A Two Step Approach to Reduced Intensity Allogeneic Hematopoietic Stem Cell Transplantation for High Risk Hematologic Malignancies
Phase: PHASE2
Conditions: Acute Myeloid Leukemia With FLT3/ITD Mutation, Acute Myeloid Leukemia With Gene Mutations, Acute Myeloid Leukemia With Inv(3) (q21.3;q26.2); GATA2, MECOM, Acute Myeloid Leukemia With t(6;9) (p23;q34.1); DEK-NUP214, Aplastic Anemia, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, BCR-ABL1 Positive, Chronic Myelomonocytic Leukemia, Follicular Lymphoma, Hodgkin Lymphoma, Myelodysplastic Syndrome, Myelofibrosis, Myeloid Leukemia, Non-Hodgkin Lymphoma, Plasma Cell Myeloma, Polycythemia Vera, Recurrent Adult Acute Myeloid Leukemia, Recurrent Childhood Acute Myeloid Leukemia, Refractory Acute Myeloid Leukemia, Secondary Acute Myeloid Leukemia, Therapy-Related Acute Myeloid Leukemia
Sponsor: Sidney Kimmel Comprehensive Cancer Center at Thomas Jefferson University

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
1
 
 
 
A Two Step Approach to Reduced Intensity Allogeneic Hematopoietic Stem Cell 
Transplantation for High Risk Hematologic Malignancies  
 
 
 
Principal Investigator 
Dolores Grosso, DNP, CRNP  
 
Co-Principal Investigator 
Usama Gergis, MD 
 
Co-Investigators 
S. Onder Alpdogan, MD 
Matthew Carabasi, MD 
 
Joanne Filicko-O’Hara, MD 
Margaret Kasner, MD 
William O’Hara, PharmD 
Ubaldo Martinez-Outschoorn, MD 
John Wagner, MD 
Neil Palmisiano, MD 
Pierluigi Porcu, MD 
 
Biostatistics 
Inna Chervoneva, PhD 
 
Radiation Oncology 
Wenyin Shi,MD 
 
Thomas Jefferson University (TJU) 
Blood and Marrow Transplantation Program 
Division of Medical Oncology, Department of Medicine 
Suite 320, 834 Chestnut Street 
Philadelphia, PA 19107 
 
 
 
 
 
 
 

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
2
 
 
CONTENTS 
 
 
 
1.0      Objectives  
 
 
 
 
 
 
p  3 
 
2.0  
Introduction and Rationale 
 
 
 
 
p  3 
 
3.0      Patient and Donor Selection 
 
 
 
 
p 16 
 
4.0     Informed Consent  
 
 
 
 
 
p 19 
 
5.0  
Treatment Plan 
 
 
 
 
 
 
p 20 
 
6.0  
Study Measurements 
 
 
 
 
 
p 27 
 
7.0  
Supportive Care 
 
 
 
 
 
 
p 30 
 
8.0  
Drug Information  
 
 
 
 
 
p 32 
 
9.0  
Patient Safety 
 
 
 
 
 
 
p 37 
 
10.0 Statistical Analysis 
 
 
 
 
 
p 38 
 
11.0    References 
 
 
p 40  
 
12.0 Appendices  
 
 
 
 
 
 
p 48 
 
 

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
3
 
 
1.0    
Objectives 
 
Thirty-four patients in the TJU Blood and Marrow Transplantation Program were 
treated on the research protocol, A Two Step Approach To Reduced Intensity 
Allogeneic Hematopoietic Stem Cell Transplantation for Hematologic Malignancies 
from HLA Partially-Matched Related Donors (TJU 2 Step RIC, IRB # 06U.328), 
closed to enrollment as of December, 2010. While treatment on this protocol has 
resulted in durable responses for many older patients and younger, heavily 
pretreated patients with hematological malignancies, relapse-related mortality 
after hematopoietic stem cell transplantation (HSCT) was the major cause of 
mortality in this trial, primarily affecting those patients with disease at the time of 
transplant.  
 
The objective of this study is to decrease post HSCT relapse rates in patients with 
high risk hematological diseases characterized by the presence of disease at the 
time of HSCT or by high risk features such as adverse cytogenetics. A strategy of 
immunological reduction of disease within the conditioning regimen will be 
employed to achieve this end. The specific objectives are: 
 
Primary Objective 
 
1.   
To compare the rate of disease-free survival (DFS) at 1 year post HSCT in 
patients undergoing HSCT treated on this successor TJU 2 Step RIC 
haploidentical regimen with that of the initial 2 Step RIC regimen  
 
Secondary Objectives 
 
2.  
To assess the 100 day regimen-related mortality (RRM) in patients 
undergoing HSCT on this treatment protocol.  
 
3. 
To determine the incidence and severity of graft-versus-host disease 
(GVHD) in patients undergoing treatment on this regimen  
 
4.  
To evaluate engraftment rates and lymphoid reconstitution in patients 
treated on this trial. 
 
5. 
To assess overall survival at 1 and 3 years past HSCT in patients treated 
on this trial. 
 
 
2.0  
Introduction and Rationale 
 
Allogeneic 
HSCT 
is 
a 
curative 
therapy 
for 
many 
disorders 
of 
lymphohematopoiesis.1-5  While allogeneic transplants are often associated with 
lower rates of relapse than autografts or conventional dose treatment, this 

 
 
 
12D.501 Protocol v5.2 
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4
 
 
advantage is partially offset by higher regimen related mortality.6-24 Much of this 
increase can be traced to the toxicities of the conditioning regimen, GVHD, and 
the immunosuppressive measures required for the prevention and/or treatment of 
GVHD.24-30  For years it has been understood that conditioning regimen intensity 
is not the primary basis of long-term disease control after allogeneic HSCT.  Donor 
lymphocytes can also eradicate host tumor cells through graft versus tumor (GVT) 
effects, even in the absence of overt GVHD.31 In AML and CML for example, 
relapse rates are higher in recipients of twin transplants than in GVH-free 
recipients of matched sibling grafts.32  Moreover, in CML, many patients who 
relapse after BMT can be rendered disease free through the infusion of additional 
lymphocytes from the marrow donor without any additional chemoradiotherapy.33-
37  
 
Development of less intensive conditioning regimens 
 
With the recognition that GVT effects, not conditioning regimen intensity are 
responsible for long term control of many disease states, transplant regimens that 
are not lethally myeloablative (NM HSCT) have been developed over the last 
decade.  These approaches do not rely dose intensity to eradicate malignancy. 
Rather they use immunosuppressive agents, irrespective of their anti-neoplastic 
properties, to facilitate donor lymphoid and stem cell engraftment.  The donor 
lymphoid elements then destroy the residual normal and in some cases malignant 
lymphohematopoietic elements allowing the transition to donor chimerism.  These 
regimens rely less heavily on the conditioning regimen for disease control by 
exploiting the graft versus tumor effects of the donor immune system.  They are 
associated with less treatment-related mortality38 and have allowed older and 
heavily pretreated patients who otherwise would not tolerate the rigors of a fully 
myeloablative HSCT, to undergo transplant successfully.39  Nonmyeloablative 
(NM) HSCT has been dramatically effective in CML, chronic lymphocytic leukemia 
(CLL), and follicular lymphoma in its original application and has proven efficacious 
in other diseases as well.40-43  
 
A spectrum of intensity exists among the different NM regimens. Many of the non-
myeloablative regimens are minimally myelosuppressive, while others are more 
immunosuppressive and are associated with prompter engraftment of donor cells 
than their less intensive counterparts.  The former approach has been associated 
with less TRM but with incomplete initial chimerism and increased rates of 
relapse.44-46  The latter approach, alternately referred to as “reduced intensity” 
(RIC) HSCT, has been associated with more TRM but less relapse.47, 48  A NM 
HSCT approach that is not an “either/or” proposition has not been clearly identified.  
 
Approaches to Alternative Donor Reduced Intensity Allogeneic HSCT 
A barrier to the application of NM transplantation in hematologic malignancies is 
the availability of donors. Only 30% of patients in North America or Europe who 
may benefit from allogeneic HSCT will have an HLA-matched sibling donor. This 
percentage is lower for the older patient who may benefit from NM HSCT, but 

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
5
 
 
whose HLA-identical sibling donor may be too old or have comorbidities that would 
preclude their use as a donor.  Registries can provide a matched unrelated 
allogeneic stem cell graft for an additional 30 % of patients. However this is not an 
option for patients who do not have a match in the registry, or whose disease status 
precludes them from waiting to identify an appropriate unrelated donor. In this 
setting, it is easier and faster to identify a partially HLA-matched (haploidentical) 
family member as a stem cell donor. There is little data regarding the use of 
reduced intensity approaches in haploidentical HSCT, although a few recent trials 
have formed the basis for a growing application of this therapeutic modality.     
 
RIC Approaches in Haploidentical HSCT 
Ciurea et al.49 treated 22 patients (median age 36 years) with AML/MDS on a RIC 
T-cell depleted HSCT regimen consisting of fludarabine, melphalan, thiotepa and 
antithymocyte globulin (ATG) followed by mobilized stem cells from a 
haploidentical donor (>2 antigen mismatch). While no patients with advanced 
disease at transplant survived, 5 of 12 (42%) with better risk disease have become 
long-term survivors. NRM at 100 days in this very young sample group was 18%.  
Bethge and colleagues50 infused a CD3/CD19 depleted peripheral blood product 
from haploidentical donors into 29 patients (median age 42 years), primarily with 
acute leukemia, after conditioning with fludarabine, thiotepa, melphalan, and OKT-
3.  Overall survival was 31%, with 7 deaths due to infection, 1 from GVHD, and 12 
due to relapse, median follow-up of 241 days (range, 112–1271). The NRM at day 
100 for this younger aged population was 20%.  Dodero et al.51 conditioned 28 
patients (median age 38 years) primarily with advanced lymphoproliferative 
disorders with thiotepa, fludarabine, cyclophosphamide, and 2 Gy total body 
irradiation followed by an infusion of a T cell depleted stem cell product from a 
haploidentical donor.  After transplant, the patients were given CD 8+ depleted T 
cells from their donors to support immune reconstitution. Six of 28 patients (21%) 
died of NRM at a median of 250 days post HSCT. The 2-year cumulative incidence 
of nonrelapse mortality was 26% and the 2-year overall survival (OS) was 44%, 
with a better outcome for patients with chemosensitive disease (OS of 75%).  
These trials are proof of principal that RIC haploidentical HSCT is a feasible 
approach to transplant and when applied to patients with earlier stage disease 
results in better survival rates.   
 
T-Cell Depletion in Haploidentical HSCT 
Early transplant approaches utilizing haploidentical donors employed soy bean 
agglutinin, E-rosetting and later T10B9 to deplete the donor product of T cells to 
avoid the catastrophic GVHD which would have occurred as a result of crossing 
the major histocompatibility barrier in this type of HSCT. Transplantation with 
products T cell depleted to this degree is associated with post-transplant 
immunodeficiency52-55 and mortality from infection and relapse.56-63  More recently, 
techniques have been developed to avoid profound T cell depletion of the donor 
product in order to avoid the negative consequences associated with this type of 
approach.   

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
6
 
 
 
In two of the RIC trials discussed on page 5, strategies to attenuate rather than 
completely deplete T cells were employed to augment immune reconstitution post 
HSCT and resulted in highly acceptable rates of GVHD and infectious mortality. 
Another successful methodology used to avoid T cell depletion in RIC 
haploidentical HSCT was developed by the transplant group at Hopkins who 
employed cyclophosphamide (CY) both as part of the conditioning regimen and to 
tolerize T-cells for GVHD prophylaxis.  
 
