A Two Step Approach To Matched-Sibling Allogeneic Hematopoietic Stem Cell Transplantation for High-Risk Hematological Malignancies
Phase: PHASE2
Conditions: Hematological Malignancies, Leukemia, Lymphoma, Multiple Myeloma, Hodgkin's Disease
Sponsor: Sidney Kimmel Comprehensive Cancer Center at Thomas Jefferson University

Blood and Marrow Transplant Program 
 
Version 7.1 
Thomas Jefferson University Hospital 
September 13, 2017 
1 
 
 
A Two Step Approach To Matched-Sibling Allogeneic Hematopoietic Stem Cell Transplantation 
for High-Risk Hematological Malignancies 
 
Principal Investigator 
Neal Flomenberg, MD 
 
Co-Principal Investigator 
Dolores Grosso, DNP, CRNP 
 
Co-Investigators 
Matthew Carabasi, MD 
Joanne Filicko-O’Hara, MD 
Margaret Kasner, MD 
William O’Hara, Pharm-D 
John L. Wagner, MD 
S. Onder Alpdogan 
Beth Colombe, MD 
Ubaldo Martinez-Outschoorn, MD 
Neil Palmisiano, MD 
Sameh Gaballa, MD 
Pierlugi Porcu, MD 
 
Radiation Oncologists 
Maria-Werner-Wasik, MD 
Wenyin Shi, MD 
 
Biostatistics: 
Benjamin Leiby, Ph.D. 
 
 
Thomas Jefferson University 
Blood and Marrow Transplantation Program 
Division of Medical Oncology, Department of Medicine 
834 Chestnut St, Suite 320t 
Philadelphia, PA 19107 
 
Protocol Written By: 
Dolores Grosso, CRNP 

 
 
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Thomas Jefferson University Hospital 
 
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September 13, 2017 
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CONTENTS 
 
 
 
1.0      Objectives 
 
 
 
 
 
 
 
Page 3 
 
2.0  
Introductions and Rationale 
 
 
 
 
Page 3 
 
3.0      Patient Selection 
 
 
 
 
 
 
Page 14 
 
 
 
 
 
4.0     Informed Consent 
 
 
 
 
 
 
Page 16 
 
5.0  
Treatment Plan 
 
 
 
 
 
 
Page 17 
 
6.0  
Study Measurements 
 
 
 
 
 
Page 23 
 
7.0  
Supportive Care 
 
 
 
 
 
 
Page 25 
 
8.0  
Drug Information 
 
 
 
 
 
 
Page 27 
 
9.0  
Patient Safety 
 
 
 
 
 
 
Page 30 
 
10.0 
Statistical Measures  
 
 
 
 
 
Page 31 
 
11.0      References 
 
 
Page 34 
 
 
12.0 
Appendices 
 
 
 
 
 
 
 
Page 43 

 
 
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2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
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1.0 
Objectives 
 
This research protocol has been developed for patients undergoing matched-sibling 
hematopoietic stem cell transplant (HSCT).  The patients who are treated according to 
this 2 step allogeneic HSCT protocol will receive cyclophosphamide to induce in-vivo 
tolerization of both autologous and allogeneic lymphocytes, followed by an allogeneic 
CD34-selected HSCT. The primary research questions relate to immune reconstitution, 
incidence of GVHD, and relapse in patients who receive lymphocyte treatment of this 
type in allogeneic HSCT and how it impacts overall survival.  Our objectives are: 
 
Primary Objective 
1. 
To determine the overall survival in patients undergoing allogeneic HSCT on this 
protocol at one year post HSCT. 
 
Secondary Objectives 
2. 
To determine the incidence and severity of GVHD in patients undergoing 
allogeneic HSCT on this protocol. 
 
3. 
To assess the pace of lymphoid recovery in patients undergoing allogeneic HSCT 
on this protocol. 
 
2.0  
Introduction and Rationale 
 
High-dose chemoradiotherapy followed by hematopoietic allogeneic stem cell 
transplantation (HSCT) is a potentially curative modality for a variety of hematologic 
disorders, including acute and chronic leukemia, myelodysplastic syndrome (MDS), 
multiple myeloma (MM), and lymphoma that are incurable with conventional dose 
chemotherapy1. There are three major therapeutic components of a conventional 
allogeneic HSCT. The first component is the use of a high-dose myeloablative-
conditioning regimen to eradicate the underlying malignancy and to suppress the host 
immune system in preparation to receive the donor stem cell graft. This is followed by 
the infusion of donor stem cells to both rescue the host from the lethality of the 
conditioning regimen as well as to eliminate residual tumor cells and host resistance to 
donor stem cells by graft-versus-tumor reactions (GVT). The third component is pre-
grafting T-cell depletion of donor stem cells or post-grafting immunosuppression to 
regulate the development of graft-versus-host disease (GVHD).2,3  The consequence to 
one degree or another, of all types of transplants is a period of immune system 
vulnerability either from post-grafting immunosuppressant medication or the removal of 
T-cells from the donor inoculum.  During this period, patients are at risk for infection and 
relapse.  Patients undergoing matched sibling allogeneic HSCT have the lowest rates of 
GVHD, and consequentially can be removed from immunosuppressive medications 
sooner.  Decreased incidence of GVHD and more rapid immune reconstitution contribute 

 
 
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2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
to greater overall survival rates after matched sibling HSCT as compared to transplants 
utilizing alternate donor sources.  
 
 Overall Survival in Matched-Sibling Transplantation 
 
Although overall survival rates after matched-sibling allogeneic HSCT are superior to 
matched unrelated donor (MUD) HSCT, and partially-matched HSCT, there continues to 
be obstacles to long-term survival in this patient group. Age > 40 – 50 years at the time of 
HSCT4,5 and consequently older sibling donor age,6 disease type,7 advanced disease,4,5 
patient comorbidities,6,8,9,10 and an ECOG performance score of > 1 at the time of 
transplant6,9 can have adverse effects on survival after HSCT regardless of donor source.  
 
Marks et al11 analyzed 298 adult patients with acute lymphoblastic leukemia (ALL) in 
first or second remission (CR1 or CR2).  The patients were conditioned with 
cyclophophamide and total body irradiation (Cy/TBI) or etoposide and TBI.  At one year, 
the best overall survival rate was 80% in patients transplanted in CR1 with 
TBI/etoposide. For patients transplanted in CR2, the best one year overall survival was 
77% in the Cy/TBI group.  For patients transplanted in CR1, the best leukemia free 
survival (LFS) rate for one year was in patients conditioned with TBI/etoposide, and was 
79%.  For patients transplanted in CR2, the best LFS rate one year was 68%, and was 
achieved in the group of patients receiving TBI/etoposide.   The major single cause of 
death in this study was disease recurrence.  GVHD, infection, interstitial pneumonia, and 
organ failure were other major causes of treatment failure. In addition, the presence of 
cytogenetic abnormalities, including t (9:22), was associated with a greater risk of 
relapse.  
 
Lee et al12 examined survival rates in patients with acute myelogenous leukemia (AML) 
in first remission after matched-sibling HSCT.  The patients were stratified by the dose of 
CD34+ cells they received at transplant (<2.5 x 106/kg versus >2.5 x 106/kg).  At one 
year, patients receiving the higher cell dose had an overall survival rate of over 80% and 
a LFS rate of 80%. Patients transplanted with the lower CD34+ cell dose had an overall 
survival rate of over 75% at one year, and a LFS rate of over 65% at one year. In contrast 
to this study, Cook et al13 examined the survival of 44 patients post matched-sibling 
HSCT who had a history of primary induction failure AML.  The study patients obtained 
first remission only after 2 to 5 courses of chemotherapy.  In this higher-risk group of 
patients with AML, the one year overall survival rate was 67%, with adverse cytogenetics 
being significantly associated with poorer survival.  GVHD, infection and pulmonary 
toxicity were the major sources of treatment-related death. 
 
Runde et al14 analyzed the outcomes of 131 MDS patients reported to the Chronic 
Leukemia Working Party of the European Group for Blood and Marrow Transplantation 
(EBMT).  All of the patients were treated with bone marrow from matched-sibling donors 
and the majority (70%) received a TBI based conditioning regimen. T-cell depletion was 

 
 
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2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
the GVHD prophylaxis in 12%.  The majority of patients had low or high-risk MDS. Low 
risk MDS was defined as refractory anemia or refractory anemia with ringed sideroblasts. 
Higher-risk MDS was defined as refractory anemia with excessive blasts, blasts in 
transformation, or secondary AML.  Four patients had chronic myelomonocytic leukemia 
(CMML).  Median patient age was 33 years.  At one year, the best disease-free survival 
DFS was about 60% in the low-risk MDS patients.  Overall survival at one year was 
about 60% for patients transplanted within 4 months of diagnosis, but only about 30% for 
patients transplanted between 4 and 12 months after diagnosis.  In the other disease 
stages, DFS at one year was around 50% or lower. Two of the 4 patients with CMML 
died within 2 months of HSCT. The third patient with CMML died at 24 months, and the 
fourth patient was alive and disease-free at 62 months.  Pretransplant diagnosis was 
significantly associated with outcome, and acute GVHD and infection were the major 
causes of treatment related mortality. Two of the four CMML patients died of relapsed 
disease.  In a large multicenter retrospective study of patients with MDS, Guardiola et 
al15 compared the outcomes of patients undergoing matched-sibling HSCT who received 
either peripheral blood progenitor cells (PBPC) versus bone marrow from their donors.  
The International Prognostic Score (IPS) for the majority of patients in this study was 
intermediate-2 or higher, and less than half the patients received pretransplant 
chemotherapy. Most patients were older than 35 years.  Conditioning regimens were 
variable with slightly less than half the patients receiving a TBI-based regimen.  The 
overall survival at one year for patients receiving PBPC versus bone marrow grafts was 
60% or greater for both groups, and the event free survival at one year was 60% and 45% 
respectively. Although the use of PBPCs was associated with a 10% increased early 
treatment mortality due to acute GVHD, the 2-year outcome was significantly better with 
the use of a PBPC graft. 
 
Several studies have examined the outcome of patients with myeloproliferative disorders, 
excluding CML, after HSCT. In a retrospective study from the EBMT and Fred 
Hutchinson Cancer Center, Guardiola et al16 examined the outcomes of 61 patients, the 
majority of whom underwent a matched-sibling HSCT (6 patients received matched 
unrelated donor grafts), with agnogenic myeloid metaplasia (AMM).  Patients without 
extensive marrow fibrosis had a one year overall survival of 85%, in contrast to 55% in 
patients with more significant marrow fibrosis.  GVHD was a significant contributor to 
transplant-related mortality. Daly et al17 examined the outcome of 25 patients with AMM 
and essential thrombocytosis (ET) who underwent HSCT mostly with Cy/TBI 
conditioning. The stem cell source was mostly bone marrow; with half of the patients 
receiving cells from a matched sibling donor (most other patients received cells from a 
matched unrelated donor).  The one year non-relapse mortality rate was 48.3% for this 
group.  Although source of stem cells was not found to have a significant effect on 
outcome, it is conceivable that the overall survival in this study was poorer than the one 
in the Guardiola study because the use of unrelated donors was associated with a higher 
incidence of GVHD.  Like the Guardiola study, GVHD was a contributor to toxicity, and 
was significantly associated with prior splenectomy.  Finally, Kerbauy et al18 reported on 

 
 
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2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
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the outcomes of 43 patients with CMML undergoing HSCT at the Fred Hutchison Cancer 
Center.  About half of the patients received a TBI-based conditioning regimen, and about 
half the patients received progenitor cells from a matched-sibling donor.  Older age and 
poorer performance status were associated with increased mortality. Overall survival at 
one year was close to 60% for those with a better performance status and only about 25% 
for the rest.  Patients who received unrelated donor grafts were the largest group alive at 
the time of publication, although stem cell source was not reported to have an impact on 
overall survival.  GVHD, multi-organ failure and pulmonary toxicity were the major 
causes of death. 
 
