Peripheral Blood Stem Cell Allotransplantation For Hematological Malignancies Using Ex Vivo CD34 Selection - a Platform For Adoptive Cellular Therapies
Phase: PHASE1
Conditions: MDS (Myelodysplastic Syndrome), Myeloproliferative Disorder, Lymphoma, Non-Hodgkin, ALL (Acute B-Lymphoblastic Leukemia), AML (Acute Myelogenous Leukemia
Sponsor: National Heart, Lung, and Blood Institute (NHLBI)

Protocol # 13-H-0144 
Sawa Ito, MD 
Version date: April 12, 2018 
 
 
1
CLINICAL RESEARCH PROJECT 
 
 
 
 
Protocol #13-H-0144 
 
 
 
 
 
 
 
 
IDE 15632 
 
 
 
Product: Miltenyi CliniMACS® CD34Selection System 
IDE Holder: A. John Barrett, M.D. 
Date:     April 12, 2018 
 
To: 
Richard Cannon, MD, Chair, NHLBI IRB 
 
Title: Peripheral blood stem cell allotransplantation for hematological malignancies using ex vivo 
CD34 selection – a platform for adoptive cellular therapies. 
 
Other identifying words: AML, ALL, CLL, CML, MDS, MPD, NHL 
 
Principal Investigator:   
*Sawa Ito, M.D. H.B., NHLBI (E) 
 
 
 
326-5233 
Bldg 10, CRC 3-5330 
 
 
 
 
Medically Responsible Investigator:  
*Richard Childs, MD, HB, NHLBI (E)  
 
             594-8008 
Bldg 10, CRC 3-5332 
 
Associated Investigators: 
David Stroncek, M.D., DTM, Clinical Center (E) 
496-9702 
Bldg 10, 1C7111 
Pawel Muranski, MD, NHLBI (V) 
                                       240-688-3538 Columbia University, NY 
Deborah Citrin, MD, ROB, NCI (E) 
 
 
 
496-5457 
Bldg 10, CRC 3B43C  
Eleftheria Koklanaris, RN, Research Nurse, HB, NHLBI (E) 
594-4709 
Bldg 10, CRC 3-2473  
Colin Wu, PhD, Biostatistician, DECA/OBR, NHLBI (E) 
435-0440 
RKL2, Room 8218 
Xin Tian, PhD, Biostatistician, DECA/OBR/NHLBI (E)  
435-1298 
RKL2, Room 8217 
A. John Barrett, M.D., M.S.  HB, NHLBI (V)     
402-4170     
Bldg 10, CRC 3-5322   
 
Note: *Investigators designated to obtain informed consent from research subjects. 
 
Collaborators: 
Catherine Bollard, MD, Children’s National Medical Center, Washington DC 
Kim Green, PhD, NIAD 
 
 
Subjects in Study: 
 
Number 
 
Sex 
 
Age range 
 
     Subjects: 
 
 up to 192 
 
either  
Recipients: 10-80 inclusive 
 
 
 
      (recipients & donors) 
 
 
Donors:  > 2 - <80 
Project involves ionizing radiation?  
Yes (medically indicated) 
Off-site project? 
 
 
No 
Multi-institutional project? 
 
No 
DSMB Involved? 
 
 
Yes 
Tech Transfer?  
 
 
Yes 
 
 
 
 
PRECIS   
 

Protocol # 13-H-0144 
Sawa Ito, MD 
Version date: April 12, 2018 
 
 
2
Peripheral blood stem cell transplant research carried out by the NHLBI BMT Unit focus on transplant 
techniques designed to decrease graft versus host disease (GVHD), increase the graft-versus-leukemia 
(GVL) effect and reduce the risk of post-transplant graft rejection.  
 
Through incremental transplant clinical trials we have shown that by controlling the stem cell (CD34+ 
cell) and T lymphocyte (CD3+ cell) dose, severe GVHD can be reduced whilst beneficial GVL effects 
can be preserved. We found that T cell depleted transplants using the Nexell/Baxter Isolex 300i system 
and subsequently, the Miltenyi CliniMACS® CD34+ system to obtain high CD34+ doses depleted of 
lymphocytes were safe to administer and associated with less severe acute GVHD and promising response 
rates and overall survival. Our previous trials have helped us to create the transplant environment 
(significant lymphodepletion and minimal post-transplant immunosuppression) that make for an ideal 
platform for adoptive cellular immunotherapy. Adoptive cell transfer is the passive transfer of immune 
cells, into a new recipient host with the goal of transferring the immunologic functionality and 
characteristics into the new host.  
 
This protocol is designed to evaluate the safety and efficacy of the Miltenyi CliniMACS® CD 34 
selection system in HLA-matched sibling allogeneic peripheral blood stem cell transplant. The 
manipulation of the graft is the primary research intervention, subject to IDE# 15632, and all other 
aspects of clinical management on this protocol are standard care. The target CD34+ dose range will be 
>3 x 106/kg and the target CD3+ dose range will be 5 x 104/kg to 1 x 106/kg. Once we demonstrate 
adequacy of this platform for engraftment and absence of significant GVHD in ten consecutive recipients, 
we will seek IRB permission to proceed with planned adoptive cellular therapies. 
 
The protocol will accrue up to 96 transplant recipients aged 10-80 with a hematological malignancy and 
their HLA-matched sibling donors, in whom allogeneic stem cell transplantation from an HLA-matched 
sibling would be routinely indicated. Diagnostic categories will include acute and chronic leukemia, 
myelodysplastic syndromes, lymphomas, multiple myeloma and myeloproliferative syndromes.  
 
Subjects will receive a myeloablative conditioning regimen of cyclophosphamide (120 mg/kg total), 
fludarabine (125 mg/m2 total) and total body irradiation (1200 cGy with lung shielding to 600 cGy), 
followed by an infusion of a stem cell product selected for CD34+ progenitors using the Miltenyi 
CliniMACS® system.  Older subjects will receive a lower dose of irradiation (800 or 600 cGy based on 
age) to reduce the regimen intensity. 
 
The overall objective is to assess the feasibility of using this system as a platform for cellular 
immunotherapy initiatives. The primary study endpoint will be overall survival at day +200. Stopping 
criteria for safety will monitor non-relapse mortality at day +200. Secondary endpoints will be standard 
transplant outcome variables such as non-hematologic toxicity, incidence and severity of acute and 
chronic GVHD and relapse of disease.  
 
 
 

Protocol # 13-H-0144 
Sawa Ito, MD 
Version date: April 12, 2018 
 
 
3
TABLE OF CONTENTS 
1.0 
OBJECTIVES ................................................................................................................ 4 
2.0 
BACKGROUND ............................................................................................................ 4 
3.0 
DISTINCTIVE FEATURES OF THIS PROTOCOL ................................................................ 8 
4.0  
STUDY DESIGN ......................................................................................................... 10 
5.0  
ELIGIBILITY ASSESSMENT .......................................................................................... 10 
6.0 
CLINICAL EVALUATION OF THE TRANSPLANT RECIPIENT ............................................ 12 
7.0 
TREATMENT PLAN .................................................................................................... 15 
8.0 
DONOR EVALUATION, STEM CELL COLLECTION AND PROCESSING  PLAN ................... 18 
9.0 
MANAGEMENT OF COMPLICATIONS ......................................................................... 21 
10.0 
ANCILLARY LABORATORY RESEARCH STUDIES ........................................................... 23 
11.0 
BIOSTATISTICAL CONSIDERATIONS ........................................................................... 25 
12.0 
DATA AND SAFETY MONITORING ............................................................................. 31 
13.0 
HUMAN SUBJECT PROTECTIONS ............................................................................... 40 
14.0 
PHARMACEUTICALS.................................................................................................. 49 
15.0 
REFERENCES ............................................................................................................. 55 
16.0 
APPENDICES ............................................................................................................. 56 
APPENDIX A: NHLBI HEMATOLOGY BRANCH LABORATORY RESEARCH STUDIES -2/5/2013 ... 56 
APPENDIX B: GVHD scoring scales ........................................................................................ 60 
 
 
 
 
SEE ALSO NIH BMT CONSORTIUM SUPPORTIVE CARE GUIDELINES 
[http://intranet.cc.nih.gov/bmt/] NHLBI, IRB reviewed January, 2012. 

Protocol # 13-H-0144 
Sawa Ito, MD 
Version date: April 12, 2018 
 
 
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1.0 
OBJECTIVES 
 
1.1 
To determine the safety and efficacy of using the Miltenyi CliniMACS CD34 selection system for 
graft manipulation in HLA-matched sibling allogeneic stem cell transplantation. 
 
1.2  To characterize the feasibility of using the Miltenyi CliniMACS CD34selection system as a 
platform for adoptive cellular immunotherapy in HLA-matched sibling stem cell transplantation. 
 
2.0 
BACKGROUND 
 
2.1 
Allogeneic Stem Cell Transplantation 
 
Allogeneic stem cell transplantation has long been recognized as a curative treatment for a variety 
of hematologic malignant diseases (Armitage 1994). Over time this technique has improved mainly 
through significantly diminished non-relapse mortality. One notable advance has been the use of 
reduced-intensity transplants for toxicity reduction and the use of T cell depletion for graft versus 
host disease (GVHD) control. However, these transplants often fail to control the hematological 
malignancy (Georges and Storb 2003, Marmont, et al 1991, Platzbecker, et al 2004).  Relapse of 
disease is now the main problem (Barrett and Battiwalla 2011). Data from the Center for 
International Bone Marrow Transplant Research (http://www.cibmtr.org/) on over 30,000 
transplants for hematological malignancies performed worldwide over the last 30 years shows that 
relapse rates have remained unchanged (20% for early disease, 40% for intermediate disease, and 
60% for advanced disease subjects).  Disease-free survival follows the same trend (65% for early 
disease, 40% for intermediate disease, and 20% for advanced leukemia). 
 
2.2 
Complications of Allogeneic Stem Cell Transplantation 
 
Death after stem cell transplantation can be categorized as relapse and non-relapse mortality 
(NRM). NRM includes GVHD, infection and organ toxicity.  
 
2.2.1 Relapse 
 
The curative potential of allogeneic stem cell transplantation is largely dictated by the stage of the 
disease (see 2.1 above) and disease free survival for advanced hematological malignancies has not 
changed significantly in recent years. To prevent relapse, subjects receive a chemo-radiotherapy 
conditioning regimen which is as intensive as they can tolerate. Based on previously reported 
experience for the treatment of elderly subjects with stem cell allografts we will use a reduced 
intensity conditioning regimen with fludarabine (Flu), cyclophosphamide (Cy) and reduced-dose 
total body irradiation (TBI) for subjects 55 years and older of age (Khoury et al. 2001, Weisser et 
al. 2004). The use of reduced-intensity conditioning for elderly subjects is a broadly accepted 
standard of care treatment and will not be part of the scientific questions addressed in this research 
protocol. In addition, there is a powerful immune response (graft-versus-leukemia, GVL effect) 
from the donor’s T cells and natural killer cells. However because transplants also cause GVHD, it 
is necessary to give immunosuppression after the graft which reduces but does not eliminate 
GVHD, but decreases the GVL effect. 
 
2.2.2 Graft Versus Host Disease 
 
Graft-versus-host disease (GVHD) represents a major complication of allogeneic bone marrow 
transplantation (BMT) or peripheral blood stem cell transplantation (PBSCT), leading to significant 

Protocol # 13-H-0144 
Sawa Ito, MD 
Version date: April 12, 2018 
 
 
5
transplantation associated morbidity and mortality. Despite prophylaxis with cyclosporine (CSA) or 
methotrexate (MTX) or both, acute GVHD occurs in 30% to 50% of subjects receiving transplants 
from HLA-identical siblings. Long term survival in subjects developing severe acute GVHD has 
generally been less than 30% (Barrett 1993, Barrett 1997, Beatty, et al 1991, Storb, et al 1986, 
Weisdorf, et al 1990).   
 
2.2.3 Infection 
 
 
Following allogeneic stem cell transplantation, subjects are at increased risk of bacterial, fungal and 
viral infections.  Pre-emptive antiviral agents, antibiotic fever regimens, and availability of effective 
new antifungal agents have greatly improved the management of infection after transplant. 
Infectious death now mainly occurs when infection complicates GVHD in the setting of intense 
immunosuppression. 
 
2.2.4 Other complications 
 
 
Some subjects die from major organ failure due to conditioning regimen toxicity and to the 
consequence of pre-transplant treatments for their malignancy. Depending on the underlying 
transplantation approach (e.g. conditioning regimen) the transplant can fail to engraft in up to 5% of 
cases.    
 
2.3 
  Ex Vivo T Cell Depletion 
 
 
T cell depletion is a technique where lymphocytes are removed from allografts. T cell depleted 
transplantation can prevent GVHD but results in delayed immune recovery which increases the 
risk of viral infections (HSV, CMV or EBV), graft rejection as well as relapse of the malignant 
disease (Appelbaum 2001, Barrett 1993, Barrett 1997, Barrett and Malkovska 1996, Goldman, et 
al 1988, Marmont, et al 1991).  
 
Prior Hematology Branch protocols utilized the Nexell/Baxter Isolex 300i system to obtain high 
CD34+ doses depleted of lymphocytes to a fixed CD3+ T cell dose. The use of the cell separator 
and the monoclonal antibodies was covered by IDE 8139 and the protocol series 99-H-0046, 02-
H-0211, 03-H-0192, 03-H-0209 and 04-H-0112.  Between 1997 and 2004, 138 subjects were 
treated on these protocols (Montero, et al 2006).They received a myeloablative T cell depleted 
peripheral blood stem cell transplant (PBSCT) from an HLA-identical sibling donor. The T-cell 
dose was adjusted to 0.2-1x105 CD3+ cells/kg.  The CD34+ dose was 2.7–16 x106/kg.  Subjects 
with less than grade II acute GVHD received 1 or 2 donor lymphocyte infusions (DLI) of 107 
CD3+ cells/kg between days 45 and 100. Subjects were designated as standard (n=77) or high 
(n=61) relapse risk according to their pre-transplant characteristics. Overall survival (OS), relapse 
free survival, relapse and non-relapse mortality (NRM) were 58%, 46%, 40% and 20%, 
respectively, after a median follow up of 4 years. Fifty-three (39%) and 21(15%) subjects 
developed grade II-IV and III-IV acute GVHD.  Forty-two (36%) had limited and 29 (25%) 
extensive chronic GVHD. In multivariate analysis, disease risk was an independent factor for OS 
and relapse, the day 30 lymphocyte count (LC30) for OS and NRM, and chronic GVHD for OS 
and relapse. In summary, these results show promising overall survival with low non-relapse 
mortality. 
 
In 2006, we utilized the Miltenyi CliniMACS immunomagnetic selection system to select for 
CD34+ cells (IND 13058)achieving ~4-log CD3+ lymphocyte depletion on protocol # 06-H-
0248(Battiwalla, et al 2011). At last analysis, the protocol continues to exceed its primary 
endpoint goal of day 200 overall survival >75%. Fifty-five subjects with hematologic 

Protocol # 13-H-0144 
Sawa Ito, MD 
Version date: April 12, 2018 
 
 
6
malignancies underwent allogeneic hematopoietic stem cell transplantation (HSCT) with a graft 
from their HLA-identical siblings. The median age was 43 years (range 13-68), 25/55 were 
males. Transplant indications were AML(27), ALL(14), acute biphenotypic leukemia(2), 
MDS/MPD(6), NHL/CLL(4), CMMoL(1) and CML(1). 47% were standard risk and 53% were at 
high risk for relapse. Subjects received myeloablative conditioning with cyclophosphamide (60 
mg/kg/dose x 2), fludarabine (25 mg/m2/dose x 5) and total body irradiation (12 Gy in 8 fractions, 
lungs shielded to 6Gy). Fifteen subjects, who were 55 years of age or older, received 4 Gy 
divided in 8 fractions without lung shielding. G-CSF mobilized peripheral blood grafts from the 
donor were CD34+ selected (Miltenyi CliniMACS), with infusion of a target CD34+ dose of 
6x106/kg and a fixed CD3+ dose of 5x104/kg. Low-dose cyclosporine (100-200ng/mL) till day 21 
was the only GVHD prophylaxis. Delayed lymphocyte add back (5x106 CD3+/kg) was given at 
day 90 if subjects had no significant GVHD. CD3+ and myeloid chimerism analyses were 
performed with early lymphocyte add-back in cases with falling chimerism. Day 200 overall 
survival (the primary study endpoint) was 86.7%. One subject, who was postpartum, failed to 
engraft and required a second transplant. Thirty seven out of 39 subjects achieved complete 
(>95%) donor myeloid chimerism by day14. The median times to complete donor CD3+ 
chimerism were day30 for 12 Gy subjects and >6 months for 4 Gy subjects (Mann Whitney test, 
p=0.004). The incidence of acute GVHD grade II, III and IV were 29%, 18% and 0%, 
respectively for all subjects and significantly lower for those who did not receive the routine DLI. 
The incidence of chronic limited or extensive GVHD was 34 and 11%, respectively. At a median 
follow up of 2.5 years, Kaplan-Meier estimates of relapse, NRM and OS were 31%, 37% and 
50% respectively without any significant difference based on conditioning intensity.  
 
Figure 1: Overall survival, relapse and NRM observed in subjects treated on protocol 06-H-0248 
 
Outside the NIH, equally encouraging results with ex vivo T cell depletion using CD34+ depletion 
have just been reported from the multicenter Clinical Trials Network (CTN) protocol #0303 
(Devine, et al 2011). A total of 44 subjects with AML in CR1 (n = 37) or CR2 (n = 7) with a 
median age of 48.5 years (range, 21-59 years) received myeloablative chemotherapy and 
fractionated total body irradiation (1375 cGy) followed by immunomagnetically selected CD34-
enriched, T cell depleted allografts from HLA-identical siblings. No pharmacologic GVHD 
prophylaxis was given. All subjects engrafted. The incidence of acute GVHD grade II-IV was 
22.7%, and the incidence of extensive chronic GVHD was 6.8% at 24 months. The relapse rate for 
subjects in CR1 was 17.4% at 36 months. With a median follow-up of 34 months, DFS for all 
subjects was 82% at 6 months, and DFS for subjects in CR1 was 72.8% at 12 months and 58% at 
36 months. 
 
In 2012, protocol# 06-H-0248 was replaced by a more complicated ex-vivo graft manipulation 
protocol 12-H-0028, which also utilized the Miltenyi CliniMACS platform and combined CD34+ 
progenitor selection with depletion of CD3+ (T-lymphocytes) responsible for acute GVHD as well 

Protocol # 13-H-0144 
Sawa Ito, MD 
Version date: April 12, 2018 
 
 
7
as depletion of CD19+ (B-lymphocytes) responsible for chronic GVHD. This method was designed 
to preserve NK cells and CD34+ cells, while achieving a comparable degree (~ 3.5 log) of CD3+ 
depletion. Preservation of NK cells in protocol 12-H-0028 was intended to provide the theoretical 
advantage of reducing viral infections and relapse without impacting GVHD. We found that target 
cell doses were attained, however there was an unexpected increase in early acute GVHD when a T 
cell addback of 50x104CD3+/kg was given (Table 1). In contrast, in subsequent subjects with a T 
cell addback as low as 5 x 104 CD3+/kg, (equating with the CD3+ dose in the 06-H-0248 protocol), 
subjects experienced low donor chimerism including outright engraftment failures despite all 
subjects having received myeloablative conditioning. 
 
5
58
MDS
YES
600
06/19/12
50
8.76
No
No
None
NA
6
53
AML
NO
1200
07/03/12
50
6.5
No
No
1
18
7
55
MDS
YES
600
08/14/12
5
4.54
Yes
Yes
None
NA
8
28
AML
NO
1200
08/28/12
5
7.1
No
No
3
17
9
33
CML
YES
1200
09/16/12
5
6.75
Yes
Yes
None
NA
10
38
CML
YES
1200
10/16/12
5
6.6
Yes
No
1
38
11
40
AML
NO
1200
10/23/12
5
6.15
No
No
None
NA
 
The reason for these outcomes on 12-H-0028 is unclear, but cannot be simply explained on the 
basis of CD3+ dose alone. More problematic is that the dynamic range of CD3+ dosing is 
unacceptably narrow (1-log), and not controlled by adjusting the CD3+ addback dose. In summary, 
while overall survival on 12/16/2012 was 100% for the cohort of 13 subjects transplanted, the 
unpredictability of outcome, characterized on the one hand by early significant acute GVHD and on 
the other hand by poor engraftment make 12-H-0028 unacceptable for continued development as a 
platform for adoptive cellular immunotherapy.  
 
The current protocol will revert to positive CD34+ selection as used in the 06-H-0248 protocol with 
minor further improvements based on experience with this protocol and 12-H-0028.  
 
2.4 
 SCIENTIFIC AND CLINICAL JUSTIFICATION 
 
The focus of the NHLBI Hematology Branch allogeneic PBSCT program is to improve the 
treatment of hematologic and non-hematologic malignant diseases using transplant approaches that 
minimize GVHD, non-relapse mortality and increase the GVL effect.  
 
