Clinical trial NCT01858740
Conditions: Accelerated Phase Chronic Myelogenous Leukemia, BCR-ABL1 Positive Acute Biphenotypic Leukemia Acute Leukemia of Ambiguous Lineage Acute Undifferentiated Leukemia Adult Acute Lymphoblastic Leukemia in Remission Adult Acute Myeloid Leukemia in Remission Blast Phase Chronic Myelogenous Leukemia, BCR-ABL1 Positive Childhood Acute Lymphoblastic Leukemia in Remission Childhood Acute Myeloid Leukemia in Remission Chronic Myelogenous Leukemia, BCR-ABL1 Positive Myelodysplastic Syndrome With Excess Blasts-1 Myelodysplastic Syndrome With Excess Blasts-2 Recurrent Adult Acute Lymphoblastic Leukemia Recurrent Adult Acute Myeloid Leukemia Recurrent Childhood Acute Lymphoblastic Leukemia Recurrent Childhood Acute Myeloid Leukemia Refractory Adult Acute Lymphoblastic Leukemia Refractory Childhood Acute Lymphoblastic Leukemia
Sponsor: Fred Hutchinson Cancer Center
Start date: 2014-04-10; Completion date: 2023-07-30
Enrollment: 20 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This phase II trial studies how well T cell depleted donor peripheral blood stem cell transplant works in preventing graft-versus-host disease in younger patients with high risk hematologic malignancies. Giving chemotherapy and total-body irradiation before a donor peripheral blood stem cell transplant helps stop the growth of cancer cells. It may also stop the patient's immune system from rejecting 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. Removing a subset of the T cells from the donor cells before transplant may stop this from happening.

Detailed description:
OUTLINE:

CONDITIONING REGIMEN: Patients undergo total body irradiation (TBI) twice daily (BID) on days -10 to -7, receive thiotepa intravenously (IV) over 4 hours on days -6 and -5 and fludarabine phosphate IV over 30 minutes on days -6 to -2.

TRANSPLANT: Patients undergo CD34+ enriched, CD45RA+ T cell-depleted allogeneic PBSCT on day 0.

POST-TRANSPLANT IMMUNOSUPPRESSION: Patients receive tacrolimus IV continuously or orally (PO) every 12 hours beginning on day -1 and continuing through day 50 with taper. Patients also receive methotrexate IV on days 1, 3, 6, and 11.

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

Arms: arm 1: CONDITIONING REGIMEN: Patients undergo TBI BID on days -10 to -7, receive thiotepa IV over 4 hours on days -6 and -5 and fludarabine phosphate IV over 30 minutes on days -6 to -2.

TRANSPLANT: Patients undergo CD34+ enriched, CD45RA+ T cell-depleted allogeneic PBSCT on day 0.

POST-TRANSPLANT IMMUNOSUPPRESSION: Patients receive tacrolimus IV continuously or PO every 12 hours beginning on day -1 and continuing through day 50 with taper. Patients also receive methotrexate IV on days 1, 3, 6, and 11.
Interventions: intervention 1: Allogeneic Hematopoietic Stem Cell Transplantation intervention 2: Fludarabine Phosphate intervention 3: Laboratory Biomarker Analysis intervention 4: Methotrexate intervention 5: Peripheral Blood Stem Cell Transplantation intervention 6: T Cell-Depleted Hematopoietic Stem Cell Transplantation intervention 7: Tacrolimus intervention 8: Thiotepa intervention 9: Total-Body Irradiation
Intervention descriptions: intervention 1: Undergo CD45RA+ T cell-depleted allogeneic peripheral blood stem cell transplant intervention 2: Given IV intervention 3: Correlative studies intervention 4: Given IV intervention 5: Undergo CD45RA+ T cell-depleted allogeneic peripheral blood stem cell transplant intervention 6: Undergo CD45RA+ T cell-depleted allogeneic peripheral blood stem cell transplant intervention 7: Given IV or PO intervention 8: Given IV intervention 9: Undergo TBI

Protocol documents:
Prot_SAP_000.pdf: 
 
 
Official Title: 
A Phase II Study of Selective Depletion of CD45RA+ T Cells 
from Allogeneic Peripheral Blood Stem Cell Grafts for the 
Prevention of GVHD in Children 
NCT Number: 
NCT01858740 
Document Type:  
Study Protocol and Statistical Analysis Plan 
Date of the 
Document: 
March 7, 2022 
 
