A Phase II Pediatric Study of a Graft-VS.-Host Disease (GVHD) Prophylaxis Regimen With no Calcineurin Inhibitors After Day +60 Post First Allogeneic Hematopoietic Cell Transplant for Hematological Malignancies
Phase: PHASE2
Conditions: Hematologic Malignancy, Myeloid Malignancy
Sponsor: St. Jude Children's Research Hospital

Official Study Title: A PHASE II PEDIATRIC STUDY OF A GRAFT-VS.-HOST 
DISEASE (GVHD) PROPHYLAXIS REGIMEN WITH NO CALCINEURIN INHIBITORS 
AFTER DAY +60 POST FIRST ALLOGENEIC HEMATOPOIETIC CELL TRANSPLANT 
FOR HEMATOLOGICAL MALIGNANCIES 
 
CTG Number: NCT05579769 
 
Document Date: 09/22/2023 
 

Revision 0.3 dated: 07/27/2023 
Protocol document date: 07/27/2023
St. Jude
CNI60
Initial version, dated: 02/01/2022 (IRB Approved: 09/26/2022) Activated: 11/04/2022 
Revision 0.1, dated 17 Jan 2023 (IRB Approved: 02/07/2023) Activated: 02/14/2023 
Revision 0.2, dated 17 Mar 2023 (IRB Approved: 04/19/2023) Activated: 06/23/2023 
Revision 0.3, dated 27 Jul 2023 (IRB Approved: 09/22/2023) Activated:10/10/2023
A PHASE II PEDIATRIC STUDY OF A GRAFT-VS.-HOST DISEASE (GVHD) 
PROPHYLAXIS REGIMEN WITH NO CALCINEURIN INHIBITORS AFTER 
DAY +60 POST FIRST ALLOGENEIC HEMATOPOIETIC CELL 
TRANSPLANT FOR HEMATOLOGICAL MALIGNANCIES
Principal Investigator
Ashok Srinivasan, MD1
Co-Investigators
Stephen Gottschalk, MD1
Brandon Triplett, MD1
Renee Madden, MD1
Amr Qudeimat, MD1
Aimee Talleur, MD1
Ewelina Mamcarz, MD1
Akshay Sharma, MD1
Swati Naik, MBBS1
Ali Suliman, MD1
Paulina Velasquez, MD1
Kim Nichols, MD2
Rebecca Epperly, MD1
Shane Cross, PharmD4
Cheng Cheng, PhD3
Kristine Crews, PharmD4
Diana Wu, PharmD4
Matthew Krasin, MD5
John C. Panetta, PhD4
Caitlin Zebley, MD, PhD1
Ben Youngblood, PhD6
Paul Thomas, PhD6
Senthil Bhoopalan, MBBS, PhD1
Esther Obeng, MD, PhD2
Sophia Folk, PNP1 
1Department of Bone Marrow Transplantation and Cellular Therapy
2Department of Oncology
3Department of Biostatistics
4Department of Pharmacy and Pharmaceutical Sciences
5Department of Radiation Oncology
6Department of Immunology
St. Jude Children's Research Hospital
262 Danny Thomas Place
Memphis, Tennessee  38105-3678
Telephone:  (901) 595-3300
St. Jude Children's Research Hospital
IRB NUMBER: 22-1023
IRB APPROVAL DATE: 09/22/2023

Revision 0.3 dated: 07/27/2023 
Protocol document date: 07/27/2023
Contents of this document may not be extracted without permission from the Principal Investigator.
Protocol Summary
Protocol MNEMONIC and Title: CNI60 - A Phase II Pediatric Study of a Graft-vs.-
host disease (GVHD) prophylaxis regimen with no calcineurin inhibitors after day +60 
post first allogeneic hematopoietic cell transplant for hematological malignancies. 
Principal Investigator: Ashok Srinivasan, MD
Brief Overview: The primary objective of the study is to estimate the incidence of severe 
acute Graft-vs.-Host disease (saGVHD) using a prophylaxis regimen with no calcineurin 
inhibitors after day +60 post a first allogeneic Human Leukocyte antigen (HLA)-matched 
sibling or unrelated donor Hematopoietic cell transplant (MSD/MUD HCT) for 
hematological malignancies. Secondary objectives are focused on determining the 
cumulative incidence of relapse, non-relapse mortality (NRM), chronic GVHD, and 
overall survival (OS). Exploratory objectives are focused on evaluating the 
pharmacokinetic/pharmacodynamic (PK/PD) profiles of ruxolitinib, fludarabine, and  
rabbit anti-thymocyte globulin (rATG), and to assess immune reconstitution in HCT 
recipients.
The central hypothesis of the study is that it may be possible to safely stop calcineurin 
inhibitors (CNIs) early on day +60 post allogeneic hematopoietic cell transplantation by 
adding ruxolitinib after engraftment. This may decrease the incidence of saGVHD within 
the first 100 days post-transplant while reducing toxicity from prolonged use of CNIs. We 
expect that the performed PK/PD tests will have a broad impact on other transplant 
protocols that utilize these agents. Finally, we expect that the performed immune 
reconstitution studies will inform future transplant studies.
Intervention: The primary interventions are allogeneic progenitor cell transplantation for 
treatment of hematologic malignancy and subsequent administration of selected agents 
for prevention of GVHD.
Brief Outline of Treatment Plan: Children 12 years and older will be eligible for the 
study. Only bone marrow donors will be used for this trial.  Human leukocyte antigen 
(HLA) identical sibling donors will be considered first followed by histocompatible 
relatives or unrelated donors matched at 12 of 12 HLA alleles.
We propose to employ two preparative regimens based on the underlying hematological 
malignancy. For hematological malignancies of the lymphoid lineage we will use a 
standard preparative regimen consisting of Total Body Irradiation and cyclophosphamide 
(TBI/Cy), unless TBI is contraindicated. For myeloid  malignancies we will use thiotepa, 
busulfan, and fludarabine (TBF), a preparative regimen that has been associated with a 
reduced risk of relapse and trend for improved survival with comparable NRM in 
comparison to busulfan and cyclophosphamide (BuCy), our current regimen. All HCT 
recipients will receive cyclosporine in combination with methotrexate and ruxolitinib as 
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Protocol MNEMONIC and Title: CNI60 - A Phase II Pediatric Study of a Graft-vs.-
host disease (GVHD) prophylaxis regimen with no calcineurin inhibitors after day +60 
post first allogeneic hematopoietic cell transplant for hematological malignancies. 
GVHD prophylaxis. Recipients of MUD HCT will receive rATG for additional 
immunosuppression as is standard for unrelated donor transplants
Study Design: Phase II
Sample Size: We anticipate 32 enrollees to yield 30 evaluable research participants.
Data Management: Data management and statistical analysis will be provided locally by 
the Department of Bone Marrow Transplantation and Cellular Therapy and the 
Department of Biostatistics at St. Jude Children’s Research Hospital
Human Subjects: The risks to participants are primarily related to the conditioning 
regimen and post-transplant complications. The proposed regimen and donor cells may 
induce serious and possibly fatal disorders such as GVHD, veno-occlusive disease, and 
post-transplant lymphoproliferative disease. Because of the required conditioning, 
recipients may be at high-risk for serious and possibly life-threatening infection, bleeding, 
and anemia. Adverse events will be treated, monitored, and reported as per the Department 
of Bone Marrow Transplantation and Cellular therapy SOP for recipients of allogeneic 
HCT.
St. Jude Children's Research Hospital
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TABLE OF CONTENTS
1.0
OBJECTIVES...................................................................................................................1
1.1
Primary objectives ...................................................................................................1
1.2
Secondary objective.................................................................................................1
1.3
Exploratory objectives.............................................................................................1
2.0
BACKGROUND AND RATIONALE.............................................................................1
2.1
Hypothesis to be tested in this study........................................................................1
2.2
HCT donor types and conditioning regimens..........................................................1
2.3
Graft-vs.-host disease ..............................................................................................3
2.4
Relapse following HCT and strategies to decrease relapse.....................................3
2.5
Can Cyclosporine A be weaned early to reduce toxicity without increasing risk for 
GVHD?.............................................................................................................................4
2.6
Ruxolitinib for treatment of GVHD ........................................................................4
2.7
Ruxolitinib for prophylaxis of GVHD.....................................................................6
2.8    Ruxolitinib for treatment of refractory heme-malignancies ....................................6
2.9
Rationale for early discontinuation of CsA and replacement with ruxolitinib........6
2.10 Rationale for PK/PD profiling.................................................................................7
2.12 Impact of study on field of HCT..............................................................................9
3.0
PARTICIPANT ELIGIBILITY CRITERIA AND STUDY ENROLLMENT ................9
3.1
Inclusion criteria ......................................................................................................9
3.2
Exclusion criteria...................................................................................................10
3.3
Research participant recruitment and screening....................................................11
3.4
Enrollment on study at St. Jude .............................................................................11
4.0
DESIGN AND METHODS............................................................................................11
4.1
Design and study overview....................................................................................11
5.0
TREATMENT PLAN.....................................................................................................11
5.1
Treatment...............................................................................................................11
5.2
Supportive care ......................................................................................................15
5.3
Ruxolitinib dose modification ...............................................................................16
6.0
DRUG/DEVICE/BIOLOGIC AGENT INFORMATION..............................................16
6.1 Medications...............................................................................................................16
6.2 Total Body Irradiation (TBI) .................................................................................26
7.0
REQUIRED EVALUATIONS, TESTS, AND OBSERVATIONS ...............................28
7.1
Schedule of evaluations .........................................................................................28
7.2 Evaluation for chimerism and engraftment ...........................................................28
7.3 Evaluation for immune reconstitution ...................................................................28
7.4 Minimal residual disease evaluation......................................................................29
7.5
Evaluations for ruxolitinib pharmacokinetics testing............................................29
7.6
Evaluations for rATG pharmacokinetics testing ...................................................30
7.7
Evaluations for fludarabine pharmacokinetics testing...........................................30
7.8 Evaluation for ex-vivo target inhibition ................................................................30
7.9
Long-term follow-up evaluations ..........................................................................30
8.0
EVALUATION CRITERIA...........................................................................................31
8.1
Response criteria and evaluations..........................................................................31
8.2
Toxicity evaluation criteria....................................................................................31
9.0
OFF STUDY AND  OFF THERAPY CRITERIA.........................................................31
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9.1    Off-study criteria ...................................................................................................31
9.2
Off-therapy criteria ................................................................................................32
10.0
SAFETY AND ADVERSE EVENT REPORTING REQUIREMENTS.......................32
10.1 Adverse events (AEs) ............................................................................................32
10.2 Definitions .............................................................................................................32
10.3 Handling of adverse events (AEs) and deaths .......................................................33
10.4 Reporting to the CIBMTR.....................................................................................34
11.0
DATA COLLECTION, STUDY MONITORING, AND CONFIDENTIALITY..........34
11.1 Data collection.......................................................................................................34
11.2 Study Monitoring...................................................................................................35
11.3 Confidentiality .......................................................................................................36
11.4 Genomic Data Sharing...........................................................................................36
12.0
STATISTICAL CONSIDERATIONS............................................................................36
12.1 Historical St. Jude data relating to the primary endpoint ......................................36
12.2  Primary objective...................................................................................................37
12.3 Secondary objective...............................................................................................37
12.4 Exploratory objectives...........................................................................................38
12.5 Safety Monitoring..................................................................................................38
12.6 Anticipated Completion Dates...............................................................................39
13.0
OBTAINING INFORMED CONSENT.........................................................................40
13.1 Informed Consent Prior to Research Interventions ...............................................40
13.2 Consent at Age of Majority ...................................................................................40
13.3 Consent When English is Not the Primary Language ...........................................40
14.0
REFERENCES ...............................................................................................................41
APPENDIX A.............................................................................................................................46
APPENDIX B.............................................................................................................................47
APPENDIX C.............................................................................................................................54
APPENDIX D.............................................................................................................................57
APPENDIX E .............................................................................................................................58
APPENDIX F .............................................................................................................................64
APPENDIX G.............................................................................................................................65
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1.0 OBJECTIVES
1.1
Primary objectives