Ex Vivo T Cell Depletion: 
Our prior T cell depletion protocols achieved promising results for overall survival and significantly 
reduced non-relapse mortality (Battiwalla, et al 2011, Montero, et al 2006).The extramural 
published experience with CD34+ selection also shows reductions in GVHD without increase in 
relapse(Devine, et al 2011). The Isolex system employed for stem cell selection up to 2005was 
withdrawn from the market. The CliniMACSCD34 selection system preservesCD34+ cells (yield is 
reliably >50% but typically 75%), but induces a 4- log reduction in T lymphocytes (responsible for 
acute GVHD) along with a similar degree of reduction in B lymphocytes (contributes to chronic 
GVHD). We expect the final CD3+ counts at the end of the selection process to range between 
5x104/kg to 1x106/kg, providing sufficient protection from GVHD on the one hand, and yet 
providing adequate T cells for safe engraftment on the other hand. The delayed T cell add-back at 
day 90 in protocol 06-H-0248 has not been particularly beneficial in the last protocol except for 
correcting poor donor CD3 chimerism, and will be no longer be given routinely. Low dose CSA 
will continue as the only immunosuppression at least until day +21, or until engraftment is stable. 
However, CSA cessation will provide an immunosuppression-free environment for the T cells to 
expand sufficiently and develop strong GVL effects as required for relapse-control. We will select 

Protocol # 13-H-0144 
Sawa Ito, MD 
Version date: April 12, 2018 
 
 
8
subjects with hematological malignancies for whom allogeneic stem cell transplantation from a 
fully matched donor is considered a standard, uncontroversial treatment. We regularly review the 
place of stem cell allotransplantation in the treatment of hematological malignancies, particularly 
chronic myelogenous leukemia (CML), where tyrosine kinase inhibitors such as imatinib and newer 
agents have made first line transplantation obsolete. Indications for transplantation are regularly 
reviewed by the DSMB.  
 
 
3.0 
DISTINCTIVE FEATURES OF THIS PROTOCOL 
 
Distinctive features of this protocol are shown in context of serial protocol development in Table 2. 
 
Table 2: SEQUENTIAL PROTOCOL CHANGE SUMMARY TABLE 
 
 
Generation of T cell depleted stem cell product: We will continue to utilize the Miltenyi 
CliniMACS system but will revert to using CD34+ selection alone to deplete the donor stem cell 
graft of lymphocytes. The Miltenyi CliniMACS is a clinical-scale immunomagnetic system widely 
used in Europe and is available in the USA under an IND/IDE. This system uses CD34antibody-
coated magnetic bead reagent manufactured for clinical use. The National Institutes of Health 
(NIH) Department of Transfusion Medicine (DTM) Cell Processing Section has extensive 
experience in generating CD34+ enriched stem cell products. 
 
Intensity of the TBI conditioning for older subjects (≥55 years): In this protocol, we will continue to 
include older subjects. Based on previously reported experience for the treatment of the elderly we 
will use reduced intensity conditioning with Flu/Cy and reduced dose TBI for these subjects 
(Khoury et al. 2001, Weisser et al. 2004). However, in the 06-H-0248 protocol, 400 cGy TBI 
resulted in sluggish conversion to full donor CD3+ chimerism compared to 1200cGy (median 180 
days versus 30 days, p <0.001). This has often required premature DLIs to rescue failing grafts. 
 
Figure 2: Protocol 06-H-0248 – Slow donor CD3+ engraftment for older recipients conditioned with 
400cGy TBI 
Features
06-H-0248
12-H-0028
This Protocol (prior 
to 12/2015)
This Protocol (after 12-
2015)
Graft manipulation
CD34+ selection
CD 3/19 depletion and 
CD34+ selection
CD34+ selection
CD34+ selection
Dose range for CD3+ 
addback at day 0
1 to 5 x 10e4/kg
5 to 100 x 10e4/kg
5 to 100 x 10e4/kg
 0 to 100 x 10e4/kg
Conditioning TBI
<55 years
1200 cGy
1200 cGy
1200 cGy
1200 cGy
≥55 years
400 cGy
600 cGy
600 cGy
≥55 to <65 yrs =1200 cGy; 
≥65 to <75 yrs =800 cGy; 
≥75 to 80 yrs =600 cGy
Routine DLI 
at day 90
none
none
none
Platform for 
adoptive cellular 
therapy
No
Proposed but failed
Proposed
Multivirus T lymphocytes

Protocol # 13-H-0144 
Sawa Ito, MD 
Version date: April 12, 2018 
 
 
9
 
 
We will therefore increase the intensity of TBI to 600cGy for the reduced-dose cohort in this 
protocol. This increase to 600 cGy was well tolerated in subjects above 55 years in the 12-H-0028 
protocol and will continue. 
 
The TBI dose will be further increased for some of the older subjects by protocol amendment since 
12/2015 in a graded fashion. [Age ≥55 to <65 yrs =1200 cGy(with lung shielding to 600 cGy); ≥65 
to <75 yrs = 800 cGy (with lung shielding to 600 cGy); ≥75 to 80 yrs =600 cGy] The rationale is to 
reduce the risk of relapse (which is usually lethal), currently at 58% in subjects over age 55. TBI at 
600cGy has been extremely well tolerated in these subjects over age 55 and the TRM rate is 
actually 0% at the time of this amendment, which makes room for increasing TBI dose. The 
justification is based upon the relatively lower toxicity of our TBI approach, measures taken to 
reduce TBI toxicity, existing practice at other centers and evolving evidence to maximize 
conditioning intensity when possible. 
 
Avoidance of Routine Donor Lymphocyte Infusion (DLI):  Before PBSCT, the donor will undergo 
leukapheresis in order to collect lymphocytes for possible future transfusion to the recipient. The 
leukapheresis collection will be divided and stored as 2 or more products in DTM. In our previous 
protocols, we had gradually postponed a DLI till day 90. The extramural experience with T 
lymphocyte depletion has avoided delayed DLI entirely (Devine, et al 2011).  Additionally, both 
acute and chronic GVHD were increased in frequency and grade in 06-H-0248 transplant recipients 
who received DLIs. The requirement for DLI has been dropped since protocol 12-H-0028. 
Similarly, in this protocol, donor lymphocytes will be collected and stored in advance but only 
administered for standard clinical indications: for relapse, falling or delayed chimerism.  
 
Platform for adoptive immunotherapy strategies: Our goal for advancing allogeneic HSCT 
strategies is to develop a T cell depletion platform for adoptive cellular immunotherapy to minimize 
GVHD and optimize antiviral and antimalignancy immune responses. In sequential clinical trials at 
NIH we have used ex-vivo T cell depletion to develop an HSCT with minimal graft versus host 
disease (GVHD) prophylaxis comprising low dose cyclosporine alone till day +21. Tcell depletion 
creates the lymphodepleted environment for preferential homeostatic expansion of adoptively 
infused cells, while minimization of post-transplant immunosuppression promotes expansion of the 
adoptively transfused cells. The adoptive transfer concept will be explored in this protocol, once 10 
consecutive subjects have enrolled and demonstrated satisfactory outcomes (full engraftment in at 
least 9 subjects and acute GVHD of - grade II-IV<40%), Section 11.5. This criterion has been met 

Protocol # 13-H-0144 
Sawa Ito, MD 
Version date: April 12, 2018 
 
 
10
as of May, 2014 and formal IRB approval will permit utilization of this protocol as a transplant 
platform for additional immunotherapy protocols: adoptive cellular immunotherapy against 
malignancy and viral infections, cancer vaccines, donor IL-2 derived cellular products, bone 
marrow stromal cell delivery and donor vaccinated cellular products. 
 
4.0  
STUDY DESIGN 
 
 
This is a phase I-II clinical trial designed to evaluate the safety and efficacy of the CliniMACS 
CD34 selection system as a T cell depletion technique and, as a primary endpoint, to determine 
overall survival at day +200 after transplantation. Recipients will receive a myeloablative 
conditioning regimen of cyclophosphamide, fludarabine and total body irradiation, followed by an 
infusion of a stem cell product prepared using the Miltenyi CliniMACS system. 
 
Figure 3: Schema 
 
5.0  
ELIGIBILITY ASSESSMENT 
 
 
Subjects in remission as well as subjects with primary induction failure or refractory disease will be 
considered for inclusion. At the discretion of the Principal Investigator (PI), subjects may continue 
standard of care treatment options to control their baseline disease burden up to the start of the 
protocol as detailed in section 7.1. 
 
 
5.1 
Inclusion Criteria-Recipient 
 
5.1.1 
Ages 10-80 years inclusive  

Protocol # 13-H-0144 
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Version date: April 12, 2018 
 
 
11
 
5.1.2 
Any one of the following hematologic conditions, confirmed by pathology, meeting a 
standard indication for allogeneic stem cell transplant: 
 
5.1.2.1 Chronic myelogenous leukemia (CML): Subjects under the age of 21 in chronic 
phase OR Subjects ages 10-80 in chronic phase who have failed or are intolerant to 
treatment with second generation tyrosine kinase inhibitors OR Subjects ages 10-80 
in accelerated phase or blast transformation.  OR 
5.1.2.2 Acute lymphoblastic leukemia (ALL): any of these categories: Adult ALL including 
standard risk; Pediatric ALL in first remission with high-risk features (presenting 
leukocyte count >100,000/cu mm, karyotypes t (9; 22), t4, t19, t11, biphenotypic 
leukemia). All second or subsequent remissions, primary induction failure, partially 
responding or untreated relapse. OR 
5.1.2.3 Acute myelogenous leukemia (AML): AML in first remission - except AML with 
good risk karyotypes: AML M3 (t15; 17), AML M4Eo (inv 16), c-kit unmutated 
AML t (8; 21). All AML in second or subsequent remission, primary induction 
failure and resistant relapse. OR 
5.1.2.4 Myelodysplastic syndromes(MDS): any of these categories - refractory anemia with 
transfusion dependence, refractory anemia with ANC<500/PL, refractory anemia 
with excess of blasts, transformation to acute leukemia, chronic myelomonocytic 
leukemia, atypical MDS/myeloproliferative syndromes.OR 
5.1.2.5 Myeloproliferative disorders including atypical (Ph-negative) chronic myeloid and 
neutrophilic leukemias, progressing myelofibrosis, and polycythemia vera, essential 
thrombocythemia either in transformation to acute leukemia or with progressive 
transfusion requirements or pancytopenia. OR 
5.1.2.6 Chronic lymphocytic leukemia refractory to fludarabine treatment and with bulky 
progressive disease or with thrombocytopenia (<100,000 / Pl) or anemia (<10g/dl) 
not due to recent chemotherapy. OR 
5.1.2.7 Non-Hodgkin’s lymphoma including Mantle cell lymphoma relapsing or refractory to 
standard of care treatments. OR 
5.1.2.8 Multiple myeloma, Waldenstroms macroglobulinemia, unresponsive or relapsed 
following standard of care treatments. OR 
5.1.2.9 Hodgkin's Lymphoma relapsing following an autologous transplant. OR 
5.1.2.10 Other rare hematologic malignancies for which hematopoietic stem cell 
transplantation has been performed and offers a durable remission or as the only 
option for potential for cure.  
• 
Chemotherapy-resistant multisystem Langerhans cell histiocytosis (MSLCH) 
especially involving organs like the bone marrow, liver, spleen, and lungs 
• 
Aggressive systemic mastocytosis, and mast cell leukemia (MCL) in first CR 
(CR1) 
• 
Hypereosinophilic syndrome who have failed imatinib therapy or FIP1L1-
PDGFRa-negative patients who develop end-organ dysfunction 
• 
Adult T-cell leukemia/lymphoma at first diagnosis 
• 
Refractory or disseminated nasal-type extranodal NK/T-lymphoma or aggressive 
Natural killer cell leukemia/lymphoma 
• 
Mycosis fungoides and Sézary syndrome after failure of two or three initial 
therapies 
• 
Primary or relapsed refractory Angioimmunoblastic T-cell lymphoma at first 
diagnosis 
• 
Hepatosplenic T-cell lymphoma (gamma/delta T-cell lymphoma) at first 
diagnosis 

Protocol # 13-H-0144 
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Version date: April 12, 2018 
 
 
12
• 
T-cell prolymphocytic leukemia at first diagnosis 
• 
Subcutaneous panniculitic T-cell lymphoma at first diagnosis 
• 
Hematodermic neoplasm (blastic natural killer cell lymphoma or 
Blasticplasmacytoid dendritic cell neoplasm) at first diagnosis 
5.1.3 
HLA identical (6/6) related donor. 
 
5.1.4 
For adults: ability to comprehend the investigational nature of the study and provide 
informed consent.  For minors: written informed consent from one parent or guardian.  
Informed oral assent from minors: the process will be explained to the minor on a level of 
complexity appropriate for their age and ability to comprehend. 
 
5.2 
Exclusion Criteria – Recipient (any of the following) 
 
5.2.1 
Major anticipated illness or organ failure incompatible with survival from transplant 
5.2.2 
Severe psychiatric illness or mental deficiency sufficiently severe as to make compliance 
with the transplant treatment unlikely and making informed consent impossible. 
5.2.3 
Positive pregnancy test for women of childbearing age 
5.2.4 
DLCO adjusted for Hb and ventilation< 50% predicted 
5.2.5 
Left ventricular ejection fraction < 40% (evaluated by ECHO) or < 30% (evaluated by MUGA) 
5.2.6 
AST/SGOT > 10 x ULN  
5.2.7 
Total bilirubin  > 5 x ULN  
5.2.8 
Estimated GFR < 15 mL/min 
5.2.9 
Recipients who have active infections with HIV or active hepatitis C (HCV)  
 
5.3 
Inclusion criteria- Donor  
 
5.3.1 
Related donor, HLA identical (6/6) with recipient 
5.3.2 
Weight > 18 kg  
5.3.3 
Age > 2 or <80 years old 
5.3.4 
For adults: ability to comprehend the investigational nature of the study and provide 
informed consent.  For minors: written informed consent from one parent or guardian and 
informed assent: The process will be explained to the minor on a level of complexity 
appropriate for their age and ability to comprehend. 
 
5.4 
Exclusion Criteria - Donor  (any of the following) 
 
5.4.1 
Pregnant or breast-feeding.  Lactating donors are permitted provided breast milk is discarded 
during the days filgrastim (G-CSF) is given 
5.4.2 
Unfit to receive G-CSF and undergo apheresis (abnormal blood counts, history of stroke, 
uncontrolled hypertension) 
5.4.3 
Sickling hemoglobinopathy including HbSS, HbSC 
5.4.4 
Donors who are positive for HIV, active hepatitis B (HBV), hepatitis C (HCV) or human T-
cell lymphotropic virus (HTLV-I/II)  
5.4.5 
Severe psychiatric illness or mental deficiency sufficiently severe as to make compliance 
with the donation process unlikely, and making informed consent impossible. 
 
 
6.0 
CLINICAL EVALUATION OF THE TRANSPLANT RECIPIENT 
 

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13
Samples will be ordered and tracked through the CRIS order sets.  Should a CRIS screen not be 
available, the NIH form 2803-1 will be completed and will accompany the specimen and be filed 
in the medical record. 
 
6.1 
Pre-Study Evaluation (within 3 months of transplant) 
 
 
 
Transplant recipients may also be screened on the Hematology Branch screening protocol (97-H-
0041 or its replacement) or in the patient’s NIH medical record. 
x 
History and physical exam, waist circumference 
x 
HLA-A, B &DR-B1 typing, KIR typing (no time frame) 
x 
Bone marrow aspirate and biopsy. Chromosome analysis, if clinically indicated. Minimal 
residual disease markers and any other disease-appropriate staging (such as a CT-PET 
scan) 
x 
BCR-ABL  (CML, and Ph+ ALL only) or other disease-specific molecular test 
x 
Serum HbsAg, anti-HBs, anti-HBc, anti-HCV, HAV, anti-HIV, anti-HTLV-1, anti CMV, 
anti-EBV, anti-Toxoplasma, RPR, PPD 
x 
PCR: blood for CMV/EBV, Toxoplasma, HHV6, BK virus, adenovirus and urine for 
adeno/BK virus  
x 
Red cell ABO, Rh, antibody screen,  
x 
HLA alloimmunization screen (where clinically indicated)  
x 
Coagulation Screen, Fibrinogen 
x 
CBC with differential 
x 
Reticulocyte count 
x 
Acute care (Na, K, Cl, CO2, Creatinine, Glucose, and Urea Nitrogen) panel 
x 
Hepatic (Alk Phosphatase, ALT, AST, Total Bilirubin, and Direct Bilirubin) panel 
x 
Mineral (Phosphorus, Magnesium, Albumin, and Calcium) panel 
x 
Lipid panel 
x 
LDH 
x 
CK 
x 
Uric acid 
x 
Ferritin, Iron, Transferrin 
x 
Urinalysis (UA) 
x 
Quantitative immunoglobulins 
x 
Lymphocyte Immunophenotyping (CD4, CD8, B, NK) 
x 
Endothelial damage markers (vWF panel/FactorVIII activity, thrombomodulin, P-
Selectin, Plasminogen Activation Inhibitor-Type 1, Pro BNP, haptoglobin, endothelin 1)   
x 
Vitamin D levels (1,25 Dihydroxy and 25 Hydroxy) 
x 
Pre albumin 
x 
C-reactive protein 
x 
Pregnancy test (serum or urine) 
x 
STR profile (chimerism) 
x 
Thyroid function, adrenal function, gonadal function, cortisol  
x 
Growth hormone, growth chart, bone age (prepubertal children) 
x 
Chest radiograph, pulmonary function tests including Vital capacity, FEV-1, DLCO 
x 
Sinus CT scan and other body CT scans as medically required 
x 
Cardiac function: EKG, ECHO, and/or MUGA scan 
x 
Nutritional assessment, as needed 
x 
Dental evaluation, as needed 
x 
Social work consult 
x 
Ophthalmology consultation as needed 
x 
Gynecology consultation as needed 

Protocol # 13-H-0144 
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14
x 
The possibilities and limitations of semen storage for males and in vitro                  
fertilization and storage of zygotes for females will be discussed with subjects as 
appropriate 
x 
Durable power of attorney form completed 
 
x 
Semen analysis (optional) 
x 
Bone Mineral Density (optional) 
x 
Health Related Quality of Life (PROMIS 29-Profile v1.0, PROMIS v1.0-Applied 
Cognition-General Concerns-Short Form, FACT-BMT, Sexual Functioning 
Questionnaire (SFQ) [Screening questions 1-3 and subscales: Medical Impact, 
Satisfaction and Problems, gender specific versions]). HRQOL is optional for the subject. 
x 
All subjects age 50 or greater  
 
Cardiac Stress test (stress nuclear perfusion imaging or treadmill stress test) 
 
Cardiac consultation  
 
6.2 In-Patient Monitoring (Day -8 to discharge - approximately day 30)  
 
As the subjects' condition stabilizes and they approach hospital discharge this monitoring 
frequency may change as clinically indicated. These are only guidelines, the nature of clinical 
care and exact schedule of laboratory and questionnaire testing for these complex transplant 
subjects will depend on the clinical status and PI discretion.  
 
x 
CBC with differential (daily) 
x 
Acute care panel (daily) 
x 
Hepatic panel (daily) 
x 
Mineral panel (daily) 
x 
Protein, Total (twice weekly) 
x 
LDH (daily) 
x 
CK (twice weekly) 
x 
Uric Acid (twice weekly) 
x 
Temperature, pulse, blood pressure, respiratory rate, weight, (daily) 
x 
Reticulocytes (twice weekly)  
x 
Prealbumin (twice weekly) 
x 
CMV/EBV, Toxoplasma, HHV6, BK, adenoviral surveillance in blood and adeno/BK in 
urine (weekly when counts allow)  
x 
C-reactive protein (weekly)  
x 
Urine for viral cytopathic change (weekly) 
x 
Lymphocyte Immunophenotyping (CD4, CD8, B, NK) (weekly) 
x 
Coagulation screen (if clinically indicated) 
x 
BCR-ABL (day 30 and as clinically indicated, CML and Ph+ ALL patient only) 
x 
Stool for C. difficile (when clinically indicated) 
x 
Bone marrow aspirate or peripheral blood to quantitate engraftment in subjects without 
evidence of hematologic recovery (as needed) 
x 
Drug levels where appropriate (e.g., gentamicin, vancomycin, cyclosporine, 
voriconazole) (frequency variable) 
 
6.3 
Follow Up: After Initial Inpatient Hospitalization to Referral Back to Home Physician 
(typically 3-4 months) 
 
Weekly (+/- 3 days): these are only guidelines, the nature of clinical care and exact schedule of 
laboratory testing for these complex transplant subjects will depend on the clinical status and PI 
discretion.  

Protocol # 13-H-0144 
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Version date: April 12, 2018 
 
 
15
x 
CBC with differential 
x 
Acute care panel 
x 
Hepatic panel  
x 
Mineral panel  
x 
Protein, Total 
x 
LDH 
x 
CK 
x 
Uric Acid 
x 
Reticulocyte count 
x 
Temperature, pulse, blood pressure, respiratory rate, weight,  
x 
C-reactive protein 
x 
Coagulation screen (if clinically indicated) 
x 
Serum CMV/EBV, BK, adenoviral surveillance & Urine BK, adenoviral surveillance. 
x 
Urine for viral cytopathic change (weekly) 
x 
Lymphocyte Immunophenotyping (CD4, CD8, B, NK) (weekly) 
• 
Vitamin D levels (1,25 Dihydroxy and 25 Hydroxy) (monthly) 
x 
Drug levels as appropriate (i.e. cyclosporine) 
 
Frequency as indicated: these are only guidelines, the nature of clinical care and exact schedule of 
laboratory testing for these complex transplant subjects will depend on the clinical status and PI 
discretion.  
x 
BCR-ABL (day 60, 90 and +120 or as clinically indicated +/- 7 days, CML and Ph+ ALL 
subjects only) 
x 
Chimerism profile (on days 14, 21, 28, 42, 60, 90 and as clinically indicated  +/- 7 days) 
 
6.4 
Follow up after returning home 
 
Subjects and/or their referring physician will remain in contact with the NIH and encouraged to 
send the results of laboratory testing at least every 3 months (+/- 2 weeks) for 200 days. At 3 
years, subjects will be offered enrolment in the long-term follow-up protocol 05-H-0130 and 
communication with the patient and the referring physician is continued. 
 
