Clinical trial NCT00919503
Conditions: Non-Neoplastic Hematologic and Lymphocytic Disorder
Keywords: nonmalignant diseases nonmalignant inherited disorders primary immunodeficiency diseases primary immune deficiency disorders chronic granulomatous disease IPEX syndrome hemophagocytic lymphohistiocytosis Wiskott Aldrich Syndrome bone marrow failure syndromes Shwachman Diamond Syndrome Dyskeratosis Congenita Diamond Blackfan Anemia inborn errors of metabolism metabolic diseases hemoglobinopathies sickle cell disease thalassemia reduced intensity transplantation hematopoietic cell transplantation bone marrow transplantation umbilical cord blood transplantation
Sponsor: Fred Hutchinson Cancer Center
Start date: 2009-07-31; Completion date: 2020-06-10
Enrollment: 98 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This phase II clinical trial studies how well treosulfan and fludarabine phosphate with or without low dose radiation before donor stem cell transplantation works in treating patients with nonmalignant (noncancerous) diseases. Hematopoietic cell transplantation has been shown to be curative for many patients with nonmalignant (noncancerous) diseases such as primary immunodeficiency disorders, bone marrow failure syndromes, hemoglobinopathies, and inborn errors of metabolism (metabolic disorders). Powerful chemotherapy drugs and/or radiation are often used to condition the patient before infusion of the new healthy donor cells. The purpose of the conditioning therapy is to destroy the patient's abnormal bone marrow which doesn't work properly in order to make way for the new healthy donor cells which functions normally. Although effective in curing the patient's disease, many hematopoietic cell transplantation regimens use intensive chemotherapy and/or radiation which can be quite toxic, have significant side effects, and can potentially be life-threatening. Investigators are investigating whether a new conditioning regimen that uses less intensive drugs (treosulfan and fludarabine phosphate) with or without low dose radiation results in new blood-forming cells (engraftment) of the new donor cells without increased toxicities in patients with nonmalignant (noncancerous) diseases.

Detailed description:
OUTLINE:

CONDITIONING REGIMEN: Patients receive treosulfan intravenously (IV) over 2 hours on days -6 to -4 and fludarabine phosphate IV over 1 hour on days -6 to -2. Patients receive anti-thymocyte globulin IV over 4-6 hours on days -4 to -2. Patients undergoing umbilical cord blood transplantation will also receive low dose total-body irradiation on day -1.

TRANSPLANTATION: Patients receive either bone marrow, peripheral blood stem cells (PBSC), or umbilical cord blood (UCB) from the donor on day 0. The use of either bone marrow, PBSC, or umbilical cord blood will depend on the donor status.

IMMUNOSUPPRESSION: Patients receive a combination of immunosuppressive medications to try and prevent graft-versus-host disease. There are 2 regimens depending on the donor.

Regimen A: Patients undergoing bone marrow or PBSC transplantation receive tacrolimus daily from day -1 to 50 followed by a taper until day 180 in the absence of GVHD. Patients also receive methotrexate IV on days 1, 3, 6, and 11.

Regimen B: Patients undergoing UCB transplantation receive cyclosporine on days -3 to 100 followed by a taper until day 180 in the absence of GVHD. Patients also receive mycophenolate mofetil on days 0 to 40 followed by a taper until day 96 in the absence of GVHD.

After completion of study treatment, patients are followed up periodically for 5 years.

Arms: arm 1: CONDITIONING REGIMEN A : Patients receive treosulfan IV on days -6 to -4 and fludarabine phosphate IV on days -6 to -2. Patients receive anti-thymocyte globulin IV on days -4 to -2. Patients undergoing umbilical cord blood transplantation will also receive low dose total-body irradiation on day -1.

TRANSPLANTATION: Patients receive either bone marrow, peripheral blood stem cells (PBSC), or umbilical cord blood from the donor on day 0. The use of either bone marrow, PBSC, or umbilical cord blood will depend on the donor status.

Patients undergoing bone marrow or PBSC transplantation receive tacrolimus IV continuously or PO twice daily on days -1 to 50 followed by a taper until day 180 in the absence of GVHD. Patients also receive methotrexate IV on days 1, 3, 6, and 11. arm 2: CONDITIONING REGIMEN B: Patients receive treosulfan IV on days -6 to -4 and fludarabine phosphate IV on days -6 to -2. Patients receive anti-thymocyte globulin IV on days -4 to -2. Patients undergoing umbilical cord blood transplantation will also receive low dose total-body irradiation on day -1 .

TRANSPLANTATION: Patients receive either bone marrow, peripheral blood stem cells (PBSC), or umbilical cord blood from the donor on day 0. The use of either bone marrow, PBSC, or umbilical cord blood will depend on the donor status.

Patients undergoing UCB transplantation receive cyclosporine IV over 1 hour every 8-12 hours on days -3 to 100 followed by a taper until day 180 in the absence of GVHD. Patients also receive mycophenolate mofetil IV or PO every 8 hours on days 0 to 40 followed by a taper until day 96 in the absence of GVHD.
Interventions: intervention 1: Allogeneic Bone Marrow Transplantation intervention 2: Anti-Thymocyte Globulin intervention 3: Cyclosporine intervention 4: Fludarabine Phosphate intervention 5: Laboratory Biomarker Analysis intervention 6: Methotrexate intervention 7: Mycophenolate Mofetil intervention 8: Peripheral Blood Stem Cell Transplantation intervention 9: Tacrolimus intervention 10: Total-Body Irradiation intervention 11: Treosulfan intervention 12: Umbilical Cord Blood Transplantation
Intervention descriptions: intervention 1: Infused IV intervention 2: Given IV intervention 3: Given IV or PO intervention 4: Given IV intervention 5: Correlative studies intervention 6: Given IV intervention 7: Given IV or PO intervention 8: Infused IV intervention 9: Given IV or PO intervention 10: Undergo total body irradiation intervention 11: Given IV intervention 12: Single or double unit umbilical cord blood transplant, infused IV

Protocol documents:
Prot_SAP_000.pdf: 
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1 
Scott Baker 
FRED HUTCHINSON CANCER RESEARCH CENTER 
UNIVERSITY OF WASHINGTON SCHOOL OF MEDICINE 
1. TITLE:
Allogeneic hematopoietic cell transplantation for patients with 
nonmalignant inherited disorders using a Treosulfan based 
preparative regimen 
Protocol Number: 
2256 
Current Version: 
04/06/2020 
Prior Version(s): 
12/18/2019 
IND Number: 
147602 
Investigational Agent: 
Treosulfan 
Study Regimen: 
Fludarabine, Treosulfan, and Thymoglobulin-based conditioning 
regimen followed by marrow, PBSC, cord blood, or combination of 
bone marrow and cord blood transplant, followed by total body 
irradiation (for cord blood recipients only) 
Principal Investigator: 
Lauri M Burroughs, MD, Associate Professor, 
Fred Hutch Clinical Research Division 
Associate Professor, Pediatrics, 
University of Washington School of Medicine 
IND Sponsor: 
Fred Hutchinson Cancer Research Center 
1100 Fairview Ave N, M/S LM-200 
Seattle, WA 98109-1024 
(O): 206-667-2731 
(F): 206-667-6068 
ISIOC@fredhutch.org 
Medical Monitor: 
K. Scott Baker, MD
Professor, Fred Hutch Clinical Research Division
(O): 206-667- 5594
ksbaker@fredhutch.org
Authorized Sponsor Representative: 
Name (typed or printed):  
K. Scott Baker, MD
Institutional Title: 
Professor, ISIOC Medical Monitor 
Institution: 
Fred Hutchinson Cancer Research Center 
Digitally signed by Scott Baker 
Date: 2020.04.16 09:09:15 -07'00' 
Signature: 
IRO REC'D 04/28/2020
FHCRC IRB Approval  
05/21/2020 
Document Released Date


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Signature Page: 
 
Investigator Statement: 
 
I have carefully read Protocol 2256 entitled “Allogeneic hematopoietic cell transplantation for 
patients with nonmalignant inherited disorders using a Treosulfan based preparative regimen” 
version date 04/06/2020. 
 
I agree to carry out my responsibilities in accordance with the Protocol, applicable laws and 
regulations (including 21 CFR Part 312), Good Clinical Practice: Consolidated Guidance (ICH-E6), 
and applicable policies of Fred Hutch. 
 
 
Principal Investigator: 
 
Name (typed or printed): 
Lauri M Burroughs, MD 
 
 
Institution and Address: 
Fred Hutchinson Cancer Research Center 
 
1100 Fairview Avenue N, M/S D5-280 
 
Seattle, WA  98109 
 
 
Phone Number:  206-667-2396 
 
Signature:_________ 
 
Date:  04/16/2020 
 


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2. 
INTRODUCTION 
 
Hematopoietic cell transplantation (HCT) has been used to treat life-threatening non-malignant 
disorders, including certain inherited disorders, by providing normal cells or cellular proteins to 
replace or substitute for defective components.  Until recently conventional myeloablative 
regimens; primarily busulphan and cyclophosphamide, have been used to establish hematopoietic 
cell grafts. The significant toxicities associated with conventional regimens often preclude 
transplantation for those severely affected patients with end organ dysfunction or serious 
infections. In a broader sense, the risks associated with conventional approaches are deemed 
unacceptable for many patients with chronic disorders who might otherwise be cured with HCT. In 
the past 5-10 years, encouraging results have been reported using nonmyeloablative conditioning 
regimens for patients with nonmalignant disorders.1-3 Although decreased regimen related toxicities 
have been seen; graft rejection and/or low donor chimerism with inadequate disease responses 
have been seen for certain groups of patients. Thus, there is a need to explore other preparative 
regimens with the goal of decreasing regimen related toxicities and mortalities while preserving 
engraftment and disease response. 
 
Treosulfan-based regimens are currently being evaluated in Europe as a potential alternative to 
conventional HCT in patients with malignant and nonmalignant diseases and preliminary results 
are very encouraging. This busulfan analog has several attractive characteristics including less 
erratic pharmacokinetics and decreased regimen related toxicities compared to busulfan. The 
primary objective of this clinical trial is to evaluate, within the limits of a phase II study, the 
preliminary efficacy as defined by engraftment, of a regimen consisting of treosulfan and 
fludarabine followed by allogeneic HCT in patients with nonmalignant inherited disorders. 
 
3. 
BACKGROUND 
 
3A. Conventional Marrow Transplantation for Treatment of Non-Malignant Disorders 
 
1) Primary Immunodeficiency diseases. 
 
HCT is effective in the treatment of a variety of primary immunodeficiency disorders, by 
restoring normal cells or cellular proteins to the deficient host to correct the underlying genetic 
deficits. Multiple studies have demonstrated the effectiveness of HCT using myeloablative 
conditioning for the treatment of patients with primary immunodeficiency disorders (excluding 
SCID).4-9 Diagnosis, donor type, age at HCT and pre-HCT co-morbid conditions; in particular 
infections; were important factors that impacted survival. Death from transplant related causes 
including infections, GVHD and end organ toxicity were major problems that limited the 
success of this approach. Therefore, strategies aimed at decreasing toxicity are needed. 
 
2) Metabolic storage diseases. 
 
Marrow transplantation has been used to treat patients with metabolic storage disorders by 
using transplanted hematopoietic cells to provide a source of deficient enzyme. Conventional 
transplantation results in cure for a substantial number of patients with certain disorders, 
although TRM and disease progression continue to be problematic. The largest group of 
patients studied are those with mucopolysaccharidosis type 1, wherein successful marrow 
transplantation results in significant reduction of storage material in liver, tonsils, conjunctiva, 
CSF, and urine, improvement of CNS findings on MRI, normalization of hearing, improved left 
ventricular function and resolution of arrhythmia.10,11 Significant amelioration of disease 
symptoms has been associated with partial as well as complete enzyme replacement.11 


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Peters, et al. reported outcome for 54 patients with Hurler syndrome treated with conventional 
marrow transplantation from HLA-identical related (n=28) or haploidentical grafts (n=26; 15/26 
received T-cell deplete grafts). Patients were conditioned with chemotherapy with or without 
TBI.10 Among the 26 evaluable recipients of HLA-identical related grafts, 22 (85%) achieved 
full donor (n=15) or mixed donor-host (n=7) chimerism, and 4 (15%) failed to engraft. Among 
the 26 recipients of HLA-haploidentical grafts, 16 (62%) achieved full donor chimerism, 1 (4%) 
achieved mixed donor-host chimerism, and 9 (35%) failed to engraft. Twenty patients (37%) 
died of transplant related causes primarily cardiac (n=8) or GVHD +/- infection (n=8). More 
recently, Souillet et al. reported the outcome of 27 patients with Hurlers syndrome who 
received related (n=10) or unrelated (n=17) T-replete grafts (n=26) from primarily bone 
marrow (n=25) following conditioning with busulfan/ cyclophosphamide alone (n=27) or 
combined with ATG (n=20).12 Of the 27 patients, 23 engrafted (full donor n=11, mixed n=10) 
and 4 had primary graft failure. Importantly, disease responses, specifically improvement in 
enzyme activity levels, cognitive function and clinical improvement in somatic disease were 
seen in both the fully engrafted and mixed chimera patients. Staba et al. reported on 20 
patients with Hurlers syndrome who received busulfan/cyclophosphamide and ATG followed 
by unrelated donor cord blood grafts.13 With a median follow up of 905 days, 17 children were 
alive with an event free survival of 85%. All living recipients had complete donor chimerism, 
normal enzyme activity levels and demonstrated improvement in neurocognitive performance 
and decreased somatic features of Hurler’s syndrome. 
 
Marrow transplantation has been used to treat other metabolic storage disorders, including 
Sanfilippo A, Sanfilippo B, Hunter syndrome, metachromatic leukodystrophy, adrenal 
leukodystrophy, Krabbe’s disease, and Neiman-Pick disease.14-16 Potential for reversing or 
arresting disease course has depended upon early transplantation, before onset of significant 
neurologic impairment. Morbidity and mortality from transplant conditioning regimens have 
contributed to treatment failure. 
 
3) Thalassemia and other hemoglobinopathies. 
 
Several studies have demonstrated efficacy of conventional marrow transplantation using 
HLA-identical sibling donors17-25 and unrelated donors26-28 for cure of thalassemia and other 
hemoglobinopathies. Stable mixed chimerism has been documented in a number of patients 
after conventional regimens and has been associated with cure of disease.20,29 Mortality 
resulted from infection, pneumonitis, graft rejection, and GVHD. Again, pre-transplant patient 
characteristics were the most important factors that predicted outcome after HCT. Patients 
with liver fibrosis or hepatomegaly had a 2-fold increase in risk of mortality. Older age was 
also associated with worse outcome. Recently, Iannone et al. published results on 7 patients 
with sickle cell disease (n=6) or thalassemia (n=1) who received bone marrow or peripheral 
blood stem cell grafts from HLA-identical siblings following conditioning with 200 cGy TBI and 
fludarabine [30 mg/m2 x 3 days (n=2) or 5 days (n=5)] alone or combined with ATG (n=2).30 
Although this regimen resulted in minimal toxicity, 6 of the 7 patients lost their grafts following 
tapering of the immunosuppression. These results suggest that donor engraftment is more 
difficult to achieve among patients with hemoglobinopathies than what has been found in 
patients with malignancies. One possible explanation for this is that patients with 
hemoglobinopathies may have been more sensitized to donor minor histocompatibility 
antigens by blood transfusions. Therefore, in order to improve the engraftment rate among 
high-risk patients with thalassemia and sickle cell disease, a more aggressive conditioning 
regimen may be required in order to overcome these barriers. 


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4) Osteopetrosis. 
 
Osteopetrosis is an autosomal recessive disease resulting from lack of osteoclast resorption of 
bone that causes in marrow failure and nerve entrapment, and early death. Normally 
functioning osteoclasts can be restored by HCT, resulting in amelioration of disease 
symptoms.31 Partial as well as full chimerism has been associated with establishment of 
osteoclast function.32 Studies have shown 40-80% disease-free survivals for recipients of 
HLA-matched related grafts, and 30-40% for recipients of unrelated grafts.31-34 Treatment 
failure is primarily due to transplant related complications such as infection, GVHD, or toxicity 
in particular hepatic veno-occlusive disease.35 Development of a conditioning regimen with 
decreased toxicity in particular hepatic toxicity would be appealing in this group of patients. 
 
5) Hemophagocytic lymphohistiocytosis (HLH). 
 
Marrow transplantation is the only reported cure for hemophagocytic lymphohistiocytosis 
(HLH), characterized by progressive organ infiltration of lymphocytes and macrophages 
associated with hemophagocytosis. Marrow transplantation has been successful in treating 
both the inherited form, known as familial erythrophagocytic lymphohistiocytosis (FEL), as well 
as the sporadic form. Henter et al. reported on the outcomes of 65 patients conditioned with 
primarily busulfan, cyclophosphamide, and etoposide +/- ATG.36 At a median follow up of 3.1 
years, the 3 year probability of overall survival was 62%. Twenty (31%) patients died of 
transplant related causes prior to day 100. Recently, Baker et al. reported on the outcomes of 
91 patients with HLH who were conditioned primarily with BU/CY/Etoposide/ATG followed by 
unrelated bone marrow (86%) grafts.37 The 1 and 5 year overall survivals were 52% and 45% 
respectively after HCT. Day 100 TRM was 33% and was primarily from infections +/-GVHD 
(n=13) or organ toxicity (n=17).  These results support the need for new studies with 
potentially lower transplant related mortality. 
 
6) Inherited Marrow Failure Syndromes. 
 
Inherited bone marrow failure syndromes are genetic disorders that may present at birth or 
later in life and are characterized by defective single or multi-lineage hematopoiesis with many 
patients progressing to develop severe aplastic anemia (SAA) and they are often associated 
with congenital defects. Patients are at risk for significant infections due to neutropenia; 
however many patients also have an additional underlying immunodeficiency. In addition, 
patients with bone marrow failure syndromes are at particularly increased risk for developing 
malignancies; in particular myelodysplastic syndrome (MDS), acute myeloid leukemia, and 
solid tumors. For those patients who develop SAA, the primary curative therapy is 
hematopoietic cell transplantation (HCT). In addition, although not the primary goal for 
patients with bone marrow failure associated SAA, HCT has been used to try and prevent the 
development of MDS and myeloid leukemias. 
 
a. Dyskeratosis congenita 
 
Dyskeratosis congenita (DC) is a rare inherited multi-system disorder characterized by the 
diagnostic triad of reticulated hyperpigmentation of the skin, dystrophic nails, and oral 
leukoplakia. In addition, several somatic manifestations are seen including skeletal, dental, 
gastrointestinal, genitourinary, pulmonary, immunological and ocular abnormalities.38 
Autosomal dominant and recessive forms have been described; however over 75% of the 
cases are an X-linked recessive pattern. The genes identified have been found to encode 
products involved in telomere maintenance. The primary causes of death are from bone 
marrow failure culminating in SAA. A recent analysis of data from the dyskeratosis 
congenita registry found the probability of developing bone marrow failure [one or more 


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peripheral cytopenia(s)] by age 40 to be around 94%.38 In addition to death from infections 
as a result of bone marrow hypoplasia, a large number of patients die from infections 
resulting from cellular and humoral abnormalities of the immune system. Specifically, 
studies have found abnormal immunoglobulin production, reduced B and or T lymphocyte 
numbers and reduced T-cell function in patients with DC.39 Other causes of death include 
myeloid and solid tumor malignancies as well as pulmonary failure. 
 
Therapy has consisted of androgens, prednisone, splenectomy and hematopoietic growth 
factors; however none of these has resulted in a cure of the underlying hematopoietic 
disorder. The only definitive treatment for those patients who develop bone marrow failure 
is HCT; however prior studies have demonstrated poor survival due to both early and late 
complications following HCT.40 Langston et al published on 8 patients with DC and aplastic 
anemia who received bone marrow grafts following conditioning with either 
cyclophosphamide (140-200 mg/kg) +/- ATG (HLA-matched related, n=6) or 
Cyclophosphamide (120 mg/kg) and 1200 cGy TBI (HLA-matched unrelated, n=2).41 At the 
time of publication, 7 of the 8 patients died of overwhelming fungal infections (n=3; day +7, 
+13, +14), acute and chronic interstitial lung disease (n=3; day 70, 8 years, 20 years), and 
1 patient died of acute GVHD. The remaining alive patient has since died of colorectal 
cancer. Rocha et al published on 5 patients with aplastic anemia associated DC who 
received HLA-matched related donor grafts following primarily cyclophosphamide (120-200 
mg/kg) with irradiation (7 Gy TBI or 6 Gy TAI).42 All patients engrafted; however 4 of the 5 
died of transplant related causes including late veno-occlusive disease (n=2), thrombotic 
microangiopathic arterial syndrome (n=1), and invasive aspergillosis (n=1). Others have 
also reported poor outcomes due to significant transplant related complications.38,40,42 
 
Unfortunately, the outcomes of allogeneic HCT for patients with SAA associated DC are 
significantly worse than that seen for patients with SAA alone or even for patients with other 
marrow failure syndromes such as Fanconi anemia. One possible explanation is the fact 
that patients with DC often have significant underlying comorbidities as a result of their 
disease, such as pulmonary disease, which likely accounts for the higher incidences of 
mortality seen in this patient population. Therefore, due to the high incidences of mortality 
associated with more ablative preparative regimens, strategies aimed at reducing toxicities 
are indicated as well as the use of preparative regimens that do not use drugs that are 
known to be toxic to the lungs such as busulfan. 
 
Several case reports have recently been published using various reduced intensity or 
nonmyeloablative approaches. Dror et al reported on 2 patients with DC who underwent 
HCT secondary to severe cytopenias.43 The preparative regimen consisted of fludarabine 
(30mg/m2/day x 5 days), cyclophosphamide (60 mg/kg/day x 2 days), and ATG (40 mg/kg x 
4 days) followed by HLA matched unrelated donor bone marrow grafts. Both patients 
engrafted and are alive with full donor chimerism 15 and 16 months after HCT. Ayas et al. 
recently published 1 case of a 5 year old boy with DC who received cyclophosphamide (15 
mg/kg x 4 days) and ATG (13 mg/kg x 4 days) followed by HLA-matched related graft.44 He 
engrafted and had minimal early toxicity. At 6 months post HCT he had 55% and 100% 
donor chimerism in both the lymphoid and myeloid compartments, respectively. Gungor et 
al reported on the treatment of a 10 year old female with DC who had neutropenia, 
thrombocytopenia and combined immunodeficiency with recurrent bacterial pulmonary 
infections and disseminated refractory molluscum contagiosum skin infections.45 This 
patient was conditioned with the nonmyeloablative preparative regimen developed at the 
hutch [fludarabine (30mg/m2/day x 3 days) and 2 Gy TBI] followed by G-CSF mobilized 
peripheral blood stem cell (PBSC) graft. This patient is alive and well 2 years after HCT 


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with 100% donor chimerism in myeloid and lymphoid lineages and normal T and B cell 
function. Importantly she has no pulmonary dysfunction. 
 