To estimate the incidence of severe acute GVHD (saGVHD) using a 
prophylaxis regimen with no calcineurin inhibitors after day +60 post first 
allogeneic Human Leukocyte antigen (HLA)-matched sibling or unrelated 
donor HCT for hematological malignancies.
1.2
Secondary objective
1.2.1 Determine the cumulative incidence of relapse, NRM, chronic GVHD, and OS 
in study participants at one year post-transplant.
1.3
Exploratory objectives
1.3.1 To evaluate the pharmacokinetic/pharmacodynamic (PK/PD) profiles of 
ruxolitinib, fludarabine, and rATG.
1.3.2
To assess immune reconstitution in study participants within the first year post-
HCT.
2.0 BACKGROUND AND RATIONALE
2.1
Hypothesis to be tested in this study
While outcomes of MSD/MUD HCTs have improved, severe acute GVHD (saGVHD), 
defined as grade II-IV GVHD, remains a significant clinical problem. CNIs (for 
example, cyclosporine (CsA) or tacrolimus) in combination with methotrexate are 
commonly used for GVHD prophylaxis. However, their use can be associated with 
significant side effects, and are only effective in a subset of patients. Further, CNIs 
inhibit the graft-vs.-leukemia (GVL) effect increasing the risk of relapse which occurs 
in a quarter of  recipients and is the most common cause of death post-HCT for children 
with hematological malignancies.
The central hypothesis is that it may be possible to safely stop CNIs early on day +60 
post allogeneic HCT by adding ruxolitinib after engraftment on day +40. CNIs are 
currently stopped on day +100. Early discontinuation of CsA will help decrease 
toxicities. Replacement with ruxolitinib may decrease the incidence of saGVHD within 
the first 100 days post-transplant as ruxolitinib has potent effects in treating GVHD. 
Ruxolitinib also does not impair the GVL effect; this may decrease the risk of relapse.    
Ruxolitinib is approved for treatment of saGVHD in children 12 years of age and older, 
hence only children in this age group will be eligible to participate in the study.
2.2
HCT donor types and conditioning regimens
2.2.1
Matched sibling and matched unrelated donor HCT for hematological 
malignancies
Most patients referred for allogeneic HCT lack an HLA-MSD, thus an 
alternative HLA-MUD must be identified. As advances in supportive care and 
donor selection have improved, HCT outcomes using MUD have been 
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compared to those using MSD. A single center retrospective analysis of 87 
children with ALL in second complete remission (CR2) showed comparable 
outcomes with MSD and MUD HCT.1 Transplant outcomes for children with 
T-ALL in CR2 as reported by the Center for International Blood and Marrow 
Transplant Research (CIBMTR) showed no difference in relapse, GVHD or 
disease-free survival rates based on graft source.2 A multi-center retrospective 
analysis from Europe analyzing the influence of stem cell source on transplant 
outcomes for children with AML showed no difference in relapse, GVHD, 
NRM or OS based on donor source.3 The addition of rATG to GVHD 
prophylaxis with MUD donors has resulted in comparable GVHD rates between 
the 2 groups.4  Hence MSD and MUD HCT will be analyzed together in this 
study.
2.2.2   Total body irradiation/cyclophosphamide preparative regimen for ALL
A CIBMTR analysis of children with ALL receiving MSD HCT compared 
outcomes with TBI/Cy (451 patients) and busulfan/cyclophosphamide (BuCy; 
176 patients). The 3-year probability of leukemia-free survival was 
significantly higher in the TBI/Cy group. The risk of relapse was similar in the 
2 groups, but NRM was higher in children receiving BuCy.5 Similar inferior 
outcomes of BuCy conditioning compared to TBI/Cy were reported in a 
prospective trial conducted by the Pediatric Blood and Marrow Transplant 
Consortium (PBMTC).6 A prospective phase III, multicenter, randomized 
controlled trial from Europe was stopped prematurely owing to significantly 
improved OS with TBI-based compared to chemotherapy-based conditioning 
in 417 children with high risk ALL.7 Hence, we selected TBI/Cy for children 
with lymphoid malignancies.
2.2.3   Thiotepa, busulfan and fludarabine (TBF) preparative regimen for AML
The combination of intravenous fludarabine and busulfan (FluBu) is considered 
to be the standard conditioning regimen for older patients with AML.8 A 
prospective multicenter randomized study by the Italian Bone Marrow 
Transplant Group (GITMO), comparing FluBu with BuCy in older patients 
with AML in remission, showed the cumulative incidence of relapse at 5 years 
to be 38% in both groups. However, NRM was reduced to 8% in the FluBu arm 
compared to 17% with BuCy.9 PBMTC reported favorable results with a FluBu 
regimen with a NRM of 11% and relapse rate of 43%.10 
Bartelink et al. in a retrospective European study found FluBu to be a less toxic 
regimen for children with AML with lower rates of infection, lung injury, veno-
occlusive disease, and shorter duration of neutropenia.11 Historically, our rates 
of NRM and relapse with the BuCy regimen using a bone marrow graft are 11% 
and 28%, respectively.
A modified FluBu regimen has been evaluated in which one day of busulfan 
was omitted, and thiotepa was added (TBF).12 In a European multicenter 
retrospective comparison of FluBu and TBF in adults with AML in remission, 
TBF reduced the risk of relapse, and there was a trend for improved survival 
with comparable NRM.13 Augmentation of thiotepa to myeloablative FluBu for 
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adults and children with AML reduced relapse rates from 46% to 14% without 
additional toxicity in a single-center retrospective study.14
Thus, we selected this conditioning regimen for children with myeloid or 
undifferentiated leukemia. Our hypothesis is that substituting FluBu for BuCy 
will decrease the NRM, and adding thiotepa will decrease relapse rates. 
Busulfan targeting will continue to be in the myeloablative range.
 2.3 Graft-vs.-host disease
2.3.1   GVHD after myeloablative conditioning for hematological malignancies 
and prophylactic regimens
While HCT is a curative modality for children with certain hematological 
malignancies, acute GVHD limits its efficacy ultimately preventing broader 
application.15 
This 
life-threatening 
complication 
occurs 
when 
immunocompetent T-cells in the graft recognize the recipient (host) as foreign 
and mount an immune response to eliminate foreign antigen-bearing cells. 
Acute GVHD prophylaxis with CsA, and methotrexate is used in the majority 
of patients receiving allogeneic HCT based on a randomized trial showing 
efficacy.16 Addition of pan T-cell depleting reagent rATG to the standard 
prophylaxis further decreased risk of acute GVHD in a subset of patients.17
2.3.2
Limitations of calcineurin inhibitors for GVHD prophylaxis 
A third of our patients still develop acute GVHD with CNI-containing regimens 
and a fifth have saGVHD. Furthermore, some patients have contraindications 
to the use of CNIs, such as renal/hepatic dysfunction, veno-occlusive disease, 
thrombotic microangiopathy,18 and posterior reversible encephalopathy 
syndrome.19 CNI replaced by other agents for GVHD prophylaxis such as 
sirolimus in these situations increases the risk of veno-occlusive disease.20 CNIs 
increase the risk for viral reactivation post -HCT.21 This results in significant 
morbidity and mortality. 
CNIs are metabolized by CYP 3A 4/5 and are also a substrate of P-glycoprotein. 
Hence interactions with azoles which are commonly used for antifungal 
prophylaxis, may result in erratic levels. Further, its pharmacokinetics are 
complex and highly variable resulting in wide inter-and-intra-patient 
variability. Variability in serum concentrations have been associated with 
increased risk for GVHD and CNI-therapy-related toxicities. CNIs are highly 
lipophilic, hence dosing in obese patients risks renal dysfunction owing to high 
levels or increased risk of acute GVHD with subtherapeutic levels Thus, CNIs 
are not ideal agents to prevent GVHD, and there is a need to establish more 
effective and better tolerated GVHD prophylactic regimens.
2.4
Relapse following HCT and strategies to decrease relapse
CNIs can also inhibit the Graft-vs.-Leukemia (GVL) activity of the transplanted 
immune cells, contributing to relapse which occurs in a fourth of our patients. The 
outcome after relapse is poor.22 A second allogeneic HCT is a treatment option. Second 
transplantations are, however, more problematic than first ones owing to increased 
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NRM with cumulative organ toxicity and an even higher risk of a subsequent relapse. 
Hence GVHD prophylactic agents which do not inhibit the GVL effect are desirable.  
2.5
Can Cyclosporine A be weaned early to reduce toxicity without increasing risk for 
GVHD?
In a retrospective study by Storb et al. 103 adults with hematological malignancies 
were transplanted with MSD (92 patients), or mis-matched related donors (11 
patients).23 GVHD prophylaxis was with CsA and methotrexate. By day +60 patients 
who never had acute GVHD, or whose acute GVHD had resolved, were randomized to 
have CsA stopped (52 patients) or continued for 180 days (51 patients). With a median 
follow up of 9.3 years after transplant, patients in whom CsA was discontinued on day 
+60 had a significantly more rapid onset, but not a significantly higher overall 
incidence of chronic GVHD than those in whom the drug was stopped on day +180 (p 
= 0.26). NRM was comparable in both arms. They concluded that CsA can safely be 
discontinued in patients who never had evidence of acute grade II-IV GVHD by day 
+60. Patients who have acute grade II-IV GVHD prior to day +60 on our study will 
not be eligible for early CsA wean (Sec 5.1).
It must be noted that this study was performed more than 30 years ago and in an adult 
population at high risk for chronic GVHD (43-54%) compared to 4% in our historical 
cohort of children. GVHD rates have decreased substantially in the past 2 decades.24 
Further, all our patients will be fully matched at a much higher level of resolution.
In a retrospective study by Zhao et al. 10 adults with hematological malignancies who 
were intolerant to CNIs after allogeneic HCT received ruxolitinib to replace CNI as 
GVHD 
prophylaxis. 
Reasons 
for 
CNI 
replacement 
included 
thrombotic 
microangiopathy, renal dysfunction, hepatic injury and CNI-induced pain syndrome. 
The median interval from HCT to ruxolitinib was 35 days (range 28-45 days). After 
ruxolitinib replacement, toxicities resolved rapidly, and only 1 patient developed grade 
II skin GVHD within 100 days.25
2.6
Ruxolitinib for treatment of GVHD
The Janus Kinase (JAK)-signal transducer and activator of transcription (STAT) 
pathway is now recognized as an evolutionary conserved signaling pathway employed 
by diverse cytokines, interferons, growth factors and related molecules.26
JAK kinases have several domains. Ruxolitinib competitively inhibits the ATP-binding 
site of the kinase domain of JAK1 and JAK2. This was the first Jakinib approved by 
the FDA in 2011 for the treatment of myelofibrosis. It is an oral, reversible, class I 
inhibitor. Peak plasma concentrations are achieved within one hour after administration 
and decline in a monophasic or biphasic manner with a mean terminal half-life of 2.3 
hours. Ruxolitinib selectively inhibits JAK1 and JAK2 with IC50 values of 3.3 and 2.8 
nM, respectively.27
2.6.1
Ruxolitinib for treatment of GVHD in adults
saGVHD is a life-threatening complication after allogeneic HCT. 
Glucocorticoids are the choice for induction of remission. Patients with 
glucocorticoid-refractory (SR) aGVHD have long-term survival rates of only 
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5% to 30%.28 While SR aGVHD is usually severe in degree, not all cases of 
saGVHD are steroid-refractory. Cytokines play a major role in aGVHD and a 
common denominator of many cytokines is the downstream activation of 
JAK1/2. Inhibition of JAK1/2 was tested as an approach to interfere with 
aGVHD in different mouse models and found to have activity.29-31 Based on a 
retrospective multicenter survey where ruxolitinib showed promise in patients 
who had failed multiple previous therapies for SR aGVHD,32 prospective trials 
were initiated including a single-arm phase II study (REACH1) showing 
activity of ruxolitinib leading to FDA approval for SR aGVHD.33, 34 To clarify 
the question of whether ruxolitinib was superior to best available therapy 
(BAT), a multicenter randomized phase III trial was performed (REACH2).35 
The primary study objective was met: the ORR at day 28 was statistically 
significantly higher with ruxolitinib than with BAT (62% vs 39%; odds ratio, 
2.64; 95% CI, 1.65-4.22; P < .001). Ruxolitinib is now being considered the 
gold standard in SR aGVHD.36 
     2.6.2     Ruxolitinib for treatment of GVHD in children
Ruxolitinib use in children was associated with an ORR of 45% in a 
retrospective study of 11 children with SR aGVHD. Ruxolitinib was 
administered orally at 5 mg twice daily for children ≥25 kg or 2.5 mg twice 
daily if <25 kg, and escalated weekly as tolerated to a maximum of 10 mg twice 
daily. 
Adverse 
events 
included 
transaminitis, 
neutropenia, 
and 
thrombocytopenia which were reversible with discontinuation of therapy.37 
Infectious complications were observed, and all were successfully treated with 
antimicrobial therapy. 
In another retrospective study including 13 children with SR aGVHD, ORR 
was 77%. Dosing being similar, only one patient developed thrombocytopenia 
which was reversible.38 Cytomegalovirus reactivation was successfully treated 
without discontinuation of ruxolitinib. Responders included those with sa gut 
GVHD suggesting good absorption and bioavailability in the setting of diarrhea 
and gastrointestinal bleeding. 
A multicenter, retrospective study of 29 children who received ruxolitinib for 
SR aGVHD reported an ORR of 72.4%.39 No cytopenias requiring ruxolitnib 
dose adjustment occurred and viral infections were noted in 41.4% of patients. 
The authors reported a wide range for ruxolitinib dosing, however, they did not 
find a correlation with dose and response in their study.39 
In a prospective study of 17 children with SR aGVHD the ORR was 75%. 
Dosage was 10 mg twice daily for children >40 kg and 0.15 mg/kg twice daily 
for children weighing <40 kg.40 More than half the patients had hematologic 
toxicity with this higher dosing regimen. A phase III trial of ruxolitinib for SR 
aGVHD in children is ongoing (NCT03491215). Ruxolitinib is currently FDA 
approved for treatment of SR aGVHD in children 12 years of age and older. 
We will use the dose recommended for treatment (i.e., 5 mg twice daily).
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2.7
Ruxolitinib for prophylaxis of GVHD
2.7.1
Biology
Ruxolitinib prevents GVHD via suppression of inflammatory cytokine 
production. In murine GVHD models of fully MHC-mismatched HCT, TNF-α 
and IL-12 were significantly reduced in mice treated with ruxolitinib compared 
with vehicle.41 Ruxolitinib also impairs differentiation of CD4+ T cells into 
IFN-γ and IL-17A producing cells,41 modulates chemokine receptor 
expression,29 increases FoxP3+ regulatory T cells,28 and promotes Th2 
differentiation. All these immunologic effects of ruxolitinib reduce GVHD, 
while preserving the GVL effect.28,29
 2.7.2 Clinical experience 
In a prospective study by Zhang et al. after alternative donor HCT for 
hematological malignancies, early administration of ruxolitinib as GVHD 
prophylaxis after engraftment in an experimental group of 41 adults 
significantly decreased the incidence of aGVHD compared to a control group 
of 16 patients who received post-transplant cyclophosphamide, Fresnius ATG, 
CNI and mycophenolate mofetil for prophylaxis. There was no adverse effect 
on hematopoiesis.42
In the REACH-1 phase II study investigating ruxolitinib for treatment of SR 
aGVHD, approximately half the patients were rescued, however, a substantial 
proportion of patients had limited benefit from ruxolitinib treatment, showing 
no clinical response and a very bad outcome. Our hypothesis is that early 
“prophylactic” use of ruxolitinib will target kinase activity and block the 
inflammatory cascade leading to T-cell activation and GVHD. 
 2.8    Ruxolitinib for treatment of refractory heme-malignancies 
Oncogenic IL-7Rα gain of function mutations are found in 9% of individuals with T-
ALL. This drives constitutive signaling via JAK1 and promotes cell transformation and 
tumor formation.43 In vitro, treating a transformed cell line with ruxolitinib inhibited 
ligand-independent signaling and induced cell death. Transfer of the transformed cell 
line into mice resulted in aggressive leukemia. Mice treated with ruxolitinib showed a 
potent therapeutic benefit with reduction of leukemic burden and extension of 
survival.44 In a phase II study of ruxolitinib in 18 adults with relapsed/refractory 
leukemias, ruxolitinib showed modest antileukemic activity with 3 of the patients with 
post-myeloproliferative neoplasms showing a significant response.45 A GVHD 
prophylactic agent which has anti-leukemic activity may thus be more effective in 
preventing relapse than one which inhibits GVL activity of the transplanted immune 
cells.     
2.9
Rationale for early discontinuation of CsA and replacement with ruxolitinib
The rationale for early discontinuation of CsA by day +60 rather than the standard 
practice of discontinuation by day +100 is supported by the retrospective study by Storb 
et al. who concluded that early withdrawal of CsA was safe.
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The rationale for early discontinuation of CsA after engraftment and replacement with 
ruxolitinib is supported by the retrospective study by Zhao et al. and the prospective 
study by Zhang et al. detailed above where this practice  allowed resolution of toxicities 
due to CsA without increasing risk of GVHD.
Our hypothesis is that early discontinuation of CsA will preserve renal function, 
decrease risk of viral reactivation, thrombotic microangiopathy, veno-occlusive 
disease, and inhibition of the GVL effect. Further, patients will receive standard GVHD 
prophylaxis early after HCT with close monitoring of CsA levels early after transplant. 
Optimum CsA levels early after transplantation has been shown to be important in 
reducing the risk of GVHD.46  Replacement with ruxolitinib on day +40 will remove 
its potential for serious hematopoietic toxicity such as delayed engraftment; 
preservation of the GVL effect and activity against ALL may improve outcomes.
2.10 Rationale for PK/PD profiling 
2.10.1 Ruxolitinib profiling
Pharmacodynamics of ruxolitinib will be evaluated by inhibition of pSTAT 3 
and/or 5. Studies in healthy adult volunteers and children with solid tumors have 
shown good correlation with ruxolitinib plasma concentrations and inhibition 
of pSTAT 3 and/or 5.47, 48 However, no PK/PD data exist for children post-HCT 
and there is a need to perform the proposed studies to help define an optimal 
exposure range to prevent GVHD and minimize hematologic toxicity as well as 
correlate exposure with pSTAT 3 and/or 5 inhibition.  
2.10.2 rATG profiling
rATG has been incorporated into conditioning regimens to prevent GVHD after 
HCT.49-51 Pre-treatment with rATG in a phase III, multicenter, open-label, 
randomized controlled trial in adults undergoing HCT led to a significant 
proportion of patients having freedom from immunosuppressive treatment, 
including steroid usage, at 12 months.52 rATG is recommended for addition to 
preparative HCT regimens when using unrelated donors.52  However, the use 
of weight-based dosing may result in rATG overexposure in heavier patients in 
the setting of lymphopenia resulting in higher NRM and lower OS in adults.53 
rATG-induced T-cell depletion is unpredictable and may contribute to delayed 
immune reconstitution and increase both relapse and NRM.54 The optimum 
range of rATG exposure after HCT with a non-myeloablative conditioning 
regimen in a retrospective adult study was determined to be 60-95 AU per 
day/mL. rATG exposure after HCT was shown to be the best predictor for 5-
year OS. Below-optimum exposure increased NRM, whereas above optimum 
exposure led to higher relapse-related mortality.54 Lower rATG exposure prior 
to HCT has also been shown to be associated with inferior relapse-free survival, 
whereas lower rATG exposure after HCT was associated with increased acute 
GVHD in adults undergoing myeloablative allogeneic HCT.49 These studies 
suggest that both patient weight and absolute lymphocyte count (ALC) prior to 
rATG infusion may affect rATG PK and exposure.49,54
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An association was found between rATG exposure and CD4+ immune 
reconstitution in a multicenter retrospective PD analysis in children.50 rATG 
exposure after HCT was predictive of successful immune reconstitution at day 
+100 with exposure exceeding 100 AU per day/mL noted to have decreased 
immune reconstitution in recipients of a bone marrow or peripheral blood HCT. 
This finding is consistent with the optimal range suggested in the previously 
described retrospective adult study. Pre-transplant rATG exposure was 
associated with both acute and chronic GVHD as well as graft failure with 
improved outcomes with an exposure of at least 40 AU per day/mL.50 A 
population PK model of rATG in children receiving allogeneic HCT revealed 
that a cumulative dosing regimen of 10 mg/kg resulted in higher exposure in 
children with a higher bodyweight and/or lower ALC pre-rATG infusion, which 
was consistent with adult PK studies.51
We will prospectively study rATG PK/PD to characterize exposure in our 
patient population to correlate with transplant-related outcomes (e.g. GVHD, 
immune reconstitution, relapse, NRM) and to identify patient-specific 
parameters that affect exposure and may be used to inform individualized or 
optimized dosing in future protocols.
2.10.3 Fludarabine profiling
Fludarabine is the most frequently used agent in conditioning regimens for 
allogeneic HCT. Despite the use of body surface area-based dosing, high 
variability in fludarabine exposure has been demonstrated.55, 56 A retrospective 
analysis between fludarabine exposure and clinical outcomes was conducted in 
a study with adults and children undergoing myeloablative conditioning with a 
FluBu regimen.56 The relationship between fludarabine exposure and 2-year 
EFS was evaluated. The optimal fludarabine exposure was determined to be a 
cumulative AUC of 20 mg*h/L ± 5 in this analysis. EFS was lower in the 
overexposed group due to higher NRM associated with impaired immune 
reconstitution. The risks of graft failure were increased in the underexposed 
group. No relationship with relapse was found. This analysis suggests 
individualized fludarabine dosing based on weight and renal function or 
therapeutic drug monitoring may improve EFS.55, 56 
A prospective, multicenter study characterizing the PK/PD of fludarabine in 
children underdoing HCT with a fludarabine containing regimen was 
conducted.57 The highest OS and EFS were observed in patients with a 
cumulative exposure ranging from 15 mg*h/L to 19 mg*h/L, which is 
consistent with the previously described study.56, 57
We will prospectively study fludarabine PK/PD to characterize the exposure in 
our patient population and correlate exposure with transplant-related outcomes. 
These evaluations will help validate these relationships and help define the 
optimal exposure in children undergoing HCT as well as identify patient-
specific parameters that affect exposure and may be used to inform 
individualized or optimized dosing in future protocols. We hypothesize that 
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survival after HCT may be improved with individualized dosing of fludarabine 
in future studies. 
2.11  Risk-benefit analysis of the study
      Early withdrawal of CsA has been shown to be safe and may prevent toxicities from its 
prolonged use. Adding ruxolitinib on day +40 removes its potential for serious 
hematopoietic toxicity such as delayed engraftment. Additionally, ruxolitinib has been 
found have a good safety profile in the REACH studies and is FDA approved for 
treatment of SR aGVHD in children 12 years of age and older. Its potent activity 
in preventing GVHD may reduce risk of saGVHD. Since it does not impair the GVL 
effect and has anti-leukemic effect for some heme malignancies, its use early post-HCT 
may decrease the risk for relapse, the most important cause of death after HCT for 
hematological malignancies.
2.12 Impact of study on field of HCT
Determining the efficacy and safety of a regimen with CsA, methotrexate and 
ruxolitinib, with/without rATG as GVHD prophylaxis will pave the way for a future 
randomized controlled trial comparing this regimen with the standard regimen for 
GVHD prophylaxis using CsA and methotrexate with or without rATG.  In addition, 
the proposed  PK/PD studies for ruxolitinib, fludarabine, and rATG will have a broad 
impact on other transplant protocols. Individualized dosing may maximize efficacy and 
minimize adverse effects leading to a decrease in NRM and improved OS. Likewise, 
we expect that the gained insights from the conducted immune reconstitution studies 
will inform future transplant studies.
3.0 PARTICIPANT ELIGIBILITY CRITERIA AND STUDY 
ENROLLMENT 
According to institutional and NIH policy, the study will accession research participants 
regardless of gender and ethnic background. Institutional experience confirms broad 
representation in this regard. However, pregnant and lactating females are excluded from 
participation as the short and long-term effects of the preparative agents, cellular infusion, 
and GVHD prophylaxis on a fetus and a nursing child through breast milk are not entirely 
known at this time.
3.1
Inclusion criteria 

Diagnosis: 
o
Patients with high risk acute lymphoblastic leukemia in first remission. 
Examples include, but are not limited to, patients with certain leukemic cell 
cytogenetic findings (e.g. t(9;22) or t(4;11)); delayed response to induction 
chemotherapy; re-emergence of leukemic blasts by MRD (at any level) in 
patients previously MRD negative; persistently detectable MRD at lower 
levels; early T-cell precursor (ETP) ALL.
o
Patients with acute lymphoblastic leukemia beyond first remission. 
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o
Patients with Hodgkin’s disease beyond first remission or with refractory 
disease.
o
Patients with chronic myelogenous leukemia.
o
Patients with primary or secondary myelodysplastic syndrome.
o
Patients with Non-Hodgkin’s lymphoma beyond first remission or with 
refractory disease.
o
Patients with de novo acute myeloid leukemia in or beyond first remission 
or with relapsed or refractory disease, or myeloid sarcoma (extra-medullary 
AML).
o
Patients with secondary acute myeloid leukemia.
o
NK cell lymphoblastic leukemia in any CR.
o
Biphenotypic, bilineage, or undifferentiated leukemia.
o
Juvenile Myelomonocytic Leukemia (JMML).

Patients must have a related or unrelated donor matched at 12 of 12 HLA alleles.

Patients must have a Karnofsky/Lansky score of 70 or higher.

Patients must be 12 years of age or older.

Patients must have a shortening fraction >26% or left ventricular ejection 
fraction >40%.

Patients must have bilirubin less than or equal to 2.5 mg/dL and alanine 
aminotransferase (ALT) less than or equal to 5 times the upper limit of normal.

Patients must have creatinine clearance, or a glomerular filtration rate (GFR), 
greater than 70 mL/min/1.73m2.

Patients must be free of severe infection that upon determination of principal 
investigator precludes BMT.

Patients must have FVC >50% predicted OR, if unable to perform pulmonary 
function testing, must maintain pulse oximetry oxygen saturation >92% on 
room air.

Female patients of childbearing age must have a negative pregnancy test.

All patient with prior evidence of CNS leukemia must be treated and be in CNS 
CR.
3.2 Exclusion criteria

Patients who have undergone prior HCT.

Patients who have a peripheral blood stem cell graft source.

Patients who have a non-permissive mismatch at the DPB1 allele.

Patients who are HIV positive.

Patients positive for Hepatitis B surface antigen (HBsAg).

Patients positive for Hepatitis C.

