Conditions: Acute Leukemia Chronic Lymphocytic Leukemia Chronic Myelogenous Leukemia, BCR-ABL1 Positive Diffuse Large B-Cell Lymphoma Follicular Lymphoma Graft Versus Host Disease Mantle Cell Lymphoma Marginal Zone Lymphoma Myelodysplastic Syndrome Myelofibrosis Myeloproliferative Neoplasm Small Lymphocytic Lymphoma
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 5
Healthy volunteers: no

Brief summary:
This phase II trial studies how well tacrolimus, bortezomib, and anti-thymocyte globulin (thymoglobulin) work in preventing low toxicity graft versus host disease (GVHD) in patients with blood cancer who are undergoing donor stem cell transplant. Tacrolimus and anti-thymocyte globulin may reduce the risk of the recipient's body rejecting the transplant by suppressing the recipient's immune system. Giving bortezomib after the transplant may help prevent GVHD by stopping the donor's cells from attacking the recipient. Giving tacrolimus, bortezomib, and anti-thymocyte globulin may be a better way to prevent low toxicity GVHD in patients with blood cancer undergoing donor stem cell transplant.

Detailed description:
PRIMARY OBJECTIVES:

I. To determine a composite end point of alive and severe acute GVHD free at 6 months following human leukocyte antigen (HLA) matched related or unrelated donor hematopoietic peripheral blood transplant in patients with hematologic malignancies who receive the immunosuppressive combination tacrolimus, bortezomib, anti-thymocyte globulin (TBT) as GVHD prophylaxis.

II. To determine the safety of this combination in the first six months post-transplant.

SECONDARY OBJECTIVES:

I. To determine the cumulative incidence of grade III-IV aGVHD.

II. To determine incidence and severity of chronic GVHD.

III. To determine disease relapse or progression overall and disease free survival at one year.

OUTLINE:

Patients receive tacrolimus intravenously (IV) on day -3 through day 180. Patients may receive tacrolimus orally (PO) later at the doctor's discretion. Patients receive anti-thymocyte globulin IV on days -3, -2, and -1 and bortezomib IV on day 0 and day 3. Patients undergo allogeneic bone marrow transplant on day 0.

After completion of study treatment, patients are followed up for 6 months and then periodically for up to 2 years.

Arms:
arm 1: Patients receive tacrolimus IV on day -3 through day 180. Patients may receive tacrolimus PO later at the doctor's discretion. Patients receive thymoglobulin IV on days -3, -2, and -1 and bortezomib IV on day 0 and day 3. Patients undergo allogeneic bone marrow transplant on day 0.

Interventions: intervention 1: Thymoglobulin intervention 2: Bortezomib intervention 3: Tacrolimus
Intervention descriptions: intervention 1: Given IV intervention 2: Given IV intervention 3: Given IV and PO

Protocol document:
Prot_SAP_000.pdf: 
Winship Cancer Center  
Blood and Marrow Stem Cell Transplantation Protocol 
 
Protocol Date:   
Current Submission:   
  
 
Phase II Trial of Low Toxicity GVHD Prevention and Enhanced Immune Recovery 
with Tacrolimus, Bortezomib and ThymoglobulinTBT 
 
Principal Investigator: 
Zaid Al-Kadhimi, MD 
 
Co-Principal Investigator: 
Edmund Waller 
Amelia Langston  
 
External collaborator: 
Jeffrey Miller, MD, University of Minnesota 
 
BMT Immunotherapy Winship Cancer Center/ Emory University 
 
Co-Investigators: 
 
 
Statistician 
Zhengjia Chen  
 
BMT Transplant Coordinators 
 
Data Manager: 
 
Transplant Data Managers 
 
 


Protocol Synopsis 
 
Study Design:The study is designed as a Phase II, single center trial of a low toxicity immune 
prophylaxis combination of tacrolimus(Tac), bortezomib and thymoglobulin (Thymo) (TBT) 
given as GVHD prophylaxis for allogeneic peripheral hematopoietic stem cell transplant (allo-
HSCT) recepients. 
 
Primary Objective: 
 
1- To determine a composite end point of alive and severe acute GVHD free at 6 months 
following HLA matched related or unrelated donor hematopoietic peripheral blood transplantion 
in patients with hematologic malignancies who receive the immunosuppressive combination 
TBT as GVHD prophylaxis. 
2- To determine the safety of this combination in the first six months post-transplant. 
 
 
Secondary Objectives: To determine: incidence and severity of aGVHD (grade II-IV), time to 
platelet and absolute neutrophil recovery (engraftment), the cumulative incidence of grade III-IV 
aGVHD, days 30, 60 , 90, 180, natural killer cell and gamma delta T-cell recovery and 
phenotype, Thymo pharmacokinetics and it’s correlation with IL-15 levels and natural killer (NK) 
cell and gamma delta (γδ) T-cell recovery on day 30 post-transplant, donor cell engraftment, 
incidence of infections-including CMV and EBV reactivation including lymphoproliferative 
disorders PTLD, incidence of veno-occlusive disease, incidence and severity of chronic GVHD, 
rates of Grade ≥ 3 non-hematologic toxicity according to the Common Terminology Criteria for 
Adverse Events (CTCAE) Version 4.0, non-relapse mortality (NRM), disease relapse or 
progression overall and disease free survival at one year, immunosuppression-free survival at 
one year, and KPS both pre-transplant and at various points post-transplant.   
 
 
 
Eligibility Criteria: Eligible patients are between 18 and 75 years undergoing allo-HSCT for 
treatment of acute leukemia, chronic myeloid leukemia, myelodysplasia, myelofibrosis, chronic 
lymphocytic leukemia/small lymphocytic lymphoma, chemosensetive lymphoma; and multiple 
myeloma who are eligible for an allogeneic transplant. Patients must have an HLA matched 
(8/8) related or unrelated donor, who is willing to donate peripheral blood stem cells and meet 
institutional criteria for donation. Patients are eligible to receiving a reduced intensity 
conditioning (RIC) or myeloablative conditioning (MAC) regimens.  
 
Treatment Description:Patients will receive a combination of triple immune suppressive 
medications TBT as follows: Tacrolimus IV infusion to start on day -3, bortezomib 1.3 mg/m2 IV 
on days 0, and +3 post-transplant, and thymoglobulin IV infusions on days -3 0.5 mg/kg,-2 (2 
mg/kg), and -1 (2.5 mg/kg).     
 
Accrual Objective:The clinical trial will enroll 38 patients over 24 months.  
 
Study Duration:Patients will be followed for 6 months and 2 years following allo-HSCT. 
 
 
 
 
 
 
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TABLE OF CONTENTS 
 
 
 
 
 
 
      PAGE 
 
 
 
 
1. 
Hypothesis   
 
 
 
 
 
 
 
 5 
 
2. 
Objectives 
 
 
 
 
 
 
 
 
 5 
 
3. 
Background and Rationale  
 
 
 
 
 
 5 
 
4.  
Experimental plan  
 
 
 
 
 
 
 13 
 
 
5. 
Eligibility  
 
 
 
 
 
 
 
            15  
 
6. 
 Drug information     
 
 
  
             
 
 16 
 
7. 
Donor and Graft selection  
 
 
 
 
 
 19  
 
8. 
Pre transplant conditioning  
 
 
 
 
 
 20 
 
9. 
Allogeneic stem cell infusion 
 
 
 
 
           22 
 
10. 
Anti-Infective prophylaxis   
 
 
 
 
 
 22 
 
11. 
GVHD Prophylaxis                
 
 
 
 
 
 23 
 
12. 
Immuno-correlative and Pharmacokinetic studies 
 
 
 26 
 
13. 
Study procedures. Screening & baseline evaluation 
 
 
 28 
 
14. 
Study procedures. Maintenance phase.  
 
 
 
 28 
 
15. 
GVHD  
 
 
 
 
 
 
 
 
 29 
 
16.  
NCI Toxicities 
 
 
 
 
 
 
 
 30 
 
17.  
Statistical Considerations  
 
 
 
 
 
 30 
 
18. 
Data & Safety monitoring  
 
 
 
 
 
 32 
 
19. 
Removal of patients from study 
 
 
 
 
 
 34 
 
20. 
Ethics  
 
 
 
 
 
 
 
 
 35 
 
21. 
References  
 
 
 
 
 
 
 
 36 
 
 
 
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4 


 
 
1.0    HYPOTHESES:  
 
 
1.1 We hypothesize that the combination of TBT will provide effective prevention of 
 
aGVHD in patients with hematologic malignancies undergoing allo-HSCT. 
1.2 The TBT combination is safe and does not add to the preparative regimen related 
 
toxicity. 
1.3 The TBT regimen will result in an enhanced mean recovery of natural killer (NK) 
 cells compared to historical controls at day 30 post-transplant. 
1.4 Post-transplant Thymo area under the curve (AUC) correlate with higher IL-15 level 
peak post-transplant and with higher NK/ γδ T-cell recovery on day 30 post-
transplant. 
  
2.0    OBJECTIVES: 
 
Primary:  
 
2.1  To determine a composite end point of alive and severe acute GVHD free at 6 
months following HLA matched related or unrelated donor hematopoietic peripheral 
blood transplant in patients with hematologic malignancies who receive the 
immunosuppressive combination TBT as GVHD prophylaxis. 
2.2  To determine the safety of this combination in the first six months post-transplant. 
 
 
Secondary: 
To determine: incidence and severity of aGVHD (grade II-IV), time to platelet and absolute 
neutrophil recovery (engraftment), the cumulative incidence of grade III-IV aGVHD, days 
30, 60 , 90, 180, natural killer cell and gamma delta T-cell recovery and phenotype, 
Thymo pharmacokinetics and it’s correlation with IL-15 levels and natural killer (NK) cell 
and gamma delta (γδ) T-cell recovery on day 30 post-transplant, donor cell engraftment, 
incidence of infections-including CMV and EBV reactivation including lymphoproliferative 
disorders (PTLD), incidence of veno-occlusive disease, incidence and severity of chronic 
GVHD, rates of Grade ≥ 3 non-hematologic toxicity according to the Common 
Terminology Criteria for Adverse Events (CTCAE) Version 4.0, non-relapse mortality 
NRM, disease relapse or progression overall and disease free survival at one year, 
immunosuppression-free survival at one year, and KPS both pre-transplant and at 
various points post-transplant.   
 
 
 
3.0 
BACKGROUND AND RATIONALE: 
 
3.1 Graft versus host disease continues to be a major limitation of allo-HSCT outcomes  
Graft versus host disease (GVHD) is the major complication of allogeneic hematopoietic stem 
cell transplantation (allo-HSCT) and a leading cause of morbidity and mortality post-transplant. 
Factors which influence the incidence and severity of GVHD include the use of: peripheral blood 
stem cells, unrelated donors, and transplants from human leukocyte antigen (HLA) mismatched 
donors.1-4 Methods to decrease the incidence of GVHD have relied primarily on pharmacologic 
immunosuppression which at most centers consists of a combination of methotrexate (MTX) 
and a calcineurin inhibitor (CNI) either cyclosporine A (CSA) or tacrolimus (Tac). In spite of the 
use of these agents, the rate of grade II-IV aGVHD ranges from 35-50% with HLA matched 
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sibling donors (SD) and up to 70% in unrelated donors (UD).1-3 Additionally, MTX is associated 
with significant mucositis, delayed engraftment, pulmonary, renal and hepatic toxicities.4-7 At 
Winship cancer center, Langston et al. reported grade II-IV aGVHD 44%, III-IV aGVHD 
incidence of 23% with an NRM of about 38% at 36 months in patients who underwent allo-
HSCT from UD using RIC and Tac/MTX or Tac/mycophenolate mofetel (MMF). Intermediate 
dose Thymo (6mg/kg) was added for recipients of HLA 7/8 matched donors8. Therefore, safer 
and more efficacious agents for the prevention of GVHD are needed for improved patient 
outcomes. 
 
3.2 Intermediate dose Thymoglobulin® in GVHD prevention  
3.2.1 Intermediate doses of thymoglobulin (Thymo) when combined with CNI have showed 
encouraging results in improving the rates of both acute and chronic GVHD as well as NRM   
Devillier et al. reported on 206 patients who were transplanted from an HLA matched donor (SD 
& UD) with a combination of Tac and Thymo (5mg/kg total dose). Transplant outcomes were as 
follows: aGVHD II-IV was 23%, III-IV was 9%, NRM 22% at 4 years.9  The same group showed 
that ATG dose < 6mg/kg was associated with less risk of relapse in RIC AML patients.10 Mohty 
et al. recently reported on a large phase II trial of 80 elderly or less fit patients who underwent 
transplant from HLA matched (C mismatch was allowed) using an intermediate intensity 
preparative regimen (3 days of busulfan at 130mg/m2 and fludarabine) with ATG total dose of 
5mg/kg(on days -2, -1) and Tac alone or with Tac+ mycophenolate (for C antigen mismatch).  
He showed a grade III-IV aGVHD incidence of 7.5%, cGVHD of 33% and NRM of 11% at 2 
years.11  
 
3.2.2 Thymoglobulin: Pharmacology 
Thymoglobulin® is a purified, pasteurized, gamma immune globulin, obtained by immunization 
of rabbits with human thymocytes (Thymoglobulin® Prescribing Information).  This 
immunosuppression product, a polyclonal antibody, contains cytotoxic antibodies of defined 
specificity against functional molecules on T lymphocytes. To minimize the risk of transmission 
of infective agents from this biological product, viral testing is performed during production, and 
a viral inactivation step (pasteurization, i.e., heat treatment of the active ingredient at 60˚C/10 
hours) is an integral part of the Thymoglobulin manufacturing process.  The potency of the 
product is determined by lymphocytotoxicity assay.  In each lot, antibody activity against human 
red blood cells and platelets is analyzed and determined to be within acceptable limits before 
release.  Additionally, before release for clinical use, each lot of Thymoglobulin is tested to 
ensure its ability to inhibit E-rosette formation between human peripheral lymphocytes and 
sheep red blood cells in vitro.  Only sterile lots that test negative for anti-human serum protein 
antibody, anti-glomerular basement membrane antibody, fibroblast toxicity and pyrogens are 
released. 
 
