A Reduced Intensity Conditioning Regimen With CD3-Depleted Hematopoietic Stem Cells to Improve Survival for Patients With Hematologic Malignancies Undergoing Haploidentical Stem Cell Transplantation
Phase: PHASE2
Conditions: Leukemia, Acute Lymphocytic (ALL), Leukemia, Myeloid, Acute(AML), Leukemia, Myeloid, Chronic(CML), Juvenile Myelomonocytic Leukemia (JMML), Hemoglobinuria, Paroxysmal Nocturnal (PNH), Hodgkin Lymphoma, Lymphoma, Non-Hodgkin (NHL), Myelodysplastic Syndrome (MDS)
Sponsor: St. Jude Children's Research Hospital

HIFLEX 
Initial version, dated: 03-01-07 (IRB approval: 10-23-07) Activation Date: 11-29-07 
Amendment 1.0 August 01, 2008 (IRB approval: October 28, 2008) Activated: 12/08/08 
Amendment 2.0 December 31, 2008 (IRB approval: February 10, 2009) Activated: 2/17/09  
Revision 2.1 March 17, 2009 (IRB approved: March 18, 2009) Activated: 03/20/09 
Revision 2.2 April 24, 2009 (IRB approved: April 27, 2009) Activated: May 7, 2009 
Amendment 3.0 October 30, 2009 (CPSRMC approved: December 15, 2009) 
Amendment 3.0 January 7, 2010 Appendix E (IRB approved: January 12 2010)  
Revision 3.1 May 12, 2010 (IRB approved: May 28, 2010) Activation Date: June 10, 2010  
Amendment 4.0 September 27, 2010 CTSRMC Resubmission Date: December 28, 2010 
(IRB approval:  February 8, 2011) Activation Date: February 17, 2011 
Amendment 5.0 July 19, 2011 (IRB Approved: October 11, 2011) 
Amendment 6.0 May 10, 2012 (IRB Approved: August 1, 2012) Activated: August 22, 2012 
Revision 6.1, May 30, 2013 (IRB Approved July 9, 2013) Activated: 
Revision 6.2, December 24, 2014 (IRB Approved: March 2, 2015) Activated: 
Un-numbered Revision, November 18.2016  
 
A REDUCED INTENSITY CONDITIONING REGIMEN WITH CD3-DEPLETED 
HEMATOPOIETIC STEM CELLS TO IMPROVE SURVIVAL FOR PATIENTS                        
WITH HEMATOLOGIC MALIGNANCIES UNDERGOING HAPLOIDENTICAL                  
STEM CELL TRANSPLANTATION 
IDE #9162 
Principal Investigators 
 
Brandon Triplett, MD1 
 
Sub-Investigators 
Shane Cross, Pharm.D4 
William Janssen, PhD1 
Randall Hayden, MD3  
Sheila Shurtleff , PhD3 
Amr Qudeimat, MD1 
Ashok Srinivasan, MD1 
Terrence Geiger, MD, PhD3 
Guolian Kang, PhD2 
 
Ewelina Mamcarz, MD1 
 
 
 
 
Department of Bone Marrow Transplantation and Cellular Therapy1 
Department of Biostatistics2 
Department of Pathology3 
Department of Pharmaceutical Sciences4 
Department of Therapeutics, Production and Quality 5 
St. Jude Children’s Research Hospital 
262 Danny Thomas Place 
Memphis, TN  38105-3678 
(901) 595-3300 
This is a confidential research document.  No information may be extracted without permission of the principal investigator. 

 
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TABLE OF CONTENTS 
Study Summary 
 
1.0 
Objectives   ………………………………………………………………….. 
1 
2.0 
Background and Rationale   ………………………………………………… 
1 
3.0 
Research Participation Eligibility Criteria   ………………………………… 
18 
4.0 
Treatment Plan   …………………………………………………………….. 
20 
5.0 
Drug and Device Information   ……………………………………………... 
27 
6.0 
Required Observations and Evaluations   …………………………………… 
35 
7.0 
Evaluation Criteria   ………………………………………………………… 
38 
8.0 
Off-study and Off-therapy Criteria   ………………………………………… 
38 
9.0 
Reporting Criteria   ………………………………………………………….. 
40 
10.0 
Statistical Considerations   ………………………………………………….. 
43 
11.0 
Data Acquisition and Quality Assurance   ………………………………….. 
50 
12.0 
Obtaining Informed Consent   ……………………………………………… 
51 
13.0 
References   …………………………………………………………………. 
52 
 
 
Checklists 
 
 
Appendices 
Informed Consent Statements 
 
 
Investigators Brochures 
 
 
 
 
 

 
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   STUDY SUMMARY 
Allogeneic hematopoietic stem cell transplantation (HSCT) has improved the outcome for 
patients with high-risk hematologic malignancies. However, many patients do not have a 
matched sibling donor available or are unable to identify an acceptable unrelated donor in a 
timely manner. Therefore, many of these patients are unable to undergo allogeneic 
transplantation, which is the preferred or sole curative treatment for their disorder. 
Haploidentical (HAPLO) HSCT, using a mismatched family member, is another transplantation 
option for these patients. While this method of transplantation has proven curative in many 
patients, there are significant transplant-related issues that require further study.   
Graft-versus-host disease (GVHD), infections due to delayed immune reconstitution, regimen-
related toxicity, posttransplant lymphoproliferative disorder (PTLPD), and relapse have been and 
continue to be barriers to successful haploidentical transplantation, resulting in death or a poor 
quality of life in transplant recipients. To reduce the incidence of severe GVHD, patients 
undergoing HAPLO HSCT must receive grafts depleted of T lymphocytes.  In prior studies at 
our institution, we demonstrated that HAPLO HSCT with CD34+ selected or CD3-depleted 
grafts is feasible, resulting in prompt engraftment with low rates of PTLPD, which has been 
treatable in the majority of cases.   
In our first clinical trial, HAPSCT, we used a myeloablative total body irradiation (TBI)-based 
regimen for children and young adults with high-risk hematologic malignancies undergoing 
HAPLO HSCT.  All participants engrafted promptly and survival rates were promising, but 
regimen-related toxicity and disease recurrence were significant problems. Immune 
reconstitution was delayed, and viral reactivation occurred commonly.  A concurrent trial 
(REFSCT) using a reduced intensity conditioning regimen for patients undergoing second 
allogeneic HSCT or with refractory disease, demonstrated prompt engraftment with low rates of 
severe GVHD and regimen-related toxicity. When compared with the high-risk patients 
undergoing HAPLO HSCT, the refractory group had more rapid immune reconstitution with 
lower rates of viral reactivation.  Based on these observations, our next generation of clinical 
trials, HAPREF, used a reduced intensity regimen for both patient populations (high-risk and 
second HSCT/refractory) combined with a higher CD3+ graft content.  However, in this latter 
study intentionally employing grafts with higher CD3+ graft content to increase anti-leukemia 
effects, we observed that the higher CD3+ graft content was associated with higher rates of 
overall grade III-IV acute GVHD.  
In this clinical trial, we aim to improve event-free survival by reducing GVHD and regimen-
related mortality by increasing the tempo of immune reconstitution using a reduced intensity-
conditioning regimen.  Transplant recipients will receive a mismatched family member donor 
graft partially depleted of T lymphocytes using the investigational CliniMACS device and the 
anti-CD3 antibody, with a limited number of CD3 cells in the graft.  For participants who meet 
the maximum CD3 dose on the first day of collection, the second day of collection will be 
depleted of T lymphocytes by CD34+ selection using an anti-CD34 antibody and the 
CliniMACS system.  This product manipulation will be used to derive an allogeneic graft with an 
acceptable CD3 content and maximal CD34 content from cytokine-mobilized peripheral blood 
stem cell products. A reduced intensity-conditioning regimen without TBI will be employed in 
an effort to reduce regimen-related toxicity and mortality. 

 
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This protocol will describe event-free, disease-free, and overall survival following HAPLO 
HSCT with a graft partially depleted of CD3+ cells using the CliniMACS system.  Secondly, this 
trial will define a platform for performing HAPLO HSCT safely so that future studies of 
posttransplant immunomodulation designed to improve immune reconstitution and to generate 
anti-leukemia effects can be employed.  Additional goals of this protocol are to estimate the rates 
of acute and chronic GVHD, graft failure, and peri-transplant morbidity and mortality in this 
group of research participants.  We will also explore defined biologic markers as predictive 
factors for the development of acute and chronic GVHD in these participants.  Ultimately, this 
study will serve as a foundation on which to build future studies of immunomodulation and 
immune reconstitution in the posttransplant setting with the ultimate goal of identifying 
strategies that significantly decrease disease recurrence, enhance immune reconstitution, and 
improve overall treatment outcome for patients with high-risk hematologic malignancies 
undergoing HAPLO HSCT. 
 
Identification of the St. Jude protocols referred to throughout document: 
HAPSCT 
Title: Haploidentical Stem Cell Transplantation Utilizing Purified CD34+ Hematopoietic Cells for 
Patients with Hematological Malignancies 
Total number of transplant recipients = 27  
 
Arms/strata:  
Arm A =  CD34 positive selection - N = 7 
Arm B = CD3 negative selection - N=20 
Date of first transplant:  05/2002 
Date of last transplant: 06/2005 
 
REFSCT 
Title: Haploidentical Stem Cell Transplantation Utilizing T-Cell Depletion as Therapy for 
Patient with Refractory Hematological Malignancies 
Total number of transplant recipients = 25 
Arms/strata:  
Not applicable, 1 arm/not stratified 
Date of first transplant:  06/2003   
Date of last transplant: 07/2005 
 
HAPREF 
Title:  Haploidentical Hematopoietic Stem Cell Transplantation Utilizing Partial T-Cell 
Depletion as Immunotherapy for Hematologic Malignancies.   
Total number of transplant recipients = 17  
Arms/strata Arm A  = High-Risk Disease - N = 9   Arm B  = Refractory Disease - N = 8 
Date of first transplant:  09/2005                         Date of last transplant: 03/2006 
 
 

 
  
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1.0 OBJECTIVES  
1.1 Primary objective:   
1.1.1 To assess if the event-free survival at one-year posttransplant for research 
participants with high-risk hematologic malignancies can be improved following 
HAPLO HSCT using a graft depleted of CD3+ cells ex vivo and a reduced 
intensity-conditioning regimen. 
1.2 Secondary objectives:  
1.2.1.. To estimate the one-year overall survival (OS) and disease-free survival (DFS) for 
research participants who receive this study treatment. 
1.2.2  To estimate the cumulative incidence of relapse for research participants who 
receive this study treatment. 
1.2.3  To estimate the rate of overall grade III-IV acute GVHD, and the rate and severity 
of chronic GVHD in research participants. 
1.2.4  To estimate the incidence of non-hematologic regimen-related toxicity and 
regimen-related mortality in the first 100 days posttransplant. 
1.3 Exploratory objectives: 
1.3.1  To explore the biologic significance of soluble interleukin-2 receptor and 
immunologic state (quantitative lymphocyte studies, V beta spectratyping, TREC 
assay) to predict the development of acute and chronic GVHD in these research 
participants. 
1.3.2 To measure the pharmacokinetics of Campath-1H in pediatric HAPLO HSCT 
recipients 
2.0 BACKGROUND AND RATIONALE  
2.1 Introduction 
Allogeneic HSCT is curative for many childhood hematologic malignancies that fail 
standard dose chemotherapy.  Unfortunately, many patients do not have an HLA-
identical sibling donor or an appropriate matched unrelated donor (MUD) identified.   
Furthermore, regimen-related toxicity, acute and chronic GVHD, prolonged immuno-
deficiency, and disease recurrence remain significant causes of morbidity and mortality 
among patients who do undergo transplant.  Thus, therapies that reduce regimen-related 
toxicity, GVHD, and relapse while promoting immune reconstitution may improve 
disease-free survival and quality of life, allowing this treatment to be extended to all 
patients who require it.  Nearly all patients have a readily available mismatched family 
member donor. However, T-cell depletion of these grafts, predominantly by negative 
selection strategies, has been necessary for successful transplantation. Even so, severe 
GVHD, infections, and PTLPD are significant problems.    
Preliminary data suggest that grafts depleted of CD3+ cells engraft promptly in patients 
receiving stem cell grafts from alternate donors (unrelated or HAPLO family member 
donors). Therefore, for patients lacking a matched sibling or an available unrelated donor, 
the transplant program at St. Jude Children’s Research Hospital (St. Jude) plans to utilize 

 
  
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grafts containing large numbers of hematopoietic progenitor cells that are depleted of T 
lymphocytes.  The graft will be engineered to the target values using the CliniMACS 
system to specifically deplete CD3+ T cells using the OKT-3 antibody.  This protocol 
will also address specific issues of engraftment, GVHD, chimerism, regimen-related 
toxicity, disease recurrence, and posttransplant cellular therapies based on chimerism 
status. 
2.2 Indications for HSCT 
Many childhood leukemias that cannot be cured with chemotherapy alone can be 
effectively treated by allogeneic bone marrow transplantation.1,2 Moreover, for patients 
with chronic myeloid leukemia (CML), allogeneic HSCT is the only modality of treatment 
that is curative. Several studies have demonstrated that transplantation from a matched 
sibling donor reduces the risk of relapse in patients with acute myeloid leukemia (AML) in 
1st or 2nd remission and in patients with acute lymphoblastic leukemia (ALL) in 2nd 
remission.  However, although effective at eradicating disease in this context, improvements 
in survival are limited by the morbidity and mortality associated with the procedure.3-14 
Nevertheless, when marrow transplantation is undertaken using an HLA-matched sibling as 
the donor, multi-institutional studies have demonstrated that the procedure is curative in 60-
80% of patients with CML, 50% of patients with standard or high-risk leukemia (AML or 
ALL in 1st remission), 30% of patients with myelodysplasia, and 20-30% of patients with 
poor-risk leukemia (AML or ALL in second or subsequent remission, AML or ALL in 
relapse and secondary leukemia).10,12,15-17, These data have been corroborated by our single 
institution studies at St. Jude, with standard-risk recipients achieving an 81% 2-year disease-
free survival (DFS), and a 30% DFS being observed in recipients at high-risk of relapse.18  
Patients with non-Hodgkin lymphoma or Hodgkin disease who recur after autologous 
HSCT or who have refractory disease are generally not curable with autologous 
transplantation, salvage chemotherapy, or radiation therapy alone. In addition, some 
studies demonstrate high rates of morbidity and mortality in lymphoma patients 
undergoing allogeneic HSCT after failing previous autologous HSCT.19 These patients 
typically require more aggressive novel therapies such as allogeneic HSCT, which takes 
advantage of an allogeneic graft vs. lymphoma effect, while significantly reducing 
regimen-related toxicities. Preliminary studies in these patient populations are promising, 
suggesting that following allogeneic HSCT, even those with refractory and multiply 
relapsed disease can be long-term survivors.20-24 Allogeneic HSCT also allows the use of 
other novel therapeutic maneuvers such as the withdrawal of immunosuppression after 
allogeneic HSCT and donor lymphocyte infusions (DLIs or therapeutic cell, T-cell 
infusions) to enhance the graft vs. lymphoma effect. 
2.3 Hematopoietic stem cell transplantation using alternate donors 
Therapeutic options for the majority of patients with leukemia requiring allogeneic HSCT 
have focused on the use of two groups of alternate donors, given that only approximately 
30% of patients have a suitable matched sibling donor.25 HAPLO donors share a 
genotypically identical HLA-haplotype and possibly additional phenotypic identity on the 
unshared haplotype with the recipient.26Alternatively, the identification and utilization of 
unrelated donor bone marrow grafts, termed matched unrelated donors (MUD), who share 
phenotypic or genotypic HLA identity or near-identity with the recipient has been supported 

 
  
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by the establishment of the National Marrow Donor Program (NMDP)27However, many 
patients do not have an appropriate unrelated donor identified or formally requested for 
donation in a timely manner.  Thus, HAPLO donors are a viable alternative since these 
family members are highly motivated, easily accessible, and readily available for most 
patients.  
Transplantation with unmanipulated alternate donor stem cell grafts result in a significant 
increase in the incidence of GVHD and graft rejection (a form of host-versus-graft or HvG 
disease) when compared to recipients of matched sibling donor grafts, particularly with 
respect to HAPLO grafts. Several factors influence graft rejection including the degree of 
immunosuppression of the recipient, the genetic disparity between donor and recipient, and 
the decreasing numbers of CD3+, CD34+, and total nucleated cells/kg in the graft.  In 
contrast, the incidence of GVHD is not only associated with the degree of donor-recipient 
genetic disparity but also with the intensity of conditioning, the recipient's age, increasing 
numbers of CD3+ cells and decreasing numbers of CD34+ cells.28 Finally, this complex 
relationship is further compounded by the high-risk nature of the leukemias treated with this 
approach, as well as with the risk of increasing peri-transplant toxicity as it relates to the 
intensity of pre-transplant therapy.27-30 
2.4 The use of T-cell depleted allografts by negative selection 
Several approaches have been developed to reduce the incidence of GVHD in the HAPLO 
donor setting.  Because larger numbers of T cells (>3 x 106 CD3+ cells/kg) have resulted in 
grade III-IV acute GVHD with significant morbidity and mortality,28 investigators have 
utilized methods to remove T cells from grafts.  Methods of T-cell depletion are based 
predominantly on negative selection strategies.  These include physical methods such as T-
cell specific lectins in association with sheep RBC rosetting or counterflow elutriation.31-
33An alternate approach is the use of T-cell specific monoclonal antibodies in association 
with complement, immunotoxins or magnetic beads.34-45These approaches in reducing T cell 
number have resulted in an approximately 26 - 35% disease-free survival in high-risk 
leukemia, with a 16-39% incidence of grade II-IV GVHD and a 5-10% non-engraftment 
rate.27,46-48However, despite the use of various preparative regimens in this patient group, 
there have been no demonstrable differences in outcome by the type of regimen employed.  
Overall, 30-45% of patients succumb to regimen-related toxicity with an additional 30-40% 
dying from progressive disease or recurrence.26-28,46-53 
Our institutional experience at St. Jude with T-cell depleted MUD-HSCT (HUD, 
MISMUD) demonstrate that outcomes following MUD-HSCT (including both 5/6 and 
6/6 MUD as a single group) are similar to those from HLA-identical siblings.18 These 
studies were based on the infusion of an allograft depleted of T cells for unrelated donors, 
primarily utilizing a CD6/CD8 monoclonal antibody/complement cocktail. We observed 
a significant reduction in the incidence of GVHD-related complications utilizing this 
approach, the incidence of grade II-IV acute GVHD being 22% with a 19% incidence of 
chronic. Coincidentally, our graft failure incidence remains quite low (less than 5%) in 
recipients of alternate donor grafts.54  However, at our institution, HAPLO donor product 
recipients continued to have inferior overall survival (OS) and DFS when compared to 
MUD recipients even when adjusted for the number of recipients with high-risk disease, 
reflecting the historical observation that donor source is related to recipient outcome.55 

 
  
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In addition to regimen-related toxicity and disease recurrence, the use of T-cell depleted 
grafts is associated with higher rates of graft failure when compared to HSCT utilizing 
unmanipulated grafts. Indeed, this complication, coupled with a high incidence of 
GVHD, has hindered the use of the most available group of donors: mismatched family 
members.  Previous efforts to overcome this barrier have focused on the eradication of 
residual host-immunocompetent cells, the probable mediators of rejection. These include 
highly immunosuppressive chemo-radiotherapy conditioning regimens and/or the in vivo 
administration of ATG or monoclonal antibodies.56-59However, these approaches increase 
the risk of posttransplant infectious complications.  An alternate strategy is derived from 
studies by Handgretinger and other investigators, suggesting that increasing numbers of 
CD34+ hematopoietic cells may enable the recipient to better tolerate the genetic disparities 
between self and donor.60These results, coupled with studies in the autologous HSCT setting 
which demonstrate that large numbers of CD34+ cells can be harvested by peripheral blood 
apheresis, suggest a new approach to achieving stable engraftment with a reduced incidence 
of peri-transplant toxicity. 
2.5.Experience at St. Jude using allogeneic HSCT for patients with high-risk hematologic 
malignancies 
Between the years 1993 to 2000, 249 infants, children and young adults with high-risk 
hematologic malignancies underwent allogeneic HSCT at St. Jude (Laura Bowman, 
unpublished data).  Donor sources are listed below: 
Donor Source 
N 
Matched sibling donor 
84 
Unrelated donor matched at 6/6 HLA loci 
86 
Unrelated donor matched at 5/6 HLA loci 
48 
Mismatched family member  
31 
It has been the policy of the Transplant Program at St. Jude that children should receive 
grafts from an HLA-identical sibling when one is available. Eighty-four of these patients 
received HLA-identical sibling grafts during the time period.  For patients lacking a 
matched sibling, a search for a MUD was performed.  During this time period, acceptable 
unrelated donors were those matched at 5 or 6 HLA loci.  Of these, 86 and 48 patients 
received a 6/6 and 5/6 unrelated donor graft, respectively.  These patients received a graft 
depleted of T cells using CD6/CD8 monoclonal antibodies plus complement.  In 
recipients of MUD grafts matched at 6 HLA loci, grade 0, I, II, III, and IV acute GVHD 
occurred in 34, 38, 7, 3, and 4 patients, respectively. Chronic GVHD was seen in 9 of 86 
evaluable patients.  In patients with lymphoid and myeloid malignancies, the overall 
survival at one year is 49.2 + 10.1% and 55.7 + 6.3%, respectively, for recipients of 6/6 
MUD grafts. 
For recipients of marrow matched at 5/6 HLA loci, grade 0, I, II, III, and IV acute GVHD 
occurred in 15, 19, 3, 5, and 6 patients, respectively.  Chronic GVHD was seen in 9 of 48 
evaluable patients.  In patients with lymphoid and myeloid malignancies, the overall 
survival at one year is 36.4 + 13.0%% and 37.8 + 7.7%, respectively.  Thus, the 5/6 

 
  
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MUD transplant recipients had an inferior survival as compared to the 6/6 MUD 
transplant patients. 
During this time period, 31 patients with high-risk hematologic malignancies underwent 
allogeneic HSCT from HAPLO donors. All patients received the same graft 
manipulation, conditioning regimen and GVHD prophylaxis as those receiving unrelated 
donor grafts.  Fourteen patients had no acute GVHD, 6 experienced overall acute grade I, 
8 grade II, none with grade III, and 3 with grade IV.  Four of 16 evaluable patients had 
chronic GVHD.  Of the 8 patients transplanted from donors matched at 3/6 HLA loci, 
only 1 survives.  Of the 8 receiving grafts from donors matched at 4/6 HLA loci, 3 
survive.  Of 15 patients transplanted from donors matched at 5/6 HLA loci, 5 survive. 
Patients in the mismatched family member recipient group who have lymphoid 
malignancies have an overall survival of 22.2 + 13.9% at one year posttransplant; those 
with myeloid malignancies have an overall survival of 34.5 + 9.9% at this same one year 
posttransplant time period.    
It is important to note several facts about the patient population undergoing HAPLO 
HSCT at St. Jude between the years of 1993 to 2000.  These patients had high-risk 
hematologic malignancies and had been heavily pretreated and many had refractory 
disease.  Patients and their health care providers often chose to delay transplant as long as 
possible due to concerns over morbidity and mortality in those patients having only a 
HAPLO donor.  In addition, patients who had medical conditions or infections, which 
made allogeneic HSCT with traditional methodologies too risky, were not offered this 
potentially curative procedure.  Lastly, it is important to realize that patients who did not 
have an unrelated donor identified and confirmed through a registry search within a 90 
day period were unlikely to have one identified by prolonging the time required for the 
donor search.  Therefore, the number of patients who actually underwent HAPLO HSCT 
from 1993 to 2000 is an underestimation of the potential population who would benefit 
from this approach.  Thus, all patients who could potentially be cured by HSCT at our 
institution during this time period were not able to proceed to allogeneic HSCT if their 
only donor were a HAPLO.  Furthermore, from the donor-recipient match distribution, it 
is evident that this is a selected population with fewer patients transplanted with donors 
matched at 3 HLA loci and a preponderance of those transplanted matching at 4 or 5 
HLA loci.   
2.6 Allogeneic peripheral blood stem cell (PBSC) transplantation utilizing unmanipulated 
grafts 
Initial studies of allogeneic PBSC transplantation have focused predominantly on matched 
sibling allografts. The first report of allogeneic PBSC transplantation from a matched sibling 
was published in 1989.61 Since that time, reports of syngeneic and matched-sibling PBSC 
transplants, utilizing unmanipulated grafts, have demonstrated a similar outcome to that 
observed for patients undergoing allogeneic bone marrow transplant (BMT).61-65However, 
several added benefits appear to be inherent to allogeneic PBSC grafts.  These include more 
rapid engraftment and immune reconstitution, decreased treatment-related mortality and the 
ability to deliver a larger hematopoietic stem cell dose as measured by the number of CD34+ 
cells/kg infused, when compared to other forms of graft manipulation.66-72 In addition, recent 
data suggests that the number of CD34+ cells infused can be related to both the rate of 
engraftment and peritransplant toxicity.73However, GVHD is a concern with this approach 

 
  
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with the unmanipulated graft containing a 10-100 fold higher number of CD3+ cells/kg.  
Although no significant increase in acute GVHD in patients receiving matched sibling donor 
grafts has been observed, an increased incidence of chronic GVHD has been reported in this 
population.68,74,  Finally, a low number of CD3+ cells in the graft has been identified as the 
most important factor for graft failure following PBSCT in this sibling donor product 
recipient population.75 
2.7 CD34+ positive selection strategies 
The CD34 antigen, a cell surface glycoprotein expressed on a small fraction of bone marrow 
cells, serves as a surrogate marker for the hematopoietic stem cell.  This population contains 
cells capable of long-term hematopoietic reconstitution of all hematopoietic lineages.76-
81Several approaches have been utilized to perform CD34+ selection. Two of these 
approaches, the Baxter Isolex and CellPro Ceprate systems, have proven invaluable for 
CD34+ cell selection and tumor purging of autologous grafts.81-83However, these 
approaches lack the ability to exclude sufficient T cells to allow graft infusion without 
further graft manipulation.84In contrast, the Miltenyi CliniMACS CD34+ selection system 
allows the isolation of CD34+ cells in a single procedure with minimal contamination by 
mature T- and B cells.85,86In over 70 grafts prepared for infusion, this has resulted in a 
median CD34+ purity of 97% with a yield of 71%, and an associated 99-99.9% depletion 
of T cells.85,86In contrast, these results are unattainable with other CD34+ selection 
systems. Thus, the reproducible purification of large numbers of CD34+ cells permits a 
precise titration of the number of CD34+ and CD3+ cells in the graft, allowing the 
infusion of a defined graft in all patients. 
2.8 Preliminary studies of CD34+ based allografts obtained from alternate donors 
Studies of HSCT in the HAPLO setting suggest that removal of T cells reduces the 
incidence of GVHD significantly.  To date, only small studies utilizing CD34+-selected 
HAPLO grafts have been performed.  However, the results are informative and suggest 
that this approach may be highly beneficial.  In patients with high-risk leukemia, Aversa 
and colleagues have reported successful long-term engraftment with minimal GVHD 
utilizing CD34+ enriched grafts, purified utilizing positive (CD34+) and negative (lectin 
agglutination) selection.84A disease-free survival of 30% was reported in this study, with 
a similar incidence of relapse and a 40% incidence of transplant-related mortality. 
The Tuebingen group (Handgretinger and colleagues, Tuebingen, Germany) has focused 
their efforts on optimizing the CliniMACS CD34+ positive selection system. Utilizing 
predominantly HAPLO grafts and megadose cell therapy (>1 x 107 CD34+ cells/kg) to 
overcome engraftment resistance, they have reported similar preliminary results in a 
pediatric population.60,85  They initially studied the utility of highly purified grafts 
prepared from PBSC products obtained from HAPLO parental donors in a population of 
patients with high-risk leukemia who lacked matched sibling or matched unrelated 
donors. Using megadoses of CD34+ cells (20.7 + 9.8 x 106 CD34+ cells/kg), the 
Tuebingen group observed a low incidence of graft failure (7%), a median time to 
engraftment of 11 days, and no severe primary acute grade III-IV GVHD in 39 patients 
treated. Of note, since acute GVHD was not observed in the first 7 patients treated, the 
subsequent 32 patients received no pharmacologic GVHD prophylaxis.  

