Clinical trial NCT00358657
Conditions: Immunodeficiency Syndrome Severe Aplastic Anemia Genetic Disorder
Sponsor: Fred Hutchinson Cancer Center
Start date: 2006-05-24; Completion date: 2019-05-25
Enrollment: 14 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This phase I/II trial studies the side effects of fludarabine phosphate, cyclophosphamide and total-body irradiation followed by donor bone marrow transplant and cyclophosphamide, mycophenolate mofetil, tacrolimus, and sirolimus in treating patients with primary immunodeficiency disorders or noncancerous inherited disorders. Giving low doses of chemotherapy and total-body irradiation before a bone marrow transplant helps prepare the patient's body to accept the incoming donor's bone marrow and decrease the risk that the patient's immune system will reject the donor's stem cells. When the healthy stem cells from a donor are infused into the patient they may help the patient's bone marrow make stem cells, red blood cells, white blood cells, and platelets. Sometimes the transplanted cells from a donor can make an immune response against the body's normal cells called graft versus host disease. Giving cyclophosphamide, mycophenolate mofetil, tacrolimus, and sirolimus after the transplant may help decrease this from happening.

Detailed description:
PRIMARY OBJECTIVE:

I. Determine safety of nonmyeloablative conditioning and hematopoietic cell transplantation (HCT) from human leukocyte antigen (HLA)-haploidentical related donors for patients with nonmalignant inherited disorders who do not have an HLA-matched related or unrelated donor.

SECONDARY OBJECTIVES:

I. Determine whether nonmyeloablative conditioning and HCT from an HLA-haploidentical related donor graft can establish mixed chimerism (\> 5% cluster of differentiation \[CD\]3+ donor T-cell chimerism) in patients with nonmalignant inherited disorders.

II. Transplant related mortality at day 100.

III. Incidence and severity of graft-versus-host disease (GHVD).

IV. Immune reconstitution.

V. Infections during the first 200 days after HCT.

OUTLINE:

NONMYELOABLATIVE CONDITIONING REGIMEN: Patients receive fludarabine phosphate intravenously (IV) over 1 hour on days -6 to -2; cyclophosphamide IV over 1 hour on days -6 and -5; and undergo total body irradiation on day -1.

TRANSPLANTATION: Patients undergo allogeneic bone marrow transplant on day 0.

POST-TRANSPLANT IMMUNOSUPPRESSION: Patients receive cyclophosphamide IV over 1 hour on days 3 and 4, and mycophenolate mofetil orally (PO) every 8 hours on days 5-30 then twice daily (BID) to day 40, and then if there is no evidence of active GVHD and donor engraftment is \> 95% (or by principal investigator \[PI\] approval) taper until approximately day 96, or faster at discretion of PI. Patients also receive tacrolimus IV continuously over 22-24 hours starting on day 5 post-transplant and continue on tacrolimus through day 100 followed by a taper to approximately day 180 if there is no evidence of GVHD and their graft is doing well. Patients may convert to oral tacrolimus given BID or three times daily (TID) when the patient is able to take medications orally and has a therapeutic drug level. In addition, patients will receive sirolimus PO beginning on day 5 through day 180 followed by a taper to approximately day 210 if there is no evidence of GVHD and their graft is doing well.

After completion of study treatment, patients are followed up at 6, 12, 18, and 24 months, and then annually for 5 years.

Arms: arm 1: See Detailed Description
Interventions: intervention 1: Allogeneic Bone Marrow Transplantation intervention 2: Cyclophosphamide intervention 3: Fludarabine Phosphate intervention 4: Laboratory Biomarker Analysis intervention 5: Mycophenolate Mofetil intervention 6: Nonmyeloablative Allogeneic Hematopoietic Stem Cell Transplantation intervention 7: Sirolimus intervention 8: Tacrolimus intervention 9: Total-Body Irradiation
Intervention descriptions: intervention 1: Undergo allogeneic bone marrow transplantation intervention 2: Given IV intervention 3: Given IV intervention 4: Correlative studies intervention 5: Given PO intervention 6: Undergo allogeneic bone marrow transplantation intervention 7: Given PO intervention 8: Given IV or PO intervention 9: Undergo total-body irradiation

Protocol documents:
Prot_SAP_000.pdf: 
 
Protocol 2032.00  
 
 
 
FRED HUTCHINSON CANCER RESEARCH CENTER 
UNIVERSITY OF WASHINGTON SCHOOL OF MEDICINE 
SEATTLE CHILDREN’S 
Current Version: 09/22/2020 
Previous Version: 02/12/2019 
 
1. PROTOCOL 2032: HLA-haploidentical related marrow grafts for the treatment of primary 
immunodeficiencies and other nonmalignant disorders using conditioning with low-dose 
cyclophosphamide, TBI and fludarabine and postgrafting cyclophosphamide. 
Investigator 
Professional Title 
Area Code (206) 
Kanwaldeep K Mallhi, M.D 
 
Assistant Member, FHCRC 
Assistant Professor of Pediatrics, UW  
 
667-2014 
Lauri M. Burroughs, M.D. 
Associate Member, FHCRC 
Associate Professor of Pediatrics, UW 
667-2396 
Paul Carpenter, MD 
 
Member, FHCRC 
Professor of Pediatrics, UW 
 
667-3786 
Brenda M. Sandmaier, M.D. 
Member, FHCRC 
Professor of Medicine, UW  
 
667-4961 
Ralph Ermoian, M.D. 
Assistant Professor, Radiation Oncology, UW 
598-4100 
K. Scott Baker, M.D. 
 
Rainer Storb, M.D. 
Member, FHCRC  
Professor of Pediatrics, UW 
 
Member, FHCRC 
Professor of Medicine, UW 
667-5594 
 
667-4407 
 
 
 
 
Biostatistician: 
 
 
 
 
Wendy Leisenring, ScD (206) 667-4374 
 
 
 
 
 
 
 
 
 
Member, FHCRC, Affiliate Professor of Biostatistics, UW  
 
 
Research Staff: 
 
 
 
 
Courtney Vandervlugt, Research Coordinator (206) 667-7385 
 
 
 
 
 
 
 
 
 
 
Jenny Tseng, Research RN (206) 667-7115, pager (206) 314-1739 
 
 
 
 
 
 
 
 
Additional Performance Sites: 
Vanderbilt University Medical Center 
James Connelly, MD 
 
 
 
 
 
 
 
 
 
 
 
 


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2. 
INTRODUCTION 
Hematopoietic cell transplantation (HCT) is often the only curative therapy for patients with non-malignant 
disorders including immunodeficiencies and other inherited diseases. Unfortunately, less than 30% of 
children with life-threatening nonmalignant disorders will have a matched family donor[1]. While unrelated 
registries have expanded donor options, patients who are not of Caucasian ethnicity have difficulty finding 
donors matched for HLA-A, B, C, DRB1 and DQB1 alleles[2-5]. Therefore, the ability to use related, HLA-
haploidentical donors for allogeneic HCT of patients with nonmalignant disorders would provide almost all 
patients a chance at a potential cure.  Several studies have demonstrated HCT using related HLA-
haploidentical grafts to be a viable treatment option for patients with immunodeficiency [6-14] and other 
nonmalignant disorders [15-18]. However, inferior survivals were seen for recipients of HLA-haploidentical 
grafts compared to recipients of HLA-matched related or unrelated grafts. Several problems were seen 
including graft failure, delayed immune reconstitution, increased infections, and other significant toxicities 
including GVHD [10,19]. Therefore, developing safe and effective approaches to HCT from HLA-
haploidentical related donors is an important goal in order to increase the number of patients eligible for HCT. 
A nonmyeloablative approach has several advantages over an ablative approach including decreased 
regimen related toxicity and recovery of autologous hematopoiesis in the event of graft rejection.  
 
It is important to extend the option of nonmyeloablative HCT for potential therapy of patients with 
nonmalignant disorders to patients who do not have an HLA-matched donor. Almost all patients would have 
a related donor identical for one HLA haplotype (haploidentical) and mismatched at HLA-A, -B or -DR of the 
unshared haplotype. In this protocol, we will use a combination of immunosuppressive agents including 
cyclophosphamide administered before and after HCT to facilitate engraftment and to remove highly 
alloreactive T-cell clones presumably involved in GVHD. Feasibility of this approach has been demonstrated 
in a mouse model, a Phase I clinical trial for patients with hematological malignancies, and preliminary results 
from a phase II study at the FHCRC and collaborating centers (see below). Encouraged by these early 
results, a Phase I-II study is warranted to determine the safety and efficacy of such approach in patients with 
nonmalignant inherited disorders. 
 
3. BACKGROUND 
3A. Haploidentical Transplantation for patients with Nonmalignant Disorders. 
1) Immunodeficiency disorders 
Transplantation with haploidentical donors greatly expanded transplant options for patients with 
immunodeficiencies and other inherited disorders. The largest experiences reported are for patients 
with severe combined immunodeficiency disorders (SCID). In the 1980’s, several studies 
demonstrated successful immune reconstitution following transplantation with T-cell depleted 
haploidentical grafts in patients with SCID [6-8,20,21]. Since that time, several other studies have 
demonstrated HCT with haploidentical grafts to be a viable treatment option for patients with 
immunodeficiency [9-14] and other nonmalignant disorders who did not have HLA-matched related 
or unrelated donors [15,16,16-18].  
Fischer et al. compared the outcome of patients with SCID who received either HLA-identical related 
bone marrow grafts (n=70) versus HLA-nonidentical related T-cell depleted bone marrow grafts 
(n=100) and found significantly lower overall survivals for patients who received nonidentical versus 
identical grafts (52% versus 76%, p=0.01) [9]. Pretransplant lung infections were a negative risk factor 
for survival for recipients of nonidentical grafts. Sustained engraftment was significantly better for 
those patients who received conditioning prior to HLA-nonidentical marrow grafts versus those 
patients who did not (86% vs. 50%, p<0.01). Graft-versus-host disease (GVHD) was the primary 
cause of death in the HLA-nonidentical recipients.  


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Buckley et al. reported on the outcome of 89 infants with SCID who received T-cell depleted HLA-
haploidentical parenteral marrow (n=77) or HLA-identical marrow (n=12) grafts without conditioning 
or GVHD prophylaxis [10]. GVHD developed in 28 of the 77 infants (36%) given T-cell depleted 
haploidentical marrow and 6 of the 12 HLA-identical marrow recipients (50%), and was in general 
mild (<grade II) and not responsible for any deaths. Twenty of the 89 patients (22%) received 
“booster” transplants due to poor B- or T-cell function or problems with engraftment. Although not 
clearly detailed, at least 15 of the 20 patients who required booster transplants were patients who 
received haploidentical grafts. With a median follow up of 5.6 years, 72 of the total 89 patients (81%; 
100% HLA-identical, 78% haploidentical) were alive. The most common causes of death were viral 
infections. Of the 81 patients, infants <3.5 months of age had superior survivals (95%) compared to 
patients who were 3.5 months or older (76%). T-cell immune recovery was significantly delayed in 
recipients of T-cell depleted marrow (3-4 months) compared to HLA-identical marrow recipients (2 
weeks).  Other studies have also found a delay in immune reconstitution in patients with 
immunodeficiency disorders who received HLA nonidentical T-cell depleted bone marrow 
transplantation [19]. 
Filipovich et al. summarized the experience of the International Bone Marrow Transplant Registry and 
the National Marrow Donor Program for 170 patients with Wiskott-Aldrich Syndrome who received 
transplantation with HLA-identical sibling donors (n=55), unrelated donors (n=67), and other related 
relatives (n=48) [12]. The 5-year probabilities of survival by donor type were 87% for HLA-identical 
sibling recipients, 71% for unrelated donor recipients, and 52% for recipients of “other” related donors. 
A higher percentage of patients who received grafts from HLA-identical sibling or unrelated donors 
were cured of their disease (both 75%) versus patients who received grafts from other related donors 
(57%). The incidence of grade II-IV acute GVHD was 16%, 56%, and 30% for recipients of related, 
unrelated and “other” related donors, respectively. More recently, Dal-Cortivo et al. reported on the 
outcome of 40 patients with primary immunodeficiencies (SCID n=32, other n=8) conditioned with 
busulfan and cyclophosphamide followed by T-cell depleted HLA-haploidentical grafts. Mixed or full 
donor chimerism (exact numbers not given) was achieved in 24 of the 38 evaluable patients. Grade 
II-IV GVHD occurred in 11/24 (46%) of patients. Overall survival was 21/40 (53%), with infection being 
the primary cause of death (8 of 19 deaths) [13]. Klein et al. reported on 19 patients with major 
histocompatibility complex class II deficiency, who received HLA-identical BMT (n=7) or HLA-
haploidentical BMT (n=12). Four of the 8 HLA-identical recipients and 4 of the 12 haploidentical 
recipients were cured (n=3) or had improvement (n=1) of their disease with mixed of full donor 
chimerism [14].  
Buckley et al. summarized the outcome of patients with immunodeficiencies who received bone 
marrow transplantation from HLA-identical, unrelated donors, or haploidentical donor grafts [11] 
(Table 1). When comparing overall survivals, patients who received HLA-identical grafts had superior 
survivals (79%) compared to patients who received unrelated donor grafts (65%) or haploidentical 
donor grafts (55%).  
 
 
 
 
 
 
 
 
 


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Table 1: Outcome of patients with Immunodeficiency Disorders who received HLA-identical donor, 
HLA-unrelated donor or Haploidentical donor grafts 
Type of Immune Defect 
HLA-Identical Donor 
Unrelated Donor 
Haplo Donor 
 
# survived/ 
# transplanted 
# survived/ 
# transplanted 
# survival/  
# transplanted 
SCID 
105/125 
12/17 
260/425 
Wiskott-Aldrich Syndrome 
57/65 
20/29 
20/58 
Combined Immune Deficiency 
10/13 
0/2 
12/35 
Omenn Syndrome 
9/12 
3/6 
8/24 
Leukocyte Adhesion Defect 
6/8 
2/2 
14/20 
MHC Class II antigen deficiency 
4/8 
2/2 
7/20 
Chediak-Higashi Syndrome 
10/12 
5/5 
1/5 
DiGeorge Syndrome 
2/3 
0/0 
0/3 
Chronic Granulomatous Disease 
8/12 
2/3 
0/0 
PNP Deficiency 
2/5 
0/1 
2/2 
Cartilage-hair Hypoplasia 
3/3 
0/1 
1/3 
Hyper-IgM Syndrome 
2/3 
1/1 
1/3 
Immunodeficiency with Albinism 
3/6 
0/0 
0/0 
X-linked lymphoproliferative disease 
3/5 
0/1 
0/0 
Ataxia Telangiectasia 
0/1 
0/2 
0/0 
Common variable immunodeficiency 
0/4 
0/0 
0/0 
Capping Deficiency 
1/1 
0/0 
3/3 
IL-2 Deficiency 
0/0 
0/0 
2/2 
Neutrophil Disorders 
2/2 
0/0 
0/0 
ZAP70 Deficiency 
0/0 
0/0 
1/1 
Chronic Mucocutaneous candidiasis 
0/0 
0/0 
0/1 
Neutrophil Actin Dysfunction 
1/1 
0/0 
0/0 
All defects 
228/289 (79%) 
47/72 (65%) 
332/605 (55%) 
 
2) Other inherited disorders 
 
 
Data on patients who were transplanted with haploidentical grafts for inherited disorders other 
than immunodeficiency disorders is limited. Peters, et al. reported outcome for 54 patients with Hurler 
syndrome treated with conventional marrow transplantation from HLA-identical related (n=28) or 
haploidentical donors grafts (n=26). Patients were conditioned with chemotherapy with or without TBI 
[15]. T-cell depleted grafts were given to 15 recipients of haploidentical marrow, and the remaining 
patients received T-cell replete grafts. Among the 26 evaluable recipients of HLA-identical related 
grafts, 15 (58%) achieved full donor chimerism, 7 (27%) achieved mixed donor-host chimerism, and 
4 (15%) failed to engraft. Among the 26 recipients of haploidentical grafts, 16 (62%) achieved full 
donor chimerism, 1 (4%) achieved mixed donor-host chimerism, and 9 (35%) failed to engraft. With 
a median follow up of 7.3 years for the HLA-identical recipients and 4.6 years for the recipients of 
haploidentical grafts, the overall actuarial probability of survival at 5 years was 75% for the HLA-
identical recipients and 53% for the haploidentical recipients. Grade II-IV acute and extensive chronic 
GVHD occurred in 29% and 0% of HLA-identical recipients and 46% and 19% of recipients of 
haploidentical grafts. Death from GVHD occurred in 6 patents (HLA-identical n=1, haploidentical n=5). 
Enzyme levels were assessed in 28 patients (20 HLA-identical, 8 haploidentical) surviving > 1 year. 
Among the 10 recipients of homozygous normal donor marrow, levels of leukocyte -L-iduronidase 
normalized in 6 and were between 25-100% in 3. Among 18 recipients of heterozygous carrier donor 
marrow, 15 achieved carrier level of enzyme activity and 3 achieved levels in the lower limit of carrier 
range. Neurologic outcome was favorably associated with transplantation before age 24 months and 
achievement of normal enzyme levels.  Kapelushnik et al. reported on 1 patient with Hurler’s 
syndrome who received a T-cell depleted 3/6 matched haploidentical PBMC graft from his sister 
following conditioning with fludarabine (30 mg/m2/day; days –10 to –5), busulfan (4 mg/kg/day; days 
–7 to –6), ATG (10 mg/kg/day; days –4 to –1) and 750 cGy TBI (day –1) [16]. The patient fully 


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engrafted, did not have significant GVHD, and had correction of  -L-iduronidase levels at time of last 
follow up; 30 months after HCT. Although these data are limited, the results are encouraging for the 
use of haploidentical donors for patients with Hurlers when a related or unrelated donor is unavailable. 
Extending potential stem cell sources to include haploidentical grafts is particularly important for 
patients with hurlers syndrome due to only 30-40% of the patients who need HCT have related donors 
and the length of time required to find an unrelated donor may be prohibitively long, particularly for 
patients with rapidly progressive disease.  
 
Osteopetrosis is an autosomal recessive disease resulting from lack of osteoclast resorption of bone, 
resulting in marrow failure and nerve entrapment, and early death. Normally functioning osteoclasts 
can be restored by HCT, resulting in amelioration of disease symptoms [2]. Schulz et al. recently 
reported on the outcome of 7 patients with Osteopetrosis who received T-cell depleted HLA-
haploidentical grafts following conditioning with Busulfan, Thiotepa and either cyclophosphamide 
(n=5) or fludarabine (n=2) [17]. Five of the 7 patients had stable donor engraftment. Of the 2 patients 
with graft failure, 1 died and the other patient engrafted following transplantation with a second graft 
from the same donor without conditioning. Three patients did not have any significant complications; 
however, 4 patients had veno-occlusive disease and respiratory failure. With a median follow up of 
47 (range, 25-60) months, 5 patients are alive with stable to improved disease. Although the number 
of patients treated in this study was small, these results are encouraging; however, transplant related 
toxicity was high. Therefore, exploring a nonmyeloablative approach for those patients who do not 
have HLA-matched related or unrelated donors is warranted.  
Caillat-Zucman et al. reported on the outcome of 94 children who received HCT from HLA-identical 
sibling donors (n=31), unrelated donors (n=23) or haploidentical donors (n=40) for primary 
immunodeficiency disorders (n=64), metabolic diseases (n=12) or nonmalignant hematological 
diseases (n=18) [18]. Patients primarily received conditioning with busulfan and cyclophosphamide. 
All recipients of HLA-haploidentical marrow received either T-cell depleted or positive selection of 
CD34+ cells for GVHD prevention. Of the 90 patients evaluable for engraftment, 83 (92%) engrafted 
and there was no significant difference in the engraftment rate between the 3 groups. With a median 
follow up for the living recipients of 63 (range, 41-90) months, superior survivals were seen for 
recipients of HLA-identical grafts (80.6%) compared to recipients of unrelated grafts (63%) or 
haploidentical grafts (48%). Infections were the primary cause of death in all patients. 
These studies demonstrate myeloablative HCT with HLA-haploidentical grafts to be a viable 
treatment option for patients with nonmalignant disorders who do not have HLA-matched related or 
unrelated donors. However, inferior survivals were seen for recipients of HLA-haploidentical grafts.  
Death from transplant related toxicities primarily infections and GVHD were significant problems. 
Therefore, strategies aimed at reducing transplant related toxicities without compromising 
engraftment are needed.  
 