The Use of Cyclophosphamide (CY) for T cell Tolerization in Haploidentical 
NM HSCT 
One of the earliest human NM trials using haploidentical donors and CY 
tolerization was conducted by O’Donnell and colleagues and published in 2002.64 
In this less intensive NM regimen, ten patients were treated with CY, total body 
irradiation and fludarabine.  In the second group of treated patients, CY was given 
before transplant as part of the conditioning regimen and after the infusion of the 
donor product to establish bidirectional T cell tolerization and avoid rejection.  
Mycophenolate mofetil and tacrolimus were added on day 4. Two of the ten 
patients rejected the graft.  At the time of the publication, 6/8 patients were alive 
and 4/8 were without evidence of disease (range 157-423 days).  Of the 4 without 
evidence of disease, 2 had grade II GVHD, and 2 had no GVHD at all.  The authors 
reported excellent immune reconstitution, and the primary causes of treatment 
failure were rejection and relapsed disease.  A later publication by the same 
group65 reported on outcomes of a larger sample size of 67 patients with advanced 
hematological malignancies treated with this same approach. Again, mortality from 
GVHD and infection was very low at 3% and 6% respectively. The primary cause 
of treatment failure was relapsed disease. Interestingly, patients with lymphoid 
disease fared better in terms of event free survival as did a subgroup of patients 
with Hodgkin disease analyzed separately.66 These trials demonstrated the 
efficacy of CY tolerization for avoiding significant rates of GVHD and infection post 
haploidentical HSCT. Relapsed disease, whether due to the CY tolerization 
combined with a NM conditioning regimen or the late stage of disease of the 
patients treated or both, was a significant barrier to long term survival.   
 
Jefferson 2 Step Approach to RIC HSCT 
Based on murine studies and human clinical trials with CY, as well as promising 
results from our myeloablative trial using CY tolerization, we developed an RIC 
approach to haploidentical HSCT. The conditioning regimen is better characterized 
as a reduced intensity one in that the chemoradiotherapy is more intensive than 
the approach initially developed at Hopkins. We reasoned that because the 
patients being treated at our institution were primarily older or more heavily 
pretreated, they required a higher degree of regimen intensity to control the 
resistant diseases associated with this type of patient population. 
 

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
7
 
 
 
A Two Step Approach to Reduced Intensity Allogeneic Hematopoietic Stem Cell 
Transplantation for Hematologic Malignancies from HLA Partially-Matched 
Related Donors (TJU 2 Step RIC, IRB # 06U.328) began enrollment in 2006 and 
met its accrual goal in 2010.  This protocol was specifically designed to treat older 
patients (> 66 years) or patients who underwent previous transplants and could 
not tolerate the rigors of a second myeloablative conditioning regimen.  In this 2 
step transplant regimen, patients received fludarabine and either ARA-C or 
Thiotepa for 4 days.  The patients then received one day of TBI (2 Gy), followed 
by an exact dose of 2.0 x 10e8 of their donor’s T cells (step 1 of the transplant).  
After the donor T cell infusion (DLI) the patients consistently developed fevers and 
in many cases a skin rash and diarrhea, consistent with an immune reaction.  On 
the 3rd day after the DLI the patients received the first of two daily doses of CY in 
order to tolerize the reactive lymphocytes. Twenty-four hours after the last dose of 
CY, the patients received a stem cell product from their donor (step 2 of the 
transplant).  The conditioning regimen is shown in Table 1. 
 
Table 1 
2 Step Reduced Intensity HSCT-Conditioning Regimen  
 
In general, patients received Thiotepa if there was evidence of an active 
malignancy where therapy with an alkylating agent was thought to be more 
beneficial than therapy with an anti-metabolite.  We considered this especially 
important in our patient population which contains a preponderance of patients 
with acute leukemia who have already received front-line therapy with ARA-C.  
ARA-C was given to patients who did not have evidence of disease at the time of 
HSCT or had diseases in which ARA-C is thought to be beneficial such as CLL or 
follicular NHL. Therefore, the conditioning regimen was developed in this manner 
to allow for patient-specific adjustments within a consistent regimen.  Patients were 
treated with ARA-C versus Thiotepa based on defined programmatic guidelines 
which were part of a greater policy governing patient placement on the various TJU 
2 Step protocols.  
 
 
Fri 
-12 
Sat 
-11 
Sun 
-10 
Mon 
-9 
Tues 
-8 
Wed 
-7 
Thur 
-6 
Fri 
-5 
Sat 
-4 
Sun 
-3 
Mon 
-2 
Tues 
-1 
Wed 
0 
A
M 
Admit 
Fludara 
30  
mg/m2 
Fludara 
30  
mg/m2 
Fludara 
30  
mg/m2 
Fludara 
30  
mg/m2 
Rest 
TBI 
2 Gy 
Rest 
Rest 
CY 
60 
mg/k
g 
CY 
 60 
mg/
kg 
Rest 
 
CD- 
34+ 
PBSC 
Infu-
sion 
P
M 
 
Ara-C 
2 
gm/m2 
 
OR 
Thiotep
a 
5 mg/kg 
Ara-C 
2 
gm/m2 
 
OR 
Thiotep
a 
5 mg/kg 
Ara-C 
2 
gm/m2 
 
OR 
Thiotep
a 
5 mg/kg 
Ara-C 
2 
gm/m2 
 
OR 
No 
Chemo-
therapy 
 
DLI 
 
 
 
 
Start 
FK 
506 
& 
MMF 
→ 

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
10
 
 
was performed by a third party and not the primary team.  This “modified KPS” was 
stringently applied during the latter half of the trial resulting in the selection of 
patients who were better able to tolerate the rigors of HSCT. Consequently, the 
application of this screening resulted in a decrease in mortality related to toxicity 
such that by the end of the trial, relapsed disease and not toxicity was the major 
cause of death. (See Figure 2). In addition, assessment of organ function based 
on the hematopoietic cell transplant comorbidity index (HCT CI)69 scores was 
added although this assessment has not yet proven to be as predictive as the KPS, 
at least for older patients, in the original trial.   
 
Based on these adjustments, a second generation RIC 2 Step HSCT regimen was 
opened in 2011 with an identical conditioning regimen to that of the initial trial (TJU 
IRB #11D.247). The primary difference in the trials is that only patients with better 
risk disease meeting stringent KPS and HCT CI criteria are eligible. The scientific 
aim of the successor trial is to demonstrate that despite older age, patients in good 
health with responsive disease can be safely transplanted using the 2 Step 
approach. Of 10 patients who have undergone haploidentical HSCT on this 
successor trial (1 to 12 months follow-up), no patients have died, reflecting a 
dramatic improvement in toxicity based on only more targeted screening and 
earlier treatment with HSCT prior to the development of comorbid conditions or 
resistant disease. 
 
Unfortunately, a large group of patients who could potentially benefit from RIC 
HSCT in the initial trial had resistant diseases at HSCT. This group includes 
patients with high-risk diseases associated with aging.70  To address this issue, an 
additional second generation 2 Step RIC was opened for patients with disease at 
HSCT with the strategy of substituting Melphalan (Mel) for CY to tolerize T cells. It 
was hypothesized that Mel would be less cardiotoxic in older patients and for many 
diseases, have more anti-tumor activity. The use of Mel in conditioning regimens 
have been associated with excellent rates of DFS in MDS, AML and AML arising 
from MDS, secondary leukemia,71-77  myelofibrosis with transformation to acute 
leukemia,49  CML,78   ALL,79-81 high-risk CLL,82  Hodgkin Disease,83  and NHL.84-87  
In 2010, A Two Step Approach to Reduced Intensity Allogeneic Hematopoietic 
Stem Cell Transplantation for Hematologic Malignancies Using Melphalan for T 
Cell Tolerization was opened (TJU IRB #10D.535). This phase I/II trial utilized a 
duplicate 
conditioning 
regimen 
to 
the 
initial 
2 
Step 
RIC 
approach 
(fluadarabine/thiotepa/TBI), except for a substitution of Mel 70 mg/m2/day x 2 days 
for CY 60 mg.kg/day x 2 days for T cell tolerization. The dose of Mel was to be 
optimized based on outcomes in the phase I part of the trial. Eight patients were 
treated prior to its closure in mid-2012 for excessive toxicity. The initial Mel dose 
of 70 mg/m2/day x2 resulted in acceptable rates of GVHD and robust immune 
reconstitution. Unfortunately, there was an increased incidence of gut and hepatic 
toxicity at this dose. Based on these findings, the dose of Mel was decreased to 
60 mg/ m2/day x2. In addition, cytarabine was substituted for thiotepa in the 
condition regimen to prevent the additive effects of two alkylating agents. Both 

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
11
 
 
patients treated on this updated regimen experienced excessive gut toxicity AND 
grade 4 GVHD and the protocol was closed.  Because higher doses of Mel are 
routinely used in autologous HSCT without this high grade of toxicity, it was 
hypothesized that the proximity of Mel to the cytokine storm associated with the 
alloreaction was primarily responsible for the high degree of gut injury, although 
the substitution of cytarabine for thiotepa successfully resulted in much less 
hepatic toxicity in these two patients. The trial was ultimately closed because the 
higher dose of Mel required to tolerize T cells and the lower dose of Mel required 
to avoid excessive toxicity were mutually exclusive. Table 3 summarizes the 
outcomes of patients on this trial. To date 33% of the patients undergoing 
haploidentical HSCT on this trial are alive without evidence of disease. This figure 
is close to the DFS of the initial trial and therefore does not represent improvement, 
especially in the context of the short follow-up time (2-12 months). 
 
Table 3 
Patient 
Melphalan 
Dose 
Donor 
Source 
100 Day 
Grades 4-5 
Regimen 
Related 
Toxicity 
100 Day  
Grade 3-4 
GVHD 
Outcome 
#1  
70 mg/m2 
Matched 
Related  
No 
No 
Alive and Well 
1 year post HSCT 
#2  
70 mg/m2 
Haplo 
Related 
* Grade 4 
Respiratory 
Failure 
No 
Alive and Well 
10 months post 
HSCT 
#3  
70 mg/m2 
Haplo 
Related 
No 
No 
Alive and Well 8 
Months post HSCT 
(In Rehab) 
#4  
70 mg/m2 
Haplo 
Related 
Not evaluable 
Not 
evaluable 
Died early in 
course from 
neutropenic sepsis 
#5  
70 mg/m2 
Haplo 
Related 
*Grade 4 Gut 
Toxicity 
No 
Alive 6 months 
after HSCT, 
recently readmitted 
for pneumonia and 
possible drug 
toxicity 
Melphalan Dose Reduced Based on Two Grade 4 CTCAE Toxicities 
#6  
60 mg/m2 
Matched 
Related 
No  
(81 days after 
HSCT Bili rose 
to grade 4 
levels not 2° 
regimen) 
Grade 4 
Gut 
Died of gram 
negative sepsis 95 
days after HSCT 
Remove 1 Alkylator from Regimen  
#7  
60 mg/m2 
Haplo 
Related 
*Grade 4 Gut 
Toxicity 
Grade 4 
Gut 
Died 2 months after 
HSCT of infection 

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
12
 
 
# 8  
60 mg/m2 
Haplo 
Related 
*Grade 4 Gut 
Toxicity 
Grade 4 
Gut 
2 months after 
HSCT and stable, 
being treated for 
gut GVHD 
 
 
While agents that are preferentially cytotoxic to activated T cells, such as thiotepa 
could also be substituted for CY in the 2 Step RIC approach to increase anti-tumor 
activity, little data exists regarding appropriate dosing of these other agents in the 
context of T cell tolerization. Therefore, other strategies are needed to decrease 
relapse rates in RIC HSCT for high risk patients. To address this issue, we 
hypothesized that immunological reduction of tumor burden during conditioning 
may represent a potent new strategy to address this issue. 
 
Immunological Tumor Reduction at the Time of Conditioning 
After treatment with 2 x 108/kg T cells in the DLI, all patients undergoing 
haploidentical HSCT on the TJU 2 Step approach experience high fevers, and in 
many cases rash and diarrhea, within 24 hours of the infusion which universally 
abates after the second dose of CY or Mel. This “alloreaction” is essentially an in-
vivo mixed lymphocyte reaction with symptoms consistent with those reported by 
Colvin et al.88 who demonstrated antitumor effects in patients receiving 
haploidentical T cells at the same doses used in the 2 Step trial, but who were 
without long-term engraftment. Skin and gut biopsies performed in two different 
patients at the time of the alloreaction were consistent with GVHD in the initial 
myeloablative TJU 2 Step trial, contrary to the lack of evidence for GVHD in the 
Colvin et al. study. Whether this difference is due to histopathological findings that 
differ in a setting of engraftment versus rejection, or the avoidance of type 2 
polarization of T cells in the 2 Step approach versus the Colvin et al. approach is 
not known. However, based on our finding of graft versus host responses and the 
association of anti-tumor effects with haploidentical DLI in the Colvin et al. study, 
we hypothesize that the post DLI alloreaction may enhance the GVT effects of the 
regimen and furthermore, that the timing of the alloreaction could be optimized to 
increase these GVT effects.  
 
