Clinical trial NCT01690520
Conditions: Acute Biphenotypic Leukemia Acute Lymphoblastic Leukemia in Remission Acute Myeloid Leukemia in Remission Chronic Myelogenous Leukemia Myelodysplastic Syndrome
Sponsor: Nohla Therapeutics, Inc.
Start date: 2012-12-11; Completion date: 2020-05-29
Enrollment: 163 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This randomized phase II trial studies how well donor umbilical cord blood transplant with or without ex-vivo expanded cord blood progenitor cells works in treating patients with acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, or myelodysplastic syndromes. Giving chemotherapy and total-body irradiation before a donor umbilical cord blood transplant helps stop the growth of cancer cells. It may also stop the patient's immune system from rejecting the donor's cells. When the healthy stem cells and ex-vivo expanded cord blood progenitor cells are infused into the patient they may help the patient's bone marrow make stem cells, red blood cells, white blood cells, and platelets. It is not yet known whether giving donor umbilical cord blood transplant plus ex-vivo expanded cord blood progenitor cells is more effective than giving a donor umbilical cord blood transplant alone.

Detailed description:
PRIMARY OBJECTIVES:

I. Compare the time to neutrophil engraftment (absolute neutrophil count \[ANC\] \>= 500) in patients receiving a standard of care myeloablative cord blood transplant (CBT) augmented with an off-the-shelf pre-expanded and cryopreserved cord blood product to those who do not receive the product.

SECONDARY OBJECTIVES:

I. Provide initial data on clinical and economic benefit, such as time to platelet engraftment, duration of initial hospitalization, transplant-related mortality (TRM), death without engraftment, and incidence of severe infections in the first 100 days post-transplant.

II. The kinetics of immune system recovery will also be evaluated in both arms.

OUTLINE: Patients are randomized to 1 of 2 treatment arms.

Standard of Care Arm:

CONDITIONING REGIMEN: One of two possible conditioning regimens is chosen by the attending physician: Patients receive fludarabine phosphate intravenously (IV) over 30 minutes on days -8 to -6, cyclophosphamide IV on days -7 to -6., and undergo high dose total-body irradiation (TBI) twice daily (BID) on days -4 to -1 OR, patients receive fludarabine phosphate IV over 30-60 minutes on days -6 to -2, cyclophosphamide IV on day -6, thiotepa IV over 4 hours on days -5 to -4, and undergo middle intensity TBI once daily (QD) on days -2 to -1.

TRANSPLANT: Patients undergo single-unit or double-unit unmanipulated umbilical cord blood (UCB) transplant on day 0.

GRAFT-VERSUS-HOST DISEASE (GVHD) PROPHYLAXIS: Patients receive cyclosporine IV over 1 hour BID (adults) or thrice daily (TID) (children) or orally (PO) on days -3 to 100 with taper beginning on day 101. Patients also receive mycophenolate mofetil (MMF) IV TID on days 0-7 then may receive MMF PO TID. Patients remain on MMF TID for a minimum of 30 days, and then may begin taper if there is no evidence of GVHD and are well-engrafted from one donor unit.

Experimental Arm:

CONDITIONING REGIMEN: Patients receive the conditioning regimen chosen by the attending physician as in Standard of Care Arm.

TRANSPLANT: Patients undergo single-unit or double-unit unmanipulated UCB transplant on day 0. Patients also receive an infusion of ex vivo-expanded cord blood progenitors at least 4 hours after completion of UCB transplant.

GVHD PROPHYLAXIS: Patients receive cyclosporine IV or PO and mycophenolate mofetil IV or PO as in Standard of Care Arm.

After completion of study treatment, patients are followed up periodically for 2 years.

Arms: arm 1: CONDITIONING REGIMEN: One of two possible conditioning regimens is chosen by the attending physician: Patients receive fludarabine phosphate IV over 30 minutes on days -8 to -6, cyclophosphamide IV on days -7 to -6., and undergo high dose TBI BID on days -4 to -1 OR Patients receive fludarabine phosphate IV over 30-60 minutes on days -6 to -2, cyclophosphamide IV on day -6, thiotepa IV over 4 hours on days -5 to -4, and undergo middle intensity TBI QD on days -2 to -1.

TRANSPLANT: Patients undergo single-unit or double-unit unmanipulated UCB transplant on day 0.

GVHD PROPHYLAXIS: Patients receive cyclosporine IV over 1 hour BID (adults) or TID (children) or PO on days -3 to 100 with taper beginning on day 101. Patients also receive MMF IV TID a day on days 0-7 then may receive MMF PO TID. Patients remain on MMF TID for a minimum of 30 days, and then may begin a taper if there is no evidence of GVHD and are well-engrafted from one donor unit. arm 2: CONDITIONING REGIMEN: Patients receive the conditioning regimen chosen by the attending physician as in Standard of Care Arm.

TRANSPLANT: Patients undergo single-unit or double-unit unmanipulated UCB transplant on day 0. Patients also undergo infusion of ex vivo-expanded cord blood progenitor cell infusion at least 4 hours after completion of UCB transplant.

GVHD PROPHYLAXIS: Patients receive cyclosporine IV or PO and mycophenolate mofetil IV or PO as in Standard of Care Arm.
Interventions: intervention 1: Cyclophosphamide intervention 2: Cyclosporine intervention 3: Ex Vivo-Expanded Cord Blood Progenitor Cell Infusion intervention 4: Fludarabine Phosphate intervention 5: Mycophenolate Mofetil intervention 6: Thiotepa intervention 7: Total-Body Irradiation intervention 8: Umbilical Cord Blood Transplantation
Intervention descriptions: intervention 1: Given IV intervention 2: Given IV or PO intervention 3: Given IV intervention 4: Given IV intervention 5: Given IV or PO intervention 6: Given IV intervention 7: Undergo high dose or middle intensity TBI intervention 8: Undergo single-unit or double-unit unmanipulated umbilical cord blood transplant

Protocol documents:
Prot_000.pdf: 
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FRED HUTCHINSON CANCER RESEARCH CENTER 
UNIVERSITY OF WASHINGTON SCHOOL OF MEDICINE 
SEATTLE CHILDREN’S 
Current version: 3/27/19 
 
Previous version: 8/27/18 
 
Title of Protocol: 
Multi-center, open-label randomized study of single or double myeloablative cord blood 
transplantation with or without infusion of off–the-shelf ex vivo expanded cryopreserved cord blood 
progenitor cells in patients with hematologic malignancies 
 
Investigators List: 
Filippo Milano, MD, PhD 
Assistant Member, FHCRC  
Assistant Professor, UW 
Principal Investigator (FHCRC) 
206-667-5925 
Colleen Delaney, MD, MSc 
Affiliate Member, FHCRC 
Associate Professor, Dep’t of Pediatric 
Hematology/Oncology , UW 
206-667-1385 
 
Ann Dahlberg, MD 
Assistant Member, FHCRC 
Assistant Professor, Dept’t of Pediatric 
Hematology/Oncology, UW 
206-667-1959 
Shelly Heimfeld, PhD 
Affiliate Member, FHCRC 
206-667-4004 
Rachel Salit, MD 
Assistant Member, FHCRC  
206-667-1317 
Corinne Summers, MD 
Assistant Member, FHCRC 
Assistant Professor, UW 
206-667-3119 
Biostatistician: 
Wendy Leisenring, ScD  
Member, FHCRC 
206-667-4374 
Research Staff: 
Nancy Anderson 
Research Nurse 
206-667-6264 
Connie Nakano 
Lead Regulatory Affairs Associate  
206-667-3011 
Denise Ziegler 
Study Coordinator 
206-667-5762 
Mary Joy Lopez 
Data Coordinator 
206-667-6139 
 
BB-IND 14184 (IND Sponsor: Nohla Therapeutics, Inc.) 
 
Coordinating Center: 
Fred Hutchinson Cancer Research Center  
Additional Performance Sites: 
Site 
Investigator 
Phone Number 
City of Hope Cancer Center 
Chatchada Karanes, MD 
Clinical Professor of Hematology/HCT, COH 
626-359-8111 ext. 
62691 
Cleveland Clinic 
Rabi Hanna, MD 
Director, Pediatrics Bone Marrow Transplantation 
Program 
216-444-0663 
Duke University 
Joanne Kurtzberg, MD 
Professor of Pediatrics and Pathology, Duke  
919-668-1100 
University of Colorado 
Jonathan Gutman, MD 
Assistant Professor of Medicine, UCD 
720-848-0644 


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Affiliate Investigator, FHCRC 
Dana-Farber Cancer 
Institute/Boston Children’s 
Hospital   
Christine Duncan, MD, MSc 
Attending Physician of Medicine, Hematology-
Oncology, Boston Children’s Hospital 
Attending Physician of Pediatric Oncology, DFCI 
617-632-6255 
Stanford University 
Andrew Rezvani, MD 
Assistant Professor of Medicine (Blood and 
Marrow Transplantation) at the Stanford 
University Medical Center 
650-725-4077 
Icahn School of Medicine at 
Mount Sinai   
Alla Keyzner, MD 
Assistant Professor of Medicine  
Department of Medicine, Hematology/Medical 
Oncology, Adult Hematopoietic Stem Cell 
Transplant Program at Icahn School of Medicine 
at Mount Sinai 
646-942-8201 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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TABLE OF CONTENTS 
 
1.0 
INTRODUCTION 
 
2.0 
BACKGROUND 
2.1 
Study Disease 
2.2 
Preclinical Data 
2.3 
Clinical Data to Date 
2.4 
Investigational Agent (or Regimen) 
2.5 
Agent Rationale (or Regimen Rationale) 
2.6 
Other Treatments in the Study 
2.6 
Risks/Benefits 
 
3.0 
STUDY OBJECTIVES 
3.1 
Primary Objectives  
3.2 
Secondary Objectives 
 
4.0 
STUDY DESIGN 
4.1 
Description of Study 
4.2 
Endpoints 
 
5.0 
SUBJECT SELECTION 
5.1 
Inclusion Criteria 
5.2 
Exclusion Criteria 
 
6.0 
CORD BLOOD GRAFT SELECTION 
6.1 
Inclusion Criteria 
6.2 
Exclusion Criteria 
 
7.0 
INFORMED CONSENT OF SUBJECT AND DONOR 
 
8.0 
SUBJECT REGISTRATION 
 
9.0 
PLAN OF TREATMENT 
 
10.0 
EVALUATION 
10.1 
Pre-transplant Evaluation 
10.2 
Evaluation on the morning of the cord blood infusions (Day 0) 
10.3 
Evaluation during infusion of Ex Vivo Expanded Cord Blood Progenitor Cell Product  
10.4 
24 hours post completion of expanded cord blood progenitor product  
10.5 
Patient Evaluations During Therapy Until Engraftment (Through Day 30) 
10.6 
Patient Evaluations from Engraftment through Day 100, 6 Months, 1 Year and 2 Years 
 
10.7 
Evaluation of In Vivo Persistence of Expanded and Unmanipulated Cord Blood Cells:  
Chimerism Studies/UCB Engraftment Evaluation 
10.8 
Residual/Recurrent Disease Evaluation 
10.9 
Immune Reconstitution after UCBT 
10.10 Study Evaluation Windows 
 
11.0 
DRUGS, IRRADIATION AND EXPANDED CORD BLOOD CELL ADMINISTRATION 
TOXICITIES AND COMPLICATIONS 
11.1 
Cyclophosphamide (and/or other conditioning agent) Administration 


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11.2 
Total Body Irradiation 
11.3 
Infusion of Unmanipulated and Expanded Cord Blood Units 
11.4 
G-CSF 
11.5 
Cyclosporine 
11.6  MMF 
 
12.0 
GUIDELINES FOR ADVERSE EVENT REPORTING 
12.1 
Adverse Event Reporting/ Institutional Policy 
12.2 
Study-Specific Adverse Event Capture 
 
13.0 
ASSESSMENT OF DISEASE RESPONSE 
 
14.0 
DATA AND SAFETY MONITORING PLAN 
 
15.0 
DATA MANAGEMENT/CONFIDENTIALITY 
 
16.0 
STATISTICAL CONSIDERATIONS 
 
17.0 
TERMINATION OF THE STUDY 
 
REFERENCES 
 
APPENDICES 
 
Appendix A: 
Performance Status Criteria 
Appendix B 
FHCRC Standard Practice Guideline for Infusion of Cryopreserved Products  
Appendix C: 
GVHD Assessment 
Appendix D: 
Comorbidity Index 
Appendix E: 
Potential Adverse Events Associated or Expected with Hematopoietic Cell Transplantation 
Appendix F: 
FHCRC IRB Policies  
Appendix G: 
Schedule of Study Evaluations  
 
 
 
 


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1.0 INTRODUCTION 
  
Delayed myeloid engraftment is a known risk factor for CBT recipients and is associated with the low TNC and 
CD34+ cell doses provided in a single or double CB graft. In fact, a recent analysis of adult single CBT (sCBT) 
recipients demonstrated that infused CD34+ cell dose is the most important predictor of myeloid engraftment [6]. 
Furthermore, Brunstein/Delaney et al have shown that non-relapse mortality (NRM) is highest in double CBT 
(dCBT) recipients when compared to matched and mismatched unrelated donor recipients (figure 1 to the left) [7]. 
The majority of the NRM occurred within the first 100 
days post transplant with infection being the most 
common cause of death. Importantly, an analysis of 
the risk factors for NRM among dCBT recipients 
revealed a higher risk in patients with delayed myeloid 
recovery (time to ANC >500/µl) if the recovery was ≥ 
26 days, the median time to engraftment in dCBT 
recipients. However, when the analysis of risk factors 
for NRM was restricted to include only those dCBT 
recipients engrafting before day 26, no difference 
could be found between the donor sources, 
emphasizing the important contribution of delayed 
engraftment to increased risk of NRM. Moreover, an 
ANC of >100 on any given day post stem cell 
transplant has been previously shown to be a critical 
threshold for a decreased risk of mortality before day 
100 post transplant [8, 9]. Thus, the significant delay 
in myeloid recovery that is observed in CBT recipients remains a critical barrier to successful outcomes in the 
CBT setting. This project aims to overcome this critical problem by further development of an ex vivo expanded 
cell therapy already shown to be capable of providing rapid early myeloid recovery post CBT.  
 
Using an engineered form of the Notch ligand Delta1 for the ex vivo generation of increased numbers of CB 
CD34+ stem and progenitor cells, we have previously demonstrated that infusion of our expanded, partially HLA-
matched cell product results in a significant reduction in the median time to achieve an absolute neutrophil count 
of 500/l to just 14.5 days as compared to a median time of 25 days (p=0.002) in a concurrent cohort of 29 
patients undergoing identical treatment but with two non-manipulated CB units. Thus, with established proof of 
principal, our goal was further development of our cellular therapy as an economically and clinically feasible 
product with ready patient access for evaluation in pivotal clinical efficacy trials.  
 
Critical to this, and a focus of was the question of whether HLA-matching is required for safe infusion of our 
product which is devoid of T cells. Without the need for HLA matching, fresh CB units can be collected in 
collaboration with accredited public CB banks for immediate ex vivo expansion and the final product 
cryopreserved for future, but immediate, on demand use. In contrast to our initial trial using at least partially 
HLA-matched CB units for expansion followed by immediate infusion, patient access to the newly proposed 
expanded CB product would be dramatically enhanced as all of the expanded products banked would be 
potentially available for any given patient, regardless of HLA typing, race/ethnicity or location of the patient. 
Moreover, the ability to create an off-the-shelf universal donor expanded cell therapy is not only important to the 
project proposed herein aimed at shortening the duration of severe neutropenia post CBT, but is likely to enhance 
more broad areas of investigation outside of stem cell transplantation, e.g., as a way of providing temporary 
myeloid engraftment for treatment of chemotherapy induced severe neutropenia or accidental radiation exposure.  
 
To first address the question of safety when infusing an off-the-shelf product, we carried out a pilot study, 
FHCRC Protocol #2378, Infusion of Off–the-Shelf Ex Vivo Expanded Cryopreserved Cord Blood Progenitor Cells 
to Augment Single or Double Myeloablative Cord Blood Transplantation in Patients with Hematologic 
Malignancies. As discussed more below, no safety issues have been observed, and the ability of the expanded cell 
product to contribute to early myeloid engraftment has been demonstrated. We are now ready to determine the 
clinical efficacy of this product in a randomized multi-center trial comparing the time to engraftment in patients 
Years post-transplantation
Cumulative Incidence
Non-Relapse Mortality
by Donor Type
SIB
P < 0.01
MMUD
MUD
DUCB
0.0
0.2
0.4
0.6
0.8
1.0
0
1
2
3
4
5
Years post-transplantation
Cumulative Incidence
Non-Relapse Mortality
by Donor Type
SIB
P < 0.01
MMUD
MUD
DUCB
0.0
0.2
0.4
0.6
0.8
1.0
0
1
2
3
4
5
Figure 1. Risk of non-relapse mortality is highest among 
double CBT recipients. Non-relapse mortality after double CBT 
(DUCB), matched unrelated donor (MURD), mismatched unrelated 
donor (MMURD) and matched related donor (SIB) transplant.  


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receiving a myeloablative single or double CBT with or without infusion of a non-HLA matched pre-expanded 
cryopreserved cord blood progenitors.  
 
2.0 BACKGROUND 
  
2.1 
Study Disease 
 
Cord blood is an effective and widely used source of stem cells for patients undergoing hematopoietic stem cell 
transplantation for hematologic malignancies.  Most patients now receive two units of CB derived from different 
donors to better ensure provision of adequate stem cell numbers for reliable donor engraftment. However, with 
this modest two-fold increase provided by the second CB unit, the time to donor engraftment is still relatively 
delayed, averaging more than three weeks to achieve adequate numbers of myeloid cells. This leaves patients 
susceptible to infection and associated morbidity and mortality. Thus, although CB offers some distinct 
advantages over conventional stem cell sources as it is readily available with no donor attrition and allows 
reduced stringency in HLA matching without an increase in GVHD thereby increasing the donor pool especially 
for minority or mixed ethnicity patients, the increased risks of delayed engraftment or graft failure and early 
transplant related mortality that is associated with the low cell doses in a CB graft remains a barrier to the more 
wide spread use of this stem cell source. To address this, our laboratory has investigated the role of the Notch 
signaling pathway in regulating ex vivo expansion of hematopoietic stem/progenitor cells with the goal of 
generating increased numbers of progenitor cells capable of rapid repopulation in vivo to improve the kinetics of 
hematopoietic recovery following CBT.  
 
Notch mediated ex vivo expanded cord blood progenitor cells 
 
Using purified CD34+ cord blood progenitor cells cultured in the presence of an engineered form of the Notch 
ligand Delta1, we have developed a cellular therapy product that may provide rapid temporary myeloid 
engraftment for prevention of infection and that may potentially facilitate host hematopoietic recovery following 
high dose therapy. Initial clinical data from our Phase I trial have demonstrated rapid recovery of myeloid cells in 
hematopoietic cell transplant patients who received partially HLA-mismatched expanded cells along with a 
second, partially HLA-mismatched but non-manipulated cord blood unit (FHCRC Protocol 2044). We now 
propose to extend the indication for this cellular based therapy to overcome infectious complications and 
prolonged neutropenia seen in patients undergoing myeloablative conditioning regimens prior to transplant with 
one or two partially mismatched non-manipulated units. These “off-the-shelf”, non HLA-matched expanded cord 
blood progenitors are capable of at least temporarily rescuing hematopoiesis until engraftment can take place. 
 
In preparation for trial using a pre-expanded and cryopreserved product, the methods for selecting and culturing 
CD34+ cells from fresh cord blood units and cryopreserving the final expanded product for future use as an “off-
the-shelf”, non-HLA matched product have been optimized. We have generated pre-clinical data demonstrating 
the ability to cryopreserve the expanded cell product without loss of in vivo repopulating ability in an 
immunodeficient mouse model. In addition, work done in a murine model has demonstrated the ability of murine 
hematopoietic progenitors that have been expanded in the presence of Notch ligand to provide short-term 
engraftment when transplanted into major H-2 mismatched recipients, and in separate studies, to facilitate 
recovery of autologous residual stem cells remaining after radiation.  
  
2.2 
Preclinical Data  
 
Below, preclinical data supporting the use of ex vivo expanded and cryopreserved cells is presented. 
 
In vivo repopulating ability is retained following cryopreservation of the expanded cell product  
A number of validation experiments have been done to optimize cryopreservation methods of the expanded cell 
product and to determine whether in vivo repopulating ability is retained when the expanded product is 
cryopreserved for future infusion. Using an immunodeficient mouse model, we evaluated the ability of ex vivo 
expanded cryopreserved progenitor cells to engraft. As shown below, initial experiments compared in vivo 
repopulating ability of expanded cells that were directly infused into immunodeficient mice upon harvest versus 
those that were harvested post expansion and cryopreserved for future use. There were no significant differences 


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observed in the in vivo repopulating ability of cells that were cultured, cryopreserved (in standard “Allo” media 
used for hematopoietic cell cryopreservation in the SCCA Cellular Therapy Lab), and then thawed prior to 
transplant when compared to expanded progenitor cells that were harvested at the end of culture and freshly 
infused (Figure 1a). Additional experiments have confirmed that repopulating ability of the expanded cell product 
is retained following cryopreservation. As shown in Figure 1b, all mice that received expanded cryopreserved 
cells engrafted (defined >0.5% human CD45 in the marrow) with an average overall human engraftment of 8% at 
2 weeks post infusion and 7% at 4 weeks post infusion. Lastly, we have compared various thawing methodologies 
(thaw and wash, thaw and dilute with dextran/HSA and thaw and directly infuse) and have also not seen a 
significant difference in the three methods evaluated (Figure 1c). Thus, to avoid cell losses, a thaw and dilute 
protocol will be used.  
 
Cultured vs
Cultured and Cryopreserved
Fresh
Frozen - Allo Med.
0
5
10
15
20
25
p=0.7211
Cultured
and infused
Cultured and
cryopreserved
Human CD45% in Marrow
 
Engraftment of Cryopreserved
Expanded  Cord Blood Progenitor Cells
2 Weeks
4 Weeks
0
10
20
30
40
Human CD45% in Marrow
 
 
Figure 1: Cryopreservation of ex vivo expanded cord 
blood progenitors does not impair in vivo 
repopulating ability. CD34+ cord blood progenitor cells 
were enriched and placed into culture with Delta1ext-IgG. 
Overall human engraftment as measured by human CD45 
in the marrow of recipient mice is shown on the y axis. 
The solid lines represent the mean level of human 
engraftment. (a) Cells infused immediately post culture 
compared with harvested cells that were cryopreserved 
prior to infusion. Results shown are at 4 weeks post 
infusion. (b) Ex vivo expanded and cryopreserved 
progenitor cells were thawed and infused. The figure 
represents the combined results of two experiments. (c) 
The expanded progeny derived from expansion of 
CD34+ progenitors obtained from a single cord blood 
unit were divided into equal groups and cryopreserved 
per standard practice. Three methods of thaw prior to 
infusion were then compared for in vivo repopulating 
ability: thaw and wash, thaw and dilute (albumin/dextran 
dilution), thaw and direct infusion.  
 
 
 
Infusion of mismatched ex vivo expanded and cryopreserved hematopoietic progenitors after radiation exposure 
results in temporary donor hematopoietic recovery and prolongs survival in a murine model 
Relevant to the proposed clinical trial, preliminary studies with a murine model have also shown that expanded 
numbers of progenitor cells derived from murine hematopoietic progenitors (LSK cells) are capable of providing 
short term engraftment when transplanted in an H-2 mismatched recipient. In this preclinical animal model, LSK 
cells were isolated by flow cytometry from whole bone marrow (BM) of Ly5a mice (H-2b, CD45.1) and placed in 
culture in the presence of immobilized Notch ligand. Serum free conditions were used, consisting of Iscove’s 
medium supplemented with cytokines mSCF, hFlt-3 ligand, hIL-6, and hIL-11. After 14 days, expanded cells 
Cryopreserved Expanded CB Progenitors:
Methods of thaw and infuse
Allo 
Allo 
Allo 
0
5
10
15
Thaw and Wash        Thaw and Dilute       Thaw and Infuse
        p=0.6
        p=0.8
        p=0.9
Human CD45% in Marrow
a) 
b) 


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were harvested and cryopreserved in 90% FBS + 10% DMSO. On the day of transplant, frozen cells were thawed, 
washed, and resuspended in PBS + 1% FBS.  
 
To determine the ability of the expanded cells to contribute to in vivo hematopoietic recovery and provide 
radioprotection in a MHC mismatched model, Balb-c recipient mice (H-2d, CD45.2) were treated with single 
dose TBI at 7, 7.5 and 8 Gy followed by infusion of either normal saline solution (NS) or 5 x 106 expanded cells 
per mouse. The results presented herein represent the combined data from 3 consecutive experiments, including a 
total of 150 mice for the generation of survival curves and an additional 54 mice to assess donor engraftment. All 
animals were observed for 30 days to assess survival. Moribund animals that met specific criteria established by 
the IACUC were euthanized and counted as an experimental death. Mice contributing to assessment of donor 
engraftment were sacrificed and peripheral blood and BM collected at 1, 2, and 3 weeks after infusion to 
determine the in vivo contribution of donor hematopoiesis as determined by flow cytometry. 
 
As shown in figure 2 below, 97% of mice that received NS after a single dose of 8 Gy died in contrast to only 
52% mortality in the group treated with 5 x 106 expanded cells (despite major MHC differences) (p<0.0001).  
Similarly, at a dose of 7.5 Gy, the survival rate at 30 days was 25% and 70% for mice that received NS and 
expanded cells, respectively (p=0.0004). When the TBI doses was reduced to 7 Gy, all mice infused with 
expanded cells survived with only one death in the NS group (p=0.22) (Figure 2). Of note, no deaths were 
observed after 30 days. Evaluation of donor derived hematopoiesis demonstrated in vivo persistence of the 
mismatched expanded cells through day 28, with peak engraftment occurring before day 14. The percent of donor 
cells in the peripheral blood one week following infusion ranged between 50 to 95% in all mice that received 
expanded cells, with higher donor engraftment correlating with increased dose of TBI. The level of donor 
engraftment then decreased over time, but in the surviving mice remained persistent to levels ranging from 10 to 
30%. Importantly, no signs of graft versus host disease (GVHD) were observed despite the full-mismatch between 
the recipient and donor product.  
 
 
These studies demonstrated that ex vivo expanded and cryopreserved murine hematopoietic progenitors can be 
infused safely and effectively to provide temporary donor derived hematopoiesis and prolong survival after acute 
ionizing radiation. Importantly, these cells can be ex vivo expanded and cryopreserved for future use and retain 
repopulating ability. Furthermore, in vivo transplantation studies demonstrate that short-term engraftment without 
manifestations of GVHD can be attained across major H-2 barriers, supporting the use of this product as a 
universal or third party donor product. 
 
2.3 Initial Clinical Information 
 
Proof of Principal: Preliminary Results of a Phase I CBT Trial Using Ex Vivo Expanded Cord Blood Progenitors 
A phase I trial involving transplantation of a non-manipulated partially HLA-matched unit along with CB 
progenitors from a second partially HLA-matched CB unit that have undergone Notch-mediated ex vivo 
expansion (FHCRC Protocol 2044) has been conducted. The primary objective was evaluation of safety of 
TBI 7 Gy + Deltaext-IgG 
TBI 8 Gy + Deltaext-IgG 
TBI 7.5 Gy + Deltaext-IgG 
TBI 7 Gy + NS 
TBI 7.5 Gy + NS 
TBI 8 Gy + NS 
Figure 2. Survival curve of mice irradiated with 
single dose TBI at 7, 7.5 and 8 Gy and treated with 
either 5 x 106 cryopreserved Delta expanded cells 
(solid lines) or saline solution (dashed lines). 


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infusing the ex vivo expanded CB progenitors, while secondary objectives included evaluation of the kinetics and 
durability of hematopoietic reconstitution and the relative contribution to engraftment as provided by the 
expanded and non-manipulated CB units.  
Sixteen patients with high risk acute leukemia in morphologic remission at the time of transplant have been 
enrolled with a median age of 28.3 years (range, 3.6 to 43.3) and median weight of 66 kilograms (range, 16.5 to 
84.7).  Based on pioneering studies by Wagner and colleagues at the University of Minnesota demonstrating 
safety of double CB unit infusion, patients received a myeloablative preparative regimen (1320 cGy TBI, 
120mg/kg cytoxan and 75mg/m2 fludarabine) followed by infusion of one non-manipulated and one ex vivo 
expanded CB graft. Cord blood donors were chosen on the basis of cell dose and HLA, with all units being ≥ 4/6 
matched to the recipient (intermediate resolution at HLA-A and B, and high resolution at DRB1) and at least 3/6 
matched to each other. Additional criteria for donor CB selection included a minimum requirement of a total 
nucleated cell (TNC) dose in the non-manipulated graft of at least 2.5 x 107 TNC/kg (based on pre-
cryopreservation numbers), independent of the match grade (HLA) of the unit to the recipient. All patients 
received prophylaxis for graft-versus-host-disease (GVHD) consisting of cyclosporine and mycophenolate mofetil 
beginning on day -3. 
 
