Post Transplant CD34+ Selected Stem Cell Infusion to Augment Graft Function in Children With Primary Immunodeficiency Diseases and Bone Marrow Failure Syndromes
Phase: PHASE2
Conditions: Waning Donor Chimerism, Waning Immune Function, Primary Immunodeficiency Disease(s), Bone Marrow Failure
Sponsor: Children's Hospital Medical Center, Cincinnati

 
 
COVER PAGE FOR CLINICALTRIALS.GOV
 
 
 
Protocol Title: 
Post transplant CD34+ Selected Stem Cell Infusion to 
Augment Graft Function in Children with Primary Immunodeficiency 
Diseases and Bone Marrow Failure Syndromes
 
 
 
NCT Number: NCT01856582  
 
 
Document Version Date: 
01/10/2018  

CCHMC IRB # 2010-2344 
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Post transplant CD34+ Selected Stem Cell Infusion to Augment Graft 
Function in Children with Primary Immunodeficiency Diseases and 
Bone Marrow Failure Syndromes. 
 
 
 
PRINCIPAL INVESTIGATOR: 
Rebecca Marsh, MD 
 
Professor, Division of Hematology/Oncology 
 
Blood and Marrow Transplant Program 
 
Cincinnati Children's Hospital Medical Center, MLC-7015 
 
Cincinnati, Ohio 45229-3039 
 
(513) 803-1139 
 
Email: Rebecca.Marsh@cchmc.org  
 
 
CO-INVESTIGATORS:    
Jacob Bleesing, MD, PhD  
 
 
Stella M Davies MB BS, PhD, MRCP  
 
 
Michael Jordan, MD 
 
 
Parinda Mehta, MD 
 
 
Ashish Kumar, MD 
 
 
Michael Grimley, MD 
 
 
Kasiani Myers, MD 
 
 
Sharat Chandra, MD 
 
 
Pooja Khandelwal, MD 
 
 
Thomas Leemhuis, PhD 
 
 
 
ABSTRACT 
 
 
The purpose of this study is to investigate the usefulness of infusing purified CD34+ cells of 
donor origin in order to augment graft function in response to declining chimerism after 
initially performing an allogeneic hematopoietic stem cell transplant (HSCT) for children with 
primary immunodeficiency diseases. This protocol will be utilized for patients with waning 
mixed donor chimerism that is inadequate for correction of clinical condition or disease for 
which stem cell transplant was performed, or for augmentation of immune function.  An 
infusion of selected CD34+ stem cells will be given without any preparative regimen.  As the 
children eligible for this protocol have reduced immune function and pre-existing donor 
chimerism, we hypothesize that stem cells will be able to engraft and the infusion will 
augment graft function.  This therapy serves as an alternative to a second stem cell 
transplant that is known to be associated with significant morbidity and mortality.  CD34+ 
stem cells will be collected from the donor used for initial stem cell transplant.  Cells will be 
T-cell depleted (TCD) by performing a CD34 selection using the CliniMACS device (Miltenyi 
Biotec)  in order to prevent development of new or exacerbation of existing graft versus host 
disease (GVHD), as avoidance of GVHD in nonmalignant diseases is desirable.  There is 
sufficient data showing that mixed donor chimerism is adequate for reverting disease 
phenotype in certain primary immunodeficiencies.  Observations from Europe and CCHMC 
show that donor chimerism might be boosted by CD34+ stem cell infusion alone without any 
specific preparative regimen.  This therapy is likely to be associated with low toxicity due to 
the absence of a preparative regimen and lack of exposure to fresh donor cells capable of 
initiating GVHD, and offers potential significant benefit. 

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I. PURPOSE OF STUDY 
 
 
Hypothesis 
 
 
Mixed or full donor chimerism that is adequate for disease control will be achieved with 
selected CD34+ stem cell infusion without preparative regimen with minimal or no 
therapy related toxicity or GVHD. 
 
 
Objectives 
 
1. Assess the toxicity of infusion of donor stem cells in augmentation of graft function in 
previously transplanted children with immune deficiencies, and bone marrow failures. 
2.   Assess the efficacy of infusion of donor stem cells in augmentation of graft function   
in previously transplanted children with immune deficiencies, and bone marrow 
failures. 
Endpoints 
 
The primary endpoint of the study is augmentation of graft function.  Graft function will 
be measured in two ways.  Firstly the percentage of donor chimerism, and secondly 
measured improvement in immune function; improvement in numbers of circulating white 
blood cells and/or platelets, and/or clearance of opportunistic viral infections. 
 
 
Improvement in immune function studies compared to baseline levels can be 
documented at 3, 6, and 12 months.  Immune function studies will include 
quantitation of T and B cells and functional studies (mitogens and immunoglobulin 
levels).  In the case of Wiskott-Aldrich syndrome, improvement of platelet count to 
>20,000/L on 2 separate occasions at least three months apart will be considered 
successful graft augmentation.  Children requiring graft augmentation for a specific 
limited immune defect, e.g. inability to clear papilloma virus will also be assessed by 
clinical response of the infection; resolution or >50% improvement in infection will be 
considered successful graft augmentation.  
 
The secondary endpoint is to determine the frequency and characteristics of potential 
infusion-related toxicity.  Experience at our center, using CD34+ selected cells has 
shown infusion-related toxicity has occurred at an incidence of < 10% (zero toxicity in 9 
patients participating in CCHMC IRB study # 2008-0777).  While there is little 
expectation of infusion-related toxicity, the incidence of infusion-related reactions will be 
recorded and evaluated. 
 