Due to early and late complications some of which is disease related following more 
aggressive preparative regimens, the outcome following HCT for DC has been poor. 
Although the number of patients reported in these case reports is small, the results are 
encouraging and support further investigation of HCT using reduced intensity preparative 
regimens for patients with DC. 
 
b. Shwachman-Diamond Syndrome 
 
Shwachman Diamond syndrome (SDS) is a rare autosomal recessive disease 
characterized by metaphyseal dysostosis, exocrine pancreatic dysfunction, growth 
retardation and/or short stature and varying degrees of bone marrow dysfunction. The 
primary causes of death are hemorrhage and infections due to associated hematological 
abnormalities such as marrow aplasia, neutropenia, and MDS/acute leukemia. Similar to 
patients with DC, patients with SDS also have abnormalities in their immune system. 
Specifically, defects in neutrophil mobility, migration and chemotaxis as well as B and T cell 
abnormalities have been described.46-48 
 
Although supportive measures such as transfusions, pancreatic enzymes, antibiotics and 
G-CSF have been used, the only definitive therapy for marrow aplasia and/or MDS/AML is 
HCT. However, poor outcomes with HCT have been reported due to excessive cardiac and 
other organ toxicities which are felt to be due to the preparative regimen.49,50 Cesaro et al. 
recently reported on registry data from the European Group for Blood and Bone Marrow 
Transplantation (EBMT) on 26 patients with SDS and severe bone marrow abnormalities.51 
Various preparative regimens were used however, the majority contained busulphan (54%) 
followed by primarily T-cell depleted unrelated donor bone marrow grafts. Five (19%) 
patients failed to engraft. The incidence of grade III-IV and chronic GVHD were 24% and 
29%, respectively. With a median follow up of 1.1 years, the 1 year overall survivals were 
65%. Day 200 and 1 year transplant-related mortalities were 35% for both and were 
primarily due to infections +/- GVHD (n=5) or major organ toxicities (n=3). Based on data 
demonstrating that bone marrow cells of SDS patients have an abnormal susceptibility to 
Fas-mediated apoptosis,52 the authors speculated that SDS patients are more susceptible 
to major organ toxicity and infections and as a result higher transplant related mortalities 
when more aggressive preparative regimens are used. For this reason, the authors 
concluded that future studies should evaluate reduced intensity preparative regimens for 
this group of patients. 
 
3B. Myeloablative HCT for Treatment of Nonmalignant Disorders - Seattle Experience 
 
1) Immunodeficiency disorders. 
 
From 1980 onward, efforts were focused on developing a myeloablative conditioning regimen 
sufficient for engraftment of marrow from related, and eventually unrelated, donors for 
treatment of nonmalignant disorders, including immunodeficiency disorders. Busulfan and 
cyclophosphamide were the backbone of the regimen, which eventually included anti- 
thymocyte globulin (ATG). Twenty-four patients with primary immunodeficiency disorders 
were transplanted at the FHCRC between March 1980 and August 2005. Currently, 14 of 24 
patients are alive; while 10 patients died from infections +/- GVHD (n=6), veno-occlusive 
disease (n=2), multi-organ failure (n=1), and graft rejection with subsequent disease 


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recurrence (n=1). Day 100 and 200 TRM were 29% for both. With a median follow up for the 
living recipients of 11.3 (range, 2.4-26.1) years, the 12-year overall survival, event free survival 
and TRM were 58%, 58% and 38% respectively. Further analysis suggested that tolerance of 
the conditioning regimen depended upon diagnosis. Among the 11 patients with WAS, 10 are 
surviving a median of 8 years (range 2-26) years after HCT with disease resolution while one 
patient died of infection. In comparison, 4 of the 13 patients with other immunodeficiency 
disorders survive long-term. TRM by day 100 for the latter group was 46%, compared to 10% 
for the WAS patients. These analyses show that, while conventional HCT was an effective 
therapy for patients with immunodeficiency disorders, early TRM was prohibitive among the 
subgroup of patients with diagnoses other than WAS. For the most part, the high day 100 
TRM can be explained by the presence of infections or co-morbidities at time of HCT. 
 
2) Other inherited disorders. 
 
Fifty-three patients with metabolic storage diseases (n=10), red cell disorders (n=26), 
Osteopetrosis (n=8), and HLH/Gricellis syndrome (n=9) were transplanted at the FHCRC 
between October 1981 and October 2007. Day 100 and 200 TRM were 21% and 25%, 
respectively. The 15-year overall and event free survival and TRM for the entire group was 
50%, 45% and 27%, respectively after HCT. An analysis of a subgroup of patients (n=27 
metabolic storage diseases, osteopetrosis and HLH) demonstrated that 37% of the patients 
died by day 200 of transplant related causes. Therefore, efforts at decreasing the toxicity of 
the conditioning regimen thereby decreasing TRM without compromising engraftment are 
needed for patients with nonmalignant disorders. 
 
3C. Treosulfan 
 
1) Mechanism of action, activity, pharmacokinetics and toxicity. 
Treosulfan (Ovastat®, L-treitol-1,4-bis-methanesulfonate, dihydroxybusulfan, TREO; Medac, 
Hamburg, Germany) is a prodrug of 2 biologically active epoxy-derivatives and a water-soluble 
intravenous Busulfan analog approved for therapy of advanced ovarian carcinoma in Europe. 
Under physiologic conditions, Treosulfan is non-enzymatically, in a pH-dependent manner, 
spontaneously activated into the monoepoxide((2S,3S)-1,2-epoxybutane-3,4-diol-4-methane- 
sulfonate, S,S-EBDM), which converts to (2S,3S)-1,2:3,4-diepoxybutane (S,S-DEB). The 
formed epoxy-transformers are responsible for cross-linking of DNA molecules and cytotoxicity 
in rapidly proliferating cells, both malignant and non- 
malignant, such as normal hematopoietic cells.53 
Contrary to Busulfan, Treosulfan does not require 
enzymatic activation, thus bypassing hepatic 
metabolism. The hydroxyl groups in positions 2 and 
Figure 1. Molecular structures of 
busulfan and tresoulfan 
3 of the molecule account for differences in 
pharmacological activity between Treosulfan and 
Busulfan (Figure 1). Busulfan directly alkylates 
nucleophilic centers, while Treosulfan induces alkylation of nucleophilic centers by 
intramolecular epoxide formation. Clinical pharmacokinetic studies have demonstrated that 
Treosulfan has a similar t½ to Busulfan (1.8 – 2 hours) and a higher, dose-independent, 
cumulative renal excretion (50% vs. 20%).54,55 Pharmacokinetic studies of both single and 
multiple intravenous infusions of Treosulfan have demonstrated low inter-patient and inter-day 
variability. There is a high linear correlation between the area under the curve and Treosulfan 
dose (r2=0.9227), which compares favorably with that of oral and intravenous Busulfan.56,57 On 
CH3O2SO-CH2CH2CH2CH2-OSO2CH3 Busulfan 
CH3O2SO-CHCH2CH2CH-OSO2CH3 
Treosulfan 
OH 
OH 


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preclinical studies in mice, rats, dogs and primates, Treosulfan resulted in at least 10 times 
lower acute and chronic toxicity than Busulfan [unpublished data, Investigator’s Brochure]. 
 
The pronounced effect of Treosulfan on primitive and committed hematopoietic stem cells and 
its immunosuppressive effects have been demonstrated in allogeneic murine transplant 
models.53 At doses 80-88% lower than those used for humans in clinical trials, Treosulfan was 
at least as effective as Busulfan in depleting hematopoietic cell subsets (Figure 2). Treosulfan 
was capable of inducing full donor engraftment and immune tolerance across MHC barriers. 
Marrow suppression is the limiting toxicity for conventional chemotherapy with Treosulfan at 
doses over 10 g/m2. With autologous stem cell rescue, the Treosulfan dose could be 
escalated up to 47 g/m2 before dose-limiting toxicity including mucositis, diarrhea, dermatitis or 
metabolic acidosis was observed.58 
 
2)  Initial phase I/II studies of Treosulfan -containing regimens for allogeneic HCT – 
European Experience 
 
2a. Treosulfan and fludarabine 
Fifty-six patients, median age 50 (range 18-66) years, with varied hematologic malignancies 
received three doses of 10, 12 or 14 g/m2  Treosulfan and 5 doses of 30 mg/kg FLU 
followed by HLA-identical sibling (n=28) or matched unrelated (n=28) marrow or PBSC.59 
GVHD prophylaxis consisted of cyclosporine with or without thymoglobulin (unrelated 
recipients only). All patients engrafted, with a median time to neutrophil engraftment of 14 
(range 10-23) days. Acute GVHD of grades II-IV and extensive chronic GVHD were 
observed in 24/56 (43%) and 8/42 (19%) evaluable patients, respectively. Significant grade 
3 toxicities included elevation of bilirubin or liver transaminases (n=7), diarrhea (n=3), 
mucositis (n=2), dyspnea (3) and elevation of creatinine (n=1). Eight (15%) patients died 
from relapse. Eleven (20%) patients died from non-relapse causes, 8 from infection, 2 from 
GVHD with or without infection, and 1 from RRT (myocardial infarction). The 1-year DFS is 
53%, with a median follow-up of 21 months. These results suggest that the combination of 
Treosulfan /FLU may result in improved RRT and NRM and similar survival compared to 
Treosulfan /CY. Recently several other groups have published encouraging results in 
patients with hematologic malignancies using a Treosulfan/fludarabine based conditioning 
regimen.60-62 
 
2b. Experience with use of Treosulfan in children 
The experience with this drug in patients of pediatric age is limited. Wachowiak and 
colleagues conducted a pilot study of Treosulfan -containing preparative regimens for 
children. Eight children considered at high risk for toxicity from conventional regimens, of 
median age 11 (4-13) years, and diagnosis of AML (n=3), ALL (n=1), MDS (n=1), 
histiocytosis (n=1), Wiskott-Aldrich syndrome 
(n=1) or adrenoleukodystrophy (n=1) were 
studied. They received three doses of 10 
g/m2 Treosulfan in combination with other 
drugs including FLU, CY, melphalan or 
etoposide, followed by HLA-identical sibling 
marrow (n=7) or cord blood (n=1). GVHD 
prophylaxis was with cyclosporine only. All 
patients engrafted, with a median time to 
neutrophil engraftment of 19 (range 10-22) 
days. Acute grade II GVHD was observed in 
3 (38%) patients. Extensive chronic GVHD 
was observed in 1 (13%) patient. Severe or 


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fatal toxicities were not observed. Three patients developed mild (Bearman grade I) 
mucositis or transient elevation of liver transaminases (n=2). Two (25%) patients relapsed. 
The remaining 6 patients are alive and disease-free, with a median follow-up of 12 months. 
 
More recently, Sauer63 et al. published on 3 patients with Shwachman-Diamond Syndrome 
who received treosulfan (12 g/m2/d x 3), fludarabine (30 mg/m2/d x 6), and Melphalan (140 
mg/m2/d x 1) in combination with CAMPATH-1H (0.1 mg/kg x 2) in 2 cases and ATG in one 
case followed by HLA-identical sibling (n=1), HLA-matched unrelated donor (n=1) or 9/10 
matched unrelated cord blood graft. All patients engrafted at 24, 31, and 26 days after HCT 
and had 100% donor chimerism at last follow up.  Two of the 3 patients are alive at 9 and 
20 months after HCT. The third patient who received a cord blood graft died 3 months after 
HCT of IPS. Greystoke and colleagues recently published a retrospective analysis of 32 
pediatric patients undergoing HCT for nonmalignant diseases including primary 
immunodeficiency (n=18), metabolic disorders (n=9), osteopetrosis (n=4) and thalassemia 
(n=1).64 Patients received a total treosulfan dose of 36 g/m2  (n=6) or 42 g/m2  (n=26) given 
in three divided doses on consecutive days. A variety of other conditioning agents were 
used depending on the stem cell source and donor type including fludarabine (150 mg/m2) 
Cyclophosphamide (200 mg/m2), plus or minus Campath or ATG. HLA-matched or 
mismatched related or unrelated bone marrow (n=17), peripheral blood (n=9), and cord 
blood (n=5) grafts were used as the stem cell source. Post HCT immunosuppression 
included Cyclosporine alone or combined with Mycophenolate Mofetil, methylprednisolone, 
or methotrexate. Twenty eight patients (87.5%) established donor cell engraftment; 
however, 4 patients have required additional transplantation procedures to maintain 
adequate donor derived hematopoiesis. With a median follow up of 471 days, 27 (84%) of 
the patients survive.  One patient died early (day +4) due to sinusoidal obstructive 
syndrome and four other deaths were late due to disease progression, GVHD or infection. 
The incidence of GVHD was very low with 6 (19%) patients developing acute grade II (n=4) 
or grade III (n=2) acute GVHD and 4 (13%) or patients developing chronic GVHD. 
Although multiple different donors and conditioning regimens were used, these results are 
encouraging in pediatric patients with nonmalignant disorders. 
 
Bernardo et al recently published on 20 patients with Thalassemia who were conditioned 
with Thiotepa (8 mg/kg; day -7), Treosulfan (14 g/m2; days -6 to -4), and fludarabine (40 
mg/m2; days -6 to -3) followed by HLA matched related (n-3) or unrelated (n=17) bone 
marrow grafts. GVHD prophylaxis consisted of Cyclosporine and methotrexate. 
Pretransplantation ATG (10 mg/kg; days -5 to -3) was administered to all the unrelated graft 
recipients. Two patients experienced secondary graft failure. Cumulative incidence of TRM 
was 5% and the 2 year probability of survival and thalassemia-free survival was 95% and 
85%, respectively. 
 
2c. Phase I/II Study of Treosulfan and fludarabine in the United States- 
Protocol 1931 -Preliminary Results 
Forty-six patients, median age 41 years (5-59 years), with acute myeloid leukemia in first 
remission (n=22), second remission (n=9), partial remission or relapse (n=5) or ALL in 
second remission (n=2) or myelodysplastic syndrome (n=8) have been enrolled on study. 
Eight of the AML patients had secondary (treatment-induced) AML and 5 patients had failed 
a previous transplant. Most patients had underlying conditions that made them poor 
candidates for conventional myeloablative regimens. All patients received therapy as per 
protocol with treosulfan (5 at dose level 1 or 12 g/m2/day, and 41 at dose level 2 or 14 
g/m2/day) on days -6 to -4 and fludarabine (30 mg/m2/day) on days -6 to -2. Patients 
received HLA-matched sibling grafts (n=1 marrow, n=23 PBSC), or unrelated grafts (n=6 
marrow, n=16 PBSC) on day 0. Neutrophil engraftment occurred in 100% of 43 evaluable 
patients at a median of 17 (range, 5-30) days after HCT. Acute graft-versus-host disease 


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(GVHD) has been observed in 25 of 43 evaluable patients (grade I-II). Chronic GVHD has 
been observed in 13 of 35 evaluable patients. Dose limiting toxicity has been observed in 1 
of 46 patients (Table 1). There have been 9 reportable serious adverse events, 2 of which 
were deemed study drug related. One patient died of systemic aspergillosis on day 10. Ten 
patients have relapsed at a median of 76 (26-184) days, of which 7 have died of disease 
recurrence. Thirty-six patients are alive and disease-free, with a median follow-up of 204 
(range, 10-711) days after HCT. 
 
Table 1: Outcomes Summary 
Probability (%) 
100-day 
200-day 
1-year 
OS 
93 
81 
76 
EFS 
81 
75 
72 
Relapse 
17 
23 
26 
TRM 
2 
2 
2 
 
2d. Treosulfan Pharmacokinetics 
Studies have demonstrated highly predictable PK in adult patients; however, limited data 
are available for pediatric patients. In addition, very little data is available on the relationship 
between treosulfan exposure and HCT outcomes including early toxicities and engraftment. 
Van der Stoep recently published on treosulfan pharmacokinetics and the drug’s 
relationship with regimen-related toxicity and early clinical outcome in 77 pediatric patients 
with hematological malignancies (n=12) or non-malignant diseases (n=65) who underwent 
HCT. Conditioning consisted of treosulfan, fludarabine, +/- thiotepa (n=52) followed by HLA- 
matched sibling n=27, matched URD (>9/10; n=36), or haploidentical (n=14) grafts. 
Hematopoietic cell sources varied and included bone marrow (n=50), PBSC (n=20), cord 
blood (n=6), and bone marrow plus cord blood (n=1). Van der Stoep and colleagues 
reported low intra-patient variability (13.9%) but high inter-patient variability [56%; <1-year 
old, (n=12) and 33%; ≥1 to 21 years of age (n=65)] among 77 pediatric patients. This is in 
contrast to that seen in adult patients where there is low intra- and low inter-patient 
variability. In addition, high treosulfan exposure (>1650 mg*h/L) was associated with an 
increased risk of developing grade 2 or higher mucositis [OR 4.40; 95% CI 1.19–16.28, 
p=0.026], increased risk of skin toxicity, including erythematous rash and skin exfoliation, 
[OR 4.51; 95% CI 1.07-18.93; p=0.040], and increased risk of developing multiple toxicities 
[OR 4.52; 95%CI 1.32-15.53, p=0.016] compared to the lower exposure (<1350 mg*h/L) 
group. The authors highlighted that their study included a heterogeneous group of diseases 
and additional data in more homogeneous and single disease groups with longer follow up 
are needed in order to better understand the relationship between AUC and long-term 
outcomes. We plan on collecting PK samples on patients enrolled on this clinical trial in 
order to gain a better understanding whether the exposure to treosulfan AUC for pediatric 
patients with nonmalignant diseases impacts engraftment and both acute and long-term 
toxicities.  This information will help guide future studies using treosulfan-based 
conditioning in patients with non-malignant diseases. 
 
3D. Fludarabine 
Fludarabine (Fludara) is a purine analog that inhibits lymphocyte proliferation, promotes 
lymphocyte apoptosis, and is effective in the treatment of lymphoid and myeloid 
malignancies.65-67 Fludarabine is phosphorylated intracellularly in several steps to its active 
form 2-fluoroadenosine arabinoside triphosphate, which acts by inhibiting DNA synthesis.65,66 
Fludarabine induces immunosuppression and long-lasting lymphopenia reducing the 
incidence of GVHD and facilitating engraftment along histocompatibility barriers when used in 
combination with other chemotherapeutic agents or low-dose TBI.66,68 


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3E. Stored cord blood use 
Historically, bone marrow, peripheral blood, and cord blood have all been used as stem cell 
sources for transplantation. It is becoming more common for parents to store the cord blood 
of a sibling, particularly if that sibling does not have the same genetic disease and is a match 
to the patient. If a patient has an HLA-matched sibling donor and the cord blood has been 
stored, we may combine the bone marrow and cord blood in order to try and improve the 
transplant outcomes.  Soni et al. 69  recently published on 13 patients with 
hemoglobinopathies who received an HLA-matched sibling cord blood and marrow grafts and 
demonstrated that this strategy was feasible and safe in this small cohort. Furthermore, this 
combined graft source may potentially result in faster neutrophil recovery, decreased GVHD, 
and decreased transplant related mortality compared to either cord blood or bone marrow 
alone.69 
 
3F. Prioritization of Peripheral Blood Stem Cells 
Less intense conditioning regimens have led to improved survival and lower transplant 
related toxicities and mortality in patients with nonmalignant diseases, particularly those with 
co-morbid conditions. However, less intense regimens have been associated with mixed 
donor/host chimerism or graft rejection.70,71 In addition, many patients with nonmalignant 
disease have increased barriers to engraftment due to characteristics of their underlying 
disease, which pose further challenges to sustained engraftment, particularly following 
reduced intensity conditioning. Finally, amelioration of many nonmalignant diseases requires 
high-level multi-lineage donor chimerism. 
 
Thus, the goal of this study for patients with nonmalignant diseases is to develop a regimen 
that overcomes the barriers of engraftment without increasing the toxicities and mortality of 
the transplant procedure. Results of the current trial using a reduced toxicity/intensity regimen 
of Treosulfan, fludarabine, and rabbit ATG are encouraging, with excellent overall survival 
and very low transplant-related mortality. However, mixed donor/host chimerism remains a 
challenge, particularly in patients with diseases that have barriers to engraftment. Several 
clinical trials have shown that in the setting of reduced intensity or nonmyeloablative 
conditioning, engraftment is superior when the graft is composed of peripheral blood stem 
cells (PBSC) compared to bone marrow. 72 Slatter et al. recently published on 160 pediatric 
patients with primary immunodeficiency diseases who received Treosulfan, fludarabine +/- 
Campath (n=154) followed by unrelated or related donor grafts. The initial stem cell source 
was bone marrow; however due to a high proportion of patients with poor engraftment or low- 
level donor chimerism, the trial transitioned to PBSC grafts. Results show a significant 
association between high-level donor myeloid chimerism (>95% donor) and use of PBSCs 
without an increased risk of significant GVHD. 73 Although the number of recipients of PBSC 
enrolled on protocol 2256 (Regimen A) has been low (n=10), we have not seen an increased 
risk for GVHD in recipients of PBSC vs bone marrow to date. Therefore, going forward in this 
clinical trial, PBSCs will be prioritized whenever feasible with the goal of improving donor 
engraftment, in particular CD33 myeloid engraftment. 


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4. 
OBJECTIVES 
 
A. Primary Objectives: 
The primary objective of this clinical trial is to evaluate, within the limits of a phase II study, 
the preliminary efficacy as defined by engraftment, of a regimen consisting of treosulfan and 
fludarabine followed by allogeneic HCT in patients with nonmalignant inherited disorders. 
 
B. Secondary Objectives: 
1. Incidence of non-relapse mortality 200 days and 1 year post-HCT 
2. Incidence of grade II-IV acute GVHD 
3. Incidence of chronic GVHD – as defined as those patients requiring systemic 
immunosuppression 
4. Donor chimerism on days +28 and +100 
5. Assess disease response following HCT 
6. Immune reconstitution following HCT 
7. Incidence of infections 
8. Overall survival 
 
C. Exploratory Objectives: 
1. Pharmacokinetic parameters of treosulfan and treosulfan monoepoxide 
5. 
PATIENT SELECTION 
5A. Inclusions 
1. Age < 50 years with nonmalignant disease treatable by allogeneic HCT. 
2. Patients with a known nonmalignant disease that is not clearly defined will need to be 
discussed with the protocol PI (Dr. Lauri Burroughs) and potentially the 
Nonmalignant board to determine if they are eligible for HCT on this study. 
 
5B. Exclusions: 
1. 
Patients with Idiopathic Aplastic Anemia and Fanconi Anemia. (Patients with 
Aplastic Anemia associated with PNH or inherited marrow failure syndromes, except 
Fanconi Anemia, will be allowed). 
2. 
Patients with the following organ dysfunction: 
a. With impaired cardiac function as evidenced by ejection fraction < 35% (or, if 
unable to obtain ejection fraction, shortening fraction of <26%) or cardiac 
insufficiency requiring treatment or symptomatic coronary artery disease. 
Patients with a shortening fraction < 26% may be enrolled if approved by a 
cardiologist. 
b. With impaired pulmonary function as evidenced by DLCO < 50% of predicted 
(or, if unable to perform pulmonary function tests, then O2 saturation < 92% on 
room air). 
c. With impaired renal function as evidenced by creatinine-clearance < 50% for 
age, weight, height or serum creatinine > 2X upper normal limit or dialysis- 
dependent. 
d. With evidence of synthetic dysfunction or severe cirrhosis requiring deferral of 
conditioning as recommended by a gastroenterology specialist. 


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3. 
Patients with an active infectious disease requiring deferral of conditioning; as 
recommended by an Infectious Disease specialist. 
4. 
Patients who are positive for human immunodeficiency virus (HIV). 
5. 
Females who are pregnant or breast-feeding. 
6. 
Patients with a known hypersensitivity to treosulfan and/or fludarabine. 
7. 
Receiving another experimental drug within 4 weeks of initiation of conditioning (day 
-6) unless approved by the PI. 
 
6. 
DONOR SELECTION 
Regimen A: Bone marrow or Peripheral Blood or HLA-matched sibling bone marrow 
combined with HLA-matched sibling cord blood 
 
A. Inclusions: Donors must be: 
1. 
HLA-identical related donors or 
2. 
Unrelated donors matched for HLA-A, B, C, DRB1, and DQB1 or mismatched for a 
single allele at HLA-A, B, C, DRB1 or a single DQB1 antigen or allele mismatch by 
high resolution DNA typing. 
3. 
PBSC is the preferred cell source (when feasible) for fully matched donors. PBSC 
may also be used for a mismatched donor following discussion with the PI. Bone 
marrow is allowed when PBSC is not feasible or as determined by the PI. 
4. 
HLA-matched sibling bone marrow in combination with HLA-matched sibling umbilical 
cord blood if the HLA-matched sibling umbilical cord blood was collected and stored. 
The HLA-matched sibling bone marrow and cord blood would be matched for HLA-A, 
B, C, DRB1, and DQB1. 
 