In a report from the International Bone Marrow Transplant Registry (IBMTR), Freytes et 
al19 examined the outcomes of 114 patients with low, intermediate, and high-grade Non-
Hodgkin Lymphoma (NHL) or Hodgkin Lymphoma (HL) who underwent allogeneic 
HSCT for relapse disease after autologous transplant.  Sixty-one percent of the patients 
received stem cells from a matched-related donor.  The one year overall survival for this 
group of patients was close to 43% and was significantly better than patients who 
received grafts from other sources. TBI-based conditioning regimens and complete 
remission at the time of HSCT were also significantly associated with a better outcome. 
Peniket et al20 reported on an EBMT registry matched study that compared outcomes of 
1185  allogeneic stem cell transplants to 14,687 autologous transplants from 1982 to 1998 
in patients with low grade NHL, intermediate grade NHL, high-grade NHL, Burkitt’s 
lymphoma, and Hodgkin disease.  In each disease subcategory, more than 81% of the 
donors were matched-siblings. The majority of patients in the allogeneic arm had 
advanced disease.  A matched analysis showed that overall survival was superior in the 
autologous arm for every disease category despite better relapse rates in many categories 
of lymphoma.  Poorer outcomes in the allogeneic arms were attributed to advanced 
disease stage and chemo-resistance at the time of HSCT, grades III and IV GVHD, and 
treatment related mortality.  Overall survival at 1 year was about 60% for low grade 
lymphoma, about 50% for the other lymphomas, and about 40% for Hodgkin disease. 
 
Akpek et al21 examined the long-term results of 157 patients with relapsed Hodgkin 
disease who underwent autologous or matched-sibling allogeneic transplant using various 
conditioning regimens.  Fifty-three patients received matched-sibling allografts and had a 
42% probability of survival at one year and a 36% probability of event-free survival at 
one year.  GVHD was the most common cause of treatment-related mortality, and having 
disease that was still sensitive to chemotherapy was associated with a better event free 
survival.  
 
Several large studies have examined outcomes of patients after matched-sibling HSCT 
with heterogeneous diagnoses.  Retrospective data reported to the IBMTR and EBMTR 
was analyzed by Champlin et al22 for patients undergoing matched sibling HSCT for 
ALL, AML, and CML.  The purpose of the study was to compare outcomes for 536 
patients receiving bone marrow versus 288 patients receiving blood stem cells from their 

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
donors.  All patients with acute leukemia were in first or second CR, and patients with 
CML were in chronic or accelerated phase.  The patients received a variety of 
conditioning regimens, with 40% of the patients receiving TBI.  The probability of LFS 
for patients with acute leukemia in first CR at one year was 70% with blood stem cells 
and 61% with bone marrow.  Probability of LFS among patients with acute leukemia in 
2nd CR was 77% with blood stem cells and 57% with bone marrow.  The probability of 
LFS among patients with CML transplanted in chronic phase was 63% with blood stem 
cells and 74% with bone marrow.  Patients with CML transplanted in accelerated phase 
or second chronic phase had a probability of LFS at one year of 68% with blood stem 
cells and 23% with bone marrow.   For patients with advanced disease, blood stem cell 
products were associated with a better outcome. 
 
The Stem Cell Trialists’ Collaborative Group23 performed an individual patient data 
meta-analysis of nine randomized trials involving 1,111 adult patients undergoing HSCT 
who received peripheral blood stem cells versus bone marrow from their donors.  All of 
the studies required an HLA-matched sibling donor except for one of the smaller studies 
(N=60) which allowed a one mismatched family donor.  Diagnoses included both early 
and late stage AML, ALL, CML, myeloma, NHL, Hodgkin disease, MDS, and 
myelofibrosis.  Disease-free survival (DFS) at one year was between 60 and 70% for both 
stem cell and bone marrow recipients.  In early stage disease, one year overall survival 
for both groups was between 75 and 80%.  Patients who had later stage disease had a one 
year overall survival of about 58% for blood stem cell recipients versus 48% for bone 
marrow recipients.  This was related to a higher incidence of relapse among patients 
receiving stem cells from bone marrow.  At 3 years, relapse rates, DFS, and OS were 
improved for patients with late stage disease receiving blood stem cell transplants.  The 
use of blood stem cells was associated with a significantly increased risk of acute and 
extensive chronic GVHD. 
 
Finally, Alyea et al24 examined the outcomes of 152 patients over the age of 50 
undergoing HLA-matched HSCT who received a myeloablative versus a non-
myeloablative transplant.  There were 81 patients in the myeloablative arm, 64% of 
whom had matched-sibling donors.   Diagnoses included AML, CML, ALL, MDS, NHL, 
and CMML.  Patients had early and late stage disease, and almost all of the patients 
received CY/TBI for conditioning.  Overall survival at one year was 38%, and 
progression-free survival at one year was 36%.  GVHD, pulmonary complications, and 
infection were major contributors to transplant-related mortality.    
 
Based upon the results discussed above, younger patients with early stage leukemia can 
expect a one year DFS of 70% to 80%.  Older patients, patients in later stage remission or 
relapse, patients with lymphoma, later stage MDS or myelofibrosis have much lower 
survival rates and should be considered a high-risk group.  With the rare exception of 
disease status, none of these adverse risk characteristics can be changed. As reflected in 
the above literature review, one year DFS percentages for these higher-risk patients are 

 
 
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Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
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between 35-60%. As opposed to pre-transplant risk factors, post-transplant complications 
have the potential to be avoided or abrogated with improved conditioning regimens.  
Three major sources of post-HSCT mortality are infection, relapse and GVHD. All three 
of these complications are to some extent related to T-cell function post-transplant.  New 
methods of transplantation that lead to earlier lymphoid recovery without significant 
GVHD post HSCT may attenuate these complications and result in better outcomes.  At 
one year post transplant, a regimen that could improve DFS percentages in high-risk 
patients to at least 60% would demonstrate efficacy in terms of decreasing treatment-
related mortality, early infectious death, early relapse, and acute GVHD.   
 
Strategies to Improve HLA-Matched Related HSCT 
 
If there was a mechanism to manipulate the donor lymphocyte content of the graft in such 
a way that the donor lymphocytes were made tolerable to the host, morbidity from 
GVHD would likely decrease without affecting the benefits of lymphoid reconstitution 
relating to infection and relapse.  There is data regarding the immune modulating effects 
of many drugs, such as cyclophosphamide (CTX) and GM-CSF that may be exploited to 
create immune tolerance of donor lymphocytes in the transplant inoculum.  CTX has 
been a backbone of allogeneic conditioning for years, however most of its use has been 
prior to the infusion of donor cells, and thus tolerizing effects on donor T lymphocytes 
have not been realized.  An HSCT regimen that allows donor cells to be given in two 
steps, the lymphocyte infusion and the stem cell infusion, would provide an opportunity 
to tolerized donor lymphocytes without affecting donor stem cells. 
 
Cyclophosphamide  
CTX is an alkylating agent used extensively in the treatment of malignancies in both 
transplant and non-transplant settings.  Athough CTX has direct effects on tumor cells, its 
modulation of T-cell responses to tumor have also documented.  CTX was shown to 
cause immunologically mediated regression of CTX-resistant L5178Y lymphoma in mice 
by eliminating tumor-induced suppressor T-cells which resulted in an increased number 
of Lyt-2+ T-cells capable of augmenting immunity and causing tumor regression.25.26  
The direct cytotoxic effect of CTX on malignant cells, as well as the adoptive 
immunotherapeutic properties of CTX against tumor cells, make it an effective anti-
cancer therapy. 
 
CTX is also known to be preferentially cytotoxic to activated T-cells and to induce clonal 
deletion of these cells in the periphery and thymus.27 It is this aspect of CTX’s 
immunomodulatory effects that may prove beneficial in HSCT.    
 
Much of what is known about the immunomodulatory effects of CTX come from murine 
systems of CTX-induced tolerance.  In one such model, called a “cells-followed-by-CTX 
system,”28-30 CTX is given 1 to 3 days after antigenic stimulation with allogeneic cells 
(usually spleen cells).  In this type of system, tolerance to tumor, skin, or other solid 

 
 
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Thomas Jefferson University Hospital 
 
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September 13, 2017 
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organs can be induced.31 The infusion of allogeneic cells results in a proliferative 
response in mature T and B cells after exposure to alloantigen.  The DNA in the 
proliferating cells is especially sensitive to CTX, therefore alloreactive clones are 
preferentially destroyed, including donor cells which might mediate GVHD and host cells 
which might mediate rejection.32 In the cells-followed-by CTX murine system, 
intrathymic chimerism also occurs when donor type cells arising from stem cells 
contained in the infused spleen or marrow cells 33 are regenerated.  As a result, 
subsequent intrathymic clonal deletion of donor-antigen reactive T-cells occurs.  Thus, 
alloantigenicity and hematopoietic regenerative capability are necessary for long-lasting 
tolerance.33,34 One possible difficulty in applying this type of immunological approach to 
human matched-sibling HSCT is that in the murine haploidentical models tolerance 
induction relies on a disparity between donor and host lymphocytes.34-38 In the matched 
sibling setting, this disparity is not as great and therefore the activation and subsequent 
deletion of reactive lymphocytes may not be as profound. The effectiveness of CTX 
given after lymphocytes to induce tolerance in a human matched-sibling setting is 
unknown. 
 
The establishment of tolerance to allografts with the use of CTX has been demonstrated 
by many other investigators.  Allogeneic tolerance was established in diabetic mice when 
simultaneous transplantation of islet cell and bone marrow was performed just after 
treatment with fludarabine and cyclophophamide.39 Zhang et al, 40  demonstrated CTX 
induced tolerance in C3H mice to skin and heart allografts from H-2 matched AKR mice 
after priming the recipient mice with donor spleen cells followed by intraperotineal CTX.  
In this study, the clonal destruction of MIs-1a-reactive CD4+ Vbeta 6+ T-cells in the 
periphery, and the clonal deletion of Vbeta 6+ thymocytes were observed. Luznik et al, 41 
demonstrated that the dose of TBI in allogeneic marrow transplantation in mice could be 
reduced from 500 cGy to < 200 cGy with the addition of post-transplant CTX.  Animals 
that were conditioned with TBI alone or given post-transplant CTX alone failed to 
achieve engraftment and contained host anti-donor cytotoxic T-cells.   
 