 
 
 
II. SIGNIFICANCE OF STUDY IN RELATION TO HUMAN HEALTH 
 
Primary or congenital immunodeficiency diseases result from inborn genetic defects of 
the immune system frequently manifesting itself as recurrent and chronic infections and 

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commonly lead to lethal complications.  Currently, more than 100 immunodeficiency 
diseases have been identified and classified according to the area of immune system 
that is compromised.  Most of these have been molecularly defined and are due to single 
gene defects. 
 
Allogeneic hematopoietic stem cell transplantation is the only curative approach to date 
for most of the lethal immunodeficiency diseases including severe combined 
immunodeficiencies (SCID), T-cell immunodeficiencies, Wiskott-Aldrich syndrome, 
Chediak-Higashi syndrome and others, where it restores immune function and 
decreases risk of long term complications such as malignant transformation.  
 
Although stem cell transplant from HLA (human leukocyte antigen) identical sibling 
donor is the most preferable and effective treatment in congenital immunodeficiency 
diseases, the majority of these patients do not have an available sibling donor and 
alternative approaches such as T-cell depleted haploidentical parental marrow and 
matched unrelated donor marrow are used as the only curative option with variable 
results.  As reported by European study from 37 centers in 18 countries (1968-1999), in 
SCID patients 3 year survival with sustained engraftment was significantly better after 
HLA-identical than after mismatched transplantation (77% versus 54%) and improved 
over time 1,2.  The outcome for patients with Wiskott-Aldrich syndrome after 
transplantation, as reported by International Bone Marrow Registry and/or National 
Marrow Donor Program is significantly influenced by donor type showing the 5 year 
probability of survival of 87% with HLA-identical sibling donors, 52% with other related 
donors and 71% (p=0.0006) with unrelated donors 3. 
 
In patients with nonmalignant diseases avoidance of graft versus host disease (GVHD) is 
most desirable.  In some cases use of alternative donor transplant necessitates 
aggressive graft T-cell depletion that is needed to reduce GVHD.  Severe T depletion 
predisposes these patients to somewhat delayed or incomplete immune recovery and 
unacceptably high rate of graft failure. 
 
Unlike in malignant diseases where fully ablative conditioning is required to eliminate the 
tumor, most primary immunodeficiency diseases could be cured with low but sustained 
levels of donor chimerism 4.  Compartment specific chimerism after HSCT is commonly 
reported in SCID with T cells being of donor origin while B, NK and myeloid cells are 
either from recipient or mixed origin 5.  For patients with Chediak-Higashi Syndrome 
(CHS) 12-36% of donor NK cells and T lymphocytes expressing normal CHS1usually is 
sufficient to treat hematologic manifestations of CHS 6,7.  Early mixed chimerism post-
transplant is commonly reported in Wiskott-Aldrich syndrome.  Chimerism may 
eventually increase to full donor hematopoiesis in the peripheral compartment and 
persistent donor chimerism markedly decreases long-term risk of lymphoproliferative 
disease 8,9. 
 
Even though low levels of mixed donor chimerism can be enough to revert disease 
phenotype, graft failure, Epstein-Barr virus lymphoproliferative disease (EBV-LPD), and 
severe viral infections remain major obstacles to successful transplantation of children 
with immune deficiencies.  Sometimes repeated stem cell transplants are necessary to 
achieve adequate and stable immune recovery 10,11.  Multiple stem cell infusions are not 
infrequently needed in SCID patients, particularly if grafts are given without preparative 
regimen. Persistent post-transplant thrombocytopenia has been reported in some patients 

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with Wiskott-Aldrich syndrome where low platelet counts were associated with waning 
mixed chimerism in hematopoiesis exhibiting a high percentage of recipient cells.  
 
The use of second allogeneic stem cell transplant in non-malignant inherited diseases, is 
usually associated with significant risks of morbidity and mortality 12.  Occurrence of acute 
and chronic GVHD, which is considerably higher in HLA-related mismatched and 
unrelated stem cell transplants, is another limiting factor in these circumstances.  
 
Taking these facts into consideration, there is a need to develop an alternative method to 
boost waning donor mixed chimerism in this group of patients, to the level adequate for 
disease control, without causing excessive toxicities or GVHD.  
 
Based on European and local (CCHMC) data (see below) waning mixed chimerism after 
stem cell transplant for certain primary immunodeficiency diseases may be reversible by 
infusion of large dose of selected CD34+ stem cells from the original donor without using 
any preparative regimen.  The infusion toxicity is anticipated to be negligible with a  CD34 
selected product, and the likelihood of GVHD is very low due to the efficacy of the T-cell 
depletion procedure used.  Due to recipients’ immune status and a fact that they are 
already living with mixed donor chimerism, we believe that increased engraftment can be 
achieved without the use of a preparative regimen 13. 
 
 
III. PREVIOUS WORK DONE IN THIS AREA 
 
Three patients with primary congenital immunodeficiency diseases have been treated 
using above proposed therapy with promising results. 
 