3.2.3 Thymoglobulin: Mechanism of action 
The in vitro mechanism of action by which polyclonal anti-lymphocyte preparations suppress 
immune responses is not fully understood.  Thymoglobulin includes antibodies against T-cell 
markers such as CD2, CD3, CD4, CD8, CD11a, CD18, CD25, CD44, CD45, HLA-DR, HLA 
Class I heavy chains and ß2 microglobulin 12-16 as well as dendritic cells adhesion molecules17 
and B-cell markers such as CD20.12,18  In vitro, Thymoglobulin (concentrations >0.1 mg/ml) 
mediates T-cell suppressive effects via inhibition of proliferative responses to several mitogens, 
with post-transcription blockade of interferon gamma and CD25 synthesis.19 The in vivo 
mechanism of action of Thymoglobulin is also not fully understood.  Possible mechanisms by 
which Thymoglobulin may induce immunosuppression in vivo include T cell clearance from the 
circulation and modulation of T-cell activation, homing and cytotoxic activities. T-lymphocyte 
depletion is promptly observed in transplant patients following administration of 
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Thymoglobulin.20  This may result from the complement dependent lysis in the intravascular 
space or the opsonization and subsequent phagocytosis by macrophages of antibody-coated T 
cells.19  Monitoring reveals that T lymphocyte depletion in peripheral blood persists for several 
days to several months following cessation of Thymoglobulin therapy.21 
 
Thymoglobulin is a potent immunosuppressive agent that demonstrates a rapid and profound 
effect resulting in lower white blood cells (WBC), primarily from a decrease in T-cell counts.  The 
magnitude and duration of lymphopenia have been studied.  Reductions of circulating 
lymphocytes by 83% to 92% from pretreatment values were seen after a single dose of 
Thymoglobulin and were sustained throughout the daily dosing period in 4 clinical pharmacology 
studies.  The recovery from Thymoglobulin-induced lymphocyte depletion was gradual, 
beginning soon after cessation of therapy, with most recovery by 3 months.  Recovery may take 
longer than 3 months, but persistent lymphocyte depletion that has been seen but has not 
resulted in any morbidity.  T-cell subsets determined by flow cytometry also demonstrate similar 
dramatic decreases.  In a phase III randomized trial, T-cell monitoring was performed in 26 
patients.  T-cell depletion was greater in the Thymoglobulin patients as compared with the 
Atgam® (Pharmacia-Upjohn, Kalamazoo, MI) patients. T-cell depletion lasted for at least 90 
days after the cessation of therapy.  At that time, T-cell counts remained 40% lower in 
Thymoglobulin-treated patients than in Atgam-treated patients. 21Thymoglobulin also induces 
apoptosis in all B-cell lineages, particularly at higher doses.19 
 
3.2.4 Thymoglobulin Pharmacokinetics: 
General PK: Only few studies evaluated the full pharmacokinetics of rabbit anti-thymocyte 
globulin R-ATG in stem cell transplant.22-25 These studies have demonstrated few important 
observations about R-ATG PK: (1)Total R-ATG (measured by ELISA) have a longer half-life 
than active R-ATG (capable of binding human lymphocytes and measured by flowcytometry). 
(2)One pediatric study showed a constant half-life and linear correlation between dose (7.5-20 
mg/kg) and maximum concentration (C max), whereas higher doses (30-40mg/kg) accumulated 
in the body.24 (3)Clearance of active R-ATG is variable with a median half-life of about 6 days 
for the active form and about 14 days for total R-ATG. Sub-therapeutic levels of active R-ATG 
can be detected in a median of about 15 days. (4) R-ATG clearance has a bi-phasic clearance 
pattern consistent with an initial fast clearance of target bound antibody followed by a slower 
degradation of unbound targets. (5)ATG-F PK in one study had a slightly shorter half-life of the 
active component (4 days) compared to R-ATG.26 (6)Two studies showed that ATG (R-ATG and 
ATG-F) persist post-transplant with variable reactivity to different lymphoid subsets compared to 
fresh R-ATG. There was less avidity to NK cells compared to other immune cells.23,27 This may 
be explained in part by faster recovery of NK cells which leads to faster clearance of the active 
component of the antibody.   
Factors affecting PK-The ATG PK is variable depending on multiple factors: Host factors 
including size or body weight, lymphocyte counts at the time of ATG administration, and the 
presence of anti ATG anti bodies. Transplant factors include: ATG dose, type and Timing of 
infusion. Admiral et al. proposed a R-ATG PK model in which higher weight and lower 
lymphocyte counts pre ATG can predict higher ATG exposure.25 What is not known today is 
which weight dosing formula would produce a consistent & predictable ATG PK. All therapeutic 
trials have used total body weight.  Since being overweight does not increase the size of the 
lymphoid tissue in the body, then theoretically this will lead to higher ATG exposure and 
potentially worse side effects. The other unknown is the effect of graft lymphocyte content on 
the ATG clearance. The presence of anti-Thymoglobulin anti-bodies will accelerate the 
clearance and reduce GVHD prevention efficacy.28 The last factor is timing of ATG infusion in 
relation to the day of transplant. Early pre transplant ATG infusion (day -10 or -7) can lead to 
effective host T-cell and APC depletion with little graft T-cell depletion. Which can lead to robust 
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engraftment and possibly more GVHD.22 While late ATG infusion (closer to day 0 or after) can 
lead to more graft T-cell depletion, less GVHD, poor immune reconstitution and possibly more 
infections and relapses post-transplant.      
3.2.5  Thymoglobulin: Safety profile 
Thymoglobulin presents a consistent AE profile, with AEs that are generally manageable or 
reversible. Adverse events with Thymoglobulin occur with similar or less frequency than those 
reported with other polyclonal anti-T-cell agents.  The most frequently reported AEs include 
fever, chills, leukopenia, thrombocytopenia, headache, diarrhea and abdominal pain.  In the US 
Phase III controlled clinical trial (n=163) comparing the efficacy and safety of Thymoglobulin and 
Atgam for the treatment of acute renal allograft rejection, AEs (using the COSTART system) 
were similar in both treatment groups and were generally manageable or reversible.21  Below is 
an overview of the safety profile of Thymoglobulin. 
3.2.5.1  Overview of Safety 
Total patient exposure worldwide since 1984 when Thymoglobulin was first licensed in France, 
is approximately 2.1 million vials, equivalent to over 54,000 courses. Approximately 60% of 
patients can be expected to experience adverse reactions with the use of Thymoglobulin.  
These usually occur after the first infusion.   The mechanism of some of those adverse reactions 
is probably related to cytokine release.  Pre-medication with corticosteroids and antihistamines 
and a decrease in the infusion rate may enable the incidence and severity of certain adverse 
reactions to be reduced.  While anaphylactic hypersensitivity reactions are rare, delayed allergic 
responses in the form of serum sickness have been reported.  Infection and PTLD are 
associated with use of immunosuppressive agents, including Thymoglobulin.  The adverse 
events seen typically with Thymoglobulin can be grouped.   
Adverse events reported during and after Thymoglobulin infusions: 
The most common systemic adverse reactions frequently reported in clinical trials which present 
are fever (63%), chills (57%), pain (46%) headache (40%), abdominal pain (38%), diarrhea 
(37%), hypertension (37%), nausea (37%), peripheral edema (34%), dyspnea (28%), asthenia 
(27%), tachycardia (26%). Hypotension, vomiting and malaise have also been commonly 
reported.  The likely mechanism of action for these events is the release of cytokines during the 
first dose administration, particularly if appropriate pre-medications were not administered. Local 
adverse reactions such as pain at the infusion site and peripheral thrombophlebitis have also 
been reported.   
Rare delayed allergic reactions such as serum sickness (fever, pruritus, rash associated with 
joint and muscle pain) may occur 7 to 15 days post-treatment initiation. Immediate serious 
allergic reactions or anaphylaxis are exceptional.  
Adverse events associated with undesirable cross-reacting antibodies: 
Adverse reactions associated with the presence of antibodies inducing cross-reactions such as 
leukopenia (57%) and thrombocytopenia (37%), have been reported during and subsequent to 
treatment with rabbit anti-human thymocyte globulin.  The reactions may emerge during the first 
2 days of treatment or after the end of treatment.  The mechanism of action likely causing these 
effects involves the presence of antibodies cross-reacting with shared antigens of lymphocytes, 
neutrophils and platelets, or additive effects from concomitant medication.  Monitoring of the 
white blood cell and platelet counts enables the severity and frequency of such reactions to be 
reduced. 
Adverse effects associated with over-immunosuppression: 
Adverse effects associated with over-immunosuppression including infectious complications 
(bacterial, fungal, viral and protozoal) and malignancies (particularly lymphoproliferative 
syndrome) have been reported.  It is important to note that concomitant or previous 
immunosuppressive treatments may contribute to the over-immunosuppression observed.  
Appropriate anti-infective prophylaxis will significantly reduce the incidence of infections; EBV 
monitoring and pre-emptive treatment will possibly decrease the incidence of PTLD.  
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3.2.5.2. Particular comments related to Adverse Events Seen in Hematology 
Some transient abnormalities of liver function tests have been described in patients with aplastic 
anemia treated by Thymoglobulin or other ATG’s.  It is not clear whether these abnormalities 
are related to the disease and pre-existing liver dysfunction, or to the treatment.29 When 
Thymoglobulin is used during conditioning prior to hematopoietic stem cell transplantation (allo-
HSCT) or in the first 3 months after the transplantation to treat acute GVHD, the ability of the 
patient to form antibodies against rabbit immunoglobulins is abolished.  Serum sickness thus, 
does not appear to occur, however alert observation is recommended.  Although an initial 
study30 noted an increase in CML relapse with Thymoglobulin compared to other T depletion 
agents when dosed at 10mg/kg, subsequent studies with lower doses did not have an increase 
in CML relapse.  No increase in relapse was seen in other studies or other disease cohorts.31 
The incidence of PTLD is mainly influenced by the HLA disparity, the T-cell depletion and both 
the occurrence of and the treatment of acute GVHD.32 PTLD cases have rarely been reported in 
patients who were conditioned with Thymoglobulin: In the Karolinska Institute, Sweden, 
Remberger diagnosed no cases of PTLD in the 61 MUD BMT patients who received 
Thymoglobulin 2 mg/kg/day from day –5 to –1 prior to transplant.30 At City of Hope, there were 
no cases of PTLD in the 20 UD patients who received thymoglobulin 7.5 mg/kg, despite a 
relatively high rate of EBV reactivation (6/20). Treatment with rituximab was successful in 
eliminating EBV reactivation in all patients.33  
 
3.3 The benefit of bortezomib in GVHD prophylaxis  
 
3.3.1 In the allogeneic allo-HSCT context, a Phase I/II clinical trial of a regimen of bortezomib 
plus standard tacrolimus/methotrexate for GVHD prophylaxis after fludarabine/busulfan-based 
RIC allo-HSCT with 1-2 locus HLA-mismatched donors (HLA-A, -B, -C; -DQB1, -DRB1).  
Bortezomib MTD was 1.3 mg/m2 administered on Days +1, +4 and +7 after stem cell infusion.  
Phase I results indicated minimal toxicity (rapid engraftment, zero non-relapse mortality in the 
first year) and excellent acute GVHD control with the bortezomib-based regimen.34  Phase I/II 
results demonstrated that the grade II-IV acute GVHD rate at Day 180 was 22% and no patients 
developed grade IV acute GVHD. Additionally, 2-year probabilities of non-relapse mortality and 
relapse were 11% and 38% respectively in a population receiving HLA-mismatched unrelated 
donor allo-HSCT. Furthermore, a significant improvement in day 30 NK recovery was observed 
compared with previous Tac/Sirolimus cohort.35 The same GVHD prevention regimen was 
tested in the context of full intensity preparative regimen with similar low rates of GVHD and 
NRM.36 Al-Homsi et al. showed effective GVHD prevention by combining post-transplant 
cyclophosphamide with 2 doses of bortezomib 1.3 mg/m2 on days 0 and +3 post-transplant.37  
 
 
3.3.2 Bortezomib pharmacology 
Bortezomib for Injection is a small-molecule proteasome inhibitor developed by Millennium 
Pharmaceuticals, Inc., (Millennium) as a novel agent to treat human malignancies.  Bortezomib 
is currently approved by the United States Food and Drug Administration (US FDA) for the 
treatment of patients with multiple myeloma (MM).  It is also indicated for the treatment of 
patients with mantle cell lymphoma (MCL) who have received at least 1 prior therapy.   
 
Bortezomib Mechanism of Action 
 
By inhibiting a single molecular target, the proteasome, bortezomib affects multiple signaling 
pathways.  The antineoplastic effect of bortezomib likely involves several distinct mechanisms, 
including inhibition of cell growth and survival pathways, induction of apoptosis, and inhibition of 
expression of genes that control cellular adhesion, migration, and angiogenesis.  Thus, the 
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mechanisms by which bortezomib elicits its antitumor activity may vary among tumor types, and 
the extent to which each affected pathway is critical to the inhibition of tumor growth could also 
differ.  Bortezomib has a novel pattern of cytotoxicity in National Cancer Institute (NCI) in vitro 
and in vivo assays. 38 In addition, bortezomib has cytotoxic activity in a variety of xenograft 
tumor models, both as a single agent and in combination with chemotherapy and radiation.39-51 
Notably, bortezomib induces apoptosis in cells that over express bcl-2, a genetic trait that 
confers unregulated growth and resistance to conventional chemotherapeutics.52 
 
The mechanisms of action leading up to apoptosis have been more clearly defined and include 
initiation of the unfolded protein response and direct/indirect effects on various molecular targets 
including cell cycle control proteins p27 and p21, cyclins, signal transduction molecules, 
transcription factors c-jun and HIF1-α, tumor suppressor protein p53, angiogenesis factors, and 
many others.  Bortezomib is thought to be efficacious in multiple myeloma via its inhibition of 
nuclear factor κB (NF-κB) activation, its attenuation of interleukin-6 (IL-6)-mediated cell growth, 
a direct apoptotic effect, and possibly anti-angiogenic and other effects.53-60 
 
Bortezomib is thought to be efficacious in multiple myeloma via its inhibition of nuclear factor κB 
(NF-κB) activation, its attenuation of interleukin-6 (IL-6) mediated cell growth, a direct apoptotic 
effect, and possibly anti-angiogenic and other effects.53 Bortezomib also has immunomodulatory 
effects relevant to allogeneic allo-HSCT.  The proteasome, acting via NF-kB, plays an important 
role in cytokine signaling and the generation of cell mediated immune responses via T cell 
activation, proliferation and apoptosis. 61-63 Bortezomib also attenuates TLR4 mediated antigen-
presenting cell activation, with reduced cytokine production and immunostimulatory activity.64 In 
the allogeneic setting, bortezomib preferentially and specifically depletes alloreactive T 
lymphocytes.65 It however spares human regulatory T cells (Treg) that act to suppress 
inappropriate immune responses underlying GVHD.66 Given the immunomodulatory effects of 
bortezomib on APCs, alloreactive T lymphocytes and Tregs, as well as its anti-tumor effects and 
its lack of hematopoietic stem cell toxicity, it is an attractive candidate for control of GVHD after 
allogeneic transplantation. 
 