CTX-induced tolerance in rat transplantation has also been demonstrated when CTX is 
given after allogeneic stimulation.  Okano, S et al, 42 gave Lewis recipients spleen and 
bone marrow cells from Dark Agouti (DA) donors followed by CTX 2 days later.  DA 
livers were then grafted on d+25.  Rats survived for more than 165 days with donor-
specific tolerance confirmed by second skin grafts.  In other studies with rats, fetal small 
bowel, heart, and kidney allografts were successfully placed with the establishment of 
CTX-induced donor tolerance. 43-45  
 
In human T-cells responding to LCL cell lines, CTX induced apoptosis and modulation 
of activation and effector functions.  Strauss et al, 27 examined the mechanisms of action 
of various immunosuppressive agents on LCL line 721.221, CIR, and the mouse 
mastocytoma cell line P1.HTR.  In these studies, CTX was found to induce massive 
apoptosis in activated T-cells.  In the same study, CTX was found to accelerate CD95-

 
 
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mediated apoptosis in human T-cells and suppress activation of naïve T-cells at the 
initiation step of an allo-response without affecting cytokine production. This finding 
suggests that CTX may induce deletion of antigen-specific T-cells.  
 
In human clinical treatment, CTX has been used successfully with and without other 
immunosuppressive agents to treat a variety of autoimmune disorders.  CTX has selective 
immune effects in multiple sclerosis by suppressing IL-12 and Th1-type responses and is 
effective in worsening MS that has an inflammatory component due to its ability to ablate 
autoaggressive lymphocytes.46  Traynor et al used CTX in the setting of autologous HSCT 
to obtain remission in patients with end-stage lupus.47 In this study, patients had 
spontaneous decline or normalization of the T-cell activation marker, CD 69+ after the 
therapy without an excessive rise of this marker upon stimulation.  CTX has also been 
used successfully to treat acquired hemophilia,48  rheumatoid arthritis, 49 and autoimmune 
hepatitis.50 High-dose CTX has also been used in the treatment of aplastic anemia 
because of its ablative effect on activated lymphocytes.  In this setting hematopoietic 
stem cells are spared because they contain aldehyde dehydrogenase, an enzyme which 
degrades cyclophosphamide thus providing partial protection to the stem cells from the 
cytotoxic effects of CTX.51 
 
Data and experience with CTX-induced tolerance in human allogeneic transplantation is 
limited. Luznik et al, 52 reported on 46 patients who received high dose CTX after 
matched related and unrelated HSCT as the sole agent for GVHD prophylaxis. The 
cumulative incidence of grades III-IV GVHD in this group was only 9%.   In partially-
matched related HSCT, O’Donnell et al, 53 reported on 10 patients receiving CTX after 
the infusion of donor cells.  Although follow up was short, four patients were alive 
without significant GVHD.   
 
These human studies provide proof of principal of the tolerizing effects of CTX.  In both 
studies, CTX was given after the infusion of the donor product.  One potential 
deleterious effect of this method is the effect that CTX may have on the progenitor cells 
contained in the product.  Aldehyde dehydrogenase contained in progenitor cells is 
thought to be protective against the effects of CTX, and unquestionably, count recovery 
is possible after high doses of CTX.  However there is data that demonstrates both 
negative effects of CTX on progenitor cells and variable patient response to the effects of 
CTX.  Studies examining the effects of 4-hydroperoxycyclophosphamide (4HC) on 
human progenitor cells in marrow purging demonstrate dose-related inhibition of 
hematopoietic colony formation, 54 significantly increased times to leukocyte recovery 
after transplant, 55 and interpatient variability to the effects of 4HC on CFU-GM 
recovery. 56 In addition to the short term effects of CTX on progenitor cells, this 
cytotoxic agent has also been implicated in the development of serious late effects on 
hematopoietic progenitor cells, including acute leukemia 57 and MDS.  Le Beau et al 58 
reviewed the clinical course of 63 patients with therapy-related MDS or AML. Twenty-
one of these patients developed their secondary malignancy after chemotherapy alone.  

 
 
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Seven of the 21 patients had received therapy with CTX, including one patient who had 
received CTX alone.  In contrast, secondary lymphomas have been far less common.   
 
Based on these studies, an optimal method to establish donor tolerance without exposing 
donor progenitor cells to CTX in allogeneic HSCT may be to establish a 2-step transplant 
process.  In this scenario, donor lymphocytes would be given first, followed three days 
later (after activation) by CTX similar to the “cells-followed-by-CTX” model discussed 
above.  As a second step, CD34+ selected progenitor cells would be infused after CTX 
has been metabolized.  Thus the benefit of CTX elimination of alloreactive lymphocytes 
could be realized, while the hematopoietic progenitors would be spared CTX exposure.  
Here at Jefferson, this 2 step model of HSCT has been introduced for patients undergoing 
partially-matched related HSCT.  Of the 7 patients treated so far, there has only been one 
case of serious (grade III) GVHD, which as promptly responded to steroids.  
 
Growth Factors 
Another potential strategy to manipulate donor lymphocytes is through the use of growth 
factors which are known to affect their cytokine secretion profile as they mature after 
recognizing antigen. This has been demonstrated in CD4+ and CD8+ subsets of T-
lymphocytes as well as in NK-cells in humans and murine models.59-62 The 
differentiation takes place either towards a type-1 or a type-2 cytokine secretion profile. 
G-CSF (granulocyte-colony stimulating factor) has been shown to skew lymphocytes 
toward a type 2 secretion profile whereas GM-CSF (granulocyte-macrophage-colony 
stimulating factor) skews them toward a type 1 profile.  
  
It has been shown for CD4+ and CD8+ subsets of T-cells that type-1 cytokine-secreting 
cells (termed Th1 & Tc1 cells) predominantly secrete -interferon and mediate and 
escalate GVHD by recruiting secondary effector cells that include cytotoxic T 
lymphocytes (CTLs), NK-cells and macrophages that kill host cells. In addition, these 
type-1 cytokine-secreting T-cells directly mediate host cell apoptosis by utilizing the 
perforin-granzyme pathway as well as by engaging the Fas (CD95) receptor on the host 
target cell membrane. On the other hand, type-2 cytokine secreting cells (Th-2 and Tc-2) 
that predominantly secrete IL-4, IL-5 and IL-10 tend to inhibit GVHD.63  
 
 
G-CSF  
Peripheral blood stem cell products from granulocyte-colony stimulating factor-
mobilized donors contain a ten-fold higher dose of T-cells than products obtained from 
bone marrow harvest.  Despite this difference in T-cell content, the rates of acute GHVD 
are the same for recipients of PBSC products as they are for those who receive marrow 
products.  Arpinati et al 64 enumerated dendritic cell subsets in peripheral blood products 
from normal donors treated with G-CSF versus bone marrow products from unstimulated 
donors.  They demonstrated that DC-2 dendritic cells were found in much higher 
numbers in the G-CSF –mobilized product, and that this resulted in polarization of T-
cells toward a Th2 response.  DC1 content was the same for both products.  Franzke and 

 
 
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September 13, 2017 
Version 7.1 
 
colleagues 65 showed that a G-CSF receptor can be induced on T-cells, and have 
functional activity, when T-cells are stimulated with G-CSF.  Thus G-CSF’s effects on 
polarization of T-cells towards a Th-2 subtype is both direct and indirect.  Th-2 cells are 
associated with less cytotoxic activity and proliferative response than Th-1 cells.  It is this 
imbalance in Th-1 and Th-2 subsets that is thought to be responsible for the mitigation of 
GVHD despite the use of these T-cell rich grafts.   
 
In the setting of allogeneic HSCT, one could argue that the use of a G-CSF-primed donor 
product may be optimal to decrease the incidence of GVHD.  The use of this product is a 
double-edged sword however.  Volpi and colleagues, 66 compared the lymphocyte 
profiles of 2 groups of patients post T –cell depleted partially-matched related donor 
HSCT.  The first group contained 36 patients who received post-transplant G-CSF.  The 
second group (43 patients), were not given G-CSF at any time. The investigators found 
that the patients receiving post-transplant G-CSF had impaired production of interleukin 
12 by dendritic cells and delayed recovery of T-cells with functional Th1 reactivity. This 
impairment of functional immune recovery was long-lasting and was expected due to the 
use of G-CSF.  Although not reaching statistical significance, the group receiving post-
transplant G-CSF had an infection-related mortality rate of 35% as compared to the 25% 
infection-related mortality rate of the group that did not receive G-CSF.  GVHD was not 
significantly affected in either group.  In HSCT, early immune function is dependent 
upon the expansion of a donor lymphocyte population infused with the graft until de novo 
production of naïve T-cells occurs.  Skewing the available T-cell population towards a 
Th2 subtype may magnify defects in reconstitution Therefore, it may be reasonable to 
assume that the benefits of using G-CSF to decrease GVHD in this setting may be 
outweighed by the risks of infection related to the lack of T-cell cytotoxic activity.    
 
 
GM-CSF 
GM-CSF induces the growth and differentiation of several different cell lines including 
those of neutrophils, eosinophils, and monocytic colonies. 67 The use of GM-CSF has 
been shown to decrease the duration of severe neutropenia, and consequently decrease 
fatal infections after AML induction therapy. 68 In allogeneic transplantation, GM-CSF 
therapy significantly shortened the duration of neutropenia, decreased length of stay, and 
reduced the use of antimicrobials. 69   
 
Whereas G-CSF does not have a significant effect on monocytes or macrophages, GM-
CSF has a stimulatory effect on Th1 lymphocyte-inducing dendritic cells which 
facilitates cell-mediated immune responses.  It is this property of GM-CSF that seems to 
convey superior infectious protection versus G-CSF, beyond that of simply decreasing 
the period of neutropenia. 70 For example, Peters et al 71  found that patients undergoing 
high-dose chemotherapy with or without auto PBSC who received filgrastim or no 
growth factor had a 4.2 times greater risk of developing a fungal infection than those 
patients who received GM-CSF.  Because GM-CSF is known to enhance the functional 

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
effects of monocytes, macrophages and dendritic cells, 72 its use has also been associated 
with anti-tumor activity both alone 73 and in combination with monoclonal antibodies74,75 
and vaccine therapy.76   
 
In the setting of allogeneic HSCT, the use of GM-CSF in donor PBSC mobilization 
would be expected to produce a skewing of TH1/Th2 balance of the donor lymphocytes 
contained therein toward a TH1 pattern, reciprocal to G-CSF’s effects.  Presumably, this 
would result in a greater ability of donor lymphocytes to mount an inflammatory 
response which may help fight infection.  However, a GM-CSF induced imbalance may 
favor the development of GVHD.  Therefore, the avoidance of any growth factor effect 
during collection of the lymphocytic portion of the transplant product may be the most 
beneficial option.  Post-transplant, the use of GM-CSF appears to convey a degree of 
infectious protection and anti-tumor activity that is not afforded by G-CSF by virtue of its 
stimulation and enhancement of cell lines other than neutrophils.  Therefore, in patients 
receiving allogeneic HSCT, post-transplant GM-CSF may help reduce the morbidity 
associated with this type of procedure. 
 