 
  
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Graft failure appeared to be related to ATG-containing regimens and in recipients given 
fewer than 1.0 x 107 CD34+ cells/kg. Two of these patients re-engrafted after a second 
infusion of a higher dose of HAPLO CD34+ cells following the administration of the pan 
T cell antibody OKT3 and methlyprednisolone.  In this cohort, 33% of patients have 
relapsed and 10 patients (25%) have died of regimen-related toxicity. Of these 10 deaths, 
5% were related to veno-occlusive disease (VOD), 18% due to viral and fungal 
infections, and 2.5% due to non-engraftment.  Interestingly, neither this cohort, nor 
subsequent patients reported below have experienced Epstein-Barr virus (EBV) PTLPD, 
a known complication of T-cell depletion methodologies.  This observation is consistent 
with reports of others that this complication is related, at least in part, to the number of 
mature B cells in the infused graft. 
Between 1995 and 2000, the Tuebingen group provided 48 children and adolescents with 
HAPLO grafts processed on the CliniMACS system.  Thirty-eight of these patients had 
hematologic malignancies and will be discussed more fully in this section (Rupert 
Handgretinger, unpublished data).  Of these 38, 6 patients with ALL in relapse at the time 
of transplant are excluded to more accurately make comparisons with St. Jude data as 
these patients are not transplanted at St. Jude.   Diagnoses included ALL (5 in CR1, 4 in 
CR2, 5 in > CR3), NHL (1 in CR2, 1 in CR3, 2 with persistent disease), AML (1 in CR1, 
1 in CR2, 4 with disease); MDS (4 with disease), and CML (4 chronic phase). No patient 
developed acute GVHD of greater than grade II and none experienced chronic GVHD.  
Twelve of these patients are alive disease-free. Twelve died of relapse, 4 of infection, 2 
of regimen-related toxicity, and 1 of an accident while in remission.  Patients with 
lymphoid malignancies (ALL, NHL) have an overall survival of 47.7 + 12.2% at one-
year posttransplant; those with myeloid malignancies (CML, AML, MDS) have an 
overall survival of 24.5 + 10.6% at the one-year posttransplant time period. 
Several important facts must be noted with this Tuebingen experience. This patient group 
was indeed high-risk with the majority having sustained several prior relapses or having 
active disease at transplant, particularly in those with myeloid diseases.  All patients for 
whom allogeneic HSCT was indicated underwent the procedure; no patients were denied 
HSCT due to lack of a donor.  Furthermore, all patients received HAPLO grafts, with 
donor and recipients matched at 3/6 HLA loci. 
Historically, the most powerful prognostic features of patients undergoing allogeneic 
HSCT have included leukemia type, disease status at time of HSCT, and donor stem cell 
source.  When comparing overall survival rates in the patients treated at Tuebingen and at 
St. Jude, several important differences are noted. First, the methods for graft T-cell 
depletion were different. Second, all patients eligible for transplantation underwent this 
procedure in Germany, while all referred patients at St. Jude did not.  Third, disease 
status at the time of transplant was significantly different between Tuebingen and St. 
Jude, possibly due to differences in patient referral patterns and treatment philosophies. 
For example, in Tuebingen, patients with AML in first CR were not referred for 
allogeneic HSCT from alternate donors. At St. Jude, patients with myeloid malignancies 
were referred for transplantation when deemed high-risk, including those in first 
remission. The converse was true for patients with lymphoid malignancies. Patients at St. 
Jude were referred for HSCT much later than patients in Tuebingen.  These differences in 
disease status at the time of transplant were quite significant.  In fact, they were 

 
  
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sufficiently poor enough to obscure effects of donor source, particularly in the lymphoid 
disease group. However, for the proposed trial we anticipate that referral patterns will 
reflect those historically observed at St. Jude. 
Based on these encouraging preliminary data, participants in this trial undergoing 
HAPLO-HSCT will receive partially depleted hematopoietic grafts from HAPLO family 
member donors depleted of T cells using negative selection.  Intravenous Campath-1H 
will be administered in the conditioning regimen to enhance immunosuppression and 
decrease the incidence of graft rejection.  In patients experiencing mixed chimerism or 
graft failure, withdrawal of immunosuppression (if applicable) with DLIs and CD34+ 
boosts/repeat stem cell infusions, respectively, will be utilized to restore hematologic and 
immunologic function. 
2.9 Evaluation and treatment of mixed chimerism and graft failure after CD34+-enriched 
allografts 
Mixed chimerism (10%-95% donor engraftment), graft failure (<10% donor engraftment) 
and disease recurrence remain common problems after HSCT. The ability to detect these 
complications and to treat them effectively is an important goal. Preliminary data suggests 
that monitoring hematopoietic chimerism in a serial fashion may provide a guide for 
therapeutic intervention.87-89For example, in 55 allograft recipients, those with complete 
donor chimerism experienced a 67% relapse free survival rate.  Of these 55, those with 
increasing donor chimerism experienced 100% survival while recipients with decreasing 
donor chimerism demonstrated a 10% survival rate.89   In a similar group of 32 patients 
receiving allografts for malignant diseases, the risk of developing relapse or graft failure 
was significantly increased in recipients with mixed chimerism when compared with 
those with full donor chimerism (p<0.0005).87 
One approach to treating mixed chimerism is the use of posttransplant therapeutic cell, T 
cell infusions, so called DLIs. This approach has been used extensively by others, and us, 
and has been successful as a therapeutic strategy.90In a pilot feasibility study, the 
Tuebingen group treated 21 patients considered to be at high-risk of relapse or who 
showed transient host hematopoietic recovery at various times posttransplant with a CD3 
dose between 2.5 x 104 to 1 x 105 CD3+/kg.  These infusions were performed once in 9 
patients, twice in 7, three times in 4 and four times in 1 patient.  Five patients developed 
grade I-II acute GVHD; only 1 patient developed grade IV disease. Chronic GVHD was 
induced in 1 patient. It is important to note that the incidence of GVHD was related to T 
cell dose, with those receiving less than 3 x 104 CD3+ cells/kg being less likely to 
experience this complication. 
This preliminary experience suggests that the related complication, graft failure, may 
respond to immunosuppressive reconditioning regimens and infusion of a second CD34+-
enriched allograft. Of 9 pediatric stem cell recipients receiving highly purified CD34+ 
grafts, 3 did not engraft and 6 rejected their graft after 21 days.  The 4 patients receiving 
grafts from an unrelated donor were reconditioned with fludarabine (40 mg/m2/day for 5 
days), cyclophosphamide (60 mg/kg/dose) for 1 day and ATG.  The 5 recipients of 
HAPLO related transplants received methlyprednisolone (20 mg/kg/day for 4 days) and 
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Successful engraftment was observed in 7 of the 9 patients.  The other 2 patients received 
a third transplant and 1 engrafted.91 
In addition, preliminary data from the Tuebingen group also suggest that CD34+ boosts 
may be clinically useful in patients with poor hematologic and immune reconstitution 
(i.e. CD4+ < 50/uL) who also have infectious complications.  In 4 such patients who 
received boosts, all exhibited a rapid immunological reconstitution accompanied by 
clinical improvement.  One was noted to have a subsequent increase in the lymphocyte 
populations with simultaneous clearing of CMV infection; in the others there were no 
influence on lymphocyte recovery (R. Handgretinger, unpublished data).  Other 
investigators have confirmed that stem cell boosts restore trilineage hematopoiesis and 
immune recovery in patients with malignant and non-malignant disorders after 
HSCT.92,93 
2.10 Use of T-cell depleted HSCT grafts 
Historically, the transplant program at St. Jude has used CD6/CD8 monoclonal antibodies 
with complement to deplete T lymphocytes from HAPLO and MUD bone marrow grafts.  
This result is a graft with a median of 5 x 105 T cells/kg. This antibody is no longer 
available from the manufacturer.  Additionally, antibody-complement depletion 
methodologies do not adequately deplete peripheral blood stem cell grafts (which have a 
log more T cells than bone marrow) to achieve this target number of CD3 cells.  
Therefore, we have chosen OKT3 (Orthoclone, Muronomab-CD3), a murine monoclonal 
antibody comprising a purified IgG immunoglobulin that recognizes the CD3 antigen, as 
a new agent for T-cell depletion by negative selection.  We feel that OKT3 is an ideal 
agent for T-cell depletion since it is an antibody with a narrow specificity and can be used 
with either peripheral blood or bone marrow grafts.94 
Other investigators have used OKT3 for ex vivo T-cell depletion of mismatched family 
member bone marrow grafts. 48,94 Thirty-one patients were treated in this study with 
donor grafts containing a median of 1.06 x 108 nucleated cells/kg (range, 0.32-3.21 x 108 
nucleated cells/kg) and 4.27 x 104 CD3+ cells/kg (range, 0.61-75.5 x 104 CD3+cells/kg).  
The median log T-cell depletion was 2.48 (range, 1.44-3.15). The study population also 
included an additional 35 patients receiving grafts T-cell depleted with T10B9 
methodology.  For the entire patient group, the estimated probability of engraftment was 
0.96.  Acute GVHD grade II-IV was observed in 24% and acute grade III-IV in 10%. The 
estimated probability of acute GVHD was not affected by degree of mismatch. The DFS 
at 3 years was 26% and the estimated probability of relapse at 3 years was 41%.  
OKT3 methodology has been used in published studies for the ex vivo T-cell depletion of 
grafts from mismatched family members. Furthermore, Dr. Handgretinger has used this 
technology in Tuebingen in the mid-1990s. At that time, only the OKT3 
antibody/microbead conjugates were available and he used his own magnetic system (not 
the CliniMACS device) for the depletion.  In 3 patients, bone marrow from unrelated 
donors was depleted.  The log depletion was higher than with PBSC, since the starting 
number of T cells in bone marrow is lower.  A median log depletion of 4 was achieved.  
All 3 patients engrafted an ANC of 1000/mm3 at 13, 14, and 16 days.  No acute or 
chronic GVHD was observed in the absence of GVHD prophylaxis. Two of the patients 
currently survive and are in remission; 1 patient has since relapsed. 

 
  
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A problem of the OKT3 depletion methodology is that these grafts contain more B-
lymphocytes than those grafts obtained by CD34+ positive selection.  EBV-PTLPD, 
which is a potentially life-threatening complication, occurs when EBV, latent in B- cells, 
reactivates. 
In this HIFLEX trial, we plan to T-cell deplete hematopoietic stem cell grafts from 
HAPLO donors using the CliniMACS system with the anti-CD3 antibody OKT3 labeled 
to magnetic microbeads. Mononuclear cells from mobilized peripheral blood (or 
alternatively bone marrow) from HAPLO donors will be incubated with the CliniMACS 
T cell CD3 microbeads. The depletion will be performed using the automated 
CliniMACS device with the ‘Depletion’ software. Our institution has performed 190 CD3 
depletion processes on peripheral blood hematopoietic grafts, with a mean 4.15 log 
depletion of CD3+ cells.  Post-depletion, the CD3+ content is 0.011%, CD19+ content is 
10.84%, and the percentage CD34 recovery is 71.79.  We have used this methodology 
regardless of graft source (bone marrow or PBSC).   
To achieve the desired dose of HSC, it is anticipated that the HAPLO donors will 
undergo two sequential collections by apheresis. In the unusual situation of the target 
dose of T cells is reached from the first CD3+ depleted HSC product, the second product 
will be T-cell depleted using positive selection of CD34+ cells.   
Several approaches have been developed to reduce the incidence of GVHD in the HAPLO 
donor setting.  Because larger numbers of T cells (>3 x 106 CD3+ cells/kg) has resulted in 
overall grade III-IV acute GVHD with significant morbidity and mortality22 many 
investigators have utilized methods to remove T cells from grafts. These methods of T-cell 
depletion are based predominantly on negative selection strategies.  These include physical 
methods such as T-cell specific lectins in association with sheep RBC rosetting or 
counterflow elutriation.31-33,95 An alternate approach is the use of T-cell specific monoclonal 
antibodies in association with complement, immunotoxins or magnetic beads.35-45,96 These 
approaches have resulted in an approximately 26-35% disease-free survival in high-risk 
leukemia and other high risk malignancies, with a 16-39% incidence of grade II-IV GVHD 
and a 5-10% non-engraftment rate.20,21,38,39,42-46 However, despite the use of various 
preparative regimens in this group, there have been no demonstrable differences in outcome 
by the type of regimen employed.  Overall, 30-45% of transplant recipients succumb to 
regimen-related toxicity with an additional 30%-40% dying from progressive disease or 
recurrence. 20,21,38,39,42-46 
Our institutional experience at St. Jude with CD6/CD8 T-cell depleted MUD-HSCT 
(HUD, MISMUD) demonstrate that outcomes following MUD-HSCT (including both 5/6 
and 6/6 MUD as a single group) are similar to those from HLA-identical siblings.18 These 
studies were based on the infusion of an allograft depleted of T cells for unrelated donors, 
primarily utilizing a CD6/CD8 monoclonal antibody/complement cocktail.  We observed 
a significant reduction in the incidence of GVHD-related complications utilizing this 
approach, the incidence of acute grade II-IV being 22% with a 19% incidence of chronic 
GVHD. Coincidentally, our graft failure incidence remains quite low (less than 5%) in 
recipients of alternate donor grafts.54 However, at our institution, HAPLO recipients 
continued to have inferior OS and DFS when compared to MUD recipients even when 
adjusted for the number of transplant recipients with high-risk disease, reflecting the 
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In our transplant clinical trial for patients with high-risk hematologic malignancy 
(HAPSCT), we enrolled 27 transplant recipient participants, distributed between two 
types of T-cell depletion:  positive selection with the CD34 antibody and negative 
selection with OKT3.  Of the 20 evaluable research participants receiving OKT3-depleted 
grafts, 12 have died (4 relapse and 8 regime-related toxicities). Six experienced acute 
GVHD, of the six 2 were overall grade III-IV.  Chronic GVHD occurred in 4 participants 
(2 limited, 2 extensive).  In addition, 2 participants developed PTLPD. 
The Transplant Faculty have extensive experience with the proposed reduced intensity 
conditioning regimen as it has been used in our institutional REFSCT protocol, 
(Haploidentical Stem Cell Transplantation utilizing T-Cell Depletion as Therapy for 
Patients with Refractory Hematological Malignancies). From this protocol, we have 
gained substantial knowledge in respect to both the preparative regimen and the negative 
graft selection methodology that is, in part, employed in this study.  Research participants 
received fludarabine 200 mg/m2, melphalan 60-120 mg/m2 depending on marrow aplasia, 
thiotepa 10 mg/kg, escalating and de-escalating doses of OKT3, and a negatively 
selected, CD3 depleted, mobilized stem cell graft from a HAPLO donor.  GVHD 
prophylaxis consisted primarily of mycophenolate mofetil (MMF). 
Conditioning and GVHD prophylaxis have been well tolerated by these REFSCT 
research participants, a population who has been heavily pre-treated prior to initiation of 
this transplant procedure. The 25 evaluable participants experienced a mean time to 
neutrophil engraftment (ANC greater than 500/mm3) of 10.3 days (range 7-12 days) and 
platelet engraftment (greater than 20,000/mm3 without transfusion support for the 
preceding 7 days) of 17.7 days (range 12 - 36 days). Two participants experienced non-
engraftment with 1 having autologous reconstitution and the other requiring the infusion 
of their previous unrelated donor’s stored stem cells.  A total of 4 participants did not 
experience platelet engraftment prior to their deaths.    
In this REFSCT trial, 14 transplant recipients died of primary/progressive disease. No 
unexpected AEs have been noted.  Severe toxicities have been limited.  Clinically 
significant NCI grade IV and/or fatal AEs that occurred included dilated cardiomyopathy, 
sepsis and multi-organ system failure. One recipient developed severe toxic epidermal 
necrolysis (TEN) at 91 days posttransplant. Four participants experienced clinically 
significant neuropathy.  However, 3 of the 4 had either a history of neurological 
complications with prior therapy or central nervous system (CNS) disease involvement. 
The fourth patient was found to have brain infiltrates (consistent with fungal infection) 
believed to be the etiology of the neuropathy.  This patient was also heavily sedated for a 
prolonged period of time, further confounding assessment.   
Only 2 REFSCT research participants experienced overall grade III acute GVHD.  
Fourteen have experienced overall grade I-II acute GVHD; all responsive to therapy. Five 
participants have experienced extensive chronic GVHD (skin, oral, and/or gut) 
(institutional data, unpublished).   
Since 2002, 27 children and young adults with hematologic malignancies underwent 
HAPLO HSCT on the HAPSCT trial (4 in 2002, 3 in 2003, 12 in 2004 and 8 in 2005).  
Since 2003, 25 research participants with refractory hematologic malignancies or 
requiring another allogeneic HSCT underwent HAPLO HSCT on the REFSCT trial (6 in 

 
  
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2003, 16 in 2004, 3 in 2005).  These enrollment data demonstrate that accrual to these 
institutional trials has improved over time. 
Our subsequent trial, HAPREF (Haploidentical Hematopoietic Stem Cell Transplantation 
Utilizing Partial T-Cell Depletion as Immunotherapy for Hematologic Malignancies) was 
then activated in 2006.  In this trial, 5 of 9 research participants enrolled in the high-risk 
group (Group A) developed overall grade III-IV acute GVHD.  At the same time point, 4 
of the 8 research participants enrolled in the refractory Group B had also developed 
overall grade III-IV acute GVHD. These incidences of GVHD in both groups met the 
specified criterion for stopping accrual and reevaluation of the study.  We concluded that 
the CD3+ dose used in this study was associated with excessive rates of acute GVHD.  
For this reason, we altered the T cell (CD3+) content of the stem cell graft, as this is 
strongly correlated with GVHD risk.  The CD3+ cell dose was reduced to 1.0 x 105 
CD3+/kg in the high-risk Group A. This specified  CD3+ dose is still higher than the 
median CD3+ graft content in the prior HAPSCT (2 x 104 CD3+/kg) and REFSCT (4 x 
104 CD3+/kg) clinical trials, theoretically allowing a more robust graft-versus-leukemia 
effect in this new trial.   
2.11 Importance of donor selection. 
There are several NK inhibitory receptors that recognize MHC class I molecules.  Some 
of these receptors (KIRs) recognize specific determinants shared by certain class I alleles, 
and are clonally distributed among NK cells.  Therefore, in the NK repertoire, some NK 
cells recognize and are blocked by specific class I alleles. Consequently, these NK cells 
can mount alloreactions against an allogeneic target if the target does not express the 
class I allele that blocks them. Ruggeri et al.97 showed these reactions greatly improve 
outcomes of HAPLO transplantation.  For poor risk AML research participants (mostly ≥ 
3rd complete remission or in relapse), the event free survival at 5 years was 5% if 
transplanted from HAPLO donors unable to mount NK alloreactions, but was 60% if 
transplanted from donors who do mount NK alloreactions.  A recent study by the same 
group demonstrates that alloreactive NK cells may ablate leukemic cells, recipient T cells 
that cause rejection, and recipient dendritic cells that may trigger GVHD.98 
Interest in the role of NK cells in HSCT developed from the observation of “hybrid 
resistance”, in which lethally irradiated F1 hybrid mice reject parental strain bone marrow 
yet tolerate other parental grafts.99Although the explanation of “hybrid resistance” is still 
debated, there is agreement that it is primarily mediated by NK cells.  The authors stated 
that resting host-derived NK cells were capable of mediating resistance to both 
autologous and allogeneic marrow grafts; in addition, the transfer of activated donor NK 
cells mediated beneficial effects in allogeneic and syngeneic marrow transplantation.  
This observation and the generally recognized anti-tumor properties of NK cells led to 
exploratory studies to define the activity and manipulate the response of NK cells in 
transplant HSCT participants.   
The Tuebingen group has reported the use of a low dose regimen of IL-2 posttransplant 
in leukemic research participants with high-risk for relapse. While the NK activity was 
low before starting the IL-2 treatment, a considerable increase could be measured during 
the low dose IL-2 treatment.  After 7 weeks of therapy, the NK activity declined to 
starting levels prior to the IL-2 therapy.100 

 
  
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Since it has been shown that NK cells are able to lyse fresh leukemic target cells 101and 
that allogeneic NK cells can control leukemia when given early posttransplant,102 we are 
interested in the reconstitution of NK-cell subsets and their anti-leukemic cytotoxicity 
after HSCT. Since the cytotoxic function of NK cells is regulated via KIRs,103 we are 
especially interested in the expression of these KIRs on the NK cells posttransplant.  
KIRs are receptors for HLA class I antigens and binding of these receptors to their 
corresponding ligands results in inhibition of cytotoxicity and other cell functions. 104 
While the cDNA of a number of KIRs is known, only few can be identified via 
monoclonal antibodies and flow cytometry.  Among the identifying receptors are CD94, 
CD158a, CD158b and DX9; we plan to analyze this surface expression on CD56+ NK 
cells periodically after HSCT. 
From our institutional HAPSCT, MUDSCT and REFSCT protocols as well as from 
published data,105it is known that the first cells to reconstitute the immune system 
posttransplant are donor NK cells. Published studies from our institution show that NK-
cell alloreactivity in haploidentical related donor-recipient pairs is associated with 
decreased relapse rates.105,106  We propose to confirm the relationship between NK-cell 
activity and disease recurrence in a larger number of patients.   
HAPLO donors will be selected preferentially for KIR ligand mismatches in the graft-
versus-host direction.  Selection for KIR mismatching is based on HLA typing as 
performed by serological and high-resolution molecular techniques.  We will select the 
family donor with the most NK alloreactivity when possible.     
2.12 Conditioning regimen considerations.  
Patients undergoing HSCT require sufficient conditioning to prevent rejection, while 
allowing engraftment of donor hematopoietic cells to provide an antileukemic effect 
without increasing the risk of regimen-related toxicity. 
The most commonly used conditioning regimens include combinations of busulfan and 
cyclophosphamide or cyclophosphamide and TBI.  Our approach for this protocol will 
differ in that it prescribes a reduced regimen including melphalan, fludarabine, thiotepa 
and Campath-1H.   TBI can increase the risk of acute organ toxicities and is also 
associated with an increased risk of second cancers and long-term complications, 
especially in children.107-110 Avoidance of TBI may also decrease thymic damage and 
allow more rapid immune reconstitution posttransplant.111The use of busulfan is 
associated with risk for veno-occlusive disease of the liver (VOD).112Cyclophosphamide 
is an effective antileukemic drug that the majority of this patient population has already 
received in the past without benefit.  Our institutional experience with HSCT using a 
non-TBI containing regimen for children and young adults with relapsed/refractory 
malignancy indicates that it permits engraftment with an acceptable risk of regimen 
related toxicity.113 
We hypothesize that an immunoablative regimen will be better tolerated and will also 
provide an antileukemic effect. Data from Aversa et al and our institution113,114 have 
demonstrated that fludarabine-based conditioning regimens can be used safely instead of 
TBI in HAPLO HSCT.  In our institutional regimen of fludarabine, thiotepa, and 
melphalan, we reduced the dose of melphalan by 15% to decrease the gastrointestinal 
side effects.  For those who had not recovered from the toxicities of prior therapy at the 

 
  
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time of HSCT, we may further reduce the melphalan dose by 50% to lower the risk of 
unacceptable toxicity and mortality.   
In the REFSCT trial, using melphalan, fludarabine, and thiotepa, 2 of 25 enrolled 
transplant recipients developed peripheral neuropathy with white matter changes in the 
brain on MRI.  One of the 2 progressed to a vegetative state and died of sepsis.  Prior to 
HSCT, this participant had a history of severe vincristine-induced neuropathy, which had 
required him to be wheelchair bound. The second had persistent CNS leukemia and 
received intensive intrathecal therapy to induce remission prior to enrollment on the 
REFSCT protocol.  This participant is steadily improving with supportive care and 
therapy and is an outpatient.  Because no definitive etiology was found in either case and 
because fludarabine may cause neurologic abnormalities when administered at high 
doses, we have reduced the dose of fludarabine in this trial.     
Lastly, regarding conditioning regimen considerations, T-cell reconstitution, as measured 
by CD3 quantitation in peripheral blood and based on T-cell receptor excision circles 
(TREC), was found to be more rapid in the more heavily pre-treated patient population 
on REFSCT trial than those treated on the HAPSCT trial.115  
2.13 Use of Campath-1H in place of OKT3 in the conditioning regimen 
We have been notified by Centocor Ortho Biotech, Inc that the manufacture and 
subsequent availability of OKT3 will cease in 2010.  Therefore, an alternative agent is 
required for use in this protocol.  The two agents which are most similar to OKT3 are 
ATG and Campath-1H. Campath-1H is chosen for Amendment 3.0 because ATG was 
found to be insufficient for engraftment of haploidentical graft and was associated with 
high risk of transplanted related mortality (TRM). Even in infants in whom megadoses of 
stem cells can be ensured, the rejection rate and TRM was still too high with ATG (1/8 
engraftment failure rate and 3/8 TRM in our prior infant haploidentical transplant study – 
INFBMT). Furthermore, we have previously demonstrated that NK cells were highly 
susceptible to lysis by ATG, even more so than T cells.116 Because NK cells are the key 
mediators of graft-versus-leukemia effects, ATG should be avoided in our transplant 
setting.    
Campath-1H is a humanized monoclonal antibody with specificity for CD52.117Because 
Campath-1H is a recombinant monoclonal antibody with a consistent quality, there is not 
the lot to lot variability as is seen with ATG.  Campath-1H has been shown to result in 
preferential immunodepletion of T cells, as CD52 is expressed on several leucocyte 
populations, but is most heavily expressed on T cells.  CD34+ cells are largely unaffected 
by Campath-1H; thus making it an attractive candidate in bone marrow transplant 
settings.  It has been used successfully in preparative regimens for MSD and MUD HCT, 
and in the doses used, Campath-1H has shown greater immunosuppressive effects in the 
form of decreased GVHD and delayed immune reconstitution.117 
More recently Campath-1H has been incorporated successfully into haploidentical 
HCT.118,119 Because Campath-1H has a long half life of approximately 5 days in 
children,120we will begin Campath-1H two weeks prior to graft infusion to maximize its 
effect on residual patient cells and engraftment, and minimize its effect on the graft and 
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2.14 Rationale for the measurement of Campath-1H pharmacokinetics 
The pharmacokinetics (PK) of Campath-1H remain incompletely understood.  Although 
Campath-1H was originally developed as a lymphocyte-depleting agent for HSCT, the 
majority of available PK data comes from adult chronic lymphocytic leukemia (CLL) 
patients.121 Published PK data in children is limited, and involves significantly different 
dosing than this study.120,122 
Combining study outcomes to date, the largest factor in clearance of Campath seems to 
be the burden of CD52.(Mould 2007)  As such, Campath seems to have two phases of 
clearance: an initial rapid clearance when lymphoid tissues are being targeted and 
depleted, and a subsequent slow clearance when lymphoid tissues have been depleted. 
The half-life of Campath has been estimated to be as long as 15 to 21 days in this slow 
phase.119  In T cell replete (unmanipulated) HSCT, Campath may remain at therapeutic 
levels as far out as 60 days post-HSCT.125   Because the haploidentical grafts are heavily 
T cell depleted, the lymphoid burden (and consequently the CD52 burden) is predicted to 
be lower for the initial post-transplant period in our patients.  Therefore, we propose to 
monitor Campath levels out to 16 weeks post-HSCT. 
The PK of Campath-1H is of particular importance in HSCT as it can mediate depletion 
of both host and graft lymphocytes, thereby impacting both rejection and GvHD.  Too 
much exposure to Campath can delay necessary immune reconstitution and increase 
infection risk, while too little may increase the risk of both rejection or GvHD. 
2.15 Minimal residual disease (MRD) 
Approximately 30 to 40 % of patients ultimately relapse after HSCT, despite the fact that 
the vast majority of patients are in clinical remission immediately before and after HSCT. 
Alternative approaches are needed to detect the residual leukemic cells that are below the 
limits of detection by standard morphologic examination. This is clinically important as 
studies confirm that, at least in CML, posttransplant relapse can be cured by interventions 
such as DLI, and the chance of cure is higher when the patients are still in MRD status.  
By using flow cytometry and polymerase chain reaction (PCR) amplification of antigen 
receptor genes in tandem, investigators at our institution have been able to conduct MRD 
studies in 80 consecutive ALL cases.  Results of St. Jude institutional studies have shown 
that detection of MRD by immunologic techniques at any point in the treatment course is 
a powerful predictor of relapse in children with ALL.123-127However, other studies 
suggest that eradication of all acute leukemia cells may not be a prerequisite for cure.123-
127  
Similar to conventional-dose therapy, controversy also exists on the implication of MRD 
in the setting of HSCT.  Unlike CML, there is a paucity of data on the natural history of 
AML and ALL patients who have MRD after HSCT, and how pre-transplant MRD levels 
influence posttransplant outcomes.125,128,129It is unclear whether they are also at greater 
risk of relapse, and whether further pharmacological or immunologic therapy indeed 
prolongs survival and increases cure rates.  Thus, for those enrolled in this protocol who 
are unable to proceed to posttransplant immunomodulatory protocols, we will gather the 
MRD information together with hematopoietic chimerism in a descriptive manner to 
study the relationship between MRD and chimerism in this large cohort of patients. The 

