Clinical trial NCT03018223
Conditions: Non-Hodgkin's Lymphoma Acute Leukemia in Remission Chronic Myeloid Leukemia Primary Myelofibrosis Chronic Myelomonocytic Leukemia Myelodysplastic Syndromes Hodgkin Lymphoma Multiple Myeloma
Keywords: Acute leukemia in CR Chronic myeloid leukemia Non-Hodgkin lymphoma in high risk Graft vs. Host Disease GVHD Hematopoietic cell transplant (HCT)
Sponsor: H. Lee Moffitt Cancer Center and Research Institute
Start date: 2017-01-31; Completion date: 2021-03-18
Enrollment: 32 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The purpose of this study is to find out if a combination of drugs (these are called: cyclophosphamide, sirolimus, and mycophenolate mofetil) will protect participants better against graft vs. host disease (GVHD) after receiving a hematopoietic cell transplant from a related partially matched (haploidentical) donor. As part of the treatment for their blood cancer, participants need a hematopoietic cell transplantation (HCT) to improve their chances of cure. In any HCT, after the stem cell infusion is given, a combination of drugs is needed to prevent GVHD and facilitate acceptance of the graft.

Detailed description:

Arms: arm 1: Pre-HCT Conditioning, HCT, GVHD Prophylaxis.

1. Conditioning regimen: To reduce heterogeneity, two commonly used myeloablative (MAC) and reduced intensity (RIC) regimens are permitted on this trial. Myeloablative conditioning: fludarabine, busulfan. Reduced intensity conditioning: fludarabine, cyclophosphamide, total body irradiation.
2. Peripheral blood hematopoietic cell transplantation
3. Graft vs. Host Disease (GVHD) prevention treatment: cyclophosphamide, mycophenolate mofetil, sirolimus.
4. Growth factor support: G-CSF
Interventions: intervention 1: Fludarabine intervention 2: Busulfan intervention 3: Cyclophosphamide intervention 4: Total body irradiation (TBI) intervention 5: Peripheral Blood Hematopoietic Cell Transplantation (HCT) intervention 6: Sirolimus (SIR) intervention 7: Mycophenolate mofetil (MMF) intervention 8: Granulocyte-colony stimulating factor (G-CSF)
Intervention descriptions: intervention 1: Myeloablative conditioning: 40 mg/m\^2 daily for 4 days. Dose will be adjusted for estimated creatinine clearance. Reduced intensity conditioning: 30 mg/m\^2 daily on days -6, -5, -4, -3 and -2. Dose will be adjusted for estimated creatinine clearance. intervention 2: Myeloablative conditioning: IV dosing targeted for a daily total area under curve (AUC) 5300 mmol\*min/L for 4 days. Busulfan AUC will be pharmacokinetically targeted. An AUC 3500 mmol\*min/l may be considered in patients over 60 years of age or with multiple comorbidities. Chemotherapy may start on day -6 or day -5 depending on the day of admission (-6 for Wednesday admission, -5 for Sunday admission). intervention 3: Reduced intensity conditioning: 14.5 mg/kg/day on days -6, -5. GVHD prophylaxis: 50 mg/kg ideal body weight (IBW) daily dose will be given on days +3 and +4 post-transplant as an IV infusion over 1-2 hours. intervention 4: Reduced intensity conditioning: 200 centigray (cGy) on day -1. intervention 5: On day 0, patients will receive a peripheral blood hematopoietic cell graft. intervention 6: GVHD prophylaxis: SIR will be administered as a 9 mg oral loading dose on day +5, followed by maintenance. SIR levels will be monitored and maintenance dosing adjusted as needed for a target trough level 8 to 14 ng/ml, per Moffitt BMT program standard practice. In the absence of acute GVHD, sirolimus taper will start on day +90 (+/- 10 days) and it is suggested to finish by day +180. intervention 7: GVHD prophylaxis: MMF will start on day +5 at a dose of 15 mg/kg every 8 hours IV with the maximum daily dose not to exceed 3 gm. MMF will be changed to orally (PO) and discontinued on day +35 (without taper) in the absence of acute GVHD. intervention 8: Growth factor support: G-CSF will be given beginning on day 5 at a dose of 5 mcg/kg/day (rounding to the nearest vial dose), until absolute granulocyte count (ANC) is \> 1,000/mm\^3 for three consecutive days. G-CSF may be given IV or subcutaneously.

Protocol documents:
Prot_SAP_000.pdf: 
 
A Calcineurin Inhibitor-Free GVHD Prevention Regimen After Related 
Haploidentical Peripheral Blood Stem Cell Transplantation 
 
 
NCT030108223 
 
Version 6.0 
July 13, 2018 


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A Calcineurin Inhibitor-Free GVHD Prevention Regimen After Related Haploidentical 
Peripheral Blood Stem Cell Transplantation 
 
 
Principal investigator:  
Nelli Bejanyan, MD 
 
Sub-Investigators: 
 
Melissa Alsina, MD 
Lia Elena Perez, MD 
Brian Betts, MD 
Marco Davila, MD 
Linda Kelley, Ph.D. 
Farhad Khimani, MD 
Aleksandr Lazaryan, MD, PhD 
Hien Liu, MD 
Fred Locke, MD 
Asmita Mishra, MD 
Taiga Nishihori, MD 
Leonel Ochoa, MD 
Michael Nieder, MD 
Joseph Pidala, MD, PhD 
Omar Castaneda Puglianini, MD 
Michael Jain, MD 
 
 
 
 
Biostatistician:  
 
 Xuefeng Wang, PhD 
 
 
H. Lee Moffitt Cancer Center & Research Institute 
Blood and Marrow Transplant Program 
12902 Magnolia Drive 
Tampa, FL 33612 
Telephone:  (813) 745-2557 / Fax: (813) 745-8468 
 
 


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Table of contents 
  
1. Study schema……………………………………………………………..3 
2. Background and rationale………………………………………………...6 
3. Trial objectives…………………………………………………………..12 
a. Primary objective………………………………………………...12 
b. Secondary objectives…………………………………………….12 
4. Trial Endpoints…………………………………………………………..12  
5. Patient population………………………………………………………..14 
a. Inclusion criteria…………………………………………………14 
b. Exclusion criteria………………………………………………...15 
c. Donor selection…………………………………………………..15 
6. Study design and treatment plan…………………………………………18 
7. Data collection and safety monitoring……………………………………20 
8. Patient enrollment and evaluation………………………………………..20 
9. Statistical Considerations and sample size……………………………….24 
10. Tolerance biomarker assessment…………………………………………24 
11. References………………………………………………………………..29 
Apendix A - Data collection and safety monitoring……………………...31 
Apendix B - Management of Cytokine Release Syndrome (CRS) after 
 Haploidentical transplantation…………………………………………..36 
 


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1. Study schema  
Title  
A calcineurin inhibitor-free GVHD prevention regimen after related 
haploidentical peripheral blood stem cell transplantation 
Study type 
Single institution phase II interventional 
Objectives 
Primary objective is to estimate the cumulative incidence of grade II-IV 
acute GVHD by day 100 after related haploidentical peripheral blood stem 
cell transplantation using the GVHD prophylaxis regimen PTCy, sirolimus 
and mycophenolate mofetil. 
Secondary objectives are: 
- 
To determine the cumulative incidence of chronic GVHD by 1 year. 
- 
To examine additional transplantation outcomes including 
malignancy relapse, overall survival, progression-free survival, non-
relapse mortality, and engraftment and immune reconstitution.  
- 
To examine peripheral blood markers of immune tolerance 
development. 
- 
To evaluate the impact of the use of haploidentical related donors in 
the access of ethnic/race minority patients to allogeneic 
transplantation.   
Sample size 
32 evaluable patients (pending statistics) 
Study duration  
3 years 
Patient selection 
criteria 
Inclusion Criteria: 
- 
Age: Must be older than 18 years, no upper age limit. 
- 
Karnofsky performance status > 80% for full intensity, > 60% for 
reduced intensity conditioning. 
- 
Adequate organ function: LVEF > 45%, FEV1/FVC/aDLCO > 50%, 
AST/ALT<2 times ULN, estimated creatinine clearance >50 ml/min. 
- 
Signed informed consent. 
- 
Included diseases: Acute leukemia in first or subsequent CR, CML, 
primary myelofibrosis, CMML, Intermediate-2 or high risk MDS, 
NHL in high risk CR1, PIF or beyond CR1 if in PR (SD accepted if 
no mass>3 cm), HD beyond CR1 if in PR (SD accepted if no mass>3 
cm), MM in CR/VGPR. 