  
Strategy For the Current Protocol - A Two-Step Approach to Partially-Matched Related 
Donor HSCT 
In the following protocol, we seek to decrease complications of matched sibling HSCT in 
high-risk patient groups and increase disease-free and overall survival.  In order to 
accomplish this, patients on this protocol will receive their transplant in 2 steps.  Many of 
these patients have been heavily pretreated and may be chemotherapy-resistant.  
Therefore, a TBI-based regimen will be used in this protocol.  The combination of TBI 
and cyclophosphamide is one of the most durable and successful transplant conditioning 
regimens and will be used as the basis of this therapy.  TBI will be administered first in 
this protocol.   Next, a fixed dose of donor lymphocytes, collected prior to any donor 
growth factor exposure, will be infused just after TBI in order to avoid graft rejection and 
post-transplant infection.  The starting dose of lymphocytes will be 2 X 10e8.  This dose 
is based upon our already established experience with this type of transplant in partially-
matched related HSCT and as of this writing, has successfully obtained engraftment in 7 
patients.  Because the donor will have lymphocyte collection prior to G-CSF 
administration, the infused lymphocytes will express a balanced cytokine profile which 
will hopefully allow a better immune response to infection.  Three days after the donor’s 
lymphocytes are infused we will attempt to establish donor-host tolerance, minimizing 
the risks of both graft rejection and severe GVHD by administering two doses of 
cyclophosphamide to the patient.  After collection of the lymphocytes, donors will then 
begin priming with G-CSF, in preparation for a PBSC collection which will allow a 
higher CD34+ content than that obtainable through marrow harvest.  The last step of the 
transplant process, the infusion of donor progenitor cells, will occur two days after 
cyclophosphamide.  In this way, donor progenitor cells will not be exposed to the toxic 
effects of CTX.  These PBSC will be CD34+ selected to remove contamination by any 

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
additional T cells which would not be in vivo treated with CTX by virtue of their infusion 
with the CD34+ cells after CTX therapy.   
 
An important aspect of this two step approach is that it separates the dosing of T cells and 
hematopoietic stem cells in ways not possible with other approaches.  Normally these two 
cellular populations are contained in the same transplant product and the doses of these 
cells cannot be independently controlled.  This two step approach allows us to give the 
entire innoculum of hematopoietic stem cells, but to vary the T cell dose using the 
method of continuous reassessment to define the optimal T cell dose which will 
maximize engraftment while minimizing GVHD.  This will hopefully allow the outcome 
of therapy to become more reproducible one patient to the next.   
 
In summary, this two-step transplant process will: 
 
Allow us to define and then consistently utilize a fixed, optimized dose of 
lymphocytes 
Avoid the effects of G-CSF on donor lymphocytes 
Promote the establishment of donor-recipient tolerance by in vivo CTX treatment 
Increase the amount of donor progenitor cells collected by using PBSC 
mobilization as opposed to OR harvest 
Prevent the exposure of donor progenitor cells to cyclophosphamide 
 
This is a phase II protocol with the primary objective being an increase in disease-free 
survival at on year in a high-risk patient population. Any patient who has failed standard 
treatment for their disease may be treated on this protocol.  However, only patients 
designated as high-risk will be assessed in terms of efficacy of this treatment. 
 
3.0   
Patient Selection 
 
 
Inclusion Criteria 
 
1) Any patient with a hematologic or oncologic diagnosis in which allogeneic 
HSCT is thought to be beneficial, and in whom front-line therapy has already 
been applied. Patients will be considered high-risk if they have any of the 
following: 
a. Age > 50 years 
b. ECOG Performance status of  <2  
c. Acute leukemia: requiring more than one chemotherapy regimen to 
obtain 1st CR; second or greater CR, 1st relapse; any ph+ ALL 
d. CML 2nd chronic phase, accelerated phase, or blastic phase 
e. MDS with IPS of Intermediate 2 or greater 
f. Any myeloproliferative disorder 
g. Hodgkin lymphoma: relapsed, refractory, or primary induction failure 

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
h. Non-Hodgkin lymphoma: relapsed, refractory, primary treatment 
failure, or not eligible for an autologous HSCT 
i. Other conditions not listed will be assessed as high-risk by the PI 
    
2) Patients must have a related donor who is either HLA-identical or a one 
antigen mismatch at the HLA- A; B; C; and DR loci.  
 
3) Patients must adequate organ function: 
 
 
 
 
a. LVEF of >45% 
 
b. DLCO (adjusted for hemoglobin) >45% of predicted   
 
c. Adequate liver function as defined by a serum bilirubin <1.8, AST or 
ALT < 2.5X upper limit of normal 
 
d. Creatinine clearance of > 60 ml/min 
 
4) Patients must be willing to use contraception if they have childbearing 
potential 
 
5) Able to give informed consent  
 
 
 
Patient Exclusion Criteria 
 
1)  ECOG performance status of 3 or 4. 
 
2) HIV positive 
 
3) Active involvement of the central nervous system with malignancy 
 
4) Psychiatric disorder that would preclude patients from signing an informed 
consent 
  
5) Pregnancy  
 
6) Patients with life expectancy of < 6 months for reasons other than their 
underlying hematologic/oncologic disorder.   

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
 
 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 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 
 
Patients referred for the trial will have their eligibility criteria verified. On 
meeting the eligibility for the trial as outlined, informed consent will be obtained 

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
using forms approved by the Thomas Jefferson University Hospital 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 after the consent interview.   
 
5.0  
 
Treatment Plan 
 
 
 
Proposed Schema   - Patient 
 
-9 
Tues 
-8 
Wed 
-7 
Thu 
-6 
Fri 
-5 
Sat 
-4 
Sun 
-3 
Mon 
-2 
Tues 
-1 
Wed 
0 
Thu 
AM 
TBI 
TBI 
TBI 
TBI 
Rest 
Rest 
Cy 60 
mg/kg 
Cy 60 
mg/kg 
Tacrolimus 
&MMF* 
 
CD 34+ 
selected 
HSCT 
PM 
TBI 
TBI 
TBI 
TBI 
DLI 
 
 
 
 
 
 
Proposed Schema   - Donor 
 
-7 
Thu 
-6 
Fri 
-5 
Sat 
-4 
Sun 
-3 
Mon 
-2 
Tue 
-1 
Wed 
AM Lymphocyte 
Collection 
Lymphocyte 
Collection 
(If Needed) 
Neup** 
Neup 
Neup 
Neup 
PBSC 
Collection 
Neup 
PBSC 
Collection 
PM 
 
 
Neup 
Neup 
Neup 
Neup 
 
*Mycophenolate Mofetil (MMF) 
**Neupogen (Neup) 
 
There should be no administration of agents that suppress lymphocyte 
reactivity from admission until day -1 in this protocol.  This includes steroids, 
calcineurin inhibitors, MMF, or monoclonal antibodies that affect lymphocyte 
number or function.  If patients have previously required steroids as a 
premedication for transfusion, they may receive a dose of steroid equivalent to 5 
mg of prednisone through day -7. After day -7, the majority of TBI is complete.  
At this time, the immune system response to alloantigens should be somewhat 
attenuated.  Diphenhydramine and meperidine may be used if necessary.  Any use 
of steroids from day -6 through day 0 should not be administered without 
approval from the PI. 
 
5.1 TBI 
 
 
 
TBI will be administered twice daily for 4 days (8 fractions) on days -9 through -
6. 
TBI will be utilized for all patients eligible for this protocol unless they have 
received prior irradiation.  Prior irradiation will be evaluated by the radiation 

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
oncologist to define eligibility for this TBI schedule. In addition there may be 
technical or patient related factors which will require some minor modification in 
the TBI technique utilized. Selected patients may require local boosting of certain 
organ sites prior to conditioning therapy. Deviations from the guidelines 
described here may only be performed with the approval of the radiation 
oncologist and the investigators. See Appendix A for radiation guidelines. 
 
 
5.2  Donor Lymphocyte Infusion 
 
The dose of the donor lymphocyte infusion (DLI) will be based on CD3+ T cells 
per kilogram of recipient body weight. 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 
donor T-cells will be collected prior to the use of G-CSF for progenitor cell 
collection.  An exact dose of 2 X 10e8/kg lymphocytes will be infused.  Donors 
will be apheresed for lymphocytes on days -7 and -6. 
 
For the donor lymphocyte apheresis, total blood volumes to be processed will be 
determined using the following calculation: 
 
Recipient weight in kg:                    __________kg 
Multiply by desired CD3+ cells/kg:  x __________x10(7)/kg 
Total CD3+ cells requested:          = __________x10(7) 
Multiply by 2                              x 2 
TOTAL mononuclear cells (TMC)     = __________x10(7) 
Divided by 100 x 10(&) TMC/L        = _____ Liters processed 
 
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.  
 
The following guidelines should be used to calculate the correct volume of blood 
to be obtained from the donor to achieve the target T-cell dose.  
 
An aliquot of the apheresis product will be assessed for CD3 content by flow 
cytometry. The following cell panel will be used:  

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
 
 
ITC  
PE  
gG1  
IgG1  
gG1  
IgG2a  
D45  
CD14 
+ CD13  
D3  
CD4  
D3  
CD8  
D3  
CD16 
+ CD56  
D3  
CD19  
D4  
CD8  
 
A gate is drawn around the entire CD45+ population. %WBC/total events = the 
percentage of CD45+ cells within this gate corrected for the isotype control. CD3 
percentages are calculated, corrected for the isotype control, based on the total 
white cell (CD45+) gate, not based on a “lymphocyte gate”. There are 4 CD3 
counts performed in the panel. The two median values are averaged to determine 
the final raw CD3 count. The raw CD3 count is then corrected for any counted 
events which are not WBC (i.e. CD45-), as follows:  
 
Corrected %CD3 = (raw CD3 count)/ (%WBC/total events).  
Total T-cells required for the initial infusion = (2x10
8 
T-cells/kg) * (Weight in kg) 
T-cells/ml of product  
T-cells/ml of product = (WBC) * (Corrected %CD3)  
Volume to be infused = (Total T-cells required for the initial infusion)/(T-cells/ml 
of product)  
 
Lymphocyte apheresis will be performed at Thomas Jefferson University Hospital 
or the American Red Cross, by trained apheresis personnel using standard 
techniques. No hematopoietic growth factors will be administered to apheresis 
donors prior to lymphocyte collection. The donor will have venous catheters 
placed in each arm for the purposes of undergoing leukopheresis. Leukocyte 
collections will be performed using a standard apheresis machine such as the 

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
Cobe Spectra apheresis instrument (Cobe Laboratories Inc., Lakewood, CO).  
 
During the infusion, the patient will be monitored for any untoward reactions. 
Each infusion will take place in the Bone Marrow Transplant Unit. Donor 
lymphocyte infusions will be 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.  
 
 
5.3 Cyclophosphamide 
 
 
 
CTX 60 mg/kg IV over 2 hours 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 beginning with CTX on day -3.  Day –1 is a day of 
rest. 
 
Voriconazole can block the conversion of CTX to its active metabolite, 4-
hydroxycyclophosphamide.  For this reason, no voriconazole will be 
administered to any patient from admission (or the beginning of 
conditioning) until day -1.  Voriconazole may be started on day -1. There are 
no restrictions on the use of ambisome.  
 
5.4 Collection and Infusion of Progenitor Cells 
The dose of progenitor cells will be based on CD 34+ cells per kilogram of 
recipient body weight. Donors will begin G-CSF, 5µg/kg bid, on day -5. The 
donor will return for neupogen-primed progenitor cell collection on days -2 and -
1.  Each day, 18-27 liters will be processed.  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 dose of CD34+ cells/kg 

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
is not to exceed 10 X10e6/kg.  A minimum dose of 2.0 X 10e6/kg should be 
collected.  Failure to meet this target may result in a third day of collection.   
 
In our experience, the ideal amount of T-cells left in the CD34+ product is no 
greater than 5x104/kg, so that every effort will be made to keep T-cell amounts to 
below this threshold.  It is recognized that because of donor heterogenicity, 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 CD 34+ 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. The donor will have venous catheters placed in each arm for 
the purposes of undergoing leukopheresis. Leukocyte collections will be 
performed using a standard apheresis machine such as the Cobe Spectra apheresis 
instrument (Cobe Laboratories Inc., Lakewood, CO).  
 