 
  
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knowledge gained from this study should allow their future application to guide 
therapeutic interventions. 
2.16 Rationale for decreasing CD3+ graft content 
Due to the unacceptable rate of overall grade III-IV acute GVHD in the HAPREF trial 
(8/17, 47%) we have decreased the T cell content of the stem cell graft in this study, as 
we believe this was strongly correlated with GVHD risk. The CD3+ dose of 1.5 x 105 
CD3+/kg used in the HAPREF study has been decreased to 1.0 x 105 CD3+/kg.  In 62 
research participants who received a negative selected product in the 3 recent HAPLO 
HSCT protocols (HAPSCT [N = 20], REFSCT [N = 25], and HAPREF [n = 17]), one 
participant (3%) receiving a CD3 dose of < 1.5 x 105 CD3+ cells/kg experienced overall 
grade III-IV acute GVHD compared to 36% of those with a dose > 1.5 x 105 CD3+ 
cells/kg (p=0.0002, Table 1).   
Table 1:  Overall acute GVHD rate in relation to CD3 dose for recipients of negative selected 
donor products in HAPSCT, REFSCT, and HAPREF (N = 62) 
 
(N = 62) 
Acute GVHD 
Overall grade 0 - II 
Overall grade III – IV 
Total CD3 x 105/kg 
N 
N 
< 1.5 × 105/kg 
29 
1 
≥1.5 × 105/kg 
20 
12 
Total 
49 
13 
Table 2: Overall acute GVHD rate in relation to participant age, donor product cell dose, and 
neutrophil (ANC) and platelet engraftment for recipients of negative selected donor products 
enrolled in HAPSCT, REFSCT and HAPREF (N = 62) 
 
Acute GVHD 
Grade 0-2 
Grade 3-4 
N 
Mean 
Std 
Median 
Min 
Max 
N 
Mean 
Std 
Median 
Min 
Max 
Age at HSCT 
49 11.45 
5.65 
11.95 
2.83 
26.55 13 12.89 
6.08 
15.20 
2.67 
20.71 
Total CD34 cells 10^6/Kg 49 15.22 
11.65 
11.80 
1.73 
42.96 13 11.48 
7.72 
9.85 
2.23 
26.05 
Total CD3 Cells x10^6/Kg 49 
0.12 
0.10 
0.14 
0.01 
0.45 
13 
0.16 
0.04 
0.15 
0.10 
0.25 
Days to ANC >=500 
45 12.62 
6.04 
11.00 
7.00 
48.00 13 10.77 
1.42 
11.00 
9.00 
15.00 
Days to Platelets >=20K 
41 17.88 
4.84 
17.00 
12.00 36.00 12 15.67 
2.27 
15.50 
12.00 
19.00 
Time to Relapse 
20 133.20 144.54 
99.00 
26.00 714.00 2 264.00 149.91 
264.00 
158.00 370.00 
p = 0.0201 
 

 
  
HIFLEX 17 
Revision 6.1 dated: 12/24/2014 
    
          IRB Approval date: 12/06/2016 
Protocol document date: 11/18/2016 
 2.17 Chimerism analysis 
Serial analysis of chimerism in peripheral blood and marrow of transplant recipients is 
extremely important to diagnose graft rejection, a significant problem after T-cell 
depleted grafts from mismatched donor-recipient pairs used for transplantation.130,131 
Because the initial signs and symptoms of graft rejection can be non-specific, the St. Jude 
Transplant Service presently measures peripheral blood chimerism weekly on all 
allogeneic transplant recipients.  The definitive test for graft failure is chimerism 
performed on marrow or peripheral blood—reported as the percentage of cells of donor 
origin and the percentage of recipient origin. Presently chimerism is a DNA-based 
analysis using variable number of tandem repeats (VNTR) and the testing takes at least 
48 to 72 hours to perform.   
2.18 Predictive factors to identify risk for non-relapse morbidity and mortality 
Non-relapse morbidity and mortality (NRM) is an important cause of transplant failure.  
NRM includes illness and death resulting from infection, acute and chronic GVHD, and 
organ failure not due to the underlying disease. Although HLA disparity is a major 
factor-identifying patients at risk for GVHD, other non-HLA factors have been 
demonstrated to be important in regimen-related toxicities.  The inflammatory state of the 
patient before, during and shortly after conditioning may establish an environment in the 
transplant recipient that results in ongoing tissue damage and atypical antigen expression, 
leading to GVHD or other transplant toxicities.132 The majority of studies of predictive 
factors for NRM have been performed in recipients of matched sibling or unrelated donor 
grafts; nearly all studies have been done in recipients of non-T-cell depleted grafts. For 
example, in animal studies, elevated tumor necrosis factor levels at day +7 posttransplant 
have been shown to contribute to the pro-inflammatory effects of GVHD.132Soluble 
interleukin-2 receptor has been shown in animal and human studies to be elevated during 
GVHD.133These two cytokines are important to study as there are established assays 
available and there are medications to block or inactivate each of these cytokines.  
Therefore, if elevated levels of these cytokines early posttransplant are demonstrated to 
be predictive of subsequent NRM, then early intervention may be indicated.   
 
  
2.19 Rationale for present study 
Haploidentical HSCT is complicated by the need for T-cell depletion of grafts due to the 
unacceptably high rates of severe acute GVHD when non-T-cell depleted products are 
used.  However, this graft processing may lead to graft failure and delayed immune 
reconstitution.  Our institutional experience suggests that a reduced intensity-conditioning 
regimen that omits total body irradiation and ATG may lead to a more rapid immune 
reconstitution and fewer infectious complications.  Furthermore, a reduction in the CD3+ 
content of the graft will still allow engraftment, with more immunotherapeutic cells 
(monocytes, natural killer cells) being infused in the graft, potentially resulting in a graft-
versus-malignancy effect and providing cells to fight infection, while potentially 
lowering the risk of GVHD observed when grafts of higher CD3+ content are infused.  
Therefore, in this trial we will employ a reduced intensity-conditioning regimen of 
fludarabine, melphalan and thiotepa with a graft depleted of T cells using the anti-CD3 
monoclonal antibody OKT3 on the CliniMACS system.  Conditioning regimens that 
utilize this backbone, while avoiding TBI and ATG, enable a more rapid immune 

 
  
HIFLEX 18 
Revision 6.1 dated: 12/24/2014 
    
          IRB Approval date: 12/06/2016 
Protocol document date: 11/18/2016 
reconstitution in our institutional HAPLO HSCT trials.  In total, we aim to improve 
overall survival using a multimodal approach to reduce GVHD, enhance immune 
reconstitution, and reduce non-relapse mortality, ultimately establishing a platform on 
which to build posttransplant immunomodulatory strategies. 
3.0 PROTOCOL ELIGIBILITY CRITERIA 
3.1 Inclusion criteria (transplant recipient): 
3.1.1  Patients less than or equal to 21 years old; may be greater than 21 years old if a 
current St. Jude patient or previously treated St. Jude patient within 3 years of 
completion of prior treatment.  
3.1.2  Has one of the following diagnoses:  
3.1.2.1 ALL high risk in second remission.  Examples include: relapse on therapy, 
first remission duration of less than or equal to 30 months, or relapse 
within 12 months of completing therapy. 
3.1.2.2 ALL in third or subsequent remission.  
3.1.2.3 ALL high-risk in first remission.  Examples include: induction failure, 
minimal residual disease greater than or equal to 1% marrow blasts by 
morphology after induction, persistent or recurrent cytogenetic or 
molecular evidence of disease during therapy requiring additional therapy 
after induction to achieve remission. 
3.1.2.4 High-risk AML in first remission.  Examples include: monosomy 7, M6, 
M7, t(6;9), FLT3-ITD, or patients who have greater than or equal to  25% 
blasts by morphology after induction or who do not achieve CR after 2 
courses of therapy (includes myeloid sarcoma). 
3.1.2.5 Relapsed or persistent AML (less than or equal to 25% blasts in marrow 
by morphology). 
3.1.2.6 AML in second or subsequent morphologic remission (includes myeloid 
sarcoma). 
3.1.2.7 CML in first chronic phase with detectable molecular or cytogenetic 
evidence of disease despite medical therapy; or CML with a history of 
accelerated or blast crisis, now in chronic phase; or unable to tolerate 
tyrosine kinase inhibitor therapy. 
3.1.2.8 Juvenile myelomonocytic leukemia (JMML). 
3.1.2.9 Myelodysplastic syndrome (MDS). 
3.1.2.10 Therapy related (secondary) AML, ALL, or MDS. 
3.1.2.11 Hodgkin lymphoma after failure of prior autologous HSCT or unsuitable 
for autologous HSCT. 
3.1.2.12 Non-Hodgkin lymphoma (NHL) in CR2 or subsequent. 
3.1.3 Has not received a prior allogeneic hematopoietic stem cell transplant. 
3.1.4  Does not have a suitable HLA-matched sibling donor available for stem cell 
donation. 

 
  
HIFLEX 19 
Revision 6.1 dated: 12/24/2014 
    
          IRB Approval date: 12/06/2016 
Protocol document date: 11/18/2016 
3.1.5  Does not have a suitable cord blood product or volunteer MUD available in the 
necessary time for stem cell donation (refer to subsection 4.1 for definition of 
“necessary time”).  
3.1.6  Has a suitable HLA partially matched family member available for stem cell 
donation.  
3.1.7  Cardiac shortening fraction greater than or equal to 25%. 
3.1.8  Creatinine clearance or glomerular filtration rate (GFR) greater than or equal to 
40 ml/min/1.73 m2. 
3.1.9. Forced vital capacity (FVC) greater than or equal to 40% of predicted value or a 
pulse oximetry value of greater than or equal to 92% on room air.  
3.1.10  Direct bilirubin less than or equal to 3 mg/dl. 
3.1.11  Serum glutamic pyruvic transaminase (SGPT) less than 3 times the upper limit of 
normal for age. 
3.1.12  Karnofsky or Lansky (age-dependent) performance score of greater than or equal 
to 50 (See Appendix A). 
3.1.13 No known allergy to murine products or human anti-mouse antibody (HAMA) 
results within normal limits. 
3.1.14  Not pregnant (confirmed by negative serum or urine pregnancy test within 14 days 
prior to enrollment).  
3.1.15  Not breast feeding. 
3.2 Inclusion criteria (stem cell donor): 
3.2.1  Partially HLA matched family member. 
3.2.2 At least 18 years of age. 
3.2.3  Human immunodeficiency virus (HIV) negative. 
3.2.4  Not pregnant (confirmed by negative serum or urine pregnancy test within 7 days 
prior to enrollment).  
3.2.5  Not breast feeding. 
 3.3 Inclusion criteria (transplant recipient - stem cell boost) 
3.3.1  Has experienced one of the following disorders posttransplant: graft failure, graft 
rejection, delayed hematopoietic and/or immune reconstitution. (Definitions 
provided in Sections 4.7 and 7.0 Evaluation Criteria).  
3.4 Gender and minorities 
According to institutional and NIH policy, this study will accession patients regardless of 
gender and ethnic background providing all inclusion/exclusion criteria have been met. 
Institutional experience confirms broad representation in this regard.   
 

 
  
HIFLEX 20 
Revision 6.1 dated: 12/24/2014 
    
          IRB Approval date: 12/06/2016 
Protocol document date: 11/18/2016 
4.0 TREATMENT PLAN 
GENERAL STUDY TREATMENT PLAN SCHEMA 
Day 
Transplant Recipient Activities 
Donor Participant Activities 
Day -14 Initiate conditioning regimen 
Day -5 
 
Begin 6 days of HSC mobilization. 
Day -1 
 
Undergo day 1of apheresis.  
Day  0 
HSC infusion #1 
Undergo day 2 of apheresis. Donor stem 
cells from day 1 of apheresis are 
processed using CliniMACS system. 
Day +1 
HSC infusion #2                          
Donor stem cells from day 2 of apheresis 
are processed using CliniMACS system. 
4.1 Donor selection 
Patients with either a suitable HLA-identical sibling MUD, or umbilical cord graft 
product, available in a timely manner will not be eligible for enrollment.  The time period 
for consideration and identification of a suitable unrelated volunteer donor or cord blood 
graft will be defined as a minimum of 90 days from the time of obtaining the patient’s 
HLA typing specimen until enrollment is requested.  Patients who have no potential cord 
blood or volunteer unrelated donor identified in the registries on the initial search will 
proceed to HAPLO transplant providing all inclusion/exclusion criteria met. Those whose 
potential cord product or unrelated donors are completely typed and found not suitable 
and for whom no other suitable cord product or donors remain on the registries list may  
proceed to HAPLO transplant when indicated, even if prior to the 90 day timeframe. 
This protocol will use HAPLO donors only.   If more than one HAPLO family member 
donor is acceptable, then donor selection will be based on the largest KIR repertoire 
mismatch between donor and host, donor-recipient infectious disease markers, and donor 
health screen and examination.  
Donor eligibility for stem cell collection will be determined through the use of the 
guidelines outlined in 21 CFR 1271 and the draft Guidance for Industry: Eligibility 
Determination for Donors of Human Cells, Tissues, and Cellular and Tissue-Based 
Products (HCT/Ps).  Potential donors will undergo a screening process which will include at 
least a physical examination, history and testing for relevant communicable diseases.  The 
physical examinations to evaluate donor candidacy will be conducted by a non-transplant 
physician (St. Jude or non-St. Jude). A repeat physical exam is required prior to the boost 
stem cell collection process if there is no change in the health status of the donor and the 
initial physical exam was conducted greater than 180 days prior to the start of the boost stem 
cell collection process.   
If a donor is determined to be ineligible, the donor is not automatically excluded.  Part 21 
CFR 1271.65 (b)(1)(i) allows for the use of ineligible donors who are first or second degree 
blood relatives. In this situation, the physician will document the necessity of using the 
ineligible donor by providing a statement of "Urgent Medical Need" as explained in the 21 
CFR 1271.3 (u).  The cell therapy products will be labeled as required in 21 CFR 1271.65 

 
  
HIFLEX 21 
Revision 6.1 dated: 12/24/2014 
    
          IRB Approval date: 12/06/2016 
Protocol document date: 11/18/2016 
(b)(2).  Standard donor testing will include for HIV-1 Ag, anti-HIV1, anti-HIV2, anti-
HTLV-I, anti-HTLV-II, HBsAg, anti-HBc, anti-HCV, anti-CMV, and serologic tests for 
syphilis using FDA licensed test kits.  Recipients or their legal guardians will be 
informed of the use of an ineligible donor.   
4.2 Donor mobilization and donor stem cell graft collections  
A mobilization regimen of granulocyte colony stimulating factor (G-CSF) will be used to 
obtain a PBSC product from the donor.  The desired target goal will be ≥ 5 - 10 x 106 
CD34+ cells/kg, representing the total number infused in the transplant infusion 
irrespective of the number of days. This number of cells will be necessary to provide an 
adequate graft following the various ex vivo manipulations for prompt reconstitution.  
The donor mobilization guidelines are as follows:  
        
Days 
Mobilization regimen 
 
1, 2, 3, and 4 
G-CSF 10 mcg/kg/day subcutaneous. 
        
5 and 6  
G-CSF 10 mcg/kg/day subcutaneous. Apheresis in St. Jude Donor Room.   
It is anticipated that apheresis will be performed for a minimum of two days.  Additional 
days of apheresis will be done at the transplant or apheresis physician’s discretion.  
However, some donors may be able to provide the needed graft stem cell count after only 
one day of apheresis. If the donor is unwilling or unable to complete the mobilization 
process or apheresis procedure, a bone marrow product may be used.  The bone marrow 
product will be processed using the same cell selection methodology on the CliniMACS 
device.  Every effort will be made to infuse a fresh stem cell product; however, a frozen 
product may be infused when necessary. 
All PBSC products will be collected as per American Association of Blood Banks 
(AABB) guidelines. Donors will be monitored during the time period of the mobilization 
and apheresis procedure with appropriate laboratory evaluation (see Appendix D).   
4.3 Graft preparation 
All cell processing will take place in the Human Applications Laboratory (HAL) in the 
Department of Therapeutics Production and Quality (TPQ) of St. Jude using established 
Standard Operating Procedures. The hematopoietic stem cell graft product will be T-cell 
depleted using the investigational CliniMACS device and CD3 Microbead reagent 
(OKT3 antibody conjugated iron-dextran beads) as directed by the manufacturer 
(Miltenyi Biotech). Briefly, hematopoietic progenitor cells collected by apheresis (HPC-
A) from the mobilized donor will be initially assessed in the HAL and stored overnight at 
4oC.  The next morning, the HPC-A product will be washed to remove platelets and 
adjusted to an appropriate cell concentration for incubation with the CliniMACS CD3 
Microbead reagent in the manufacturer provided media.  The cells will be washed to 
remove unbound microbeads.  The cells will be applied to the CliniMACS device and the 
depletion will be performed using the "Depletion 2.1" software as described by the 
manufacturer.  After depletion is complete, the cells will be washed and resuspended in 
an infusion grade solution.  The graft product will be enumerated and assessed for viable 

 
  
HIFLEX 22 
Revision 6.1 dated: 12/24/2014 
    
          IRB Approval date: 12/06/2016 
Protocol document date: 11/18/2016 
stem cell (CD34+) and T cell content (CD3+) by flow cytometry.  The HPC-A graft 
product will be infused fresh after completion of release testing and evaluation.   
Under the unusual situation that the target number of CD3+ cells is reached on the first 
day of T-cell depletion, then the graft collected on subsequent days will be processed 
using CD34+ selection on the CliniMACS device. In rare circumstances, the product may 
be cryopreserved for later infusion to the recipient. The processing of the graft on the 
CliniMACS for CD34+ selection is similar to the processing steps for CD3+ depletion. In 
this case, the cells are positively selected with the “CD34 Enrichment” program on the 
CliniMACS after labeling with CD34 Microbead reagent per manufacturer’s instructions. 
Cell dose parameters for the primary HSC infusion donor graft are as follows.  The cell 
doses noted are defined as the total CD3+ and CD34+ counts contained in the one donor 
stem cell product, irrespective of the number of infusions/days needed for the product to 
be administered (meaning that one final donor product is generally divided into 2 or 3 
separate daily infusions. Thus, the doses below indicate the total donor product cell 
count). 
• 
The target HSC dose is 10 x 106 CD34+ cells/kg.  The minimum cell dose will be 
2 x 106 CD34+ cells/kg with a maximum of 100 x 106 CD34+ cells/kg.   
• 
The total T cell dose content in this primary infusion donor product will be < 1.0 
x 105 CD3+ cells/kg and ≥ 0.01 x 105 CD3+ cells/kg.  
Typically, the total graft will be provided with two sequential T-cell depleted HPC-A cell 
infusions. In rare situations, the minimum T cell dose may be achieved by the addition of 
T cells to the final graft product to reach the minimum dose of ≥ 0.01 x 105 CD3+ 
cells/kg. 
4.4 Quality assurance of cellular product 
Quality assurance for cell products is overseen by the TPQ Quality Assurance division, 
which authorizes release of all products. Only trained stem cell processors will process 
the cell products. Special training will be provided to all personnel operating the 
CliniMACSTM system. A labeling and product tracking system is in place to ensure that 
the correct cells are infused into the research participant. 
Assays of cell numbers and immunophenotyping will be performed both before cell 
processing and at critical stages of the process.  These values will be recorded according 
to standard operating procedures of the Human Applications Laboratory.  All products 
will be tested for viability, sterility (culture and gram stain), and the presence of 
endotoxin. Culture and endotoxin results are not available before infusion of fresh cell 
products.  If the gram stain is positive, the research participant/parent and/or guardian 
will be informed of this event and of the risks of proceeding prior to infusion.  Positive 
results will be investigated as per the variance procedures of the Human Applications 
Laboratory. The IRB and FDA will be notified, if at any time after infusion, cell product 
has been determined to be contaminated or endotoxin results exceed release limits.   
4.5 EBV PTLPD prophylaxis 
A dose of rituximab will be administered within approximately 24 hours prior to the stem 
cell transplant infusion.134-136This is provided as part of the pre-transplant preparative 

 
  
HIFLEX 23 
Revision 6.1 dated: 12/24/2014 
    
          IRB Approval date: 12/06/2016 
Protocol document date: 11/18/2016 
regimen to prevent EBV PTLPD.  This intervention has been chosen because of studies 
indicating that the EBV DNA level in the peripheral blood is suggestive of PTLPD. 
Rituximab has been demonstrated to be an effective therapy in PTLPD. The current 
standard clinical practice within the Division of Transplant is to administer rituximab 
when peripheral blood EBV DNA levels exceed 2000 copies/microgram.  This is done 
regardless if the patient has concurrent clinical signs and symptoms in order to prevent an 
onset and/or progression of this  PTLPD disorder.  The medication will continue at the 
discretion of the treating physician as clinically indicated based on ongoing EBV copy levels 
and clinical assessment.  
4.6 Preparative regimen –primary infusion 
Recipients will receive the following standard conditioning regimen: 
Day(s)  
       Treatment 
Days -14 to -11 
Campath-1H intravenous (dosing table below) 
-9 
Fludarabine 40mg/m2 intravenous every 24 hrs (dose 1 of 5) 
-8 
Fludarabine 40mg/m2 intravenous every 24 hrs (dose 2 of 5) 
-7 
Fludarabine 40mg/m2 intravenous every 24 hrs (dose 3 of 5) 
-6 
Fludarabine 40mg/m2 intravenous every 24 hrs (dose 4 of 5) 
-5 
Fludarabine 40mg/m2 intravenous every 24 hrs (dose 5 of 5) 
-4 
Thiotepa 5 mg/kg intravenous every 12 hrs 
-3 
Melphalan 60 mg/m2 intravenous (dose 1 of 2) 
-2 
Start MMF 600 mg/m2/dose intravenous twice a day 
-2 
Melphalan 60 mg/m2 intravenous (dose 2 of 2) 
-1 
Rituximab 375 mg/m2 intravenous  
  0  
HSC infusion #1  on Day 0 HSC infusion #2 on Day +1 if subsequent infusion 
days may be needed 
+6 
Start daily G-CSF 5 mcg/kg/day subcutaneous or intravenous until ANC           
> 2,000/mm3 for 2 consecutive days and then as clinically indicated.            
 
Notes:  
• A reduced dose of fludarabine (i.e. 30 mg/m2/day for 5 days) will be administered to 
participants with a history of significant neurologic disease, CNS-directed therapy or 
toxicity such as CNS irradiation, grade III-IV chemotherapy-induced neurotoxicity, 
and/or those receiving CNS radiation with this transplant. 
• Participants in aplasia at the beginning of the conditioning regimen will receive a 50% 
reduction in melphalan dose as follows: 60 mg/m2/day on day -3 and then day -2 no 
melphalan. 
• MMF may be taken orally when recipient is able to tolerate solid or liquid formula.  

 
  
HIFLEX 24 
Revision 6.1 dated: 12/24/2014 
    
          IRB Approval date: 12/06/2016 
Protocol document date: 11/18/2016 
• G-CSF may be administered for longer time periods if there are medical concerns 
regarding weak graft function or if there is an evident or possible (documented) 
concern for the existence of an active infection. 
• Clinically indicated changes in dosing/days and supportive medications may be done 
for safety reasons when indicated such as in the case of allergic reactions, hypotensive 
reaction, etc.  These changes will be reported as clinically indicated and in the 
continuing reviews where applicable.  
• Although Campath-1H may be given subcutaneously for the first two doses, the final 
two doses must be given intravenously.  Otherwise the dose outlined below in section 
4.6.1 cannot be changed. 
4.6.1 Preparative regimen – intravenous Campath-1H schedule 
 
Day 
Treatment 
Participant less than 15 kg 
Participant greater than or equal to 15 kg 
-14 
Campath -1H 
 
 
1 mg 
2 mg 
-13 
Campath -1H 
10 mg 
10mg/m2 
-12 
Campath -1H 
10 mg 
15mg/m2 
-11 
Campath -1H 
10 mg 
20mg/m2 
4.7 Additional stem cell administration (“stem cell boost”) 
Infusions of hematopoietic progenitor cells from the original donor may be performed for 
participants when clinically indicated for such conditions as graft failure, graft rejection, 
delayed hematopoietic recovery, or delayed immune reconstitution.  Should the originally 
selected donor be unwilling or unable to donate cells following enrollment, an alternative 
haploidentical donor will be considered for the purposes of rescue. The boost will be 
processed on the CliniMACSTM device using CD3 depletion methodology.  The boost target 
doses will be > 10 x 106 CD34+ cells/kg with a CD3+ cell/kg dose of ≤ 1.0 x 105 CD3+ 
cells/kg.  These doses represent the total number of cells infused with the boost process, 
irrespective of the number of days the infusion.   
Research participants for whom a graft processed with CD3+ depletion may not be 
clinically appropriate, such as in the case of active bronchiolitis obliterans organizing 
pneumonia or active acute GVHD, a graft from the original donor processed on the 
CliniMACSTM device using CD34+ selection will be infused. The boost target dose for 
these participants is >10 x 106 CD34+cells/kg with a CD3+ cell/kg dose of ≤ 5.0 x 104 
CD3+ cells/kg. 
In the event of graft failure or graft rejection, the following regimen will be provided 
prior to infusion of the stem cell boost product and a separate informed consent will be 
required.  This regimen will be used regardless of the selection methodology used for the 
boost donor product. 
 