Patients with latent tuberculosis with positive TB IFN gamma release assay.
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3.3
Research participant recruitment and screening
This study will be posted on http://clinicaltrials.gov. Potential transplant recipients are 
typically referred from their primary clinical service at St. Jude, from the Physician 
Referral Office for non-St. Jude patients, or are patients currently being treated by the 
Department of BMTCT. Each patient considered for transplantation is registered on the 
“Transplant List,” an ongoing list used to provide the clinical service with information 
regarding new patients for consultation and under consideration for transplantation and 
the status of those undergoing pre-evaluation. The patient remains on this list until a 
decision has been confirmed for acceptance or rejection based on several factors, 
primarily completion of prior therapy and/or clinical status. The proposed patient 
schedules on this form are often subject to change due to various reasons such as family 
travel issues, donor availability for procurement of progenitor cells, recipient clinical 
status, etc. This list is a confidential document, updated and maintained by the 
Department of BMTCT Coordinators.
3.4
Enrollment on study at St. Jude
A member of the study team will confirm potential participant eligibility as defined in 
Sections 3.1-3.2, and register the participant in OnCore.  A research participant-specific 
consent form and assent document (where applicable) will be generated. The entire 
signed consent/assent form(s) must be scanned into the Electronic Health Record 
(EHR) by the study team designee.
4.0 DESIGN AND METHODS
4.1
Design and study overview
This is a phase-II study with development of saGVHD within 100 days post-HCT as 
the primary endpoint. Proportion (probability) of aGVHD and saGVHD events within 
100 days post-HCT, will be estimated. GVHD will be defined using consensus criteria.
5.0 TREATMENT PLAN
5.1
Treatment
Donor Selection
Bone marrow grafts will only be used. The graft will not be T-cell depleted. The donor 
should be sufficiently healthy not to be at increased risk from the marrow harvest. 
Therefore donors will undergo evaluation for suitability and eligibility for collection in 
accordance with departmental guidelines and as outlined in 21 CFR 1271 and agency 
guidance. HLA-identical sibling donors will be considered first followed by 
histocompatible relatives or unrelated donors (matched at 12 of 12 HLA-A, B, C and 
DRB1, DRB3/4/5, and DQB1 alleles). Permissive DPB1 mismatched grafts will be 
included. Unrelated grafts will be obtained through the NMDP or other cooperative 
registries.
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Preparative Regimens
We have selected TBI/Cy as the backbone conditioning regimen for patients with 
lymphoid malignancies, and thiotepa, busulfan, and fludarabine (TBF) for patients with 
myeloid, bi-phenotypic, bilineage or undifferentiated leukemia. In addition, patients 
will receive the TBF regimen for whom TBI is not appropriate.  
TBI/Cy preparative regimen
The TBI/Cy preparative regimen is summarized in Table 1. The dose of 
cyclophosphamide will be 60 mg/kg/day for 2 days. TBI, total dose 12 Gy, will be 
delivered in 8 fractions of 150 cGy each, two fractions (fxn) per day (typically 
separated by 6 hours).  The dose rate will be <24 cGy/min. Partial pulmonary 
shielding will be used.  Males with lymphoid lineage leukemia will receive an 
additional 200 cGy x 2 (400 cGy) testicular boost concurrent with TBI.  Patients 
who receive a bone marrow product from MSD will not receive rATG. They will 
have a rest day on Day -1. 
Table 1: TBI/Cy preparative regimen
DAY
MEDICATION
DOSE
DOSE #
-7
TBI
150 cGy x 2 fxn BID (<24 cGy/min)
1 & 2 of 8
-6
TBI
150 cGy x 2 fxn BID (<24 cGy/min)
3 & 4 of 8
-5
TBI
150 cGy x 2 fxn BID (<24 cGy/min)
5 & 6 of 8
-4
TBI
150 cGy x 2 fxn BID (<24 cGy/min)
7 & 8 of 8
Cyclophosphamide
60 mg/kg/day IV
1 of 2
-3
Rabbit ATG*  
1 mg/kg/day IV
1 of 3
Cyclophosphamide
60 mg/kg/day IV
2 of 2
-2
Rabbit ATG*   
3 mg/kg/day IV   
2 of 3
-1
Rabbit ATG*   
3 mg/kg/day IV 
3 of 3
0
Bone Marrow infusion
1 of 1
*Only for MUD HCT
Thiotepa, Busulfan and Fludarabine (TBF) preparative regimen
The TBF preparative regimen is summarized in Table 2. The dose of thiotepa will 
be 5 mg/kg/day for 2 days. This will be followed by busulfan given at a dose of 3.2 
mg/kg/day and fludarabine 50 mg/m2/day for 3 days. Patients who receive a bone 
marrow product from a MSD will not receive rATG. They will have a rest day on 
Day -1. 
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 Table 2: TBF preparative regimen
DAY
MEDICATION
DOSE
DOSE #
-6
Thiotepa
5 mg/kg/day IV
1 of 2
-5
Thiotepa
5 mg/kg/day IV
2 of 2
Busulfan
3.2 mg/kg/day IV 
1 of 3
-4
Fludarabine
50 mg/m2/day IV
1 of 3
Busulfan*
3.2 mg/kg/day IV 
2 of 3
Fludarabine
50 mg/m2/day IV
2 of 3
-3
Rabbit ATG**  
1 mg/kg/day IV
1 of 3
Busulfan*
3.2 mg/kg/day IV 
3 of 3
Fludarabine
50 mg/m2/day IV
3 of 3
-2
Rabbit ATG**  
3 mg/kg/day IV
2 of 3
-1
Rabbit ATG**   
3 mg/kg/day IV 
3 of 3
0
Bone Marrow infusion
1 of 1
*Dosing of intravenous busulfan may be based on targeted levels (cumulative AUC goal 
of 75 mg*h/L ± 5).
**Only for MUD HCT  
Graft-versus-Host Disease Prophylaxis
The regimen for GVHD prophylaxis will include the combination of CsA, methotrexate 
and ruxolitinib. rATG will be included for patients receiving a MUD HCT.
Cyclosporine will be initiated on day -1 at 3 mg/kg continuous intravenous infusion 
and adjusted to maintain a goal steady state concentration within 250-350 ng/mL. Once 
stable CsA concentrations are achieved on continuous infusion, CsA may be 
transitioned to intermittent administration every 12 hours (either IV or PO) to maintain 
a goal trough concentration within 175-250 ng/mL. For patients without GVHD, CsA 
will be tapered beginning day +42 and discontinued by day +60. Patients who have 
evidence of grade II-IV acute GVHD before day +60 will not be eligible for early CsA 
wean. Most likely, these patients may also require additional immunosuppressive 
agents and steroids as outlined in our SOP 20.01 “Acute GVHD”.   
Methotrexate will be given at a dose of 10 mg/m2/dose intravenously for four doses 
given on days +1, +3, +6, and +11. Leucovorin rescue will be administered starting 
approximately 12 hours after each dose of methotrexate per institution standard.
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Ruxolitinib will be administered beginning day +40 at 5 mg twice daily. For patients 
without GVHD, ruxolitinib will be tapered beginning at approximately day +100 (+/- 
1 week) over 2 weeks. The ruxolitinib dose will be reduced by 50% to 5 mg daily for 
2 weeks, prior to discontinuation. 
If at any point prior to initiation of ruxolitinib or during ruxolitinib treatment, whether 
still on CsA or not, the patient develops evidence of grade II-IV GVHD; the patient 
will be considered off therapy (but remain on study) and receive immunosuppressive 
therapy as outlined by SOP 20.01 “Acute GVHD”.
Cellular Infusion Procedures and Monitoring
For the proper infusion procedures and monitoring of the HPC product please refer to 
BMTCT SOP 40.02 “Hematopoietic Progenitor Cell Infusion – FRESH (Allogeneic): 
IV Push and IV Drip” or SOP 40.03 “Hematopoietic Progenitor Cell Infusion – 
FROZEN: IV Push”. Please note that all relevant SOPs can be found on the BMTCT 
Clinical Transplant Program intranet page: https://home.stjude.org/bmt/Pages/policies-
transplant-program.aspx 
Importantly, during the cellular infusions, monitoring of vital signs, breath sounds, 
heart rate, pulse oximetry, and I/O will be done per the established transplant nursing 
procedure, as well as appropriate Department of BMTCT SOPs, then documented on 
the Cellular Product Infusion Record. If a reaction is suspected at any time during the 
infusion, the nurse will 1. Stop the infusion, 2. Notify the Attending Transplant 
Physician immediately, 3. NOT discard the product until physician orders are given. 
Proper documentation (symptoms of patient, vital signs, actions taken, outcome, and 
follow-up) will be completed in the Cellular Product Infusion Record.
General Treatment Related Comments

The bone marrow infusion may be delayed by approximately 24 hours in order to 
accommodate collection with the donor and/or HAL as well as the research 
participant clinical condition.

The term “day” or “daily” used in tables is an approximate term meaning that these 
medications noted will be administered approximately every 24 hours. The drug 
administration timing may be modified by approximately +/- 4 hours or as clinically 
indicated such as to accommodate surgical procedures, radiation therapy, 
administration of other necessary medications, blood product delivery, or 
procedures (such as a needed CT scan). The term “day” or “daily” refers to a general 
24-hour period.

Dosing for the medications busulfan, cyclophosphamide, fludarabine and thiotepa 
may be modified for research recipients based upon actual body weight or adjusted 
ideal body weight when clinically indicated. Dose rounding at St. Jude will be 
allowed according to institutional policy (See St. Jude Policy # 20.109, Institutional 
Policy and Procedure Manual).  

Criteria for medication calculations based on body weight/body surface area and 
other medication related information can be found in the St. Jude Formulary 
http://www.crlonline.com/crlsql/servlet/crlonlineor 
the 
St. 
Jude 
Dept 
of 
Pharmaceutical Sciences intranet website https://home.stjude.org/pharmaceutical-
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services/Pages/default.aspx. Medication doses may be rounded to the nearest 
integer or to the nearest appropriate quantity when clinically or pharmaceutically 
indicated as per the MD and PharmD. 
Quality Assurance of Cellular Products
Quality assurance for cell products is overseen by the TPQ Quality Assurance division, 
which authorizes release of all products. Only trained cell processors will process the 
cell products. A labeling and product tracking system is in place to ensure that the 
correct cells are infused into the research participant.
Assays of cell numbers and immunophenotyping will be performed both before cell 
processing and at critical stages of the process. These values will be recorded according 
to SOP of the HAL. All products will be tested for viability and sterility (culture and 
Gram stain). Culture results are not available before infusion of cell products. If the 
gram stain is positive, the research participant/parent and/or guardian will be informed 
of this event and of the risks of proceeding prior to infusion. Positive results will be 
investigated as per the variance procedures of the HAL. The IRB and FDA will be 
notified, if at any time after infusion, cell product was determined to be contaminated.
5.2
Supportive care
Antimicrobial prophylaxis: All patients are required to start anti-pneumocystis jiroveci 
pneumonia (PJP), anti-fungal and anti-viral prophylaxis post-HCT as per BMTCT 
SOP. Acyclovir prophylaxis will be given to HSV and VZV seropositive patients. 
Surveillance for Epstein Barr virus (EBV), adenovirus (ADV), and cytomegalovirus 
(CMV) and pre-emptive treatment for viral reactivation will be done as per BMTCT 
SOP.
Initiation of azole prophylaxis: Micafungin prophylaxis may be replaced with azoles 
after neutrophil engraftment for better coverage against Aspergillus spp. Azoles will be 
used after day +8 in patients with prior history of proven or probable invasive fungal 
infections.
Use of growth factors: Use of GCSF is not recommended except after day +21 when a 
single dose not exceeding 2.5 mcg/kg may be given after rounding off if the absolute 
phagocytic count (APC) is >500 cells/mm3, and ANC is <500 cells/mm3 to mobilize 
cells into the mature compartments.
Hemorrhagic cystitis prophylaxis: The medication cyclophosphamide is known to 
increase the risk of hemorrhagic cystitis.  Mesna (60 mg/kg; divided into 5 doses) will 
be administered approximately 15 minutes prior to each dose of cyclophosphamide and 
approximately 3, 6, 9, and 12 hours after each dose of cyclophosphamide. Mesna dose 
and administration schedule may vary based on physician or PharmD recommendation. 
Seizure prophylaxis: The medication busulfan is known to increase the risk of seizures.  
Levetiracetam 10 mg/kg/dose every 12 hours (max 1000 mg/dose) starting 24 hours 
prior to the first dose of busulfan and continuing until at least 48 hours after the last 
dose will be prescribed as seizure prophylaxis for the transplant recipients receiving 
busulfan.
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Pre-medication for rATG: Reactions to rATG administration are well documented and, 
in the most severe cases, include anaphylaxis. Pre-medications, including an 
antihistamine, a corticosteroid, and an antipyretic, will be given to prevent such severe 
reactions. NOTE: Patients receiving rATG on the TBF regimen will NOT be able to 
receive acetaminophen as a pre-medication due to interference with busulfan clearance.
5.3
Ruxolitinib dose modification
Concomitant CYP3A4 inhibitors/inducers: reduce ruxolitinib dose by 50% when 
administered with strong CYP3A4 inhibitors (e.g., voriconazole, posaconazole, etc.); 
use of CYP3A4 inducers should be avoided if possible.
Hematologic toxicity: 
ANC <500 cells/mm3 after day +40: reduce ruxolitinib dose by 50%*.
Hemoglobin less than 7gm% after day + 40 reduce ruxolitinib dose by 50%.
Platelet count less than 20,000 after day +40: reduce ruxolitinib dose by 50%.
Renal dysfunction: CrCl 15-59 mL/min: reduce ruxolitinib dose by 50%; Avoid use if 
CrCl < 15 mL/min and not on dialysis
Liver dysfunction: total bilirubin >3 x ULN: reduce ruxolitinib dose by 50% until 
recovery.
*A reduction in ruxolitinib dose by 50% is 5mg once daily.
Additionally, we will include stopping rules for ruxolitinib administration for the 
following reasons:   

ANC remains <500 cells/µL 2 weeks after the dose has been reduced by 50%.

Secondary graft failure due to other causes with ANC <500 cells/µL and bone 
marrow cellularity <5%

Uncontrolled infection
6.0 DRUG/DEVICE/BIOLOGIC AGENT INFORMATION 
6.1 Medications
Anti-thymocyte globulin (rabbit) (Thymoglobulin®, rabbit ATG)
Source & 
Pharmacology
Anti-thymocyte globulin is a purified, pasteurized, gamma immune 
globulin, obtained by immunization of rabbits with human thymocytes.  
This immunosuppressive product contains cytotoxic antibodies directed 
against antigens expressed on human T-lymphocytes.  The mechanism of 
action by which polyclonal anti-lymphocyte preparations suppress 
immune responses is not fully understood.  Possible mechanisms by 
which anti-thymocyte globulin may induce immunosuppression in vivo 
include: T-cell clearance from the circulation and modulation of T-cell 
activation, homing, and cytotoxic activities.  Anti-thymocyte globulin  
(rabbit) includes antibodies against T-cell markers such as CD2, CD3, 
CD4, CD8, CD11a, CD18, CD25, CD44, CD45, HLA-DR, HLA Class 1 
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heavy chains, and 2 microglobulin. T-cell depletion is usually observed 
within a day from initiating anti-thymocyte globulin therapy.  
Formulation 
and Stability
Anti-thymocyte globulin (refrigerated) is available as sterile, lyophilized 
powder to be reconstituted with sterile diluent (both lyophilized powder 
and diluent should be at room temperature before reconstitution).  
Reconstituted solutions provide a final concentration of 5 mg/mL x 5 mL.  
The product must be further diluted in normal saline prior to 
administration.  Infusion solutions may be administered over 2 to 6 hours 
depending on dose and should be prepared immediately prior to 
administration.
Supplier
Commercially available
Toxicity 
Information
Frequently reported events include fever, chills, leukopenia, pain, 
headache, abdominal pain, diarrhea, hypertension, nausea, 
thrombocytopenia, peripheral edema, dyspnea, asthenia, hyperkalemia, 
tachycardia.  The most serious toxicity is that of anaphylaxis.  The full 
dose must be administered over at least 4 hours and the patient pretreated 
with antihistamine, corticosteroid, and antipyretic.  Supportive medical 
resources must be readily available for patient management. Anaphylaxis 
precludes further administration of the drug.  
Dosage and 
Route of 
Administration
1 mg/kg test dose followed by 2 additional consecutive days of 3 mg/kg, 
(7 mg/kg total dose); intravenous.
Busulfan (Myleran, Busulfex)
Source & 
Pharmacology 
Busulfan is a polyfunctional alkylating agent. It interferes with the normal 
function of DNA by alkylating intracellular nucleophiles and cross linking 
DNA strands.   It is cell cycle phase non-specific. It is well absorbed orally 
and is metabolized by the liver.  Drugs that induce hepatic metabolism 
(e.g., phenytoin) increase clearance and those that inhibit hepatic 
metabolism (e.g., itraconazole) may decrease clearance.  The plasma half 
life is  2.5 hours in adults, but children may have higher clearances.
Formulation 
and Stability
Busulfan is available as a 2 mg tablet and as a solution for intravenous 
administration.
Supplier
Commercially available.
Toxicity 
Information
Acute dose limiting toxicity is myelosuppression including leukopenia, 
thrombocytopenia, and anemia.  This effect is delayed with a nadir of 14-
21 days.  Some patients may develop bone marrow fibrosis.  Nausea and 
vomiting are generally mild.  Other GI symptoms include diarrhea, 
anorexia and associated weight loss.  Seizures are associated with high 
doses. 
 