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Exclusion criteria: 
- 
Uncontrolled active bacterial, viral, fungal infection. 
- 
Prior alloHCT. 
- 
Patient unwilling or unable to comply with study requirements. 
- 
Patient with active, progressive or advanced disease based on 
diagnosis. 
Donor selection 
criteria 
In the absence of an allele level matched (8/8 HLA A, B, C and DR) sibling 
or unrelated donor, all potential related haploidentical donors will be typed. 
(See algorithm in page 16). A haploidentical donor will share at least one 
haplotype with the patient. 
A haploidentical donor will be excluded if the recipient has HLA antibodies 
against the donor HLA. 
Among several potential haploidentical donors, in order of priority: Matched 
CMV IgG serologic status between donor and recipient, ABO matched > 
minor ABO mismatch > major ABO mismatch, younger donor, male donor. 
Treatment plan 
Conditioning regimens: 
Myeloablative conditioning: Fludarabine 30 mg/m2 IV daily for 4 days. Dose 
will be adjusted for estimated creatinine clearance (see SOP BMT-G-115). 
Busulfan IV dosing targeted for a daily total AUC 5300 mmol*min/L for 4 
days.. Busulfan AUC will be pharmacokinetically targeted.  AUC 3500 may 
be used in patients over 60 years of age or with HCT-CI of 2 or more. 
Chemotherapy may start on day -6 or -5 depending on admission day. 
Reduced intensity conditioning: Fludarabine: 30 mg/m2 daily on days -6, -5, 
-4, -3 and -2. Dose will be adjusted for estimated creatinine clearance (see 
SOP BMT-G-115). Cyclophosphamide 14.5 mg/kg/day on days -6, -5 and 
total body irradiation 200 cGy on day -1. 
Graft: 
Donor peripheral blood hematopoietic cells for a target yield of 5 x 106 
CD34+ cells/kg, minimal accepted number is 2 x 106 CD34+ cells/kg 
recipient IBW, maximum accepted 10 x 106 CD34+ cells/kg. 
GVHD prophylaxis: 
 Cyclophosphamide (50 mg/kg IBW daily dose) will be given on days 
+3 and +4 post-transplant as an IV infusion over 1-2 hours (see BMT-


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G-119). 
 Sirolimus (SIR) will be administered as a 9 mg oral loading dose on 
day +5, followed by maintenance 4 mg daily, target level 8 to 14 
ng/ml. In the absence of acute GVHD, sirolimus taper will start on 
day +90 (+/- 10 days) and it is suggested to finish by day +180.  
 Mycophenolate mofetil (MMF) will start on day +5 at a dose of 15 
mg/kg every 8 hours IV with the maximum daily dose not to exceed 3 
gm. MMF will be changed to PO when feasible and discontinued on 
day +35 (without taper) in the absence of acute GVHD. 
Growth factor support: 
G-CSF IV or SQ will start on day +5, at 5 mcg/kg/day, until ANC > 
1,000/mm3 for 3 consecutive days. 
Supportive care: 
Will follow established SOP BMT G-132. Given higher fevers and potential 
risk of cytokine release syndrome (CRS) associated with the use of PBSC 
grafts, patients will be monitored for CRS and if present will be treated 
according to published guidelines. (Appendix B) 
Study 
procedures 
baseline 
All clinic assessments, labs, procedures needed to assess eligibility are 
clinical standard of care evaluation done pre-transplantation.  
Collection of baseline data 
Research data collection on baseline patient, disease, remission status, 
transplantation characteristics and collect baseline immune deficiency panel 
(IMDFP Tregs) 
Statistical 
considerations 
With a total of 32 evaluable patients enrolled, we will have 90% power (α = 
0.1) to demonstrate a reduction from 40% to 20% in cumulative incidence of 
grade II-IV acute GVHD. Approximately 40 patients will be accrued to reach 
32 evaluable.  Any enrolled, but non-evaluable subject will be replaced in 
case there was a clinical indication to modify or change the transplant 
conditioning regimen. The cumulative incidence of acute and chronic GVHD 
will be estimated, considering malignancy relapse and non-relapse death as 
competing risk events.  
 
 


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2. Background and rationale 
Hematopoietic cell transplantation (HCT) from HLA identical related or unrelated donors or 
from umbilical cord blood units can cure malignant and non-malignant hematologic, immune 
and metabolic disorders. Major limitations for wider use of HCT remain timely identification of 
suitable donors and risk of transplant related morbidity and mortality.  
Donor availability and race/ethnic background  
The likelihood of finding an optimal donor varies with racial and ethnic background. According 
to data from the NMDP the highest probability is among whites of European descent at 75%, 
with a much lower probability in ethnic minorities such as African Americans at 19% or 
Hispanics at 34%. Optimal cords are only found in a small proportion of patients, 17% for white 
Europeans, 1% for African Americans and 5% for Hispanics. By using suboptimal donors and 
suboptimal cord-blood units, a donor may be found in 90% of patients.1 However, the use of 
suboptimal donors (mismatched adult or cord-blood units) is associated with a higher rate of 
complications (graft failure, graft vs. host disease, infection) and lower survival. Patients from 
ethnic/race minorities more frequently suffer this burden.2 In addition, once unrelated adult 
donors are identified, donor attrition decreases the number of donors actually available and 
willing to donate. Donor attrition is more common in donors from ethnic/race minorities.3 
We previously performed a comprehensive analysis of unrelated donor searches (n=531) 
conducted for new HCT consult patients seen in the Moffitt BMT Program (March 2006 to 
December 2009). Among other major findings, this analysis demonstrated the following: (1) The 
likelihood of finding a suitable (7/8 or 8/8, i.e. single mismatch or full match at HLA-A, -B, -C, 
or –DRB1) unrelated donor was significantly dependent on race/ethnicity; all non-Caucasian 
groups had lower rates of successful donor identification. (2) Among those with a suitable donor, 
race/ethnicity remained a significant obstacle to actual HCT utilization. The other strongest 
factor in this domain was disease progression, which speaks to the adverse impact of prolonged 
wait from donor search to transplant.4  


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These data speak to the need for prompt and successful donor identification for racial/ethnic 
minorities (hence the rationale for rapid identification of nearly universally available 
haploidentical donors), and also support the overall feasibility of our planned trial: Non-
Caucasian groups made up 26% of our published consecutive series of unrelated donor searches, 
and current Moffitt BMT Program data suggests that figure has increased to 29%. With our 
current allogeneic HCT activity (approximately 195 allogeneic HCT procedures done per year, 
with approximately double that seen in initial consult – expected rate of attrition around 50% 
with current donor selection practices), 29% of these numbers would result in over 50 minority 
allogeneic HCT procedures per year (or > 100 minority allogeneic HCT consults). Our proposed 
study would be highly feasible under these terms, but would become even more feasible if the 
option of haploidentical transplant was implemented much earlier in the donor search algorithm 
for minority groups. Importantly, increasing numbers of consults done in Puerto Rico are 
expected to continue to increase the proportion of minorities in our overall consultation and 
transplant populations.  
Haploidentical related donors 
HLA haplotypes (HLA-A, HLA-B, HLA-C, HLA-DR) are tightly linked genes that are jointly 
inherited. A potential related donor that shares one haplotype with the recipient (4/8 identical), 
usually a sibling, a parent or a child is available in over 95% of patients. Haploidentical related 
donors in general, are rapidly identified, readily available and highly motivated to donate. Such 
donors can be selected at the time siblings are HLA typed and before an unrelated donor search 
is formally started. Furthermore, haploidentical related donors are available for any patient 
independent of ethnic/racial background.5 
Haploidentical Transplantation (HaploT)  
The fundamental obstacle of crossing the HLA barrier in HaploT arises from the intense 
bidirectional responses from T-cells reacting to allogeneic HLA molecules resulting in 
overwhelmingly high incidence of GVHD and graft rejection.6 The first effective approach used 
to overcome these limitations was developed by Aversa et al. with the use of megadoses of 
CD34+ cells and profound T-cell depletion of the graft. Although effective at decreasing graft 
failure and facilitating engraftment, this strategy led to slow immune reconstitution and as a 