 

 
  
HIFLEX 25 
Revision 6.1 dated: 12/24/2014 
    
          IRB Approval date: 12/06/2016 
Protocol document date: 11/18/2016 
4.7.1 Preparative regimen - stem cell boost: 
Day 
Regimen  
-10 
TLI (2Gy per fraction once a day) (TLI 1 of 4) 
-9 
TLI (2Gy per fraction once a day) (TLI 2 of 4) 
-8 
TLI (2Gy per fraction twice a day) (TLI 3 of 4 and 4 of 4) 
-7 
Fludarabine (40mg/m2 intravenous every 24 hrs) (dose 1 of 3) 
-6 
Fludarabine (40mg/m2 intravenous every 24 hrs) (dose 2 of 3) 
-5 
Fludarabine (40mg/m2 intravenous every 24 hrs) (dose 3 of 3) 
-4 
Cyclophosphamide (50mg/kg intravenous once per day) (dose 1 of 3) 
-3 
Cyclophosphamide (50mg/kg intravenous once per day) (dose 2 of 3) 
-3 
Rabbit ATG (TEST DOSE 1mg/kg intravenous) (test dose 1 of 1) 
-2 
Cyclophosphamide (50mg/kg intravenous once per day) (dose 3 of 3) 
-2 
Rabbit ATG (3mg/kg intravenous) (standard dose 1 of 2) 
-1 
Rabbit ATG (3mg/kg intravenous) (standard dose 2 of 2) 
 0 
HSC boost infusion (may be infused over more than 1 day) 
Boost regimen related notes: 
• Mesna will be administered intravenously for prevention of hemorrhagic cystitis from the 
medication cyclophosphamide.  In general, mesna is administered at 12.5 mg/kg/dose 
prior to cyclophosphamide and at approximately 3, 6, and 9 hours post cyclophosphamide 
infusion.  Mesna dose and administration schedule may vary based on physician 
recommendation.    
• Rituximab 375 mg/m2/dose will be administered intravenously within approximately 24 
hours of the prior to boost infusion for PTLPD prophylaxis.  
Notes: The following apply to all medication and infusion regimens noted in this protocol 
(primary /boost regimen/mobilization, etc):  
• The stem cell infusion(s) may be delayed by approximately 24 hours in order to 
accommodate stem cell collection with the donor, the Blood Donor Center and/or Human 
Applications Laboratory as well as the recipient research participant clinical condition.  
• The term “every” used in this regimen is an approximate term meaning that these 
medications noted will be administered approximately “every” 12 hours.  The drug 
administration timing in the case of “every 12 hours” may be modified by approximately 
+/- 4 hours or as clinically indicated such as in the case of surgical procedures or to 
accommodate other necessary medication, blood product delivery, dialysis or emergency 
diagnostic procedures.  

 
  
HIFLEX 26 
Revision 6.1 dated: 12/24/2014 
    
          IRB Approval date: 12/06/2016 
Protocol document date: 11/18/2016 
• The term “day” does not refer to an absolute calendar day.  It refers to an approximate, 
general 24 hour time period.   
• Medication dosing may be modified for research recipients based upon actual body 
weight or adjusted ideal body weight when clinically indicated. Criteria for medication 
calculations based on body weight/body surface area can be found in any version of the 
St. Jude Formulary. Medication doses may be rounded to the nearest integer or to the 
nearest appropriate quantity when clinically or pharmaceutically indicated by the 
physician. 
4.8 GVHD prophylaxis for the stem cell boost  
We propose to T-cell deplete hematopoietic stem cell grafts from HAPLO donors using 
the CliniMACS system. Transplant recipients receiving a CD3+ depleted graft for an 
additional stem cell boost infusion will receive MMF prophylaxis beginning on day –2.  
Research participants who receive ≤ 2.5 x 104 CD3+ cells/kg will have MMF 
discontinued on the last day of stem cell infusion.  In general, for research participants 
without GVHD, MMF will be discontinued by approximately day + 60. Research 
participants who require an additional stem cell boost using a CD34+ positive selection 
methodology will not require administration of GVHD prophylactic medication. 
4.9 Donor Lymphocyte Infusions (DLIs)/therapeutic cell, T cell infusions  
DLIs may be administered from the original HSC donor for decreasing donor chimerism, 
serious viral infections or reactivations, or evidence of recurrent disease (including MRD or 
frank relapse). The DLI collected may be collected as a whole blood unit donation or by 
leukapheresis.  If the DLI is collected by standard phlebotomy, the volume to be collected 
would be approximately 300 ml whole blood.  If the DLI is collected by leukapheresis, 
the amount to be processed would be approximately 2 total blood volumes. 
Prior to DLI administration, the immunosuppression should be withdrawn and the recipient 
should have no active serious GVHD.  The initial dose will be 2.5 x 104 CD3+/kg.  
Subsequent doses may be escalated to 5 x 104 CD3+/kg and then 1 x 105 CD3+/kg if no 
moderate or severe GVHD occurs with the prior DLIs. No prophylactic immunosuppression 
will be administered with the DLI.  The typical initial dose escalation for patients on this 
protocol is presented in the following table: 
DLI DOSE AND SCHEDULE 
DLI 
Dose(104 CD3+/kg) 
Comments 
Initial Dose 
2.5 
Approximately 2-4 week 
intervals 
If no moderate or severe 
GVHD 
Dose #2 
5 
Dose #3 
10 
Although this algorithm will be appropriate for a majority, the treating transplant 
attending physician may alter the dose and/or interval of DLI based on response to 
previous DLI, the severity of the clinical situation, and the condition of the patient.  There 
is a low threshold for providing an initial dose of 2.5 x 104 CD3+/kg.  DLI at this dose 
level have not been associated with GVHD.  Therefore, an initial dose may be given for 
any chimerism less than 100% in a patient who is off immune suppression.  The risk of 

 
  
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Revision 6.1 dated: 12/24/2014 
    
          IRB Approval date: 12/06/2016 
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GvHD increases with increasing doses.  In the last 5 years, no patient has developed 
moderate or severe GVHD after DLI dosed at 5 x 104 CD3+/kg, though mild GVHD has 
occasionally occurred.  Doses of 10 x 104 CD3+/kg should be considered to carry a risk of 
severe GVHD.  Therefore dose escalation to this level must incorporate the appropriate 
risk/benefit analysis by the treating transplant physician. 
5.0 DRUG AND DEVICE INFORMATION  
5.1 Fludarabine (Fludara) 
Source and Pharmacology: Fludarabine phosphate is a synthetic purine nucleoside 
analog.  It acts by inhibiting DNA polymerase, ribonucleotide reductase and DNA 
primase by competing with the physiologic substrate, deoxyadenosine triphosphate, 
resulting in inhibition of DNA synthesis.  In addition, fludarabine can be incorporated 
into growing DNA chains as a false base, thus interfering with chain elongation and 
halting DNA synthesis.  Fludarabine is rapidly dephosphorylated in the blood and 
transported intracellularly via a carrier-mediated process.  It is then phosphorylated 
intracellularly by deoxycytidine kinase to the active triphosphate form.  Approximately 
23% of the dose is excreted as the active metabolite in the urine (with dosages of 18-25 
mg/m2/day for 5 days).  Renal clearance appears to become more important at higher 
doses, with approximately 41-60% of the dose being excreted as the active metabolite in 
the urine with dosages of 80-260 mg/m2. 
Formulation and Stability:  Fludarabine is supplied in single-dose vials containing 50 mg 
fludarabine as a white lyophilized powder and 50 mg of mannitol.  The intact vials should 
be stored under refrigeration.  Each vial can be reconstituted by adding 2 ml of sterile 
water for injection resulting in a final concentration of 25mg/ml. Because the 
reconstituted solution contains no antimicrobial preservative, the manufacturer 
recommends that it should be used within 8 hours of preparation.  The solution should be 
further diluted in 5% dextrose or 0.9% NaCl prior to administration. 
 
Supplier: Commercially available. 
 
Toxicity: The major dose-limiting toxicity of fludarabine is myelosuppression.  Nausea 
and vomiting are usually mild.  Side effects reported commonly include anorexia, fever 
and chills, alopecia and rash.  Neurotoxicity can be manifested by somnolence, fatigue, 
peripheral neuropathy, mental status changes, cortical blindness and coma and is more 
common at high doses.  Neurotoxicity is usually delayed, occurring 21-60 days after the 
completion of a course of therapy and may be irreversible.  Side effects reported less 
commonly include diarrhea, stomatitis, increased liver function tests, liver failure, chest 
pain, arrhythmias and seizures.  Pulmonary toxicity includes allergic pneumonitis 
characterized by cough, dyspnea, hypoxia and pulmonary infiltrates.  Drug induced 
pneumonitis is a delayed effect, occurring 3-28 days after the administration of the third 
or later course of therapy.  Administration of corticosteroids usually results in resolution 
of these symptoms. 
Route of Administration: Intravenous. 
 
 

 
  
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5.2 Thiotepa (Thioplex by Immunex) (TESPA, TSPA) 
Source and Pharmacology: Thiotepa is a cell-cycle nonspecific polyfunctional alkylating 
agent.  It reacts with DNA phosphate groups to produce cross-linking of DNA strands 
leading to inhibition of DNA, RNA and protein synthesis.  Thiotepa is extensively 
metabolized in the liver to metabolites that retain activity, primarily triethylene-
phosphoramide (TEPA).  The main route of elimination is via the urine, mainly as 
metabolites; the elimination half-life of the thiotepa is 2.5 hours, and that of TEPA is 17.6 
hours. 
Formulation and Stability: Thiotepa is supplied in single-use vials containing 15 mg of 
lyophilized thiotepa, 80 mg NaCl and 50 mg NaHCO3.  The intact vials should be stored 
under refrigeration and protected from light.  Each vial should be reconstituted with 1.5 
ml of sterile water for injection to yield a concentration of 10 mg/ml.  Further dilution 
with sterile water for injection to a concentration of 1 mg/ml yields an isotonic solution; 
if larger volumes are desired for intracavitary, intravenous infusion, or perfusion therapy, 
this solution may then be diluted with 5% dextrose or 0.9% NaCl containing solutions.  
The 10 mg/ml reconstituted solution is chemically stable when stored in the refrigerator 
for up to 5 days, however, it is recommended that solutions be prepared just prior to 
administration since they do not contain a preservative.  Reconstituted solutions should 
be clear to slightly opaque: the solutions may be filtered through a 0.22 micron filter to 
eliminate haze. 
Supplier:  Commercially available; manufactured by Immunex. 
Toxicity: Dose limiting toxicity is myelosuppression.  The leukocyte nadir may occur at 
any time from 10 to >30 days.  Other toxicities include pain at the injection site, nausea 
and vomiting, anorexia, mucositis, dizziness, headache, amenorrhea, interference with 
spermatogenesis, and depigmentation with topical use.  Allergic reactions, including skin 
rash and hives, have been reported rarely.  Rare cases of apnea, hemorrhagic cystitis, and 
renal failure have occurred.  Thiotepa is mutagenic, carcinogenic, and teratogenic in 
animals.  Pregnancy category D.  
Route of Administration: Thiotepa will be administered as an intravenous infusion. 
5.3 Melphalan (L-phenylalanine mustard, phenylalanine mustard, L-PAM, L-sarcolysin, 
Alkeranâ) 
 
Source and Pharmacology: Melphalan, a derivative of nitrogen mustard, is a bifunctional 
alkylating agent.  Its chemical name is 4-[bis(2-chloroethyl)amino]-L-phenylalanine, and 
it has a molecular weight of 305.20.  Melphalan is active against tumor cells that are 
actively dividing or at rest.  Its cytotoxicity is thought to be due to inter-strand cross-
linking with DNA, probably by binding at the N7 position of guanine.  Melphalan is 
highly protein bound and does not penetrate well into the cerebral spinal fluid. 
Elimination half-life after intravenous administration in adults is approximately 75 
minutes. Elimination appears to be primarily by chemical hydrolysis, but caution should 
be used in patients with renal impairment.  Plasma concentrations of melphalan after oral 
administration are highly variable, possibly due to incomplete absorption, variable “first 
pass” hepatic metabolism or rapid hydrolysis. Area under the plasma concentration-time 

 
  
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Revision 6.1 dated: 12/24/2014 
    
          IRB Approval date: 12/06/2016 
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curves for orally administered melphalan is approximately 60% of intravenously 
administered melphalan in adult studies.  
Formulation and Stability: Available as 2 mg tablets for oral administration. This 
medication is stable at room temperature until expiration date on the packaging.  
Intravenous formulation is supplied as 50 mg freeze dried glass vial. Each 50 mg vial is 
supplied in a carton containing a 10 ml vial of sterile diluent. Lyophilized melphalan 
should be stored at controlled room temperature and protected from light. Each vial is 
marked with its expiration date. The melphalan for injection must be reconstituted 
immediately prior to infusion by rapidly adding the contents of the diluent vial (10 ml) to 
the freeze dried powder with a 20 gauge or larger sterile needle and immediately shaking 
vigorously until a clear solution is obtained.  This results in a 5 mg/ml solution.  The dose 
should then be diluted in 0.9% sodium chloride for injection to a final concentration of 
not greater than 0.45 mg/ml. The resulting admixture should be infused over a minimum 
of 15 minutes.  The infusion should be completed within 60 minutes of 
reconstitution.  Do Not Refrigerate the Reconstituted Melphalan.   
Supplier: Commercially available. 
  
Toxicity: Melphalan is cytotoxic and caution should be used in handling and preparing 
the solution or administering the tablets. Use of gloves is recommended, and if contact 
with skin or mucosa occurs, immediately wash thoroughly. Second cancers such as acute 
non-lymphocytic leukemia, myeloproliferative syndrome, and carcinoma have been 
reported in patients taking melphalan alone or in combination with other chemotherapy or 
radiation. Melphalan causes suppression of ovarian function in premenopausal women, 
with a significant number of patients having amenorrhea. Testicular suppression 
(reversible and irreversible) has been reported.  The most common adverse reaction is 
myelosuppression. Irreversible bone marrow failure has been reported. Gastrointestinal 
side effects reported include nausea/vomiting, diarrhea and oral mucosa ulceration.  
Hepatic toxicity has occurred, including veno-occlusive disease.  Acute hypersensitivity 
reactions occur in about 2.4% of patients, and can include anaphylaxis. Hypersensitivity 
reactions were characterized by urticaria, pruritus, and edema.  Some patients exhibited 
tachycardia, bronchospasm, dyspnea and hypotension that responded to antihistamines 
and corticosteroids. Other side effects that have been reported include skin ulceration or 
necrosis at injection site, vasculitis, alopecia, hemolytic anemia, pulmonary fibrosis, and 
interstitial pneumonitis.  
   Route of Administration: Intravenous 
5.4 Mycophenolate mofetil (MMF, CellCept) 
Source and Pharmacology: Mycophenolate mofetil is hydrolyzed to mycophenolic acid 
(MPA), an immunosuppressive agent. MPA inhibits B and T-cell proliferation, T-cell 
synthesis, and antibody secretion by potent, noncompetitive reversible inhibition of 
inosine monophosphate dehydrogenase (IMPDH) in the purine biosynthesis pathway. 
Inhibition of IMPDH results in a depletion of guanosine triphosphate and 
deoxyguanosine triphosphate, important intermediates in the synthesis of lymphocyte 
DNA, RNA, proteins and glycoproteins. Oral formulations of mycophenolate mofetil are 
rapidly and extensively absorbed when given on an empty stomach. Food and aluminum 
and magnesium-containing antacids decrease absorption of MMF.  MMF is rapidly 

 
  
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Revision 6.1 dated: 12/24/2014 
    
          IRB Approval date: 12/06/2016 
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hydrolyzed to the active metabolite (MPA) after oral or intravenous administration.  Free 
MPA is conjugated in the liver by glucuronyl transferase to inactive mycophenolic acid 
glucuronide (MPAG) that is excreted in the urine and feces.  Time to peak plasma 
concentration is 0.8 – 1.3 hours, and the mean elimination half-life is 17.9 hours. 
Enterohepatic recirculation of MPA contributes to plasma concentrations. Administration 
of cholestyramine interrupts the enterohepatic recirculation and can decrease 
bioavailability by as much as 40%.  Patients with renal insufficiency have increased 
plasma concentrations of MPA and MPAG.  Acyclovir and ganciclovir may compete 
with MPAG for renal tubular secretion, resulting in increased plasma concentrations of 
both drugs.   
Formulation and Stability: Mycophenolate mofetil is commercially available as 250 mg 
capsules, 500 mg tablets, 200 mg/ml powder for oral suspension, and 500 mg vials of 
powder for injection.  
Supplier: Hoffmann La Roche, Inc.    
Toxicity: Adverse events seen in patients taking mycophenolate mofetil include 
hypertension, hypotension, peripheral edema, leukopenia, anemia, thrombocytopenia, 
hypochromic anemia, leukocytosis, headache, insomnia, dizziness, tremor, anxiety, 
paresthesia, hyperglycemia, hypercholesterolemia, hypokalemia, diarrhea, hyperkalemia, 
hypophosphatemia, constipation, nausea, vomiting,  anorexia, abdominal pain, dyspepsia, 
urinary burning or frequency, renal tubular necrosis, hematuria, increase serum creatinine 
and BUN, a variety of infections due to immunosuppression, rash, acne, ocular changes 
(cataracts, blepharitis, keratitis, glaucoma, and macular abnormalities) occasional leg 
cramps or pain, bone pain, myalgias, and hand cramps.  Intravenous infusions have been 
reported to cause thrombosis and phlebitis. There have been occasional reports of 
gastrointestinal hemorrhage.  High dose therapy with mycophenolate in adults with 
psoriasis has been associated with the following neoplasms: adenocarcinoma of the breast 
and colon, basal cell carcinoma, carcinoma of the gallbladder, histiocytic lymphoma, 
glioblastoma multiforme, and squamous cell carcinoma of the epiglottis.   
Administration: Oral or intravenous. 
5.5 Rituximab (Rituxan ™) 
 
Source and Pharmacology: Rituximab is a murine /human chimeric monoclonal antibody.  
It is specific for the CD20 antigen located on B cells. It has been shown to mediate 
complement-dependent tumor cell lysis and antibody-dependent cellular cytotoxicity. 
Direct binding to the CD20 antigen is thought to play a role in inhibition of cell growth.  
Rituximab is administered intravenously. The mean serum half-life after a single 
intravenous dose of 375 mg/m2 is 59.8 hours (range 11.1-104.6 hours).  
 
Formulation and Stability: Rituximab is available as 100 mg/10 ml single-use and 500 
mg/50 ml single-use vials. Each vial also contains sodium chloride 9 mg/ml, sodium 
citrate 7.35 mg/ml, polysorbate 80 0.7 mg/ml and water for injection. Rituximab for 
injection concentration must be diluted with 5% Dextrose or 0.9% NaCl prior to 
administration.  After dilution, unused drug is stable for 24 hours when refrigerated (2-8 
degrees Celsius) and 12 hours at room temperature. Vials should be protected from direct 
sunlight.  

 
  
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Revision 6.1 dated: 12/24/2014 
    
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Supplier:  Commercially available. 
 
Toxicity: Hypersensitivity reactions may occur; therefore, premedication with 
acetaminophen and diphenhydramine should be considered before each infusion.  The 
most common toxicities are infusion related and may include chills, fever, headache, 
nausea, vomiting, angioedema (13%), hypotension (10%), bronchospasm (8%), and 
arrhythmias. Other possible adverse reactions include thrombocytopenia, myalgias, 
arthralgias, asthenia, and throat irritation.  
 
Route of Administration: Intravenous.  Do not administer as an intravenous push or 
bolus.   
5.6 Alemtuzumab (Campath-1H, Campath) 
Source and Pharmacology:  Alemtuzumab is an unconjugated, humanized monoclonal 
antibody against the CD52 antigen, which is expressed on the surface of B-lymphocytes, 
T-lymphocytes, monocytes, and platelets but not hematopoietic stem cells. It has 
preferential effects in the blood and bone marrow as opposed to the spleen or lymph 
nodes. Alemtuzumab is associated with profound T-lymphocyte depletion. It mediates the 
lysis of lymphocytes via complement and antibody dependent cell mediated cytotoxicity 
mechanisms. Alemtuzumab is associated with the release of tumor necrosis factor (TNF), 
interleukin-6, and interferon gamma. 
Formulation and Stability:  Alemtuzumab is available as a clear, colorless, isotonic, 
sterile phosphate buffered saline solution at 10 mg/ml in 3 ml ampules. It is stable at least 
12 months if refrigerated and protected from light. After dilution in NS or 5% dextrose, 
alemtuzumab is physically and chemically stable at room temperature for up to 24 hours. 
The manufacturer recommends use within 8 hours of preparation due to absence of a 
preservative. At concentrations above 20 mcg/ml, there was no detectable loss of drug 
associated with IV administration sets. A sterile, low-protein binding, non-fiber releasing 
5 micron filter should be use when removing the medication from the ampule. The 
medication ampule should not be shaken. The admixture should be mixed by gently 
inverting the bag containing the final solution.  
Toxicity: Infusion related reactions occur in most patients, and commonly include rigors, 
fever, headache, nausea, vomiting, diarrhea, rash, pruritis, dyspnea and hypotension. Also 
reported are chills, abdominal and back pain, bronchospasm, angioedema, and 
tachyarrhythmias. These reactions are most common during the first week of treatment, 
and usually improve with subsequent doses. Other expected adverse events are anemia, 
neutropenia, thrombocytopenia, prolonged and profound lymphopenia, and bacterial and 
opportunistic infections. Also reported are fatigue, hypertension, angina and myocardial 
infarction, peripheral vasoconstriction, anorexia, constipation, dyspepsia, liver function 
abnormality, DIC, hemolysis, eosinophil disorder, bleeding (GI, gum, ecchymosis), 
myalgia, arthritis, bone pain, hypotonia, tremor, tumor lysis syndrome, acidosis, 
dizziness, confusion, somnolence, peripheral neuropathy, cerebral hemorrhage, speech 
disorder, paresthesia, syncope, depression, aphasia, cough, pleural effusion, pulmonary 
edema, interstitial pneumonitis, facial flushing, diaphoresis, urticaria, oliguria, polyuria, 
urinary retention, urinary tract infection, impotence, tinnitus, and  injection site reactions 
if given SC.  

 
  
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Revision 6.1 dated: 12/24/2014 
    
          IRB Approval date: 12/06/2016 
Protocol document date: 11/18/2016 
Supplier: Campath is produced by Ilex Oncology Services and Millennium 
Pharmaceuticals and distributed by Berlex Laboratories.  It has been designated an 
orphan drug for the treatment of chronic lymphocytic leukemia and is commercially 
available.  
Dosage and Route of Administration: Alemtuzumab is associated with infusion related 
reactions, especially with the first dose. The severity of these reactions can be decreased 
by premedication with diphenhydramine and acetaminophen 30 minutes prior to infusion, 
and by starting with a low dose and gradually increasing to the target dose. The dose 
should be mixed in NS or 5% dextrose at a concentration greater than 20 mcg/ml and 
administered over 2 hours through a 0.22 micron filter. Alemtuzumab can be 
administered subcutaneously.  
5.7 Cyclophosphamide (Cytoxan) 
 
Source and Pharmacology: Cyclophosphamide is a nitrogen mustard derivative.  It acts as 
an alkylating agent that causes cross-linking of DNA strands by binding with nucleic 
acids and other intracellular structures, thus interfering with the normal function of DNA. 
It is cell cycle, phase non-specific. Cyclophosphamide is well absorbed from the GI tract 
with a bioavailability of >75%.  It is a prodrug that requires activation. It is metabolized 
by mixed function oxidases in the liver to 4-hydroxycyclo-phosphamide, which is in 
equilibrium with aldophosfamide. Aldofosfamide spontaneously splits into nitrogen 
mustard, which is considered to be the major active metabolite, and acrolein. In addition, 
4-hydroxycy-clophosphamide 
may 
be 
enzymatically 
metabolized 
to 
4-
ketocyclophosphamide and aldophosfamide may be enzymatically metabolized to 
carboxyphosphamide that is generally considered inactive.  Cyclophosphamide and its 
metabolites are excreted mainly in the urine.  Dose adjustments should be made in 
patients with a creatinine clearance of <50 ml/min.     
 
Formulation and Stability: Cyclophosphamide is available in vials containing 100, 200, 
500, 1000 and 2000mg of lyophilized drug and 75 mg mannitol per 100 mg of 
cyclophosphamide.  Both forms of the drug can be stored at room temperature. The vials 
are reconstituted with 5, 10, 25, 50 or 100 ml of sterile water for injection, respectively, 
to yield a final concentration of 20 mg/ml.  Reconstituted solutions may be further diluted 
in either 5% dextrose or 0.9% NaCl containing solutions.  Diluted solutions are 
physically stable for 24 hours at room temperature and 6 days if refrigerated, but contain 
no preservative, so it is recommended that they be used within 24 hours of preparation. 
 
Supplier:  Commercially available 
 
Toxicity: Dose limiting toxicities of cyclophosphamide includes bone marrow 
suppression and cardiac toxicity.  Cardiac toxicity is typically manifested as congestive 
heart failure, cardiac necrosis or hemorrhagic myocarditis and can be fatal.  Hemorrhagic 
cystitis may occur and necessitates withholding therapy. The incidence of hemorrhagic 
cystitis is related to cyclo-phosphamide dose and duration of therapy. Forced fluid intake 
and/or the administration of mesna decreases the incidence and severity of hemorrhagic 
cystitis.  Other toxicities reported commonly include nausea and vomiting (may be mild 
to severe depending on dosage), diarrhea, anorexia, alopecia, immunosuppression and 
sterility.  Pulmonary fibrosis, SIADH, anaphylaxis and secondary neoplasms have been 
reported rarely.    

 
  
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   Route of Administration: intravenous infusion  
5.8 Mesna (Mesnex) 
 
Source and Pharmacology: Mesna is a synthetic sulfhydryl (thiol) compound. Mesna 
contains free sulfhydryl groups that interact chemically with urotoxic metabolites of 
oxaza-phosphorine derivatives such as cyclophosphamide and ifosfamide. Oral 
bioavailability is 50%. Upon injection into the blood, mesna is oxidized to mesna 
disulfide, a totally inert compound.  Following glomerular filtration, mesna disulfide is 
rapidly reduced in the renal tubules back to mesna, the active form of the drug.  Mesna 
and mesna disulfide are excreted primarily via the urine. 
 