The first patient, treated in Europe, received a single HLA antigen-mismatched (HLA-A in 
HvG-direction) stem cell transplant from his father as initial treatment for Wiskott-Aldrich 
syndrome at the age of 2 years.  The patient received 2.2x108 MNC/kg of unmanipulated 
bone marrow after a conditioning regimen with busulfan and cyclophosphamide.  Acute 
GVHD was successfully treated with prednisone without occurrence of any chronic GVHD.  
The patient’s immunodeficiency improved but thrombocytopenia persisted.  Only 20% of 
cells were documented to be of donor origin.  At the age of 12 years the patient was 
administered a boost of positively selected stem cells without any conditioning regimen 
for correction of persistent bleeding tendency. Stem cells from the primary donor (father) 
were selected with anti CD133-coated microbeads using a CliniMACS device (Milteny-
Biotec). The graft consisted predominantly of CD133/CD34 double-positive cells with a 
total dose of 8.3x106/kg stem cells with 8000 residual T cells/kg.  Although immune 
suppression was avoided, no signs of GVHD occurred.  The platelet count increased to 
100,000 three weeks after stem cell boost and the recovery was sustained.  The patient 
had no therapy related toxicities. Chimerism analysis showed decreased recipient cells 
(exact percentage is not reported). 
 
The second patient with Wiskott-Aldrich syndrome received HLA-matched unrelated 
donor bone marrow transplant as a primary therapy at age of 10 month, after conditioning 
regimen with busulfan, cyclophosphamide and ATG.  The child experienced  
graft failure with significant decline in donor chimerism (2-4%), clinically evident as 
persistent thrombocytopenia.  The patient’s immune function studies showed decreased 
absolute numbers of all lymphocyte subsets with normal NK and cytotoxic T-cell function 

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and normal proliferation to mitogens.  The patient was receiving steroid therapy for colitis.  
At the age of 3 years and 10 month the child was referred to CCHMC for therapy.  The 
child  received a boost of Isolex selected CD34+ stem cells (4.9x106 CD34/kg) from his 
original donor without any preparative regimen or immunosuppression, as an outpatient 
procedure.  There was one episode of fever 2 days after stem cell infusion due to a 
reaction to a platelet transfusion.  The patient did not experience any other therapy related 
toxicities or signs of GVHD.  Two and a half months after stem cell infusion, donor 
chimerism has increased from 2% to 10% with a sustained increase in platelet count to 
20-25,000 (previously 8,000) without any episodes of bleeding.  
 
The third patient is a boy with X-linked severe combined immunodeficiency (X-SCID) 
which received a haploidentical bone marrow transplant from his father at the age of 7 
months with immunological recovery (80% donor chimerism) and clinical wellness lasting 
for 7 years.  Over the past 2 years the patient has experienced debilitating skin warts due 
to papilloma virus unresponsive to topical medications and -interferon.  This complication 
has been recognized in significant number of BMT survivors for SCID and can be 
associated with later skin cancer as observed in several patients in Europe.  The inability 
to clear the papilloma virus appears to be due to a specific defect in immune repertoire.  
The child was referred to CCHMC for further management.  The patient received an Isolex 
selected T-cell depleted CD34+ stem cells infusion (13.6x106 CD34/kg) from his primary 
donor (father) without any preparative regimen in attempt to stabilize T cell and NK cell 
function and to control papilloma virus infection.  The patient   had no infusional reactions, 
nor did he experience any toxicity for two month period after stem cell infusion.  It is too 
early to evaluate efficacy of the procedure in this child.   
 
In summary, all three patients with primary immunodeficiencies and post-transplant graft 
failure received highly T-cell depleted, selected CD34+stem cells that were infused 
without any preparative regimen or additional immunosuppression.  None of these 
patients have had any infusion reactions, treatment related toxicities or GVHD. The first 
patient with Wiskott-Aldrich syndrome, as reported from Europe, had significant and 
stable platelet recovery at the time of reporting.  The second patient with Wiskott-Aldrich 
syndrome, treated at CCHMC, shows steady improvement in platelet count and donor 
chimerism.  It is anticipated that his platelet counts will improve with time, perhaps over a 
period as long as one year.  While no adverse effects or toxicities were observed in the 
patient with SCID treated at our institution, it is too early to evaluate efficacy. 
 
Most recently, the transplant group from Newcastle-upon-Tyne, UK have published a 
series of twenty boosts in nineteen patients with transplant for primary immunodeficiency 
with improvement in chimerism and/or graft function observed in 12/19. 
 
 
IV. 
RESEARCH PLAN 
 
1a. 
How many subjects will be studied? 
 
We plan to enroll 45 patients on this study (10 in strata I, II, and IV, 15 in stratum 
III).  This number is considered sufficient to exclude major frequent toxicity, and 
will allow evaluation of efficacy. 
 
1b. 
How will subjects be selected for this study? 

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Subjects will be selected from the population of patients who have undergone 
 
allogeneic hematopoietic stem cell transplant. 
 
Patients will be assigned to four strata according to the underlying disease (due 
to different disease pathogenesis) and expected end-points after stem cell 
infusion. Strata are as follows: 
 
1. Stratum I: patients with Wiskott-Aldrich syndrome (thrombocytopenia) 
2. Stratum II: patients with SCID (immune defect) 
3. Stratum III: other patients (rare immune diseases) 
4. Stratum IV: patients with bone marrow failure and other related syndromes. 
 