Sixteen days prior to the stem cell infusion date, the unit selected for ex vivo expansion was thawed, clinical 
grade CD34+ cell selection was performed and cultures initiated in the presence of Notch ligand. On the day of 
transplant, the cultures were harvested and infused four hours after infusion of a non-cultured, unrelated donor CB 
graft. At harvest, there was an average fold expansion of CD34+ cells of 184 (range 41-471) and an average fold 
expansion of total cell numbers of 800 (146 - 1496). Of note, there were no mature T cells infused with the 
expanded graft. Despite cell losses that occur with CD34 cell selection, in all cases the absolute number of CD34+ 
cells at the end of culture greatly exceeded the pre-cryopreservation CD34 cell number. The infused CD34+ cell 
dose derived from the expanded CB graft averaged 12.5 x106 CD34/kg (range 0.93 to 49 x106, median 8.3 x 106).  
 
The kinetics of hematopoietic recovery and the relative contribution of the expanded and non-manipulated CB 
grafts to engraftment were determined beginning on day +7 post transplant. Time to an initial ANC ≥ 500/l was 
evaluable in 14 out of the first 16 patients. In these 14 patients, the time to achieve an absolute neutrophil count of  
≥ 500/l has been shortened significantly, with a median time of 11 days (range, 6 to 34 days). This compares 
quite favorably with a median time of 25 days (range, 14 to 48 days, p=0.004) in patients undergoing double CB 
transplantation at our institution with an identical conditioning and post-transplant immune suppression regimen 
(Figure 3). One patient did experience primary graft 
rejection due to residual host CD8+ T cell recognition of 
both the non-manipulated and expanded progenitor CB 
grafts [2]. Of note, no infusional toxicities or other 
safety concerns have been observed. Median follow-up 
time for this set of 16 patients is currently 289 days 
(range 54-1987), and 8 out of 16 patients remain alive 
with no evidence of disease, and sustained complete 
donor engraftment. GVHD has been evaluable in 14 out 
of 16 patients, and 12 of the 14 were diagnosed with 
grade II acute GVHD and 2 with grade III. All patients 
responded to therapy. No chronic extensive GVHD has 
been observed and three patients have been diagnosed 
with chronic limited GVHD.  
 
 
 
 
 
Figure 3. Culture of CB progenitors with Delta1ext-IgG 
results in more rapid neutrophil recovery in a 
myeloablative double CBT setting The individual and 
median times (solid line) to ANC of ≥ 500/µl for patients 
receiving double unit CBT with two non-manipulated units 
(“conventional”) versus with one ex vivo expanded unit and 
one non-manipulated unit (“expanded”) is presented. 
 
Median Time to Initial ANC >500
0
10
20
30
40
50
Conventional
dCBT
N=29
Expanded +
unmanipulated
N=14
p<0.0001
25 days
11 days
Days post transplant


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Infusion of third party donor mobilized peripheral blood stem cells to augment CBT  
The infusion of ex-vivo expanded cord blood progenitor cells independent of HLA-matching as an “off-the-shelf” 
product (third party donor product) to overcome delayed hematopoietic recovery in CBT is not dissimilar to an 
approach taken by MN Fernandez et al., who have demonstrated the safety of infusing T cell depleted third party 
donor mobilized peripheral blood stem cells as a means to augment the low cell dose in recipients of a single-unit 
CBT [4]. The third party donor cells were haploidentical in 60% of patients (n=18), but shared no haplotype in 
40% (N=12). Using this approach, the incidence of severe GVHD was not increased (and no biopsies obtained for 
GVHD had detectable DNA from the third party donor) and all patients engrafted with no deaths prior to ANC 
recovery. The third party donor products infused did contain mature T cells, but were T cell depleted products. 
Importantly, in the trial proposed herein, CD34+ CB progenitors that have been ex vivo expanded in the presence 
of Notch ligand do not contain mature T cells as evidenced by immunophenotyping of the harvested product, 
which should allow for their safe use in a HLA-mismatched setting. 
 
Preliminary Results from a Pilot Study using Off-the-Shelf Expanded Cord Blood Progenitors (FHCRC #2378):  
Having previously demonstrated a significant reduction in ANC recovery time in patients receiving these partially 
HLA-matched expanded cells in a myeloablative CBT, we are now evaluating whether this product could also 
support rapid myeloid recovery when used as a non-HLA matched, off-the-shelf product. In this pilot study, 
patients received either a single or double ablative CBT plus infusion of progenitors that have been previously 
expanded from a fresh CB unit and then cryopreserved for future use. Thirteen patients were enrolled in this pilot 
study (see Table 1 for patient/graft characteristics). Importantly, no infusional toxicities have been observed and 
no serious adverse events have been attributed to the off-the-shelf non-HLA matched expanded cord blood 
progenitor product. All (n=13) patients have engrafted. The median time to achieve an ANC of 100/l and 500/l 
was 12 (range 7 to 23) and 20 days (range 9 to 31), respectively as compared to 19 (range 11 to 37) (p=0.0001) 
and 25 days (range 14 to 45) 
(p=0.004) in a concurrent cohort of 
patients receiving ablative double 
CBT without the expanded cells. 
Donor(s)/host chimerism studies 
were performed weekly from day 7 
to 28 on peripheral blood flow sorted 
into myeloid and lymphoid fractions. 
Similar to our initial expansion trial 
using partially HLA-matched 
expanded CB cells, early (day 7) 
myelomonocytic (CD33 and CD14) 
recovery is almost entirely (98–
100%) due to cells arising from the 
expansion product. Cells derived 
from the expansion product are no 
longer detected at day 14 in all but 2 
patients, which is similar to our 
previous trial in which contribution 
to donor engraftment in any cell 
fraction from the expanded unit was 
no longer detected by day 14 in half 
of the patients and only two patients had persistence of the expanded cells beyond day 21. Of note, the expanded 
cells have not been associated with increased incidence or severity of acute GVHD or with development of  
alloimmunization. One patient with relapsed disease at the start of conditioning had persistent disease post-
transplant and has since died secondary to relapse. Another patient relapsed and is currently being treated. These 
promising results warrant evaluation in a randomized phase II study to assess clinical efficacy of this non-HLA 
matched, off-the-shelf expansion product.  
 
Table 1: Unit and Patient Characteristics 
Total Enrolled N=13 
Patient Characteristics 
Gender, No (%) 
 
Male 
7 (54%) 
Female 
6 (46%) 
Age in years, median  (range) 
22 (5 to 45) 
Weight in kilograms, median (range) 
67 (22 to 84) 
Diagnosis, No (%) 
 
 
ALL 
6 (46%) 
AML 
6 (46%) 
MDS 
1 (8% 
Follow-up in days, median (range) 
180 (16 to 385) 
Unit Characteristics 
Number of Unmanipulated Donors, No (%) 
 
1 
2 (15%) 
2 
11 (85%) 
HLA match unmanipulated Donors, No (%) 
 
4/6 
8 (62%) 
5/6 
5 (38%) 
Infused Cell Doses (pre-freeze) 
 
Total Unmanipulated TNC/kg x 107 
6.2 (4.3 to 17) 
Total Unmanipulated CD34/kg x 106 
0.3 (0.09 to 1) 
Expanded Product TNC/kg x 107 
5.8 (2.2 to 10)  
Expanded Product CD34/kg x 106 
6 (3.1 to 11) 
 


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Engraftment Kinetics and Donor Chimerism: As in our previous trial, the kinetics of hematopoietic recovery and 
the relative contribution of the expanded and unmanipulated grafts to engraftment are determined beginning on 
day +7 post transplant. All patients treated to date have engrafted. It has been previously demonstrated that an 
ANC ≥100 is a critical threshold to reduce the risk of death prior to day 100 post hematopoietic cell transplant [8]. 
Furthermore, in our own single center analysis of severe neutropenia following CBT, modeling both ANC <100 
and time to engraftment as a time-dependent covariate correlates significantly with both day 200-TRM and 
overall survival (unpublished data). In this analysis of 88 patients undergoing CBT, at any given time point, an 
ANC <100 is associated with a 4.77-fold increase in the risk of overall mortality compared to an ANC >100 (1.74 
– 13.11, p=0.002) and an 8.95-fold increase in risk of day 200 transplant related mortality (TRM) (2.59-30.89, 
p=0.00095). This is similar to findings when modeling the time to engraftment (ANC >500), such that 
engraftment at a specific time point is associated with a 0.23-fold risk of death as compared to lack of engraftment 
at this time (0.08 – 0.62, p=0.004) and a 0.11-fold risk of day 200 TRM (0.03 – 0.38, p=0.0005). We therefore 
evaluated the time to achieve an ANC ≥100 as well as the time to achieve ANC ≥500 in patients undergoing 
myeloablative dCBT, comparing a concurrent cohort of patients receiving a myeloablative dCBT, our previous 
trial using partially matched expanded CB units and the initial four patients on this pilot study.  
 
As seen in figure 4 below, the time to achieve an ANC of 100, perhaps the more critical threshold as a threshold 
for decreased risk of mortality, continues to be significantly shorter in patients receiving our off-the-shelf 
expanded cell product as compared to conventional dCBT recipients not receiving expanded cells and is similar to 
what we observed in our previous CB expansion trial using partially matched freshly expanded cord blood 
progenitors for infusion. With respect to the time to achieve an ANC ≥ 500, the first four patients treated with off-
the-shelf progenitors are all evaluable, with a median time to ANC ≥500 of 20 days. Importantly, the results 
achieved to date compare quite favorably with that seen in 2044 and are supportive for the evaluation of clinical 
efficacy of this approach compared to conventional CBT in the proposed herein.   
 
 
 
 
 
0
10
20
30
40
50
Conventional
dCBT
N=29
Expanded +
unmanipulated
N=14
Off-the-shelf +
unmanipulated
N=13
p<0.0001
25 days
20 days
11 days
Time to Achieve an ANC  500
p=0.01
p=0.009
Days post transplant
Figure 4: Time to Neutrophil Recovery of 100 and 500 after Myeloablative CBT with and without expanded 
cells. The time to achieve an ANC of ≥ 100 and 500 in days post transplant is compared in three groups. 
“Conventional” dCBT recipients (N=29) are those patients receiving a double unmanipulated CB graft with no 
expanded cell infusion. “Expanded” recipients are patients receiving one unmanipulated graft and one expanded 
graft, both at least 4/6 HLA-matched to the recipient (N=14). “Off-the-shelf” recipients are patients receiving at 
least 4/6 HLA-matched unmanipulated CB unit(s) with the addition of a non-HLA matched and off-the-shelf
0
10
20
30
40
Median Time to Achieve an ANC >100
Conventional
dCBT
N=29
Expanded +
unmanipulated
N=12
Off-the-shelf +
unmanipulated
N=13
19 days
7 days
12 days
p=0.4
p=0.0002
p=0.0003
Days post Transplant


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In order to determine the contribution to donor engraftment in the first month post transplant as derived from the 
multiple donors and host, recipient peripheral blood is collected weekly from day 7 to 28. The peripheral blood 
sample is sorted by flow cytometry into lymphoid and myeloid cell fractions (CD33, CD14, CD56, CD3 and 
CD19) followed by DNA extraction and determination of donor/host chimerism by amplified fragment length 
polymorphism. An obvious limitation to this approach is the potential lack of sufficient numbers of circulating 
cells, particularly 7 days after receiving high dose TBI, to enable flow sorting into the various lineages and DNA 
extraction for donor analysis. However, in the 28 total patients who have received ex vivo expanded cells (either 
fresh or cryopreserved), day 7 chimerism analysis in all cell fractions, except CD19, was possible in all but 2 
patients. Of the two patients that were not evaluable for day 7 chimerism, both received fresh partially matched 
expanded cell grafts; one patient experienced host mediated graft failure and the other did not have any 
contribution to donor engraftment from the expanded cells in the setting of early onset HHV6 viremia.  
This is in striking contrast to our attempts to collect day 7 donor chimerism data from patients undergoing 
conventional myeloablative dCBT, where essentially the only chimerism data reliably obtained at this early time 
point is CD3 chimerism, likely partially reflecting the CD3 cells infused with the graft. However, similar to our 
initial expansion trial using partially matched expanded CB cells, determination of day 7 peripheral blood 
chimerism has been possible on all cell fractions except CD19, suggesting that despite low overall counts as 
reported in the daily clinical CBCs, there are higher absolute numbers of circulating cells in patients receiving ex 
vivo expanded cell products. Again, early myelomonocytic recovery in patients receiving expanded products is 
almost entirely (98 – 100%) due to circulating cells as contributed from the off-the-shelf product. However, by 
day 14, the off-the-shelf product is no longer detected by peripheral blood chimerism analysis in all but two 
patients who have since converted to 100% donor from an unmanipulated unit (see Figure 5 below). This is not 
dissimilar to our previous trial in which contribution to donor engraftment in any cell fraction from the expanded 
unit was no longer detected by day 14 in 5 of 9 evaluable patients. Only two patients had persistence of the 
expanded cells beyond day 21, but contribution to CD3 engraftment was never observed from the expanded cell 
graft. One of the two patients died at approximately 6 months post transplant from sepsis/multi-system organ 
failure, still with engraftment from both her unmanipulated and expanded units and the other patient converted to 
100% donor engraftment from the unmanipulated unit at approximately one year post transplant.  
Sorted Peripheral Blood Chimerism Day 7
CD33
CD56
CD3
0
20
40
60
80
100
CD33/CD14
% Off-the-shelf donor
Sorted Peripheral Blood Chimerism Day 14
CD33
CD56
CD3
0
20
40
60
80
100
CD33/CD14
% Off-the-shelf donor
Figure 5. Peripheral blood chimerism day 7 and 14, FHCRC Protocol 2378 
 
2.4 
Investigational Agent 
Biological Representation 
The ex vivo expanded cord blood product (“Delta1-cultured Cryopreserved Umbilical Cord Blood Cells”, or 
DCC-UCB), is derived by culture of enriched CD34+ progenitor cells isolated from a single donor umbilical cord 
blood (UCB) unit in the presence of recombinant cytokines and the Notch ligand Delta1ext-IgG for a period of 14 to 
16 days. At the end of the culture period, the resulting cell population that makes up the DCC-UCB cells is 
heterogeneous, consisting of CD34+ progenitor cells and more mature myeloid and lymphoid precursors, as 
evidenced by flow cytometric analysis for the presence of CD34, CD7, CD14, and CD56 antigens. Typical flow 
cytometry characterization of DCC-UCB cells is presented in Table 2 below.  
 
 


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Table 2: DCC-UCB Cell Phenotype (N= 80)  
 
  
Median 
Percent 
Range  
CD34 
10.4 
4.9 
- 
24.6 
CD7 
8.5 
2.3 
- 
25.2 
CD14 
7.0 
0.9 
- 
22.2 
CD15 
39.3 
0.3 
- 
65.5 
CD34+/56+ 
2.6 
0.2 
- 
9.2 
CD3-/CD16+/56+ 
4.8 
0.7 
- 
13.0 
CD20 
0.1 
0.0 
- 
10.2 
CD3+/CD4+ 
0.3 
0.0 
- 
2.9 
CD3+/CD8+ 
0.0 
0.0 
- 
0.5 
TNC Fold Expansion 
1018 
213 
- 
3333 
CD34 Fold Expansion 
129 
23 
- 
392 
 
 
There is significant increase of CD34+ and total cell numbers during the culture period, ranging from 23 to 392 
fold expansion of CD34+ cells and 213 to 3333 fold expansion of total cell numbers (N= 80 individual cord blood 
units, processed per defined clinical expansion procedures). Notably, there is a lack of T cells as measured by 
immunophenotyping. Functionally, this expanded cord blood product is capable of multi-lineage human 
hematopoietic engraftment in a NOD/SCID mouse model. 
 
2.5 
Regimen Rationale 
 
As discussed above, it is important to note that the expanded cell product contains no mature T cells, and 
therefore should be able to be infused in a recipient who has undergone an intensive preparative regimen. As in 
the previous trial, we do not anticipate that the expanded unit will be the long term engrafting unit, due to likely 
eventual (or immediate) rejection mediated by the non-manipulated unit(s). No safety issues infusing these 
products have been identified in a previously conducted pilot study, and we are therefore now evaluating the 
clinical efficacy of this product in a randomized multi-center trial comparing the time to engraftment in the 
patients undergoing myeloablative CBT with or without infusion of this off-the-shelf product.  
 
This clinical trial will allow variation in preparative regimen (see Section 9.2) but will have identical regimen for 
GVHD prophylaxis. The protocol allowed conditioning regimens are:  
 
1. High dose TBI + Fludarabine + cyclophosphamide 
2. Middle intensity TBI + Fludarabine + cyclophosphamide + thiotepa 
 
The additional regimen (middle intensity TBI + Fludarabine + cyclophosphamide + thiotepa) was chosen in 
addition to the more conventional high dose TBI regimen that was used in our pilot study based on the extensive 
work published by Dr. Juliet Barker at the Memorial Sloan Kettering Cancer Center10.This novel regimen was 
originally developed as an alternative intensive conditioning regimen aimed at reducing the increased risk of 
lethal regimen-related organ toxicity that has been associated with the more conventional high dose TBI-based 
regimen used for myeloablative CBT (using 13.2 Gy TBI). In fact, the known risk of end organ toxicity with high 
dose TBI has led to restriction of this regimen to those 45 years and younger. Although there are 
nonmyeloablative conditioning regimens that have been used in the CBT setting for those patients older than 45 
years, nonmyeloablative conditioning itself is limited by increased risk of graft rejection and relapse in this 
setting. Thus, there was a significant need to develop a well-tolerated middle intensity, near myeloablative 
regimen to address the limitations of the high dose TBI and nonmyeloablative regimens. This is particularly true 


2603.00 
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for those patients older than 45 years or younger patients with significant pre-existing co-morbidities precluding 
them from the conventional high dose TBI regimen.  
 
Dr. Barker published on this regimen for the first time in 2013 which presented results for 30 patients with acute 
leukemia and MDS, with the majority having acute leukemia (n=26) [10].  The median age was 56 years (range 
18 to 69).  The overall incidence of engraftment was 97% (1 graft failure), with a median time to engraftment of 
26 days (range, 13 to 43). 
 
Expanding our clinical trial to allow the option of this new conditioning regimen as an alternative to the 
conventional high dose TBI regimen, with corresponding expanded age range, is strongly motivated by arguments 
for the importance of generalizability and for the ability to pool results: 
 
• 
The results of the Barker trial reveal that there is a promising alternative to high dose TBI based 
conditioning that expands the benefits of a conditioning regimen to a wider cohort, in particular to 
patients 50 years and older who are otherwise reasonably fit. Furthermore, it is thought to represent a 
promising alternative to high dose TBI based conditioning. 
 
• 
The Barker trial results reveal that time to median engraftment using this regimen in an adult population 
(18 – 69 years) is nearly identical to the setting of CBT using the high dose TBI based regimen currently 
in use in Protocol 2603. Furthermore, the reported rate of TRM at day 180 was 20%, similar to the 
conventional high dose TBI regimen.  Therefore, it appears that there is a similar unmet need across these 
settings to reduce median time to engraftment that is a median of 26 days across these settings. Data 
showing that engraftment times are similar to the conventional high dose TBI regimen are presented in 
Figure 6 below.  
 
• 
Importantly, it is reasonable that efficacy of the 
experimental cell therapy product would be similar 
across these conditioning regimen. Specifically, the 
experimental cell therapy product under investigation in 
this trial is thought to have a mechanism of action that 
is independent of the conditioning regimen. This is 
based on preliminary efficacy data demonstrating a 
reduction of infections and bridging hematopoiesis even 
in patients receiving this product to mitigate 
chemotherapy induced neutropenia (i.e., in the non-
transplant setting).11  
Figure 6. Time to median engraftment using   
middle-intensity regimen (Ponce D, et al, BBMT 2013)  
 
For these reasons, the inclusion of this new middle-intensity regimen and the inclusion of patients up to age 65 
enables a clinically well motivated generalization of the assessment of the experimental cell therapy product, and 
it is scientifically well motivated to use of the same primary endpoint of engraftment across these settings. The 
use of the same secondary endpoints across these settings also is well motivated. 
 
The regimen selection for individual patients is flexible to allow for attending physician and site preference. There 
are some absolute criteria, for example, that patients between the ages of 46 and 65 would have to go on the 
middle-intensity regimen. However, outside of this, the decision of which conditioning regimen would be based 
on the underlying disease, presence of MRD, age, co-morbidities and attending preference. Furthermore, there is 
also a move away from high dose TBI by some physicians/centers in younger patients to avoid the long term 
sequelae of high dose TBI. Because both regimens result in an increased risk of delayed hematopoietic recovery 
and early transplant related mortality, there will be other patient specific factors that lead to the specific regimen 
choice by the physician. In general, it is anticipated that younger patients would continue to accrue to the high 
dose TBI regimen, especially those with minimal residual disease and the middle-intensity regimen would accrue 
those patients between 46 and 65 years of age and patients for whom the attending physician feels this is the more 
appropriate regimen.   


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Supportive care will be in accordance with institutional guidelines to our previous cord blood expansion protocols 
(FHCRC #2044 and #2378). As in FHCRC Protocols 2044 and 2378, stopping rules for GVHD, infusional 
toxicity and graft failure will also be in place. The major differences are donor selection for the expanded cell 
product and the use of one or two unmanipulated units for the unmanipulated cell graft (allowing for a single or 
double cord blood transplant with infusion of expanded cryopreserved cord blood progenitor cells as adjunctive 
treatment).  
 
In this study, we will choose the unmanipulated CB donors per a CB selection algorithm (see Section 6.1) 
allowing a single or double non-manipulated cell graft, and randomize the patients then to receive the additional 
off-the-shelf product or not.  
 
2.6 
Risks/Benefits 
 
Potential Risks 
As an off-the-shelf, non-HLA matched cellular therapy, theoretical concerns regarding the infusion of expanded 
cells exist, including the possibility of infusional toxicity, transfusion associated GVHD, alloimmunization, and 
immediate rejection of the expanded product. There have been no infusional toxicities associated with the 
infusion of total nucleated cells (TNC) ranging in dose from 2.8x107/kg to 13.9x107/kg (CD34+ dose ranging from 
0.12x106/kg to 13.4x106/kg) in our current Phase I clinical trial. In our pilot study, no infusional toxicities were 
observed in the first four patients either, with TNC up to 9 x 107/kg from the expanded product. Patients enrolled 
in this protocol will have been treated with a standard of care myeloablative preparative regimen; however, the 
expanded cell product will be an off-the-shelf product selected without respect to HLA. Transfusion associated 
GVHD is unlikely as the expanded product has been CD34+ selected (and therefore T cell depleted) and ex vivo 
expanded prior to cryopreservation and no mature T cells are generated in culture. Alloimmunization is a definite 
possibility, but because the expanded cells will come from only one donor at a time, if alloimmunization 
develops, it will be to only a limited number of HLA antigens and this will be monitored. Lastly, cord blood 
donor selection for the unmanipulated units for all patients enrolled in this protocol will follow the current 
umbilical cord blood graft selection algorithm (Section 6.1) that is based on both HLA-typing and cell dose, with 
1 or 2 cord blood units selected to achieve the required cell dose. Thus, all patients will have an adequate graft to 
undergo a cord blood transplant even without the expanded cells.  The expanded unit, in this case, is used to 
augment this conventional graft with the goal of providing rapid but transient myeloid recovery. If there is near 
immediate immune-mediated rejection of the expanded cell product, it is anticipated that this would not impact 
patient safety above and beyond the risk inherent in conventional myeloablative cord blood transplantation.  
 
Potential Benefits 
Extensive research has been done to define the optimal conditions necessary for ex vivo expansion of 
hematopoietic stem cells (HSC), and various expansion techniques have been developed for this purpose [5]. 
Despite a lack of convincing data that pluripotent stem cells increase in number and retain an ability to engraft 
and sustain multi-lineage hematopoiesis in a myeloablated recipient following ex vivo expansion, we have focused 
on the use of an extrinsic regulator of stem cell fate (Notch) to manipulate cell fate decisions ex vivo and have 
convincingly demonstrated that Notch-mediated expansion of CB stem/progenitor cells results in a dramatic 
expansion of hematopoietic precursors capable of at least short term myeloid engraftment when infused in a 
clinical cord blood transplant setting Thus, the preliminary results from our Phase I trial provide the first evidence 
that a key regulator of cell fate, the Notch pathway, can be used in a clinically compliant manner to generate a 
safe and potentially efficacious cell therapy product.  
 
Currently, our focus is on overcoming the delay in engraftment experienced by patients undergoing a cord blood 
transplant, with the goal of improving patient outcomes. The use of CB as a source of hematopoietic stem cells for 
transplantation offers many advantages, especially for those patients of minority or mixed ethnicity background 
(and 30% of Caucasians) who cannot identify a suitably matched adult volunteer donor. However, as discussed 
above, having already demonstrated the ability of this expanded product to engraft at least temporarily in a 
myeloablative cord blood transplant setting, a critical question becomes whether this product which is devoid of 
mature T cells can be safely infused in a non-HLA matched setting. While this should be possible due to the lack 
of mature T cells in the expanded cell population, especially in a myeloablated host, there is also the potential for 


2603.00 
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rejection of the infused expanded product prior to therapeutic benefit. If we can demonstrate the safety of this 
approach, the potential impact is tremendous, not just for patients undergoing cord blood transplantation but also 
for individuals who experience chemotherapy or radiation induced aplasia.  
 
3.0 
  STUDY OBJECTIVES 
 
3.1 
Primary Objectives  
 
Compare the time to neutrophil engraftment (ANC ≥500) in patients receiving a standard of care myeloablative 
CBT augmented with an off-the-shelf pre-expanded and cryopreserved cord blood product to those who do not 
receive the product.  
 
3.2 
Secondary and Exploratory Objectives 
 
Provide initial data on clinical and economic benefit, such as time to platelet engraftment, duration of initial 
hospitalization, transplant-related mortality (TRM), death without engraftment, and incidence of severe infections 
in the first 100 days post transplant. The kinetics of immune system recovery will also be evaluated in both arms.  
 
4.0  STUDY DESIGN 
 
4.1 
Description of Study 
 
This Phase II trial is a multi-center, open-label randomized study of single or double myeloablative cord blood 
transplantation with or without infusion of off–the-shelf ex vivo expanded cryopreserved cord blood progenitor 
cells in patients with hematologic malignancies 
 
Primary Endpoint 
 
Time to engraftment (ANC ≥500) in both arms (standard myeloablative CBT with and without off-the-shelf 
expanded cord blood progenitors). 
 
4.2 
Secondary Endpoints 
1. Platelet engraftment  (20k) 
2. Incidence of infectious complications in the first 100 days post transplant 
3. Overall Survival 
4. Non-relapse mortality 
5. Acute and chronic GVHD. 
 
4.3 
Exploratory Endpoints 
1. In vivo persistence of the ex vivo expanded cord blood product 
2. Duration of initial hospitalization 
3. Grade ≥ 3 infusional toxicity 
4. Graft failure:  Primary and secondary (see protocol section 13.0 for definition of graft failure) 
5. Immune reconstitution: TCR sequencing (see protocol section 10.9) 
 
5.0 
  PATIENT SELECTION 
 
5.1    Inclusion Criteria  
 
5.1.1 
Age Criteria 
1. High dose TBI regimen: 6 months to ≤ 45 years 
2. Middle intensity TBI regimen: 6 months to ≤ 65 years 
 


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Conditioning regimen selection should be based on the underlying disease, presence of MRD, 
age, co-morbidities, attending physician, and site preference. Conditioning regimen will not 
require stratification of the randomization due to heterogeneity in the cohort of eligible patients. 
 
5.1.2  
Disease Criteria 
A.  Acute Myeloid Leukemia, including Biphenotypic Acute Leukemia or Mixed-Lineage Leukemia 
1. All patients must have AML that is considered best treated by stem cell transplant by the referring 
physician and the attending transplant physician. 
2. All patients must be in CR as defined by < 5% blasts by morphology/flow cytometry in a 
representative bone marrow sample with cellularity > 15% for age.  
3. Patients in which adequate marrow/biopsy specimens cannot be obtained to determine remission 
status by morphologic assessment, but have fulfilled criteria of remission by flow cytometry, 
recovery of peripheral blood counts with no circulating blasts, and/or normal cytogenetics (if 
applicable) may still be eligible. Reasonable attempts must be made to obtain an adequate 
specimen for morphologic assessment, including possible repeat procedures. These patients must 
be discussed with the Principal Investigator, Filippo Milano (206-667-5925 or pager, 206-314-
1037) prior to enrollment.  
B.  Acute Lymphoblastic Leukemia, including Biphenotypic Acute Leukemia or Mixed-Lineage Leukemia 
1. High risk CR1 [for example, but not limited to: t(9;22), t(1;19), t(4;11) or other MLL  
rearrangements, hyplodiploid] or HR as defined by referring institution treatment protocol, 
greater than 1 cycle to obtain CR;  CR2 or greater. 
2. All patients must be in CR as defined by <5% blasts by morphology/flow cytometry in a 
representative bone marrow sample with cellularity ≥ 15% for age. 
3. Patients in which adequate marrow/biopsy specimens cannot be obtained to determine remission 
status by morphologic assessment, but have fulfilled criteria of remission by flow cytometry, 
recovery of peripheral blood counts with no circulating blasts, and/or normal cytogenetics (if 
applicable) may still be eligible. Reasonable attempts must be made to obtain an adequate 
specimen for morphologic assessment, including possible repeat procedures. These patients must 
be discussed with the Principal Investigator, (206-667-5925 or pager, 206-314-1037) prior to 
enrollment.  
C.  
Chronic myelogenous leukemia excluding refractory blast crisis. To be eligible in  
 first chronic phase (CP1) patient must have failed or be intolerant to tyrosine kinase inhibitor 
therapy. 
D.   
Myelodysplasia (MDS) IPSS Int-2 or High risk (i.e., RAEB, RAEBt) or refractory anemia with 
severe pancytopenia or high risk cytogenetics. Blasts must be < 10% by a representative bone 
marrow aspirate morphology. 
 