3B. Haploidentical Transplantation Using a Nonmyeloablative Approach 
 
The primary problem of HCT from related, HLA-haploidentical donors is controlling the potent 
alloreactivity of host T-cells that can lead to rejection of the graft and of donor T-cells that can lead to 
severe GVHD. Many approaches to this problem are possible. Protocol 2032 will incorporate a 
combination of immunosuppressive agents in a strategy designed to achieve control of host and donor 
immune responses with the objective of safe donor engraftment. The platform for this protocol is the 
approach to nonmyeloablative HCT pioneered by Storb and colleagues at the FHCRC which utilizes an 
immunosuppressive regimen that includes 2 Gy TBI before transplant (with or without fludarabine) to 
reduce the risk of graft rejection and post grafting MMF and CSP to control both GVHD and residual HVG 
reactions [22]. This regimen has been proven to be minimally toxic, well tolerated, and leads to successful 
engraftment in a high percentage of patients with malignant and nonmalignant hematologic diseases who 
received HLA-matched related and unrelated grafts [23,24]. However, additional immunosuppression will 
be needed with this regimen to achieve the objective of safe, nonmyeloablative HCT from HLA-


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haploidentical donors [25,26]. Based on the studies of investigators at Johns Hopkins Medical School 
and the FHCRC (see below), this protocol will provide additional immunosuppression with the alkylating 
agent cyclophosphamide, before and after transplant, aimed at removing alloreactive host and donor T-
cell clones presumably involved in both rejection and GVHD. 
 
3C. Rationale for the Use of Cyclophosphamide 
Cyclophosphamide was introduced into the clinical practice of marrow transplantation in 1969/1970, and 
has remained a major component of many transplantation regimens. Historically, due to its 
immunosuppressive abilities it was used pre HCT to suppress HVG reactions and improve engraftment 
[27-29]. The use of cyclophosphamide post HCT was deterred due to concerns that it might be 
myelotoxic, however, it is now clear that a single low dose of CY does not cause myeloablation [30]. The 
immunosuppressive rather than myelosuppressive effects of cyclophosphamide are due to its unique 
pharmacology. Specifically, hematopoietic stem cells express high levels of aldehyde dehydrogenase, 
an enzyme responsible for cellular resistance to cyclophosphamide. However, B-cells, T-cells and natural 
killer cells express low levels of this enzyme and are very sensitive to the cytotoxic properties of 
cyclophosphamide [31]. Therefore, the use of a single dose of cyclophosphamide early after HCT during 
the activation of alloreactive effector cells, results in cytotoxic activity against both HVG and GVH 
reactions. Studies have demonstrated that post transplant cyclophosphamide results in destruction of 
antigen-stimulated, proliferating T-cells (alloactivated) in the peripheral blood [32] and intrathymic clonal 
deletion of reactive T-cells [32-34], thereby improving engraftment and decreasing GVHD.  
 
3D. Methodology of Using Cyclophosphamide post HCT 
1) Pre-clinical data: Results in a Mouse Model of nonmyeloablative HCT using Haploidentical 
donors 
Luznik et al. published his results of transplantation using nonmyeloablative conditioning followed by 
Haploidentical grafts in the mouse model [35]. In this model, pre-transplant immunosuppression 
consisted of fludarabine and low-dose total body irradiation (TBI, 200 cGy). High-dose 
cyclophosphamide was then given on day +3 post-HCT to remove highly alloreactive T-cells of the 
donor as described by Nomoto’s group (for a review, see Mayumi, H., et al) [36]. Successful induction 
of stable, mixed chimerism was achieved across total MHC disparity in congenic mice (6/6 H-2 
mismatch, no minor histocompatibility differences) and in haploidentical strain combinations (3/6 H-2 
mismatch, minor histocompatibility differences) with a decreased incidence of GVHD compared to a 
set of control mice that were not given cyclophosphamide after HCT. Another set of control mice not 
given a marrow graft recovered autologous hematopoiesis.  
 
2) Results of a Phase 1 Trail Using Cyclophosphamide pre and post HCT.  
Based on the data of the effect of cyclophosphamide on prevention of rejection and induction of donor 
tolerance in the mouse model, a phase I clinical trial was conducted [37]. The major objective of this 
clinical trial was to determine the minimum dose of pre-transplantation cyclophosphamide required 
for prevention of allograft rejection. In addition, to reduce the risk of GVHD, all patients received 
immunosuppression after HCT with high-dose cyclophosphamide plus the combination of 
mycophenolate mofetil (MMF) and a calcineurin-inhibitor, tacrolimus. Two cohorts of patients existed. 
The first cohort (n=3) did not receive any pre-transplantation cyclophosphamide. The second cohort 
(n=10) received 14.5 mg/kg cyclophosphamide on days -6 and -5. All patients received fludarabine 
(30 mg/m2/day; days -6 to -2) and 200 cGy TBI (day -1) followed by HLA-mismatched (up to 3 HLA 
antigens) related marrow allografts. In addition, all patients received 50mg/kg cyclophosphamide on 
day +3. The first 9 patients received MMF (15 mg/kg twice daily; days 4-35) and tacrolimus (initial 
dose 1 mg daily; days 4-50). Due to 3 of the first 6 patients developing GVHD after day 100, tacrolimus 
was extended beyond day 90. Median CD34 and CD3 cell doses were 5.3 +/- 1.5 x 106 and 3.2 +/- 


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0.6 x 107 cells/kg, respectively. Of the patients in cohort 1 (no pre-HCT cyclophosphamide), 1 patient 
engrafted but died of relapsed disease and the remaining 2 patients did not engraft. One patient had 
autologous recovery and the other patient died of infection during aplasia. Both patients who rejected 
had a long history of transfusion-dependence ( 1 yr) and, in retrospect, one patient was found to be 
cross-match positive with her donor prompting a revision of the eligibility criteria to exclude such 
patients subsequently. Of the 10 patients in cohort #2, sustained donor cell engraftment occurred in 
8 patients. Two patients failed to engraft and both had MDS, a long history of transfusion dependence 
and no prior chemotherapy; known risk factors for graft rejection. Six patients developed acute GVHD 
(Grade II n=3, grade III n=3) and 1 patient developed limited chronic GVHD. An additional patient 
developed grade III GVHD after donor lymphocyte infusion for relapsed AML. With a median follow 
up for the living recipients of 284 (range 185-423) days, 6 of the 10 patients were alive. Immune 
reconstitution was evaluated in 6 patients in cohort 2 and demonstrated recovery of CD4 counts to 
>200 cells/ml by day 40 in 4/6 patients and recovery of NK cells to pretransplantation levels by day 
40 in 5/6 patients tested. 
The level of immunosuppression used in this protocol before and after HCT showed promise in 
performing nonmyeloablative HCT from related, HLA-haploidentical donors. Transplant-related 
toxicity was relatively low in an older group of patients with advanced hematologic malignancies who 
were transplanted with allografts from related, HLA-haploidentical donors containing >107 CD3+ 
cells/kg. Based on these results a phase II study was developed at the FHCRC to evaluate the 
efficacy and safety of this regimen in patients with high-risk hematologic malignancies.  
 
3) Nonmyeloablative Hematopoietic Stem Cell Transplantation for Patients with High-Risk 
Hematologic Malignancies using Related, HLA-Haploidentical Donors: A Phase II Trail of 
Combined Immunosuppression Before and After Transplantation – Preliminary Results 
(protocol 1667)  
Twenty five patients with high-risk hematologic malignancies (AML (n=6), MDS or MDS/AML (n=5), 
CML (n=2), ALL (n=2), NHL (n=2), HD (n=7) and MM (n=1)) who were ineligible for conventional 
transplant and did not have an HLA-matched related or unrelated donor were entered onto this phase 
II clinical study (protocol 1667). (Table 2) Patients received Cyclophosphamide (14.5 mg/kg days -6 
and -5), Fludarabine (30 mg/m2/day, days -6 to -2), and 200 cGy TBI (day –1) followed by bone 
marrow grafts on day 0 from HLA-mismatched related donors (8/10 mismatch (n=4), 7/10 mismatch 
(n=3), 6/10 mismatch (n=4) and 5/10 mismatch (n=14)) followed by postgrafting immunosuppression 
with MMF and Tacrolimus. In addition, all patients received 50mg/kg/dose cyclophosphamide on day 
+3. The primary endpoints of this study are efficacy (true engraftment rate of 85% with >50% donor 
chimerism at day 84) and safety (<35% grade III-IV GVHD and <25% day 200 non-relapse mortality). 
Median age of patients was 44 (range 20-67) years. The median CD34 and CD3 cell doses were 5.3 
(2.7-12.8) x 106 and 4.9 (2.0-7.2) x 107 cells/kg, respectively. Median neutrophil recoveries occurred 
at 13 (range, 11-42) days after transplant. Three patients (14%) rejected their grafts at days 12, 13, 
and 42 after transplant and all had autologous hematopoietic recovery. In addition, all three patients 
received grafts from 5/10 HLA mismatched related donors (GVHD mismatched vector (n=1), bi-
directional mismatched vector (n=2)). Of the remaining 22 patients, 19 were evaluable for engraftment 
at day 84. Of the 19 patients, 18 had full donor CD3 and CD33 chimerism defined as donor chimerism 
>95%. Only < grade II acute GVHD was seen (12 patients (67%) grade II, 3 patients (19%) grade I). 
Five patients (20%) developed extensive chronic GVHD. With a median follow up of the living 
recipients of 10 (range, 3-26) months, 14 patients have died from relapse (n=10), transplant related 
toxicities (infections, n=2), secondary AML (n=1), or unknown causes (n=1).  
Despite the low incidence of graft rejection (3/25 = 12%), protocol 1667 will be amended to increase 
the pre-transplant cyclophosphamide dose from 14.5 mg/kg (days -6 and -5) to 25 mg/kg (days -6 
and -5) with the goal of increasing host immunosuppression and further decreasing the risk of graft 
rejection.  


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Table 2: Characteristics and outcome of 25 patients who received HCT with nonmyeloablative conditioning 
followed by HLA-haploidentical grafts for high-risk hematologic malignancies. 
Pt # 
Diagnosis 
Age 
HLA-
Mismatch/ 
Donor 
Status at 
HCT 
D84 
chimerism 
CD3/CD33 
Sustained 
Engraft-
ment 
GVHD 
F/U 
(mos) 
Cause of 
Death 
Acute 
(Grade) 
Ext 
Chronic  
1 
AML 
42 
5/10 sib 
CRp 
100/100 
+ 
II 
- 
†6 
Relapse 
2 
AML 
33 
5/10 parent 
CR2 
N/A 
- 
- 
- 
†7 
Relapse 
3 
AML 
67 
7/10 child 
CR3 
N/A 
+ 
II 
- 
†3 
Relapse 
4 
AML 
35 
5/10 parent 
CRp 
100/100 
+ 
- 
- 
†4 
Relapse 
5 
AML 
51 
5/10 child 
Ref 
100/100 
+ 
- 
- 
†8 
Relapse 
6 
AML 
60  
5/10 child 
CRt 
100/100* 
+ 
- 
- 
>3 
 
7 
MDS/AML 
60 
5/10 child 
CRp 
100/100 
+ 
II 
- 
†22 
Relapse 
8 
MDS/AML 
60 
5/10 sib 
CR1 
N/A 
+ 
- 
N/A 
†1 
Infection 
9 
MDS/AML 
62 
8/10 sib 
CRp 
97/100 
+ 
II 
- 
†4 
Relapse 
10 
MDS/AML 
34 
5/10 sib 
CR2 
N/A 
- 
N/A 
N/A 
>7 
 
11 
MDS 
37 
5/10 parent 
CRp 
100/100 
+ 
- 
+ 
>3 
 
12 
Ph+ ALL 
36 
5/10 sib 
MRD 
N/A 
- 
N/A 
N/A 
>16 
 
13 
ALL 
51 
6/10 child 
Ref 
100/100 
+ 
II 
+ 
>10 
 
14 
CML 
66 
8/10 child 
AP 
92/2 
+ 
II 
+ 
†10 
Infection 
15 
CML 
51 
6/10 child 
Ref 
100/100 
+ 
II 
- 
>13 
 
16 
MM 
63 
6/10 sib 
Ref 
100/100 
+ 
II 
+ 
†8 
Relapse 
17 
NHL 
59 
6/10 sib 
Ref 
100/100 
+ 
II 
- 
†5 
2° AML 
18 
NHL 
44 
7/10 child 
CR 
100/100 
+ 
II 
- 
†6 
Unknown 
19 
HD 
26 
5/10 parent 
Ref 
N/A 
+ 
- 
N/A 
†1 
Relapse 
20 
HD 
20 
7/10 parent 
SD 
100/100 
+ 
- 
- 
>26 
 
21 
HD 
33  
5/10 parent 
Ref 
100/100 
+ 
- 
- 
>22 
 
22 
HD 
34 
8/10 sib 
Ref 
100/95 
+ 
- 
- 
†12 
Relapse 
23 
HD 
51 
8/10 sib 
CR 
100/100 
+ 
II 
+ 
>11 
 
24 
HD 
31  
5/10 parent 
Relapse 
100/100 
+ 
II 
- 
>4 
 
25 
HD 
26 
5/10 parent 
SD 
100/100 
+ 
- 
- 
>3 
 
AML = acute myelogenous leukemia, CML = chronic myelogenous leukemia, CR = complete remission, CRp = complete remission 
(<5% blasts) with pancytopenia or thrombocytopenia, Ext chronic = extensive chronic GVHD, F/U = follow up, HD = Hodgkin 
disease, MDS = myelodysplastic syndrome, MM = multiple myeloma, MRD = minimal residual disease, N/A = not applicable, NHL 
= non Hodgkin Lymphoma, Ph+ ALL = Philadelphia chromosome positive acute lymphoblastic leukemia, Ref = refractory, SD = 
stable disease, > alive, † dead 
* Patient #6 day 56 peripheral blood chimerism was 100% CD3, 100% CD33. The day 84 bone marrow was not sorted but was 
100% donor. 
Although relapse related mortality in this group of patients with high-risk hematologic malignancies 
was high (10/14 patients), only 2 patients died from transplant related causes. This is particularly 
relevant for patients with nonmalignant diseases where death from relapse is not an issue and a low 
incidence of transplant related mortality is desirable. In addition, although preliminary, > 75% of the 
patients engrafted and there was a low incidence of grade III-IV (0%) and extensive chronic GVHD 
(20%). Again, this is particularly appealing for patients with nonmalignant diseases who do not need 
a graft versus leukemia effect from GVHD. Encouraged by both the phase I and II clinical studies for 
patients with hematologic malignancies, a Phase I-II study is warranted to determine the safety and 
efficacy of this approach in patients with nonmalignant disorders. Similar to what will be done for 
patients with hematologic malignancies; protocol 2032 will use a higher dose of pre-transplant 
cyclophosphamide with the goal of improving donor engraftment and decreasing graft rejection. 
Specifically, patients will receive 25 mg/kg (days -6 and -5) instead of 14.5 mg/kg (days -6 and –5).  
We have enrolled 5 patients with nonmalignant diseases on protocol 2032 to date, and have observed 
a high incidence of both acute grades II (n=3) or III (n=1) GVHD and chronic GVHD (n=4). A phase 
II trial randomized three-arm trial of post-transplant immune suppression following nonmyeloablative 
conditioning for recipients of unrelated PBSC grafts showed that the addition of sirolimus to a regimen 


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of tacrolimus and MMF was superior in preventing acute GVHD [Kornblit, B, et al, 2014 
Haematologica, In Press]. Specifically, the incidence of grades II-IV acute GVHD were 64% for the 
arm that included tacrolimus and MMF given similarly to the current 5 patients, compared to 51% for 
the arm with a lengthend course of MMF, compared to 46% for the arm that included sirolimus in 
addition to tacrolimus and MMF (hazard ratio 0.59, p=.02). Since relapse is not a concern for patients 
with nonmalignant diseases, and consequently there is no benefit for GVHD, the protocol was revised 
to include sirolimus in addition to tacrolimus and MMF for GVHD prophylaxis.  
 
4. OBJECTIVES 
It is important to extend the option of nonmyeloablative HCT for potential therapy of patients with 
nonmalignant disorders to patients who do not have HLA-matched related or unrelated donors. Almost all 
patients would have a related donor identical for one HLA haplotype (haploidentical) and mismatched at 
HLA-A, B or DR of the unshared haplotype. The central problem of HCT from related, HLA-haploidentical 
donors is controlling the potent alloreactivity of host T-cells, which can lead to rejection of the graft and of 
donor T-cells, which can lead to severe toxicity from GVHD. Many approaches to this problem are possible. 
This protocol will incorporate a combination of immunosuppressive agents in a strategy designed to achieve 
control of host and donor immune responses with the objective of safe donor engraftment. Specifically, 
cyclophosphamide will be administered before and after HCT to facilitate engraftment and to remove highly 
reactive T-cell clones presumably involved in GVHD.  
 
4A. Primary Objective 
1) Safety 
 
Determine safety of nonmyeloablative conditioning and HCT from HLA-haploidentical related 
donors for patients with nonmalignant inherited disorders who do not have an HLA-matched 
related or unrelated donor. 
4B. Secondary Objectives 
1) Efficacy 
 
Determine whether nonmyeloablative conditioning and HCT from an HLA-haploidentical related 
donor graft can establish mixed chimerism (>5% CD3+ donor T-cell chimerism) in patients with 
nonmalignant inherited disorders. 
2) Transplant related mortality at day 100  
3) Incidence and severity of GHVD  
4) Immune Reconstitution 
5) Infections during the first 200 days after HCT 
 
 
 
5. PATIENT SELECTION 
5A. Patient Inclusions 
1) Age < 55 years with primary immunodeficiency disorder or other nonmalignant inherited disease 
(except fanconi anemia) treatable by allogeneic HCT.  
2) Patients with pre-existing medical conditions or other factors that renders them at high risk for 
regimen related toxicity or ineligible for conventional myeloablative HCT and who do not have 
HLA-matched related or unrelated donors.  
3)  Patients with a related donor who is identical for one HLA haplotype. 


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4)  Acquired Aplastic anemia:  SAA is defined as follows: 
 
Bone marrow cellularity < 25%, or marrow cellularity < 50% but with < 30% residual 
hematopoietic cells. 
 