Bortezomib Clinical Pharmacokinetics and Pharmacodynamics 
 
The clinical pharmacology characterization of bortezomib has been determined from Phase 1 
studies in subjects with solid tumors and hematological malignancies, and confirmed in Phase 2 
studies in subjects with multiple myeloma. 
 
Bortezomib demonstrates multi-compartmental pharmacokinetics. Following intravenous 
administration of 1.0 mg/m2 and 1.3 mg/m2 dose, the mean first-dose maximum observed 
plasma concentrations of bortezomib were 57 and 112 ng/mL, respectively in 11 patients with 
multiple myeloma and creatinine clearance values >50 mL/min participating in a 
pharmacokinetics study.  In subsequent doses, mean maximum observed plasma 
concentrations ranged from 67 to 106 ng/mL for the 1.0 mg/m2 dose and 89 to 120 ng/mL for 
the 1.3 mg/m2 dose.  The mean elimination half-life of bortezomib upon multiple dosing ranged 
from 40 to 193 hours.  Bortezomib is eliminated more rapidly following the first dose.  Mean 
Total Body Clearances were 102 and 112 L/h following the first dose for doses of 1.0 mg/m2 and 
1.3 mg/m2, respectively, and ranged from 15 to 32 L/h following subsequent doses for doses of 
1.0 and 1.3 mg/m2, respectively.  Clinical experience has shown that the change in clearance 
does not result in overt toxicity from accumulation in this multi-dose regimen in humans. 
 
In subjects with advanced malignancies, the maximum pharmacodynamic effect (inhibition of 
20S activity) occurred within 1-hour post dose.  At the therapeutic dose of 1.3 mg/m2 in subjects 
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10 


with multiple myeloma, the mean proteasome inhibition at 1-hour post dose was approximately 
61%. 
 
The time course of proteasome inhibition in subjects is characterized by maximum inhibition 
observed within the first hour after administration, followed by partial recovery of proteasome 
activity over the next 6 to 24 hours to within 50% of the pretreatment activity.  On the Day 1, 4, 
8, and 11 schedule variable (10%–30%) levels of proteasome inhibition have been observed at 
next scheduled dosing.  In theory, this advantage allows cells to recover proteasome activity for 
normal cellular housekeeping functions between doses. 
 
The relationship between bortezomib plasma concentrations and proteasome inhibition can be 
described by a maximum effect (Emax) model.  The Emax curve is initially very steep, with small 
changes in plasma bortezomib concentration over the range of 0.5 to 2.0 ng/mL relating to large 
increases in the percent inhibition (0–60%).  After that, a plateau occurs where marginal 
increases of proteasome inhibition are observed in spite of large changes in plasma bortezomib 
concentrations.67 
 
Clinical Experience with Bortezomib: To date, more than 436,000 patients have been treated 
with bortezomib, including patients treated through Millennium-sponsored clinical trials, 
Investigator-Initiated Studies, the US NCI Cancer Therapy Evaluation Program (CTEP), and 
with commercially available drug.  Bortezomib has been commercially available since 13 May 
2003. 
 
3.3.4 Bortezomib and Graft-versus-Host-Disease: Preclinical Data 
 
In murine models of severe acute GVHD involving fully-HLA mismatched myeloablative allo-
HSCT, bortezomib administered early after stem cell infusion protected against GVHD without 
impairing engraftment. 68,69 In a model intended to induce less severe GVHD, mice receiving 
bortezomib had 100% survival with no animal developing GVHD, while control animals all 
succumbed to GVHD prior to Day 50 post-transplantation.68 Futrthermore administration of 
bortezomib beyond day 5 post-transplant resulted in acceleration of gut aGVHD in a CD4 
dependent mechanism through activation of TLR4 and IL-1β.70-72.    
 
 
 
3.4  Immunologic changes post allogeneic HCT. 
3.4.1  Immune reconstitution NK and Gamma Delta T-cells. Following hematopoietic stem 
cell transplant (allo-HSCT), there is a prolonged period of profound immune deficiency, which 
includes defects in thymopoiesis. This immune deficiency contributes to the high incidence of 
opportunistic infection, which continues for years after HCT (40, 41).73,74 In the early post 
transplantation period, most peripheral blood lymphocytes are NK cells which can mediate 
cytotoxicity without prior sensitization and may be responsible for early GVL effects.75,76 Savani 
et al. reported that patients who had higher(>150/mm3) NK cell counts on day 30 after 
transplantation achieved rapid molecular remission in CML and significantly improved transplant 
outcomes not only by reduced relapse but also by reduced NRM and GVHD.77 Ruggeri et al. 
demonstrated that NK cell allo-reactivity in haplo-identical transplants not only induced a strong 
GVL effect, but also prevented GVHD reaction through killing of host antigen presenting cells.78. 
Furthermore, haploidentical transplants with α/β T-cell receptor +CD19 B cell depletion, resulted 
in enrichment of γδ T-cells & NK cell early after transplant with low incidence of GVHD and 
infections.79 Godder et al. showed that higher γδ T-cells in the 1st year post haploidentical 
transplant correlated with less risk of relapse.80 Both NK and γδ T-cells where shown to acquire 
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a mature phenotype upon activation with (cytomegalovirus viremia). These activated innate 
immune cells were capable of killing viral infected targets as well as leukemic blasts, 
demonstrating their GVL effect.80-83.  Finally Koreth et al. showed in his Tac/MTX/ bortezomib 
combination a significant improvement in day 30 NK cell recovery compared with a retrospective 
Tac/sirolimus cohort.35 
 
3.4.2  GVHD prevention agents with favorable innate immune cell reconstitution and 
function. 
Animal models suggest that CNI has a more selective immune suppressor pressure on T-cells 
than NK & gamma delta T cells.84-86   Furthermore, Bosch et al. compared two cohorts of 
patients (pts) with Tac/MTX vs. Tac/MTX/Thymo and showed enhanced day 30 NK recovery in 
the second group of pts.87  It is believed that T-cell depletion provides space for faster innate 
immune cell reconstitution from CD34+ stem cells than adaptive immune cells. Pical-Izard et al. 
evaluated 
NK 
cell 
reconstitution 
and 
phenotype 
after 
RIC 
conditioning 
regimen 
(Fludarabine/Busulfan) and Tacro/Thymo GVHD prophylaxis. She reported a mean NK cell of 
115/mm3 (confidence interval 75-145) at day 30 post allo-HSCT. She also showed that earlier 
development of mature NK cell phenotype markers (CD 56 dim, CD107) correlated with better 
survival and less relapse.88 Bortezomib has shown pre-clinical evidence of tumor cell 
sensitization to NK cell activity through multiple potential mechanisms, including the induction of 
expression and activity of: DR 5, NKG2D ligands, Caspase 8, DNAM-1, and down regulation of 
HLA class I expression on target tumor cells.89-94 Furthermore, Koreth et al. showed in his 
Tac/MTX/ bortezomib combination a significant improvement in day 30 NK cell recovery 
compared with retrospective Tac/sirolimus cohort. He showed a median day 30 post-transplant 
NK cell of 120/mm3 for RIC and 252/mm3 for MA preparative regimen.35,36 
 
3.4.3 ATG pharmacokinetics and IL-15 levels post-transplant: 
IL-15 is one of the main drivers for NK cell/ γδ T-cells homeostasis and expansion in vivo.95-97 
Boyiadzis et al. showed in a group of patients undergoing allo-HSCT with pre-transplant induced 
lymphopenea and RIC preparative regimen, a significant rise in IL-15 level at the peak of 
lymphopenia which was on day 0 of transplant. Furthermore, he demonstrated an inverse 
correlation between the rapid drop of IL-15 level at day 14 with the expansion of NK cells on day 
30 post-transplant.98 Admiraal et al. showed that higher Thymo AUC post-transplant (≥100 
AU × day/mL; p=0·0024) Correlated with more graft T-cell depletion and less CD4 reconstitution 
post-transplant.99  Therefore, it is likely that the more intense lymphodepletion caused by Thymo 
will lead to higher IL-15 levels and hence better recovery of NK/ γδ T-cells on day 30 post-
transplant.       
 
3.5  Study rationale. 
This proposal combines the three least toxic and most effective GVHD prevention agents with 
favorable impact on NK cell reconstitution.  Both Tac and Thymo have been extensively used in 
GVHD prevention before. Bort has been safely combined with Tac/MTX in >100 patients in 
multiple phase I-II trials with suggestions of efficacy. Furthermore, Bort is being tested in a large 
national randomized phase II trial with Tac/MTX, which just finished accrual. There is no 
perceived overlap in toxicity/side effects between the three agents Bort, Thymo and Tac.  The 
goal is to add no extra toxicity from the GVHD regimen to the preparative regimen toxicity, 
leading to low NRM. Additionally the enhanced innate immune cell reconstitution will help in 
preventing infection and relapse, hence improving overall survival. The rationale behind the 
bortezomib timing is to match the timing schedule by Al-Homsi et al. and to avoid the remote 
chance of gut GVHD exacerbation when bortezomib given late post-transplant in animal models 
as described above. 70-72       
 
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12 


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4.3  Number of Patients 
Up to thirty eight patients will be enrolled on this study. 
 
4.4  Study Duration 
The duration of active patient participation is 24 months. The primary endpoints will be 
assessed during the first 6 months post-transplant. We expect 24 months to completion of 
patient enrollment and 30 months to study completion for the primary endpoints. For secondary 
endpoint analyses, patients will be followed for up to two years post-transplant. 
 
4.5  Definitions: 
4.5.1  Acute and Chronic GVHD: 
Acute GVHD usually develops within the first 6 months post-transplantation and appears as a 
characteristic dermatitis often accompanied by cholestasis and enteritis and based on NIH 
definition of aGVHD.100  Initial symptoms of chronic GVHD frequently include nausea, and 
anorexia with ocular and oral sicca.  Rash characteristically appears with changes in skin 
pigment progressing to sclerosis and contractures. Other organs may be involved.  Symptoms 
may mimic those seen in patients with scleroderma and other autoimmune disorders. Chronic 
GVHD typically does not occur until three months after transplantation. The modified Keystone 
Conference 1994 will be used to grade acute GVHD (Appendix A). The Chronic GVHD NIH 
Consensus Project will be used for grading severity of chronic GVHD (Appendix G). 
4.5.3  Clinical Response, Relapse and Residual Disease 
Clinical response is a decrease in the amount of disease that was present before the 
conditioning therapy, by whatever objective measurements were used to determine extent of 
disease such as physical examination; radiographic/nuclear scans or bone marrow/cytogenetic 
evaluation. 
The term relapse is used to describe the recurrence of malignancy after stem cell 
transplantation, utilizing the techniques employed pre-transplant to determine extent of disease.  
The time to relapse is the time to the first observation of radiographic and/or 
histologic/cytogenetic change which results in characterization as relapse.  Molecular relapse 
(as diagnosed for example by the presence of bcr-abl transcripts by RT-PCR analysis on repeat 
testing) will be documented but not be included in the overall figures for relapse.  For example: 
Acute Leukemia:  Relapse will be diagnosed when leukemic blasts (>5%) are documented in 
the blood or bone marrow after transplantation or when there is detection of disease at an 
extramedullary site.  The diagnosis of hematologic relapse will be supported by the 
reappearance of host cells using short tandem repeats (STR) or other method (e.g., FISH) and 
confirmed by the reappearance of cytogenetic abnormalities previously documented before 
transplantation (if applicable). 
Lymphoma: The IWG-PET based response criteria will be used for lymphomas (Appendix G) 
Response criteria for other diseases will be used when available. 
Residual disease is defined as failure to eradicate original disease without prior documentation 
of remission. 
4.5.4  Disease-Free Survival 
Disease-free survival is defined as the minimum time interval from the day of transplant to -
relapse, -death, or -last contact. For patients with documented residual disease at the start of 
the study, the time interval will be considered 0 days from transplant. 
4.5.5  Cause of death 
When defining the cause of death, physicians should classify deaths occurring from infection 
and from multiple organ failure in the presence of grade ≥ II acute GVHD (Glucksberg), as being 
caused by acute graft versus host disease.  Similarly deaths occurring in the presence of graft 
failure or secondary rejection, should be classified as being due to graft failure, and deaths in 
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the presence of relapse of the primary malignancy as being due to relapse (even if other 
pathology contributing to the death is present). 
4.5.6  Non relapse mortality (NRM) 
This is defined as death in continuous or complete remission, measured from the date of 
transplant to the date of death. 
 
5.0 
PATIENT ELIGIBILITY 
5.1   Study Population 
All patients age ≥18 who present with hematological malignancies and are scheduled for 
peripheral blood HCT from an HLA-matched or -mismatched unrelated donor will be screened 
for eligibility.  
5.2   Inclusion Criteria 
1. Age 18-75 years 
2. Patients with acute leukemia, chronic myelogenous leukemia, myeloproliferative disorder 
and myelodysplasia with no circulating blasts and with less than 5% blasts in the bone 
marrow within 4 weeks of the start of transplant conditioning regimen. 
3. Patients with chronic lymphocytic leukemia/small lymphocytic lymphoma; follicular, 
marginal zone, diffuse large B-cell or mantle cell lymphoma with chemo-sensitive 
disease at time of transplant. 
4. Patients must have a related or unrelated peripheral blood stem cell donor. Sibling donor 
must be a 6/6 match for HLA-A and -B at intermediate (or higher) resolution, and -DRB1 
at high resolution using DNA-based typing, and must be willing to donate peripheral 
blood stem cells and meet institutional criteria for donation. Unrelated donor must be 8/8 
match at HLA-A, -B, -C and –DRB1 at high resolution using DNA-based typing. 
Unrelated donor must be willing to donate peripheral blood stem cells and be medically 
eligible to donate stem cells according to NMDP criteria. 
5. Cardiac function: Ejection fraction >40% 
6. Estimated creatinine clearance greater than 50 mL/minute (using the Cockcroft-Gault 
formula and actual body weight) 
7. Pulmonary function: DLCO ≥40% (adjusted for hemoglobin) and FEV1≥50%  
8. Liver function: total bilirubin < 1.5x the upper limit of normal and ALT/AST < 2.5x the upper 
normal limit. Patients who have been diagnosed with Gilbert’s Disease are allowed to 
exceed the defined bilirubin value of 1.5x the upper limit of normal. 
9. Female subjects (unless postmenopausal for at least 1 year before the screening visit, or 
surgically sterilized), agree to practice two effective methods of contraception or agree to 
complete abstain from heterosexual intercourse from the time of signing the informed 
consent through 12 months post-transplant. 
10. Male subjects (even if surgically sterilized), of partners of women of childbearing potential 
must agree to practice effective barrier contraception or abstain from heterosexual 
intercourse from the time of signing the informed consent through 12 months post-
transplant. 
11. Signed informed consent. 
 