7.0 
TREATMENT PLAN 
 
 
7.1 
Pre Transplant Treatment of Hematological Malignancy  
 
Following enrollment on the Branch Standard of Care protocol (94-H-0010 or its replacement), 
subjects may receive any clinically indicated pre-transplant antineoplastic therapy according to 
standard indications to control their primary disease and increase the chance of successful 
transplantation. The PI will decide on the details of the treatment necessary in the individual case. 
 
7.2 
Apheresis of Transplant Recipient for Lymphocytes 
 
            With Amendment Q, Sept 2017, apheresis will not be performed on transplant recipients. 
One 1 x 1010 cell apheresis of the recipient will be collected between day 80-180 post-transplant 
to study post-transplant immune function. 
 
Research apheresis may be substituted by a 100 ml blood sample if apheresis is not feasible in 
terms of timing, recipient availability, scheduling constraints or the clinical condition of the 
recipient (blood counts and performance) at the discretion of the PI. 
 

Protocol # 13-H-0144 
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Version date: April 12, 2018 
 
 
16
7.3 
Central Venous Line Placement (See NIH BMT Consortium Supportive Care Guidelines) 
 
7.4 
Infection Prophylaxis and Treatment (See NIH BMT Consortium Supportive Care Guidelines) 
Voriconazole or posaconazole will be the preferred antifungal prophylaxis, to begin on day +1. 
 
7.5 
Fever Regimen (See NIH BMT Consortium Supportive Care Guidelines) 
 
7.6 
Preparative Regimen  
 
x 
Fludarabine (Flu): 25 mg/m2/dose IV infusion once daily over approximately 30 minutes 
(e.g. 20 to 60 minutes)  on Days -8, -7, -6, -5, and -4 (total dose of 125 mg/m2). Dose 
adjustment for reduced creatinine clearance is required per the product label. 
 
x 
Total body irradiation (TBI): 
 
Energy 
All recipients should be treated with a linear accelerator using energies higher than 4MV. 
 
Timing 
It is anticipated that TBI will be delivered on days -7, -6, -5, -4  
 
Technique 
TBI will be delivered with lateral fields using extended SAD values of 600cm.  For subjects 
receiving 1200 cGy, partial transmission lung blocks will be used combined with AP/PA 
Mediastinal Boost fields using full blocks in the lung area.  Tissue compensators will be used 
as appropriate for all recipients.  Gonadal shielding will not be used. 
 
Dose/Fractionation 
Recipients less than 65 years of age will be treated with TBI to a total dose of 1200 cGy 
delivered in 150 cGy fractions twice daily, at least 6 hours apart (eight total treatments in four 
days).  The mediastinal boost fields will be delivered at a dose of 75cGy per fraction for a 
total dose of 600cGy, also at least six hours apart. The median lung dose goal is 600 cGy 
from the total treatment. 
 
Recipients 65 years to <75 years will receive TBI to a total dose of 800 cGy delivered in 100 
cGy fractions bid, at least 6 hours apart (eight total treatments in four days).  The mediastinal 
boost fields will be delivered at a dose of 50 cGy per fraction for a total dose of 400cGy.  The 
median lung dose goal is 600 cGy from the total treatment. 
 
Recipients 75 years and older will receive TBI to a total dose of 600 cGy delivered in 75 cGy 
fractions bid, at least 6 hours apart (eight total treatments in four days).  No mediastinal boost 
field will be used. 
 
Dose Modifications 
Occasionally, the total dose/technique of TBI may require modifications due to patient factors 
(unexpected or serious (grade 4-5) adverse events, serious medical illnesses not conducive to 
stable patient transfer, patient refusal, etc.) or treatment factors (linear accelerator machine 
offline, etc.) Modifications to the radiation treatment will be at the discretion of the treating 
radiation oncologist and will be discussed with the PI.   
 

Protocol # 13-H-0144 
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Version date: April 12, 2018 
 
 
17
x 
Cyclophosphamide (Cy) 60 mg/kg/dose IV infusion administered over approximately 1 to 2 
hours  once daily for 2 doses on Days -3 and -2 (total dose of 120 mg/kg).  Infusion rate may be 
extended if necessary for infusion associated toxicities. Mesna and intravenous hydration will be 
given concurrently with the cyclophosphamide doses as based on current NIH BMT Consortium 
Supportive Care Guidelines. 
 
7.7 
Stem Cell Transplantation Day 0  
 
The target and acceptable range of cell doses (given in terms of recipient body weight) are: 
 
CD34+ cells 
 
 
Target dose:  
6 x 106/kg recipient weight 
 
 
Acceptable range: maximum- None; minimum- 3 x 106/ kg  
 
 
CD3+ cells 
(T cell add back)   
Target dose: Based upon our experience with previous protocols 06-H-0248 and 
12-H-0028, for relatively chemo-naïve subjects (e.g. MDS, CML), and for those who 
receive 600cGy TBI, the target dose will be 5 x 105CD3+/kg. All other subjects will 
receive 5 x 104 CD3+/kg. Target doses may be further individualized based upon 
engraftment kinetics as the protocol progresses. As of amendment dated July 1, 2016, 
subjects receiving adoptive cell therapy such as multiviral T lymphocytes between days 0 
to 7 post graft will not require the CD3+ addback. 
 
Acceptable range: maximum 1 x 10e6 CD3+/kg; minimum 0 CD3+/ kg  
 
The source of CD3+ cells will be an aliquot of the G-CSF mobilized apheresis product. 
 
If the final CD3+ dose is greater than 1 x 106/kg this is designated a technical failure of the 
selection process. Recipients will be transplanted with this final product (and additional 
methotrexate GVHD prophylaxis- Section 8.5) but their outcome will be analyzed separately. 
 
The ex vivo processing and required product safety testing is detailed in section 8.5. 
 
7.8 
GVHD prophylaxis 
Cyclosporine (CSA) will be initiated at a dose of 1.5 mg/kg IV every twelve hours day –6 to day 
+21 to reduce the risk of graft failure. (Change to equivalent oral dose when able to tolerate with 
dose adjusted to achieve levels 100-200 ng/ml).  The initial cyclosporine dose (1.5 mg/kg IV 
every 12 hours) will be based on the recipient’s actual body weight unless the recipient is obese 
(body mass index > 35); in which case the cyclosporine dose will be based on an adjusted / 
practical body weight (mid-point between ideal body weight and actual body weight; see NIH 
BMT Consortium Supportive Care Guidelines). Cyclosporine may continue beyond day 21 if 
there is risk of incomplete donor CD3+ engraftment. Cyclosporine may be substituted at the 
discretion of the PI based upon tolerance or potential toxicity. 
 
Subjects who have a technical failure of graft processing and receive a CD3+ dose >1 x 106/kg, 
will receive additional methotrexate GVHD prophylaxis at a dose of 5mg/m2 which will be given 
by IV infusion over 15 minutes post-transplant on days 1,3 and 6. Methotrexate dose adjustments 
for renal or hepatic dysfunction will be based on NIH BMT Consortium Supportive Care 
Guidelines. 
 
 

Protocol # 13-H-0144 
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Version date: April 12, 2018 
 
 
18
7.9  
Transfusion Support:  Filtered and irradiated blood products as needed. HLA-alloimmunized 
recipients will be identified by history and confirmed by lab testing. Premedications will be given 
as clinically required. 
 
 
7.10  
Nutrition:  Parenteral nutrition will be instituted as clinically necessary. 
 
7.11 
Hospital Discharge:  The recipient will be in the hospital for about 3-6 weeks and will be 
discharged when clinically indicated to follow up as an outpatient.  
 
7.12 
Donor lymphocyte Infusion (DLI) 
Donor lymphocyte infusions will not be routinely administered at a fixed time point in this 
protocol. At the discretion of attending physician up to 5x 108 CD3+ cells/kg DLI can be given at 
any time post-transplant for standard clinical indications: in overwhelming viral infections, falling 
donor chimerism, delayed chimerism or in subjects who appear to be at risk of disease relapse, or 
are in frank relapse. The ex vivo processing and required product safety testing is detailed in 
section 8. 
 
7.13 
Antiviral Cellular Therapy 
With amendment version 6-5-14, subjects may qualify to participate in a concurrent protocol for 
testing the safety and efficacy of transfer of multiviral specific T lymphocytes in reducing 
common post-transplant viral infections.   
 
7.14 
Misc. Supportive Care  
Palifermin may be selectively used for subjects at high risk of mucositis (such as those receiving 
a cranial irradiation boost) and ursodiol will be routinely used as prophylaxis against hepatic 
complications. Other aspects of supportive care will follow NIH BMT Consortium Supportive 
Care Guidelines. 
 
8.0 
DONOR EVALUATION, STEM CELL COLLECTION AND PROCESSING 
 
PLAN 
 
 
8.1 
Pre-Study Consult and Evaluation 
 
Donors may also be screened on the Hematology Branch screening protocol (97-H-0041 or its 
replacement). 
x 
HLA, -A -B –C -DR typing, KIR typing 
x 
Confirm HLA compatibility with patient (6/6 HLA match). 
x 
Rule out sickling hemoglobinopathies including HbSS and HbSC by history and peripheral blood 
smear. Donors with HbAS are acceptable 
x 
History and physical examination 
x 
Chest X-ray in donors with underlying pulmonary disease or history of smoking 
x 
HbsAg, anti-HBc, anti-HCV, anti-HIV, anti-HTLV, anti-CMV, RPR, anti- T. cruzi, West Nile 
virus NAT (nucleic acid test) and human transmissible bovine spongiform encephalopathy (BSE 
is done by questionnaire of associated risk factors) 
x 
Red blood cell ABO group, Rh type, antibody screen 
x 
CBC with differential, coagulation screen, Acute Care, Mineral, Hepatic, and Other (Total 
Protein, CK, Uric Acid, and LD panel), pregnancy test (females of child bearing potential) 
x 
Profile STR (chimerism) 

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19
x 
Fit to donate: Orientation - visit to Department of Transfusion Medicine - inspection of veins to 
determine the need for a central line for apheresis. It is estimated that about 50% of the donors 
will require apheresis catheter placement to successfully complete apheresis.  
x 
Counseling on filgrastim (G-CSF) mobilization and donation of leukocytes 
 
8.2  
Pre-consent Evaluation and Concurrent Care of Minor Donors (donors less than age 18 
only) 
For donors less than age 18, a social worker and/or mental health specialist (psychologist or 
psychiatrist) will meet with the minor prior to the assent process to confirm willingness to 
participate. For donors less than age 18, a separate pediatric provider who is charged solely to 
consider the health and welfare of the minor donor will be provided. This practitioner will serve 
as the donor’s health care provider and advocate during the minor’s participation on the clinical 
trial. 
 
8.3 
Donor Lymphocyte Collection – Pre-transplant 
 
One 10-15 liter apheresis will be collected prior to or 2 or more days after filgrastim (G-CSF) 
mobilization and stem cell collection, to obtain 1 x 1010 lymphocytes for cryopreservation of DLI 
(at least one aliquot of 5 x 106/kg, additional aliquots for clinical use, and an aliquot for in vitro 
laboratory research studies [section 10.0]). If a central catheter is necessary, please refer to NIH 
BMT Consortium Supportive Care Guidelines. 
 
8.4 
Donor Mobilization with G-CSF 
After medical evaluation and clearance for suitability as an allogeneic donor by the BMT service 
in consultation with DTM, the donor will undergo mobilization with filgrastim (G-CSF) as an 
outpatient. Filgrastim (G-CSF) will be administered based on body weight (see below) for up to 6 
days, subcutaneously.  Filgrastim (G-CSF) will be administered according to a vial-based 
algorithm to reduce wastage, improve donor compliance, and optimize CD34 yields. The doses 
for days 1-4 may be given at any time of day, but the doses for days 5 and 6 must be given very 
early in the morning, at least one hour prior to starting apheresis.  Predictable side effects of 
filgrastim (G-CSF), including headache, bone pain, and myalgia, will be treated with 
acetaminophen or ibuprofen or oxycodone. Prophylactic treatment of these side effects with the 
same medications may be elected.  Other side effects will be evaluated and treated accordingly. 
 
Donor Wt           Total daily filgrastim (G-CSF) Dose (range) 
19-30 kg 
 300 mcg (10.0 to 15.8 mcg/kg) 
31-37 kg 
 480 mcg (10.0 to 13.0 mcg/kg) 
38 - 48 kg 
600 mcg (12.5 to 15.8 mcg/kg) 
49 - 56 kg 
780 mcg (13.9 to 15.9 mcg/kg) 
57 - 60 kg 
900 mcg (15.0 to 15.8 mcg/kg) 
61 - 67 kg 
 960 mcg (14.3 to 15.7 mcg/kg) 
68 - 108 kg         1080 mcg (10.0 to 15.9 mcg/kg) 
> 109 kg             1200 mcg (11.0 or less) 
 
8.5 
Peripheral Blood Stem Cell Collection &Ex Vivo Processing   
 
PBSC apheresis will be done on days 5 and 6 of filgrastim (G-CSF), i.e., after the 5th and 6th 
doses of filgrastim (G-CSF) (which are given in the early morning).  All apheresis procedures 
will be done using a 2-armed approach or by temporary central venous catheter using the Cobe 
Spectra or other blood separator in use in DTM. If a central catheter is necessary, please refer to 
NIH BMT Consortium Supportive Care Guidelines. 

Protocol # 13-H-0144 
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Version date: April 12, 2018 
 
 
20
 
Anticoagulation will be accomplished with ACD-A.  If the donor is small or intolerant to ACD-
A, and the adverse citrate effects cannot be controlled by usual means (slowing flow rate, oral or 
IV calcium), consideration will be given to using heparin anticoagulation.   
 
Donors will receive divalent cation prophylaxis to prevent citrate toxicity during apheresis, in 
accordance with standard DTM policies. The volume processed per apheresis procedure will be 
determined by DTM medical staff on the day of apheresis, based on peak CD34 cell mobilization 
response to GCSF and the CD34 cell dose needed, based on kilogram weight of recipient.  This 
will range from 15 to 30 liters processed per day for up to 2 days, not to exceed a total of 60 liters 
over 2 days.  In donors weighing less than 40 kg, a maximum of 8 blood volumes will be 
processed per day, for up to 2-3 days. 
 
The second day apheresis collection will not be required if the CD34+ dose is 4 x106/kg or more 
after the first day of collection, processing and selection.  
 
The aim is to provide a CD34+ selected graft with a minimum of 3x 106/kg and a maximum of 10 
x 106/kg CD34+ cells within the constraint of a maximum CD3+ cell dose of 1 x 106kg.  
 
If the minimum CD34+ cell dose is not achieved after the first apheresis and if it is predicted, 
based on the circulating CD34+ cell count prior to the first apheresis and the CD34+ yield in the 
apheresis component, that the minimum cell dose will not be achievable with a second 
consecutive apheresis, then plerixafor may be added to a sixth dose of filgrastim prior to the 
second procedure. The dose of plerixafor is 0.24 mg/kg SC given at 10 PM the evening prior to 
the second apheresis.  The sixth dose of filgrastim should be given in the early AM, at least one 
hour prior to the start of the second apheresis procedure.  
 
If the minimum CD34+ cell dose has not been achieved after one cycle of mobilization, a 2nd 
mobilization with up to two full apheresis collections to achieve the minimum post-selection 
CD34+ cell dose may be performed after an interval of at least 14 days.  
 
In the event insufficient number of CD34+ cells are mobilized to allow for efficient selection by 
the Miltenyi system during the first round of apheresis, subsequent courses of mobilization will 
only be processed through the Miltenyi system if the minimum target of total CD34+ cell dose (3 
x 106/kg) is predicted to be obtained following Miltenyi selection.  If the post Miltenyi processing 
total CD34+ cell dose is predicted to be less than the minimum total target CD34+ cell dose, the 
CD34 positively selected cells from the first apheresis procedure will be combined with the 
CD34+ unselected cells collected following subsequent mobilization procedures so that the 
minimum target stem cell dose is provided.  
 
Because the unselected CD34+ fraction (containing the required additional stem cell number) will 
retain all lymphocytes, the graft will be considered a T-replete stem cell graft with associated 
increased risk of GVHD.    This technical failure of the mobilization process will be reported to 
the IRB and the FDA. The recipient will be followed for safety reason per protocol.  The recipient 
will be replaced and the data analyzed separately.   
 
Methotrexate in the Stem Cell Recipient: In accordance with standard practice of T-replete 
stem cell transplantations, in the event of a technical failure with a decision to proceed with a T-
replete graft off study, methotrexate 5mg/m2 will be given post-transplant on days 1,3 and 6 in 
addition to the cyclosporine already described in the protocol (Ringden et al., 1993).  
 

Protocol # 13-H-0144 
Sawa Ito, MD 
Version date: April 12, 2018 
 
 
21
Stem cell products will be cryopreserved after each collection and stored until transfusion into the 
recipient (see NIH Clinical Center, DTM, Cell Processing Section’s SOPs).  From experience 
with over 100 stem cell processing procedures, we anticipate that in most cases both the CD34+ 
cell target and the CD3+ T cell target will be achieved.  In the majority of donors, peripheral 
blood stem cell processing with the Miltenyi system will result in doses of CD3+ cells well below 
5 x 104/kg. In such cases the T cell dose will be adjusted to ensure that at least 5 x 104/kg are 
transfused with the allograft. The source of CD3+ T cells for the addback will be an aliquot from 
G-CSF mobilized lymphocytes. 
 
The erythrocyte content of the apheresis product is approximately 1.0 ml of packed RBCs per 
liter processed.  Therefore the typical RBC content of a 25-liter PBPC product would be about 25 
ml. CD34 positive selection procedures results in elimination of RBCs and thawing will also lyse 
residual donor RBCs. Donor plasma will also be eliminated by the processing procedures. 
Therefore, minor ABO or other RBC incompatibility between donor and recipient, will require no 
specific processing or precautions during processing of the PBSC product.  PBSC products will 
be cryopreserved after processing. 
 
All products will be prepared for infusion by standard operating procedures of the DTM Cell 
Processing Section. Details for cell processing for this protocol are in the Protocol Specific 
Instruction (PSI) SOP document in Cell Processing. Detailed information is also provided by the 
Drug Master File BB MF 11054 “Facility, Operational and Quality Systems for Manufacture of 
HCT/Ps” of the DTM Cell Processing section (FDA registration #1174694).  This includes the 
section’s quality program, facilities, environmental control and monitoring, operational control 
systems and aseptic processing, equipments, supplies and eligibility, manufacturing systems, 
process development, process validation, process control and change control, product evaluation 
and lot release, storage, product labeling, label controls and tracking, product receipt and 
distribution, final product preparation, issue and administration, sterility testing, management of 
positive sterility tests, environmental impact and good laboratory practice statements.  
 
8.6 
Donor Monitoring and Follow Up Plan: 
 
Donors will be contacted by our clinical staff within 48 hours after their cell collection. Adverse 
events will be documented and appropriate therapy will be provided either through the home 
physician or at the NIH. Donors will go off study one week after completion of their last 
apheresis collection or after resolution of any serious adverse events related to the collection, 
whichever is later. If additional cellular products are required at a later stage from the same 
donor, they will donate under the standard care protocol 94-H-0010. 
 
9.0 
MANAGEMENT OF COMPLICATIONS 
 
The major complications are reactivation of cytomegalovirus (CMV), acute and chronic GVHD, 
leukemic relapse, and graft failure. Subjects with these complications will be treated as follows: 
9.1 
Viral Reactivation: Will be managed according with standard antivirals according to NIH BMT 
Consortium Supportive Care Guidelines. 
 
Overwhelming Viral Infection: Additional unscheduled add-back of lymphocytes (a bulk DLI, 
please see section 7.12), may be given at any time if an indication arises such as viral infections, 
EBV lymphoproliferative disorder. Add-back may be given in the presence of GVHD, if the 
investigator considers the risk from overwhelming viral infection to outweigh the risk of 
exacerbating GVHD.  
 

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9.2 
Acute GVHD (See NIH BMT Consortium Supportive Care Guidelines). GVHD markers have 
been recently validated in the clinical setting and will be utilized to guide therapy when available. 
Subjects developing steroid resistant or refractory GVHD may also be offered treatment with 
bone marrow stromal cells (MSCs) or other clinical trials. 
 
9.3 
Chronic GVHD (See also NIH BMT Consortium Supportive Care Guidelines) 
x 
Cyclosporine at standard dose. 
x 
Prednisone dosed according to severity. 
x 
Change to alternate day steroid and cyclosporine therapy when response is established. 
x 
Non-responding subjects may be treated with other standard of care therapies including 
but not limited to PUVA, extracorporeal photopheresis, low-dose IL2, mycophenolate, 
azathioprine, photopheresis, daclizumab, infliximab, rituximab, MSCs (on clinical trial), 
tyrosine kinase inhibitors or thalidomide at the discretion of the attending physician.   
 
9.4 
Graft Failure:  Failure to achieve a neutrophil count of 200/μl by day 21 post-transplant and the 
aplastic appearance of a marrow sample taken 21-28 days after transplant will be deemed primary 
graft failure (also known as failure to engraft).  
 