Two out of three of the following (in peripheral blood): neutrophils < 0.5 x 109/L; 
platelets < 20 x 109/L; reticulocytes < 20 x 109/L. 
 
SAA diagnostic criteria may be applied to assessment at initial diagnosis or follow-up 
assessments.  
 
 
5B. Patient Exclusions 
1) Fanconi Anemia 
2) Suitably HLA- matched related or unrelated donors 
3)  Patients with metabolic storage diseases who have severe CNS involvement of disease, defined 
as IQ score < 70.  
4) Patients with the following organ dysfunction: 
 
a. Cardiac: 
i)  Cardiac ejection fraction < 30% (or, if unable to obtain ejection fraction, shortening fraction 
< 26%) on MUGA scan or cardiac echo, symptomatic coronary artery disease, other 
cardiac failure requiring therapy. Patients with a history of, or current cardiac disease 
should be evaluated with appropriate cardiac studies and/or cardiology consult. Patients 
with a shortening fraction of <26% must be seen by cardiology for approval.  
ii) Poorly controlled hypertension despite anti-hypertensive medications.  
b. Hepatic: Patients with clinical or laboratory evidence of liver disease will need to be evaluated 
for the cause of the liver disease, its clinical severity in terms of liver function and the degree 
of portal hypertension. Patients will be excluded if they are found to have: fulminant liver 
failure, cirrhosis of the liver with evidence of portal hypertension, bridging fibrosis, alcoholic 
hepatitis, esophageal varices, a history of bleeding esophageal varices, hepatic 
encephalopathy, uncorrectable hepatic synthetic dysfunction evidenced by prolongation of the 
prothrombin time, ascites related to portal hypertension, bacterial or fungal liver abscess, 
biliary obstruction, chronic viral hepatitis with total serum bilirubin > 3mg/dl, or symptomatic 
biliary disease.  
5) Positive for human immunodeficiency virus (HIV). 
6) Females who are pregnant (-HCG+) or breast-feeding. 
7) Fertile men or women who are unwilling to use contraceptives during HCT and up to 12 months 
post-treatment. 
8) Patients with fungal pneumonia with radiological progression after receipt of amphotericin 
formulation or mold-active azoles for greater than 1 month will not be eligible for this protocol 
(either regimen A or B).  
 
6. DONOR SELECTION – See Standard Practice Manual  
6A. Donor Inclusions 
1) Related donors who are identical for one HLA haplotype.   
2) Bone marrow will be the only allowed stem cell source. 
6B. Donor Exclusions 
1) Donor-recipient pairs in which the HLA-mismatch is only in the HVG direction. Patients are 
homozygous and donor is heterozygous. 


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2) Donors who are not expected to meet the minimum target dose of marrow cells (1 x 108 nucleated 
cells/kg recipient Ideal Body Weight) 
3) HIV-positive donors 
4) A positive anti-donor cytotoxic cross match is absolute donor exclusion. 
5) Donor < 6 months old and > 75 years old. 
 
7. INFORMED CONSENT  
The outpatient 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 
for marrow donors. 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 fludarabine, low dose TBI, Cyclophosphamide, immunosuppressive drugs and 
allogeneic HCT will be discussed as objectively as possible. Discussion of potential complications should 
include graft rejection, GVHD, infections, and death. It should be explained that patients offered this protocol 
have an inherited disorder and are at high risk of early transplant mortality from conventional allogeneic HCT 
due to associated co-morbidities.  
 
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. A summary of the conference will be dictated for the medical record detailing what was 
covered. 
 
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 H 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 or her staff at (206) 667-7385 or (206) 667-4916..  
 
 
 


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9. PLAN OF TREATMENT  
9A. Outline of Treatment Plan (Scheduling is Shown in Table 3) 
Days -6, -5 
Fludarabine 30 mg/m2 IV once per day 
Based on estimated creatinine clearance 
Cyclophosphamide 25mg/kg IV once per day 
Mesna (dosed at 100% cyclophosphamide dose) 
 
Days -4 to -2 
Fludarabine 30 mg/m2 IV once per day  
Based on estimated creatinine clearance 
 
Day -1 
 
200 cGy TBI at 6-15 cGy per min 
 
Patients with acquired aplastic anemia, sickle cell disease, thalassemia, HLH, or other non-
malignant disease at high risk for graft rejection (as discussed at the pediatric non-malignant 
arrival meeting, non-malignant board,  patient care conference, or with study PI’s) will receive 
400 cGy TBI (2 doses of 200 cGy) at 6-15 cGy per min. 
 
Day 0 
Infuse marrow allograft 
 
Day +3, +4 
Cyclophosphamide 50 mg/kg (First dose must be administered 48-72 hr post-BMT) 
Mesna (100% cyclophosphamide dose) 
 
Day +5 
Tacrolimus: 
0.03 mg/kg/day continuous IV infusion over 22-24 hours and continue until day + 100 with taper 
through day +180. 
 
Tacrolimus may be converted from IV to oral (twice daily or three times daily dosing per standard 
practice) when the patient is able to take medications orally and has a therapeutic drug level.   
 
MMF: 15 mg/kg orally every 8 hours and continue until day +30; then BID to day +40 and then if 
there is no evidence of active GVHD and donor engraftment (CD3) are >95% (or approved by the 
PI) the MMF may be tapered by roughly 10% per week and discontinued around day +96.  MMF 
can be tapered faster if there is evidence of count suppression following discussion with the PI.   
 
Sirolimus:  
 
≤1.5 m2:  1 mg/m2 orally every day until day +180 then taper until approximately day +210.   
 
 
>1.5 m2: 
2 mg orally every day until day +180 then taper until approximately day +210.  
 
G-CSF  
(5 g/kg/day) IV or SC, continue until ANC>500/l x 3d 
Note: Patients with sickle cell disease will not receive G-CSF. 
 
 


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Table 3:  Outline of Treatment Plan 
Day 
Treatment Plan  
-6 to -5 
CY, 25 mg/kg/day (with Mesna), Fludarabine, 30 mg/m2/day (based on estimated creatinine clearance) 
-4 to -2 
Fludarabine, 30 mg/m2/day (based on estimated creatinine clearance) 
-1 
200 cGy TBI at 6-15 cGy/min 
Or 
400 cGy TBI (2 doses of 200 cGy) for patients with Aquired Aplastic Anemia, sickle cell disease, 
thalassemia, HLH, or other non-malignant diseases at high risk for graft rejection (as discussed at the 
pediatric non-malignant arrival meeting, non-malgnant board, patient care conference, or with study 
PI’s) at 6-15 cGy/min 
0 
Infuse Marrow allograft  
+3, +4 
CY, 50 mg/kg/day (with Mesna) 
+5 
Start G-CSF 5 g/kg/day and continue until absolute neutrophil count > 500/l x 3 days. Patients with 
sickle cell disease will not receive G-CSF. 
Start MMF (15 mg/kg orally every 8 hours) and continue until day +30; then BID until day +40 and then 
if there is no evidence of active GVHD and donor engraftment (CD3) are >95% (or approved by the 
PI) the MMF may be tapered by roughly 10% per week and discontinued around day +96.  MMF can 
be tapered faster if there is evidence of count suppression following discussion with the PI.    
Start Tacrolimus 0.03 mg/kg/day IV and continue until day +100 with taper through day +180.  
Tacrolimus may be converted from IV to oral (twice daily or three times daily dosing per standard 
practice) when the patient is able to take medications orally and has a therapeutic drug level.   
Start Sirolimus 2 mg (>1.5 m2) or 1 mg/m2 (≤1.5 m2) orally per day and continue until day +180 then 
taper to day +210. 
 
9B. Indwelling central venous catheter 
Placement of a double lumen central venous catheter will be required for administration of IV medications 
and transfusion of blood products. 
 
10. CONDITIONING REGIMEN 
Menstruating female patients should be placed on an anti-ovulatory agent prior to initiating the conditioning 
regimen. 
 
10A. Fludarabine 
1) Fludarabine dose is based on estimated creatinine clearance: 
 
Adults: creatinine clearance may be estimated by the Cockcroft Formula: 
CCr = (140 – age) x IBW (kg) x 0.85 (for women) 
PCr x 72 
 
Pediatrics (Age <17): creatinine clearance may be estimated by the Swartz formula or 
appropriate calculation for pediatric patients 
 
2) Fludarabine is administered by IV infusion over 1 hour on days -6 to -2 
If estimated creatinine clearance is less than 60 ml/min, obtain a formal clearance with a 24 hour 
urine collection, iothalamate or equivalent study. 


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Creatinine Clearance ml/min 
Daily Fludarabine Dose (mg/m2) 
> 60 
30 
46-60 
24 
31-45 
22.5 
21-30 
19.5 
<20  
15 
 
** The dose of fludarabine is based on M2, the calculation is always based on the Actual 
Body Weight (See appendix L) 
 
10B. Cyclophosphamide (See Appendix K) 
 
1)   Cyclophosphamide will be administered at a dose of 25 mg/kg/day as an IV infusion over 1 hour 
on days -6 and -5 (Total dose = 50mg/kg) 
 
2) The dose should be based on the Adjusted Body Weight if the patient’s Actual Body Weight is > 
100% of the Ideal Body Weight. If the Ideal Body Weight (IBW) > Actual Body Weight, then Actual 
body weight will be used. 
 
3) Please see appendix K for mesna and fluid administration guidelines.  
 
 
10C. Total body irradiation (See Appendix M for guidelines for TBI administration) 
200 cGy TBI will be given on day –1 at a rate of 6-15 cGy per min per TBI administration guidelines 
(Appendix M).  Patients with acquired aplastic anemia, sickle cell disease, thalassemia, or other 
non-malignant disease at high risk for graft rejection (as discussed at the non-malignant board) will 
receive 400 cGy TBI (2 doses of 200 cGy) on day -1 at a rate of 6-15 cGy per min per TBI 
administration guidelines (Appendix M) 
 
11. BONE MARROW INFUSION 
Donor bone marrow will be harvested with a target yield of 4 x 108 nucleated cells/kg recipient IBW. The 
minimum acceptable yield should be 1 x 108 nucleated cells/kg recipient IBW. The CD34 and CD3 
composition of allografts will be determined by flow cytometry.  
See Appendix A for ABO Incompatibility guidelines.     
 
12. POST-TRANSPLANT IMMUNOSUPPRESSION 
Immunosuppression to permit engraftment and provide GVHD prophylaxis will be performed with MMF and 
Tacrolimus. MMF and Tacrolimus treatment are described below and are depicted in Table 3 
 
12A. Cyclophosphamide:  
Day +3 and +4: Cyclophosphamide [50mg/kg/day Adjusted Body Weight, unless Ideal Body Weight 
>Actual Body Weight, then Actual Body Weight] will be given on days +3 and +4 after transplant.  (See 
Appendix K) The first dose of Cyclophosphamide (day +3) will be given within 48-72 hr of marrow 
infusion. Cyclophosphamide will be given as an IV infusion over 1 hr with Mesna and appropriate 
hydration.  (See Appendix K) Monitoring of urine output and for hematuria will be performed similarly. 


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12B. Tacrolimus  
Day +5: Starting on day +5, tacrolimus will be given at a dose of 0.03 mg/kg/day continuous infusion over 
22-24 hours. Tacrolimus is dosed based on adjusted body weight. If actual weight is less than adjusted 
body weight; dosing should be based on actual weight.Tacrolimus can be changed to a PO dosing 
schedule (Q12 or Q8 dosing per standard practice) once oral medications are tolerated and once a 
therapeutic level (5-10 ng/ml) is achieved. Serum levels of tacrolimus should be measured on day +8 
and then twice weekly thereafter and the dose adjusted accordingly to maintain a level of 5-10 ng/ml. 
Tacrolimus will be tapered after day +100 by 5% per week, provided there is no evidence of GVHD. In 
addition, in the case of <95% CD3 donor chimerism, continuation of therapeutic tacrolimus beyond day 
100 should be determined in consultation with the PI.  The combination of sirolimus and tacrolimus can 
lead to certain toxicities that are increased when the levels of tacrolimus and sirolimus are high.  Once a 
patient has stable donor engraftment, the combined targeted level of sirolimus and tacrolimus should not 
exceed 16 ng/mL and the tacrolimus level should be <8 ng/mL.   
12C. MMF 
Day +5: Starting on day +5, MMF will be given orally at a dose of 15 mg/kg based on adjusted body 
weight every 8 hours. MMF will be switched to BID dosing on day +30 and BID to day +40 and then if 
there is no evidence of active GVHD and donor engraftment (CD3) are >95% (or approved by the PI) the 
MMF may be tapered by roughly 10% per week and discontinued around day +96.  MMF can be tapered 
faster if there is evidence of count suppression following discussion with the PI.  Myfortic may be used in 
place of MMF as clinically indicated. 
12D. Sirolimus 
Day +5: Starting on day +5, Sirolimus will be given orally at a dose of 2 mg (for patients >1.5 m2) or 1 
mg/m2 (for patients ≤1.5 m2) daily through day +180. Dosing should be adjusted to maintain a target blood 
level of 3-12 ng/mL. Sirolimus should be tapered at day +180 by 25% per week for 4 weeks and 
discontinued around day +210, provided there is full donor chimerism.  In the case of donor chimerism 
<95% CD3 or treatment for GVHD, continuation of sirolimus should be determined in consultation with 
the PI.  The combination of sirolimus and tacrolimus can lead to certain toxicities that are increased when 
the levels of tacrolimus and sirolimus are high.  Once a patient has stable donor engraftment, the 
combined targeted level of sirolimus and tacrolimus should not exceed 16 ng/mL and the tacrolimus level 
should be <8 ng/mL.   
 
13. GUIDELINES FOR TACROLIMUS DOSE ADJUSTMENT AND MONITORING  
13A. If there is nausea and vomiting which prevents oral intake at any time during tacrolimus treatment, 
the drug should be given intravenously at the appropriate dose that was used to obtain a therapeutic 
level. (IV: PO ratio = 1: 4).  
13B.  Whole blood trough levels of tacrolimus (i.e., just prior to the next dose) should be obtained on Day 
+8 and then twice weekly until the taper is initiated, unless high levels (>20 ng/ml) are detected or toxicity 
is suspected, in which case more frequent monitoring will be performed as clinically indicated.  The dose 
should be adjusted accordingly to maintain a level of 5-10 ng/ml.  
13C. Dose reductions should only be made if Tacrolimus toxicity is present or levels exceed 10ng/ml in 
the absence of toxicity or as clinically indicated. Dose reductions of Tacrolimus for high levels without 
toxicity should be conservative, e.g. roughly 25%, to avoid inadequate immunosuppression. If creatinine 
is greater or equal to 2 times the baseline levels then the tacrolimus dose will be reduced by roughly 
25%.  
13D.  Blood pressure, renal function tests (creatinine, BUN), electrolytes and magnesium need to be 
followed at least two to three times per week while receiving tacrolimus at full dose and then twice weekly 
or per attending until tacrolimus is discontinued. 


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13E. Tacrolimus levels should be performed more frequently when 1) drug is converted from oral to IV 
or IV to oral, 2) dose adjustments are made due to levels outside the therapeutic range, or 3) voriconazole 
(see table below) is initiated or withdrawn 4) if toxicity is suspected. Steady state levels will not be 
achieved for at least 72 hours after any change in dosing, i.e. levels determined earlier may not reflect 
an accurate steady state concentration. 
13F. Patients requiring hemodialysis should have tacrolimus levels maintained in the therapeutic range 
(5 to 15 ng/ml). 
13G.   
Decrease Tacrolimus Levels 
Increase Tacrolimus Levels 
Dilantin 
Steroids 
Phenobarbital 
Fluconazole 
Carbamazepine 
Ketoconazole 
Rifamycin 
Itraconazole 
Caspofungin 
Voriconazole 
 
Cimetidine 
 
Macrolide antibiotics 
 
Calcium channel blockers 
 
Danazol 
 
metoclopramide 
 
** When initiating therapy with voriconazole in patients already receiving tacrolimus, it is 
recommended that the tacrolimus dose be reduced to one-third of the original dose, (a 67% reduction) 
and followed with frequent monitoring of the tacrolimus blood levels. Increased tacrolimus levels have 
been associated with nephrotoxicity. When voriconazole is discontinued, tacrolimus levels should be 
carefully monitored and the dose increased as necessary. 
 
13H. Grapefruit and grapefruit juice affect metabolism of tacrolimus and should be avoided. Oral 
tacrolimus should be taken consistently with or without food.  
 
14. GUIDELINES FOR MYCOPHENOLATE MOFETIL DOSE ADJUSTMENT AND MONITORING 
14A. Initiating MMF therapy: Oral administration of MMF will be at 15 mg/kg orally every 8 hours (45 
mg/kg/day) starting the evening of day +5. If there is nausea and vomiting at any time preventing the oral 
administration of MMF, MMF should be administered intravenously at the appropriate dose.  Myfortic may 
be used in place of MMF as clinically indicated. 
14B. Maintaining MMF: Markedly low (<40%) donor T-cell chimerism after HCT may indicate impending 
graft rejection. MMF should be continued at full dose or, if the MMF taper has been initiated, reinstitution 
of full dose MMF should occur if clinically feasible. If MMF has been discontinued, MMF should be 
reinitiated at full dose or as clinically indicated. 
 
15. GUIDELINES FOR MMF DOSE ADJUSTMENT DUE TO DRUG TOXICITY  
15A. If in the clinical judgment of the attending physician the observed toxicity is related to MMF 
administration, a dose adjustment may occur. The discontinuation of MMF at any point should be 


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discussed with the Study PI and should be documented in the permanent medical record and all Case 
Report Forms (CRF) 
15B.  Gastrointestinal Toxicity. Severe gastrointestinal toxicities such as gastrointestinal hemorrhage 
have been very rare after nonmyeloablative HCT. In the event of gastrointestinal toxicity that requires 
medical intervention including medication for control of persistent vomiting or diarrhea that is considered 
to be due to MMF after day 28, a 20% dose reduction will be made or the drug may be given IV. If severe 
refractory diarrhea or overt gastrointestinal bleeding occurs, MMF may be temporarily stopped. The MMF 
should be restarted at 20% reduced dose when the underlying toxicity subsides. 
15C. Neutropenia. Based on previous experience in patients after nonmyeloablative HCT, dose 
adjustments are likely to occur because of hematopoietic adverse effects, in particular neutropenia. A 
thorough evaluation of neutropenia should occur including peripheral blood chimerism studies, marrow 
aspiration (if clinically indicated) and review of marrow suppressive medications. If all other potential 
causes of marrow toxicity are ruled out, dose adjustments will only be made for severe, 
prolongedneutropenia (ANC <500/l for 5 days or more) that persists after day +21 post-transplant. Dose 
reductions should be conservative (20%). After day +21, the use of G-CSF will be permitted for severe 
neutropenia as clinically indicated. MMF may be tapered early following discussion with the protocol PI.  
The discontinuation of MMF at any point earlier than planned should be discussed with the Study PI and 
should be documented in the permanent medical record and all Case Report Forms (CRF). 
 