Hypothesis for the Current 2nd GenerationTrial-Immunological Reduction of 
Tumor During Conditioning Through Optimization of DLI Timing for Patients 
with High-Risk Disease at HSCT 
A potentially important difference between remission and relapsed patients 
undergoing transplant is the percentage of GVT versus GVH reactive T cells that 
are likely to be rapidly activated in vivo.  In both remission and relapsed patients, 
the majority of GVH reactive T cells are likely to encounter an antigen presenting 
cell capable of activating them, thus rendering them more susceptible to the 
tolerogenic effects of CY.  In the remission patient, with a small tumor burden, 
many GVT reactive T cells may not encounter a tumor target in the first few days 
after infusion and therefore will potentially avoid activation and tolerization by CY.  

 
 
 
12D.501 Protocol v5.2 
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13
 
 
This creates a preferential reduction in GVH versus GVT when CY is administered.  
As the tumor burden progressively increases, more and more GVT reactive T cells 
will encounter tumor cells during the first few days after infusion, thus becoming 
activated and subsequently eliminated by CY as well.  The larger the tumor burden 
at the time of lymphocyte administration, the more the potential of CY to blunt the 
GVT effect, ultimately eliminating the differential impact compared to GVHD which 
may occur in remission patients. This phenomenon is diagramed in Figure 3.  
 

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
15
 
 
Figure 4 
* Counts are AM counts before that day’s therapy 
** Counts for ALL patients stayed at 0.0 or 0.1 until engraftment about 10 days later.   
 
Therefore, in this current, 2nd generation trial, the DLI is given on the 4th day after the TBI at 
the time of the immune system nadir. This timing difference will potentially result in a 
decrease in the quantity of tumor cells available to react with GVT reactive T cells. Therefore, 
there will be less activated GVT reactive T cells available for elimination by CY. Ten patients 
have been treated to date on this 2nd generation protocol.  In Figure 5, the median white cell 
counts of these 10 patients have been added to the schema to demonstrate that the DLI is 
now successfully given closer to the immune system nadir.  
 
Figure 5 
* Counts are AM counts before that day’s therapy 
** Counts for ALL patients stayed at 0.0 or 0.1 until engraftment about 10 days later.   
 
The median age of the 10 patients treated to date on the current, 2nd generation 
trial is 60 years old, and all have had resistant disease (AML-4, MDS-2, Hodgkin-
1, NHL-1, Ph+ALL-1, Myeloma-1) at the time of HSCT. While follow-up time on 
this protocol is very short, we are pleased with the probability of OS rate thus far 
 
-11 
-10 
-9 
-8 
-7 
-6 
-5 
-4 
-3 
-2 
-1 
0 
A
M 
Fludara 
30  
mg/m2 
Fludara 
30  
mg/m2 
Fludara 
30  
mg/m2 
Fludara 
30  
mg/m2 
Rest 
TBI 
2 Gy 
Rest 
Rest 
CY 
60 
mg/kg 
CY 
 60 
mg/kg 
Rest 
 
CD- 
34+ 
PBSC 
Infu-
sion 
P
M 
Thiotepa 
5 mg/kg 
Thiotepa 
5 mg/kg 
Thiotepa 
5 mg/kg 
 
 
DLI 
↓ 
 
 
 
 
Start 
FK 
506 
& 
MMF 
→ 
Median White Cell Counts of 26 Patients Treated To Date 
 
3.95 
3.95 
2.5 
2.05 
1.45 
0.85 
0.6 
0.25 0.1 
0.1 
** 
** 
 
 
-15 
 
-14 
 
-13 
 
-12 
 
-11 
 
-10 
 
-9 
 
-8 
 
-7 
 
-6 
 
-5 & 
-4 
 
-3 
 
-2 
 
-1 
 
0 
A
M 
Fludar 
30  
mg/m2 
Fludar 
30  
mg/m2 
Fludar 
30  
mg/m2 
Fludar 
30  
mg/m2 
Rest 
TBI 
2 Gy 
Rest 
Rest 
Rest 
 
Rest 
 
CY 
60 
mg/kg 
CY  
60 
mg/kg 
 
Stem 
Cell 
Infus 
P
M 
Thio 
5  
mg/kg 
Thio 
5 
mg/kg 
Thio 
5  
mg/kg 
 
 
 
 
 
 
DLI 
↓ 
 
 
 
 
→ 
Median White Cell Counts of 10 Patients Treated To Date* 
 
4.9 
3.95 
3.2 
2.25 
1.95 
0.95 
0.7 0.4 
0.2 0.05 
** 
** 
 
 
 

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
16
 
 
which is 77% in this older, higher risk population. There has been 1 death from 
relapse and 1 death from toxicity.  
 
April 19, 2014 Update 
 
The Substitution of Alkylating Agents 
Thiotepa has been used in the 2 step RIC approach for approximately 5 years 
and in cancer chemotherapy for over 50 years. It was designated as an orphan 
drug in 2007, and a critical shortage of Thiotepa was identified in 2013, 89 
originating from market forces which have affected many oncology medications.90 
The drug is no longer manufactured in the United States91 and purchasing the 
drug from overseas has been associated with increasing cost.92 Therefore, the 
current expense of the drug makes its use in HSCT at TJUH no longer feasible, 
and a drug substitution in the regimen is required. The toxicities of the regimen to 
date have been minimal, allowing the assessment of the primary scientific 
question: Do the extra days in the regimen result in greater control of malignancy 
and by extension disease free survival? Therefore, when substituting for 
Thiotepa, the goal is to come as close as possible to the original regimen as both 
the toxicity rate and the ability to deliver the DLI at the immune system nadir 
have been achieved, at least in the first 10 patients treated on this study. 
Towards that end, we will use Busulfan 3.2 mg/m2 IV daily x 2 days. This drug 
has been used in HSCT for over 30 years and is commonly used in RIC HSCT.93-
95 The majority of patients being enrolled on this trial will have AML or MDS. The 
IV formulation of the drug was compared to total body irradiation for conditioning 
in patients with AML and found to have excellent activity in the disease.96 A 
comprehensive review of recent data regarding Busulfan in HSCT was written by 
Champlin in 201397  and supports the efficacy of the drug in this setting. It serves 
as a reasonable substitute for Thiotepa because like Thiotepa, its efficacy in 
hematopoietic diseases and HSCT, as well as its side effect profile are well 
known. Because there are only two low doses of Busulfan will be used in this 
regimen, dosing based pharmacokinetic analysis is not necessary.  
 
We will use the dose of Busulfan that is most commonly used with Fludarabine in 
other RIC regimens. Typically, 3-4 doses of Busulfan are administered in these 
approaches.98-101  CY is an additional alkylating drug used in the 2 step regimen 
to further treat malignancy and tolerize lymphocytes.  Other regimens using 
Busulfan do not typically contain a second alkylator.  Therefore, we will 
administer only 2 doses of Busulfan.  Thiotepa is an alkylating agent that was 
successfully paired with CY, and so this will be a class for class substitution.  
   
In the proposed approach, patients will receive Fludarabine, Busulfan, and TBI 
on a similar schedule to that of the patients treated on the 2 step trials using 
Thiotepa. Therefore, based on the median white counts of the patients on the 
approach using Thiotepa in the conditioning regimen, it is estimated that the 

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
17
 
 
median WBC will be 0.05 at the time of the T cell infusion in the proposed trial 
using Busulfan.  See Table 6. 
 
 
 
 
 
Table 6 
 
The updated regimen is shown is section 5.0. 
 
The earlier administration of the conditioning regimen does not affect the principles 
of T cell tolerization as outlined by Mayumi et al. in the “cells-followed-by-CY 
system,” 102-104 in which CY is given 1 to 3 days after antigenic stimulation with 
allogeneic cells resulting in the preferential bidirectional destruction of alloreactive 
clones leading to T cell tolerization. In this protocol as in all of the other 2 Step 
approaches, the CY will be given two days after allogeneic stimulation with the DLI 
satisfying the requirement for alloantigenicity to establish tolerization.   
 
In addition to offering this therapy to patients with disease at the time of HSCT who 
have haploidentical donor options, two other groups of patients will be eligible for 
this trial.  
 
Patients with high-risk disease, without evidence of disease at the time of HSCT, 
who have haploidentical donors and would benefit from a deeper reduction of 
tumor undetectable by current methodology, will be offered therapy on this high-
risk protocol. In addition, the historical number of patients with available matched 
sibling donors presenting for RIC HSCT at our institution is very small precluding 
our ability to develop effective clinical trials for this group.  Thus, patients with 
matched sibling donors who have evidence of disease at HSCT or who have high-
risk disease and would potentially benefit from a deeper reduction of tumor, 
undetectable by current methodology will be offered therapy on this high-risk 
protocol. In this matched related donor group, alloreactivity of T cells is potentially 
based on minor histocompatibility differences between donor and recipient.  
 
 
-15 
 
-14 
 
-13 
 
-12 
 
-11 
 
-10 
 
-9 
 
-8 
 
-7 
 
-6 
 
-5 & 
-4 
 
-3 
 
-2 
 
-1 
 
0 
A
M 
Fludar 
30  
mg/m2 
Fludar 
30  
mg/m2 
Fludar 
30  
mg/m2 
Fludar 
30  
mg/m2 
Rest 
TBI 
2 Gy 
Rest 
Rest 
Rest 
 
Rest 
 
CY 
60 
mg/kg 
CY  
60 
mg/kg 
 
Stem 
Cell 
Infus 
P
M 
 
Bu 
3.2 
mg/kg 
Bu 
3.2 
mg/kg 
 
 
 
 
 
 
DLI 
 
 
 
 
→ 
Median White Cell Counts of 10 Patients Treated To Date* 
 
4.9 
3.95 
3.2 
2.25 
1.95 
0.95 
0.7 0.4 
0.2 0.05 
** 
** 
 
 
 

 
 
 
12D.501 Protocol v5.2 
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18
 
 
 
Because the outcomes of the current trial will be compared to that of the first 2 
Step RIC trial in which only haploidentical donors were used, the outcomes of the 
patient group undergoing HSCT from haploidentical donors (2, 3, or 4 antigen 
mismatches in the GVH direction) will be exclusively used in the analysis of 
outcomes for the statistical ends of the trial. The outcomes of patients undergoing 
matched related donor HSCT will be reported descriptively.  
 