B. Exclusions: Ineligible donors will be those: 
1. 
Deemed unable to undergo marrow harvesting or PBSC mobilization and 
leukapheresis. 
2. 
HIV-positive. 
3. 
With active infectious hepatitis. 
4. 
Females with a positive pregnancy test. 
5. 
HLA-matched sibling cord blood exclusions: 
a. Any cord blood units that have not passed donor screening for infectious disease 
markers as recommended by the National Marrow Donor Project (NMDP) will not 
be used unless a waiver is signed by the clinical attending allowing use of cord 
blood unit. Cord blood units are presumed to be CMV negative regardless of 
serologic testing due to passive transmission of maternal CMV antibodies. 
 
Regimen B: Unrelated Umbilical Cord Blood will be allowed if an HLA-matched related 
or unrelated donor is not identified or available 
 
A. Unrelated Umbilical Cord Blood Inclusions: 
 
1. 
Unit selection is based on the cryopreserved total nucleated cell (TNC) dose and 
matching at HLA-A, B antigen level and DRB1 allele level typing. While HLA-C 
antigen/allele level typing is not considered in the matching criteria, if available, may 
be used to optimize unit selection. 
2. 
The patient and the cord blood unit(s) must be matched for at least 4 of 6 loci as 
defined above. 
3. 
Selection of two UCB units is allowed to provide sufficient cell dose (see 3d below 
for algorithm to determine single versus double unit transplant). When multiple units 
are selected, the following rules apply: 


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a. The UCB unit with the least HLA disparity (with the patient) will be selected 
first (i.e., selection priority is 6/6 match >5/6 match>4/6 match). Additional UCB 
units then may be selected to achieve the required cell dose, as outlined below 
(3e). If a second unit is required, this unit will be the unit that most closely HLA 
matches the patient and meets minimum size criteria outlined below of at least 
1.5 x 107 TNC/kg (i.e. a smaller more closely matched unit will be selected over 
a larger less well matched unit as long as minimum criteria are met). 
b. Each UCB unit MUST contain at least 1.5 x 107 TNC per kilogram recipient 
weight. 
c. The total cell dose of the combined units must be at least 3.0 x 107 TNC per 
kilogram recipient weight. 
d. Algorithm for determining single versus double cord blood transplant: The donor 
selection algorithm has been designed to favor single cord HCT to decrease the 
risk of GVHD in the nonmalignant setting while attempting to not compromise 
engraftment. 
 
Single Unit 
Allowed for: 
Multiple Units 
Required for: 
Match Grade 
TNC Dose 
TNC Dose 
6/6 
≥ 3.0 x 107/kg 
< 3.0 x 107/kg 
5/6 
≥ 4.0 x 107/kg 
<4.0 x 107/kg 
4/6 
≥ 6.0 x 107/kg 
<6.0 x 107/kg 
 
4. 
General Comments: 
a. Units will be selected first based on the TNC dose and HLA matching. 
b. CD34+ cell dose will not be used for unit selection unless 2 units of equal HLA- 
match grade and similar TNC dose (± 0.5 x 107 TNC/kg) are available. In this 
case, the unit with the larger CD34+ cell dose (if data available) should be 
selected. 
c. A UCB unit that is 5/6 mismatched but homozygous at the locus of mismatch 
should be chosen over a 5/6 unit with bidirectional mismatch even if the latter 
unit is larger (has more cells). This also applies to 4/6 units. This is only 
applicable to choosing units within a given match grade. 
d. Within an HLA match grade, the unit containing the greatest number of cells will 
be chosen. If there are two units of equivalent cell dose (± 0.5 x 107 TNC/kg) 
within a match level, choose the unit with match by higher resolution molecular 
typing, if known. 
e. Other factors to be considered: 
1. 
Within the same HLA match grade, matching at DR takes preference. 
2. 
Cord blood banks located in the United States are preferred. 
3. 
The youngest unit would be preferred. 
 
B. Unrelated UCB Unit Exclusions: 
1. 
Any cord blood units with <1.5 x 107 total nucleated cells per kilogram recipient 
weight. 
2. 
Any cord blood units that have not passed donor screening for infectious disease 
markers as recommended by NMDP will not be used unless a waiver is signed by 
the clinical attending allowing use of cord blood unit. Cord blood units are 
presumed to be CMV negative regardless of serologic testing due to passive 
transmission of maternal CMV antibodies. 


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7. INFORMED CONSENT 
 
The attending physician will conduct a conference with the patient and family to discuss this 
study and alternative treatments available. In a separate conference, the risks of the donation 
procedure will be outlined to the donor and/or his parent/guardian(s), including the risks of 
anesthesia, bleeding, and infection. The goals of the study, requirement for data collection, and 
requirement for release of medical records will be discussed with the patient and/or his/her 
parent/guardian. All potential risks associated with the use of treosulfan, fludarabine, low dose 
TBI (cord only), immunosuppressive drugs and allogeneic HCT will be discussed as objectively 
as possible. Discussion of potential complications should include graft rejection, GVHD, 
infections, and death. 
 
Informed consent from the patient and donor will be obtained using forms approved by the 
Institutional Review Board of the Fred Hutchinson Cancer Research Center. Informed consent 
will be obtained by the Principal Investigator, Co-Investigator, or Attending Physician who is 
familiar with the study but not necessarily an investigator. When patients are less than 18 years 
of age, consent will be obtained from parents or legal guardian(s). All patients enrolled at 
collaborating centers will engage in institution-specific informed consent conferences after 
completion of the pre-transplant evaluation. Informed consent from the donor and patient will be 
obtained using a form approved by the Institutional Review Board for each treatment center. 
 
8. PROTOCOL REGISTRATION 
 
8A. FHCRC Patients: Eligible patients will be identified by the Clinical Coordinator’s Office who 
will register patients with the Registration office (206-667-4728) between 8:30 am and 4:00 PM, 
Monday through Friday. After hours, the Registration Office can be reached by paging (206)- 
995-7437 
 
8B. Collaborating Institutions: Eligible patients will be identified by the principal investigator of 
the collaborating institution who will register the patient with the FHCRC Registration Office. 
Registration will include completion of the eligibility checklist and demographic form (Appendix 
F of protocol). This form and a copy of the signed informed consent will be faxed to the Trial 
Coordinator (206-667-4427). Questions regarding eligibility or protocol information should be 
directed to the Principal Investigator, Lauri Burroughs (206) 667-2396. 


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9. PLAN OF TREATMENT/CONDITIONING REGIMEN: 
Regimen A: For patients receiving marrow, PBSC or HLA-matched sibling bone marrow 
combined with HLA-matched sibling cord blood 
Day 
Treatment 
Dose 
-6 
Treosulfan 
Treosulfan PK samplingb 
Fludarabine 
14 grams/m2/day IVa 
30 mg/m2/day IVa 
-5 
Treosulfan 
Treosulfan PK samplingc 
Fludarabine 
14 grams/m2/day IVa 
30 mg/m2/day IVa 
-4 
Treosulfan 
Fludarabine 
Rabbit Antithymocyte globulin (ATG; Thymoglobulin) 
14 grams/m2/day IVa 
30 mg/m2/day IVa 
1.0 mg/kg/day IVd 
-3 
Fludarabine 
Rabbit Antithymocyte globulin (ATG; Thymoglobulin) 
30 mg/m2/day IVa 
2.5 mg/kg/day IVd 
-2 
Fludarabine 
Rabbit Antithymocyte globulin (ATG; Thymoglobulin) 
30 mg/m2/day IVa 
2.5 mg/kg/day IVd 
-1 
Reste 
0 
Marrow or PBSC infusion or HLA-matched sibling bone marrow combined with 
HLA-matched sibling cord blood 
a For patients >120% ideal body weight (IBW) BSA will be calculated using adjusted weight 
b Research Treosulfan PK sampling (7 timepoints): All patients, see section 14B.7 for details 
c Research Treosulfan PK sampling (7 timepoints): Only in patients who are < 4 years of age at time of 
start of conditioning, see section 14B.7 for details. 
d Actual body weight 
e Additional rest days may be added given delays that may occur with stem cell collection, transportation 
or processing. The actual day of stem cell infusion will remain “Day 0” even if 1-2 days later than 
originally scheduled 
 
Regimen B: For patients receiving unrelated umbilical cord blood 
Day 
Treatment 
Dose 
-6 
Treosulfan 
Treosulfan PK samplingb 
Fludarabine 
14 grams/m2/day IVa 
30 mg/m2/day IVa 
-5 
Treosulfan 
Treosulfan PK samplingc 
Fludarabine 
14 grams/m2/day IVa 
30 mg/m2/day IVa 
-4 
Treosulfan 
Fludarabine 
Rabbit Antithymocyte globulin (ATG; Thymoglobulin) 
14 grams/m2/day IVa 
30 mg/m2/day IVa 
1.0 mg/kg/day IVd 
-3 
Fludarabine 
Rabbit Antithymocyte globulin (ATG; Thymoglobulin) 
30 mg/m2/day IVa 
2.5 mg/kg/day IVd 
-2 
Fludarabine 
Rabbit Antithymocyte globulin (ATG; Thymoglobulin) 
30 mg/m2/day IVa 
2.5 mg/kg/day IVd 
-1 
Recipients with underlying T-cell primary 
Immunodeficiency or 
All other recipients 
TBI 200 cGy 
TBI 300 cGy 
0 
Unrelated umbilical cord blood infusion 
a For patients >120% ideal body weight (IBW) BSA will be calculated using adjusted weight 
b Research Treosulfan PK sampling (7 timepoints): All patients, see section 14B.7 for details 
c Research Treosulfan PK sampling (7 timepoints): Only in patients who are < 4 years of age at time of 
start of conditioning, see section 14B.7 for details. 
d Actual body weight 


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Conditioning regimen. 
 
The regimen will consist of: 
 
1. Treosulfan. 
 
Treosulfan will be administered intravenously over 120 minutes for three consecutive 
days (day –6 to –4). Use actual weight to calculate BSA. For patients >120% of ideal 
weight, use adjusted weight to calculate BSA. Optional pharmacokinetic evaluation will 
be performed on days -6 (all patients) and -5 (only in patients who are < 4 years of age 
at time of start of conditioning; see Section 14B.7). 
 
Treosulfan will be supplied to investigators by Medac GmbH, Hamburg Germany under 
an IND held by Fred Hutchinson Cancer Research Center (IND 72,479). The drug will 
be delivered by courier to each participating centers’ pharmacy as a clinical 
investigational product with a study-specific label on the glass vial, as approved by the 
FDA. Treosulfan is available as a white crystalline powder in vials of 1000 mg and 5000 
mg. The dry product must be stored at room temperature and has a shelf life of 5 years. 
TREO must be dissolved in 20 ml (1 g vial) or 100 ml (5g vial) of 0.45% sodium chloride 
solution, for injection for a final concentration of 50 mg/ml. The 0.45% sodium chloride 
must be warmed to 25-30ºC (not higher) using a warmer or water bath. The 
reconstituted solution should remain at room temperature and be used the day of 
preparation. The reconstituted solution should not be stored in a refrigerator because 
crystallization may occur at low temperatures. Acidic media do not influence the stability 
of treosulfan. In alkaline and neutral solutions treosulfan decomposes to 
methanesulfonic acid and threitol. The reconstituted solution has a physicochemical 
stability of at least 1 day when stored at room temperature in the original glass bottle. 
 
There have been isolated reports of seizure in infants after combined administration of 
Treosulfan with Fludarabine or cyclophosphamide. Therefore, seizure prophylaxis 
during Treosulfan administration should be considered for patients ≤ 1 year of age. We 
recommend the following: for patients ≤ 1 year begin levetiracetam (Keppra) at 10 
mg/kg every 12 hours to start the evening prior to Treosulfan administration and 
continue until 24 hours after the infusion. 
 
2. Fludarabine. 
 
Fludarabine will be administered intravenously at a dose of 30 mg/m2/day over 60 
minutes for five consecutive days (day –6 to –2). On days –6 to –4, fludarabine will be 
given after the treosulfan dose is given. For patients > 120% of ideal weight, BSA will be 
calculated using adjusted weight. Fludarabine (Fludara, Berlex Laboratories, Inc., 
Richmond, CA) is commercially available in the U.S. Details of the product’s description, 
preparation; storage and stability are found in the drug’s package insert. 
 
3. Rabbit Anti-thymocyte globulin (ATG; Thymoglobulin®) 
 
Thymoglobulin will be administered intravenously over three consecutive days (days -4, 
-3, -2) at a dose of 1.0 mg/kg on day -4, and then 2.5 mg/kg on days -3 and -2 for a total 
dose of 6.0 mg/kg. Thymoglobulin will be given intravenously over a minimum of 6 
hours for the first two doses and 4 hours for the subsequent dose per institutional 
standard practice. Doses should be based on actual body weight. 


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Anti-Human Thymocyte Globulin (ATG; Sangstat Medical Corp., Fremont, CA) is an 
immunosuppressant which alters the function of or eliminates T-cells and NK cells. 
Rabbit anti-thymocyte globulin (Thymoglobulin®) is a polyclonal immunoglobulin 
mixture (IgM/IgG) raised in rabbits against human T thymocytes which reduces all 
circulating lymphocytes or lymphocyte subsets. ATG is used in preparation for 
hematopoietic cell transplant (HCT), as immunosuppressive therapy in aplastic anemia 
or MDS, and for GVHD treatment. 
 
Preparation 
Thymoglobulin® is provided in 25mg vials by Sangstat. Thymoglobulin® vials should 
be reconstituted with 5ml sterile water to a concentration of 5mg/ml. It can then be 
further diluted with D5W or NS to a volume of 50ml per vial. Diluted drug should be 
used immediately. 
 
Skin Test 
Skin tests are not required prior to Thymoglobulin® administration. 
 
Administration 
Thymoglobulin® should be administered alone and not run piggyback with any other 
solution. A 0.22 micron in-line filter should be used during administration. The first and 
second dose of Thymoglobulin® should be infused over 6 hours, and subsequent doses 
should be infused over 4 hours. 
 
Pre-Meds: 
Acetaminophen, diphenhydramine, and 1 mg/kg of methylprednisolone should be given 
before every dose of Thymoglobulin®. 
 
Significant Allergic Reaction Precautions 
Have recommended medications for significant allergic reactions available at the 
bedside in case of allergic reaction: 
 
Medication Recommendations For Significant Allergic Reactions 
 
Pediatric: 
Adults > 40 kg 
Epinephrine 
(1:1000 dilution) 
[1 mg/ml] 
0.01 mg/kg IM 
(Every 20 minutes as needed). 
Maximum dose 0.3 mg 
0.3 ml SC 
(every 20 minutes as needed) 
Diphenhydramine 
1 mg/kg IV (max 50 mg) 
50 mg IV 
Hydrocortisone 
2 mg/kg IV (max 250 mg) 
250 mg IV 
 
4. Total Body Irradiation (TBI) 
 
A.Cord Blood Recipients Only: 
Patients with an underlying T-cell immunodeficiency will receive 200 cGy TBI on day -1. 
All other patients will receive 300cGY TBI on day – 1. See institutional standard 
practice guidelines for radiation (Appendix B). 


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10. COLLECTION OF HEMATOPOIETIC STEM CELLS/INFUSION 
 
Related donors will be collected at each participating center and unrelated donors will be 
collected through the NMDP or other donor collection centers. 
 
A. 
PBSC: Is the preferred stem cell source for fully matched donors. PBSC may also be 
used for a mismatched donor following discussion with the PI. PBSC collection and 
infusion will be according to institutional standard practice guidelines. 
 
B. 
Bone Marrow: Donors will undergo standard bone marrow harvest following each center’s 
standard practice. The marrow will be infused according to each center’s standard practice 
guidelines. 
C. 
Unrelated Umbilical Cord Blood: will be used if an HLA-matched related or 
unrelated donor is not identified or available (see matching criteria section 6) 
 
1. Procedures for requesting, receiving and characterizing the cord blood unit for infusion 
will be according to institutional standard practice. 
2. Pre-infusion hydration should be performed per institutional guidelines. 
3. For FHCRC patients, the cord blood unit should be thawed and infused per FHCRC 
standard practice guidelines. Cord blood products should be infused without delay as 
soon as the product arrives on the unit. 
4. The infusion should take no longer than 30 minutes per unit. Pre-medications prior to 
cord blood infusion will be per institutional standard practice guidelines. Under no 
circumstances is the cord blood to be irradiated. No medications or fluids should be 
given piggyback through the catheter lumen that is being used for cord blood infusion. 
5. The product is infused via IV drip directly into the central line according to standard 
practice with gravity filtered tubing. 
6. Vital signs should be monitored before beginning the infusion and periodically during 
administration. Notify the attending physician, fellow, PA, or NP immediately if the 
patient exhibits signs or symptoms of a reaction. 
7. Benadryl, epinephrine, and hydrocortisone should be available at the bedside for 
emergency use if necessary. Oxygen with nasal prongs for standby use should be 
present in the room. 
8. If the patient is a double cord blood recipient, the two units may be given consecutively 
with no wait between infusions of the units. The start and stop time of each unit should 
be recorded on the infusion record. 
 
D. Combined bone marrow and cord blood from the same HLA-matched sibling donor: 
At the discretion of the PI, bone marrow and cord blood cells from the same HLA–matched 
sibling may be used together for the transplant in cases where such cord blood cells were 
previously stored and found to be qualified for use as a donor for stem cell transplant. 
1. The HLA-matched sibling umbilical cord blood unit will be deemed qualified for use in 
combination with bone marrow when the matched sibling cord blood unit meets the 
eligibility criteria outlined in section 6 A. 
2. The HLA-matched sibling bone marrow will be infused first followed by HLA-matched 
sibling cord blood cells. The attending physician will determine the appropriate timing 
of infusion of the HLA-matched cord blood unit following completion of the HLA- 
matched sibling bone marrow graft. However, infusion of the HLA-matched sibling 
cord blood unit will not begin until any acute toxicities from the bone marrow 
infusion have been controlled. In addition, the attending physician or Principal 
investigator may determine that infusion of the HLA-matched sibling cord blood 


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is not clinically feasible or safe depending on the clinical circumstances and 
therefore may decide to NOT infuse the HLA-matched sibling cord blood. 
The HLA-matched sibling cord blood unit must undergo rigorous donor screening and 
testing as well as evaluation for infectious disease markers as is required to release an 
unrelated donor cord blood unit for clinical use. If the related donor cord blood unit was 
processed and stored by a FACT accredited cord blood bank, then as standard 
practice, the documents verifying donor eligibility are available for review prior to 
shipment of the unit and will accompany the unit. If the cord blood unit was processed 
and stored by a bank that is NOT FACT accredited, documents regarding verification 
of HLA typing, cell count (including total nucleated cell count and CD34 if available), 
pre-cryopreservation viability, and infectious disease testing will be requested and 
reviewed prior to release of this cord blood unit for clinical use. 
3. Procedures for infusion of the HLA-matched sibling bone marrow and HLA-matched 
sibling cord blood cells will follow institutional standard practice guidelines for each cell 
product. 
4. Procedures for requesting, receiving and characterizing the cord blood unit for infusion 
will be according to institutional practice. 
5. Pre-infusion hydration should be performed per institutional guidelines. 
6. Conditioning and GVHD prophylaxis will be per regimen A. 
7. Patients receiving HLA-matched sibling bone marrow and HLA-matched sibling cord 
blood products may have infusions that go over 2 days. Day 0 will be considered the 
day the 2nd product finishes and administration of methotrexate will be adjusted 
accordingly. 
8. For FHCRC patients, the cord blood unit should be thawed and infused per FHCRC 
standard practice guidelines. Cord blood products should be infused without delay as 
soon as the product arrives on the unit. Outside centers should follow institutional 
standard practice guidelines. 
9. The cord blood infusion should take no longer than 30 minutes. Pre-medications (if 
any) prior to cord blood infusion will be at the discretion of the attending. Under no 
circumstances is the cord blood to be irradiated. No medications or fluids should be 
given piggyback through the catheter lumen that is being used for cord blood infusion. 
10. The product is infused via IV drip directly into the central line according to standard 
practice with gravity filtered tubing. 
11. Vital signs should be monitored before beginning the bone marrow and cord blood 
infusion and periodically during administration. Notify the attending physician, fellow, 
PA or NP immediately if the patient exhibits signs or symptoms of a reaction. 
12. Benadryl, epinephrine, and hydrocortisone should be available at the bedside for 
emergency use if necessary. 
13. Oxygen with nasal prongs for standby use should be present in the room. 
 
11. IMMUNOSUPPRESSION: 
 
Regimen A: Bone Marrow or PBSC or recipients of HLA-matched sibling bone marrow 
combined with HLA-matched sibling cord blood 
 
All patients who receive bone marrow or peripheral blood stem cells will receive 2 agents for 
GVHD prophylaxis as follows: 


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A. Tacrolimus 
 
1. 
Tacrolimus should be initiated per Standard Practice Guidelines as an IV continuous 
infusion on day –1 at a dose of 0.03 mg/kg/day. Dosing should be based on adjusted 
body weight. If actual weight is less than adjusted body weight, dosing should be 
based on actual weight. The same lumen of the central catheter should be used for all 
tacrolimus infusions. Conversion to the oral formulation (IV:PO ratio 1:4) should be 
made when oral feeding is established. Oral tacrolimus is available in 0.5, 1 and 5 mg 
capsules and as a compounded suspension of 1 mg/mL. Oral tacrolimus should be 
given in two divided daily doses every 12 hours. If the patient vomits within one hour of 
taking the medication, repeat the dose. If vomiting persists, the medication should be 
given intravenously at the appropriate dose (PO:IV ratio 4:1). Beverages containing 
the enzyme bergamottin (grapefruit juice, Sunny Delight, Fresca and Squirt) should be 
avoided. If possible, oral tacrolimus should be taken on an empty stomach at a 
consistent time each day. 
 
2. 
In the absence of GVHD, we recommend that the tacrolimus be tapered starting on 
day +50. In the absence of GVHD, the dose should be tapered by 5% each week for 
liquid and 20% each month for capsules. In the presence of GVHD, or if the patient is 
receiving corticosteroids or other therapy for GVHD, tacrolimus should not be tapered 
at day +50, and dosing should be maintained to target therapeutic levels. Long-term 
administration of tacrolimus will be per the recommendations of the Long-term Follow- 
up Department (LTFU). 
 
3. 
Tacrolimus levels: Tacrolimus levels should be maintained in the range of 5-15 ng/ml. 
In patients who are also taking sirolimus, it is generally recommended that tacrolimus 
trough levels not exceed 10ng/ml. Tacrolimus levels will be performed weekly starting 
on day +2 through day +50 per Standard Practice Guidelines, see Calcineurin Inhibitor 
Therapy (Cyclosporine, Tacrolimus) section. To avoid contamination, all levels should 
be drawn from the central catheter port opposite the lumen used to infuse tacrolimus. 
If a tacrolimus taper is initiated at day +50, and the patient has adequate oral fluid 
intake and stable volume status, checking of blood levels may generally be 
discontinued once tacrolimus has been reduced by 25%. If a tacrolimus taper is not 
initiated, levels should be performed weekly through day +100. Tacrolimus levels 
should be performed more frequently 1) when the medication is converted from oral to 
IV or IV to oral; 2) when dose adjustments are made due to levels outside the 
therapeutic range; or 3) if toxicity is suspected. Steady state levels will not be achieved 
for at least 72 hours after any change in dosing, so levels obtained earlier than this 
may not reflect an accurate steady state concentration. 