Other 
side 
effects 
include 
liver 
dysfunction, 
skin 
hyperpigmentation, skin rash, gynecomastia, sterility, cataracts, and 
alopecia.  Secondary cancers have occurred.  “Busulfan lung”, manifested 
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by diffuse interstitial pulmonary fibrosis, persistent cough, fever, rales, and 
dyspnea may occur, most commonly after high doses or prolonged therapy.
Dosage and 
Route of 
Administration
Based on targeted levels (cumulative AUC of 75 mg*h/L ± 5), initial dose 
at 3.2 mg/kg/day; intravenous.
Cyclophosphamide (Cytoxan)
Source & 
Pharmacology
Cyclophosphamide is a nitrogen mustard derivative.  It acts as an alkylating 
agent that causes cross-linking of DNA strands by binding with nucleic 
acids and other intracellular structures, thus interfering with the normal 
function of DNA. It is cell cycle phase non-specific. Cyclophosphamide is 
well absorbed from the GI tract with a bioavailability of >75%.  It is a 
prodrug that requires activation. It is metabolized by mixed function 
oxidases in the liver to 4-hydroxycyclo-phosphamide, which is in 
equilibrium with aldophosfamide. Aldofosfamide spontaneously splits into 
nitrogen mustard, which is considered to be the major active metabolite, 
and acrolein. In addition, 4-hydroxy-cyclophosphamide may be 
enzymatically 
metabolized 
to 
4-keto-cyclophosphamide 
and 
aldophosfamide 
may 
be 
enzymatically 
metabolized 
to 
carboxyphosphamide 
that 
is 
generally 
considered 
inactive.  
Cyclophosphamide and its metabolites are excreted mainly in the urine. 
Dose adjustments should be made in patients with a creatinine clearance of 
<25 mL/min.
Formulation 
and Stability
Cyclophosphamide is available in vials containing 100, 200, 500, 1000, and 
2000 mg of lyophilized drug and 75 mg mannitol per 100 mg of 
cyclophosphamide. Both forms of the drug can be stored at room 
temperature. The vials are reconstituted with 5, 10, 25, 50, or 100 mL of 
sterile water for injection, respectively, to yield a final concentration of 20 
mg/mL.  Reconstituted solutions may be further diluted in either 5% 
dextrose or 0.9% NaCl containing solutions. Diluted solutions are 
physically stable for 24 hours at room temperature and 6 days if 
refrigerated, but contain no preservative, so it is recommended that they be 
used within 24 hours of preparation.
Supplier
Commercially available
Toxicity 
Information
Dose limiting toxicities of cyclophosphamide include myelosuppression 
and cardiac toxicity. Cardiac toxicity is typically manifested as congestive 
heart failure, cardiac necrosis, or hemorrhagic myocarditis and can be fatal.  
Hemorrhagic cystitis may occur and necessitates withholding therapy. The 
incidence of hemorrhagic cystitis is related to cyclophosphamide dose and 
duration of therapy. Forced fluid intake and/or the administration of mesna 
decreases the incidence and severity of hemorrhagic cystitis.  Other 
toxicities reported commonly include nausea and vomiting (may be mild to 
severe 
depending 
on 
dosage), 
diarrhea, 
anorexia, 
alopecia, 
immunosuppression, and sterility.  Pulmonary fibrosis, SIADH, 
anaphylaxis, and secondary neoplasms have been reported rarely.   
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Dosage and 
Route of 
Administration
60 mg/kg for 2 consecutive days, (120 mg/kg total dose); intravenous.
Cyclosporine (Gengraf)
Source & 
Pharmacology
Cyclosporine (cyclosporin A) is a cyclosporin immunosuppressive agent 
produced as a metabolite of the fungus species Aphanocladium 
album or Beauveria nivea. The drug is a nonpolar, cyclic polypeptide 
antibiotic consisting of 11 amino acids. Cyclosporine is one of several 
biologically active antibiotics (cyclosporins) produced by these 
fungi; cyclosporin A and C are the major metabolites.
Cyclosporine occurs as a white or essentially white, finely crystalline 
powder. The drug is relatively insoluble in water, having an aqueous 
solubility of 0.04 mg/mL at 25°C, and is generally soluble in lipids and 
organic solvents, having a solubility of more than 80 mg/mL in alcohol 
at 25°C. The potency of cyclosporine is determined on the anhydrous 
basis; each mcg of cyclosporine is defined as the activity (potency) 
contained in 1.0173 mcg of the FDA’s cyclosporine master standard.
Formulation 
and Stability
Cyclosporine concentrate for injection occurs as a clear, faintly 
brownish-yellow solution. Cyclosporine concentrate for injection is a 
sterile solution of the drug in polyoxyl 35 castor oil (Cremophor® EL, 
polyethoxylated castor oil) with 32.9% (v/v) alcohol. At the time of 
manufacture, the air in the ampuls of cyclosporine concentrate for 
injection is replaced with nitrogen. The concentrate for injection 
contains no more than 42 USP endotoxin units per mL.
Cyclosporine also is commercially available as a modified, nonaqueous 
liquid formulation of the drug that immediately forms an emulsion in 
aqueous fluids; the formulation is available as an oral solution for 
emulsion and as oral 25- and 100-mg liquid-filled soft gelatin capsules 
containing the oral solution for emulsion. When exposed to an aqueous 
environment, the oral solution for emulsion forms a homogenous 
transparent emulsion with a droplet size smaller than 100 nm in 
diameter; as a result, the formulation has been referred to as an oral 
solution for microemulsion. In this formulation, the molecular structure 
of cyclosporine is unaltered, and aqueous dilution results in formation of 
an emulsion without reprecipitation of the drug. Cyclosporine is 
dispersed in a mixture of propylene glycol (hydrophilic solvent) and 
corn oil monoglycerides, diglycerides, and triglycerides (lipophilic 
solvent); when dispersed, polyoxyl 40 hydrogenated castor oil serves as 
a surfactant, and d,l-α-tocopherol is present as an antioxidant. 
Cyclosporine oral solution and liquid-filled capsules also contains 
dehydrated alcohol in a maximum concentration of 11.9% (v/v).
Supplier
Commercially available.
Toxicity 
Information
The principal adverse reactions of cyclosporine therapy are renal 
dysfunction, tremor, hirsutism, hypertension, and gum hyperplasia. 
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Hypertension, which is usually mild to moderate, may occur in 
approximately 50% of patients following renal transplantation and in 
most cardiac transplant patients. The pathologic changes resembled 
those seen in the hemolytic-uremic syndrome and included thrombosis 
of the renal microvasculature, with platelet-fibrin thrombi occluding 
glomerular capillaries and afferent arterioles, microangiopathic 
hemolytic anemia, thrombocytopenia, and decreased renal function. 
Similar findings have been observed when other immunosuppressives 
have been employed post-transplantation. Hypomagnesemia has been 
reported in some, but not all, patients exhibiting convulsions while on 
cyclosporine therapy. Although magnesium-depletion studies in normal 
subjects suggest that hypomagnesemia is associated with neurologic 
disorders, multiple factors, including hypertension, high dose 
methylprednisolone, hypocholesterolemia, and nephrotoxicity associated 
with high plasma concentrations of cyclosporine appear to be related to 
the neurological manifestations of cyclosporine toxicity.
Dosage and 
Route of 
Administration
3 mg/kg; dose will be adjusted to maintain a steady concentration 
between 250-350 ng/mL or trough concentration between 175-250 
ng/mL when transitioned to intermittent dosing.
Fludarabine (Fludara)
Source & 
Pharmacology 
Fludarabine phosphate is a synthetic purine nucleoside analog.  It acts by 
inhibiting DNA polymerase, ribonucleotide reductase, and DNA primase 
by competing with the physiologic substrate, deoxyadenosine triphosphate, 
resulting in inhibition of DNA synthesis. In addition, fludarabine can be 
incorporated into growing DNA chains as a false base, thus interfering with 
chain elongation and halting DNA synthesis.  Fludarabine is rapidly 
dephosphorylated in the blood and transported intracellularly via a carrier-
mediated process. It is then phosphorylated intracellularly by 
deoxycytidine kinase to the active triphosphate form.  Approximately 23% 
of the dose is excreted as the active metabolite in the urine (with dosages 
of 18-25 mg/m2/day for 5 days). Renal clearance appears to become more 
important at higher doses, with approximately 41-60% of the dose being 
excreted as the active metabolite in the urine with dosages of 80-260 
mg/m2.
Formulation 
and Stability
Fludarabine is supplied in single-dose vials containing 50 mg fludarabine 
as a white lyophilized powder and 50 mg of mannitol.  The intact vials 
should be stored under refrigeration. Each vial can be reconstituted by 
adding 2 mL of sterile water for injection resulting in a final concentration 
of 25 mg/mL. Because the reconstituted solution contains no antimicrobial 
preservative, the manufacturer recommends that it should be used within 8 
hours of preparation. The solution should be further diluted in 5% dextrose 
or 0.9% NaCl prior to administration.
Supplier
Commercially available.
Toxicity 
Information
The major dose-limiting toxicity of fludarabine is myelosuppression.  
Nausea and vomiting are usually mild.  Side effects reported commonly 
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include anorexia, fever and chills, alopecia, and rash.  Neurotoxicity can 
be manifested by somnolence, fatigue, peripheral neuropathy, mental 
status changes, cortical blindness, and coma and is more common at high 
doses.  Neurotoxicity is usually delayed, occurring 21-60 days after the 
completion of a course of therapy and may be irreversible.  Side effects 
reported less commonly include diarrhea, stomatitis, increased liver 
function tests, liver failure, chest pain, arrhythmias, and seizures.  
Pulmonary toxicity includes allergic pneumonitis characterized by cough, 
dyspnea, hypoxia, and pulmonary infiltrates.  Drug induced pneumonitis is 
a delayed effect, occurring 3-28 days after the administration of the third 
or later course of therapy. Administration of corticosteroids usually results 
in resolution of these symptoms.
Dosage and 
Route of 
Administration
50 mg/m2 daily for 3 consecutive days (150 mg/m2 total dose) intravenous.
Mesna (Mesnex)
Source & 
Pharmacology 
Mesna is a synthetic sulfhydryl (thiol) compound. Mesna contains free 
sulfhydryl groups that interact chemically with urotoxic metabolites of 
oxaza-phosphorine derivatives such as cyclophosphamide and ifosfamide. 
Oral bioavailability is 50%. Upon injection into the blood, mesna is 
oxidized to mesna disulfide, a totally inert compound. Following 
glomerular filtration, mesna disulfide is rapidly reduced in the renal tubules 
back to mesna, the active form of the drug.  Mesna and mesna disulfide are 
excreted primarily via the urine.
Formulation 
and Stability
Mesna is available in 2 mL, 4 mL and 100 mL amps containing 100 mg/mL 
of mesna solution. The intact vials can be stored at room temperature.  
Mesna may be further diluted in 5% dextrose or 0.9% NaCl containing 
solutions.  Diluted solutions are physically and chemically stable for at 
least 24 hours under refrigeration.  
Supplier
Commercially available
Toxicity 
Information
Mesna is generally well tolerated. Nausea and vomiting, headache, 
diarrhea, rash, transient hypotension, and allergic reactions have been 
reported.  Patients may complain of a bitter taste in their mouth during 
administration. Mesna may cause false positive urine dipstick readings for 
ketones.  
Dosage and 
Route of 
Administration
Intravenous. Mesna is dosed based on the cyclophosphamide dose and 
generally administered in fractionated doses at approximately 20% of the 
total cyclophosphamide dose.  It is generally given prior to and again at 3, 
6, 9 and 12 hours following each dose of cyclophosphamide.
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Methotrexate
Source & 
Pharmacology 
A folate analogue which reversibly inhibits dihydrofolate reductase, the 
enzyme that reduces folic acid to tetrahydrofolic acid. Inhibition of 
tetrahydrofolate formation limits the availability of one carbon fragments 
necessary for the synthesis of purines and the conversion of deoxyuridylate 
to thymidylate in the synthesis of DNA and cell reproduction. The 
polyglutamated metabolites of MTX also contribute to the cytotoxic effect 
of MTX on DNA repair and/or strand breaks. MTX cytotoxicity is highly 
dependent on the absolute drug concentration and the duration of drug 
exposure. MTX is actively transported across cell membranes. At serum 
methotrexate concentrations exceeding 0.1 μmol/mL, passive diffusion 
becomes a major means of intracellular transport of MTX. The drug is 
widely distributed throughout the body with the highest concentration in 
the kidney, liver, spleen, gallbladder, and skin. Plasma concentrations 
following high dose IV MTX decline in a biphasic manner with an initial 
half-life of 1.5-3.5 hours and a terminal half-life of 8-15 hours. About 50% 
is bound to protein. After oral administration, approximately 60% of a 30 
mg/m2 dose is rapidly absorbed from the GI tract, with peak blood levels 
at 1 hour. At doses >30 mg/m² absorption decreases significantly. Even at 
low doses, absorption may be very erratic, varying between 23% and 95%. 
The elimination of MTX from the CSF after an intrathecal dose is 
characterized by a biphasic curve with half-lives of 4.5 and 14 hours. After 
intrathecal administration of 12 mg/m², the lumbar concentration of MTX 
is ~100 times higher than in plasma (ventricular concentration is ~ 10% of 
lumbar concentration). MTX is excreted primarily by the kidneys via 
glomerular filtration and active secretion into the proximal tubules. Renal 
clearance usually equals or exceeds creatinine clearance. Small amounts 
are excreted in the feces. There is significant entero-hepatic circulation of 
MTX. The distribution of MTX into third-space fluid collections, such as 
pleural effusions and ascitic fluid, can substantially alter MTX 
pharmacokinetics. The slow release of accumulated MTX from these third 
spaces over time prolongs the terminal half-life of the drug, leading to 
potentially increased clinical toxicity.
Formulation 
and Stability
Methotrexate for Injection is available as a lyophilized powder for 
injection in 1000 mg vials. The powder for injection contains 
approximately 7 mEq sodium in the 1000 mg vial. Methotrexate for 
Injection is also available as a 25 mg/mL solution in 2, 4, 8, 10, and 40 mL 
preservative free vials and 2 and 10 mL vials with preservative. The 2, 4, 
8, 10, and 40 mL solutions contain approximately 0.43, 0.86, 1.72, 2.15, 
and 8.6 mEq sodium per vial, respectively. The preserved vials contain 
0.9% benzyl alcohol as a preservative. Sterile methotrexate powder or 
solution is stable at 20º-25ºC (68º-77ºF); excursions permitted to 15º-30ºC 
(59º- 86 Fº). Protect from light.
Supplier
Commercially available
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Toxicity 
Information
The dose limiting toxicities of methotrexate are generally bone marrow 
suppression, ulcerative stomatitis, severe diarrhea, or acute nephrotoxicity.  
Toxicities reported frequently include nausea and vomiting, diarrhea, 
anorexia, alopecia, hepatic toxicity, and alopecia.  Less common side 
effects include blurred vision, photosensitivity, anaphylaxis, headache, 
pneumonitis, skin depigmentation or hyperpigmentation, rash, vasculitis, 
and encephalopathy. During high-dose methotrexate therapy, most patients 
experience a transient decrease in GFR, but renal failure can occur, 
particularly if the patient does not receive urinary alkalinization and 
aggressive hydration  before, during, and after receiving high dose 
methotrexate.  Leucovorin rescue should be initiated within 48 hours of 
starting high-dose methotrexate and adjusted based on MTX levels to 
prevent bone marrow toxicity and mucositis.  Leucovorin may also be 
necessary after IT administration, especially if IT methotrexate therapy is 
given to patients with renal dysfunction.  Patients with Down Syndrome 
have a tendency to have delayed methotrexate clearance and a greater risk 
of toxicity, despite increased leucovorin rescue.   
Dosage and 
Route of 
Administration
10 mg/m2/dose for four doses given on days +1, +3, +6, and +11; 
intravenous. Doses may be omitted based on clinical status of patient (e.g., 
liver dysfunction, mucositis). Each case should be discussed with PI.
Ruxolitinib (Jakafi®)
Source & 
Pharmacology 
Ruxolitinib (INCB018424 phosphate, INC424, ruxolitinib phosphate) 
represents a novel, potent, and selective inhibitor of JAK1 (Janus kinase 1) 
(inhibition concentration 50% [IC50]=3.3 ± 1.2 nM) and JAK2 (IC50=2.8 
± 1.2 nM) with modest to marked selectivity against TYK2 (tyrosine kinase 
2) (IC50=19 ± 3.2 nM) and JAK3 (IC50=428 ± 243 nM), respectively.
Formulation 
and Stability
Ruxolitinib (Jakafi®) is commercially available in the US in 5, 10, 15, 20, 
and 25 mg strength tablets. The tablet contains the active ingredient and 
may include the following commonly used excipients: microcrystalline 
cellulose, lactose, stearic acid, magnesium stearate, colloidal silicone 
dioxide, sodium starch glycolate, povidone, and hydroxylpropyl cellulose. 
All excipients are of US and EuPh compendial grade.  The 5 mg (free base 
equivalent) and 25 mg (free base equivalent) tablets are packaged in HDPE 
bottles. Ruxolitinib has been shown to be stable for up to 6 months at 40°C 
and up to 24 months when stored at 25°C. Ruxolitinib may be taken either 
with food or without food. Qualified personnel, familiar with procedures 
that minimize undue exposure to themselves and the environment, should 
undertake the preparation, handling, and safe disposal of the 
chemotherapeutic agent in a self-contained and protective environment.
Supplier
Commercially available
Toxicity 
Information
The following adverse events were reported as common (occurring in at 
least 1%) or very common (occurring in at least 10%) side effects in 
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patients who were treated with ruxolitinib for myelofibrosis (MF) or 
polycythemia vera:
Very common (at least 10%): Anemia, thrombocytopenia, bruising, 
neutropenia, 
raised 
ALT 
and 
AST, 
hypercholesterolemia, 
hypertriglyceridemia, dizziness, headache, urinary tract infections, and 
weight gain, peripheral edema, hypertension, hypokalemia.
Common (more than 1% but less than 10%): Flatulence, constipation, 
herpes zoster, hypertension.
Non-melanoma skin cancers (NMSCs), including basal cell, squamous 
cell, and a rare and aggressive type of skin cancer called Merkel Cell 
Carcinoma has been reported in patients who took ruxolitinib. It is 
unknown whether this was due to ruxolitinib treatment, as many of these 
patients had been either diagnosed with non-melanoma skin cancer in the 
past or had previously been treated with hydroxyurea which is associated 
with multiple types of skin cancers.
Uncommon: (occurring in less than 1% of patients): These events were 
uncommon but have occurred in patients with MF during ruxolitinib 
treatment and are potentially serious.
Tuberculosis (TB) has occurred in a small number of patients (less than 
1%) with MF who were treated with ruxolitinib, but it is not known 
whether this was due to MF, ruxolitinib, or other factors that are known 
to increase the risk of tuberculosis (such as diabetes, bronchitis, asthma, 
smoking, emphysema, or steroid use.
About one week following interruption or discontinuation of ruxolitinib, 
some patients with MF experienced a return of symptoms (such as 
fatigue, bone pain, fever, itching, night sweats, weight loss, or an 
enlarged spleen). There have been cases of MF patients stopping 
ruxolitinib during another ongoing illness who became more severely ill, 
but it was not clear whether stopping ruxolitinib therapy contributed to 
the patients’ conditions worsening.
A rare disease called progressive multifocal leukoencephalopathy (PML) 
has been reported during ruxolitinib treatment for MF. PML comes from 
a viral infection that causes brain damage and can be fatal. It is unknown 
whether this was due to ruxolitinib treatment since PML has occurred in 
patients with blood cancers, including MF, who were not treated with 
ruxolitinib.
The effect of ruxolitinib on viral replication in patients with chronic 
hepatitis B virus is unknown.
Dosage and 
Route of 
Administration
Oral. See Treatment and Dose Modifications sections of protocol. 
Ruxolitinib tablets should be administered orally BID, approximately 
every 12 hours, continuously. Ruxolitinib may be taken with or without 
food. If a patient vomits within 30 minutes following a dose of ruxolitinib, 
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the dose should be repeated. If dose is vomited after more than 30 minutes 
following a dose of ruxolitinib, the dose will not be repeated.
For patients unable to ingest tablets, ruxolitinib can be administered 
through a nasogastric tube (8 French or greater) as follows:
Suspend one tablet in approximately 40 mL of water with stirring for 
approximately 10 minutes
Within 6 hours after the tablet has dispersed, the suspension can be 
administered through a nasogastric tube using an appropriate syringe
The tube should be rinsed with approximately 75 mL of water. The effect 
of tube feeding preparations on ruxolitinib exposure during administration 
through a nasogastric tube has not been evaluated.
Thiotepa (Triplex by Immunex) (TESPA, TSPA)
Source & 
Pharmacology 
Thiotepa is a cell-cycle nonspecific polyfunctional alkylating agent.  It 
reacts with DNA phosphate groups to produce cross-linking of DNA 
strands leading to inhibition of DNA, RNA and protein synthesis.  
Thiotepa is extensively metabolized in the liver to metabolites that retain 
activity, primarily triethylene-phosphoramide (TEPA).  The main route of 
elimination is via the urine, mainly as metabolites; the elimination half-life 
of the thiotepa is 2.5 hours, and that of TEPA is 17.6 hours.
Formulation 
and Stability
Thiotepa is supplied in single-use vials containing 15 mg of lyophilized 
thiotepa, 80 mg NaCl and 50 mg NaHCO3.  The intact vials should be 
stored under refrigeration and protected from light.  Each vial should be 
reconstituted with 1.5 mL of sterile water for injection to yield a 
concentration of 10 mg/mL. Further dilution with sterile water for injection 
to a concentration of 1 mg/mL yields an isotonic solution; if larger volumes 
are desired for intracavitary, intravenous infusion, or perfusion therapy, 
this solution may then be diluted with 5% dextrose or 0.9% NaCl 
containing solutions. The 10 mg/mL reconstituted solution is chemically 
stable when stored in the refrigerator for up to 5 days, however, it is 
recommended that solutions be prepared just prior to administration since 
they do not contain a preservative.  Reconstituted solutions should be clear 
to slightly opaque: the solutions may be filtered through a 0.22 micron 
filter to eliminate haze.
Supplier
Commercially available; manufactured by Immunex
Toxicity 
Information 
Dose limiting toxicity is myelosuppression. The leukocyte nadir may occur 
at any time from 10 to 30 days. Other toxicities include pain at the injection 
site, nausea and vomiting, anorexia, mucositis, dizziness, headache, 
amenorrhea, interference with spermatogenesis, and depigmentation with 
topical use.  Allergic reactions, including skin rash and hives, have been 
reported rarely. Rare cases of apnea, hemorrhagic cystitis, and renal failure 
have occurred. Thiotepa is mutagenic, carcinogenic, and teratogenic in 
animals. Pregnancy category D. 
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Dosage and 
Route of 
Administration
5 mg/kg daily for 2 consecutive days, (10 mg/kg total dose); intravenous.
6.2 Total Body Irradiation (TBI)
Total body Irradiation (TBI) will be used in this clinical trial as a part of a 
conditioning regimen for patients with lymphoid malignancies. 
6.2.1
Treatment Planning
Planning will be based on measurements obtained in the treatment room at the 
time of the “in room simulation”.   The following will be recorded:

Patient thickness at umbilicus

Distance from the source to the patient surface

Distance from beam central axis to umbilicus

Testicular treatment cutout size (may also be obtained during treatment) 
and testicular thickness or depth