16. GUIDELINES FOR SIROLIMUS DOSE ADJUSTMENT AND MONITORING 
16A. Sirolimus should be given at least 4 hours after an oral dose of tacrolimus as concurrent administration 
leads to elevation of the sirolimus levels. To minimize variability of exposure to sirolimus, the drug 
should be taken consistently with or without food. Grapefruit juice should not be administered with 
sirolimus or used for dilution. The dosage should be replaced if the patient  vomits within 15 minutes of 
taking a dose. 
16B. Whole blood trough levels of sirolimus (i.e., just prior to the next dose) should be obtained on Day +8 
and then twice weekly until day +35, and then weekly until the taper is initiated. More frequent levels 
may be indicated if high levels (>12 ng/ml) are detected or toxicity is suspected. 
16C. Concommittant antifungal therapy: Follow Standard Practice Antifungal Therapy Guidelines for dosing 
reductions for patients receiving azole antifungal therapy. 
 
16D. Severe neutropenia or thrombocytopenia. The combination of sirolimus and tacrolimus may result in an 
increased risk of sirolimus toxicity such as anemia, diarrhea, hypokalemia, and/or thrombocytopenia. 
Dose adjustments should be made for severe neutropenia and thrombocytopenia that persists after 
day 21 post-transplant, or if the neutropenia is unresponsive to G-CSF. Dose reductions of sirolimus of 
approximately 50% should occur. If neutropenia persists despite dose reduction, sirolimus should be 
held until blood counts recover to ANC>1500/L.  
   
16. GROWTH FACTOR SUPPORT 
Patients will receive G-CSF at 5 g/kg/day IV or SC starting at day +5 and continuing until the ANC >500/L 
for 3 days. Patients with sickle cell disease will not receive G-CSF. 
 
 
 
 


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17. INFECTION PROPHYLAXIS AND THERAPY 
17A. Patients will receive prophylaxis for PCP, VZV, and HSV, S. pneumoniae and Candida as per the 
standard practice guidelines of the individual institution. Recommendations are listed in Appendix B of 
protocol. 
The highlights of the recommendations are: 
1) Fluconazole until day +75.  
2) Acyclovir until day +365 (To be continued beyond day +365 until all immunosuppression is 
discontinued in patients with chronic GVHD). 
3) Sulfamethoxazole/trimethoprim should be started at day 30 only if the ANC is >500 x 3 
consecutive days, and the patient should continue on sulfamethoxazole/trimethoprim until 
discontinuation of immunosuppressive drugs  
4) Immunoglobulin (IVIG): see standard practice  
a. Immunodeficiency disorders: all patients with primary Immunodeficiencies should receive 
IVIG every month regardless of serum IgG level until they have been off immunosuppression 
for 3 months. 
b. Other inherited disorders: see standard practice. 
18. EVALUATION OF CHIMERISM 
18A. 
Definitions: For the purposes of this protocol, the following definitions will apply 
1) Mixed chimerism: the detection of donor T-cells (CD3+), as a proportion of the total T-cell 
population, respectively, of greater than 5% and less than 95% in the peripheral  blood. 
2) Full donor chimerism: > 95% donor CD3+ T-cells.  
3) Donor engraftment: mixed or full donor chimerism will be evidence of donor engraftment 
4) Increasing donor chimerism: a 20% absolute increase in the CD3+ T-cell chimerism compared 
to the previous months chimerism evaluation.  
5) Decreasing donor chimerism: a 20% absolute decrease in the CD3+ T-cell chimerism 
compared to the previous months chimerism evaluation. 
6) Low donor chimerism: <40% CD3+ T-cells after HCT on two consecutive evaluations within a 
4-week period. The two evaluations must be at least 14 days apart. Low donor chimerism should 
always be confirmed with repeat blood T-cell and granulocyte  chimerism analysis. A DNA-
based assay that compares the profile of amplified fragment length polymorphisms (ampFLP) (or 
VNTR analyses or FISH studies) of sorted peripheral blood CD3+ T-cells will be used to quantitate 
chimerism. The same assay should be used for a given patient for repeated studies of chimerism. 
This DNA-based assay (or VNTR and FISH analyses) will also be performed on bone marrow 
aspirates. Therapeutic decisions (i.e. donor lymphocyte infusion) will be made based on the 
results of sorted T-cell studies of peripheral blood. 
7) Rejection: the inability to detect, or less than 5% donor T-cells (CD3) as a proportion of  the 
total T-cell population, respectively, after nonmyeloablative HCT.  
8)  Graft failure: grade IV thrombocytopenia and neutropenia after day 21 that lasts > 2 weeks and 
is refractory to growth factor support.  
18B. 
Evaluation of Chimerism: 
Patients will be evaluated for peripheral blood chimerism on days +28, +56 and +84 post-transplant. 
Unsorted (whole) bone marrow chimerism will be done on Day +84, and 1 year. In the absence of any 
further immunotherapy, peripheral blood chimerism studies will then be performed at 6, 12, 18, and 24 
months post-transplant, then yearly for 5 years post-transplant or more often if clinically indicated.  


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18C. 
Continuation of Immunosuppression: 
If a patient has low donor chimerism, immunosuppression should be continued or re-instituted at full 
dose.  
18D. 
Discontinuation of Immunosuppression: 
Immunosuppression should be discontinued as per protocol unless the patient develops GVHD or has 
falling donor chimerism.  
18E. 
Treatment of Graft Failure/Rejection or Mixed Chimerism with Persistent Disease: 
1) Patients with graft failure or rejection (<5% donor T-cells measured from peripheral blood sample) 
may be eligible for second marrow transplant, using protocol of institutional preference. At time of 
second transplant, patients will be scored as graft failure and followed as a separate group for 
accumulation of descriptive outcome data.  
2) Patients with mixed chimerism (defined as 5-95% donor T-cells measured from peripheral blood 
sample) with persistent life threatening disease are eligible for a second nonmyeloablative HCT. 
Patients with amelioration of disease symptoms in presence of mixed donor chimerism should not 
be given second HCT.  
 
19. PATIENT AND DONOR CLINICAL AND LABORATORY EVALUATIONS 
19A. 
HLA- typing of Patient and Potential Donors (Pre-transplant Evaluation) – See Standard 
Practice Manuel 
1) As broad a range of potential donors as possible should be typed. Included would be parents, 
siblings, eligible children and spouses. 
2) Serotyping (HLA-A, B, C) and DNA typing (HLA-A, B, C, DRB1, DQB1) of patient and donor. 
3) Leukocyte and/or florescence activated cell sorter cross match between the patient and donor. 
4) Blood samples should be sent to: Clinical Immunogenetics Lab for HLA-typing (green top tube, 
10 cc) 
19B. 
Donor 
Donors will be evaluated according to Standard Practice Guidelines and should include:  
1) Complete history and physical examination prior to and the day after bone marrow harvest to 
include the following: 
a. Medical problems, including pulmonary and upper airway disease, cardiovascular disease, 
diabetes, arthritis, or abnormalities of the spine. 
b. Last menstrual period 
c. Previous exposure to anesthetics, and family history of anesthesia-associated complications 
d. Blood transfusions 
e. Medications 
f. Vaccinations 
g. Allergies 
h. Height and weight 
 
2) Lab tests:  
a. CBC with differential including platelet counts on day –2 and day 0. 
b. Serum sodium, potassium, Chloride, CO2, Glucose, Bun, Creatinine, Calcium, Magnesium, 
Phosphorus, Uric acid, LDH, alkaline phosphatase, Total bilirubin, AST, ALT and albumin 


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c. Hepatitis screen, CMV, syphilis, HIV and HTLV I serologies (tests must be completed within 
30 days of transplantation) 
d. 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. 
e 
Female donors of menstruation age: serum HCG 
f. Sickle cell analysis for any patients of African-American ethnicity  
3) A heparinized blood sample (green top tube, 10 cc) as a donor reference for subsequent 
determination of donor chimerism should be sent to the Cytogenetics Lab if the donor-recipient pair 
is sex-mismatched or to the Clinical Immunogenetics Lab if sex-matched. Label “Protocol 2032” 
 
19C. Patient 
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 pretransplant workups may be used for eligibility determination if conducted 
within an appropriate window prior to screening) 
 
1) Pre-transplant Baseline Evaluation 
a. History: A complete history with full details of the patient’s prior treatment and response 
including the following: 
i) Hematologic findings at diagnosis (including biochemical markers, cytogenetic and 
molecular markers, disease process, and immunologic criteria) 
ii) Transfusion history (including type of donor, i.e. random or family donor 
iii) Current medical problems 
iv) Current medications 
v) Female patients who have gone through puberty – pregnancy history, menstrual history, 
and date of last sexual intercourse 
b. Physical Exam: Careful physical examination 
c. Laboratory evaluation: 
i) CBC with differential 
ii) BUN/Creatinine 
iii) Total bilirubin, fractionated bilirubin, alkaline phosphatase, AST, ALT, GGT 
iv) ABO/Rh typing, direct coombs 
v) Quantitative serum immunoglobulins (trough level if on IVIG if possible) 
vi) Pre-transplant viral testing 
 
 
 
 
 
 
 
 
 
 
 
 


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Pre Transplant 
Viral Testing 
All 2032 
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 
 
viii)  
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 and sent 
to: 
A) 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).  
B) Clinical chimerism studies post-transplant should only be sent to the relevant 
laboratory that performed the chimerism analysis pre-transplant. 
 
 
 
 
d.   See staging tables for further evaluation 
 
e. Radiological evaluation: The following studies should be obtained prior to the start of 
the transplant 
 
i) CXR-PA and lateral view (Other x-rays as clinically indicated) within approximately 1 
month of HCT as clinically indicated.  If a CT scan of the chest has been done a CXR 
is not needed. 
ii) CT scan of the chest, abdomen and pelvis within approximately 30 days of HCT if 
clinically indicated 
iii) EKG within approximately 3 months of HCT if clinically indicated 
iv) Echo or MUGA for calculation of cardiac ejection fraction within approximately 3 
months of HCT, if clinically indicated.  If unable to obtain ejection fraction, then please 
obtain shortening fraction calculation.  


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f. Bone marrow evaluation: 
If clinically indicated, a bone marrow aspirate should be obtained within approximately 4 
months of start of transplant conditioning for morphology, flow cytometry, and cytogenetics.   
We recommend that all patients with bone marrow failure disorders, aplastic anemia, and 
hemophagocytic lymphohistiocytosis have a bone marrow aspirate and biopsy within 
approximately 1 month of start of transplant conditioning for morphology, flow cytometry, 
cytogenetics, and MDS FISH panel (bone marrow failure and aplastic anemia) in order to 
assess disease status prior to HCT.  
 
2) Post HCT Evaluation:  
Note: Day + 28, 56, 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. 
 
Once patients are discharged they should be evaluated at least twice weekly in the out patient 
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 low-dose regimens.  
 
a. History and physical exam: 
i) 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.  
ii) Weekly weights 
 
b. Laboratory Evaluation: 
i) 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. 
ii) Electrolyte panel and serum BUN/creatinine should be checked 3 times a week until 
day 28 and then twice weekly while on full dose Tacrolimus or per attending until 
Tacrolimus is discontinued. 
iii) Calcium, magnesium, phosphate should be checked twice weekly until day 28 and then 
weekly. 
iv) Serum albumin should be checked weekly. 
v) 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. 
vi) 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 
vii) Iron studies: Ferritin level, binding capacity including serum iron, total iron binding 
capacity, direct and % transferring saturation checked on Day + 84, and 365 and yearly x 
5 years if clinically indicated.  
viii) Quantitative Immunoglobulins per standard practice.  
 
ix) Infection Monitoring: Infection monitoring per institutional standard practice, although 
the following is recommended 
A) CMV surveillance: Per institutional standard practice 
 


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B) Adenovirus surveillance: 
All Patients 
Adenovirus PCR weekly until day 100.  Monitoring beyond day 100 should 
continue if the patient’s absolute lymphocyte count < 300 cells/microliter or 
as clinically indicated.   
 
C) EBV lymphoproliferative syndrome surveillance: 
 
All Patients 
If stable copies by PCR 
Weekly until day 100 
If rising copies by PCR 
Twice weekly until day 100 
 
x) Serum triglyceride levels (fasting) should be drawn every two weeks through day 56, then 
monthly while on sirolimus, or more often if clinically indicated.  
 
xi) Cholesterol level (fasting) should be drawn around day 84 while on sirolimus, or more 
often if clinically indicated. 
 
xii) Haptoglobin every other week until Day +56, then as clinically indicated. Evaluation of 
schistocytes weekly with CBC through Day + 56. 
 
xiii) Disease Specific analysis, per staging table  
 
d.   See staging tables for further evaluation  
 
e. Radiology evaluation:  Two view chest x-ray (PA and lateral) as clinically indicated 
 
3) 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 Tacrolimus and MMF taper, instructions should be provided for 
preventing and detecting GVHD  as per standard practice of collaborating institution.  
GVHD evaluation guidelines are as follows: 
i) 
History and physical exam 
ii) 
CBC, serum IgG, total bilirubin, alkaline phosphatase, ALT and AST 
 
iii) GVHD evaluation (histopathology) 
A)  Oral mucosal biopsy – 3 mm incisional biopsy (if deemed necessary by oral medicine) 
 
B)  Skin biopsy - 3 mm punch biopsy from affected area or posterior iliac crest 
iv) Schirmer’s tear test if clinically indicated: if the Schirmer’s test is abnormal (<15 mm OS 
or OD) include ophthalmologic evaluation 
v) 
Pulmonary function test 


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vi) Oral medicine evaluation 
vii) Dietitian assessment 
viii) Gynecological assessment (adult female) 
ix) Staging workup, as indicated by staging table and text 
 
See section 20.G for diagnosis and treatment guidelines of acute and chronic GVHD 
 
19D. Additional evaluation guidelines following 100 Days post HCT 
1) First month – weekly evaluations by referring physician, including physical examination and 
weights (children <17 years – record height and weight every 3 months 
2) Weekly - CBC, BUN, creatinine, ALT, AST, Alkaline phosphatase, Total bilirubin and direct 
bilirubin. If stable, monthly evaluations as above for 2 years, then annually 
3) Per institutional standard practice, immunization antibody titers should be obtained at 
approximately 1 year (if patient on IVIG, do not obtain): including Hepatitis B, Tetanus, Pertussis, 
Pneumococcal, Haemophilus Influenzae Type b, and polio. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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STAGING TABLE 
 
ALL DISEASES 
 
Pre 
Day 
+28 
Day 
+56 
Day 
+84 
Day 
+180 
Day 
+365 
18 
months 
24 
months 
Years 
3-5 
BMA w/ pathology, flow 
cytometry, cytogenetics, 
FISH1 and chimerism7 
(recommended) 
X1 
 
X8 
 
X8 
 
X8 
 
X1 
 
X1 
 
PB chimerism9 
X2 
X3 
X3 
X3,4 
X3,4 
X3,4 
X3,4 
X3,4 
X5 
Quantitative 
Immunoglobulins (Serum 
Troughs)6 
X 
X 
X 
X 
 
X 
 
 
 
1 If clinically indiated 
2 For storage  
3 CD3, CD33, CD56 
4 CD19 recommended for PID patients or as clinically indicated 
5 CD3 and CD33. In addition, we recommend that CD56 chimerism be sent if mixed chimerism was 
present at the previous timepoint or as clinically indicated.  We recommend that CD19 chimerism also be 
sent if the patient has a primary immunodeficiency disorder and mixed chimerism was present at the 
previous time point or as clinically indicated. 
6 Per standard practice.  
7  Bone marrow aspirate (unsorted) to Clinical Chimerism Lab for chimerism studies on around day +84 and 1 year after 
HCT. 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). 
8 Recommend for patients with acquired Aplastic anemia: Bone marrow aspiration and biopsy on Days +28, 84 and 
365 or as clinically indicated: send for pathology, flow cytometry, cytogenetics, and for chimerism analysis.  
9 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- 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. Outside institutions may use VNTR analysis (sex- matched transplants) or sex chromosome FISH-
analysis (sex-mismatched transplants). 


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DISEASE SPECIFIC STAGING 
 
Primary Immunodeficiency Diseases 
The following tests are to be done for all primary immunodeficiency diseases 
1 All patients with primary immunodeficiency disorders should be referred to infectious disease for consultation prior to 
transplantation if they have not been seen by infectious disease. If infectious disease team determines a consult is not 
needed, an ID consult does not need to occur.  
 
2 We recommend for patients with primary immunodeficiency disorders (or as clinically indicated) that patients undergo 
an evaluation of lymphocyte phenotype and function if it has not already been done: Of note, patients with chronic 
granulomatous disease and leukocyte adhesion deficiency do not need lymphocyte phenotype or function testing. In 
addition, patients with HLH do not need lymphocyte function testing: 
3 Lymphocyte phenotype (subsets): 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 the Cell Marker Lab for recommendations regarding the total blood 
volume needed to complete this study. Sample should be obtained around day +84. Repeat around 12 months and 
then annually x 5 years post HCT if clinically indicated. 
4 Lymphocyte function: For FHCRC patients send 10 ml in 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 a concern, please 
call the Cell Marker Lab for recommendations regarding the total blood volume needed to complete this study. For 
outside centers send to appropriate lab. Samples should be obtained around day+84. Repeat around 12 months and 
then annually x 5 years if clinically indicated.  
5 If not already completed pre conditioning 
6 As clinically indicated 
7 As directed by immunology 
 
Pre 
Day 
+28 
Day 
+56 
Day 
+84 
Day 
+180 
Day 
+365 
Yearly x5 
 
Consultation with Pediatric Immunologist 
X5 
 
 
X 
 
X 
X7 
Infectious Disease Referral 
X1,5 
 
 
 
 
 
 
Lymphocyte (phenotype) subsets2,3 
X5 
 
 
 
X 
 
X 
X6 
Lymphocyte Function Analysis2,4 
X5 
 
 
X 
 
X 
X6 
Quantitative Immunoglobulins (serum 
troughs) 
X 
X 
X 
X 
 
X 
X6 


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Disease Specific Tests: The following tests are to only be done if the patient has the specific primary 
immunodeficiency disease listed below. 
1 If not already completed pre conditioning 
2 If most recent result abnormal 
3 Send 5 ml blood in heparin (green top) to Seattle Children’s Cell Marker Lab 
4 Send 10mL blood in EDTA tube to Immunology Diagnostic Laboratory Seattle Children’s Research Institute 1900 9th 
Avenue Seattle 98101, attn. IDL lab (206-987-7442) 
5 Send 5-10 ml blood (green top) to Immunology Diagnostics Laboratory Seattle Children’s Research Institute 1900 9th 
Avenue Seattle 98101, attn. IDL lab (206-987-7442). 
6 Send 5-10 ml blood in heparin (green top)  to Immunology Diagnostics Laboratory Seattle Children’s Research Institute 
1900 9th Avenue Seattle 98101, attn. IDL lab (206-987-7442). 
7 Send 1-2 ml blood in heparin (green top) to Seattle Children’s Cell Marker Lab or appropriate outside lab 
8 Send 5 ml blood in EDTA (purple top) to Genedx (www.genedx.com) 
9 SCIDs patients only: 5 cc blood in Na Heparin – sort (UWMC Hematopathology), then FISH vs. VNTR.  
A. For male patients, send 5 ml blood in heparin to flow cytometry lab for sorting T cells and then 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). 
B. For female patients, send 5 ml blood in heparin from patient to flow cytometry lab for sorting T cells for then 
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). In addition, from mother send 5 ml blood in EDTA (purple top) to Clinical Immunogenetics 
Laboratory. 
 