Five patients will be enrolled into each stratum for evaluation of graft 
augmentation.  If none of the five patients per stratum show any graft 
augmentation after one year, accrual to that particular stratum will be closed due 
to lack of efficacy.  Patients will continue to be enrolled into other strata until 
criteria for five patients per stratum are fulfilled.  If one out of five patients per 
stratum will meet listed criteria for graft augmentation, the next five patients may 
be enrolled to that stratum.  If graft augmentation is seen in 1 of 5 or 2 of 10 
cases in any stratum the strategy will be considered worthy of further 
investigation. 
 
Since initiation of the study, it has been noted that stratum III encompasses a 
wide variety of diseases and therefore the majority of subjects enrolled have 
been to this stratum.  As at least one subject has met the criteria for improved 
graft augmentation, the target enrollment for stratum III will be increased to 15 to 
accommodate this large group of patients. 
 
If the infusion of purified CD34+ donor cells does not increase graft 
augmentation, patient will be removed from study and treated according to 
standard procedures. 
 
There are no specific plans at this time to treat the patient with an additional 
boost of CD34+ cells if graft augmentation does not occur on study. However, 
this decision will be made on a per case basis as clinically indicated. 
 
2a. 
Inclusion Criteria  
 
To be eligible for this protocol, patients must have the following: 
 
1. Primary immunodeficiency (e.g. SCID, Wiskott-Aldrich and/or other more 
rare conditions), or a bone marrow failure syndrome with prior allogeneic 
stem cell transplant. 
2. Waning donor chimerism or immune function that is inadequate to correct 
their disease or clinical condition, for which primary transplant was given, 
as determined by their attending physician. 
3. Available primary donor. 
4. Must not have other organ dysfunction deemed by the attending 
physician to preclude this procedure. 

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5. Age < 35 years at time of transplant 
6. One of the following must be true: 
 
 
Patients must have evidence of persistent or recurrent 
immunodeficiency or thrombocytopenia. 
-OR- 
 
Primary immunodeficiency disease with known potential to 
progress to malignant condition if untreated. 
-OR- 
 
Debilitating secondary disease known to be a consequence of 
inadequate immune response to known agent or pathogen, 
uncontrollable by other available medical therapies (e.g. third 
patient described on page 5). 
 
2b. 
Exclusion Criteria 
 
1. Absence of an available original donor 
2. Failure to sign consent form, or inability to undergo informed consent      
 
process 
3. Pregnant or lactating female 
4. Uncontrolled GVHD 
 
3. 
Randomization 
 
Not applicable. 
 
4. 
Procedures  
 
Pre-infusion evaluation:   
 
Patients enrolled on this protocol will be evaluated for disease status and organ 
function prior to stem cell infusion, required testing as follows: 
 
1. Peripheral Donor Chimerism Studies 
2. CBC with differential and platelets 
3. Immunological studies including but not limited to absolute numbers of all 
lymphocyte subsets NK, T, B lymphocyte function, proliferation to 
mitogens, immunoglobulins 
4. Urine pregnancy tests for girls of childbearing age 
5. Renal and liver function tests 
 
 
 
Treatment Plan:  
 
 
Treatment Scheme 
 
This treatment will involve these steps: 

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1. Pre-infusion evaluation 
2. Collection of Granulocyte Colony Stimulating Factor (GCSF) mobilized 
peripheral blood stem cells from same donor used for first transplant as 
per standard procedures 
3. Selection of CD34+ stem cells and T-cell depletion using CliniMACS 
4. Infusion of stem cells into recipient 
5. Continue monitoring 
 
Schedule*:  
 
Pre-infusion 
Clinic visit and evaluation of peripheral blood counts 
 
Day  0 
Infusion of CD34+ selected stem cells 
Day  7 
Clinical assessment 
3, 6, 12 
months and 
then yearly for 
up to 3 years 
post infusion 
Clinical assessment 
Donor chimerism studies 
Immunological studies 
*For details see Required Observations, page 10. 
 
Additional testing such as disease specific markers (CD132, WASP) or more 
frequent testing might be needed at attending physician discretion if indicated by 
patient’s clinical condition. 
Preparative therapy:  
 
No preparative therapy or additional immunosuppressive therapy will be given in 
preparation to stem cell infusion. 
 
Stem Cell Infusion: 
 
Stem Cells will be administered per standard institutional protocols. 
 
Immune Suppression after stem cell infusion: 
 
No additional immune suppression will be given after stem cell infusion.  The 
stem cell product will be highly T-cell depleted, reducing risk of GVHD.  In 
addition the patient is already a mixed chimera (has both donor and own cells) 
and likely tolerant of donor cells.  Occurrence of GVHD in these settings is 
unlikely. 
 
 
Support therapy: 
 
Any infusion related reactions will be treated with supportive therapy. 

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 
Patients with fever (temperature of 38.0°C or above) will be treated in 
compliance with institution guidelines in regards to disease risk and immune 
status of the patient.  
 
Blood products may be administered according to standard of care and 
institutional guidelines for immunodeficient patients if needed for medical 
condition or prophylaxis. 
 
Patients may remain on current medication that they are taking for their 
medical problems or infection prophylaxis as per attending physician 
decision. 
 
If acute or chronic GVHD occurs, patients will be treated according to 
standard of care. 
 
 
 
Stem Cell Processing:  
 
The CD34+ cells will be separated by CliniMACs cell purification protocols which are not 
yet licensed in the US. This protocol will be submitted to the FDA under an existing IND. 
 