Gantry and couch coordinates

PA film for design of pulmonary shields (shown below and with 
verification image)
CT 
6.2.2
Target Volume
The target for this treatment is the whole body including the head.  
Partial transmission lung shields are used from the posterior direction to bring 
the lung dose to approximately a mean dose of 1000 cGy.
Electron cutots for testicular irradiation (semi-custom) will be designed for 
males.   
6.2.3
Target Dose
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1200 cGy total dose delivered 150 cGy per treatment fraction delivered BID 
over 4 days with 6 MV photons. 
 Dose rate should be < 10 cGy/min in patients treated at extended SSD (450-
500 cm) in the TBI couch and will be less than 15 cGy/min in young children 
and infants treated at extended SSD (200-220 cm) on the floor.  
Intra-fraction interval should be 6 hours.
Custom posteriorly placed (PA only) partial transmission lung shields (blocks) 
will be used to reduce the average dose to the lung to approximately 
1000 cGy total dose. 
An additional 400 cGy supplemental testicular radiation delivered in 2 
fractions of 200 cGy delivered for males with lymphoid lineage leukemia.   
6.2.4
Treatment Technique
Immobilization and Position
TBI rotational couch: Patient lays prone and supine in a clock bag on the TBI 
rotational couch with alpha cradle on the posterior and anterior of patient.  
Head turned to left, arms down by sides, fingers spread out, and legs relaxed 
and straight. TBI rotational couch rotated to face beam.
TBI drop-off board on floor (patients < 100 cm tall): Patient lays prone and 
supine on TBI drop-off board, with arms away from body and by sides with 
fingers spread, legs frogged, head turned to left. Baby blanket roll under feet 
for prone position.
Beam Configuration – Open fields are delivered to the patient AP/PA by 
changing the orientation of the patient relative to the treatment beam.
Testicular treatment (males with lymphoid lineage leukemia) will be delivered 
with an en face electron field using standardized cutouts and an appropriate 
beam energy to cover the testes by 90% dose. 
6.2.5
Dose Calculation
The monitor units per treatment field are manually calculated for each patient 
using the following equation.
Where d1/2 = the mid-plane depth = separation/2, MU = monitor unit, TMR = 
tissue maximum ratio, SSD = source to surface distance, OAR = off-axis ratio, 
Sc = collimator scatter factor, Sp = patient scatter factor.
The dose rate required on the linear accelerator is manually calculated to 
achieve the prescribed dose rate.
6.2.6
Supplemental site of irradiation
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Patients with CNS involvement from leukemia, persistent choloroma, gross 
testicular involvement or other sites of disease may require supplemental 
irradiation.  Dosing and timing should be selected in consultation between the 
radiation oncologist and bone marrow transplant physician. 
7.0 REQUIRED EVALUATIONS, TESTS, AND OBSERVATIONS
7.1
Schedule of evaluations
All evaluations for these participants will be carried out as outlined in Appendix E, 
and guided by the Standard Operating Procedures (SOPs) of the St. Jude Children's 
Research Hospital, Department of BMTCT, for recipients of allogeneic stem cell 
transplantation.  Copies of these SOPs and ongoing updates can be found at the 
following site: Please note that all relevant SOPs can be found on the BMT&CT 
Clinical Transplant Program intranet page:
https://home.stjude.org/bmt/Pages/policies-transplant-program.aspx
Furthermore, to accommodate the research studies, flexibility in the date is allowed 
without a deviation from protocol.  The degree of flexibility in the timing is also 
provided in Appendix E.  Other studies, or additional routine studies may be obtained 
as needed for good clinical care.  
7.2 Evaluation for chimerism and engraftment
Evaluation for chimerism and engraftment will be performed on bone marrow or 
peripheral blood samples according to the timelines noted in Appendix E.  Bone 
marrow chimerism studies will be conducted on or about the following time points: day 
+21, day +100, and year one post-transplant. However, for research participants who 
have less than 100% donor chimerism at or about day +21 post-transplant, a repeat 
bone marrow study (to include chimerism) may be performed as indicated.  
The time to neutrophil engraftment will be recorded.  Neutrophil engraftment will be 
defined as the first of 3 consecutive days of an absolute neutrophil count (ANC) greater 
than or equal to 500/mm3. 
If there is an initial decrease in donor chimerism to less than 90% at any time on 
peripheral blood studies, a bone marrow examination will subsequently be performed 
within approximately two weeks to confirm this initial decline. In addition, chimerism 
analysis may be performed in subsets of lymphocytes, granulocytes, and monocytes for 
research participants with increasing host chimerism until the research participant 
attains 100% donor chimerism. Chimerism studies will be performed in the St. Jude 
Department of Pathology using standard techniques.
7.3 Evaluation for immune reconstitution
Performed according to the schedule outlined in Appendix E until the immune 
parameters recover to normal level or donor pattern: 
7.3.1 
Lymphocyte subsets study: Flow cytometry enumeration.
7.3.2
Quantitative immunoglobulins: IgG, IgM, and IgA levels.
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7.3.3
CNI60 Lymphocyte Research Lab, CNI60 Phenotype Research Lab and CNI60 
pSTAT 3/5 Assay Research Lab:  Correlative studies will be performed in the 
Immune Monitoring and Flow Cytometry cores of the BMTCT Department and 
through the Center of Translational Immunology and Immunotherapy (CeTI2), 
which is a joint endeavour between the BMTCT and Immunology Departments. 
Studies are focused on characterizing the reconstituted immune cells, and may 
include flow cytometry, functional assays, and cytokine/chemokine, T cell 
receptor (TCR) diversity or TREC analyses. In addition, sequencing studies 
(genetic test) will be performed to 
i)
evaluate and track immune cells, e.g. single cell RNA sequencing (sc 
RNA-seq), single cell ATAC-sequencing (sc ATAC-seq),
ii) analyze the clonal structure and specificity of immune cells, e.g. sc 
TCR-seq, single nucleotide polymorphism (SNP) analysis,
iii) analyze the immune cell methylome, e.g. whole genome bisulfite 
sequencing (WGBS), sc ATAC-seq.
All of these assays may not be performed on every specimen as a limited 
number of cells will be available. 
7.4 Minimal residual disease evaluation
Minimal residual disease (MRD) assays in BM by immunologic and molecular 
methods will be performed for those research participants who have had this test 
performed during prior therapy for their disease at St. Jude only or those for who 
samples of diseased marrow were available to identify a leukemic marker for MRD 
testing.  MRD assays may be performed more frequently in participants with increasing 
host chimerism.  Tests will be performed in the appropriate St. Jude laboratories.
7.5
Evaluations for ruxolitinib pharmacokinetics testing
Ruxolitnib PK testing will be completed in the PK Shared Resource at St. Jude. PK 
testing on ruxolitinib will be obtained on HCT recipients to evaluate drug exposure and 
correlate the exposure with transplant-related outcomes. 2 mL of peripheral blood will 
be drawn into a heparinized (dark green top) tube at the following timepoints: pre-dose 
and 0.5, 1, 2, 4, and 8 hours post-dose on day +40 and +47 and pre-dose and 2 hours 
post-dose on days +54 and +6147 (total samples = 16). NOTE: Do NOT give the next 
dose until the 8-hour sample is collected. Sample collection date may be adjusted ±2 
days as needed (such as to accommodate blood volume concerns, line access issues, 
etc.). Ideally, ruxolitinib PK samples should be obtained on the same day and same 
time as pSTAT3/5 activity if needing to adjust collection days. Additionally, leftover 
PK samples from days +54 and +61 will be utilized for pSTAT 3/5 testing (no 
additional blood draws will be required from the patient).  The pharmacokinetics of 
ruxolitinib will be evaluated using nonlinear mixed-effects modeling approaches.
Detailed instructions for collection, handling and shipping of PK samples can be found 
in Appendix G: CNI60 Data Collection, Specimen Transmittal Forms.
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7.6
Evaluations for rATG pharmacokinetics testing
rATG PK testing will be completed in the PK Shared Resource at St. Jude. PK testing 
on rabbit ATG will be obtained on HCT recipients to evaluate drug exposure and 
correlate the exposure with transplant-related outcomes. We will evaluate whether 
covariates such as weight and lymphocyte count are related to rATG exposure. 2 mL 
of peripheral blood will be drawn into a heparinized (dark green top) tube at the 
following timepoints: pre- and post- rATG infusions on day -3, -2, and -1 and on days 
0, +3, +7, +14, +21 (total samples = 11).51, 58 Sample collection date may be adjusted 
±2 days as needed (such as to accommodate blood volume concerns, line access issues, 
etc.).
Detailed instructions for collection, handling and shipping of PK samples can be found 
in Appendix G: CNI60 Data Collection, Specimen Transmittal Forms.
7.7
Evaluations for fludarabine pharmacokinetics testing
Fludarabine PK testing will be completed in the PK Shared Resource at St. Jude. PK 
testing on fludarabine will be obtained on HCT recipients to evaluate drug exposure 
and correlate the exposure with transplant-related outcomes. We will evaluate whether 
covariates such as weight and renal function are related to fludarabine exposure. 2 mL 
of peripheral blood will be drawn into a heparinized (dark green top) tube at the 
following timepoints: 0.5, 2, 7, 24 hours after fludarabine infusion on day -44 (total 
samples = 4).59 Sample collection date may be adjusted ±2 days as needed (such as to 
accommodate blood volume concerns, line access issues, etc.). These coincide with the 
peak concentration (at the end of infusion) along with samples either before or after the 
inflection in the pharmacokinetic curve. The pharmacokinetics of fludarabine will be 
evaluated using nonlinear mixed-effects modeling approaches. 
Detailed instructions for collection, handling and shipping of PK samples can be found 
in Appendix G: CNI60 Data Collection, Specimen Transmittal Forms.
7.8 Evaluation for ex-vivo target inhibition
Patient’s plasma/whole blood will be used to determine if circulating levels of 
ruxolitinib inhibit pSTAT 3 and/or 5 ex vivo. Samples will be obtained on day +40 and 
day +47 before and after dosing with ruxolitinib.  Leftover PK samples from days +54 
and +61 will be utilized for pSTAT 3/5 testing (no additional blood draws will be 
required from the patient).  This will allow us to assess the level of inhibition of JAK-
STAT pathway by ruxolitinib at these time points and allow us to trend over a 3 week 
period.
7.9
Long-term follow-up evaluations
In general, recipients of allogeneic HCT at St. Jude are seen at least annually until 10 
years post-transplant in the Department of BMTCT outpatient clinic or in the after 
completion of therapy (ACT) clinic. For the purpose of this study, research participants 
will be followed to year 1 post-transplantation..
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8.0 EVALUATION CRITERIA
8.1
Response criteria and evaluations
8.1.1 Hematologic Recovery Criteria 
Neutrophil recovery will be defined as the first of 3 consecutive tests performed 
on different days of an ANC ≥500/mm3 with evidence of donor cell engraftment.
Platelet recovery will be defined as the first of 3 consecutive tests performed on 
different days of a platelet count ≥20,000/mm3 with no platelet transfusions in 
the preceding 7 days.
8.1.2 Graft Failure Criteria
Primary graft failure will be defined as an ANC never meeting or exceeding 
500/mm3 for 3 consecutive tests performed on different days and no evidence 
of donor chimerism (<5%) by  day +30 post-HCT.
Secondary graft failure will be defined as a decline in ANC to <500/mm3 with a 
decline in donor chimerism to <5% in research participants with prior 
engraftment.
8.1.3 Relapse criteria
Relapse is defined as excess of 5% blasts in bone marrow by morphology confirmed 
by flow cytometry analysis.
8.2
Toxicity evaluation criteria
Adverse event (AE) monitoring for on-study research participants will be assessed using 
the NCI Common terminology Criteria for Adverse Events Version 5.0. The standard 
procedures for adverse event collection and monitoring are noted in Appendix D. An 
exception will be made with evaluating, staging and grading of GVHD, which will be 
evaluated using consensus criteria which are provided for both acute and chronic GVHD 
in Appendices B and C respectively.
9.0 OFF STUDY AND  OFF THERAPY CRITERIA
9.1    Off-study criteria
Recipient research participants will remain on-study until one of the following 
occurs:

Death.

Lost to follow-up, unable to be contacted and/or effectively monitored by the 
Principal Investigator (PI) and/or designees.

Noncompliance, participant misses so many appointments that the data cannot be 
used in the study.
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
Study evaluations are complete.  One year after HPC infusion (i.e. has completed 
the year +1 post-primary transplant evaluation) 

Discretion of the Study PI, including but not limited to:
o
PI decides continuing in the study would be harmful.
o
A treatment is needed that is not allowed on this study.
o
Donor unable to provide the cell product required for recipient to undergo 
the study procedure.
o
Participant’s condition gets worse or a change in health status which renders 
the study interventions medically unsafe or not in the participant’s best 
interest.
o
New information is learned that a better treatment is available, or that the 
study is not in the participant’s best interest.
9.2    Off-therapy criteria
Recipient research participants will remain on-study, but considered off-therapy, if one 
of the following occurs:

Requires conventional chemotherapy for confirmed (generally >5%) disease 
relapse: epigenetic or targeted therapy, immune therapy, and low dose 
lymphodepleting chemotherapy with DLI, even if molecularly detectable 
disease is present, would not trigger this off-therapy criterion.

Experiences graft failure/rejection, requiring non-protocol therapy.

Positive pregnancy test post cellular product infusion.

Development of unacceptable toxicity during treatment.

Grade II-IV GVHD prior to day +100 post-HCT.
10.0 SAFETY AND ADVERSE EVENT REPORTING REQUIREMENTS
10.1 Adverse events (AEs)
Adverse events will be monitored from the start of transplant conditioning throughout 
the first year post-transplant.  Participants will be instructed to report all AEs during 
the study and will be assessed for the occurrence of AEs throughout the study by the 
assigned personnel within the Department of Bone Marrow Transplantation and 
Cellular Therapy Clinical Research Office (BMTCT CRO).  
10.2 Definitions
Adverse event (AE): Any untoward medical occurrence associated in a study 
participant after the start of transplant conditioning and until the participant completes 
study interventions or meets any of the defined off-study criteria.  Adverse Events will 
be graded by the NCI CTC AE version 5.0; and will be documented and reported per 
guidance defined in Appendix D.
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Serious adverse event (SAE)  Any adverse event temporally associated with the 
subject’s participation in research that meets any of the following criteria:
results in death;
is life-threatening (places the subject at immediate risk of death from the event 
as it occurred);
requires inpatient hospitalization or prolongation of existing hospitalization;
results in a persistent or significant disability/incapacity;
results in a congenital anomaly/birth defect; or
any other adverse event that, based upon appropriate medical judgment, may 
jeopardize the subject’s health and may require medical or surgical intervention 
to prevent one of the other outcomes listed in this definition.
Unanticipated problem (UP)  An event which was not expected to occur and which 
increases the degree of risk posed to research participants.  Such events, in general, 
meet all of the following criteria:

unexpected

related or possibly related to participation in the research, and

suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was 
previously known or recognized.  An unanticipated problem involving risk to 
subjects or others may exist even when actual harm does not occur to any 
participant.
10.3 Handling of adverse events (AEs) and deaths 
Recording of Adverse Events and Serious Adverse Events:  All NCI Grade III-V 
adverse events from the start of conditioning and throughout the first year post HCT, 
regardless of their relationship to the treatment given will be collected in the study 
database. GVHD events will be recorded on an ongoing basis regardless of stage or 
grade using the criteria defined in Appendix B and C, and will not be graded according 
to NCI criteria.
Reporting Adverse Events and Serious Adverse Events:  The St. Jude PI, upon 
awareness of an event, will determine the seriousness of AEs and ensure that all related 
or possibly related AND unexpected SAEs, and all UPs are entered into the electronic 
submission system (iRIS) within 10 days.  All recorded AEs, serious or not, will be 
recorded in a log, spreadsheet, or report and submitted to the St. Jude IRB at the time 
of continuing review.  The St. Jude Regulatory Affairs Office, upon receipt, reports 
SAEs on study to the FDA within mandated regulatory timelines per 21 CRF 312 or 
812. 
Reporting of unanticipated problems:  The St. Jude PI will refer to St. Jude Human 
Research Protection Program (HRPP) Policy 01.720 for specifics on the reporting of 
unanticipated problems to the St. Jude IRB.  The St. Jude IRB reports UPs to BIMO as 
per 21 CFR 56.  The UP link follows:  https://home.stjude.org/hrpp/Policies/01-
720.pdf#search=unanticipated.
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Reporting of deaths:  When an unexpected death that is related or possibly related to 
the study treatment occurs, the PI should report it to the Director of Human Subject’s 
Protection immediately, by phone: (901) 595-4359, cell: (901) 336-2894, fax: (901) 
595-4361, or e-mail: hsp-1@stjude.org.  A reportable event entry into iRIS should 
follow within 48 hours of notification of the event.
10.4 Reporting to the CIBMTR
The Transplant Program at St. Jude is required by the federal government to report 
transplant information to the Center for International Blood and Marrow Transplant 
Research (CIBMTR). The CIBMTR is a research partnership of the International Bone 
Marrow Transplant Registry, the National Marrow Donor Program (NMDP), and the 
Foundation for the Accreditation of Cellular Therapy (FACT). This organization is 
responsible for the collection and maintenance of a standardized data warehouse 
registry of autologous and all allogeneic (related and unrelated donor) transplants 
performed in the United States.
The Office of General Counsel, U.S. Department of Health and Human Services, had 
deemed the CIBMTR not a covered entity under the Privacy Rule (45 CFR 164.512), 
45 CFR Parts 160 and 164, and the Health Insurance Portability and Accountability 
Act (HIPPA) of 1996. For this reason, the submission and disclosure of certain 
protected health information (PHI), including that required for CIBMTR, is allowable 
without the individual’s authorization (i.e. consent is waived) when such disclosure is 
made to public health authorities authorized by law for the purpose of preventing or 
controlling disease, injury, or disability.
Data resulting from this transplant procedure will be sent for general registry purposes 
to comply with the federal government requirements. This information for both donor 
and recipient is submitted using a unique participant identification number.  The 
information submitted for haploidentical recipients is less extensive than recipients of 
other donor products. For this reason, variables submitted may include but are not 
limited to the transplant recipient’s date of birth, country/state of current residence, 
diagnosis, basic lympho-hematopoietic reconstitution (e.g. date of ANC and platelet 
engraftment), post-HCT disease status, and basic AEs (e.g. GVHD- yes or no), survival 
status, date/cause of death.
11.0 DATA COLLECTION, STUDY MONITORING, AND CONFIDENTIALITY
11.1 Data collection 
Data from the participant’s source record will be entered directly into a secure study-
specific database (Trial Master). This will be completed by the assigned personnel 
within the BMTCT CRO and will be supervised by the PI. Instructions for data entry 
are outlined in the database.
The PI and designees will be responsible for the review of data for accuracy and 
completeness.  Protocol-specific data will be recorded in the electronic database within 
2 to 4 weeks of availability within the source record. All questions will be directed to 
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the PI or designee and will be reviewed at regularly scheduled working meetings 
(monthly). 
Regular summaries of toxicity and protocol events will be generated for the PI and the 
Department of Biostatistics to review.
11.2 Study Monitoring
This study is considered moderate risk for monitoring purposes. Protocol and 
regulatory compliance, including essential regulatory documentation, will be assessed 
as well as the accuracy and completeness of data points related to the primary study 
objective semi- annually. If the study design has strata, accrual will be tracked 
continuously.   The first two enrollees and then 10 % of participants will be monitored 
semi-annually. 
 