Pre 
Day 
+28 
Day 
+56 
Day 
+84 
Day 
+180 
Day 
+365 
Yearly x5 
Leukocyte Adhesion defect  
 
 
 
 
 
 
 
Analysis of CD18 expression by flow 
cytometry3 
 
X1 
 
 
 
X 
 
X 
X2 
Wiskott-Aldrich Syndrome 
 
 
 
 
 
 
 
Gene Sequencing4 
 
X1 
 
 
 
 
 
 
 
Determination of WASP expression by 
flow cytometry5  
 
X1 
 
 
 
X 
 
X 
X2 
IPEX 
 
 
 
 
 
 
 
Determination of FOXP3 expression by 
flow cytometry6 
 
X1 
 
 
 
X 
 
X 
X2 
Chronic Granulomatous Disease 
 
 
 
 
 
 
 
Neutrophil Oxidative Burst7  
X1 
 
 
 
X 
 
X 
X2 
Genetic analysis of cytosolic components 
of NADPH oxidase system8  
 
X1 
 
 
 
 
 
 
 
CD40LD 
 
 
 
 
 
 
 
Determination of CD40 Ligand 
expression by flow cytometry6 
 
X1 
 
 
 
X 
 
X 
X2 
SCID 
 
 
 
 
 
 
 
Maternal T Cell Engraftment9 
X 
 
 
 
 
 
 


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Other Disease Specific Tests:  
 
Pre 
Day +28 
Day +56 
Day +84 
Day +365 
Yearly x5 
Sphingolipidoses 
 
 
 
 
 
 
Consultation with Metabolic Disorders 
specialist 
X3 
 
 
X 
X 
X 
Nerve Conduction studies 
X3 
 
 
 
X 
 
Consultation with Neurologist 
X3 
 
 
X 
X 
X 
MRI of Head 
X3 
X 
X 
X 
X 
X 
Mucopolysaccharidoses 
 
 
 
 
 
 
Disease specific enzyme level1 
X3 
X 
X 
X 
X 
X 
Consultation with Pediatric Specialist in 
Metabolic Disorders 
X3 
 
 
X 
X 
X 
Marrow, skin, liver, and/or gut biopsy as 
indicated according to disease for 
determination of storage material 
X3 
 
 
X 
X 
 
 MRI of Head if indicated 
X3 
 
 
X 
X 
 
LP for storage determination if indicated 
X3 
 
 
X 
X 
 
Osteopetrosis 
 
 
 
 
 
 
Bone biopsy for pathology2 
X3 
 
 
X 
X 
X 
Head MRI 
X3 
 
 
X 
X 
X 
Aquired Aplastic Anemia 
 
 
 
 
 
 
Flow cytometry of peripheral blood to 
assess for absence of PIG1-associated 
surface proteins characteristic of 
paroxysmal nocturnal hemoglobinuria. 
X3 
 
 
 
 
 
Peripheral blood for mitomycin C and 
DEB chromosome breakage analyses to 
assess for Fanconi anemia. 
X3 
 
 
 
 
 
Hemophagocytosis 
Lymphohistiocytosis (HLH) 
 
 
 
 
 
 
Lymphocyte phenotype (subsets) 
X3 
 
 
X 
X 
X4 
Soluble Interleukin-2 
Receptor/Interleukin 2 
X3 
 
 
X 
X 
 
Sickle Cell Disease 
 
 
 
 
 
 
Percent Hemoglobin S 
X5 
 
 
X 
 
 
Dyskeratosis Congenita 
 
 
 
 
 
 
Telomere length analysis 
X3 
 
 
 
 
 
 
 


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Pre 
Day +28 
Day +56 
Day +84 
Day +365 
Yearly x5 
Other Diseases 
 
 
 
 
 
 
Send disease specific markers after 
consultation with a specialist in the 
disorder. 
 
X3 
 
 
 
 
 
1 For FHCRC patients send to Seattle Children’s laboratory. 
2 For FHCRC patients send to Seattle Children’s pathology. 
3 Disease specific testing does not need to be done pre HCT if it has already been performed pre start of conditioning. 
4 If abnormal at previous interval or if clinically indicated. 
5 On arrival and then within 1 week prior to start of conditioning.  
 
 
 
20. DRUGS, IRRADIATION, COMPLICATIONS INCLUDING GVHD AND TOXICITIES: 
20A. 
Cyclophosphamide 
1) Description: 
Cyclophosphamide is an alkylating agent which prevents cell division primarily by cross-linking 
DNA strands. Cyclophosphamide is cell cycle non-specific. Cyclophosphamide is not stem cell 
toxic. 
 
2) Storage and Administration: 
Cyclophosphamide for injection is commercially available in 2000 mg vials, which are 
reconstituted with 100 ml sterile water for injection. The concentration of the reconstituted product 
is 20 mg/ml. The calculated dose will be diluted further in 250-500 ml of Dextrose 5% in water. 
Each dose will be infused over 1-2 hr (depending on the total volume). 
 
3) Side Effects and Toxicity: 
Clinical toxicities of cyclophosphamide include alopecia, nausea and vomiting, headache and 
dizziness, 
hemorrhagic 
cystitis, 
cardiotoxicity, 
immunosuppression, 
myelosuppression, 
pulmonary fibrosis, increased hepatic enzymes and syndrome of inappropriate anti-diuretic 
hormone (SIADH). 
20B. 
Mesna 
1) Description: 
Mesna is a prophylactic agent used to prevent hemorrhagic cystitis induced by the 
oxazaphosphorine (cyclophosphamide and iphosphamide). It has no intrinsic cytotoxicity and no 
antagonistic effects on chemotherapy. Mesna binds with acrolein, the urotoxic metabolite 
produced by the oxazaphosphorine, to produce a non-toxic thioether and slows the rate of 
acrolein formation by combining with 4-hydroxy metabolites of oxazaphosphorine. 
 
2) Storage and Administration: 
 
Mesna is commercially available in 200 mg, 400 mg and 1000 mg vials containing a  100mg/ml 
solution. Each dose of mesna will be diluted further in 50 ml of normal saline to be infused over 
15 min. Mesna dose will be based on the cyclophosphamide dose being given. The total daily 
dose of mesna is equal to 100% of the total daily dose of cyclophosphamide. 
 


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3) Side Effects and Toxicity: 
At the doses used for uroprotection mesna is virtually non-toxic. However, adverse effects, which 
may be attributable to mesna, include nausea and vomiting, diarrhea, abdominal pain, altered 
taste, rash, urticaria, headache, joint or limb pain, hypotension and fatigue. 
 
 
20C. 
Fludarabine (side effects): 
1) Description:  
Fludarabine’s active metabolite 2-fluoro-ara-A is an antimetabolite that inhibits DNA primase, 
DNA polymerase alpha and Ribonucleotide nuclease.  
2) Dosage and Administration: Fludarabine monophosphate is commercially available as a 50 
mg/vial, which is reconstituted with 2 ml of sterile water, resulting in a 25mg/ml solution. The 
desired dose is further diluted to concentrations of 0.04-1 mg/ml in normal saline or 5% dextrose 
(50-100ml) for injection and will be administered by IV infusion over 30 minutes or longer. 
Fludarabine will be administered by IV infusion over 1 hr in a dose of 30 mg/m2/day on days -6 to 
-2.  
3) Side Effects and Toxicities: The dose of fludarabine used in this protocol is nonmyeloablative; 
however, it can cause severe immunosuppression particularly in the CD4+ T cell compartment. 
Immunosuppression increases the risk of infection, which can be life threatening. In addition, 
clinical toxicities of fludarabine monophosphate include: myelosuppression, primarily 
lymphopenia and granulocytopenia, alopecia, rash, dermatitis, nausea, vomiting, anorexia, 
stomatitis, diarrhea, somnolence, fatigue, peripheral neuropathy, mental status changes, cortical 
blindness, hepatocellular toxicity with elevation in serum transaminases, neurotoxicity and 
interstitial pneumonitis.  These effects are reversible when the drug is discontinued. 
Immunosuppression observed with the use of fludarabine increases the risk of infection which 
can be life-threatening. 
 
20D. 
TBI (side effects) 
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 dose of TBI (200 cGy or 400 cGy 
for patients with Acquired Aplastic Anemia, sickle cell disease, thalassemia, or other non-
malignant disease at high risk for graft rejection (as discussed at the non-malignant board or the 
patient care conference)) used in this protocol is about one-sixth (200 cGy) to one-third (400 cGy) 
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.  
 
20E. 
Tacrolimus 
See sections 12 and 13. Pages 16-18 for information about administration and dosage adjustments. 
(See Standard Practice Manual) 
1) Introduction 
Tacrolimus, also known as FK-506, is a macrolide immunosuppressive agent. Tacrolimus inhibits 
lymphocytes by forming a complex with FKBP-12, calcium, and calmodulin, leading to the 
decrease in the phosphatase activity of calcineurin. Calcineurin mediates the first intracellular 
signal required for T-cell activation after antigen recognition by the T-cell receptor. This drug is 
used with corticosteroids for prophylaxis of organ rejection in patients receiving allogeneic liver 
transplants and for prophylaxis of GVHD in the setting of HCT. It is also used for 


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immunosuppression after kidney, cardiac, pancreas, pancreatic islet cell and small bowel 
transplantation. This drug is well-absorbed orally. It is metabolized in the liver by unknown 
mechanisms, but demethylation and hydroxylation have been proposed based on in vitro studies. 
The metabolized products are excreted in the urine. 
 
2) Administration 
a. Oral 
i) Tacrolimus capsules – 0.5 mg, 1 mg or 5 mg capsules . (There is currently no oral solution 
available.)  
ii) For better absorption, it is recommended that Tacrolimus capsules be taken on an empty 
stomach. 
iii) Tacrolimus should not be taken with grapefruit juice as it may increase blood levels. 
iv) If patient vomits within one hour of oral administration, repeat dose. 
v) If vomiting persists, switch to IV administration. 
b. Intravenous 
i) Sterile solution of 5 mg/ml ampules in polyoxyethylated castor oil (Cremophor FCL). 
ii) Diluted in D5W in glass or other non-PVC container. 
iii) Final dilution volume: 50-250 ml dependent upon patient size and Tacrolimus dose. 
vi) Infusion time: Standard – 22-24 hours (continuous infusion). 
c. Conversion from IV to PO dosing of tacrolimus. 
Patients should be converted to an oral dose at 4 times the IV dose to be given in divided 
(Q12 hour) doses.  For children aged < 6 years old who have sub-therapeutic levels, the dose 
interval may need to be reduced to every 8 hours.   
 
3) 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 – Arthralgia. 
 
 
 
20F. 
Mycophenolate Mofetil (MMF) – side effects: 
See section 14 for information about administration and dosage adjustments. 
 


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1)  Description 
MMF is the morpholinylethylester of mycophenolic acid (MPA) and reversibly inhibits inosine 
monophosphate dehydrogenase, particularly the type II isoform that 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. 
 
2) 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.  
 
3) Side Effects and Toxicity 
a)  
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). Dose adjustments are described in Sections 14 & 15. 
 
20G. 
Sirolimus 
a) 
Formulation, Storage, and Administration: Sirolimus is supplied as an oral solution 
(Rapamune Oral Solution) 1 mg/mL or as 1 mg tablets. Rapamune Oral Solution pouches 
should be stored protected from light and refrigerated at 2-8oC. If necessary, the patient may 
store the pouches at room temperatures upt to 25oC for a short period of time (no longer than 
30 days). The tablets should be stored at 20-25oC and be protected from light. Refer to section 
12D for dosing instructions. To minimize variability of exposure to sirolimus, this drug should 
be taken consistently with or without food. Grapefruit juices should not be administered with 
sirolimus or used for dilution. If patients are receiving Rapamune Oral Solution, the dose 
should be mixed well with 60 mL of water or orange juice and taken immediately. It is 
recommended that the container be refilled with a minimum of 120 mL of water or orange 
juice, mixed well, and this rinse solution should be swallowed. 
b) 
Adverse Reactions: The incidence of adverse reactions was determined in two randomized 
double-blind multicenter controlled trials in which 499 renal transplant recipients received 
Rapamune oral solution 2 mg/day and 477 received 5 mg/day. Specific adverse reactions 
associated with the administration of Rapamune oral solution included hypocholesterolemia, 
hyperlipidemia, hypertension, and rash. At the higher dose of 5 mg, these adverse effects 
included anemia, arthralgia, diarrhea, hypokalemia, and thrombocytopenia. Additional 
toxicities from studies in stem cell transplantation include: hemolytic uremic syndrome, 
seizures, and neutropenia.  
c) 
Clinically Significant Inducers or Inhibitors of the Cytochrome P450 Enzyme System: 
Sirolimus levels may increase in patients taking fluconazole, itraconazole, cyclosporine, 


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methylprednisolone, or tacrolimus. Other medications that may affect metabolism of sirolimus 
are listed below: 
 
Agents likely to 
increase sirolimus 
levels 
Agents which may  
increase sirolimus 
levels 
Agents likely to 
decrease sirolimus 
levels 
Agents which may 
decrease sirolimus 
levels 
Diltiazem 
Nicaripine 
Verapamil 
Erythromycin 
Ketoconazole 
Voriconazole 
Clarithromycin 
Cimetidine 
Carbamazepine 
Phenobarbital 
Phenytoin 
Rifampin 
 
Primidone 
Valproic acid 
Rifabutin 
 
d) 
Management of Toxicieis:  
Severe neutropenia or thrombocytopenia: Refer to section 16D.  
Hyperlipidemia: Sirolimus is known to cause elevations in serum cholesterol and triglyceride 
levels. Serum triglyceride levels should be drawn every two weeks through day 56, then 
monthly while on sirolimus, or more often if clinically indicated. Cholesterol levels will be drawn 
at the Discharge evaluation (around day 80). In 10-20% of patients, the triglyceride leel 
exceeds 1000 mg/dL. To avoid complications due to pancreatitis, patients should be treated 
with gemfibrozil or atorvastatin for triglyceride levels >800 mg/dL. 
 
 
20H. 
GVHD 
The major toxicity of T-replete HCT from related, HLA-haploidentical donors is GVHD. In 
nonmyeloablative HCT from related, HLA-haploidentical donors the incidence of GVHD Grade II was 
>90% (50% Grades III and IV) in studies described by Sykes and co-workers [38,39] using a different 
conditioning and immunosuppressive regimen. Overall mortality was 75%. However, preliminary results 
of the Phase II clinical trial (protocol 1667) discussed above in Section E. #3 (pages 8-9) show that only 
< acute Grade II GVHD was observed in 12/25 patients and none of the patients have died as a result of 
GVHD or its therapy.  
1) Diagnosis: Skin involvement will be assessed by biopsy with percentage of body surface area 
involved recorded. GI symptoms suspicious for GVHD will be evaluated by biopsy as indicated. 
Acute GVHD and chronic GVHD will be graded according to established criteria (Appendices C 
and D). 
2) Recommended Treatment: 
a. Patients Developing Grade II-IV Acute GVHD and Tacrolimus has started to taper: 
i) If acute GVHD develops during Tacrolimus taper, reinitiate tacrolimus at the last dose prior 
to the initiation of the taper. If off tacrolimus, reinstitute Tacrolimus at 0.06 mg/kg PO q12 
hours. If there is concern of GI absorption, use IV route (0.03 mg/kg/d continuous IV 
infusion over 22-24 hours, pediatric patients or 0.015 mg/kg every 12 hours, adult 
patients).  
ii) Prednisone (2 mg/kg/day) should be added if severe GVHD occurs and/or no GVHD 
response occurs by 72 hours after initiation of Tacrolimus. Patients who respond to 
steroids after 10 to 14 days of treatment, should begin a 6 week taper. A faster prednisone 
taper may be initiated if clinically indicated.   
b) Patients developing acute GVHD  grade II on full dose Tacrolimus and full dose MMF: 


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i.) Prednisone (2 mg/kg/day) or intravenous equivalent is to be added. Patients who respond 
to steroids after 10 to 14 days of treatment, should begin a 6 week taper. A faster 
prednisone taper may be initiated if clinically indicated.   
ii.) When steroids are tapered to 0.5 mg/kg PO QD then an MMF taper may be initiated. In 
the absence of GVHD flare, the MMF and prednisone tapers should continue until 
completion. If nausea and/or vomiting prevent the oral administration of MMF, MMF should 
be administered intravenously at 15 mg/kg q 8hrs. 
iii) Patients are eligible for institutional trials of GVHD therapy. 
c) Patients with Chronic Extensive GVHD: 
i) Tacrolimus dosing: 
Adults: 0.01-0.02 mg/kg PO q12hrs 
Pediatric doses for patients < 17 years of age: 0.05-0.08 mg/kg PO q12hrs. 
ii) Prednisone (1mg/kg/day qd.) 
Taper of prednisone as per recommendations of the Long Term Follow Up (LTFU) at 
FHCRC and collaborating institutions standard practice. Patients can also be treated on 
other protocols eligible at that time. Patients off immunosuppression who develop 
concurrent manifestations of GVHD that satisfy criteria for  grade II acute GVHD (e.g. 
erythematous rash, diarrhea, hyperbilirubinemia) and are pathognomonic of clinical 
extensive chronic GVHD (e.g. lichenoid oral changes, ocular sicca, scleroderma, 
bronchiolitis obliterans, contractures), should receive prolonged immunosuppressive 
therapy similar to that for chronic extensive GVHD. 
 
iii) Antibiotic prophylaxis with daily Bactrim and anti-viral prophylaxis with acyclovir.  
20I. 
Myelosuppression: 
1) Grade IV myelosuppression will be defined as a decrease in ANC to <500/L and/or platelet count 
to  20,000/L.  
2) Myelosuppression may occur in this patient population for a number of reasons such as direct 
toxic effect of drugs (MMF, ganciclovir etc.), rejection, and relapse.  
3) Patients with myelosuppression may be managed as follows:  
a) Suspected MMF toxicity: refer to section 15, Guidelines for  MMF dose adjustment above 
for management recommendations.  
b) Suspected ganciclovir toxicity: consider changing to foscarnet.  
Thrombocytopenic patients will receive platelet transfusion as per standard care. 
 
 
 
 
21. PROTOCOL DEVELOPMENT AND SPECIAL CONSIDERATIONS 
 
PROJECTED TARGET ACCRUAL 
ETHNIC AND GENDER DISTRIBUTION CHART 
 
All ethnic groups and both genders will be enrolled according to the patient distribution in the participating 
institutions, as outlined in the following Chart. Skewing of gender may occur if a preponderance of X-linked 
syndromes are enrolled. 


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22. DATA AND SAFETY MONITORING PLAN AND ADVERSE EVENT REPORTING 
22A. 
Monitoring the progress of trials and the safety of participants 
The principal investigators (PI) monitor multi-institutional clinical trials, with oversight by 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.  
 
Serious adverse events are reported to the trial coordinator, the study nurse or directly to the PI. The trial 
coordinators at collaborating centers or the local PIs will fax an official report of a serious adverse event 
to the coordinating center (FHCRC) within ten days. The serious adverse event report is reviewed by PI. 
If the serious adverse event meets the FHCRC criteria for reporting then an official signed report is 
submitted to the FHCRC Institutional Review Office (IRO). All deaths, regardless of the cause, are 
reported to the IRB. Multi-center protocols have a DSMB responsible for monitoring patient safety on this 
clinical trial. 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 as well 
as 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.  
 
