on a continuous basis for any of these events and standard 
prophylaxis for graft versus host disease, infections, organ damage is provided through 
our standard operating procedures. 
Furthermore, the risk of organ damage is minimized by pre transplant organ function 
testing, such as echo, pulmonary function tests and laboratory testing for renal and 
hepatic function. 
9. 
CORRELATIVE/SPECIAL STUDIES 
 
9.1 
Laboratory Correlative Studies 
Bone marrow samples (3 to 5 mL) will be obtained by the Investigator, or co-
investigator, within 10 days of the first 5-azacytidine dose and on the day after the last 


 
 
19
 
dose of 5-azacytidine (day -6).  Samples will be processed in the laboratory of Dr. Joseph 
Scandura at Weill Medical College of Cornell University.  
These samples will serve to measure global gene methylation status through 
HIST1H2AA methylation evaluation. 
10. 
MEASUREMENT OF EFFECT 
 
Disease-free survival is the primary endpoint. Bone marrow biopsies at days +30, 
+100, +180, +365 and then annually will serve as the means to detect recurrent 
disease. As the patients will be monitored after stem cell transplantation on a very 
regular basis (at least weekly for the first 3 months), it will be at the investigators 
discretion to perform a bone marrow biopsy outside of these time points if a change 
in blood counts raises the suspicion of recurrence or graft failure. Relapse will be 
recorded by the day of initial detection of malignant cells, if these cells were on 
subsequent testing confirmed to be increasing in number. The diagnosis of disease 
recurrence will be based on clinical and pathological criteria. 
 
Toxicity will be scored according to the Bearman criteria,(25, 26) (Appendix B) and 
acute GVHD will be scored according to the criteria proposed by Przepiorka et 
al.(24) Chronic GVHD will be scored according to appendix D. Limited Chronic 
GVHD is defined as GVHD with limited skin involvement only or presenting with 
liver function abnormalities only. All other presentations of chronic GVHD are 
defined as extensive and will require treatment. 
 
High risk extensive chronic GVHD is characterized by the presence of 
thrombocytopenia (<100,000/mm3). (12) 
 
Myeloid engraftment will be defined as the first day in which the ANC is > 500/mm3 
for three consecutive days. Cytogenetic and chimerism studies will be performed to 
confirm donor origin. 
  
Platelet engraftment will be defined as the first day the platelet count is > 
20,000/mm3 without transfusion support for seven consecutive days.  
 
Failure to engraft will be defined as lack of evidence of hematopoietic recovery 
(ANC < 500/mm3 and platelet count < 20,000/mm3 ) by day +35, confirmed by a 
biopsy revealing a marrow cellularity < 5%. Graft failure will be defined as initial 
myeloid engraftment by day +35, documented to be of donor origin, followed by a 
drop in the ANC to < 500/mm3 for more than three days, independent of any 
myelosuppressive drugs, severe GVHD, CMV, or other infection.  
 
Graft rejection will be defined as graft failure with documentation of return of 
recipient hematopoiesis as determined by cytogenetic and/or chimerism studies. 
 


 
 
20
 
11. 
DATA REPORTING / REGULATORY CONSIDERATIONS 
11.1 Data Collection 
 
The data collection plan for this study is to utilize REDCap to capture all treatment, 
toxicity, and efficacy data for all enrolled patients.  
11.1.1 
REDCap 
 
REDCap (Research Electronic Data Capture) is a free data management software 
system that is fully supported by the Weill-Cornell Medical Center CTSC.  It is a tool 
for the creation of customized, secure data management systems that include Web-
based data-entry forms, reporting tools, and a full array of security features including 
user and group based privileges, authentication using institution LDAP system, with a 
full audit trail of data manipulation and export procedures.  REDCap is maintained on 
CTSC-owned servers that are backed up nightly and support encrypted (SSL-based) 
connections.  Nationally, the software is developed, enhanced and supported through 
a multi-institutional consortium led by the Vanderbilt University CTSA. 
 
 
11.1.2 Regulatory Considerations 
 
All protocol amendments and consent form modifications will be made by the 
Principal Investigator. 
12. 
STATISTICAL CONSIDERATIONS 
 
12.1 Statistical Design 
 
The primary endpoint will be disease-free survival (DFS), as measured from the 
start of the treatment to the date of either documentation of disease recurrence or death.  
The diagnosis of disease recurrence will be based on clinical and pathological criteria.  
All patients will be observed for a minimum of one year.  The 1-year DFS for patients in 
the study cohort has been estimated to be 35%; therefore, the target 1-year DFS for the 
regimen under evaluation will be 50%.  We will define “evaluable” patients as patients 
who met eligibility requirements, have initiated therapy, and were not removed from the 
study for non-compliance or patient withdrawal within the first 12 months.  Sample size 
recommendations for the phase II design are determined according to A’Hern’s exact 
single-stage phase II design (28).  We project a DFS at 1 year of 35%, below which the 
regimen will be unacceptable, and a DFS at 1 year of 50%, above which the regimen will 
be considered worthy of further exploration.  The null hypothesis that the 1-year DFS 
proportion is less than or equal to 35% will be tested against the alternative hypothesis 
that the 1-year DFS proportion is greater than or equal to 50%. 
 