Formulation and Stability: Mesna is available in 2 ml, 4 ml and 100 ml amps containing 
100 mg/ml of mesna solution.  The intact vials can be stored at room temperature.  Mesna 
may be further diluted in 5% dextrose or 0.9% NaCl containing solutions.  Diluted 
solutions are physically and chemically stable for at least 24 hours under refrigeration.   
 
Supplier:  Commercially available 
 
Toxicity:  Mesna is generally well tolerated.  Nausea and vomiting, headache, diarrhea, 
rash, transient hypotension and allergic reactions have been reported.  Patients may 
complain of a bitter taste in their mouth during administration. Mesna may cause false 
positive urine dipstick readings for ketones.   
Dosage and Administration: Mesna is generally dosed at approximately 25% of the 
cyclo-phosphamide dose.  It is normally ordered to be given intravenously prior to and 
again at 3, 6 and 9 hours following each dose of cyclophosphamide. 
5.9 Anti-thymocyte globulin (Rabbit) (Thymoglobulin, Rabbit ATG) 
 
Source & Pharmacology: Anti-thymocyte globulin is a purified, pasteurized, gamma 
immune globulin, obtained by immunization of rabbits with human thymocytes.  This 
immunosuppressive product contains cytotoxic antibodies directed against antigens 
expressed on human T-lymphocytes.  The mechanism of action by which polyclonal 
antilymphocyte preparations suppress immune responses is not fully understood.  
Possible mechanisms by which anti-thymocyte globulin may induce immunosuppression 
in vivo include: T-cell clearance from the circulation and modulation of T-cell activation, 
homing and cytotoxic activities.  Anti-thymocyte globulin (rabbit) includes antibodies 
against T-cell markers such as CD2, CD3, CD4, CD8, CD11a, CD18, CD25, CD44, 
CD45, HLA-DR, HLA Class 1 heavy chains, and β2 microglobulin. T-cell depletion is 
usually observed within a day from initiating anti-thymocyte globulin therapy.   
Formulation and Stability: Anti-thymocyte globulin (refrigerated) is available as sterile, 
lyophilized powder to be reconstituted with sterile diluent (both lyophilized powder and 
diluent should be at room temperature before reconstitution). Reconstituted solutions 
provide a final concentration of 5mg/ml x 5ml.  The product must be further diluted in 
normal saline prior to administration.  Infusions should be prepared immediately prior to 
administration.  
Supplier:  Commercially available. 
Toxicities: Frequently reported events include fever, chills, leukopenia, pain, headache, 
abdominal pain, diarrhea, hypertension, nausea, thrombocytopenia, peripheral edema, 

 
  
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dyspnea, asthenia, hyperkalemia, tachycardia.  The most serious toxicity is that of 
anaphylaxis.  Supportive medical resources must be readily available for patient 
management anaphylaxis.  Anaphylaxis precludes further administration of the drug.  The 
dose must be administered over at least 4 hours and the patient pretreated with 
antihistamine, corticosteroid, and antipyretic. 
Route of administration:  Intravenous. Test doses may be infused over 1 to 2 hours. The 
first full dose should be infused over a minimum of 6 hours. If tolerated, subsequent 
doses may be infused over 4 hours. 
5.10 G-CSF (Filgrastim, Neupogen)  
Source and Pharmacology:  G-CSF (granulocytic colony stimulating factor), is a 
biosynthetic hematopoietic agent that is made using recombinant DNA technology in 
cultures of Escherichia coli.  G-CSF stimulates production, maturation and activation of 
neutrophils.  In addition, endogenous G-CSF enhances certain functions of mature 
neutrophils, including phagocytosis, chemotaxis and antibody--dependent cellular 
cytotoxicity. 
Formulation and Stability:  G-CSF is supplied in vials containing 300 mcg and 480 mcg 
of G-CSF at a concentration of 300mcg/ml. The intact vials should be stored under 
refrigeration.  The vials can be left out of refrigeration for 24 hours, but should be 
discarded if left at room temperature for longer periods of time.  G-CSF can be drawn up 
into tuberculin syringes for administration and stored under refrigeration for up to 7 days 
prior to usage.  G-CSF can be further diluted for intravenous infusion in 5% dextrose.  Do 
not dilute in saline---precipitate may form. If the final concentration of this product is 
<15 mcg/ml, it is recommended that albumin be added to a final concentration of 2mg/ml 
(0.2%) to minimize adsorption of the drug to infusion containers and equipment.   
Supplier:  Commercially available. 
Toxicity: G-CSF causes marked leukocytosis.  Adverse reactions reported commonly 
include bone pain, thrombocytopenia, diarrhea, nausea, rash, alopecia, fever, anorexia 
and pain or bruising at the injection site.  Allergic reactions, MI, atrial fibrillation, and 
splenomegaly have been reported rarely. G-CSF is contraindicated in research 
participants with allergy to E. coli derived products. 
Route of administration: Intravenous or subcutaneous. 
5.11 CliniMACSTM System using CD3+ depletion 137 
The mechanism of action of the CliniMACS Cell Selection System is based on magnetic-
activated cell sorting (MACS).  The CliniMACS device is a powerful tool for the 
isolation of many cell types from heterogeneous cell mixtures, (e.g. apheresis 
products).  These can then be separated in a magnetic field using an immunomagnetic 
label specific for the cell type of interest, such as CD3+ human T cells. 
The cells to be isolated are specifically labeled with super-paramagnetic particles by an 
anti-body directed toward a cell surface antigen.  After magnetic labeling, the cells are 
separated using a high-gradient magnetic separation column as described below.  The 
magnetically labeled cells are retained in the magnetized column while the unlabeled 
cells flow through the column for collection.  The retained cells are eluted by removing 

 
  
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the magnetic field from the column, washing the cells out and collecting them in a 
separate container from the unlabeled cells. 
The super-paramagnetic particles are small in size (about 50 nm in diameter) and are 
composed 
of 
iron 
oxide/hydroxide 
and 
dextran 
conjugated 
to 
monoclonal 
antibodies.  These magnetic particles form a stable colloidal solution and do not 
precipitate or aggregate in magnetic fields.  The OKT3 antibody used in this system is 
highly specific for CD3+ cells. High-gradient MACS technology has been shown to 
achieve rapid and highly specific depletion of large numbers of CD3+ cells from bone 
marrow, cord blood, and normal peripheral blood mononuclear cells.  
The CliniMACS device incorporates a strong permanent magnet and a separation column 
with a ferromagnetic matrix to separate the cells labeled with the magnetic particles. The 
high-gradient system allows the application of strong magnetic forces and a rapid de-
magnetization. Small ferromagnetic structures, such as the column matrix, placed in a 
magnetic field concentrate this homogenous field and thereby produce high magnetic 
gradients. In their immediate proximity, the ferromagnetic structures generate magnetic 
forces 10,000-fold greater than in the absence of those structures enabling the retention of 
magnetically labeled cells. After removing the column from the magnet, the rapid de-
magnetization of the column matrix allows the release of retained cells.  
The CliniMACS device is comprised of a computer controlled instrument incorporating a 
strong permanent magnet, a closed-system sterile tubing set containing columns with a 
coated ferromagnetic matrix and a paramagnetic, cell specific, labeling reagent. The 
instrument will separate the cells in a fully automated process yielding a cell population 
highly depleted of CD3+ cells. The CliniMACS device is not licensed by the FDA and 
therefore is investigational. 
5.12 CliniMACSTM System for CD34+ positive selection 138 
The CliniMACS device has separate programs that allow cell selection procedures 
optimized for either a depletion (e.g. CD3 depletion) or a selection of a target cell 
population (e.g. CD34+ cells). The basic mechanism is the same for either application; 
target cells are "tagged" with super-paramagnetic particles and eventually separated from 
the unlabeled cells using the CliniMACS device as described above. The desired target 
cells can either be infused or discarded appropriately.  In this protocol, if required, 
CD34+ hematopoietic stem cells from the original donor will be positively selected with 
the CliniMACS and used as the graft. 
6.0 REQUIRED OBSERVATIONS/EVALUATIONS 
6.1 Pre/peri/post-transplant evaluations 
All pre/peri/posttransplant and long-term follow-up evaluations for these participants will 
be carried out as outlined in Appendix D, and guided by the Standard Operating 
Procedures (SOPs) of the St. Jude Children's Research Hospital, Department of BMTCT, 
for recipients of allogeneic stem cell transplantation.  Copies of these SOPs and ongoing 
updates 
can 
be 
found 
at 
the 
following 
site: 
http://home.web.stjude.org/bone_marrow/clinicalHome.shtml. 

 
  
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Furthermore, to accommodate the research studies, flexibility in the date is allowed 
without a deviation from protocol.  The degree of flexibility in the timing is also provided 
in Appendix D.    
6.2 Long-term follow-up evaluations 
In general, recipients of allogeneic HCT at St. Jude are seen at least annually until 10 
years posttransplant in the Department of BMTCT outpatient clinic. For the purpose of 
this study, research participants will be followed to year 5 post-transplantation (refer to 
Appendix D for long-term follow-up requirements). At the 1 year post-transplantation 
time point, all study participants will be eligible for enrollment in the institutional long-
term follow-up protocol for children and young adults who have received stem cell 
transplantation at St. Jude Children’s Research Hospital (BMTFU protocol). 
6.3 Evaluation for chimerism and engraftment 
Evaluation for chimerism and engraftment will be performed on bone marrow or 
peripheral blood samples according to the timelines noted in Appendix D. However, for 
research participants who have less than 95% donor chimerism at/about day +21 
posttransplant, a repeat bone marrow study (to include chimerism) may be performed 
approximately 1 week after the initial procedure, at the discretion of the treating 
physician.  The time to neutrophil and platelet engraftment will be recorded. Neutrophil 
engraftment will be defined as the first of 3 consecutive days of an absolute neutrophil 
count (ANC) > 500/mm3. Time to platelet engraftment will be designated as the time to 
platelet count exceeding 20,000/mm3 and 50,000/mm3 without a platelet transfusion in 
the preceding 7 days.  Patients requiring platelet transfusions to maintain platelet counts 
above the previously defined values will not be evaluable for these outcomes. 
Chimerism studies will be performed by VNTR methodology performed in Molecular 
Pathology with a time to result of approximately 48 hours.  Additional bone marrow 
and/or peripheral blood chimerism studies may be performed throughout the course of 
this study when clinically indicated. Chimerism studies derived from bone marrow may 
be used in lieu of a specified peripheral blood sample if a bone marrow sample is 
available.  In the event of graft failure/rejection, subsequent chimerism studies may be 
held, as they would not be clinically indicated at that time. 
If there is an initial decrease in donor chimerism to less than 90% at any time on 
peripheral blood studies, a bone marrow examination will subsequently be performed 
within approximately 2 weeks to confirm this initial decline.  In addition, chimerism 
analysis will be performed in subsets of lymphocytes, granulocytes, and monocytes for 
research participants with increasing host chimerism until the research participant attains 
≥ 95% donor chimerism. Chimerism studies will be performed in the St. Jude Department 
of Pathology using standard DNA techniques [i.e. VNTR (variable number tandem 
repeat)] and/or FISH (fluorescent in-situ hybridization) analysis.  Chimerism studies are 
reported in the database as donor percentages. 
6.4 Evaluation for immune reconstitution 
Research participants will have immune reconstitution studies of lymphocyte subsets (i.e. 
cells, B cells, and NK cells).  These studies may be performed more often than what is 
outlined in Appendix D depending on the clinical status of the research participant.   

 
  
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Immune analysis as described (Mnemonic listed in 6.4.1 through 6.4.4) will be performed 
according to the schedule outlined in Appendix D until the immune parameters recover to 
normal level or donor pattern:  
6.4.1  Lymphocyte subsets study: Flow cytometry enumeration.   
6.4.2 
 VBETA/TREC Research: Thymic output and T cell repertoire.   
6.4.3 
 Lymphocyte Phenotypes Research: T cell and NK cell number and function. 
6.4.4  
 Quantitative immunoglobulins: IgG, IgM, and IgA levels.  
6.4.5  Soluble interleukin-2 receptor (sIL-2r) and tumor necrosis factor (TNF-alpha, 
TNFR1): Testing will be performed weekly starting on day -7 and then weekly 
until on or about day +100 posttransplant. 
6.5 General viral surveillance 
Serial PCR testing for CMV, adenovirus, and EBV will be done weekly to day 100 and 
then on an as needed basis.  In research participants with progressive or active infection, 
samples may be obtained more frequently (St. Jude test mnemonic BMTPCR).   
6.6 Evaluations for Campath-1H pharmacokinetics (optional) 
   Campath-1H pharmacokinetics will be measured on up to 15 recipients.  The participants 
will be divided into 3 cohorts, with up to 5 recipients in each: 1) less than 15kg; 2) 
greater than 15kg and less than 1m2; 3) greater than 1m2.  The following time points will 
be used for each patient: Day -10, Day -8, Day -3 before transplant, and then at weeks 1, 
2, 4, 8, and 16 after transplant.  Approximately 2mL of peripheral blood will be drawn at 
each time point. The samples will be stored frozen and will be shipped in dry ice, in 
batches, every 6 months, to Genzyme for analysis. 
The responsibilities and procedures for PK sample collection, submission, and analysis 
are outlined in APPENDIX F.  
6.7 Minimal residual disease (MRD) evaluation  
 
MRD assays in peripheral blood and/or bone marrow by immunologic and molecular 
methods will only be performed for those research participants who have had this test 
performed during prior therapy for their disease at St. Jude or for those who have samples 
of diseased marrow available to identify a leukemic marker for MRD testing. Bone 
marrow and/or peripheral blood will be obtained from the research participants at specific 
time points (refer to Appendix D for schedule of MRD testing).  MRD assays may be 
performed more frequently in research participants with increasing host chimerism. We 
will apply immunologic and molecular methods, as previously described.123,124 Tests will 
be performed in the appropriate St. Jude laboratories.   
6.8 Research tests on haploidentical donor (optional) 
Donors will be offered the option for participation in research studies of immune 
reconstitution of T cells, B cells, and NK cells.  These tests will be obtained after consent 
and preferably prior to growth factor administration.  Lymphocyte subset analysis of the 
donor appears to allow for the prediction of the reconstitution of the lymphocyte subsets 
in the research participant after transplantation.  Data in larger donor/research participant 

 
  
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pairs will help to verify these observations. A list of these optional research studies are 
noted in Appendix D and detailed below: 
6.10.1  Lymphocyte subsets study: Flow cytometry enumeration.  
6.10.2  VBETA/TREC Research: Thymic output and T cell repertoire.   
6.10.3 Lymphocyte Phenotypes Research: T cell and NK cell number and function. 
6.10.4 Soluble interleukin-2 receptor (sIL-2r) and tumor necrosis factor (TNF-alpha, 
TNFR1) 
  7.0 EVALUATION CRITERIA 
7.1 Adverse event (AE) monitoring for on-study research participants will be assessed using the 
NCI Common Toxicity Criteria Version 3.0.  The specific criteria for AE monitoring are 
noted Section 9.0 and in Appendix C.   
7.2 GVHD scoring (acute and chronic) will be evaluated and graded using the criteria found 
in Appendix B of this protocol. 
7.3 Performance status will be assessed by Karnofsky/Lansky Performance Scores (age-
dependent) (see Appendix A). 
7.4 Hematologic recovery posttransplant will be determined using the engraftment criteria as 
follows: (1) neutrophil engraftment will be defined as the first of 3 consecutive days of an 
ANC ≥ 500/mm3 with evidence of donor cell engraftment; (2) platelet engraftment will be 
defined as platelet count ≥ 50,000/mm3 with no platelet transfusions in the preceding 7 days. 
7.5 Primary graft failure will be defined as an ANC never meeting or exceeding 500/mm3 for 
3 consecutive days and no evidence (<10%) of donor chimerism by day +21 
posttransplant.  
7.6 Secondary graft failure or graft rejection will be defined as no evidence (<10%) of donor 
chimerism in research participants with prior neutrophil engraftment.  
7.7 Mixed hematopoietic chimerism will be defined as between 10% and 95% donor 
chimerism in the absence of immunosuppressive therapy. 
8.0 OFF-STUDY AND OFF-THERAPY CRITERIA 
 
8.1 Transplant recipient research participants will remain on-study until one of the following 
occurs (i.e. off-study criteria): 
  8.1.1 Withdrawal from protocol. Research participants or legal guardian(s) may 
withdraw consent to participate in this study at any time.  Physician may 
withdraw participant if at any time the treatment is deemed unsafe or not in the 
participant’s best interest. 
8.1.2 Death. 

 
  
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8.1.3 Donor unable to provide the stem cells/stem cell doses required for intended 
recipient to undergo primary stem cell transplant.   
8.1.4 Requires an additional transplant procedure using a different allogeneic donor 
(meaning a donor other than the HAPLO donor used for this protocol procedure).   
8.1.5 Unable to be contacted and/or effectively monitored by the principal investigator 
(PI) and/or designees for follow-up (lost to follow-up). 
8.1.6 Five years post primary stem cell infusion (i.e. has completed the year +5 post 
primary transplant evaluation). 
8.2 Transplant recipient research participants will remain on-study but considered off-therapy 
if one of the following occurs (i.e. off-therapy criteria): 
8.2.1 Requires additional chemotherapy for recurrent disease. 
8.2.2 Experiences graft failure or rejection despite receiving one stem cell boost 
infusion. 
8.2.3 Experiences graft failure or rejection despite receiving more than two donor 
lymphocyte infusions within the first 100 days post primary transplant infusion.  
8.2.4 Noncompliance with protocol medications/administrations and/or required 
follow-up evaluations as judged by the PI.  
8.2.5 Pregnancy. 
8.2.6 Donor has been removed from study after the primary HSCT has been 
administered (criteria for donor removal for study – see below) and the recipient 
requires a stem cell boost.  Meaning donor unable to provide the additional cells 
required for the boost infusion if needed.  
Notes:  
• 
Off-therapy transplant recipient research participants will be followed for disease 
status, survival status (cause of death /date of last follow-up) and GVHD only 
until the time an off- study criterion is met. 
8.3 Donor research participants will remain on study until one of the following occurs:  
8.3.1 Donor research participant withdrawal. Donor research participants may withdraw 
their consent to participate at any time. 
8.3.2 Off-study date will be determined once the PI has established that the transplant 
recipient does/will not require the primary transplant infusion, or an additional 
infusion for the purpose of this protocol.   
8.3.3 Seven days post final cell collection procedure.   
8.3.4 Day of transplant recipient death.  
8.3.5 Pregnancy.  
8.3.6 Development of a health disorder, including a clinically significant risk 
for/positive testing for communicable disease, which in the opinion of the PI 

 
  
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would render the donor ineligible to serve (or continue to serve) as a stem cell 
donor.  
8.3.7 Death. 
8.3.8 Unable to be contacted and/or effectively monitored by PI and/or designees for 
follow-up (lost to follow-up). 
8.3.9 Noncompliance with protocol mobilization medications and/or apheresis 
procedure(s) and/or required testing as judged by the PI.  
9.0 REPORTING CRITERIA  
9.1 Reporting Adverse Experiences and Deaths to St. Jude IRB 
The principal investigator is responsible for promptly reporting to the IRB any adverse 
events that are unexpected/unanticipated, serious, and that may represent potential harm or 
increased risk to research participants.  When an unexpected death occurs, the PI should 
report it to the Director of Human Subject’s Protection immediately, by phone: (901) 595-
4359, cell: (901) 336-2894, fax: (901) 595-4361, or e-mail: hsp-1@stjude.org.  A 
reportable event entry into TRACKS should follow within 48 hours of notification of the 
event. 
Serious, unexpected, and related or possibly related events must be reported within 10 
business days of notification of the event.  At the same time, the investigator will notify the 
study sponsor and/or the FDA, as appropriate.  All other SAEs, including expected death, 
and all captured AEs will be reported to the IRB at the time of the continuing reviews, with 
the following exceptions: 
• Any grade III-IV infusion reactions will be reported as soon as possible but every 
effort should be made to assure reporting is no more than within 10 business days of 
the event. 
• Any episodes of overall grade III or IV acute GVHD in participants will be reported 
to the IRB as soon as possible but no more than within 10 business days of the PI’s 
confirmation of the diagnosis/grade of the event. 
• Clinical diagnosis of PTLPD will be reported to the IRB as soon as possible but no 
more than within 10 days of the PI’s determination of the disorder. 
For this research study, recipient participants will be followed for all NCI Grade III-V 
adverse events from the start of conditioning and throughout the first year post HCT, 
regardless of their relationship to the treatment given.  In addition, clinically significant 
NCI Grade I-II adverse events that are judged to be related/possibly related may be 
collected per the discretion and judgment of the PI.  Examples of “clinically significant 
Grade I-II adverse events could include, but are not limited to: events meeting criteria for 
SAE, infections requiring oral systemic therapy, VOD or hemorrhagic cystitis. GVHD 
events will be recorded on an ongoing basis regardless of stage or grade using the criteria 
defined in Appendix B, and will not be graded according to NCI criteria. 
With regard to the haploidentical donor participants, they will be followed for all SAEs and 
any clinically significant AEs, per the judgment of the PI, that are deemed related to the 
mobilization and/or apheresis procedure from the time mobilization with growth factors 

 
  
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begins, until 7-days post last day of the final apheresis procedure.  If the transplant 
recipient requires a second HSC infusion, meaning that the donor is required to undergo the 
mobilization and apheresis procedure again, collection of this donor safety data will restart 
upon the initiation of the subsequent mobilization procedure and continue until 7-days post 
the last day of this apheresis procedure. Timelines for reporting of these donor events to the 
institutional and federal governing agencies will be according to the same timelines utilized 
for the recipients.  A listing of the captured donor safety data will be provided in a separate 
table from the transplant recipients within each respective continuing review report.   
The following definitions apply with respect to reporting adverse experiences:  
Serious adverse event – any adverse event temporally associated with the participant’s 
participation in research that meets any of the following criteria: 
• results in death; 
• is life-threatening (places the participant at immediate risk of death from the event as 
it occurred); 
• requires inpatient hospitalization or prolongation of existing hospitalization 
• results in a persistent or significant disability/incapacity; 
• results in a congenital anomaly/birth defect; or 
• any other adverse event that, based upon appropriate medical judgment, may 
jeopardize the participant’s health and may require medical or surgical intervention to 
prevent one of the above outcomes. 
Unexpected adverse event – any adverse event meeting any of the following criteria: 
• an event for which the specificity or severity is not consistent with the protocol 
related documents, including the applicable investigator brochure, IRB approved 
consent form, IND/IDE application or any of the product labeling or package inserts; 
• an event for which the observed rate of occurrence is significantly increased above 
what is expected or credible baseline rate for comparison; 
• an event for which the occurrence is not consistent with the expected natural 
progression of any underlying disease, disorder, or condition of the participant(s) 
experiencing the adverse event and the participant’s predisposing risk factor profile 
for the adverse event. 
The principal investigator is responsible for reviewing the aggregate toxicity reports and 
reporting to the IRB if the frequency or severity of serious toxicities exceed those expected 
as defined in the protocol or based on clinical experience or the published literature.  Any 
proposed changes in the consent form or research procedures resulting from the report are 
to be prepared by the study team and submitted with the report to the IRB for approval. 
9.2 Reporting requirements to the St. Jude Institutional Biosafety Committee (IBC) 
Continuing review reports will be sent to the St. Jude IBC on at least an annual basis 
using the most current version of the continuing review form found on the Institutional 
Biosafety Committee website. The safety reports, sent to the IRB for both the donors and 
stem cell recipients, will be simultaneously forwarded to the St. Jude IBC. Therefore, 
reporting for safety events to this committee will be according to the same timelines as 
reporting to the IRB.  As per the direction of the IBC, only those protocol revision and 

 
  
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amendments that are directly related to the CliniMACS processing and related reagent(s) 
will require review and consideration by the IBC.  Other revisions/amendments will be 
noted in the IBC continuing review report.  
9.3 Reporting requirements to the FDA 
Any unexpected fatal or unexpected life-threatening event judged by the PI to possibly be 
due to the study treatment, will be reported to the FDA by telephone or fax as soon as 
possible but no later than 7 calendar days after notification of the event and followed by a 
written safety report as complete as possible within 8 additional calendar days (i.e. full 
report 15 calendar days total after notification of event).    
Unexpected, non-fatal and non-life-threatening SAEs, which occur in on-study 
participants during the time periods specified in Section 9.1 that are considered due to or 
possibly due to the investigational agent, will be reported to the FDA by written safety 
report as soon as possible but no later than 15 calendar days of the notification of the 
occurrence of the event. Expected SAEs, even unexpected fatal SAEs, considered by the 
PI to be not related to the study, will be reported to the FDA in the annual review report.  
A summary of the safety of DLIs will also be provided to the FDA in the annual review 
report. 
9.4 Summary of reporting mechanism by the St. Jude Office of Regulatory Affairs  
Copies of all correspondence to the St. Jude IRB, including SAE reports, are provided to 
the St. Jude Office of Regulatory Affairs. All FDA related correspondence and reporting 
will be conducted through the Regulatory Affairs Office.  Adverse event reporting and 
annual reporting will be in accord with the FDA Title 21 CFR312.32 and Title 21 
CFR312.33, respectively. The Regulatory Affairs Office can be reached at 901-595-2347 
(secondary contact: St. Jude Vice President of Clinical Trials Administration 901-595-
2876). 
9.5 Continuing review reports  
Continuing review reports of protocol progress and summaries of AEs will be filed with 
the St. Jude IRB, IBC and FDA at least annually.    
9.6 Reporting to the St. Jude Data Safety Monitoring Board 
This study has been referred to the St. Jude Data and Safety Monitoring Board (DSMB) 
for regular monitoring.  The DSMB is charged with advising the Director and other 
senior leaders of St. Jude on the safety of clinical protocols being conducted by St. Jude 
investigators and on their continuing scientific validity.  DSMB monitoring and review 
for this study will be conducted in accordance with the NCI guidance for DSMBs on an 
approximate semiannual basis.  The St. Jude DSMB is responsible for ongoing review of 
the protocol and related information such as enrollment, issues related to participant 
safety (specifically toxicities and the risk:benefit ratio of the trial), interim analyses, and 
the overall study conduct necessary to accomplish the primary protocol objectives.  This 
includes evaluation of the accrual rate, adherence to the study design, outcome measures, 
and review of protocol related primary outcome data. The PI will meet with DSMB 
during the semiannual visits to review the information submitted and discuss the status of 
the protocol.  The DSMB may recommend that the trial be modified, suspended to 
accrual, and/or stopped based on their review.   