Peripheral blood stem cells (PBSC) with a target of ≥10x106/kg CD34+ cells will be 
requested.  All CD34+ stem cells will be infused if more than 10x106/kg CD34+ stem 
cells are collected.  The maximum number of CD34+ cells that will be infused will be 
defined by the maximum T-cell (CD3+) dose.  Stem cells will be depleted of T-cells using 
a CliniMACS CD34+ cell selection device and infused into the patient with a maximum T-
cell dose of 5 x 104/kg.  The minimum number of CD34+ cells that will be infused is 
1x106/kg. Stem cell processing will occur at Hoxworth Blood Center per standard 
operating procedures.  If T-cell depletion yields a product that does not meet the release 
parameters, the product will not be infused, although a portion of the product may be 
given if the T cell content of the entire product exceeds the specified maximum dose.  
(See the table below.)  As the patient has not received a preparative regimen, there will 
be no negative consequences. 
 
Product Release Parameters 
 
Attribute 
Test Method 
Specification 
Volume 
Measured 
≤ 20 mL/kg 
Total Cell Count 
Hemacytometer 
Report Value 
Viability 
7AAD 
≥ 80% 
% CD34+ 
Flow Cytometry 
≥ 70% 
Viable CD34+/Kg 
Flow Cytometry 
≥ 106/kg 
% CD3+ 
Flow Cytometry 
Report Value 
Viable CD3+/Kg 
Flow Cytometry 
≤5 x 104/kg 
Gram Stain 
Gram Stain 
Negative 
 
 
 
 
 
Safety of Product 
 

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Unrelated donor evaluation will be completed per the relevant unrelated donor 
registry standards (additional testing at discretion of physician and 
NMDP/registry donor center). 
 
Related donors will be evaluated per donor standard operating procedures. As 
part of this research we will collect clinical data to ensure eligibility. 
 
 
Product testing 
 
The following tests will be performed on each product produced to assure product 
safety, purity, and potency: 
 
Pre CD34 Selection (T cell Depletion):  
Total nucleated cell count 
Viability 
CD 34+ cells/kg of recipient 
CD 3+ cells/kg 
total volume collected 
sterility (bacterial and fungal culture) 
 
Post CD34 Selection (T cell Depletion): 
Total nucleated cell count 
Viability 
Viable CD 34+ cells/kg of recipient 
Viable CD 3+ cells/kg of recipient 
total volume for infusion 
endotoxin assay (Limulus Amebocyte Lysate Test or equivalent) 
stat gram stain 
sterility (bacterial and fungal culture) 
 
Culture of the product: 
If the culture of the product (either fungal or bacterial) becomes positive following 
infusion of the cells, antibiotics or antifungal medications will be administered to 
the recipient.  The patient/guardian and treating physician will be informed.  
 
Endotoxin testing: 
An endotoxin assay will be performed on the post-selection product within 24 hrs 
of infusion, and the patient’s physician will be notified if more than 5 EU/kg was 
infused into the recipient.  
 
Required Observations: 
 
1. Clinical Assessment 
An assessment of the patient will be done on Day 7 (+/- one day), at three 
months (+/- four weeks), and at or around six and twelve months. This will 
include a complete physical exam  and CBC. 
 
 
 
2. Donor chimerism studies 

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Peripheral blood donor chimerism studies will be performed only for subjects 
for whom the CD34 product has been provided for inadequate donor 
chimerism.  Chimerism studies will be performed at or around three, six and 
twelve months, then annually for up to 3 years after the stem cell infusion.  
Thereafter, chimerism studies will be performed as clinically indicated.  Donor 
chimerism analysis will be done on peripheral blood using standard FISH or 
VNTR analysis. 
 
3. Immunological studies 
 
Appropriate immunologic studies relevant to indications for CD34 infusion will 
be performed for stratum II and stratum III patients (see strata on page 6) at 
three months (+/- four weeks) and at or around six, twelve months and 
annually for up to 3 years post infusion. These tests will include, but not be 
limited to, lymphocyte subsets, T cell activation and NK function.  
 
 
Additional testing like disease specific markers (CD132, WASP) or more 
frequent testing might be needed at attending physician discretion if indicated 
by patient’s clinical condition. 
 
5. Blood Specimens 
 
 
Not applicable 
 
6. Other Previously Approved Research Studies In Which The Projected Patient 
Population May Also Be Involved (include what precautions will be taken to 
avoid any cumulative risk, e.g., repeated blood sampling, radiation exposure, 
etc.) 
  
 
 
Patients eligible for this study may be eligible for other open therapeutic studies at 
CCHMC.  Study options will be discussed and decided on a case-by-case basis. 
 
7. Data Analysis/Statistical Considerations  
 
Primary Endpoints 
The primary endpoint of the study is augmentation of graft function.  Graft function 
will be measured in two ways: firstly the percentage of donor chimerism, and 
secondly immune function.  
Successful augmentation of graft function will be achieved if: 
 
Donor chimerism is doubled compared to the value immediately pre-infusion at 3, 
6, and 12 months, -OR- 
 
Improvement in immune function studies compared to baseline levels can be 
documented at 3, 6, and 12 months.  Example of immune function studies which 
would be used would include quantitation of T and B cells and functional studies 
(mitogens and Immunoglobulin levels).  In the case of Wiskott-Aldrich syndrome, 
improvement of platelet count to >20,000/L without transfusions on 2 separate 
occasions at least three months apart will be considered successful graft 
augmentation.  Children requiring graft augmentation for a specific limited 
immune defect, e.g. inability to clear papilloma virus will also be assessed by 

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clinical response of the infection; resolution or >50% improvement in infection will 
be considered successful graft augmentation.  
 