The PI and study team are responsible for protocol and regulatory compliance, and for 
data accuracy and completeness.  The study team will meet at appropriate intervals to 
review case histories or quality summaries on participants and retain copies of the 
minutes which are signed by the PI.
Clinical Trials Operations (CTO) will verify informed consent documentation and 
eligibility status for 50% of  trial participants within 5 days of enrollment. Additionally, a 
quality review will be performed by CTO personnel on 100% of St. Jude participants’ 
informed consent forms to assure completeness.  The Clinical Research Monitor 
(CRM) will verify the informed consent and eligibility processes of all non-St. Jude 
participants during routine monitoring intervals (every 6 months).   
Overall study conduct, compliance with primary objectives, age of majority consenting, 
safety assessments and reporting, and the timeliness and accuracy of database entries 
are monitored routinely.  Study documents routinely monitored on selected participants 
include medical records, database entries, study worksheets, and case report forms.  
Study documents are monitored for participant status, demographics, staging, subgroup 
assignment, treatments, investigational drug accountability, evaluations, responses, 
participant protocol status, off-study and off-therapy criteria, and for all other specifics 
as detailed in a separate study-specific monitoring plan. The frequency and intensity 
of monitoring will adapt over time, based on study progress, site compliance, accrual, 
participants on active treatment, rate of adverse events, and monitoring findings thus 
allowing refocusing on critical study risks based on findings.  Once all participants 
selected for monitoring have completed treatment and are in long term follow-up, the 
monitoring frequency may be changed to annual monitoring until a new participant 
enrolls.  Once a site is closed to accrual and participants selected for monitoring have 
completed treatment, the monitoring will be conducted annually.
   
The recording and reporting of Adverse Events, Serious Adverse Events (SAEs), and 
Unanticipated Problems (UPs) to include type, grade, attribution, duration, timeliness 
and appropriateness will be reviewed by the Monitor/ CRM. The CRM will generate a 
follow-up letter which is shared with the Principal Investigator (PI), study team and 
the Internal Monitoring Committee (IMC).
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Continuing reviews by the Institutional Review Board (IRB) and Scientific Review 
Committee (CT-SRC) will occur at least annually.  In addition, unanticipated problems 
are reviewed in a timely manner by the IRB.
11.3 Confidentiality
Unique study numbers will be used in place of an identifier such as a medical record 
number. These numbers will be used to identify any data that is released to persons or 
agencies outside of the study team.  No research participant names will be recorded on 
the data collection forms. The list containing the study number and the medical record 
number will be maintained in a password protected file that is accessible only to study 
team members. 
The medical records of study participants may be reviewed by the St. Jude IRB, FDA, 
clinical research monitors, auditors, etc.
11.4 Genomic Data Sharing
Genomic and epigenomic data may be shared through the St. Jude Cloud, the Database 
for Genotypes and Phenotypes (dbGAP) and the Gene Expression Omnibus (GEO), 
which are both run by the NIH, and the Sequence Read Archive (SRA). Prior to 
submitting data, data will be stripped of identifiers such as name, date of birth, medical 
record number, and any other information that could be used to identify participants 
and will be fully de-identified by standards consistent with the Common Rule and 
HIPAA. The genotype data will be made publicly available no later than six months 
after completion of sequencing and analysis for all patients on the study, or the date of 
initial publication, whichever comes first.
12.0 STATISTICAL CONSIDERATIONS
This is a phase II study with development of  saGVHD within 100 days post-HCT as the 
primary endpoint.  
12.1 Historical St. Jude data relating to the primary endpoint
We have been accruing 15-20 patients each year for the past 30 years on the BEAL1 
clinical practice plan for treatment of patients with hematological malignancies with 
MSD or MUD HCT using the same eligibility criteria. This experience indicates that it 
is logistically feasible to enroll 30 evaluable patients in 4 years.
A retrospective study of 255 patients with ALL and AML who underwent HCT at St. 
Jude between January 2000 through December 2015 on BEAL1 practice plan with a 
bone marrow graft was evaluated for OS. NRM and relapse rates. The treatment plan 
consisted of conditioning with TBI/Cy for patients with ALL and Bu/Cy for patients 
with AML. GVHD prophylaxis was with a calcineurin inhibitor and either 
mycophenolate mofetil or methotrexate. Patients who had an unrelated donor in 
addition received rATG.
The mean age at HCT was 9.89 years, approximately 60% were 12 years of age or 
older, approximately 38% were diagnosed with ALL and 43% with AML, 72% of 
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patients were in remission prior to HCT, 38% received a MSD graft, and 62% a MUD 
graft. Approximately 92% of patients engrafted neutrophils at 28 days, 33% had acute 
GVHD, 19% had saGVHD, and 4% had chronic GVHD. The OS was 65%, 28% 
patients relapsed, 24% patients died due to relapse, and 11% from NRM. 
12.2  Primary objective
To estimate the incidence of severe acute GVHD (saGVHD) using a prophylaxis 
regimen with no calcineurin inhibitors after day +60 post first allogeneic Human 
Leukocyte antigen (HLA)-matched sibling or unrelated donor HCT for hematological 
malignancies.
We regard any patient experiencing any competing event (except death of any non-
GVHD-cause within 100 days) that renders the patient nevaluable for GVHD, as 
unevaluable for the primary endpoint and GVHD monitoring (Table 3 below); such 
patients will be replaced. Death of any cause within 100 days will be considered as a 
failure for both the primary endpoint and monitoring. 
We regard any patient experiencing toxicity from CNI requiring replacement of CNI 
with another GVHD prophylaxis agent other than ruxolitinib as inevaluable for the 
primary endpoint, such patients will be replaced. However, patients switched to 
ruxolitinib before Day +60 will be considered evaluable.
GVHD will be defined using Children’s Oncology Group (COG) Committee consensus 
guidelines (Appendix B). Proportion (probability) of sa GVHD defined as Gr II-IV 
GVHD events within 100 days post-HCT will be estimated by sample proportions and 
exact 95% binomial-based confidence interval. Patients who are alive but drop out 
within 100 days due to events unrelated to saGVHD are considered as unevaluable and 
will be replaced. Notably from our experience, the probability of dropout during this 
period is below 1%. Historically, the saGVHD proportion is 0.19; if the proposed 
regimen reduces the saGVHD proportion to 0.10 (0.05) then with n=30 evaluable 
patients the estimation accuracy as represented by the half-length of the 95% 
confidence interval is expected to be 0.11 (0.07).  
12.3 Secondary objective
12.3.1 Estimate the cumulative incidence of relapse, NRM, chronic GVHD, and OS 
in study participants at one year post-transplant.
Cumulative incidence (probability) function of relapse, NRM, chronic GVHD 
will be estimated by the Kalbfleisch-Prentice method. For each endpoint 
(relapse, NRM, chronic GVHD) deaths and drop outs not related to the 
endpoint are treated as competing risks. For OS, the survival probability 
function will  be estimated by Kaplan-Meier estimate with standard error 
estimated according to Peto’ method.60 Subset analyses in patients with 
lymphoid and myeloid malignancies respectively will also be conducted. 
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12.4 Exploratory objectives
12.4.1 To evaluate the PK/PD profiles of ruxolitinib, fludarabine, and rATG.
PK.PD parameters estimated for each individual patient will be summarized 
across the study cohort using descriptive statistics including mean, standard 
deviation, and the five number summary (minimum, 3 quartiles, and 
maximum). Subset analyses in patients with lymphoid and myeloid 
malignancies respectively will also be conducted.
12.4.2 To assess immune reconstitution in study participants within the first year 
psot-HCT
Biomarkers of immune reconstitution will be summarized using descriptive 
statistics including mean, standard-deviation, and the five number summary 
(minimum, 3 quartiles, and maximum) or sample proportions, as appropriate 
for the data type. Subset analyses in patients with lymphoid and myeloid 
malignancies respectively will also be conducted.
12.5 Safety Monitoring
To ensure that the proposed therapy does not significantly increase saGVHD that the 
historical control rate of 19%, the following safety stoppint rule will be followed after 
the evaluation of 5, 15, 25 and 30 patients. A trial will be suspended if we observe the 
incidence of saGVHD of  ≥ 2/5, 4/15, 6/25 or 7/30. 
The stopping rule is obtained based on Bayesian toxicity monitoring developed by Lee 
J, 
Kuo 
Y-K, 
Liu 
D 
and 
Chen 
N 
at 
MD 
Anderson: 
https://biostatistics.mdanderson.org/shinyapps/BTOX/
The parameter specifications are: the maximum acceptable saGVHD rate of 19%, a 
Beta(0.5,0.5) prior distribution, and the probability threshold of 0.7 (i.e., the trial is 
suspended if the posterior probability of saGVHD rate being greater than 19% is at 
least 0.7). 
Toxicity Stopping Boundaries
Table SB1: Toxicity Stopping Boundaries
# Patients (inclusive)
# saGVHD’s considered excessive
Actions
5
≥2
Early stopping
15
≥4
Early stopping
25
≥6
Early stopping
30
≥7
Reach to Nmax
This stopping rule has reasonable operating characteristics as illustrated in the table 
and the figures below.
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Operating characteristics (stopping probabilities) at each interim
Interim
Cum.     
(n)
Suspend if cum # saGVHD 
reaches
Postulated true saGVHD rate
0.28
0.25
0.20
0.15
0.10
1
5
≥2
0.43
0.37
0.26
0.16
0.08
2
15
≥4
0.27
0.24
0.17
0.09
0.03
3
25
≥6
0.12
0.12
0.09
0.05
0.01
4
30
≥7
0.04
0.04
0.04
0.02
0.00
≤6  saGVHDs, continue to 
full accrual
0.14
0.23
0.44
0.68
0.87
12.6 Anticipated Completion Dates
Anticipated Primary Completion Date: 4 years and 6 months from start of  the study.  
Anticipated Study Completion Date: 5 years from the start of the study.
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13.0 OBTAINING INFORMED CONSENT
13.1 Informed Consent Prior to Research Interventions 
The PI or physician sub-investigator will conduct the signature authorization portion 
of the consent process. Authorization for the recipient procedure should be conducted 
in the presence of an independent witness, such as a nurse from the St. Jude Department 
of Nursing or the St. Jude Institutional Review Board Ombudsperson/Patient Advocate, 
as applicable and available to serve as a witness.
13.2 Consent at Age of Majority
The age of majority in the state of Tennessee is 18 years old.  Research participants 
must be consented at the next clinic visit after their 18th birthday.
13.3 Consent When English is Not the Primary Language
When English is not the patient, parent, or legally authorized representative’s primary 
language, the Social Work department will determine the need for an interpreter.  This 
information documented in the participant’s medical record.  Either a certified 
interpreter or the telephone interpreter’s service will be used to translate the consent 
information.  The process for obtaining an interpreter and for the appropriate use of an 
interpreter is outlined on the Interpreter Services, OHSP, and CTO websites.  Please 
refer to HRPP Policy 01.724 Informed Consent Involving Participants with Limited 
English Proficiency for more details.
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14.0 REFERENCES
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12.
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19.
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21.
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23.
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24.
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on acute and chronic GVHD in children undergoing HLA-identical sibling bone marrow 
transplantation for acute leukemia: implications for prophylaxis. Biol Blood Marrow 
Transplant 2018; 24:521-528.
25.
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Ruxolitinib as Graft-versus-Host Disease Prophylaxis for Patients after Allogeneic Stem 
Cell Transplantation. Biol Blood Marrow Transplant 2020; 26(5): e128-e133. 
26.
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STAT pathway: impact on human disease and therapeutic intervention. Annu Rev Med 
2015; 66: 311-328. e-pub ahead of print 2015/01/15; doi: 10.1146/annurev-med-051113-
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27.
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excretion, and pharmacokinetics of [14C]INCB018424, a selective Janus tyrosine kinase 
1/2 inhibitor, in humans. Drug Metab Dispos 2010; 38(11): 2023-2031. 
28.
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of a phase 3 clinical trial of inolimomab for the treatment of primary steroid refractory 
aGVHD. Blood Adv 2019; 3(2): 184-186. 
29.
Choi J, Ziga ED, Ritchey J, Collins L, Prior JL, Cooper ML et al. IFNγR signaling mediates 
alloreactive T-cell trafficking and GVHD. Blood 2012; 120(19): 4093-4103. 
30.
Spoerl S, Mathew NR, Bscheider M, Schmitt-Graeff A, Chen S, Mueller T et al. Activity 
of therapeutic JAK 1/2 blockade in graft-versus-host disease. Blood 2014; 123(24): 3832-
3842.
31.
Carniti C, Gimondi S, Vendramin A, Recordati C, Confalonieri D, Bermema A et al. 
Pharmacologic Inhibition of JAK1/JAK2 Signaling Reduces Experimental Murine Acute 
GVHD While Preserving GVT Effects. Clin Cancer Res 2015; 21(16): 3740-3749. 
32.
Zeiser R, Burchert A, Lengerke C, Verbeek M, Maas-Bauer K, Metzelder SK et al. 
Ruxolitinib in corticosteroid-refractory graft-versus-host disease after allogeneic stem cell 
transplantation: a multicenter survey. Leukemia 2015; 29(10): 2062-2068. 
33.
Jagasia M, Perales MA, Schroeder MA, Ali H, Shah NN, Chen YB et al. Ruxolitinib for 
the treatment of steroid-refractory acute GVHD (REACH1): a multicenter, open-label 
phase 2 trial. Blood 2020; 135(20): 1739-1749. 
34.
Przepiorka D, Luo L, Subramaniam S, Qiu J, Gudi R, Cunningham LC et al. FDA Approval 
Summary: Ruxolitinib for Treatment of Steroid-Refractory Acute Graft-Versus-Host 
Disease. Oncologist 2020; 25(2): e328-e334. 
35.
Zeiser R, von Bubnoff N, Butler J, Mohty M, Niederwieser D, Or R et al. Ruxolitinib for 
Glucocorticoid-Refractory Acute Graft-versus-Host Disease. N Engl J Med 2020; 382(19): 
1800-1810. 
36.
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versus-host disease. Blood Adv 2020; 4(15): 3789-3794. 
37.
Khandelwal P, Teusink-Cross A, Davies SM, Nelson AS, Dandoy CE, El-Bietar J et al. 
Ruxolitinib as Salvage Therapy in Steroid-Refractory Acute Graft-versus-Host Disease in 
Pediatric Hematopoietic Stem Cell Transplant Patients. Biol Blood Marrow Transplant 
2017; 23(7): 1122-1127. 
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38.
González Vicent M, Molina B, González de Pablo J, Castillo A, Díaz M. Ruxolitinib 
treatment for steroid refractory acute and chronic graft vs host disease in children: Clinical 
and immunological results. Am J Hematol 2019; 94(3): 319-326. 
39.
Laisne L, Neven B, Dalle JH, Galambrun C, Esvan M, Renard C et al. Ruxolitinib in 
children with steroid-refractory acute graft-versus-host disease: A retrospective 
multicenter study of the pediatric group of SFGM-TC. Pediatr Blood Cancer 2020; 67(9): 
e28233. 
40.
Moiseev IS, Morozova EV, Bykova TA, Paina OV, Smirnova AG, Dotsenko AA et al. 
Long-term outcomes of ruxolitinib therapy in steroid-refractory graft-versus-host disease 
in children and adults. Bone Marrow Transplant 2020; 55(7): 1379-1387. 
41.     Quintás-Cardama A, Vaddi K, Liu P, Manshouri T, Li J, Scherle PA et al. Preclinical 
characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications 
for the treatment of myeloproliferative neoplasms Blood 2010; 115(15):3109-3117.
42.
Zhang B, Chen L, Zhou J, Zu Y, Gui R, Li Z et al. Ruxolitinib early administration reduces 
acute GVHD after alternative don or hematopoietic stem cell transplantation in acute 
leukemia. Sci Rep 2021; 11(1): 8501.
43.
Zenatti PP, Ribeiro D, Li W, Zuurbier L, Silva MC, Paganin M et al. Oncogenic IL7R gain-
of-function mutations in childhood T-cell acute lymphoblastic leukemia. Nat Genet 2011; 
43(10): 932-939. 
44.
Senkevitch E, Li W, Hixon JA, Andrews C, Cramer SD, Pauly GT et al. Inhibiting Janus 
Kinase 1 and BCL-2 to treat T cell acute lymphoblastic leukemia with IL7-Rα mutations. 
Oncotarget 2018; 9(32): 22605-22617. 
45.
Eghtedar A, Verstovsek S, Estrov Z, Burger J, Cortes J, Bivins C et al. Phase 2 study of 
the JAK kinase inhibitor ruxolitinib in patients with refractory leukemias, including 
postmyeloproliferative neoplasm acute myeloid leukemia. Blood 2012; 119(20): 4614-
4618.
46.
Byrne JL, Stainer C, Hyde H, Miflin G, Haynes AP, Bessell EM and Russell NH. Low 
incidence of acute graft-versus-host disease and recurrent leukaemia in patients undergoing 
allogeneic haemopoietic stem cell transplantation from sibling donors with methotrexate 
and dose-monitored cyclosporin A prophylaxis. Bone Marrow Transplantation 
1998;22:541-545.
47.
Loh ML, Tasian SK, Rabin KR, Brown P, Magoon D, Reid JM et al. A phase 1 dosing 
study of ruxolitinib in children with relapsed or refractory solid tumors, leukemias, or 
myeloproliferative neoplasms: A Children's Oncology Group phase 1 consortium study 
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48.
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pharmacodynamics, and safety of orally dosed INCB018424 phosphate in healthy 
volunteers. J Clin Pharmacol 2011; 51(12): 1644-1654. 
49.
Jamani K, Dabas R, Kangarloo SB, Prokopishyn NL, Luider J, Dharmani-Khan P et al. 
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50.
Admiraal R, van Kesteren C, Jol-van der Zijde CM, Lankester AC, Bierings MB, Egberts 
TCG et al. Association between anti-thymocyte globulin exposure and CD4+ immune 
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reconstitution in paediatric haemopoietic cell transplantation: a multicentre, retrospective 
pharmacodynamic cohort analysis. The Lancet Haematology 2015; 2(5): e194-e203.
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et al. Population pharmacokinetic modeling of Thymoglobulin((R)) in children receiving 
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individualized dosing. Clin Pharmacokinet 2015; 54(4): 435-446. 
52.
Walker I, Panzarella T, Couban S, Couture F, Devins G, Elemary M et al. Pretreatment 
with anti-thymocyte globulin versus no anti-thymocyte globulin in patients with 
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donors: a randomised, controlled, open-label, phase 3, multicentre trial. The Lancet 
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53.
Scordo M, Bhatt V, Hilden P, Smith M, Thoren K, Cho C et al. Standard Antithymocyte 
Globulin Dosing Results in Poorer Outcomes in Overexposed Patients after Ex Vivo 
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Transplant 2019; 25(8): 1526-1535. 
54.
Admiraal R, Nierkens S, de Witte MA, Petersen EJ, Fleurke G-j, Verrest L et al. 
Association between anti-thymocyte globulin exposure and survival outcomes in adult 
unrelated haemopoietic cell transplantation: a retrospective, pharmacodynamic cohort 
analysis. The Lancet Haematology 2017; 4(4): e183-e191. 
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Langenhorst JB, Dorlo TPC, van Maarseveen EM, Nierkens S, Kuball J, Boelens JJ et al. 
Population Pharmacokinetics of Fludarabine in Children and Adults during Conditioning 
Prior to Allogeneic Hematopoietic Cell Transplantation. Clin Pharmacokinet 2019; 58(5): 
627-637.
56.
Langenhorst JB, van Kesteren C, van Maarseveen EM, Dorlo TPC, Nierkens S, Lindemans 
CA et al. Fludarabine exposure in the conditioning prior to allogeneic hematopoietic cell 
transplantation predicts outcomes. Blood Adv 2019; 3(14): 2179-2187. 
57.
Ivaturi V, Dvorak CC, Chan D, Liu T, Cowan MJ, Wahlstrom J et al. Pharmacokinetics 
and Model-Based Dosing to Optimize Fludarabine Therapy in Pediatric Hematopoietic 
Cell Transplant Recipients. Biol Blood Marrow Transplant 2017; 23(10): 1701-1713. 
58.
Call SK, Kasow KA, Barfield R. et al. Total and active rabbit antithymocyte globulin (r 
ATG; Thymoglobulin) pharmacokinetics in pediatric patients undergoing unrelated donor 
bone marrow transplantation. Biol Blood Marrow Transplant 2009; 15:274-278.
59.
Salinger DH, Blough DK, Vicini P et al. A limited sampling schedule to estimate individual 
pharmacokinetic parameters of fludarabine in hematopoietic cell transplant patients. Clin 
Cancer Res 2009;15:5280-5287.
60.       Peto R.1973. Experimental survival curves for interval-censored data. Applied Statistics. 
1973;22:86-91.
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APPENDIX A
KARNOFSKY PERFORMANCE STATUS SCALE
≥16 YEARS OLD
Score
General Description
100
Normal. No complaints. No evidence of disease.
90
Able to carry on normal activity. Minor signs or symptoms of disease.
80
Normal activity with effort. Some signs or symptoms of disease.
70
Care of self.  Unable to carry out normal activity or to do active work.
60
Requires occasional assistance, but is able to care for most of his needs.
50
Requires considerable assistance and frequent medical care.
40
Disabled.  Requires special care and assistance.
30
Severely disabled. Hospitalization is indicated although death is not imminent.
20
Hospitalization necessary, very sick, active support treatment necessary.
10
Moribund. Fatal processes progressing rapidly.
0
Dead.
LANSKY PERFORMANCE STATUS SCALE
<16 YEARS OLD
Score 
General Description
100
 Fully active, normal
90 
Minor restrictions in physically strenuous activity
80
Active, but tires more quickly
70
Both greater restriction of and less time spent in play activity
60
Up and around, but minimal active play; keeps busy with quieter activities
50
Gets dressed but lies around much of the day, no active play but able to participate in 
all quiet play and activities
40
Mostly in bed; participates in quiet activities
30
In bed; needs assistance even for quiet play
20
Often sleeping; play entirely limited to very passive activities
10
No play; does not get out of bed
0
Unresponsive
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APPENDIX B
COG STEM CELL COMMITTEE CONSENSUS GUIDELINES FOR ESTABLISHING 
ORGAN STAGE AND OVERALL GRADE OF ACUTE GRAFT VERSUS HOST 
DISEASE (GVHD)
Table 1 outlines standard criteria for GVHD organ staging. However, confounding clinical 
syndromes (such as non-GVHD causes of hyperbilirubinemia) may make staging GVHD in a 
given organ difficult. In addition, timing of organ specific symptoms affects whether that 
symptom is more or less likely to be true GVHD. Please refer to Tables 2 and 3 to assist you in 
deciding whether to attribute these clinical findings to GVHD, especially in situations where a 
biopsy is not possible. For additional help, please see the text which follows the tables. Table 4 
reviews the approach to assessing GVHD as acute, chronic, or the overlap between the two.
Finally, engraftment syndrome will be reported separately from the GVHD scoring presented 
below.
Engraftment Syndrome
A clinical syndrome of fever, rash, respiratory distress, and diarrhea has been described, just 
prior to engraftment in patients undergoing unrelated cord blood and mismatched 
transplantation. If, in the judgment of the treating physician, a patient experiences this 
syndrome, details of the event will be recorded in the medical record.
Modified Glucksberg Staging Criteria for Acute Graft versus Host Disease
Table 1:  Organ Staging (See tables and text below for details)
Stage
Skin
Liver (bilirubin)
Gut (stool output/day)
0
No GVHD rash
<2 mg/dL
Adult: <500 mL/day
Child: <10 mL/kg/day
1
Maculopapular rash
<25% BSA
2-3 mg/dL
Adult: 500-999 mL/day
Child: 10-19.9 mL/kg/day.
Or persistent nausea, vomiting, or 
anorexia, with a positive upper GI 
biopsy.
2
Maculopapular rash
25-50% BSA
3.1-6 mg/dL
Adult: 1000-1500 mL/day
Child: 20-30 mL/kg/day
3
Maculopapular rash
>50% BSA
6.1-15 mg/dL
Adult: >1500 mL/day
Child: >30 mL/kg/day
4
Generalized
erythroderma plus bullous 
formation and 
desquamation >5% BSA
>15 mg/dL
Severe abdominal pain with or 
without ileus, or grossly bloody stool 
(regardless of stool volume).
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Overall Clinical Grade (based on the highest stage obtained): 
Grade 0: No stage 1-4 of any organ
Grade I: Stage 1-2 skin and no liver or gut involvement
Grade II: Stage 3 skin, or Stage I liver involvement, or Stage l GI 
Grade III: Stage 0-3 skin, with Stage 2-3 liver, or Stage 2-3 GI 
Grade IV: Stage 4 skin, liver or GI involvement
Table 2 Evaluating Liver GVHD in the Absence of Biopsy Confirmation (See Table 3.0 below)
Establishing liver GVHD with no skin or GI GVHD
No Skin/GI  GVHD
Day 0-35
Assume no liver GVHD, unless proven by biopsy
No Skin/GI  GVHD 
Day 36-100
If NO other etiology identified, 
NO improvement with stopping 
hepatotoxic medications/TPN: 
Stage as liver GVHD
If other etiology identified or improves 
with stopping hepatotoxic drugs/TPN:
Do not stage as liver GVHD
Establishing liver GVHD with skin or GI GVHD and other cause of hyperbilirubinemia
Skin and/or GI GVHD 
present
Worsening bilirubin level 
(includes worsening just prior 
to onset of skin or GI tract 
GVHD) OR stable elevated 
bilirubin despite resolution of 
non-GVHD cause of increased 
bilirubin:
Stage as liver GVHD
Stable or improving bilirubin after 
diagnosis of skin or GI GVHD, 
irrespective of treatment:
Do not stage as liver GVHD
For GI staging: The “adult” stool output values should be used for patients >50 kg in weight.  Use 
3 day averages for GI staging based on stool output. If stool and urine are mixed, stool output is 
presumed to be 50% of total stool/urine mix (see 3.2 below).  For Stage 4 GI:  the term “severe 
abdominal pain” will be defined as:
a) Pain control requiring institution of opioid use, or an increase in on-going opioid use, PLUS
b) Pain that significantly impacts performance status, as determined by the treating MD.
If colon or rectal biopsy is +, but stool output is < 500 mL/day (< 10 mL/kg/day), then consider as 
GI stage 0.
There is no modification of liver staging for other causes of hyperbilirubinemia.
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Changing liver GVHD stage with other cause of hyperbilirubinemia
Skin and GI GVHD 
stable, improving, or 
absent
Liver GVHD staging is carried forward without increase in stage until 
other disease process resolves (e.g., if Thrombotic Thrombocytopenic 
Purpura (TTP) is diagnosed in the presence of stage 2 liver GVHD, the 
liver GVHD stage 2 is carried forward despite rising bilirubin level until 
TTP is resolved.  If there is no liver GVHD – stage 0 – and new onset 
TTP, the stage 0 is carried forward until TTP is resolved).
Skin and/or GI GVHD 
worsening
Liver GVHD is staged according to the Glucksberg criteria. The 
elevated bill is attributed to GVHD alone.
Thus, when skin or GI GVHD is worsening, there is no downgrading of 
liver GVHD staging for other causes of hyperbilirubinemia. (e.g., if 
TTP is diagnosed in the presence of stage 2 liver GVHD and worsening 
skin or GI GVHD, the liver is staged according to the actual bilirubin 
level even if some of the rise in bilirubin is attributed to TTP).
Similarly, even if there is no liver GVHD at onset of a new process, 
(such as TPN cholestasis), but skin or GI GVHD worsen during that 
process,  then liver GVHD is diagnosed and staged according to the 
height of the bilirubin.
There is one exception to this: the diagnosis of TTP, with high LDH 
and unconjugated bilirubin precludes the diagnosis and staging of new 
liver GVHD in the absence of a confirmatory liver biopsy.
Table 3 Evaluating GI GVHD in the Absence of Biopsy Confirmation (See Table 4.0 below)
Establishing GI GVHD with new onset diarrhea and no skin or liver GVHD
No skin/liver GVHD 
Day 0 through engraftment
Assume no GI GVHD, unless proven by biopsy
No skin/liver GVHD 
engraftment through Day 100
NO other etiology of diarrhea 
identified:
Stage as GI GVHD
Any other etiology of diarrhea 
identified:
Do not stage as GI GVHD
Establishing GI GVHD with pre-existing diarrhea and skin or liver GVHD
Skin and/or liver GVHD 
present
Worsening diarrhea (includes 
worsening just prior to onset of 
skin or liver GVHD) OR 
persistent diarrhea despite 
resolution of non-GVHD cause:
Stage as GI GVHD
Improving diarrhea after the 
diagnosis of skin or liver 
GVHD (irrespective of 
treatment) OR persistent 
diarrhea without resolution of 
underlying non-GVHD cause:
Do not stage as GI GVHD
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Category
Time of Symptoms 
after HCT or DLI
Presence of Acute 
GVHD features
Presence of Chronic 
GVHD  features
Acute GVHD
Classic acute GVHD
<100 d
Yes
No
Persistent, recurrent, 
or late-onset acute 
>100 d
Yes
No
Chronic GVHD
Classic chronic GVHD
No time limit
No
Yes
Overlap syndrome
No time limit
Yes
Yes
Differentiating Acute GVHD, Chronic GVHD, and Overlap Syndrome:
There is often confusion differentiating acute from chronic GVHD, especially in the setting of 
reduced intensity transplants, DLI and new prophylactic treatments. The NIH Working Group 
recently published new classifications for GVHD:
Table 4 Acute GVHD, Chronic GVHD, and Overlap Syndrome