With respect to safety, patients are monitored for the development of GVHD, myelosuppression, 
infections and rejection. All patients, regardless of diagnosis, will be considered in the safety analysis. 
GVHD events will be closely monitored and severity of GVHD graded. Formal stopping rules for GVHD 
grade IV and day-100 mortality are provided. 
 
 
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. This documentation is 
extracted by the study nurse within 140 days +/- after HCT via chart review and collection of copies of 
source documents and entered into a hard copy or electronic Case Report Form (CRF). Thus, multiple 
TARGETED / PLANNED ENROLLMENT: Number of Subjects 20 
 Ethnic Category 
 Sex / Gender  
 
Females 
Males 
Total 
Hispanic or Latino 
0 
1 
1 
Not Hispanic or Latino 
7 
12 
19 
Ethnic Category Total of All Subjects* 
7 
13 
20 
Racial Categories 
American Indian/Alaska Native 
0 
0 
0 
Asian 
0 
1 
1 
Native Hawaiian or Other Pacific Islander 
0 
0 
0 
Black or African American 
0 
1 
1 
White 
7 
11 
18 
Racial Categories: Total of All Subjects* 
7 
13 
20 


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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. 
 
 
All centers have an IRB that reviews the protocol and that the local PIs contact when an adverse event 
on the protocol occurs. Most of the centers have internal auditing mechanisms that assure accurate 
assessment of clinical outcomes. Clinical outcome data are summarized and transmitted from 
collaborating centers as case report forms (CRFs).  CRFs from external sites are verified and signed by 
the local investigators at that site. When possible, primary source documents regarding patientoutcomes 
are collected with patients’ names removed and replaced by Unique Patient Numbers (UPNs). The CRFs 
are generated from the collaborating centers at defined time points (100 days, 6 months, and yearly).   
22B. 
Plans for assuring compliance with requirements regarding the reporting of adverse 
events 
The adverse event reporting in these multi-institution clinical trials will follow the FHCRC Guidelines for 
serious adverse event (SAE) reporting (see Appendix E of the protocol). These guidelines detail the 
expedited reporting requirements and definitions of particular events. All SAEs that meet expedited 
reporting are reported to the IRO within 10 days of the investigator, trial coordinator, or research nurse 
upon learning of the event. The IRO must receive a completed SAE report form, signed by the PI, within 
10 calendar days. For patients being cared for at the FHCRC, health care providers communicate with 
the PI, trial coordinator or research nurses as events occur triggering subsequent reporting. For patients 
not being cared for at FHCRC the outside facilities communicate with the PI, trial coordinator, or research 
nurse for these reporting purposes. All serious adverse events are reported to the IRB at the time of 
annual renewal and at the biannual mixed chimerism meeting. 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 subjects protection. 
 
 
22C. 
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 uses commercial agents and there is no associated Investigational New Drug 
(IND) or Investigational Device Exemption (IDE). Any temporary or permanent suspension, as 
determined by the PI, IRB, or DSMC, of this clinical research trial will be reported to the NCI grant program 
director by the PI.  
 
22D. Plans for assuring data accuracy and protocol compliance 
This protocol has a DSMB that is responsible for reviewing protocol data and safety endpoints. The DSMB 
meets on a twice yearly basis, reviews a report of appropriate endpoint data, and compiles a report that 
is submitted to the Institutional Review Board, DSMC Chairman, and Protocol Office. 
 
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 one of the study nurses at 100 
days after HCT via chart review and entered into an electronic Case Report Form (CRF). The study 
nurses also continue to follow patients after day 100, review source documentation, and complete CRFs 


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at 6 month and then yearly intervals per protocol. The CRFs are printed directly from the database, and 
the PI reviews the CRFs and the primary source documents for data accuracy. When 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. The study has dedicated nurses who at a 
minimum 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 
nurses are responsible for review and maintenance of all patient records to ensure data integrity and 
protocol adherence. 
 
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. 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 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. Queries are sent to the collaborating investigators if CRFs are inaccurate or incomplete.  
 
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.  
 
23. 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, keyentry 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. 
 
24. STATISTICAL CONSIDERATIONS AND TERMINATION OF THE STUDY  
We plan to enroll 20 patients on this study. The primary endpoint of this study is to determine safety.  We will 
consider this study a success if we do not reach the stopping rules.  All patients, regardless of diagnosis, will 
be considered in the safety analysis. The study will be suspended and referred to the DSMB for consideration 
of results and appropriate modification or termination of the study if there is sufficient evidence that the 
probability of transplant related mortality (death not due to the underlying disease) by day 100 exceeds 25% 
or that the probability of grades III-IV acute GVHD exceeds 25%. This will be deemed to occur if the lower 
level of a one-sided 80% confidence interval for either proportion exceeds 25% and will be evaluated after 5 
patients have been followed for at least 100 days. Operationally, the study will be suspended if: 3 out of the 
first 5, 5 out of the first 10, 6 out of the first 15, or 8 of the first 20 patients die before day 100 or develop 
grade III or IV acute GVHD before diagnosis of chronic GVHD.  (See appendix C for grading of acute 
GVHD).  In addition, this study will be suspended and referred to the DSMB for consideration of results and 
appropriate modification or termination of the study if there is sufficient evidence that the probability of graft 
rejection/graft failure by day 84 exceeds 20%.  (See Section 18 for definitions of graft rejection and graft 
failure) This will be considered true if the lower bound of a one-sided 80% confidence interval exceeds 20%.  
Operationally, the study will be suspended if: 3 out of the first 5, 4 out of the first 10, 5 out of the first 15, or 
6 out of the first 20 patients have graft rejection or fail to engraft.   


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Therefore, stopping rules will be imposed for: 
 
 
Day 84 Graft rejection/graft failure > 20% (3/5, 4/10, 5/15, 6/20). 
 
Day 100 TRM >25% (3/5, 5/10, 6/15, 8/20) 
 
Grade III/IV acute GVHD preceding diagnosis of chronic GVHD >25% (3/5, 5/10, 6/15, 8/20) 
 
In addition, at the conclusion of the study, all unexpected toxicities will be summarized and reported.  The 
following adverse outcomes will be monitored carefully throughout the study: 
1.  Assessment of Adverse events - Adverse events will be assessed using an adapted version of the 
Common Toxicity Criteria (Appendix J). 
2. Infections. Every effort will be made to obtain tissue documentation of infection as consistent with 
appropriated medical management.  For details on grading infections see Appendix J.  Death due to 
infection will be scored as TRM, including for patients with primary immunodeficiency disorders. 
 
As a secondary endpoint we will also evaluate the proportion of patients who achieve >5% donor T-cell 
chimerism by day 84.  From this study we will obtain a preliminary estimate of incidence of successful T-
cell chimerism for use in designing future studies. Although not definitive, for the endpoint of mixed 
chimerism given the small number of patients, we shall consider this study a success if the observed rate 
of successful establishment of mixed chimerism (>5% donor T-cell chimerism) is 80% or greater (16/20). If 
the true rate is 90%, then the probability that 16 or more patients among 20 will develop mixed chimerism 
is approximately 93%. If the true rate is 70%, 60%, or 50% then this probability is 38%, 17%, or  5%, 
respectively.  
 
 
In June 2016 the Data Safety Monitoring Board met to discuss the possibility of the study having 
met stopping rules based on the fact that 5 patients out of the first 10 patients developed grade III 
or IV acute GVHD.  Per the protocol the study will be suspended if 3 out of first 5, 5 out of the first 
10, 6 out of the first 15, or 8 out of the first 20 develop grade III or IV acute GVHD.  
 
The DSMB met and made the following recommendation: The DSMB members agreed that current 
protocol findings met criteria for convening the DSMB. The DSMB members noted that, while there is an 
apparent relatively high proportion of patients with Grade 3-4 GVHD, there has been a very low rate of non-
relapse mortality with only 1 death. The DSMB members agreed to continue accrual on the protocol with 
the requirement that subsequent patients be enrolled no sooner than 100 days after the previous patient’s 
transplant day 0. If an additional patient develops Grade 3-4 acute GVHD, then the DSMB should be 
reconvened and will likely result in study closure. 
 
 


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25. REFERENCES 
Reference List 
  1.   Rao K, Amrolia PJ, Jones A, Cale CM, Naik P, King D, Davies GE, Gaspar HB, Veys PA.  Improved survival after 
unrelated donor bone marrow transplantation in children with primary immunodeficiency using a reduced-
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R, Vossen J.  European experience of bone-marrow transplantation for severe combined 
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haploidentical transplantation in children with primary immunodeficiencies have the potential to exploit 
donor NK cell alloreactivity?  Bone Marrow Transplant 34: 945-947, 2004. 


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  14.   Klein C, Cavazzana-Calvo M, Le Deist F, Jabado N, Benkerrou M, Blanche S, Lisowska-Grospierre B, Griscelli 
C, Fischer A.  Bone marrow transplantation in major histocompatibility complex class II deficiency: a 
single-center study of 19 patients.  Blood 85: 580-587, 1995. 
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PA, Twist C, Nachman JB, Hale GA, Harris RE, Rozans MK, Kurtzberg J, Grayson GH, Williams TE, 
Lenarsky C, Wagner JE, Krivit W.  Hurler syndrome: II. Outcome of HLA-genotypically identical sibling 
and HLA-haploidentical related donor bone marrow transplantation in fifty-four children. The Storage 
Disease Collaborative Study Group.  Blood 91: 2601-2608, 1998. 
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stem cell transplantation in Hurler syndrome.  J Pediatr Hematol Oncol 22: 433-436, 2000. 
  17.   Schulz AS, Classen CF, Mihatsch WA, Sigl-Kraetzig M, Wiesneth M, Debatin KM, Friedrich W, Muller SM.  HLA-
haploidentical blood progenitor cell transplantation in osteopetrosis.  Blood 99: 3458-3460, 2002. 
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Casanova JL, Fischer A, Cavazzana-Calvo M.  Impact of HLA matching on outcome of hematopoietic 
stem cell transplantation in children with inherited diseases: a single-center comparative analysis of 
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  19.   Haddad E, Landais P, Friedrich W, Gerritsen B, Cavazzana-Calvo M, Morgan G, Bertrand Y, Fasth A, Porta F, 
Cant A, Espanol T, Muller S, Veys P, Vossen J, Fischer A.  Long-term immune reconstitution and outcome 
after 
HLA-nonidentical 
T-cell-depleted 
bone 
marrow 
transplantation 
for 
severe 
combined 
immunodeficiency: a European retrospective study of 116 patients.  Blood 91: 3646-3653, 1998. 
  20.   O'Reilly RJ, Keever CA, Small TN, Brochstein J.  The use of HLA-non-identical T-cell-depleted marrow transplants 
for correction of severe combined immunodeficiency disease.  Immunodeficiency Reviews 1: 273-309, 
1989. 
  21.   Namdemanee A, Sniecinzki IJ, Schmidt CA, Dagis AC, O'Donnell MR, Snyder DS, Parker PM, Stein AS, Smith 
EP, Arturo M, Stepan DE, Somlo G, Margolin KA, Woo D, Niland JC, Forman SJ.  High-Dose therapy 
followed by autologous peripheral-blood stem-cell transplantation for patients with hodgkin's disease and 
non-hodgkin's lymphoma using unprimed and granulocyte colony-stimulating factor-mobilzed peripheral-
blood stem cells.  J Clin Oncol 12: 2176-2186, 1994. 
  22.   Storb R, Yu C, Wagner JL, Deeg HJ, Nash RA, Kiem H-P, Leisenring W, Shulman H.  Stable mixed hematopoietic 
chimerism in DLA-identical littermate dogs given sublethal total body irradiation before and 
pharmacological immunosuppression after marrow transplantation.  Blood 89: 3048-3054, 1997. 
  23.   Maris MB, Niederwieser D, Sandmaier BM, Storer B, Stuart M, Maloney D, Petersdorf E, McSweeney P, Pulsipher 
M, Woolfrey A, Chauncey T, Agura E, Heimfeld S, Slattery J, Hegenbart U, Anasetti C, Blume K, Storb 
R.  HLA-matched unrelated donor hematopoietic cell transplantation after nonmyeloablative conditioning 
for patients with hematologic malignancies.  Blood 102: 2021-2030, 2003. 
  24.   Woolfrey AE, Nash RA, Sanders JE, Ochs HD, Thomson B, Storb R.  A nonmyeloablative regimen for induction 
of multi-lineage hematopoietic mixed donor-host chimerism in nonmalignant disorders.  Blood  98 (Part 
1): 784a, #3390, 2000.(Abstract) 
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TR.  Mixed lymphohaemopoietic chimerism and graft-versus-lymphoma effects after non-myeloablative 
therapy and HLA-mismatched bone-marrow transplantation.  Lancet 353: 1755-1759, 1999. 
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Sachs DH, Sykes M.  Haploidentical donor bone marrow transplantation (BMT) for advanced hematologic 
malignancy (HM) following non-myeloablative preparative therapy: role of in vivo T-cell depletion with 


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anti-thymocyte globulin or anti-CD2 monoclonal antibody therapy (medi-507).  Blood  96 (Part 1): 841a, 
#3633, 2000.(Abstract) 
  27.   Santos GW, Sensenbrenner LL, Burke PJ, Mullins GM, Blas WB, Tutschka PJ, Slavin RE.  The use of 
cyclophosphamide for clinical marrow transplantation.  Transplant Proc 4: 559-564, 1972. 
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in man following cyclophosphamide.  Transplant Proc 3: 400-404, 1971. 
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Aplastic anaemia treated by marrow transplantation.  Lancet 1: 284-289, 1972. 
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high-dose cyclophosphamide without stem-cell rescue for refractory, severe autoimmune disease.  Ann 
Intern Med 129: 1031-1035, 1998. 
  31.   Jones RJ, Barber JP, Vala MS, Collector MI, Kaufmann SH, Ludeman SM, Colvin OM, Hilton J.  Assessment of 
aldehyde dehydrogenase in viable cells.  Blood 85: 2742-2746, 1995. 
  32.   Eto M, Mayumi H, Tomita Y, Yoshikai Y, Nishimura Y, Maeda T, Ando T, Nomoto K.  Specific destruction of host-
reactive mature T cells of donor origin prevents graft-versus-host disease in cyclophosphamide-induced 
tolerant mice.  J Immunol 146: 1402-1409, 1991. 
  33.   Mayumi H, Himeno K, Tokuda N, Nomoto K.  Drug-induced tolerance to allografts in mice. VII. Optimal protocol 
and mechanism of cyclophosphamide-induced tolerance in an H-2 haplotype-identical strain combination.  
Transplant Proc 18: 363-369, 1986. 
  34.   Tomita Y, Yoshikawa M, Zhang QW, Shimizu I, Okano S, Iwai T, Yasui H, Nomoto K.  Induction of permanent 
mixed chimerism and skin allograft tolerance across fully MHC-mismatched barriers by the additional 
myelosuppressive treatments in mice primed with allogeneic spleen cells followed by cyclophosphamide.  
J Immunol 165: 34-41, 2000. 
  35.   Luznik L, Jalla S, Engstrom LW, Iannone R, Fuchs EJ.  Durable engraftment of major histocompatibility complex-
incompatible cells after nonmyeloablative conditioning with fludarabine, low-dose total body irradiation, 
and posttransplantation cyclophosphamide.  Blood 98: 3456-3464, 2001. 
  36.   Mayumi H, Umesue M, Nomoto K.  Cyclophosphamide-induced immunological tolerance: an overview (Review).  
Immunobiology 195: 129-139, 1996. 
  37.   O'Donnell PV, Luznik L, Jones RJ, Vogelsang GB, Leffell MS, Phelps M, Rhubart P, Cowan K, Piantados S, 
Fuchs EJ.  Nonmyeloablative bone marrow transplantation from partially HLA-mismatched related donors 
using posttransplantation cyclophosphamide.  Biol Blood Marrow Transplant 8: 377-386, 2002. 
  38.   Tyms AS, Davis JM, Jeffries DJ, Meyers JD.  BWB759U, an analogue of acyclovir, inhibits human 
cytomegalovirus in vitro.  Letter.  Lancet 2: 924,1984. 
  39.   Cheever MA, Greenberg PD, Gillis S, Fefer A.  Specific adoptive therapy of murine leukemia with cells secondarily 
sensitized in vitro and expanded by culture with interleukin 2. In: Fefer A, Goldstein AL (eds):  Progress 
in Cancer Research and Therapy.  The Potential Role of T Cells in Cancer Therapy.  New York:  Raven 
Press, p. 127-146, 1982. 
 
 
 
 
 


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APPENDICES – TABLE OF CONTENTS 
 
 
APPENDIX A   
 
ABO INCOMPATIBILITY  
 
APPENDIX B   
 
INFECTIOUS DISEASE GUIDELINES 
 
APPENDIX C  
 
ACUTE GRAFT-VERSUS-HOST DISEASE GRADING 
 
APPENDIX D  
 
CHRONIC GRAFT-VERSUS-HOST DISEASE GRADING 
 
APPENDIX E 
STUDY COORDINATOR’S MANUAL INCLUDING PROCEDURE FOR REPORTING 
ADVERSE EVENTS 
APPENDIX F 
ADVERSE EVENT REPORT 
APPENDIX G  
 
NOTICE OF DEATH FORM 
 
APPENDIX H  
 
PATIENT DEMOGRAPHICS AND REGISTRATION FORM 
 
APPENDIX I  
 
OUTSIDE CENTER CORE CASE REPORT FORM 
 
APPENDIX J  
 
ADAPTED COMMON TOXICITY CRITERIA 
 
APPENDIX K  
 
CYCLOPHOSPHAMIDE ADMINISTATION GUIDELINES 
 
APPENDIX L  
 
WEIGHT CALCULATIONS FOR DRUG DOSING 
 
APPENDIX M  
 
TOTAL BODY IRRADIATION GUIDELINES  
 
APPENDIX N  
 
 COORDINATING CENTER FUNCTIONS 
 
APPENDIX O  
 
 HEMATOPOIETIC CELL TRANSPLANT-COMORBIDITY INDEX (HCT-CI) 
 
 


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APPENDIX A 
 
 
ABO INCOMPATIBILITY 
 
Red Blood Cell - Incompatibility (Major): 
Occasional patients may have antibodies directed against red blood cell antigens found on the donor's cells. 
These are generally ABO or Rh antigens, although incompatibility with other red cell antigens identified by 
donor-recipient crossmatch may occur. Although the volume of red blood cells (RBC) in most PBMC products 
will only be 2-5% of the product volume before infusion, the small quantity may cause a hemolytic transfusion 
reaction. According to the FHCRC policy it is generally acceptable to infuse a volume of about 10ml RBCs 
per product. If the recipient shows an anti-donor titer of  1:32 or the RBC volume is greater than 10ml (or > 
20ml in two products combined) the PBMC components should be RBC depleted by Starch Sedimentation 
(flowsheet below). Refer to the Clinical Coordinator's Patient Information Sheet for instructions regarding 
management of a specific patient.  
Post transplant blood component support will be according to Standard Practice Guidelines. 
 
Timing: Every attempt should be made to infuse red cell depleted PBMC products within 2 hours of depletion. 
Expected Results: Red blood cell depleted PBMC products will contain < 10ml of red blood cells and  90% 
nucleated cell recovery. 
 