 
Delayed (secondary) graft failure after initial achievement of ANC recovery to >500/PL for three 
consecutive occasions is a recognized risk from T cell depleted transplantation. In such cases, loss 
of donor CD3+ chimerism precedes loss of donor myeloid chimerism. Donor CD3+ chimerism 
will be carefully monitored and falling donor CD3+ chimerism will be treated with escalating 
dose DLI +/- prior conditioning. 
Stem cell rescue/boost from a CD34+ selected graft from the original donor may be necessary in 
cases of graft failure. This is permissible under the protocol after suitable preconditioning, 
typically fludarabine +cyclophosphamide + either ATG or Campath. However, stem cell boost 
for primary graft failure will declare the subjects as having reached an end of study criterion, and 
the subjects will at that point be taken off study and be offered a standard care protocol or an 
experimental protocol if available. Note: Stem cell boosts for secondary graft failure or other 
cytopenias will not represent an end of study event. 
 
9.5 
Relapse of Hematologic Malignancy: Markers for minimal residual disease will be 
incorporated, where available, into the management of recipients. Standard approaches to 
minimal residual disease monitoring include flow cytometry, PCR and array-based 
techniques, which are unique to each type of malignancy.   
 
As the mortality for recipients relapsing before day +200 is very high, only salvage therapy is 
available. Any relapse will be considered a protocol end of study criterion. Subjects relapsing will 
be declared off-study and offered a standard care protocol or an experimental protocol for 
relapsed malignancy if available. 
x 
Subjects with progressive disease after transplant requiring intervention or who are deemed to 
have relapsed will be managed as detailed in section 11.6.2.  
 
x 
Subjects with detectable disease may receive pre-transplant chemotherapy as clinically indicated 
to decrease disease burden and increase the chance of successful transplantation as detailed 
section 7.1).  The PI will decide on continuing the pre transplant therapy or initiating similar post-
transplant antineoplastic therapy (such as rituximab for CLL, imatinib for CML, sorafenib for 
Flt3-ITD AML or other standard care treatments) on an individual basis (especially in the setting 
of detectable disease at or after transplant). 
 

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9.6 
Pulmonary Engraftment Syndrome:  Subjects who develop pulmonary engraftment syndrome 
(most likely 10-14 days post-transplant) will be treated with steroids.  
 
9.7 
Delayed or Falling Donor Chimerism: This will be detected by serial chimerism testing. DLI, 
with or without pretreatment by additional immunosuppressive drugs, may be given in the setting 
of poor chimerism, falling chimerism or delayed chimerism which places the subject at increased 
risk of relapse. Subjects showing poor engraftment unresponsive to standard treatments may be 
offered treatment with bone marrow stromal cell infusions on another applicable NHLBI 
protocol. 
 
9.8 
Organ failure: Subjects developing organ failure (pulmonary, GI, GU or hepatic) unresponsive 
to standard treatments may be offered treatment with bone marrow stromal cell infusions on 
clinical trial. 
 
9.9 
Reactions to the graft and ABO incompatibility: Instances of infusional reactions and ABO 
incompatibility are expected to be rare given the manipulation of the stem cell graft. For 
management please refer to NIH BMT Consortium Supportive Care Guidelines. 
 
9.10 
Endothelial Damage Syndromes: These comprise a spectrum of overlapping disorders such as 
transplant microangiopathy, posterior reversible encephalopathy syndrome, diffuse alveolar 
hemorrhage, idiopathic pneumonitis, sinusoidal occlusive syndrome/veno-occlusive disease and 
delayed cardiovascular disease. Management includes removal of triggers (calcineurin 
inhibitor/sirolimus) and defibrotide according to NIH BMT Consortium Supportive Care 
Guidelines.  
 
9.11 
Cytokine release syndrome/cytokine storm: This typically occurs early (engraftment 
syndrome) but may occur beyond 1 year. A triggering event such as viral infection or PTLD may 
be identifiable. The diagnosis is typically one of exclusion and the differential diagnosis includes 
infections and GVHD. Capillary leak (with anasarca, hypoalbuminemia, pre-renal azotemia, 
effusions, lung infiltrate) is a distinctive manifestation and may respond to IL6 blockade. Other 
findings are non-specific- cytopenias, coagulopathy, high fevers, transaminitis, delirium, 
cardiopulmonary, rash and organ failure. Characterization could involve CRP, ferritin, 
triglyceride, soluble CD25 receptor, IL1/TNF-a/IFN-g/IL6 levels, endothelial damage markers, 
coagulation parameters, imaging, marrow/skin biopsy. The severity of inflammation and clinical 
features may mimic that of secondary Hemophagocytic Lymphohistiocytosis(HLH) / 
Macrophage Activation Syndrome (MAS) and there may even be features of vasculitis on tissue 
biopsy.  Management is supportive with anti-inflammatory therapy and treatment of an 
underlying trigger. 
 
10.0 
ANCILLARY LABORATORY RESEARCH STUDIES 
 
10.1 
Research Sample Collection 
 
With Amendment Q, Sept 2017, research sample collection will cease. 
 
During the course of participating on this study, specimens will be collected for correlative 
studies as follows:  
 
10.1.1   Blood Sample from the recipient   
 
Regular samples for research studies of engraftment, cytokines, lymphocyte subsets, malignancy 
antigen expression, minimal residual disease, graft-versus-leukemia effects will be collected as 

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24
below. Sampling may be cancelled at the discretion of the PI based upon the availability of the 
patient and the clinical status. The overall composition of the types and number of tubes used to 
collect research blood may be subject to alteration from time to time as long as there is no 
increase in the volume of blood or the timing of collection. All research studies will conform to 
those listed in the Appendix A, list of IRB approved Hematology Branch Research Studies. Note: 
per NIH guidelines, the amount of blood that may be drawn from adult patients and volunteers 
for research purposes shall not exceed 10.5 mL/kg or 550 mL, whichever is smaller, over any 
eight week period.  For pediatric patients, no more than 5 mL/kg may be drawn for research 
purposes in a single day, and no more than 9.5 mL/kg may be drawn over any eight-week period. 
 
x 
Preconditioning: 100 ml peripheral blood draw any time before starting conditioning 
(this blood draw replaces an apheresis session). 
x 
*Weekly peripheral blood draw (interval +/- 4days): Not to exceed 40 ml starting 
after day+ 14 through day+120or weight-based maximum. [* not collected on weeks 
of 120 mL collection] 
x 
Days 30, 60, 90,120 blood draw (+/- 7 days):  Not to exceed 120 ml or weight-based 
maximum 
x 
6 month interval visits (+/- 3months): Not to exceed120 ml or weight-based 
maximum 
 
10.1.2 Apheresis: One apheresis collection of approximately 1010 leukocytes in a volume of 200 
ml, between 80-180 days post-transplant (when feasible per the PI).   
 
10.1.3 Blood sample from the donor (Optional). 30 ml peripheral blood day 14, 21, 30 and 60 
(when available). Donor leukocytes obtained at leukapheresis additional to those 
required for T cell add-back will be used for research studies investigating graft-versus-
leukemia effects and testing new T cell depletion techniques.  The recipient's leukemia 
cells and normal lymphocytes will serve as targets for all subsequent testing of donor-
anti host responses.  
 
10.1.4 Patient Bone Marrow Samples: An extra volume (up to 25 ml) of bone marrow aspirate 
will be collected for research at the same time as the pre transplant evaluation and when 
clinically indicated for monitoring minimal residual disease. The cells will be used to 
investigate lymphocyte interactions with bone marrow progenitor cell proliferation. No 
marrow aspirates solely for research purposes are planned. 
 
10.2 
Collection, Storage and Disposition of Samples 
Intended use: These specimens will not be read by a pathologist or used for diagnostic purposes.   
These studies will not be used in assessing the primary endpoint but are undertaken for 
descriptive or exploratory ancillary research, and have been approved by the NHLBI IRB and are 
listed in the Appendix A of the protocol.  Research data is the confidential property of the 
generating party, hence all such information shall not be disclosed outside of NIH for any purpose 
other than as described in section 12.6.  
Tracking:  Samples will be ordered and tracked through the CRIS Research Screens.  Should a 
CRIS screen not be available, the NIH form 2803-1 will be completed and will accompany the 
specimen and be filed in the medical record.  Samples will be processed and stored in the 
laboratory of Dr. John Barrett and will not be sent outside NIH without IRB notification and an 
executed MTA.  

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25
Storage:  Research samples will be stored with identifiers in the secure laboratory of the Dr. John 
Barrett using the NHLBI’s BSI tracking system. 
End of study procedures:  Samples from consenting subjects will be stored until they are no 
longer of scientific value or if a subject withdraws consent for their continued use, at which time 
they will be destroyed.  
Loss or destruction of samples:  Should we become aware that a major breech in our plan for 
tracking and storage of samples has occurred, the IRB will be notified. 
 
11.0 
BIOSTATISTICAL CONSIDERATIONS 
 
11.1 
Primary Endpoint and Primary Hypothesis 
 
The primary endpoint of this protocol is to determine the rate of overall survival at 200 day using 
the Miltenyi CliniMACS CD34selection system. Thus, the primary endpoint is all cause mortality 
at day 200, and the proportion of subjects who have survived at day 200 will be used to determine 
the sample size. We have consistently used the day 200 overall survival endpoint for serial T cell 
depletion trials in the Hematology Branch, enabling us to identify potential problems early. The 
primary hypothesis is that the 200-day survival rate for subjects treated with allogeneic transplant 
using the Miltenyi CliniMACS CD34selection system will exceed 75%. 
 
11.2      Secondary Endpoints 
 
Secondary endpoints will include standard transplant outcome variables such as relapse, non-
relapse mortality, disease free survival, engraftment kinetics of donor neutrophils, platelets and 
lymphocytes, incidence and severity of acute GVHD and chronic GVHD (limited and extensive) 
and GVHD-free survival.  Technical failure rates and the use of DLIs will be tracked as will 
concurrent participation on adoptive antiviral/antimalignancy cellular therapy protocols. 
Outcomes of transplants in subjects with advanced age will be tracked. 
 
Subjects who co-enroll onto any antiviral adoptive cellular therapy protocol (under IND# 16061), 
will continue to have their primary and secondary endpoints measured on 13-H-0144 and will not 
be “removed”. Also, since this is an objective of the 13-H-0144 protocol, we will compare 
primary and secondary endpoints in patients receiving the antiviral adoptive cellular therapy 
versus those who do not for various reasons such as: a. belonging to the first cohort of ~11 
subjects who did not receive antiviral cellular therapy  b. refusal to receive antiviral 
lymphocytes  c. death prior to administration of antiviral lymphocytes or  d. technical failure of 
manufacture and/ or release of the antiviral lymphocyte product. We also have the option of 
including in this comparison subjects on our prior CD34+ selected transplant trial (concluded 
protocol 06-H-0248, under this same IND), none of whom received antiviral lymphocytes. 
 
11.3 
Sample Size 
 
Existing result in the literature suggests that the 200-day survival rate for subjects treated with T-
cell depleted PBSCT should be at least 75%, and our treatment would be determined to be 
ineffective if the 200-day survival rate under this protocol is not higher than 75%. Our experience 
in protocol 06-H-0248 also suggests that the 200-day survival rate may be as high as 86%. Thus, 
we consider here the Simon’s “Optimum Two-Stage” design for testing the null hypothesis H0: 
“the 200-day survival rate is at or below 75%” versus the alternative H1:“the 200-day survival 

Protocol # 13-H-0144 
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26
rate is 86% or higher” with 0.05 significance level and 0.80 power. This design has the advantage 
of allowing us to stop the study early at the first stage if the true 200-day survival rate is likely to 
be 75% or lower.  Based on this design, we will accrue 31 patients in the first stage, and the trial 
will be continued to the second stage if 25 or more patients are to survive to day 200. Otherwise, 
the trial will be stopped and the treatment will be determined ineffective, i.e., the null hypothesis 
H0 will be accepted. If the trial goes on to the second stage, a total of 96 patients will be accrued, 
and the treatment will be determined ineffective, i.e. the null hypothesis H0 will be accepted, if 
the total number of patients to survive to day 200 is less than or equal to 78. If the treatment is 
actually not effective, i.e., the null hypothesis is true; there is a 0.046 probability of concluding it 
is, i.e., accepting the alternative hypothesis. On the other hand, if the treatment is actually 
effective, we have a 20% probability of concluding that it is not. The computation is carried out 
using the R Package “clinfun” version 1.0.5.  
 
11.4     Methods of Statistical Analysis  
 
Although the primary endpoint for this protocol occurs at 200 days, recipients will be followed up 
for 36 months on protocol and then indefinitely on the long term follow up protocol (05-H-0130) 
so that the incidence of chronic GVHD, long-term disease-free and overall survival can be 
estimated.  At the completion of follow-up for all subjects, time-to-event distributions (disease-
free survival, non-relapse mortality and overall survival) will be estimated using the Kaplan-
Meier method and 95% confidence intervals for the median time to events will be calculated (T. 
Therneau and P. Grambsch, “Modeling Survival Data, Extending the Cox Model”, Ch. 2, 2000, 
Springer: New York). When deemed appropriate, regression models in survival analysis, such as 
the Cox Proportional Hazard model, competing risk models and frailty models, will be used to 
evaluate the effects of covariates, such as age, treatment group, disease, baseline history of prior 
transplant, use of DLI in response to infection on time-to-event distributions (T. Therneau and P. 
Grambsch, “Modeling Survival Data, Extending the Cox Model”, Ch. 3&8, 2000, Springer: New 
York). Logistic regression models (or other generalized linear models when deemed appropriate) 
will be used to evaluate the effects of covariates on the event rates of graft failure, chronic 
GVHD, leukemia relapse, NRM and other secondary endpoints.  
 
Subjects will not be replaced however their outcome will be analyzed separately if they receive a 
final product that is designated a technical failure of the selection process (i.e. if the final CD3+ 
dose is greater than 5 x 105/kg CD3+ after reducing the CD34+ dose to 3 x 106/kg) (see section 
7.7) 
 
11.5     Dose Limiting Toxicity, Interim Analyses and Stopping Rules 
 
Dose limiting toxicity is defined in terms of non-relapse mortality (NRM), a useful composite and 
early endpoint for overall safety. For safety NRM at day +200 will be monitored. To account for 
relapse post-transplant we will also analyze overall survival at day +200. Overall survival reflects 
death from relapse and NRM. Subtracting NRM from overall survival provides an estimate of 
relapse since most subjects die after relapse of disease.  Although, not all subjects destined to 
relapse will have done so by day +200, most relapses do occur by then. The early estimate of 
relapse on day +200 will particularly detect a tendency towards precocious relapse caused by the 
protocol.   
 
Interim analysis for optimization of engraftment and GVHD prior to any adoptive cellular 
immunotherapy 
 

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27
The intended use of this trial is as a stable, low-GVHD platform for adoptive immunotherapy. 
Engraftment and GVHD rates will be analyzed on an ongoing basis. Since lymphocyte depletion 
increases the risk of slow donor CD3+ engraftment, adjustments in the dose of T cells added back 
on day 0 may be necessary. Once the T cell adjustment is finalized, the last block of 10 
consecutive subjects will be analyzed. Future adoptive cellular transfer protocols will not start 
until the cohort of 10 consecutive recipients meet the following criteria:  
x 
Primary graft failure (ANC<200/μL at day 21) in no more than 1 subject AND 
x 
Complete (>95%) donor CD3+ lymphocyte chimerism achieved at no later than a 
median of 180 days post-transplant AND 
x 
Acute GVHD (grade II-IV) <40%AND 
x 
Formal IRB approval prior to adoptive cellular therapies. 
 
As of May, 2014, this set of criteria has been successfully satisfied in the first 10 subjects  
 
Parameter
Study results in 
first 10 patients
Criterion for success
Primary graft failure rate
(ANC<200/μL at day 21) 
 ZERO
no more than 1 subject
Complete (>95%) donor CD3+ 
lymphocyte chimerism (median)
37 days
no later than 180 days
Acute GVHD (grade II-IV) 
30%
<40%
 
 
With amendment version 6-5-14, this protocol will now permit enrolled subjects to participate in 
adoptive cellular therapy protocols.” 
 
Interim Analysis for Futility 
 
One interim analysis is planned based on overall survival after the first 31 subjects have been 
transplanted, section 11.3. In order to proceed to the second stage, 25 out of these 31 subjects 
need to survive beyond day 200. 
 
Stopping Rules for Safety (Early Stopping Rule- Non-relapse mortality at day 200 & Late 
Stopping Rule for Disease free survival at 2 years) 
 
To prevent the possibility of having an unacceptable rate in early non-relapse mortality (NRM) 
for the study, NRM at day 200 will be monitored as a safety endpoint. Early stopping of the study 
will be considered if the numbers of NRM cross some established stopping boundaries. We adopt 
the Bayesian approach of Geller et al. (2004) (Nancy L. Geller, Dean Follmann, Eric S. Leifer, 
and Shelly L. Carter, “Design of Early Trials in Stem Cell Transplantation: A Hybrid Frequentist-
Bayesian Approach” in Advances in Clinical Trial Biostatistics (Editor: Nancy L. Geller), 2004. 
New York: Marcel Dekker, Inc.) that formally incorporates our “prior” expectations about these 
rates. Based on experience with similar protocols, we assume that the 200-day NRM rate should 
not be higher than 20%.  A stopping boundary is reached if the true rate exceeds the above 
anticipated rate with probability .90 or greater. We take our prior distribution to be the beta 
distribution with (alpha, beta) = (2, 8), so that alpha+beta= 10 is approximately 10% of the total 

Protocol # 13-H-0144 
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28
sample size. In order to prevent premature termination of the study, we will start safety 
monitoring when there are 4 deaths.  The stopping boundaries are given in the table 3 below.  
 
Table 3: Early stopping rule for safety based on NRM at day 200 
 
Number of 
transplant 
recipients 
“Stop” if # of NRM by day-200 
reaches or exceeds 
≤7 
4 
≤11 
5 
≤15 
6 
≤19 
7 
≤23 
8 
≤27 
9 
≤31 
10 
≤36 
11 
≤40 
12 
≤44 
13 
≤48 
14 
≤53 
15 
≤56 
16 
≤61 
17 
≤66 
18 
≤70 
19 
≤74 
20 
≤79 
21 
≤83 
22 
≤87 
23 
≤92 
24 
≤96 
25 
 
We investigated the performance of the above stopping rule by a simulation study. In each 
simulation run, we generated a study with 96 independent Bernoulli trials, each had a probability 
p for having NRM and q=1-p for not having NRM, and compared the NRM outcomes with the 
above stopping boundary to determine whether the study was stopped. We repeated the 
simulation 100,000 times and computed the proportion of stopped studies (i.e. “number of 
stopped studies”/100,000) which were stopped using the above stopping rule. The following 
Table 4 summarizes the proportions of stopped studies under a number of p values: 
 
Table 4: Proportion of stopped studies for different p values 
Prob of NRM = p 
5% 
10% 
15% 
20% 
25% 
 
30% 
 
35% 
Proportion of Stopped 
Studies 
0.03% 
0.7% 
5.7% 
26.2% 
64.2% 
 
90.8% 
 
98.9% 
Average # of Subjects 
Needed 
95.98 
95.42 
91.83 
79.71 
57.66 
 
36.54 
 
23.30 
Average # of NRM 
4.79 
9.53 
13.78 
15.94 
14.43 
10.96 
8.15 
 
These results suggest that the above stopping rule has a low probability of stopping a study when 
the proportion of NRM is below the benchmark value of 20%, while the probability of stopping a 
study is high when the true proportion of NRM exceeds this benchmark value. Based on these 
results, we believe that this Bayesian stopping rule has satisfactory statistical properties.  

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29
 
Our past protocol 06-H-0248 suggests that the probability of serious late complications (defined 
as relapse or death, or 1-Disease-free survival) at 2 years is 49.5%.  We quantify this experience 
by establishing a stopping boundary table for “2-year Relapse-or-Mortality” (RM) using a 
Bayesian approach based on the beta prior distribution with parameters (a, b) = (2.94, 3.06).  We 
will immediately report to the DSMB and seriously consider stopping the study if the posterior 
probability that the underlying rate of “2-year Relapse-or-Mortality” exceeding 49% is at least 
80%.  The parameter are chosen so that the mean a / (a + b) = 49% as the expected proportion of 
SAEs and the relative weight we place on our prior opinion is about 10% of the weight we will 
place on the results of the new study of sample size 96.  The stopping boundary is summarized in 
Table 5 and early stopping of the study will be considered if the boundary is reached. 
Table 5: Late stopping rule for safety based upon 2 year Relapse-or-Mortality  
 
# of transplant 
recipients 
Stop if # of recipients with relapse 
or mortality at 2 years reaches 
≤ 6 
5 
≤ 8 
6 
≤ 10 
7 
≤ 12 
8 
≤ 14 
9 
≤ 16 
10 
≤ 17 
11 
≤ 19 
12 
≤ 21 
13 
≤ 22 
14 
≤ 25 
15 
≤27 
16 
≤29 
17 
≤31 
18 
≤33 
19 
≤35 
20 
≤37 
21 
≤38 
22 
≤40 
23 
≤42 
24 
≤44 
25 
≤46 
26 
≤48 
27 
≤50 
28 
≤52 
29 
≤54 
30 
≤56 
31 
≤58 
32 
≤60 
33 
≤62 
34 
≤64 
35 
≤66 
36 
≤67 
37 

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30
# of transplant 
recipients 
Stop if # of recipients with relapse 
or mortality at 2 years reaches 
≤69 
38 
≤71 
39 
≤73 
40 
≤75 
41 
≤77 
42 
≤79 
43 
≤81 
44 
≤83 
45 
≤85 
46 
≤87 
47 
≤89 
48 
≤91 
49 
≤93 
50 
≤95 
51 
≤96 
52 
 
 
Table 6: Proportion of stopped studies for different probabilities of Relapse/Mortality 
Prob of RM = p 
30% 
40% 
50% 
55% 
58% 
 
60% 
 
65% 
Proportion of Stopped 
Studies 
2.8% 
15.5% 
58.7% 
84.1% 
93.4% 
96.9% 
99.8% 
Average # of Subjects 
Needed 
93.57 
83.55 
54.60 
36.23 
27.17 
22.4 
14.73 
Average # of RM 
28.07 
33.42 
27.28 
19.91 
15.75 
13.5 
9.58 
 
We conducted a simulation study to evaluate the statistical properties of the stopping boundaries of Table 
5 and summarized the simulation results in Table 6. Similar to Table 4, the simulation results in Table 6 
suggest that the above stopping rule has a low probability of stopping a study when the proportion of RM 
is below the benchmark value of 49.5%, while the probability of stopping a study is high when the true 
proportion of RM exceeds 49.5%.  
 