 
Secondary Endpoints 
 
Infusional Toxicity: Infusion of stem cells could result in infusion reaction presenting 
as shortness of breath, fever, or anaphylaxis.  To reduce the risk of infusion reaction, 
CD34+ stem cells will be highly T-cell depleted.  While we have the expectation of a 
low occurrence of this toxicity, we will monitor for the frequency and characteristics of 
infusion related toxicities.    
 
Statistical Analysis 
 
We expect the frequency of adverse events in this study to be low (likely less than 
5% events).  We will therefore use all 45 patients to determine the frequency of 
adverse events as our ability to correctly define the frequency of events in any one 
stratum of 10 cases is low. 
 
Descriptive statistics will be compiled on responding and non-responding cases, 
including patient and graft characteristics that might influence response.  It is 
recognized that this portion of the analysis is exploratory due to the small sample 
size, but these data will be used to inform the design of future studies. 
                  Stopping Rules 
 
The primary endpoint of this study is qualitative graft augmentation.  Five patients will 
be enrolled into each stratum for evaluation of graft augmentation.  If none of the five 
patients per stratum show any graft augmentation, accrual to that particular stratum 
will be closed due to lack of efficacy.  Patients will continue to be enrolled into other 
strata until criteria for five patients per stratum are fulfilled.  If one out of five patients 
per stratum will meet listed criteria for graft augmentation, an additional five patients 
may be enrolled to that stratum. 
 
      Any serious unexpected adverse events (grade 4 toxicity per Common Terminology 
Criteria for Adverse Events version 3.0) will lead to suspension of study enrollment 
for determination of attribution of the event to study procedure.  The principal 
investigator will review and evaluate these events in relation to the study procedures 
and outcomes before further patients are enrolled.  Any fever occurring within twelve 
hours of the stem cell infusion requiring hospital admission will be considered an 
expected serious adverse event.  This event will require prompt reporting to the 
Institutional Review Board (IRB), but will not require suspension of the study.  In the 
event of documented graft versus host disease, grade II or above at any site, the 
study will be suspended to assess and evaluate the situation and to reconsider study 
procedures.  The IRB may determine that the study should be terminated based on 
their review of reported SAEs. 
 
 
9. Facilities Utilized In the Study 

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The stem cell infusion will be performed at Children’s Hospital of Cincinnati Medical 
Center. 
 
V. POTENTIAL BENEFITS 
 
There may be no benefit from participating in this study.  However, the information 
learned from this study may help make better the health care that is given to patients 
with primary immunodeficiency disorders in the future. 
 
This CD34+ stem cell infusion procedure may be able to increase donor chimerism, 
resulting in correction of immunodeficiency, thrombocytopenia or enzyme deficiency 
sufficient to reverse present disease or clinical condition.  If this happens, the patient will 
no longer require transfusions of platelets and have less risk of infection.  This procedure 
may also be able to decrease the risk of malignant transformations that potentially occur 
in some of untreated primary immunodeficiencies.  These outcomes would be a benefit 
to participants.  
  
 
VI. 
POTENTIAL RISKS, DISCOMFORTS, INCONVENIENCES AND PRECAUTIONS 
 
1. Known or potential risks, discomforts, or inconveniences 
 
Infusion reactions:  Infusion reactions are unlikely because of the highly purified 
nature of the product.  However there is a potential risk of reaction including fever, 
chills, anaphylaxis, rash, shortness of breath.  Transfusions also carry the risk of 
hepatitis, human immunodeficiency virus and viral pneumonia due to 
cytomegalovirus. 
 
Graft versus host disease (GVHD):  GVHD is unlikely as a low number of T-cells will 
remain after cell selection and the recipients already have stable mixed chimerism 
with cells from their donors.  Acute GVHD can cause a skin rash, liver dysfunction, 
and/or enteritis, and is potentially fatal.  Chronic GVHD is a multi-organ autoimmune 
disease, which may resolve over several months or years or lead to death, 
commonly from infection. 
 
2. Precautions that will be taken to monitor and avoid the above mentioned risks, 
discomforts, and inconveniences, including methods for detecting adverse 
events 
 
Infusion reactions: The risk of transfusion reaction will be minimized by appropriate 
testing of the donor and products, and by use of pre-medication.  Pre-medications 
will be given according to standard practice.  Patients will be monitored according to 
CCHMC guidelines during stem cell infusion for any infusion reactions.  To reduce 
the risk of product infusion, CD34+ stem cells will be highly T-cell depleted.  Should 
an infusion reaction occur it will be treated with supportive therapy according to 
standard transplant procedure.  
 
Graft versus host disease:  The risk of developing a new case of GVHD is 
anticipated to be very low.  No GVHD prophylaxis will be given.  Any occurring 

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GVHD would be treated according to accepted standards. 
 
3. The method of monitoring study conduct. 
 
All grade 3 or greater adverse events occurring during this study will be recorded on 
the case report forms.  The principal investigator will review each event and assess 
its relationship to study events as to whether the event is unrelated, unlikely related, 
possibly related, probably related, or definitely related to the study device.  The 
CTCAE Version 3.0 will be used to evaluate these adverse events.   
 