5.1.3  
Organ Function and Performance Status Criteria 
A. Performance status score 
1. Karnofsky (≥ 16 years old) ≥ 70 or ECOG 0-1 
2. Lansky (<16 years old) ≥ 60 
B. Renal Function    
1. Adults:  Calculated creatinine clearance must be > 60 mL and serum creatinine ≤ 2 mg/dL.   
2. Children (<18 years old): Calculated creatinine clearance must be > 60 mL/min   
C. Hepatic Function 
1. Total serum bilirubin must be <3mg/dL unless the elevation is thought to be due to Gilbert’s 
disease or hemolysis.  
2. Transaminases must be < 3x the upper limit of normal per reference values of referring institution 
D. Pulmonary function 
1. DLCO corrected >60% normal 
2. For pediatric patients unable to perform pulmonary function tests, O2 saturation >92% on room 
air 


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3. May not be on supplemental oxygen.  
E. Cardiac function 
1. Left ventricular ejection fraction >45% OR 
2. Shortening fraction > 26% 
F. Ability to understand and the willingness to sign a written informed consent document 
 
 
5.2   
Exclusion Criteria 
A. Uncontrolled viral or bacterial infection at the time of study enrollment 
B. Active or recent (prior 6 month) invasive fungal infection without ID consult and approval 
C. History of HIV infection 
D. Pregnant or breastfeeding 
E. Prior myeloablative transplant containing full dose TBI (greater than 8 Gy). 
F. CNS leukemic involvement not clearing with intrathecal chemotherapy and/or cranial radiation prior 
to initiation of conditioning. Diagnostic lumbar puncture is to be performed as per section 9.7.  
G. Patients > 45 years: comorbidity score of 5 or higher 
 
6.0  Cord Blood Graft Selection 
 
6.1  Unmanipulated Unit(s)   
CB donor selection will be based on institutional guidelines and in general should be selected to optimize both 
HLA match and cell dose. Additionally, CB grafts shall consist of one or two CB donors based on, but not 
exclusively determined by, cell dose (TNC/kg and CD34/kg), HLA matching and disease status and indication for 
transplant. Attending preference will be allowed for single versus double unit as well as the degree of 
mismatching based on patient specific factors, as long as the following minimum criteria are met: 
 
A. HLA matching: 
i. 
Minimum requirement: The CB graft(s) must be matched at a minimum at 4/6 HLA-A, B, DRB1 
loci with the recipient. Therefore 0-2 mismatches at the A or B or DRB1 loci. based on 
intermediate resolution A, B antigen and DRB1 allele typing for determination of HLA-match is 
allowed. 
ii. HLA-matching determined by high resolution typing is allowed per institutional guidelines as 
long as the minimum (A.i., above) are met.  
B. Selection of two CB units is mandatory when a single cord blood unit does not meet the following in 
the table below.  
 
 
 
 
 
 
If two CB units are used the total cell dose of the combined units must be at least 3.0 x 107 TNC 
per kilogram recipient weight based on pre-cryopreservation numbers, with each CB unit 
containing a MINIMUM of 1.5 x 107 TNC/kg.  
 
C. The minimum recommended CD34/kg cell dose should be 2 x 105 CD34/kg, total dose from a single or 
combined double.  
D. The unmanipulated CB unit(s) will be FDA licensed or will be obtained under a separate IND, such as 
the National Marrow Donor Program (NMDP) Protocol 10-CBA conducted under BB IND-7555 or 
another IND sponsored by (1) a participating institution or (2) an investigator at FHCRC or one of the 
participating institutions.  
 
Single Unit Allowed for: 
Match Grade TNC Dose 
6/6 
≥ 2.5 x 107/kg 
5/6, 4/6 
≥ 4.0 (± 0.5) x 107/kg 


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E. FHCRC only: Up to 5% of the unmanipulated cord blood product (s), when ready for infusion, may be 
withheld for research purposes as long as thresholds for infused TNC dose are met.  These products will 
be used to conduct studies involving the kinetics of engraftment and immunobiology of double cord 
transplantation. 
 
6.2  Ex Vivo Expanded Unit (DCC-UCB - Investigational Agent) 
The expanded cord blood progenitors to be used for this trial will be selected from a bank of previously 
expanded cord blood progenitors (per IND 14184) that have been cryopreserved for future clinical research 
use at FHCRC. Each individual progenitor cell product is derived from a single cord blood unit (donor) that 
undergoes CD34 selection (and is therefore a T cell depleted product) and is then placed into culture and ex 
vivo expanded in the presence of Notch ligand (see background). The final product is the total progeny 
derived from the starting CD34+ cell population which is harvested on day 14 – 16 of culture and then 
cryopreserved. The fresh cord blood units used for expansion are obtained through collaborations with 
accredited public cord blood banks. These public cord blood banks provide appropriately screened cord blood 
units meeting donor eligibility criteria per 21 CFR 1271.  
Product selection will be performed for each enrolled patient by the FHCRC PI or qualified designee and 
product will be shipped to the participating site for outside patients. Refer to shipping details and handling 
instructions in the FHCRC Protocol 2603: Receipt and Handling of NLA101/DCC-UCB (Delta1-Cultured 
Cryopreserved Umbilical Cord Blood Cells) Information Sheet (June 2017). Selection of the expanded cord 
blood progenitor product will be based on the following:  
A. Panel Reactive Antibody (PRA) is to be performed on all enrolled patients, and product will be 
 
selected based on the specificity of donor directed antibodies when present as possible. PRA negative  
patients may receive any product that fits cell dose restrictions. HLA matching will not be considered 
outside of PRA+ patients.  
B.  Cell Doses: 
1.  Infused TNC/kg cell dose will not exceed 1 x 108 TNC/kg recipient body weight based on final 
viable TNC count at the time of cryopreservation.  
2.  CD34 cell dose: No upper limit will be placed on the CD34 cells/kg infused.  
3.  All expanded products are evaluated by immunophenotyping for the presence of CD3+ cells prior 
to freezing. While there has been no convincing evidence of a CD3+ cell population, if a product 
has evidence of a T cell (CD3+) population, this product will not be used unless the dose of CD3+ 
cells is <5x105 CD3 cells/kg (recipient weight) based on pre-freeze assessment of CD3 content in 
the final product.  
4. 
The expected volume of the ex vivo expanded product is 120 mL after thaw and dilute 
methodology. 
 
7.0  INFORMED CONSENT OF SUBJECT AND DONOR 
 
The patient and donor units will be completely evaluated. The protocol will be discussed thoroughly with the 
patient and family, including requirement for data collection and release of medical records and all known risks to 
the patient will be described. The procedure and alternative forms of therapy will be presented as objectively as 
possible and the risks and hazards of the procedure explained to the subject or, in the case of minors, to the 
subject's responsible family members. Consent will be obtained using forms approved by the Institutional Review 
Board. A summary of the conference will be dictated for the medical record detailing what was covered. 
 
8.0  SUBJECT REGISTRATION 
 
Informed consent must be signed prior to the performance of any study related procedures or assessments. 
 
FHCRC patients: Eligible subjects will be identified and registered into the system by the Clinical Coordinators 
Office (CCO) (Intake Office) and assigned a UPN (Unique Patient Number). The CCO will register the subject on 
to the protocol through the Data Management Office. 
 


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Collaborating institutions: Eligible patients will be identified by the principal investigators of the collaborating 
institutions who will register the patient with the FHCRC Coordinating Center. Registration will include 
completion of the eligibility checklist/demographic form provided in the CRF packet. This form will be submitted 
to the Coordinating Center along with source documents and eligibility will be confirmed by the Coordinating 
Center PI or qualified designee before enrollment occurs.  Questions regarding eligibility or protocol information 
should be directed to Filippo Milano, MD (206-667-5925). 
 
9.0  TREATMENT PLAN 
 
9.1 
Treatment Schema 
This schema is a pictorial overview only; please see narrative Treatment and Evaluation sections of the protocol for 
specifics and allowable deviations from time frames shown.  
 
Pre-transplant Determination of Eligibility and Randomization 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Potential Patient 
Identified &
CB Search Done
Determination 
of 
Eligibiity
Obtain Patient
Consent 
and/or Assent
RANDOMIZATION
+/- Off-the-shelf 
expanded progenitor cells
No
Assess for 
alternative 
protocol
Yes
Treatment per protocol
Potential Patient 
Identified &
CB Search Done
Determination 
of 
Eligibiity
Obtain Patient
Consent 
and/or Assent
RANDOMIZATION
+/- Off-the-shelf 
expanded progenitor cells
No
Assess for 
alternative 
protocol
Yes
Treatment per protocol


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Treatment Schema   
 
 
9.2  
Conditioning Regimens 
 
There are two conditioning regimens allowed per protocol. They are:  
 
1. Regimen A: High dose TBI + Fludarabine + cyclophosphamide 
2. Regimen B: Middle intensity TBI + Fludarabine + cyclophosphamide + thiotepa 
 
REGIMEN A: High Dose TBI regimen (included ages: 6 months through 45 years old) 
 
Fludarabine total 75 mg/m2 (25 mg/m2/day IV x 3 days, days -8 to -6), Cyclophosphamide total 120 mg/kg (60 mg/kg IV x 2 
days, days -7 to -6), High Dose TBI total 1320 cGy (165 cGy BID, total 8 fractions, days -4 to -1) 
 
Day 
Preparative Regimen 
Supportive Care/Other 
-8 
Fludarabine 25 mg/m2 IV over 30 minutes 
 
-7 
Fludarabine 25 mg/m2 IV over 30 minutes  
Cyclophosphamide 60 mg/kg IV 
 
-6 
Fludarabine 25 mg/m2 IV over 30 minutes  
Cyclophosphamide 60 mg/kg IV 
 
-5 
Rest 
-4 
TBI 165 cGy twice daily 
 
-3 
TBI 165 cGy twice daily 
Begin CSA (as per 
Section 9.3A) 
-2 
TBI 165 cGy twice daily 
 
-1 
TBI 165 cGy twice daily 
 
0 
EXPERIMENTAL ARM 
UCBT: Unmanipulated unit(s) 
(umbilical cord blood #1 
and/or #2), followed by 
STANDARD OF CARE ARM 
 
Begin MMF  (as per 
Section 9.3B) 


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infusion of ex vivo expanded 
cell product at a minimum of 4 
hours after completion* of last 
unmanipulated unit 
UCBT: Unmanipulated unit(s) 
(umbilical cord blood #1 and/or 
#2) 
+1 
 
Begin G-CSF (as per 
Section 9.4A) 
 * See 9.6B.3 
 
A. Fludarabine: 25 mg/m2/day IV over 30 minutes x 3 days (days -8 to -6); total dose 75 mg/m2. For 
patients > 120% of ideal weight, BSA will be calculated using adjusted weight.  
B. Cyclophosphamide  
1. Cyclophosphamide: 60 mg/kg/Day IV x 2 days (Days -7 and -6); total dose 120mg/kg. 
2. Preparation, administration and monitoring will be according to Institutional Guidelines. If 
patient’s actual weight is >100% of IBW, adjusted body weight should be used for 
calculating initial doses based on per kilogram weight, per Institutional Guidelines. MESNA 
will be given for bladder prophylaxis according to Institutional Guidelines. Continuous 
bladder irrigation is an alternative for bladder prophylaxis at the attending physician’s 
discretion.  
3. Cyclophosphamide administration and hydration guidelines for outside centers will be 
reviewed and approved by FHCRC PI if varying significantly from the above. Monitoring for 
toxicities will be according to Institutional Guidelines. 
C. Total Body Irradiation: TBI 165 cGy will be given twice daily for a total dose of 1320 cGy (days -4 to 
–1). 
 
REGIMEN B:  Middle Intensity TBI regimen (included ages: 6 months through 65 years old) 
 
Fludarabine total 150 mg/m2 (30mg/m2 IV x 5 days, days -6 to -2), Cyclophosphamide total 50 mg/kg IV x 1 day, day - 6),  
Thiotepa total 10 mg/kg (5 mg/kg/day x 2 days, -5 to -40), TBI total 400 cGy (200 cGy/day x 2 days, -2 to -1)  
 
* See 9.6B.3 
 
Day 
Preparative Regimen 
Supportive Care/Other 
-6 
Fludarabine 30 mg/m2 IV over 30-60 minutes 
Cyclophosphamide 50 mg/kg IV  
 
-5 
Fludarabine 30 mg/m2 IV over 30-60 minutes 
Thiotepa 5 mg/kg IV over 4 hours 
 
-4 
Fludarabine 30 mg/m2 IV over 30-60 minutes  
Thiotepa 5 mg/kg IV over 4 hours 
 
-3 
Fludarabine 30 mg/m2 IV over 30-60 minutes 
Begin CSA (as per Section 9.3A) 
-2 
Fludarabine 30 mg/m2 IV over 30 minutes 
    TBI 200 cGy once daily 
 
-1 
TBI 200 cGy once daily 
 
0 
EXPERIMENTAL ARM 
UCBT: Unmanipulated unit(s) 
(umbilical cord blood #1 
and/or #2), followed by 
infusion of ex vivo expanded 
cell product at a minimum of 4 
hours after completion* of last 
unmanipulated unit 
STANDARD OF 
CARE ARM 
 
UCBT: Unmanipulated 
unit(s) (umbilical cord 
blood #1 and/or #2) 
Begin MMF (as per Section 9.3B) 
+1 
 
Begin G-CSF (as per Section 
9.4A) 


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A. Fludarabine: 30 mg/m2/day IV over 30 minutes x 5 days (days -6 to -2); total dose 150 mg/m2. For 
patients > 120% of ideal weight, BSA will be calculated using adjusted weight 
B. Thiotepa: 5 mg/kg IV x 2 days (days -5 to -4); total dose 10 mg/kg. For subjects > 125% of ideal 
weight, BSA will be calculated using adjusted weight. 
C. Cyclophosphamide:  
a. 50 mg/kg IV x1 day (day -6) 
b. Preparation, administration and monitoring will be according to Institutional Guidelines. If 
patient’s actual weight is >100% of IBW, adjusted body weight should be used for calculating 
initial doses based on per kilogram weight, per Institutional Guidelines. MESNA will be given for 
bladder prophylaxis according to Institutional Guidelines. Continuous bladder irrigation is an 
alternative for bladder prophylaxis at the attending physician’s discretion 
c. Cyclophosphamide administration and hydration guidelines for outside centers can be according 
to Institutional Guidelines. Monitoring for toxicities will be according to Institutional Guidelines 
           D. Total Body Irradiation: TBI 200 cGy will be given once daily for a total dose of 400 cGy (days -2 to –1) 
9.3  Immunosuppressive Therapies  
All patients will receive GVHD prophylaxis with 2 drugs as follows: 
A. Cyclosporine (CSA) 
1. Patients will receive cyclosporine (CSA) therapy beginning Day -3 maintaining a level of >200 
ng/mL. For adults the initial dose will be 2.5 mg/kg IV over 1 hour every 12 hours. For children 
< 40 kg the initial dose will be 2.5 mg/kg IV over 1 hour every 8 hours. For outside centers, 
initial CSA administration can be according to Institutional Guidelines. 
2. Dose adjustments will be made on the basis of toxicity and CSA levels with a targeted trough 
level of 200-400ng/mL. Once the patient can tolerate oral medications and has a normal gastro-
intestinal transit time, CSA will be converted to an oral form. Refer to Institutional Guidelines 
for conversion from IV to PO dosing, CSA dosing will be monitored and altered as  clinically 
appropriate 
3. Initial cyclosporine dose is calculated using actual body weight except for those patients who 
are greater than 100% ideal body weight in which case calculation of dose using adjusted 
body weight is recommended. 
4. Patients will receive CSA until Day +100.  If there is no GVHD, the dose will be tapered 10% 
per week beginning on Day +101, to discontinue no sooner than 6 months post transplant. 
 
B. Mycophenolate mofetil (MMF) 
1. All patients will begin mycophenolate mofetil (MMF) on Day 0, starting approximately 4 - 6 
hours after infusion of the cord blood unit(s) is completed OR after infusion of the expanded 
product is completed for patients randomized to this arm. All patients will receive MMF at 
the dose of 15 mg/kg (based on adjusted weight) every 8 hours with a maximum of 1 
gram/dose. If actual body weight is < ideal body weight, calculation based on actual weight is 
allowed. Rounding of the dose to the nearest 250 mg capsule size is also allowed.  
2. Use IV route between days 0 and +7; then, if tolerated, may change to PO beginning on day 
+8 three times a day.  
3. All patients will remain on three times a day as permitted after day 7 for a minimum of 30 
days. At day +30 or 7 days after engraftment (defined as 1st day of 2 consecutive days of 
absolute neutrophil count [ANC] ≥ 0.5 x 109 /L), whichever day is later, if there is no 
evidence of acute GVHD and donor CD3 engraftment is at least 50% from one donor, 
taper MMF to BID. At day +45 (or 15 days after engraftment if engraftment occurred > day 
+30), if there continues to be no evidence of acute GVHD and donor engraftment has been 
achieved (defined as <10% host in CD3, CD33 and CD56), taper MMF over the next two to 
three weeks.    
4. If there is no donor engraftment, do not stop MMF. If there is no evidence of donor 
engraftment on the day +28 bone marrow biopsy, notify the PI, Filippo Milano (pager: 206-
314-1037).   


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9.4  Growth Factor 
A. All patients will receive G-CSF 5 mcg/kg/day IV (pediatric patients) or SC (adult patients) (dose 
rounded to vial size) based on the actual body weight beginning on Day +1 after UCB infusion.  G-
CSF will be administered daily until the ANC exceeds 2.5 x 109/L for three consecutive days and then 
discontinued.  If the ANC decreases to ≤1.0 x 109/L, G-CSF will be reinstituted and titrated to 
maintain an ANC >1.0 x 109/L. 
 
9.5  Supportive Care 
A. Patients will receive transfusions, infection prophylaxis (bacterial, fungal, viral), and other therapy 
(including GVHD) according to Institutional Guidelines. 
 
9.6  Cord Blood Infusion ( FHCRC - see also Protocol Specific Infusion Orders)   
A. Infusion of unmanipulated (freshly thawed) cord blood unit(s) 
1. The unmanipulated (freshly thawed) cord blood unit(s) will be infused first.  
2. Procedures for requesting, receiving and characterizing the cord blood unit(s) for infusion 
will be according to Institutional Guidelines. 
3. Pre-infusion hydration should be performed per institutional guidelines.  
4. Procedures for infusion of the cord blood unit(s) will be according to Institutional Guidelines. 
See Appendix B for FHCRC Standard Practice Guideline for Infusion of Cryopreserved Cord 
Blood for procedures at FHCRC and suggested guidelines for Participating Centers. 
5. If the patient is a recipient of a double cord blood graft, the two unmanipulated units should 
be given consecutively with no wait between infusion of the units. However, infusion of the 
second unit will not begin until any acute toxicities from the first unit have been 
controlled. The start and stop time of each unit should be recorded on the infusion record. 
6. FHCRC: If the cord blood unit(s) fail to pass inspection or if there is insufficient information 
to verify the cell product for the patient, notify the Cellular Therapy Lab ((206) 606-1200) 
and the PI (Filippo Milano, (206) 667-5925, pager (206) 314-1037 immediately).  
 
B. Infusion of expanded, cryopreserved cord blood progenitor cells 
1. Dosage and selection of expanded product: See Section 6.2 B 
2. FHCRC patients: please see protocol specific infusion orders. Infuse product as provided by 
Cellular Therapy. 
3. Infusion time of ex vivo expanded cell product is ideally approximately 4 hours after 
completion of infusion of last unmanipulated unit. Variation is allowable for the infusion 
time of ex vivo expanded cell product, provided that any toxicities related to infusion of the 
last unmanipulated product have resolved. The acceptable window of infusion time is 3 hours 
30 minutes to 24 hours following completion of the last unmanipulated unit. 
The patient should be premedicated with diphenhydramine and acetaminophen.  
Additional diphenhydramine, epinephrine and hydrocortisone should be available at the 
bedside for emergency if necessary. If diphenhydramine and acetaminophen have been 
previously administered as premedication for the unmanipulated unit(s), repeated dosing 
is at provider discretion.   
4. Oxygen with nasal prongs for standby use should be present in the room. 
5. Filtration: Filter through a blood component administration filter set by gravity.  If 
institutional guidelines include pump infusion use, please infuse by syringe pump only (no 
roller set infusions). 
6. The goal rate for infusion of the expanded cells is no longer than 30 minutes, as clinically 
possible. Infuse at a rate of 3–5 ml/min for the first 4 minutes. If tolerated, increase rate to 
“wide open” as tolerated. No medications or fluids should be given piggyback through the 
catheter that is being used for the expanded cell infusion.  
7. Monitoring of immediate toxicities: See Section 11.3. 
 
 


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9.7  CNS Prophylaxis and Testicular Irradiation 
A. All patients with ALL will undergo a diagnostic lumbar puncture during the pre-admission work-up 
following institutional guidelines for diagnostic tap alone or with prophylactic therapy. AML patients 
previously demonstrated to be CNS negative prior to pre-transplant work-up may follow institutional 
guidelines for work-up of potential CNS disease, however it is strongly recommended that all AML 
patients undergo a diagnostic lumbar puncture during pre-transplant evaluation to rule out CNS 
involvement. For patients with CML and MDS, lumbar puncture and prophylactic therapy are performed 
per institutional guidelines.  
B.   No dose of intrathecal therapy is to be given less than 72 hours before marrow/stem cell infusion. 
Beginning on day 32 post transplant, patients will receive intrathecal (IT) Methotrexate as indicated, per 
institutional guidelines. 
C.   The CNS prophylaxis regimen may be modified by the attending physician in consultation with the 
clinical coordinator as clinically indicated (i.e., IT methotrexate can be omitted in patients with a low risk 
of CNS relapse or in patients who have a high risk of leukoencephalopathy). 
D. FHCRC: Testicular irradiation will be performed per standard practice. Outside centers: testicular 
irradiation will be performed as possible and per institutional guidelines. 
 
9.8  Management of Pre-Engraftment Immune Reactions 
A. A well-recognized clinical entity consisting of skin rash, fever, and, often, loose stools and respiratory 
distress has been noted to occur prior to engraftment among cord blood patients, generally between 
Days +7 and +21.  This clinical syndrome likely involves cytokine activation, and though clinically 
similar to acute or hyperacute graft versus host disease, it appears to be a distinct entity, “pre-
engraftment syndrome.”   This syndrome is often controlled with brief steroid bursts, thus avoiding a 
commitment to extended steroid exposure. Patients should be monitored carefully for this syndrome. 
 
If patients have moderate to severe symptoms as described above and alternative etiologies (i.e., 
infection) have been excluded or are being appropriately evaluated, recommendations for management 
are: 
1. For patients not on steroid therapy when the syndrome occurs:  methylprednisolone should be 
given at 1 mg/kg IV q day for three days.  If symptoms have abated, steroids should be 
stopped.  If symptoms persist, 1 mg/kg methylprednisolone can be continued through six days 
then stopped if symptoms have abated.  If symptoms persist for more than six days, the 
patient should be considered to have acute/hyperacute GVHD and should be treated with 
prolonged steroids as deemed appropriate. 
 
2. For patients already on steroids for other reasons when the syndrome 
occurs:  methylprednisolone should be given at a dose of 3-5 mg/kg IV (max dose 500 mg) q 
12 hours x 48 hours, followed by a rapid taper to 1 mg/kg IV q 12 hours.  Patients should be 
weaned after response as tolerated. 
 
10.0  EVALUATION (in addition to procedures and evaluations listed below, additional clinical evaluations as 
directed by the clinical team may be captured for research purposes)  
 
10.1  Pre-Transplant Evaluation 
 
FHCRC Patients: Refer to FHCRC/SCCA Standard Practice Manual for Pre-Transplant Evaluation 
Guidelines for Allogeneic Transplant. (results of tests and/or procedures conducted as per standard of 
care for pre-transplant workups may be used for eligibility determination if conducted within an 
appropriate window prior to screening) 
 
Fred Hutch and Outside Patients: Upon approval of protocol version 3.12.19, please refer to Appendix G 
for an updated schedule of study evaluations for 1 and 2-year. 
 
In addition to Standard Practice Guidelines complete the following protocol specific requirements: 


2603.00 
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A. Bone marrow aspirate (and biopsy as clinically indicated), CBC with differential, comprehensive 
metabolic panel, and creatinine clearance within 30 days of start of conditioning. 
B. MUGA or echocardiography with measurement of the left ventricular ejection fraction (LVEF) or left 
ventricular shortening fraction.   
C. Chest CT without contrast to exclude occult infection prior to transplant within 30 days of start of 
conditioning.   
D. Pulmonary function tests; pediatric pre-transplant evaluation qualification:  see section 5.1.3D 
E. Viral screening including CMV PCR 
F. DNA specimen from cord blood unit(s) for chimerism studies (FHCRC patients: submitted to CIL) 
G. Panel Reactive Antibody (PRA) per Institutional Guidelines 
H. Pregnancy test per Institutional Guidelines in females of childbearing potential. 
 
10.2   Evaluation on the day of cord blood infusions (Day 0) 
A. Physical exam and review of systems by provider 
B. Weight 
C. CBC with differential 
D. Renal and hepatic function: NA, K, CL, CO2, GLU, BUN, CRE, CA, P, ALB, MG, ALT, AST, 
ALK, BILIT/D, TP, ALB, LDH   
E. Complete urinalysis only on patients receiving the expanded cord blood progenitor cell product 
 
10.3   Evaluation during infusion of Ex Vivo Expanded Cord Blood Progenitor Cell Product  
A. Patient must be monitored during infusion as per institutional guidelines. 
B. For any changes in cardiac or respiratory status, notify physician.  
C. Obtain and record vital signs including temperature, BP, HR, respirations,O2 saturation at the 
following approximate time points: 
 
Pre-infusion 
1 hour after the start of infusion 
15 minutes after the start of infusion 
2 hours after the start of infusion 
30 minutes after the start of infusion 
4 hours after the start of infusion 
45 minutes after the start of infusion 
24 hours after the start of infusion 
D. Please also refer to Section 11.3B 
 
10.4 
Evaluation approximately 24 hours post completion of infusion of expanded cord blood progenitor 
product 
A. CBC with differential 
B. Renal and hepatic function: NA, K, CL, CO2, GLU, BUN, CRE, CA, P, ALB, MG, ALT, AST, 
ALK, BILIT/D, TP, ALB, LDH  
C. Complete urinalysis 
 
10.5  
Patient Evaluations during Therapy until Engraftment (Or Through Day 30, Whichever Occurs 
First)  
A. Physical exam, interval history and vital signs daily while in the hospital.  After discharge, vital signs 
twice weekly until engraftment.   
B. Complete blood count with leukocyte differential daily (+/- 4 hour window) until the absolute 
neutrophil count (ANC) > 5x108/L for 2 consecutive measurements. For outside centers, complete 
blood count with leukocyte differential requirement can be according to Institutional Guidelines.  
C. Serum electrolytes, BUN, creatinine, glucose every other day at a minimum until engrafted.  
D. Hepatic function panel at least three times weekly or as clinically indicated. 
E. CMV monitoring per Institutional Guidelines. 
F. Urinalysis as clinically indicated. 
G. Chest radiographs as clinically indicated. 
H. Peripheral blood donor chimerism. See Section 10.7 for details. 