Scoring of acute GVHD may need to occur past day 100.  In particular, patients should 
continue to be scored for acute GVHD when classic acute GVHD symptoms 
(maculopapular rash, nausea, vomiting, anorexia, profuse  diarrhea - particularly if 
bloody and ileus) persist past day 100 or if identical symptoms previously scored as acute 
GVHD resolve and then recur within 30 days during immunosuppression taper but past 
day 100.

Those patients being scored as having acute GVHD should NOT have diagnostic or 
distinctive signs of chronic GVHD.

Patients with both acute and chronic symptoms should be diagnosed as having 
Overlap Syndrome and scored according to their chronic GVHD score.
Further Explanation of Criteria presented in Tables 2 and 3
1.0 Assessment of Skin GVHD
1.1 Presence or Absence of Skin GVHD: Skin GVHD will be considered present if a rash 
characteristic of acute GVHD develops after allogeneic marrow transplantation involving 
more than 25% of the body surface not clearly attributable to causes such as drug 
administration or infection.  The extent of the body surface area involved can be estimated by 
the "Rule of Nines". In estimating the extent of skin GVHD, the area involved is calculated 
for individual anatomic areas, such as the arm or leg, and then the total is derived from a 
simple summation. Areas that are non-blanching should not be considered involved 
regardless of the overlying color of the rash (red, brown, etc.). Limited distribution erythema 
(with the exception of palms and soles) in the absence of associated rash elsewhere on the 
body will not be considered GVHD.
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2.0 Assessment of Liver GVHD
2.1 Assessing for the Presence or Absence of Liver GVHD
A. Hyperbilirubinemia (total bilirubin ≥2.0 mg/dL) in the absence of other signs of acute 
GVHD in the skin or GI tract:
i)
Day 0-35: If hyperbilirubinemia alone is present with no other signs of acute GVHD 
in other organ systems, acute GVHD will not be diagnosed based solely on laboratory 
abnormalities.  
Acute GVHD will be diagnosed if findings on histopathology studies of liver from a 
biopsy or autopsy are confirmatory.
ii) Day 35-100: If hyperbilirubinemia (must be conjugated bilirubin) is not improving or 
is exacerbated (especially if serum alkaline phosphatase is increased), in the absence 
of acute GVHD in other organ systems, no other etiologies are identified, and does 
not improve with discontinuation of hepatotoxic drugs, acute GVHD will be 
diagnosed.  However, it is distinctly unusual to develop ascites or a coagulopathy in 
the early stages of acute GVHD of the liver alone. In the absence of histopathology 
studies of liver from a biopsy or autopsy specimen, ascites or a coagulopathy  
secondary  to liver dysfunction  will be considered  to indicate  the  presence  of  
another  disease process  (e.g., veno-occlusive disease). Recommended  non-invasive 
studies to define an etiology for hyperbilirubinemia are:
a.
Imaging of liver (CT or ultrasound)
b. Hepatitis screen (only if ALT is elevated)
c.
PT
d. Blood cultures
e.
Review of medication list for potentially hepatotoxic drugs
f.
Review of risk factors for viral liver infection (HSV, CMV, VZV, adenovirus, 
EBV, HBV, and HCV)
g. Hemolysis screen
B. Pre-existing hyperbilirubinemia clearly attributed to an etiology other than acute GVHD 
in the presence of signs of acute GVHD in other organ systems.
i)
If pre-existing non-GVHD liver disease (documented clinically, by lab assessment, or 
by imaging studies) is stable or improving at the onset of signs of acute GVHD in 
other organs, then acute GVHD of the liver will not be considered to be present 
unless proven by liver biopsy or autopsy.
ii)
If hyperbilirubinemia worsens several days before or at the time of onset of signs of 
acute GVHD in other organ systems, GVHD will be considered to be present unless 
histopathology studies of liver are available and negative on a biopsy during that time 
interval or autopsy results exclude GVHD.
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iii)
If hyperbilirubinemia persists and is not improving after resolution of a pre-existing 
non-GVHD liver disease process (e.g., localized infection of liver, systemic sepsis, 
biliary tract obstruction) when signs of acute GVHD are present in other organ 
systems or no other intervening cause has been diagnosed, then acute GVHD will be 
considered to be present in the absence of a new, clearly identifiable cause of non-
GVHD liver disease or unless a liver biopsy or autopsy specimen is negative.
C. Prior acute GVHD in liver with new onset of a disease process that exacerbates pre-
existing or recently resolved hyperbilirubinemia:
i)
If an etiology other than acute GVHD is clearly identified as causing or exacerbating 
hyperbilirubinemia and acute liver GVHD has been diagnosed and has been stable, 
improving, or resolved, then the liver will not be restaged for acute GVHD until the 
resolution or stabilizing of the concurrent disease process (i.e., the liver stage prior to 
the onset of the new disease process will be carried forward until the new disease 
process resolves). Example: Acute GVHD of the liver and gut is diagnosed on day 20. 
Treatment of acute GVHD results in falling bilirubin levels to liver stage 1. Sepsis or 
TTP develops with transient worsening of the hyperbilirubinemia. The liver stage is 
not increased, despite a higher bilirubin level, because the cause of worsening 
hyperbilirubinemia is attributed to sepsis or TTP.
ii) If an etiology other than acute GVHD is clearly identified as causing or exacerbating 
hyperbilirubinemia in the presence of already worsening acute liver GVHD or 
GVHD of the skin or GI tract is simultaneously worsening, then the liver GVHD will 
be staged according to the actual bilirubin level, even though another cause of 
hyperbilirubinemia is present.
3.0 Assessment of GVHD of the Gastrointestinal Tract
3.1 Assessing for the Presence or Absence of GVHD of the Gastrointestinal Tract
A. Diarrhea (≥500 mL/day in adults or >10 mL/kg in pediatric patients) in the absence of 
other signs of acute GVHD in other organ systems
i)
Day 0-engraftment: If diarrhea alone is present without other signs of acute GVHD in 
other organ systems, acute GVHD will not be considered present. Diarrhea will be 
attributed to acute GVHD if histopathology studies of gastrointestinal tract from a 
biopsy or autopsy are diagnostic.
ii)
Engraftment-day 100: If diarrhea persists and is not improving, is exacerbated, or 
develops de novo in the absence of acute GVHD in other organ systems, 
histopathology studies of gut biopsies or from autopsy specimens are not available, 
and no other etiologies are clearly identified, acute GVHD will be considered to be 
the cause. A stool specimen should be examined to rule out infectious causes (e.g., 
rotavirus, adenovirus, and C. difficile toxin). It is recommended, if at all possible, that 
biopsies be obtained for diagnostic purposes.
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B. Pre-existing diarrhea clearly attributed to an etiology other than acute GVHD in the 
presence of signs of acute GVHD in other organ systems:
i)
If pre-existing diarrhea caused by a process other than GVHD has been documented 
clinically or by lab assessment and is stable or improving at the onset of signs of 
acute GVHD in the skin or liver, then acute GVHD of the intestine will not be 
considered to be present in the absence of biopsy confirmation or autopsy report.
ii)
If diarrhea or gastrointestinal symptoms are already present, but worsen significantly 
at the time of onset  of signs of acute GVHD in the skin or liver, GVHD will be 
considered present, unless biopsy or autopsy are negative.
iii)
If diarrhea persists after resolution of a pre-existing disease process with signs of 
acute GVHD present in other organ systems, GVHD will be considered present, 
unless biopsy or autopsy are negative.
C. Prior or present acute GVHD in other organ systems with new onset of diarrhea:
If diarrhea is clearly attributable to an etiology other than acute GVHD (e.g., infection) 
and a history of acute GVHD exists or acute GVHD is present in other organ systems and 
is stable, then the gastrointestinal tract will not be evaluable for acute GVHD until the 
resolution or stabilizing of the other disease process (e.g., infection) in the absence of 
biopsy or autopsy confirmation. 
D. Persistent anorexia, nausea or vomiting in the absence of signs of acute GVHD in other 
organ systems:
Persistent anorexia, nausea or vomiting in the absence of other known causes of these 
symptoms will be considered stage I acute GVHD if confirmed by endoscopic biopsy.
If a biopsy is not possible (e.g. secondary to thrombocytopenia) but the clinical findings 
are compatible with acute GVHD, then the patient will be treated and recorded as having 
acute GVHD.
3.2 Staging of the Gastrointestinal Tract for the Severity of Acute GVHD
The severity of gastrointestinal tract GVHD will be staged according to modified Glucksberg 
criteria. To minimize errors caused by large day-to-day variation, diarrhea volume is 
measured as an average over 3 days and reported as the volume in milliliters per day. When 
urinary mixing is noted the stool volume will be considered half of the total volume unless 
nursing staff is able to give a better estimate from direct observation. Abdominal cramps are 
considered significant for staging if the severity results in a clinical intervention (e.g. 
analgesia, fasting, etc.). Blood in the stools is considered significant if the blood is visible or 
hematochezia/melena is present and not clearly attributed to a cause other than GVHD (e.g., 
epistaxis/hemorrhoids).
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APPENDIX C
CRITERIA FOR GRADING CHRONIC GVHD GRADE
Score 0
Score 1
Score 2
Score 3
Performance Score:
________
KPS  ECOG  LPS
Asymptomatic 
and fully active 
(ECOG 0: KPS 
or LPS 100%)
 Symptomatic, fully 
ambulatory, restricted 
only in physically 
strenuous activity (ECOG 
1, KPS or LPS 80-90%)
Symptomatic, 
ambulatory, 
capable of self-
care, >50% of 
waking hours out 
of bed (ECOG 2, 
KPS or LPS 60-
70%)
Symptomatic limited 
self-care, >50% of 
waking hours in bed 
(ECOG 3-4, KPS or 
LPS <60%)
SKIN
Clinical features:
Maculopapular rash
Lichen 
planus-like 
features
Papuloquamous lesions 
or ichthyosis
Hyperpigmentation
Hypopigmentation
Keratosis pilaris
Erythema
Erythroderma
Poikiloderma
Sclerotic features
Pruritus
Hair involvement
Nail involvement
% 
BSA 
Involved 
_______
 No symptoms
<18% BSA with 
disease signs but NO 
sclerotic features
 19-50% BSA 
OR involvement 
with superficial 
sclerotic features 
“not hidebound” 
(able to pinch)
>50% BSA OR deep 
sclerotic features 
“hidebound” (unable to 
pinch) OR impaired 
mobility, ulceration or 
severe pruritus
MOUTH
No symptoms
 Mild symptoms with 
disease signs but NOT 
limiting oral intake 
significantly
 Moderate 
symptoms with 
disease signs 
WITH partial 
limitation of oral 
intake
 Severe symptoms 
with disease signs on 
examination WITH 
major limitation of oral 
intake
EYES
Mean tear test (mm):
>10
6-10
≥5
Not done
No symptoms
 Mild dry eye 
symptoms not affecting 
ADL (requiring 
eyedrops) ≤3 x per day) 
OR    asymptomatic signs 
of 
keratoconjunctivitissiccca
 Moderate dry 
eye symptoms 
partially affecting 
ADL (requiring 
drops ≥3x per day 
or punctual plugs), 
WITHOUT vision 
impairment
 Severe dry eye 
symptoms significantly 
affecting ADL (special 
eyeware to relieve pain ) 
OR unable to work 
because of ocular 
symptoms OR loss of 
vision cause by 
keratoconjunctivitissicca
GI Tract
No symptoms
 Symptoms such as 
dysphagia, anorexia, 
nausea, vomiting, 
abdominal pain or 
diarrhea without 
significant weight loss 
(<5%)
 Symptoms 
associated with 
mild to moderate 
weight loss) 5-
15%)
 Symptoms 
associated with 
significant weight loss 
>15%, requires 
nutritional supplement 
for most calorie needs 
OR esophageal dilation
LIVER
 Normal LFT
 Elevated Bilirubin, 
AP*, AST or ALT <2 x 
ULN
 Bilirubin >3 
mg/dl or Bilirubin, 
enzymes 2-3 x 
ULN
 Bilirubin or enzymes 
> 5 x ULN
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APPENDIX C (continued)
CRITERIA FOR GRADING CHRONIC GVHD GRADE
SCORE 0
SCORE 1
SCORE 2
SCORE 3
Lungs†
FEV1 ___________
DLCO __________
 No 
symptoms
 FEV1 >80% 
OR
LFS=2
 Mild symptoms 
(shortness of breath after 
climbing one flight of 
steps)
 FEV1 60-79% 
OR
LFS 3-5
 Moderate 
symptoms 
(shortness of breath 
after walking on flat 
ground)
 FEV1 40-59% 
OR
LFS 6-9
Severe symptoms 
(shortness of breath 
at rest; requiring 02
 FEV1 ≥39% OR
LFS 10-12
JOINTS AND 
FASCIA
 No 
symptoms
 Mild tightness of 
arms or legs, normal or 
mild decreased range of 
motion (ROM) AND not 
affecting ADL
 Tightness of 
arms or legs OR 
joint contractures, 
erythema thought 
due to fasciitis, 
moderate decrease 
ROM AND mild to 
moderate limitation 
of ADL
Contractures 
WITH significant 
decrease or ROM 
AND significant 
limitation of ADL 
(unable to tie shoes, 
button shirts, dress 
self etc.)
GENITAL TRACT
 No 
symptoms
 Symptomatic with 
mild signs on exam AND 
no effect on coitus and 
minimal discomfort with 
gynecologic exam
 Symptomatic 
with moderate signs 
on exam AND with 
mild dyspareunia or 
discomfort with 
gynecologic exam
 Symptomatic 
WITH advance 
signs (stricture, 
labial agglutination 
or severe ulcerations 
AND severe pain 
with coitus or 
inability it insert 
vaginal speculum
Other indicators, clinical manifestations or complications related to chronic GVHD (check all that apply and assign a 
score to its severity (0-3) based on its functional impact where applicable (none = 0, mild = 1, moderate =2, severe = 3.
Esophageal stricture or web ____
Pericarial Effusion  ____
Pleural Effusion(s) ____
Ascites (serositis) ____
Nephrotic syndrome ____
Peripheral Neuropathy ____
Myasthenia Gravis ____
Cardiomyopathy ____
Eosinophilia > 500/µl ____                        
Polymyositis  ____    Cardiac conduction defects ____         Coronary artery involvement ____
Platelets <100,000/µl ____                     Progressive Onset  ____
Other: Specify:      ________________________________________________________________________________
Organ scoring of chronic GVHD.
* AP may be elevated in growing children, and not reflective of liver dysfunction.   
† Pulmonary scoring should be performed using both the symptom and pulmonary function testing (PFT) scale whenever 
possible. When discrepancy exists between pulmonary symptoms or PFT scores, the higher value should be used for final 
scoring. Scoring using the Lung Function Score (LFS) is preferred, but if DLCO is not available, grading using FEV1 should be 
used.  The LFS is a global assessment of lung function after the diagnosis of bronchiolitis obliterans has already been established. 
The percent predicted FEV1 and DLCO (adjusted for hematocrit but not alveolar volume) should be converted to a numeric score 
as follows: >80% = 1; 70-79% = 2; 60-69% = 3; 50-59% = 4; 40-49% = 5; <40% = 6.  The LFS = FEV1 score + DLCO score, 
with a possible range of 2-12.  GVHD indicates graft versus host disease, ECOG, Eastern Cooperative Oncology Group, KPS, 
Karnofsky Performance Status; LPS, Lansky Performance Status; BSA, body surface area; ADL, activities of daily living; LFTs, 
liver function tests; AP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ULN, upper 
limit of normal.
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APPENDIX C (continued)
CRITERIA FOR GRADING CHRONIC GVHD GRADE
GLOBAL GRADING OF CHRONIC GVHD84
*Determined by adding the total number of organs receiving score > 0 using Figure 1, 
Appendix C.
**Defined as the maximum score given to any organ system amongst all organs scored using 
Figure 1, Appendix C. 
Final Grade
Number of 
Organs/Sites*
Maximum Organ 
Score**
Lung Score
Mild
1 - 2
1
0
3 or more
1
Moderate
At least 1
2
1
Severe
At least 1
3
2 - 3
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APPENDIX D
CRITERIA FOR ADVERSE EVENT (AE) EVALUATION AND REPORTING
The St. Jude Department of BMTCT Clinical Research Office standard operating procedure for 
the documenting and reporting of adverse (SOP 10 Documenting and Reporting of Adverse Events 
https://home.stjude.org/bmt/Policies/10.pdf) will provide guidance on the evaluation, collection 
and reporting of adverse events for this clinical trial. The current version of this document, as well 
as 
ongoing 
updates, 
can 
be 
located 
at 
the 
following 
website: 
http://home.stjude.org/bmt/Pages/policies-research.aspx
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APPENDIX E
Recommended routine clinical testing and evaluation schedule for Transplant Recipient
STANDARD OF CARE 
STUDIES
SAMPLE
VOLUME
PRE
MONTH 1
MONTH 2
MONTH 3
MONTH 6
MONTH 12
Pregnancy Test
PB
2 mL
X
As clinically indicated
Physical Exam
N/A
N/A
X
Weekly
X
X
GVHD Assessment
N/A
N/A
Weekly
CBC with diff.
PB
0.5-2 mL
X
Daily until engrafted, then weekly
X
X
Chemistry
PB
0.25-2 mL
X
Weekly
X
X
Lipid Panel
PB
1-2mL
X
Viral surveillance
(BMTPCR)
PB
4 mL
X
Weekly
As clinically indicated
PB
1-2 mL
Weekly upon engraftment
X
X
Chimerism
BM
2 mL
X
X
X
Disease Status 
Evaluation
N/A
N/A
X
X
X
X
MRD Bone Marrow      
BM
3 mL
X
X
X
X
Lymphocyte Subset 
Study
PB
2.5-4 mL
X
X
X
X
X
X
Quantitative 
Immunoglobulins
PB
2 mL
X
X
X
Total CLINICAL blood sample volumes per time 
point
19 mL
17 mL
12 mL
17 mL
10 mL
19 mL