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4. 
Tacrolimus Toxicities: 
 
Common, 
No Specific Therapy 
Common, 
Intervention Usual 
Uncommon, 
May Require 
Intervention 
Uncommon or 
Serious, Intervention 
Usual 
• 
Hypertension 
(controlled) 
• 
Tremor (mild) 
• 
Hyperlipidemia 
• 
Hypertension 
(poorly controlled) 
• 
Tremor (severe) 
• 
Hyperkalemia 
• 
Hypomagnesemia 
• 
Elevated Creatinine 
• 
Elevated BUN 
• 
Anorexia, nausea, 
vomiting 
• 
Diarrhea 
• 
Anemia 
• 
Back pain 
• 
Rash, pruritis 
• 
Atelectasis 
• 
Insomnia 
• 
Asthenia 
• 
Hyperglycemia 
• 
Hyperbilirubinemia 
• 
Elevated 
Transaminases 
• 
Pleural effusion 
• 
Coma, delirium 
• 
Hemolytic Uremic 
Syndrome 
• 
Dysarthria 
• 
Seizures 
• 
Hallucinations 
 
B. Methotrexate 
 
1. 
Administration: 
 
a. In adults and children > 10 kg, methotrexate should be administered as an IV push 
at a dose of 15 mg/m2 on day +1 after transplant and at 10 mg/m2 on days +3, +6 
and +11. Calculation of m2 will be per Standard Practice Guidelines. 
 
b. Infants who weigh < 10 kg should have methotrexate dosed on a per kilogram 
(actual weight) basis rather than per meter squared. Therefore, the methotrexate 
dose is 0.5 mg/kg on Day + 1 and 0.33 mg/kg on Days +3, +6 and +11. 
 
c. The first dose of methotrexate should be given approximately 24 hours after 
completion of the initial stem cell infusion, but no sooner than 24 hours later. 
 
2. 
Toxicities: In this setting, mucositis is the primary toxicity related to methotrexate. In 
the setting of renal or hepatic dysfunction, where there is impaired clearance of the 
medication, methotrexate may contribute to delayed neutrophil engraftment. 
Methotrexate may cause an elevation in serum transaminase. 
 
3. 
Dose adjustments - Every attempt should be made to administer methotrexate as 
prescribed, as dose reduction or omission may increase the risk of acute GVHD. 
Patients at greatest risk for methotrexate -related toxicities are those with either 
decreased renal function or significant fluid collection (i.e. ascites, pleural effusions, 
etc.), where methotrexate can accumulate and, thus, delay clearance. Renal 
dysfunction or third space fluid accumulation are not strict contraindications to the 
administration of methotrexate, although dose reduction (75–50%) and rescue with 
leucovorin (citrovorum factor) should be considered. Similarly, while one may want to 
discontinue methotrexate in patients who require intubation because of severe 
mucositis, generally mucositis should not be a contraindication for the use of 
methotrexate. 
 
4. 
Methotrexate levels should be monitored in patients at risk for the development of 
toxicity. Acceptable methotrexate levels at 24 hours or later after the administration of 


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Figure 3. Methotrexate (MTX) plasma levels and leucovorin (citrovorum) “rescue”. 
 
 
methotrexate are <0.04 x 10-6 molar (see Figure 3). If toxic levels are present, 
leucovorin should be given per our Standard Practice Guidelines. The standard dose 
of leucovorin is 10 mg/m2 IV every 6 hours. 
 
 
Regimen B: Unrelated Umbilical Cord Blood: 
 
All recipients of unrelated umbilical cord blood will receive prophylaxis for GVHD with 2 drugs 
as follows: 
 
A. 
Cyclosporine A 
1. 
Patients will receive cyclosporine A (CSA) therapy beginning on Day -3 maintaining a 
trough level between 200 and 400 ng/mL by HPLC analysis (250 and 500 ng/ml by 
immunoassay). For adults the initial dose will be 2.5 mg/kg IV over 1 hour every 12 
hours. For children < 40 kg the initial dose will be 2.5 mg/kg IV over 1 hour every 8 
hours. The initial dose will be based on adjusted body weight. 
2. 
Dose adjustments will be made on the basis of toxicity and low CSA levels with 
trough level of <200 mg/L (HPLC). Once the patient can tolerate oral medications, 
CSA will be converted to an oral form. CSA dosing will be monitored and altered as 
clinically appropriate. 
3. 
Patients will receive CSA until Day +100. If there is no evidence of GVHD, the dose 
will then be tapered 10% per week starting on Day +101 and discontinued no sooner 
than 6 months post-transplant. 
4. 
Drugs that may affect CSP levels are: 


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Decrease CSP levels 
Increase CSP levels 
Phenytoin 
Phenobarbital 
Carbamazepine 
Primidone 
Rifampicin 
Nafcillin 
Octreotide 
Sulfonamides 
Trimethoprim 
Metoclopramide 
Erythromycin 
Ketoconazole 
Acetazolamide 
Fluconazole* 
Colchicine 
Itraconazole* 
Fluoroquinolones 
Voriconazole 
Imipenem 
Caspofungin 
Azithromycin 
Diltiazem 
Clarithromycin 
Verapamil 
Doxycycline 
Nicardipine 
Nifedipine 
Alcohol 
*Discontinuation of fluconazole or itraconazole may lower CSP levels, and if used for 
antifungal prophylaxis, then changes in these drugs should be avoided during the first 2 
months post-transplant. 
 
B. 
Mycophenolate mofetil (MMF) 
 
1. 
MMF will be administered at 15 mg/kg every 8 hours with a maximum of 1 gram/dose 
beginning the evening of day 0 (i.e. first dose to follow 4-6 hours after UCBT). The 
dose will be based on adjusted body weight. 
2. 
MMF will be given every 8 hrs daily until day 40 post-transplant and then in the 
absence of GVHD, tapered by 12%/week with MMF discontinued after day + 96. 
3. 
Markedly low (<40%) donor T cell chimerism after UCBT may indicate impending 
graft rejection. MMF should be continued at full dose or, if MMF taper has been 
initiated, reinstitution of full dose MMF should occur. If MMF has been discontinued, 
MMF should be reinitiated at full dose. 
 
12. SUPPORTIVE CARE 
 
All supportive care will be provided per each center’s standard practice guidelines and will be 
dictated by the physician and transplant team caring for the patient. There are no restrictions 
or limitations to supportive care measures. Some Fred Hutch guidelines are included in 
Appendix B. 
13. POST–HCT GROWTH FACTORS 
Regimen A: Bone Marrow or PBSC - 
Patients who receive bone marrow or PBSC should in general not receive post-transplant 
growth factors during the first 3 weeks after HCT. Growth factors should not be given unless 
neutropenia develops or persists past day 21 post-transplant (ANC <500/L). 
 
Regimen B: Unrelated Umbilical Cord Blood – 
Patients who receive unrelated umbilical cord grafts will be started on G-CSF support at 
5mcg/kg (IV/SQ; round to vial size) daily starting day +1 following unrelated umbilical cord 
blood infusion until ANC > 2500/μL for 2 consecutive days. Once a patient has met these 
criteria, the ANC will be monitored and G-CSF restarted if ANC falls to < 1000/L. 
 
Note: We will not give G-CSF to those patients who receive both bone marrow and cord blood 
from an HLA-matched sibling donor. 


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14. PATIENT AND DONOR CLINICAL AND LABORATORY EVALUATIONS 
 
14A. Donor Evaluations: Please refer to the instructions below and Standard Practice Manual 
guidelines or institutional guidelines: 
 
1. 
HLA-matched related bone marrow or peripheral blood donor: 
Related donors will undergo standard evaluation for allogeneic stem cell donation, 
which should include but not limited to: 
a. 
Complete history and physical examination to include the following: 
i) 
Medical problems, including pulmonary and upper airway disease, 
cardiovascular disease, diabetes, arthritis, or abnormalities of the spine. 
ii) 
Last menstrual period 
iii) 
Previous exposure to anesthetics, and family history of anesthesia- 
associated complications 
iv) 
Blood transfusions 
v) 
Medications 
vi) 
Vaccinations 
vii) Allergies 
viii) Height and weight 
 
b. 
Lab tests: 
i) 
CBC with differential including platelet counts. 
ii) 
Serum sodium, potassium, Chloride, CO2, Glucose, Bun, Creatinine, 
Calcium, Magnesium, Phosphorus, Uric acid, LDH, alkaline phosphatase, 
Total bilirubin, AST, ALT and albumin 
iii) 
Hepatitis screen, CMV, syphilis, HIV and HTLV I serologies (tests must be 
completed within roughly 30 days of transplantation) 
iv) 
ABO Rh blood typing. If the donor has antibodies against red cell antigens 
of the recipient, the titers will be determined. Cytotoxic cross match 
between patient and donor (HLA Laboratory) will be performed. 
v) 
Female donors of menstruation age: serum HCG 
vi) 
HLA typing - See Standard Practice Manual 
A) Serotyping (HLA-A, B, C) and DNA typing (HLA-A, B, C, DRB1, DQB1 
of patient and donor. 
B) Leukocyte and/or florescence activated cell sorter cross match between 
the patient and donor. 
vii) Sickle cell analysis for any patients of African-American ethnicity per 
standard practice 
c. 
The donor will be reevaluated with a directed history and physical examination 
the day after the bone marrow harvest is completed. 
d. 
Attainment of 10 cc heparinized blood sample for subsequent determination of 
donor chimerism to the cytogenetics lab or the Clinical Immunogenetics Lab as 
outlined in section 14B3k. 
2. 
HLA-matched unrelated bone marrow or PBSC donor: 
Unrelated donors will undergo evaluation for allogeneic hematopoietic cell donation 
at the collection center by NMDP standard. The attending physician of the collection 
center will review the results of the donor evaluation. Evaluations typically include: 
a. Complete history and physical examination. 
b. Lab tests: 
i) CBC with differential and platelet counts 
ii) Serum sodium, potassium, Chloride, CO2, Glucose, Bun, Creatinine, Calcium, 
Magnesium, Phosphorus, Uric acid, LDH, alkaline phosphatase, Total bilirubin, 


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AST, ALT and albumin 
iii) 
Hepatitis screen, CMV, syphilis, HIV and HTLV I serologies 
iv) 
ABO Rh blood typing. If the donor has antibodies against red cell antigens of the 
recipient, the titers will be determined. Cytotoxic cross match between patient 
and donor (HLA Laboratory) will be performed. 
c. The donor will be reevaluated the day after the marrow aspiration. CBC with 
platelet count should be checked. 
 
14B. Recommended Patient Evaluation: 
 
Refer to FHCRC/SCCA Standard Practice Manual for Pre-Transplant Evaluation Guidelines for 
Allogeneic Transplant as clinically indicated (results of tests and/or procedures conducted as per 
standard of care for pre-transplant workups may be used for eligibility determination if conducted 
within an appropriate window prior to screening) 
 
Recommended Pre-transplant Baseline Evaluation 
 
1. History: A complete history (approximately 1 month prior to conditioning) with full 
details of the patient’s prior treatment and response including the following: 
a) Hematologic findings at diagnosis (including biochemical markers, cytogenetic and 
molecular markers, disease process, and immunologic criteria) 
b) Transfusion history (including type of donor, i.e. random or family donor) 
c) Current medical problems 
d) Current medications 
e) Female patients who have gone through puberty – pregnancy history, menstrual 
history, and date of last sexual intercourse 
f) Karnofsky/ Lansky scoring (Appendix A) 
g) Hematopoietic Cell Transplant-Comorbidity Index (HCT-CI) scoring (Appendix H) 
 
2. Physical Exam: Careful physical examination 
 
3. Laboratory evaluation (Approximately 1 month prior to conditioning): 
a) CBC with differential 
b) Reticulocyte count if clinically indicated 
c) BUN/Creatinine 
d) Total bilirubin, fractionated bilirubin, alkaline phosphatase, AST, ALT, GGT 
e) ABO/Rh typing, direct coombs 
f) Quantitative serum immunoglobulins 
g) Ferritin – for patients with a history of transfusions, bone marrow failure, or 
hemoglobinopathies 
h) Pre-transplant viral testing: 


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Pre Transplant 
Viral Testing 
All 2256 
Patients 
Patients with 
Primary Immune 
Deficiency 
Disorders 
Patients with 
Antibody 
Deficiencies 
or on IVIG 
If Clinically 
Indicated 
Hepatitis Screen 
X 
CMV Serology 
X 
VZV Serology 
X 
HSV Serology 
X 
Toxoplasma 
Serology 
X 
Anti HIV Serology 
X 
Hepatitis B PCR 
X 
Hepatitis C PCR 
X 
HIV PCR or p24 
antigen testing 
X 
CMV PCR 
X* 
EBV PCR 
X* 
X* 
Adenovirus PCR 
X* 
X* 
VZV PCR 
X 
HSV PCR 
X 
Toxoplasma PCR 
X 
*Approximately 1 month prior to conditioning 
 
i) 
Urine analysis 
j) 
As a pre-transplant reference for subsequent determination of donor chimerism 10 cc of 
heparinized peripheral blood from the patient (and the donor) will be drawn. For FHCRC 
patients: Send to Clinical Immunogenetics Lab (206-288-7700; SCCA G7-200) for a 
DNA-based assay that compares the profile of amplified fragment length polymorphisms 
(ampFLP) of the patient and donor. Outside institutions may use VNTR analysis (sex- 
matched transplants) or sex chromosome FISH-analysis (sex-mismatched transplants). 
 
4. See Recommended Disease Staging Tables for further evaluations 
 
5. Radiological evaluation: The following studies should be obtained prior to the start of 
conditioning. 
a) CXR-PA and lateral view (Other x-rays as clinically indicated) within approximately 1 month 
of HCT or as clinically indicated. If a CT scan of the chest has been done a CXR is not 
required. 
b) CT scan of the chest, abdomen and pelvis within approximately 1 month of HCT if clinically 
indicated 
c) EKG within approximately 3 months of HCT. We recommend that all patients with 
Shwachman Diamond Syndrome have an EKG within approximately 1 month of HCT. 
d) Echo or MUGA for calculation of cardiac ejection fraction within approximately 3 months of 
HCT. If unable to obtain ejection fraction, then please obtain shortening fraction calculation. 
We recommend that all patients with Shwachman Diamond Syndrome have an ECHO or 
MUGA within approximately 1 month of HCT. 
e) Pulmonary function tests if clinically indicated within approximately 1 month of HCT if older 
than age 6 and able to have pulmonary function testing done: This is particularly relevant 
for patients with Dyskeratosis Congenita and Shwachman Diamond Syndrome. 
f) Renal Ultrasound: We recommend that all patients with Dyskeratosis Congenita and 
Shwachman Diamond Syndrome have had a renal ultrasound pre HCT if this has not 
already been performed. 


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6. Bone marrow evaluation: 
A bone marrow aspirate and biopsy should be obtained within approximately 4 weeks of start of 
transplant conditioning for morphology, flow cytometry, and cytogenetics unless clinical 
concerns. Patients with bone marrow failure disorders should also have the MDS FISH panel sent 
on their bone marrow in addition to morphology, flow cytometry, and cytogenetics. If a patient’s 
transplant is delayed, the attending should discuss with the PI as to whether a bone marrow 
aspirate needs to be repeated. 
 
7. Pharmacokinetic (PK) evaluation: 
PK sampling and analysis will be done on days -6 (all patients) and -5 (patients aged <4 years 
at start of conditioning) in patients who agree to participate in the PK research studies. Same 
sample will be used for both treosulfan and treosulfan monoepoxide quantitation (samples will 
be split in PK Lab during analysis process). 
a. Day -6, n=7 samples (all patients), with window as noted below 
1. 
Pre-infusion (within 10 minutes before starting treosulfan infusion) 
2. 
End-of-infusion (window: end of infusion +5 minutes) 
3. 
End-of-infusion + 20 minutes (window: +/- 5 minutes) 
4. 
End-of-infusion + 40 minutes (window: +/- 5 minutes) 
5. 
3.5 hours after start of infusion (window: +/- 10 minutes) 
6. 
6 hours after start of infusion (window: +/- 10 minutes) 
7. 
9 hours after start of infusion (window: +/- 10 minutes) 
b. Day -5, n=7 samples (only for patients < 4 years of age at the time of start of conditioning), 
with window as noted below 
8. 
Pre-infusion (within 10 minutes before starting treosulfan infusion) 
9. 
End-of-infusion (window: end of infusion +5 minutes) 
10. End-of-infusion + 20 minutes (window: +/- 5 minutes) 
11. End-of-infusion + 40 minutes (window: +/- 5 minutes) 
12. 3.5 hours after start of infusion (window: +/- 10 minutes) 
13. 6 hours after start of infusion (window: +/- 10 minutes) 
14. 9 hours after start of infusion (window: +/- 10 minutes) 
 
Recommended Post HCT Evaluation: 
 
Once patients are discharged, they should be evaluated at least twice weekly in the outpatient 
department until day 28 and then weekly. Patients with GVHD or graft rejection should have more 
frequent follow up. Please refer to instructions below and in the Standard Practice Manual for 
guidelines regarding evaluation of patients following HCT. 
 
Note: We recommend the following studies be done following HCT and would optimally like 
all Day + 28, 84, etc. evaluations to be performed. This is a recommended evaluation 
schedule. The clinical team can decide to not do these studies if clinically indicated. 
 
1. History and physical exam: 
a) 
Once outpatient: clinic evaluation twice weekly until day 28, then weekly. More frequent 
evaluations are indicated for patients with signs of GVHD or graft failure. 
b) 
Weekly weights 
 
2. Laboratory Evaluation: 
a) CBC daily from day 0 until ANC > 500/uL for 3 days after nadir reached. Thereafter, 
a CBC should be checked three times a week until day +28. After day +28, a CBC 
should be checked twice weekly until 2 months post-transplant and later if clinically 
indicated. Daily platelet counts if the platelet count is <20,000/l. 


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b) Electrolyte panel and serum BUN/creatinine should be checked 3 times a week until 
day 28 and then twice weekly while on full dose Cyclosporine/Tacrolimus or per 
attending until Cyclosporine/Tacrolimus is discontinued. 
c) Calcium, magnesium, phosphate should be checked twice weekly until day 28 and 
then weekly. 
d) Serum albumin should be checked weekly. 
e) 
Hepatic function including AST, ALT, alkaline phosphatase, and total and direct 
bilirubin should be checked two times a week until day +28 and then every week. 
f) 
Patients on steroids should have surveillance blood cultures checked two times a 
week until steroids have been tapered to less than 0.5 mg/kg/day 
g) 
Iron studies: Ferritin level, binding capacity including serum iron, total iron binding 
capacity, direct and % transferring saturation checked around Day + 84, and 365 and yearly 
x 5 years as clinically indicated. 
h) 
Quantitative Immunoglobulins per standard practice. 
i) 
Infection monitoring per institutional standard practice, although the following 
is recommended. 
 
i. CMV surveillance: 
CMV status pre HCT 
Post HCT CMV PCR monitoring (All 
patients due to ATG administration and 
per standard practice) 
Bone Marrow 
Recipients 
+ 
Peripheral 
Blood 
Recipients 
Recipient and donor 
CMV 
Negative (-) pre HCT 
CMV PCR weekly up to 6 months post last ATG 
dose, or Absolute lymphocyte count 
>300 cells/microliter 
Recipient and/or donor CMV 
Positive (+) pre HCT 
CMV PCR weekly until off ISP. However, if off ISP 
prior to 6 months post-transplant then continue CMV 
PCR weekly monitoring until 6 months post last ATG 
dose, or Absolute lymphocyte count 
>300 cells/microliter 
Any patient treated for CMV 
before day 100 
CMV PCR weekly until off ISP. However, if off ISP 
prior to 6 months post-transplant then continue CMV 
PCR weekly monitoring until 6 months post last ATG 
dose, or Absolute lymphocyte count 
>300 cells/microliter 
Cord Blood 
Recipients 
Recipient 
CMV Negative (-) 
CMV PCR weekly up to 6 months post last ATG 
dose, or Absolute lymphocyte count 
>300 cells/microliter 
Recipient 
CMV Positive (+) 
CMV PCR 
Day 0-100 twice weekly, 
Day 101 - 1 year once weekly 
For those patients receiving both bone marrow and cord blood, we recommend CMV 
monitoring according to institutional standard practice for bone marrow recipients or for 
bone marrow recipients as detailed above. 


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ii. 
Adenovirus surveillance: 
Post HCT adenovirus PCR monitoring (all patients due to ATG 
administration) 
Adenovirus PCR weekly up to 6 months post last ATG dose, or Absolute 
lymphocyte count 
>300 cells/microliter 
 
iii. EBV lymphoproliferative syndrome surveillance: See section 16. EBV Monitoring and 
Treatment for details on treatment of EBV reactivation. 
Post HCT EBV PCR monitoring (all patients due 
to ATG administration) 
If stable copies by PCR 
EBV PCR weekly up to 6 months post last ATG 
dose, or Absolute lymphocyte count >300 
cells/microliter 
If rising copies by PCR 
EBV PCR twice weekly up to 6 months post last 
ATG dose, or Absolute lymphocyte count >300 
cells/microliter 
 
 
j) 
Patients should be assessed for the need of IVIG monitoring and replacement therapy per 
Institutional Guidelines 
 
For FHCRC patients: blood to clinical flow lab for sorting, then to the chimerism lab for 
quantifying of peripheral blood. Send to Clinical Immunogenetics Lab (206-288-7700; SCCA 
G7-200) for a DNA-based assay that compares the profile of amplified fragment length 
polymorphisms (ampFLP) of the patient and donor: 
 
Outside institutions may use VNTR analysis (sex- matched transplants) or sex 
chromosome FISH-analysis (sex-mismatched transplants). 
 
3. Bone marrow evaluation: 
All patients should have a bone marrow aspiration and biopsy for morphology, flow cytometry, and 
cytogenetics (and FISH if clinically indicated) on approximately day +84 and 1 year post-transplant. 
 
4. See Recommended Disease Staging Tables for further evaluation 
 
5. Radiology evaluation: Two view chest x-ray (PA and lateral) as clinically indicated 
 
6. Additional evaluation at day 80-100: 
Please refer to instructions below and Standard Practice Manual Guidelines or institutional 
guidelines. 
a. A patient with an uncomplicated HCT would be discharged after the day +80-100 workup 
and screening for chronic GVHD are completed and analyzed. Since the patient may be 
discharged prior to starting the Tacrolimus and MMF taper, instructions should be provided for 
preventing and detecting GVHD as per standard practice of collaborating institution. 


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GVHD evaluation guidelines are as follows (also see Appendix C & Appendix D): 
a) History and physical exam 
b) CBC, serum IgG, total bilirubin, alkaline phosphatase, ALT and AST 
c) GVHD evaluation as directed by the attending physician 
d) Pulmonary function test (if old enough to perform this test) 
e) Oral medicine evaluation 
f) Dietitian assessment 
g) Gynecological assessment (adult female) 
h) Staging workup, as indicated by staging table and text 
 
7. Additional evaluation guidelines following 100 Days post HCT 
a) First month – weekly evaluations by referring physician, including physical examination and 
weights (children <17 years – record height and weight every 3 months 
b) Weekly - CBC, BUN, creatinine, ALT, AST, Alkaline phosphatase, Total bilirubin and direct 
bilirubin. If stable, monthly evaluations as above for 2 years, then annually 
c) Following discharge from the transplant center, we would like to track the patient’s medical 
condition for the rest of their life in conjunction with their primary care physician and the 
guidance of the Long-Term Follow-Up department. 
 
 
RECOMMENDED DISEASE STAGING TABLES 
 
Timing of studies: Post-transplant time points represent guidelines for recommended 
evaluations. Due to numerous factors influencing scheduling (patient and provider availability, 
testing services limitation, as well as other factors) variation in evaluation performance dates is 
anticipated and acceptable to the protocol. The following is the recommendation; however, as 
stated above, we recognize that patients may follow up outside these windows (e.g., within +/- 
7 days of time points < Day 100; +/- 30 days for time points > Day 100 to 1 year and +/- 3 
months for time points > 1 year). 