The DSMB will also evaluate all treatment related serious adverse events (TRSAEs) and will have access 
to treatment allocation. The DSMB may recommend early study termination of the trial if other 
unforeseen adverse events necessitate this decision.    
 
11.6   Off Study Criteria 
 
 
11.6.1 Withdrawal per subject choice 
 
Recipients and their donors will be given ample opportunity to withdraw from the study prior 
to admission for conditioning and transplantation. Should they wish to withdraw prior to 
transplant they will be replaced in order to maintain appropriate statistical power to evaluate 
primary endpoints. After the transplant, the risks of withdrawal will be carefully explained. If 
the recipient chooses to withdraw before the primary endpoint of the study is reached, this 
recipient will be followed in the safety analysis. 
 
Without compromising their participation in the treatment protocol recipients and donor may 

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31
at any time withdraw from participation in the ancillary laboratory research aspects of the 
protocol. Research data and samples collected prior to withdrawal of consent may still be 
used for analysis. 
 
11.6.2 Withdrawal by physician decision  
 
Recipients with disease relapse will be offered co-enrollment onto the standard of care protocol 
on which they may receive tapering of immunosuppression, donor lymphocyte transfusions, 
immunostimulation (i.e. interferon-alpha, IL-2, GM-CSF, rituximab, imatinib and/or 
chemotherapy) with or without stem cell rescue, or enrollment in phase I/II trials, or they may 
be referred back to their referring physician (whichever is in the best interest of the patient). 
Data collection to include outcome and survival will continue, however complications 
following standard of care therapies will be reported per the standard of care protocol.   
 
Recipients who fail to achieve hematological recovery (primary graft failure) will be 
offered co-enrollment onto the Branch’s standard of care protocol where they may be treated 
with further immunosuppression, growth factors or a second stem cell transplant using 
unmanipulated G-CSF mobilized, donor stem cells or they may be referred back to their 
referring physician whichever is in the best interest of the patient. Data collection to include 
outcome and survival will continue, however complications following standard of care 
therapies will be reported per the standard of care protocol.   
 
If the target dose of CD34+ and CD3+ cells cannot be achieved after processing all the 
collections, the recipient will go off study (technical failure) and be offered the choice of 
participating in other protocols or referral back to their home physician. In this event, the 
recipient will be replaced in order to maintain appropriate statistical power to evaluate 
primary endpoints. 
 
Subjects with a significant decline in performance status (e.g. life-threatening GVHD) which 
negates further treatment on protocol will be offered co-enrollment onto the standard of care 
protocol or referred back to their home physician depending on what is in the best interest of 
the patient. In such cases, typically at end of life, where it is impossible to follow protocol 
mandated investigations and treatments, the patient will remain on protocol for collection of 
endpoints but the standard care provided will not be considered a protocol deviation. Data 
collection to include outcome and survival will continue, however complications following 
standard of care therapies will be reported per the standard of care protocol. 
 
11.6.3 Completion of the study. 
 
Following the completion of 200 days of follow up care the recipient and donor will go 
off study. At 3 years after transplantation, the subjects will  be offered the opportunity to 
enroll on the Late Effects of Transplant protocol “Long-term evaluation and follow up 
care of patients treated with allogeneic stem cell transplants” (05-H-0130). This late 
effects protocol is designed to evaluate the very late consequences of transplant treatment 
and, if necessary, manage transplant recipients suffering from iatrogenic consequences of 
treatment. Recipients opting not to continue follow up care on the Late Effects protocol 
will be referred back to their primary care physician. 
 
12.0 
DATA AND SAFETY MONITORING 
 

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12.1 
Safety Monitoring 
 
Principal Investigator: Accrual and safety data will be monitored by the PI. The protocol will be 
continuously evaluated for any unusual or unpredicted complications with the aim of detecting 
and preventing unacceptable increase in morbidity and mortality over and above that anticipated 
from unmanipulated bone marrow transplants (see stopping rules in section 11.5). 
 
NHLBI IRB: Prior to implementation of this study, the protocol and the proposed recipient and 
donor consent and assent forms will be reviewed and approved by the properly constituted 
Institutional Review Board (IRB) operating in accordance with 45 CFR 46.  The IRB will 
conduct continuing annual review of accrual and safety data. IRB approval will be obtained for 
all amendments to the protocol or informed consent for as long as the protocol is open to accrual 
or follow up of subjects.  
 
DSMB: The NHLBI Data Safety and Monitoring Board (DSMB) will review the protocol at the 
regularly scheduled six or twelve month intervals as applicable.  A progress report will be 
forwarded to the DSMB at these times. The DSMB may recommend early termination of the 
study for considerations of safety and efficacy.  
 
Miltenyi Biotec, Inc.: An annual progress report, any amendments to the protocol, and any 
change in the status of the protocol will be forwarded to Miltenyi Biotec, Inc. to 
 
 
 
Tara Clark, General Manager US Clinical Operations 
 
 
Miltenyi Biotec, Inc. 
 
 
85 Hamilton Street Cambridge, MA 02139- 4524 
 
FDA: An annual progress report, any amendments to the protocol, and any change in the status of 
the protocol will be forwarded to FDA to:   
 
 
 
 
U.S. Food and Drug Administration 
Center for Biologics Evaluation and Research  
Document Control Center 
10903 New Hampshire Avenue 
WO71, G112 
Silver Spring, MD 20993-0002 
12.2 
Adverse Event Reporting 
 
Adverse events used to evaluate the safety of this protocol regimen will be collected to include 
any unfavorable and unintended signs (including abnormal laboratory findings), symptoms and/or 
diseases (i.e. incidence of GVHD, graft failure, regimen related toxicities, or infectious 
complications) which either occur during the study, having been absent at baseline or if present at 
baseline, appear to worsen with the following exceptions.  The AEs will be attributed (unrelated, 
unlikely, possibly, probably or definitely) to study medication and/or disease and graded by 
severity utilizing CTC version 3.0.  A copy of the criteria can be down-loaded from the CTEP 
home page at http://ctep.cancer.gov/reporting/ctc.html. 
 
Adverse events considered possibly, probably, or definitely related to investigational agents 
administered as adoptive cell therapies in conjunction with this protocol will be reported only 
under the clinical protocol for the stated cell therapy. 
 
12.2.1 Definitions 

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33
 
Adverse Event (AE):  Any untoward medical occurrence in a human subject, including any 
abnormal sign (e.g., abnormal physical exam or laboratory finding), symptom, or disease, 
temporally associated with the subject’s participation in the research, whether or not considered 
related to the subject’s participation in the research. 
 
An abnormal laboratory value will be considered an AE if the laboratory abnormality is 
characterized by any of the following: 
 
x 
Results in discontinuation from the study 
x 
Is associated with clinical signs or symptoms 
x 
Requires treatment or any other therapeutic intervention 
x 
Is associated with death or another serious adverse event, including hospitalization 
x 
Is judged by the Investigator to be of significant clinical impact 
x 
If any abnormal laboratory result is considered clinically significant, the investigator will 
provide details about the action taken with respect to the test drug and about the patient’s 
outcome. 
 
Serious Adverse Event (SAE):  A serious adverse event that: 
x 
results in death; 
x 
is life-threatening (places the subject at immediate risk of death from the event as it 
occurred); 
x 
results in in-patient hospitalization or prolongation of existing hospitalization; 
x 
results in a persistent or significant incapacity; 
x 
results in a congenital anomaly/birth defect; or 
x 
based upon appropriate medical judgment, may jeopardize the subject’s health and may 
require medical or surgical intervention to prevent one of the other outcomes listed in this 
definition. 
 
Serious event: An event is serious if it meets the definition of a serious adverse event (above) or if 
it requires immediate corrective action by a PI and/or IRB to protect the safety, welfare or rights 
of subjects. 
 
Unanticipated Problem (UP):  Any incident, experience, or outcome that meets all of the 
following criteria: 
1. unexpected in terms of nature, severity, or frequency in relation to 
a. the research risks that are described in the IRB-approved research protocol and 
informed consent document; Investigator’s Brochure or other study documents; 
and 
b. the characteristics of the subject population being studied; and 
2. related or possibly related to participation in the research; and 
3. places subjects or others at a greater risk of harm (including physical, psychological, 
economic, or social harm) than was previously known or recognized. 
 
Unanticipated Problem that is not an Adverse Event:  An unanticipated problem that does not fit 
the definition of an adverse event, but which may, in the opinion of the investigator, involves risk 
to the subject, affect others in the research study, or significantly impact the integrity of research 
data.  For example, report occurrences of breaches of confidentiality, accidental destruction of 
study records, or unaccounted-for study drug. 
 

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Protocol Deviation (PD):  Any change, divergence, or departure from the IRB approved research 
protocol. 
 
Event Characterization and Reporting to the IRB and Clinical Director (CD) 
Approved by HSRAC on September 30, 2013 
Date effective: October 28, 2013 
 
All adverse events occurring during the study, including those observed by or reported to the 
research team, will be recorded.  Serious unanticipated problems, and serious protocol deviations, 
will be reported to the IRB and Clinical Director as soon as possible but not more than 7 days 
after the PI first learns of the event.  Not serious unanticipated problems will be reported to the 
IRB and Clinical Director as soon as possible but not more than 14 days after the PI first learns of 
the event.  Not serious protocol deviations will be reported to the IRB as soon as possible but not 
more than 14 days after the PI first learns of the event.  SAEs that do not meet the criteria of 
Unanticipated Problem (UP) must be reported to the IRB Chair and Clinical Director within 14 
days of learning of the event. 
 
Deaths will be reported to the Clinical Director within 7 days after the PI first learns of the event. 
 
 
 
12.3 
Adverse Events (AEs)–Transplant Recipients  
 
The following are expected outcomes for the transplant recipient and will be documented in 
the subjects medical record but not reported to IRB unless they meet the criteria for an 
SAE as defined in Section 12.2.1 
 
x 
Renal insufficiency 
x 
Hepatic insufficiency 
x 
Transient cardiac arrhythmias 
x 
Transient cardiac insufficiency 
x 
Pulmonary insufficiency 
x 
Neutropenia and its complications 
x 
Thrombocytopenia and its complications 
x 
Anemia and its complications 
x 
Transfusion reactions 
x 
Treatable infections from bacteria, viruses, protozoa and fungi 
x 
Late effects of transplant regimens including: chronic fatigue, cataracts, infertility, 
growth  impairment, hypothyroidism, bone complications, dental caries, cardiovascular, 
delayed infections, subsequent neoplasms 
x 
Headache, insomnia, psychosis, mood changes, disorientation, seizures from metabolic 
imbalance 
x 
Nausea, vomiting, diarrhea, mucositis, weight loss, dry mouth, hiccoughs, constipation 
x 
Well-characterized drug reactions - allergic manifestations, "red man" syndrome, steroid 
effects 
x 
Well-characterized drug side effects from drugs used routinely in transplant recipients 
(e.g.; preparative regimen chemotherapy, immunosuppressive drugs, antimicrobials)  
x 
Common side effects of antiemetics, analgesics, anti-inflammatory agent and known 
complications of steroid therapy 
x 
Complications from intravenous catheters, thrombotic occlusion, infection, local 
reactions, cardiac arrhythmia  

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35
x 
Expected adverse events related to investigational reagents and transplant drugs are listed 
in section 13.3. 
 
The following are expected transplant outcomes that will be reported in summary form at 
the time of continuing review but will not be reported to IRB at each occurrence unless they 
meet the criteria of an SAE as defined in section 12.2.1: 
 
x Acute graft-versus-host disease 
x Chronic graft-versus-host disease 
x Graft failure / graft rejection 
x Veno-occlusive disease 
x Hemorrhagic cystitis 
x Cytomegalovirus reactivation or disease 
x EBV reactivation or disease 
x Autoimmune phenomenon 
x Fungal infections 
x Disease relapse or progression 
 
12.4 
Adverse Events - Donors 
 
The following are expected outcomes for the donor that will not be reported to the IRB 
unless they meet the criteria of an SAE as defined in section 12.2.1: 
 
• Common side effects of filgrastim (G-CSF) administration (bone pain, fatigue, 
arthralgias, headache, insomnia, fever, worsening of pre-existing skin rashes, increases of 
alkaline phosphatase, lactate dehydrogenase and/or uric acid levels, elevated blood 
leukocyte count, or thrombocytopenia) 
• Hypotension during apheresis 
• Hospital admission to safeguard a catheter 
 
The following are expected outcomes for the donor that will be reported in summary form 
at the time of continuing review but will not be reported to IRB at each occurrence unless 
they meet the criteria of an SAE as defined in section 12.2.1:   
  
• 
Ischemic chest pain during filgrastim (G-CSF) administration 
• 
Splenic enlargement 
• 
Cutaneous vasculitis 
• 
Bone pain, muscle aches or headaches requiring narcotic analgesics 
 
12.5 
Reporting of SAEs 
 
All serious adverse events will be reported to the Principal Investigator  
Minoo Battiwalla, M.D., M.S. 
Bldg 10, CRC Room 5-3581 
Phone: 301-827-0939 
 
Fax: 301-827-3228 
 
The Principal Investigator, Minoo Battiwalla, M.D., M.S. shall submit to the sponsor, A. John 
Barrett, M.D. a report of any unanticipated adverse device effect occurring during an 

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36
investigation as soon as possible, but in no event later than 10 working days after he first learns of 
the effect. 
 
IRB:  Suspected Unexpected Serious Adverse Reaction (SUSAR) and any unanticipated adverse 
device effectobserved during the clinical trial for which there is a relationship with the use of the 
Miltenyi CliniMACS CD34Selection System and/or its components or the conduct of the study 
will be reported to the IRB as soon as possible, but in no event later than 10 working days, except 
for a life threatening or fatal outcome which will be reported within 7 days. 
 
Miltenyi Biotec, Inc.: Reports of SUSARs and any unanticipated adverse device effectobserved 
during the clinical trial and for which there is a relationship with the use of the Miltenyi 
CliniMACS CD34Selection System and/or its components will also be forwarded as soon as 
possible to Miltenyi Biotec.  Miltenyi will be provided with a copy of the annual FDA progress 
report which includes a summary of all SAEs occurring in this clinical trial.  
 
 
 
Tara Clark, General Manager US Clinical Operations 
 
 
and Norman Pilon, PhD  
 
 
Miltenyi Biotec, Inc. 
 
 
85 Hamilton Street  
 
 
Cambridge, MA 02139- 4524 
 
FDA: 
IDE Unanticipated Adverse Device Effects Report (Refer to 21 CFR 812.50): The IDE 
sponsor, A. John Barrett, M.D., will report SUSARs and any unanticipated adverse device effect 
observed during the clinical trial for which there is a relationship with the use of the Miltenyi 
CliniMACS CD34 Selection System and/or its components on the conduct of the study to the 
FDA as soon as possible, but in no event later than 10 working days after the sponsor learns of 
the effect. Thereafter, the sponsor shall submit such additional reports concerning the effects as 
FDA requests. 
 
FDA Annual Reports (Refer to 21 CFR 812.150) 
The study sponsor will submit progress reports at regular intervals and at least annually to the 
FDA. All communications to the FDA will be submitted to: 
 
U.S. Food and Drug Administration 
Center for Biologics Evaluation and Research  
Document Control Center 
10903 New Hampshire Avenue 
WO71, G112 
Silver Spring, MD 20993-0002 
 
DSMB 
SUSARs and any unanticipated adverse device effectobserved during the clinical trial and for 
which there is a relationship with the use of the Miltenyi CliniMACS® CD34Selection System 
and/or its components or the conduct of the study will also be reported within 24 hours to the 
Data and Safety Monitoring Board (DSMB). A summary of all SAEs and AEs will be included 
for review at the regularly scheduled DSMB meeting. 
 
 
 
 

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12.6 
Data Management 
 
The PI will be responsible for overseeing entry of data into an in-house password protected 
electronic system and ensuring data accuracy, consistency and timeliness. Laboratory values from 
referring home physicians will be entered into the system. The principal investigator, associate 
investigators/research nurses and/or a contracted data manager will assist with the data 
management efforts to ensure that data is verifiable and evaluable.   
 
Research data will be prospectively collected by authorized Investigator personnel and entered 
into a NHLBI Database.  
 
The database will maintain complete data records on each research subject. Subjective and 
objective patient experiences during the duration of the study will be documented in the patient 
medical record notes. These protocol notes will serve as the primary source material from which 
data will be collected and research analyses will be performed. Any pertinent supplementary 
information obtained from outside laboratories, outside hospitals, radiology reports, laboratory 
reports, or other patient records will be used as additional source for data collection. 
 
Neither individual personal identifiers nor the key linking coded data to individuals will be 
released to Miltenyi Biotec, Inc. without prior IRB approval and an executed MTA or CTA. 
 
End of study procedures:  Data will be stored in locked cabinets and in a password protected 
database until it is no longer of scientific value. Identifiable data will not be sent outside NIH 
without prior IRB approval or appropriate conditions for disclosure outlined in the executed MTA 
or CTA. After closure of this protocol at this time as all primary endpoints for this study have been 
met, all remaining samples and data will be transferred to protocol 17- H-N001 “Retrospective Data 
and Biospecimen analysis for Allogenic Stem Cell Transplantation Protocols”. The project covers 
existing data review and biospecimen analysis from allo-SCT protocols performed at the 
Hematology Branch, NHLBI, NHLBI.  
 
Loss or destruction of data:  Should we become aware that a major breech in our plan to protect 
patient confidentiality and trial data has occurred, the IRB will be notified. 
 
CIBMTR: For the purposes of quality assurance (i.e. accreditation of the Transplant program), 
data will be released to the Center for International Blood and Marrow Transplant Research 
(CIBMTR) in accordance with federally mandated policies and procedures. 
 
Publication Policy: Given the research mandate of the NIH, patient data including the results of 
testing and responses to treatment will be entered into an NIH-authorized and controlled research 
database. Any future research use will occur only after appropriate human subject protection 
institutional approval such as prospective NIH IRB review and approval or an exemption from 
the NIH Office of Human Subjects Research Protections (OHSRP). 
 
All of the following elements will be recorded in the database and subject to updates by the PI: 
 
A. Subject Enrollment  
x 
Date of birth, age, gender, race, ethnicity, MRN 
x 
Height, Weight, Performance Status (ECOG AND Karnofsky) 
x 
Disease diagnosis, subdiagnosis, date of original diagnosis  
x 
Stage at diagnosis and at study entry  

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x 
HCT-CI score, EBMT score pre transplant 
x 
Disease morphology (marrow) at diagnosis, at last progression and at study entry along 
with dates 
x 
Cytogenetics/FISH at diagnosis, at last progression and at study entry along with dates 
x 
Molecular testing at diagnosis, at last progression and at study entry along with dates 
x 
Prior therapy for disease 
x 
Number of prior transplants for recipient 
x 
Recipient: PPD test results, serologies for CMV, EBV, Hepatitis A,B,C, HIV, HTLV, 
Toxoplasma, RPR. 
x 
Recipient: Echocardiogram (LVEF), Pulmonary Function Testing 
x 
Donor and recipient: CBC and comprehensive metabolic panel, ferritin, CRP. 
x 
Donor/and recipient : HLA typing, KIR typing 
x 
Donor/and recipient : Date of Informed Consent signature, consent version and date of 
registration  
x 
Donor/and recipient : CMV serostatus, ABO-RH types, 
x 
Donor: gender, gravida//parity if female, relationship to recipient, date of birth, age, 
gender, race, ethnicity, MRN 
x 
Donor: serologies for CMV, EBV, Hepatitis A,B,C, HIV, HTLV, Toxoplasma, RPR, 
T.cruzii, W. Nile. 
 
B. Cellular product administration 
x 
Date of stem cell infusion (transplant date) 
x 
CD34+ dose (x10e6/kg), CD3+ dose (x10e4/kg) 
x 
Given per protocol: G-CSF mobilized PBPC,  CD34+/kg is  >3x10e6 on D0, CD3+/kg is 
5x10e4 to 1x10e6 on D0, Fludarabine [25 mg/m2/d x 5 on days -8 to -4], Cytoxan [60 mg/kg 
x2 on days -3 and -2], Total Body Irradiation [<55yrs: 8 x 1.5 Gy, BID days -7 to -4 w lungs 
shielded OR ≥55 yrs 8 x 0.75 Gy& no lung shielding], CSA from D-6 to D+21 
x 
Post-transplant therapy (Imatinib or other TKI, FGF, Palifermin) 
x 
Cyclosporine duration- at least 21 days. 
 