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I. Bone marrow aspirate (+/- biopsy, as clinically indicated) on Day 28 for disease restaging 
(morphology, immunophenotyping, cytogenetics and molecular studies as indicated) and assessment 
of donor engraftment. FHCRC patients: bone marrow specimen, unsorted, to CIL for chimerism 
studies.  
J. Quantitative immunoglobulin levels (IgG, IgA, IgM) at Day 28 
K. As possible, total T lymphocytes and subset enumeration at Day 28 
L. Panel Reactive Antibody (PRA) upon count recovery on patients who received the expanded cells. 
Please check with research staff (PI or Research Nurse (Nancy Anderson) or Research 
Coordinator (Denise Ziegler)). Research staff will notify clinical staff to initiate the orders. 
PRAs will be charged to research budget.  
M. GVHD evaluation (Appendix C) weekly or as clinically indicated 
N. Other tests (e.g., x-rays and tumor markers) as clinically appropriate for assessment of underlying 
malignancy on Day 28 
O. See also Section 11.3B 
 
10.6 
Patient Evaluations from Engraftment through Day 100, 6 Months, 1 Year and 2 Years 
A. Physical examinations at least weekly until discharge, then as clinically indicated during first 100 
days, 6 months, 1 year and 2 years  
B. Vital signs twice weekly until discharge 
C. Karnofsky/ECOG/Lansky performance status (Appendix A) once between Day 80 and Day 100, 6 
months, 1 year and 2 years 
D. Complete blood count with leukocyte differential at least weekly and/or as clinically indicated 
through Day 100 or until discharge then at 6 months, 1 year and  2 years  
E. Serum electrolytes, BUN, creatinine, glucose twice weekly or as clinically indicated through day 
+100  
F. Hepatic function panel at least weekly through Day 100 or until discharge and then as clinically 
indicated 
G. CMV monitoring per Institutional Guidelines 
H. Chest radiographs, EKG and pulmonary function tests as clinically indicated 
I. Bone marrow aspirate (+/- biopsy as clinically indicated) on Day 80 and 1 year for assessment of 
donor engraftment and disease restaging. FHCRC patients: bone marrow aspirate submitted, 
unsorted, to CIL for chimerism studies. 
J. Peripheral blood for chimerism studies. See Section 10.7 for details. 
K. Quantitative immunoglobulin levels (IgG, IgA, IgM) at Day 56, 80, 6 months, 1 year and 2 years 
L. As possible, total T lymphocytes and subset enumeration on Day 56, 100, 6 months, 1 year and 2 
years   
M. GVHD evaluation (Appendix C) weekly and/or as clinically indicated through Day 100 (or longer if 
clinically indicated), then at 6 months, 1 year and 2 years  
N. Autopsy report, if available, if death occurs before the 2 year follow-up 
O. See also Section 11.3B 
 
10.7 
Evaluation of In Vivo Persistence of Expanded and Unmanipulated Cord Blood Cells:  Chimerism  
Studies/UCB Engraftment Evaluation   
Chimerism studies will be performed as outlined below. Subsequently, if the patient's peripheral blood 
counts drop after an initial recovery, the peripheral blood and bone marrow should again be evaluated 
unless a cause has been determined (e.g., use of Ganciclovir for treatment of CMV).  Patients diagnosed 
with graft failure as defined in section 13.0 must be reported to the Principal Investigator (Filippo Milano, 
206-667-5925/pgr 206-314-1037). 
A. Bone marrow aspirate (unsorted) for chimerism studies at day +28, +80 and 1 year. Day +80 may be 
performed on Day +100 at outside centers as per institutional guidelines.  After 1 year, bone marrow 
chimerism is to be performed as clinically indicated. Send to Clinical Immunogenetics Laboratory. 
B. Bone marrow aspirate for morphology and immunophenotyping by flow cytometry at day +28, +80 
and 1 year.  Day +80 may be performed on Day +100 at outside centers as per institutional guidelines. 
After 1 year, bone marrow evaluation is to be performed as clinically indicated.  


2603.00 
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C. Peripheral blood donor chimerism: Day +7, +14 and +21* 
Peripheral blood is to be collected and cell sorted for CD3+, CD33+, CD14+, and CD56+, as possible at 
outside centers) and donor/host chimerism determined. 
Peripheral blood donor chimerism: Day +28**, +42*, +56, +80, +180, 1 and 2 years: 
Peripheral blood is to be collected and cell sorted for CD3+, CD33+, CD14+, CD19+ (or 20+) and 
CD56+ (as possible at outside centers) and donor/host chimerism determined. Day +80 may be 
performed at Day +100 at outside centers as per institutional guidelines. After 1 year, peripheral 
blood chimerism is to be sorted for CD3+, CD33+, and CD56+ only, unless otherwise clinically 
indicated.  
 
*Draw peripheral blood for chimerism at Day +21 and Day +42 only if previous chimerism time 
point did not show at least 95% donor engraftment from a single cord blood unit.  
**Day +28 chimerism may be skipped IF the patient had Day 21 chimerisms and it showed at least 
95% donor engraftment from a single cord blood unit.  
 
10.8   Residual/Recurrent Disease Evaluation 
A. Patients will be evaluated routinely for evidence of recurrent malignancy as per Institutional 
Guidelines on day +28 and day +80.  If at any time the attending physician suspects recurrent disease, 
additional analyses will be performed as clinically indicated. 
 
10.9   Immune Reconstitution  
 
Clinical Studies: As possible, quantitative immunoglobulin levels (IgG, IgA, IgM) will be assessed at 
pre-conditioning, Day 28, 56, 80 or 100 (per institutional practice), 6 months, 1 year and 2 years. As 
possible, total T lymphocytes and subset enumeration (lymphocyte panel) will be performed at Day 28, 
56, 100, 6 months, 1 year and 2 years.                                          
 
Research Studies: Upon approval of this protocol modification, all research blood collections will 
discontinue. Adaptive Biotechnologies will continue to conduct TCR studies (immunophenotypic 
evaluation of immune reconstitution) using previously collected de-identified samples and data.  
 
10.10 Study Evaluation Windows 
The target dates for post-transplant study evaluations are outlined in the table below. In certain clinical 
circumstances (e.g., relapse, clinical status or terminal illness) study tests may be omitted at the 
physician’s or PI’s discretion.  
 
Evaluation Timepoint Post Transplant 
Window 
24 hrs post infusion of expanded cell product 
± 2 hours 
Day +7 
± 2 days 
Day +14 
± 3 days 
Day +21 
± 3 days 
Day +28 
± 3 days 
Day +42 
± 7 days 
Day +56 
 ± 7 days 
Day +80 
± 7 days 
Day +100 
± 7 days 
Day +180 
± 30 days 
1 year 
* 
2 year 
* 
* Every effort will be made to complete the 1 year and 2 year evaluations as close to 
these dates as possible, taking into consideration patient’s circumstances at these time 
points. 
 
 
 


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11.0  DRUGS, IRRADIATION AND CORD BLOOD INFUSION: TOXICITIES AND  
 COMPLICATIONS 
 
11.1 Cyclophosphamide and Other Conditioning Agents 
Common 
Occurs in 21-100 people out of 100 
Less Frequent 
Occurs in 5-20 people out of 100 
Uncommon 
Occurs in  <5 people out of 
100 
Nausea/vomiting 
Mucositis 
Sterility 
Severe suppression of blood counts 
Diarrhea 
Fluid weight gain or edema 
Alopecia 
Hemorrhagic cystitis 
Cardiomyopathy 
Skin rash 
SIADH (Syndrome of 
Inappropriate Anti-diuretic 
Hormone) 
 
Fludarabine 
 
 
Common 
Occurs in 21-100 people out of 100 
Less Frequent 
Occurs in 5-20 people out of 
100 
Uncommon 
Occurs in <5 people out of 
100 
Severe suppression of blood counts 
Diarrhea  
Anorexia 
Mucositis 
Nausea/vomiting 
Stomatitis 
Osteoporosis 
Dysuria 
Chills 
Fever 
GI bleeding 
Peripheral edema 
Neurotoxicity 
Agitation and confusion 
Blurred vision 
Peripheral neuropathy 
Hearing loss  
Headache 
Cerebellar syndrome 
Blindness 
Coma  
Weakness 
Depression 
Insomnia 
Hemorrhagic cystitis (except 
in FA) 
Abnormal renal function test 
Autoimmune hemolytic 
anemia 
Deep venous thrombosis 
Aneurysms 
Pruritic skin rash 
Abnormal liver 
function/Liver failure 
Constipation 
Transient ischemic attack 
Dysphagia 
Myalgia 
Arthralgia 
Renal failure 


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Thiotepa 
 
Likely (Over 10%) 
Less Likely (1-10%) 
Rare (Less than 1%) 
• 
Low white blood cell count with 
an increased risk of infection 
(from bacteria, fungi or viruses) 
• 
Low platelet count with increased 
risk of bleeding 
• 
Anemia 
• 
Nausea/vomiting  
• 
Diarrhea  
• 
Anorexia (loss of appetite) 
• 
Mouth ulcers  
• 
Sores in mouth or on lips  
• 
Missing or stopping of menstrual 
periods in women 
 
• 
Skin rash 
• 
Change in skin coloring 
• 
Fatigue, weakness 
• 
Dizziness 
• 
Headache 
• 
Permanent sterility (inability 
to have children) 
• 
Allergic reactions during 
infusion (fever, chills, 
itching, hives, flushing, 
rash, shortness of breath, 
wheezing, chest tightness, 
muscle stiffening)  
• 
Confusion 
• 
Seizures 
• 
Liver damage 
• 
Secondary cancers  
 
11.2 Total Body Irradiation 
See Institutional Guidelines for recommendations and technical parameters for administration, toxicity and 
complications of TBI by linear accelerator. 
  
Common 
Occurs in 21-100 people out of 
100 
Less Frequent 
Occurs in 5-20 people out of 100 
Uncommon 
Occurs in <5 people out of 100 
Nausea and vomiting 
Diarrhea 
Cataracts 
Sterility 
Endocrinopathies 
Growth failure 
Intestinal cramps 
Mucositis 
Parotitis 
Interstitial Pneumonitis 
Generalized mild erythema 
Veno-occlusive disease 
 
Dysphagia 
Vertebral deformities 
Nephropathy 
11.3 Infusion of Unmanipulated and Expanded Cord Blood Units 
 
A. Unmanipulated Cord Blood Units 
Potential toxicities: Although the cord blood cells are thawed per standard institutional procedures prior 
to infusion, potential toxicities associated with the infusion include DMSO toxicity and side effects from 
red cells. Allergic reactions to the thawing diluent (especially dextran) have also been reported. DMSO 
toxicity and side effect of red cells may include changes in heart rate, rhythm or function, changes in 
blood pressure, fever, chills, sweats, nausea/vomiting, diarrhea, abdominal cramping, headache, allergic 
reaction, presence of DMSO taste and odor, hemoglobinuria, and acute renal failure.   
 
 
B. Expanded Cells 
 
 
Please refer to Section 9.6 for infusion guidelines 
1. Immediate toxicities: Immediate toxicities (those occurring either during or within the first 24 hours 
following the infusion of the expanded cord blood unit) might occur. These symptoms could include 
fever, chills, fatigue, dyspnea, chest tightness or myalgias; alteration in vital signs such as 
hypotension, tachycardia, tachypnea, or hypoxemia; and skin changes such as erythema, urticaria, or 
other rash. Such symptoms will be managed by acetaminophen, diphenhydramine, intravenous fluids 


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and supplemental oxygen. Severe toxicity, including but not limited to, fever (>38.50C), hypotension 
(adults: systolic BP <90 mm Hg and > 20 mm Hg below baseline), tachycardia (adults: HR >130), 
tachypnea (adults: RR >32) or hypoxemia (arterial O2 saturation <90%) will be evaluated and 
managed by the clinical team. The infusion may be terminated by the team if deemed clinically 
necessary, followed by supportive medical care.  Termination of the infusion for pediatric patients 
will be at the treating physician’s discretion.  If the patient does not respond adequately to supportive 
care alone, methlyprednisolone will be administered at 2mg/kg IV every 12 hours for 2 total doses.  
2. Delayed toxicities:  It is possible that infusion of the expanded cells may lead to graft failure or 
increased rates of acute or chronic GVHD. As such, there are stopping rules in place for these 
possible delayed toxicities (Section 16). Patients with potential graft failure will be treated per 
institutional guidelines.  Patients with acute or chronic GVHD will be treated as directed by their 
primary clinicians and/or per recommendations of GVHD specialists as per institutional practice.  
 
11.4 Granulocyte Colony Stimulating Factor (GCSF)  
  
Bone pain 
Insomnia 
Headaches 
Dyspnea 
Body aches 
Rash 
Fatigue  
Edema 
Nausea/vomiting 
 
 
11.5 Cyclosporine  
   
Nephrotoxcity 
Thrombotic thrombocytopenic purpura 
Seizures 
Electrolyte imbalances 
Hypertension 
Paresthesias/neuropathy 
Hirsutism 
Gingival hyperplasia 
Increased risk of relapse 
Increased risk of opportunistic infection 
 
11.6 
Mycophenolate Mofetil (MMF) 
MMF is supplied in 250 mg hard gelatin capsules or intravenous formula.  Capsules can be stored at room 
temperature. 
A. Precautions: Mycophenolate mofetil has not extensively been previously studied in patients after 
marrow transplantation. Previous clinical studies in patients after renal allografting suggested that the 
principal adverse reactions associated with the administration of MMF include diarrhea, leukopenia, 
sepsis, vomiting and a higher incidence of certain viral infections (CMV, VZV, Herpes Simplex). 
Patients will be monitored for the development of these complications. 
 
B. Adverse Events: MMF may be associated with vomiting and diarrhea, decline in hematocrit and white 
blood cell count, and infection. In the setting of marrow transplantation, several etiologic factors may 
contribute to alterations in G.I. and hematologic parameters. MMF dose adjustments will be made if 
clinically indicated if in the opinion of the attending physician, no other cause is thought to be 
responsible for the abnormality. These adjustments should be discussed with the PI. Dose adjustments 
are described in Section 9.3. 
 
  
Potential Toxicities Associated with Mycophenolate Mofetil (MMF) 
Pancytopenia 
Hypertension 
Headache 
Dizziness 
Insomnia 
Hyperglycemia 
Electrolyte imbalances 
Rash 
Leg cramps/bone pain 
Nausea/diarrhea 
Spontaneous abortion 
Birth defects 


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Progressive multifocal 
leukoencephalopathy 
 
 
 
 
12.0  GUIDELINES FOR ADVERSE EVENT REPORTING 
 
12.1  Adverse Event Reporting/Institutional Policy 
The following guidelines are the minimum Cancer Consortium IRB adverse event (AE) reporting guidelines. 
Protocol-specific additional reporting requirements for adverse events are addressed in Section 12.2. 
 
In accordance with institutional policy, all adverse events which in the opinion of the principal investigator are 
unexpected and related or possibly related to the research and serious or suggest that the research places research 
participants or others at greater risk of physical or psychological harm than was previously known or recognized 
are to be reported to the IRB within 10 calendar days of learning of the problem. 
 
Definitions: 
 
Adverse Event - Any untoward medical occurrence in a patient or clinical investigation subject administered a 
pharmaceutical product, medical treatment or procedure and which does not necessarily have to have a causal 
relationship with this treatment. An adverse event can therefore be any unfavorable and unintended sign 
(including an abnormal laboratory finding, for example), symptom, or disease temporally associated with the use 
of a medicinal product, medical treatment or procedure whether or not considered related to the medicinal 
product.  
 
Life-threatening Adverse Event – Any adverse event that places the patient or subject, in view of the 
investigator, at immediate risk of death from the reaction.  
 
Unexpected Adverse Event – An adverse event is “unexpected” when its nature (specificity), severity, or 
frequency are not consistent with (a) the known or foreseeable risk of adverse events associated with the research 
procedures described in the Protocol-related documents, such as the IRB-approved research protocol, informed 
consent document and other relevant sources of information such as product labeling and package inserts; and are 
also not consistent with (b) the characteristics of the subject population being studied including the expected 
natural progression of any underlying disease, disorder or condition any predisposing risk factor profile for the 
adverse event. 
Serious Adverse Event (SAE) – Any adverse event occurring that results in any of the following outcomes: 
• 
death  
• 
a life-threatening adverse event (real risk of dying) 
• 
inpatient hospitalization or prolongation of existing hospitalization (see section 12.2)  
• 
a persistent or significant disability/incapacity 
• 
a congenital anomaly 
• 
requires intervention to prevent permanent impairment of damage 
 
Attribution - The following are definitions for determining whether an adverse event is related to a medical 
product, treatment or procedure: 
 
• 
An adverse event is “related or possibly related to the research procedures” if in the opinion of the 
principal investigator, it was more likely than not caused by the research procedures. 
• 
Adverse events that are solely caused by an underlying disease, disorder or condition of the subject or by 
other circumstances unrelated to either the research or any underlying disease, disorder or condition of the 
subject are not “related or possibly related.”  
• 
If there is any question whether or not an adverse event is related or possibly related, the adverse event 
should be reported. 


2603.00 
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12.2 Protocol –Specific Collection, Grading and Reporting of Adverse Events   
Patients enrolled in this study are receiving myeloablative pre-transplant treatments and other transplant-related 
procedures that are generally associated with high rates of “expected” adverse events. Refer to Appendix D for a 
list of potential adverse events associated or expected with hematopoietic cell transplantation. Toxicities will be 
graded using the NCI Common Terminology Criteria for Adverse Events (CTCAE) Version 4.0. The scale in its 
entirety can be found at: http://www.acrin.org/Portals/0/Administration/Regulatory/CTCAE_4.02_2009-09-
15_QuickReference_5x7.pdf 
 
All patients undergoing allogeneic transplantation using any investigational or standard myeloablative approach 
are expected to experience multiple grade 1-3 adverse events. It is expected that local PIs will be reviewing these 
AEs as documented by signing off on CRFs. Grade <4 hematologic toxicity is expected and will be recorded as 
time to engraftment. All AEs/SAEs will be followed until resolution or until the PI attributes the AE/SAEs to a 
cause other than the study drug or assess them as chronic or stable and captured in protocol specific case report 
forms:  
 
• 
Non-hematologic adverse events of ≥ grade 3  
• 
Grade ≥4 blood and lymphatic system disorder-related adverse events occurring between Day 43 and 
Day 100  
• 
Serious adverse events as defined above   
 
The following events are not identified as AEs in this study: 
• 
Disease progression or relapse. However, clinical events associated with progression/relapse may be 
reportable as AEs. 
• 
Hospitalization for the purpose of facilitating stem cell transplant, for protocol-scheduled procedures, 
blood product transfusions, or for social reasons (i.e., awaiting transport home) will not be considered 
a serious adverse event. Previously, hospitalization occurring at any time pre- or post-transplant were 
considered a SAE only if it was unexpected or the duration of the hospital stay was unexpected per 
the definition of “unexpected adverse event” above.  As of this protocol amendment, hospitalizations 
occurring at any time pre- or post-transplant are considered an SAE regardless of meeting the 
“unexpected adverse event” definition above and will be collected and reported retroactively and 
prospectively for all study patients. Retroactive collection of expected hospitalizations will not follow 
the protocol SAE reporting timelines. 
• 
Medical or surgical procedures in and of themselves, including those that require hospitalization (e.g., 
surgery, endoscopy, biopsy procedures) are not considered AEs. However, an event or condition 
requiring such procedures may be an AE. 
• 
All patients undergoing hematopoietic stem cell transplant are expected to have ≤ Grade 4 
pancytopenia as an intended therapeutic effect. These hematologic AEs will therefore be tracked and 
recorded between day 0 and 43 only as time to recovery of blood counts/engraftment. 
• 
Abnormal laboratory values will be identified and recorded as AEs only if clinical intervention is 
required as a result. 
 
When a grade 3 adverse event increases in severity to grade 4 or above, the event will be captured at its highest 
grade. If a patient experiences relapse or graft failure and goes on to further treatment off protocol, adverse events 
will no longer be collected with the exception of death. 
After day 100 patients will be tracked for progression and survival as well as secondary endpoints. 
Graft versus Host Disease assessment, performed by the clinical team as part of routine care, will be reviewed 
once weekly through Day +100.  After Day +100, GVHD data will be captured at Day +180, 1 year, then once 
yearly through 2 years after Day 0. GVHD data is captured in a GVHD-specific CRF.  
Relapse, graft failure and death data will be captured as they occur in CRFs specific to those events. If a patient 
experiences relapse or graft failure and goes on to further treatment off protocol, adverse events will no longer be 
collected with the exception of death. 


2603.00 
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Coordinating Center IRB reporting 
In accordance with FHCRC institutional policy, AEs from any participating site that affect the risk assessment of 
the trial as a whole (indicates that the research places research participants or others at a greater risk of harm) and 
those occurring at FHCRC as a participating site which in the opinion of the FHCRC principal investigator meet 
all three of the following criteria will be reported to the Coordinating Center IRB within 10 calendar days of 
learning of the problem: 
Expedited reporting criteria:  
A. unexpected  
B. related or possibly related to the research  
C. serious or suggests that the research places research participants or others at greater risk of physical or 
psychological harm than was previously known or recognized 
 
In addition, severe infusion reactions related to the expanded cellular product requiring termination of the infusion 
will be reported to the Coordinating Center IRB within 10 calendar days of learning of the problem. 
 
All other SAEs and deaths, not meeting the expedited reporting criteria, will be reported to the IRB as part of the 
annual continuation review report to the IRB. The FHCRC principal investigator will comply with IRB Policy 1.1 
on Reporting Obligations for Principal Investigators. The FHCRC PI will report all serious adverse events to the 
IND Sponsor (Nohla Therapeutics, Inc.) within the timeline described in the table below.   
 
FDA reporting (IND Sponsor Responsibilities) 
 
The IND Sponsor assumes responsibility for IND safety reporting to the FDA and participating investigators, in 
accordance with regulations under 21 CFR 312.32. Each SAE report received from the PI will be evaluated by the 
IND Sponsor who will assess the seriousness of the event, the expectedness of the event, and the relationship to 
participation in the study. For regulatory reporting purposes, the IND Sponsor will determine expectedness 
relating to the investigational agent using safety information specified in the investigator brochure and consent 
form. An event will be classified as related if either the investigator or the IND Sponsor determines that the event 
may be related to the investigational agent. 
 
For determination of IND safety reporting, AE attribution will be assessed according to the suspected adverse 
reaction definition described in 21 CFR 312.32 as an AE for which there is a reasonable possibility that the 
investigational agent caused the adverse event where “reasonable possibility” means there is evidence to suggest a 
causal relationship between the investigational agent and the AE. Suspected adverse reactions that are serious, 
related, and unexpected will be reported to the FDA as an IND safety report, in accordance with regulations under 
21 CFR 312.32. 
 
IND safety reports will be submitted to the FDA by the IND Sponsor for serious, unexpected suspected expanded 
cell product adverse reactions within 15 calendar days of the IND Sponsor determining that the information 
requires reporting. The IND Sponsor will submit all unexpected fatal or life-threatening events associated with the 
expanded cell product to the FDA fax or phone within seven days of initial receipt of information from the PI. 
Safety reports will also be submitted for severe infusion reactions associated with the expanded cellular product 
requiring termination of the infusion. IND safety reports will be forwarded by the IND Sponsor to the 
Coordinating Center. The PI will distribute the IND safety reports to sites and submit IND safety reports to the 
FHCRC IRB, consistent with the IRB policy. Participating site investigators will be responsible for local IRB 
submission of trial-wide safety reports. 
 
All SAEs must be reported to the IND Sponsor from the time of the investigational agent infusion until SAE 
duration as outlined in the protocol. SAEs that occur after the study-specific informed consent is signed but prior 
to the first dose of the investigational agent will be collected only if they are considered by the investigator to be 
causally related to the study required procedures. SAEs will be reported by the FHCRC PI or Research Nurse to 
the IND Sponsor according to the following timelines described in the table below: 


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Classification 
Reporting 
Time 
Reporting 
Action 
FHCRC PI and 
Research Nurse Contact 
Information 
IND Sponsor Contact 
Information 
SAE 
Fatal or 
life-
threatening
1 
Within 24 
hours of 
research 
team2 
awareness 
Email 
notification to 
IND Sponsor 
(via completed 
SAE Reporting 
Form signed by 
PI or 
designated sub-
Investigator) to 
FHCRC PI or 
Research Nurse 
 
 
FHCRC PI email: 
fmilano@fredhutch.org 
Research Nurse email: 
nlanders@fredhutch.org 
 
IND Sponsor email: 
safety@nohlatherapeutics
.com 
 
All SAEs 
Within 3 
business 
days of 
research 
team2 
awareness 
Submit 
completed IND 
SAE Reporting 
Form signed by 
PI or 
designated sub-
Investigator to 
FHCRC PI or 
Research Nurse 
 
 
 
FHCRC PI email: 
fmilano@fredhutch.org 
Research Nurse email: 
nlanders@fredhutch.org 
 
IND Sponsor email: 
safety@nohlatherapeutics
.com 
 
 
Non-serious AE 
Per CRF 
completion 
guidelines 
Record on 
appropriate CRFs 
 
 
N/A 
 
N/A 
 
1 Includes severe infusion reactions associated with the expanded cellular product requiring termination of the 
infusion. 
2 Research team is defined as the individuals listed on the delegation of authority log. Physicians listed on the 
study’s delegation of authority log as transplant service attending physician’s delegated authority to administer 
informed consent will not be considered part of the research team unless additional responsibilities related to the 
conduct of the study have been delegated to them by the Principal Investigator.  
 
NHLBI reporting (FHCRC only) 
 
Any event or problem that is (1) unexpected, (2) possibly, probably, or definitely related to study participation, 
and (3) one of the following will be reported to the NHLBI Program Official within the noted timeframe: fatal, 
life-threatening, or serious (report within 7 days); suggests greater risk of harm to study participant(s) than was 
previously known or recognized (report within 30 days). 
 
Participating Centers 
 
Adverse events meeting expedited reporting criteria (see Coordinating Center IRB reporting section for criteria) 
must be reported to the Coordinating Center within 3 working days (Monday-Friday) of learning of the event and 
to local IRBs per institutional policy. The report must include the date, severity and duration of the event, the 
relationship to the study, the treatment given and eventual outcome. If the event is ongoing, follow-up information 
must be submitted as soon as the relevant information is available.  
 
Adverse events that must be recorded per Section 12.2 that do not meet FHCRC expedited reporting criteria will 
be reported to the Coordinating Center  at the regularly scheduled CRF intervals (see Section 14.0). 
 


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Participating centers must also report (1) any other unanticipated problems or events that place research 
participants or others at greater risk of harm (e.g. breach of confidentiality) or (2) instances of serious or 
continuing noncompliance with the IRB-approved protocol to the FHCRC Coordinating Center within 5 working 
days of the local PI learning of the event. If there is uncertainty regarding whether an event requires expedited 
reporting to the Coordinating Center, the event should be reported. The following noncompliance events must be 
reported to the Coordinating Center on an expedited basis whether or not the local PI considers them serious: 
enrolling a patient who does not meet eligibility criteria, failure to obtain informed consent for research 
procedures, and administering study drug/product at a dose that is not IRB-approved. A written report must be 
submitted to the FHCRC Coordinating Center with the following information: a description of the event, 
assessment of whether the event placed a participant or others at increased risk of harm, whether re-consenting 
has or will occur, whether changes to study documents are required, the plan of action to prevent similar 
occurrences in the future, and whether the event has been reported to the local IRB on an expedited basis. A case 
report form is provided for this purpose. Unanticipated problems that do not increase risk and minor 
noncompliance may be reported at the regular CRF timepoints. A link to FHCRC IRB policies, including those 
regarding unanticipated problems and noncompliance, is provided in Appendix E. 
 
13.0 ASSESSMENT OF DISEASE RESPONSE 
 
A. Primary Graft Failure:  Patients will be considered primary graft failure/rejection provided they meet any 
criteria listed below: 
i. Absence of 3 consecutive days with neutrophils ≥500/ul combined with host CD3 peripheral blood 
chimerism ≥ 50% by day +42 
ii. Absence of 3 consecutive days with neutrophils ≥500/ul under any circumstances at day 55 
iii. Death after day 28 with neutrophil count <100/ul without any evidence of engraftment  
(< 5% donor CD3) 
iv. Primary autologous count recovery with < 5% donor CD3 peripheral blood chimerism at count 
recovery and without relapse 
B. Secondary graft failure/rejection: Decline of neutrophil count to <500/ul with loss of donor chimerism after 
day 55. 
C. Inadequate graft function: Patients who have met the definition of engraftment as defined above will be 
considered to have inadequate graft function if by day 80 they continue to require GCSF to maintain an 
ANC>1000 and/or they remain platelet transfusion dependent in the absence of GVHD or infectious 
complications (e.g., clinically significant infections/sepsis, BK viruria).  
D. GVHD will be graded according to Appendix C. 
E. Kinetics of Engraftment: the kinetics and durability of hematopoietic reconstitution will be assessed and the 
relative contribution to engraftment of the expanded cord blood product and the unmanipulated unit(s) will be 
determined by frequent peripheral blood donor chimerism assays (as in Section 10.6).  
 
14.0 DATA AND SAFETY MONITORING PLAN 
 
This is a multi-institution Phase 2 clinical trial that is monitored by the principal investigator (PI) and an 
independent Data and Safety and Monitoring Board (DSMB). The PI of the study will have primary responsibility 
for ensuring that the protocol is conducted as approved by the FHCRC Scientific Review Committee and 
Institutional Review Board. The PI will ensure that the Fred Hutch/UW Cancer Consortium Data and Safety 
Monitoring Plan (DSMP) is followed, that all data required for oversight of monitoring are accurately reported to 
the DSMB, that all adverse events are reported according to the protocol guidelines, and that any adverse 
reactions reflecting patient safety concerns are appropriately reported.  
 
Under the provisions of the Fred Hutch/UW Cancer Consortium Data and Safety Monitoring Plan (DSMP), the 
Cancer Consortium Clinical Research Support Office provides monitoring for quality process and compliance by 
qualified monitors unaffiliated with the conduct of the study. Monitoring visits occur at specified intervals while 
accrual is ongoing. The scope of monitoring is specified in the Cancer Consortium DSMP:  


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http://www.cancerconsortium.org/content/dam/consortium/Resource-Documents/Data-Safety-
Monitoring/Fred%20Hutch%20Consortium%20DSMP_Nov20_2015%20Final_Signed.pdf 
Nohla Theraputics, as the IND Sponsor, will have access to participants’ complete medical records, study 
treatment accountability records, and other source documents as needed to appropriately monitor the trial, in 
addition to, site regulatory files. Data from collaborating centers will be summarized and transmitted as paper case 
report forms (CRFs), then entered by Coordinating Center staff into the Fred Hutch research database. Case report 
forms (CRFs) must be submitted to the Fred Hutch coordinating center for the following time points: 
approximately day + 28 , +100, and +180 post transplant and at 1 year and 2 year follow up.  CRFs are expected 
to be submitted as expeditiously as possible after the relevant time points (within 28 days). When possible, 
primary source documents regarding patient outcomes are collected from the collaborating centers with the case 
report forms to allow verification of protocol compliance, data accuracy and completeness and full and timely 
reporting of safety data.  
 