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ALL DISEASES 
Pre 
Day 
-6 
Day 
-5 
Day 
+28 
Day 
+56 
Day 
+84 
Day 
+180 
Day 
+365 
18 
months 
24 
months 
Years 
3-5 
History and physical 
exam1 
X2 
Laboratory Evaluation 
(See baseline and post- 
HCT Laboratory 
sections) 
X 
Reference for 
subsequent 
determination of donor 
chimerism3 
X 
BMA w/ pathology, flow 
cytometry, cytogenetics, 
and FISH (see baseline 
and post-HCT bone 
marrow sections) 
X4 
X5 
X5 
PB chimerism6 
X7 
X8 
X8, 9 
X8, 9 
X8, 9 
X8, 9 
X8, 9 
X10 
Radiological evaluation 
(see baseline and post- 
HCT radiological 
evaluation sections) 
X 
Iron Studies (Ferritin, 
Iron, Total Iron Binding 
Capacity, Transferrin, 
Transferrin Saturation) 
X11 
X 
X 
X 
X 
Optional Research: 
Treosulfan PK 
Evaluations 
X12 
X13 
1 Once outpatient, twice weekly until day +28, then weekly. More frequent evaluations are indicated for 
patients with signs of GVHD or graft failure. Weekly weights. 
2 Including hematologic findings at diagnosis (including biochemical markers, cytogenetic and molecular 
markers, disease process, and immunologic criteria), transfusion history (including type of donor, i.e. 
random or family donor), current medical problems, current medications, and for female patients who have 
gone through puberty – pregnancy history, menstrual history, and date of last sexual intercourse 
3 10 cc of heparinized peripheral blood from the patient (and the donor) will be drawn. For FHCRC patients: 
Send to Clinical Immunogenetics Lab (206-288-7700; SCCA G7-200) for a DNA-based assay that 
compares the profile of amplified fragment length polymorphisms (ampFLP) of the patient and donor. 
Outside institutions may use VNTR analysis (sex- matched transplants) or sex chromosome FISH-analysis 
(sex-mismatched transplants). 
4 A bone marrow aspirate should be obtained within approximately 4 weeks of start of transplant 
conditioning for morphology, flow cytometry, cytogenetics unless clinical concerns. Patients with bone 
marrow failure disorders should have the MDS FISH panel sent on their bone marrow in addition to 
morphology, flow cytometry, and cytogenetics. If a patient’s transplant is delayed, the attending should 
discuss with the PI as to whether a bone marrow aspirate needs to be repeated 
5 All patients should have a bone marrow aspiration and biopsy for morphology, flow cytometry, and 
cytogenetics (and FISH if clinically indicated) on approximately day +84 and 1 year post-transplant. 
6 For FHCRC patients: blood to clinical flow lab for sorting, then to chimerism lab for quantifying of peripheral 
blood. Send to Clinical Immunogenetics Lab (206-667-7700; SCCA G7-200) for a DNA-based assay that 
compares the profile of amplified fragment length polymorphisms (ampFLP) of the patient and donor: 


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Outside institutions may use VNTR analysis (sex- matched transplants) or sex chromosome FISH- 
analysis (sex-mismatched transplants). 
7 For DNA storage only 
8 CD3, CD33, CD56 
9 CD19 recommended for PID patients or as clinically indicated 
10 CD3 and CD33 only. If mixed chimerism present at previous timepoint then CD56 recommended. For PID 
patients CD19 recommended if mixed chimerism present at previous timepoint or as clinically indicated. 
11 We recommend that patients with a history of transfusions, bone marrow failure disorders, or 
hemoglobinopathies have a ferritin checked pre-transplant. 
12 1 cc of blood will be drawn at 7 timepoints for treosulfan PK evaluations. PK sampling kit 
will be provided by SCCA Pharmacokinetics Lab, 206-606-7389 (after hours 206-994-5942): All patients 
13 1 cc of blood will be drawn at 7 timepoints for treosulfan PK evaluations. PK sampling kit will be provided 
by SCCA Pharmacokinetics Lab, 206-606-7389 (after hours 206-994-5942): only in patients who are < 4 
years of age at time of start of conditioning. 


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Primary Immunodeficiency Diseases 
 
The following tests are recommended for all primary immunodeficiency diseases 
Pre 
Day 
+28 
Day 
+56 
Day 
+84 
Day 
+365 
Yearly x5 
Consultation with Immunologist 
(recommend) 
X1 
X 
X 
X11 
Consultation with Infectious Disease 
(recommend)12 
X1 
Lymphocyte (phenotype) subsets3, 13 
X1 
X 
X 
X2, 5 
Lymphocyte function analysis4, 13 
X1 
X 
X 
X2, 5 
Quantitative Immunoglobulins (Serum 
troughs) 
X 
X 
X 
X 
X 
X2 
Disease Specific Tests: 
The following tests are only recommended if the patient has the specific primary immunodeficiency disease 
listed below: 
Chronic Granulomatous Disease 
• Neutrophil Oxidative Burst6 
X1 
X2 
X2 
5 
• Genetic analysis of cytosolic 
components of NADPH oxidase 
system7 
X1 
Leukocyte Adhesion defect 
• Analysis of CD 18 expression8 
X1 
X2 
X2 
5 
Wiskott-Aldrich Syndrome 
• Gene sequencing1, 9 
X1 
• Determination of WASP expression 
by flow cytometry10 
X1 
X 
X 
5 
IPEX 
• Determination of FOXP3 
expression by flow cytometry10 
X1 
X2 
X2 
5 
CD40LD 
• Determination of CD40 ligand 
expression by flow cytometry10 
X1 
X2 
X2 
5 
1 If not already completed pre-conditioning 
2 As clinically indicated 
3 2-3 ml ETDA Seattle Children’s - Cell Marker Lab (206) 987-2560 for flow cytometry to determine number 
of CD3, CD4, CD8, CD19/20, and CD16/56 cells. For patients <20 kg where blood volume is concern, 
please call Cell Marker lab for recommendations. Of note, patients with chronic granulomatous disease 
and leukocyte adhesion deficiency should not have lymphocyte phenotype testing. 
4 10 ml in Na Heparin; to Seattle Children’s Cell Marker Lab (206) 987-2560 for proliferation to mitogens 
(PHA & CD3 only) testing. For patients <20 kg where blood volume is concern, please call Cell Marker lab 
for recommendations. For outside centers send to appropriate lab. Of note, patients with chronic 
granulomatous disease, leukocyte adhesion deficiency, and HLH should not have lymphocyte function 


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testing. 
5 If most recent result abnormal 
6 1-2 ml in heparin to Seattle Children’s Cell Marker Lab (206-987-2560) or appropriate outside lab 
7 5 ml blood in EDTA (purple top) to Genedx (www.genedx.com) 
8 5ml in heparin to Seattle Children’s Cell Marker Lab 
9 Send 10mL blood in EDTA tube to Immunology Diagnostic Laboratory Seattle Children’s Research Institute 
1900 9th Avenue Seattle 98101 
10 Send 5-10 ml (green top) to Immunology Diagnostics Laboratory, Seattle Children’s Research Institute 
1900 9th Avenue Seattle 98101 
11 As directed by immunology 
12 If the infectious disease team determines that a formal consult is not needed, an infectious disease consult 
does not need to occur. 
13 If blood volume is an issue, the priority for tests is as follows (we recommend that these blood draws be 
spaced out during the pre-transplant evaluation period. If not all tests are able to be performed due to 
issues regarding blood volume contact Dr. Burroughs at (206) 667-2396) 
1. Lymphocyte phenotype 
2. Lymphocyte function 
 
Marrow Failure Syndromes 
 
The following tests are recommended for all marrow failure syndromes 
Pre 
Day 
+28 
Day 
+56 
Day 
+84 
Day 
+365 
Yearly x5 
• Quantitative Immunoglobulins 
X2 
X 
X 
X 
X 
X1 
• Consultation with Marrow 
Failure Specialist/Hematology 
(recommend) 
X2 
X 
X 
• Appropriate genetic testing 
X2 
Disease Specific Tests: 
The following tests are only recommended if the patient has the specific marrow failure syndrome listed 
below: 
Dyskeratosis Congenita 
• Telomere length analysis 
X2 
1 If clinically indicated 
2 If not already completed pre-conditioning 


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Other Diseases 
 
Pre 
Day +28 
Day +56 
Day +84 
Day 
+365 
Yearly 
X5 
Mucopolysaccharidoses 
• 
Disease specific enzyme level 
X2 
X 
X 
X 
X 
X 
• 
Consultation with Pediatric 
Specialist in Metabolic Disorders 
X2 
X 
X 
X 
• 
Marrow, skin, liver, and/or gut 
biopsy as indicated 
X2 
X 
X 
• 
MRI of Head if indicated 
X2 
X 
X 
• 
LP for storage determination if 
indicated 
X2 
X 
X 
Sphingolipidoses 
• 
Consultation with Pediatric 
Specialist in Metabolic Disorders 
X2 
X 
X 
X 
• 
Nerve Conduction studies 
X2 
X 
• 
Consultation with Pediatric 
Neurologist 
X2 
X 
X 
X 
• 
MRI of Head 
X2 
X 
X 
X 
X 
X 
Osteopetrosis 
• 
Bone biopsy for pathology1 
X2 
X 
X 
X 
• 
Head MRI 
X2 
X 
X 
X 
Hemophagocytosis Lymphohistiocytosis (HLH) 
• 
Lymphocyte phenotype (subsets)5 
X2 
X 
X 
X3 
• 
Soluble Interleukin-2 Receptor/ 
Interleukin 2 
X2 
X 
X 
Sickle Cell Disease 
• 
% Hemoglobin S 
X6 
X 
X 
Other Diseases 
• 
Disease specific markers4 
X2 
1 For FHCRC patients send to CHMC pathology. 
2 If not already completed pre conditioning 
3 If abnormal at the previous interval or if clinically indicated. 
4 Send after consultation with a specialist in the disorder 
5 For FHCRC patients, send 2-3 ml in EDTA to Seattle Children’s Cell Marker Lab (206) 987-2560 for flow 
cytometry to determine number of CD3, CD4, CD8, CD19/20, and CD16/56 cells. For patients <20 kg where 
blood volume is a concern, please call Seattle Children’s Cell Marker Lab for recommendations regarding the 
total blood volume needed to complete this study. 
6 On arrival and then within 1 week prior to start of conditioning. 


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15. DRUGS AND GRAFT ADMINISTRATION - TOXICITIES AND COMPLICATIONS. 
 
15A. Treosulfan 
 
Treosulfan Toxicities in Adults 
Very Common (10% or 
Greater) 
Common (1% - 9%) 
Uncommon (0.1% 
- 0.9%) 
Rare/unknown 
frequency 
Infections (bacterial, viral, 
fungal) 
Sepsis, septic shock 
Hyperglycemia 
Treatment-related 
second malignancy 
Pancytopenia 
Hypersensitivity 
Confusional state 
Acidosis 
Febrile neutropenia 
Decreased appetite 
Glucose tolerance 
impaired 
Leukopenia 
Insomnia 
Peripheral sensory 
neuropathy 
Electrolyte imbalance 
Neutropenia 
Headache 
Hematoma 
Agitation 
Thrombocytopenia 
Dizziness 
Hypotension 
Encephalopathy 
Anemia 
Cardiac arrhythmias 
(atrial fibrillation, sinus 
tachycardia) 
Pneumonitis 
Intracranial 
hemorrhage 
Stomatitis/mucositis 
Hypertension 
Pleural effusion 
Extrapyramidal 
disorder 
Diarrhea 
Flushing 
Pharyngeal, lung, 
or laryngeal 
inflammation 
Syncope 
Nausea 
Dyspnea 
Cough 
Paresthesia 
Vomiting 
Epistaxis 
Laryngeal pain 
Dry eye 
Abdominal pain 
Oral pain 
Hiccups 
Cardiac arrest 
Asthenic conditions 
(fatigue, asthenia, 
lethargy) 
Gastritis 
Mouth 
hemorrhage 
Cardiac failure 
Bilirubin increased 
Dyspepsia 
Abdominal 
distension 
Myocardial infarction 
Constipation 
Esophageal or 
gastrointestinal 
pain 
Pericardial effusion 
Dysphagia 
Dry mouth 
Embolism 
Maculo-papular rash 
Veno-occlusive 
liver disease 
Hemorrhage 
Purpura 
Hepatotoxicity 
Oropharyngeal pain 
Erythema 
Erythema 
multiforme 
Hypoxia 
Palmar-plantar 
erythrodysesthesia 
syndrome 
Dermatitis 
acneiform 
Dysphonia 
Pruritus 
Rash 
Gastrointestinal 
hemorrhage 
Alopecia 
Hyperhidrosis 
Neutropenic colitis 
Pain in extremities 
Non-cardiac chest 
pain 
Esophagitis 
Back pain 
Pain 
Anal inflammation 
Bone pain 
Pulmonary 
hemorrhage 
Mouth ulceration 
Arthralgia 
Hepatic failure 
Myalgia 
Hepatomegaly 
Acute kidney injury 
Hepatic pain 
Hematuria 
Generalized erythema 


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Treosulfan Toxicities in Adults 
Very Common (10% or 
Greater) 
Common (1% - 9%) 
Uncommon (0.1% 
- 0.9%) 
Rare/unknown 
frequency 
Edema 
Dermatitis 
Pyrexia 
Skin necrosis or ulcer 
Chills 
Skin hyperpigmentation 
ALT increased 
Dry skin 
AST increased 
Muscular weakness 
GGT increased 
Renal failure 
Blood alkaline 
phosphatase increased 
Cystitis 
CRP increased 
Dysuria 
Weight decreased 
Injection site reaction 
Weight increased 
Feeling cold 
Blood creatinine 
increased 
Blood LDH increased 
 
Treosulfan Toxicities in Children 
Very Common (10% or Greater) 
Common (1% - 9%) 
Uncommon/rare/unknown 
frequency 
Infections (bacterial, viral, fungal) 
Oropharyngeal pain 
Treatment-related second 
malignancy 
Pancytopenia 
Epistaxis 
Febrile neutropenia 
Leukopenia 
Dysphagia 
Alkalosis 
Neutropenia 
Oral pain 
Electrolyte imbalance 
Thrombocytopenia 
Dermatitis exfoliative 
Hypomagnesemia 
Anemia 
Maculopapular rash 
Headache 
Stomatitis/mucositis 
Rash 
Paresthesia 
Diarrhea 
Erythema 
Seizure 
Nausea 
Pain of skin 
Conjunctival hemorrhage 
Vomiting 
Skin hyperpigmentation 
Dry eye 
Abdominal pain 
Alopecia 
Capillary leak syndrome 
Pruritus 
Transaminases (ALT/AST) 
increased 
Hypertension 
Pyrexia 
Bilirubin increased 
Hypotension 
Abnormal creatinine 
Hypoxia 
Edema 
Neutropenic colitis 
Abnormal heartbeat, heart 
failure 
Anal inflammation 
Dyspepsia 
Proctitis 
Gastrointestinal pain 
Constipation 
Veno-occlusive liver disease 
Hepatomegaly 
Hepatotoxicity 
Skin ulcer 
Erythema multiforme 
Urticaria 
Dermatitis bullous 
Dermatitis acneiform 
Palmar-plantar 
erythrodysesthesia syndrome 


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Treosulfan Toxicities in Children 
Very Common (10% or Greater) 
Common (1% - 9%) 
Uncommon/rare/unknown 
frequency 
Dermatitis diaper 
Pain in extremities 
Acute kidney injury 
Renal failure 
Noninfective cystitis 
Scrotal erythema 
Chills 
Fatigue 
Pain 
GGT increased 
Pulmonary hemorrhage 
Pericardial effusion 
Pleural effusion 
Cerebral hemorrhage 
Lung failure or pneumonia 
 
Treosulfan may affect fertility in men and women. 
 
15B. Fludarabine 
 
1. Constitutional: Fever/chills, malaise, headache, myalgia, myelosuppression, 
immunosuppression, reduced performance status, allergic reactions (rare). 
2. Cardiovascular: Hypotension, cardiac arrhythmia, cardiomyopathy, cardiac failure (all 
rare). 
3. Dermatologic: Alopecia, rash. 
4. Gastro-intestinal: Mucositis, anorexia, nausea, vomiting, diarrhea, GI bleeding (rare). 
5. Hematologic/Immunologic: Myelosuppression, immunosuppression. 
6. Hepatic: Elevation of liver transaminases and/or bilirubin; rarely ascites. 
7. Neurologic: Paresthesia, weakness or CNS demyelination (all rare). 
8. Ophthalmologic: Blurred vision, diplopia, photophobia (all rare). 
9. Pulmonary: Dyspnea or pneumonitis (all rare). 
 
15C. Rabbit Anti-Thymocyte Globulin (Thymoglobulin®) 
 
1. Chills and Fever 
Chills and fever commonly occur in patients receiving Thymoglobulin®. Minor toxicities 
can usually be managed by symptomatic treatment and temporary slowing of the 
infusion. If during infusion the patient develops fever or chills or both, the patient should 
be medicated with diphenhydramine 25-50 mg I.V. (pediatric 1 mg/kg) and an 
antipyretic, e.g. acetaminophen, pediatric 10-15 mg/kg/dose, max dose 650mg orally 
every 4 hours as needed. For severe fever and chills, meperidine 25-50 mg (pediatric 1 
mg/kg) I.V. every 4-6 hours as need may also be required. 
 
2. Pruritus 
Itching and erythema occasionally develop. Symptoms are generally controlled with 
diphenhydramine. 


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3. Respiratory Distress 
Respiratory distress may be a sign of significant allergic reaction or anaphylaxis. 
Infusion should be discontinued. If reaction persists, diphenhydramine, epinephrine or 
hydrocortisone or all three should be administered. 
 
4. Hypotension 
Hypotension occurs rarely and may be a sign of significant allergic reaction or 
anaphylaxis. Discontinue infusion and treat accordingly. 
 
5. Serum Sickness 
More common with ATGAM®, it is a syndrome of fever, arthralgias, and rash. Usually 
occurs with some delay after initial ATG administration. Treat with steroids. 
 
6. Leukopenia, Neutropenia, or Thrombocytopenia 
Neutropenia and thrombocytopenia may initially occur. Leukopenia is the expected 
consequence of ATG, ATGAM® therapy. 
 
7. Other 
Pain in chest, flank or back may be a sign of significant allergic reaction or anaphylaxis 
or hemolysis. Infusion should be discontinued. Further work up and therapy as clinically 
indicated. 
 
15D. TBI – Regimen B only Unrelated Cord blood grafts 
 
TBI given at high doses in conventional transplants may cause nausea, vomiting, 
diarrhea, temporary hair loss, and painful swelling of the salivary glands for a few days. 
TBI may destroy normal bone marrow cells in addition to the cancer cells. *The doses of 
TBI (200 cGy and 300 cGy) used in this protocol is about one-sixth and one-fourth, 
respectively, of that used in conventional transplant protocols, and severe acute side 
effects have so far not been observed. TBI has been associated with causing sterility 
and there is a risk of major genetic damage to any children conceived after 
transplantation. There is a risk that a small percentage of patients may develop a 
secondary cancer resulting from this treatment. 
 
15E. Toxicities associated with marrow/PBSC infusion 
The following are potential side effects associated with the transplant in general: 
 
1. Constitutional: Fever/chills, malaise, fatigue, headache, pain, weakness. 
2. Cardiovascular: Edema, cardiac dysrhythmia, hypertension, thrombosis, cardiac 
failure. 
3. Dermatologic: Alopecia, rash, pruritus, skin depigmentation or hypopigmentation, 
photosensitivity. 
4. Gastro-intestinal: Anorexia, nausea, vomiting, diarrhea, mucositis, dry mouth. 
5. Hematologic/Immunologic: Myelosuppression, immunosuppression, 
infections/sepsis, hemorrhage, acute and chronic GVHD, graft failure. 
6. Hepatic: Elevation of liver transaminases and/or bilirubin, VOD, hepatic failure. 
7. Metabolic: Electrolyte abnormalities, hyperglycemia. 
8. Neurologic: Dizziness, confusion, anxiety, depression; rarely seizures, 
encephalopathy, demyelination. 
9. Pulmonary: Cough, dyspnea, interstitial pneumonitis. 
10. Renal/Bladder: Elevation of BUN and/or creatinine, hemorrhagic cystitis, renal 
failure. 


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15F. Toxicities associated with umbilical cord blood infusion (both related and 
unrelated) 
Potential toxicities associated with the infusion include DMSO toxicity and side effects 
from red cells. DMSO toxicity and side effect of red cells may include changes in heart 
rate or rhythm, changes in blood pressure, fever, chills, sweats, nausea/vomiting, 
diarrhea, abdominal cramping, headache, allergic reaction, presence of DMSO taste 
and odor, hemoglobinuria, and acute renal failure. 
 
15G. Toxicities associated with immunosuppression 
 
Regimen A: bone marrow or peripheral blood recipients or recipients of HLA- 
matched sibling bone marrow combined with HLA-matched sibling cord blood: 
 
1. Tacrolimus 
Side effects are generally reversible and may include: 
a) 
Renal – Rise in serum creatinine, hemolytic uremic syndrome. 
b) 
Neurological – Peripheral: paresthesia, tremor. Central: seizures, 
headache, insomnia, dizziness, depression, confusion, hallucinations, 
psychosis, myoclonus, neuropathy, agitation. 
c) 
Gastrointestinal – Nausea, vomiting, anorexia, constipation, diarrhea. 
d) 
Cardiovascular – Hypertension, myocardial hypertrophy. 
e) 
Endocrine – Hyperglycemia, hyper/hypokalemia, hypophosphatemia, 
hypomagnesemia. 
f) 
Integument – Itching, rash. 
g) 
Hematologic – Leukocytosis, thrombocytopenia, leukopenia, anemia, 
PTLD, thrombotic microangiopathy. 
h) 
Liver – Abnormal liver function tests. 
i) 
Ocular – Blurred vision, photophobia. 
j) 
Respiratory – Pleural effusion, atelectasis, cough, dyspnea. 
k) 
Musculoskeletal – Arthralgias. 
 
2. Methotrexate 
Toxicities: In this setting, mucositis is the primary toxicity related to methotrexate. In 
the setting of renal or hepatic dysfunction, where there is impaired clearance of the 
medication, methotrexate may contribute to delayed neutrophil engraftment. 
Methotrexate may cause an elevation in serum transaminase. 
 
Regimen B: Unrelated umbilical cord blood recipients 
 
3. Cyclosporine 
See section 11 for information about administration and dosage adjustments. Side 
effects are generally reversible, and may include renal insufficiency, 
hypomagnesemia, paresthesias, tremor, seizures, visual disturbances, paresis, 
disorientation, depression, confusion, somnolence, coma, nausea, hypertension, 
hemolytic-uremic syndrome, hyperglycemia, gynecomastia, and hypertrichosis. (See 
Standard Practice Manual) 
 
4. Mycophenolate Mofetil (MMF) – See section 11 for information about 
administration and dosage adjustments. 
 
a) Description 
MMF is the morpholinylethylester of mycophenolic acid (MPA) and reversibly 
inhibits inosine monophosphate dehydrogenase, particularly the type II isoform that 


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is more prominent in activated lymphocytes. As a result of the inhibition of de novo 
purine synthesis, proliferation of B and T lymphocytes is blocked and antibody 
production is inhibited. 
 
b) Storage and Administration 
MMF is available in an oral and an intravenous formulation. The oral formulation is 
supplied in 250mg hard gelatin capsules and can be stored at room temperature. 
MMF for IV administration is supplied as a lyophilized powder in a glass vial 
containing the equivalent of 500mg. 
 
c) Side Effects and Toxicity 
Precautions: MMF has been studied extensively among patients after 
nonmyeloablative HCT. Previous clinical studies in patients after allografting 
suggest that the principal adverse reactions associated with the administration of 
MMF include nausea, vomiting, neutropenia, diarrhea, and on one occasion 
bloody diarrhea. In the setting of marrow transplantation, several etiologic factors 
may contribute to alterations in gastrointestinal and hematologic parameters. 
MMF has an increased incidence of digestive system adverse events, including GI 
tract ulceration, and hemorrhage (3% of patients receiving MMF). GI tract 
perforations have rarely been observed. Most patients in these studies were also 
on other drugs known to be associated with these complications. Up to 2% of 
patients receiving MMF for prevention of rejection developed severe neutropenia 
(ANC <500). The development of neutropenia may be related to MMF itself, 
concomitant medications, viral infections or some combination of these causes. 
MMF dose adjustments will be made if clinically indicated if in the opinion of the 
attending physician, no other cause is thought to be responsible for the 
abnormality. These adjustments should be discussed with the principal 
investigator and documented in the medical records and the clinical reporting form 
(CRF). 
 