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consequence high mortality due to opportunistic infection and relapse.7  More recently, T-cell 
replete HaploT emerged as an attractive option owing to the development of stronger and more 
effective GVHD-preventive strategies namely the use of a short course of cyclophosphamide 
early after graft infusion.8,9  
George Santos demonstrated that a short course of high dose cyclophosphamide (PTCy) soon 
after bone marrow transplantation in rodents targeted and depleted donor and host alloreactive T-
cells.10 This model was subsequently translated to human HCT. The John Hopkins and the Fred 
Hutchinson Cancer Research Center assessed the use of a non-myeloablative conditioning 
followed by PTCy (50 mg/kg days +3, +4) and mycophenolate mofetil/tacrolimus prophylaxis 
after a haploidentical marrow graft infusion in 210 patients with acute leukemia.  Engraftment 
was sustained at 87%; grade 2-4 aGVHD occurred in 27% and cGVHD in 15%. Cumulative 
incidence of relapse and non-relapse mortality (NRM) were 55% and 18% respectively. This low 
NRM was counterbalanced by a significant relapse rate in this high risk population.11 This 
experience has been duplicated by others and has compared favorably with historical data using 
HLA-identical sibling and unrelated donors.12-16 Importantly, higher rates of GVHD have been 
seen when peripheral blood stem cells are used for transplantation (an overall observation that 
recapitulates extensive evidence in the setting of matched sibling and matched unrelated donor 
transplantation).17-20 
A Treg-favoring GVHD prophylaxis 
Our group has previously shown that a sirolimus (SIR) based GVHD prophylaxis leads to 
significantly less grade 2-4 acute GVHD compared to the standard of care 
methotrexate/tacrolimus in adult recipients of HLA identical sibling and unrelated donors. The 
main benefit was in gastrointestinal GVHD. As acute GVHD remains a significant cause of early 
transplant associated morbidity and mortality, effective acute GVHD prevention is an important 
clinical goal. Furthermore, SIR-based GVHD prophylaxis also led to a lower incidence of 
moderate-severe chronic GVHD.  Because chronic GVHD remains the main cause of late 
morbidity and mortality after HCT, this represents an advance on HCT. In addition, sirolimus 
treated patients had a significantly greater proportion of regulatory T cells (Treg) among the 
CD4+ cells in the peripheral blood, and isolated Treg were functional.21,22 


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Calcineurin-inhibitor (CNI) based GVHD prophylaxis has been commonly used as GVHD 
prevention strategy on HaploT. However, CNI negatively affect regulatory T-cells which appear 
to be critical to establish tolerance after HCT, in particular if using peripheral blood stem cell 
grafts. In contrast, SIR fosters post-transplantation Treg recovery which has been shown to be 
central in tolerance development after transplantation. Besides its Treg promoting ability and its 
pleiotropic immunosuppressive properties, SIR has been shown to have an antiviral and 
antitumor activity.23 
Therefore, SIR-based GVHD prophylaxis is a desirable approach after HaploT to further 
improve acute and chronic GVHD prevention and to facilitate tolerance. Recently, Cieri et al. 
published a clinical study on 40 patients using a sirolimus based approach, a myeloablative 
conditioning and peripheral blood stem cell grafts followed by PTCy. In their hands, it was a safe 
and effective approach, with a rapid recovery of T cell compartment and in particular of Tregs. 
In particular, the cumulative incidence of grade II-IV acute GVHD in this trial was only 15%, 
while prior trials (using predominantly tacrolimus and MMF on the backbone of PTCy) have 
rather reported ≥ 40% grade II-IV acute GVHD when using peripheral blood as graft source. 
This major difference in outcome forms the key rationale for our testing this regimen, and 
provides an estimated effect size for powering our trial.24 
We want to further expand this experience using both, myeloablative and reduced intensity 
conditioning and peripheral blood stem cell grafts to allow our patients to have a safe and 
comprehensive approach that would be applicable within a large range of age and comorbidities. 
In addition, this approach would mitigate the impact of race and ethnicity in the availability of 
suitable donors and would allow more patients to access HCT.  
Tolerance associated gene-expression after HCT 
Clinical transplantation tolerance is broadly defined as the absence of ongoing immunologic 
injury due to donor-recipient incompatibility without ongoing immunosuppressive therapy (IS). 
Clinical judgment does not accurately identify the development of immune tolerance after HCT, 
and therefore IS taper and discontinuation is empiric and frequently complicated by GVHD. In 
previous investigation, Pidala, and colleagues examined peripheral blood transcriptional markers 
in tolerant and non-tolerant HCT recipients with the goals of developing an accurate phenotypic 


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classifier and dissecting biologic mechanisms of immune tolerance following HCT. The total 
study sample included 15 HCT recipients who demonstrated sustained immune tolerance, 17 
HCT recipients with established chronic GVHD on immune suppression, and 10 healthy control 
subjects. Comprehensive data on patient characteristics, prior and current GVHD activity and IS 
therapy were collected. Peripheral blood mononuclear cells (PBMC) were isolated from a single 
time point freshly obtained peripheral blood sample to (1) characterize immune cell populations 
by surface phenotype, and (2) extract RNA for microarray analysis using the Affymetrix Human 
U133 plus 2.0 array. Time from HCT to sample collection did not differ between the tolerant and 
non-tolerant groups (median 38.5 vs. 39.5 months, p =0.97). There were no significant 
differences between groups in total CD4+ T cells, total CD8+ T cells, αα or αβ CD8+ cells, 
memory or effector CD8+, regulatory T cells (CD4+CD25+CD127low), total CD14+ 
monocytes, total CD19+ B cells, plamacytoid or monocytoid dendritic cells, NK, or NKT cells. 
Significance analysis of microarray (SAM) was used to identify genes differentially expressed 
between phenotypic groups. A final tolerance gene set included 281 probe sets that distinguished 
the tolerant group from both non-tolerant and controls. A final non-tolerant gene set included 
122 probe sets that distinguished non-tolerant patients from both the tolerant group and healthy 
controls. Functional Ontology Enrichment (MetaCore by GeneGo) identified enriched process 
networks including NK cell cytotoxicity, antigen presentation, lymphocyte proliferation, and cell 
cycle and apoptosis. An accurate classifier (>90% accuracy, correctly classifying 14/15 tolerant 
cases and 15/17 non-tolerant cases) was developed only utilizing 20 probe sets. Differential 
expression of highly discriminative genes was confirmed using NanoString nCounter 
technology. In total, these data demonstrate that differential gene expression in PBMC can 
provide insight into the biology of immune tolerance after HCT, and can be used to develop a 
classifier with high degree of accuracy to identify tolerant patients.25 We now propose to use this 
technology to prospectively investigate tolerance development following PTCy/SIR/MMF 
therapy in the setting of HLA-haploidentical peripheral blood stem cell transplantation. 
Summary rationale:  
Racial/ethnic minorities suffer disparate access to transplant, largely driven by failure to identify 
suitable adult unrelated donors. This represents a particular shortcoming of our current practices 
at Moffitt, given the diversity of our usual patient population seen for BMT consultation. This 


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innovative trial will directly address this need through efficient identification of haploidentical 
donors for such patients. While this overall work-flow enhances access to HCT, it also makes 
original investigation possible to optimize outcome of haploidentical transplantation. We will 
examine a novel GVHD prevention strategy to prevent morbidity and death associated with 
GVHD, and expand current scientific knowledge in this area through correlative science focused 
on the immunobiology of GVHD and transplantation tolerance. 
 