This protocol has a dedicated independent DSMB responsible for monitoring patient safety, and assessing the 
safety and efficacy of the interventions during the trial. The DSMB meets at six-month intervals and all outcome 
data is reviewed including all adverse events. The DSMB determines whether the trial has met any stopping rules 
and reviews any patient safety problems potentially necessitating discontinuation of the trial. A report from the 
DSMB is submitted to the NHLBI Program Official, the FHCRC IRB and to the study coordinators/local PIs of 
this protocol. The DSMB will discontinue the review of outcomes when this protocol is closed to accrual. 
 
15.0 DATA MANAGEMENT/CONFIDENTIALITY 
 
Study data will be recorded in a password-protected research database.  Case report forms are generated from the 
database for review by the principal investigator and study monitors. Clinical Statistics maintains a subject 
database at Fred Hutch to allow storage and retrieval of subject data collected from a wide variety of sources. The 
investigator will ensure that data collected conform to all established guidelines for coding, collection, key entry 
and verification. Each subject is assigned a unique patient number to assure subject confidentiality. Subjects will 
not be referred to by this number, by name, or by any other individual identifier in any publication or external 
presentation. The licensed medical records department, affiliated with the institution where the subject receives 
medical care, maintains all original inpatient and outpatient chart documents. Subject research files are scanned 
and stored in a secure database (OWL). OWL records are maintained by the Fred Hutch data abstraction staff. 
Access is restricted to personnel authorized by the Division of Clinical Research. 
 
16.0 STATISTICAL CONSIDERATIONS 
 
Projected Target Accrual 
ETHNIC AND GENDER DISTRIBUTION CHART 
TARGETED / PLANNED ENROLLMENT: Number of Subjects 
Ethnic Category 
Sex / Gender 
Females 
Males 
Total 
Hispanic or Latino 
15 
18 
33 
Not Hispanic or Latino 
64 
63 
127 
Ethnic Category Total of All Subjects* 
79 
81 
160 
Racial Categories 
 
American Indian / Alaska Native 
4 
2 
6 
Asian 
15 
12 
27 
Native Hawaiian or Other Pacific Islander 
1 
4 
5 
Black or African American 
8 
7 
15 
White 
51 
56 
107 
Racial Categories: Total of All Subjects* 
79 
81 
160 


2603.00 
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A Statistical Analysis Plan (SAP) has been developed for this protocol, and the analyses and concepts laid out in 
the SAP take precedence over anything listed in the protocol in the case where there are discrepancies. 
The primary objective of this randomized phase II open-label, multi-center study  is to compare the hazard of 
engraftment (defined as the first of 2 consecutive days in which ANC≥500) among recipients of single or double 
CBT without ex vivo expanded off-the-shelf CB progenitor cells to that among recipients of expanded off-the-
shelf CB progenitor cells in patients with hematologic malignancies. Time to engraftment is associated with day-
200 NRM and overall mortality in recipients of CBT (see preliminary data), and as such we view this as a 
reasonable endpoint for the current purposes, as opposed to NRM or OM, endpoints that would be considered for 
a primary endpoint in a subsequent Phase III trial should the current trial prove to be potentially efficacious, 
primarily with respect to engraftment. Secondary endpoints aimed at further assessment of economic and clinical 
benefit for the current trial include time to platelet recovery, duration of initial hospitalization, NRM, OS, GVHD, 
and severe infection. In exploratory analyses, other factors such as such as HLA-matching, cellular composition, 
cell dose and in vivo functional assays in an immunodeficient mouse model will also be examined in an effort to 
begin to understand factors that predict in vivo persistence and/or rapid engraftment of our off-the-shelf product 
infused into patients. In addition, an exploratory subgroup analysis based on conditioning regimen will be 
conducted, allowing us to examine separately the difference in outcome between the expanded arm compared to 
the non-expanded among patients who received high-dose TBI and among those who receive middle intensity 
TBI.  
 
We propose to randomize 80 patients per arm based on the following considerations, and randomization will be 
stratified on the number of cord blood units using a permuted block design with random block sizes. Shown in the 
figure below is the observed time to engraftment for patients who have received non-expanded CB (via protocol 
2010) at our Center. A piece-wise exponential curve was fit to these data, this fitted curve (in red) serving as the 
assumed-true time-to-engraftment distribution for patients who receive a single or double CBT without ex vivo 
expanded off-the-shelf CB progenitor cells. The median time to engraftment associated with this assumed-true 
distribution is 25 days (the observed median time to engraftment in this group is also 25 days).  Shown in blue is 
the assumed-true time-to-engraftment distribution for the group to receive ex vivo expanded cells, the true median 
time being 17.9 days.  We feel that this distribution is achievable, as it is consistent with what has been observed 
in Protocol 2044, where the median time to engraftment was 17 days. 
 
We have chosen to use the log-rank test, as 
this not only considers when patients 
engraft, but time that patients have been 
followed without engraftment (primarily due 
to death without engraftment), and failure to 
account for the non-engrafters (particularly 
if the rate in the two arms appears to be 
different) could be misleading (for example, 
using the t-test among engrafters).  We have 
also done extensive simulation studies that 
show the log-rank test and Gray’s test 
(where deaths without engraftment are 
treated as competing-risk failures) have very 
similar power (with the log-rank test slightly 
higher) under a wide variety of scenarios.  
With 80 patients per arm and the above assumed-true time-to-engraftment distributions, we’ll have approximately 
87% power to observe a statistically significant difference (at the two-sided level of .05) in engraftment rates 
(power estimated from 5,000 simulations). As a secondary analysis of engraftment, we shall also compare groups 
using Gray’s test.  We shall also compare the risk of the competing risk (death without engraftment) between 
groups using both the log-rank and Gray’s test.  The log-rank test compares the cause-specific hazards of 
“failure”, while Gray’s test compares the cause-specific cumulative incidence function of “failure”.  Although we 
expect that patient and treatment characteristics will be well-balanced between study arms due to random 
allocation of patients, but in the event that there is some imbalance, we will carry out analyses comparing hazards 


2603.00 
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using Cox proportional hazards regression and competing risks hazard regression models adjusted for relevant 
factors.  
 
An interim analysis for superiority will be conducted when 80 patients have been enrolled and followed 
sufficiently long to assess their time to engraftment. An O’Brien-Fleming boundary will be used at this interim 
analysis in order to maintain most of our type I error rate of .05 at the final analysis. In particular, regarding the 
primary endpoint of time to engraftment (ANC > 500), if there is a statistically significant difference in favor of 
the experimental regimen at the two-sided significance level of .0054, the boundary would be crossed at that 
interim analysis. The final analysis after 80 patients per arm are enrolled (should the trial run to completion) will 
be conducted at a significance level of .0492 (per the O’Brien-Fleming boundary). To enable the trial to provide 
more reliable benefit would occur only if the O’Brien-Fleming boundary also is crossed for the endpoint of 
overall survival, where full information on survival will be assumed to be 35 deaths.  
 
Although not the primary objective of this study, we will continue to monitor for safety of infusion of non-HLA 
matched ex vivo expanded cryopreserved cord blood progenitors as adjunct therapy in the context of conventional 
single or double cord blood transplant. The endpoints to be used for this purpose include severe (Grades 3-4) 
acute GVHD, Grade >3 infusion toxicity and graft failure (see Section 13). It is not our objective to show that the 
rate of failure for these safety endpoints is less than that seen in conventional cord blood transplant with 
unmanipulated units, but rather that the rate is consistent with acceptable values. Towards this end, we have 
implemented stopping rules for each of these complications.   
 
The study will be suspended pending further review if there is sufficient evidence to suggest that the true 
probability of severe GVHD exceeds 25%, the true probability of  Grade >3 infusion toxicity exceeds 10%, or the 
true probability of graft failure exceeds 15% in the group of patients randomized to receive expanded cells.  
Sufficient evidence will be taken to be an observed failure rate whose lower one-sided 90% confidence limit 
exceeds the thresholds listed above (25% for GVHD, 10% for infusion toxicity, 15% for graft failure).  The 
observed failure rates will be examined after every 10th enrolled patient becomes evaluable. Operationally, these 
limits will be met if any of the following is observed. 
 
Grade 3-4 acute GVHD:  5/10, 9/20, 12/30, 15/40, 17/50, 20/60, 23/70, 26/80, 29/90, 32/100, 34/110, 
37/120, 40/130, 43/140, 45/150, 48/160 
Grade >3 Infusion Toxicity:  3/10, 5/20, 6/30, 7/40, 9/50, 10/60, 11/70, 13/80, 14/90, 15/100, 16/110, 
17/120, 18/130, 20/140, 21/150, 22/160 
Graft Failure:  4/10, 6/20, 8/30, 10/40, 12/50, 14/60, 15/70, 17/80, 19/90, 21/100, 22/110, 24/120, 26/130, 
27/140, 29/150, 31/160 
 
The table below summarizes the probability of each of these limits being met (latter 3 columns of table) by 
40,70,100 or 130 patients under a variety of assumed-true failure probabilities (2nd column of table). For 
example, the probability of suspending the trial due to excess GVHD at or before 40 patients are accrued is 0.95 if 
the true probability of GVHD is 0.40; the probability of suspending the trial due to excess graft failure at or before 
70 patients is 0.97 if the true probability of graft failure is 0.30. Probabilities of suspension were estimated from 
5,000 simulations. 
 
 
Probability of Stopping from Listed Event  
Number Pts 
True Prob 
of Failure 
Grade 3-4 
 acute GVHD 
 
Grade >3 
 Infusion 
Toxicity 
Graft 
Failure 
40 
.20 
.04 
 
 
70 
.20 
.05 
 
 
100 
.20 
.05 
 
 
130 
.20 
.06 
 
 
40 
.40 
.76 
 
 
70 
.40 
.95 
 
 
100 
.40 
.98 
 
 


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40 
 
130 
.40 
.99 
 
 
 
 
 
 
 
40 
.05 
 
.02 
 
70 
.05 
 
.02 
 
100 
.05 
 
.02 
 
130 
.05 
 
.02 
 
40 
.20 
 
.75 
 
70 
.20 
 
.90 
 
100 
.20 
 
.95 
 
130 
.20 
 
.99 
 
 
 
 
 
 
40 
.10 
 
 
.03 
70 
.10 
 
 
.03 
100 
.10 
 
 
.03 
130 
.10 
 
 
.03 
40 
.30 
 
 
.85 
70 
.30 
 
 
.97 
100 
.30 
 
 
.99 
130 
.30 
 
 
1.00 
 
If any of the above stopping rules are met at any time, the study will be suspended pending review by a DSMB for 
a recommendation regarding termination or continuation of the trial.   
 
17.0 TERMINATION OF STUDY 
 
The PI or IND  Sponsor may terminate the study at any time. The IRB and FDA also have the authority to 
terminate the study should it be deemed necessary. 
 


2603.00 
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REFERENCES 
 
 
1. Delaney C, Heimfeld S, Brashem-Stein C, Voorhies H, Manger R and Bernstein ID. Notch-mediated 
Expansion of Human Cord Blood Progenitor Cells Capable of Rapid Myeloid Reconstitution. Nature 
Medicine, 2010 Feb;16(2):232-6. 
2. Gutman JA, Turtle CJ, Manley TJ, Heimfeld S, Bernstein ID, Riddell SR, Delaney C. Single-unit 
dominance after double-unit umbilical cord blood transplantation coincides with a specific CD8+ T-cell 
response against the nonengrafted unit. (Plenary paper) Blood, 2010 Jan 28;115(4):757-65. 
3. Nitsche A, Fleischmann J, Klima KM, Radonić A, Thulke S, Siegert W. Inhibition of cord blood cell 
expansion by human herpesvirus 6 in vitro. Stem Cells Dev 13, 197-203 (2004). 
4. Martín-Donaire T, Rico M, Bautista G, Gonzalo-Daganzo R, Regidor C, Ojeda E, Sanjuán I, Forés R, 
Ruiz E, Krsnik I, Navarro B, Gil S, Magro E, Millán I, Sánchez R, Pérez-Sanz N, Panadero N, García-
Marco JA, Cabrera R, Fernández MN, Immune reconstitution after cord blood transplants supported by 
coinfusion of mobilized hematopoietic stem cells from a third party donor, Bone Marrow Transplant 2009 
Aug;44(4):213-25.   
5. Delaney C, Bernstein I, Hematopoietic Stem Cell Expansion, in Thomas’ Hematopoietic Cell 
Transplantation 4th Edition, ed. Appelbaum FR, Forman SJ, Negrin RS, Blume KG, Wiley-Blackwell 
Publishing, 2009. 
6. Sanz J, Montesinos P, Martinez D, Lorenzo I, Palau J, Larrea L, Planelles D, Martin G, Jarque I, De La 
Rubia J, Moscardo F, Martinez J, Sanz MA, Sanz GF. CD34+ Cell Dose Is the Major Cell Dose 
Determinant of Myeloid Engraftment After Single Umbilical Cord Blood Transplantation (sUCBT) for 
Adult Patients with High-Risk Hematologic Malignancies. Blood, Nov 2010; 116:1339. 
7. Brunstein C, Gutman J, Weisdorf C, Woolfrey A, DeFor T, Gooley T, Verneris M, Appelbaum F, 
Wagner J, Delaney C. Allogeneic hematopoietic cell transplantation for hematological malignancy: 
relative risks and benefits of double umbilical cord blood. Blood, 2010 Nov 25; 115(22):4693-9. 
8. Offner F, Schoch G, Fisher LD, Torok-Storb B, Martin PJ. Mortality Hazard Functions as Related to 
Neutropenia at Different Times after Marrow Transplantation. Blood. 1996 Nov 15; 88(10):4058-62.  
9. Dahlberg A, Milano F, Gooley T, Delaney C, The Risk of Day 100 Mortality Following Umbilical Cord 
Blood Transplantation Is Significantly Higher in Patients Who Do Not Achieve An ANC of 100 by Day 
+16 Post Transplant Blood Nov 2011, Vol 118; 3033. 
10. Ponce D, Sauter C, Devlin S, Lubin M, Gonzales A, Kernan N, Scaradavou A, Giralt S, Goldberg J, 
Koehne G, Perales M, Young J, Castro-Malaspina H, Jakubowski A, Papadopoulos E, Barker J. A Novel 
Reduced-Intensity Conditioning Regimen Induces a High Incidence of Sustained Donor-Derived 
Neutrophil and Platelet Engraftment afterDouble-Unit Cord Blood Transplantation. BBMT, Feb 2013; 
19: 799-803. 
11. Delaney C, Milano F, Cicconi L, Othus M, Becker PS, Sandhu V, Nicoud I, Dahlberg A, Bernstein ID, 
Appelbaum FR, Estey EH. Infusion of a non-HLA-matched ex-vivo expanded cord blood progenitor cell 
product after intensive acute myeloid leukaemia chemotherapy: a phase 1 trial. Lancet Haematol. 2016 
Jul;3(7):e330-9.  
 
 
 


2603.00 
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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. 
 
 
 
Lansky Play Performance Scale   
Score 
Description 
100 
Fully active, normal 
90 
Minor restrictions in physically strenuous activity 
80 
Active, but tires more quickly 
70 
Both, greater restrictions of, and less time spend in play activities 
60 
Up and around, but minimal active play, keeps busy with quieter activities 
50 
Gets dressed but lies around much of the day, no active play; able to participate in all 
40 
Mostly in bed; participates in quiet activities 
30 
In bed; needs assistance even for quiet play 
20 
Often sleeping; play entirely limited to very passive activities 
10 
Unresponsive 
0 
Dead 


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Appendix B 
 
 
FHCRC Standard Practice Guideline: “Infusion - Cryopreserved Hematopoietic Progenitor Cells, 
Marrow, Apheresis, or Cord Blood (HPC, Marrow, HPC, Apheresis, HPC, Cord Blood), or 
Therapeutic Cells (TC, Apheresis)” 
 
 
This document is provided as a suggested guideline for Participating Centers. 
 
 
 
Cryopreserved 
Infusion rev102611  
 
 
 


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Appendix C - GVHD Assessment 
 
 
ACUTE GVHD GRADING SCALE 
 
Severity of Individual Organ Involvement 
System 
Severity 
Skin 
 
+1 
maculopapular eruption involving less than 25% of the body surface 
 
 
+2 
maculopapular eruption involving 25-50% of the body surface 
 
 
+3 
generalized erythroderma 
 
 
+4 
generalized erythroderma with bullous formation + desquamation 
 
 
 
 
Liver 
 
+1 
bilirubin (2.0-3.0 mg/100 ml) 
 
 
+2 
bilirubin (3-5.9 mg/100 ml) 
 
 
+3 
bilirubin (6-14.9 mg/100 ml) 
 
 
+4 
bilirubin > 15 mg/100 ml 
 
 
 
 
Gut 
 
+1 
≤ 1000 ml of liquid stool/day* (≤ 15ml of stool/kg/day)†  
 
 
+1 
Nausea or vomiting or anorexia 
 
 
+2 
>1,000 ml of stool/day* (> 15ml of stool/kg/day)† 
 
 
+3 
>1,500 ml of stool/day* (> 20ml of stool/kg/day)† 
 
 
+4 
2,000 ml of stool/day* (≥ 25ml of stool/kg/day)† 
*In the absence of infectious/medical cause 
†For pediatric patients 
 
Overall Grade 
(Maximum grade) 
 
I 
+1 to +2 skin rash. No gut or liver involvement. 
 
II 
+1 to +3 skin rash  or 
 
 
+1 gastrointestinal involvement and/or +1 liver involvement 
 
III 
+2 to +4 gastrointestinal involvement and/or  
 
 
+2 to +4 liver involvement with or without a rash 
 
IV 
Pattern and severity of GVHD similar to grade 3 with extreme constitutional 
symptoms or death. 
 
 
 
 
Reference: From “Graft-vs-host disease” Sullivan, Keith M. Hematopoietic Cell Transplantation Ed: D.  
Thomas, K. Blume, S. Forman, Blackwell Sciences; 1999, pages 518-519 
 


2603.00 
45 
 
 
ACUTE GVHD ASSESSMENT 
 
Staging by Individual Organ Involvement 
 
SKIN: 
measured by rash first appearing generally between 10 and 70 days after transplant. (excludes 
rashes of known viral or other origin) 
 
Stage 
Description 
1 
Maculopapular rash <25% BSA 
2 
Maculopapular rash 25 – 50% BSA 
3 
Generalized erythroderma  
4 
Generalized erythroderma with 
bullous formation and desquamation 
 
LIVER*: measured by total serum bilirubin 
 
Stage 
Description 
1 
2.0 – 2.9 mg/dL 
2 
3.0 – 5.9 mg/dL 
3 
6.0 – 14.9 mg/dL 
4 
> 15.0 mg/dL 
 
GUT**: includes only diarrhea occurring after Day +21 
 
Score 
Adult 
Pediatric*** 
1 
upper GI (anorexia, nausea, 
vomiting) with diarrhea of <1000 
mL/day 
upper GI (anorexia, nausea, 
vomiting) with diarrhea of <555 
mL/m2/day 
2 
1000 – 1499 mL/day diarrhea 
556-833 mL/m2/day diarrhea 
3 
> 1500 mL/day diarrhea 
>833 mL/m2/day diarrhea 
4 
severe abdominal cramping, bleeding or ileus caused by GVHD 
 
* In cases where another cause of hyperbilirubinemia antedated the onset of rash, the liver score 
should be decreased by one stage. 
** In cases where peak GI symptoms are exacerbated by a cause other than GVHD, the gut score 
should be decreased by one stage. 
*** Pediatric patients <17 years of age 
 
 


2603.00 
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ACUTE GVHD ASSESSMENT 
 
Overall Grade 
 
The determination of an overall GVHD grade should be based on the organ stage, response to treatment 
and whether GVHD was a major cause of death. 
 
Overall 
Grade 
 Organ Stage 
Qualifying Conditions 
Additional Qualifying Conditions 
I 
Stage 1 -2 skin  
No liver or gut 
Indicates that the prophylactic 
immunosuppressive regimen was not 
sufficient to prevent all manifestations of 
aGVHD. 
II 
Stage 3 skin  
 
or  
Stage 1 liver  
 
or  
Stage 1 gut 
N/A 
Indicates that the prophylactic 
immunosuppressive regimen was not 
sufficient to prevent all manifestations of 
aGVHD, but glucocorticoid treatment 
after the onset of GVHD was generally 
sufficient to control the disease. 
III 
Stage 4 skin  
 
or  
Stage 2-4 liver  
 
or  
Stage 2-4 gut  
 
without GVHD as a 
major contributing cause 
of death 
 
 
Indicates that the prophylactic 
immunosuppressive regimen was not 
sufficient to prevent all manifestations of 
aGVHD and that additional treatment 
after the onset of GVHD did not readily 
control the disease. 
IV 
Stage 4 skin  
 
or  
Stage 2-4 liver  
 
or  
Stage 2-4 gut  
 
with GVHD as a major 
contributing cause of 
death 
 
 
GVHD was resistant to both the 
prophylactic immunosuppressive 
regimen and any additional treatment 
after the onset of the disease. 
 
References:  
 
1. Leisenring, WM, Martin, PJ, Petersdorf, EW, et al. An acute graft-versus-host-disease activity 
index to predict survival after hematopoietic cell transplantation with myeloablative conditioning 
regimens, Blood, 2006:108: 749-755. 
2. Przepiorka D, Weisdorf D, Martin PJ, et al. 1994 Consensus Conference on Acute GVHD 
Grading. Bone Marrow Transplant. 1995:15: 825-828. 
 
 


2603.00 
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Chronic graft-versus-host disease grading* 
 
In all cases, concomitant processes (i.e. infections or drug reactions) must be ruled out. Karnofsky or 
Lansky Clinical Performance scores, 60%, > 15% weight loss, and recurrent infections are usually signs 
of clinical extensive chronic GVHD. Abnormalities that could indicate chronic GVHD are categorized 
by organ systems as listed below. 
 
Skin 
Erythema, dryness, pruritus, pigmentary changes (i.e. hyperpigmentation, vitiligo), 
mottling, papulosquamous plaques, nodules, exfoliation, macular-papular or 
urticarial rash, scleroderma, morphea (one or several circumscribed, indurated and 
shiny lesions) 
Nails 
Ridging, onychodystrophy, onycholysis 
Hair 
Premature graying, (scalp hair, eyelashes, eyebrows), thinning scalp hair, alopecia, 
decreased body hair 
Mouth 
Dryness, burning, gingivitis, mucositis, striae, atrophy, erythema, lichenoid 
changes, ulcers, labial atrophy or pigmentary changes, tooth decay, tightness 
around the mouth 
Eyes 
Dryness, burning, blurring, gritty eyes, photophobia, pain 
Vagina/vulva 
Dryness, dyspareunia, stricture or stenosis, erythema, atrophy or lichenoid changes 
not included 
Liver 
Elevated liver function tests not due to other causes (alkaline phosphatase  3x 
upper limit of normal, AST or ALT 4x upper limit of normal or total serum 
bilirubin  2.5; in the absence of chronic GVHD involving other organs, liver 
biopsy is required to confirm diagnosis) 
Lung 
Bronchiolitis obliterans (see diagnostic indicators), cough, wheezing, dyspnea on 
exertion, history of recurrent bronchitis or sinusitis 
GI 
Anorexia, nausea, vomiting, weight loss, dysphasia, odynophagia, malabsorption 
Fasciitis 
Stiffness and tightness with restriction of movement, occasionally with swelling 
pain, cramping, erythema and induration, most commonly affecting forearms, 
wrists and hands, ankles, legs, and feet, inability to extend wrists without flexing 
the fingers or the elbows, contractures 
Serositis 
Chest pain or cardiopulmonary comprise due to pericarditis or pleuritis 
Muscle 
Proximal muscle weakness, cramping 
Skeletal 
Arthralgia of large proximal girdle joints and sometimes smaller joints 
 
________________ 
* From Standard Practice Guidelines for “Chronic Graft-versus-Host Disease Classification at the time 
of presentation” developed by Long Term Follow-Up at the FHCRC 
 
 
 
 
 
 


2603.00 
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Laboratory testing and diagnostic indicators of chronic GVHD* 
Eye 
Schirmer’s test with a mean value  5mm at 5 minutes, or symptomatic with 
values of 6-10mm or keratitis detected by slit lamp examination 
Liver 
Elevated liver function tests not due to other causes (see definition of clinical 
limited and extensive chronic GVHD) 
Lung 
New obstructive lung defect defined as FEV1 < 80% of predicted with either an 
FEF 25-75 <65% of predicted or RV >120% of predicted, or a decrease of 
FEV1/FVC by > 12% within a period of less than 1 year. A diagnosis of 
bronchiolitis obliterans requires negative microbiological tests from 
bronchoalveolar lavage and evidence of air trapping by high resolution end-
expiratory and end-inspiratory CAT scans o the chest. A thoracoscopic lung 
biopsy may be necessary in order to confirm the diagnosis of bronchiolitis 
obliterans in patients who have obstructive lung disease without air trapping 
when chronic GVHD involving other organs is absent 
Esophagus 
Esophageal web formation, stricture or dysmotility demonstrated by barium 
swallow, endoscopy or manometry 
Muscle 
Elevated CPK or aldolase, EMG findings consistent with myositis 
Blood 
Thrombocytopenia (usually 20,000-100,000/l), eosinophilia, 
hypogammaglobulinemia, hypergammaglobulinemia, and autoantibodies occur 
in some cases 
________________ 
* From Standard Practice Guidelines for “Chronic Graft-versus-Host Disease Classification at the time 
of presentation” developed by Long Term Follow-Up at the FHCRC 
 


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Appendix D - The Hematopoietic Cell Transplant-Comorbidity Index (HCT-CI) 
 
Instructions: Circle applicable scores and provide actual value or cause of' co-morbidity. 
Comorbidities  
Definitions 
 
HCT-CI 
weighte
d scores  
Actual Lab 
Values/Comments 
Arrhythmia 
Atrial fibrillation or flutter, sick sinus  syndrome, 
and ventriuclar arrhythmias 
1 
 
Cardiac 
Coronary artery disease, congestive heart 
failure, myocardial infarction, or EF<50% 
1 
 
Inflammatory 
bowel disease 
Crohn's disease or ulcerative colitis 
1 
 
Diabetes* 
Requiring treatment with insulin or oral 
hypoglycemics, but not diet alone 
1 
 
Cerebro-
vascular disease 
Transient ischemic attack or cerebro-vascular a 
ccident 
1 
 
 
 
Psychiatric 
Disturbance 
Depression anxiety requiring psychiatric consult 
or treatment 
1 
 
Hepatic -mild* 
Chronic hepatitis, Bilirubin >ULN- 1.5 X ULN,or 
AST/ALT >ULN-2.5XULN 
1 
 
 
Obesity* 
Patients with a body mass index > 35kg/ m2 
1 
 
 
 
Infection* 
Requiring continuation of anti-microbial 
Treatment after day 0 
1 
 
 
 
Rheumatologic 
SLE, RA, polymyositis, mixed CTD 
Polymyalgia rheumatica 
2 
 
 
Peptic ulcer* 
Requiring treatment 
2 
 
Moderate/severe 
renal* 
serum creatinine>2mg/dl, on dialysis, or prior 
renal transplantation 
2 
 
 
Moderate 
pulmonary* 
DLCO and/or FEV, >65%-80% or 
Dispend on slight activity 
2 
 
 
Prior solid tumor Treated at any time point in the patient's past 
history, excluding non-melanoma skin cancer 
3 
 
 
 
Heart valve 
disease* 
Except mitral valve prolapse 
3 
 
 
Severe 
pulmonary* 
DLCO and/or FEV1 <65% or 
dyspnea at rest requiring oxygen 
3 
 
Moderate/severe 
Hepatic 
Liver cirrhosis, Bilirubin>1.5XULN or 
AST/ALT>2.5XULN 
3 
 
 
 
 
Please provide         Karnofksy performance Score (KPS) 
=______________% 
Total 
Score 
=______  
 
Completed by (Print ): _____________________________________   Date: ________________ 
 


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Signature: ________________________________________________ 
*Comorbidity is currently active or patient requires medical treatment + 
One or more vessel-coronary artery stenosis, requiring medical treatment, stent, or bypass graft  
EF indicates ejection fraction; ULN, upper limit of normal; SLE, systemic lupus erythematosus;  RA, 
rheumatoid arthritis; CTD, connective tissue disease; 
DLCO, diffusion capacity of carbon monoxide; FEV1, forced expiratory volume in one second; AST, 
aspartate aminotransfcrase; ALT, alanine aminotransferas 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Appendix E  
Potential Adverse Events Associated or Expected with Hematopoietic Cell Transplantation 
 
1. Graft versus host disease:  GVHD is a major toxicity associated with the infusion of allogeneic donor stem 
cells.  GVHD may be acute or chronic and may affect multiple organ systems, including the skin, liver, and GI 
tract. 
 