 


Protocol 2660.00 
Page 1 of 57 
FRED HUTCHINSON CANCER RESEARCH CENTER 
UNIVERSITY OF WASHINGTON SCHOOL OF MEDICINE 
SEATTLE CHILDREN’S 
 
Current Version: 01/25/2022 
Previous Version: 01/20/2017 
 
1. 
Title of Protocol: A Phase II Study of Selective Depletion of CD45RA+ T Cells from 
Allogeneic Peripheral Blood Stem Cell Grafts for the Prevention of GVHD in Children 
 
 
 
 
 
 
Investigator 
Professional Title 
 
Telephone number 
Marie Bleakley, MD, PhD (PI) 
Assistant Professor, FHCRC 
Assistant Professor of Pediatrics, UW 
(206) 667-6572 
(206) 469-4487 (pager) 
Stanley Riddell, MD 
Member, FHCRC 
Professor of Medicine, UW 
(206) 667-5249 
Shelly Heimfeld, PhD 
Ann Woolfrey, MD 
Member, FHCRC 
Member, FHCRC 
Professor of Pediatrics, UW  
(206) 667-4004 
(206) 667-4453 
 
 
Biostatistician 
 
 
Ted Gooley, PhD 
Member, FHCRC 
Affiliate Professor, UW 
 
 
 
Radiation Oncologist 
 
 
Ermoian, Ralph P 
 
Assistant Professor of Radiation 
Oncology 
(206) 598-4100 
 
Research Staff 
 
 
Barbara Hilzinger , RN 
Research Nurse 
(206) 667-2303 
(206) 340-3035 (pager) 
(206) 667-6056 (fax) 
 
 
Emergency (24 hour) Phone: (206) 987-4536; Fax: (206) 667-6056 
IRO received 02/28/22
FHCRC IRB Approval  
MAR 07 2022 
Document Released Date


Protocol 2660.00 
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Table of Contents 
 
2. Introduction ...................................................................................................................................................4  
 
3. Background ....................................................................................................................................................5 
 
4. Objectives ......................................................................................................................................................13  
4A. Primary  ...................................................................................................................................................13  
4B. Secondary  ...............................................................................................................................................13  
 
5. Patient Selection ............................................................................................................................................14 
5A. Inclusions ................................................................................................................................................14  
5B. Exclusions ...............................................................................................................................................14 
 
6. Donor Selection .............................................................................................................................................14  
6A. Inclusions ................................................................................................................................................15 
6B. Exclusions ...............................................................................................................................................15  
 
7. Evaluation and counseling of patient  ............................................................................................................15 
 
8. Protocol registration ......................................................................................................................................15  
 
9. Plan of treatment ............................................................................................................................................16 
9A. Conditioning regimen ..............................................................................................................................16 
9B. CNS prophylaxis and testicular irradiation..............................................................................................17 
9C. Collection of G-CSF mobilized PBSC ....................................................................................................17 
9D. PBSC graft engineering/cell selection .....................................................................................................17  
9E. PBSC infusion .........................................................................................................................................19 
9F. Post transplant immunosuppression .........................................................................................................20 
9G. Use of hematopoietic growth factors…………………………………………………………………...21 
 
10. Evaluation ....................................................................................................................................................21 
10A. Donor evaluation ...................................................................................................................................21 
10B. Patient pre-transplant evaluation ...........................................................................................................21 
10C. Patient post-transplant evaluation guidelines ........................................................................................22 
  
11. Irradiation, drugs, and PBSC administration - toxicities and complications ...............................................25 
11A. Total body irradiation ............................................................................................................................25  
11B. Thiotepa .................................................................................................................................................25 
11C. Fludarabine ............................................................................................................................................26 
11D. PBSC infusion .......................................................................................................................................26 
11E. Tacrolimus .............................................................................................................................................26 
11F. Methotrexate …………………………………………………………………………………………..26 
 
12. Protocol enrollment and special considerations ...........................................................................................27 
12A. Projected target accrual .........................................................................................................................27 
12B. Infection prophylaxis .............................................................................................................................28 
12C. Ursodeoxycholic acid prophylaxis of hepatic complications ................................................................28  
12D. Management of acute and chronic GVHD ............................................................................................28 
12E. Management of delayed engraftment ....................................................................................................29 
12F. Management of relapse ..........................................................................................................................29 
 