3. Trial objectives 
a. Hypothesis: 
A calcineurin inhibitor-free GVHD prevention regimen with the addition of sirolimus to 
post-transplant cyclophosphamide and mycophenolate mofetil will allow adequate 
prevention of acute GVHD in the setting of haploidentical peripheral blood 
hematopoietic cell transplant. 
b. Primary objective:  
To estimate the cumulative incidence and severity of  acute GVHD by day 100 after 
related haploidentical peripheral blood stem cell transplantation using the GVHD 
prophylaxis regimen PTCy, sirolimus and mycophenolate mofetil (MMF). 
c. Secondary objectives:  
1. To determine the cumulative incidence and severity of chronic GVHD by 1 
year. 
2. To examine additional transplantation outcomes including malignancy 
relapse, overall survival, progression-free survival, non-relapse mortality, and 
engraftment and immune reconstitution.  
3. To examine peripheral blood markers of immune tolerance development. 
4. To evaluate the impact of the use of haploidentical related donors in the 
access of ethnic/race minority patients to allogeneic transplantation.   


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4. Trial endpoints 
a. Primary endpoint: 
Cumulative incidence of grade II-IV acute GVHD by day 100 after HCT. Acute 
GVHD organ staging and assessment of overall grade will use standard consensus 
criteria.26 
b. Secondary endpoints: 
1. Cumulative incidence and severity of chronic GVHD by 1 year. 
- 
Chronic GVHD diagnosis and severity scoring will follow NIH 
Consensus guidelines.27  
2. Evaluate following outcomes at 100 days and 1 year: 
- 
Overall survival defined as time from transplant to death or last 
follow-up.  
- 
Progression-free survival defined as the minimum time interval from 
transplant to relapse/recurrence, to death or to last follow-up. 
- 
Cumulative incidence of non-relapse mortality. 
- 
Cumulative incidence of relapse/progression: Defined as hematologic 
relapse or any unplanned intervention to prevent progression of 
disease in patients with evidence (molecular, cytogenetic, flow 
cytometric, radiographic) of malignant disease after transplantation. 
3. Rate of infectious complications: Data on all infections grade III and IV according 
to standard criteria (CTCAE v 4.0) will be collected.  
4. Time to hematopoietic recovery (ANC and platelets) defined as the first of 3 
consecutive days with ANC above 500 cells/uL and the first of 3 consecutive days 


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with a platelets count of 20,000/uL or higher (without transfusions in preceding 7 
days). 
5. Donor chimerism (CD3, CD33, unsorted BM) at day 30, 90, 180, and 365.  
6. Immune reconstitution 
1. Will be collected at baseline, day 30, 90, 180 and 365. 
2. This will be assessed through collection of a routinely available 
clinical laboratory immune deficiency panel. 
7. Serial research blood sample collection for tolerance biomarker studies will be 
collected at day 21, 90, and day 180. 
8. Detailed race/ethnicity data will be collected on patients getting haploidentical 
donors or unrelated donors. 
 
5. Patient population 
a. Patient inclusion criteria 
1. Age: Patient must be older than 18 years. There will be no upper limit of age. 
Comorbidities and vital organ function will define eligibility criteria. 
2. Karnofsky performance status: Full intensity conditioning, 80-100%; reduced 
intensity conditioning, 60-100%. 
3. Vital organ function: 
- 
Cardiac: Left ventricular ejection fraction must be > 45% assessed 
by MUGA scan or echocardiogram. No myocardial infarction 
within 6 months of transplant evaluation. 
- 
Pulmonary: FEV1, FVC, and adjusted DLCO must be  ≥ 50% of 
predicted values. 


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Liver: Transaminases (AST, ALT) < 2 times upper limit of normal 
values. 
- 
Kidney: Estimated creatinine clearance ≥ 50 cc/min. 
4. Signed informed consent. 
5. Included disease conditions and remission status 
a. Acute leukemia in CR1 or second/subsequent CR. 
b. Chronic myeloid leukemia, primary myelofibrosis, chronic 
myelomonocytic leukemia..  
c. Int-2 or high risk MDS 
d. Hodgkin lymphoma beyond CR1 with chemosensitive disease, SD 
may be included if no mass>3 cm.  
e. Non-Hodgkin lymphoma in high risk CR1 or subsequent CR (by 
clinical, cytogenetic or molecular criteria), PIF or relapsed with 
chemosensitive disease. SD may be included if no mass>3 cm. 
f. Multiple myeloma in CR/VGPR. 
 
6. Patient will be excluded if there is: 
a. Active bacterial, viral, fungal infection not controlled with 
appropriate antimicrobial therapy. 
b. Prior allogeneic HCT. 
c. Patients unwilling to comply with study requirements. 
d. Patients with active, progressive or advanced disease based on 
diagnosis. 


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b. Donor selection: 
1. Per MCC BMT program practices, an allele level matched (8/8 HLA A, B, 
C and DR) sibling or unrelated donor is preferred. If a matched donor is 
not found, mismatched unrelated or haploidentical donors may be 
considered. See suggested algorithm for donor selection on page 17.  
2. If a haploidentical donor is considered, parents, children,  full siblings and 
in selected cases, extended family, will have high resolution typing at the 
MCC HLA laboratory. A familiar haploidentical donor is chosen among 
those who share at least one HLA-A, B, C, DRB1 and DQB1 haplotype 
with the patient.  
3. Patient will be screened for antibodies targeting mismatched HLA 
antigens in potential haploidentical donors (donor specific antibodies, 
DSA). Antibody screen and confirmatory testing using Luminex single-
antigen-bead test will be done. 
4. Among several potential donors, will choose in order of priority: 
a. Matched CMV IgG serologic status between donor and recipient. 
b.ABO-matched donor preferred, then minor ABO mismatch, then 
major ABO mismatch. 
c. Younger donor preferred: child, then sibling, and then parent 
d.For male recipient, male donor will be preferred. Avoid mother 
as a donor unless no other choices. 
 
 
 
 
 


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Candidate for allo-HCT 
Typing potential 
sibling donors 
HLA identical 
donor identified 
Yes 
PROCEED TO BMT 
Unrelated donor 
search 
8/8 HLA A, B, C, DR 
unrelated donor match 
Haploidentical 
donors  
Donor identified 
PROCEED TO BMT 
Yes 
No 
No 
Yes 
7/8 mismatched 
unrelated donor  
Donor selection for allogeneic HCT  


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6. Study design and treatment Plan 
This will be a single arm, prospective, single institution interventional trial. 
a. Conditioning regimen 
Several transplant conditioning regimens are routinely employed in standard practice. To 
reduce heterogeneity, two commonly used myeloablative (MAC) and reduced intensity 
(RIC) regimens are permitted on this trial. The intensity of the conditioning regimen will 
be decided by primary investigator based on performance status, vital organ function and 
comorbidities: 
1. Myeloablative conditioning: 
- 
Fludarabine: 40 mg/m2 daily for 4 days. Dose will be adjusted for estimated 
creatinine clearance (see SOP BMT-G-115). 
- 
Busulfan: IV dosing targeted for a daily total AUC 5300 mmol*min/L for 4 
days. Busulfan AUC will be pharmacokinetically targeted (see SOP BMT-G-
111). An AUC 3500 mmol*min/l may be considered in patients over 60 years 
of age or with multiple comorbidities. Chemotherapy may start on day -6 or 
day -5 depending on the day of admission (-6 for Wednesday admission, -5 
for Sunday admission). 
 