3.0   Patient and Donor Selection 
 
 
Patient Selection 
 
 
Inclusion Criteria 
 
1) By definition, patients with hematological malignancies or dyscrasias 
that require HSCT as part of cure-directed therapy are by definition high-
risk and can be treated on this protocol. Examples of high risk patients 
include but are not limited to: .    
 
a. 
Acute myeloid leukemia with high risk features as defined by:  
 
Age greater than or equal to 60 
Secondary AML (prior therapy or hematologic malignancy) 
Normal cytogenetics but FLT3/ITD positive  
Any relapse or primary refractory disease 
Greater than 3 cytogenetic abnormalities or any one of the 
following cytogenetic abnormalities: 
-5/del(5q), -7/del(7q), Abn(9q),(11q),(3q),(21q),(17p),t(6;9), 
t(6;11), t(11;19), +8,del(12p),inv(3),t(10;11),-17, 11q 23 
 
Any single autosomal monosomy105 
 
b. 
Acute lymphoid leukemia in 1st or 2nd morphological remission. 
ALL with any morphological evidence of disease will not be 
eligible.  
c. 
Myelodysplasia (MDS) other than refractory anemia (RA), 
refractory anemia with rare sideroblasts (RARS), or isolated 5q- 
syndrome subtypes.   
d. 
Hodgkin’s or Non-Hodgkin’s lymphoma in 2nd or greater remission 
or with persistent disease.   
e. 
Myeloma with evidence of persistent disease after front-line 
therapy.  
f. 
Chronic myeloid leukemia (CML) resistant to signal transducer 
inhibitor (STI) therapy 
g. 
Myelofibrosis and CMML  

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
19
 
 
h. 
Essential Thrombocytopenia or Polycythemia Vera with current or 
past evidence of evolution to acute leukemia  
i. 
Patients with CLL, follicular NHL, or other lymphoid malignancies 
who have highly adverse cytogenetics (such as p53 deletion), are 
chemo-insensitive, are not responsive to highly effective novel 
treatments such as CART or Ibrutinib, or who have transformed 
disease 
j. 
Any hematological malignancy or dyscrasia not cited above which 
is thought to be high-risk with increased chance of post HSCT 
relapse.  
k. 
Any patient who has an aggressive disease that would normally be 
treated on a myeloablative study, but is prevented from doing so 
by factors in their past medical history. Examples are patients with 
previous treatment with radiation therapy precluding TBI, or a past 
history of myeloablative therapy, precluding a 2nd myeloablative 
regimen.  
l.  
Patients with aplastic anemia may be treated on this protocol, with 
outcomes reported descriptively.  
 
2) Patients must have a related donor who is at least a 2-4/8 antigen 
mismatch at the Human Leukocyte Antigen (HLA)-A; B; C; DR loci. 
Patients with only a 1 out of 8 mismatch in the GVH direction will be 
classified in the matched related category 
 
3) Patients must have adequate organ function: 
 
 
 
 
a. Left ventricular end diastolic function (LVEF) of >50% 
 
b. Diffusion Lung Capacity of Oxygen (DLCO) >50% of predicted 
corrected for hemoglobin 
 
c. Adequate liver function as defined by a serum bilirubin <1.8, 
aspartate aminotransferase (AST) or alanine aminotransferase 
(ALT) < 2.5X upper limit of normal 
 
d. Creatinine Clearance of ≥ 60 mL/min 
 
4) Patients must have adequate KPS and HCT-CI scores:  
 
a) Patients < age 60 years must have a KPS of ≥80% and an HCT-
CI score of 5 or less 
 
b) Patients aged 60 to 65 years must have a KPS of ≥80% and an 
HCT-CI score of 4 or less 
 
c) Patients aged 66 to 69 years must have a KPS of 90% and an 
HCT-CI score of 3 or less 

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
20
 
 
 
d) Patients aged 70 years or more must have a KPS of 90% and an 
HCT-CI score of 2 or less 
 
(Patients with greater than the allowable HCT-CI points for age can be enrolled for 
trial with approval of the PI and at least 1 Co-I not on the primary care team of the 
patient). This is an adjustment to account for healthy patients who meet the spirit 
of this protocol but have histories that result in higher than guideline HCT-CI points. 
An example is a patient with a solid tumor malignancy in their remote history (adds 
3 points to HCT-CI total) where the treatment for the malignancy occurred years 
to decades before and there has been complete recovery of toxicities 
 
5) Patients must be willing to use contraception if they have childbearing 
potential 
 
6)  Patient or patient’s guardian is able to give informed consent 
 
 
 
Exclusion Criteria 
 
 
1)  HIV positive 
 
2)  Active involvement of the central nervous system with malignancy.  This 
can be documented as a normal neurological exam and/or a negative CSF 
analysis 
 
3)  Pregnancy  
 
4) Patients with life expectancy of < 6 months for reasons other than their 
 
  underlying hematologic/oncologic disorder   
 
5) Patients who have received alemtuzumab or ATG within 8 weeks of the 
transplant admission. 
 
6) Patients with evidence of another malignancy, exclusive of a skin 
cancer that requires only local treatment, should not be enrolled on 
this protocol   
 
Donor Selection 
All donors are selected and screened for their ability to provide adequate infection-
free apheresis products for the patient in a manner that does not put the donor at 

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
21
 
 
risk for negative consequences. Donor selection will be in compliance with 21 CFR 
1271 and TJU BMT Program SOP CP: P009.03.  
 
Specifically, donors will be tested, using the appropriate FDA-licensed and 
designated screening tests, for:  
1. HIV, type 1 
2. HIV, type 2  
3. HBV (HBsAg, anti-HBc IgG and IgM)  
4. HCV  
5. Treponema pallidum  
6. Human T-lymphotropic virus, types I and II  
7. Cytomegalovirus  
8. West Nile Virus  
9. Trypanosoma cruzi 
 
As per the Jefferson Blood Donor Center Quality Plan, all allogeneic donor testing 
samples (including HPC donors) will be sent to a laboratory that is FDA and CLIA 
licensed. Agreements/contracts for these services will be developed according to 
TJUH policies and all pertinent regulatory requirements will be retained by the 
Blood Bank. 
 
Additional donor testing may be performed as required to assess the possibility of 
transmission of other infectious and non-infectious diseases. 
 
TJUH HPC transplant personnel will discuss the potential for disease transmission 
from donor to recipient (i.e. the purpose of infectious disease testing) during the 
donor evaluation. 
 
Infectious disease testing must be completed by the time of the recipient’s 
transplant admission date. 
 
As per FACT guidelines, pregnancy will be assessed during the initial donor 
evaluation and just prior to the initiation of the recipient’s conditioning regimen in 
female donors of childbearing age.  
 
4.0 
Informed Consent 
Upon meeting the eligibility criteria for the trial, informed consent will be obtained 
using forms approved by the TJUH Institutional Review Board and following 
guidelines related to the use of human subjects in research. The risks and hazards 
of the procedure, as well as alternative forms of therapy will be presented to the 
patient in detail.  Patients will receive a signed copy of the consent form.  In 
addition, donors will be asked to sign consent after they have been fully informed 
about the procedures and risks of donating. 

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
22
 
 
 
 

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
23
 
 
5.0  
 
Treatment Plan 
 
 While the days of radiation and drug administration are fixed, the exact timing of these treatments on 
the day they are due is not specified because of expected variations in clinical care. 
  Treatment Schema 
 
Patient Schedule 
Bu=Busulfan, Fludar=fludarabine, TBI=total body irradiation, DLI= donor 
lymphocyte infusion (2 x 108 T cells/kg recipient weight), CY=cyclophosphamide, 
Tacro=tacrolimus, MMF=mycophenolate mofetil 
 
Donor Schedule 
 
Wed  
-7 
Thur  
-6  
Fri 
-5 
Sat 
-4 
Sun 
-3 
Mon 
-2 
Tues 
-1 
AM 
Lymphocyte 
Collection 
Lymphocyte 
Collection 
G-
CSF 
G-
CSF 
G-
CSF 
G-CSF  
PBSC 
Collection 
G-CSF 
PBSC 
Collection 
PM 
 
 
G-
CSF 
G-
CSF 
G-
CSF 
G-CSF 
 
G-CSF=Granulocyte Stimulating Factor (Neupogen, Filgrastim) 
 
a.There should be no administration of agents that suppress lymphocyte 
reactivity from day-15 until day -1 in this protocol.  This includes steroids, 
calcineurin inhibitors, MMF, or monoclonal antibodies that affect lymphocyte 
number or function.  Diphenhydramine and meperidine may be used if necessary 
for transfusion reactions after day -12.  Any use of steroids from day -15 through 
day -1 should not be administered without approval from the PI. 
 
b. Patients will not receive azole drugs, Acetaminophen, Metronidazole, or 
any drug inhibiting Busulfan metabolism from d-15 through d-12.  
 
c. Voriconazole is prohibited until d-1 due to its interaction with 
cyclophosphamide.  
 
 The absence of prohibited drugs (5.0 a, b, c) in the medical record serves as 
documentation that they were not given. 
 
 
 
-15 
 
-14 
 
-13 
 
-12 
 
-11 
 
-10 
 
-9 
 
-8 
 
-7 
 
-6 
 
-5 & -4 
 
-2 &-3 
 
-1 
 
0 
 
Fludar 
30  
mg/m2 
Fludar 
30  
mg/m2 
Fludar 
30  
mg/m2 
Fludar 
30  
mg/m2 
Rest 
TBI 
2 Gy 
Rest 
Rest 
Rest 
 
Rest 
 
CY 
60 
mg/kg 
 
Stem 
Cell 
Infus 
 
 
Bu 
3.2 
mg/kg 
Bu 
3.2 
mg/kg 
 
 
 
 
 
 
DLI 
 
 
Tacro 
& 
MMF 
→ 

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
24
 
 
All chemotherapy and HPC doses in this protocol are to be based on dosing weight 
(40% the difference between actual and ideal weight). 
 
 
5.1  
Administration of Fludarabine and Busulfan 
 
Fludarabine is administered for 4 days on (days -15 through – 12) at a dose of 30 
mg/m2 IV daily for 4 days.  Creatinine must be checked prior to each dose of 
fludarabine.  If renal insufficiency develops, the attending physician must be 
notified in cases where a dose adjustment needs to be made. 
 
Busulfan is administered for 2 days on days -14 and -13 at a dose of 3.2 
mg/kg/day IV.99-101, 106  The infusion can be started upon the completion of the 
fludarabine.99, 101, 106 
 
PK levels are not required for Busulfan dosing based on the low dose and low 
number of administrations of the drug.  
 
Seizure prophylaxis is required with the use of Busulfan. The recommended 
schedule is: 
 
Clonazepam 0.5 mg and Levetiracetam 500 mg, both drugs administered orally 
and given BID, beginning the evening prior to the first Busulfan dose and ending 
the morning after the last dose of Busulfan, days -15 through -12.107-109 
 
If patients cannot tolerate Levetiracetam, the suggested alternate regimen is: 
 
Lorazepam 0.02/kg (max 2 mg) orally or intravenously every 6 hours starting 30 
minutes prior to the first dose of Busulfan. and continuing for 4 doses after the 2nd 
dose of Busulfan. The dosage can be reduced by 20-50% (to the nearest 0.5 mg) 
if the patient experiences excessive sedation.110 
 
Day -11 is a rest day.  
 
5.2   
TBI 
2 Gy of TBI will be administered on day -10. At this low dose, there is almost no 
clinical scenario in which this small dose of radiation would be associated with 
added toxicity from prior radiation. However, all patients will be evaluated by a 
radiation oncologist in preparation for radiation treatment. See Appendix A for 
radiation guidelines. 
 
Days 9- to -7 are days of rest. 
5.3             Donor Lymphocyte Infusion  
 

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
25
 
 
CD3+ T cell and progenitor cell doses and cyclophosphamide dosing will be based 
on adjusted dosing weight (40% the difference between actual and ideal body 
weight + the actual body weight).  The dose of the donor lymphocyte infusion (DLI) 
will be based on CD3+ T cells per kilogram of recipient adjusted body weight. Donor 
lymphocytes will be collected prior to the use of G-CSF for progenitor cell 
collection.   
 
The goal of the first day of donor lymphocyte collection is to process a blood 
volume that is both safe for the donor as well as to obtain the prescribed dose of 
CD3+ T cells/recipient kg.  Approximately 18-27 liters will be processed the first 
day of donor lymphocyte collection. If a second day of collection is needed, the 
volume processed will be based on the amount of T cells required to meet the T 
cell target.  
 
DLI specimen handling and labeling conventions will be performed in accord with 
the relevant AABB (American Association of Blood Banks) and/or FACT 
(Foundation for Accreditation for Cell Therapy) regulations and guidelines. All DLI 
specimens must be appropriately labeled in accord with these standards to be 
accepted by the Processing Laboratory. A valid prescription and request form must 
be submitted by the requesting physician.  
 
Determination of the targeted T cell dose from the apheresis product is as follows: 
 
Total T-cells required for the initial infusion = (2x108 T-cells/kg) * (Weight in kg) 
  
Panel: 
          
FITC 
PE 
PE-Cy7 
APC 
APC-H7 
Tube1 
  
CD19 
CD16+56 
CD3 
CD45 
Tube2 
CD8 
  
CD4 
CD3 
CD45 
Tube3 
TCR-ab 
TCR-gd 
  
CD3 
  
  
CD3 count is calculated directly with single-platform flow cytometry. Reported CD3 
absolute count is the mean from 3-tube counts 
 
All donors will be apheresed for lymphocytes on day -7.  If the target number of 
CD3+ T cell lymphocytes, 2 x 108/recipient kg is not obtained, apheresis will be 
repeated on day -6. 
 