2. Opportunistic infections, including viral and fungal infections, can result in severe pulmonary, neurologic, 
hepatic and other organ dysfunction, and possible death. 
 
3. Gastrointestinal toxicity. Nausea and vomiting can be anticipated during the entire course of ablative therapy. 
Mucositis and diarrhea should be expected. Prednisone can cause GI bleeding. 
 
4. Cardiac toxicity. Cardiotoxicity (congestive heart failure, pericardial effusion, EKG changes) is uncommonly 
associated with the chemotherapy agents and TBI used in the regimen and these sequelae may prove lethal. 
 
5. Pulmonary toxicity. Diffuse interstitial pneumonitis of unknown etiology and diffuse alveolar hemorrhage 
occurs with some regularity after BMT and interstitial fibrosis occurs much more rarely. Both are well-described 
complications of intensive chemotherapy and TBI regimens and may prove lethal. 
 
6. Hepatic toxicity. Veno-occlusive disease of the liver is a common toxicity of high-dose chemoradiotherapy and 
may result in death. Cyclosporine may cause elevation of ALT/AST. 
 
7. Renal dysfunction. Chemoradiotherapy may uncommonly cause renal dysfunction. More commonly, 
nephrotoxicity results from cyclosporine and generally responds to dose reduction. Rarely, idiopathic or 
calcineurin inhibitor-associated hemolytic-uremic syndrome may occur and may be progressive and fatal. A 
syndrome of moderate renal insufficiency and hemolysis has been seen 5-7 months post HSCT after intensive 
multi-agent conditioning plus TBI. 
 
8. Hemorrhagic cystitis, manifested either as gross or microscopic hematuria, is a common toxicity after high-
dose chemoradiotherapy, but usually associated with regimens that include cyclophosphamide. Hemorrhagic 
cystitis may predispose to a long-term increased risk of bladder cancer. 
 
9. Central nervous system toxicity. Radiation and chemotherapy can cause CNS toxicity, including seizures, 
depressed mental status, or leukoencephalopathy. Calcineurin inhibitors can cause seizures or other CNS toxicity. 
 
10. Marrow aplasia. Severe neutropenia, thrombocytopenia, and anemia, is expected to occur for a period of 7 to 
42 days following infusion of marrow. Transfusion of platelets and red blood cells is expected as supportive care. 
Transfusion of blood products may be associated with acquisition of HIV or a hepatitis virus. Neutropenia may 
increase the risk for acquiring serious infection. Thrombocytopenia may increase the risk of life-threatening 
hemorrhage. Hemorrhagic or infectious complications during the expected period of aplasia may result in death. 
 
11. Miscellaneous. Alopecia and sterility are expected complications of the program as a whole. Cataract 
development is possible after TBI and/or steroids. Deficiencies of growth hormone, thyroid hormone, and sex 
hormones are possible after TBI. Calcineurin inhibitors can cause transient gingival hyperplasia, tremor, seizure, 
hypertension, headache, dysesthesia and hirsutism. Steroid therapy can also contribute to fluid retention, easy 
bruising, hypertension, aseptic necrosis of bone and increased susceptibility to infection. MMF can cause 
spontaneous abortions and birth defects. Hospitalization during conditioning and recovery period is expected to be 
5-9 weeks in duration. 
 
 


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Appendix F - FHCRC IRB Policies   
 
 
IRB Policy Web Page: http://extranet.fhcrc.org/EN/sections/iro/irb/policy/index.html 
 
Adverse Events and Noncompliance are addressed in the following policies: 
 
Policy 1.9: “Noncompliance” 
 
Policy 1.11: “Reporting Obligations for Principal Investigators” 
 
Policy 2.6:  “Unanticipated Problems Involving Risks to Subjects or Others” 
 
 
 
 


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Appendix G Schedule of Study Evaluations 
 
 
Effective as of this amendment, the schedule of activities that occurred prior to one year are shown in the 
prior protocol version. Every effort will be made to complete the 1 and  2 year evaluations as close to these 
dates as possible, taking into consideration  the patient’s circumstances.  

SAP_001.pdf: 
Statistical Analysis Plan 
  
 
This document is proprietary and confidential to Nohla Therapeutics  
 
Page 1 of 55 
Sponsor Name: 
Nohla Therapeutics 
Protocol Number and Title: 
 
2603.00 Multi-center, open-label randomized 
study of single or double myeloablative cord 
blood transplantation with or without infusion of 
off–the-shelf ex vivo expanded cryopreserved 
cord blood progenitor cells in patients with 
hematologic malignancies 
Original Protocol Version, 
Date: 
Version 1, 05SEP2012 
Protocol Amendment 
Number, Date 
Amendment 1, 02NOV2012; Amendment 2, 
09MAY2013; Amendment 3, 16SEP2013; 
Amendment 4, 18DEC2013; Amendment 5, 
13JAN2014; Amendment 6, 13MAY2014; 
Amendment 7, 31JUL2014; Amendment 8, 
26JAN2015; Amendment 9, 19MAR2015; 
Amendment 10, 13APR2015; Amendment 11, 
11JUN2015; Amendment 12, 07OCT2015; 
Amendment 13, 07DEC2015; … Amendment 22, 
23AUG2017; Amendment 23, 20SEP2017; 
Amendment 24, 24APR2018; Amendment 25, 
24AUG2018 
Syneos Health Project Code: 
1011511 
SAP Version: 
Final Version 3.0  
SAP Version Date:  
26FEB2019 
 
Notice of Confidential and Proprietary Information: 
The information contained in this document is confidential belonging to Nohla Therapeutics. Acceptance of 
this document constitutes agreement by the recipient that no information contained herein will be published 
or disclosed without prior written authorization from an official of Nohla Therapeutics.  However, this 
document may be disclosed to appropriate Institutional Review Board and Ethics Committees or duly 
authorized representatives of a national regulatory authority under the condition that they are requested to 
keep it confidential. In the event of an actual or suspected breach of this obligation, Syneos Health should be 
notified promptly. 


Statistical Analysis Plan 
Nohla Therapeutics Protocol #2603 
Final Version 3.0 
Syneos Health #1011511 
 
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Revision History 
 
Version # 
Date 
(dd-mmm-yyyy) 
Document Owner 
Revision Summary 
1 
11MAY2018 
Ted A. Gooley 
Initial Release Version 
1.1 
04JUN2018 
K Kwarteng 
Update version 0.1 to full clinical study report SAP. 
2 
24JUL2018 
K Kwarteng 
Update based on sponsor comments 
3 
26FEB2019 
T Marmon 
Update to remove safety and efficacy infection endpoints 
based on blinded review of the data, as reliable analyses 
for these infection endpoints were deemed impossible, 
and the study was terminated. 
 
 




Statistical Analysis Plan 
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TABLE OF CONTENTS 
 
 
GLOSSARY OF ABBREVIATIONS ................................................................. 8 
 
PURPOSE ...................................................................................................... 11 
 
Responsibilities ..................................................................................................................... 11 
 
Timing of Analyses ............................................................................................................... 11 
 
INFORMATION FROM THE STUDY PROTOCOL ...................................... 12 
 
Study Objectives ................................................................................................................... 12 
 
Primary Objective .............................................................................................................. 12 
 
Secondary and Exploratory Objectives ................................................................................. 12 
 
Brief Description of Study Design ......................................................................................... 12 
 
Background ....................................................................................................................... 12 
 
Planned Study Duration ...................................................................................................... 13 
 
Subject Selection ................................................................................................................... 13 
 
Inclusion Criteria ............................................................................................................... 13 
 
Exclusion Criteria .............................................................................................................. 15 
 
Determination of Sample Size ............................................................................................... 15 
 
Treatment Assignment and Blinding ..................................................................................... 16 
 
Administration of Study Treatment ...................................................................................... 17 
 
Study Procedures and Flowchart .......................................................................................... 19 
 
ENDPOINTS .................................................................................................. 21 
 
Primary Efficacy Endpoint ................................................................................................... 21 
 
Secondary Efficacy Endpoints .............................................................................................. 21 
 
Exploratory Efficacy Endpoints ............................................................................................ 21 
 
Safety Endpoints ................................................................................................................... 22 
 
ANALYSIS SETS ........................................................................................... 23 


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Screened Set ......................................................................................................................... 23 
 
Safety Set .............................................................................................................................. 23 
 
Intent-to-Treat or Full Analysis Set ...................................................................................... 23 
 
Modified Intent-to-Treat Set ................................................................................................. 23 
 
Per Protocol Set .................................................................................................................... 23 
 
GENERAL ASPECTS FOR STATISTICAL ANALYSES ............................... 24 
 
General Methods .................................................................................................................. 24 
 
Key Definitions ..................................................................................................................... 25 
 
Baseline ............................................................................................................................ 25 
 
Protocol Day ...................................................................................................................... 25 
 
Study Day ......................................................................................................................... 25 
 
Completed Study ................................................................................................................ 25 
 
Partial Dates ......................................................................................................................... 26 
 
Visit Windows....................................................................................................................... 27 
 
Examination of Subgroups .................................................................................................... 27 
 
STUDY POPULATION .................................................................................. 28 
 
Subject Disposition ............................................................................................................... 28 
 
Protocol Deviations ............................................................................................................... 28 
 
DEMOGRAPHIC, OTHER BASELINE CHARACTERISTICS AND 
MEDICATIONS ............................................................................................. 29 
 
Demographic and Other Baseline Characteristics ................................................................. 29 
 
Recipient Demographic ...................................................................................................... 29 
 
Donor Demographic ........................................................................................................... 29 
 
Significant Pre-Transplant Medical History .......................................................................... 30 
 
Significant Pre-Transplant Treatment/Disease History ........................................................... 30 
 
Disease Characteristics at Diagnosis .................................................................................... 30 
 
Transplant-Related Information ........................................................................................... 30 
 
Performance Status ............................................................................................................. 31 
 
Cardiovascular and/or Pulmonary Function Tests (PFT) ........................................................ 31 
 
Conditioning Regimen ........................................................................................................ 31 
 
Disease Evaluation at Time of Transplant ........................................................................ 31 
 
Donor Cell Panel ........................................................................................................... 32 
 
Medical History and Concomitant Diseases ........................................................................... 32 


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Medications .......................................................................................................................... 32 
 
EFFICACY ..................................................................................................... 34 
 
Adjustments for Covariates .................................................................................................. 34 
 
Handling of Dropouts or Missing Data .................................................................................. 34 
 
Interim Analyses and Data Monitoring ................................................................................. 34 
 
Interim Analysis ................................................................................................................. 34 
 
Safety Monitoring Committee ............................................................................................. 34 
 
Multicenter Studies ............................................................................................................... 35 
 
Use of an “Efficacy Subset” of Subjects ................................................................................. 35 
 
Multiple Comparisons/Multiplicity ....................................................................................... 35 
 
Primary Efficacy Analysis..................................................................................................... 35 
 
Secondary Efficacy Analyses ................................................................................................. 36 
 
Key Secondary ................................................................................................................... 36 
 
Other Secondary Efficacy Analyses ..................................................................................... 37 
 
Exploratory Efficacy Analyses .............................................................................................. 38 
 
Exploratory Efficacy Endpoint 1: In Vivo Persistence of the Ex-Vivo Expanded Cord Blood 
Product 39 
 
Exploratory Efficacy Endpoint 2: Duration of Hospitalization ................................................ 40 
 
Exploratory Efficacy Endpoint 3: Intensive Care Unit-Free Days ........................................... 40 
 
Exploratory Efficacy Endpoint 4: Relapse/Recurrence of the Underlying Malignancy .............. 40 
 
Exploratory Efficacy Endpoint 5: Disease-Free Survival ....................................................... 40 
 
Exploratory Efficacy Endpoint 6: GVHD-free, Relapse-free Survival (GRFS) ......................... 40 
 
Exploratory Efficacy Endpoint 7: Grade ≥ 3 Infusion Toxicity ............................................... 41 
 
Exploratory Efficacy Endpoint 8: Graft Failure ..................................................................... 41 
 
Exploratory Efficacy Endpoint 9: Immune Reconstitution ..................................................... 41 
 
Exploratory Efficacy Endpoint 10: Time to Platelet Engraftment ≥50,000/µL ..................... 41 
 SAFETY ......................................................................................................... 43 
 
Extent of Exposure and Follow-Up ....................................................................................... 43 
 
Exposure ....................................................................................................................... 43 
 
Follow-up ..................................................................................................................... 43 
 
Treatment Compliance ......................................................................................................... 43 
 
Adverse Events ..................................................................................................................... 44 
 
Laboratory Evaluations ........................................................................................................ 46 
 
Performance Status .............................................................................................................. 47 


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 CHANGES FROM PROTOCOL PLANNED ANALYSES .............................. 48 
 REFERENCES ............................................................................................... 49 
 PROGRAMMING CONSIDERATIONS ......................................................... 50 
 
General Considerations ........................................................................................................ 50 
 
Table, Listing, and Figure Format ........................................................................................ 50 
 
General ......................................................................................................................... 50 
 
Headers ......................................................................................................................... 50 
 
Display Titles ................................................................................................................ 51 
 
Column Headers ............................................................................................................ 51 
 
Body of the Data Display ............................................................................................... 51 
 
Footnotes ...................................................................................................................... 53 
 QUALITY CONTROL .................................................................................... 55 
 
 
 
 


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GLOSSARY OF ABBREVIATIONS 
Abbreviation 
Description 
AE 
Adverse Event 
ALL 
Acute Lymphocytic Leukemia 
AML 
Acute Myeloid Leukemia 
ANC 
Absolute Neutrophil Count 
ATC 
Anatomical Therapeutic Chemical 
BAL 
Biphenotypic Acute Leukemia 
BM 
Bone Marrow 
CB 
Cord Blood 
CBT 
Cord Blood Transplant 
CI 
Confidence Interval 
CML 
Chronic Myeloid Leukemia 
CNS 
Central Nervous System 
CR 
Complete Response 
CR1 
First Complete Response 
CR2 
Second Complete Response 
CRF 
Case Report Form 
CSR 
Clinical Study Report 
CTCAE 
Common Terminology Criteria for Adverse Events 
DLCO 
Carbon Monoxide Diffusing Capacity (Lung Function) 
DSMB 
Data Safety Monitoring Board 
ECOG 
Eastern Cooperative Oncology Group 
EXP 
Experimental Arm 
FAB 
French-American-British Classification System 
FAS 
Full Analysis Set 
FHCRC 
Fred Hutchinson Cancer Research Center 
GRFS 
Graft-versus-Host Disease-free, Relapse-Free Survival 
GVHD 
Graft-versus-Host Disease 
HCT 
Hematopoietic Cell Transplant 
HCT-CI 
Hematopoietic Cell Transplantation–Specific Comorbidity Index 


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Abbreviation 
Description 
HLA  
Human leukocyte Antigen 
ICH 
International Council for Harmonization 
IgG 
Immunoglobulin G 
IgM 
Immunoglobulin M 
IPSS 
International Prognostic Scoring System 
IRB 
Institutional Review Board 
Max 
Maximum 
MedDRA 
Medical Dictionary for Regulatory Activities 
MDS 
Myelodysplastic Syndrome 
Min 
Minimum 
mITT 
Modified Intent to Treat 
MLL 
Mixed-Lineage Leukemia 
N/A 
Not Applicable 
NIH 
National Institutes of Health 
NRM 
Non-relapse Mortality 
PCR 
Polymerase Chain Reaction 
PI 
Principal Investigator 
PPS 
Per-Protocol Set 
RAEB 
Refractory Anemia with Excess Blasts 
RAEBt 
Refractory Anemia with Excess Blasts in Transformation 
REFR 
Refractory 
REL 
Relapse 
Rh 
Rhesus 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SI 
Standard International System of Units 
SOC 
Standard of Care 
SOP 
Standard Operating Procedure 
SS 
Safety Set 
TBI 
Total-body Irradiation 
TCR 
T-cell Receptor 


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Abbreviation 
Description 
TEAE 
Treatment Emergent Adverse Event 
TKI 
Tyrosine Kinase Inhibitors 
TLF 
Table, Listing and Figure 
TREC 
T-cell Receptor Excision Circles 
TRM 
Transplant-related Mortality 
WHO 
World Health Organization 
 


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PURPOSE 
The purpose of this Statistical Analysis Plan (SAP) is to ensure that the data listings, 
summary tables and figures which will be produced to support the completion of the 
Clinical Study Report (CSR), and the statistical methodologies that will be used, are 
complete and appropriate to allow valid conclusions regarding the study objectives 
according to International Council for Harmonization (ICH) E91, ICH E32 and ICH E63. 
This analysis plan is based on the latest version of both the protocol and case report forms.  
Any ad hoc or unplanned analyses performed and not identified in this SAP will be 
documented in the CSR.   
 
RESPONSIBILITIES 
Syneos Health will perform the statistical analyses and is responsible for the production 
and quality control of all tables, listings and figures (TLFs) under the direction of Nohla 
Therapeutics.   
 
TIMING OF ANALYSES 
The analyses for this study will be performed as follows: 
• An interim analysis was performed when 80 subjects were enrolled and followed 
sufficiently long to assess their time to engraftment. An O’Brien-Fleming boundary 
was used at this interim analysis in order to maintain most of our type I error rate of 
0.05 at the final analysis. A two-sided significance level of 0.0054 was used at this 
interim analysis, consistent with an O’Brien-Fleming boundary. 
• A primary analysis will be performed after all subjects have completed 100 days 
follow-up post- Hematopoietic Cell Transplant (HCT). The clinical data cut date 
will take place 100 days beyond HCT for the last subject randomized.  The analysis 
of the primary endpoint of ANC engraftment and the key secondary endpoints will 
be conducted at the time of the database lock for the primary analysis, ensuring 
complete capture of a minimum of 100 days and maximum of 2 years follow-up for 
each subject.  
• Additional ad hoc analyses will be performed as needed. Any subsequent analyses 
of the primary and key secondary endpoints would be viewed as supportive 
analyses.  
• A final supportive efficacy analysis is planned after all subjects have 2 years of 
follow-up or have discontinued the study. 


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INFORMATION FROM THE STUDY PROTOCOL 
 
STUDY OBJECTIVES 
 
Primary Objective 
Compare the time to neutrophil engraftment (first of two consecutive days of an absolute 
neutrophil count [ANC] ≥500/µL) in subjects receiving a standard-of-care myeloablative 
cord–blood transplant (CBT) augmented with an off-the-shelf pre-expanded and 
cryopreserved cord-blood product to those who do not receive the product.  
 
 
Secondary and Exploratory Objectives 
Provide initial data on clinical and economic benefit, such as time to platelet engraftment, 
duration of initial hospitalization, transplant-related mortality (TRM), and death without 
engraftment.  The kinetics of immune system recovery will also be evaluated in both arms. 
 
 
BRIEF DESCRIPTION OF STUDY DESIGN 
This is a Phase II multi-center, open-label, randomized study to compare outcomes 
following use of single or double myeloablative cord blood transplantation with or without 
infusion of off–the-shelf ex vivo expanded cryopreserved cord blood progenitor cells in 
subjects with hematologic malignancies. 
 
 
Background 
Cord blood (CB) is an effective and widely used source of stem cells for patients 
undergoing hematopoietic stem cell transplantation for hematologic malignancies.  Most 
patients now receive two units of CB derived from different donors to better ensure 
provision of adequate stem cell numbers for reliable donor engraftment. However, with 
this modest two-fold increase provided by the second CB unit, the time to donor 
engraftment is still relatively delayed, averaging more than three weeks to achieve 
adequate numbers of myeloid cells. This leaves patients susceptible to infection and 
associated morbidity and mortality. Thus, although CB offers some distinct advantages 
over conventional stem cell sources as it is readily available with no donor attrition and 
allows reduced stringency in human leukocyte antigen (HLA) matching without an 
increase in graft-versus-host disease (GVHD) thereby increasing the donor pool especially 
for minority or mixed ethnicity patients, the increased risks of delayed engraftment or graft 
failure and early transplant related mortality that is associated with the low cell doses in a 
CB graft remains a barrier to the more wide spread use of this stem cell source. To address 
this barrier, research has investigated the role of the Notch signaling pathway in regulating 
ex vivo expansion of hematopoietic stem/progenitor cells with the goal of generating 
increased numbers of progenitor cells capable of rapid repopulation in vivo to improve the 
kinetics of hematopoietic recovery following CBT.  This expansion is studied in this 
randomized Phase II clinical trial. 


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Planned Study Duration 
A total of 160 subjects were enrolled into the study across 8 participating centers. Subjects 
enrolled to the study will be followed for 2 years post-HCT.   
 
 
SUBJECT SELECTION 
All subjects who sign the informed consent form, and have met all eligibility criteria, as 
confirmed by the Principal Investigator (PI) of the coordinating center, will be enrolled.  
 
Inclusion Criteria 
Ability to understand and the willingness to sign a written informed-consent document 
 
1.   Subject age 
 High-dose total-body irradiation (TBI) regimen: 6 months to ≤ 45 years 
 Middle-intensity TBI regimen: 6 months to ≤ 65 years 
 
2.   Diseases 
A.  Acute Myeloid Leukemia (AML), including Biphenotypic Acute Leukemia (BAL) or 
Mixed-Lineage Leukemia (MLL) 
 All subjects must have AML that is considered best treated by stem cell 
transplant by the referring physician and the attending transplant physician. 
 All subjects must be in complete response (CR) as defined by < 5% blasts by 
morphology/flow cytometry in a representative bone marrow sample with 
cellularity > 15% for age.  
 Subjects in which adequate marrow/biopsy specimens cannot be obtained to 
determine remission status by morphologic assessment, but have fulfilled criteria 
of remission by flow cytometry, recovery of peripheral blood counts with no 
circulating blasts, and/or normal cytogenetics (if applicable) may still be eligible. 
Reasonable attempts must be made to obtain an adequate specimen for 
morphologic assessment, including possible repeat procedures. These subjects 
must be discussed with the Principal Investigator prior to enrollment.  
B.  Acute Lymphocytic Leukemia (ALL), including BAL or MLL 
    High-risk first complete response (CR1) [for example, but not limited to: t(9;22), 
t(1;19), t(4;11) or other MLL  rearrangements, hyplodiploid] or HR as defined by 
referring institution treatment protocol, greater than 1 cycle to obtain CR;  CR2 or 
greater. 
    All subjects must be in CR as defined by <5% blasts by morphology/flow 
cytometry in a representative bone marrow sample with cellularity ≥ 15% for age. 


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    Subjects in which adequate marrow/biopsy specimens cannot be obtained to 
determine remission status by morphologic assessment, but have fulfilled criteria 
of remission by flow cytometry, recovery of peripheral blood counts with no 
circulating blasts, and/or normal cytogenetics (if applicable) may still be eligible. 
Reasonable attempts must be made to obtain an adequate specimen for 
morphologic assessment, including possible repeat procedures. These subjects 
must be discussed with the Principal Investigator prior to enrollment.  
C.  
Chronic myeloid leukemia (CML) excluding refractory blast crisis. To be  
          eligible in first chronic phase (CP1) subject must have failed or be  
          intolerant to tyrosine kinase inhibitor therapy. 
D.   
Myelodysplasia (MDS) International Prognostic Scoring System (IPSS) Int-2 or 
High risk (i.e., refractory anemia with excess blast [RAEB], refractory anemia with 
excess blast in transformation [RAEBt]) or refractory anemia with severe 
pancytopenia or high-risk cytogenetics. Blasts must be < 10% by a representative 
bone marrow aspirate morphology. 
 
3.   Organ Function and Performance Status Criteria 
A. Performance status score 
 Karnofsky (≥ 16 years old) ≥ 70 or Eastern Cooperative Oncology Group 
(ECOG) 0-1 
 Lansky (<16 years old) ≥ 60 
B. Renal Function    
 Adults:  Calculated creatinine clearance must be > 60 mL and serum creatinine ≤ 
2 mg/dL.   
 Children (<18 years old): Calculated creatinine clearance must be > 60 mL/min   
C. Hepatic Function 
 Total serum bilirubin must be <3mg/dL unless the elevation is thought to be due 
to Gilbert’s disease or hemolysis.  
 Transaminases must be < 3x the upper limit of normal per reference values of 
referring institution 
D. Pulmonary function 
 
Carbon Monoxide Diffusing Capacity (Lung Function), DLCO corrected >60% 
normal 
 For pediatric subjects unable to perform pulmonary function tests, O2 saturation 
>92% on room air 
 May not be on supplemental oxygen.  
E. Cardiac function 
 Left ventricular ejection fraction >45% OR 
 Shortening fraction > 26% 
 


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 Exclusion Criteria 
A. Uncontrolled viral or bacterial infection at the time of study enrollment 
B. Active or recent (prior 6 month) invasive fungal infection without ID consult and 
approval 
C. History of HIV infection 
D. Pregnant or breastfeeding 
E. Prior myeloablative transplant containing full dose TBI (greater than 8 Gy) 
F. Central nervous system (CNS) leukemic involvement not clearing with intrathecal 
chemotherapy and/or cranial radiation prior to initiation of conditioning. Diagnostic 
lumbar puncture is to be performed as per Section 9.7 of the study protocol. 
G. Subjects ≥ 45 years; comorbidity score of 5 or higher. 
 
DETERMINATION OF SAMPLE SIZE 
The clinical trial will randomize approximately 80 subjects per arm for a total of 160 
subjects. The primary endpoint in this clinical trial, and hence the endpoint upon which the 
sample size/power are based, is ANC engraftment.  This endpoint is a time-to-event 
outcome, with the occurrence of the event being the first of 2 consecutive days on which 
ANC≥500. Subjects who die without engraftment are included in the analysis as non-
events at infinity.   
 
To derive sample size, assumed-true distributions for engraftment were derived from data 
from previous studies for each of the arms, assuming piecewise exponential distributions 
for each arm.  Each assumed-true distribution consisted of 9 windows, with the “hazard” of 
engraftment assumed to be constant within a window, but different across the windows. In 
general, the hazards of engraftment within each window were chosen so that the 
probability of engraftment at the end of the window matched the observed probability of 
engraftment at the corresponding time in the appropriate treatment group.  The choice of 9 
windows was somewhat arbitrary, but many choices were examined until the “fit” of the 
assumed-true distribution appeared to match the observed data.  The observed data and the 
fit to the observed data, i.e., the assumed-true distributions, are shown in the figure below, 
which displays the observed time to engraftment for subjects who had received non-
expanded CB (via protocol 2010) at FHCRC.  
 
A piece-wise exponential curve was fit to these data, this fitted curve (in red) serving as the 
assumed-true time-to-engraftment distribution for subjects who receive a single or double 
CBT without ex vivo expanded off-the-shelf CB progenitor cells. The median time to 
engraftment associated with this assumed-true distribution is 25 days (the observed median 


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time to engraftment in this group is also 25 days).  Shown in blue is the assumed-true time-
to-engraftment distribution for the group to receive ex vivo expanded cells, the true median 
time being 17.9 days.  This distribution is believed to be achievable, as it is consistent with 
what has been observed in previous studies using an expanded product at FHCRC. 
 
The log-rank test was chosen, as 
this not only considers when 
subjects engraft, but time that 
subjects have been followed 
without engraftment. With 80 
subjects per arm and the above 
assumed-true time-to-engraftment 
distributions, there will be 
approximately 87% power to 
observe a statistically significant 
difference (at the two-sided level 
of 0.05) in engraftment rates 
(power estimated from 5,000 
simulations).  Treating deaths without engraftment as non-events as infinity (rather than 
censoring) provides approximately 83% power. 
 
TREATMENT ASSIGNMENT AND BLINDING 
Approximately 160 subjects who meet inclusion/exclusion criteria will be enrolled and 
randomized in the study over multiple investigative sites. At the time of randomization, 
eligible subjects will be randomized into one of the following two treatment groups in a 
1:1 ratio: 
 
Experimental Arm: This arm will undergo Umbilical cord transplant (CBT) which consists 
of unmanipulated unit(s) [umbilical cord blood #1 or #2] followed by infusion of ex vivo 
expanded product at a minimum of 4 hours after completion of last unmanipulated unit. 
Standard of Care (SOC) Arm: This arm will only undergo CBT which consists of 
unmanipulated unit(s) [umbilical cord blood #1 or #2]. 
 
The randomization will be done using a permuted block design with random block sizes 
and stratified on the number of cord blood units (1 vs. 2) to ensure balance in the number 
of subjects receiving 1 and 2 unexpanded CB units in each arm. 
 
A central randomization list will be used to assign subjects to treatment.  
This is an open-label study because it is not possible to blind investigators or study 
subjects. The study endpoints are largely objective and clinically determined, but to reduce 
the potential for bias and maintain the study integrity Syneos Health will not provide 
Nohla Therapeutics with any analyses of aggregate data by treatment group. For all 


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statistical deliverables such as SDTM/ADaM, analysis outputs for dry-runs, prior to the 
completion of the primary analysis, dummy treatment assignments will be used.  
 
 
ADMINISTRATION OF STUDY TREATMENT 
The unmanipulated units and the ex vivo expanded unit will be selected for CBT according 
to protocol Sections 6.1 and 6.2. The treatment schema at pre- and post-transplants are 
provided below. 
 


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STUDY PROCEDURES AND FLOWCHART 
Details of the visit structure and any time windows applied to these visits according to the 
schedule of visits in the protocol are provided below.  
 