2. Reduced intensity conditioning: 
- 
Fludarabine: 30 mg/m2 daily on days -6, -5, -4, -3 and -2. Dose will be 
adjusted for estimated creatinine clearance (see SOP BMT-G-115). 
- 
Cyclophosphamide 14.5 mg/kg/day on days -6, -5. 
- 
Total body irradiation 200 cGy on day -1. 
 
 


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b. Peripheral Blood Hematopoietic Cell Transplantation 
On day 0, patients will receive a peripheral blood hematopoietic cell graft. Donor 
peripheral blood hematopoietic cells will be harvested for a target yield of 5 x 106 CD34+ 
cells/kg, maximum accepted number is 10 x 106 CD34+ cells/kg,  minimal accepted 
number is 2 x 106 CD34+ cells/kg recipient IBW. Total amount of T-cells in the graft will 
be noted. 
c. GVHD prophylaxis 
1. Cyclophosphamide 50 mg/kg IBW daily dose will be given on days +3 and +4 
post-transplant as an IV infusion over 1-2 hours. Hydration will start 2 hours prior 
and Mesna will be given in divided doses IV, pre- and post-cyclophosphamide 
according to MCC guidelines (BMT-G-119). 
2. Sirolimus (SIR) will be administered as a 9 mg oral loading dose on day +5, 
followed by maintenance. SIR levels will be monitored and maintenance dosing 
adjusted as needed for a target trough level 8 to 14 ng/ml, per Moffitt BMT 
program standard practice (BMT SOP #103). In the absence of acute GVHD, 
sirolimus taper will start on day +90 (+/- 10 days) and it is suggested to  finish by 
day +180. 
3. Mycophenolate mofetil (MMF) will start on day +5 at a dose of 15 mg/kg every 8 
hours IV with the maximum daily dose not to exceed 3 gm. MMF will be changed 
to PO and discontinued on day +35 (without taper) in the absence of acute 
GVHD. 
d. Growth factor support 
G-CSF will be given beginning on day 5 at a dose of 5 mcg/kg/day (rounding to the 
nearest vial dose), until absolute granulocyte count (ANC) is > 1,000/mm3 for three 
consecutive days. G-CSF may be given IV or subcutaneously. 
 
 


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e. Supportive Care 
Supportive care, including management of Non-infectious fever, transfusion, infection 
prevention, and nutritional support will be managed according to established SOP BMT 
G-132. As the proportion of T-cells in a peripheral blood graft may be 5 to 10 times 
higher than in bone marrow, there is evidence suggesting that higher fever and possibly 
cytokine release syndrome (CRS)  may happen in these patients shortly (hours to a few 
days) after graft infusion.29 Patients will be monitored for CRS and may be treated with 
tocilizumab according to published guidelines. (Please see appendix B). 
 
7.  Data collection and safety monitoring 
Procedures on this trial will follow the Data & Safety Monitoring Plan (DSMP) adopted by the 
H. Lee Moffitt Cancer Center & Research Institute. This ensures that all clinical research 
conducted or coordinated by the Cancer Center is scientifically well designed, responsibly 
managed, appropriately reported and protects the rights and welfare of human participants. The 
methods and amount of monitoring required are dictated by the degree of risk involved to the 
individual participants and the complexity of the clinical research. Other entities that will assume 
responsibility for data and safety monitoring include: Principal Investigators (PI); Scientific 
Review Committee (SRC); Protocol Monitoring Committee (PMC); Research Compliance 
Division (RCD) of the Cancer Center’s Corporate Compliance Office; Institutional Review 
Board (IRB). Full details of this DSMP are provided in Appendix A.  
  
8. Patient enrollment and evaluation 
a. Baseline evaluation 
The baseline study visit encompasses the following: 
Eligibility determination (review inclusion and exclusion criteria). 
a. All clinic assessments, labs, procedures needed to assess eligibility are clinical 
standard of care evaluation done pre-transplantation.  


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b. Collection of baseline data 
i. Research data collection on baseline patient, disease, remission 
status, transplantation characteristics. 
c. Collect baseline immune deficiency panel (IMDFP Tregs). 
 
 
b. Post-transplant evaluations 
Post-transplantation study visits occur on day 30, 90, 180, and at 1 year (with acceptable 
windows as outlined in the study calendar).  
All clinic assessments and laboratory studies needed to inform study endpoints captured 
at these time points are part of clinical standard of care (with exception of underlined 
research samples). 
1. Day 21 study visit 
- 
collect day 21 research blood sample  
 
2. Day 30 study visit 
- 
Capture ANC/PLT engraftment data, day 30 disease response 
assessment and donor chimerism, and any occurrence of acute GVHD 
- 
monitor AE/SAE throughout observation period 
- 
Collect day 30 immune deficiency panel IMDFP Tregs. 
 
3. Day 90 study visit 
- 
Capture acute GVHD data, day 90 disease response assessment and 
donor chimerism, and death events. 
- 
Capture initiation of sirolimus taper. 


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- 
monitor AE/SAE throughout observation period 
- 
Collect day 90 immune deficiency panel IMDFP Tregs 
- 
collect day 90 research blood sample  
 
4. Day 180 study visit 
- 
Capture acute and chronic GVHD data, day 180 disease response 
assessment and donor chimerism, mortality events 
- 
Capture time of sirolimus discontinuation 
- 
monitor AE/SAE throughout observation period 
- 
Collect day 180 immune deficiency panel IMDFP Tregs 
- 
collect day 180 research blood sample 
 
5. 1 year study visit 
- 
capture chronic GVHD data, 1 year response assessment and donor 
chimerism, mortality events 
- 
monitor AE/SAE throughout observation period 
- 
Collect 1 year immune deficiency panel IMDFP Tregs 
c- Relapse 
Relapsed patients will be considered off-trial and will be followed only for date of death. 
 
 


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Table 8.1 Summary of patient clinical assessment 
Study assessments 
Baseline 
21 days 
(+/-3) 
  
30 days 
 (+/-7) 
90days 
(+/-10) 
180 days 
(+/-14) 
365 days 
(+/-21) 
Baseline pre-transplant assessment: 
- History, physical exam, weight,  
Karnofsky performance status, 
infectious disease markers, EKG, 
LVEF, pulmonary function tests 
- Disease staging 
- Pregnancy test  
X 
 
 
 
 
 
Neutrophil and platelet engraftment 
 
 
X 
 
 
 
Donor chimerism 
 
 
X 
X 
X 
X 
Acute GVHD  
 
 
X 
X 
X 
X 
Chronic GVHD  
 
 
 
X 
X 
X 
Disease response  
 
 
X 
X 
X 
X 
Death 
 
 
X 
X 
X 
X 
AE/SAE  
X 
 
X 
X 
X 
X 
IDP-Treg 
X 
 
X 
X 
X 
X 
Research blood sample 
 
    X 
 
X 
X 
 
 
Notes: 
 
 
 
 


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9. Statistical considerations 
The primary objective of this single-arm phase II trial is to estimate the cumulative incidence of 
grade II-IV acute GVHD by day 100 after transplantation. The historical benchmark for grade II-
IV acute GVHD in this setting (related haploidentical transplantation, peripheral blood stem cell 
graft, conventional immune suppression prophylaxis including PTCy, tacrolimus and MMF) is ≥ 
40%. The preliminary data from Cieri and colleagues suggests that a novel approach (above 
treatment program, but use of sirolimus and MMF) results in only 15% grade II-IV acute GVHD 
by day 100 post-HCT. With a total of 32 evaluable patients enrolled, we will have > 90% power 
(α = 0.1) to demonstrate a reduction from 40% to 20% in our trial. With an anticipated accrual of 
1-2 patients per month, our planned accrual timeline is consistent with the overall grant period. 
Approximately 40 patients will be accrued to reach 32 evaluable. Any enrolled, but non-
evaluable subject will be replaced in case there was a clinical indication to modify or change the 
transplant conditioning regimen. 
The cumulative incidence of acute and chronic GVHD will be estimated, considering 
malignancy relapse and non-relapse death as competing risk events. Overall and progression-free 
survival will be estimated using the Kaplan-Meier method. Stratified survival curves will be 
compared using the log-rank test. Patient characteristics and other factors of interest will be 
associated with overall and progression-free survival using Cox proportional hazard model. 
Rates of other complications will be estimated with allied confidence interval.  
 