Lymphocyte apheresis will be performed at Thomas Jefferson University Hospital 
or the American Red Cross, by trained apheresis personnel using standard 
techniques and equipment.  
 
Patients will receive 2 x 108/kg T cells on day -6. During the infusion, the patient 
will be monitored for any untoward reactions. Donor lymphocyte infusions will be 

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
26
 
 
administered by nursing staff experienced in the administration of blood products.  
 
DLI must NOT be irradiated. DLI should NEVER be administered through a 
leukocyte depletion (PALL) filter. If blood filtration is necessary, the filter should be 
a standard blood product filter with pore size of at least 170 microns.  
 
Days -5 and -4 are rest days. 
 
 
 
 
5.4  
Cyclophosphamide 
 
 
 
CY 60 mg/kg IV will be administered on days –3 and –2 of the conditioning 
regimen. Mesna 60 mg/kg continuous IV infusion over 24 hours X 2 doses will be 
infused on days –3 through –2.   
 
Day –1 is a day of rest. 
 
 
 
5.5  
Collection and Infusion of Progenitor Cells (PBSCs) 
Donors will begin G-CSF, 5µg/kg bid, on day -5. Adjunctive or alternate white cell 
stimulators such as Pegfilgrastim and/or Plerixafor are acceptable for use. 
Progenitor cell collection will occur on days -2 and -1.  Approximately 18 to 27 liters 
will be processed on the first day of donor collection. The volume processed on 
the second day of collection will be based on the amount of CD34+ cells required 
to meet the CD34+ cell target. Specific guidelines regarding the reduction of 
volume processed the second day based on the collection totals of the first day is 
contained in BMT SOP CP:P022.08. CD34+ cell enrichment will be performed via 
the closed system method using the CliniMACS® CD34 Reagent System (Miltenyi 
Biotec Inc., Auburn, CA). The CliniMACS system utilizes super-paramagnetic 
particles composed of iron oxide and dextran conjugated to monoclonal antibodies. 
These antibodies bind to target cells with the corresponding cell surface antigen 
(in this case, CD34). After magnetic labeling, the cells are separated using a high-
gradient magnetic separation column. The magnetically labeled cells are retained 
in the column and separated from the unlabeled cells. Removing the magnetic field 
from the separation column elutes the retained cells. Eluted cells will be 
characterized using fluorescent-activated cell sorting (FACS) analysis. All 
procedures will be performed in a sterile environment with strict adherence to all 
applicable regulations regarding the processing and use of human stem cells. The 
use of this device will conform to TJU BMT Laboratory standard operating 
procedures. 
 
The target dose of donor PBSCs to be infused into the recipient is between 3 –5 
x 106 CD34 cells/kg of recipient dosing body weight. The acceptable minimum 
infusion target of PBSCs will be 1 x 106 CD34 cells/kg.  Recipients will receive no 

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
27
 
 
more than 10 x 106 CD34 cells/kg, the maximum dose. If less than 50% of the 
minimum acceptable CD34 cells/kg  target dose is obtained after the first 
collection, Plerixafor,12 mg may be administered subcutaneously the evening 
prior to the second collection.  Because the meaningful dose of T cells has 
already been collected and infused by this time, Plerixafor would have not 
polarization effects on T helper cells.  
 
In our experience, the ideal amount of T-cells left in the PBSC product is no greater 
than 5x104/kg, so that every effort will be made to keep T-cell amounts to below 
this threshold. In over 100 2 Step HSCT procedures, approximately 1% of products 
contained greater than this amount of T cells. In addition, the amount over the 
targeted minimal dose was negligible. It is recognized that because of donor 
heterogeneity, every product will have varying percentages of cells.  Thus, patients 
will be advised during the informed consent process that an excess amount of 
residual T-lymphocytes in the PBSC product may increase the risk of GVHD. 
 
Progenitor cell apheresis will be performed at Thomas Jefferson University 
Hospital or the American Red Cross, by trained apheresis personnel using 
standard techniques and equipment.  
 
Handling and labeling of the progenitor cell product will be performed in accord 
with the relevant AABB (American Association of Blood Banks) and/or FACT 
(Foundation for Accreditation for Cell Therapy) regulations and guidelines. All 
donor specimens must be appropriately labeled in accord with these standards to 
be accepted by the Processing Laboratory. A valid prescription and request form 
must be submitted by the requesting physician.  
The donor PBSC product is infused UNFILTERED or through a filter of at least 170 
micron size intravenously through a central catheter. PBSCs should only be 
piggybacked through normal saline and not other intravenous solutions. 
Contingency plans for an inadequate collection of progenitor cells via apheresis or 
non-viable donor cells will be made according to institutional policies. All donors 
will be available for a third day of progenitor cell apheresis and will be given extra 
neupogen in case there is a need for a third collection day. 
 
During the infusion, the patient will be monitored for any untoward reactions. Each 
infusion will be administered by nursing staff experienced in the administration of 
blood products. PBSC products must NOT be irradiated.  PBSC products should 
NEVER be administered through a leukocyte depletion (PALL) filter. If blood 
filtration is necessary, the filter should be a standard blood product filter with pore 
size of at least 170 microns.  
Significant red cell incompatibility between donor and recipient will be managed 
according to standard operating procedure, CL: Ppp033, of the Thomas Jefferson 

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
28
 
 
University Hospital Blood and Marrow Transplant Processing Lab.  Pre-
medications (if any) prior to PBSC infusion will be at the discretion of the physician.  
Diphenhydramine, epinephrine, and hydrocortisone should be available for 
emergency use if necessary. Oxygen with nasal cannula should be immediately 
available. 
 
 
5.6  
GVHD Prophylaxis 
 
Tacrolimus will be started on day -1.  Tacrolimus dose titration will occur to target 
a goal level of 7 ng/ml +/- 2. It is recognized that there may be values beyond this 
target range due to interpatient variability.   
 
MMF will be dosed at 1 gram IV BID beginning on day -1. 
 
The tacrolimus taper can be initiated by day + 42 in the absence of concern for 
GVHD or interference with a GVHD plan of care that was developed prior to day 
+42. Because of the variability in patient outpatient office visit times and the need 
for GVHD assessment, it is not mandatory that the taper begins exactly day on 
+42. 
 
MMF will be discontinued beginning at day +28 +/- 3 days in the absence of 
GVHD.   
 
Tacrolimus and MMF may be discontinued earlier if there is count suppression 
from the drugs or other unforeseen circumstances in which the drug is felt to be 
deleterious to the plan of care, such as infection, count suppression, drug side 
effects, or a need for alternate GVHD treatment.   
 
The BMTU attending physician may change these GVHD prophylaxis guidelines 
if clinically indicated.  
 

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
29
 
 
6.0 
 
Study Measurements** 
The table below outlines the measurements and time points specific to this study. Only 
the day +28 studies are mandatory. The other elements are recommended. The 
attending physician may perform assessments/labs more or less frequently based on 
the patient’s unique course.   
 
 
 
 
 
Baseline 
assessment  
During 
conditioning 
After 
Condition
-ing 
through 
Day + 28 
Days 28-90 
Days 90-
180 
 
Day 180 
 
Days 180- 
365 
History and 
physical with vital 
signs, including 
SPO2.  
Assessment of 
infectious signs, 
pregnancy test 
for females of 
childbearing 
potential done on 
baseline 
assessment 
X 
Every 1-2 
days 
Daily if in 
hospital 
weekly 
until day 
28 after 
discharge 
Monthly 
As  
clinically 
indicated 
 
 
 
 
 
 
 
 
 
As clinically 
indicated 
Laboratory 
Studies* 
X 
Every 1-2 
days 
Daily if in 
hospital 
weekly 
until day 
28 after 
discharge 
Twice 
monthly  or 
as clinically 
indicated 
As  
clinically 
indicated 
 
 
 
As  
clinically 
indicated 
Quantitative 
cytomegalovirus
CMV by 
polymerase chain 
reaction PCR 
 
Weekly or as 
clinically 
indicated 
Weekly 
until 
discharge 
or as 
clinically 
indicated 
Twice 
monthly or 
as clinically 
indicated 
As  
clinically 
indicated 
 
 
Monthly or 
as clinically 
indicated 
Viral throat 
gargle/sputum 
culture and 
sensitivity C&S 
 
If respiratory 
symptoms 
If 
respiratory 
symptoms 
If 
respiratory 
symptoms 
If respiratory 
symptoms 
 
If respiratory 
symptoms 
Stool culture (cx), 
viral screening & 
cx  & fungal cx 
If clinically 
indicated 
If clinically 
indicated 
If clinically 
indicated 
If clinically 
indicated 
If clinically 
indicated 
 
If clinically 
indicated 

 
 
 
12D.501 Protocol v5.2 
(17June2022) 
 
30
 
 
 
Baseline 
assessment  
During 
condition-
ing 
Day + 
28 
 
Days 28-90 
 
Days 90-
180 
 
Day 180 
 
Days 180- 
365 
GVHD 
Assessment 
Presence and 
degree of skin 
rash, presence 
and amount of 
diarrhea, LFT’s 
N/A 
Daily after 
engraftment 
until 
discharge 
and then 
weekly as 
indicated 
X 
Twice 
monthly 
 
As 
clinically 
indicated 
 
 
 
 
 
As 
clinically 
indicated 
Chimerism/ 
Disease 
Assessment  
 
 
 
 
 
 
 
 
 
Peripheral blood 
for CD3+ 
chimerism & 
Buffy coat 
chimerism  
 
 
X 
Twice 
monthly until 
>95% donor 
chimerism  
Once d+90 
X 
As clinically 
indicated 
Bone marrow 
exam 
(morphology, 
flow cytometry, 
cytogenetics, 
buffy coat 
chimerism) 
 
 
X 
 
Day +90 
Marrow 
 
 
Day +180 
Marrow 
Is optional 
 
 
 
Day +270 
Marrow 
Is optional 
 
 
 
Day +365 
Marrow 
Is optional 
 
Immune 
Reconstitution 
Studies 
 
 
 
 
 
 
 
Flow cytometry 
for lymphocyte 
subsets 
 
 
 
X 
 
Monthly 
Monthly 
 
X 
 
 
Quarterly  
Radiographic 
Studies 
In applicable 
situations for 
disease staging 
 
X 
 
 
 
Day +90 
or as 
clinically 
indicated 
 
Day +365 
or as 
clinically 
indicated 
**Laboratory studies include a complete blood count with differential, 
comprehensive metabolic panel, lactic and GVHD prophylaxis drug levels 
when applicable  
The day +28 peripheral blood, marrow studies, and IRP can be obtained within 
1 week before day 28 (i.e day +21 through day +28) and within 2 weeks after day 
+28 (i.e. day 28 through day +42) to account for scheduling factors and failed 
testing.  
The formal endpoint of this study for efficacy is 1 year post HSCT. Therefore 
patients will not be followed for this study after this time. However, outcomes for 
patients undergoing HSCT at TJUH are followed programmatically beyond this 
study indefinitely. 
 
 
6.1  
Hematopoietic engraftment. Will be defined as:   

 
 
 
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31
 
 
 ANC >/= 0.5x10e9/L for at least 3 days 
 Platelet engraftment >20,000 with no transfusions X 7 days. 
 
 
6.2  
Toxicity Criteria.  
 
Regimen-related toxicity will be graded according to the NCI Common Toxicity 
Criteria, version 4.0.  These criteria can be found on the Thomas Jefferson 
University Hospital BMT WEB site available via the TJUH Intranet. 
 