 
5.3  Exclusion Criteria 
1. Prior allogeneic transplant 
2. Karnofsky Performance Score < 70% 
3. Active CNS involvement by malignant cells 
4. Patients with uncontrolled bacterial, viral or fungal infections (currently taking medication 
and with progression or no clinical improvement) at time of enrollment. 
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5. Patients with transformed lymphoma (e.g., Richters transformation arising in follicular 
lymphoma or chronic lymphocytic leukemia). 
6.  
7. Patients seropositive for the human immunodeficiency virus (HIV). 
8. Patient with active Hepatitis B or C. 
9. Patients with hypersensitivity to bortezomib, boron or mannitol. 
10. Patients with > grade 2 sensory peripheral neuropathy. 
11. Myocardial infarction within 6 months prior to enrollment or New York Heart Association 
(NYHA) Class III or IV heart failure (see Appendix D), uncontrolled angina, severe 
uncontrolled ventricular arrhythmias, or electrocardiographic evidence of acute ischemia 
or active conduction system abnormalities.  Prior to study entry, any ECG abnormality at 
screening must be documented by the investigator as not medically relevant 
12. Female patients who are lactating or pregnant 
13. Patients with a serious medical or psychiatric illness likely to interfere with participation in 
this clinical study  
14. Patients with prior malignancies, except resected basal cell carcinoma or treated cervical 
carcinoma in situ.  Cancer treated with curative intent > 5 years previously will be 
allowed.  Cancer treated with curative intent < 5 years previously will not be allowed 
unless approved by the Protocol Officer or one of the Protocol Chairs.  
 
6.0  
DRUG INFORMATION 
6.8  Bortezomib  
6.8.1 Mechanism of action: see section 3.3.2 
6.8.2 Toxicity: The most common Bortezomib side effects include: 
 
Hematologic: anemia, neutropenia, thrombocytopenia, leucopenia, lymphopenia
 
Neurologic: asthenia, dizziness, anxiety, syncope, headache, insomnia, fever, 
 
rigors, chills, sensory peripheral neuropathy, and leukoencephalopathy, including 
 
reversible posterior leukoencephalopathy syndrome 
 
Pulmonary: cough, dyspnea, pleural effusion, pneumonitis, interstitial pneumonia,
 
edema acute respiratory distress syndrome (ARDS) 
 
Cardiovascular: hypotension, tachycardia, atrial fibrillation, palpitation, congestive
 
heart failure, bradycardia, atrial flutter, atrioventricular block, arrhythmia, cardiac
 
failure, cardiac arrest, pericardial effusion, pericarditis 
 
Infectious: 
reactivations 
of 
herpes 
zoster, 
opportunistic 
infections
 
Gastrointestinal: weight loss, decreased appetite, anorexia, constipation,
 
dehydration, diarrhea, heartburn, dyspepsia, stomatitis, nausea, vomiting, ileus,
 
GI perforation, acute pancreatitis 
 
Metabolic: 
hyperglycemia, 
hypoglycemia, 
hyponatremia, 
hypokalemia,
 
hypercalcemia  
 
Renal: renal failure 
 
Neuromuscular and skeletal: arthralgias, back pain, bone pain, muscle cramp 
 
and myalgias 
 
Miscellaneous: rash, hemorrhage, blurred vision, deafness, hepatitis, 
 
Hyperbilirubinemia 
 
Other medical events of interest that are considered not causally related to
 
bortezomib include hepatic failure and QT prolongation.  Fatal outcomes have
 
been reported. 
 
Women of childbearing potential should avoid becoming pregnant while being 
treated with bortezomib.  Genotoxicity testing has shown that bortezomib is 
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negative in the in vitro Ames assay and in the in vivo micronucleus assay, but it 
is a clastogen in the in vitro chromosomal aberration assay. 
 
Additional details on the potential risks of bortezomib may be found in the current 
Investigator’s Brochure. 
 
Bortezomib Precautions and Restrictions 
It is not known what effects bortezomib has on human pregnancy or development 
of the embryo or fetus.  Therefore, female patients participating in this study 
should avoid becoming pregnant, and male patients should avoid impregnating a 
female partner.  Non-sterilized female patients of reproductive age and male 
patients should use effective methods of contraception through defined periods 
during and after study treatment as specified below. 
 
Female patients must meet 1 of the following: 
• 
Postmenopausal for at least 1 year before the screening visit. Postmenopausal is 
defined as the time after which a woman has experienced twelve (12) 
consecutive months without a menstrual period.   
• 
Surgically sterile 
• 
If they are of childbearing potential (i.e., not postmenopausal or surgically sterile), 
agree to practice 2 effective methods of contraception from the time of signing 
the informed consent form through 12 months post-transplant, or agree to 
completely abstain from heterosexual intercourse. It is strongly recommended 
that at least 1 of these 2 methods be ‘highly effective’ (see Table 2.6). 
Table 2.6 Methods of Contraception 
Highly Effective Methods 
Other Effective Methods (barrier 
methods) 
Intra-uterine devices (IUD) 
Latex condom 
Hormonal 
contraceptives 
(birth 
control 
pills/oral 
contraceptives, 
injectable 
contraceptives, 
contraceptive 
patches, 
or 
contraceptive implants) 
Diaphragm 
with 
spermicide 
Cervical 
cap 
Sponge 
If one of the highly effective methods cannot be used, using 2 effective methods 
at the same time is recommended. 
 
 
6.8.3 Packaging, Formulation, Preparation and Storage  
VELCADE (bortezomib) for Injection is supplied as individually cartoned 10 mL 
vials 
containing 3.5 mg of bortezomib as a white to off-white cake or powder 
 
Unopened vials may be stored at controlled room temperature 25º C (77º F); 
excursions permitted from 15 to 30º C (59 to 86º F) [see USP Controlled Room 
Temperature]. Retain in original package to protect from light 
 
Unopened vials of VELCADE are stable until the date indicated on the package 
when stored in the original package protected from light. VELCADE contains no 
antimicrobial preservative. When reconstituted as directed, VELCADE may be 
stored at 25ºC (77ºF); excursions permitted from 15 to 30ºC (59 to 86ºF) [see 
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USP Controlled Room Temperature]. Reconstituted VELCADE should be 
administered within eight hours of preparation. The reconstituted material may be 
stored in the original vial and/or the syringe prior to administration. The product 
may be stored for up to three hours in a syringe, however total storage time for 
the reconstituted 
material must not exceed eight hours when exposed to normal indoor lighting. 
 
Prior to use, the contents of each vial must be reconstituted with 3.5 mL of 
normal (0.9%) saline, Sodium Chloride Injection, USP. The reconstituted product 
should be a clear and colorless solution. Parenteral drug products should be 
inspected visually for particulate matter and discoloration prior to administration 
whenever solution and container permit. If any discoloration or particulate matter 
is observed, the reconstituted product should not be used. 
 
 
6.8.4 Supplier: Commercially available 
 
6.9  Tacrolimus (FK-506, Prograf®) 
6.9.1 Mechanism of Action: Calcineurin inhibitor. Immunosuppressive agent that 
interferes with IL-2-mediated T-cell activation. 
6.9.2 Toxicity: The most commonly reported toxicities are renal insufficiency, tremors, 
hypomagnesemia, hypertension, hyperglycemia and seizures. 
6.9.3 Packaging and Formulation: Supplied as a sterile solution in 1-mL ampoules 
containing the equivalent of 5 mg of anhydrous tacrolimus per mL, in boxes of 10 
ampoules. Also available as 1 mg and 0.5 mg capsules for oral administration. 
6.9.4  Storage and Stability: Store between 5°C and 25°C (41°F and 77°F).  Prograf 
capsules are stored at controlled room temperature, 15°C-30°C (59°F-86°F).  
6.9.5  Administration: Tacrolimus injection must be diluted with 0.9% Sodium Chloride 
Injection or 5% Dextrose Injection to a concentration between 0.004 mg/mL and 
0.02 mg/mL prior to use. Diluted infusion solution should be stored in glass or 
polyethylene containers and should be discarded after 24 hours. The diluted 
infusion solution should not be stored in a PVC container due to decreased 
stability and the potential for extraction of phthalates. In situations where more 
dilute solutions are utilized (e.g., pediatric dosing, etc.), PVC-free tubing should 
likewise be used to minimize the potential for significant drug adsorption onto the 
tubing. Parenteral drug products should be inspected visually for particulate 
matter and discoloration prior to administration, whenever solution and container 
permit. Due to the chemical instability of tacrolimus in alkaline media, Prograf 
injection should not be mixed or co-infused with solutions of pH 9 or greater (e.g., 
ganciclovir or acyclovir). 
6.9.6 Supplies: Commercially available. 
 
6.10   Anti-Thymocyte Globulin, Rabbit (Thymoglobulin®)  
6.10.1 Mode of action. See section 2.3.2 
6.10.2 Toxicity. See section 2.3.3-2.3.5 
6.10.3 Packaging and Formulation.  Each vial of Thymoglobulin is supplied as 25mg 
lyophilized powder, packaged together with 5ml of diluent (sterile water for 
injection).  Allow Thymoglobulin and diluent vials to reach room temperature 
before constituting the lyophilized powder with 5ml of diluent.  Rotate the vial 
gently until the powder is complete dissolved.  When the 25mg vial is 
reconstituted with 5ml of diluent, each ml contains 5mg of thymoglobulin.  
Visually inspect the solution for particular matter.  If some particulate matter 
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remains, continue to gently rotate the vial until the Thymoglobulin is completely 
dissolved.  Should particulate matter persist, discard this vial.  Thymoglobulin is 
then further diluted with D5W or NS to a concentration of approximately 
0.5mg/ml.  Attach a 0.22 micron filter to the infusion bag. 
6.10.4 Storage and Stability: Intact vials are stored under refrigeration between 2-8°C 
(36°F-46°F) and protected from light. Do not freeze. Thymoglobulin should be 
used within 24 hours once reconstituted with 5 ml of diluent. Thymoglobulin 
further diluted in 0.9% saline or 5% dextrose must be used immediately.  Any 
unused drug remaining after infusion must be discarded.  The following is a 
summary of in-house stability data for Thymoglobulin at various conditions (data 
on file with sponsor): 
 
- For vials containing the unreconstituted lyophilized powder, the product is 
stable for 36 months at 5º + 3ºC (41º+ 4ºF) and 12 months at 37ºC (98.6ºF). 
- Reconstituted product is stable for 24 hours at room temperature 20º to 25ºC 
(68º to 77º F).  
- Reconstituted product further diluted in NS or D5W is stable for 24 hours at 
room temperature 20ºC (68ºF).freezing of Thymoglobulin is not recommended.  
A frozen product should not be used and should be discarded. 
6.10.5 Administration: Infuse the first dose over a minimum of 6 hours, and subsequent 
 
doses over a minimum of 4 hours via a 0.22 micron in-line filter. 
6.10.6  Supplies: Thymoglobulin® is commercially available.  
 
 
 
7.0 DONOR  AND GRAFT SELECTION     
7.1 
In selecting a donor, priority should be given to matched related donors and then 
matched unrelated donors at  the A, B, C and DRB1 loci using high resolution typing. Donors 
may not have a single mismatch at the allele or antigen level. The use of  mismatched donors 
in which disparity is only in the host versus graft direction (because of recipient 
homozygosity) is discouraged because of the potentially heightened risk for graft 
rejection. Extended typing (e.g. DQB1 and DPB1) can be performed according to 
institutional practices and use these results in  selecting 
donors; 
however, 
it 
is 
recommended that this extending typing be used only to select between donors who are 
equally well matched with the recipient at the A, B, C and DRB1.  
 
7.2 
Donors will undergo G-CSF mobilization according to local institutional and donor center 
 
practices.  PBSC will be collected by apheresis according to local institutional guidelines. 
 
Plasma and red cell depletion are allowed for volume reduction or ABO incompatibility 
 
but any other form of graft manipulation (including ex-vivo T cell depletion) is not  
 
permitted. 
 
7.4 
The target stem cell dose is between 2 x 106/kg and 10 x 106/kg (actual body weight) 
 
CD34+ cells. The maximum CD34+ cell dose is 10 x 106/kg.    
 
7.5 
Up to two leukapheresis procedures may be performed to obtain the minimum CD34+ 
 
cell target.  If, after two leukapheresis procedures, fewer than 2 x 106/kg CD34+ cells 
 
have been collected, transplant centers will have the discretion to continue PBSC cell 
 
harvesting or to proceed to bone marrow harvesting to obtain sufficient cells.  If bone 
 
marrow harvesting is needed in order to meet the desired cell dose, the transplant center 
 
needs to notify the Protocol Coordinator, Chairs or Officer.   
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7.6 
If more than 10 x 106/kg CD34+ stem cells are collected, the excess will either be  
 
discarded or cryopreserved for future use, but will not be administered to the patient. 
 
 
 
8.0 PRE-TRANSPLANT CONDITIONING:   
 
Treating physicians may select one of four conditioning approaches (outlined below) for use 
and should choose the regimen that he or she thinks is best suited for the patient and the 
patient’s disease.  Patients who have had prior autologous allo-HSCT, however, must be 
conditioned with melphalan and fludarabine. 
 