The above-indicated follow-up regimen for these evaluations is guided by the SOPs of the Department of BMTCT, for recipients of allogeneic stem cell transplantation. As 
these evaluations are considered standard clinical care (non-research), variations in frequency (more or less frequent) of these evaluations can occur due to the participant’s 
current clinical condition and will not be noted as protocol deviations.

Disease status evaluations/BM testing results obtained prior to enrollment may be used for the baseline/pre-infusion assessments.

Lymphocyte subset studies may be omitted without variance when the absolute lymphocyte count (ALC) is zero.

In the event of graft failure/rejection, bone marrows, chimerisms and several applicable immune studies would no longer be clinically indicated and these studies may be held.
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APPENDIX E (continued)
Immune reconstitution testing and evaluation schedule for Transplant Recipient
RESEARCH 
STUDIES
SAMPLE
VOLUME
CONTAINER
PRE
DAY +40
DAY +47
MONTH 3
MONTH 6
MONTH 12
CNI60 Lymphocyte 
Research Lab
PB
15 mL
Yellow ACD
X
X
X
X
CNI60 Phenotype 
Research Lab
PB
5 mL
Yellow ACD
X
X
X
X
CNI60 pSTAT 3/5 
Assay Research Lab
PB
5 mL
Yellow ACD
0800 (pre 
Ruxolitinib) x1
1200 (4H post 
Ruxolitinib) x1
0800 (pre 
Ruxolitinib) x1
1200 (4H post 
Ruxolitinib) x1
Total RESEARCH blood sample volumes per time point
20 mL
10 mL
10 mL
20 mL
20 mL
20 mL

CNI60 Lymphocyte Research Lab, CNI60 Phenotype Research Lab and CNI60 pSTAT 3/5 Assay Research Lab results will be maintained in the Immune Monitoring Core 
database.

For these research studies, the posted volumes are the minimum volumes required to perform the respective protocol evaluations, unless otherwise specified.
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APPENDIX E (continued)
Proposed PK sampling schedule calendar for TBI/Cy regimen
Drug
Time
Sample
Volume
Day -3
Day -2
Day -1
Day 0
Day 
+3
Day 
+7
Day 
+14
Day 
+21
Day 
+40
Day 
+47
Day 
+54
Day 
+61
rATG
(test dose)
0800-1000
0800 
(pre)
1000 
(post)
rATG
(full dose)
0800-1400
2 mL
(1 mL min)
0800 
(pre)
1400 
(post)
0800 
(pre)
1400 
(post)
0800
0800
0800
0800
0800
Ruxolitinib
0800/2000
Dark 
Green 
(sodium 
heparin)
2 mL
(1 mL min)
0800 
(pre)
0830
0900
1000
1200
1600
0800 
(pre)
0830
0900
1000
1200
1600
0800
1000
0800
1000
MSD (no rATG)
12 
mL
12 
mL
4 mL
4 mL
Total PK Sample 
Volume/day
MUD (rATG)
4 mL
4 mL
4 mL
2 mL
2 mL
2 mL
2 mL
2 mL
12 
mL
12 
mL
4 mL
4 mL
*Minimal volumes are posted in parentheses when applicable.

Leftover PK samples from days +54 and +61 will be utilized for pSTAT 3/5 testing (no additional blood draws will be required from the 
patient).
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APPENDIX E (continued)
Proposed PK sampling schedule calendar for TBF regimen
Drug
Time
Sample
Volume
Day -4
Day -3
Day -
2
Day -
1
Day 
0
Day 
+3
Day 
+7
Day 
+14
Day 
+21
Day 
+40
Day 
+47
Day 
+54
Day 
+61
Busulfan
0200-
0500
2 mL
(1 mL 
min)
0500 
(EOI)
0600
0800
1000
0500 
(EOI)
0600
0800
1000
Fludarabine
0530-
0600
2 mL
0600 
(EOI)
0800
1300
0530 
(24 hr)
rATG
(test dose)
0800-
1000
0800 
(pre)
1000 
(post)
rATG
(full dose)
0800-
1400
2 mL
(1 mL 
min)
0800 
(pre)
1400 
(post)
0800 
(pre)
1400 
(post)
0800
0800
0800
0800
0800
Ruxolitinib
0800-
2000
Dark 
Green 
(sodium 
heparin)
2 mL
(1 mL 
min)
0800 
(pre)
0830
0900
1000
1200
1600
0800 
(pre)
0830
0900
1000
1200
1600
0800
1000
0800
1000
Busulfan
8 mL
8 mL
MSD (no rATG)
6 mL
2 mL
12 
mL
12 
mL
4 
mL
4 
mL
Total PK Sample 
Volume/day
MUD (rATG)
6 mL
6 mL 
4 mL
4 mL
2 mL
2 mL
2 mL
2 mL
2 mL
12 
mL
12 
mL
4 
mL
4 
mL
*Minimal volumes are posted in parentheses when applicable.
St. Jude Children's Research Hospital
IRB NUMBER: 22-1023
IRB APPROVAL DATE: 09/22/2023

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
Leftover PK samples from days +54 and +61 will be utilized for pSTAT 3/5 testing (no additional blood draws will be required from the 
patient).
St. Jude Children's Research Hospital
IRB NUMBER: 22-1023
IRB APPROVAL DATE: 09/22/2023

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APPENDIX E (continued)
Research Study Evaluation Target Windows
Several laboratory tests can only be processed on weekdays; therefore, if the scheduled evaluation 
falls on a weekend, or during a holiday period, an adjustment in the follow-up visit is expected and 
would not be noted as a protocol variation. Additionally, in order to accommodate such logistical 
constraints, evaluation/collection dates of all protocol assessments (unless otherwise specified), 
may be performed within a reasonable window of the intended date following the guidelines 
provided in the table below:
If the Planned Evaluation 
Time Point is:
Window
Weekly
+3 Days
Month 1
Week 2 to Week 6
Month 2
Week 7 to Week 11
Month 3
Week 12 to Month 4
Month 6
Month 5 to Month 7
Month 9
Month 8 to Month 10
Month 12
Month 10 to Month 14
St. Jude Children's Research Hospital
IRB NUMBER: 22-1023
IRB APPROVAL DATE: 09/22/2023

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Protocol document date: 07/27/2023
APPENDIX F
The St. Jude Department of BMTCT Clinical SOPs for standard of care for all allogeneic stem cell 
infusion recipients and stem cell donors will provide guidance on the evaluation, ongoing clinical 
care and follow up for this clinical trial. The current versions of these SOPs, as well as ongoing 
updates, of these documents can be located at the following website: http://home.web.stjude.org 
/bone_marrow/clinicalHome.shtml.   
 
St. Jude Children's Research Hospital
IRB NUMBER: 22-1023
IRB APPROVAL DATE: 09/22/2023

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Revision 0.3 dated: 07/27/2023 
Protocol document date: 07/27/2023
APPENDIX G
Pharmacokinetic Data Collection & Specimen Transmittal Forms
CNI60: Ruxolitinib Pharmacokinetic Data Collection & Specimen Transmittal Form
Complete collection and dosing data at time of administration. Email completed forms to 
claire.mills@stjude.org  and PKNurses@stjude.org. For questions, call PK Nurses at 901-595-2482.
Patient Name:
SJ MRN:
Hospital/Clinic:
DOB:
Contact Name:
Contact Phone:
Contact Email:
Subject BSA (m2): _________ Ruxolitinib Dose (mg/m2): _________ Ruxolitinib Actual Dose mg): 
__________
Collection and Processing of Peripheral Blood Samples:  
Whole blood (2 mL) will be drawn into a green top sodium heparin tube. Centrifuge samples and place 
plasma only into two 2ml screw top tubes.  Samples should be frozen at -80°C; overnight refrigeration 
prior to centrifugation is acceptable for late collections. 
Ruxolitinib Day +40 (+2 days) PK: 
Date:  _____________   Ruxolitinib Dose Administration Time: _____________
Protocol Time Point
Date Sample Collected
Time Sample Collected
Pre
30 min (+/- 15 min)
1 hr (+/- 15 min)
2 hr (+/- 15 min)
4 hr (+/- 15 min)
8 hr (+/- 15 min)
Ruxolitinib Day +47 (+/- 2 days) PK:
Date:  _____________   Dose Administration Time: _____________
Protocol Time Point
Date Sample Collected
Time Sample Collected
Pre
30 min (+/- 15 min)
1 hr (+/- 15 min)
2 hr (+/- 15 min)
4 hr (+/- 15 min)
8 hr (+/- 15 min)
St. Jude Children's Research Hospital
IRB NUMBER: 22-1023
IRB APPROVAL DATE: 09/22/2023

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Protocol document date: 07/27/2023
Ruxolitinib Day +54 (+/- 2 days) PK: 
Date:  _____________   Ruxolitinib Dose Administration Time: _____________
Protocol Time Point
Date Sample Collected
Time Sample Collected
Pre
2 hr  (+/- 15 min)
Ruxolitinib Day +61 (+/- 2 days) PK:
Date:  _____________   Dose Administration Time: _____________
Protocol Time Point
Date Sample Collected
Time Sample Collected
Pre
2 hr  (+/- 15 min)
CNI60: rATG Pharmacokinetic Data Collection & Specimen Transmittal Form
St. Jude Children's Research Hospital
IRB NUMBER: 22-1023
IRB APPROVAL DATE: 09/22/2023

CNI60
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Revision 0.3 dated: 07/27/2023 
Protocol document date: 07/27/2023
Complete collection and dosing data at time of administration. Email completed forms to 
claire.mills@stjude.org  and PKNurses@stjude.org. For questions, call PK Nurses at 901-595-2482.
Patient Name:
SJ MRN:
Hospital/Clinic:
DOB:
Contact Name:
Contact Phone:
Contact Email:
Subject BSA (m2): _______________
 
Collection and Processing of Peripheral Blood Samples:  
Whole blood (2 mL) will be drawn into a green top sodium heparin tube. Centrifuge samples at 2000 
rpm for 10 minutes at 4Co within 4 hours of collection. Place plasma only into two 2ml screw top tubes. 
Samples should be frozen at -80°C within 30 minutes of separation. 
Dose Day
rATG Dose 
(mg/kg IV)
rATG Actual 
Dose (mg)
Infusion Start 
Time
Infusion Stop 
Time
Date Sample 
Collected
Time Sample Collected
Day -3
Pre:
Pre (- 15 min):
EOI:
EOI (+ 15 min):
Day -2
Pre:
Pre (- 15 min):
EOI:
EOI (+ 15 min):
Day -1
Pre:
Pre (- 15 min):
EOI:
EOI (+ 15 min):
Day 0
Pre:
Pre (- 15 min):
EOI:
EOI (+ 15 min):
Day +3
(+/- 2 days)
Pre:
Pre (- 15 min):
EOI:
EOI (+ 15 min):
Day +7
(+/- 2 days)
Pre:
Pre (- 15 min):
EOI:
EOI (+ 15 min):
Day +14
(+/- 2 days)
Pre:
Pre (- 15 min):
EOI:
EOI (+ 15 min):
Day +21
(+/- 2 days)
Pre:
Pre (- 15 min):
EOI:
EOI (+ 15 min):
CNI60: Fludarabine Day 1 Pharmacokinetic Data Collection & Specimen Transmittal Form
St. Jude Children's Research Hospital
IRB NUMBER: 22-1023
IRB APPROVAL DATE: 09/22/2023

CNI60
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Revision 0.3 dated: 07/27/2023 
Protocol document date: 07/27/2023
Complete collection and dosing data at time of administration. Email completed forms to 
claire.mills@stjude.org  and PKNurses@stjude.org. For questions, call PK Nurses at 901-595-2482.
Patient Name:
SJ MRN:
Hospital/Clinic:
DOB:
Contact Name:
Contact Phone:
Contact Email:
Subject BSA (m2): __________ Fludarabine Dose (mg/m2/day IV): __________ Fludarabine Actual 
Dose (mg): _________
Collection and Processing of Peripheral Blood Samples:  
Whole blood (2 mL) will be drawn into a green top sodium heparin tube. Centrifuge samples and place 
plasma only into two 2ml screw top tubes. Samples should be frozen at -80°C within 30 minutes of 
collection; however, overnight refrigeration prior to centrifugation is acceptable for late collections. 
Fludarabine Day -4 (-2 days) PK:
Date:  _____________   
Fludarabine Infusion Start Time: _____________ Fludarabine Infusion Stop Time: _____________ 
Protocol Time Point
Date Sample Collected
Time Sample Collected
30 min (+/- 5 min):
2 hr (+/- 15 min):
7 hr (+/- 15 min):
24 hr (+/- 15 min):
**PRIOR to start of infusion
St. Jude Children's Research Hospital
IRB NUMBER: 22-1023
IRB APPROVAL DATE: 09/22/2023