 
  
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9.7 Data submission to Miltenyi Biotec 
Clinical and safety related data will be provided to Miltenyi Biotec, the manufacturer of 
the CliniMACS system. Data will include but is not limited to the transplant research 
participant’s age and diagnosis, donor product(s) related information including HAPLO 
donor type (e.g. parent, sibling), the stem cell mobilization, selection, and infusion 
procedure. Outcome data including lymphohematopoietic reconstitution, immunological 
response, disease response and transplant complications will be shared with Miltenyi 
Biotec.  Representatives from Miltenyi Biotec will be able to review the research 
participant’s (donor and transplant research recipient) laboratory and medical record for 
data verification purposes.  Copies of all reports to the governing regulatory bodies will 
also be accessible to Miltenyi Biotec upon request.   
In the event that the protocol is placed on a clinical hold by the PI or governing 
regulatory authorities (IRB and/or FDA), representatives from Miltenyi Biotec will be 
notified as soon as possible.   
9.8 Reporting to bone marrow transplant registry  
The Transplant Program at St. Jude is required by the federal government to report 
transplant information to the Center for International Blood and Marrow Transplant 
Research (CIBMTR). The CIBMTR is a research partnership of the International Bone 
Marrow Transplant Registry, the National Marrow Donor Program (NMDP), and the 
Foundation for the Accreditation of Cellular Therapy (FACT). The CIBMTR is 
responsible for the collection and maintenance of a standardized database of all 
autologous and allogeneic (related and unrelated donor) transplants performed in the 
United States.   
Data sent to the CIBMTR will include but is not limited to the transplant recipient’s date 
of birth, country/state of current residence, diagnosis, previous medical therapy including 
prior chemotherapy regimens and doses. Other information will include the relationship 
of the donor to the transplant recipient (i.e. parent, sibling), HLA type, and donor 
product(s) related information including the stem cell mobilization, selection and infusion 
procedure. Outcome data including short- and long-term lymphohematopoietic 
reconstitution, immunological response, disease response and transplant complications 
will be shared with the CIBMTR.  Donor participant information will also be included 
and will be more limited to information such as age in years, relationship to recipient, and 
infectious disease testing. Information for both donor and recipient is submitted using a 
unique research participant number.  
10.0 STATISTICAL CONSIDERATIONS 
This primary focus of this protocol is to improve the outcome for the patients receiving CD3-
depleted allogeneic stem cell graft obtained from HAPLO family member donors to treat 
patients who lack a matched sibling donor or a MUD.  The approach adopted in this protocol 
is derived from the experiences gained from three previous institutional protocols HAPSCT, 
REFSCT and HAPREF.  
In HAPSCT research participants with high-risk risk hematologic malignancies received a 
conditioning regimen containing TBI, ATG, cyclophosphamide and thiotepa.  Cyclosporine 
was used for GVHD prophylaxis. On the other hand, REFSCT enrolled research participants 

 
  
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with refractory disease or who required second (or subsequent) allogeneic HSCT to receive a 
reduced intensity conditioning regimen of melphalan, fludarabine and thiotepa.  MMF was 
used as the GVHD prophylaxis.  Following the completion of both trials and the analysis of 
data revealed that the research participants treated on REFSCT has a more rapid immune 
reconstitution, a lower rate of viral reactivation and a lower regimen-related toxicity as 
compared to those on HAPSCT.  Both trials had low rates of overall grade III-IV acute 
GVHD.  
Because of the promising results of REFSCT, a trial (HAPREF) was initiated to treat both 
groups of research participants.  These participants with hematological malignancies were 
treated on two strata: Arm A-high-risk (“like HAPSCT”), and Arm B-refractory and/or 
second (or subsequent) transplant (“like REFSCT”). All participants received a non-TBI 
based less intensive conditioning regimen, the same GVHD prophylaxis but a higher CD3+ 
cell dose in the graft (the primary transplant infusion donor product) with the expectation that 
it would significantly reduce the relapse rate.  However, most likely, this higher T cell dose 
resulted in significantly higher grade III-IV acute GVHD in both strata and the protocol was 
closed after reaching the stopping rules.  
Thus, in proposing a new trial for the high-risk patient population (i.e. those treated on 
HAPSCT and those in Arm A of the HAPREF protocol), a major change from HAPREF 
protocol has been made to reduce the total T cell dose in this primary donor graft product 
from 1.5 x 105 to a total dose of ≤ 1.0 x 105 cells/kg.  The justification for the choice of this T 
cell dose is based on the analysis of previous protocols.  We analyzed the data of the 
recipients of negative selected donor products in the HAPSCT trial (N = 20), all participants 
in the REFSCT (N = 25) and HAPREF studies (N = 17 total for both arms) to better 
understand the relationship between total T cell dose in the primary donor product infused 
and incidence of acute GVHD.  The findings are reported in the following Table 3: 
Table 3. Overall acute GVHD, relapse, graft failure/boost, and DLIs for recipients of 
negative selected donor products in HAPSCT, REFSCT, and HAPREF (N = 62) 
 
Primary Transplant 
Infusion CD3 cells 
Overall Grade 
Acute GVHD  Relapse? 
Graft 
Failure 
Receive 
Boost within 
100 days 
Receive 
DLI within 
100 days? 
Total 
0-II 
III-IV 
No Yes No 
Yes 
No 
Yes 
No 
Yes 
N 
N 
N 
N 
N 
N 
N 
N 
N 
N 
N 
20 
1 
14 
7 
19 
2 
19 
2 
16 
5 
21 
<=1.0 x 105 cells/kg 
> 1.0 x 105 cells/kg 
29 
12 
27 
14 
39 
2 
38 
3 
37 
4 
41 
Total 
49 
13 
41 
21 
58 
4 
57 
5 
53 
9 
62 
Overall acute GVHD: p-value = 0.0445; Relapse: p-value =1.0; Graft failure: p-
value=0.5987; DLIs: p-value = 0.25 
It is seen from the above table (Table 3) that 1 of the 21 (5%) participants who received a 
total T- cell dose of ≤1.0 x 105 developed grade III-IV overall acute GVHD, whereas 12 of 
the 41 (29%) who received the total T cell dose of  >1.0 x 105 had grade III-IV. This was 

 
  
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significant with an exact p-value, based on binomial distribution, of .0445.  However, a lower 
rate of occurrence of grade III-IV acute GVHD would be suggestive of a higher relapse rate 
and it was seen that relapse rate was 7/21 (33%) and 14/41(34%) in the two T cells groups.  
The exact p-value of 1.0 suggests that there is no significant difference in the incidence of 
relapse between those who received a lower T cell dose and those who received a higher T 
cell dose.  In addition, the p-value for the incidence of graft failure and the need for DLIs for 
the two groups do not appear to be significantly different with p-values of 0.5987 and 0.25, 
respectively. Thus, in this new protocol with the modifications suggested (for details see 
Section 4.0) it is expected that the overall grade III-IV acute GVHD rate would be 
significantly reduced.  Further, with the use of non-TBI based and less myeloablative 
conditioning, we expect to reduce regimen-related toxicity, have faster immune 
reconstitution and also have a lower infection rate. Thus, with this approach we expect to 
significantly improve the outcome in terms of event free survival (EFS) in transplant 
recipients in this trial.  
A similar analysis for the research participants with these high-risk hematologic 
malignancies treated in Arm A of HAPREF (N = 9) and those who received negatively 
depleted graft on the HAPSCT (N = 20) suggested similar findings as depicted in the 
following table:  
Table 4.  Acute GVHD, relapse, graft failure and additional cell infusion(s) by total T cell 
dose in HAPREF Arm A and recipients of negative selected products in HAPSCT (N = 29)  
Primary Transplant 
Infusion CD3 cells 
Overall 
Grade Acute 
GVHD 
Relapse? 
Graft 
Failure? 
Receive 
boost within 
100 days? 
Receive 
DLI within 
100 days? 
Total 
0-II 
III-IV No 
Yes 
No 
Yes 
No 
Yes 
No 
Yes 
N 
N 
N 
N 
N 
N 
N 
N 
N 
N 
N 
<=1.0 x 105 cells/kg 
10 
1 
9 
2 
11 
0 
9 
2 
7 
2 
11 
> 1.0 x 105 cells/kg 
12 
6 
14 
4 
17 
1 
15 
3 
17 
3 
18 
Total 
22 
7 
23 
6 
28 
1 
24 
5 
24 
5 
29 
Overall acute GVHD: p-value =0.2021; Relapse: p-value = 1.0; Graft failure: p-value=1.0; 
DLIs:  p-value = 1.0 
It is seen from the above table that 1 out of 11 patients (9%) who received a total T cell dose 
of ≤1.0 x 105 cells/kg developed grade III-IV overall acute GVHD, whereas 6 of the 18 
(33%) who received the total T cell dose of > 1.0 x 105 cells/kg had grade III-IV. This was 
not statistically significant with an exact p-value, based on binomial distribution, of 0.2021 
but was suggestive of the fact that patients who received a higher T cell dose are likely to 
have more GVHD. Also, the relapse rate was 2/11 (18%) and 4/18 (22%) in the two T cells 
groups. There does not appear to any statistically significant difference in the incidence of 
relapse, incidence of graft failure or need for DLIs with exact p-values of 1.0 for all 3 
comparisons. Because of the small sample sizes the results should be interpreted with 

 
  
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caution. However, from the analyses presented above it is clear that only 1 participant who 
received a total T cell dose of ≤ 1 x 105 developed this acute GVHD and that did not seem to 
affect the relapse rate or graft failure rate. Thus, we expect that administering a total T cell 
dose of ≤1 x 105 cells/kg should be relatively safe and should help in improving the outcome 
of the research participants on this protocol.  
Further, focusing only on these 29 participants with high-risk hematologic malignancies who 
received negatively depleted grafts on HAPSCT and Arm A of HAPREF, it is seen that one-
year EFS estimates are 40.0%±10.3% and 55.6%±16.6%, respectively.  The Kaplan-Meier 
plots along with the survival estimates are provided in the tables below (Tables 5 and Table 
6):  
 
 
Table 5. Event-free survival in HAPSCT negative selection recipients and HAPREF Arm A 
participants (N = 29 total) 
 
Table 6. Event-free survival in HAPSCT negative selection recipients and HAPREF Arm A 
participants (N = 29 total) 
HAPSCT (N=20) 
HAPREF (N=9) 
Time in 
Months 
Risk Fail Cens 
Prob 
SE 
Time in 
Months 
Risk Fail Cens 
Prob 
SE 
0 
20 
1 
0 
0.950 0.048 
0 
9 
1 
0 
0.889 0.099 
2 
19 
1 
0 
0.900 0.065 
2 
8 
1 
0 
0.778 0.130 
4 
18 
3 
0 
0.750 0.094 
4 
7 
0 
0 
0.778 0.130 
5 
15 
2 
0 
0.650 0.103 
5 
7 
2 
0 
0.556 0.151 
6 
13 
3 
0 
0.500 0.107 
6 
5 
0 
0 
0.556 0.151 
7 
10 
1 
0 
0.450 0.106 
7 
5 
0 
0 
0.556 0.151 
Event-free survival in HAPSCT negative selection and HAPREF Arm A participants 
0
0.
1 
0.
2 
0.
3 
0.
4 
0.
5 
0.
6 
0.
7 
0.
8 
0.
9 
1
0 
5
10
15 
20
25
30 
35 
Time in Months
Probability
HAPSCT (N=20)
HAPREF (N=9)

 
  
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HAPSCT (N=20) 
HAPREF (N=9) 
Time in 
Months 
Risk Fail Cens 
Prob 
SE 
Time in 
Months 
Risk Fail Cens 
Prob 
SE 
8 
9 
1 
0 
0.400 0.103 
8 
5 
0 
0 
0.556 0.151 
11 
8 
0 
0 
0.400 0.103 
11 
5 
0 
1 
0.556 0.166 
13 
8 
0 
0 
0.400 0.103 
13 
4 
0 
1 
0.556 0.185 
14 
8 
0 
0 
0.400 0.103 
14 
3 
0 
1 
0.556 0.214 
16 
8 
0 
0 
0.400 0.103 
16 
2 
0 
2 
0.556 0.370 
18 
8 
1 
1 
0.343 0.105 
 
 
 
 
 
 
19 
6 
0 
1 
0.343 0.113 
 
 
 
 
 
 
20 
5 
0 
2 
0.343 0.139 
 
 
 
 
 
 
25 
3 
0 
1 
0.343 0.160 
 
 
 
 
 
 
27 
2 
0 
1 
0.343 0.197 
 
 
 
 
 
 
31 
1 
0 
1 
0.343 0.278 
 
 
 
 
 
 
It may be noted that as of this time few research participants have follow-up of more than 
two years on HAPREF protocol and, hence, the estimate obtained may not be very reliable. 
Furthermore, since the HAPREF protocol was closed prematurely, it makes sense to assess 
the effectiveness of the proposed treatment plan by comparing the outcome (EFS) of this 
protocol to the outcome of HAPSCT.  We would consider the findings to be of significant 
merit if we can demonstrate an improvement of at least a 20% in one-year EFS.  The best 
approach would be to use the methods for analyzing survival data and propose the sample 
size based on comparing the EFS of the current protocol to the historical control group 
(HAPSCT).  However, there is no good way to incorporate interim analysis to monitor for 
inferior EFS on the current protocol.  
It may further be noted that on HAPSCT protocol there was no research participant censored 
within one-year of follow-up.  Thus, with no censoring expected during one-year time period 
we can approximate the EFS at one-year using a Binomial distribution. That is, each research 
participant will be followed for one-year and will be counted as a success if the research 
participant survived without any events (death due to any cause or relapse).  Denote by P the 
proportion of participants surviving at one-year without these events mentioned. Then using 
a more flexible phase II design such as the one proposed by Chen and Ng, we can assess if 
the proportion of participants surviving is in the desired range and if a larger clinical trial is 
warranted to validate the findings.139 
Then setting up the problem in the binomial framework and assuming the proportion of 
research participants surviving without relapse at one-year on HAPSCT protocol to be 
roughly 40%, we would like to see if that proportion could be improved to 60%, i.e. an 
increase of 20%. Then using the optimal flexible phase II design proposed by Chen and Ng 
to detect an improvement of 20%, with 80% power and type I error control at level α=.05, 
we would enroll 12 to 19 participants in the first phase and 44 - 51 in the second.139 
Because Amendment 3.0 includes a substantial change in conditioning (with the replacement 
of OKT3 by Campath-1H), we will restart counting of enrollment beginning from the first 

 
  
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patient enrolled since amendment 3.0. All the patients enrolled before Amendment 3.0 will 
be reported separately.  The enrollment goal remains the same for this new cohort of patients 
as originally written, as does the statistical design. 
The flexible stopping rules are provided in the following tables:  
Table 7. Stopping rules for lack of efficacy (unacceptable lower rate of one-year event-free 
survival) based on the two-stage design   
Reject the treatment plan if the number of research participants surviving at one-year 
P0 
P1 
( ≤ ri/ni ) 
( ≤ Rj/Nj ) 
AEN(P0) 
APET(P0) 
0.40 
0.60 
5/12-13, 6/14, 7/15-16, 
8/17-19 
22/44-45, 23/46-47, 
24/48-49, 25/50, 26/51 
25.01 
0.71 
Note: AEN(P0) denotes the minimum average expected sample size under P0; APET(P0) 
denotes the probability of early termination at stage I under P0 
From the above Table 7 it is clear that the proposed stopping rules provide more flexibility 
since we can enroll research participants in a range of 12 - 19 and stop the trial if the number 
of participants surviving without any event is fewer than those listed in the table. For 
example, if at any point we see seven or more (≥ 7) relapses and/or deaths within one-year in 
the first 15- 16 then we would halt the enrollment and consider modifying the treatment plan 
and amending the protocol. The above stopping rule provides more flexibility in enrolling 
research participants and not requiring the study to be closed while the research participants 
are being followed for one-year. We expect to enroll roughly 12 - 15 participants per year 
with one-year of follow-up.  The expected duration of the study will be about 5 years.  
However, there still exists a possibility that once 19 research participants have been enrolled 
we may have to temporarily close the trial to accrual while the research participants are being 
followed for one year.  Only participants who initiate protocol therapy will be considered for 
the stopping rules.  A participant who enrolls and subsequently dies prior to beginning 
protocol treatment would not count toward the efficacy stopping rule. 
In addition, we will closely monitor for acute GVHD and TRM (Transplant Related 
Mortality as defined as death while in remission) for at least 100 days post-transplant to 
ensure and enhance safety of participants.  Initially the stopping rule for toxicity excluded 
recipients who received more than 2 DLI in the first 100 days, as this was considered to be 
rare.  However, after amendment 3.0 substituted Campath for OKT3 (due to OKT3 being no 
longer available), it became clear after the first 14 patients, that DLI were required more 
frequently.  In addition, 4 of the first 14 patients evaluable at day 100 had developed Grade 
III-IV GVHD – and all of them had received multiple escalating DLI for post-transplant 
complications (see Section 4.9).  Importantly, the use of DLI as described in this protocol has 
been largely successful (manuscript in preparation).  So, continued use of DLI remains 
integral therapy on this protocol.  Therefore with amendment 6.0, the toxicity stopping rules 
will include all patients who received protocol therapy (including DLI).  Although the 4 
patients above all developed grade III-IV GVHD within 100 days, the use of DLI can extend 
the time at which acute GVHD will occur.  Therefore, any GVHD that occurs within the first 
100 days post-transplant or within 100 days of their last cell therapy (including any stem cell 
boost and/or DLI) will count towards the GVHD stopping rule. 

 
  
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The initial stopping rule for toxicity included both TRM and GVHD together in the same 
monitoring rule.  The stopping rule was based on exact upper 95% Blyth-Still-Casella 
confidence bounds that the combined rate of Grade III-IV GVHD and TRM was less than 
40%. However with amendment 6.0, we have chosen to separate GVHD and TRM into 
separate stopping rules to allow more specific categorical assessment of each of these 
important toxicities.  The stopping rules will each be based on the minimum exact lower 95% 
Blyth-Still-Casella confidence bounds that the rate of the specific toxicity (Grade III-IV 
GVHD and TRM respectively) do not exceed 20%.  These current stopping rules are in place 
such that if there is statistical evidence that the rate of either toxicity is in excess of 20%, 
then the trial will be closed to further accrual.   
Since we plan to treat 51 research participants, we will monitor the acute GVHD and TRM 
rate by setting up 4 interim evaluation time points as noted in Tables 8 and 9 below:  
Table 8. Stopping rules for toxicities based on incidence of  TRM 
Number of 
participants enrolled 
Number of TRM observed ≥ 
Minimum Exact Lower 
Confidence Bounds 
19 
7 
0.209 
30 
11 
0.221 
40 
13 
0.204 
51 
16 
0.208 
Based on the above Table 8, if we observe 7 incidences of TRM within the first 100 days 
post-transplant in the first 19 evaluable research participants treated, then we will halt the 
trial and consider amending the protocol.   
Table 9. Stopping rules for toxicities based on incidence of GVHD 
Number of 
participants enrolled 
Number of overall grade III-IV 
acute GVHD observed ≥ 
Minimum Exact Lower 
Confidence Bounds 
19 
7 
0.209 
30 
11 
0.221 
40 
13 
0.204 
51 
16 
0.208 
Again, based on the above Table 9, if we observe 7 incidences of acute grade III-IV GVHD 
within the first 100 days post-transplant (or 100 days after the last cellular infusion) in the 
first 19 evaluable research participants treated, then we will halt the trial and consider 
amending the protocol.  Again, it may be noted that as part of the treatment plan, some 
transplant recipients may require donor lymphocyte infusions (DLIs) or additional “boost” 
infusions (see Section 4.7 and 4.9 for additional details).   If this happens within 100 days 

 
  
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post transplantation, then those research participants will have their evaluation period for 
acute grade III-IV GVHD extended up to 100 days post last cellular therapy (any stem cell 
boosts and/or DLI).  It may be noted that the above stopping rules are “ad hoc” in nature. 
However, these are justified on the basis of stopping the trial if there is evidence to support a 
rate of acute grade III-IV GVHD or TRM higher than 20%.  Also, the proposed stopping 
rules were deemed reasonable in PI’s evaluation.   
However, in estimating the incidence of overall grade III-IV acute GVHD or TRM, the 
estimates may be biased, as there are competing risks such as death due to causes other than 
acute GVHD or the transplant.  Once the study is completed we will use more appropriate 
approaches, e.g. those based on Gray’s approach to estimate the incidence of overall grade 
III-IV acute GVHD and provide confidence intervals.140 
Secondary Objectives:  
Objective 1.2.1 Once the data is collected the Kaplan-Meier estimates along with their 
confidence intervals will be provided for EFS, OS, and DFS.  The terms EFS, DFS and OS 
are further defined below: LFU stands for “last follow-up date.” 
EFS = min(LFU, date of relapse, date of death due to any cause) - date of transplant and all 
participants surviving at the time of analysis would be considered as censored. 
DFS = min(LFU, date of relapse, date of death due to relapse) - date of transplant and all 
participants surviving at the time of analysis and those who die due to other causes will be 
censored at the time of their event.  
OS = min(LFU, date of death) – date of transplant and all patients surviving at the time of 
analysis will be considered as censored.   
A historical comparison with the HAPSCT protocol will also be undertaken to see if the 
outcome on the current therapy has improved.  Further a Cox’s proportional hazards model 
will be used to assess the affect of several characteristics of the graft and disease status on the 
outcome measures such as EFS, DFS and OS.141 
Objective 1.2.2 and 1.2.3.  The estimates of cumulative incidence of relapse, acute and 
chronic GVHD will be estimated using Prentice’s approach and compared with the historical 
HAPSCT data using the approaches proposed in Gray and Fine and Gray.140, 142,143,157   
Objective 1.2.4.  The estimates of non-hematologic regimen related toxicity and regimen 
related mortality in first 100 days for the 2 groups will be estimated and compared using 
methods for comparing 2 bionomial distributions discussed in Agresti.144 
Objective 1.3.1. The biologic significance of soluble interleukin-2 receptor and immunologic 
state to predict the development of acute and chronic GVHD will be evaluated using the 
method proposed in Fine and Gray and implemented using an in house SAS Macro.143 
11.0 DATA ACQUISITION AND QUALITY ASSURANCE MONITORING   
11.1 Enrollment on study 
After verification of subject eligibility (donor and transplant recipient), potential 
participants will be registered by completing the eligibility checklist and faxing the 

 
  
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completed Eligibility Checklists to the Central Protocol and Data Monitoring Office 
(CPDMO) at 595-6265. Follow with a phone call to (901) 595-2568 to ensure that the fax 
has been received. Eligibility will be reviewed and entered into the institutional database, 
and a research participant-specific consent form will be generated.  The consents, 
protocol, and protocol standard order set will be delivered to the area designated on the 
Eligibility Checklist(s). The signed consent forms must be faxed to the CPDMO for entry 
into the centralized database.  
Central Protocol Office Hours.  The CPDMO is staffed 7:30 am to 6:00 pm CST, 
Monday through Friday (excluding holidays).  A staff member is available by pager 8:00 
am to 5:00 pm on weekends and weekday holidays.  To reach the person on call during 
these hours, call 901-595-3578, wait for the prompt, and enter pager #2173, followed by 
the telephone number to which the call should be returned. 
 11.2 Data submission 
The St. Jude Cancer Center Clinical Research Associates and Research Nurses with the 
Division of Bone Marrow Transplant and Cellular Therapy will be responsible for 
protocol compliance assurance/monitoring as well as clinical and safety data collection. 
The PI will be responsible for review of case report forms for accuracy and completeness 
prior to entry into the secure divisional database. Data such as laboratory values will also 
be transferred directly into the database from the institutional Millennium data system.    
11.3 Quality assurance monitoring 
This protocol will be monitored for safety and data as per the St. Jude institutional Data 
Safety Monitoring Plan, dated August 17, 2001.  Source document verification of 
eligibility for all patients will be performed within 2 weeks of completion of enrollment. 
This will include verification of appropriate documentation of consent. Monitoring of 
timeliness of adverse and serious AE reporting will be done as events are reported.  
Monitoring of modified Clinical Data Update System (CDUS) elements, AE reporting, 
data required for primary objectives, and compliance with the conduct of the protocol 
will be performed on a semiannual (every 6 months) or an as needed basis. 
12.0 OBTAINING INFORMED CONSENT 
The ongoing informed consent process will be carried out per the policies and procedures put 
forth in the St. Jude Investigator’s Handbook for Clinical Research (http://home.web.stjude. 
org/ clinical_research/administration/doc/handbook.pdf). The PI or physician sub-
investigator will conduct the signature authorization portion of the consent process.  
Authorization for the recipient procedure will be conducted in the presence of an independent 
witness such as the St. Jude Ombudsperson/Research Participant Advocate or designee, a 
professional staff member from the Department of Nursing, or Social Work. 
 

 
  
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 13.0 REFERENCES 
1. 
Thomas ED, Storb R, Clift RA, et al. Bone-marrow transplantation (second of two parts). 
N Engl J Med. Apr 24 1975;292(17):895-902. 
2. 
Thomas ED, Buckner CD, Banaji M, et al. One hundred patients with acute leukemia 
treated by chemotherapy, total body irradiation, and allogeneic marrow transplantation. 
Blood. Apr 1977;49(4):511-533. 
3. 
Appelbaum FR, Fisher LD, Thomas ED. Chemotherapy v marrow transplantation for 
adults with acute nonlymphocytic leukemia: a five-year follow-up. Blood. Jul 1988;72(1): 
179-184. 
4. 
Santos GW. Marrow transplantation in acute nonlymphocytic leukemia. Blood. Aug 15 
1989;74(3):901-908. 
5. 
Butturini A, Gale RP. Chemotherapy versus transplantation in acute leukaemia. Br J 
Haematol. May 1989;72(1):1-8. 
6. 
Allogeneic bone marrow transplantation for leukaemia in Europe. Report from the 
Working Party on Leukaemia, European Group for Bone Marrow Transplantation. 
Lancet. Jun 18 1988;1(8599):1379-1382. 
7. 
Transplant or chemotherapy in acute myelogenous leukaemia. International Bone 
Marrow Transplant Registry. Lancet. May 20 1989;1(8647):1119-1122. 
8. 
McGlave PB, Haake RJ, Bostrom BC, et al. Allogeneic bone marrow transplantation for 
acute nonlymphocytic leukemia in first remission. Blood. Nov 1988;72(5):1512-1517. 
9. 
Brochstein JA, Kernan NA, Groshen S, et al. Allogeneic bone marrow transplantation 
after hyperfractionated total-body irradiation and cyclophosphamide in children with 
acute leukemia. N Engl J Med. Dec 24 1987;317(26):1618-1624. 
10. 
Clift RA, Buckner CD, Thomas ED, et al. The treatment of acute non-lymphoblastic 
leukemia by allogeneic marrow transplantation. Bone Marrow Transplant. Oct 
1987;2(3):243-258. 
11. 
Barrett AJ, Horowitz MM, Gale RP, et al. Marrow transplantation for acute 
lymphoblastic leukemia: factors affecting relapse and survival. Blood. Aug 1 
1989;74(2):862-871. 
12. 
Herzig RH, Bortin MM, Barrett AJ, et al. Bone-marrow transplantation in high-risk acute 
lymphoblastic leukaemia in first and second remission. Lancet. Apr 4 1987;1(8536):786-
789. 
13. 
Ramsay NK, Kersey JH. Indications for marrow transplantation in acute lymphoblastic 
leukemia. Blood. Feb 15 1990;75(4):815-818. 
14. 
Sanders JE, Thomas ED, Buckner CD, Doney K. Marrow transplantation for children 
with acute lymphoblastic leukemia in second remission. Blood. Jul 1987;70(1):324-326. 
15. 
Goldman JM, Gale RP, Horowitz MM, et al. Bone marrow transplantation for chronic 
myelogenous leukemia in chronic phase. Increased risk for relapse associated with T-cell 
depletion. Ann Intern Med. Jun 1988;108(6):806-814. 
16. 
Thomas ED, Clift RA. Indications for marrow transplantation in chronic myelogenous 
leukemia. Blood. Mar 1989;73(4):861-864. 
17. 
Appelbaum FR, Storb R, Ramberg RE, et al. Treatment of preleukemic syndromes with 
marrow transplantation. Blood. Jan 1987;69(1):92-96. 
18. 
Hongeng S, Krance RA, Bowman LC, et al. Outcomes of transplantation with matched-
sibling and unrelated-donor bone marrow in children with leukaemia. Lancet. Sep 13 
1997;350(9080):767-771. 