8.1 Total body irradiation (TBI) based conditioning: 
 
Centers may employ the myeloablative TBI-based regimens they ordinarily employ for 
patients with hematologic malignancies as long as these regimens meet the following 
criteria: 
 
a) The total dose of TBI is at least 900 cGy and not more than 1400 cGy. 
b) It is administered in no less than 6 fractions. 
c) It includes one or two chemotherapy agents (e.g. cyclophosphamide, 
etoposide, thiotepa) 
 
  
 
 
Some degree of lung shielding during TBI administration is encouraged, using an 
electron boost to the chest wall to preserve full dosing to the ribs.  
 
 
Cranial and testicular boosts may be administered prior to the start of 
conditioning at the discretion of the treating physician. 
 
 
 
TBI VP16 (Etoposide): 
 
TBI  
 
 
 
 
 
 
 
 
days -7 to -4 
 
VP16 60mg/kg IV 
 
 
 
 
 
 
day -3 
 
TBI/Cy (cyclophosphamide) 
 
TBI   
 
 
 
 
 
 
 
 
days -8 to -5 
 
Cy 60mg/kg 
 
 
 
 
 
 
 
days -3 & -2 
 
 
8.2 Busulfan and Cyclophosphamide: 
 
In recognition of the fact that varying doses of cyclophosphamide are used in 
combination with high dose busulfan, centers may use the regimen they ordinarily 
employ for patients with myeloid malignancies as long as the regimen meets the 
following criteria: 
 
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a) The initial regimen (i.e. before adjustments are made for pharmacokinetic 
testing results) includes the equivalent* of sixteen, ≥0.8 mg/kg IV doses (q 6 
hours) of busulfan, administered q 6 hours.  
b) The regimen uses two daily 60 mg/kg doses or four daily 50 mg/kg doses of 
cyclophosphamide. 
 
 
Cyclophosphamide Ideal Body Weight Dose Adjustments should follow institutional 
practices.  
 
Initial busulfan dosing may be based on actual or adjusted-ideal body weight based 
on the discretion of the treating physician.  
 
Busulfan pharmacokinetic testing: Pharmacokinetic testing is recommended, but 
should be performed according to institutional standards. Pre-transplant test dosing 
may be used. 
 
*Modifications in busulfan administration: The busulfan administration may be 
modified according to institutional practices as follows: 
 
1) Initial doses as high as 1.2 mg/kg q 6 hr (or 4.8 mg/kg/day see #2) may be used for 
younger children. 
2) Daily doses (e.g. 3.2 mg/kg/day or 130 mg/m2 rather than 0.8 mg/kg/day) may be 
used in place of q 6hr dosing. 
3) The dosing may be adjusted based on the results of pharmacokinetic testing. This 
can involve a change in the size of the individual doses or for patients who are 
getting q 6hr dosing where the pharmacokinetic testing result exceeds the upper 
limit of the targeted range, the targeted total dose (e.g. 16 * targeted per dose AUC) 
may be apportioned into 14 or 15 doses rather than 16 as part of the adjustment.   
 
Hydration and MESNA administration: The administration of hydration and 
cyclophosphamide to prevent hemorrhagic cystitis from cyclophosphamide will follow 
institutional practices. 
 
Seizure prophylaxis: An anti-convulsant will be administered according to institutional 
guidelines to prevent busulfan-induced seizures. 
 
 
8.3 Melphalan and Fludarabine Reduced Intensity Conditioning RIC: 
 
Fludarabine 
 
25 mg/m2/dose IV qd (5 doses) 
 
Days -6 to -2 
Melphalan*  
 
140 mg/m2/dose IV 
  
 
   
Day -2 
 
 
Fludarabine and Melphalan should each be infused over 30 minutes. 
Alternatively Melphalan can be given as 140mg/m2 as a single dose on day -2. 
Alternatively Fludarabine can be given as 30 mg/m2 daily X 4 days from day -5 to -2 
 
Fludarabine and melphalan dosing will be based on actual body weight. 
 
 
8.4 Busulfan and Fludarabine myeloablative conditioning MAC: 
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Fludarabine 
40 mg/m2/dose IV qd-4 doses 
 
 
 
Days -5 to -2 
Busulfan 
0.8 mg/kg/dose IV q6H-16 doses or the equivalent*  
Days --5 to -2 
 
  
Fludarabine should each be infused over 30 minutes and busulfan over two 
 
 
hours for the q6 hour dosing and over 3 hours for the daily dosing. 
 
 
Fludarabine dosing will be based on actual body weight.  Initial busulfan dosing may be 
based on actual or adjusted-ideal body weight based on the discretion of the treating 
physician.    
 
Busulfan pharmacokinetic testing: Pharmacokinetic (PK) testing is recommended, but 
should be performed according to institutional standards. Pre-transplant test dosing may 
be used or PK from the first infusion of buslfan can be used to adjust the remaining 
doses . The recommended final daily AUC range is close to 5000. 
 
*Modifications in busulfan administration: The busulfan administration may be 
modified according to institutional practices as follows: 
 
1) Daily doses (e.g. 3.2 mg/kg/day or 130mg/m2/day rather than 0.8 mg/kg/day) may 
be used in place of q 6hr dosing. 
2) The dosing may be adjusted based on the results of pharmacokinetic testing. This 
can involve a change in the size of the individual doses or for patients who are 
getting q 6hr dosing where the pharmacokinetic testing result exceeds the upper 
limit of the targeted range, the targeted total dose (e.g. 16 * targeted per dose AUC) 
may be apportioned into 14 or 15 doses rather than 16 as part of the adjustment. 
 
Seizure prophylaxis: An anti-convulsant will be administered according to institutional 
guidelines to prevent busulfan-induced seizures. 
 
 
 
9. Allogeneic Stem Cell Infusion:  
 
PBSC will be administered on Day 0 to all patients according to individual institutional 
 
guidelines after appropriate processing and quantification has been performed by the 
 
local laboratory. Stem cells are administered through an indwelling central venous 
 
catheter. If infusion occurs over two days, Day 0 is the day the last infusion is completed. 
 
10.0   Anti-Infective Prophylaxis 
10.1 
It is mandatory once weekly CMV PCR screening beginning around day 0 and until day 
 
120 post-transplant be performed and preemptive therapy be given for positive cultures, 
 
according to institutional guidelines.  
10.2 
EBV monitoring by PCR is also mandatory at weekly intervals from day 0 to around day 
 
100; treatment with Rituximab 375 mg/m2 shall be given for PCR levels of >1000  
 
genome copies/ml. EBV PCR will be repeated once a week and a second dose of 
 
Rituximab can be considered if there was no drop in EBV PCR.  
10.3 
Antibacterial prophylaxis will be administered based on institutional standards of care. 
 
The following guidelines are recommended at physician discretion.  
10.4  If solumedrol is used for engraftment syndrome or acute skin graft versus host disease 
 
in the first 30 days post-transplant, the recommended dose is 1 mg/kg/day and the 
 
recommended taper would be 50% down every three days.  
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11.0  GVHD Prophylaxis 
The GVHD prophylactic regimen will consist of the following:  
Tacrolimus 0.03 mg/kd/d CIV, beginning day -3 (IBW) 
Thymoglobulin 0.5 mg/kg day-3 (adjusted body weight ABW) 
Thymoglobulin 2 mg/kg day -2 
Thymoglobulin 2.5 mg/kg day -1  
Bortezomib 1.3 mg/m2 IV day 0 (after the graft),and  day +3. (ABW) 
 
 
11.1     Tacrolimus 
11.1.1  Tacrolimus Dosing 
Tacrolimus will be administered intravenously at a dose of 0.03 mg/kg (ideal body 
weight) q 24h by continuous infusion starting on Day –3. Intravenous tacrolimus will be 
discontinued once the patient starts eating and the drug will then be given orally at a 
dose of approximately 4 times the intravenous dose.  
11.1.2   Dose Modifications of Tacrolimus.   
If vomiting occurs within twenty minutes after an oral dose of tacrolimus, the dose should 
be repeated. Anti-nausea medications may be given as needed.  Intractable nausea and 
vomiting may require intravenous administration of tacrolimus at 1/4 of the oral dose.  
Tacrolimus may cause impaired renal function, hyperbilirubinemia, increased serum 
transaminase levels, hypertension, tremor, and seizures. Impaired renal function may 
require tacrolimus dose reductions.  Severe toxicities may require temporary suspension 
or discontinuation of treatment.  
 
Tacrolimus dose adjustments should not be based exclusively on serum levels.  
Tacrolimus levels should be used as a guide in conjunction with clinical observations of 
the biologic effects of the drug, i.e., toxicity and immunosuppression. 
Suggested Tacrolimus Dose Adjustment Guidelines 
 
Plasma Levels 
Toxicity 
Grade 
Tacrolimus 
Dose 
<5ng/ml 
0 
Increase 50% 
5-12ng/ml 
0,1 
No change 
“           “ 
II 
Decrease 25% 
“           “ 
III 
Decrease 50% 
“           “ 
IV 
Stop 100% 
>15ng/ml 
0 
Decrease 
25% 
every 3-4 days 
>20ng/ml 
0 
Stop 100% 
 
 
11.1.4 Tacrolimus Blood Levels 
Tacrolimus levels should be measured at least weekly during the first 50 days post-
transplant. Target levels of 5-12 ng/ml are acceptable in patients who manifest no 
evidence of toxicity or GVHD.  While the patients are getting Tacrolimus continuous I.V 
infusion the levels are checked on Monday, Wednesday, Friday and PRN with am labs. 
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When the patients switch to oral Tacrolimus trough levels will be checked.  Trough 
plasma levels should be drawn 10-12 hours after the last dose if possible. 
11.1.5   Drug Interactions 
Drugs that may increase tacrolimus levels: Fluconazole, Voriconazole, posaconazole 
Itraconazole, 
Ketoconazole, 
erythromycin, 
H2 
blockers, 
Verapamil, 
Diltiazem, 
nicardipine, danazol, bromocriptine, metoclopramide, methylprednisolone, somatostatin 
(octreotide). 
Drug that may decrease tacrolimus levels: Rifampin, Phenobarbital, phenytoin, 
carbamazepne, Octreotide (may lower serum levels by decreasing intestinal absorption 
of the oral drug). 
Drugs that may result in additive nephrotoxicity: Aminoglycosides, Amphotericin B, 
Acyclovir, Furosemide, TMP-SMX, and grape fruit juice.  These drugs are to be used 
with caution and close attention in anticipation of tacro level change. 
 
11.2 
Bortizomib 
11.2.1 Bortezomib will be administered at the dose of 1.3 mg/m2 (based upon actual body 
weight) as an approximately 3-5 second IV push on Days +0 (after graft infusion),and + 
1post transplant. There must be at least 72 hours between each dose of bortezomib. 
Subcutaneous administration of bortezomib is not allowed on this protocol. 
11.2.2 Bortezomib dose modifications: 
Before each drug dose, the patient will be evaluated for possible toxicities that may have 
occurred after the previous dose(s).  Toxicities are to be assessed according to the NCI 
Common Toxicity Criteria for Adverse Events (CTCAE), Version 4.0 
Neuropathic pain and peripheral sensory neuropathy are to be managed as described in 
Table 2.4b. 
 
Table: Management of Patients With VELCADE-Related Neuropathic Pain and/or Peripheral 
Sensory or Motor Neuropathy  
Severity of Peripheral Neuropathy Signs 
and Symptomsa  
Modification of Dose and Regimen 
Grade 1 (asymptomatic; loss of deep 
tendon reflexes or parasthesias) without 
pain or loss of function 
No action 
Grade 1 with pain or Grade 2 (moderate 
symptoms; limiting instrumental Activities 
or Daily Living [ADL]b) 
Reduce VELCADE to 1.0 mg/m2 
Grade 2 with pain or Grade 3 (severe 
symptoms; limiting self care ADLc) 
Withhold 
VELCADE 
therapy 
until 
toxicity 
resolves. When toxicity resolves reinitiate with a 
reduced dose of VELCADE at 0.7 mg/m2 
(missed dose days will not be made up). 
Grade 4 (life-threatening consequence; 
urgent intervention indicated) 
Discontinue VELCADE 
Source:  VELCADE USPI issued January 2012. 
Abbreviations: ADL = activities of daily living 
a   Grading based on NCI Common Terminology Criteria for Adverse Events (CTCAE) v4.0. 
b   Instrumental ADL: refers to preparing meals, shopping for groceries or clothes, using 
telephone, managing money, etc 
c   Self care ADL: refers to bathing, dressing and undressing, feeding self, using the toilet, 
taking medications, and not bedridden. 
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11.2.4 Patients with mild hepatic impairment (bilirubin ≤ 1.5 × ULN) do not require dose 
adjustment.    If a patient develops moderate or severe hepatic impairment with bilirubin 
≥ Grade 2 (> 1.5 -3.0 X ULN) after exposure to Bortezomib, the dose should be held until 
the toxicity returns to < Grade 2. Restarting Bortezomib must be done at the next lower 
dose level as below.  
Once bortezomib is reduced for any toxicity, the dose may not be re-escalated. 
If after bortezomib has been held, the toxicity does not resolve, then bortezomib must be 
discontinued. 
If the toxicity resolves, as described above, bortezomib may be restarted at the same 
schedule the patient was on prior to holding therapy, and the dose must be reduced by 
approximately 25% as follows: 
• 
If the patient was receiving 1.3 mg/m2, reduce the dose to 1 mg/m2. 
• 
If the patient was receiving 1 mg/m2, reduce the dose to 0.7 mg/m2. 
 
11.3 
Thymoglobulin 
11.3.1 Thymoglobulin Pre-medication 
One hour prior to the first Thymoglobulin dose, solumedrol 0.5 mg/kg will be given 
intravenously; 3 hours past the start of Thymoglobulin, solumedrol 0.5 mg/kg will be 
repeated intravenously.  Acetaminophen 650mg PO, and diphenhydramine 50mg IV 
should be given within 30 minutes prior to thymoglobulin infusion.  
 
It is recommended to consider B1 blockers (metoprolol or atenolol) to start one 
day before the Thymo infusion and continue until after day 0 of transplant to 
protect patients at risk for CAD from Thymo induced tachycardia.  
 