C. Engraftment 
x 
ANC and platelet recovery 
x 
Primary or secondary graft failure 
x 
Time to donor CD3+ chimerism >95% 
x 
Time to donor myeloid chimerism >95% 
x 
Engraftment Syndrome requiring steroids 
 
D. GVHD 
x 
Acute GVHD: maximum grade (CIBMTR definition), date of first onset 
x 
Chronic GVHD: Maximum grade (Shulman criteria), date of first onset 
x 
Last date of systemic immunosuppression 
 
E. Infectious complications 
x 
Date of first CMV >500 copies 
x 
CMV organ disease- site/date 
x 
First CMV/ EBV reactivation requiring treatment (site/date) 
x 
Proven viral hemorrhagic cystitis (type Adeno/BK, date) 
x 
Fungal infections- types, site, date 
x 
Blood stream/vital organ/severe manifestation/opportunistic organism infection 

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F. Second malignancy 
x 
Date, type, EBV positivity, donor vs recipient origin cell 
 
G. Severe pulmonary toxicity 
x 
Defined as requiring mechanical ventilation, hypoxia with SaO2<90% on RA, Any BAL 
or lung biopsy, any lung GVHD (COP or BOS). 
x 
Date, type and therapy. 
 
H. Other toxicity 
x 
Suspected Thrombotic microangiopathy and date 
x 
Veno occlusive disease and date 
x 
Other serious complication (CTCS-AE grade4 and above), type, date, treatment. 
 
 
I. Donor cellular Infusions 
x 
Type of cells, Cell dose, indication, pretreatment regimen, Acute GVHD date, Acute 
GVHD maximum grade 
 
J. Malignant disease evaluation: 
x 
First relapse/progression: date, molecular staging, cytogenetic/FISH staging, 
Morphology, Imaging staging with dates. 
x 
All restaging evaluations: molecular staging, cytogenetic/FISH staging, Morphology, 
Imaging staging with dates. 
x 
Additional antimalignancy treatment- chemotherapy, immunotherapy, donor cellular 
infusions, radiation 
 
K. Vital outcomes; 
x 
Last contact date 
x 
Alive/dead, date of death. 
x 
Subject accrued but did not start conditioning 
x 
End of study: date, reason, comments 
x 
Cause of death, date of death, subtype of non-relapse mortality 
x 
Autopsy findings 
 
L. Adverse Events  
x 
Regimen Related Toxicity (Bearman scale) 
x 
Serious Adverse Events 
 
12.7       Protocol Monitoring 
 
As per ICH-GCP 5.18 and FDA 812.46 clinical protocols are required to be adequately 
monitored by the study sponsor. The monitoring of this study will be conducted by Clinical  
Research Associates (CRAs)/Monitors employed by an independent contract organization 
working under an agreement with NHLBI to monitor aspects of the study in accordance 
with the appropriate regulations and the approved protocol. The objectives of a monitoring 
visit will be: 1) to verify the existence of signed informed consent form (ICF) and 
documentation of the ICF process for each monitored subject; 2) to verify the prompt and 
accurate recording of all monitored data points, and prompt reporting of all SAEs; 3) to 
compare abstracted information with individual subjects’ records and source documents 

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(subject’s charts, laboratory analyses and test results, physicians’ progress notes, nurses’ 
notes, and any other relevant original subject information); and 4) to help ensure 
investigators are in compliance with the protocol. The monitors also will inspect the 
clinical site regulatory files to ensure that regulatory requirements (Office for Human 
Research Protections-OHRP), FDA and applicable guidelines (ICH-GCP) are being 
followed. During the monitoring visits, the investigator (and/or designee) and other study 
personnel will be available to discuss the study progress and monitoring visit. 
 
The investigator (and/or designee) will make study documents (e.g., consent forms and 
pertinent hospital or clinical records readily available for inspection by the local IRB, the 
FDA, the site monitors, and the NHLBI staff for confirmation of the study data. 
 
13.0 
HUMAN SUBJECT PROTECTIONS 
 
The investigators will protect the rights and welfare of human research subjects set forth 
in 45 C.F.R. Part 46 and 21 C.F.R Part 50, Protection of Human Subjects. 
 
13.1 
Rationale for Subject Selection 
 
This protocol is open to males and females from all ethnic and racial groups. Distribution of 
transplant recipients on prior hematology branch protocols 06-H-0248 and 12-H-0028 was as 
follows:  
x 
by disease: 100% neoplastic conditions. 41% AML; 29% ALL; 17% 
MDS/MPD/CMMoL; 7% NHL/CLL; 4% CML; 1% biphenotypic acute leukemia. 
x 
by gender: 50.7% females; 49.3% males 
x 
by age: median age 42, range 8 to 69. 
x 
by race: 5.3% Asian, 9.3% Black, 37.3%, White, 48% Unknown or other. 
x 
by ethnicity: Hispanic or Latino 53.3%; Non-Hispanic 46.6%. 
 
Recruitment:  The study will be listed on clinicaltrials.gov, clinical center research studies, 
PDQ, Aplastic Anemia Foundation, and the NHLBI patient recruitment websites.  If recruitment 
goals are not met, a recruitment plan will be developed by the Clinical Center Office of Patient 
Recruitment. 
 
Competition with other Branch transplant protocols:  
 
03-H-0170:  Non-myeloablative Allogeneic Peripheral Blood Mobilized Hematopoietic Precursor 
Cell Transplantation for Hematologic Malignancies in High Risk Subjects and in Subjects with 
Debilitating Hematologic Diseases 
 
02-H-0250: A Phase I/II Study of HLA-matched Mobilized Peripheral Blood Hematopoietic 
Stem Cell Transplantation for Advanced Mycosis Fungoides/Sezary Syndrome Using 
Nonmyeloablative Conditioning with Campath-1H. 
 
08-H-0186: Safety and the Anti-Tumor Effects of Escalating Doses of Adoptively Infused Ex 
Vivo Expanded Autologous Natural Killer (NK) Cells Against Metastatic Cancers or 
Hematological Malignancies Sensitized to NK-TRAIL Cytotoxicity with Bortezomib 
 
Reimbursement for protocol participation, travel, food, and lodging  
 

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There is no payment or compensation to subjects for their participation in this protocol. 
 
Reimbursement for travel, food, and lodging will be consistent with NHLBI DIR Travel and 
Lodging Compensation of Clinical Research Subjects policy or institutional guidelines.  
 
13.2 
Participation of Children  
 
13.2.1 As recipients: 
 
Pediatric subjects under 10 years of age are excluded from participating in the protocol because in 
children less than age 10, the risk of severe GVHD (one of the secondary endpoints of the study) 
is substantially less than that experienced in transplant recipients over the age of 10.  It is 
therefore, not appropriate to expose children under the age of 10 to an experimental protocol 
designed to evaluate the incidence and severity of acute GVHD after transplant. 
 
13.2.2 As donors: 
 
We are excluding from participation as donors, children who weigh <18 kg and are < 2 years of 
age. The risks of the apheresis procedure are related to the weight of the child, more precisely 
his/her extracorporeal volume, which is weight-dependent.  The risks have to do with (1) need for 
a central line, (2) need for an allogeneic red cell prime, (3) need for systemic heparinization 
because subject is too small to get citrate:     
 
25 kg:  the procedure and associated risk is the same as that in an adult, however a central 
line is often needed (at the discretion of the apheresis department). 
 
19 to 25 kg:  A central line is usually required (at the discretion of the apheresis department). 
With concurrent magnesium and calcium infusion, children may be safely anti-coagulated with 
citrate. 
 
Two apheresis procedures will be performed in minor donors - one for the collection of 
lymphocytes and the other for the collection of stem cells.  Collecting stem cells and lymphocytes 
in two apheresis sessions is widely practiced in donors of all ages including children (Elmaagcli 
et al 2003, Barge et al. 2003, Reviewed in Baron and Beguin 2002) DLIs may be given post-
transplant to treat relapse, to treat recurrent infection, or to promote engraftment of stem cells 
when at risk of rejection after a SCT regardless of how the stem cells are prepared. 
 
13.2.3  As participants in research studies:   
 
Pediatric donors may participate only in those laboratory studies that the IRB finds involves no 
greater than minimal risk to children provided that adequate provisions are made for soliciting the 
assent of the children and the permission of their parents or guardians. 
 
13.3  
Hazards and Discomforts 
 
 
13.3.1  The recipient 
 
Related to the transplant procedure: 
 
Blood and marrow stem cell transplantation is a major procedure, which entails serious 
discomforts and hazards for the patient. The mortality from the transplant procedure itself may be 

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42
as high as 40%. It is therefore only appropriate to carry out the procedure with its experimental 
component in the context of a life-threatening condition and with full informed consent from the 
patient and donor and immediate family. The major hazards (GVHD, infection, graft rejection, 
and leukemic relapse) have already been mentioned. Some poorly understood post transplant 
complications can lead to multiorgan failure including: inflammatory states such as a cytokine 
release syndrome and the endothelial damage syndromes (transplant microangiopathy, posterior 
reversible encephalopathy syndrome, diffuse alveolar hemorrhage, idiopathic pneumonitis, 
sinusoidal occlusive syndrome/veno-occlusive disease and delayed cardiovascular disease). 
Inevitably after transplantation, blood counts will decrease with an associated requirement for red 
blood cell and platelet transfusions, as well as a need for empiric antimicrobial use to prevent 
infectious complications.  At the time of count recovery, there is also a small risk of pulmonary 
engraftment syndrome, manifested by non-infectious fever, pulmonary infiltrates, and dyspnea, 
which can be treated effectively with prompt administration of corticosteroids.  The major 
discomforts during the post-transplant period are those of nausea, mucositis, anorexia, diarrhea, 
fever, malaise, and intolerance of the isolation period.  Possible organ toxicities related to the 
high-dose preparative regimen include hepatic veno-occlusive disease and interstitial 
pneumonitis, which can be fatal. 
 
The procedure will render the patient infertile.  Because of this, we will discuss the possibility of 
sperm banking or egg retrieval with the subject before undergoing the transplant.  There is no effect 
of the treatment on male hormone production or potency.  Women stop ovulating and menstruating 
after the treatment, which also reduces estrogen production to low levels, inducing premature 
menopause.  This is treated by hormone replacement therapy, which is important in order to 
conserve the body’s store of calcium, amongst other reasons.  In children before puberty, the 
treatment produces infertility, but with a small possibility of delayed recovery (about 10 years 
later).  Puberty may be delayed, but often progresses normally.  Although the whole transplant 
procedure may be accompanied by a stunting of growth, normal growth is subsequently resumed in 
the majority of children.  In only a few cases, is it necessary to give growth hormone, testosterone 
(male hormone) or estrogen (female hormone) to complete normal growth and sexual development. 
 
Allogeneic SCT is increasingly being offered to older individuals (> age 70) with the realization 
that chronological age alone is not as important as comorbidities, performance status or disease 
risk. Careful selection of patients with advanced age will mitigate the risk of complications. 
Nevertheless, older individuals will need to be carefully counseled prior to transplant regarding 
higher risks from transplant related mortality. 
 
With amendment in April 2017, the protocol will no longer exclude subjects with prior allogeneic 
transplantation. In order to minimize risk and maximize benefit, candidates with a prior transplant 
will be carefully selected; they will be excluded if their prior transplant involved fully 
myeloablative conditioning or if their malignant disorder is highly refractory and deemed unlikely 
to benefit from an allotransplant approach. Analysis of patients on this protocol is standardized by 
disease risk index and comorbidity index, that will allow merging of the data of subjects with and 
without prior transplantation. 
 
Related to the apheresis procedure: 
 
The apheresis procedures will be performed in accordance with standard apheresis donation 
policies and procedures operative in DTM and will be in compliance with the Blood Donor 
Standards of the American Association of Blood Banks and the rules and regulations of the Food 
and Drug Administration. Adverse reactions to apheresis procedures are rare, but include: 
 

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x 
Pain and hematoma at the needle placement site 
x 
Vasovagal episodes, characterized by transient hypotension, dizziness, nausea and rarely, 
syncope are seen in less than 2% of the procedures. Hypotension secondary to volume 
depletion is known for the rare potential for a cerebrovascular or cardiovascular event.  
x 
Cutaneous or circumoral parasthesias, chills, nausea, heartburn and rarely muscle spasms 
may result from the use of citrate anticoagulant used to prevent clotting in the 
extracorporeal circuit. Citrate reactions are usually relieved by slowing the rate of the 
anticoagulant infusion and by administering oral calcium carbonate tablets or with 
intravenous calcium gluconate. 
 
Prior to each apheresis, the potential risks associated with the procedure will be explained to the 
patient and a separate informed consent obtained. 
 
Related to the CliniMACS CD34 Reagent Systems:  
 
Theoretical risks to the patient could include system failure, user error, or patient reaction to 
selected product components (i.e. residual amounts of unbound CliniMACS CD 34 Reagent, Iron 
dextran, or human anti-mouse antibodies). For full text see Investigator’s brochure for Miltenyi 
CliniMACS CD34+ system, Version 6, dated 5/27/2008, section 6, Summary and Guidance for the 
Investigator). 
 
Related to the common transplant related drugs and radiotherapy. 
 
 
 
Cyclosporine: CSA is metabolized primarily in liver but the major toxicity is renal. Side effects 
include renal impairment, reversible renal insufficiency, hemolytic uremic syndrome, elevated 
bilirubin and transaminases that normalize with continued administration or reduced dose, 
hypertrichosis, headaches, nausea, gingival hypertrophy, parasthesias (painful hands and feet), 
hypertension, hypomagnesium, bilirubinemia, hypertrichosis, nausea, tremor, and seizure.  An 
extremely rare complication of cyclosporine is blindness, which may be irreversible. Posterior 
Reversible Encephalopathy Syndrome (PRES) is an increasingly recognized neurologic disorder 
seen in 1% of subjects on cyclosporine which manifest with acute to subacute hypertension 
and/or seizures. In the event of hypertension, subjects will be prescribed 1 or more medications to 
control blood pressure in an effort to decrease the risk of this complication. 
 
Cyclophosphamide: immediate: tingling and metallic taste, nausea and vomiting, ADH-like 
effect, cardiotoxic at high doses (>70 mg/kg). Rare - pulmonary toxicity, urticaria and flushing, 
mucositis. Delayed: marrow suppression, nausea, mucositis, rash, hemorrhagic cystitis, 
myocardial damage, alopecia, infertility. 
 
Fludarabine: myelosuppression, fever and chills, nausea and vomiting, malaise, fatigue, anorexia, 
weakness, and rarely hemolysis and pulmonary toxicity, hemolytic anemia and interstitial 
pneumonitis. Serious opportunistic infections have occurred in CLL subjects treated with fludarabine.   
 
Methotrexate (used in the event of technical failure of graft processing resulting in CD3+ cells>1 
x 106/kg): mucositis, nausea, dizziness, neutropenia, thrombocytopenia, malaise, fatigue, fever, 
melena, headaches, blurred vision, rashes, alopecia, elevated liver functions,  
Hematologic: myelosupression (leukopenia [nadir 7 days] thrombocytopenia, anemia) 
Hepatic: acute (elevated transaminases) and chronic (fibrosis and cirrhosis) hepatic toxicity. 
Chronic toxicity has generally occurred after prolonged use (generally 2 years or more) and 
after a total dose of at least 1.5 grams. 

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Urogenital:  severe nephropathy or renal failure, azotemia, cystitis, hematuria; defective 
oogenesis or spermatogenesis, transient oligospermia, menstrual dysfunction and vaginal 
discharge; infertility, abortion, fetal defects. With high doses of methotrexate, close attention 
to renal function including adequate hydration, and urine alkanization are essential for safe 
administration.  
Gastrointestinal: gingivitis, pharyngitis, stomatitis, anorexia, nausea, vomiting, diarrhea, 
hematemesis, melena, gastrointestinal ulceration and bleeding, enteritis. Should be used with 
extreme caution in the presence of peptic ulcer disease or ulcerative colitis. Therapy may be 
discontinued if ulcerative stomatitis or other severe GI adverse reactions occur. 
Pulmonary: interstitial pneumonitis deaths have been reported, and chronic interstitial obstructive 
pulmonary disease has occasionally occurred. Pulmonary symptoms or a nonspecific 
pneumonitis may be indicative of a potentially dangerous lesion and require interruption of 
treatment and careful investigation; infection needs to be excluded. This lesion can occur at 
all dosages. 
Skin: erythematous rashes, pruritis, urticaria, photosensistivty, pigmentary changes, alopecia, 
ecchymosis, telangiectasia, acne, furunculosis. 
Central Nervous System: headaches, drowsiness, blurred vision. There have been reports of 
leukoencephalopathy following intravenous administration of methotrexate to subjects who 
have craniospinal irradiation. Aphasia, hemiparesis, paresis, and convulsions have also 
occurred following administration of methotrexate. Following low doses, occasional subjects 
reported transient subtle cognitive dysfunction, mood alteration, or unusual cranial 
sensations. 
Other: opportunistic infections, arthralgia/myalgia, loss of libido/impotence, diabetes, and 
osteoporosis. A few cases of anaphylactoid reactions have been reported. 
 
 
Related to antimicrobials in general: Allergic reactions, renal impairment (gentamicin, 
vancomycin, amphotericin, acyclovir), "red man" syndrome (vancomycin), hepatic damage 
(acyclovir, rifampin), nausea, anorexia, abdominal discomfort, pancytopenia, neutropenia, 
peripheral neuropathy, renal damage (Ganciclovir) (reversible on stopping drug). 
 
Related to the total body radiation: side effects of radiation have been well described. The most 
common include nausea and mucositis. Mucositis is associated with bacteremias and sepsis. There 
also exists a risk of hypothyroidism, cataracts, interstitial pneumonitis, nephropathy, and an 
unspecified long term risk of developing secondary malignancies. Importantly, the majority of the 
nonneoplastic effects were sub clinical and/or reversible.  
 
13.3.2 The Donor 
 
Related to filgrastim (G-CSF). The hazard to the donor is low. The discomfort from G-CSF 
mobilization and leukapheresis for collection of blood stem cells are probably lower than those 
associated with marrow harvesting. G-CSF has been given to large numbers of normal donors 
without major side effects or long-term consequences. The immediate side effects of G-CSF are 
bone pain, fatigue, insomnia, myalgia and headache. These are usually mild and are self-limiting. 
Reversible thrombocytopenia, with platelet counts falling to the region of 100,000 /cu mm is 
frequent. Although regarded as safe and well tolerated, G-CSF administration could result in 
splenic rupture, a potentially life-threatening complication. Up to now five cases of splenic 
rupture have been reported in healthy donors (Becker et al. 1997, Falzetti et al. 1999, Balaguer et 
al. 2004, Dincer et al. 2004, Nuamah et al. 2006). Donors will be asked to avoid vigorous 
activities and to report any left upper abdominal and/or shoulder pain to the research team or the 
on-call physician for the NIH Department of Transfusion Medicine at 301-496-1211.  

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Subjects with ongoing ischemic heart disease have been reported to have angina seemingly 
temporally-related to G-CSF administration and apheresis (Kopp, et al 2007, Vij, et al 1999). In 
addition, a rare occurrence of pulmonary hemorrhage has been reported in a healthy donor who 
was a cigarette smoker and had underlying pulmonary disease (Kopp, et al 2007). Finally, donors 
with sickle cell trait have been reported to develop vaso-occlusive crises when treated with G-
CSF, necessitating special precautions (Kang, et al 2002). 
 
 
Related to central line placement. It is estimated that about 50% of the donors will require 
intravenous line placement to successfully complete apheresis. Intravenous line placement in the 
central vein using an apheresis catheter carries a small risk of bleeding, bruising or pain and a very 
low risk of accidental injury to the adjacent artery and nerve. Central line placement in the upper 
circulation carries a small risk of puncture of a pneumothorax. Some subjects may experience a 
vasovagal reaction (lightheadedness, or, rarely, fainting due to temporary lowering of blood 
pressure).Using only experienced staff for the procedure minimizes these risks. 
 
In exceptional instances the donor does not mobilize well and is required to receive plerixafor or 
to donate PBPC a second time. Plerixafor is an FDA approved mobilizing agent and would have a 
standard indication in this setting. The risks of plerixafor are mainly GI upset. There is no 
additional risk to the donor from a second PBPC donation.  
 
Related to Apheresis: see section 13.3.1. 
 
13.4 
Research Risks in Relation to Research Benefits  
 
13.4.1 For Adult Transplant Subjects 
 
Allogeneic stem cell transplantation is the treatment of choice for many subjects with leukemia 
and other hematologic malignancies, however it entails serious discomforts and hazards for the 
patient (section 13.3). -The investigational component of this transplant protocol aims to decrease 
non-relapse mortality and improve overall survival by reducing GVHD and the exposure to 
prolonged immunosuppression. This approach is theoretically likely to increase the risk of 
infections, relapse/progression and slow donor lymphoid engraftment 
 
Clinically, this approach is ethically acceptable because we will target a patient group with lethal 
hematologic malignancies and no other curative treatment options who have made an informed 
decision to opt for a procedure which they understand offers a chance of prolonged disease-free 
survival but with a risk of death from treatment failure. Implicit in this decision is the opportunity 
to weigh all treatment options after full information has been given to them by the NIH team and 
the referring physician.  
 
Therefore for adult transplant recipients on this protocol, the research involves greater than 
minimal risk to subjects with the prospect of direct benefit (45 CFR 46.102). 
 
Exclusion of pregnant women: The exclusion of pregnant women is justified on medical grounds 
as pregnancy is an absolute contraindication for myeloablative doses of chemo-radiation.  
 