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ENDPOINTS 
 
PRIMARY EFFICACY ENDPOINT 
Time to engraftment, defined as the first of 2 consecutive days with ANC ≥ 500/µL. 
 
 
SECONDARY EFFICACY ENDPOINTS 
1. Platelet engraftment, defined as the first of 7 consecutive days with platelets ≥ 
20,000/µL without transfusions 
2. Severe (grades III-IV) acute graft-versus-host disease (GVHD) 
3. Overall Survival 
4. Non-relapse mortality (hereafter referred to as transplant-related mortality, TRM) 
5. Chronic GVHD defined by the 2014 NIH criteria 
 
 
EXPLORATORY EFFICACY ENDPOINTS  
1. In vivo persistence of the ex vivo expanded cord blood product 
2. Duration of initial hospitalization 
3. Number of intensive care unit (ICU)-free days within the first 100 days post HCT 
4. Relapse/recurrence of the underlying malignancy, defined as follows:  
a. For subjects with a pre-transplant diagnosis of AML or ALL, relapse is defined 
as the presence of ≥ 5% leukemic blasts in the marrow or peripheral blood by 
morphology and/or flow cytometry. Extramedullary disease that requires 
initiation of induction chemotherapy, such as presence of leukemic blasts in the 
CNS or testicles, will also be considered relapsed disease. Similarly, subjects 
will also be considered to have relapsed disease if at any time point post-
transplant they require initiation of chemotherapy to treat the underlying 
malignancy, even with < 5% blasts in the bone marrow or peripheral blood. 
b. For subjects with a pre-transplant diagnosis of Myelodysplastic Syndrome 
(MDS), recurrent/relapsed disease is defined as the presence of abnormal 
findings in the peripheral blood or bone marrow sufficient to diagnose MDS per 
French American System (FAB) or World Health Organization (WHO) 
classifications and requiring systemic therapy, or the presence of >10% 
leukemic blasts in the peripheral blood or bone marrow. The use of single agent 
chemotherapy (such as Azacitidine) as prophylaxis against disease recurrence 
will not be an indication of relapsed disease.  
c. For subjects with pre-transplant diagnosis of  CML, relapsed disease is defined 
as leukemic blast crisis and/or accelerated phase with ≥ 5% leukemic blasts in 
the bone marrow or peripheral blood. Subjects with evidence of molecular or 


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cytogenetic disease alone is not considered disease relapse unless the disease is 
refractory to Tyrosine Kinase Inhibitors (TKIs).   
5. Disease-free survival 
6. GVHD-free, relapse-free survival (GRFS), with GVHD defined as either severe acute 
or chronic GVHD as defined by the 2014 National Institutes of Health (NIH) criteria  
7. Common Terminology Criteria for Adverse Events (CTCAE) Grade ≥ 3 infusional 
toxicity, defined as grade 3+ events within the first 24 hours after completion of 
infusion of expanded cells 
8. Graft failure:  Primary and secondary  
9. Immune reconstitution: kinetics of immune system recovery as measured by T and B 
cell subsets, T-cell Receptor Excision Circles (TREC), and T-cell Receptor (TCR) 
sequencing  
10. Time to platelet engraftment of 50,000/µL, defined as the first of at least 2 separate days 
with platelets ≥50,000/µL that span a period of at least 7 days without intervening 
platelet transfusion. 
 
 
SAFETY ENDPOINTS  
Safety assessments will include incidences of adverse events (AEs), serious adverse events 
(SAEs), performance status, and laboratory values results (complete blood count, blood 
chemistry, immunoglobulins, and lymphocyte panel). 
 


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ANALYSIS SETS 
 
SCREENED SET 
The Screened Set will include all subjects screened. This set will be used for the 
summaries of subject disposition.  
 
 
SAFETY SET  
The Safety Set (SS) will be defined as all randomized subjects who undergo HCT. Subjects 
will be analyzed according to treatment they actually received.  The SS will be used for all 
analyses of safety endpoints.  
 
 
INTENT-TO-TREAT OR FULL ANALYSIS SET  
The Intent-to-Treat Set or Full Analysis Set (FAS) will be defined as all randomized 
subjects regardless of whether they receive assigned treatment. Subjects will be analyzed 
according to randomized treatment.  The FAS will be used for supportive analyses of 
primary, secondary and exploratory endpoints and for the presentation of subjects in all 
subject listings. 
 
 
MODIFIED INTENT-TO-TREAT SET 
The Modified Intent-to-Treat (mITT) Set will be defined as all randomized subjects who 
undergo HCT and received assigned treatment per randomization arm. Subjects will be 
analyzed according to randomized treatment.  The mITT Set will be used as the primary 
analysis set for all analyses of primary, secondary and exploratory endpoints.  
 
 
 
PER PROTOCOL SET  
The Per Protocol Set (PPS) will include all subjects who are randomized, underwent HCT, 
have sufficient data to assess the primary efficacy endpoint, and who have no important 
protocol deviations (IPDs). Subjects will be analyzed according to treatment they actually 
received.   
 
The important protocol deviations will be identified and defined prior to the primary 
analysis data lock and primary analysis. Any additional protocol deviations between 
primary analysis and final analysis will be identified and defined prior to the final data lock 
and analysis (after all subjects have 2 years of follow-up). The Nohla Therapeutics study 
team will identify and approve all IPDs categories and determinations.  The IPD 
specifications will be defined prior to the database lock for primary analysis.  The protocol 
deviations SAS datasets SDTM.DV and ADAM.ADDV will then be generated.  


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GENERAL ASPECTS FOR STATISTICAL ANALYSES 
 
GENERAL METHODS 
This SAP provides the formal specification of principal analyses to be conducted in this 
study. In the event of any discrepancies between this document and the study protocol, 
interpretation of results should be based on the specifications in the SAP.  The protocol 
would be amended only if there would be changes to a principal feature of trial design, 
conduct or analyses. 
Summaries and statistical analyses of study data will be performed by Syneos Health. 
Validated results will be reviewed by Consultant Biostatistician and clinicians and 
biostatistician at Nohla Therapeutics. All analyses will be performed by treatment group 
[Experimental Arm (EXP), Standard of Care Arm (SOC)]. Subject listings of all data from 
the case report forms (CRFs) as well as relevant derived variables will be presented.  
All analyses will be implemented using SAS Version 9.4 or more recent version. The latest 
MedDRA version at the time of statistical analyses would be used to code medical history 
and adverse events datasets. Prior and concomitant medications will be coded using the 
latest version of the World Health Organization Drug Dictionary (WHO-DD) at the time of 
analysis. 
Unless otherwise specified, continuous variables will be summarized by presenting the 
number of non-missing observations, mean, standard deviation, median, minimum and 
maximum, and distribution according to a clinically relevant discretization.  Categorical 
variables will be summarized by presenting the number of subjects and percentage for each 
category.   
Unless otherwise noted, all formal analyses of the primary and key secondary endpoints 
will be conducted in a manner to preserve the experimental two-sided 0.05 error rate, and 
two-sided 95% confidence intervals (CI) will be calculated.  The interim analysis used 
0.0054 alpha, so if the primary endpoint is statistically significant at the two-sided alpha 
level of 0.0492 (per the O’Brien-Fleming boundary), then formal comparisons will be 
conducted for the first 2 of the 5 secondary endpoints – platelet engraftment and severe 
acute GVHD.  Using a Bonferroni adjustment for multiplicity, statistical significance for 
each of the first 2 secondary endpoints analyses will be based on an alpha of 0.0246 
(=0.0492/2).  The other 3 secondary endpoints (overall survival, TRM, and chronic 
GVHD) which may require longer follow-up, will be treated similar to exploratory 
endpoints, with reporting of nominal p-values for descriptive purposes only.    
If major imbalances (due to chance alone) are identified across treatment groups for factors 
known to influence outcome, supportive analyses will be conducted adjusting for these 
imbalances using appropriate regression techniques (linear for continuous, logistic for 
binary, and Cox for time-to-event outcomes).  Sensitivity analyses will be conducted in an 
exploratory manner to examine the consistency of effects across various subgroups as 


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defined in Section 6.5.  When parametric analyses are conducted, in the event of highly 
skewed data, sensitivity analyses will be conducted using, for example, a logarithmic 
transformation to reduce skewness.   
P-values will be presented for the pre-specified analyses of primary, secondary, and 
exploratory endpoints conducted as specified in Sections 9.7, 9.8, and 9.9, respectively.  P-
values will be rounded to 3 significant digits. 
All efficacy analyses will be conducted from date of HCT, with the mITT population as 
the primary population.  Timing of assessments will be stated relative to date of HCT (e.g., 
100 days of follow-up following HCT).  Sensitivity analyses will be conducted for 
engraftment endpoints from date of randomization using the full analysis population. 
 
 
KEY DEFINITIONS 
 
Baseline 
Baseline is the last measurement taken prior to or on the day of HCT, unless otherwise 
specified. 
 
 
Protocol Day 
Per the study protocol, Protocol Day 0 is regarded as the date of Hematopoietic Cell 
Transplant (HCT).   
 
 
Study Day 
The reference date is the day of HCT, considered as Protocol Day 0. Study day will be 
calculated as (date of assessment – date of HCT + 1) if assessment date is on or after the 
date of HCT; or date of assessment – date of HCT, if assessment date is prior to the date of 
HCT. Therefore, Protocol Day 0 is equal to Study Day 1.    
 
 
Completed Study 
Study completion for the primary analysis is met if one of the following apply: 
• A subject has completed the two-year follow up visit 
• A subject remains in follow-up (i.e., has not withdrawn) at the time of the clinical 
cut date 
• A subject has died prior to the clinical cut date  
 
Study completion for the final analysis is met if one of the following apply: 
• A subject has completed the two-year follow up visit 
• A subject has died prior to the clinical cut date  
 


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PARTIAL DATES  
Data points that appear to be spurious will be investigated and will not be excluded from 
the listings. Influential cases will be handled in an appropriate statistical manner.  
All dates recorded on the Adverse Events (AEs) and prior/concomitant medication CRF 
should be complete. However, if there are records for which complete start or stop dates 
could not be obtained following best effort attempts for query resolution, partial or 
incomplete start/end dates in the AEs or prior/concomitant medication datasets will be 
imputed to help determine treatment-emergent adverse events (TEAE) and (prior or 
concomitant) medications.  
For incomplete start dates, use the following imputation rules: 
• Missing day - Impute the 1st of the month unless month is same as month of HCT 
then impute using the day of HCT.  
• Missing day and month – impute 1st January unless year is the same as year of 
HCT then impute using the month of HCT for missing month, and the first day of 
the month of HCT for missing day.  
• Completely missing – Impute HCT date unless the end date suggests it could have 
started prior to this in which case impute the 1st January of the same year as the 
start date.   
 
When imputing a start date, ensure that the new imputed date is sensible i.e. is prior to the 
end date of the AE or medication. 
 
For incomplete concomitant data end dates only (not applicable to AE datasets), use the 
following imputation rules:  
• Missing day - Impute the last day of the month   
• Missing day and month – Impute 31st December and reconcile with the 
discontinuation date or death date. If the imputed date with 31st December is greater 
than the discontinuation date or date of death, then use the discontinuation date. For 
subjects who have not discontinued, check if the imputed date with 31st December 
is greater than their last available visit date, if so then use their last available visit 
date.  
• Completely Missing – Determine if the medication is still ongoing before imputing 
a date and when the medication started in relation to date of HCT. If the ongoing 
flag is not missing, then assume that the medication is still being taken (i.e. do not 
impute a date). If the medication has stopped and start date is prior to HCT date 
then impute the HCT date, if the medication started on or after HCT date then 
impute a date that is after the last available visit date. 
• Should any of the previous stop dates created come before a start date, either a 
complete date or an imputed one, use the start date instead of the date that would 
otherwise be created. 
    


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VISIT WINDOWS 
Details of the planned protocol-specified visits and visit windows are given in the protocol 
and in Section 3.7. 
No derivation of analysis windows are planned for the study regarding data collected 
outside the protocol-specified visit windows. Study data will be analyzed according to the 
scheduled visits reported. 
Where assessments are carried out more than once at a visit or within the same window, 
data and timings of all assessments would be reported in the data listings, including 
assessments that may have occurred from unscheduled visits. However, for data analysis in 
the tables, the closest value to the protocol-specified visit will be used. If there is a tie in 
determining the closest value, then the data value just prior to the protocol-specified visit 
will be used. 
 
EXAMINATION OF SUBGROUPS 
Subgroups will be defined only if there are a sufficient number of subjects in each category 
of the subgroup (e.g., >5 subjects per treatment group). 
Subgroup analyses will be descriptively conducted to assess treatment effects and include, 
but are not limited to, the following:  
• Age groups of < 18 versus 18-65 years;  
• Weight (below the median for the FAS versus above the median for the FAS) 
• Receipt of single versus double CBT;  
• FHCRC versus non-FHCRC sites;  
• Conditioning regimen (High intensity:  fludarabine, cyclophosphamide, total body 
irradiation versus Mid Intensity:  fludarabine, cyclophosphamide, total body 
irradiation, Thiotepa, Other); and  
• Underlying disease (AML, ALL, CML, MDS, Other).  
The primary and key secondary efficacy endpoints will be analyzed for the subgroups 
using the mITT Set.  Forest plots will be presented. In addition, select safety analyses will 
also be analyzed by subgroup using the SS as specified in Section 10. Subgroup 
characteristics will be summarized by treatment group. During the CSR preparation, 
additional subgroup analyses may be performed. Those analyses will be considered as ad 
hoc analyses and will be presented and discussed in the CSR. 


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STUDY POPULATION 
 
SUBJECT DISPOSITION  
The following subject data will be presented: 
• Number of subjects screened, signed informed consent (overall only); 
• Number of subjects screened failed and reasons for screen failure (overall only); 
• Number and percentage of subjects randomized in each analysis set (SS, FAS, 
mITT, PPS), by treatment group and overall; 
• Number and percentage of subjects completed or discontinued study, by primary 
reason for study discontinuation for FAS and mITT Set, by treatment group; and 
• Number and percentage of subjects excluded from PPS by reason for exclusion 
defined in Section 5.5, by treatment group for FAS. 
A subject listing for early discontinuations and reasons for discontinuation will be 
provided for the FAS.   
 
PROTOCOL DEVIATIONS 
Protocol deviations will be collected by study monitors. Sufficient detail on the types of 
protocol deviations may include but are not limited to  
 Subject did not meet all of the inclusion or exclusion criteria 
 Subject used excluded medications during the study 
 No post-HCT efficacy data 
 Any known major protocol non-compliance 
Important protocol deviations that could potentially affect the efficacy or safety 
conclusions of the study will be identified prior to database lock. Important protocol 
deviations will be summarized by deviation category and treatment group using the FAS 
and mITT Set.  
All protocol deviations will be presented in a data listing. 
In addition, prior to database lock, a data listing will be prepared for sponsor review in 
order to determine the subjects to exclude from the PPS, as well as categorize each 
protocol deviation criteria as not important or important. 
 


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DEMOGRAPHIC, OTHER BASELINE CHARACTERISTICS 
AND MEDICATIONS 
 
DEMOGRAPHIC AND OTHER BASELINE 
CHARACTERISTICS 
Age (years) will be calculated as the number of years between the date of birth and the date 
of randomization. 
Age at randomization = floor (randomization date – date of birth +1)/365.25 and truncated 
to complete years. 
If height is recorded in inches (in), then height will be summarized in centimeters (cm) 
using the following conversion: 
 
Height (cm) = Height (in) x 2.54 
If weight is recorded in pounds (lb), then weight will be summarized in kilograms (kg) 
using the following conversion: 
 
Weight (kg) = Weight (lb) x 0.4536 
All demographic and baseline characteristics will be summarized by treatment group using 
descriptive statistics for the FAS, mITT Set, and SS. No formal hypothesis testing will be 
performed to compare differences between treatment groups.  
 
Recipient Demographic  
The following demographic variables for the recipient will be summarized: 
• Age (years) at time of randomization, sex, ethnicity, race, height (cm), and 
weight (kg). 
 
 
Donor Demographic 
The following demographic variables for the donor will be summarized: 
• Cord blood storage (years) at time of transplantation as determined by donor 
age, and donor sex. 
 


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Significant Pre-Transplant Medical History 
The number and percentage of subjects with previous and active medical or surgical 
conditions at screening will be summarized and presented by MedDRA system organ class 
and preferred term.   
 
 
Significant Pre-Transplant Treatment/Disease History 
The following will be summarized: 
• Number and percentage of subjects with each type of previous therapy. 
• Result/Outcome for each previous therapy [Complete Response (CR), Partial 
Response (PR), Stable Disease (SD), Progressive Disease (PD), Relapse (REL) 
and Refractory (REFR)]. 
• Duration of time since last treatment prior to randomization (months). 
 
 
Disease Characteristics at Diagnosis 
The following disease characteristics as recorded on the CRF will be summarized: 
• Diagnosis (AML, ALL, CML, MDS, other) 
• Duration of disease prior to randomization (months) defined as (randomization 
date – date of diagnosis)/(365.25/12) 
• Cytology Results 
• Morphology Results 
• Flow 
• Disease specific characteristics for AML, ALL, CML, MDS, other 
 
 
Transplant-Related Information 
The following transplant-related information will be summarized: 
• Number and percentage of subjects who underwent previous transplants 
• Recipient viral serology status [Cytomegalovirus , Herpes Simplex Virus, 
Varicella Zoster Virus, antibody titer-A [Immunoglobulin G (IgG), 
Immunoglobulin M (IgM)], antibody titer-B (IgG, IgM) 
• Recipient Viral Reactivations: Virus, reactivation site, first Polymerase Chain 
Reaction (PCR) copy # (unit), max PCR copy # 


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• ABO compatibility for each recipient/donor pair (A-A, A-<specify per data>, 
B-B, B-<specify per data>, AB-AB, AB-<specify per data>, and O-O, O-
<specify per data>) 
• Recipient and Donor Rhesus (Rh) 
• Recipient Panel reactive antibody [class I (%), class II (%)] 
• HLA Subject/Donor and [HLA match (A, B, DR), Locus of mismatch (C if 
available), and locus of mismatch (DQB1 if available) for Unit #1 and Unit #2 
respectively] 
• Hematopoietic Cell Transplantation –Specific Comorbidity Index (HCT-CI) 
• Abnormal donor findings, (Yes/No) 
• Donor red cell depleted, (Yes/No)  
 
 
Performance Status 
The following will be summarized: 
• Type of performance status assessment (ECOG, Lansky, Karnofsky) 
• Results 
 
 
Cardiovascular and/or Pulmonary Function Tests (PFT) 
The following pre-transplant cardiovascular and/or PFT results will be summarized: 
• PFT Tests (DLCO and Oxygen saturation) 
• Echocardiogram/Multigated Acquisition (Ejection fraction and fractional 
shortening) 
 
 
Conditioning Regimen 
The following conditioning regimen variables will be summarized: 
• Treatment (Fludarabine, Cyclophosphamide, TBI, Thiotepa, Other) 
• Dose (reported as total dose) and units 
 
 
Disease Evaluation at Time of Transplant 
The following variables will be summarized: 


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• Weight (kg) 
• Disease status at transplant [CR1, CR2, >= CR3, PR, CP1 (CML only), CP2 s/p 
AP (CML only), CP2 s/p BC (CML only), CP>=3 (CML only), AP1 (CML 
only), AP2 s/p BC (CML only), AP >=3 (CML only), CR (CML only), Other 
• Disease Response status at transplant (complete remission, partial remission, 
stable disease, progressive disease) 
• Minimal residual disease (Yes/No/Unknown/Not applicable) 
• Cytology results 
• Morphology 
• Flow 
• FISH 
• Molecular characterization 
• Imaging scan type [Cat scan (CT), X-ray, Magnetic Resonance Imaging (MRI)] 
and results 
 
 
Donor Cell Panel 
The following variables will be summarized: 
• Test results [CD34 (×10^6), CD34kg (×10^6), TNC (×10^7), TNCkg (×10^7), 
TVOL], viability (%) for Unit #1, Unit #2, and expanded product. 
 
 
MEDICAL HISTORY AND CONCOMITANT DISEASES 
Significant pre-transplant medical history will be collected. The number and percent of 
subjects with each medical history condition will be summarized by MedDRA system 
organ class and preferred term using the mITT Set by treatment group. All medical history 
data will be listed. 
 
MEDICATIONS 
Medications received and stopped before HCT day will be considered as prior medications. 
Medications that stopped or are ongoing after HCT through to the end of study or early 
termination will be considered as concomitant medications. The medications will be 
summarized for each treatment group by therapeutic subgroup (ATC 2nd level) and 
preferred WHO-DD name for the mITT Set. Subjects taking the same medication multiple 
times will be counted once per medication.  


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Handling missing or incomplete start or stop date in defining concomitant medications is 
described in Section 6.3.  
All medication data will be listed, sorted by investigative site treatment group, subject 
number, start and stop date.  Information listed will include medication, indication, total 
daily dose, dose units, frequency and route of administration. 
Descriptive statistics of the number of antibiotic-free days within the first 100 days for 
subjects will also be provided. 
 


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EFFICACY 
All efficacy analyses will be conducted using the mITT Set as the primary analysis. If the 
total FAS or PPS differ from the mITT Set by more than 10 subjects, efficacy analyses will 
be repeated using these analysis sets for relevant primary, key secondary, other secondary 
efficacy endpoint analyses, and select exploratory efficacy endpoints. Analyses using the 
FAS will use the date of randomization, instead of date of HCT, as the reference date. All 
efficacy endpoints, recorded and derived, will be presented in data listings. The analyses of 
primary and key secondary endpoints will be repeated for the subgroups as specified in 
Section 6.6. 
 
ADJUSTMENTS FOR COVARIATES 
N/A.  
 
HANDLING OF DROPOUTS OR MISSING DATA 
No imputation will be done for missing efficacy data. Observed data will be used for 
analyses. 
 
INTERIM ANALYSES AND DATA MONITORING 
 
Interim Analysis  
For the primary endpoint of time to engraftment (ANC ≥ 500), an interim analysis for 
superiority was conducted when 80 subjects have been enrolled and followed sufficiently 
long to assess their time to engraftment. An O’Brien-Fleming boundary was used for the 
interim analysis in order to maintain most of our type I error rate of 0.05 at the final 
analysis. In particular, if there is a statistically significant difference in favor of the 
experimental regimen at the two-sided significance level of 0.0054, the boundary was to be 
considered crossed at the interim analysis. At the primary analysis, analysis of this 
endpoint will be conducted at a two-sided significance level of 0.0492 (per the O’Brien-
Fleming boundary).  
 
To enable the trial to provide more reliable evidence with respect to other outcomes, 
stopping for superiority at the interim analysis as defined above was to occur only if the 
O’Brien-Fleming boundary also is crossed for the endpoint of overall survival, but at the 
overall two-sided 0.01 level of significance.   
 
 
Safety Monitoring Committee  
A separate Data Safety Monitoring Board (DSMB) will receive deliverables based on 
actual treatment assignments. Additional details on the role of the DSMB are provided in a 
separate charter. 
 


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MULTICENTER STUDIES 
The randomization is not stratified by site. Likewise, analyses of efficacy data will not be 
stratified by study site. The number and percentage of subjects randomized by study site 
will be summarized by treatment group and for all subjects. 
 
USE OF AN “EFFICACY SUBSET” OF SUBJECTS 
N/A 
 
MULTIPLE COMPARISONS/MULTIPLICITY 
The interim analysis used 0.0054 alpha, so for the final analysis of the primary and key 
secondary efficacy analyses, the overall 2-sided level of significance will be alpha=0.0492. 
The hypothesis testing of key secondary endpoints will be conducted by splitting the 
0.0492 alpha equally among the 3 key secondary endpoints (0.0164), provided the primary 
efficacy endpoint comparison is statistically significant at an alpha level 0.0492. If this 
comparison is not statistically significant, then the comparison of key secondary efficacy 
endpoints will be considered nominal, descriptive and exploratory. This procedure controls 
the study-wise type I error. 
 
PRIMARY EFFICACY ANALYSIS 
The primary endpoint of ANC engraftment is assessed daily until a subject engrafts, rejects 
their graft, or reaches day 55 following HCT, whichever comes first.  Per convention in 
HCT, ANC engraftment is defined as the first of 2 consecutive days in which neutrophil 
count ≥500/µL. 
The analysis of the primary endpoint (ANC engraftment) will be conducted by treating 
engraftment as a time-to-event outcome from date of HCT.  A stratified log-rank test will 
be used for the primary analysis of this endpoint, with stratification on number of cord-
blood units (1 vs. 2), reflecting the randomization stratification variable.  Subjects who die 
without engraftment will be included in the analysis as non-events at infinity (rather than 
censoring). Subjects who reject the graft or reach day 55 following HCT without 
engraftment will be censored at day 55.   
Kaplan-Meier (KM) estimates of the distribution of the time-to-event will be tabulated and 
graphed by treatment group. The tabulation will include the KM estimate of the medians, 
25th and 75th quartiles, and corresponding 95% CIs, if estimable. The tabular and graphical 
summaries will include the at-risk counts for every visit. The number and percent of 
subjects censored and with events will be presented. The hazard ratio and 95% CI will be 
determined based on a Cox regression model stratified by randomization stratification 
variable to estimate the magnitude of the effect. 


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The primary analysis will be conducted in days, from date of HCT, but a sensitivity 
analysis will be conducted from date of randomization.  A sensitivity analysis will also be 
done by censoring deaths without engraftment at time of death. 
 
SECONDARY EFFICACY ANALYSES 
The secondary endpoints include: 
1. Platelet engraftment, defined as the first of 7 consecutive days with platelets ≥ 
20,000/µL without transfusions 
2. Severe (grades 3 or higher) acute graft-versus-host disease (GVHD) 
3. Overall Survival 
4. Transplant-related mortality(TRM) 
5. Chronic GVHD defined by the 2014 NIH criteria 
 
The first 2 secondary endpoints listed above (platelet engraftment, severe acute GVHD) 
will be formally compared between treatment groups and are therefore considered “key” 
secondary endpoints as described below in Section 9.5.1.  The remaining 3 secondary 
endpoints will be considered “other” secondary endpoints as described below in Section 
9.6. 
 
Key Secondary 
The key secondary endpoints include: 
1. Platelet engraftment, defined as the first of 7 consecutive days with platelets ≥ 
20,000/µL without transfusions 
2. Severe (grades 3 or higher) acute graft-versus-host disease (GVHD) 
 
The endpoints listed above (platelet engraftment, severe acute GVHD) will be formally 
compared between treatment groups, provided the primary endpoint of ANC engraftment 
is deemed to be statistically significantly different between treatment groups (at the two-
sided significance level of 0.0492) as noted above in Section 6.1.  In such case, we shall 
conduct formal analyses where the two-sided significance level of 0.0492 will be shared 
equally among these three secondary endpoints that will undergo formal comparison.  
Therefore, the two-sided level of significance for these formal comparisons will be 0.0246.  
The key secondary endpoints of platelet engraftment and severe acute GVHD will be 
conducted from time of HCT, with a sensitivity analysis conducted from date of 
randomization.   


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9.8.1.1. 
Key Secondary Efficacy Endpoint 1: Platelet Engraftment 
The secondary endpoint platelet engraftment is assessed and analyzed in the same manner 
as the primary endpoint of ANC engraftment (Section 9.7). Platelet engraftment, per 
convention in HCT, is defined as the first of seven consecutive days in which platelets 
≥20,000/µL in the absence of platelet transfusions.  Subjects who reject the graft or do not 
achieve engraftment by day 100 will be censored at that date. 
9.8.1.2. 
Key Secondary Efficacy Endpoint 2: Severe (grade 3 or 4) Acute GVHD 
GVHD is defined according to the National Institutes of Health (NIH) dictionary of cancer 
terms as a disease caused when cells from a donated stem cell graft attack the normal tissue 
of the transplant subject. Symptoms include jaundice, skin rash or blisters, a dry mouth, or 
dry eyes. 
To reduce potential bias, a single central reader, Dr. Paul Martin will grade all subjects for 
acute GVHD.  Every effort will be made to shield Dr. Martin from information that may 
contain reference to treatment assignment. The grading is based on data collected within 
the first 100 days following HCT and is assessed per the acute GVHD assessment outlined 
in protocol Appendix C.  Dr. Martin has been the sole grader of acute GVHD at FHCRC 
for more than 20 years.   
Severe (grades 3 or 4) acute GVHD will be analyzed using the Cochran-Mantel-Haenszel 
test (stratified on number of cord-blood units) to compare the proportion of subjects with 
severe GVHD between treatment groups.  The number and percentage, with associated 
two-sided exact (Clopper-Pearson) 95% CIs, of subjects with and without severe GVHD 
will be presented by treatment group and an odds ratio calculated (SOC arm/experimental 
arm). 
 
 
Other Secondary Efficacy Analyses 
The other secondary endpoints include: 
1. Overall Survival 
2. Transplant-related mortality(TRM) 
3. Chronic GVHD defined by the 2014 NIH criteria 
 
Other secondary endpoints will be compared in a manner consistent with exploratory 
endpoints as they may require longer follow-up time. Nominal p-values will be provided 
for descriptive and exploratory purposes only. The endpoints of overall survival, TRM, and 
chronic GVHD will be conducted in days using date of HCT, with minimum follow-up of 
100 days and maximum follow-up of 2 years after HCT.  Sensitivity analyses will be 
conducted using date of randomization and the FAS.   