11.3.2   Thymoglobulin Dosing 
The total dose chosen for this protocol is 5 mg/kg because of a high rate of EBV 
reactivation with a recent experience (personal Communication from City of Hope) using 
7.5 mg/kg. Thymoglobulin will be dosed according to an escalated regimen to minimize 
infusional toxicities, as follows: 0.5 mg/kg on day –3, 2 mg/kg on day –2, and 2.5 mg/kg 
day –1.  
11.3.3   Thymoglobulin Administration 
After the patient is pre-medicated, Thymoglobulin will be infused using a 0.22-micron in-
line filter.  Thymoglobulin should not be co-administered with blood products or other 
biologic agents.  All Thymoglobulin doses will be infused over 6 hours.  Avoid 
simultaneous infusion of Thymoglobulin with any other solution, particularly lipids, in the 
same infusion line.  Patients will be observed for AEs during all Thymoglobulin infusions.  
All patients will receive the second Thymoglobulin dose approximately 24 hours (no 
sooner than 12 hours and no greater than 36 hours) after the start of the first 
Thymoglobulin dose.  Subsequent doses should be administered at similar time intervals 
unless dose interruption is required (see below).  
11.3.4 Thymoglobulin Discontinuation or Interruption Guidelines 
Thymoglobulin treatment may be discontinued or interrupted at any time for the 
following reasons: 
a). Patient refuses to continue treatment 
b). Infusion of study drug related SAE 
Thymoglobulin may be re-instituted if, in the judgment of the investigator, the primary 
clinical cause(s) for dose adjustment have been resolved. 
 
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The initial dose of Thymoglobulin may be associated with a reaction typically associated 
with fever and chills commonly referred to as a cytokine release syndrome.  Often such 
symptoms are mild and can be reduced by decreasing the rate of infusion so that the 
drug is administered over 12 hours.  Therapy such as additional Benadryl, 
corticosteroids, or adrenergic agents may be required.  For severe cytokine release 
symptoms such as dyspnea or symptoms of capillary leak syndrome, infusion should be 
interrupted while appropriate clinical therapy is administered.  Clinical therapy for 
cytokine release symptoms includes additional corticosteroids and antihistamines.  
Additional resuscitative measures for severe symptoms or anaphylaxis (e.g. 0.3 ml 
aqueous epinephrine (1:1000 dilution), pressor agents, oxygen, intravenous fluids and 
airway management) should be provided as clinically indicated. 
Hypersensitivity type III, such as arthralgia, myalgia, skin rash or other symptoms of 
immune complex disease may occur, and usually responds within 24-48 hours to 100mg 
of IV hydrocortisone or oral prednisone equivalent (20mg).  If, in the opinion of the 
physician, additional therapy is required for treatment of immune complex symptoms, 
additional corticosteroid therapy (e.g. a prednisone taper over the next 7-10 days) may 
be given as necessary.  Ranitidine 150mg BID or other H-2 blockade or antacid 
prophylaxis should be given while patients are on corticosteroid therapy and for 2 weeks 
thereafter. 
11.3.5   Guidelines for Tapering of Immunosuppression 
In general, no evidence of GVHD should be present and the following guidelines should 
be observed. Tacrolimus taper commenced after 12 weeks, with the goal of having the 
patient no longer receiving immune suppression by 6 months in the absence of GVHD 
 
 
 
12.0 
Immuno-correlative and pharmacokinetic studies: 
This section is for immunologic correlative studies in patients who participate in the protocol. 
12.1 
Correlative immunologic study objectives: 
In this study, we plan to evaluate the following immunologic assays (biomarkers) to better 
understand mechanisms involved in graft-versus-host disease and immune reconstitution in 
allogeneic donor transplants performed in this protocol with the introduction of a new GVHD 
prophylaxis combination of tacrolimus, bortezomib, and thymo. These markers are: 
(1) NK cell recovery and phenotype post-transplant. CD3, CD56, KIR (four antibodies one 
color), NKG2A, NKG2C, & functional phenotype (TNFα, CD107, IFNγ after exposure to 
K562). 
(2) (2) γδ T-cells recovery and phenotype post-transplant. CD3, Gamma delta Pan TCR 
 
T cell immunophenotyping and absolute subset number quantification will be performed in bulk 
for each patient using PBMC samples isolated and frozen at each time point.  Absolute 
leukocyte counts will be determined on each sample and will be used to convert the percentage 
of each subset in samples determined below into absolute cell numbers of each subset.  
Percentage of each subset in samples will be determined using multi-parameter flow cytometry. 
The mean numbers and phenotype of NK cell and γδ T-cells at days 30, 60,100,180 post-
transplant will be compared with historical published French NK cell reconstitution data with 
tacrolimus/Thymo,88 in addition to our local NK/ γδ T-cells reconstitution numbers in the first 3 
months after transplant. 
  
12.3 Pharmacokinetic studies  
Plasma samples were (10 ml in red top tube) obtained daily from the day preceding Thymo 
administration (day-4) to day +4 post-transplantation, then on days+7,+14,and 30+/-2 post-
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transplant. Plasma will be frozen at -80°for future active R-ATG and IL-15 level measurements. 
Measurement of active R-ATG (the fraction which retains the capacity to bind to human 
lymphocytes) will be done according to the methods described by Regan et al.101. The IL-15 
QuantiGlo ELISA (R&D Systems) will be used to measure IL-15 level. 
12.4   Immunocorrelative Studies  
Patients will have blood draws at 30 mls into CPT tubes Becton Dickinson Vacutainer Systems 
Franklin Lakes, NJ) on days +14,+30 (+/-2),+60(+/-4),+100 (+/-7),+180(+/-7) post-transplant.   
PBMCs were isolated from CPT tubes after centrifugation. PBMCs will be used for 
immunophenotyping studies. 
 
Immunologic correlative and pharmacokinetic study calendar 
 
 
All samples will be sent to the Dr Waller’s lab for processing, then, saved for immunologic 
assays as above.   
 
12.5  Maintenance of Patient related biological material and records.  
Dr. Al-Kadhimi (pager number 71101) will be contacted to pick up and start processing all 
patients’ blood samples.  Each sample will have a unique identifying number that will link it to 
the patient’s name and medical record number as well as the date of collection. The samples 
will be stored for a total of 5 years to give ample time after finishing patient accrual to carry out 
the experiments outlined in the methods and procedures section.  Beyond 5 years from the date 
of starting patient accrual the samples will be destroyed by thawing and bleaching.  The cost of 
storing and maintaining the samples will be covered by Dr. Al-Kadhimi’s startup funds. If for any 
unforeseeable reason the PI runs out of funds to maintain and store these patients’ samples or 
the PI leaves the institution or the study, Dr. Al-Kadhimi will identify another investigator to 
assume responsibility of the protocol, the maintenance and storage of patient’s samples, 
otherwise the samples will be destroyed.   
 
13.0      Study Procedures: SCREENING AND BASELINE EVALUATION 
A flow chart of study procedures and assessments is provided below.   
  
The transplant patients that meet the study eligibility criteria and are consented for the study will 
undergo baseline evaluations. Much of the information listed below will be obtained at the time 
of determining eligibility/screening prior to enrollment on the protocol. 
The following baseline evaluations should represent those assessments/labs performed within 4 
weeks prior to the start of the conditioning regimen. 
• 
History, physical examination, height and weight. 
• 
KPS (See Appendix F). 
• 
Donor and recipient serological status for HIV, CMV, HSV, HCV Ab, and 
HBV, including Hepatitis B core antibody (HbcAb), Hep B surface 
antibody (HepBsAb) and Hepatitis B surface antigen (HbsAg). 
Test Time 
point 
Pre 
Thymo 
Post Thymo  
Days-4, -3,-2,-1, 
0,+1,+2,+3,+4 
Day+7 Day + 
1 4 
 
Day + 
30 
(+/-2) 
Day + 
60 
(+/-4) 
Day + 
100 
(+/- 7) 
Day + 
180 
(+/- 7) 
Active 
Thymo 
X 
X 
X 
X 
X 
X 
 
 
IL-15 
X 
X 
X 
X 
X 
X 
 
 
NK cell 
 
 
 
X 
X 
X 
X 
X 
γδ T-cells 
 
 
 
X 
X 
X 
X 
X 
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• 
Serum or urine pregnancy test (females only). 
• 
CBC with differential, platelet count, basic metabolic panel (creatinine, 
glucose, Na, K, CI, HCO3, calcium, blood urea nitrogen), liver function 
tests (Bilirubin, AST, ALT, alkaline phosphatase, LDH), cholesterol and 
triglycerides  
• 
EKG 
• 
Pulmonary function tests, including DLCO, FEV1 and FVC 
• 
Bone marrow aspirates for pathology and cytogenetics 
• 
Chest-X-Ray  
• 
MUGA or Echocardiogram 
 
14.0 
STUDY PROCEDURES: MAINTENANCE PHASE/Post-Transplant PERIOD 
The following procedures will be performed post-transplantation, according to Appendix E: 
 
• 
History and physical examination to assess GVHD and other morbidity weekly 
through       first discharge after transplant, then around day 30, 90, 180 and 360 
post-transplant 
• 
Record specific adverse events and opportunistic infections 
• 
KPS Performance Status (see Appendix F) 
• 
Disease specific re-staging based on institutional standards 
• 
Perform laboratory evaluations weekly while in the hospital, then at day 30, 90, 
60, 100, and 180 post transplant: 
o CBC (hemoglobin, hematocrit, WBC with differential and platelet count) 
o Basic metabolic panel: creatinine, glucose, Na, K, CI, HCO3, calcium, blood urea nitrogen (BUN) 
o Liver function tests (bilirubin, AST, ALT, alkaline phosphatase, LDH) 
o Tacrolimus level once or twice/week 
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o Note any serologic change (CMV and EBV) 
 
 
1Clinical Assessment at screening. 
2 Serum chemistries to include creatinine, glucose, sodium (Na), potassium (K), chloride (CI), 
bicarbonate (HCO3), calcium, blood urea nitrogen (BUN)/Urea. 
3 Liver function tests: bilirubin, aspartate aminotransferase (AST), alanine aminotransferase 
(ALT), alkaline phosphatase, LDH 
4 At least three times a week during hospitalization 
5 Liver function tests will be done as clinically indicated based on KCI BMT standards   
6Tacrolimus levels to be checked 3 times per week and sirolimus levels two times a week and at 
every clinic visit as per standard operating procedure for the Blood and Marrow Transplant 
Program. 
7 Disease restaging to be done at discretion of physician in keeping with standard of care.   
8 It is mandatory once or twice weekly CMV shell vial culture or PCR screening beginning 
around day 21 and until day 100 post-transplant be performed. EBV monitoring by PCR is also 
mandatory at weekly intervals from day 21 to around day 100. 
15.0 
GVHD 
15.1 
Diagnosis 
No skin biopsy is required in any patient, but it is recommended in patients with only skin 
manifestations.  A gastro-intestinal tract biopsy is also recommended in patients diagnosed with 
upper gastro-intestinal acute GvHD alone. Appropriate biopsies are also recommended in 
patients diagnosed with aGvHD, but without skin signs. 
15.2 
Staging and Grading 
Study Calendar 
 
Baseline 
Study Day 
Prior to 
start 
Post- 
Transplant 
Hospitalization 
30 
60 
100 
180 
Window Days for Study Visit 
-30 days  
(varies) 
+/- 
4 
+/- 
7 
+/-
10 
+/-  
20 
Study Month 
- 
- 
1 
2 
3 
6 
Informed Consent 
X 
 
 
 
 
 
Inclusion/Exclusion Criteria, including organ 
function/staging 
X 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
Concomitant Medications 
 
X 
X 
X 
X 
X 
Clinical Assessment & KPS 
Adverse Events 
Opportunistic Infections 
Assessment of Engraftment/GVHD 
Assessment for Patient/Graft Survival 
X1 
X 
X 
X 
X 
X 
Hematology (complete blood count (CBC) 
with differential platelets) 
X 
X 
X 
X 
X 
X 
BMP2 
X 
X4 
X 
X 
X 
X 
Liver function tests3 
X 
X5 
X 
X 
X 
X 
Pregnancy Test 
X 
 
 
 
 
 
Tacrolimus Trough Level 
NA 
X6 
X 
X 
X 
X 
Disease restaging7  
X 
X 
CMV and EBV screening8 
 
      X 
X 
X 
X 
X 
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For acute GVHD, the clinical stage of the three organs involved will be assessed, so that an 
overall grade according to modified Keystone criteria cab be obtained. See Appendix A. For 
chronic GVHD, the NIH consensus Project (Appendix G) will be used for grading and severity. 
15.3 
Outcome of acute GVHD 
This will be defined as follows: 
Complete response – clinical stages in all organ systems are zero. 
Partial response – at least one organ system has improved by at least one stage, and none of 
the other organ systems have worsened. 
Mixed response – at least one organ system has improved by at least one stage and at least 
one organ system has worsened by at least one stage. 
Stable disease – no change in the clinical stage of any organ system from baseline. 
Progressive disease – at least one organ system has worsened by at least one stage without 
improvement of any other organ systems from baseline. 
Recurrence – complete or partial response followed by recurrence of aGvHD requiring an 
increase of systemic methylprednisolone or equivalent by at least 2 mg/kg/day, or the 
introduction of another immunosuppressant agent. 
15.4 
Treatment of acute GVHD 
If acute GVHD grade II or greater develops during therapy, it will be treated with 
methylprednisolone 1-2 mg/kg/d, or per active protocols. Treatment with tacrolimus should 
continue. For steroid refractory GVHD, treatment will be at the discretion of the investigator.  
 
16.0 
NCI Toxicities 
Toxicities will be recorded using the NCI CTCAE 4.0 Scale. The NCI CTCAE 4.0 can be found 
at http://ctep.cancer.gov/reporting/ctc.html  
 
17.0 
Statistical Considerations 
17.1   Study Design:  Single Arm phase II trial 
 
17.2   Endpoint:   
17.2.1 Primary endpoints  
1- To determine a composite end point of alive and severe acute GVHD free at 6 months 
following HLA matched related or unrelated donor hematopoietic peripheral blood transplant in 
patients with hematologic malignancies who receive the immunosuppressive combination TBT 
as GVHD prophylaxis. 
 