The sample size computations were performed assuming a 10% level of significance and 
80% power.  Patients will be continuously accrued throughout the study up to a 
maximum of 49 patients.  The new regimen will be declared effective and worthy of 


 
 
21
 
further testing if 22 or more patients are disease-free after 12 months of follow-up among 
the 49 patients entered into the study.  This exact single-stage design yields a ≥ 0.80 
probability of a positive result if the true DFS proportion at 12 months is ≥ 50%.  It yields 
a ≥ 0.90 probability of a negative result if the true DFS proportion at 12 months is ≤ 35%.  
A 95% confidence interval constructed around the expected 12-month DFS proportion of 
50% can be estimated to be within ± 14.0% of the observed DFS proportion.  Assuming 
10% are unevaluable/ineligible, we anticipate that a total of 55 patients will be enrolled in 
the study. 
 
12.2     Stopping Rules for Graft Failure, Transplant-related Mortality and 
Acute Graft Versus Host Disease (Acute GVHD) 
 
Our hypothesis is that the graft failure rate is ≤5%. The stopping rule to be employed 
is if 2 of the first 7, 3 of the first 10, or 4 of the first 20 patients have graft failure, the 
trial will be terminated. 
 
The expected transplant related mortality should not exceed 25%. The trial will be 
considered for termination for safety concerns if by day +180, 4 of the first 10, 6 of 
the first 15 or 7 of the first 20 patients die of any cause other than disease relapse. 
With regard to grade 3 and 4 acute graft versus host disease, we will be operating 
under the hypothesis that the rate is ≤30%. The trial will be considered for 
termination if by day +100 4 of the first 10, 6 of the first 15, or 8 of the first 20 
patients develop grade 3-4 acute GVHD. 
 
 12.3 Sample Size/Accrual Rate 
 
The planned sample size will be 55 patients and we expect an accrual rate of 2-3 
patients per month 
 
12.4 Analysis of Endpoints 
 
Analysis Plan for Endpoints: 
 
 
 
Primary Endpoint: 
 
The primary endpoint is the DFS proportion at 1 year; A 95% confidence interval will be 
estimated for the 1-year DFS proportion via binomial proportions.  
 
 
 
 
Secondary endpoints: 
 
Secondary endpoints include graft failure, acute graft versus host disease, high risk 
extensive chronic graft versus host disease, and overall survival (OS).  Median DFS and 
OS, including survival curves, will be estimated using Kaplan-Meier methodology.  
Greenwood’s formula will be used to calculate 95% confidence intervals for the Kaplan-
Meier estimates. 
 
 
All analyses will be performed in SAS Version 9.3 (SAS Institute, Inc., Cary, North 


 
 
22
 
Carolina) and STATA Version 13.0 (Stata Corporation, College Station, Texas).  
 
 
 
 
 
 
 
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APPENDIX A 
 
 
Performance Status Criteria 
 
 
ECOG Performance Status Scale 
 
 
Karnofsky Performance Scale 
Grade 
Descriptions 
Percent 
Description 
0 
Normal activity.  Fully active, able 
to carry on all pre-disease 
performance without restriction. 
100 
Normal, no complaints, no evidence 
of disease. 
90 
Able to carry on normal activity; 
minor signs or symptoms of disease. 
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). 
80 
Normal activity with effort; some 
signs or symptoms of disease. 
70 
Cares for self, unable to carry on 
normal activity or to do active 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. 
60 
Requires occasional assistance, but 
is able to care for most of his/her 
needs. 
50 
Requires considerable assistance and 
frequent medical care. 
3 
In bed >50% of the time.  Capable 
of only limited self-care, confined 
to bed or chair more than 50% of 
waking hours. 
40 
Disabled, requires special care and 
assistance. 
30 
Severely disabled, hospitalization 
indicated.  Death not imminent. 
4 
100% bedridden.  Completely 
disabled.  Cannot carry on any 
self-care.  Totally confined to bed 
or chair. 
20 
Very sick, hospitalization indicated. 
Death not imminent. 
10 
Moribund, fatal processes 
progressing rapidly. 
5 
Dead. 
0 
Dead. 
 
 
 
 
 
 
 


 
 
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APPENDIX B 
 
WCMC IRB SAE Reporting Forms 
 
http://researchintegrity.weill.cornell.edu/institutional_review_board/forms.html