The NCI Common Toxicity Criteria can also be found at the following WEB 
address:     http://ctep.cancer.gov/reporting/ctc.html 
 
 
 
 
6.3    Disease Response: 
Disease response will be measured according to the National Comprehensive 
Cancer Network Guidelines (NCCN).  The guidelines are disease specific and the 
guidelines for each disease can be found at: 
 
http://www.nccn.org/professionals/physician_gls/f_guidelines.asp#site  
 
 
6.4  
GVHD Scoring 
GVHD will be graded according to standard criteria contained in Appendix B. 
 
 
6.5  
Adverse event reporting.   
All patients will be followed for adverse experiences (AEs) (serious and 
nonserious), regardless of relationships to study treatment, from the time of 
enrollment until d +100 after transplant. The following events are expected side 
effects of high-dose chemotherapy and transplant and will be recorded but will not 
be reported except as noted:  
 
• Alopecia, headache, dry skin 
• Emesis from chemotherapy or other agents unless refractory to 
standard supportive care, nausea, anorexia 
• Weight loss, cough, dry mouth 
• Grades 1-3 fever 
• Grades 1-3 infectious sequellae  
• Grades 1-3 electrolyte imbalances 

 
 
 
12D.501 Protocol v5.2 
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32
 
 
• Grades I-III abnormalities in alkaline phosphatase, AST and ALT 
• Neutropenia/uncomplicated neutropenic fever, grades 1-3 
infectious sequellae 
 
• Thrombocytopenia, petechiae, ecchymoses, minor vaginal 
bleeding, epistaxis, hemorrhoidal bleeding, or other similar bleeding 
events will not be reported.  (Bleeding events requiring intervention 
such as endoscopy or radiologic evaluation will be reported.) 
• Grades 1-3 Rash 
• Grades I-III Fatigue 
• Anemia 
• Grade I - III Mucositis  
• Grade I - III Diarrhea  
• Allergic or other reactions to drugs used for supportive care or 
GVHD prophylaxis unless grade 4-5  
After d+100, only AEs that are considered by the investigator to be possibly or 
probably associated with the treatment regimen will be reported.   
 
 
6.6   Reports to the Federal Drug Administration (FDA) 
All grade 3-5 infusion reactions and all unexpected SAEs as defined in 21 CFR 
312.32 will be reported to the FDA in an expedited fashion 
An annual report will be sent to the FDA regarding the progress to date of 
patients on the trial.  In the report, a separate listing of infusion toxicities and all 
biological product deviations will be included in addition to the other required 
elements. 
These requirements will end with cessation of the need for an IDE (ie CliniMacs 
approval and/or reporting through an alternate mechanism) 
 
6.7  
Study Endpoint 
 
The endpoint of this study is DFS at 1 year post HSCT.   
 
7.0 
 
Supportive Care 
 
 
 
7.1  
Avoidance of Infection 

 
 
 
12D.501 Protocol v5.2 
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33
 
 
 
Patients who are post HSCT are susceptible to infection. BMT Clinical 
Program SOPs CP:P050.01 and CP:P001.04 address infectious prophylaxis 
and management of suspected infection.  
Central venous catheters will be removed as soon as clinically manageable.  
 
It is recommended that IVIG 0.5 g/kg IV will be administered monthly post-
transplant to support immune function, until the IgG level is > 500 mg/dL on 2 
consecutive monthly measurements. The first dose will be targeted for 
administration on day +7. It is recognized that fluid overload, changes in renal 
function, or outpatient lack of coverage for the IVIG may prohibit or delay this 
therapy.  
 
 
 
7.2  
Infectious Prophylaxis-General Guidelines 
 
Patients post allogeneic HSCT will be maintained on antifungal prophylaxis, 
usually voriconazole 200 mg BID.  It is at the discretion of the treating attending 
physician to change agents as clinically indicated.   
 
Patients post allogeneic HSCT will be maintained on HSV prophylaxis, usually 
valacyclovir 500 mg daily.  It is at the discretion of the treating attending 
physician to change agents based on culture results and sensitivities.  
 
Patients post allogeneic HSCT will be maintained on Pneumocystis jiroveci 
pneumonia prophylaxis, usually TMP-SMZ DS 1 tablet daily.  It is at the 
discretion of the treating attending physician to change agents based on culture 
results or drug intolerance.  
 
 
 
7.3  
Growth Factor and Transfusion Support 
 
To prevent inadvertent lymphoid engraftment, all blood cell products must be 
irradiated. 
 
All red cell and platelet products will be leukodepleted to prevent 
alloimmunization and decrease infectious sequellae.   
 
Packed red blood cell transfusions will be given as necessary to keep the 
hemoglobin >/= 7-8g/L. 
 
Platelet transfusions will be used as needed to keep the morning count >/= 10-
20x10e9/L, with 10x10e9/L used for situations without an excessive bleeding 
risk.  
 

 
 
 
12D.501 Protocol v5.2 
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34
 
 
GM-CSF 250µg/m2 will be administered daily beginning on day +1.  GM-CSF will 
be weaned/discontinued at the discretion of the attending physician. Every effort 
should be made to keep the ANC > 1000 for all patients post allogeneic HSCT. 
G-CSF 5µg/m2 can be substituted for GM-CSF in the event of a GM-CSF 
shortage or if a patient has a deleterious reaction to GM-CSF as determined by 
the BMTU attending physician.  
 
 
Red cell growth factors are permissible after transplantation. 
 
 
8.0 
 
Drug Information and Administration 
  
 
 
8.1 Busulfan 
 
Mechanism: Busulfan is an alkylating agent which reacts with the N-7 position of 
guanosine and interferes with DNA replication and transcription of RNA. Busulfan 
has a more marked effect on myeloid cells than on lymphoid cells and is also very 
toxic to hematopoietic stem cells. Busulfan exhibits little immunosuppressive 
activity, and therefore in this protocol is given with fludarabine and TBI both of 
which have lymphopenic affects. Busulfan interferes with the normal function of 
DNA by alkylation and cross-linking the strands of DNA.  
Metabolism: Extensively hepatic; glutathione conjugation followed by oxidation 
Incompatibilties: Busulfan does not have an extensive list of medications that 
cause problematic interactions. However, there are a few drugs, commonly used 
with Busulfan that may affect its metabolism. Phenytoin may decrease the serum 
concentration of Busulfan and Azoles may decrease the metabolism of Busulfan. 
Acetominophen and Metronidazole may increase the serum concentration of 
Busulfan.  
Toxicity: Side effects of Busulfan include but are not limited to: tachycardia, 
hypertension, insomnia, anxiety, headache, fever, vomiting, mucositis, diarrhea, 
anorexia, myelosupression, hyperbilirubinemia, VOD, weakness, and arthralgias, 
Administration: Busulfan is administered for 2 days on days -14 and -13 at a dose 
of 3.2 mg/kg/day IV.(Alatrash, deLimaAndersson McCune) The infusion can be 
started upon the completion of the fludarabine. 
Reference: 
http://online.lexi.com.proxy1.lib.tju.edu/lco/action/doc/retrieve/docid/patch_f/6487
#f_adverse-reactions 
 
8.2 Cyclophosphamide 
 
Mechanism: 
A 
multistep 
process 
activates 
it 
by 
conversion 
to 
4-
hydroxycyclophosphamide by the liver microsomal oxidase system and to 
aldophosohamide by tautomerization in the peripheral tissues.  Aldophosphamide 
spontaneously degrades into acrolein and phosporamide mustard, which cause 

 
 
 
12D.501 Protocol v5.2 
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35
 
 
cellular glutathione depletion and DNA alkylation. This results in inhibition of DNA 
replication and transcription. Cells expressing high levels of aldehyde 
dehydrogenase (e.g. stem cells, L1210 leukemia cells) resist cyclophosphamide-
mediated cytotoxicity as aldophosphamide is inactivated by this enzyme. The drug 
also does not affect quiescent cells and therefore stem cells are generally 
protected, an important factor if autologous hematopoietic recovery is relied on in 
the event of graft failure.  
Metabolism: Cyclophosphamide is broken down as described above and the 
break down products are excreted by the kidneys. It is a substrate of CYP2A6 
(minor), CYP2B6 (major), CYP2C19 (minor), CYP2C9 (minor), CYP3A4 (minor); 
Note: Assignment of Major/Minor substrate status based on clinically relevant 
drug interaction potential; Inhibits CYP3A4 (weak); Induces CYP2B6 
(weak/moderate), CYP2C9 (weak/moderate). 
Incompatibilties: Phenobarbital or rifampin may increase the toxicity of 
cyclophosphamide. Concurrent allopurinol or thiazide diuretics may exaggerate 
bone 
marrow 
depression 
May 
prolong 
neuromuscular 
blockade 
from 
succinylcholine Cardiotoxicity may be additive with other cardiotoxic agents ( 
cytarabine, daunorubicin, doxorubicin).  May decrease serum digoxin levels.  
Additive bone marrow depression with other antineoplastics or radiation therapy.  
May potentiate the effects of warfarin. May decrease antibody response to live-
virus vaccines and increase the risk of adverse reactions.  Prolongs the effects of 
cocaine. 
Toxicity:  Nausea, vomiting, water retention due to inappropriate secretion of anti-
diuretic hormone (SIADH), cardiomyopathy with myocardial necrosis and 
congestive heart failure, hemorrhagic cystitis, alopecia, skin rash, pulmonary 
fibrosis, sterility and secondary malignancies. 
Administration: Patients will receive a dose of cyclophosphamide 60 mg/kg IV, on 
days –3 and -2.  The dose of cyclophosphamide will be calculated according to the 
dosing body weight. MESNA (sodium-2-mercaptoethane sulfonate) will be 
administered prior to cyclophosphamide infusion and ending approximately 24 
hours after the last dose of cyclophosphamide. The dose of MESNA will also be 
calculated based on dosing body weight.  
 
References: Skeel R & Lachant N.  Handbook of Cancer Chemotherapy, 4th Ed. 
Little, Brown & Co.: Boston. 
Information 
from 
LexiComp 
on 
line 
reviewed 
on 
7/4/12 
at 
http://online.lexi.com.proxy1.lib.tju.edu/lco/action/doc/retrieve/docid/patch_f/6674
#f_interactions 
 
8.3  
Donor Leukocyte Infusion (DLI) 
 
Administration: All patients will receive a dose of CD3+ T cells per kilogram of 
dosing body weight as outlined in the treatment design. Although unlikely, if more 

 
 
 
12D.501 Protocol v5.2 
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36
 
 
than a single apheresis of the donor is required to obtain the target cell dose, the 
white cells that are obtained from each procedure will be given to the patient on 
the same day. Details of the apheresis procedure to obtain white blood cells, 
quantification of CD3+ T cells by flow cytometry, and administration of the white 
cell product to the recipient are provided in the treatment section. All drugs that 
may cause lymphocyte suppression are held prior to lymphocyte infusion (day -6), 
through day 0 as detailed in the treatment section. Every effort will be made to 
administer the donor lymphocytes around or as close to the designated day of 
lymphocyte infusion. Moreover the viability of the lymphocytes will be tested by 
flow cytometry and the number of viable CD3+ T cells will be used to dose the DLI.  
Toxicity: GVHD, delayed myelosuppression, infusion reactions. 
 
 
 
8.4  
Fludarabine 
 
Mechanism: Fludarabine phosphate is fluorinated nucleotide and analog of 
antiviral agent vidarabine, that is relatively resistant to adenosine deaminase 
deamination. It is actively dephosphorylated to 2-fluoro-ara-A and phosphorylated 
further by deoxycytidine kinase to 2-fluoro-ara-ATP, then acts by inhibiting DNA 
polymerase alpha, ribonucleotide reductase and DNA primase resulting in DNA 
synthesis inhibition. 
Metabolism: Renal Excretion  
In a pharmacokinetic study of patients treated with fludarabine for rheumatoid 
arthritis, the mean total clearance was 14.01 L/hr following a dose of 20 
mg/m(2)/day, and 13.4 L following a dose of 30 mg/m(2)/day (Knebel et al, 1998). 
The median total body clearance was 9.6 L/hr after intravenous or subcutaneous 
fludarabine 30 mg/m(2) for 3 days in 5 patients with lupus nephritis (Kuo et al, 
2001). 
Incompatibilities: Fludarabine has drug interactions with several vaccines and its 
simultaneous use with Rotavirus vaccine is contraindicated. 
Toxicities: Common: Endocrine/Metabolic: Shivering, Gastrointestinal: Loss of 
Appetite, Nausea, Vomiting, Neurologic: Asthenia, Other: Fatigue, Malaise, 
Serious: Cardiovascular: Edema (frequent), Dermatologic: Aplasia of skin (rare), 
Hematologic: Autoimmune Hemolytic Anemia, Graft versus host disease, 
Transfusion-associated, with non-irradiated blood (rare), Myelosuppression 
(frequent), Neurologic: Neurotoxicity, Respiratory: Pneumonia (frequent), Other: 
Fever (frequent), Infectious disease. 
Administration: In this protocol, Fludarabine is administered for 4 days on (days -
15 through – 12) at a dose of 30 mg/m2 IV daily for 4 days.  Creatinine should be 
checked prior to each dose of fludarabine.  If renal insufficiency develops, the 
attending physician must be notified in cases where a dose adjustment needs to 
be made. 
 