10. Tolerance biomarker assessment 
Sample collection: Research blood samples will be drawn at day 21, day 90, and day 180 after 
HCT. These time points are highly feasible, and chosen for the following reasons: (1) The day 


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90 sample will serve as the primary single time point predictor for subsequent complete IS 
discontinuation, as we expect most patients would have stopped MMF by that point, and would 
be in the midst of SIR taper to discontinuation. The day 21 sample permits an earlier single time 
point assessment to determine whether findings at day 90 are present at earlier post-HCT time 
points. (2) The inclusion of both day 21 and day 90 samples permits a secondary approach of 
examining change in gene expression from day 21 to 90 as a predictor of subsequent tolerant vs. 
not tolerant phenotype; we expect this will be highly informative of evolution of tolerance 
mechanisms over time. (3) These single time point samples are also positioned for prediction of 
GVHD syndromes as major clinical indicator of alloreactivity. Day 21 samples will predate the 
majority of acute GVHD events, and day 90 is ideal for predictive analyses for development of 
chronic GVHD. Finally, the day 180 sample should capture the majority of subjects at time of 
immune suppression discontinuation. This sample will be used as predictor at time of immune 
suppression stop for subsequent sustained tolerance vs. development of GVHD after immune 
suppression discontinuation. Sample characteristics:  10mL peripheral blood will be drawn at 
each time point. Based on expected yield of 0.5-1ug RNA/mL whole blood, total RNA isolated 
will be more than sufficient for planned mRNA profiling. RNA will be isolated (total RNA, 
inclusive of small RNA to permit future microRNA studies), and RNA integrity confirmed. 
 
Clinical phenotypes: The tolerant phenotype of interest in the primary analysis (inclusive of 
both single time-point prediction and longitudinal change) is immune tolerance as defined by 
complete discontinuation of IS and absence of GVHD by the end of study follow up period. In 
contrast, non-tolerant is defined by either/both continued IS or GVHD. As secondary objectives 
day 21 and day 90 samples will be examined for prediction of alloreactivity (acute and chronic 


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GVHD, respectively). Definitions of acute and chronic GVHD will follow standard consensus 
criteria.26,27 
 
C1.3. Methodology and Analyses 
 
Identify transcriptional markers predictive of immune tolerance development 
We have performed power calculations based on a two-sided two-sample t-test (PASS software) to 
estimate the required sample size for the initial discovery cohort. We examined power according to 
the proportion of differentially expressed probe sets at an effect size of 2 with a false discovery rate 
of 10%. With n=15 vs. n=15 in the respective phenotypic groups, we have > 90% power to detect 
differential expression of as low as 1% of the total probe sets tested. As our prior SAM two-group 
comparison (see preliminary data) showed n=655 (~1% of total probes) differentially expressed, 
n=30 will be used for discovery.   
 
We will identify differentially expressed genes that distinguish tolerant vs. non-tolerant patients 
using microarray (Affymetrix Human U133 plus 2.0 array). Scanned output files will be 
analyzed by robust multi-array average analysis (RMA).30 The Significance Analysis of 
Microarrays (SAM) technique will be employed to identify differentially expressed genes 
between groups,31 using 10% FDR, and ≥ 1.5 fold difference in mean expression values. 
Functional Ontology Enrichment (MetaCore by GeneGo) with 5% FDR filter will be used to 
identify enriched pathways and cellular process networks. We will confirm candidate markers 
identified using NanoString nCounter technology.  
 


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Investigate longitudinal change in transcriptional markers for prediction of immune 
tolerance development   
 
Approach: We will employ linear models for microarray data with a FDR of 10% for this 
analysis examining change in gene expression (day 21 to day 90) to subsequent tolerant vs. non-
tolerant phenotype.32,33 As a secondary approach, we will also use generalized linear mixed 
effect modeling to identify genes with longitudinally different change between groups. A 
predictive model based on genes identified above will be developed using the machine learning 
technique, including the PCA method and support vector machine. The final predictive model 
will take into account both the identified gene signature, and also significant clinical variables.  
 
 
Examine predictive markers of acute and chronic GVHD 
 
Approach: We will study day 21 and day 90 samples for prediction of acute and chronic 
GVHD, respectively. This will provide key information for the following: (1) Differentially 
expressed genes predictive of GVHD will be compared to those segregating the ultimate tolerant 
and non-tolerant phenotype to address the extent to which the tolerant transcriptional program 
reflects absence of GVHD-associated determinants vs. induction of a specific tolerance-inducing 
program. Shared and unique genes among these two gene lists will be determined, mapped to 
biologic pathways, and compared to prior data. (2) GVHD predictive biomarkers published to 
date have not been studied in this clinical context (HLA-disparate haploidentical transplants 


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using PTCy/SIR/MMF). We anticipate unique findings in this setting that will contribute to 
mechanistic understanding of failure/GVHD development in this setting. 
 
 
11. References 
1. 
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2. 
Lee SJ, Klein J, Haagenson M, et al. High‐resolution donor‐recipient HLA matching contributes to 
the success of unrelated donor marrow transplantation. Blood. 2007;110(13):4576‐4583. 
3. 
Lown RN, Marsh SG, Switzer GE, Latham KA, Madrigal JA, Shaw BE. Ethnicity, length of time on 
the register and sex predict donor availability at the confirmatory typing stage. Bone Marrow 
Transplant. 2014;49(4):525‐531. 
4. 
Pidala J, Kim J, Schell M, et al. Race/ethnicity affects the probability of finding an HLA‐A, ‐B, ‐C 
and ‐DRB1 allele‐matched unrelated donor and likelihood of subsequent transplant utilization. Bone 
Marrow Transplant. 2013;48(3):346‐350. 
5. 
Ciurea SO, Champlin RE. Donor selection in T cell‐replete haploidentical hematopoietic stem cell 
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6. 
Bayraktar UD, Champlin RE, Ciurea SO. Progress in haploidentical stem cell transplantation. Biol 
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7. 
Aversa F, Terenzi A, Tabilio A, et al. Full haplotype‐mismatched hematopoietic stem‐cell 
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2005;23(15):3447‐3454. 
8. 
Luznik L, O'Donnell PV, Symons HJ, et al. HLA‐haploidentical bone marrow transplantation for 
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Luznik L, O'Donnell PV, Fuchs EJ. Post‐transplantation cyclophosphamide for tolerance induction 
in HLA‐haploidentical bone marrow transplantation. Semin Oncol. 2012;39(6):683‐693. 
10. 
Owens AH, Jr., Santos GW. The effect of cytotoxic drugs on graft‐versus‐host disease in mice. 
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11. 
Kasamon YL, Luznik L, Leffell MS, et al. Nonmyeloablative HLA‐haploidentical bone marrow 
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outcome. Biol Blood Marrow Transplant. 2010;16(4):482‐489. 
12. 
Bashey A, Zhang X, Sizemore CA, et al. T‐cell‐replete HLA‐haploidentical hematopoietic 
transplantation for hematologic malignancies using post‐transplantation cyclophosphamide results in 
outcomes equivalent to those of contemporaneous HLA‐matched related and unrelated donor 
transplantation. J Clin Oncol. 2013;31(10):1310‐1316. 
13. 
Bashey A, Solomon SR. T‐cell replete haploidentical donor transplantation using post‐transplant 
CY: an emerging standard‐of‐care option for patients who lack an HLA‐identical sibling donor. Bone 
Marrow Transplant. 2014;49(8):999‐1008. 
14. 
Ciurea SO, Mulanovich V, Saliba RM, et al. Improved early outcomes using a T cell replete graft 
compared with T cell depleted haploidentical hematopoietic stem cell transplantation. Biol Blood 
Marrow Transplant. 2012;18(12):1835‐1844. 