16. EBV Monitoring and Treatment 
Patients will have EBV by PCR plasma samples collected once a week until day 100 following 
transplantation. If there is documented evidence of rising copy number of EBV by PCR, monitoring 
will increase to twice per week through day 100 following HCT. EBV monitoring by PCR will be done 
every other week from Day 101 until Month 6. If there is evidence of a rising copy number, the 
frequency of the monitoring will be increased to once or twice a week as clinically indicated according 
to institutional standard. 
 
A) 
Patients who develop a viral load of > 1000 copies per mL plasma will receive further 
individualized monitoring, evaluation by Transplant Infectious Disease service according to 
institutional practice and/or therapy as per institutional standards and Infectious Disease 
recommendations. 
B) 
If the patient develops increasing viral load, it is suggested that he or she be treated with either 
preemptive Rituximab 375 mg/m2 IV weekly x 4 weeks or an EBV reactivation protocol 
according to local institution practice and ID recommendation. It is suggested that subjects 
who develop EBV reactivation and/or asymptomatic increasing viral load by PCR undergo flow 
cytometry of the peripheral blood and CT or MRI imaging of chest, abdomen, and pelvis to 
assess the development of lymphadenopathy, organomegaly, or other evidence of the 
development of PTLD. Whenever possible, the clinical diagnosis of PTLD should be confirmed 
by tissue biopsy. 
 
17. DATA AND SAFETY MONITORING PLAN AND ADVERSE EVENT REPORTING 
17A. Monitoring the Progress of Trials and the Safety of Participants 


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The principal investigators (PI) monitor multi-institutional clinical trials, with oversight by the 
Medical Monitor, a Data Safety and Monitoring Board (DSMB), the Data and Safety Monitoring 
Committee (DSMC) and the Institutional Review Board (IRB). The PI reviews outcome data for 
each individual patient on an ongoing basis. 
 
This multi-center protocol has a DSMB responsible for monitoring patient safety by reviewing 
protocol data and safety endpoints. The DSMB meets twice a year and all outcome data is 
reviewed including all adverse events reported to the coordinating center (FHCRC) along with 
those officially reported to the FHCRC IRO. The DSMB confirms that the trial has not met any 
stopping rules and reviews any patient safety problems necessitating discontinuation of the trial. 
A report from the DSMB is submitted to the FHCRC IRB, the IND sponsor, the DSMC Chairman, 
Protocol Office, and the trial coordinators/local PIs of this protocol. The DSMB will discontinue 
the review of outcomes when this protocol is closed to accrual. Furthermore, the FHCRC also has 
a DSMC that reviews the progress of the protocol with respect to the monitoring plan at the time 
of each annual renewal. As with initial review, annual IRB review and approval is also required. 
 
Flow of information concerning clinical trial participants originates with the clinicians and nurses 
in the clinic or referring clinicians at other institutions and is transmitted to the FHCRC Trial 
Coordinator. At the FHCRC, health care providers and rotating attending physicians assess 
patients and record their observations regarding toxicity and response outcomes in the medical 
record. Thus, multiple health care providers provide independent observations and participate in 
monitoring this trial. The PI may be a clinician for some patients entered on this trial. However, 
assessments are the sum total of the primary health care provider (fellow or physician 
assistant), floor or outpatient nurse and the PI or other attending clinician involved with the 
patient averting possible conflict of interest having the PI as the attending clinician for protocol 
patients. If determination of adverse events is controversial, co-investigators will convene on an 
ad hoc basis as necessary to review the primary data and render a decision. 
 
17B. Adverse Event Definitions 
 
a. Adverse Event 
An Adverse Event (AE) is any untoward medical occurrence in a clinical investigation subject 
administered a medicinal product; the event does not necessarily have a causal relationship 
with study drug administration or usage. An adverse event can therefore be any unfavorable 
and unintended sign (including an abnormal laboratory finding), symptom, or disease 
temporally associated with the use of a medicinal product, whether or not considered related to 
the medicinal product. 
 
b. Serious Adverse Event 
A serious adverse event (SAE) is defined as an untoward medical occurrence that results in 
any of the following outcomes: 
• 
Death. 
• 
Life-threatening situation (ie, with an immediate risk of death from the event as it 
occurred but not including an event that, had it occurred in a more serious form, might 
have caused death). 
• 
In-patient hospitalization or prolongation of existing hospitalization. Inpatient 
hospitalization comprises formal admission to a hospital for medical reasons, for any 
length of time, whether or not hospitalization extends overnight. However, hospital 
admissions for administration of the study drug, procedures required by the study 
protocol, or tumor-related diagnostic procedures are not considered serious. 


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• 
Persistent or significant disability/incapacity or substantial disruption of the ability to 
conduct normal life functions. 
• 
Congenital anomaly/birth defect. 
• 
An important medical event that requires intervention to prevent one of the above 
outcomes. 
 
c. Unexpected Adverse Event 
An unexpected adverse event is defined as an event that has a nature or severity, or frequency 
that is not consistent with the applicable investigator brochure. “Unexpected,” as used in this 
definition, refers to an adverse drug experience that has not been previously observed and 
reported rather than an experience that has not been anticipated based on the pharmacological 
properties of the study drug. 
17C. 
Monitoring and recording AEs 
 
Adverse events will be collected and graded using the modified (for HCT) NCI Common 
Toxicity Criteria (Appendix G of the protocol). All grade 3 and 4 adverse events (or highly 
unusual grade 2 adverse events), which occur from the start of study treatment (pre-transplant 
conditioning) through day 100 post-transplant, will be assessed by the investigator or qualified 
designee and recorded in the CRF, whether or not attributed to the study intervention. Adverse 
events that occur between the start of conditioning and day 30 will be assessed separately to 
identify early regimen related toxicities.  The investigator should attempt to establish a 
diagnosis of the event on the basis of signs, symptoms and/or other clinical information. In such 
cases, the diagnosis should be documented as the adverse event and/or serious adverse event 
and not described as the individual signs or symptoms. The following information should be 
recorded: 
• 
Description of the adverse event using concise medical terminology 
• 
Description as to whether or not the adverse event is serious 
• 
The start date (date of adverse event onset) 
• 
The stop date (date of adverse event resolution) 
• 
The severity (grade) of the adverse event 
• 
A description of the potential relatedness of the adverse event to study drug or a study 
procedure 
• 
The action taken due to the adverse event 
• 
The outcome of the adverse event 
 
The adverse event reporting in this multi-institution clinical trial will follow both the sponsor and 
IRB guidelines for serious adverse event (SAE) reporting. The classification of an adverse event 
determines what reporting procedure to follow for reporting the adverse event. (See Table 2 
and 3). 


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17D: Grading of the severity of an Adverse Event 
 
Adverse events will be graded according to CTCAE criteria. If a CTCAE criterion does not exist, 
the investigator should use the grade or adjectives: Grade 1 (mild), Grade 2 (moderate), Grade 
3 (severe), Grade 4 (life-threatening), or Grade 5 (fatal) to describe the maximum intensity of the 
adverse event. 
 
Attribution of Adverse Event 
 
Association or relatedness to the study agent will be assessed by the investigator as follows: 
 
• 
Definite: The event follows a reasonable temporal sequence from exposure to the 
investigational agent, has been previously described in association with the investigational 
agent, and cannot reasonably be attributed to other factors such as the patient’s clinical 
state, other therapeutic interventions or concomitant medications; AND the event 
disappears or improves with withdrawal of the investigational agent and/or re-appears on 
re-exposure (e.g., in the event of an infusion reaction). 
• 
Probable: The event follows a reasonable temporal sequence from exposure to the 
investigational agent and has been previously been described in association with the 
investigational agent OR cannot reasonably be attributed to other factors such as the 
patient’s clinical state, other therapeutic interventions or concomitant medications. 
• 
Possible: The event follows a reasonable temporal sequence from exposure to the 
investigational agent, but could be attributable to other factors such as the patient’s clinical 
state, other therapeutic interventions or concomitant medications. 
• 
Unlikely: Toxicity is doubtfully related to the investigational agent(s). The event may be 
attributable to other factors such as the patient’s clinical state, other therapeutic 
interventions or concomitant medications. 
• 
Unrelated: The event is clearly related to other factors such as the patient’s clinical state, 
other therapeutic interventions or concomitant medications. 
 
For general AE assessment, an AE is considered related if it is assessed as definitely, probably, 
or possibly related; unrelated if it is assessed as unlikely related or unrelated. For determination 
of IND safety reporting, AE attribution will be assessed according to the suspected adverse 
reaction definition described in 21 CFR 312.32 as an AE for which there is a reasonable 
possibility that the drug caused the adverse event where “reasonable possibility” means there is 
evidence to suggest a causal relationship between the drug and the AE. Suspected adverse 
reactions that are both serious and unexpected will be reported to the FDA as an IND safety 
report, in accordance with regulations under 21 CFR 312.32. 
 
17E: Adverse Event Reporting Period 
 
All grade 3 and 4 adverse events (or highly unusual grade 2 adverse events), which occur from 
the start of study treatment (pre-transplant conditioning) through day 100 post-transplant, will be 
assessed by the investigator or qualified designee and recorded in the CRFs, whether or not 
attributed to the study intervention. AEs with an onset date prior to the start of study treatment 
will not be recorded, except in the case of clinically significant worsening of the AE during the 
specified monitoring time frame. The end of the adverse event reporting period occurs through 
day 100 post-transplant, or when any ongoing drug-related adverse events and/or serious 
adverse events have resolved or become stable. A subject withdrawn from the study because of 
an adverse event must be followed until the clinical outcome from the adverse event is 
determined. 


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All adverse events reportable to the IRB will be collected according to current reporting FHCRC 
IRB Policies for Reportable Events. Definitions, instructions and forms associated with 
reportable events can be found on the FHCRC’s Institutional Review Office (IRO) extranet 
website: http://extranet.fhcrc.org/EN/sections/iro/irb/ae.html. All investigators must report 
adverse events to their respective IRB as locally mandated. 
 
All events which in the opinion of the PI are (1) unexpected, and (2) related or possibly related 
to the research and (3) serious or suggest that the research places research participants or 
others at a greater risk of physical or psychological harm than was previously known or 
recognized will be submitted to the IRB within 10 calendar days (see Tables 2, 3, and 4 below). 
In addition, all serious adverse events are reported to the IRB at the time of annual renewal. The 
PI for a study is responsible for this reporting and the IRO assures adverse event reporting on 
an annual basis. The PI in the annual application for grant continuation will summarize reports of 
toxicities. Furthermore, stopping rules and interim analysis provides an additional safeguard for 
adverse event analysis and reporting in this protocol. All collaborating PIs have fulfilled all NIH 
requirements for training in human subject’s protection. 
 
The FHCRC PI and research staff will meet regularly to review all reported events. If the event 
meets FHCRC IRB current reporting obligations, it will be sent to the FHCRC IRB. After the 
FHCRC IRB has reviewed the event report it will be disseminated to all participating 
sites/investigators, the DSMB, and the study sponsor. 
 
When a patient experiences disease recurrence or graft failure and goes on to further treatment 
off protocol, adverse events will no longer be collected with the exception of death. 
 
Patients enrolled in this study are receiving treatments that are generally associated with high 
rates of expected adverse events (outlined in Appendix I). 
 
17.F. Reporting to Coordinating Center and Fred Hutch IRB 
 
For patients being cared for at the FHCRC, heath care providers communicate with the PI or 
research staff as events occur triggering subsequent reporting. For patients not being cared 
for at the FHCRC the outside facilities communicate with the PI or research study staff for 
these reporting purposes. 
 
As FHCRC is the coordinating center, all reportable events will be collected by the FHCRC PI 
and/or study staff for submission to FHCRC IRB. All reportable events should be submitted to 
the coordinating center on the relevant FHCRC IRB forms. (See Tables 2 and 3 for FHCRC 
IRB Reporting Policies and Forms for Reporting) 
 
Table 2 FHCRC IRB Policies for Reportable Events (Relevant FHCRC policies include, but 
are not limited to the following documents. Please also refer to the FHCRC IRO website.) 


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IRB 
Policy 2.6 
Unanticipated Problems 
Involving Risks to Subjects 
or Others (Adverse Events) 
http://extranet.fhcrc.org/EN/sections/iro/irb/ae.html 
IRB 
Policy 1.9 
Noncompliance with the 
Office of the Director’s 
Human Research Protection 
Program Policy 
http://extranet.fhcrc.org/EN/sections/iro/irb/ae.html 
IRB 
Policy 
1.11 
Reporting Obligations for 
Principal Investigators 
http://extranet.fhcrc.org/EN/sections/iro/irb/policy/ 
IRB 
Policy 2.2 
Continuing Review 
http://extranet.fhcrc.org/EN/sections/iro/irb/policy/index.html 
IRB 
Policy 
1.13 
Investigational New Drugs 
(IND), Biologics and 
Investigational Device 
Exemptions (IDE) 
http://extranet. 
fhcrc.org/EN/sections/iro/irb/policy/index.html 
 
Table 3 Fred Hutch IRB Forms for Reporting 
 
Adverse Event Reporting Form 
http://extranet.fhcrc.org/EN/sections/iro/irb/forms/index.html 
Expedited Reporting Form for 
Unanticipated Problems or 
Noncompliance 
http://extranet.fhcrc.org/EN/sections/iro/irb/forms/index.html 
 
17.G Research Site Reporting Requirements to Sponsor 
 
Classification of an event as serious or non-serious determines the reporting procedures to 
be followed by the site for reporting the event to the Sponsor. 
 
The information in the Institution-Sponsored IND SAE Reporting Form (see Appendix E) 
must match or be reconciled with the information recorded in the adverse events section of 
the CRF and study database. For example, the same adverse event term should be used on 
both forms. 
 
All SAEs must be reported to the Sponsor from the time of study drug administration until 
SAE duration as outlined in the protocol. SAEs that occur after the study-specific informed 
consent is signed but prior to the first dose of the investigational agent will be collected only if 
they are considered by the investigator to be causally related to the study required 
procedures. SAEs will be reported by the PI or Research Nurse to the Sponsor according to 
the timelines described in Table 4. 


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Table 4: Reporting Requirements to Sponsor for Adverse Events 
 
Classification 
Reporting 
time 
Reporting 
Time 
Reporting Action 
Contact Information 
Serious 
Adverse 
Event (SAE) 
Fatal or 
life- 
threatening 
Within 24 
hours of 
research team* 
awareness 
Email notification to 
sponsor and to both the 
Sponsor’s Medical Monitor 
and the PI 
IND Sponsor’s Medical 
Monitor email: 
cdelaney@fredhutch.org 
IND Sponsor’s email: 
ISIOC@fredhutch.org 
PI’s Email: 
lburroug@fredhutch.org 
All SAEs 
Within 2 
business days 
of research 
team* 
awareness 
Submit completed IND 
SAE Reporting Form 
signed by PI or designated 
sub-Investigator to both 
the IND Sponsor and the 
PI 
IND Sponsor’s email: 
ISIOC@fredhutch.org 
PI’s Email: 
lburroug@fredhutch.org 
Non-serious Adverse 
Event 
Per CRF 
completion 
guidelines 
Record information on 
appropriate CRFs 
N/A 
*Research team is defined as the individuals listed on the delegation of authority log. Physicians listed on the 
study’s delegation of authority log as transplant service attending physicians delegated authority to 
administer informed consent will not be considered part of the research team unless additional 
responsibilities related to the conduct of the study have been delegated to them by the Principal Investigator. 
 
17H: FHCRC Sponsor Reporting Requirements 
 
The sponsor assumes responsibility for IND safety reporting to the FDA and participating 
investigators, in accordance with regulations under 21 CFR 312.32. 
Each serious adverse event report received from the investigator will be evaluated by the Medical 
Monitor who will assess the seriousness of the event (see Section 17B.b), the expectedness of the 
event (see Section 17.B.c), and the relationship to participation in the study (see Section 17.D). For 
regulatory reporting purposes, the Sponsor will determine expectedness relating to treosulfan 
using safety information specified in the treosulfan investigator brochure. An event will be classified 
as related if either the investigator or the Sponsor determines that the event may be related to the 
study drug. 
 
The Sponsor or its designee will provide all investigators with a safety letter notifying them of an 
event that meets FDA IND Safety Reporting criteria. Investigators will be requested to provide 
written notification of safety report to the FHCRC IRB as soon as is practical, consistent with IRB 
requirements. 
 
17I. Plans for Assuring that any Action Resulting in a Temporary or Permanent Suspension 
of an NCI-funded Clinical Trial is Reported to the NCI Grant Program Director Responsible 
for the grant 
This clinical research trial is associated with an Investigational New Drug (IND). Any temporary or 
permanent suspension, as determined by the PI, IRB, IND Sponsor, or DSMC, of this clinical 
research trial will be reported to the NCI grant program director by the PI. 


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17J. Plans for Assuring Data Accuracy and Protocol Compliance 
The study has dedicated research staff who follow patients to confirm eligibility; reporting of 
adverse events; reporting of events, which are part of the safety-monitoring plan, and protocol 
adherence. The PI, data coordinators, and research staff are responsible for review and 
maintenance of all patient records to ensure data integrity and protocol adherence. 
 
At the FHCRC, health care providers and rotating attending physicians assess patients and record 
their observations in the medical record. This documentation is extracted by study research staff 
via chart review and entered into an electronic Case Report Forms (CRF’s). The study staff also 
continue to follow patients after day 100, review source documentation, and complete CRFs at 6 
months, 1 year, 1.5 years, and then annually per protocol. The CRFs are printed directly from the 
database, and the PI reviews the CRFs and the primary source documents for data accuracy. After 
the CRFs are verified, they are signed by the PI. Thus, multiple health care providers provide 
independent observations and participate in assessments on this trial. 
 
Outside center clinical trial participant’s information is transmitted to the FHCRC Trial Coordinator. 
Clinical outcome data are summarized and transmitted from collaborating centers as CRFs. CRFs 
from external sites are verified and signed by the local investigators at that site. The CRFs are 
generated from the collaborating centers at protocol defined time points. When possible, primary 
source documents regarding patient outcomes are collected with patients’ names removed and 
replaced by Unique Patient Numbers (UPNs). The data are incorporated into a central database by 
the data manager. Collaborating sites send signed consents, eligibility forms, and CRFs with 
source documents demonstrating eligibility, treatment, and serious adverse events (if applicable) to 
the study staff. These are reviewed for eligibility, adherence to the protocol, accuracy, and 
completeness by the study staff. 
 
The study is monitored under the FHCRC Monitoring Plan. The FHCRC Data and Safety 
Monitoring Plan details the full scope and extent of monitoring and provides for immediate action in 
the event of the discovery of major deviations. 
 
18. RECORDS 
Clinical records will be maintained as confidentially as possible by all collaborating institutions. 
Data will be collected from the collaborating institutions and will be maintained by Clinical Statistics. 
The investigator will ensure that data collected conform to all established guidelines for coding, 
collection, key entry and verification. Each patient is assigned a unique patient number to assure 
patient confidentiality. Any publication or presentation will refer to patients by this number and not 
by name. The licensed medical records department, affiliated with the institution where the patient 
receives medical care, maintains all original inpatient and outpatient chart documents. Patient 
research files are kept in a locked room. They are maintained by the FHCRC data collection staff, 
which is supervised by an A.R.T. Access is restricted to personnel authorized by the Division of 
Clinical Research. 
 
19. STATISTICAL CONSIDERATIONS AND TERMINATION OF STUDY 
The primary objective of this clinical trial is to evaluate, within the limits of a phase II study, the 
preliminary efficacy as defined by engraftment, of a regimen consisting of treosulfan and 
fludarabine followed by allogeneic HCT in patients with nonmalignant inherited disorders. We plan 
to enroll at least 30 patients on this study. A minimum of 30 patients will be enrolled onto Arm A. 
The protocol will continue to accrue until this goal is met. There will be no set limit for Arm B: the 
cord blood arm. Analyses will be carried out separately between Arm A and B. We will be 
encouraged to continue study of this treatment if at least 27 of 30 patients engraft. Table 5 shows 
the probability of observing 27 or more successes among 30 patients for several possible true 
rates of engraftment. 


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The protocol was modified in January 2011 and September 2011. Due to a higher than anticipated 
incidence of acute grade III-IV GVHD for both arms in the first 13 patients (5/13 = 38%; 3/8 bone 
marrow recipients, 2/5 cord blood recipients) the protocol was modified to add rabbit ATG 
(thymoglobulin) to both arms initially for unrelated recipients (January 2011) and subsequently for 
the related recipients (September 2011). In addition, due to graft rejection (n=1) and low donor 
chimerism/inadequate disease response (n=1) in 2 of the 5 patients enrolled onto the cord blood 
arm, the cord blood arm was modified to increase the TBI from 200 cGy to 300 cGy TBI for non T- 
cell deficient patients. The revised protocol will reset the target accrual to at least 30 patients as of 
January 2011, with a minimum enrollment of 30 onto Regimen A (as stated above). The efficacy 
analysis will be performed separately for the initial 13 patients (Regimen A: n=8, Regimen B: n=5) 
and those accrued under the modified regimen. Only patients enrolled after this protocol 
modification will be counted towards the stopping rules. 
 
Table 5. Probability of observing 27 or more successes in 30 patients 
True rate of engraftment 
Pr(≥ 27 successes) 
70% 
0.01 
80% 
0.12 
90% 
0.65 
95% 
0.94 
 
The incidence of day 200 transplant-related mortality (TRM) will be monitored in all patients. If 
there is sufficient evidence suggesting that the true incidence of day 200 TRM exceeds 25% on 
either arm, enrollment on that arm will be suspended and the DSMB will be convened to make 
recommendations regarding protocol modification. Sufficient evidence will be an observed rate of 
day 200 TRM that corresponds to a one-sided 80% confidence interval with a lower limit greater 
than 25%. These limits will be examined after every 5th enrolled patient becomes eligible for 
evaluation. In practice, the study arm will be suspended if we observe the following number of 
TRM in study patients: 3/5, 5/10, 6/15, 8/20, 9/25, 10/30, 12/35, 13/40, 15/45 or 16/50 per disease 
subgroup per study arm (Regimen A versus Regimen B). Table 6 summarizes the operating 
characteristics of this stopping rule, for enrollment of up to 50 patients per disease subgroup per 
study arm (Regimen A versus Regimen B) as estimated from 5,000 Monte Carlo simulations. We 
will apply stopping rules to regimens A and B separately. 
 
The protocol related to regimen B was modified in April 2012. Stopping rules for excess day 200 
TRM were met in October 2011 due to 3 deaths occurring in the 5 patients enrolled since the 
January 2011 protocol modifications (1 due to multi-system organ failure; 1 due to ARDS/diffuse 
alveolar damage; and 1 due to gram positive sepsis (strep viridans) with multi-system organ failure, 
questioned possible sinusoidal obstructive syndrome/veno-occlusive disease). All deaths occurred 
before day 100, with two very early on post-transplant day 1 and day 7. However, the causes of 
death did not appear to be related to any single part of the regimen. The Data Safety Monitoring 
Board met and reviewed the data and recommended the following: The revised protocol will allow 
up to 5 more patients to be enrolled on regimen B before reconvening the DSMB, with each patient 
enrolled no sooner than 28 days following the date of transplantation (defined as the date of stem 
cell infusion) of the previous patient. The stopping rules for this arm (Regimen B) were not reset 
and will continue as stated in the protocol. The next stopping rule would be triggered at 5/10. 
Therefore, if 2 of the next 5 patients enrolled dies on Regimen B (cord blood), the stopping rule 
would be triggered and the DSMB will reconvene. 