References: MicroMedex Health Care Series, Thomson. In addition, information 
from 
LexiComp 
on 
line 
reviewed 
on 
7/4/12 
at 
http://online.lexi.com.proxy1.lib.tju.edu/lco/action/doc/retrieve/docid/patch_f/6674
#f_interactions 

 
 
 
12D.501 Protocol v5.2 
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8.5  
G-CSF (Figrastim, Neupogen) 
 
Mechanism: G-CSF is a human granulocyte colony-stimulating factor produced by 
recombinant DNA technology.  It is a glycoprotein which acts on hematopoietic 
cells by binding to specific cell surface receptors and stimulating proliferation, 
differentiation, commitment, and some end-cell functions. Activates neutrophils to 
increase migration andtoxicity. 
Metabolism: 
Absorption 
and 
clearance 
of 
G-CSF 
follows 
first-order 
pharmacokinetic modeling without apparent concentration dependence.  The 
elimination half-life in both normal and cancer patients is 3.5 hours.   
Incompatibilties: Safety and efficacy of G-CSF when used simultaneously with 
chemotherapy or radiotherapy has not been evaluated. Donors receiving either of 
these 2 modalities will not be permitted on study. 
Toxicities: Allergic reactions consisting of rash, wheezing and tachycardia.  Splenic 
rupture, ARDS, and exacerbation of sickle cell disease have been reported rarely. 
Administration: In this protocol, G-CSF will be administered to healthy donors at a 
dose of 10 µg/kg (actual weight) subcutaneously on days -5 through day -1. 
 
References: Physician’s Desk Reference, Edition 58, 2004. In addition, information 
from 
LexiComp 
on 
line 
reviewed 
on 
7/4/12 
at 
http://online.lexi.com.proxy1.lib.tju.edu/lco/action/doc/retrieve/docid/patch_f/6674
#f_interactions 
 
 
 
8.6  
GM-CSF (Sargramostim, Leukine) 
 
Mechanism: GM-CSF is a recombinant human granulocyte-colony stimulating 
factor produced by recombinant DNA technology in a yeast expression system. It 
supports survival, clonal expansion, and differentiation of hematopoietic cells.  GM-
CSF is also capable of activating mature granulocytes and macrophages, and is a 
multilineage factor with effects on the myelomonocytic, erythroid, and 
megarkaryocytic lines.  
Metabolism: GM-CSF is detected in the serum at 15 minutes after injection.  Peak 
levels occur about 1 to 3 hours after injection, and it is detectable in the serum for 
up to 6 hours after injection.  
Incompatibilities: Interactions between GM-CSF and other drugs have not been 
fully evaluated.  Drugs which may potentiate the myeloproliferative effects of GM-
CSF, such as lithium and corticosteroids, should be used with caution.  
Toxicities: Allergic and anaphylactic reactions have been reported.  A syndrome 
characterized by respiratory distress, hypoxia, flushing, hypotension, syncope and 
or tachycardia has been associated with the first administration of GM-CSF in a 
cycle.  These signs have resolved with treatment.  

 
 
 
12D.501 Protocol v5.2 
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38
 
 
Administration: In this protocol, GM-CSF will be given to the patients beginning on 
Day +1. The drug should continue until the patient has a self-sustaining ANC of ≥ 
1000.  
 
 
 
References: Physician’s Desk Reference, Edition 58, 2004. In addition, information 
from 
LexiComp 
on 
line 
reviewed 
on 
7/4/12 
at 
http://online.lexi.com.proxy1.lib.tju.edu/lco/action/doc/retrieve/docid/patch_f/6674
#f_interactions 
 
 
8.7  
Mycophenolate Mofetil (MMF) 
 
Mechanism: Inhibits the enzyme inosine monophosphate dehydrogenase, which 
is involved in purine synthesis. This inhibition results in suppression of T- and B-
lymphocyte proliferation.  
Metabolism: Following oral and IV administration, mycophenolate is rapidly 
hydrolyzed to mycophenolic acid (MPA), its active metabolite. Distribution is 
unknown. MPA is extensively metabolized; <1% excreted unchanged in urine. 
Some enterohepatic recirculation of MPA occurs. Half Life: MPA¾17.9 hr. 
Incompatibilities: Combined use with azathioprine is not recommended (effects 
unknown) · Acyclovir and ganciclovir compete with MPA for renal excretion and, in 
patients with renal failure, may increase each other's toxicity. · Magnesium and 
aluminum hydroxide antacids decrease the absorption of MPA (avoid 
simultaneous administration). Cholestyramine and colestipol decrease the 
absorption of MPA (avoid concurrent use).  Toxicity may be increased by 
salicylates. · May interfere with the action of oral contraceptives (additional 
contraceptive method should be used). · May decrease the antibody response to 
and increase risk of adverse reactions from live-virus vaccines, although influenza 
vaccine may be useful. ·When administered with food, peak blood levels of MPA 
are significantly decreased. 
Toxicities: GI: Bleeding, Diarrhea, Vomiting, Hematopoietic: Leukopenia 
Miscellaneous: Sepsis, Increased Risk of Malignancy  
Administration: In this protocol, MMF will be administered at a dose of 1 gram IV 
BID beginning on day -1.  MMF will be discontinued on day +28 in the absence of 
GVHD. MMF may be stopped earlier if there is count suppression from the drug.  
 
Reference: Information from LexiComp on line reviewed on 7/4/12 at 
http://online.lexi.com.proxy1.lib.tju.edu/lco/action/doc/retrieve/docid/patch_f/6674
#f_interactions 
 
 
8.8  
Tacrolimus  
 
Mechanism: Tacrolimus, it is a macrolide immunosuppressant. It inhibits 
lymphocytes by forming a complex with FKBP-12, calcium, calmodulin leading to 
the decrease in the phosphatase activity of calcineurin. This in turn prevents 
generation of NF-AT, a nuclear factor for initiating gene transcription for 

 
 
 
12D.501 Protocol v5.2 
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39
 
 
lymphokines like interleukin-2 and interferon-฀฀฀. This drug is used with 
corticosteroids for prophylaxis of organ rejection in patients receiving allogeneic 
liver transplants. Its use is also currently being investigated in kidney, bone 
marrow, cardiac, pancreas, pancreatic island cell and small bowel transplantation.  
Metabolism: This drug is well absorbed orally. It is metabolized in the liver by 
unknown mechanisms and demethylation and hydroxylation has been proposed 
based on in vitro studies. The metabolized products are excreted in the urine. 
Tacrolimus is a substrate of CYP3A4 (major), P-glycoprotein; Note: Assignment of 
Major/Minor substrate status based on clinically relevant drug interaction potential; 
Inhibits CYP3A4 (weak), P-glycoprotein.  
Incompatibilities: Nephrotoxic drugs, antifungals (azoles), calcium-channel 
blockers, 
cimetidine, 
danazol, 
erythromycin, 
methylprednisone 
and 
metoclopramide increase the bioavailabilty of tacrolimus. On the other hand 
phenobarbital, phenytoin, rifamycins and carbamazepine decrease tacrolimus 
levels.     
Toxicities: Adverse reactions include: tremor, headache, neurotoxicity; diarrhea, 
nausea; hypertension; TTP and renal dysfunction.     
 Administration: Tacrolimus will be started on day -1.  with  a goal target level of 
7ng/ml +/- 2 as noted in section 5.  
 
Reference: Information from LexiComp on line reviewed on 7/4/12 at 
http://online.lexi.com.proxy1.lib.tju.edu/lco/action/doc/retrieve/docid/patch_f/6674
#f_interactions 
 
9.0  
 
Patient Safety 
 
To ensure patient safety, a number of steps will be taken.   
 
The study will be monitored monthly by the Principal Investigator (PI) and the study 
medical monitor.  Monitoring reports will be submitted to the Clinical Research 
Organization (CRO), Protocol Review Committee (PRC), and the Data Monitoring 
and Safety Committee (DMSC). The PI will submit all unexpected serious adverse 
events (SAE) to the TJU IRB utilizing the electronic Sidney Kimmel Cancer Center 
Clinical Trials Adverse Event Reporting system, with hard copies also submitted 
to the Office of Scientific Affairs within 48 hours of occurrence.  Due to the nature 
of the study treatment as outlined in this protocol, expected grade 3 AE/SAEs that 
occur while receiving standard inpatient protocol treatment may be included on the 
patient’s AE log for quarterly review by the DSMC rather than be reported via the 
eSAEy System per the DSMC Plan.  It is the responsibility of the study Principal 
Investigator (PI) to report any grade 3 AE/SAE to the DSMC per the DSM Plan 
should the length of standard protocol treatment hospitalization be extended 
and/or the grade 3 AE/SAE is more acute than expected as outlined in the informed 
consent form. Unexpected deaths due related to this protocol will be reported 
within 24 hours. 
 

 
 
 
12D.501 Protocol v5.2 
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40
 
 
The medical monitor will be a TJU physician who is not a collaborator in this trial.  
The medical monitor will review all adverse events (in addition to unexpected 
adverse events), safety data and activity data observed when this trial is ongoing.  
The medical monitor may recommend reporting adverse events and relevant 
safety data not previously reported, and may recommend suspension or 
termination of the trial.  The summary of all discussions of adverse events will be 
submitted to the DSMC after completion and included in the PI's reports to the 
PRC and the TJU IRB as part of the study progress report.  The PRC, DMSB, 
and/or the TJU IRB may, based on the monitor's recommendation suspend or 
terminate of the trial. The quarterly safety and monitoring reports will include a 
statement as to whether this data has invoked any stopping criteria (dose-limiting 
toxicities) in the clinical protocol.  
 
9.1 Safety and Adverse Event Reporting 
 
Unanticipated Problems 
 
Unanticipated problems (UAPs) include, in general, any incident, experience, or 
outcome that meets the following criteria: 
• unexpected in terms of nature, severity, or frequency given (a) the 
research procedures that are described in the protocol-related documents, 
such as the IRB-approved research protocol and informed consent 
document; and (b) the characteristics of the participant population being 
studied; 
UAPs are considered to pose risk to participants or others when they suggest 
that the research places participants or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or 
recognized. 
 
Adverse Events 
An adverse event is any untoward or unfavorable medical occurrence in a human 
participant, including any abnormal sign (for example, abnormal physical exam or 
laboratory finding), symptom, or disease, temporally associated with the 
participant’s participation in the research, whether or not considered related to 
the participant’s participation in the research. 
 
Serious Adverse Events 
A serious adverse event (SAE) is one that meets one or more of the following 
criteria: 
• Results in death 
• Is life-threatening (places the participant at immediate risk of death from 
the event as it occurred) 
• Is disabling or incapacitating 
• Results in inpatient hospitalization or prolongation of existing 
hospitalization 

 
 
 
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41
 
 
• Results in a persistent or significant disability or incapacity 
• Results in a congenital anomaly or birth defect 
• An important medical event that may not result in death, be life 
threatening, or require hospitalization may be considered an SAE when, 
based upon appropriate medical judgment, the event may jeopardize the 
participant or may require intervention to prevent one of the outcomes 
listed in this definition. Potential drug induced liver injury (DILI) is also 
considered an important medical event 
• Suspected transmission of an infectious agent (e.g. pathogenic or 
nonpathogenic) via the study drug is an SAE 
• Although pregnancy, overdose, cancer and potential DILI are not always 
serious by regulatory definition, these events must be handled as SAEs 
Following the subject’s start of treatment, all SAEs, whether related or not related 
to study drug, must be collected, including those thought to be associated with 
protocol-specified procedures. All SAEs must be collected, that occur within 100 
days of discontinuation of dosing. 
A nonserious adverse event is an AE not classified as serious.  
 