Red Blood Cell - Incompatibility (Minor): 
Occasional donors may have antibodies directed against red blood cell antigens (ABO, Rh, or other antigen 
system) found on the recipient's cells. The risk of hemolysis of recipient red cells immediately after transplant 
is not of very much clinical import. Due to the high number of lymphocytes in the PBMC inoculum, recipients 
may be at much greater risk for a delayed type of hemolysis that can be severe. PBMC products contain < 
200ml of plasma according to FHCRC policy and no deleterious effects have been observed so far. However, 
if donors show an anti-recipient titer  1:256, the PBMC component should be plasma depleted (see 
flowsheet below). Refer to the Clinical Coordinator's Patient Information Sheet for instructions regarding 
management of a specific patient.  
Post transplant blood component support will be according to Standard Practice Guidelines. 
 
Timing: Every attempt should be made to infuse plasma-depleted PBMC within 2 hours of depletion. 
Expected Results: The plasma depletion should not affect the nucleated cell recovery. 
 
Red Blood Cell – Bidirectional Incompatibility: 
Patients undergoing transplants for bidirectional RBC incompatibility should be managed according to both 
algorithms shown below. Most red cell depletion techniques also deplete plasma from the PBMC component 
with no additional cell loss. Refer to the Clinical Coordinator's Patient Information Sheet for instructions 
regarding management of a specific patient.  
 
Post transplant blood component support will be according to Standard Practice Guidelines. 
 
 
 
 
 
 
 
 
Appendix A (continued) 


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ABO INCOMPATIBILITY 
 
MAJOR ABO INCOMPATIBLE 
Recipient 
anti-
Donor titer 
 1:32 
<20ml RBC total 
  Infuse without modification 
>20ml RBC total 
  RBC depletion of component 
 1:16 
  
Infuse without modification 
MINOR ABO INCOMPATIBLE 
Donor 
anti-
Recipient titer 
 1:256 
Plasma depletion of component 
 1:128 
Infuse without modification 


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APPENDIX B 
 
INFECTIOUS DISEASE GUIDELINES 
 
Herpes Simplex and Varicella Zoster Virus Prevention and TreatmentPg 
HSV VZV Prevention
and Treatment v6Jun 
CMV Prevention: Surveillance and Preemptive Therapy 8/16/17 
cmvprevention 
v16Aug17.pdf
 
CMV Disease: Diagnosis and Treatment 10/19/16 
cmvdiseasetreatme
nt v19Oct16.pdf  
Antifungal Therapy Guidelines 3/16/16 
antifungal_therapy 
v16Mar16.pdf
 
Pneumonia / Pneumocystis Carinii Prophylaxis 10/19/16 
pneumocystisjirove
ci v19Oct16.pdf
 
Antibiotic Prophylaxis for Encapsulated Bacteria in Allogeneic Patients with Chronic GvHD 
Requiring Immunosuppressive Therapy 10/19/16 
antibioticprophylax
is v19Oct16.pdf
 
Vaccinations 6/21/18 
ltfu_guidelines. 
vaccinations v12Sep 
Foscarnet 8/16/17 
foscarn 
v16Aug17.pdf
 
 


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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 sand 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 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Appendix D (continued) 


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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 
manifestations of chronic GVHD 
2. 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 the body 
surface), positive skin biopsy, and no other manifestations of chronic GVHD 
4. 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 


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 APPENDIX E 
 
Study Coordinator’s Manual 
 
I. 
     Introduction 
 
The mixed chimerism protocols have been opened to multiple sites to increase the referral base and accrual. 
Because of this expansion of collaborators, the data collection procedures are being revised. The procedure 
manual was created to assure consistency of data reporting across the centers and to assure compliance 
with regulations. General expectations of collaborators are that they will comply with appropriate regulatory 
requirements, specified protocol requirements, and provide outcome data. 
 
The manual translates working procedures for study coordination. Its goal is to describe the procedures with 
sufficient clarity to ensure that all study centers will use the same procedures and follow-up schedules for 
participant data management and reporting. Changes to the manual and relevant forms will be made as soon 
as practical and will become effective on receipt of the revised procedures at the study centers, unless 
otherwise noticed. 
 
II. 
Institutional Review Board Review of Protocols and Modifications 
 
All research protocols proposed for use that involves human subjects must be reviewed and approved by the 
Institutional Review Board (IRB) prior to implementation. New protocols will undergo review at the FHCRC 
IRB and then will be distributed to sites that wish to participate for their IRB’s review. For Centers that have 
a Federal Wide Assurance (FWA), formal collaboration includes submission of a form 310 and a copy of the 
IRB approved protocol and consent forms to the FHCRC.  For sites without a FWA, an FWA form needs to 
be filed. Once the paperwork is submitted to the Office for Human Research Protection, the approval process 
can take up to a couple of months, and must be completed before collaboration on a protocol can begin.  
 
In addition, all amendments and/or revisions to on-going, approved activities must be submitted for review 
and approved prior to implementation at an institution.  No revisions may be implemented at outside 
institutions without the prior approval of the FHCRC Principal Investigator.  The FHCRC and the local site’s 
IRB must review all protocol activities at least once annually. This must be done within 365 days of the last 
review regardless of the policies of the institution. A copy of annual renewal approvals must be received for 
collaboration to continue for the next year. 
 
III. 
Registrations 
 
Collaborating Institutions: The principal investigator of the collaborating institution who will register the patient 
with the FHCRC will identify eligible patients. Registration will include completion of the eligibility 
checklist/demographic form (Appendix H) and receipt of signed consent. Fax these documents to 206-667-
4427 prior to treatment initiation. Patients should be registered prior to treatment initiation for valid registration 
 
 
 
 
 
 
 
 


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APPENDIX E (continued) 
 
IV. 
Reporting Adverse Events 
 
The following guidelines are the minimum serious adverse event (SAE) reporting guidelines for Category 1 
and 2 studies conducted at the Fred Hutchinson Cancer Research Center. 
 
Expedited Reporting Requirements 
All unexpected and serious adverse events which may be due to study treatment or intervention must be 
reported to the FHCRC Institutional Review Office as soon as possible but within at least 10 calendar days 
of the investigator learning of the event. 
 
Definitions 
 
Adverse Event - Any untoward medical occurrence in a patient or clinical investigation subject administered 
a pharmaceutical product, medical treatment or procedure and which does not necessarily have to have a 
causal relationship with this treatment.  An adverse event can therefore be any unfavorable and unintended 
sign (including an abnormal laboratory finding, for example), symptom, or disease temporally associated with 
the use of a medicinal product, medical treatment or procedure whether or not considered related to the 
medicinal product.  
 
Life-threatening Adverse Event  – Any adverse event that places the patient or subject, in view of the 
investigator, at immediate risk of death from the reaction.  Study toxicities are graded using the adapted NCI 
Common Toxicity Criteria (where appropriate use the criteria for transplant patients.)  All Grade 4 (life-
threatening) toxicities occurring between first protocol intervention and day 200 that meet expedited reporting 
requirements must be reported as soon as possible but within at least 10 calendar days of the investigator 
learning of the event.  
 
Unexpected Adverse Event – An adverse event, the nature or severity of which is not consistent with the 
applicable product information (e.g., Investigator’s Brochure for an unapproved investigational product or 
package insert/summary of product characteristics for an approved product).  If applicable product 
information is not available, such as for studies that do not involve pharmaceutical products or devices, an 
unexpected adverse event is an adverse event that was not described in the study protocol or informed 
consent. 
 
Serious Adverse Event (SAE) – Any adverse event occurring that results in any of the following outcomes:   
 Death – first protocol intervention until day 200, regardless of cause,  
 a life-threatening adverse event (see above)  
 a persistent or significant disability/incapacity,  
 a congenital anomaly  
 requires intervention to prevent permanent impairment or damage.  
 
 
 
 
 
 
 


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APPENDIX E (continued) 
 
Hospitalization, in general, will not be considered a serious adverse event as approximately half of evaluable 
MRD patients AND the majority of evaluable URD patients receiving nonmyeloablative transplants were 
hospitalized.  Hospitalization will be considered a serious adverse event if it fulfills the criteria for a serious 
and unexpected adverse event as described above. 
 
To ensure no confusion or misunderstanding exist of the differences between the terms “serious” and 
“severe,” which are not synonymous the following note of clarification is provided: 
 
The term “severe” is often used to describe the intensity (severity) or a specific event (as in mild, 
moderate or severe myocardial infarction); the event itself, however, may be of relatively minor 
medical significance (such as severe headache).  This is not the same as “serious,” which is based 
on patient/event outcome or action criteria usually associated with events that pose a threat to a 
patient’s life or functioning.  Seriousness (not severity) serves as a guide for defining regulatory 
obligations. 
 
Attribution - The FHCRC designation for the determination of whether an adverse event is related to a 
medical product, treatment or procedure will be as follows: 
 Related – includes adverse events that are definitely, probably, or possibly related to the medical 
treatment or procedure. 
 Not Related – includes adverse events are doubtfully related or clearly not related to the medical 
treatment or procedure. 
 
 
The FHCRC Serious Adverse Event (SAE) Report Form should be completed for all adverse events that 
meet the expedited reporting requirements.   All available information should be submitted but it is acceptable 
to fax an incomplete report form at the initial report.  A completed report should be faxed as soon as possible 
but must be received within 10 calendar days. 
 
It is the responsibility of the FHCRC Principal Investigator to notify the sponsor, NIH, FDA or other agencies 
of serious adverse events as required in the protocol. 
 
Serious adverse events that do not meet the requirement for expedited reporting (not related to study 
treatment or expected) will be reported to the IRB as part of the annual renewal of the protocol. 
 
FHCRC is acting as the Coordinating Center for this multi-institutional study, and it is the responsibility of the 
FHCRC Principal Investigator (or designee) to complete the FHCRC Serious Adverse Event Report for all 
serious adverse events that meet the expedited reporting requirements that are received from the 
participating sites.   
 
Procedure for Reporting Serious and Unexpected Adverse Events from Participating Sites 
Regulations defining the responsibilities for reporting serious and unexpected adverse reactions are defined 
above.  Serious and unexpected adverse events must be reported to the FHCRC Investigator  
within 10 days of learning of the event. This includes patient deaths, regardless of cause (serious, 
unexpected, and related/possibly related), occurring from the first protocol intervention and day 200 post-
transplant procedure.  The Fred Hutch trial coordinator contact information is telephone (206) 667-7385 or 
(206) 667-4916 and  must be followed by fax   at (206) 667-4427 of a detailed written report (See  
Appendix F of protocol) within 10 working days.  The report must include the date and time of onset, 
severity and duration of the event, the relationship to the study, the treatment given and eventual outcome.  
Follow-up information to a SAE report must be submitted as soon as the relevant information is available. 
 


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APPENDIX E (continued) 
 
Obligation of Investigators 
All adverse events using the modified NCI Common Toxicity Criteria (Appendix J of the protocol) occurring 
between first protocol intervention and day 100 during the study, whether or not attributed to the study, that 
are observed by the Investigator or reported by the patient will be recorded on the Case Report Form 
(Appendix I of the protocol). Attributes will include a description, date of onset, maximum severity, and 
assessment of relationship to the study agent or other suspect agent(s). 
 
Adverse events will be graded accordingly: 0 = none, 1 = mild, 2 = moderate, 3 = severe, 4 = life threatening 
or debilitating, and 5 = fatal.   All Grade 4 (life-threatening) or Grade 5 (fatal) events on the NCI scale meet 
expedited reporting requirements. 
 
Association or relatedness to the study agent will be graded as follows: 1 = unrelated, 2 = unlikely, 3 = 
possibly, 4 = probably, and 5 = definitely related. 
 
V. 
Case Report Forms 
 
Case report forms must be completed for all patients registered onto the protocols and submitted to the 
FHCRC data coordinating center. The first case report form (days 0-100) is due on day 120. A Staging Form 
must accompany the form with the patient staging at diagnosis, transplant and day 100. Staging forms should 
also be completed with each Follow Up Form completed on day 180, 1 year, 1.5 years, 2 years, 3 years, and 
yearly thereafter. A DLI Form must be completed and submitted for every infusion given. 
 
VI. 
Protocol Monitoring 
 
As the coordinating center, FHCRC will monitor accrual at the outside institutions. The guidelines below are 
intended to guide the reviewers in their assessment of items that significantly alter the clinical effectiveness 
of the treatment or the evaluation of its toxicity. 
 
A. Registration/Randomization 
1. Patient was registered prior treatment 
2. Information given at registration represents actual data in medical records (stage, diagnosis, cell 
type, etc.) 
 
B. Informed Consent/IRB Approval Dates 
1. The consent was signed prior to registration 
2. The consent is in language was approved by the institution’s IRB. IRB approval and reapproval are 
documented including appropriate use of full-board review and proper review of appropriate 
amendments or revisions 
3. Consent was dated and has written witness signature. IRB approval was obtained prior to the patient 
signing the consent form and start of treatment. 
 
C. Patient Eligibility 
1. Eligibility criteria and exclusion criteria were met 
2. Treatment/Intervention Administration 
3. Doses were modified according to protocol 
4. Accurate documentation of drug administration 
 
D. Study Tests/Evaluation 
1. Protocol specified laboratory tests or diagnostic studies are available 
2. Appropriate record of protocol intervention is documented. 


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APPENDIX E (continued) 
 
E. Study Events/Adverse Drug Experience 
1. Serious Adverse Evens reported according to protocol specifications 
 
F. Follow-Up 
 
1. Disease status assessed according to the required protocol guidelines documenting response to 
treatment. 
2. Accurate determination of cancer progression 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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APPENDIX F 
Fred Hutchinson Cancer Research Center 
 
SERIOUS ADVERSE EVENT REPORT (SAE) Form IRO-08 
 
FHCRC IR File Number: 
 
FHCRC Protocol Number: 
 
FHCRC Unique Patient #  
 FHCRC/SCCA   
  Other 
Gender:  
 Male  
 Female 
Age:   
FHCRC Principal Investigator:  
 
 
Phone Number: 
Mailstop: 
 
Date of Report:              
 
 
 Initial Report 
 Follow-Up Report #  
 Other 
 
Date study staff became aware of event: __________ 
Date Serious Adverse Event Started: 
Date Ended:      
Or 
 Ongoing (if ongoing – must submit follow up report) 
Adverse Event: 
 
 
Describe the Serious Adverse Event including a summary of all relevant clinical information. 
(Or attach a MedWatch Form or other SAE reporting form if one has been completed.)  Use Page 2, if necessary: 
 
Outcomes Attributed to adverse event:  (Check all that apply) 
 Death            /           /     
 Life-Threatening 
 Hospitalization (initial or prolonged) 
 
 Disability 
 Congenital Anomaly 
 
Required 
intervention 
to 
prevent 
permanent   
impairment/damage 
Specify Agent(s) and/or Procedure(s) involved in this protocol: 
#1 
Pharmaceutical 
product/medical 
treatment/procedure 
#2 
Pharmaceutical 
product/medical 
treatment/procedure 
 Not Related (Unrelated, Unlikely) 
 Not Related (Unrelated, Unlikely) 
 Related (Possible, Probable, Definite) 
 Related (Possible, Probable, Definite) 
 
 
 Follow-up Report Required 
 Final Report (PI must sign final report) 
 
Report Completed by: 
 
Date: 
 
The PI has determined that the consent form must be revised: 
 Yes      
 No 
Does this study involve the deliberate transfer of recombinant DNA or DNA or RNA derived from recombinant 
DNA, into human subjects (human gene transfer)? 
 yes  
 no  If yes and the activity involves the SCCA 
outpatient clinic, a copy of this Protocol Modification Form and any supporting documents to be reviewed and 
approved, will be forwarded to the FHCRC’s Institutional Biosafety Committee (IBC) by the Protocol Office 
(Mailstop:  LM-230). 
 
Signature of Principal Investigator 
 
Date: 
 


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APPENDIX F (continued) 
Fred Hutchinson Cancer Research Center 
Clinical Research Division 
Institutional Review Office 
SERIOUS ADVERSE EVENT REPORT (SAE) Form IRO-08 
 
 
 
FHCRC IR File Number: 
 
FHCRC Protocol Number: 
 
FHCRC Unique Patient #  
Date of Report:              
Describe the Serious Adverse Event including a summary of all relevant clinical information. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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APPENDIX G 
 
NOTICE OF DEATH 
 
Patient ID:   
 
 
 
 
 
 
 
 
 
 
Date of Death:   
 
 
 
 
 
 
__ 
 
Place of Event:    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
    
 
 
Apparent cause of death (Please be specific.  Attach hospital summary or death summary when possible): 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Form completed by:  ________________________________Date:   ______________ 
 


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APPENDIX H 
Protocol 2032 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 
 
TBI DOSE LEVEL  
      
  200 cGy  
 400 cGy: 2 doses of 200 cGy for patients with acquired 
aplastic anemia, sickle cell disease, thalassemia, HLH, or 
other non-malignant disease at high risk for graft rejection 
(as discussed at the non-malignant board or the patient 
care conference). 
Signature of Local Principal Investigator:  
__________                           Date:                  
___            
(or Designee)  
Transplant Center: ___________________________________________________________________ 
 
Signature of FHCRC Principal Investigator:  
__________                           Date:                  
___            
(or Designee) 
 
Protocol 2032 Eligibility  
 
I) 
Inclusion Criteria:  
 
 


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1) Yes 
 No 
 Patient signed IRB approved consent form.  Date:    
 
 
 
 
 
 
 
 
 
 
IRB File Number:   
 
 
 
 
 
 
 
2) Yes 
 No 
  The consent was approved by your institution’s IRB on Date: ___________  
 
The following criteria questions must be marked “Yes” or  “NA” for the patient to enter on 2032. 
 
3) Yes 
 No 
 Age < 55 years with a primary immunodeficiency or other nonmalignant inherited 
disease (except fanconi anemia) treatable by allogeneic HCT.  
 
4) Yes 
 No 
 Patients with pre-existing medical conditions or other factors that render them at 
high risk for regimen related toxicity or ineligible for a myeloablative conventional 
HCT. Pre-existing condition precluding conventional myeloablative 
transplant:_________________________________________ 
    
5) Yes 
 No 
   Patient does not have an HLA-matched related or unrelated donor.  
 
6) Yes 
 No 
   NA 
   Patient with acquired aplastic anemia. 
 
Bone marrow cellularity < 25%, or marrow cellularity < 50% but with   
< 30% residual hematopoietic cells. 
 
Two out of three of the following (in peripheral blood): neutrophils       
< 0.5 x 109/L;platelets < 20 x 109/L; reticulocytes < 20 x 109/L. 
 
 
 
 
 
SAA diagnostic criteria may be applied to assessment at initial diagnosis  
 
 
 
 
 
 
or follow-up assessments.  
 
7) Yes 
 No 
   Patient with a related donor who is identical for one HLA haplotype.   
 
Patient 
 
 
 
 
 
A: ________ 
A:_________ 
C: _________ 
C: _________ 
B: _________ 
B: _________ 
DRB1: ______ 
DRB1:______ 
DQB1:_____ 
DQB1:______ 
 
 
Donor 
 
 
 
 
 
A: _________ 
A:________ 
C: _________ 
C: _________ 
B: _________ 
B: _________ 
DRB1: ______ 
DRB1:______ 
DQB1:______ 
DQB1: ______ 
 
 
 
II) 
Exclusion criteria:  
 
Each of the following questions must be marked “No” Or “NA” for the patient to enroll on 2032. 
 