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Table 6. Probability of stopping the trial for excess day 200 TRM 
Number of patients 
True rate of event 
Probability of stopping 
10 
15% 
0.03 
15 
15% 
0.04 
20 
15% 
0.04 
25 
15% 
0.05 
10 
35% 
0.32 
15 
35% 
0.49 
20 
35% 
0.54 
25 
35% 
0.63 
10 
45% 
0.57 
15 
45% 
0.78 
20 
45% 
0.84 
20 
45% 
0.91 
 
The incidence of graft rejection will also be monitored in all patients. Regimen A and B will be 
monitored/analyzed separately. If there is sufficient evidence to suggest that the true incidence of 
graft rejection exceeds within the first 365 days 10% on either arm, enrollment will be suspended 
on that arm and the DSMB will be convened to make recommendations regarding protocol 
modification and/or dose escalation. Sufficient evidence will be an observed rate of graft rejection 
that corresponds to a one-sided 80% confidence interval with a lower limit greater than 10%. 
These limits will be examined after every 5th enrolled patient becomes eligible for evaluation. In 
practice, the study arm will be suspended if we observe the following number of graft rejections in 
study patients: 2/5, 3/10, 3/15, 4/20, 5/25, 5/30, 6/35, 7/40, 7/45 or 8/50 per disease subgroup per 
study arm (Regimen A versus Regimen B). Table 7 summarizes the operating characteristics of 
this stopping rule for enrollment of up to 50 patients per disease subgroup, as estimated from 
5,000 Monte Carlo simulations. 
 
Table 7. Probability of stopping the trial for excess graft rejection 
Number of patients 
True rate of event 
Probability of stopping 
10 
5% 
0.03 
15 
5% 
0.05 
20 
5% 
0.05 
25 
5% 
0.05 
10 
20% 
0.38 
15 
20% 
0.62 
20 
20% 
0.68 
25 
20% 
0.72 
10 
30% 
0.67 
15 
30% 
0.88 
20 
30% 
0.93 
25 
30% 
0.95 
 
Therefore, stopping rules will be imposed for: 
 
Day 200 TRM > 25% (3/5, 5/10, 6/15, 8/20, 9/25, 10/30, 12/35, 13/40, 15/45, 16/50) per disease 
subgroup per study arm (Regimen A versus Regimen B). 
 
Day 365 Graft rejection (defined as CD3 T cell chimerism < 5%) > 10% (2/5, 3/10, 3/15, 4/20, 5/25, 
5/30, 6/35, 7/40, 7/45, 8/50) per disease subgroup per study arm (Regimen A versus Regimen B) 


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Day 100 Grade III/IV GVHD >15% for Arm A +rATG PBSC grafts 2/5, 3/10, 4/15, 5/20, 6/25, 7/30, 
8/35, 9/40, 10/45, or 11/50 
 
The dosing of treosulfan on protocol 2256 was modified in June 2013, specifically the dose of 
Treosulfan was decreased for smaller children. However, three of the first five patients treated with 
the reduced dose have experienced poor engraftment. Therefore the original dose of treosulfan 
was reinstituted in April 2014. The protocol accrual will continue to a total of 60 patients in Arm A 
with Treosulfan/Fludarabine/rATG, so as to ensure adequate numbers of smaller children will be 
treated at the original dose, since among the original 30 patients only a small number were <0.5 
m2 body surface area. As stated above, the initial 8 patients transplanted on Regimen A with 
Treosulfan and fludarabine only do not count towards the accrual goal of 60. Therefore the total 
number of patients accrued on Regimen A will be 68. The same stopping rules for TRM and 
rejection applied to the first 30 patients in Arm A will be applied separately to the additional 30 
patients in Arm A; however, accrual to Arm B will continue as before under the same stopping 
rules. 
 
In October/November 2015 Regimen A of the protocol was modified to increase accrual to a 
minimum of 20 patients for each subgroup of patients (for example, primary immunodeficiency 
diseases, hemophagocytic disorders, hemoglobinopathies, and marrow failure disorders) who 
receive Treosulfan, fludarabine, and rabbit ATG. The protocol will continue to accrue until a 
minimum of 20 patients are in each subgroup, in order to better evaluate the safety and 
effectiveness of the regimen in each subgroup. There is no limit to the overall accrual to Regimen 
A as long as the minimum of 20 patients per subgroup has not been reached and each subgroup 
will continue to accrue until all subgroups reach a minimum of 20 patients. We estimate the overall 
accrual will be 100-120 patients. Stopping rules for TRM and graft rejection will be applied 
separately in each disease subgroup of patients who receive Treosulfan, fludarabine, and rabbit 
ATG conditioning. There are additional patients with nonmalignant diseases that do not fit into one 
group that have enrolled onto the protocol and will continue to enroll onto the protocol such as 
metabolic disorders, autoimmune disorders, or other rare nonmalignant diseases. We will continue 
to enroll these patients but they will not count towards the stopping rules or enrollment since we do 
not anticipate very many of these patients. However, they will be reviewed by the data safety 
monitoring board. Accrual to Arm B will continue as before under the same stopping rules. 
 
In March 2017, the protocol was modified to allow the use of both bone marrow and cord blood 
from the same HLA-matched sibling donor. Specifically, at the discretion of the PI, bone marrow 
and cord blood cells from the same HLA-matched sibling donor may be used together for 
transplantation in cases where such cord blood cells were previously stored. The patients enrolled 
onto the protocol who receive both bone marrow and cord blood from the same HLA-matched 
sibling donor will be enrolled onto Regimen A of the protocol and analyzed with this group. 
 
In March 2018, the protocol was modified to prioritize PBSC (Regimen A) as the preferred stem 
cell source when feasible. This change was made with the goal of improving donor chimerism. 
PBSC has a higher risk of graft versus host disease; therefore, a stopping rule for grade III-IV 
GVHD will be added to the protocol – Regimen A only. Since bone marrow will still be allowed on 
Regimen A, we will apply this stopping rule to PBSC transplants. If there is sufficient evidence to 
suggest that the true incidence of grade III-IV GVHD at day 100 exceeds 15% (Regimen A), 
enrollment will be suspended on that arm and the DSMB will be convened to make 
recommendations regarding protocol modification. The stopping rule would be triggered if 2 of 5, 3 
of 10, 4 of 15, 5 of 20, 6 of 25, 7 of 30, 8 of 35, 9 of 40, 10 of 45, or 11 of 50 experience grade III-IV 
GVHD. This stopping rule will not be by disease subgroup but for all PBSC transplants.  The 
current PBSC transplants on the protocol will be included in this stopping rule. 


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41. 
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44. 
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Leukemia and Lymphoma. 2007;48(12):2352-2359. 
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induce durable complete chimerism and sustained remissions in patients with hematological diseases. 
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Transplant. 2017;24(3):529-536. 


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APPENDICES – TABLE OF CONTENTS 
 
 
 
APPENDIX A 
KARNOFSKY/LANSKY 
 
APPENDIX B 
STANDARD PRACTICE GUIDANCE DOCUMENTS 
APPENDIX C 
ACUTE GRAFT-VERSUS-HOST DISEASE GRADING 
APPENDIX D 
CHRONIC GRAFT-VERSUS-HOST DISEASE GRADING 
APPENDIX E 
INSTITUTION-SPONSORED IND SAE REPORTING FORM 
APPENDIX F 
PATIENT DEMOGRAPHICS AND ELIGIBILITY FORM 
APPENDIX G 
ADAPTED COMMON TOXICITY CRITERIA FOR HCT 
APPENDIX H 
HEMATOPOIETIC CELL TRANSPLANT-COMORBIDITY INDEX 
APPENDIX I 
ADVERSE EVENTS ASSOCIATED OR EXPECTED WITH HCT 


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APPENDIX A 
Karnofsky and Lansky Performance Status Scales 
Karnofsky Performance Status Scale 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
REFERENCE 
Karnofsky DA: Meaningful clinical classification of therapeutic responses to anti-cancer drugs. 
Editorial: Clin Pharmacol Ther 2:709-712, 1961. 
 
Lansky Play Performance Status Scale 
Percentage 
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 activities 
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; 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 
Unresponsive 
0 
Dead 
Percentage 
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 
Cares for self; unable to carry on normal activity or do active work 
60 
Requires occasional assistance, but is able to care for most of his/her needs 
50 
Requires considerable assistance and frequent medical care 
40 
Disabled; requires special care and assistance 
30 
Severely disabled, hospitalization indicated. Death not imminent 
20 
Very sick, hospitalization necessary, active supportive treatment necessary 
10 
Moribund, fatal processes, progressing rapidly 
0 
Dead 


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APPENDIX B 
STANDARD PRACTICE GUIDANCE DOCUMENTS 
 
The protocol references institutional standard practice for trial conduct. The standard practice 
documents utilized at Fred Hutch include the following: 
 
• 
Herpes Simplex and Varicella Zoster Virus Prevention and Treatment, 6/6/18 
• 
CMV Prevention: Surveillance and Preemptive Therapy, 08/16/17 
• 
CMV Disease: Diagnosis and Treatment, 10/19/16 
• 
Antifungal Therapy Guidelines, 03/16/16 
• 
Pneumonia/Pneumocystis Carnii Prophylaxis, 10/19/16 
• 
Antibiotic Prophylaxis for Encapsulated Bacteria in Allogeneic Patients with Chronic 
GVHD requiring Immunosuppressive Therapy, 10/19/16 
• 
Vaccinations, 6/21/18 
• 
Foscarnet, 08/16/17 
• 
TBI Adult Non Myeloablative 
• 
TBI Pediatric NON Myeloablative 
 
Most recent versions of these documents can be obtained upon request to the coordinating center. 


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APPENDIX C 
ACUTE GRAFT-VERSUS-HOST-DISEASE STAGING AND GRADING TABLES 
 
 
Clinical Stage of Acute GVHD According to Organ System 
Stage 
Skin 
Liver 
Intestine (1) 
1 
Maculopapular rash <25% 
of body surface 
Bilirubin 2-3 mg/dl 
>500-1000 mL diarrhea 
per day or (nausea, 
anorexia or vomiting with 
biopsy (EGD) confirmation 
of upper GI GVHD 
2 
Maculopapular rash 25-50% 
of body surface 
Bilirubin 3.1-6 mg/dl 
>1000 -1500 mL diarrhea 
per day 
3 
Maculopapular rash >50% 
body surface area or 
Generalized erythroderma 
Bilirubin 6.1-15 mg/dl 
>1500 mL diarrhea per 
day 
4 
Generalized erythroderma 
with bullous formation and 
desquamation 
Bilirubin >15 mg/dl 
Severe abdominal pain 
with or without ileus 
 
 
Overall Clinical Grading of Severity of Acute GVHD 
Grade 
Skin 
Liver 
GI 
I 
1-2 
0 
0 
IIA 
3 and/or 
1 and/or 
1 
IIIA,B 
4 and/or 
2-4 and/or 
2-4 
IVA,C 
4 and/or 
2-4 and/or 
2-4 
 
A. Grade II-IV GVHD with only single organ involvement should be biopsy confirmed. 
B. Non-fatal GVHD 
C. Fatal GVHD 
 
(1) For pediatric patients the following diarrhea volumes will apply: 
Stage 1 
- > 10 ml/kg/day 
Stage 2 - > 20 ml/kg/day 
Stage 3 - > 30 ml/kg/day 


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APPENDIX D 
EVALUATION OF CHRONIC GRAFT-VERSUS-HOST DISEASEa 
 
In all cases, concomitant processes (i.e. infections or drug reactions) must be ruled out. Karnofsky 
or Lansky Clinical Performance scores, 60%, > 15% weight loss, and recurrent infections are usually 
signs of clinical extensive chronic GVHD. Abnormalities that could indicate chronic GVHD are 
categorized by organ systems as listed below. 
 
Skin 
Erythema, dryness, pruritus, pigmentary changes (i.e. hyperpigmentation, vitiligo), 
mottling, papulosquamous plaques, nodules, exfoliation, macular-papular or 
urticarial rash, scleroderma, morphea (one or several circumscribed, indurated and 
shiny lesions) 
 
Nail 
Ridging, onychodystrophy, onycholysis 
 
Hair 
Premature graying, (scalp hair, eyelashes, eyebrows), thinning scalp hair, alopecia, 
decreased body hair 
 
Mouth 
Dryness, burning, gingivitis, mucositis, striae, atrophy, erythema, lichenoid changes, 
ulcers, labial atrophy or pigmentary changes, tooth decay, tightness around the 
mouth 
 
Eyes 
Dryness, burning, blurring, gritty eyes, photophobia, pain 
 
Vagina/vulva Dryness, dyspareunia, stricture or stenosis, erythema, atrophy or lichenoid changes 
not included 
 
Liver 
Elevated liver function tests not due to other causes (alkaline phosphatase  3x 
upper limit of normal, AST or ALT 4x upper limit of normal or total serum bilirubin  
2.5; in the absence of chronic GVHD involving other organs, liver biopsy is required 
to confirm diagnosis) 
 
Lung 
Bronchiolitis obliterans (see diagnostic indicators), cough, wheezing, dyspnea on 
exertion, history of recurrent bronchitis or sinusitis 
GI 
Anorexia, nausea, vomiting, weight loss, dysphasia, odynophagia, malabsorption 
Fasciitis 
Stiffness and tightness with restriction of movement, occasionally with swelling pain, 
cramping, erythema and induration, most commonly affecting forearms, wrists and 
hands, ankles, legs, and feet, inability to extend wrists without flexing the fingers or 
the elbows, contractures 
 
Serositis 
Chest pain or cardiopulmonary comprise due to pericarditis or pleuritis 
Muscle 
Proximal muscle weakness, cramping 
Skeletal 
Arthralgia of large proximal girdle joints and sometimes smaller joints 


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Appendix D (continued) 
 
Laboratory testing and diagnostic indicators of chronic GVHDa 
 
Eye 
Schirmer’s test with a mean value  5mm at 5 minutes, or symptomatic with values 
of 6-10mm or keratitis detected by slit lamp examination 
 
Liver 
Elevated liver function tests not due to other causes (see definition of clinical limited 
and extensive chronic GVHD) 
 
Lung 
New obstructive lung defect defined as FEV1 < 80% of predicted with either an FEF 
25-75 <65% of predicted or RV >120% of predicted, or a decrease of FEV1/FVC by > 
12% within a period of less than 1 year. A diagnosis of bronchiolitis obliterans 
requires negative microbiological tests from bronchoalveolar lavage and evidence of 
air trapping by high resolution end-expiratory and end-inspiratory CAT scans o the 
chest. A thoracoscopic lung biopsy may be necessary in order to confirm the 
diagnosis of bronchiolitis obliterans in patients who have obstructive lung disease 
without air trapping when chronic GVHD involving other organs is absent 
 
Esophagus 
Esophageal web formation, stricture or dysmotility demonstrated by barium swallow, 
endoscopy or manometry 
Muscle 
Elevated CPK or aldolase, EMG findings consistent with myositis 
Blood 
Thrombocytopenia (usually 20,000-100,000/l), eosinophilia, hypo- 
gammaglobulinemia, hypergammaglobulinemia, and autoantibodies occur in some 
cases 
 
 
a From Standard Practice Guidelines for “Chronic Graft-versus-Host Disease Classification at the 
time of presentation” developed by Long Term Follow-Up at the FHCRC 


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64 
 
 
 
 
Appendix D (continued) 
 
GRADING OF CHRONIC GRAFT-VERSUS-HOST DISEASE 
 
A 
Clinical limited chronic GVHD 
1. Oral abnormalities consistent with chronic GVHD, a positive skin or lip biopsy, and no other 
2. 
manifestations of chronic GVHD 
Mild liver test abnormalities (alkaline phosphatase  2x upper limit of normal, AST or ALT  
3x upper limit of normal and total bilirubin  1.6) with positive skin or lip biopsy, and no other 
manifestation of chronic GVHD 
3. Less than six papulosquamous plaques or limited skin rash or dyspigmentation (< 20% of 
4. 
the body surface), positive skin biopsy, and no other manifestations of chronic GVHD 
Ocular sicca (Schirmer’s test  5mm), positive skin or lip biopsy, and no other manifestations 
of chronic GVHD 
5. Vaginal or vulvar abnormalities with positive biopsy, and no other manifestations of chronic 
GVHD 
B. 
Clinical extensive chronic GVHD 
1. Involvement of two or more organs with symptoms or signs of chronic GVHD, with biopsy 
documentation of chronic GVHD in any organ 
2. > 15% base line body weight loss not due to other causes, with biopsy documentation of 
chronic GVHD in any organ 
3. Skin involvement more extensive than defined for limited chronic GVHD, confirmed by 
biopsy 
4. Scleroderma or morphea 
5. Onycholysis or onychodystrophy thought to represent chronic GVHD, with documentation of 
chronic GVHD in any organ 
6. Decreased range of motion in wrist of ankle extension due to fasciitis caused by chronic 
GVHD 
7. Contractures thought to represent chronic GVHD 
8. Bronchiolitis obliterans 
9. Positive liver biopsy; abnormal liver function tests not due to other causes with alkaline 
phosphatase > 2x upper limit of normal, AST or ALT > 3x upper limit of normal, or total 
bilirubin > 1.6, and documentation of chronic GVHD in any organ 
10. Pericarditis or pleuritis not due to other causes 
11. Positive upper or lower GI biopsy 
 
 
 
a From Standard Practice Guidelines for “Chronic Graft-versus-Host Disease Classification at the 
time of presentation” developed by 
Long Term Follow-Up at the FHCRC 


FHCRC 2256.00 
Current version: 04/06/2020 
65 
 
 
 
 
APPENDIX E 
Institution-Sponsored IND SAE Reporting Form 
 
 
 
 
 
ISIOC SAE 
form_v4_23May2018 


FHCRC 2256.00 
Current version: 04/06/2020 
66 
 
 
 
APPENDIX F 
Protocol 2256 Patient Demographics and Eligibility Form 
Please Fax this completed form to (206)-667-4427 for patient registration. 
Questions regarding eligibility should go to Lauri Burroughs, M.D., 206-667-2396. 
UPN:  
_ 
Patient Name: 
  
_  
_ 
(Last) 
  
_ 
(First) 
  
_ 
(MI) 
Date of 
Birth: 
  
_ /  
/  
__ Age:_  
 
(Mo) (Day) (Year) 
Gender (choose one): 
Male 
Female 
Unknown 
Diagnosis:  
_  
__ 
Planned Day 0: _  
_/_  
/ 
 
(Mo) 
(Day) (Year) 
Ethnicity (choose one): Instruct the patient to select one of the following. 
Hispanic (A person of Cuban, Mexican, Puerto Rican, South or Central American, or other Spanish 
culture or origin, regardless of race. Term “Spanish Origin” can also be used in addition to “Hispanic” 
or “Latino”.) 
Not Hispanic or Latino 
Declined to Report 
Research Subject does not know ethnicity 
Race (check all that apply): Instruct the patient to select one or more of the following. 
American Indian/Alaska Native (A person having origins in any of the original peoples of North, 
Central, or South America, and who maintains tribal affiliations or community attachment). 
Asian (A person having origins in any of the original peoples of the Far East, Southeast, Asia, or the 
Indian subcontinent including, for example, Cambodia, China, India, Japan, Korea, Malaysia, 
Pakistan, the Philippine Islands, Thailand and Vietnam). 
Black or African American (A person having origins in any of the black racial groups of Africa. 
Native Hawaiian/Pacific Islander (A person having origins in any of the original peoples of Hawaii, 
Guam, Samoa or other Pacific Islands). 
White (A person having origins in any of the original peoples of Europe, the Middle East or North 
Africa). 
Research subject does not know 
race Declined to report 


FHCRC 2256.00 
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67 
 
 
 
 
Protocol 2256 Eligibility (continued) 
 
Inclusion Criteria: 
 
1) Yes 
No 
Patient signed IRB approved consent form. Date:   
 
 
IRB File Number:   
 
 
For FH patients: 
Consent A (Regimen A: PB/BM donor) 
Consent D (Regimen B: Unrelated Cord blood donor) 
Consent C (Regimen A: HLA matched sibling bone marrow 
combined with HLA-matched sibling cord blood) 
 
2) Yes 
No 
The consent was approved by your institution’s IRB on Date:   
 
 
The following criteria questions must be marked “Yes” or “N/A” for the patient to enter on 2256. 
 
3) Yes 
No 
Age < 50 years with nonmalignant disease treatable by allogeneic HCT. 
 
4) Yes 
No 
NA 
Patient has a known nonmalignant disease that is not clearly defined and has been 
discussed and approved for enrollment with the protocol PI (Dr. Lauri Burroughs), 
and potentially by the Non-Malignant Board 
 
 
Graft Criteria: One of the following graft sources must be marked “YES” 
 
5a) Yes 
No 
Patients with HLA-matched related donor 
 
OR 
 
5b) Yes 
No 
Patient with unrelated donors matched for HLA-A, B, C, DRB1, and DQB1 or 
mismatched for a single allele at HLA-A, B, C, DRB1 or a single DQB1 antigen or 
allele mismatch by high resolution DNA typing. 
Yes 
No 
Have a negative anti-donor cytotoxic crossmatch. 
N/A 
Cytotoxic crossmatch not done as patient and donor are phenotypically identical by 
molecular methods. 
 
Fill in HLA typing for patient and HLA-matched related or unrelated donor 
Patient 
A: _  
_ A: _  
_ 
C:  
_ C:  
_ 
B: _  
 B: _  
 
DRB1:  
 DRB1:  
 
DQB1:   
 DQB1:   
 
Donor 
A: _  
_ A: _  
 C:  
_ C:  
_ 
B: _  
 B: _  
 
DRB1:  
 DRB1:  
_ DQB1:   
 DQB1:   
 
 
OR 


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Protocol 2256 Eligibility (continued) 
 
 
 
5c) Yes 
No 
Patient with unrelated umbilical cord blood graft: Unrelated UCB units will be 
selected according to current institutional standard Practice. One or 2 UCB units 
may be used to achieve the required cell dose. 
Yes 
No 
The unrelated UCB graft is matched at 4-6 HLA-A, B, DRB1 antigens with the 
recipient. This may include 0-2 antigen mismatches at the A or B or DRB1 loci. Unit 
selection based on cryopreserved nucleated cell dose and HLA-A, B, DRB1 using 
intermediate resolution A, B antigen and DRB1 allele typing. 
 
Fill in HLA typing for patient and unrelated umbilical cord unit(s) 
Patient 
A: _  
_ 
A: _  
 
C:   
 
C:   
 
B: _  
_ 
B: _  
_ 
DRB1:   
 DRB1:   
 
Unit #1 
A: _  
_ 
A: _  
 
C:   
 
C:   
 
B: _  
_ 
B:  
 
DRB1:   
 DRB1:   
 
Unit #2 N/A 
A: _  
_ 
A: _  
 
C:   
 
C:   
 
B: _  
_ 
B:  
 
DRB1:   
 DRB1:   
 
 
Exclusion criteria: 
Each of the following questions must be marked “No” Or “N/A” for the patient to enroll on 2256. 
 
6) Yes 
No 
Patients with Idiopathic Aplastic Anemia and Fanconi Anemia. (Patients with Aplastic Anemia 
associated with PNH or inherited marrow failure syndromes, except Fanconi Anemia, will be 
allowed). 
 
7) Organ dysfunction. Please check yes if patient meets any of the following: 
 
a. Yes 
No 
Impaired cardiac function as evidenced by ejection fraction < 35% (or, if unable to 
obtain ejection fraction, shortening fraction <26%) or cardiac insufficiency requiring 
treatment or symptomatic coronary artery disease. Patients with a shortening 
fraction < 26% must be seen by cardiology for approval. 
 