2- Safety defined by serious adverse events (SAE) and adverse events (AE) related to this 
immunosuppressive regimen in the first six months post-transplant 
 
17.2.2   Secondary endpoints  
The secondary endpoints for this protocol are: 
 
1- Incidence and severity of aGVHD grade II-IV. 
2- Time to platelet and absolute neutrophil recovery (engraftment) 
3- The cumulative incidence of grade III-IV aGVHD 
4- Day 30 NK, γδ T-cell recovery and phenotype  
5- Thymo pharmacokinetics and it’s correlation with IL-15 levels and natural killer (NK) cell 
and gamma delta (γδ) T-cell recovery on day 30 post-transplant 
6- Donor cell engraftment 
7- Incidence of infections-including CMV and EBV reactivation including lymphoproliferative 
disorders PTLD 
8- Incidence of veno-occlusive disease 
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9- Incidence of chronic GVHD 
10- Rates of Grade ≥ 3 non-hematologic toxicity according to the Common Terminology   
Criteria for Adverse Events (CTCAE) Version 4.0 
11- Non-relapse mortality NRM 
12- Disease relapse or progression 
13- Overall and disease free survival at one year 
14- Immunosuppression-free survival at one year 
15- KPS both pre-transplant and at various points post-transplant.   
 
 
17.3   Trial Design and Sample Size:  We will use Simon’s two stage minimax design for this 
phase 2 trial using a composite primary end point of unfavorable transplant outcomes defined 
as death from any cause or alive with a prior history of grade 3-4 aGVHD by 6 months post-
transplant. Our current institutional rate of this composite outcome at 6 months is 35% 
(10% non-fatal grade 3/4 aGvHD+ 25% 6 month mortality).  We hypothesize to reduce the 
incidence of this composite endpoint to ≤17%. We aim to accrue 17 patients for the first stage  
and 21 patients for the 2nd stage with a total of 38 patients. After the first 17 patients if we 
observe 6 or more cases of non-lethal grade 3/4 GVHD or 6 month-mortality as defined above, 
the trial will not advance to stage 2 for futility. After accruing 38 pts, if we observe 9 or more bad 
outcomes (as defined above), the trial will be deemed not promising for further clinical 
development. A sample size of 38 will achieve a power of 80% and type one error of 0.045.  
 
To test the hypothesis that this GVHD prevention regimen leads to activation of donor NK cells 
that may have potent anti-leukemic activity we will measure the number of NK cells at day 30,   
hypothesizing that the use of the TBT regimen will lead to double the number of peripheral 
blood NK cells at day 30 to 250/mm3 compared to a historical mean of 120/mm3 using 
Tacrolimus ATG.88   A sample size of 35 achieves 100% power to detect a difference of -165.0 
between the null hypothesis mean of 120.0 and the alternative hypothesis mean of 285.0 with 
an estimated standard deviation of 100.0 and with a significance level (alpha) of 0.05000 using 
a two-sided Wilcoxon test assuming that the actual distribution is normal. 
 
 
17.4  Statistical Analysis: Descriptive statistics will be mainly employed to address primary 
and secondary endpoints. The incidence of aGVHD incidence will be summarized as 
percentage and 95% confidence level will be also constructed. SAE and AE will be tabulated 
with frequency and percentage. Time to platelet and absolute neutrophil recovery will be 
presented with median and range. Other secondary endpoints, including the cumulative 
incidence of grade III-IV aGVHD, Day 30 NK, γδ T-cell recovery and phenotype, Thymo 
pharmacokinetics and it’s correlation with IL-15 levels and natural killer (NK) cell and gamma 
delta (γδ) T-cell recovery on day 30 post-transplant, donor cell engraftment, incidence of 
infections-including CMV and EBV reactivation, incidence of veno-occlusive disease, incidence 
of chronic GVHD, rates of Grade ≥ 3 non-hematologic toxicity, non-relapse mortality NRM, will 
summarized with frequency and percentage. The disease relapse or progression, overall and 
disease free survival, immunosuppression-free survival will be analyzed with Kaplan Meier 
method and Logrank test. KPS both pre-transplant and at various points post-transplant will be 
presented with frequency and percentage.   
 
 
17.5   Stopping Rules: The stopping rule for safety monitoring is based on non-relapse 
mortality at 6 months as a grade 5 SAE. Our current 1 year NRM is about 20%. For the first 
stage of the trial and considering the upper limit of the 90% confidence interval of what our 
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current experience at year, we would stop the trial if by 6 months post-transplant we observe 5 
non relapse deaths in the first 17 patients. 
 
17.6   Accrual and Study Duration: The anticipated patient’s accrual for this trial is 2 years.  
This is based on an expected transplant rate of 50 HLA matched related and unrelated 
transplant operations per year at the Winship Cancer Institute. Six months observation is 
needed for primary endpoints. Therefore, the study duration to observe the primary end point is 
18 months.   
 
18.0 
DATA AND Safety MONITORING 
18.1   Monitoring and Personnel Responsible for Monitoring 
 
The Data and Safety Monitoring Committee (DSMC) of the Winship Cancer Institute will provide 
oversight for the conduct of this study.  The DSMC functions independently within Winship 
Cancer Institute to conduct internal monitoring functions to ensure that research being 
conducted by Winship Cancer Institute Investigators produces high-quality scientific data in a 
manner consistent with good clinical practice (GCP) and appropriate regulations that govern 
clinical research.    Depending on the risk level of the protocol, the DSMC review may occur 
every 6 months or annually.  For studies deemed High Risk, initial study monitoring will occur 
within 6 months from the date of the first subject accrued, with 2 of the first 5 subjects being 
reviewed.  For studies deemed Moderate Risk, initial study monitoring will occur within 1 year 
from the date of the first subject accrued, with 2 of the first 5 subjects being reviewed.  
Subsequent monitoring will occur in routine intervals per the Winship Data and Safety 
Monitoring Plan (DSMP).  
 
The DSMC will review pertinent aspects of the study to assess subject safety, compliance with 
the protocol, data collection, and risk-benefit ratio. Specifically, the Winship Cancer Institute 
Internal Monitors assigned to the DSMC may verify informed consent, eligibility, data entry, 
accuracy and availability of source documents, AEs/SAEs, and essential regulatory documents.  
Following the monitoring review, monitors will provide a preliminary report of monitoring findings 
to the PI and other pertinent individuals involved in the conduct of the study.  The PI is required 
to address and respond to all the deficiencies noted in the preliminary report.  Prior to the 
completion of the final summary report, monitors will discuss the preliminary report responses 
with the PI and other team members (when appropriate).  A final monitoring summary report will 
then be prepared by the monitor.  Final DSMC review will include the final monitoring summary 
report with corresponding PI response, submitted CAPA (when applicable), PI Summary 
statement, and available aggregate toxicity and safety data.   
 
The DSMC will render a recommendation and rating based on the overall trial conduct.  The PI 
is responsible for ensuring that instances of egregious data insufficiencies are reported to the 
IRB.  Continuing Review submissions will include the DSMC recommendation letter.  Should 
any revisions be made to the protocol-specific monitoring plan after initial DSMC approval, the 
PI will be responsible for notifying the DSMC of such changes.  The Committee reserves the 
right to conduct additional audits if necessary.  
 
 
 
 Patient safety, study efficacy and compliance will be reviewed at the BMT working group 
meeting. The BMT working group will also oversee the conduct of this study. In addition interim 
analysis will be conducted once after 17 patients have accrued and had minimum of 6 months 
of follow up for futility assessment. The bone marrow research Team consisting of the PI, 
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Collaborating Investigators, CRA, protocol nurse, and statistician is responsible for monitoring 
the data and safety of this study, including implementation of any stopping rules for safety and 
efficacy.  
 
Scheduled meetings will occur depending upon the activity of the protocol. These meetings will 
include the Principal Investigator, data managers, and anybody deemed important to attend by 
the Principal Investigator.  
 
During these meetings the following points will be reviewed and discussed: 
 
1. Safety of protocol participants (AE reporting). 
2. Validity and integrity of the data. 
3. Enrollment rate relative to expectations and the characteristics of participants. 
4. Retention of participants and adherence to the protocol (potential or real protocol 
violations). 
5. Completeness of collected data. 
 
 
 
 
18.2  Adverse Events 
18.2.1  Reporting: In adherence to good clinical practice where the rights, safety, and well-
being of research protocol participants are protected, the timely reporting of adverse reactions  
by the sponsor-investigator in accordance to regulation of the Food & Drug Administration (Ref. 
Title 21, Code of Federal Regulations, Part 312).  Adverse reactions shall be reported to 
Institutional Review Board according to their Policies and Procedures. Completion of adverse 
reaction reporting forms provides adequate documentation of adverse events. 
 
In concert with the Blood & Marrow Stem Cell Transplant Program clinical personnel, BMT Data 
Management Research personnel  and investigator or subinvestigator shall identify and grade 
adverse actions using NCI Common Terminology Criteria for Adverse Events v 4.0 (CTAE) that 
is available at (http://ctep.cancer.gov/forms/CTCAEv4.pdf).  Data Management Research staff 
shall refer to Emory IRB for guidance in IRB reporting requirements for adverse and unexpected 
events.  All unexpected grade 3-5 AEs will be reported to Emory IRB whether it is related or 
unrelated to study intervention. Non-relapse related deaths, occurring within 30 days, as a 
grade five AE will be reported to Emory IRB as required. 
 
 
 
Study Intervention Relationship: 
The investigator will determine which events are associated with the use of the study 
intervention.  For reporting purposes, an AE should be regarded as possibly related to the use 
of the investigational product if the investigator believes: 
• 
There is a clinically plausible time sequence between onset of the AE and graft 
manipulation; and/or 
• 
There is a biologically plausible mechanism for graft manipulation to cause or 
contributing to the AE; and 
• 
The AE cannot be attributed solely to concurrent/underlying illness, other drugs, or 
procedures. 
 
18.2.2  Procedures: 
Blood & Marrow Stem Cell Transplant Program Data Management Office personnel shall: 
1. Determine the following: 
 
a. Did the Adverse Event occur within 100 days from the start of transplant conditioning?  
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b. Institutional location of the Adverse Event. 
 
c. Did death or immediate life-threatening event occur? 
 
d. Did a serious event occur – if so, was it expected? 
 
e. Can the relationship to the study intervention be ruled out? 
Based on information obtained, relevant arms of the flow chart may be employed to complete 
Emory University Adverse Reaction and Unexpected Event Form. 
Principal Investigator shall verify adverse reaction/unexpected event by signing Adverse 
Reaction and Unexpected Event Form. 
Completed Adverse Reaction and Unexpected Event Form shall be submitted to the Emory IRB 
within appropriate time frame as required. 
 
 
18.2.3  Adverse Event - An adverse event (AE) is any untoward medical experience or change 
of an existing condition that occurs during or after treatment, whether or not it is considered to 
be related to the protocol intervention. All AEs occurring during this study, whether observed by 
the physician, nurse, or reported by the patient, will be graded per NCI CTCAE version 4.0 and 
recorded on protocol-specific case report forms.   
 
18.2.4   Serious Adverse Event- A serious adverse event (SAE) is defined as any expected or 
unexpected adverse event (AE, generally equivalent to CTCAE grades 3, 4 or 5) that is related 
or unrelated to the intervention that results in any of the following outcomes: 
• 
Death 
• 
A life-threatening event 
• 
In-patient hospitalization (not required as part of the treatment) or   
• 
prolongation of existing hospitalization 
• 
A persistent or significant disability/incapacity 
• 
A congenital anomaly/birth defect 
• 
Causes cancer 
• 
Is an overdose 
Certain medical events that may not result in death, be life-threatening, or require 
hospitalization, may also be considered a serious adverse event when appropriate medical or 
surgical intervention is necessary to prevent one of the outcomes listed above. 
 
18.2.5 Unexpected Adverse Event – Any event in which the severity or specificity is not 
consistent with the risk information described in the protocol, and the event is not anticipated 
from the subject's disease history or status. 
 
18.2.6   Expected Adverse Event - Any event in which the severity or specificity is consistent 
with the risk information described in the protocol, or is anticipated based on the subject's 
medical history. 
 
18.2.7   Attribution - For reporting purposes, attribution is the assessment of the likelihood that 
an AE is caused by the research agent or protocol intervention.  The attribution is assigned by 
the Principal Investigator after considering the clinical information, the medical history of the 
subject, and past experience with the research agent/intervention.  This is recorded per 
institutional guidelines. Adverse events in one of 5 categories will be scored as the following: 
5=related, 4=probably related, 3=possibly related, 2=unlikely related and 1=unrelated.  The 
attribution is subject to change as follow-up information becomes available, and it can be 
changed by the DSMB in the process of review. 
 
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18.2.8 Outcome of Serious Adverse Events - The outcome of all SAEs will be graded as 
resolved; recovering; ongoing; resolved with sequela; fatal. 
 
18.3 Clinical Laboratory Tests and Normal Laboratory Values - Throughout the study, 
clinical laboratory tests will be performed in the clinical laboratory. 
 
18.4   Case Report Forms - Case report forms are designed to contain information necessary 
for the evaluation of the patient and investigational agent. 
 
18.5   Reporting and Recording of Data - All information required by the protocol is to be 
recorded on CRFs based on source data.  Source data is defined as all information in original 
records and certified copies of original records of clinical findings, observations, or other 
activities in a clinical trial necessary for the reconstruction and evaluations of the trial.  If source 
data are unavailable, an explanation should be given. Concomitant Medications will not be 
collected for this protocol.  Grade 1 and 2 toxicities will only be collected if they are related to 
the study drug.   
  
18.6   Records Retention - The Principal Investigator will maintain all records related to the 
study as required by regulations and institutional policy. 
 
18.7   Changes to the Protocol - All protocol amendments will be in writing and signed by the 
Principal Investigator to document his/her agreement.  The Principal Investigator will have all 
protocol amendments will be approved by the Institutional Review Board prior to their 
implementation 
 
 
19.0 
Removal of patient from study 
The investigator should withdraw a patient from the study treatment whenever: 
 
1-Continued participation is no longer in the patient’s best interests. Reasons for discontinuing 
treatment may include the occurrence of a SAE or an inter-current illness. These patients will be 
taken off treatment and followed according to the study calendar. 
2-The patient requests to end treatment. 
3-Patients who could not receive all planed Thymo/Bortezomib doses for GVHD prevention will 
be removed from the study and replaced by another patient.  
 
 
20.0 
Ethics 
20.1 
Responsibilities of the Principal Investigator (21 CFR Part 312.60)  
The Principal Investigator and his/her staff must be available to fulfill the needs of this trial.  The 
Principal Investigator ensures that the proposed trial will not be disturbed by other possible 
intervening trials.  The Principal Investigator must organize the available facilities and technical 
structures according to the nature of the trial to ensure filing of documents for the duration of the 
trial as well as after its completion. 
 