13.4.2 For Pediatric Transplant Subjects   
 
The investigational component of this transplant protocol aims to decrease non-relapse mortality 
and improve overall survival by reducing GVHD and the exposure to prolonged 

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46
immunosuppression. This approach is theoretically likely to increase the risk of infections, 
relapse/progression and slow donor lymphoid engraftment. The inclusion of children satisfies the 
criteria set forth in 45 Code of Federal Regulations 46, Subpart D: 46.405 as follows: 
 
 (a) the risk is justified by the anticipated benefit to the subjects: We are offering pediatric 
subjects with a probably lethal hematological disease, incurable with conventional treatments 
other than allogeneic stem cell transplantation, an alternative to symptomatic therapy. 
 
(b) the relation of the anticipated benefit to the risk is at least as favorable to the subjects as that 
presented by available alternative approaches. The protocol aims to decrease the risk of transplant-
related mortality, thus making more subjects candidates for potentially curative therapy and 
 
 (c) adequate provisions are made for soliciting the assent of the children and permission of their 
parents or guardians, as set forth in 46.408. 
 
Therefore for pediatric transplant recipients on this protocol, the research involves greater than 
minimal risk but presents the prospect of direct benefit to the individual subjects (45 CFR 
46.405). 
 
 
13.4.3 For Adult Donors: 
 
An HLA 6/6 identical family member will be co-enrolled into this study as a stem cell donor. The 
stem cell collection aspect of this protocol is not investigational. Despite the risks associated with 
this standard procedure (13.3.2), potential benefit does exist for family donors. The donor derives 
psychosocial benefit from donating stem cells both at the time of donation and possibly into the 
future, especially in view of the reduced life expectancy due to this disease in a family member.  
Other potential benefits include detection of illnesses, determination of blood cell counts, and 
evaluation of kidney and liver function in the potential donor at the time of screening. Therefore, 
participation as a stem cell donor on this protocol involves minimal risk to subjects. For those 
who choose to participate in the Laboratory Research Studies involving bone marrow sampling, 
the risk is greater than minimal but presents the prospect for direct benefit(45 CFR 46.102). 
 
13.4.4 For Pediatric Donors: 
 
Stem cell transplant is an accepted standard clinical intervention for the diseases under 
investigation. The donor would be donating stem cells and lymphocytes collected separately 
during two apheresis procedures to his/her family member regardless of the objectives of this 
research protocol. The stem cell and lymphocyte collection procedures are not considered part of 
the research for the donors and the risks of the stem cell and lymphocyte collection procedures 
would not be considered risks of the research for the pediatric donors. Therefore, participation as 
a stem cell donor on this protocol is considered exempt from the criteria set forth in 45 CFR 46, 
Subpart D. 
 
13.4.5 For Pediatric Donors – Healthy Volunteers- Involved in Laboratory Research Studies. 
 
The inclusion of children satisfies the criteria set forth in 45 Code of Federal Regulations 46, 
Subpart D: 46.404 as follows: 
 
(1) The research does not involve greater than minimal risk.  There is no added risk 
associated with the amount and method of collection for the additional blood for research 

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obtained concurrently with clinically indicated sampling.  We are not collecting bone marrow 
aspirate for the lab research studies. 
 
Research specimens will be stored in the PI’s laboratory. Samples will be assigned a unique 
code known only to the PI, which will serve as a link to the child’s name and clinical 
information collected as part of this research protocol.  No samples will be provided to 
investigators outside the branch without permission of the IRB, therefore confidentiality will 
be protected. 
Only those laboratory tests approved by the IRB and involving not greater than minimal risk 
to subjects will be conducted (See Appendix A). Research will not include genetic testing, 
thus there is no risk related to genetic testing.  
 
(2) adequate provisions are made for soliciting the assent of the children and permission of their 
parents or guardians, as set forth in 46.408.   
 
Therefore, participation of pediatric donors in laboratory research on this protocol does not 
involve greater than minimal risk (45 CFR 46.404). 
 
13.5 
Informed Consent Processes and Procedures 
 
Adults:  The investigational nature and research objectives of this trial, the procedure and its 
attendant risks and discomforts will be carefully explained to the subject and a signed informed 
consent document will be obtained prior to entry onto this study.  Drs. Barrett or Battiwalla or 
other senior physicians who are specifically listed as associate investigators capable of obtaining 
consent on the front page of the protocol will lead this discussion. 
 
If the patient is a minor, a minor assent will be sought.  Where deemed appropriate by the 
clinician and the child's parent or guardian, the child will also be included in all discussions about 
the trial and a minor's assent will be obtained.  The parent who signs the consent for the minor 
must be a parent or legal guardian.  The parent or guardian will sign on the designated line on the 
informed consent attesting to the fact that the child had given assent. When the assent is not age 
appropriate, the study will be explained to the child and the assent will be obtained verbally from 
the child. In cases where parents share joint legal custody in making medical decisions of their 
child (e.g. by a custody agreement or court order) both parents must give their parental 
permissions regardless of level of risk of the research. Exceptions may be made if one parent is 
deceased, becomes incompetent or is not reasonably available (e.g. in prison). 
 
If the donor is a minor, assent will not be sought until an evaluation by a social worker and 
mental health specialist (psychologist or psychiatrist) is completed to determine the minor 
donor’s willingness to participate.   As detailed above, the parent who signs the consent for the 
minor must be a parent or legal guardian.  Where deemed appropriate by the clinician, and the 
child's parent or guardian, the child will also be included in all discussions about the trial and a 
minor donor’s assent will be obtained. The investigators are requesting a waiver from the IRB to 
allow only one parent to sign the informed consent to enter a child on the protocol. The parent or 
guardian will sign on the designated line on the informed consent attesting to the fact that the 
child had given assent. In cases where parents share joint legal custody in making medical 
decisions of their child (e.g. by a custody agreement or court order) both parents must give their 
parental permissions regardless of level of risk of the research. Exceptions may be made if one 
parent is deceased, becomes incompetent or is not reasonably available (e.g. in prison). 
 
If a child (donor or transplant recipient) dissents, the IRB and Bio-ethics will be consulted. 

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48
 
If the minor subject is a female of childbearing age, she will be informed about pregnancy testing 
and will be told that if her pregnancy test is positive, we will counsel her and help her tell her 
parents or we will tell her parents with her permission. Is she does not agree she will be advised 
not to sign the assent. 
 
Decisionally impaired: The IRB and Bio-ethics Department will be consulted should there be a 
decisionally impaired patient (donor or recipient) identified as a potential candidate for 
enrollment in this protocol.  
 
 
At any time during participation in the protocol that new information becomes available relating 
to risks, adverse events, or toxicities, this information will be provided orally or in writing to all 
enrolled or prospective patient participants.  Documentation will be provided to the IRB and if 
necessary, the informed consent will be amended to reflect relevant information. 
 
Re-Consent for Minors when they reach the age of majority:  When a pediatric subject 
reaches age 18, continued participation will require re-consenting of the now adult with the 
standard protocol consent document to ensure legally effective informed consent has been 
obtained. Should sample or data analysis continue following completion of active participation 
and the subject has reached 18 years of age, we will attempt to contact the subject using the last 
known contact information to obtain consent for continued use of data or samples  collected 
during their prior visit. Given the length of time that may have transpired for some of the subjects 
since their last visit for this study, we request waiver of informed consent for those individuals 
who after good faith efforts to contact them, we are unable to contact. 
 
Requirements for Waiver of Consent consistent with 45 CFR 46.116 (d), each of which must be 
addressed in relation to the protocol: 
 
(1) The research involves no more than minimal risk to the subjects. 
a. Analysis of samples and data from this study involves no additional risks to subjects. 
(2) The waiver or alteration will not adversely affect the rights and welfare of the subjects. 
a. This is an FDA-regulated study and as such, we are mandated to retain all samples, 
once collected, regardless of the age of the subject at the time of collection.  
Retention of these samples or data does not affect the welfare of subjects.  
(3) The research could not practicably be carried out without the waiver or alteration. 
a. Considering the length of time between a minor’s enrollment and their age of 
majority, it is possible that more than a few subjects may be lost to follow up.  A 
significant reduction in the number of samples analyzed could impact the quality of 
the research.  
(4) Whenever appropriate, the subjects will be provided with additional pertinent information 
after participation. 
a. We only plan to request a waiver of reconsent for those subjects who have been lost 
to follow-up.   
 
Non-English speaking subjects: If there is an unexpected candidate for enrollment of a research 
participant for which there is no translated extant IRB approved consent document, the principal 
investigator and or those authorized to obtain informed consent will use the Short Form Oral 
Consent Process as described in MAS Policy M77-2, 45 CFR 46.117 (b) (2), and 21 CFR50.27 
(b) (a) 0. The summary that will be used is the English version of the extant IRB approved 
consent document.  
 

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We request prospective IRB approval of the use of the short form for up to a maximum of 5 
research participants in a given language and will notify the IRB at the time of continuing review 
of the frequency of the use of the Short Form.  Should we reach the threshold of 5, we will notify 
the IRB of the need for an additional use of the Short Form and we will have the consent 
document translated into the given inherent language.  
 
13.6      Conflict of Interest and Tech Transfer Agreements 
 
The Principal Investigator assured that each associate investigator listed on the protocol title page 
received a copy of the NIH’s Guide to preventing conflict of interest.  Investigators added 
subsequent to the initial circulation will be provided a copy of the document when they were 
added. Copies of the Conflict of Interest Statement were forwarded to the Clinical Director.  No 
initial members of the research team reported a potential conflict of interest.  
 
This protocol has no associated patents or CRADAs.  The protocol has the following associated 
CTA:  
x 
between Miltenyi Biotec Incorporated and the National Heart, Lung and Blood Institute, 
entered on Nov 15, 2006 by which Miltenyi agrees to make available its CliniMACS 
CD34 reagent cell selection system for clinical research purposes. 
 
This protocol has the following MTAs that is partially executed: 
 
x 
Between NHLBI and Catherine Bollard, MD, Children’s National Medical Center, 111 
Michigan Ave, NW, Washington DC, 20010, as part of a collaboration to evaluate NK 
cell phenotype and function and to determine whether the function can be improved by ex 
vivo expansion. 
x 
Between NHLBI and Pawel Muranski, MD, Columbia University Medical Center, 630 
West 168th Street, New York, NY 10032. The de-identified coded samples from healthy 
donors and healthy volunteers enrolled in 13-H-0144 study may be sent to Dr. Muranski 
to be used in development of future clinical trials.  
 
13.7 
FWA Coverage Agreement 
Dr. Muranski has recently assumed the role of Director of Cellular Immunotherapy at Columbia 
University Medical Center, NY and will be analyzing identifiable data as an Associated Investigator in 
this protocol.  Dr. Muranski’s role in the research will be limited to data analysis. An FWA coverage 
agreement to cover this activity will be executed by Dr. Muranski and Dr. Ito, once this amendment is 
approved. 
 
14.0 
PHARMACEUTICALS 
 
14.1     CliniMACSCD34 Reagent System (see Investigator’s Brochure, Version 6, dated 5/27/2008) 
 
 
14.1.1   Investigational Product: the CliniMACS® CD34 Reagent System is a medical device that 
is used in vitro to select and enrich specific cell populations. When using the CD34 Reagent, the 
system selects CD34+ cells from heterogeneous hematological cell populations for transplantation 
in cases where this is clinically indicated. The CliniMACS CD34 Reagent System is comprised of 
four primary components: 

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• CliniMACS CD34 Reagent: a sterile monoclonal antibody reagent specific for CD34+ cells  
• CliniMACS plus Instrument: a software controlled instrument that processes the blood sample 
(cell product)  
• CliniMACS Tubing Sets: single-use, sterile, disposable tubing sets with two proprietary cell 
selection columns (CliniMACS Tubing Set and CliniMACS Tubing Set LS)  
• CliniMACS PBS/EDTA Buffer: a sterile, isotonic phosphate-buffered, 1 mM EDTA, saline 
solution, used as external wash and transport fluid for the in vitro preparation of blood 
cells.  
14.1.2 Physical, Chemical and Toxicological Information 
x 
CD34 Reagent Description:  the CliniMACS CD34 Reagent is a dark amber, non-
viscous, colloidal solution containing the antibody conjugate in buffer. The conjugate 
consists of a monoclonal antibody towards the class II epitope of the human CD34 
antigen. The murine monoclonal IgG1 antibody is covalently linked to dextran beads that 
have an iron oxide/hydroxide core and are super paramagnetic. 
x 
Safety testing of the CD34 Monoclonal Antibody:  cell banking, cell culture, as well as 
subsequent purification of the antibody, follow the applicable current international 
guidelines as described in Section 4. The testing of the CD34 Master Cell Bank, the End 
of Production Cells, the CD34 mAb pooled cell culture harvest (unprocessed bulk) and 
the purified CD34 monoclonal antibody (mAb) have been completed and the purified 
CD34 mAb has been released for manufacturing of the CD34 Reagent. Additionally, the 
viral inactivation/removal steps used in the purification of the CD34 monoclonal 
antibody have been validated. 
x 
Safety testing of the CD34 Reagent: detailed toxicity studies have been undertaken to 
assess the safety of the CD34 Reagent when delivered in dosages significantly greater 
than the projected maximum dosage anticipated in clinical use. The testing was 
performed in accordance with 21 CFR §58, Good Laboratory Practices for Nonclinical 
Laboratory Studies. A summary of this testing is provided in Table 6. 
 
Table 6: Toxicity studies to assess the safety of the CD34 Reagent 
Summary of Toxicology of the CliniMACS CD34 Reagent 
Test  
Results  
Human Cryosection Cross 
Reactivity Study  
CD34 Reagent specifically reacted with cell types known to possess 
the CD34 antigen. Not considered toxicologically significant.  
Interspecies Cross Reactivity 
Study  
CD34 antibody does not cross react with non–human primate 
hematopoietic cells expressing the CD34 antigen. These species could 
be used for safety testing.  
Subchronic Toxicity  
No Toxicity  
Cardiovascular Safety Study in 
Rhesus Monkeys  
No drug-related effects on mean arterial pressure, mean right ventricle 
pressure, cardiac output, ECGs, respiration rate, heart rate or cage side 
observations were noted when escalating doses of CD34 Reagent  
Irritation  
No Irritation  
Hemocompatibility 
Compatible with human blood  
Sterility assay of final container  Reproducibly sterile product  
 
14.1.3 Safety Testing of CliniMACS® System Components (Instrument, Tubing Sets and PBS/EDTA 
Buffer) Biocompatibility Testing of the CliniMACS System components (Tubing Sets and 
PBS/EDTA Buffer) was performed according to ISO 10993. The requirements of ISO 10993 
were fulfilled for the CliniMACS CD34 Reagent System.   The CliniMACS plus Instrument has 

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51
been tested for electrical safety and the potential for fire, shock, explosion, or mechanical 
damage.  Potential safety issues have been reduced by using a design to meet European standards 
EN 60601-1. It is UL and CSA listed and approved. 
14.1.4 Overall Safety of the CliniMACS® CD34 Reagent System:  The results summarized in this 
Investigator’s Brochure support that CliniMACS CD34 Reagent System is sufficiently safe for 
clinical use with human subjects. The potential application of the CliniMACS CD34 Reagent is 
broad. Infusion of purified CD34+ cells is indicated in a number of clinical applications after 
myeloablative or lymphoablative therapy including reduction of tumor cells in the transplant and 
depletion of T cells for autologous (autoimmune diseases) and allogeneic transplantations. 
Individual risk analysis on the therapeutically used target cells isolated in conjunction with 
CliniMACS CD34 Reagent System should be addressed by each site using these cells. A European 
safety study, ACS 950101, for the CliniMACS System was published in Bone Marrow 
Transplantation 25; 243-49, February 2001. The study was designed to meet European Essential 
Requirements 3 and 14 (MDD 93/42/EEC) and was conducted per EN540 to support the CE 
Marking of the device (received December 1997).  
 
The initial clinical study with the CliniMACS System was conducted in subjects undergoing high-
dose chemotherapy for breast cancer. The purpose of the European Safety Study was to show:  
 
• Suitability of the CD34 Reagent and other CliniMACS components for selection of 
CD34+ cells with high, yield, purity viability and safety  
• CD34+ cells can safely be administered to subjects after myeloablative chemotherapy  
• Selected CD34+ cells are effective in reconstituting the hematopoietic system after 
myeloablative chemotherapy  
• Rate of device failures  
 
Cells were isolated from leukapheresis products from sixty-five subjects enrolled in the study. Fifty-
four subjects received selected CD34+ cells and fifty-two were evaluable for engraftment as 
summarized in the table below (one patient died 5 days post-transplant and prior to engraftment and 
one patient did not recover platelet counts even after back-up cells were infused). All subjects 
receiving selected cells completed 60 and 100-day follow-up after infusion, during which time their 
hematological and immune status were monitored, as was HAMA production. A summary of the 
results of the European Clinical Trial is provided below.  
 
Table 7: European Clinical Trial results 
Time to Hematological Engraftment After Infusion of CliniMACS Selected CD34+ Cells 
Time to Engraftment (Days)  
Platelets (≥ 20 x 109/L)  
Neutrophils (≥ 500/uL)  
Median (Kaplan Meier)  
SD  
Quartile range (Kaplan Meier)  
Range  
Number  
11.6  
6.05  
10.0-12.0  
8-29  
52  
9.1  
5.81  
8.0-10.0  
8-11  
52  
 
The following conclusions were made regarding this clinical study:  
 
• The CliniMACS CD34 Reagent System selects CD34+ cells from heterogeneous hematological 
cell populations. The resulting CD34+ product is of high purity (median of 96.1 %, range 27.4 

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52
– 99.4 %); the median recovery of CD34+ cells was 52.3 % (range 15.2 – 146.3%). The 
reported performance results are similar to those seen in pre-clinical studies.  
• Infusion of selected, autologous CD34+ cells after high dose chemotherapy resulted in rapid 
engraftment (see Table 2). These data are comparable to previously reported results, using bone 
marrow or peripheral blood cells as stem cell source. Following cyclophosphamide, thiotepa 
and carboplatin (CTCb), Weaver et al. reported median times to platelet and neutrophil 
engraftment of 9 days (range 0-53 days) and 9 days (range 5-26 days), respectively. Also after 
CTCb, Elias et al., reported median times to platelet and neutrophil recovery of 12 days (range 
8-134 days) and 14 days (range 10-57 days), respectively (Elias, et al 1992, Weaver, et al 1995).  
• The selection process has no discernable effect on cell viability or sterility.  
• There were no adverse events or device malfunctions reported as related to the infusion of the 
cells or use of the CliniMACS CD34 Reagent System. None of the subjects were reactive for 
HAMA post infusion. There were no reports of late engraftment failure or evidence of delayed 
immune reconstitution.  
 
14.2 
CYCLOPHOSPHAMIDE (Cytoxan, Neosar) 
Supply: Commercially available. 
Product description: Cyclophosphamide is available as a lyophilized powder for injection in 
multiple vial sizes.  
Preparation: Cyclophosphamide powder for injection should be reconstituted with sterile water 
for injection to yield a concentration of 20 mg/mL as described in the product labeling. Once 
reconstituted, the prescribed dose will be further diluted in 250 mL of 0.9% sodium chloride 
injection or 5% dextrose in water for intravenous administration over 60 minutes. 
Storage and stability: vials of cyclophosphamide are stored at room temperature. Once 
reconstituted as directed, solutions of cyclophosphamide are stable for 24 hours at room 
temperature, or 6 days when refrigerated at 2-8o C.  
Route of administration: the prescribed dose of cyclophosphamide will be diluted in an additional 
250 mL of 0.9% sodium chloride injection or 5% dextrose in water for intravenous administration 
over 60 minutes. 
Toxicities: see section 13.3.1. 
 
14.3 
CYCLOSPORINE (Gengraf, Sandimmune, Neoral) 
Supply / product description:  cyclosporine will be obtained by the NIH Clinical Center Pharmacy 
Department from commercial sources and is available in capsules (25 mg and 100 mg), USP 
[MODIFIED], oral solution (100 mg/ml), USP [MODIFIED], and as a parenteral concentrate for 
injection (50 mg/ml).  When oral capsules are prescribed for this protocol, the cyclosporine 
capsules, USP [NON-MODIFIED] should NOT be used. 
Preparation:  for parenteral doses, each milliliter of concentrate (50mg/ml) should be diluted in 20 
to 100ml of dextrose 5% in water or sodium chloride 0.9%.  Parenteral doses of cyclosporine will 
be prepared in non-PVC containers and infused with non-PVC administration sets/tubing.  Oral 
cyclosporine solution may be mixed in orange juice or other beverages, but not milk. 
Storage and stability:  capsules, oral solution, and ampules of parenteral concentrate bear expiration 
dates and are stored at room temperature and protected from light.  Cyclosporine concentrate for 
injection that has been diluted to a final concentration of approximately 2mg/ml is stable for 24 
hours in 5% dextrose or 0.9% sodium chloride injection in glass, PVC or non-PVC plastic 
containers.  To minimize the potential for absorption to PVC plastic bags and tubing as well the 
leaching of phthalate plasticizer (DEHP) into the solution, only non-PVC plastic bags and 
intravenous administration sets should be utilized. 
Administration:  cyclosporine may be given intravenously or orally. 
Toxicities: refer to section 13.3.1 

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14.4 
FLUDARABINE PHOSPHATE (Fludara) 
Supply:  commercially available. 
Product description: fludarabine phosphate is commercially available as both a lyophilized 
powder for injection in vials containing 50 mg of fludarabine phosphate with mannitol 50mg and 
sodium hydroxide for pH adjustment and a solution for injection in 2 mL vials containing 50 mg 
of fludarabine phosphate (25 mg/mL of fludarabine) with 25 mg/mL mannitol and sodium 
hydroxide for pH adjustment. 
Preparation: fludarabine lyophilized powder for injection should be reconstituted with 2 mL of 
sterile water for injection, USP to a concentration of 25 mg/mL. The prescribed dose of 
fludarabine should be diluted in 100 mL of either 0.9% sodium chloride or 5% dextrose in water 
for intravenous administration over 30 minutes. 
Storage and stability: fludarabine vials should be stored under refrigeration between 2o-8o Celsius 
(36o- 46o F).  Reconstituted fludarabine phosphate is chemically and physically stable for 24 
hours at room temperature or for 48 hours if refrigerated. The manufacturer recommends use of 
either the reconstituted powder for injection or the solution for injection (once diluted for 
administration) within 8 hours because neither product contains an antimicrobial preservative. 
Administration: the prescribed dose of fludarabine should be diluted in 100 mL of either 0.9% 
sodium chloride or 5% dextrose in water for intravenous administration over 30 minutes. 
 