 
  
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19. 
Hale GA, Tong X, Benaim E, et al. Allogeneic bone marrow transplantation in children 
failing prior autologous bone marrow transplantation. Bone Marrow Transplant. Jan 
2001;27(2):155-162. 
20. 
Peggs KS, Hunter A, Chopra R, et al. Clinical evidence of a graft-versus-Hodgkin's-
lymphoma effect after reduced-intensity allogeneic transplantation. Lancet. Jun 4-10 
2005;365(9475):1934-1941. 
21. 
Dhedin N, Giraudier S, Gaulard P, et al. Allogeneic bone marrow transplantation in 
aggressive non-Hodgkin's lymphoma (excluding Burkitt and lymphoblastic lymphoma): a 
series of 73 patients from the SFGM database. Societ Francaise de Greffe de Moelle. Br J 
Haematol. Oct 1999;107(1):154-161. 
22. 
Verdonck LF, Dekker AW, Lokhorst HM, Petersen EJ, Nieuwenhuis HK. Allogeneic 
versus autologous bone marrow transplantation for refractory and recurrent low-grade 
non-Hodgkin's lymphoma. Blood. Nov 15 1997;90(10):4201-4205. 
23. 
Dann EJ, Daugherty CK, Larson RA. Allogeneic bone marrow transplantation for 
relapsed and refractory Hodgkin's disease and non-Hodgkin's lymphoma. Bone Marrow 
Transplant. Sep 1997;20(5):369-374. 
24. 
Ratanatharathorn V, Uberti J, Karanes C, et al. Prospective comparative trial of 
autologous versus allogeneic bone marrow transplantation in patients with non-Hodgkin's 
lymphoma. Blood. Aug 15 1994;84(4):1050-1055. 
25. 
Westoff CF. Fertility in the United States. Science. Oct 31 1986;234(4776):554-559. 
26. 
Beatty PG, Clift RA, Mickelson EM, et al. Marrow transplantation from related donors 
other than HLA-identical siblings. N Engl J Med. Sep 26 1985;313(13):765-771. 
27. 
Kernan NA, Bartsch G, Ash RC, et al. Analysis of 462 transplantations from unrelated 
donors facilitated by the National Marrow Donor Program. N Engl J Med. Mar 4 
1993;328(9):593-602. 
28. 
Ferrara JaBS. The pathophysiology of acute graft-vs-host disease in a murine bone 
marrow transplant model. New York: Marcel Dekker; 1990. 
29. 
Henslee-Downey PJ, Abhyankar SH, Parrish RS, et al. Use of partially mismatched 
related donors extends access to allogeneic marrow transplant. Blood. May 15 
1997;89(10):3864-3872. 
30. 
Pietryga D, Casper J, Margolis D, et al. Bone marrow transplantation (BMT) using 
genotypically haploidentical donors. Blood. Nov 15 1996;88(10):1044-1044. 
31. 
Reisner Y, Kapoor N, Kirkpatrick D, et al. Transplantation for acute leukaemia with 
HLA-A and B nonidentical parental marrow cells fractionated with soybean agglutinin 
and sheep red blood cells. Lancet. Aug 15 1981;2(8242):327-331. 
32. 
van Dicke KAHJI, van Bekkum DW. The selective elimination of immunologically 
competent cells from bone marrow and lymphatic cell mixtures. II. Mouse spleen cell 
fractionation on a discontinuous albumin gradient. Transplantation. Jul 1968;6(4):562-
570. 
33. 
de Witte T, Hoogenhout J, de Pauw B, et al. Depletion of donor lymphocytes by 
counterflow centrifugation successfully prevents acute graft-versus-host disease in 
matched allogeneic marrow transplantation. Blood. May 1986;67(5):1302-1308. 
34. 
Okunewick J. Review of the Effects of Anti-T-Cell Monoclonal Antibodies on Major and 
Minor GVHD in the Mouse. Experimental Hematology Today. 1985. 
35. 
Vallera DA, Soderling CC, Carlson GJ, Kersey JH. Bone marrow transplantation across 
major histocompatibility barriers in mice. Effect of elimination of T cells from donor 

 
  
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grafts by treatment with monoclonal Thy-1.2 plus complement or antibody alone. 
Transplantation. Mar 1981;31(3):218-222. 
36. 
Korbling M, Hess AD, Tutschka PJ, Kaizer H, Colvin MO, Santos GW. 4-
hydroperoxycyclophosphamide: a model for eliminating residual human tumour cells and 
T-lymphocytes from the bone marrow graft. Br J Haematol. Sep 1982;52(1):89-96. 
37. 
Prentice HG, Blacklock HA, Janossy G, et al. Use of anti-T-cell monoclonal antibody 
OKT3 to prevent acute graft-versus-host disease in allogeneic bone-marrow 
transplantation for acute leukaemia. Lancet. Mar 27 1982;1(8274):700-703. 
38. 
Filipovich AH, McGlave PB, Ramsay NK, Goldstein G, Warkentin PI, Kesey JH. 
Pretreatment of donor bone marrow with monoclonal antibody OKT3 for prevention of 
acute graft-versus-host disease in allogeneic histocompatible bone-marrow 
transplantation. Lancet. Jun 5 1982;1(8284):1266-1269. 
39. 
Martin PJ, Hansen JA, Thomas ED. Preincubation of donor bone marrow cells with a 
combination of murine monoclonal anti-T-cell antibodies without complement does not 
prevent graft-versus-host disease after allogeneic marrow transplantation. J Clin 
Immunol. Jan 1984;4(1):18-22. 
40. 
Mitsuyasu RT, Champlin RE, Gale RP, et al. Treatment of donor bone marrow with 
monoclonal anti-T-cell antibody and complement for the prevention of graft-versus-host 
disease. A prospective, randomized, double-blind trial. Ann Intern Med. Jul 
1986;105(1):20-26. 
41. 
Prentice HG, Blacklock HA, Janossy G, et al. Depletion of T lymphocytes in donor 
marrow prevents significant graft-versus-host disease in matched allogeneic leukaemic 
marrow transplant recipients. Lancet. Mar 3 1984;1(8375):472-476. 
42. 
Slavin S, Or R, Weiss L, et al. Elimination of graft versus host disease in matched 
allogeneic leukemic transplant recipients using CAMPATH-1. Adv Exp Med Biol. 
1985;186:813-818. 
43. 
Trigg ME, Billing R, Sondel PM, et al. Depletion of T cells from human bone marrow 
with monoclonal antibody CT-2 and complement. J Biol Response Mod. Aug 
1984;3(4):406-412. 
44. 
Kvalheim G, Sorensen O, Fodstad O, et al. Immunomagnetic removal of B-lymphoma 
cells from human bone marrow: a procedure for clinical use. Bone Marrow Transplant. 
Jan 1988;3(1):31-41. 
45. 
Trickett AE, Ford DJ, Lam-Po-Tang PR, Vowels MR. Immunomagnetic bone marrow 
purging of common acute lymphoblastic leukemia cells: suitability of BioMag particles. 
Bone Marrow Transplant. Mar 1991;7(3):199-203. 
46. 
Munn RK, Henslee-Downey PJ, Romond EH, et al. Treatment of leukemia with partially 
matched related bone marrow transplantation. Bone Marrow Transplant. Mar 
1997;19(5):421-427. 
47. 
Fleming DR, Henslee-Downey PJ, Romond EH, et al. Allogeneic bone marrow 
transplantation with T cell-depleted partially matched related donors for advanced acute 
lymphoblastic leukemia in children and adults: a comparative matched cohort study. 
Bone Marrow Transplant. Jun 1996;17(6):917-922. 
48. 
Godder KT, Hazlett LJ, Abhyankar SH, et al. Partially mismatched related-donor bone 
marrow transplantation for pediatric patients with acute leukemia: younger donors and 
absence of peripheral blasts improve outcome. J Clin Oncol. May 2000;18(9):1856-1866. 

 
  
HIFLEX 55 
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          IRB Approval date: 12/06/2016 
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49. 
Balduzzi A, Gooley T, Anasetti C, et al. Unrelated donor marrow transplantation in 
children. Blood. Oct 15 1995;86(8):3247-3256. 
50. 
Anderson JE, Anasetti C, Appelbaum FR, et al. Unrelated donor marrow transplantation 
for myelodysplasia (MDS) and MDS-related acute myeloid leukaemia. Br J Haematol. 
Apr 1996;93(1):59-67. 
51. 
Schiller G, Feig SA, Territo M, et al. Treatment of advanced acute leukaemia with 
allogeneic bone marrow transplantation from unrelated donors. Br J Haematol. Sep 
1994;88(1):72-78. 
52. 
Nademanee A, Schmidt GM, Parker P, et al. The outcome of matched unrelated donor 
bone marrow transplantation in patients with hematologic malignancies using molecular 
typing for donor selection and graft-versus-host disease prophylaxis regimen of 
cyclosporine, methotrexate, and prednisone. Blood. Aug 1 1995;86(3):1228-1234. 
53. 
Casper J, Camitta B, Truitt R, et al. Unrelated bone marrow donor transplants for children 
with leukemia or myelodysplasia. Blood. May 1 1995;85(9):2354-2363. 
54. 
Woodard P, Benaim, E., Cunningham, J.M., Horwitz, E.M., Leung, W., Shearer, P., Hale, 
G., Bowman, L.C. Characteristics and outcome of graft failure in pediatric recipients of 
allogeneic stem cell transplants (SCT). Blood. 1999;94 (Suppl):A1478. 
55. 
Benaim E, Krance, R.A., Richardson, S., Bowman, L.D., Srivastava, D.K., Cunningham, 
J.M., Brenner, M.K., Heslop, H.E. Comparison of outcome of allogeneic transplantation 
in children with leukemia using unrelated donors or mismatched family member donors. 
Blood. 1997;90 (Suppl 1):106a. 
56. 
Cahn JY, Herve P, Flesch M, et al. Marrow transplantation from HLA non-identical 
family donors for the treatment of leukaemia: a pilot study of 15 patients using additional 
immunosuppression and T-cell depletion. Br J Haematol. Jul 1988;69(3):345-349. 
57. 
Filipovich AH, Vallera D, McGlave P, et al. T cell depletion with anti-CD5 immunotoxin 
in histocompatible bone marrow transplantation. The correlation between residual CD5 
negative T cells and subsequent graft-versus-host disease. Transplantation. Sep 
1990;50(3):410-415. 
58. 
Laurent G, Maraninchi D, Gluckman E, et al. Donor bone marrow treatment with T101 
Fab fragment-ricin A-chain immunotoxin prevents graft-versus-host disease. Bone 
Marrow Transplant. Jul 1989;4(4):367-371. 
59. 
Martin PJ, Hansen JA, Torok-Storb B, et al. Effects of treating marrow with a CD3-
specific immunotoxin for prevention of acute graft-versus-host disease. Bone Marrow 
Transplant. Sep 1988;3(5):437-444. 
60. 
Handgretinger R, Schumm M, Lang P, et al. Transplantation of megadoses of purified 
haploidentical stem cells. Ann N Y Acad Sci. Apr 30 1999;872:351-361; discussion 361-
352. 
61. 
Kessinger A, Smith DM, Strandjord SE, et al. Allogeneic transplantation of blood-
derived, T cell-depleted hemopoietic stem cells after myeloablative treatment in a patient 
with acute lymphoblastic leukemia. Bone Marrow Transplant. Nov 1989;4(6):643-646. 
62. 
Bensinger WI, Buckner CD, Rowley S, Storb R, Appelbaum FR. Treatment of normal 
donors with recombinant growth factors for transplantation of allogeneic blood stem 
cells. Bone Marrow Transplant. Mar 1996;17 Suppl 2:S19-21. 
63. 
Bensinger WI, Weaver CH, Appelbaum FR, et al. Transplantation of allogeneic 
peripheral blood stem cells mobilized by recombinant human granulocyte colony-
stimulating factor. Blood. Mar 15 1995;85(6):1655-1658. 

 
  
HIFLEX 56 
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          IRB Approval date: 12/06/2016 
Protocol document date: 11/18/2016 
64. 
Schmitz N, Dreger P, Suttorp M, et al. Primary transplantation of allogeneic peripheral 
blood progenitor cells mobilized by filgrastim (granulocyte colony-stimulating factor). 
Blood. Mar 15 1995;85(6):1666-1672. 
65. 
Schmitz N, Bacigalupo A, Labopin M, et al. Transplantation of allogeneic peripheral 
blood progenitor cells--the EBMT experience. Bone Marrow Transplant. Mar 1996;17 
Suppl 2:S40-46. 
66. 
Korbling M, Przepiorka D, Huh YO, et al. Allogeneic blood stem cell transplantation for 
refractory leukemia and lymphoma: potential advantage of blood over marrow allografts. 
Blood. Mar 15 1995;85(6):1659-1665. 
67. 
Russell JA, Brown C, Bowen T, et al. Allogeneic blood cell transplants for 
haematological malignancy: preliminary comparison of outcomes with bone marrow 
transplantation. Bone Marrow Transplant. May 1996;17(5):703-708. 
68. 
Bensinger WI, Clift R, Martin P, et al. Allogeneic peripheral blood stem cell 
transplantation in patients with advanced hematologic malignancies: a retrospective 
comparison with marrow transplantation. Blood. Oct 1 1996;88(7):2794-2800. 
69. 
Urbano-Ispizua A, Solano C, Brunet S, et al. Allogeneic peripheral blood progenitor cell 
transplantation: analysis of short-term engraftment and acute GVHD incidence in 33 
cases. allo-PBPCT Spanish Group. Bone Marrow Transplant. Jul 1996;18(1):35-40. 
70. 
Barrett AJ, Mavroudis D, Tisdale J, et al. T cell-depleted bone marrow transplantation 
and delayed T cell add-back to control acute GVHD and conserve a graft-versus-
leukemia effect. Bone Marrow Transplant. Mar 1998;21(6):543-551. 
71. 
Ottinger HD, Beelen DW, Scheulen B, Schaefer UW, Grosse-Wilde H. Improved 
immune reconstitution after allotransplantation of peripheral blood stem cells instead of 
bone marrow. Blood. Oct 1 1996;88(7):2775-2779. 
72. 
Sierra J, Storer B, Hansen JA, et al. Transplantation of marrow cells from unrelated 
donors for treatment of high-risk acute leukemia: the effect of leukemic burden, donor 
HLA-matching, and marrow cell dose. Blood. Jun 1 1997;89(11):4226-4235. 
73. 
Mavroudis D, Read E, Cottler-Fox M, et al. CD34+ cell dose predicts survival, 
posttransplant morbidity, and rate of hematologic recovery after allogeneic marrow 
transplants for hematologic malignancies. Blood. Oct 15 1996;88(8):3223-3229. 
74. 
Storek J, Gooley T, Siadak M, et al. Allogeneic peripheral blood stem cell transplantation 
may be associated with a high risk of chronic graft-versus-host disease. Blood. Dec 15 
1997;90(12):4705-4709. 
75. 
Urbano-Ispizua A, Rozman C, Pimentel P, et al. The number of donor CD3(+) cells is the 
most important factor for graft failure after allogeneic transplantation of CD34(+) 
selected cells from peripheral blood from HLA-identical siblings. Blood. Jan 15 
2001;97(2):383-387. 
76. 
Civin CI, Strauss LC, Brovall C, Fackler MJ, Schwartz JF, Shaper JH. Antigenic analysis 
of hematopoiesis. III. A hematopoietic progenitor cell surface antigen defined by a 
monoclonal antibody raised against KG-1a cells. J Immunol. Jul 1984;133(1):157-165. 
77. 
Sutherland HJ, Eaves CJ, Eaves AC, Dragowska W, Lansdorp PM. Characterization and 
partial purification of human marrow cells capable of initiating long-term hematopoiesis 
in vitro. Blood. Oct 1989;74(5):1563-1570. 
78. 
Link H, Arseniev L, Bahre O, Kadar JG, Diedrich H, Poliwoda H. Transplantation of 
allogeneic CD34+ blood cells. Blood. Jun 1 1996;87(11):4903-4909. 

 
  
HIFLEX 57 
Revision 6.1 dated: 12/24/2014 
    
          IRB Approval date: 12/06/2016 
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79. 
Shpall EJ, LeMaistre CF, Holland K, et al. A prospective randomized trial of buffy coat 
versus CD34-selected autologous bone marrow support in high-risk breast cancer patients 
receiving high-dose chemotherapy. Blood. Dec 1 1997;90(11):4313-4320. 
80. 
Yabe H, Yabe M, Hattori K, et al. Successful engraftment of allogeneic CD34-enriched 
marrow cell transplantation from HLA-mismatched parental donors. Bone Marrow 
Transplant. Jun 1996;17(6):985-991. 
81. 
Civin CI, Trischmann T, Kadan NS, et al. Highly purified CD34-positive cells 
reconstitute hematopoiesis. J Clin Oncol. Aug 1996;14(8):2224-2233. 
82. 
Eaves CJ. Peripheral blood stem cells reach new heights. Blood. Oct 1 1993;82(7):1957-
1959. 
83. 
Goldman J. Peripheral blood stem cells for allografting. Blood. Mar 15 1995;85(6):1413-
1415. 
84. 
Aversa F, Tabilio A, Velardi A, et al. Treatment of high-risk acute leukemia with T-cell-
depleted stem cells from related donors with one fully mismatched HLA haplotype. N 
Engl J Med. Oct 22 1998;339(17):1186-1193. 
85. 
Schumm M, Lang P, Taylor G, et al. Isolation of highly purified autologous and 
allogeneic peripheral CD34+ cells using the CliniMACS device. J Hematother. Apr 
1999;8(2):209-218. 
86. 
Handgretinger R, Lang P, Schumm M, et al. Isolation and transplantation of autologous 
peripheral CD34+ progenitor cells highly purified by magnetic-activated cell sorting. 
Bone Marrow Transplant. May 1998;21(10):987-993. 
87. 
Bader P, Holle W, Klingebiel T, et al. Mixed hematopoietic chimerism after allogeneic 
bone marrow transplantation: the impact of quantitative PCR analysis for prediction of 
relapse and graft rejection in children. Bone Marrow Transplant. Apr 1997;19(7):697-
702. 
88. 
Bader P, Beck J, Schlegel PG, Handgretinger R, Niethammer D, Klingebiel T. Additional 
immunotherapy on the basis of increasing mixed hematopoietic chimerism after 
allogeneic BMT in children with acute leukemia: is there an option to prevent relapse? 
Bone Marrow Transplant. Jul 1997;20(1):79-81. 
89. 
Bader P, Beck J, Frey A, et al. Serial and quantitative analysis of mixed hematopoietic 
chimerism by PCR in patients with acute leukemias allows the prediction of relapse after 
allogeneic BMT. Bone Marrow Transplant. Mar 1998;21(5):487-495. 
90. 
Benaim E HG, Srivastava DK, Cunningham JM, Horwitz EM, Heslop HE, Brenner MK, 
Krance RA, Bowman LC. Efficacy with Minimal Toxicity Associated with the 
Administration of Donor Lymphocyte Infusions in Pediatric Bone Marrow Transplant 
Recipients. Blood. 1999;94 (Suppl):A4903. 
91. 
Schlegel PG EM, Ziebach R, Theurer-Mainka U, Bader P, Niethammer D, Handretinger 
R, Lingebiel T. Reconditioning Regimens for Early Graft Failure in HLA-Nonidentical 
Transplants. Bone Marrow Transplant. 1999;23 (Suppl):S76. 
92. 
Slatter MA, Bhattacharya A, Abinun M, Flood TJ, Cant AJ, Gennery AR. Outcome of 
boost haemopoietic stem cell transplant for decreased donor chimerism or graft 
dysfunction in primary immunodeficiency. Bone Marrow Transplant. Apr 
2005;35(7):683-689. 
93. 
Larocca A, Piaggio G, Podesta M, et al. Boost of CD34+-selected peripheral blood cells 
without further conditioning in patients with poor graft function following allogeneic 
stem cell transplantation. Haematologica. Jul 2006;91(7):935-940. 

 
  
HIFLEX 58 
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          IRB Approval date: 12/06/2016 
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94. 
Champlin RE, Passweg JR, Zhang MJ, et al. T-cell depletion of bone marrow transplants 
for leukemia from donors other than HLA-identical siblings: advantage of T-cell 
antibodies with narrow specificities. Blood. Jun 15 2000;95(12):3996-4003. 
95. 
Chiang KY, Van Rhee F, Godder K, et al. Allogeneic bone marrow transplantation from 
partially mismatched related donors as therapy for primary induction failure acute 
myeloid leukemia. Bone Marrow Transplant. Mar 2001;27(5):507-510. 
96. 
Okunewick J. Review of the Effects of Anti-T-Cell Monoclonal Antibodies on Major and 
Minor GvHR in the Mouse. In Baum S ed. New York: Springer-Verlag; 1985. 
97. 
Ruggeri L, Capanni M, Urbani E, et al. Effectiveness of donor natural killer cell 
alloreactivity in mismatched hematopoietic transplants. Science. Mar 15 
2002;295(5562):2097-2100. 
98. 
Ruggeri L, Mancusi A, Burchielli E, Aversa F, Martelli MF, Velardi A. Natural killer cell 
alloreactivity and haplo-identical hematopoietic transplantation. Cytotherapy. 
2006;8(6):554-558. 
99. 
Murphy WJ, Reynolds CW, Tiberghien P, Longo DL. Natural killer cells and bone 
marrow transplantation. J Natl Cancer Inst. Sep 15 1993;85(18):1475-1482. 
100. 
Lang P, Klingebiel, T., Schumm, M., Pfeiffer, M. Demirdelen, B. Schlegel, P.G., Bader, 
P., Niethammer, D. Handgretinger, R. Interleukin 2 therapy after allogeneic 
transplantation with highly purified CD34+ stem cells. BMT. 2000;25 (Suppl 1):S64. 
101. 
Uharek L, Zeis M, Glass B, et al. High lytic activity against human leukemia cells after 
activation of allogeneic NK cells by IL-12 and IL-2. Leukemia. Nov 1996;10(11):1758-
1764. 
102. 
Zeis M, Uharek L, Glass B, et al. Allogeneic MHC-mismatched activated natural killer 
cells administered after bone marrow transplantation provide a strong graft-versus-
leukaemia effect in mice. Br J Haematol. Mar 1997;96(4):757-761. 
103. 
Lanier LL. NK cell receptors. Annu Rev Immunol. 1998;16:359-393. 
104. 
Moretta A, Moretta L. HLA class I specific inhibitory receptors. Curr Opin Immunol. Oct 
1997;9(5):694-701. 
105. 
Handgretinger R, Lang P, Schumm M, et al. Immunological aspects of haploidentical 
stem cell transplantation in children. Ann N Y Acad Sci. Jun 2001;938:340-357; 
discussion 357-348. 
106. 
Leung W, Iyengar R, Turner V, et al. Determinants of antileukemia effects of allogeneic 
NK cells. J Immunol. Jan 1 2004;172(1):644-650. 
107. 
Thomas DT, Bloom, K.G., Forma S. J. Preparative Regimens. In: Bensinger WI, 
Buckner, C.D., ed. Hematopoietic Cell Transplantation. 2nd ed: Blackwell Science; 
1999:123-134. 
108. 
Miralbell R, Sancho G, Bieri S, et al. Renal insufficiency in patients with hematologic 
malignancies undergoing total body irradiation and bone marrow transplantation: a 
prospective assessment. Int J Radiat Oncol Biol Phys. Mar 1 2004;58(3):809-816. 
109. 
Carruthers SA, Wallington MM. Total body irradiation and pneumonitis risk: a review of 
outcomes. Br J Cancer. Jun 1 2004;90(11):2080-2084. 
110. 
Girinsky T, Benhamou E, Bourhis JH, et al. Prospective randomized comparison of 
single-dose versus hyperfractionated total-body irradiation in patients with hematologic 
malignancies. J Clin Oncol. Mar 2000;18(5):981-986. 
111. 
Min D, Taylor PA, Panoskaltsis-Mortari A, et al. Protection from thymic epithelial cell 
injury by keratinocyte growth factor: a new approach to improve thymic and peripheral 

 
  
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          IRB Approval date: 12/06/2016 
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T-cell reconstitution after bone marrow transplantation. Blood. Jun 15 2002;99(12):4592-
4600. 
112. 
Ringden O, Labopin M, Tura S, et al. A comparison of busulphan versus total body 
irradiation combined with cyclophosphamide as conditioning for autograft or allograft 
bone marrow transplantation in patients with acute leukaemia. Acute Leukaemia 
Working Party of the European Group for Blood and Marrow Transplantation (EBMT). 
Br J Haematol. Jun 1996;93(3):637-645. 
113. 
Benaim E, Hale, G., Horwitz, E., Leung, W., Woodard, P., Usman, Y., Handgretinger, R. 
Reduced-intensity conditioning (RIC) in haploidentical transplantation. Blood. 2004;104 
(11):590a. 
114. 
Aversa F, Velardi A, Tabilio A, Reisner Y, Martelli MF. Haploidentical stem cell 
transplantation in leukemia. Blood Rev. Sep 2001;15(3):111-119. 
115. 
Chen X, Hale GA, Barfield R, et al. Rapid immune reconstitution after a reduced-
intensity conditioning regimen and a CD3-depleted haploidentical stem cell graft for 
paediatric refractory haematological malignancies. Br J Haematol. Nov 2006;135(4):524-
532. 
116. 
Leung W, Iyengar R, Leimig T, Holladay MS, Houston J, Handgretinger R. Phenotype 
and function of human natural killer cells purified by using a clinical-scale 
immunomagnetic method. Cancer Immunol Immunother. Apr 2005;54(4):389-394. 
117. 
Rebello P, Cwynarski K, Varughese M, Eades A, Apperley JF, Hale G. Pharmacokinetics 
of CAMPATH-1H in BMT patients. Cytotherapy. 2001;3(4):261-267. 
118. 
Kanda Y, Oshima K, Asano-Mori Y, et al. In vivo alemtuzumab enables haploidentical 
human leukocyte antigen-mismatched hematopoietic stem-cell transplantation without ex 
vivo graft manipulation. Transplantation. May 27 2005;79(10):1351-1357. 
119. 
Rizzieri DA, Koh LP, Long GD, et al. Partially matched, nonmyeloablative allogeneic 
transplantation: clinical outcomes and immune reconstitution. J Clin Oncol. Feb 20 
2007;25(6):690-697. 
120. 
Shah AJ, Kapoor N, Crooks GM, et al. The effects of Campath 1H upon graft-versus-host 
disease, infection, relapse, and immune reconstitution in recipients of pediatric unrelated 
transplants. Biol Blood Marrow Transplant. May 2007;13(5):584-593. 
121. 
Elter T, Molnar I, Kuhlmann J, Hallek M, Wendtner C. Pharmacokinetics of 
alemtuzumab and the relevance in clinical practice. Leuk Lymphoma. Dec 
2008;49(12):2256-2262. 
122. 
Angiolillo AL, Yu AL, Reaman G, Ingle AM, Secola R, Adamson PC. A phase II study 
of Campath-1H in children with relapsed or refractory acute lymphoblastic leukemia: a 
Children's Oncology Group report. Pediatr Blood Cancer. Dec 2009;53(6):978-983. 
123. 
Coustan-Smith E, Behm FG, Sanchez J, et al. Immunological detection of minimal 
residual disease in children with acute lymphoblastic leukaemia. Lancet. Feb 21 
1998;351(9102):550-554. 
124. 
Coustan-Smith E, Sancho J, Hancock ML, et al. Clinical importance of minimal residual 
disease in childhood acute lymphoblastic leukemia. Blood. Oct 15 2000;96(8):2691-2696. 
125. 
Nucifora G, Larson RA, Rowley JD. Persistence of the 8;21 translocation in patients with 
acute myeloid leukemia type M2 in long-term remission. Blood. Aug 1 1993;82(3):712-
715. 