In accordance with Title 21 CFR (Part 50), the Principal Investigator commits to obtaining the 
informed consent of the patient or guardian by signature and clearly indicates the content of the 
information given to the patient and the means by which consent is obtained. 
 
The Principal Investigator commits him/herself to conform to GCP, 21 CFR Parts 50, 54, 56 and 
312 regulations, concerning his/her duties. 
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For the entire group of persons involved in carrying out the trial (day, night and emergency 
personnel), the Principal Investigator is responsible for the following: 
 
1. 
Ensuring that the personnel are informed of the protocol used and that 
they understand the part they are responsible for implementing. 
2. 
Training personnel if necessary. 
3. 
Designating individual(s) specifically responsible for the administrative 
management of the trial. 
4. 
Ensuring that other departments or services involved in this trial are 
informed of the trial and determining with them the specific operating 
procedures necessary to conduct the trial. 
 
The Principal Investigator is responsible for ensuring that the protocol and its appendices are 
scrupulously followed, particularly when other departments are involved in the trial.  The 
Principal Investigator oversees the quality of the data collected in the case reports.  The data 
obtained during the trial are recorded directly, with all modifications of data signed, dated, and 
justified as stated above.  Modifications must conform to the procedures defined for paper and 
electronic records. 
 
 
20.2 
Institutional Review Board/Ethics Committee Approval 
Prior to implementation of this study, the research protocol and the proposed patient consent 
form must be reviewed and approved by the Winship Cancer Institute Protocol Review and 
Monitoring Committee and the Emory University Institutional Review Board (Emory IRB).  All 
amendments to the protocol must be approved by the Emory IRB. 
 
16.0     Minorities and Women 
All eligible patients from both genders and from all racial/ethnic groups will be recruited equally 
into this trial, with the only exclusionary criteria being those stated in section 6.0.  
 
 
References: 
 
 
 
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Admiraal R, van Kesteren C, Jol-van der Zijde CM, et al. Population pharmacokinetic 
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Killick SB, Marsh JC, Booth JC, Gordon-Smith EC. Liver function abnormality following 
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Remberger M, Svahn BM, Hentschke P, Lofgren C, Ringden O. Effect on cytokine 
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Finn PW, Stone JR, Boothby MR, Perkins DL. Inhibition of NF-kappaB-dependent T cell 
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64. 
Nencioni A, Schwarzenberg K, Brauer KM, et al. Proteasome inhibitor bortezomib 
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Blanco B, Perez-Simon JA, Sanchez-Abarca LI, et al. Bortezomib induces selective 
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Blanco B, Perez-Simon JA, Sanchez-Abarca LI, et al. Treatment with bortezomib of 
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Papandreou CN, Daliani DD, Nix D, et al. Phase I trial of the proteasome inhibitor 
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Sun K, Welniak LA, Panoskaltsis-Mortari A, et al. Inhibition of acute graft-versus-host 
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69. 
Vodanovic-Jankovic S, Hari P, Jacobs P, Komorowski R, Drobyski WR. NF-kappaB as a 
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Sun K, Wilkins DE, Anver MR, et al. Differential effects of proteasome inhibition by 
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Sun K, Li M, Sayers TJ, Welniak LA, Murphy WJ. Differential effects of donor T-cell 
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Niederwieser D, Gastl G, Rumpold H, Marth C, Kraft D, Huber C. Rapid reappearance of 
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Ruggeri L, Capanni M, Urbani E, et al. Effectiveness of donor natural killer cell 
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Eissens DN, Van Der Meer A, Van Cranenbroek B, Preijers FW, Joosten I. Rapamycin 
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Soriani A, Zingoni A, Cerboni C, et al. ATM-ATR-dependent up-regulation of DNAM-1 
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Boyiadzis M, Memon S, Carson J, et al. Up-regulation of NK cell activating receptors 
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101. 
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active thymoglobulin levels: effects of dose and sensitization on serum concentrations. Transpl 
Immunol. Oct 2001;9(1):29-36. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
APPENDIX A: GVHD GRADING SCALE 
 
Grading of acute GVHD will be based on the following 3 tables and recorded as I,II,III, or IV as 
defined in table 2.  (Modified Glucksberg Index). 
 
 
 
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Appendix B:   Karnofsky Performance Status 
 
DESCRIP
PERCENT (%) 
Normal; no complaints; no evidence of disease 
100 
Able to carry on normal activity; minor signs and symptoms of disease 
90 
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Normal activity with effort; some signs and symptoms of disease 
80 
Cares for self; unable to carry on normal activity or do work 
70 
Requires occasional assistance, can care for most personal needs 
60 
Requires considerable assistance and frequent medical care 
50 
Disabled; requires special care and assistance 
40 
Severely disabled; hospitalization indicated although death not imminent 
30 
Very sick; hospitalization necessary; requires active support treatment 
20 
Moribund; fatal processes progressing rapidly 
10 
Dead 
0 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
APPENDIX C:  SUGGESTED CRITERIA FOR DETERMINING THE RELATIONSHIP OF 
ADVERSE EVENTS TO STUDY DRUG 
 
All AEs will be recorded on CRFs with the date of occurrence and disappearance.  Their 
relationship to the study drug and their intensity can be rated according to the following 
guidelines*: 
 
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Unrelated 
 
This category applies to those adverse experiences that, after careful consideration are clearly 
and incontrovertibly due to extraneous causes (disease, environment, etc.) 
 
 
Unlikely (two or more) 
 
 
An AE may be considered “unlikely” if or when two of the following apply: 
 
1. 
It does not follow a reasonable temporal sequence from administration of the 
drug. 
2. 
It could readily have been produced by the patient’s clinical state, environmental 
or toxic factors, or other modes of therapy administered to the patient. 
3. 
It does not follow a known or expected response pattern to the test drug. 
4. 
It does not reappear or worsen when the drug is re-administered. 
 
Possibly (two or more) 
 
An AE may be considered “possible” if or when two of the following apply: 
 
1. 
It follows a reasonable temporal sequence from administration of the drug. 
2. 
It could readily have been produced by the patient’s clinical state, environmental 
or toxic factors, or other modes of therapy administered to the patient. 
3. 
It follows a known response pattern to the suspected drug. 
 
Probably (three or more) 
 
An AE may be considered “probable” if or when three of the following apply: 
 
1. 
It follows a reasonable temporal sequence from administration of the drug. 
2. 
It could not be reasonably explained by the known characteristics of the patient’s 
clinical state, environmental or toxic factors, or other modes of therapy 
administered to the patient. 
3. 
It disappears or decreases on cessation or reduction in dose.  There are 
important exceptions when the AE does not disappear upon discontinuation of 
the drug, yet drug-relatedness clearly exists. 
4. 
It follows a known response pattern to the suspected drug. 
 
Definitely (all four) 
An AE may be assigned an attribution of “definite” if or when all four of the following apply: 
 
1. 
It follows a reasonable temporal sequence from administration of the drug. 
2. 
It could not be reasonably explained by the known characteristics of the patient’s 
clinical state, environmental or toxic factors, or other modes of therapy 
administered to the patient. 
3. 
It disappears on cessation or reduction in dose and recurs with reintroduction or 
increase in dose of the test drug.  This is not to be construed as requiring 
reintroduction of the drug, however, “definite” attribution cannot be made unless 
the drug has been reintroduced and the adverse reaction has recurred. 
4. 
It follows a known response pattern to the suspected drug. 
 
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*These suggested criteria are intended to aid the clinician in the casuality assessment process.  
The physician should exercise his/her clinical judgment in assessing final causality to a 
suspected agent. 
 
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APPENDIX D:  DECLARATION OF HELSINKI 
 
II. 
WORLD MEDICAL ASSOCIATION 
 
DECLARATION OF HELSINKI 
 
 
Recommendation guiding physicians  
in biomedical research involving human subjects 
 
Adopted by the 18th World Medical Assembly 
Helsinki, Finland, June 1964 
 
and amended by the 
29th World Medical Assembly, Tokyo, Japan, October 1975 
35th World Medical Assembly, Venice, Italy, October 1983 
and the 41st World Medical Assembly, Hong Kong, September 1989 
 
 
Introduction 
 
It is the mission of the physician to safeguard the health of the people.  His or her knowledge 
and conscience are dedicated to the fulfillment of this mission. 
 
The Declaration of Geneva of the World Medical Association binds the physician with the words, 
“The health of my patient will be my first consideration,” and the International Code of Medical 
Ethics declares that “A physician shall act only in the patient’s interest when providing medical 
care which might have the effect of weakening the physical and mental condition of the patient.” 
 
The purpose of biomedical research involving human subjects must be to improve diagnostic, 
therapeutic and prophylactic procedures and the understanding of the aetiology and 
pathogenesis of disease. 
 
In current medical practice most diagnostic, therapeutic or prophylactic procedures involve 
hazards.  This applies especially to biomedical research. 
 
Medical progress is based on research which ultimately must rest in part on experimentation 
involving human subjects. 
 
In the field of biomedical research a fundamental distinction must be recognized between 
medical research in which the aim is essentially diagnostic or therapeutic for a patient, and 
medical research, the essential object of which is purely scientific and without implying direct 
diagnostic or therapeutic value to the person subjected to the research. 
 
Special caution must be exercised in the conduct of research which may affect the environment, 
and the welfare of animals used for research must be respected. 
 
Because it is essential that the results of laboratory experiments be applied to human beings to 
further scientific knowledge and to help suffering humanity, the World Medical Association has 
prepared the following recommendations as a guide to every physician in biomedical research 
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involving human subjects.  They should be kept under review in the future.  It must be stressed 
that the standards as drafted are only a guide to physicians all over the world.  Physicians are 
not relieved from criminal, civil and ethical responsibilities under the laws of their own countries. 
 
1. 
Basic principles 
 
 
Biomedical research involving human subjects must conform to generally accepted scientific 
principles and should be based on adequately performed laboratory and animal experimentation 
and on a thorough knowledge of the scientific literature. 
 
The design and performance of each experimental procedure involving human subjects 
should be clearly formulated in an experimental protocol which should be transmitted for 
consideration, comment and guidance to a specially appointed committee independent of the 
investigator and the sponsor provided that this independent committee is in conformity with the 
laws and regulations of the country in which the research experiment is performed. 
 
Biomedical research involving human subjects should be conducted only by scientifically 
qualified persons and under the supervision of a clinically competent medical person.  The 
responsibility for the human subject must always rest with a medically qualified person and 
never rest on the subject of the research, even though the subject has given his or her consent. 
 
Biomedical research involving human subjects cannot legitimately be carried out unless the 
importance of the objective is in proportion to the inherent risk to the subject. 
 
Every biomedical research project involving human subjects should be preceded by careful 
assessment of predictable risks in comparison with foreseeable benefits to the subject or to 
others.  Concern for the interests of the subject must always prevail over the interests of science 
and society. 
 
The right of the research subject to safeguard his or her integrity must always be respected.  
Every precaution should be taken to respect the privacy of the subject and to minimize the 
impact of the trial on the subject’s physical and mental integrity and on the personality of the 
subject. 
 
Physicians should abstain from engaging in research projects involving human subjects 
unless they are satisfied that the hazards involved are believed to be predictable.  Physicians 
should cease any investigation if the hazards are found to outweigh the potential benefits. 
 
In publication of the results of his or her research, the physician is obliged to preserve the 
accuracy of the results.  Reports of experimentation not in accordance with the principles laid 
down in this Declaration should be not accepted for publication. 
 
In any research on human beings, each potential subject must be adequately informed of 
the aims, methods, anticipated benefits and potential hazards of the trial and the discomfort it 
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may entail.  He or she should be informed that he or she is at liberty to abstain from participation 
in the trial and that he or she is free to withdraw his or her consent to participation at any time.  
The physician should then obtain the subject’s freely-given informed consent, preferably in 
writing. 
 
When obtaining informed consent for the research project the physician should be 
particularly cautious if the subject is in a dependent relationship to him or her or may consent 
under duress.  In that case the informed consent should be obtained by a physician who is not 
engaged in the investigation and who is completely independent of this official relationship. 
 
In case of legal incompetence, informed consent should be obtained from the legal guardian 
in accordance with national legislation.  Where physical or mental incapacity makes it 
impossible to obtain informed consent, or when the subject is a minor, permission from the 
responsible relative replaces that of the subject in accordance with national legislation. 
 
Whenever the minor child is in fact able to give a consent, the minor’s consent 
must be obtained in addition to the consent of the minor’s legal guardian. 
 
The research protocol should always contain a statement of the ethical considerations 
involved and should indicate that the principles enunciated in the present Declaration are 
complied with. 
 
2. 
Medical research combined with professional care (clinical research) 
 
In the treatment of the sick person, the physician must be free to use a new diagnostic and 
therapeutic measure, if in his or her judgement it offers hope of saving life, re-establishing 
health or alleviating suffering. 
 
The potential benefits, hazards and discomfort of a new method should be weighed against 
the advantages of the best current diagnostic and therapeutic methods. 
 
In any medical trial, every patient – including those of a control group, if any – should be 
assured of the best proven diagnostic and therapeutic method. 
 
The refusal of the patient to participate in a trial must never interfere with the physician-
patient relationship. 
 
If the physician considers it essential not to obtain informed consent, the specific reasons for 
this proposal should be stated in the experimental protocol for transmission to the independent 
committee (1.2) 
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The physician can combine medical research with professional care, the objective being the 
acquisition of new medical knowledge, only to the extent that medical research is justified by its 
potential diagnostic or therapeutic value for the patient. 
 
3. 
Non-therapeutic biomedical research involving human subjects (non-biomedical 
research) 
 
In the purely scientific application of medical research carried out on a human being, it is the 
duty of the physician to remain the protector of the life and health of that person on whom 
biomedical research is being carried out. 
 
The subjects should be volunteers – either healthy persons or patients for whom the 
experimental design is not related to the patient’s illness. 
 
The investigator or the investigating team should discontinue the research if in his/her or 
their judgment it may, if continued, be harmful to the individual. 
In research on man, the interest of science and society should never take precedence 
over considerations related to the well-being of the subject 
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APPENDIX E: KARNOFSKY / ECOG PERFORMANCE STATUS SCALE 
 
A. 
 
 
 
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APPENDIX F:  ASSESSMENT OF LYMPHOMA BY INTEGRATED IWC+PET 
 
 
.  
 
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APPENDIX G:  Chronic GVHD NIH Consensus Project 
 
 
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