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15. 
Raiola AM, Dominietto A, di Grazia C, et al. Unmanipulated haploidentical transplants compared 
with other alternative donors and matched sibling grafts. Biol Blood Marrow Transplant. 
2014;20(10):1573‐1579. 
16. 
Raj K. Haploidentical versus matched donor stem cell transplantations for acute myeloid 
leukemia/myelodysplastic syndrome patients. Biol Blood Marrow Transplant. 2014;20(12):1875‐1876. 
17. 
Castagna L, Crocchiolo R, Furst S, et al. Bone marrow compared with peripheral blood stem cells 
for haploidentical transplantation with a nonmyeloablative conditioning regimen and post‐
transplantation cyclophosphamide. Biol Blood Marrow Transplant. 2014;20(5):724‐729. 
18. 
Clay J, Kulasekararaj AG, Potter V, et al. Nonmyeloablative peripheral blood haploidentical stem 
cell transplantation for refractory severe aplastic anemia. Biol Blood Marrow Transplant. 
2014;20(11):1711‐1716. 
19. 
Devillier R, Granata A, Furst S, et al. Low incidence of chronic GVHD after HLA‐haploidentical 
peripheral blood stem cell transplantation with post‐transplantation cyclophosphamide in older 
patients. Br J Haematol. 2016. 
20. 
Raj K, Pagliuca A, Bradstock K, et al. Peripheral blood hematopoietic stem cells for 
transplantation of hematological diseases from related, haploidentical donors after reduced‐intensity 
conditioning. Biol Blood Marrow Transplant. 2014;20(6):890‐895. 
21. 
Pidala J, Kim J, Jim H, et al. A randomized phase II study to evaluate tacrolimus in combination 
with sirolimus or methotrexate after allogeneic hematopoietic cell transplantation. Haematologica. 
2012;97(12):1882‐1889. 
22. 
Pidala J, Kim J, Alsina M, et al. Prolonged sirolimus administration after allogeneic hematopoietic 
cell transplantation is associated with decreased risk for moderate‐severe chronic graft‐versus‐host 
disease. Haematologica. 2015;100(7):970‐977. 
23. 
Timrott K, Vondran FW, Bektas H, Klempnauer J, Jager MD. Application of allogeneic bone 
marrow cells in view of residual alloreactivity: sirolimus but not cyclosporine evolves tolerogenic 
properties. PLoS One. 2015;10(3):e0119950. 
24. 
Cieri N, Greco R, Crucitti L, et al. Post‐transplantation Cyclophosphamide and Sirolimus after 
Haploidentical Hematopoietic Stem Cell Transplantation Using a Treosulfan‐based Myeloablative 
Conditioning and Peripheral Blood Stem Cells. Biol Blood Marrow Transplant. 2015;21(8):1506‐1514. 
25. 
Pidala J, Bloom GC, Eschrich S, et al. Tolerance associated gene expression following allogeneic 
hematopoietic cell transplantation. PLoS One. 2015;10(3):e0117001. 
26. 
Przepiorka D, Weisdorf D, Martin P, et al. 1994 Consensus Conference on Acute GVHD Grading. 
Bone Marrow Transplant. 1995;15(6):825‐828. 
27. 
Jagasia MH, Greinix HT, Arora M, et al. National Institutes of Health Consensus Development 
Project on Criteria for Clinical Trials in Chronic Graft‐versus‐Host Disease: I. The 2014 Diagnosis and 
Staging Working Group report. Biol Blood Marrow Transplant. 2015;21(3):389‐401 e381. 
28. 
Sorror ML, Maris MB, Storb R, et al. Hematopoietic cell transplantation (HCT)‐specific 
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29. 
Lee DW, Gardner R, Porter DL, et al. Current concepts in the diagnosis and management of 
cytokine release syndrome. Blood. 2014;124(2):188‐195. 
30. 
Irizarry RA, Hobbs B, Collin F, et al. Exploration, normalization, and summaries of high density 
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Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing 
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32. 
Rioth MJ, Warner J, Savani BN, Jagasia M. Next‐generation long‐term transplant clinics: 
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33. 
Booth GS, Gehrie EA, Jagasia MH, Shaw BE, Savani BN. When Can You Discard Stem Cells? Biol 
Blood Marrow Transplant. 2015. 
 
 
 


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Appendix A 
Data Safety and Collection Monitoring 
a. 
Data collection  
The Principal Investigator and assigned clinical research staff (clinical research coordinator, 
research data specialist) will be responsible for maintaining all study related documents. The 
Moffitt electronic medical record (PowerChart) will serve as source document. Data collected 
will be stored in Moffitt Cancer Center’s database system, ONCORE. Identifying patient 
information will be kept confidential. Representatives of the USF IRB will have access to patient 
information as it pertains to the study. Privacy and confidentiality of the information will be 
protected to the extent provided by law.  
 
b. 
Safety monitoring  
The principal investigator will have the primary responsibility for data safety and monitoring. 
Input will be sought from sub-investigators and other members of the BMT Program concerning 
data and safety issues. The PI of the study will have primary responsibility for ensuring that the 
protocol is conducted as approved by the SRC and IRB. The PI will ensure that the monitoring 
plan is followed, that all data required for oversight of monitoring are accurately reported to a 
DSMB and/or to the PMC and IRB as required, that adverse events are reported according to 
protocol guidelines, and that any adverse actions reflecting patient safety concerns are 
appropriately reported.  
The investigators and members of the BMT Research Staff will meet regularly. The following 
data will be reviewed:  
• 
Rate of accrual  
• 
Adverse events  
• 
Protocol deviations and/or violations.  


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• 
If necessary, corrective action and/or educational programs will occur to ensure subject 
safety and data integrity. Reports to the SRC, PMC, and IRB will be submitted as required.  
c. 
Anticipated toxicity following allogeneic transplantation:  
There are numerous anticipated adverse consequences of transplantation, which include, but are 
not limited to conditioning regimen related toxicity such as severe mucositis, idiopathic 
pneumonia syndrome, hepatic veno-occlusive disease and death, early and late infectious 
complications, potentially severe or fatal acute or chronic graft vs. host disease, and relapse of 
primary disease and its complications. Thus, expected complications of transplantation will not 
be collected as adverse events on this trial.  
d. 
Informed consent  
The investigator must explain to each subject (or legally authorized representative) the nature of 
the study, its purpose, the procedures involved, the expected duration, the potential risks and 
benefits involved and any discomfort it may entail. Each subject must be informed that 
participation in the study is voluntary and that he/she may withdraw from the study at any time 
and that withdrawal of consent will not affect his/her subsequent medical treatment or 
relationship with the treating physician.  
 
e. 
Adverse event collection and reporting  
Adverse Event 
An adverse event is any unexpected medical occurrence associated with the use of a drug or 
therapy in humans, whether or not considered drug related. It can be any unfavorable and 
unintended sign (e.g., an abnormal laboratory finding), symptom, or disease, temporally 
associated with the use of a drug. Infection, acute and chronic GVHD will not be considered 
adverse events. 
 