 
  
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126. 
Roberts WM, Estrov Z, Ouspenskaia MV, Johnston DA, McClain KL, Zipf TF. 
Measurement of residual leukemia during remission in childhood acute lymphoblastic 
leukemia. N Engl J Med. Jan 30 1997;336(5):317-323. 
127. 
Knechtli CJ, Goulden NJ, Hancock JP, et al. Minimal residual disease status before 
allogeneic bone marrow transplantation is an important determinant of successful 
outcome for children and adolescents with acute lymphoblastic leukemia. Blood. Dec 1 
1998;92(11):4072-4079. 
128. 
Langebrake C, Creutzig U, Dworzak M, et al. Residual disease monitoring in childhood 
acute myeloid leukemia by multiparameter flow cytometry: the MRD-AML-BFM Study 
Group. J Clin Oncol. Aug 1 2006;24(22):3686-3692. 
129. 
Buccisano F, Maurillo L, Gattei V, et al. The kinetics of reduction of minimal residual 
disease impacts on duration of response and survival of patients with acute myeloid 
leukemia. Leukemia. Oct 2006;20(10):1783-1789. 
130. 
Bader P, Niethammer D, Willasch A, Kreyenberg H, Klingebiel T. How and when should 
we monitor chimerism after allogeneic stem cell transplantation? Bone Marrow 
Transplant. Jan 2005;35(2):107-119. 
131. 
Bader P, Kreyenberg H, Hoelle W, et al. Increasing mixed chimerism is an important 
prognostic factor for unfavorable outcome in children with acute lymphoblastic leukemia 
after allogeneic stem-cell transplantation: possible role for pre-emptive immunotherapy? 
J Clin Oncol. May 1 2004;22(9):1696-1705. 
132. 
Ewing P, Miklos S, Olkiewicz KM, et al. Donor CD4+ T-cell production of tumor 
necrosis factor alpha significantly contributes to the early proinflammatory events of 
graft-versus-host disease. Exp Hematol. Jan 2007;35(1):155-163. 
133. 
Kami M, Matsumura T, Tanaka Y, et al. Serum levels of soluble interleukin-2 receptor 
after bone marrow transplantation: a true marker of acute graft-versus-host disease. Leuk 
Lymphoma. Aug 2000;38(5-6):533-540. 
134. 
Kuehnle I, Huls MH, Liu Z, et al. CD20 monoclonal antibody (rituximab) for therapy of 
Epstein-Barr virus lymphoma after hemopoietic stem-cell transplantation. Blood. Feb 15 
2000;95(4):1502-1505. 
135. 
Baldanti F, Grossi P, Furione M, et al. High levels of Epstein-Barr virus DNA in blood of 
solid-organ transplant recipients and their value in predicting posttransplant 
lymphoproliferative disorders. J Clin Microbiol. Feb 2000;38(2):613-619. 
136. 
Kimura H, Morita M, Yabuta Y, et al. Quantitative analysis of Epstein-Barr virus load by 
using a real-time PCR assay. J Clin Microbiol. Jan 1999;37(1):132-136. 
137. 
Brochure Is. CliniMACS CD3 Reagent System. Vol Version 4.0June 10 2005. 
138. 
Brochure Is. CliniMACS CD34+ Reagent System. June 27 2005;Version 5.0. 
139. 
Chen TT, Ng TH. Optimal flexible designs in phase II clinical trials. Stat Med. Oct 30 
1998;17(20):2301-2312. 
140. 
Gray RJ. A class of K-sample tests for comparing the cumulative incidence of a 
competing risk. The Annals of Statistics. 1988;16(3):1141-1154. 
141. 
Cox DR. Regression models and life tables (with discussion). Journal of the Royal 
Statistical Society. 1972;34(2):187-220. 
142. 
Prentice RL, Kalbfleisch JD, Peterson AV, Jr., Flournoy N, Farewell VT, Breslow NE. 
The analysis of failure times in the presence of competing risks. Biometrics. Dec 
1978;34(4):541-554. 

 
  
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143. 
Fine JP, Gray, RJ. A proportional hazards model for the sub distribution of a competing 
risk. Journal of the American Statistical Association. 1999;94(446):496-509. 
144. 
Agresti A. Catergorical Data Analysis. New York: John Wiley & Sons; 1994. 
 

 
  
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APPENDIX A: 
 
KARNOFSKY PERFORMANCE STATUS SCALE 
≥ 16 YEARS OLD 
Score 
General Description 
100 
Normal. No complaints. No evidence of disease. 
90 
Able to carry on normal activity. Minor signs or symptoms of disease. 
80 
Normal activity with effort. Some signs or symptoms of disease. 
70 
Care of self.  Unable to carry out normal activity or to do active work. 
60 
Requires occasional assistance, but is able to care for most of his needs. 
50 
Requires considerable assistance and frequent medical care. 
40 
Disabled.  Requires special care and assistance. 
30 
Severely disabled. Hospitalization is indicated although death is not imminent. 
20 
Hospitalization necessary, very sick, active support treatment necessary. 
10 
Moribund. Fatal processes progressing rapidly. 
0 
Dead. 
 
 
LANSKY PERFORMANCE STATUS SCALE 
< 16 YEARS OLD 
Score  
General Description 
100 
 Fully active, normal 
90  
Minor restrictions in physically strenuous activity 
80 
Active, but tires more quickly 
70 
Both greater restriction of and less time spent in play activity 
60 
Up and around, but minimal active play; keeps busy with quieter activities 
50 
Gets dressed but lies around much of the day, no active play but able to participate in 
all quiet play and activities 
40 
Mostly in bed; participates in quiet activities 
30 
In bed; needs assistance even for quiet play 
20 
Often sleeping; play entirely limited to very passive activities 
10 
No play; does not get out of bed 
0 
Unresponsive 

 
  
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APPENDIX B 
Criteria for Acute Graft-vs.-Host Disease164 
   Organ Staging:  
Organ 
Stage 0 
Stage 1 
Stage 2 
Stage 3 
Stage 4 
Skin 
No rash 
Rash on       
< 25% body 
surface areaa 
Rash  > 25% to 
< 50%  
Rash >50% 
generalized 
erythroderma 
Plus bullae and 
desquamation 
Gastrointestinal  
Lower 
GI 
Diarrhea       
< 500 mL/day 
Diarrhea 501 
to 1000 
mL/dayc 
Diarrhea 1001 
to 1500 
mL/day 
Diarrhea >1500 
mL/day 
Severe 
abdominal pain 
with or without 
ileus 
Upper 
GI 
 
Persistent 
nausead 
 
 
 
Liver 
Bilirubin       
< 2.0 mg/dl 
Bilirubin 2.1 
to 3.0 mg/dlb 
Bilirubin 3.1 to 
6.0 mg/dl 
Bilirubin 6.1 to 
15 mg/dl 
Bilirubin 15 
mg/dl 
 
    Overall Grading for acute GVHD 
Grade 
Skin 
Liver 
Gut 
I 
Stage 1-2 
None 
None 
II 
Stage 3 or 
Stage 1 or 
Stage 1 
III 
 
Stage 2-3 or 
Stage2-4  
IVf 
Stage 4 or 
Stage 4 
 
a) Use “Rule of Nines” or burn chart to determine extent of rash. 
b) Range given as total bilirubin. Downgrade one stage if additional cause of elevated 
bilirubin is documented.  
c) Volume of diarrhea applies to adults.  For pediatric patients, the volume of diarrhea 
should be based on body surface area. Downgrade one stage if additional cause of 
diarrhea has been documented.  
d) Persistent nausea with histological evidence of GVHD in stomach or duodenum. 
e) Criteria for grading given as minimum degree of organ involvement required to confer 
that grade.  
f) Grade IV may also include lesser organ involvement but with extreme decrease in 
performance status.  
 

 
  
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APPENDIX B (continued) 
Notes:  
• 
Liver GVHD is downgraded one stage if an additional cause(s) of an elevated bilirubin is 
documented.  
• 
Gut GVHD is downgraded one stage if additional cause(s) of increased diarrhea/loose stool 
volume is documented.  
• 
Specified stool volumes noted for gut GVHD applies to adult range body weights.  For 
pediatric patients, the volume of stool should generally be based on body surface area.   
• 
Stool/urine mixture cannot be considered total stool volume. Stool volume needs to be 
unmixed stool without urine volume factored into volume total.  
• 
Abdominal pains secondary to other etiologies need to be ruled out or factored into 
abdominal GVHD assessments accordingly.  
• 
Overall clinical performance may also be considered in the clinical grading of severity 
of acute GVHD. 
 
CRITERIA FOR GRADING CHRONIC GVHD 
Chronic GVHD typically occurs after the day +100 time point or 100 days post stem cell boost 
as applicable. The diagnosis is determined by the transplant physician based on clinical 
assessment and may be supported by pathology findings.  
Staging of Chronic GVHD:  
Limited - Localized skin and/or hepatic dysfunction. 
Extensive: One or more of the following (as clinically judged by a physician and deemed as 
chronic GVHD by the PI): 
• Generalized skin involvement 
• Liver histology showing chronic aggressive hepatitis, bridging necrosis and/or cirrhosis. 
• Eye dryness with Schirmer’s test <5 mm wetting 
• Oral:  involvement of salivary glands or oral mucosa. 
• Other:  another target organ involvement.  
 
 
 

 
  
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APPENDIX C 
CRITERIA FOR ADVERSE EVENT (AE) EVALUATION AND REPORTING 
The St. Jude Department of BMTCT Clinical Research Office standard operating procedure for 
the documenting and reporting of adverse (SOP 10 Documenting and Reporting of Adverse 
Events) will provide guidance on the evaluation, collection and reporting of adverse events for 
this clinical trial.  The current version of this document, as well as ongoing updates, can be 
located at the following website: http://home.stjude.org/bmt/Pages/policies-research.aspx 
 
 

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APPENDIX D 
Recommended testing and evaluation schedule 
 
STANDARD OF 
CARE STUDIES 
SAMPLE 
VOLUME 
PRE 
MONTH 1 
MONTH 2 
MONTH 3 
MONTH 6 
MONTH 12 
Pregnancy Test 
PB 
2 ml 
X 
As clinically indicated 
Physical Exam 
N/A 
N/A 
X 
Weekly 
X 
X 
CBC with diff. 
PB 
0.5-2 ml 
X 
Daily until engrafted, then weekly 
X 
X 
Chemistry 
PB 
0.25-2ml 
X 
Weekly 
X 
X 
Viral surveillance 
(BMTPCR) 
PB 
4 ml 
X 
Weekly 
As clinically indicated 
Chimerism 
PB 
1-2 ml 
 
Weekly upon engraftment 
X 
X 
BM 
2 ml 
 
X 
 
X 
 
X 
Disease Status 
Evaluation 
N/A 
N/A 
X 
X 
 
X 
 
X 
MRD Bone 
Marrow       
BM 
3 ml 
X 
X 
 
X 
 
X 
Lymphocyte 
Subset Study 
PB 
2.5-4 ml 
X 
X 
X 
X 
X 
X 
Quantitative 
Immunoglobulins 
PB 
2 ml 
X 
 
 
X 
 
X 
 
• 
The information derived from or noted on the physical examinations, standard tests, and other assessments that comprise standard of care for recipients are not 
required to be transcribed onto case report forms and/or entered into the database. In reference to section 6.1 Evaluations, the above-indicated follow-up regimen for 
these evaluations is guided by the SOPs of the Department of BMTCT, for recipients of allogeneic stem cell transplantation. As these evaluations are considered 
standard clinical care (non-research), variations in frequency (more or less frequent) of these evaluations can occur due to the participant’s current clinical condition 
and will not be noted as protocol deviations. 
• 
Disease status evaluations/BM testing results obtained prior to enrollment may be used for the baseline/pre-infusion assessments. 
• 
Additional chimerism testing may be required as clinically indicated and described in Section 6.3. 
• 
Lymphocyte subset studies may be omitted without variance when the absolute lymphocyte count (ALC) is zero. 
• 
In the event of graft failure/rejection, the post failure/rejection bone marrow, chimerism and several applicable immune studies would not be clinically indicated and 
these studies may be held. 

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APPENDIX D (continued) 
Immune reconstitution testing and evaluation schedule for RECIPIENT 
RESEARCH 
STUDIES 
SAMPLE 
VOLUME 
PRE 
MONTH 1 
MONTH 2 
MONTH 3 
MONTH 6 
MONTH 12 
VBETA/TREC 
RESEARCH 
PB 
17 mL 
X 
 
 
X 
X 
X 
LYMPHOCYTE 
PHENOTYPES 
RESEARCH 
PB 
17 mL 
X 
X 
X 
X 
X 
X 
SOLUBLE 
INTERLEUKIN-2  
PB 
2 mL 
X 
Weekly starting DAY -7, until approximately DAY 100 
 
 
TUMOR 
NECROSIS 
FACTOR 
PB 
3 mL 
X 
Weekly starting DAY -7, until approximately DAY 100 
 
 
 
 
• 
VBETA/TREC Research and Lymphocyte Phenotypes Research results will be maintained in the Leung laboratory database. 
• 
For RESEARCH studies, the posted volumes are the minimum volumes required to perform the respective protocol evaluations. 
 
Optional Campath-1H pharmacokinetic testing schedule for RECIPIENT 
RESEARCH STUDIES 
SAMPLE 
VOLUME 
DAY -10 
DAY -8 
DAY -3 
WEEK 1 
WEEK 2 
WEEK 4 
WEEK 8 
WEEK 16 
ALEMTUZUMAB 
HIFLEX (CAMPTH-
1H) 
PB 
2 mL 
X 
X 
X 
X 
X 
X 
X 
X 

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APPENDIX D (continued) 
Research testing for DONOR 
Prior to initial stem cell collection procedure: 
OPTIONAL research immune studies testing schedule 
Evaluation 
Volume Requirement 
Flow cytometry enumeration 
Lymphocyte Subset Study = 4 mL 
Thymic output and T cell repertoire 
VBETA/TREC Research = 17 mL 
T cell and NK cell number and function 
Lymphocyte Phenotypes Research = 17 mL 
 
• All donor research testing to be collected prior to stem cell collection – preferably prior to growth factor administration.  
These optional research tests may be collected at separate times. 

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APPENDIX D (continued) 
 
Research Study Evaluation Target Windows 
Several laboratory tests can only be processed on weekdays; therefore, if the scheduled 
evaluation falls on a weekend, or during a holiday period, an adjustment in the follow-up visit is 
expected and would not be noted as a protocol variation.  Additionally, in order to accommodate 
such logistical constraints, evaluation/collection dates of all protocol assessments (required and 
optional research), may be performed within a reasonable window of the intended date following 
the guidelines provided in the table below: 
If the Planned Evaluation 
Time Point is: 
Window 
Weekly 
+ 3 Days 
Month 1 
Week 2 to Week 6 
Month 2 
Week 7 to Week 11 
Month 3 
Week 12 to Month 4 
Month 6 
Month 5 to Month 7 
Month 9 
Month 8 to Month 10 
Month 12 
Month 10 to Month 14 

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APPENDIX E 
The St. Jude Department of BMTCT Clinical SOPs for standard of care for all allogeneic stem 
cell infusion recipients and stem cell donors will provide guidance on the evaluation, ongoing 
clinical care and follow up for this clinical trial.  The current versions of these SOPs, as well as 
ongoing updates, of these documents can be located at the following website: 
http://home.web.stjude.org /bone_marrow/clinicalHome.shtml.    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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APPENDIX F 
 
Dose modification and off treatment criteria for Campath-1H toxicity 
 
Recommended pre-medication to be given with Campath-1H: 
 
First doses should be timed approximately 30 minutes prior to daily Campath-1H dose. 
Diphenhydramine: 1mg/kg IV or PO q 8hr (max dose 50mg) 
Acetaminophen: 10-15mg/kg PO q 6hr (max 4gm/day) 
Hydrocortisone: 2mg/kg IV q 6 hr (when Campath is given SQ, may use 1mg/kg q 6 hr) 
Meperidine: 0.5mg/kg IV q 4hr PRN rigors 
 
Modifications in the event of an adverse reaction during test dose administration: 
 
• If the reaction is mild (Grade 1), such that no intervention is required, and the infusion is 
not interrupted; proceed with subsequent dosing as planned.  Examples of grade 1 
reactions are: Transient flushing, rash, and/or low grade fever that resolve promptly and 
without intervention. 
• If the reaction requires therapy (such as additional antihistamine or acetaminophen) or a 
brief interruption of infusion, and responds promptly (Grade 2); proceed with dosing as 
planned.  Depending on the seriousness of the adverse reaction, consider alteration of 
premedication (e.g., change steroid to methylprednisolone 2mg/kg q 6hr), and/or a 
reduction in the planned infusion rate. 
• If the reaction is prolonged (i.e., not rapidly responsive to symptomatic medication and/or 
brief interruption of infusion) but improves prior to next dose (24hr), recurs following 
initial improvement but improves prior to next dose, or is serious (i.e. symptomatic 
bronchospasm, significant hypotension) but improves with intervention (Grade 3); notify 
the PI and make the following modifications:  At a minimum, increase dose and/or 
frequency of supportive medications (corticosteroids, antihistamines, acetaminophen), 
and ensure infusion is begun at a reduced rate.  Depending on the seriousness of the 
reaction, consider discontinuation of Campath-1H with substitution of ATG as alternative 
therapy. 
• If the reaction is life threatening (Grade 4), notify the PI, discontinue Campath-1H, and 
use ATG as alternative therapy if possible. 
 
For adverse reactions which occur during/following standard dose administration: 
 
• If the reaction is mild (Grade 1), such that no intervention is required, and the infusion is 
not interrupted; proceed with subsequent dosing as planned.  Examples of grade 1 
reactions are: Transient flushing, rash, or a low grade fever that resolve quickly without 
intervention. 
• If the reaction requires therapy (such as additional antihistamines or acetaminophen) or a 
brief interruption of infusion, and responds promptly (Grade 2); may proceed with dosing 
as planned.  Consider alteration of premedication (e.g, change to methylprednisolone), 
and/or a reduction in the planned infusion rate. 

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• If the reaction is prolonged (i.e., not rapidly responsive to symptomatic medication and/or 
brief interruption of infusion) but improves prior to next dose (24hr), recurs following 
initial improvement but improves prior to next dose, or is serious (i.e. symptomatic 
bronchospasm, significant hypotension) but improves with intervention (Grade 3); notify 
the PI and make the following modifications:  Increase dose and/or reduce interval on 
supportive medications (corticosteroids, antihistamines, acetaminophen).  Ensure infusion 
is begun at a reduced rate.  No further dose escalation is permitted, and depending on the 
seriousness of the reaction consider a reduction in subsequent doses.  
• If the reaction is prolonged and does NOT improve with intervention prior to the next 
scheduled dose (Grade 3), notify the PI, discontinue Campath-1H, and use ATG as 
alternative therapy if possible.   
• If the reaction is life threatening or requires intensive support (Grade 4), notify the PI, 
discontinue Campath-1H, and use ATG as alternative therapy if possible.  May consider 
reduction of subsequent dosing and escalation of supporting medications, ONLY if a 
lower dose was previously tolerated, AND symptoms resolve entirely prior to next dose, 
AND no suitable alternative is available. 
 
 
Substitution of ATG if Campath-1H intolerance: 
 
If the patient received less than 50% of the planned Campath-1H dose, efforts should be made 
to give ATG in substitute. 
Typically rabbit ATG should be given as 3mg/kg/day for 3 days. 
In the event the patient received less than 50% of the planned Campath dose, and ATG is 
contraindicated, the patient will be removed from study. 

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APPENDIX G 
Responsibility for Campth-1H PK collection and analysis at St. Jude  
Delegation of responsibility of PK sample collection will be as follows:  
1. Blood collection: St. Jude nurse 
2. Plasma extraction: St. Jude PK technician 
3. Freeze and store plasma: St. Jude PK technician 
4. Sample log: St. Jude PK technician  
5. Shipment to Genzyme: St. Jude PK technician (2 – 5 under the supervision of the BMT and 
CT Pharmacy sub-investigators - Dr. Cross). 
6. Bioanalysis: Genzyme 
7. Interpretation of raw data: Genzyme will provide a written report of results to be emailed to 
Dr. Brandon Triplett in pdf format. 
PK TESTING  MATERIALS AND INSTRUCTIONS FOR U.S. & CANADIAN 
SITES OR COLLECTION, PROCESSING, PACKAGING & SHIPMENT OF BLOOD 
SAMPLES  
 
 
SAMPLE COLLECTION MATERIALS 
 
Supplied by Genzyme 
 
Instructions for handling/processing  
 
2 x 5 mL serum separator tubes with gold hemoguard  
 
4 x 2 mL plastic freezer storage tubes (cryovials) 
 
6 labels for the serum separator tube(s) and cryovials  
 
2 plastic transfer pipettes  
 
4 sheets of absorbent material  
 
Rubber band  
 
1 sample-shipping container pre-labeled with “BIOHAZARD” 
 
2 cold packs 
 
Styrofoam Sample Collection Box suitable for shipping 
 
Exterior Cardboard Shipping Box 
 
Pre-addressed FedEx airway bills (one U.S. Airbill and one International Air Waybill) 
 
Antibody Testing Requisition Form 
 
Plastic bag for placement of Antibody Testing Requisition Form 
 
FedEx Diagnostic Envelope with IATA Compliance Label for placement of the box for transit 
 
Pro-Forma Invoice (for Canadian Sites Only) 
 

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Supplied by Site 
 
Freezer capable of freezing to preferably < -60° C (< -20° C  is acceptable) 
 
  
- If unavailable, see alternative option (pg 4). 
 
 
Centrifuge (capable of 2000-3100 rpm) 
 
- If unavailable, see alternative option for collection and shipment of whole blood (pg 5). 
 
Blood collection materials (gloves, alcohol swabs, tourniquets, needles) 
 
Packing tape 
Please adhere to the following instructions as samples that are mishandled may result in sample degradation.   
Samples that are incorrectly labelled will not be tested. 
Blood Collection and Processing for FROZEN SPUN SERUM SAMPLES 
Fill the 2 x 5mL (for adults) or 1 x 5mL (for pediatric patients) serum separator blood collection tube(s).  Samples should be drawn prior 
to the Campath infusion. 
Label the serum separator blood collection tube(s) using the labels provided.  Label with Genzyme Product name (i.e.- Campath), patient 
initials, patient DOB (dd/mmm/yyyy – e.g., 10/Mar/1968), sample collection date (dd/mmm/yyyy), sample collection time (24 hour clock: 
2:00 PM = 14:00 hours). 
Allow blood to clot between 30 – 60 minutes (not longer) at room temperature. 
To separate serum, centrifuge blood sample at 2°C - 8˚C (if available) at 2000-3100 rpm for 10-15 minutes. 
Transfer the cell-free serum, in approximate equal aliquots, into the 4 x 2mL freezer vials (cryovials) provided (2 cryovials for pediatric 
patients).  
Label the cryovials with the labels provided.  Label with Product name (i.e.- Campath), patient initials, patient DOB (dd/mmm/yyyy -  e.g. 
10/Mar/1968)), patient Registry ID#, sample type (e.g., serum), sample collection date (dd/mmm/yyyy), sample collection time (24 hour 
clock: 2:00 PM = 14:00 hours) and Physician name. 
Freeze all cryovials in the upright position at ≤ -60˚C (≤ -20˚C is acceptable) for a minimum of 2 hours.  Keep all vials frozen until ready 
to ship. 
 
Blood Packaging and Shipping for FROZEN SPUN SERUM SAMPLES 
Contact information:  For questions contact 1-800-745-4447 or 1-617-768-9000 (option 2) 
Samples may be shipped after a minimum of 2 hours of freezing.  Multiple patient samples may be shipped together in the same box with 
proper sample labeling only. Cryovials must remain frozen at all times. DO NOT ALLOW THE SAMPLES TO THAW during the shipping 
preparations described below. 
Samples should be shipped Monday-Thursday (Next Day Delivery) from U.S. and Canadian sites, as long as WEEKDAY DELIVERY TO 
GENZYME is assured (Canadian sites should ship Monday-Wednesday, Thursday only in an emergency). 
Complete the Campath Testing Requisition Form completely.  Retain the white copy and insert the yellow and pink copies into the plastic 
paperwork bag provided. 
When ready for shipping, remove the cryovials from the freezer and wrap each cryovial with one piece of absorbent material and bundle 
all cryovials with a rubber band. 
Place the wrapped cryovials in the sample-shipping container labeled “Biohazard.” 
Place the frozen cold packs and sample-shipping container labeled “Biohazard” (containing the cryovials) into the Styrofoam shipping 
box.  [NOTE:  Cold packs must be frozen for a minimum of 48 hours prior to shipping.] 
Place the plastic bag containing the Campath Testing Requisition Form on top of the frozen cold packs. 
Place the cover on the box making sure it is flush with the top of the wall liner, allowing the box to close completely.  If not done properly, 
the adequate temperature to maintain the frozen samples may not occur.  Close the box with packing tape. 
Place the Styrofoam shipping box into the exterior cardboard box and place the cardboard box into the supplied FedEx shipping envelope. 

HIFLEX   75 
Revision 6.1 dated: 12/24/2014 
    
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Protocol document date: 11/18/2016 
 
Blood Packaging and Shipping for FROZEN SPUN SERUM, Continued 
 Complete all the necessary information requested on the FedEx airway bill, specifically:  
• 
Complete Section 1: Enter the sender’s information (you do not need to provide a FedEx account #)  
• 
Complete Section 6: "Special Handling" by checking "No."  
NOTE: These must be completed or the package will not be delivered. 
• 
If sending from U.S.,  use the enclosed  domestic US airbill  
• 
If sending from CANADA,  use the enclosed  International airbill 
Attach the pre-addressed airway bill to the envelope provided.   
Canadian sites only: complete and include 4 copies of the Pro-Forma invoice with the FedEx airway bill. 
Call FedEx at 1-800-463-3339 to arrange sample pick-up. 
Notify Genzyme Clinical Specialty Labs of the pending shipment with  the tracking number ;via;  TEL:  800-745-4447, option 
0, ext 22449, e-mail CSLtesting@genzyme.com or FAX: 508-270-2023  . 
All U.S. and Canadian sites ship samples via FedEx to: 
 
            
Genzyme Corporation - Clinical Specialty Laboratory 
One Mountain Road 
Framingham, MA  01701-9322   USA  
Telephone:  1-508-270-2449