 
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 product is infused UNFILTERED or through a filter of at least 170 
micron size intravenously through a central catheter. Marrow should only be 
piggybacked through normal saline and not other intravenous solutions. 
Contingency plans for an inadequate collection of progenitor cells via apheresis, 
non-viable donor cells, or marrow manipulation failures 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 take place in the Bone Marrow Transplant Unit. Progenitor cell 
infusions will be administered by nursing staff experienced in the administration 
of blood products.  Progenitor cell products must NOT be irradiated.  Progenitor 
cell 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.  
 

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
Significant red cell incompatibility between donor and recipient will be managed 
according to standard operating procedure, CL: Ppp033, of the Thomas Jefferson 
University Hospital Blood and Marrow Transplant Processing Lab.  Pre-
medications (if any) prior to marrow infusion will be at the discretion of the 
physician.  
Benadryl, epinephrine, and hydrocortisone should be available for emergency use 
if necessary. Oxygen with nasal cannula should be set-up and available in the 
patient room. 
 
     5.5 GVHD Prophylaxis 
 
 The day -1 tacrolimus dose is a loading dose and will be 0.03 mg/kg IV in a 
divided dose whether the patient is on voriconazole or posaconazole. Starting on 
day 0, tacrolimus will be maintained at a dose of 0.015 mg/kg in divided doses IV 
if given simultaneously with voriconazole or posaconazole.  If the patient is not 
receiving voriconazole or posaconazole, the dose of tacrolimus will remain at 
0.03 mg/kg in divided doses IV. Tacrolimus levels will be checked daily starting 
on day 0.  Tacrolimus dosing should be titrated to maintain a target level of 
8ng/ml +/- 2.   
 
 MMF will be dosed at 1 gram IV BID beginning on day -1. MMF oral dosing will 
be initiated at least 2 to 3 days prior to discharge to assure that patients tolerate 
the oral drug prior to going home. 
 
Tacrolimus oral dosing will be initiated at least 2 to 3 days prior to discharge. 
This is to assure that stable, therapeutic levels are maintained after hospitalization.   
 
If grades II-IV GVHD develop at any time after transplant (inpatient or 
outpatient), any GVHD treatment deemed necessary by the covering attending 
physician may be utilized. 
 
Patients without active GVHD will be maintained on tacrolimus until day +42.  
Tacrolimus will be weaned starting at day +42 in the absence of GVHD (mild, 
localized skin GVHD treated with topical steroids is excluded), and completed by 
d +56 if tolerated.  Once tacrolimus levels are less that 5 ng/ml levels, they no 
longer have to be checked unless there is a clinical concern to do so.   
 
MMF will be discontinued beginning at day +28 +/- 3 days in the absence of 
GVHD.  MMF may be discontinued earlier if there is count suppression thought 
to be due to the drug. 
 

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
 
6.0 
 
Study Measurements** 
 
All post-allogeneic transplant patients have physical assessments, laboratory studies and 
pathology studies performed as per the TJUH BMT Guidelines for Post-Transplant 
Allogeneic Assessments found on the BMT link on the TJUH Intranet. 
 
The table below outlines the mandatory measurements/time points for this study. 
 
 
 
 
Day + 28 
 
 
Day +90 
 
 
Day +180 
 
 
Day + 270 
 
 
1 Year 
 
GVHD Assessment 
Presence and degree of 
skin rash, presence and 
amount of diarrhea, 
LFT’s 
 
 
 
 
X 
 
 
 
 
X 
 
 
 
 
X 
 
 
 
 
X 
 
 
 
 
X 
 
Chimerism/ 
Disease Assessment  
 
 
 
 
 
 
 
Peripheral blood for 
CD3+ chimerism & 
Buffy coat chimerism 
(and/or FISH for xx/xy 
if opposite-sex donor) 
 
 
 
 
X 
 
 
 
 
X 
 
 
 
 
X 
 
 
 
 
X 
 
 
 
 
X 
 
Bone marrow exam 
(morphology, flow 
cytometry, cytogenetics, 
BC chimerism) 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
Immune 
Reconstitution Studies 
 
 
 
 
 
 
Flow cytometry for 
lymphocyte subsets 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
 
 
 
 
 
 
 
The day +28 peripheral blood, marrow studies and the day 28 assessment can be obtained within 
1 week of day 28 (i.e. +/- 7 days) to account for scheduling factors. The day +90, +180, +270, 
and 1 year marrows can be obtained within the time period of 1 month before or 1 month after the 
targeted time to account for patient scheduling factors. This table represents a minimum 
recommended sampling and visit strategy.  
 
6.1 Hematopoietic engraftment. Will be defined as   
 ANC >/= 0.5x10e9/L for at least 3 days 
 platelet engraftment >20,000 with no transfusions X 7 days. 
 

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
6.2 Toxicity Criteria. Regimen-related toxicity will be graded according to the 
NCI Common Toxicity Criteria, version 3.0.  The NCI Common Toxicity Criteria 
can also be found at the following WEB address: 
http://ctep.cancer.gov/reporting/ctcnew.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 serious adverse 
experiences (SAEs), regardless of relationships to study treatment, from the time 
of enrollment. The following events are expected side effects of high-dose 
chemotherapy and transplant and will not be reported except as noted:  
 Alopecia, dry skin, headache 
 Emesis from chemotherapy or other agents unless refractory to standard 
supportive care, nausea, anorexia, weight loss, dry mouth 
 Neutropenia/uncomplicated neutropenic fever –Only infection associated 
with shock will be reported. Other infections will be recorded.  
 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 transfusion and/or intervention such 
as endoscopy or radiologic evaluation will be reported.) 
 Anemia 

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
 Grade I - III Mucositis 
 Grades I-III Diarrhea Allergic or other common reactions to drugs used 
for supportive care unless grade 3 or higher  
After d+100, only AEs that are considered by the investigator to be possibly 
or probably associated with the treatment regimen will be reported.   
 
The formal endpoint of this study 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. 
 
6.6   
Reports to the Federal Drug Administration (FDA) 
All grade 3-5 hematopoietic cell infusion reactions and all unexpected SAEs as defined in 
21 CFR 312.32 will be reported to the FDA in an expedited fashion 
All Unanticipated Adverse Device Effects will also be reported to the FDA within 10 
working days as defined in 21 CFR 812.150 
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. 
 
6.7  
Study Endpoint 
The endpoint of this study is OS at 1 year.   
 
7.0 
 
Supportive Care 
 
 
7.1 Avoidance of Infection 
 
Patients who are post allogeneic HSCT will follow the same guidelines as 
patients who are neutropenic until advised differently by their attending 
physician.   Infectious prophylaxis and treatment of infection will be as per the 
“TJUH Guidelines for Infectious Prophylaxis and Management of Febrile 
Neutropenia”.  These guidelines can be found on the BMT link of the TJUH 
Intranet.  
 

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
The TJUH document “Preparing To Go Home After Bone Marrow 
Transplant” will be reviewed with each patient and family ideally prior to 
admission, but before discharge.   
Central venous catheters will be removed as soon as clinically manageable.  
 
Patients who are post allogeneic HSCT may be given documentation which 
identifies them at high risk for infection and GVHD if they desire.  This 
documentation will be signed by their attending physician and may be used for 
ED visits or documentation of transplant. An example of this type documentation 
is contained in Appendix C. 
 
 
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 
valtrex 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 PCP prophylaxis, usually 
Bactrim DS 1 BID, 3x weekly.  It is at the discretion of the treating attending 
physician to change agents based on culture results, drug intolerance.  
 
Prophylactic medications may be discontinued when the patient is no longer on 
immunosuppressive medications, and the CD4 count is > 100/µl.  
 
 
7.3 Growth Factor and Transfusion Support 
 
To prevent inadvertent lymphoid engraftment, all mature blood cell products must 
be irradiated to >/=2500cGy. 
 
All red cell and platelet products will be leukodepleted to prevent 
alloimmunization and decrease infectious sequela.   
 
White cell growth factor will be administered beginning on day +1.  After white 
count recovery, discontinuation of the white cell growth factor is at the discretion 
of the attending physician; however, every effort should be made to keep the 
ANC > 1000 for all patients post HSCT.   
Red cell growth factors are permissible after transplantation. 
 
 

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
8.0 
 
Drug Information and Administration 
  
 
 
8.1 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 
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. 
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. The cyclophosphamide dose is dissolved in saline and 
administered as a 2 hour IV infusion. Patients shall receive hydration consisting 
of normal saline solution at 3 ml/kg/hour (actual weight) for 2 hours before and 8 
hours after the cyclophosphamide infusion. MESNA (sodium-2-mercaptoethane 
sulfonate) will be administered as a 60 mg/kg/continuous IV infusion over 24 
hours starting 30 minutes prior to cyclophosphamide infusion and ending 24 
hours after the last dose of cyclophosphamide. The dose of MESNA will also be 
calculated based on dosing body weight.  
 
Reference: Skeel R & Lachant N.  Handbook of Cancer Chemotherapy, 4th Ed. 
Little, Brown & Co.: Boston. 

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
 
 
8.2 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. Details of the apheresis 
procedure to obtain white blood cells, quantification of 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. 
 
 
 
8.3 G-CSF 
 
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. 
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. 
 
Reference: Physician’s Desk Reference, Edition 58, 2004. 
 
 
 
8.4 GM-CSF 
 
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 

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
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.  
Administration: In this protocol, GM-CSF will be given to the patients beginning 
on Day +1 in the PM. The drug should continue until the patient has a self-
sustaining ANC of 1500.  
 
 
Reference: Physician’s Desk Reference, Edition 58, 2004. 
 
8.5 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 +/- 3 days, 

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
in the absence of GVHD. MMF may be discontinued earlier if there is count 
suppression from the drug.  
 
 
 8.6 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 
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.  
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:  
 
 
 
1.  tremor, headache, neurotoxicity 
 
 
 
 
 
2.  diarrhea, nausea,  
 
 
 
3.  hypertension  
 
 
 
4.  TTP and renal dysfunction.     
Administration: The day -1 tacrolimus dose is a loading dose and will be 0.03 
mg/kg IV in a divided dose whether the patient is on voriconazole or 
posaconazole. Starting on day 0, tacrolimus will be maintained at a dose of 0.015 
mg/kg in divided doses IV if given simultaneously with voriconazole or 
posaconazole.  If the patient is not receiving voriconazole or posaconazole, the 
dose of tacrolimus will remain at 0.03 mg/kg in divided doses IV. Tacrolimus 
levels will be checked daily starting on day 0.  Tacrolimus dosing should be 
titrated to maintain a target level of 8ng/ml +/- 2.   
 
9.0  
 
Patient Safety 
 
To ensure patient safety, a number of steps will be taken.   
 

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
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 
Safety and Monitoring Committee (DSMC). The PI will submit all unexpected 
on-site adverse events (AE) and serious adverse events (SAE) to the TJU IRB 
utilizing the electronic 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.  Unexpected deaths due related to this 
protocol will be reported within 24 hours. 
 
In addition to the Cancer Center's DSMC, the TJU BMT program has instituted 
an internal DSMC which additionally reviews safety data.  Recommendations 
from the internal DSMC will be submitted to the Cancer Center DMSB and IRB 
with study reports.   
 