8)  Yes 
 No 
  Patients with fanconi anemia 
 
9)  Yes 
 No 
 Patients with metabolic storage diseases who have severe CNS involvement 
 
 
 
 
 
of disease, defined as IQ score < 70. 
10)    Yes 
 No 
 Patients who are positive for human immunodeficiency virus (HIV).  
11)  Yes 
 No 
 NA 
   Females who are pregnant (-HCG+) or breast-feeding. 
12)  Yes 
 No 
 NA 
 Fertile men or women who are unwilling to use contraceptives during HCT 
and up to 12 months post-treatment. 


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13)  Yes 
 No 
 Patients with fungal pneumonia with radiological progression after receipt of 
amphotericin formulation or mold-active azoles for greater than 1 month will not 
be eligible for this protocol (either regimen A or B).  
14)  Yes 
 No 
    Organ dysfunction. Please check yes if patient meets any of the following: 
a.  Yes 
 No 
 Cardiac ejection fraction < 30% (or, if unable to obtain 
ejection fraction, shortening fraction <26%) on MUGA scan 
or cardiac echo, symptomatic coronary artery disease, 
other cardiac failure requiring therapy.  Patients with a 
history of, or current cardiac disease should be evaluated 
with appropriate cardiac studies and/or cardiology consult. 
Patients with a shortening fraction <26% must be seen by 
cardiology for approval.  
 
 
 
 
Date: _____/_____/_______ 
 
 
 
Ejection Fraction Value: __________% or 
 
 
 
Shortening Fraction Value: _________% 
b.  Yes 
 No 
 
Poorly controlled hypertension despite anti-hypertensive 
medications. 
c.  Yes 
 No 
 
Patients with clinical or laboratory evidence of liver disease 
will need to be evaluated for the cause of the liver disease, 
its clinical severity in terms of liver function and the degree 
of portal hypertension. Patients will be excluded if they are 
found to have: fulminant liver failure, cirrhosis of the liver 
with evidence of portal hypertension, bridging fibrosis, 
alcoholic hepatitis, esophageal varices, a history of 
bleeding esophageal varices, hepatic encephalopathy, 
uncorrectable hepatic synthetic dysfunction evidenced by 
prolongation of the prothrombin time, ascites related to 
portal hypertension, bacterial or fungal liver abscess, 
biliary obstruction, chronic viral hepatitis with total serum 
bilirubin > 3mg/dl, or symptomatic biliary disease.  
 
 
 
 
Date: ____/_____/______ 
 
 
 
ALT: _______ U/L 
 
 
AST: _________ U/L 
 
 
 
Total Bilirubin: ___________ mg/dL Alk.phos:______ U/L 
 
 
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 I 
Outside Center Core Case Report Forms 
 
For Peripheral Blood or Bone Marrow Transplantations 
OSC CRF Version 
5.0 (2018) - PB BM.pd 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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APPENDIX J 
 
Adapted from 
COMMON TOXICITY CRITERIA (CTC) 
 
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 J (continued) 
 
APPENDIX J (continued) 
Grade 
Adverse Event 
3 
4 
 
CARDIOVASCULAR (GENERAL) cont’d. 
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 J (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). 
>10% or as ascites 
>10% or fluid retention 
resulting in pulmonary 
failure 


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APPENDIX J (continued) 
Grade 
Adverse Event 
3 
4 
 
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) 
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 
 
 
 
 
 
 
 
 
 


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APPENDIX J (continued) 
Grade 
Adverse Event 
3 
4 
GASTROINTESTINAL cont’d. 
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) 
 
 
Also consider 
Hemorrhage/bleeding with 
grade 3 or 4 thrombocytopenia, 
Hemorrhage/bleeding without 
grade 3 or 4 thrombocytopenia. 
Bleeding without perforation, 
uncontrolled by outpatient 
medical management; requiring 
hospitalization or surgery 
Perforation or bleeding, 
requiring emergency surgery 
Gastritis 
 
 
Also consider 
Hemorrhage/bleeding with  
grade 3 or 4 thrombocytopenia, 
Hemorrhage/bleeding without 
grade 3 or 4 thrombocytopenia. 
Uncontrolled by out-patient 
medical management; requiring 
hospitalization or surgery 
Life-threatening bleeding, 
requiring emergency surgery 
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 J (continued) 
Grade 
Adverse Event 
3 
4 
 
 
GASTROINTESTINAL cont’d. 
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 J (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 J (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 
 
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 
 
 


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APPENDIX J (continued) 
Grade 
Adverse Event 
3 
4 
 
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 J (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 K 
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Cyclophosphamide Guidelines 
 
Introduction  
Cyclophosphamide is frequently used as a cytotoxic agent.  Cyclophosphamide is converted to its active 
form in vivo by hepatic enzymes.  After a single dose, most of the active metabolites are degraded by tissue 
enzymes.   After high doses (> 40 mg/kg), the alkylating activity in the plasma is minimal by 24 hours.  Several 
of the metabolites appear to have toxic actions.  One of the metabolic products, acrolein (CH2=CH-CHO), is 
known to be toxic to the bladder urothelium and can cause hemorrhagic cystitis when cyclophosphamide is 
administered at high doses.  In the bladder, the free thiol groups of a drug called mesna (sodium-2-
mercaptoethanesulfonate) react with the double bond in acrolein producing an inactive compound, which is 
eliminated in the urine.  Urinary excretion of mesna is almost complete within 2-4 hours after IV 
administration.  
 
Dosing of cyclophosphamide 
Cyclophosphamide doses are specified in the protocol.  Adjusted body weight should be used for calculating 
initial doses if patient’s actual weight is > 100% of ideal body weight.  If the Ideal Body Weight  > Actual Body 
Weight, then the Actual Body Weight should be used.  Doses > 5000mg must be infused IV over 2 hours.  Lower 
doses may be administered over one hour. 
 
Note: cyclophosphamide is administered to adults as inpatients or outpatients but to children only as 
inpatients. 
 
Side Effects of Cyclophosphamide 
 
1. Fluid Retention 
Cyclophosphamide can cause an antidiuretic effect with development of inappropriate ADH 
secretion.  
 
2. Cardiomyopathy 
Within 0-10 days after high-dose CY (>100 mg/kg), a clinical syndrome of severe CHF may develop 
characterized by cardiomegaly, pericardial effusions, diffuse voltage decrease on ECG and decreased LVEF.  
Mortality is >50%.  There are nine retrospective studies of post-transplant cardiomyopathy published 
between 1976 - 2001.  In the four studies of >100 patients, incidence was 0.4 – 4%.  The relationship between 
pre-transplant LVEF or pre-existing cardiac abnormalities and post-transplant cardiomyopathy is equivocal 
but the number of patients studied has been small.  At present, cardiac status is assessed on an individual 
basis. 
References: Appelbaum, et al. Lancet (1976); Goldberg, et al. Blood 68: 1114 (1986); Cazin, et al. Cancer 
57: 2061 (1986); Bearman, et al. BMT 5: 173 (1990); Braverman, et al. J. Clin. Oncol. 9: 1215 (1991); 
Hertenstein, et al. J. Clin. Oncol. 12: 998 (1994); Lee, et al. BMT 18: 573 (1996); Fujimaki, et al. BMT 27: 
307 (2001); Murdych and Weisdorf BMT 28: 283 (2001).   
 
 
 
 
 
 
 


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 (Side Effects of Cyclophosphamide- Continued) 
 
3. Hemorrhagic Cystitis 
Cyclophosphamide can cause hemorrhagic cystitis.  To prevent the development of hemorrhagic cystitis, 
patients are kept well hydrated, and mesna therapy or bladder irrigation is used.   
 
The recommended prophylaxis is mesna therapy unless (i) contraindicated clinically, e.g., bladder outlet 
obstruction as in BPH or (ii) otherwise specified per protocol or Standard Treatment Plan. 
 
a. Bladder Protection 
 
 
Mesna   
The dose of mesna is based on the cyclophosphamide dose being given.  Use of mesna 
should be considered for all patients receiving >2 g/m2 of cyclophosphamide.  The total daily 
dose of mesna is equal to 100 % of the total daily dose of cyclophosphamide.  Mesna is 
administered in 4 divided doses IV at 30 minutes prior to start of cyclophosphamide infusion 
and then at 3, 6 and 8 hours after the start of the cyclophosphamide infusion. 
 
 
Bladder Irrigation   
o Adults > 17 years of age:  Insert a #16 French of larger three-way Foley catheter and 
administer urological saline at a rate of 500 mL/hr.  Bladder irrigation should begin 1 
hour prior to the start of the first cyclophosphamide infusion and may be discontinued 
24 hours after the last dose of cyclophosphamide providing heme testing of urine is 
negative. 
 
o Children > 12 years of age:  Assess individually to determine whether or not it is 
possible to place a #16 French three-way catheter.  If possible, administer urological 
saline at a rate of 500 mL/hr.  Bladder irrigation should begin 1 hour prior to start of 
the first cyclophosphamide infusion and may be discontinued 24 hours after the last 
dose of cyclophosphamide providing heme testing of urine is negative. 
 
o Children 1.5-12 years of age:  Unless there is an anatomic abnormality, insert a simple 
Foley catheter to straight drainage as follows: 
 
11-12 years 
#14 French with balloon 
 
8-10 years  
#12 French with balloon 
 
1.5-7 years 
#10 French with balloon 
 
(Side Effects of Cyclophosphamide- 
3. Hemorrhagic Cystitis- Continued) 
 
b.   Fluid Administration  
MESNA 
Adult Patients 
 
Outpatient  (patients who are not at risk for tumor lysis syndrome) 
 
 
Solution: NS with 20 mEq KCl/L  
 


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 
Rate of infusion: 3 ml/kg/hr 
 
 
Duration of infusion: Begin at least 2 hours prior to start of cyclophosphamide 
infusion and continue until at least 8 hours post each dose of 
cyclophosphamide  
 
Inpatient 
 
 
Solution: 
o Without sodium bicarbonate: D5 ½ NS with 20 mEq KCl/L 
o With sodium bicarbonate: D5 ¼ NS with NaHCO3 50 mEq/L plus 20 mEq 
KCl/L (recommended ONLY for patients with high tumor burden such as 
>20,000 cells/mm3 or mass lesions i.e., at risk for tumor lysis syndrome) 
 
Sodium bicarbonate is added when clinically indicated to 
promote alkalinization of urine to prevent crystallization of uric 
acid.  However, alkalinization of urine is known to increase 
formation of acrolein. 
 
 
Rate of infusion: 2 x maintenance (~3000 mL/m2/24 hours) 
 
 
Duration of infusion: Begin fluid administration 4 hours prior to the first dose of 
cyclophosphamide and continue until 24 hours after the last dose of 
cyclophosphamide is given. 
 
 (Side Effects of Cyclophosphamide- 
3. Hemorrhagic Cystitis 
b. Fluid Administration 
MESNA- Continued) 
 
Pediatric patients  
Cyclophosphamide is ONLY administered in the hospital 
 
 
Solution: 
o Without sodium bicarbonate: D5 ¼ NS with KCl 20 mEq/L 
o With sodium bicarbonate: D5 ¼ NS with NaHCO3 40 mEq/L plus KCl 20 mEq/L 
(recommended ONLY for patients with high tumor burden such as >20,000 
cells/mm3 or mass lesions or patients on allopurinol, i.e., at risk for tumor lysis 
syndrome) . 
 
Sodium bicarbonate is added when clinically indicated to promote 
alkalinization of urine to prevent crystallization of uric acid.  
However, alkalinization of urine is known to increase formation of 
acrolein. 
 
 
Rate of Infusion: 
o Patients  20 kg:  2 x maintenance rate 
o Patients < 20 kg:  1.5 x maintenance rate 
 
 
Duration of Infusion: Begin fluid administration 4 hours prior to the first dose of 
cyclophosphamide and continue until 24 hours after the last dose of 
cyclophosphamide is given. 
 


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BLADDER IRRIGATION 
 
Adult patients (see above, same as for inpatient fluid administration for Mesna) 
 
Pediatric patients (see above, same as for Mesna) 
c.  Monitoring 
MONITOR 
CHILDREN 
ADULTS 
Weight  
Twice Daily 
Twice Daily 
I&O’s 
Every 2 hours 
Every 4 hours (patients without foley 
catheter or bladder irrigation must void 
every 2 hours until at least 8 hours after 
each dose of cyclophosphamide) 
Posturals 
N/A 
BID 
Urine pH 
 
 
(only patients receiving 
NaHCO3)  Keep pH > 7 
 
(only patients receiving NaHCO3) Keep pH 
> 7 
 
Urine for red cells 
before cyclophosphamide 
and around 24 hours after 
each infusion of 
cyclophosphamide 
before cyclophosphamide and around 24 
hours after each infusion of 
cyclophosphamide  
  
(Side Effects of Cyclophosphamide- 
3. Hemorrhagic Cystitis 
c.   Monitoring- Continued) 
 
 
Urine for red cells: 
Test urine by dipstick PRIOR to administration of first dose of cyclophosphamide and once around 
24 hours after infusion of each dose of cyclophosphamide.  If positive, send urine to lab for 
complete urinalysis including microscopic evaluation. 
 
Urine output: 
o Adults:  For patients >60  kg, urine output must be at least 3 mL/kg over 2 hours before 
administering a dose of cyclophosphamide.  Urine output must be maintained at  600 
mLs for the first 4 hours post-cyclophosphamide.  If urine output < 600 ml over 4 hours, 
consider either (i) a NS fluid bolus based on current weight, I&O and postural vital 
signs or (ii) administration of furosemide (10 mg/m2 IV)  
NOTE:  furosemide should not be administered until after the last daily dose of 
mesna 
o Adults For patients <60 kg, urine output must be at least 3 mL/kg over 2 hours before 
administering a dose of Cyclophosphamide.   Urine output must be maintained at 2-3 
ml/kg/hour for the first 4 hours post-cyclophosphamide.  If urine output is less, consider 
either (i) a NS fluid bolus based on current weight, I&O and postural vital signs or (ii) 
administration of furosemide (10 mg/m2 IV) 
NOTE:  furosemide should not be administered until after the last daily dose of 
mesna 
o Children < 40 kg:  Urine output should be maintained at 2-3 mL/kg/hr until 24 hours 
after the last dose of cyclophosphamide is administered. 
 
 
 
 
If urine output in children ( 40kg) is <2-3 ml/kg/hr, give NS challenge, 10 ml/kg over 
20 minutes.  If inadequate urinary response in 2 hours, give furosemide 0.5 mg/kg IV  
 
 
 
 
NOTE:  furosemide should not be administered until after the last daily dose of 
mesna 
 
Patients without foley catheter or bladder irrigation must void every 2 hours until at least 8 
hours after each dose of cyclophosphamide. 


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 
Weight:  Both inpatients and outpatients should be weighed before each dose of 
cyclophosphamide.  Then, outpatients should be weighed after completion of mesna therapy; 
inpatients should be weighed twice daily.  If the patient’s weight is > 2 kg above the pre-
treatment weight, consideration should be given to the administration of a diuretic.  NOTE:  
furosemide should not be administered until after the last daily dose of mesna. 
 
 (Side Effects of Cyclophosphamide-Continued) 
 
4. Nausea/Vomiting/Anorexia 
(See Anti-emetics Institutional Standard Practice Guidelines) 
 
5. Other Toxicities 
 
Myelosuppression 
 
Gonadal abnormalities 
 
Alopecia 
 
Rare pulmonary toxicity 
 
 
 
Approved by Medical Executive Committee 6/11/91 (AEP.SPM) cyclophosphamide.doc 
Revised by Standard Practice Committee 6/29/94 
Approved by Medical Executive Committee 6/28/94 
Revised by the Standard Practice Committee July 29, Sept 16, 1998 
Abbreviations removed May 1, 1999 
Discussed and revision concept accepted in Clinical Advisory Committee August 2002 
Revised in Standard Practice Meeting without finalization April 24, 2002, seen in committee November 20, 2002, then with all final information 
supplied by committee members, January 28, 2003, February 18, 2003 
 
 
 
 


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Appendix L 
weight_for_drug_do
sing.pdf
 


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Appendix M 
Radiotherapy Treatment Guidelines per Standard Practice 
 
TBI_Adult_Non_Myel
oablative
TBI_Pediatric_NON_
Myeloablative
 
 
 
 
 
 


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APPENDIX N 
COORDINATING CENTER FUNCTIONS 
Outside Center – PI Communication in Hematologic Malignancies 
 
I. 
Study Management, data analysis, and Data and Safety Monitoring 
 
a. Study Management: 
i. 
Each local PI is responsible for selection, training and oversight of local study 
coordinators 
ii. 
The Coordinating Center registers subjects on the study and assigns study IDs 
iii. 
One copy of the research data is retained by the site. Another data set (identified only by 
study IDs) is transmitted to the Coordinating Center to create the master data file. All 
data are kept in locked areas and password protected databases accessible only to 
study staff 
iv. 
The quality of data is monitored in an ongoing fashion with the study team and corrective 
action plans instituted as necessary  
b. Data Analysis: 
i. 
Study staff review data for completeness as it is submitted by the sites 
ii. 
The study statistician is responsible for data cleaning and the conduct of analyses as 
outlined in the protocol and grant 
c. Data Safety and Monitoring: 
i. 
The trial coordinators at collaborating centers or the local PIs will fax an official report of 
an SAE (as defined by the protocol) to the Coordinating Center within ten days 
ii. 
The SAE report is reviewed by the Overall PI. If the SAE meets the FHCRC criteria for 
reporting then an official signed report is submitted to the IRB 
iii. 
An independent DSMB will meet at six-month intervals and all outcome data is reviewed 
including all adverse events and SAEs reported to the Coordinating Center along with 
those officially reported to the IRB 
iv. 
A report from the DSMB is submitted to the IRB as well as the trial coordinators/local PIs 
participating in the protocol 
 
II.  
Protocol and informed consent document management 
a. A master protocol is maintained by the Coordinating Center and distributed to the sites for 
customization and local IRB review 
b. All protocol and consent modifications initiated by the Coordinating Center are sent to the 
Collaborating Sites following approval by the Coordinating Center IRB, for review and 
approval by the local IRB 
c. Changes required by local IRBs are reviewed by the Coordinating Center and approved prior 
to implementation at local sites 
 
III. Assurance of local IRB OHRP-approved assurance 
a. Each site provides their OHRP assurance number and evidence of IRB certification 
b. Study staff monitor maintenance of institutional assurance and IRB certification 
 
IV. Assurance of local IRB approvals 
a. The Coordinating Center maintains copies of the most current collaborating site Consent 
Forms and IRB approval documentation 
b. No site may enroll subjects until the Coordinating Center has received confirmation of local 
IRB approval 
c. Each site is responsible for preparation and submission of their continuing reviews. Any 
changes to the protocol or consent form will be communicated to the Coordinating Center 
d. Sites are required to have active IRB approvals to participate in any study related activities 
 


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V. 
Any substantive modification by the Collaborating Institution related to risks or alternative 
procedures is appropriately justified 
a.   The Coordinating Center reviews any modifications to consent forms to ensure that site consents 
do not delete or change the basic or additional elements or alternatives required in the sample 
consent form 
 
VI. 
Informed consent is obtained from each subject in compliance with HHS regulations 
a. Subjects must provide written informed consent prior to study participation 
b. The Coordinating Center verifies eligibility and signed consent prior to assigning a study ID 
number 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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APPENDIX O 
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.