This population of patients, by nature of their various disease/conditions and due to 
effects from the original allogeneic hematopoietic stem cell transplantation (with a full 
preparative regimen), often continue to experience disease and therapy-related 
complications and are frequently hospitalized for observation and/or treatment of 
these conditions.  As these patients do not receive any preparative therapy as 
participants in this study, the risk of study-related toxicity is expected to be minimal.  
Consequently any serious adverse events, as defined by the IRB guidelines, that are 
determined to be possibly, probably or definitely related to the study device will be 
reported according to IRB reporting regulations.  However, serious adverse events 
determined to be related to the patient’s underlying disease/condition or to the first 
transplant procedure, including hospitalizations or prolongation of hospitalizations 
resulting from such events, will not require expedited reporting to the IRB.  These 
events will be reported annually with the progress report.  Please note that for the 
purpose of this study we will exclude the report of hematologic toxicities, as these 
subjects, by virtue of their underlying disease and/or condition may present with 
baseline hematologic toxicity. 
 
Although these subjects will be followed on study for up to 3 years post infusion, all 
adverse events (as described above) will be captured for the first 30 days post stem 
cell infusion.  Thereafter, only related adverse events will be recorded on case report 
forms.   
 
4. Methods for maintaining data quality and confidentiality 
 
Data quality is monitored and audited by the Center for International Blood and 
Marrow Transplant Research and the National Marrow Donor Program. 
      Confidentiality is maintained as per section VI. 
 
5. An assessment of accrual (timely enrollment) and the handling of dropouts. 
 
 
Accrual assessment will be evaluated by the principal investigator with the annual 
 
progress review.  Subjects who do not complete a full six months of follow up will not  
 
be counted in the total stratum accrual and will be replaced. 
 
 
 
6. Risk assessment recommendation 
 
 
 
Minimal risk but with direct benefit to participants 
 
 
Minimal risk but without direct benefit to participants 
 
 
More than minimal risk but with direct benefit to participants 
 
 
More than minimal risk but without direct benefit to participant 
 

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7.   Data and Safety Monitoring Plan 
 
This study does not require a DSMB or medical monitor because the patient is 
already a mixed chimera (has both donor and own cells) and likely tolerant of donor 
cells.  In addition, the patient will not receive any preparative therapy prior to stem 
cell infuse, thus decreasing the risk of toxicities by the patient. Therefore, the 
principal investigator will be responsible for monitoring the study. 
 
      The data generated during this trial will be monitored by the Principal Investigator for    
safety and compliance with protocol-specified requirements. The Principal 
Investigator will review all protocol related lab results for each patient enrolled on the 
protocol, and each patient will be examined by the Principal Investigator or the 
physician following the patient during their required follow-up visits. All planned 
testing is medically indicated for patients undergoing treatment regardless of their 
participation in this research study. The study PI and research coordinator will review 
the study progress regularly. Patients entered on the trial and adverse events will be 
reviewed to ensure that the study is implemented as outlined in the protocol.  
 
All decisions regarding study continuation, modification, or termination will be 
reported immediately or annually, as appropriate, to the IRB, FDA, and other 
appropriate agencies.  Reports for events determined by the investigator to be 
possible or definitely related to study participation and reports of events resulting in 
death should be forwarded in compliance with current IRB policy and applicable 
federal regulations. 
 
Any serious unexpected adverse events (grade 4 toxicity per Common Terminology 
Criteria for Adverse Events version 3.0) which occur during the stem cell infusion or 
within thirty days after the infusion will be expeditedly reported to and evaluated by 
the Principal Investigator, as well as the IRB (see section VI-3).  Thereafter, only 
events related to the study procedures will be captured on case report forms for each 
subject on study.   
 
 
VII. 
CONFIDENTIALITY 
 
Every effort will be made to maintain patient confidentiality.  Absolute confidentiality 
cannot be guaranteed.  Patient information may be disclosed if required by law.  The 
FDA, Hoxworth Blood Center and the Cincinnati Children’s Hospital Medical Center 
Institutional Review Board will have access to the records. 
 
All study documents will be kept in a locked cabinet in the Cancer and Blood Disease 
Institute.  Access to the files will be limited to the Principal Investigator and her 
designees only.  Study records will be maintained in the secure database that has 
restricted access to only the Principal Investigator and her designees. 
 
 
 
 

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VIII. 
PERIOD OF TIME ESTIMATED TO COMPLETE PROJECT AS DESCRIBED 
 
This protocol will remain open until the number of patients planned for accrual has been 
reached, or until the study is closed for toxicity reasons. Our target accrual is 45 cases 
on this protocol.  Estimated time for patient accrual is five years. 
 
 
IX. 
FUNDING 
  
1. Name of funding agency or private sponsors 
 
 
 
This research is conducted through the Cancer and Blood Disease Institute. 
 
2. The institution to which funding will be made 
 
 
Not applicable 
 
 
X. 
PAYMENT FOR STUDIES 
 
1. 
Third party payers 
The patient's health plan/insurance company will need to pay for all of the costs of 
treatment in this study. FDA approval for cost recovery has been granted. The 
patient will be responsible for the costs of standard medical care, all hospitalizations 
and any infusion complications. Pre-authorization for the infusion will be cleared 
with the health plan/insurance company prior to admission. 
 