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, DSMC, 
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. 
 
10.0 
       Statistical Considerations 
 
The primary outcome of interest is one year survival in the patient population 
defined as high-risk in section 3.0.  We use an exponential survival model for one 
arm, where we assume the rate of survival at one year is 60%.  Using the 
relationship




0 ln 1
t
P Failure


, 
where 
0t  
is 
one 
year, 
and 


40%
P Failure 
 at one year.  The resulting  is 1.96 (exponential parameter 
0.510).  Using a similar approach, and assuming that under the null, the probability 
of failure at 1 year is 60%, yields a 1.09.  Assuming a 2-sided test and 
alpha=0.05, yields approximately 80% power for n=45 patients and 84% power for 
n=50 patients.  The figure below illustrates the power under other sample sizes.  
Power computations were completed using PASS2002. 
 

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
Power vs N with Th0=1.1 Th1=2.0 t0=1.0 Alpha=0.05
Power
N
0.2
0.4
0.6
0.8
1.0
0
10
20
30
40
50
60
 
The trial will enroll up to 50 participants onto this trial, and participants enrolled 
will be included in the analysis. The minimum number of participants needing to 
power the trial is 45. 
 
Analysis Plan 
 
Formal sequential analysis with survival analysis is complicated.  Due to the 
relatively small sample size and anticipated number of events, we propose interim 
monitoring following approximately every 10 events using the B value approach 
(Lan and Wittes).  This will allow for monitoring of the trial without a formal 
determination of the number of analyses and timing of analyses given that accrual 
rates, event rates, and other information can only be estimated at this time.  While 
not a formal sequential procedure, it could provide evidence (either pro or con) that 
the results of the study at interim monitoring times are not likely to change even if 
the study is carried to completion. 
 
Analysis will be completed using a Kaplan-Meier plot with 95% point-wise 
confidence intervals.  One-year event rates will be estimated from this plot (the 
survival distribution function), and point-wise confidence interval at one year used 
to test the hypothesis of one-year survival exceeding 60% with this treatment.  The 
point-wise confidence interval will be computed using an arcsine-square root 
transformation, as this was shown (Borgan and Liestol) to have better properties in 
small sample sizes.  This will also allow for a Z-test which can then be 
appropriately transformed to a B score for interim monitoring of the study.   
 
Secondary Objectives 
 
A descriptional analysis will be performed to address the secondary objectives: 

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
 
 To determine the incidence and severity of GVHD in patients undergoing 
allogeneic HSCT on this protocol and 
 To assess the pace of lymphoid recovery in patients undergoing allogeneic 
HSCT on this protocol 
 
Assessment for GVHD and immune reconstitution analysis will be performed 
according the table in Section 6.0.   
 
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, we anticipate that 
the incidence of graft failure should be less than 10%, the incidence of severe 
GVHD should be less than 10%, and the non-relapse mortality should be less than 
20%.  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.    
 
References 
 
Lan KKG, Wittes J.  The B Value: a tool for monitoring data, Biometrics 44, 579-
585, 1988. 
 
Borgan O, Liestol K.  A note on confidence intervals and confidence bands for the 
survival curve based on transformations.  Scandinavian Journal of Statistics, 17:35-
41, 1990. 

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
11.0 
References 
 
1. Thomas ED: Karnofsky Memorial Lecture. Marrow transplantation for 
malignant diseases.   Journal of Clinical Oncology 1:517, 1983. 
 
2.  Weiden PL, Flournoy N, Thomas ED, Prentice R, Fefer A, Buckner CD, Storb 
R: Antileukemic effect of graft-versus-host disease in human recipients of 
allogeneic-marrow grafts. New England Journal of Medicine 300:1068, 1979. 
 
3. Weiden PL, Sullivan KM, Fournoy M, Storb R, Thomas ED: Antileukemic 
effect of chronic graft-versus-host disease: contribution to improved survival 
after allogeneic marrow transplantation. New England Journal of Medicine 
304:1529, 1981. 
 
4. van Besien K, Deeg HJ.  Hematopoietic stem cell transplantation for 
myelofibrosis.  Seminars in Oncology 32: 414-421, 2005. 
 
5. Gratwohl A, Brand R, Apperley J, Crawley C, Ruutu T, Corradini P, Carreras 
E, Devergie A, Guglielmi C, Kolb HJ, Niederwieser D.  Allogeneic 
hematopoietic stem cell transplantation for chronic myeloid leukemia in 
Europe 2006: transplant activity, long term data and current results.  An 
analysis by the chronic leukemia working party of the European group for 
blood and marrow transplantation. Haematologica 91: 513-521, 2006. 
 
6. Mehta L, Gordon L, Tallman M, Winter J, Evens A, Frankfurt O, Williams S, 
Grinblatt D, Kaminer L, Meagher R, Ginghal S.  Does younger donor age 
affect the outcome of reduced-intensity allogeneic hematopoietic stem cell 
transplantation for hematologic malignancies beneficially?  Bone Marrow 
Transplantation 38: 95-100, 2006. 
 
7.  Imamura M, Asano S, Harada M, Ikedo Y, Kato K, Kato S, Kawa K, Kojima 
S, Morishima Y, Morishita M, Nakahata T, Okamura J, Okoamoto S, 
Shiobara S, Tanimoto M, Tsuchida M, Atsuta Y, Yamamoto K, Tanaka J, 
Hamajima N, Kodera Y.  Current status of hematopoietic cell transplantation 
for adult patients with hematologic diseases and solid tumors in japan.  
International Journal of Hematology 83: 164-178, 2006. 
 
8. Alamo J, Shahjahan M, Lazarus H, de Lima M, Giralt S.  Comorbidity indices 
in hematopoietic stem cell transplantation: a new report card.  Bone Marrow 
Transplantation 3: 475-479, 2005. 
 
9. Artz A, Pollyea D, Koherginsky, Stock W, Rich E, Odenike O, Zimmerman 
T, Smith S, Godley L, Thirman M, Daugherty C, Extermann M, Larson R, van 

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
Besien K. Performance status and comorbidity predict transplant-related 
mortality after allogeneic hematopoietic cell transplantation.  Biology of 
Blood and Marrow Transplantation 12: 954-964, 2006. 
 
10. Hows J, Passweg J, Tichelli A, Locasciulli A, Szydlo R, Bacigalupo A, 
Jacobson N, Ljungman P, Cornish J, Nunn A, Bradley B, Socié G.  
Comparison of long-term outcomes after allogeneic hematopoietic stem cell 
transplantation from matched sibling and unrelated donors.  Bone Marrow 
Transplantation 38: 799-805, 2006. 
 
11. Marks D, Forman S, Blume K, Pérez W, Weisdorf D, Keating A, Gale R, 
Cairo M, Copelan E, Horan J, Lazarus H, Litzow M, McCarthy P, Schultz K, 
Smith D, Trigg M, Zhang M, Horowitz M.  A comparison of 
cyclophosphamide and total body irradiation with etoposide and total body 
irradiation as conditioning regimens for patients undergoing sibling 
allografting for acute lymphoblastic leukemia in first or second complete 
remission.  Biology of Blood and Marrow Transplantation. 12: 438-453, 2006. 
 
12. Lee S-H, Lee M, Lee J-H, Min Y, Lee K-H, Cheong J-W, Lee J, Park K, Kang 
J, Kim K, Kim W, Jung C, Choi S-J, Lee J-H, Park K.  Infused CD34+ cell 
dose predicts long-term survival in acute myelogenous leukemia patients who 
received allogeneic bone marrow transplantation from matched sibling donors 
in first complete remission.  Biology of Blood and Marrow Transplantation. 
11: 122-128, 2005. 
 
13. Cook G, Clark R, Crawley C, Mackinnon S, Russell N, Thomson K, Pearce R, 
Towlson K, Marks D.  The outcome of sibling and unrelated donor allogeneic 
stem cell transplantation in adult patients with acute myeloid leukemia in first 
remission who were initially refractory to first induction chemotherapy.  
Biology of Blood and Marrow Transplantation 12: 293-300, 2006. 
 
14. Runde V, de Witte T, Arnold R, Gratwohl A, Hermans J, van Biezen A, 
Niederwieser D, Labopin M, Walter-Noel M, Bacigalupo A, Jacobsen N, 
Ljungman P, Carreras E, Kolb H, Aul C, Apperley J.  Bone marrow 
transplantation from HLA-identical siblings as first-line treatment in patients 
with myelodysplastic syndromes: early transplantation is associated with 
improved outcome.  Bone Marrow Transplantation 21, 255-261, 1998.  
 
15. Guardiola  P, Runde V, Bacigalupo A, Ruutu T, Locatelli F, Boogaerts M, 
Pagliuca A, Cornelissen J, Schouten H, Carrera E, Finke J, Biezen A, Brand 
R, Niederwiesser D, Gluckman E, deWitte T.  Retrospective comparison of 
bone marrow and granulocyte-stimulating factor-mobilized peripheral blood 

 
 
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Blood and Marrow Transplant Program 
 
2 Step Allo BMT using matched related donors 
Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
Version 7.1 
 
progenitor cells for allogeneic stem cell transplantation using HLA identical 
sibling donors in myelodysplastic syndromes.  Blood 99: 4370-4378, 2002. 
 
16. Guardiola P, Anderson J, Bandini G, Cervantes F, Runde V, Arcese W, 
Bacigalupo A, Przepiorka D, O’Donnell M, Polchi P, Buzyn A, Sutton L, 
Cazals-Hatem D, Sale G, de Witte T, Deeg HJ, Gluckman E.  Allogeneic stem 
cell transplantation for agnogenic myeloid metaplasia: a European Group for 
Blood and Marrow Transplantation, Société Française de Greffe de Moelle, 
Gruppo Italiano per il Trapianto del Midollo Osseo, and Fred Hutchinson 
Cancer Research Center collaborative study. Blood 93: 2831-2838, 1999. 
 
17. Daly A, Song K, Nevill T, Nantel S, Toze C, Hogge D, Forrest D, Lavoie J, 
Sutherland H, Sheperd J, Hasegawa W, Lipton J, Messner H, Kiss T.  Stem 
cell transplantation for myelofibrosis: a report from two Canadian centers. 
Bone Marrow Transplantation 32: 35-40, 2003. 
 
18. Kerbauy D, Chyou F, Gooley T, Sorror M, Scott B, Pagel J, Myerson D, 
Appelbaum F, Storb R, Deeg HJ.  Allogeneic hematopoietic cell 
transplantation for chronic myelomonocytic leukemia.  Biology of Blood and 
Marrow Transplantation 11: 713-720, 2005. 
 
19. Freytes C, Loberiza F, Rizzo D, Bashey A, Bredeson C, Cairo M, Gale R, 
Horowitz M, Klumpp T, Martino R, McCarthy P, Molina A, Pavlovsky S, 
Pecora A, Serna D, Tsai T, Zhang M, Vose J, Lazarus H, van Besien K.  
Myeloablative allogeneic hematopoietic stem cell transplantation in patients 
who experience relapse after autologous stem cell transplantation for 
lymphoma: a report of the International Bone Marrow Transplant Registry.  
Blood 104: 3797-3803, 2004. 
 