Date:    _/  
/ 
 
Ejection Fraction Value:  
%   Shortening Fraction Value:  
% 
 
b. Yes 
No 
Impaired pulmonary function as evidenced by DLCO < 50% of predicted (or, if 
unable to perform pulmonary function tests, then O2 saturation < 92% on room air). 
 
Date:    _/  
/ 
 
DLCO Value:  
% O2  Saturation Value: _  
% 


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Protocol 2256 Eligibility (continued) 
 
 
 
c. Yes 
No 
Impaired renal function as evidenced by creatinine-clearance < 50% for age, weight, 
height or serum creatinine > 2X upper normal limit or dialysis-dependent. 
 
Date:  
_/  
_/   _ 
Value:  
mg/dL or CrCl:  
ml/min 
 
d. Yes 
No 
Evidence of synthetic dysfunction or severe cirrhosis requiring deferral of 
conditioning as recommended by a gastroenterology specialist. 
Date:  
/ 
_/ _ 
ALT:  
U/L 
AST:  
U/L 
Total Bilirubin:  
mg/dL 
Alk.phos:  
_ U/L 
 
8) Yes 
No 
Patients with an active infectious disease requiring deferral of conditioning; as recommended 
by an Infectious Disease specialist. 
9) Yes 
No 
Patients who are positive for human immunodeficiency virus (HIV). 
10) Yes 
No 
Females who are pregnant or breast-feeding. 
11) Yes 
No 
Patients with a known hypersensitivity to treosulfan and/or fludarabine. 
12) Yes 
No 
Receiving another experimental drug within 4 weeks of initiation of conditioning (day -6) 
unless approved by the PI.. 
 
 
TBI Dose Assignment 
 
For Regimen B patients only, one of the following must be designated. 
 
200 cGy TBI (for unrelated cord blood recipients with an underlying T-cell primary immunodeficiency) 
OR 
300 cGy TBI (for all other unrelated cord blood recipients) 
 
 
Signature of Local Principal Investigator:  
Date: 
 
(or Designee) 
 
Transplant Center  __________________________________________________  
 
 
Signature of FHCRC Principal Investigator  
Date: 
 
(or Designee) 
 
 
Signature of person completing form:  
Date:   
 
 
FHCRC Patients: 
Signature of Principal Investigator:  
Date: 
 
(or Designee) 
 
OR 
Outside Center Patients: 
 
Signature of Local Principal Investigator:  
Date: 
 
(or Designee) 
Signature of FHCRC Principal Investigator:  
Date: 
 
(or Designee) 


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APPENDIX G 
Adapted from 
COMMON TOXICITY CRITERIA (CTC) for HCT 
 
Grade 
Adverse Event 
3 
4 
ALLERGY/IMMUNOLOGY 
Allergic 
reaction/hypersensitivity 
(including drug fever) 
Symptomatic bronchospasm, 
requiring parenteral 
medication(s), with or without 
urticaria; allergy-related 
edema/angioedema 
Anaphylaxis 
Vasculitis 
Requiring steroids 
Ischemic changes or requiring 
amputation 
Allergy/Immunology – 
Other - Specify,  
) 
Severe 
Life-threatening or disabling 
BLOOD/BONE MARROW 
Hemolysis (e.g., immune 
hemolytic anemia, drug- 
related hemolysis, other) 
Requiring transfusion and/or 
medical intervention (e.g., 
steroids) 
Catastrophic consequences of 
hemolysis (e.g., renal failure, 
hypotension, bronchospasm, 
emergency splenectomy) 
For BMT studies, if 
specified in the protocol. 
For pediatric BMT studies, 
if specified in the protocol. 
>4 u pRBC in 24 hours 
>30mL/kg in 24 hours 
Hemorrhage or hemolysis 
associated with life-threatening 
anemia; medical intervention 
required to improve hemoglobin 
Hemorrhage or hemolysis 
associated with life-threatening 
anemia; medical intervention 
required to improve hemoglobin 
CARDIOVASCULAR (ARRHYTHMIA) 
Cardiovascular/Arrhythmia 
-Other (Specify,  
) 
Symptomatic, and requiring 
treatment of underlying cause 
Life-threatening (e.g., arrhythmia 
associated with CHF, hypotension, 
syncope, shock) 
CARDIOVASCULAR (GENERAL) 
Acute vascular leak 
syndrome 
Respiratory compromise or 
requiring fluids 
Life-threatening; requiring pressor 
support and/or ventilatory/support 
Cardiac-ischemia/infarction Angina without evidence of 
infarction 
Acute myocardial infarction 


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APPENDIX G continued 
 
Grade 
Adverse Event 
3 
4 
CARDIOVASCULAR (GENERAL) continued 
Cardiac left ventricular 
function 
CHF responsive to treatment 
Severe or refractory CHF or 
requiring intubation 
Cardiac troponin I (cTnI) 
Levels consistent with unstable 
angina as defined by the 
manufacturer 
Levels consistent with myocardial 
infarction as defined by the 
manufacturer 
Cardiac troponin T (cTnT) 
 0.1 - <0.2 ng/mL 
 0.2 ng/mL 
Hypotension 
Requiring therapy and 
sustained medical attention, but 
resolves without persisting 
physiologic consequences 
Shock (associated with acidemia 
and impairing vital organ function 
due to tissue hypoperfusion) 
Myocarditis 
CHF responsive to treatment 
Severe or refractory CHF 
Pericardial effusion/ 
pericarditis 
With physiologic consequences 
Tamponade (drainage or pericardial 
window required) 
Syncope (fainting) is 
graded in the 
NEUROLOGY category. 
- 
- 
Thrombosis/embolism 
Deep vein thrombosis, requiring 
anticoagulant therapy 
Embolic event including pulmonary 
embolism 
Vein/artery operative injury 
is graded as Operative 
injury of vein/artery in the 
CARDIOVASCULAR 
(GENEARL) category. 
Cardiovascular/General – 
Other 
(Specify,  
) 
Severe 
Life-threatening or disabling 


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APPENDIX G continued 
 
Grade 
Adverse Event 
3 
4 
COAGULATION 
DIC (disseminated 
intravascular coagulation) 
Also consider Platelets. 
Note: Must have increased 
fibrin split products or D-dimer 
in order to grade as DIC. 
Laboratory findings present with 
no bleeding 
Laboratory findings and 
bleeding 
Thrombotic microangiopathy 
(e.g., thrombotic 
thrombocytopenic purpura/TTA 
or hemolytic uremic 
syndrome/HUS) 
Also consider Hemoglobin, 
Platelets, Creatinine. 
Note: Must have 
microangiopathic changes on 
blood smear (e.g., 
schistocytes, helmet cells, red 
cell fragments). 
Laboratory findings present 
without clinical consequences 
Evidence of RBC destruction 
with creatinine (>3 x ULN) not 
requiring dialysis 
Laboratory findings and clinical 
consequences, (e.g., CNS 
hemorrhage/bleeding or 
thrombosis/embolism or renal 
failure) requiring therapeutic 
intervention 
Evidence of RBC destruction 
with renal failure requiring 
dialysis and/or encephalopathy 
Coagulation - Other 
(Specify,  
) 
Severe 
Life-threatening or disabling 
CONSTITUTIONAL SYMPTOMS 
Weight gain associated with 
Veno-Occlusive Disease 
(VOD) for BMT studies, if 
specified in the protocol. 
Also consider Ascites Edema, 
Pleural effusion (non- 
malignant). 
0% or as ascites 
>10% or fluid retention resulting 
in pulmonary failure 
DERMATOLOGY/SKIN 
Erythema multiforme (e.g., 
Stevens-Johnson syndrome, 
toxic epidermal necrolysis) 
Severe or requiring IV fluids 
(e.g., generalized rash or 
painful stomatitis) 
Life-threatening (e.g., exfoliative 
or ulcerating dermatitis or 
requiring enteral or parenteral 
nutritional support) 


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APPENDIX G continued 
 
Grade 
Adverse Event 
3 
4 
DERMATOLOGY/SKIN continued 
Rash/desquamation 
associated with graft versus 
host disease (GVHD) for BMT 
studies, if specified in the 
protocol. 
Symptomatic generalized 
erythroderma or symptomatic 
macular, papular or vesicular 
eruption, with bullous formation, 
or desquamation covering 
50% of body surface area 
Generalized exfoliative 
dermatitis or ulcerative 
dermatitis or bullous formation 
GASTROINTESTINAL 
Ascites(none-malignant) 
Symptomatic, requiring 
therapeutic paracentesis 
Life-threatening physiologic 
consequences 
Colitis 
Also consider 
Hemorrhage/bleeding with 
grade 3 or 4 thrombocytopenia, 
Hemorrhage/bleeding without 
grade 3 or 4 thrombocytopenia, 
Melena/GI bleeding, Rectal 
bleeding/hematochezia, 
Hypotension. 
Abdominal pain, fever, change 
in bowel habits with ileus or 
peritoneal signs, and 
radiographic or biopsy 
documentation 
Perforation or requiring surgery 
or toxic megacolon 
Diarrhea associated with graft 
versus host disease (GVHD) 
for BMT studies, if specified in 
the protocol. 
For pediatric BMT studies, if 
specified in the protocol. 
Also consider 
Hemorrhage/bleeding with 
grade 3 or 4 thrombocytopenia, 
Hemorrhage/bleeding without 
grade 3 or 4 thrombocytopenia, 
Pain, Dehydration, 
Hypotension. 
>1500mL of diarrhea/day 
>15mL/kg of diarrhea/day 
Severe abdominal pain with or 
without ileus 


74 
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APPENDIX G continued 
 
Grade 
Adverse Event 
3 
4 
GASTROINTESTINAL continued 
Duodenal ulcer (requires 
radiographic or endoscopic 
documentation) 
Uncontrolled by outpatient 
medical management; requiring 
hospitalization 
Perforation or bleeding, 
requiring emergency surgery 
Gastric ulcer 
(requires radiographic or 
endoscopic documentation) 
Bleeding without perforation, 
uncontrolled by outpatient 
medical management; requiring 
hospitalization or surgery 
Perforation or bleeding, 
requiring emergency surgery 
Also consider 
Hemorrhage/bleeding with 
grade 3 or 4 thrombocytopenia, 
Hemorrhage/bleeding without 
grade 3 or 4 thrombocytopenia. 
Gastritis 
Uncontrolled by out-patient 
medical management; requiring 
hospitalization or surgery 
Life-threatening bleeding, 
requiring emergency surgery 
Also consider 
Hemorrhage/bleeding with 
grade 3 or 4 thrombocytopenia, 
Hemorrhage/bleeding without 
grade 3 or 4 thrombocytopenia. 
Pancreatitis 
Also consider Hypotension. 
Note: Amylase is graded in the 
METABOLIC/LABORATORY 
category. 
Abdominal pain with pancreatic 
enzyme elevation 
Complicated by shock (acute 
circulatory failure) 


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APPENDIX G continued 
 
Grade 
Adverse Event 
3 
4 
GASTROINTESTINAL continued 
Mucositis 
Note: Radiation-related 
mucositis is graded as 
Mucositis due to radiation. 
Painless erythema, edema, or 
ulcers preventing swallowing or 
requiring hydration or 
parenteral (or enteral) 
nutritional support 
Severe ulceration requiring 
prophylactic intubation or 
resulting in documented 
aspiration pneumonia 
Typhlitis 
(inflammation of the cecum) 
Also consider 
Hemorrhage/bleeding with 
grade 3 or 4 thrombocytopenia, 
Hemorrhage/bleeding without 
grade 3 or 4 thrombocytopenia, 
Hypotension, Febrile 
neutropenia. 
Abdominal pain, diarrhea, 
fever, and radiographic or 
biopsy documentation 
Perforation, bleeding or necrosis 
or other life-threatening 
complication requiring surgical 
intervention (e.g., colostomy) 


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Appendix G continued 
 
Grade 
Adverse Event 
3 
4 
HEMORRHAGE 
Notes: Transfusion in this section refers to pRBC infusion. 
For any bleeding with grade 3 or 4 platelets (<50,000), always grade Hemorrhage/bleeding with 
grade 3 or 4 thrombocytopenia. Also consider Platelets, Transfusion: pRBCs, and Transfusion: 
platelets in addition to grading severity by grading the site or type of bleeding. 
If the site or type of Hemorrhage/bleeding is listed, also use the grading that incorporates the site of 
bleeding: NS Hemorrhage/bleeding, Hematuria, Hematemesis, Hemoptysis, Hemorrhage/bleeding 
with surgery, Melena/lower GI bleeding, Petechiae/purpura (Hemorrhage/bleeding into skin), Rectal 
bleeding/hematochezia, Vaginal bleeding. 
Hemorrhage/bleeding with 
grade 3 or 4 thrombocytopenia 
Also consider Platelets, 
Hemoglobin, Transfusion: 
platelets, Transfusion: pRBCs, 
site or type of bleeding. If the 
site is not listed, grade as 
Hemorrhage – Other 
(Specify site,  
). 
Note: This adverse event must 
be graded for any bleeding 
with grade 3 or 4 
thrombocytopenia. 
Requiring transfusion 
Catastrophic bleeding, requiring 
major non-elective intervention 
Hemorrhage/bleeding without 
grade 3 or 4 thrombocytopenia 
Also consider Platelets, 
Hemoglobin, Transfusion: 
platelets, Transfusion: pRBCs, 
Hemorrhage – Other (Specify 
site,  
). 
Note: Bleeding in the absence 
of grade 3 or 4 
thrombocytopenia is graded 
here only if the specific site or 
type of bleeding is not listed 
elsewhere in the 
HEMORRHAGE category. Also 
grade as Other in the 
HEMORRHAGE category. 
Requiring transfusion 
Catastrophic bleeding requiring 
major non-elective intervention 


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Appendix G continued 
 
Grade 
Adverse Event 
3 
4 
HEMORRHAGE cont’d 
CNS hemorrhage/bleeding 
Bleeding noted on CT or other 
scan with no clinical 
consequences 
Hemorrhagic stroke or 
hemorrhagic vascular event 
(CVA) with neurologic signs and 
symptoms 
Hemoptysis 
Requiring transfusion 
Catastrophic bleeding, requiring 
major non-elective intervention 
Melena/GI bleeding 
Requiring transfusion 
Catastrophic bleeding, requiring 
major non-elective intervention 
Rectal bleeding/hematochezia 
Requiring transfusion 
Catastrophic bleeding, requiring 
major non-elective intervention 
Vaginal bleeding 
Requiring transfusion 
Catastrophic bleeding, requiring 
major non-elective intervention 
Hemorrhage – Other 
(Specify site,  
) 
Requiring transfusion 
Catastrophic bleeding, requiring 
major non-elective intervention 
HEPATIC 
Bilirubin 
Bilirubin associated with graft 
versus host disease (GVHD) 
for BMT studies, if specified in 
the protocol. 
>3.0 – 10.0 x ULN 
>6 - <15 mg/100mL 
>10.0 x ULN 
>15 mg/100mL 


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Appendix G continued 
 
 
Grade 
Adverse Event 
3 
4 
INFECTION/FEBRILE NEUTROPENIA 
Febrile neutropenia (fever of 
unknown origin without clinically 
or microbiologically documented 
infection) 
Present 
Life-threatening sepsis (e.g., 
septic shock) 
Infection/Febrile Neutropenia – 
Other (Specify,  
) 
Severe 
Life-threatening or disabling 
NEUROLOGY 
Aphasia, receptive and/or expressive, is graded under Speech impairment in the NEUROLOGY 
category. 
CNS cerebrovascular 
ischemia 
Transient ischemic event or 
attack (TIA) 
Permanent event (e.g., cerebral 
vascular accident) 
Leukoencephalopathy 
associated radiological 
findings 
Severe increase in SAS; severe 
ventriculomegaly; near total 
white matter T2 hyperintensities 
or diffuse low attenuation (CT); 
focal white matter necrosis 
(cystic) 
Severe increase in SAS; severe 
ventriculomegaly; diffuse low 
attenuation with calcification 
(CT); diffuse white matter 
necrosis (MRI) 
Seizure(s) 
Seizure(s) in which 
consciousness is altered 
Seizures of any type which are 
prolonged, repetitive, or difficult 
to control (e.g., status 
epilepticus, intractable epilepsy) 
PULMONARY 
Adult Respiratory Distress 
Syndrome (ARDS) 
- 
Present 
Apnea 
Present 
Requiring intubation 
Carbon monoxide diffusion 
capacity (DLCO) 
>25 - <50% of pretreatment or 
normal value 
<25% of pretreatment or normal 
value 
FEV1 
>25 - <50% of pretreatment or 
normal value 
<25% of pretreatment or normal 
value 
Hypoxia 
Decreased O2 saturation at 
rest, requiring supplemental 
oxygen 
Decreased O2 saturation, 
requiring pressure support 
(CPAP) or assisted ventilation 


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Appendix G continued 
 
Grade 
Adverse Event 
3 
4 
RENAL/GENITOURINARY 
Creatinine 
Note: Adjust to age-appropriate 
levels for pediatric patients. 
>3.0- 6.0 x ULN 
>6.0 x ULN 
Renal failure 
Requiring dialysis, but 
reversible 
Requiring dialysis and 
irreversible 
SECONDARY MALIGNANCY 
Secondary Malignancy – Other 
(Specify type,  
) 
excludes metastasis from initial 
primary 
- 
present 


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Appendix H 
The Hematopoietic Cell Transplant-Comorbidity Index (HCT-CI) 9/7/10 
Assign scores appropriately if the patient has any of these comorbidities 
Patient  
(name), UPN  
Date  
 
 
Comorbidities 
Definitions 
HCT- 
CI 
scores 
Actual Lab 
Values/Comments 
Arrhythmia 
Atrial fibrillation or flutter, sick sinus syndrome, and 
ventricular arrhythmias requiring treatment in the patient’s 
past history 
1 
Cardiac 
Coronary artery disease†, congestive heart failure, 
myocardial infarction in patient’s past history or EF of 50% 
at time of HCT 
1 
Inflammatory 
bowel disease 
Crohn’s disease or ulcerative colitis requiring treatment in 
the patient’s past history 
1 
Diabetes 
Requiring treatment with insulin or oral hypoglycemic, but 
not diet alone, at time of HCT 
1 
Cerebro-vascular 
disease 
Transient ischemic attack or cerebro-vascular accident in 
patient’s past history 
1 
Psychiatric 
disturbance 
Depression/anxiety requiring psychiatric consult or treatment 
at time of HCT 
1 
Hepatic – mild 
Chronic hepatitis, Bilirubin >ULN- 1.5 X ULN, or AST/ALT 
>ULN-2.5XULN at time of HCT 
1 
Obesity 
Patients with a BMI of 
>35 for adults or with BMI-for-age percentile of ≥ 95th 
percentile for children at time of HCT 
1 
Infection 
Documented infection or fever of unknown etiology 
requiring anti-microbial treatment before, during and after 
the start of conditioning regimen 
1 
Rheumatologic 
SLE, RA, polymyositis, mixed CTD, polymyalgia 
rheumatica in patient’s past history 
2 
Peptic ulcer 
Requiring treatment in patient’s past history 
2 
Renal 
Serum creatinine >2 mg/dl, on dialysis, or prior renal 
transplantation at time of HCT 
2 
Moderate 
pulmonary 
DLco and/or FEV1 >65%-80% or 
Dyspnea on slight activity at time of HCT 
2 
Prior solid tumor 
Treated at any time point in the patient’s past history, 
excluding non-melanoma skin cancer 
3 
Heart valve 
disease 
At time of HCT excluding mitral valve prolapse 
3 
Severe pulmonary 
DLco and/or FEV1 65% or 
Dyspnea at rest or requiring oxygen at time of HCT 
3 
Moderate/severe 
hepatic 
Liver cirrhosis, Bilirubin >1.5 X ULN, or AST/ALT 
>2.5XULN at time of HCT 
3 
Please provide 
(KPS): 
Karnofsky Performance Score =  
% 
Total 
Score 
=  
 
Signature of 
Provider: 
†One or more vessel-coronary artery stenosis, requiring medical treatment, stent, or bypass graft. EF indicates ejection fraction; ULN, upper limit of 
normal; SLE, systemic lupus erythmatosis; RA, rheumatoid arthritis; CTD, connective tissue disease; DLco, diffusion capacity of carbon monoxide; 
FEV1, forced expiratory volume in one second; AST, aspartate aminotransferase; ALT, alanine aminotransferase. 


81 
FHCRC 2256.00 
Current version: 04/06/2020 
 
 
 
 
 
Appendix I 
Adverse Events Associated or Expected with Hematopoietic Cell Transplantation 
 
1. Graft versus host disease: GVHD is a major toxicity associated with the infusion of allogeneic donor stem 
cells. GVHD may be acute or chronic and may affect multiple organ systems, including the skin, liver, and GI 
tract. 
 
2. Opportunistic infections, including viral and fungal infections, can result in severe pulmonary, neurologic, 
hepatic and other organ dysfunction, and possible death. 
 
3. Gastrointestinal toxicity. Nausea and vomiting can be anticipated during the entire course of ablative 
therapy. Mucositis and diarrhea should be expected. Prednisone can cause GI bleeding. 
 
4. Cardiac toxicity. Cardiotoxicity (congestive heart failure, pericardial effusion, EKG changes) is 
uncommonly associated with the chemotherapy agents and TBI used in the regimen and these sequelae 
may prove lethal. 
 
5. Pulmonary toxicity. Diffuse interstitial pneumonitis of unknown etiology and diffuse alveolar hemorrhage 
occur with some regularity after BMT and interstitial fibrosis occurs much more rarely. Both are well- 
described complications of intensive chemotherapy and TBI regimens and may prove lethal. 
 
6. Hepatic toxicity. Veno-occlusive disease of the liver is a common toxicity of high-dose 
chemoradiotherapy and may result in death. Tacrolimus/Cyclosporine may cause elevation of ALT/AST. 
 
7. Renal dysfunction. Chemoradiotherapy may uncommonly cause renal dysfunction. More commonly, 
nephrotoxicity results from cyclosporine/tacrolimus and generally responds to dose reduction. Rarely, 
idiopathic or calcineurin inhibitor-associated hemolytic-uremic syndrome may occur and may be progressive 
and fatal. A syndrome of moderate renal insufficiency and hemolysis has been seen 5-7 months post HSCT 
after intensive multi-agent conditioning plus TBI. 
 
8. Hemorrhagic cystitis, manifested either as gross or microscopic hematuria, is a common toxicity after high- 
dose chemoradiotherapy, but usually associated with regimens that include cyclophosphamide. Hemorrhagic 
cystitis may predispose to a long-term increased risk of bladder cancer. 
 
9. Central nervous system toxicity. Radiation and chemotherapy can cause CNS toxicity, including seizures, 
depressed mental status, or leukoencephalopathy. Calcineurin inhibitors can cause seizures or other CNS 
toxicity. 
 
10. Marrow aplasia. Severe neutropenia, thrombocytopenia, and anemia, is expected to occur for a period of 
7 to 42 days following infusion of the graft. Transfusion of platelets and red blood cells is expected as 
supportive care. Transfusion of blood products may be associated with acquisition of HIV or a hepatitis virus. 
Neutropenia may increase the risk for acquiring serious infection. Thrombocytopenia may increase the risk of 
life-threatening hemorrhage. Hemorrhagic or infectious complications during the expected period of aplasia 
may result in death. 
 
11. Miscellaneous. Alopecia and sterility are expected complications of the program as a whole. Cataract 
development is possible after TBI and/or steroids. Deficiencies of growth hormone, thyroid hormone, and sex 
hormones are possible following conditioning. Calcineurin inhibitors can cause transient gingival hyperplasia, 
tremor, seizure, hypertension, headache, dysesthesia and hirsutism. Steroid therapy can also contribute to 
fluid retention, easy bruising, hypertension, aseptic necrosis of bone and increased susceptibility to infection. 
MMF can cause spontaneous abortions and birth defects. Hospitalization during conditioning and recovery 
period is expected.