2. 
Reimbursement to participants 
        Patients will not be reimbursed for participation in this study. 
 
 
XI. 
METHOD TO BE USED IN PROCURING CONSENT OF SUBJECTS 
 
All prospective patients will have the study explained by a member of the research team.  
All the potential hazards and possible adverse reactions will be explained to 
patient/parent. 
 
Prior to the initiation of the study, acknowledgement of the receipt of this information and 
the subject's freely tendered offer to participate will be obtained in writing from each 
subject in the study.  
 
Although many of the subjects on this study will have transplants utilizing matched 
unrelated donors provided and/or facilitated by the National Marrow Donor Program, this 
study also allows for human leukocyte antigen (HLA) compatible related donors.  In 
some cases, the related donor will be local; therefore preparation for the stem cell 
collection will be performed at CCHMC and/or other area facilities.   Prospective HLA 
compatible related donors will also need to provide signed informed consent before 
initiation of the collection procedures and associated clinical data. Prospective HLA 
compatible related donors will be no younger than 3 months of age at the time of 

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consent. A physician other than the patient’s attending physician will consult with 
the prospective donor to explain the purpose, procedures, risks, and benefits of the 
study and the collection procedures at the donor’s level of understanding. Opportunity 
will be given to consider the study and have questions answered. 
 
For those patients and families for which English is not their primary language, the short 
form consent process will be utilized at CCHMC as per CCHMC Standard Operating 
Procedures.  The consent will be administered orally by an appropriate member of the 
research team with translation by an interpreter, with time for questions and clarification. 
The interpreter will be provided with the long form version of the consent that is specific 
to the participant being consented. 
 
 
This protocol, informed consent, and any amendments to the protocol will be reviewed 
and approved by the CHMCC IRB prior to initiation.  The study will not be initiated 
without the approval of the IRB. 
 
 
XII.  
MAINTENANCE OF PROTOCOL RECORDS 
 
All study documents will be kept in a locked cabinet in the Hematology/Oncology Clinical 
Research Office.  Access to the files will be limited to the Principal Investigator and his 
designees only.  Study data will be maintained in the secure database that has restricted 
access to only the Principal Investigator and his designees.  Processing records for the 
T cell depletion procedure will be maintained at Hoxworth Blood Center. 
 
XIV. 
CRC (protocol review required by CRC Scientific Advisory Committee) for 
protocols which utilize the CRC) 
 
The CRC will not be utilized for this research study. 
 
 
XV. 
PROTOCOL REVISIONS 
 
Any changes to the protocol or informed consent document will be submitted to the 
CCHMC IRB for approval prior to the implementation of the changes. 
 
 
XVI. 
CONTINUING REVIEW 
 
This study will undergo an annual review with all requested information provided to the 
CCHMC IRB. 
 
 
 
 
 
XVII.  REFERENCES 
 

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1. Buckley RH. Treatment options for genetically determined immunodeficiency. 
Lancet. 2003;361:541-542. 
2. Antoine C, Muller S, Cant A, et al. Long-term survival and transplantation of 
haemopoietic stem cells for immunodeficiencies: report of the European experience 
1968-99. Lancet. 2003;361:553-560. 
3. Filipovich AH, Stone JV, Tomany SC, et al. Impact of donor type on outcome of bone 
marrow transplantation for Wiskott-Aldrich syndrome: collaborative study of the 
International Bone Marrow Transplant Registry and the National Marrow Donor 
Program. Blood. 2001;97:1598-1603. 
4. Marmont AM, Horowitz MM, Gale RP, et al. T-cell depletion of HLA-identical 
transplants in leukemia. Blood. 1991;78:2120-2130. 
5. van Leeuwen JE, van Tol MJ, Joosten AM, et al. Relationship between patterns of 
engraftment in peripheral blood and immune reconstitution after allogeneic bone 
marrow transplantation for (severe) combined immunodeficiency. Blood. 
1994;84:3936-3947. 
6. Haddad E, Le Deist F, Blanche S, et al. Treatment of Chediak-Higashi syndrome by 
allogenic bone marrow transplantation: report of 10 cases. Blood. 1995;85:3328-
3333. 
7. Yamazaki S, Takahashi H, Fujii H, et al. Split chimerism after allogeneic bone 
marrow transplantation in Chediak-Higashi syndrome. Bone Marrow Transplant. 
2003;31:137-140. 
8. Brunel V, Mozziconacci MJ, Sainty D, et al. Direct evidence for dissociated 
megakaryocytic chimaerism in a Wiskott-Aldrich patient successfully allografted. Br J 
Haematol. 1995;90:336-340. 
9. Neudorf S. New York Plenum Press (Press Release). 1984. 
10. Derry JM, Ochs HD, Francke U. Isolation of a novel gene mutated in Wiskott-Aldrich 
syndrome. Cell. 1994;78:635-644. 
11. Ozsahin H, Le Deist F, Benkerrou M, et al. Bone marrow transplantation in 26 
patients with Wiskott-Aldrich syndrome from a single center. J Pediatr. 
1996;129:238-244. 
12. Gluckman E. Bone marrow transplantation in children with hereditary disorders. Curr 
Opin Pediatr. 1996;8:42-44. 
13. Lang P, Klingebiel T, Schumm M, et al. Correction of persistent thrombocytopenia by 
a boost of CD133+ selected stem cells in a patient transplanted for Wiskott-Aldrich 
syndrome 10 years ago. Bone Marrow Transplant. 2004;33:879-880. 
14. 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. 2005 
April;35(7):683-9