20. Peniket A, Ruiz de Elvira M, Taghipour G, Cordonnier C, Gluckman E, de 
Witte T, Santini G, Blaise D, Greinix H, Ferrant A, Cornelissen J, Schmitz N, 
Goldstone A.  An EBMTR registry matched study of allogeneic stem cell 
transplants for lymphoma: allogeneic transplantation is associated with a 
lower relapse rate but a higher procedure-related mortality rate than 
autologous transplantation. Bone Marrow Transplantation 31: 667-678, 2003. 
 
21. Alpek G, Ambinder R, Piantadosi S, Abrams R, Brodsky R, Vogelsang G, 
Zahurak M, Fuller D, Miller C, Noga S, Fuchs E, Flinn I, O’Donnell P, Seifter 
E, Mann R, Jones R.  Long-term results of blood and marrow transplantation 
for hodgin’s lymphoma.  Journal of Clinical Oncology 19: 4314-4321, 2001. 
 
22. Champlin R, Schmitz N, Horowitz M, Chapuis B, Chopra R, Cornelissen J, 
Gale R, Goldman J, Loberiza F, Hertenstein B, Klein J, Montserrat E, Zhang 

 
 
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Thomas Jefferson University Hospital 
 
PI: N. Flomenberg, MD 
September 13, 2017 
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M, Ringdén O, Tomany S, Rowlings P, Van Hoef M, Gratwohl A.  Blood 
stem cells compared with bone marrow as a source of hematopoietic cells for 
allogeneic transplantation.  Blood 95: 3702-3709, 2000. 
 
23. Stem Cells Trialists’ Collaborative Group.  Allogeneic peripheral blood stem-
cell compared with bone marrow transplantation in the management of 
hematologic malignancies: an individual patient data meta-analysis of nine 
randomized trials.  Journal of Clinical Oncology 23: 5074-5087, 2005. 
 
24. Alyea E, Kim H, Ho V, Cutler C, Gribben J, DeAngelo D, Lee S, Windawi S, 
Ritz J, Stone J, Antin J, Soiffer R.  Comparative outcome of nonmyeloablative 
and myeloablative allogeneic hematopoietic cell transplantation for patients 
older than 50 years of age.  Blood 105: 1810-1814, 2005. 
 
25. Awwad M., North RJ. Cyclophosphamide (Cy)-facilitated adoptive 
immunotherapy of a Cy-resistant tumour. Evidence that Cy permits the 
expression of adoptive T-cell mediated immunity by removing suppressor T 
cells rather than by reducing tumour burden. Immunology. 65:87-92, 1988. 
 
26. Awwad M., North RJ. Cyclophosphamide-induced immunologically mediated 
regression of a cyclophosphamide-resistant murine tumor: a consequence of 
eliminating precursor L3T4+ suppressor T-cells. Cancer Research. 49:1649-
1654, 1989. 
 
27. Strauss G, Osen W, Debatin K-M.  Induction of apoptosis and modulation of 
activation and effector function in T cells by immunosuppressive drugs.  
Clinical & Experimental Immunology, 128: 255-266, 2002. 
 
28. Mayumi H, Umesue M, Nomoto K.  Cyclophosphamide-induced 
immunological tolerance: an overview.  Immunobiology, 195: 129-139, 1996. 
 
29. Fontalin LN, Chernyakhovskaya I, Mayumi H, Good RA.  Recent state of 
cyclophosphamide-induced skin allograft tolerance in mice.  Immunobiology 
182: 323-333, 1991. 
 
30. Mayumi H, Good RA: Long-lasting skin allograft tolerance in adult mice 
induced across fully allogeneic (multimajor H-2 plus multiminor 
histocompatibility) antigen barriers by a tolerance-inducing method using 
cyclophosphamide.  Journal of Experimental Medicine. 169: 213, 1989. 
 
31. Prigozhina TB, Gurevitch O, Zhu J, Slavin S.  Permanent and specific 
transplantation tolerance induced by a nonmyeloablative treatment to a wide 
variety of allogeneic tissues.  Transplantation 10: 1394-1399, 1997. 

 
 
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32. Maeda T, Eto M, Nishimura Y, Nomoto KE, Kong YY, Nomoto KI.  Direct 
evidence for clonal destructive of allo-reactive T cells in mice treated with 
cyclophosphamide after allo-priming.  Immunology 78: 113-121, 1993. 
 
33. Mayumi H, Good RA.  The necessity of both allogeneic antigens and stem 
cells for cyclophosphamide-induced skin allograft tolerance in mice.  
Immunobiology 178: 287-304, 1989. 
 
34. Mayumi H, Himeno K, Tanaka K, Tokuda J, Fan L, Nomoto K.  Drug-
induced tolerance to allografts in mice. IX. Establishment of complete 
chimerism by allogeneic spleen cell transplantation from donors made tolerant 
to H-2-identical recipients.  Transplantation 42: 417-422, 1986. 
 
35. Mayumi H, Himeno K, Shin T, Nomoto K.  Drug-induced tolerance to 
allografts in mice.  IV. Mechanisms and kinetics of cyclophosphamide-
induced tolerance.  Transplantation 39: 209-215, 1985. 
 
36. Mayumi H, Himeno K, Tokuda J, Fan L, Nomoto K.  Drug-induced tolerance 
to allografts in mice. X. Augmentation of split tolerance in murine 
combinations disparate at both H-2 and non H-2 antigens by the use of spleen 
cells from donors preimmunized with recipient antigens.  Immunobiology  
174: 274-291, 1987 
 
37. Mayumi H, Himeno K, Shin T, Nomoto K.  Drug-induced tolerance to 
allografts in mice.  VI. Tolerance induction in H-2 haplotype-identical strain 
combinations in mice.  Transplantation 40: 188-194, 1985. 
 
38. Mayumi H, Good RA.  Induction of tolerance across major barriers using a 
two-step method with genetic analysis of tolerance induction.  
Immunobiology 179: 86-108, 1989. 
 
39. Wu T, Levay-Young B, Heuss N, Sozen H, Kirchhof N, Sutherland DER, 
Hering B, Guo Z.  Inducing tolerance to MHC-matched allogeneic islet grafts 
in Diabetic NOD mice by simultaneous islet and bone marrow transplantation 
under nonirradiative and nonmyeloablative conditioning therapy.  
Transplantation 74: 22-27, 2002. 
 
40. Zhang QW, Tomita Y, Matsuzaki G, Yoshikawa M, Shimizu I, Nakashima Y, 
Sueishi K, Nomoto K, Yasui H.  Mixed chimerism, heart, and skin allograft 
tolerance in cyclophosphamide-induced tolerance.  Transplantation 70: 906-
916, 2000. 
 

 
 
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41. Luznik L, Engstrom LW, Iannone R, Fuchs EJ.  Posttransplantation 
cyclophosphamide facilitates engraftment of major histocompatibility 
complex-identical allogeneic marrow in mice conditioned with low-dose total 
body irradiation.  Biology of Blood & Marrow Transplantation 8: 131-138, 
2002. 
 
42. Okano S, Eto M, Tomita Y, Yoshizumi T, Yamada H, Minagawa R, Nomoto 
K, Sugimachi K, Nomoto K.  Cyclophosphamide-induced tolerance in rat 
orthotopic liver transplantation.  Transplantation 71: 44-456, 2001. 
 
43. Nakao M, Taguchi T, Ogita K, Nishimoto Y, Suita S.  Immune tolerance 
induced by donor antigen and cyclophophosphamide in rat fetal small bowel 
transplantation.  Fukuoka Igaku Zasshi-Fukuaka Acta Medica 96: 49-57, 
2005. 
 
44. Okayama J, Ko S, Kanehiro H, Kanokogi H, Hisanaga M, Ohashi K, Sho M, 
Nagao M, Ikeda N, Kanamura T, Akashi S, Nakajima Y.  Bone marrow 
chimerism and tolerance induced by single-dose cyclophosphamide.  Journal 
of Surgical Research 120: 102-110, 2004. 
 
45. Eto M, Nishmura Y, Matsuo K, Omoto K, Goto K, Kumuzawa J, Nomoto K.  
Prolongation of kidney graft survival by cyclophosphamide-induced tolerance 
in rats.  Journal of Urology 153: 1693-1696, 1995. 
 
46. Weiner HL, Cohen JA.  Treatment of multiple sclerosis with 
cyclophosphamide: a critical review of clinical and immunological effects.  
Multiple Sclerosis 8: 142-154, 2002. 
 
47. Traynor AE, Schroeder J. Rosa RM. Cheng D. Stefka J. Mujais S. Baker S. 
Burt RK.  Treatment of severe systemic lupus erythematosus with high-dose 
chemotherapy and haemopoietic stem-cell transplantation: a phase I study.  
Lancet. 356: 701-7, 2000. 
 
48. Nemes L, Pitlik E.  New protocol for immune tolerance induction in acquired 
hemophilia.  Haematologica 85: 64-68, 2000. 
 
49. Suarez-Almazor ME, Belseck E, Shea B, Wells G, Tugwell P.  
Cyclophosphamide for treating rheumatoid arthritis.  Cochrane Database of 
Systematic Reviews 4: CD001157, 2000. 
 
50. Kanzler S, Gerken G, Dienes HP, Meyer zum Buschenfelde KH, Lohse AW.  
Cyclophosphamide as alternative immunosuppressive therapy for autoimmune 

 
 
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hepatitis-report of three cases.  Zeitschrift fur Gastroenterologie 35: 571-578, 
1997. 
 
51. Brodsky RA, Sensenbrenner LL, Jones RJ.  Complete remission in severe 
aplastic anemia after high-dose cyclophosphamide without bone marrow 
transplantation. Blood 87: 491-4, 1996. 
 
52. Luznik L, Chen A, Kaup  M, Bright E, Bolanos-Meade J, Thorburn  C, Kos F, 
Hess A, Jones R, and Fuchs E.  Post-transplantation high-dose 
cyclophosphamide (Cy) is effective single agent GVHD prophylaxis that 
permits prompt immune reconstitution after myeloablative HLA matched 
related and unrelated bone marrow transplantation (BMT). 
Blood (ASH Annual Meeting Abstracts) 108: 2891, 2006.  
 
53. O’Donnell PV, Luznik L, Jones RJ, Vogelsang GB, Leffell MS, Phelps M, 
Rhubart P, Cowan K, Piantadosi S, Fuchs EJ.  Nonmyeloablative bone 
marrow transplantation from partially HLA-mismatched related donors using 
posttransplantation cyclophosphamide.  Biology of Blood & Marrow 
Transplantation 8: 377-386, 2002. 
 
54. Ratajczak MZ, Ratajczak J, Kuczynski W, Light B, Lusk EJ, Gewirtz AM.  In 
vitro sensitivity of human hematopoietic progenitor cells to 4-
hydroperoxycyclophosphamide.  Experimental Hematology 21: 1663-1667, 
1993. 
 
55. Gorin NC, Aegerter P, Auvert B, Meloni G, Goldstone AH, Burnett A, Carella 
A, Korbling M, Herve P, Marininchi D, Löwenberg R, Verdonck LF, 
dePlanque M, Hermans J, Helbig W, Porcellini A, Rizzoli V, Alesandro EP, 
Franklin IM, Reiffers J, Colleselli P, Goldman JM.  Autologous bone marrow 
transplantation for acute myelocytic leukemia in first remission: a European 
survey of the role of marrow purging.  Blood 75: 1606-1614, 1990. 
 
56. Jones RJ, Zuehlsdorf M, Rowley SD, Hilton J, Santos GW, Sensenbrenner 
LL, Colvin OM.  Variability in 4-hydroxyperoxycyclophosphamide activity 
during clinical purging for autologous bone marrow transplantation.  Blood 
70: 1490-1494, 1987. 
 