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9.8.2.1. 
Other Secondary Efficacy Endpoint 1: Overall Survival 
Overall survival will be captured until the end of follow-up for each subject, defined as 2 
years after HCT.  The analysis of overall survival will occur at the clinical cut date (100 
days after HCT for the last randomized subject) if at least 30 total deaths have occurred by 
this point. Otherwise, the analysis of overall survival will occur after the clinical cut date at 
the point once the 30th death occurs. Subjects alive at the time of analysis will be censored 
at the date last known to be alive (up to two years).  Subjects lost to follow-up prior to two 
years will be censored at their last study date known to be alive. Overall survival will be 
compared between groups using the same analysis methods specified for the primary 
endpoint (Section 9.7). 
9.8.2.2. 
Other Secondary Efficacy Endpoint 2: Transplant-Related Mortality 
(TRM), Previously Referred to as Non-relapse Mortality (NRM) 
TRM is defined as the occurrence of death without prior relapse.  Relapse is defined in 
Section 4.3 under the 4th exploratory endpoint. Subjects who relapse will be censored at the 
date of first relapse. TRM will be analyzed using the same analysis methods specified for 
the primary endpoint (Section 9.7). 
9.8.2.3. 
Other Secondary Endpoint 3:  Chronic GVHD 
The cause-specific hazards of chronic GVHD, defined according to the 2014 NIH criteria, 
will be compared between treatment groups using the stratified log-rank test, stratified for 
number of cord-blood units (1 vs. 2), reflecting the randomization stratification variable.  
Chronic GVHD will be captured for each subject until end of follow-up, defined as 2 years 
after HCT.  Deaths without GVHD will be censored at time of death.  In addition, the 
cumulative incidence functions of chronic GVHD will be compared using Gray’s test4, 5, 6, 
with death without chronic GVHD regarded as a competing-risk event5. 
 
EXPLORATORY EFFICACY ANALYSES 
The exploratory endpoints include: 
1. In vivo persistence of the ex vivo expanded cord blood product 
2. Duration of initial hospitalization 
3. Number of ICU-free days within the first 100 days post HCT 
4. Relapse/recurrence of the underlying malignancy, defined as follows:  
a. For subjects with a pre-transplant diagnosis of AML or ALL, relapse is defined 
as the presence of ≥ 5% leukemic blasts in the marrow or peripheral blood by 
morphology and/or flow cytometry. Extramedullary disease that requires 
initiation of induction chemotherapy, such as presence of leukemic blasts in the 
CNS or testicles, will also be considered relapsed disease. Similarly, subjects 
will also be considered to have relapsed disease if at any time point post-


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transplant they require initiation of chemotherapy to treat the underlying 
malignancy, even with < 5% blasts in the bone marrow or peripheral blood. 
b. For subjects with a pre-transplant diagnosis of MDS, recurrent/relapsed disease 
is defined as the presence of abnormal findings in the peripheral blood or bone 
marrow sufficient to diagnose MDS per French American System (FAB) or 
World Health Organization (WHO) classifications and requiring systemic 
therapy, or the presence of >10% leukemic blasts in the peripheral blood or 
bone marrow. The use of single agent chemotherapy (such as Azacitidine) as 
prophylaxis against disease recurrence will not be an indication of relapsed 
disease.  
c. For subjects with pre-transplant diagnosis of CML, relapsed disease is defined 
as leukemic blast crisis and/or accelerated phase with ≥ 5% leukemic blasts in 
the bone marrow or peripheral blood. Subjects with evidence of molecular or 
cytogenetic disease alone is not considered disease relapse unless the disease is 
refractory to TKIs.   
5. Disease-free survival 
6. GVHD-free, relapse-free survival (GRFS), with GVHD defined as either severe acute 
or chronic GVHD as defined by the 2014 National Institutes of Health (NIH) criteria  
7. CTCAE Grade ≥ 3 infusion toxicity, defined as grade 3+ events within the first day after 
completion of infusion of expanded cells (experimental arm only) 
8. Graft failure:  Primary and secondary  
9. Immune reconstitution: kinetics of immune system recovery as measured by T and B 
cell subsets, TREC, and TCR sequencing 
10. Time to platelet engraftment of 50,000/µL, defined as the first of at least 2 separate days 
with platelets ≥50,000/µL that span a period of at least 7 days without intervening 
platelet transfusion 
 
 
Exploratory Efficacy Endpoint 1: In Vivo Persistence of the Ex-Vivo 
Expanded Cord Blood Product 
Persistence of the ex vivo expanded product is assessed at days 28, 80, and one year for 
Bone marrow (BM) sample for Unsorted Chimerism; weekly through day 28, then days 42, 
56, 80, 100, 180, 1 and 2 years for Peripheral Blood sample for Sorted Chimerisms. 
In vivo persistence of the expanded cord blood product will be summarized using 
descriptive statistics.  Results will be presented by individual cell immunophenotype 
(CD3+, CD14+ or CD15+, CD33+, CD19+ or CD20+, CD56+). This endpoint only 
pertains to the experimental arm. 


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Exploratory Efficacy Endpoint 2: Duration of Hospitalization 
Duration of initial hospitalization is assessed daily. 
Duration of initial hospitalization will be presented, with particular attention paid to 
subjects who die without being discharged.  While this is expected to be uncommon, to 
avoid a potential artificial reduction in hospitalization length due to “early” deaths while in 
the hospital, the effect of treatment on the number of days alive and out of the hospital 
during the 100 days post HCT will also be evaluated. 
The duration of initial hospitalization will be compared between treatment groups using an 
analysis of variance (ANOVA) with fixed effects for treatment groups and randomization 
stratification. Number of days alive and out of the hospital will be analyzed in the same 
manner. 
 
Exploratory Efficacy Endpoint 3: Intensive Care Unit-Free Days  
The effect of treatment on the number of days alive and out of the ICU during the first 100 
days post HCT will be evaluated in a manner consistent with the duration of initial 
hospitalization endpoint (Section 9.9.2). 
 
Exploratory Efficacy Endpoint 4: Relapse/Recurrence of the Underlying 
Malignancy 
The cause-specific hazards of relapse will be analyzed using the same analysis methods 
specified for the primary endpoint (Section 9.7).  Relapse will be captured for each subject 
until end of follow-up, defined as 2 years after HCT.  Deaths without relapse will be 
censored at time of death.  In addition, the cumulative incidence functions6 of relapse will 
be compared using Gray’s test4.5, with death without relapse regarded as a competing-risk 
event.  Subjects without relapse will be censored at the last known date alive up to 2 years 
follow-up. 
 
Exploratory Efficacy Endpoint 5: Disease-Free Survival 
Disease-free survival is defined as survival without the occurrence of relapse.  This 
endpoint will be analyzed using the same analysis methods specified for the primary 
endpoint (Section 9.7). Subjects without relapse will be censored at the last known date 
alive up to 2 years follow-up. 
 
Exploratory Efficacy Endpoint 6: GVHD-free, Relapse-free Survival 
(GRFS) 
GVHD-free, relapse-free survival (GRFS), defined as survival without relapse, severe 
acute GVHD, or chronic GVHD, will be analyzed using the same analysis methods 
specified for the primary endpoint (Section 9.7). Subjects without failure at the time of 
database lock will be censored at the last known date alive. 


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Exploratory Efficacy Endpoint 7: Grade ≥ 3 Infusion Toxicity 
Grade 3 infusion toxicity is defined as the occurrence of grade 3 events within the first day 
following completion of the expanded cell infusion and be presented as a simple 
proportion as well as table with MedDRA coded AE terms by system organ class and 
preferred term.  This endpoint will only be described for the experimental arm. 
 
Exploratory Efficacy Endpoint 8: Graft Failure 
Graft failure is assessed daily within the first 55 days following HCT. Graft failure will be 
estimated using simple proportions, with graft failure (both primary and secondary) as 
defined below: 
 
Primary Graft Failure: Subjects may be considered primary graft failure/rejection provided 
they meet any criteria listed below: 
 Absence of 3 consecutive days with neutrophils ≥ 500/µL combined with host CD3 
peripheral blood chimerism ≥ 50% by day + 42 
 Absence of 3 consecutive days with neutrophils ≥ 500/µL under any circumstances 
at day 55 
 Death after day 28 with neutrophil count < 100 µL without any evidence of 
engraftment (< 5% donor CD3) 
 Primary autologous count recovery with < 5% donor CD3 peripheral blood 
chimerism at count recovery and without relapse 
 
Secondary Graft Failure/Rejection: Decline of neutrophil count < 500/µL with loss of 
donor chimerism after day 55 
Primary and secondary graft failure will be analyzed separately using the same analysis 
methods specified for the severe acute GVHD endpoint (Section 9.8.1.3). 
 
Exploratory Efficacy Endpoint 9: Immune Reconstitution 
Immune reconstitution will be examined pre-conditioning, days 28, 56, 80 or 100 (per 
institutional practice), 6 months, 1 and 2 years. 
 
Immune reconstitution will be assessed graphically by plotting chimerism values 
longitudinally at the time points listed above, with the “average” values obtained by fitting 
non-linear continuous curves through the longitudinal data (e.g., using restricted cubic 
splines). 
 
 
Exploratory Efficacy Endpoint 10: Time to Platelet Engraftment 
≥50,000/µL 
As another measure of evaluation of kinetics of immune reconstitution, time to platelet 
engraftment to ≥50,000/µL will be evaluated.  This endpoint is defined as the first of at 


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least 2 separate days with platelets ≥50,000/µL that span a period of at least 7 days without 
intervening platelet transfusion. This endpoint will be analyzed using the same analysis 
methods as the secondary endpoint of time to platelet engraftment to ≥20,000/µL (Section 
9.8.1.1). 
 


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SAFETY 
Safety assessments will consist of summarizing transplant and infusion details; all AEs 
collected as per the protocol, including SAEs,; and laboratory values including (complete 
blood count, blood chemistry, immunoglobulin, and lymphocyte panel).  
All safety analyses will be conducted on the Safety Set, unless specified otherwise. 
Summaries of all safety assessments will be reported at visits where data are collected. All 
safety data will be presented in individual subject data listings.   
 
EXTENT OF EXPOSURE AND FOLLOW-UP 
 
Exposure 
The following exposure variables will be summarized continuously for each treatment 
using the SS: 
 Duration of infusion (minutes) during transplant for Unit 1 and Unit 2,  
 Volume (mL) infused for Unit 1 and Unit 2,  
 Volume (mL) of ex vivo expanded product infused 
 Duration (minutes) of infusion of ex vivo expanded product 
 
 
Follow-up 
The duration of follow-up for each subject will be calculated as the length of time from 
date of HCT to date of completion/discontinuation from study in days.  Similarly, the 
duration from randomization to HCT will be calculated in days.  Both will be summarized 
by treatment group as continuous variables with descriptive statistics. 
Duration of follow-up will also be categorized as follows: 
 ≥ 6 months 
 ≥ 1 year  
 ≥ 2 years 
 
These categories are not mutually exclusive.  Counts and percentages of subjects in each of 
these categories will be summarized for each treatment group using the SS. 
 
TREATMENT COMPLIANCE  
N/A. 


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ADVERSE EVENTS  
See the protocol Section 12 for the definition of adverse events (AEs), serious AEs, 
related/unrelated AEs, and other definitions relating to AEs. See protocol appendix D for a 
list of potential adverse events associated or expected with hematopoietic cell 
transplantation. 
Adverse events (AEs) defined by study protocol will be collected on the CRF and coded 
using the MedDRA coding dictionary version 21.0 or later. Severity will be graded using 
the CTCAE Version 4.0.  
A treatment-emergent adverse event (TEAE) is any untoward medical occurrence that 
either occurs or worsens at any time on or after HCT and that does not necessarily have to 
have a causal relationship with the treatment or surgical procedure. Please see Section 6.3 
for handling missing or incomplete dates in determining whether an AE is a TEAE. For 
analyses purposes, only TEAEs occurring during the first 100 days after HCT will be 
presented in summary tables. 
All collected AEs and SAEs will be presented in individual listings. The listing will 
contain the following information: treatment group, verbatim term, system organ class, 
preferred term, related to the expanded cell product, relationship to transplant, date and day 
of onset, date and day of resolution, treatment given to treat the adverse event, the 
outcome, whether the event was an SAE, CTCAE grade, immediate toxicity to Unit 1/Unit 
2, immediate toxicity to ex vivo expanded product, and whether it is a TEAE. Listings will 
be sorted by subject identification number, onset date, system organ class, and preferred 
term. If the onset date is completely missing, then these events will be presented first. If 
the onset date is missing a month or a day, then these events will be presented before any 
complete dates. 
Attribution - The following are definitions for determining whether an adverse event is 
related to a treatment or procedure: 
• An AE is considered “related” to treatment or “related” to research procedures if in 
the opinion of the principal investigator, it was considered possibly, probably, or 
definitely related on the CRF. 
• An AE is considered “unrelated” if in the opinion of the principal investigator it 
was considered unlikely related or unrelated on the CRF. 
 
Missing relatedness will be summarized as treatment or procedure related events. Missing 
CTCAE grade will be summarized using a grade 3. 
At each level of summarization, the summaries will include the number of subjects by 
system organ class and preferred term, a subject is counted once at the system organ class 


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and once at each preferred term within the system organ class level. For summaries by 
system organ class, preferred term, and maximum relationship, a subject is counted once at 
the strongest relationship level for which the event occurred at the system organ class level 
and the highest relationship level for each unique preferred term within that system organ 
class level. Therefore, subjects may only contribute once to each preferred term and once 
to each system organ class level.   Summaries by CTCAE grade would be handled similar 
to the summaries by relationship to AE.    
Summaries presenting frequency of AEs by system organ class and preferred term will be 
ordered by overall descending frequency of system organ class and then, within a system 
organ class, by overall descending frequency of preferred term. 
An overall summary of all TEAEs will be presented by treatment group and will include 
the number and percentage of subjects experiencing the following: 
• Any TEAE 
• TEAE related to the expanded cell product 
• TEAE by strongest relationship to transplant 
• TEAE by greatest CTCAE grade 
• TEAE leading to study withdrawal 
• Treatment emergent SAE (TESAE) 
• TEAE resulting in death 
In addition to the overall AE table above, the following AE tables will also be provided. 
• TEAEs overall and by system organ class and preferred term 
• Expanded Cell Product-related TEAEs overall and by system organ class and 
preferred term 
• TEAEs by maximum CTCAE grade, overall and by system organ class and 
preferred term 
• TEAEs by maximum relationship to expanded cell product , overall and by system 
organ class and preferred term 
• TEAEs by maximum relationship to transplant, overall and by system organ class 
and preferred term 
• Serious TEAEs, overall and by system organ class and preferred term 
• TEAEs resulting in death, overall and by system organ class and preferred term 


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• TEAES involving bleeding as determined by the study team prior to database lock 
and unblinding of the sponsor for the primary analysis 
Listings will be provided for the following: 
• All AEs, with the verbatim term of non-TEAEs flagged 
• CTCAE Grade 3 or higher AEs (this is a subset of AEs where severity is marked as 
CTCAE grade 3, 4, or 5) 
• Expanded Cell Product-Related AEs 
• SAEs (this is a subset of the AEs where serious is marked as “Yes”)  
• 
AEs leading to death (This is a subset of the AEs where outcome is indicated as 
“Death” or the CTCAE grade is 5) 
 
LABORATORY EVALUATIONS 
Blood samples will be collected for laboratory safety tests as specified in the schedule of 
assessments (Section 3.7).  Due to the design of the study, local laboratories are used by 
the sites involved in the study; therefore different units of measure may be used to report 
results for the same laboratory parameter. Standard laboratory values required for the 
statistical analyses of laboratory measures will therefore be obtained from originally 
entered data values using proper conversion factors.  Laboratory testing is described below. 
Complete Blood Count (CBC) 
White blood cell count, hemoglobin, hematocrit, platelet count, neutrophils (absolute), 
lymphocytes (absolute), monocytes (absolute), eosinophils (absolute), basophils (absolute), 
neutrophils (%), lymphocytes (%), monocytes (%), eosinophils (%), and basophils (%).    
Blood Chemistry 
Sodium, potassium, chloride, carbon dioxide, glucose, blood urea nitrogen (BUN), 
creatinine, phosphate, albumin, magnesium, alanine aminotransferase (ALT), aspartate 
aminotransferase (AST), alkaline phosphatase, total bilirubin, direct bilirubin, total protein, 
and lactic dehydrogenase (LDH).    
Immunoglobulin 
Immunoglobulin A (IgA), Immunoglobulin G (IgG), and Immunoglobulin M (IgM). 
Lymphocyte Panel 
CD3, %CD3, CD4, % CD4, CD8, %CD8, CD4/CD8, CD19, %CD19, CD56, and %CD56. 
Panel Reactive Antibody (PRA) 
PRA will be collected prior to HCT, at engraftment, and at unscheduled visits. 


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Descriptive statistics by treatment group and time of assessment will be presented for each 
laboratory parameter. Changes from baseline will be presented. All laboratory values will 
be classified as low, normal, or high based on normal ranges supplied by each local 
laboratory. For purposes of analyses, laboratory results based upon Standard International 
system (SI) of units will be used. 
For each summary, the number of non-missing observations, mean, median, standard 
deviation, inter-quartile range, minimum, and maximum values will be presented by 
treatment group. 
 
PERFORMANCE STATUS 
ECOG, Lansky, and Karnofsky performance status scores will be collected and presented 
descriptively by type of performance status. 
 
 
 


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CHANGES FROM PROTOCOL PLANNED ANALYSES  
This SAP is based on the most recent version of the protocol.  The only substantive 
changes to planned protocol analyses include the addition of several exploratory endpoints 
to further evaluate the clinical and pharmacoeconomic impact of NLA101 treatment, 
consistent with the protocol-defined secondary and exploratory objectives of the study.  
These additional exploratory endpoints include comparisons of disease-free survival, 
GVHD-free, relapse-free survival, ICU-free days within the first 100 days post HCT, and 
related sensitivity analyses. 
The protocol specifies a secondary endpoint of severe bacterial and fungal infections.  
However, as the study halted prior to completion of blinded review and cleaning of this 
endpoint, any meaningful analysis is not possible as consistency standards and queries 
have not been addressed.  Therefore, this SAP has removed this endpoint from the analysis 
plan as the data would not be interpretable. 
In addition, subgroup analyses, sensitivity analyses, and study populations have been 
added in order to evaluate the robustness and generalizability of the results from the 
primary and key secondary endpoints. 


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REFERENCES  
1. Guidance for Industry Statistical Principles for Clinical Trials (ICH E9), September 
1998. 
2. Guidance for Industry Structure and Content of Clinical Study Reports (ICH E3), 
July 1996. 
3. Guidance for Industry Good Clinical Practice (ICH E6), April 1996. 
4. Fine JP, Gray RJ. (1999). A proportional hazards model for the subdistribution of a 
competing risk. J Am Stat Assoc; 94:496 –509. 
5. Gray, R. (1988). A Class of K-Sample Tests for Comparing the Cumulative 
Incidence of a Competing Risk. The Annals of Statistics, 16, 1141–1154 
6. SAS macro %CIF. http://support.sas.com/kb/45/997.html 
  
 


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PROGRAMMING CONSIDERATIONS 
The following are Syneos Health’s standard programming considerations with additional 
Nohla Therapeutics specifications.   
All TLFs, and statistical analyses will be generated using SAS® for Windows, Release 9.4 
(SAS® Institute Inc., Cary, NC, USA), unless specified otherwise. Computer-generated 
TLF output will adhere to the following specifications.   
 
GENERAL CONSIDERATIONS 
• 
One SAS program can create several outputs. 
• 
Each output will be stored in a separate file. 
• 
Output files will be delivered in Word format. 
• 
Numbering of TFLs will follow ICH E3 guidance  
 
 
TABLE, LISTING, AND FIGURE FORMAT 
 
General 
• 
All TLFs will be produced in landscape format, unless otherwise specified. 
• 
All TLFs will be produced using the Courier New font, size 9  
• 
The data displays for all TLFs will have a minimum 3/4-inch margin at the top and 3/8 of an 
inch on all other sides. 
• 
Headers and footers for figures will be in Courier New font, size 9. 
• 
Legends will be used for all figures with more than 1 variable, group, or item displayed. 
• 
TLFs will be in black and white (no color), unless otherwise specified 
• 
Specialized text styles, such as bolding, italics, borders, shading, and superscripted and 
subscripted text, will not be used in the TLFs, unless otherwise specified. On some occasions, 
superscripts 1, 2, or 3 may be used (see below). 
• 
Only standard keyboard characters will be used in the TLFs. Special characters, such as non-
printable control characters, printer-specific, or font-specific characters, will not be used. 
Hexadecimal-derived characters will be used, where possible, if they are appropriate to help 
display math symbols (e.g., μ). Certain subscripts and superscripts (e.g., cm2, Cmax) will be 
employed on a case-by-case basis. 
• 
Mixed case will be used for all titles, footnotes, column headers, and programmer-supplied 
formats, as appropriate. 
 
Headers 
• 
All output should have the following header at the top left of each page: 
<Sponsor Name> Protocol XXX (Syneos Health study number xxx)      
          
Draft/Final Run <date> 


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• 
All output should have Page n of N at the top or bottom right corner of each page. TLFs should 
be internally paginated in relation to the total length (i.e., the page number should appear 
sequentially as page n of N, where N is the total number of pages in the table). 
• 
The date output was generated should appear along with the program name as a footer on each 
page. 
 
 
Display Titles 
• 
Each TLF should be identified by the designation and a numeral. (i.e., Table 14.1.1). ICH E3 
numbering will be used. A decimal system (x.y and x.y.z) should be used to identify TLFs with 
related contents. The title is centered. The analysis set should be identified on the line 
immediately following the title. The title and table designation are single spaced. A solid line 
spanning the margins will separate the display titles from the Column headers. There will be 1 
blank line between the last title and the solid line. 
Table x.y.z 
First Line of Title 
Second Line of Title if Needed 
ITT Analysis Set 
 
 
Column Headers 
• 
Column headings should be displayed immediately below the solid line described above in 
initial upper-case characters.  
• 
In the case of efficacy tables, the variable (or characteristic) column will be on the far left 
followed by the treatment group columns and total column (if applicable). P-values may be 
presented under the total column or in separate p-value column (if applicable). Within-
treatment comparisons may have p-values presented in a row beneath the summary statistics 
for that treatment. 
• 
For numeric variables, include “unit” in column or row heading when appropriate. 
• 
Analysis set sizes will be presented for each treatment group in the column heading as (N=xx) 
(or in the row headings if applicable). This is distinct from the ‘n’ used for the descriptive 
statistics representing the number of subjects in the analysis set. 
• 
The order of treatments in the tables and listings will be by randomized treatment group with 
EXP first, followed by SOC, and then screen failures where applicable., followed by a total 
column (if applicable). 
 
 
Body of the Data Display 
13.2.5.1. 
General Conventions 
Data in columns of a table or listing should be formatted as follows: 
• 
alphanumeric values are left-justified; 
• 
whole numbers (e.g., counts) are right-justified; and 


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• 
numbers containing fractional portions are decimal aligned. 
 
13.2.5.2. 
Table Conventions 
• 
Units will be included where available. 
• 
If the categories of a parameter are ordered, then all categories between the maximum and 
minimum category should be presented in the table, even if n=0 for all treatment groups in a 
given category that is between the minimum and maximum level for that parameter. For 
example, the frequency distribution for symptom severity would appear as: 
Severity 
Rating 
N 
severe 
0 
moderate 
8 
mild 
3 
Where percentages are presented in these tables, zero percentages will not be presented and so 
any counts of 0 will be presented as 0 and not as 0 (0%). 
• 
If the categories are not ordered (e.g., Medical History, Reasons for Discontinuation from the 
Study, etc.), then only those categories for which there is at least 1 subject represented in 1 or 
more groups should be included. 
• 
An Unknown or Missing category should be added to any parameter for which information is 
not available for 1 or more subjects.  
• 
Unless otherwise specified, the estimated mean and median for a set of values should be 
printed out to 1 more significant digit than the original values, and standard deviations should 
be printed out to 2 more significant digits than the original values. The minimum and 
maximum should report the same significant digits as the original values. For example, for 
systolic blood pressure: 
N 
  XX 
Mean 
XXX.X 
Std Dev 
     X.XX 
Median 
XXX.X 
Minimum 
XXX 
Maximum 
XXX 
• 
P-values should be output in the format: “0.xxx”, where xxx is the value rounded to 3 decimal 
places. Any p-value less than 0.001 will be presented as <0.001. If the p-value should be less 
than 0.0001 then present as <0.0001. If the p-value is returned as >0.999 then present as >0.999 
• 
Percentage values should be printed to one decimal place, in parentheses with no spaces, one 
space after the count (e.g., 7 (12.8%), 13 (5.4%)). Values that round down to 0.0 would be 
displayed as '<0.1', or as appropriate with additional decimal places. Unless otherwise noted, 
for all percentages, the number of subjects in the analysis set for the treatment group who have 
an observation will be the denominator. Percentages after zero counts should not be displayed 
and percentages equating to 100% should be presented as 100%, without any decimal places.  
• 
Tabular display of data for medical history, prior / concomitant medications, and all tabular 
displays of adverse event data should be presented by the body system, treatment class, or 
system organ class with the highest occurrence in the active treatment group in decreasing 


Statistical Analysis Plan 
Nohla Therapeutics Protocol #2603 
Final Version 3.0 
Syneos Health #1011511 
 
This document is proprietary and confidential to Nohla Therapeutics 
 
Page 53 of 55 
order, assuming all terms are coded. Within the body system, drug class and system organ 
class, medical history (by preferred term), drugs (by ATC1 code), and adverse events (by 
preferred term) should be displayed in decreasing order. If incidence for more than 1 term is 
identical, they should then be sorted alphabetically.  
• 
Missing descriptive statistics or p-values which cannot be estimated should be reported as “-”. 
• 
The percentage of subjects is normally calculated as a proportion of the number of subjects 
assessed in the relevant treatment group (or overall) for the analysis set presented. However, 
careful consideration is required in many instances due to the complicated nature of selecting 
the denominator, usually the appropriate number of subjects exposed. Describe details of this 
in footnotes or programming notes. 
• 
For categorical summaries (number and percentage of subjects) where a subject can be 
included in more than one category, describe in a footnote or programming note if the subject 
should be included in the summary statistics for all relevant categories or just 1 category and 
the criteria for selecting the criteria. 
• 
Where a category with a subheading (such as system organ class) has to be split over more than 
one page, output the subheading followed by “(cont)” at the top of each subsequent page.  The 
overall summary statistics for the subheading should only be output on the first relevant page. 
 
13.2.5.3. 
Listing Conventions 
• 
Listings will be sorted for presentation in order of treatment groups as above, treatment group, 
subject number, visit/collection day, and visit/collection time. 
• 
Missing data should be represented on subject listings as either a hyphen (“-”) with a 
corresponding footnote (“- = unknown or not evaluated”), or as “N/A”, with the footnote “N/A 
= not applicable”, whichever is appropriate. 
• 
Dates should be printed in SAS® DATE9.format (“ddMMMyyyy”: 01JUL2000). Missing 
portions of dates should be represented on subject listings as dashes (--JUL2000). Dates that 
are missing because they are not applicable for the subject are output as “N/A”, unless 
otherwise specified. 
• 
Units will be included where available 
 
13.2.5.4. 
Figure Conventions 
• 
Unless otherwise specified, for all figures, study visits will be displayed on the X-axis and 
endpoint (e.g., treatment mean change from Baseline) values will be displayed on the Y-axis. 
 
Footnotes 
• 
A solid line spanning the margins will separate the body of the data display from the footnotes.  
• 
All footnotes will be left justified with single-line spacing immediately below the solid line 
underneath the data display. 
• 
Footnotes should always begin with “Note:” if an informational footnote, or 1, 2, 3, etc. if a 
reference footnote. Each new footnote should start on a new line, where possible. 


Statistical Analysis Plan 
Nohla Therapeutics Protocol #2603 
Final Version 3.0 
Syneos Health #1011511 
 
This document is proprietary and confidential to Nohla Therapeutics 
 
Page 54 of 55 
• 
Subject specific footnotes are to be avoided, where possible. 
• 
Footnotes will be used sparingly and must add value to the table, figure, or data listing. If more 
than six lines of footnotes are planned, then a cover page may be used to display footnotes, and 
only those essential to comprehension of the data will be repeated on each page.  
• 
The last line of the footnote section will be a standard source line that indicates the name of the 
program used to produce the data display, date the program was run, and the listing source (i.e., 
‘Program : myprogram.sas    Listing source: 16.x.y.z’). 


Statistical Analysis Plan 
Nohla Therapeutics Protocol #2603 
Final Version 3.0 
Syneos Health #1011511 
 
This document is proprietary and confidential to Nohla Therapeutics 
 
Page 55 of 55 
 
QUALITY CONTROL 
SAS programs are developed to produce output such as analysis data sets, summary tables, data 
listings, figures or statistical analyses. Syneos Health SOP 03.010 and 03.013 provide an overview 
of the development of such SAS programs. 
Syneos Health SOP 03.009 describes the quality control procedures performed for all SAS 
programs and output. Quality control is defined here as the operational techniques and activities 
undertaken to verify the SAS programs produce the output by checking for their logic, efficiency 
and commenting and by review of the produced output.