Nonserious Adverse Event Collection and Reporting 
The collection of nonserious AE information should begin at initiation of study 
drug. All nonserious adverse events (not only those deemed to be treatment-
related) should be collected continuously during the treatment period and for a 
minimum of 100 days following the last dose of study treatment.  
 
Safety Assessment and Follow-Up 
See Section 6.6 for information on protocol specific adverse event reporting.  
 
Recording Adverse Events 
The following subsections detail what information must be documented for each 
adverse event occurring during the time period specified in Section  
 
Relationship to Study Intervention 
The relationship to study intervention or study participation must be assessed 
and documented for all adverse events. Evaluation of relatedness must consider 
etiologies such as natural history of the underlying disease, concurrent illness, 
concomitant therapy, study-related procedures, accidents, and other external 
factors.  
The following guidelines are used to assess relationship of an event to study 
intervention: 
1. Related (Possible, Probable, Definite) 
a. The event is known to occur with the study intervention. 
b. There is a temporal relationship between the intervention and event 
onset. 
c. The event abates when the intervention is discontinued. 
d. The event reappears upon a re-challenge with the intervention. 

 
 
 
12D.501 Protocol v5.2 
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42
 
 
2. Not Related (Unlikely, Not Related) 
a. There is no temporal relationship between the intervention and 
event onset. 
b. An alternate etiology has been established. 
 
Expectedness 
The PI is responsible for determining whether an AE is expected or unexpected. 
An AE will be considered unexpected if the nature, severity, or frequency of the 
event is not consistent with the risk information previously described for the 
intervention. Risk information to assess expectedness can be obtained from 
preclinical studies, the investigator’s brochure, published medical literature, the 
protocol, or the informed consent document. 
 
Severity of Event 
Adverse events will be graded for severity according to the Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.03. 
 
Intervention 
Any intervention implemented to treat the adverse event must be documented for 
all adverse events. 
 
Safety Reporting to IRB Unanticipated Problems 
 
All incidents or events that meet criteria for unanticipated problems (UAPs) as 
defined in Section 0 
Unanticipated Problems require the creation and completion of an unanticipated 
problem report form (OHR-20).  
UAPs that pose risk to participants or others, and that are not AEs, will be 
submitted to the IRB on an OHR-20 form via the eazUP system within 10 working 
days of the investigator becoming aware of the event.  
UAPs that do not pose risk to participants or others will be submitted to the IRB 
at the next continuing review.  
 
9.2 Reporting to SKCC DSMC 
 
All AEs and SAEs, safety and toxicity data, and any corrective actions will be 
submitted to the DSMC per the frequency described in the SKCC DSMP. The 
report to the SKCC DSMC will also include any unanticipated problems that in 
the opinion of the PI should be reported to the DSMC. 
For expedited reporting requirements, see table below:   
                                           
DSMC AE/SAE Reporting Requirements 

 
 
 
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44
 
 
hypothesis will be rejected if the 95% confidence interval for year DFS rate 
computed from the estimated Kaplan-Meier survival curves will be entirely above 
0.35.   
 
 
 
10.3 Sample Size  
 
This is a one arm study utilizing new methods to improve DFS. Assuming that 62 
patients will be accrued in 8 years and then followed for 1 more year there is an 
80% power to show that 1-year DFS is greater than 35% if the true 1-year DFS is 
50% or higher (calculations are based on the assumptions of uniform accrual over 
time, no loss to follow-up, exponentially distributed death times, and use of the 
exponential MLE one-sided test with alpha=0.05).  
 
 
 
10.4 Assessment of Other Secondary Objectives 
 
The secondary objectives of overall survival, regimen related toxicity, immune 
reconstitution, incidence and degree of GVHD, and engraftment rates will be 
analyzed and reported descriptively. 
 
 
10.5 Analysis for Safety 
 
Patient outcomes are routinely monitored in an ongoing fashion for all patients on 
investigational trials, beyond their formal endpoints.  Based on prior experience 
using a two-step approach similar to that described in this trial, the incidence of 
graft failure should be less than 10%, the incidence of severe GVHD should be 
less than 20%, and the non-relapse mortality should be less than 30% at 100 days.  
If at any point incidences higher than these thresholds are seen, that would trigger 
a protocol review to assess whether there are any obvious reasons for the inferior 
outcomes observed.  Depending on the results of the review, enrollment may 
continue on a limited basis with careful further observation, the protocol may be 
revised, or the protocol may be terminated.  Incidences will be calculated after 10 
patients are treated on this trial in order to have a sufficient denominator in which 
to examine outcomes based on percentages.  
 
 
10.6    Targeted Accrual Number 
 
The small number of patients undergoing matched sibling RIC HSCT in our 
transplant program precludes a separate research protocol for that group. To 
prevent withholding of transplant therapy, these patients will be treated on this 
protocol. Only the outcomes of the patient group undergoing HSCT from 
haploidentical donors (2, 3, or 4 antigen mismatches in the GVH direction) will be 
used in the analysis of outcomes for the statistical ends of the trial. Outcomes for 
patients with matched sibling donors will be reported descriptively.  
 

 
 
 
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“Therefore, when 62 evaluable patients undergoing haploidentical HSCT are treated on 
this protocol, the study will close to accrual. Up to 10 patients may undergo matched 
related donor HSCT on this protocol, but will not count toward the statistical ends of the 
study. These patients will be reported descriptively. Highest possible accrual number 
is 72 patients.” 
. 
 
 
 
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Cancer Chemother  Pharmacol. 2012;69(1):263-272. 
107. Bubalo JS, Kovascovics TJ, Meyers G, et al. 467: Clonazepam Plus 
Levetiracetam (CL) for the Prevention of Busulfan-Induced Seizures: A Single 
Center Experience. Biology of Blood and Marrow Transplantation. 2008;14(2, 
Supplement):165. 
108. Soni S, Skeens M, Termuhlen AM, Bajwa RPS, Gross TG, Pai V. 
Levetiracetam for busulfan-induced seizure prophylaxis in children undergoing 
hematopoietic stem cell transplantation. Pediatric Blood & Cancer. 
2012;59(4):762-764. 
109. Caselli D, Rosati A, Faraci M, et al. Risk of Seizures in Children Receiving 
Busulphan-Containing Regimens for Stem Cell Transplantation. Biology of Blood 
and Marrow Transplantation. 2014;20(2):282-285. 
110. Eberly AL, Anderson GD, Bubalo JS, McCune JS. Optimal prevention of 
seizures induced by high-dose busulfan. Pharmacotherapy. 2008;28(12):1502-
1510. 
  
12.0  Appendices 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
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12.1 Appendix A Guidelines for Total Body Irradiation 
Modality  
Photon irradiation is to be used for the TBI in all patients.  
 
Energy  
A linear accelerator with energy ≥ 6 MV may be used. Dose to superficial tissues 
near skin surface will be increased by using a beam “spoiler” lucite plate close to 
the patient. Since neoplastic infiltrates may be found in the skin, it is necessary for 
the superficial dose to satisfy the same total dose requirements as other locations.  
 
Geometry  
The treatment configuration shall be such that the patient is entirely included within 
the treatment beam. It is essential that the correlation between the light field and 
the radiation field be established and verified for extended TBI distances.  
 
Dose Rate  
A dose rate of 0.05 to 0.25 Gy/minute at the prescription point shall be utilized. The 
physicist of record, involved with TBI treatments, shall be consulted to achieve 
correct range of treatment dose rate.  
 
Calibration & Beam Data Verification  
The calibration of the output of the machine, used for this protocol, shall be verified 
on a daily basis prior to start TBI treatments. All dosimetric parameters, necessary 
for the calculation of dose delivered during TBI treatments, shall be measured at 
the appropriate treatment distance. They shall be documented and made available 
for calculation of every patient treatment.  
 
Treatment Volume  

 
 
 
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The patient shall be entirely included within the treatment beam. Care should be 
taken to guarantee that all of the patient is within the 90% decrement line at each 
depth. The 90% decrement line is defined as the line in each plane perpendicular 
to the central axis connecting the points which are 90% of the central axis dose, in 
that plane.  
 
Treatment Dose  
Prescription Point  
The prescription point is defined as the midplane point along the longitudinal axis 
at the level of the umbilicus.  
 
Dose Units  
All doses shall be specified in Gray (Gy) to muscle tissue. 
 
Tissue Inhomogeneity Considerations  
No inhomogeneity corrections shall be made in the calculation of the dose to the 
prescription point.  
 
Prescription Point Dose  
 
The total dose shall be 2 Gy.   
 
Time-Dose Considerations  
 
Dose Homogeneity  
The total absorbed dose along the patient's head to toe axis (in the midplane of 
the patient) shall not deviate more than 10% from the prescribed dose.  
Treatment Technique  
Treatment Fields  
Equally weighted parallel opposed portals shall be used. AP/PA fields shall be 
used.  
Field Size  
The collimation and treatment distance shall be such that the patient will be entirely 
included within the treatment beam and that no part of the patient extends beyond 
that region. The agreement of the light field and the radiation field should be 
checked periodically for the extended TBI treatment distance.  
Treatment Position  

 
 
 
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The patient shall be treated in any position that is compatible with the homogeneity 
requirement, allowing for the reproducibility of the patient setup and dosimetry.  
Field Shaping  
Patients will be treated with open fields.  
Calculations  
Central Axis Dose  
It is recommended that the dose calculation method be based upon measurements 
that are made in a unit density phantom with the following minimum dimensions:  
Length equal to top of shoulder to the bottom of the pelvis.  
Width equal to the patient width at the level of the umbilicus.  
Thickness equal to the typical patient thickness at the umbilicus.  
All measurements should be made at the appropriate extended SSD.  
Superficial Dose  
For the radiation beam with the Plexiglas plate in place, data should be available 
demonstrating that the skin dose is within 5% of the prescribed dose.  
Quality Assurance Documentation  
For purposes of quality assurance the following must be performed on every 
patient undergoing TBI:  
A check of the monitor unit calculation by a second physicist and a radiation 
oncologist prior to first treatment.  
 
 

 
 
 
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12.2 
Appendix B GVHD Grading System Grade 
 
 
Clinical Staging of Acute Graft-Versus-Host Disease 
 
Stage 
Skin 
Liver 
Gut 
+ 
Maculopapular rash < 
25% body surface 
Bilirubin, 2-3 mg/dl 
Diarrhea, 500-1,000 
ml/day or persistent 
nausea 
++ 
Maculopapular 
rash 
25-50% body surface 
Bilirubin, 3-6 mg/dl 
Diarrhea, 1,000-
1,500 ml/day 
+++ 
Generalized 
erythroderma 
Bilirubin, 6-15 mg/dl 
Diarrhea, > 1,500 
ml/day 
++++ 
Desquamation 
and bullae 
Bilirubin, > 15 mg/dl 
Pain +/- ileus 
 
Clinical Grading of Acute Graft-Versus-Host DiseaseStage 
Overall Grade 
Skin 
Liver 
Gut  
Functional 
Impairment 
0 (none) 
0 
0 
0 
0 
I (mild) 
+ to ++ 
0 
0 
0 
II (moderate) 
+ to +++ 
+ 
+ 
+ 
III (severe) 
++ to +++ 
++ to +++ 
++ to +++ 
++ 
IV (life-
threatening) 
++ to ++++ 
++ to ++++ 
++ to ++++ 
+++ 
Tables from Glucksberg H, Storb R, Fefer A, et al. Clinical manifestations of graft-versus-
host disease in human recipients of marrow from HL-A-matched sibling donors. 
Transplantation, 18: 295-304, 1974.