57. Karp J, Sarkodee-Adoo CB.  Therapy-related acute leukemia.  Clinics in 
Laboratory Medicine 20: 71-81, 2000. 
 
58. Le Beau MM, Albain KS, Larson RA, Vardiman JW, Davis EM, Blough RR, 
Golomb HM, Rowley JD.  Clinical and cytogenetic correlations in 63 patients 
with therapy-related myelodysplastic syndromes and acute nonlymphocytic 

 
 
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leukemia: further evidence for characteristic abnormalities in chromosomes 
no. 5 and 7.  Journal of Clinical Oncology 4: 325-245, 1986. 
 
59. Mosmann TR, Cherwinski HM, Bond MW, Giedlin MA, Coffman RL. Two 
types of murine helper T-cell clones. Definition according to the profiles of 
lymphokine activities and secreted proteins. Journal of Immunology 136: 
2348-2357, 1986 
 
60. Romangnani S. T cell subsets (Th1 versus Th2). Annals of Allergy Asthma 
Immunology 85; 9-18, 2000.  
 
61. Mosmann TR, Li L, Sad S, Functions of CD8 T-cell subsets secreting 
different cytokine patterns. Seminars in Immunology 9: 87-92, 1997.  
 
62. Godfrey DI, Hammond KJ, Poulton LD, Smyth MJ, Baxter AG. NK & T cell 
facts, functions and fallacies.  Immunology Today 21: 573-583, 2000.  
 
63. Fowler DH, Breglio J, Nagel G, Eckhaus MA, Gress RE.  Allospecific CD8+ 
Tc1 and Tc2 populations in graft-versus-leukemia effect and graft-versus-host 
disease.  Journal of Immunology 157: 4811-4821, 1996. 
 
64. Arpinati M, Green CL, Heimfeld S, Heuser JE, Anasetti C.  Granulocyte-
colony stimulating factor mobilizes T helper 2-inducing dendritic cells.  Blood 
95: 2484-2490, 2000. 
 
65. Franzke A, Piao W, Lauber J, Gatzlaff P, Könecke C, Hansen W, Schmitt-
Thomsen A, Hertenstein B, Buer J, Ganser A.  G-CSF as immune regulator in 
T cells expressing the G-CSF receptor: implications for transplantation and 
autoimmune diseases.  Blood 102: 734-739, 2003. 
 
66. Volpi I, Perruccio K, Tosti A, Capanni M, Ruggeri L, Posati S, Aversa F, 
Tabilio A, Romani L, Martelli MF, Velardi A.  Postgrafting administration of 
granulocyte-colony stimulating factor impairs functional immune recovery in 
recipients of human leukocyte antigen haplotype-mismatched hematopoietic 
transplants.  Blood 97: 2514-2521, 2001. 
 
67. Metcalf D.  The molecular biology and functions of the granulocyte-
macrophage colony-stimulating factors.  Blood 67: 257-267, 1986. 
 
68. Rowe JM, Andersen JW, Mazza JJ, Bennett JM, Paietta E, Hayes FA, Oette 
D, Cassileth PA, Stadtmauer EA, Wiernik PH. A randomized placebo-
controlled phase III study of granulocyte-macrophage colony-stimulating 
factor in adult patients (> 55 to 70 years of age) with acute myelogenous 

 
 
- 42 – 
 
 
 
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leukemia: a study of the Eastern Cooperative Oncology Group (E1490). 
Blood. 86: 457-62, 1995. 
 
69. Klumpp TR. Goldberg SL. Mangan KF. Effect of granulocyte colony-
stimulating factor on the rate of neutrophil engraftment following peripheral-
blood stem-cell transplantation. Journal of Clinical Oncology. 13: 2144, 1995. 
 
70. Stull DM.  Colony-stimulating factors: beyond the effects on hematopoiesis.  
American Journal of Health-System Pharmacy 59: S12-S20, 2002. 
 
71. Peters BG. Adkins DR. Harrison BR. Velasquez WS. Dunphy FR. Petruska 
PJ. Bowers CE. Niemeyer R. McIntyre W. Vrahnos D. Auberry SE. Spitzer G. 
Antifungal effects of yeast-derived rhu-GM-CSF in patients receiving high-
dose chemotherapy given with or without autologous stem cell 
transplantation: a retrospective analysis.   Bone Marrow Transplantation 18: 
93-102, 1996. 
 
72. Schiller JH, Hank JA, Khorsand M, Storer B, Borchert A, Huseby-Moore K, 
Burns D, Wesly O, Albertini MR, Wilding G, Sondel PM. Clinical and 
immunological effects of granulocyte-macrophage colony-stimulating factor 
coadministered with interleukin 2: a phase IB study. Clinical Cancer 
Research. 2: 319-30, 1996. 
 
73. Chachoua A, Oratz R, Hoogmoed R, Caron D, Peace D, Liebes L, Blum RH, 
Vilcek J. Monocyte activation following systemic administration of 
granulocyte-macrophage colony-stimulating factor. Journal of Immunotherapy 
with Emphasis on Tumor Immunology. 15: 217-24, 1994. 
 
74. Chachoua A, Oratz R, Liebes L, Alter RS, Felice A, Peace D, Vilcek J, Blum 
RH. Phase Ib trial of granulocyte-macrophage colony-stimulating factor 
combined with murine monoclonal antibody R24 in patients with metastatic 
melanoma. Journal of Immunotherapy with Emphasis on Tumor Immunology. 
16:132-41, 1994. 
 
75. Yu AL, Batova A, Alvarado C, Rao VJ, Castleberry RP.  Usefulness of a 
chimeric anti-GD2 and GM-CSF for refractory neuroblastoma: a POG phase 
II study.  Proceedings of the American Society of Clinical Oncology 16: 513a 
(abs), 1997. 
 
76. Leong SPL, Enders-Zohr P, Zhou YM, Allen RE, Sagebiel RW, Glassberg 
AB, Hayes FA.  Active specific immunotherapy with GM-CSF as an adjuvant 
to autologous melanoma (AM) vaccine in metastatic melanoma. Proceedings 
of the American Society of Clinical Oncology 15: 1360 (abs), 1996. 

 
 
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12.0  Appendices 
 
 
12.1 
Appendix A 
Guidelines for Total Body Irradiation 
 
Modality  
Photon irradiation is to be used for the TBI in all patients. Areas beneath lung 
blocks will be supplemented with electrons to maintain the homogeneity criteria.  
Energy  
Either a linear accelerator or Cobalt source 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 allof 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.  
Diagnostic Determination  
CT scans through the chest and abdomen will be done prior to initiating 
 
irradiation. An average chest wall thickness (both anteriorly and 
posteriorly) will be calculated and used in determination of electron energy for 
supplementing the chest wall beneath the lung blocks. The abdominal scan, renal 
ultrasound, or intravenous pyelogram will be used to localize the kidneys for 
proper placement of renal shielding.  
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 12.0 – 13.5 Gy.  
 
 
 
 
 
 
 A hyperfractionated  regimen over 4 consecutive days shall be  
 
 
 
 
used.  
 
Time-Dose Considerations  
Hyperfractionation  

 
 
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For patients receiving 2 fractions per day, there is a required 
minimum time interval of 6 hours between the fractions. 
Chest Wall Supplement  
 
Supplementing the chest wall dose with electrons (both anteriorly 
and posteriorly) shall be done once a day on 2 treatment days, 
immediately preceding or following treatment to the entire body. 
The area beneath the lung blocks shall receive an additional 6.0 Gy 
to dmax in a total of 2 fractions.  
 
Total Number of Treatment Days  
There shall be a total 4 consecutive treatment days.  
Treatment Interruptions  
An interruption in the radiotherapy regimen shall not be allowed.  
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  
Customized blocking to the lungs is required. Customized blocking 
to the liver and/or kidneys is optional, at the discretion of each 
participating center with the approval of the coordinating center 
radiation oncologist.  
Patient specific, individually fabricated shielding blocks are 
required for the lung from both the anterior and posterior 
directions. A partial transmission block corresponding to a total 
dose of 8.0 Gy at midplane of the patient under the blocks shall be 
used. No corrections for inhomogeneity shall be used.  
Patient specific, individually fabricated shielding blocks are 
optional for the liver from both the anterior and posterior 
directions. A partial transmission block corresponding to a dose 
reduction to 90% of the central axis dose shall be utilized.  
Patient specific, individually fabricated shielding blocks are 
optional for the kidneys from the posterior direction only. A partial 
transmission block yielding a total dose of 10.8 Gy to the midplane 
of the kidney shall be used.  
Customized 
electron 
cut-outs 
shall 
also 
be 
constructed 
corresponding to the size of the lung block plus appropriate 
margins in all directions.  
Superficial Tissue Supplement Technique  
The portion of the chest wall shielded by the partial transmission 
lung blocks will be supplemented with customized (or shaped) low 
energy electron fields. A total of 6.0 Gy to dmax in 2 fractions will 
be given to the anterior and posterior chest wall. Electron energy 
will be determined by chest wall thickness as determined by a 
chest CT scan, with the depth of the 90% dose relative to dmax used 
to determine the electron energy. The dose prescription point will 
be at dmax.  

 
 
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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.  
Normal Tissue Sparing-Lung Dose  
Lung Dose  
Each patient must have a calculation performed which 
shows that with the lung shielding and chest wall 
supplement, the TBI delivers between 9.0 Gy and 10.0 Gy 
to the mid-lung region without inhomogeneity corrections. 
The calculation will be repeated using inhomogeneity 
corrections approved by the physicist at the coordinating 
center.  
Quality Assurance Documentation  
For purposes of quality assurance the following must be performed 
on every patient undergoing TBI:  

 
 
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A check of the monitor unit calculation by a second 
physicist and a radiation oncologist prior to first treatment.  
Simulation films documenting lung, liver and kidney 
blocks in both the anterior and/or posterior projections shall 
be taken.  
Portal films (both AP & PA) verifying the position of the 
lung, liver and kidney blocks shall be taken and must be 
approved by the supervising radiation oncologist prior to 
delivery of the first  

 
 
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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. 

 
 
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12.3  Appendix C 
Patient Identification Card 
 
Patient Name __________________ 
 
Attending Physician _______________ 
 
Allergies: _______________________________________________________________ 
 
I have undergone a related-donor bone marrow transplant on ______________.  My treating 
physician has advised me that development of an infection is life-threatening to me.  If I have a 
suspected infection (fever > 100.4F, rigors or chills), I am to receive the following as soon as 
possible: 
 
______  
ceftazidime 2 grams IV  
 
______  
ciprofloxicin 400 mg IV 
 
______  
vancomycin I gram IV.  
 
Please call 215-955-8874 for the hematology fellow on call for further instructions.  If there is 
difficulty contacting the fellow on call, please do not hesitate to call the page operator and have 
the BMTU attending physician on-call paged on my behalf for further instructions. 
 
I am also at high-risk for a condition called graft-versus-host disease (GVHD).  This condition is 
also life-threatening and is manifested by a skin rash, liver inflammation, nausea, vomiting or 
severe diarrhea.  If I report to the emergency room with these symptoms please call the 
Hematology fellow on-call for further instructions as soon as possible.   
 
Attending Signature ________________________________ 
 
 
Attending Name (printed) ______________________________