14.5 
FILGRASTIM (G-CSF, Neupogen) 
Supply: commercially available. 
Product description: filgrastim injection is available in a concentration of 300mcg/ml in 1ml 
(300mcg) and 1.6ml (480mcg) vials. 
Preparation: for subcutaneous administration, the appropriate prescribed dose is drawn up from 
the vial with no further dilution prior to administration. For intravenous administration, the 
commercial solution for injection should be diluted prior to administration. It is recommended 
that the prescribed dose be diluted with dextrose 5% in water (DO NOT DILUTE WITH 
NORMAL SALINE) to a concentration greater than 5mcg/ml. Filgrastim diluted to 
concentrations between 5 and 15mcg/ml should be protected from absorption to plastic materials 
by the addition of Albumin (Human) to a final concentration of 2mg/ml. When diluted in 5% 
dextrose or 5% dextrose plus Albumin (Human), filgrastim is compatible with glass bottles, PVC 
and polyolefin IV bags, and polypropylene syringes.   
Storage and stability: filgrastim for injection should be stored in the refrigerator at 2o to 8oC (36o 
to 46oF). Avoid shaking. 
Route of administration: subcutaneous injection or intravenous infusion over 15-30 minutes. 
 
14.6 
METHOTREXATE  
Supply: Commercially available 
Product Description: Methotrexate is available as a 25 mg/mL preservative-free isotonic solution 
for injection. 
Preparation: The desired does will be diluted in 25 to 50 ML of 5% dextrose in water of 0.9% 
sodium chloride. 
Storage and Stability: Methotrexate should be stored at room temperature and protected from 
light. Once the prepared does is diluted for administration, the solution is stable for 24 hours 
refrigerated or at room temperature when protected from light. 
Route of Administration: Methotrexate will be given as an IV infusion over 15 minutes. 
Toxicities: refer to section 13.3.1 
 
 

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55
15.0 
REFERENCES 
Appelbaum, F.R. (2001) Haematopoietic cell transplantation as immunotherapy. Nature, 411, 
385-389. 
Armitage, J.O. (1994) Bone marrow transplantation. N.Engl.J.Med., 330, 827-838. 
Barrett, A.J. (1993) Graft-versus-host disease: basic considerations. Recent Results Cancer Res., 
132, 185-195. 
Barrett, A.J. (1997) Mechanisms of the graft-versus-leukemia reaction. Stem Cells, 15, 248-258. 
Barrett, A.J. & Battiwalla, M. (2011) Relapse after allogeneic stem cell transplantation. Expert 
Rev Hematol, 3, 429-441. 
Barrett, A.J. & Malkovska, V. (1996) Graft-versus-leukaemia: understanding and using the 
alloimmune response to treat haematological malignancies. Br.J.Haematol., 93, 754-761. 
Battiwalla, M., et al. (2011) 4-Log ex-vivo T-Lymphocyte Depleted Myeloablative HLA-
Matched Sibling Transplants; a Platform for Adoptive Immunotherapy Influenced by 
Conditioning Intensity. Biology of blood and marrow transplantation : journal of the American 
Society for Blood and Marrow Transplantation, 17, S287. 
Beatty, P.G., et al. (1991) Marrow transplantation from HLA-matched unrelated donors for 
treatment of hematologic malignancies. Transplantation, 51, 443-447. 
Devine, S.M., et al. (2011) Low Risk of Chronic Graft-versus-Host Disease and Relapse 
Associated with T Cell-Depleted Peripheral Blood Stem Cell Transplantation for Acute 
Myelogenous Leukemia in First Remission: Results of the Blood and Marrow Transplant Clinical 
Trials Network Protocol 0303. Biol Blood Marrow Transplant. 
Elias, A.D., et al. (1992) Mobilization of peripheral blood progenitor cells by chemotherapy and 
granulocyte-macrophage colony-stimulating factor for hematologic support after high-dose 
intensification for breast cancer. Blood, 79, 3036-3044. 
Georges, G.E. & Storb, R. (2003) Review of "minitransplantation": nonmyeloablative allogeneic 
hematopoietic stem cell transplantation. Int.J.Hematol., 77, 3-14. 
Goldman, J.M., et al. (1988) Bone marrow transplantation for chronic myelogenous leukemia in 
chronic phase. Increased risk for relapse associated with T-cell depletion. Ann.Intern.Med., 108, 
806-814. 
Kang, E.M., et al. (2002) Mobilization, collection, and processing of peripheral blood stem cells 
in individuals with sickle cell trait. Blood, 99, 850-855. 
Kopp, H.G., et al. (2007) Granulocyte colony-stimulating factor induced pulmonary hemorrhage 
in a healthy stem cell donor. J Clin Oncol, 25, 3174-3175. 
Marmont, A.M., et al. (1991) T-cell depletion of HLA-identical transplants in leukemia. Blood, 
78, 2120-2130. 
Montero, A., et al. (2006) T Cell Depleted Peripheral Blood Stem Cell Allotransplantation with T 
Cell Add Back for Subjects with Hematological Malignancies: Effect of Chronic GVHD on 
Outcome. Biology of Blood and Marrow Transplantation, 12, 1318-1325. 
Platzbecker, U., et al. (2004) Allogeneic transplantation of CD34+ selected hematopoietic cells--
clinical problems and current challenges. Leuk.Lymphoma, 45, 447-453. 
Storb, R., et al. (1986) Methotrexate and cyclosporine compared with cyclosporine alone for 
prophylaxis of acute graft versus host disease after marrow transplantation for leukemia. 
N.Engl.J.Med., 314, 729-735. 
Vij, R., et al. (1999) Unstable angina in a peripheral blood stem and progenitor cell donor given 
granulocyte-colony-stimulating factor. Transfusion, 39, 542-543. 
Weaver, C.H., et al. (1995) An analysis of engraftment kinetics as a function of the CD34 content 
of peripheral blood progenitor cell collections in 692 subjects after the administration of 
myeloablative chemotherapy. Blood, 86, 3961-3969. 
Weisdorf, D., et al. (1990) Treatment of moderate/severe acute graft-versus-host disease after 
allogeneic bone marrow transplantation: an analysis of clinical risk features and outcome. Blood, 
75, 1024-1030 

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16.0 
APPENDICES 
 
APPENDIX A 
NHLBI HEMATOLOGY BRANCH LABORATORY 
RESEARCH STUDIES -2/5/2013 
 
DESCRIPTION OF LABORATORY STUDY BY BRANCH SECTION 
Does this test 
pose a greater 
than minimal 
risk to 
pediatric 
subjects per 45 
CFR 46.404? 
Does this test 
pose a 
greater than 
minimal risk 
to healthy 
pediatric 
donors per 45 
CFR 46.404? 
 
 
 
 
A 
Stem Cell Allotransplantation Section (Dr. A. John Barrett) 
 
 
A.1 
Measurement of lymphocyte function and immune responses directed toward 
allogeneic tissues, malignant cells, and infectious agents.  Assay of a variety of 
antigens, including standard proliferation, cytotoxicity, and intracellular cytokine  
detection including GVHD predictive markers. Measurement of antigen-specific 
responses including employment of tetramers, ELISPOT technique, gene 
amplification-based assays, and flow cytometry.  Selection of cells using 
immunomagnetic beads or flow cytometry.  Culture, expansion, and selection of 
cells. Surface marker analysis of PB MC using flow cytometry. Cytokine/chemokine 
analysis of plasma/serum samples using ELISA and/or Luminex techniques. 
 
No 
No 
A.2 
Generation of cell lines for the study of immune cell interactions with other cells.  
Transformation of B-lymphocytes using Epstein-Barr virus.  Derivation of malignant 
cell lines from patient leukemic or solid tumor samples. 
No 
No 
A.3 
Infection of cells and cell lines with recombinant genes to ascertain the effects of 
expressed molecules on immune responses and on growth and development. 
Transfection of cell lines with specific molecules to study antigen-specific responses. 
No 
No 
A.4 
Assays of peripheral blood and bone marrow progenitor cells including primitive and 
late erythroid progenitor-derived colonies, myelomonocytic colonies, and primitive 
multi- potential progenitor-derived colonies. 
No 
No 
A.5 
Injection of human cells into experimental animals to study the immune system and 
the growth of normal and malignant cells under varying conditions. 
No 
No 
A.6 
Testing of selection methods, cell isolation, and cell expansion leading to the 
development of new cell-based therapies requiring scale-up for clinical application. 
No 
No 
A.7 
Identification of individual T cell clones by their T cell receptor sequence. 
No 
No 
A.8 
Measurement of tumor and tissue specific antigens in cells of subjects and donors by 
mRNA,protein, or peptide expression in cells or fluids. 
No 
No 
A.9 
Laser capture micro dissection of cells from biopsies for GVHD to determine 
clonotypes. 
No 
No 
A.10 
DNA and RNA typing of genes that control immune responses in lymphocytes. 
 
No 
No 
A.11 
Microassay studies utilizing cellular DNA, cDNA, and RNA for neoplasia and host-
tumor interactions. 
No 
No 
A12 
Serum, blood and tissue markers of post-transplant tissue injury, particularly 
endothelial cell damage. Investigation of transplant survivors for metabolic 
derangements related to cardiac and vascular risk such as lipoprotein profiles, insulin 
No 
No 

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resistance, diabetes markers, growth hormone signaling, hypertension, renal 
dysfunction. 
 
 
 
 
B 
Molecular Hematopoiesis  Section  (Dr. Cynthia Dunbar)  
 
 
B.1 
Flow cytometric analysis of cell surface and cytoplasmic proteins, including cell 
adhesion molecules, putative retroviral receptors, and markers of differentiation, 
using bone marrow and mobilized peripheral blood cells. 
No 
No 
B.2 
Hematopoietic progenitor-derived colony ascertainment in vitro (as described above), 
and engraftment of immunodeficient mice for detection of human stem cell number 
and function. 
No 
No 
B.3 
Testing ability of hematopoietic progenitor cells to be transduced with retroviral, 
lentiviral, and novel gene transfer vectors in vitro. 
No 
No 
B.4 
Reprogramming of adult mature cells, including skin fibroblasts and blood cells, into 
induced pluripotent stem cells in vitro. 
No 
No 
 
 
 
 
C 
Cell Biology Section (Dr. Neal Young)  
 
 
C.1 
Studies of blood and bone marrow hematopoietic progenitor numbers, including 
early and late erythroid progenitors, myelomonocytic progenitors, and multi-potential 
progenitor cells.  In addition, bone marrow may be placed in long-term bone marrow 
culture to assess the function of stroma and stem cells and to assay more primitive 
progenitors, as well as organelle culture.  Whole or selected bone marrow 
populations are cultured short-term for CD34 cell expansion. 
No 
No 
C.2 
Assays of apoptosis in hematopoietic cells and their progeny, using flow cytometric 
methods such as annexin and caspase-3 staining, propidium iodide uptake, and 
mitochondrial permeability tests. 
No 
No 
C.3 
Separation and functional study of cell populations characteristic of paroxysmal 
nocturnal hemoglobinuria, identified by absence of glycosylphosphatidylinositol 
anchored proteins. 
No 
No 
C.4 
Studies of mutation rates in hematopoietic cells and in buccal mucosa cells, using 
conventional hypoxanthine phosphoribosyltransferase activity functional assays, 
sequencing of mitochondrial DNA after specific gene amplification, and 
measurement of GPI-anchored deficient cells in blood and bone marrow. 
No 
No 
C.5 
Assays of immune function of T-cells, including intracellular cytokine staining, 
ELISPOT, semiquantitative gene amplification for gamma-interferon, tumor necrosis 
factor, interleukin-2, and other cytokines, and functional assessment in co-culture 
using specific neutralizing monoclonal antibodies.  In addition, peripheral blood 
lymphocytes are subjected to spectratyping for CDR3 size distribution as well as 
nucleotide sequence of CDR3 peaks obtained. 
No 
No 
C.6 
Studies of engraftment of human normal and diseased bone marrow and peripheral 
blood in immunodeficient mice in order to determine the presence of hematopoietic 
repopulating stem cells as well as functional differences among selected populations. 
No 
No 
C.7 
Flow cytometric analysis of blood and bone marrow for lymphocyte phenotype, 
especially for evidence of activation of lymphocytes, for markers of apoptosis, and 
for antigens associated with primitive and mature hematopoietic cell populations. 
No 
No 
C.8 
Flow cytometric analysis of blood and bone marrow for hematopoietic stem cell 
progenitors and CD34 positive cells. 
No 
No 
C.9 
Studies of chromosomal instability in myelopdysplastic syndromes including BM 
cell and CD34 cell response to PAS crosslinking and examination of the cytotoxic 
effect of lymphocytes to the abnormal clone of cells. 
No 
No 
C.10 
Surface Enhanced Laser/Desorption Ionization (SELDI)  time-of-flight mass 
spectrometry (Ciphergen)  (proteomics methodology). 
No 
No 
C.11 
Mitochondrial DNA (mtDNA) sequence heterogeneity. 
No 
No 

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58
C.12 
Measurement of EBV viral load. 
No 
No 
C.13 
Measurement of EBV LMP-1 via RT-PCR for LMP-1 RNA or flow cytometry for 
LMP-1. 
No 
No 
C.14 
Outgrowth assay of EBV transformed B cells. 
No 
No 
C.15 
Quantification of serumchemokines and cytokines (e.g. SDF-1, IL-10, IL-6, CXCR4, 
CXCL12). 
No 
No 
C.16 
Quantification of EBV cytotoxic T cells (tetramerstaining). 
No 
No 
C.17 
Telomere length measurement by Southern blot, Q-PCR, flow-fish, in situ 
hybridization and STELA 
No 
No 
C.18 
Telomere repair complex gene mutations by nucleotide sequencing of some or all of 
the following:  DKC1 ,TERC, TERT, SBDS, NOp10 , NHP2. 
No 
No 
C.19 
Analysis of  inflammatory markers and/or bacterial, viral, fungal or protozoal 
elements in plasma or serum using molecular, colorimetric, enzymatic, flow 
cytometric or other assays in subjects receiving immunosuppressive therapy, 
chemotherapy and/or bone marrow transplantation. 
No 
No 
C.20 
Confocal microscopic imaging of bone marrow. 
No 
No 
C.21 
Characterization of intracellular signaling proteins by cell permeabilization and flow 
cytometry, and quantitative immunoblots. 
No 
No 
C.22 
Assays for chromosomal aneuploidy by florescence in situ hybridization (FISH) and 
other molecular techniques. 
No 
No 
C.23 
Conversion of human dermal fibroblasts into hematopoietic progenitors using Oct4 
transfection. 
No 
No 
 
C.24 
Quantification of gene expression with RNA-seq 
No 
No 
C.25 
Characterization of chromatin and promoter/enhancer landscapes with ATAC-seq 
No 
No 
C.26 
Measurement of protein markers with SomaLogic's SOMAscan assay 
No 
No 
 
 
 
 
D 
Virus Discovery Section (Dr. Neal Young) THESE ASSAYS WILL NOT BE 
PERFORMED ON SAMPLES FROM HEALTHY PEDIATRIC DONORS 
 
 
D.1 
Assays of serum, blood cells, and bone marrow cells for B19 parvovirus and possible 
B19  variants using gene amplification, cell culture, and hematopoietic colony 
inhibition assays. 
No 
N/A 
D.2 
Assays of blood, bone marrow, liver, and other tissues for potentially novel viruses, 
using a variety of techniques including RNA and DNA assays, differential display, 
gene amplification with conserved and random primers, cell culture assays, 
immunohistochemical methods, and inocculation of mice, rabbits, and monkeys, as 
well as antibody measurements. 
No 
N/A 
D.3 
Assays of blood, bone marrow, and liver for known viruses, including herpesviruses 
such as cytomegalovirus, human herpesviruses 6, 7, and 8, enteric viruses such as A-
6, circiviruses, and parvoviruses, using assays as in (2). 
No 
N/A 
D.4 
Spectra-typing of blood cells to determine response to known or putative viral 
infections. 
No 
N/A 
D.5 
HLA typing or subtyping to determine risk factors/determinants for hepatitis-AA 
studies. 
No 
N/A 
D.6 
Cytotoxic lymphocyte assays with intracellular cytokine measurement for 
determining anti-viral response and lymphocyte cloning to obtain clones with 
specific antiviral activity. 
No 
N/A 
 
 
 
 
E 
Solid Tumor Section (Dr. Richard Childs) 
 
 
E.1 
Cr51 cytotoxicity assay to evaluating killing of patient tumor cells by patient NK cell 
clones and T-cells. 
No 
No 
E.2 
ELISA for IL-12  maturity of DC's made from subjects monocytes. 
No 
No 
E.3 
ELISA for IFN ã to evaluate specificity of CTL clones. 
No 
No 

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59
E.4 
H thymidine uptake to evaluate proliferation potential of antigen specific T-cells. 
No 
No 
E.5 
PCR of STR to assess chimerism status of cellular subsets grown in-vitro or retrieved 
from subjects post-transplant. 
No 
No 
E.6 
Flow sorting of PBL and/or tissue samples to evaluate chimerism of different subsets. 
No 
No 
E.7 
Surface marker analysis of peripheral blood mononuclear cells using flow cytometry. 
No 
No 
E.8 
cDNA expression arrays to evaluate T-cells expression/gene patterns in subjects with 
GVHD and a GVT effect. 
No 
No 
E.9 
Geno typing of tumor or tissue samples by high density cDNA arrays. 
No 
No 
E.10 
VHL mutation analysis on kidney cancer tissue. 
No 
No 
E.11 
Transduction of dendritic and tissue cells with tumor antigens using plasmids, viral 
vectors and hybrid fusions. 
No 
No 
E.12 
Lasar capture microdisection of cells from tumor biopsies and tissue samples  to 
determine origin (donor vs patient). 
No 
No 
E.13 
Quantification of polyoma virus BK exposure by serology and PCR in stem cell 
transplant donors and recipients from blood and urine samples. 
No 
No 
E.14 
Quantification of polyoma virus BK specific T cells in stem cell transplant donors 
and recipients from peripheral blood samples. 
No 
No 
E.15 
Determination of origin of neovasculature endothelial cells in tumor and tissue 
samples obtained from subjects post transplant. 
No 
No 
E.16 
Quantification of lymphocyte subsets CD34 progenitors and endovasculator 
progenitors in G-CSF mobilized peripheral cell allografts. 
No 
No 
E.17 
Testing for polyoma virus BK latency in CD34 progenitors, B cells and T cells in the 
G-CSF mobilized peripheral cell allografts. 
No 
No 
E.18 
Determination of etiology of membraneous nephropathy using serum from subjects. 
No 
No 
E.19 
Serum Proteomic patterns analysis to diagnose complications related to allogeneic 
transplantation. 
No 
No 
E.20 
Determine cell origin (donor vs patient) of tissue samples using IHC, IF, sorting, and 
FISH. 
No 
No 
 
 
 
 
F 
Lymphoid Malignancies Section (Dr. Adrian Wiestner) 
 
 
F.1 
Culture of cells from research subjects to investigate molecular disease mechanisms, 
model host tumor interactions, and to test effect of drugs on cell survival and cellular 
functions. 
No 
No 
F.2 
Generation of stable cell lines for the study of hematologic malignancies. 
No 
No 
F.3 
Modifications of cells using standard expression systems or biologic molecules, e.g. 
interfering RNA, to investigate the effects of candidate genes on cellular functions. 
 
 
F.4 
Identification and monitoring of B or T cell populations as identified by flow 
cytometry and by their B cell or T cell receptor expression. 
No 
No 
F.5 
Measurement of gene expression in cells or tissues. Techniques frequently used 
include gene expression profiling on microarrays, quantitative RT-PCR, Western 
blotting, flow cytometry and ELISA assays. 
No 
No 
F.6 
Analysis of chromosomal abnormalities or mutations in malignant cells and non-
malignant cells including FISH technology and DNA sequencing. 
No 
No 
F.7 
Assays of immune function of B-cells and T-cells, including intracellular cytokine 
staining, ELISPOT, quantitative RT-PCR for cytokines or other immune regulatory 
genes. 
No 
No 
F.8 
Analysis of antibody specificities in serum and antigen specificity of the B-cell 
receptor on cells. Techniques may include expression of antibodies in phage display 
systems, generation of antibodies in cell culture systems and use of such antibodies to 
screen for cognate antigens. 
No 
No 
F.9 
Transplantation of human cells into mice (xenograft model) to study disease biology 
and to investigate the effect of experimental therapy. 
No 
No 

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60
 
 
 
 
APPENDIX B: GVHD scoring scales 
 
 
 
 
F.10 
Measurements of drug concentrations, biologic molecules and disease markers in 
blood, serum, and plasma. 
No 
No