 


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AE Grading Criteria: 
AE severity is graded on a scale from 1 to 5 according to NCI CTCAE v4.03 or current version. 
Adverse events not included in the NCI CTCAE should be recorded and graded according to the 
General Grade Definition provided below: 
 
ADVERSE EVENT GENERAL GRADE DEFINITIONS 
Grade 1 
Mild 
Asymptomatic or mild symptoms; clinical or diagnostic 
observations only; intervention not indicated 
Grade 2 
Moderate 
Minimal, local or non-invasive intervention indicated; 
limiting age-appropriate instrumental ADL* 
Grade 3 
Severe 
Severe or medically significant, but not immediately 
life-threatening; hospitalization or prolongation of 
hospitalization indicated; disabling; limiting self-care 
ADL** 
Grade 4 
Life-threatening 
Life-threatening consequences; urgent intervention 
indicated 
Grade 5 
Death related to AE Death related to AE 
 
*Instrumental ADL: Preparing meals, shopping for groceries or clothes, using the telephone, 
managing money, etc. 
**Self-care ADL: Bathing, dressing and undressing, feeding self, using the toilet, taking 
medications, and not bedridden. 
 
 


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Suspected Adverse events – a subset of Adverse Events based on causality        
 A suspected adverse reaction is any adverse event for which there is a reasonable possibility that 
the drug caused the event. Reasonable possibility means there is evidence to suggest a causal 
relationship between the drug and the event. 
 
Unexpected  
An adverse event is considered unexpected if it is not listed in the investigator brochure, occurs 
in severity greater than previously described, or - for the purpose of this protocol - if it is not in 
keeping with expected post-transplantation toxicity. 
 
Serious 
An adverse event is considered “serious” if, in the view of either the investigator or the sponsor, 
it results in any of the following outcomes: 
1. 
Death 
2. 
A life-threatening adverse event (places the subject at an immediate risk of death) 
3. 
Inpatient hospitalization or prolongation of existing hospitalization 
4. 
A persistent or significant disability or incapacitation 
5. 
A congenital anomaly or birth defect 
Additionally, events that jeopardize the subject and may require medical or surgical intervention 
to prevent one of the outcomes above may also be considered serious based on appropriate 
medical judgment. 
 


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Adverse event recording: 
Non-serious AE:  Only unexpected, grade 3-5 AE will be recorded. 
Serious AE:  All SAE will be recorded. 
 
Adverse event reporting:  
Non-serious AE: Unexpected, grade 3-5 AE will be reported to the IRB in summary form on an 
annual basis. 
SAE: The principal investigator will report each serious adverse event, regardless attribution, 
within 24 hours of learning of the occurrence. In the event that the principal investigator does not 
become aware of the serious adverse event immediately (e.g., participant sought treatment 
elsewhere), the principal investigator is to report the event within 24 hours after learning of it 
and document the time of his or her first awareness of the adverse event. The SAE report must 
include event term(s), serious criteria, and the investigator’s determination of both the intensity 
of the event(s) and the relationship of the event(s) to study drug administration. 
 
 


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Appendix B 
 
Management of Cytokine Release Syndrome (CRS) after Haploidentical transplantation 
CRS is a non-antigen specific toxicity that occurs as a result of a high level immune activation. 
This immune activation is associated with modern cellular immunotherapies and is usually 
required as a mediator for clinical benefit.  
It manifests when large numbers of lymphocytes and/or myeloid cells become activated and 
release inflammatory cytokines. It has been classically associated with the infusion of 
monoclonal antibodies like anti-CD3 (OKT3), anti-CD52 (alemtuzumab), anti-CD20 (rituximab) 
among others. It has also been reported with the infusion of bi-specific antibodies for leukemia 
(blinatumomab), haploidentical cells to patients with refractory leukemia,  adoptive 
immunotherapies for cancer and most notably following the infusion of T-cells engineered to 
express chimeric antigen receptors (CAR T-cells) to treat leukemia or lymphoma.  
Symptoms usually begin shortly after cell infusion (within 1 to a few hours) and coincide with 
peak of cytokine release. Multiple cytokines are elevated, initially TNFα, and then IFNγ, IL-1β, 
IL-2, IL-6, IL-8 and IL-10. Commonly, in severe CRS, patients will develop progressive organ 
dysfunction and will require aggressive support in intensive care unit. Emerging evidence has 
implicated IL-6 as a central mediator of toxicity in CRS as the initiator of the pro-inflammatory 
cascade.  C-reactive protein (CRP) is an acute phase reactant produced in the liver largely in 
response to IL-6, and CRP level serve as a reliable surrogate of IL-6 activity. During CRS, it 
increases by several logs. Rapid increase of CRP has been seen in patients at risk for severe 
CRS. 


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In haploidentical transplantation, patients receive a donor hematopoietic cell graft containing 
large numbers of T-cells. Upon antigen presentation, donor and host alloreactive T-cells are 
activated and enter a rapid proliferation phase. This phase is heralded by the development of high 
grade fevers in day +1 to +3 after graft infusion. Fever rapidly abates after administration of 
cyclophosphamide in days +4, +5.  
The bulk of the published data using marrow grafts suggests that this febrile period is rarely 
associated with organ dysfunction. In this protocol, we will use peripheral blood stem cell grafts, 
which in average, have 1 more log of T-cells compared to marrow. It is likely that this large 
increase in T-cells may generate a stronger T-cell activation/expansion and proportionally 
stronger cytokine release with a potential major clinical impact. Very little data is available in the 
literature on how to address this concern. As a safety measure we will incorporate an algorithm 
for the management of CRS that follows the guidelines published by Lee et al.,29 and has been 
used successfully in other settings, particularly after infusion of CAR-T cells. Table 1 
summarizes the clinical manifestations of CRS and table 2 has the algorithm for management. 
Table 1. Clinical manifestations of CRS. 
Organ system 
Symptoms 
Constitutional 
Fever + rigors, malaise, fatigue, anorexia, myalgias, arthralgias, nausea, HA 
Skin 
Rash 
Gastrointestinal 
Nausea, vomiting, diarrhea 
Respiratory 
Tachypnea, hypoxemia 
Cardiovascular 
Tachycardia, hypotension, ↑ cardiac output, ↓ cardiac output (late) 
Renal 
Azotemia 
Hepatic 
Transaminitis, hyperbilirubinemia 
Neurologic 
Headache, AMS, confusion, delirium, hallucinations, tremor, 
 seizures 
Coagulation 
Elevated D-dimer, hypofibrinogenemia 


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Table 2. Algorithm for the management of CRS 
 
Cytokine Release Syndrome 
Grading  assessment 
Extensive co-
morbidities or older 
age? 
No/Yes 
Treatment 
Grade 1:  
 Fever (defined as ≥ 38.3oC) 
 Constitutional symptoms 
N/A 
 Vigilant supportive 
care 
 Assess for infection 
 Treat fever and 
neutropenia if 
present, monitor fluid 
balance, antipyretics, 
analgesics as needed 
Grade 2: 
 Hypotension: responds to fluids or one 
low dose vasopressor 
 Hypoxia: responds to <40% O2 
 Organ toxicity: grade 2 
No 
 As above for grade 1 
 Monitor cardiac and 
other organ function 
closely  
Grade 2: 
 Hypotension: responds to fluids or one 
low dose vasopressor 
 Hypoxia: responds to <40% O2 
 Organ toxicity: grade 2 
Yes 
 As above for grade 2 
 Consider tocilizumab 
Grade 3: 
 Hypotension: requires multiple 
vasopressors or high dose 
vasopressors 
 Hypoxia: requires ≥ 40% O2  
 Organ toxicity: grade 3, grade 4, 
transaminitis 
N/A 
Grade 4 
 Mechanical ventilation 
 Organ toxicity: grade 4 excluding 
transaminitis  
N/A 


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Tocilizumab dose will be 4 mg/kg and may be repeated if clinical improvement does not occur 
on 24 to 48 hours. The goal of the treatment of CRS in haploidentical transplantation is the 
prevention of organ dysfunction, while preserving the GVHD prevention effect associated with 
the use of post-transplantation cyclophosphamide. In addition to the clinical evaluation during 
the first 3 days after graft infusion, it is recommended to follow CRP on a daily basis until day 
+5. Rapid increases in CRP should be taken in consideration in conjunction with the clinical 
information to diagnose and trigger the treatment of CRS.