13. Records ........................................................................................................................................................29 
 
14. Evaluation and statistical considerations .....................................................................................................29 
14A. Type of study.........................................................................................................................................29 
14B. Definition of endpoints ..........................................................................................................................30 
14C. Statistical analysis and stopping rules ...................................................................................................31 


Protocol 2660.00 
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15. Guidelines for reporting and tracking events ...............................................................................................33 
15A. Toxicity Grading ...................................................................................................................................33  
15B. Definitions .............................................................................................................................................33 
15C. Tracking of Events  ...............................................................................................................................33 
15D. Reporting to the FHCRC IRB ...............................................................................................................34 
15E. Reporting Requirements to FDA ...........................................................................................................34 
 
16. Data safety monitoring plan .........................................................................................................................35 
16A. Monitoring the progress of trials and the safety of participants ............................................................35 
16B. Plans for assessing data accuracy and protocol compliance ..................................................................36 
 
17. References ...................................................................................................................................................36 
 
18.  Appendices 
18A. Cell selection flow sheet and guidelines  .........................................................................................39 
18B. Product testing .................................................................................................................................41 
18C. Positive culture from donor product –action plan  ...........................................................................42 
18D. Infusion of selected cells..................................................................................................................43 
18E. Acute GVHD staging and grading assessment  ...............................................................................45 
18F. Grading of chronic graft versus host disease ...................................................................................46 
18G. Potential adverse events associated or expected with hematopoietic cell transplantation  ..............47 
18H. Karnofsky and Lanksy performance status scales  ..........................................................................48 
18I.  Weight/Adjusted body weight for drug dosing  ...............................................................................49 
18J.  Radiotherapy treatment plan ............................................................................................................50 
18K.  Guidelines for pediatric research blood draws .................................................................................55 
 
 
 
 
 
 


Protocol 2660.00 
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2. 
Introduction 
Graft versus host disease (GVHD) is a frequent complication of allogeneic hematopoietic 
cell transplantation (HCT) in adults and in children. Morbidity and mortality as a 
consequence of GVHD result from direct organ damage, and from infections and organ 
toxicity related to the use of immunosuppressive drugs to treat GVHD.[1] 
 
Despite the administration of immunosuppressive drugs early post transplant to prevent 
GVHD, following myeloablative allogeneic bone marrow transplant from unrelated 
donors in children at the FHCRC and other centers the incidence of grade II – IV acute 
GVHD is 40-90% and the incidence of extensive chronic graft versus host disease is 20-
60%.[2-7] In an analysis of data reported to the Center for International Bone Marrow 
Transplant Research (CIBMTR), Eapen and colleagues observed a rate of chronic GVHD 
of 32% amongst 116 children with acute leukemia who received bone marrow transplants 
without T cell depletion from 8/8 allele matched unrelated donors. [4] Similarly, amongst 
51 children who received myeloablative bone marrow transplants from 10/10 allele 
matched unrelated donors (URD) at FHCRC the rate of clinically extensive chronic 
GVHD was 35%.[8] & personal communication AW and TG  
 
Although bone marrow is currently the most frequently used donor stem cell source for 
pediatric HCT recipients there has been a trend toward increasing use of peripheral blood 
stem cells (PBSC) in pediatrics as well as adults. At FHCRC, of the transplants 
performed in pediatric recipients with HLA matched unrelated donors in the past ten 
years, 40% have employed PBSC and 60% have employed bone marrow as the stem cell 
source . PBSC has several advantages over BM including more rapid engraftment and a 
shorter duration of initial hospitalization, and is frequently preferred by the donor center 
or donor for feasibility reasons. However, a large randomized study demonstrated an 
increased rate of chronic GVHD was observed amongst predominantly adult patients who 
received PBSC (53%, CI 45 to 61) compared with bone marrow (41%, CI 34 to 48) from 
HLA matched (78%) or 1-2 antigen mismatched (22%) URD [9], although the rate of 
graft failure was lower amongst PBSC recipients (3% CI 1-5%) compared to bone 
marrow recipients (9% CI 6-13%). There was no significant difference in overall survival 
between the groups. There is a clear need to develop strategies to reduce the rate of 
chronic GHVD, particularly in PBSC transplants. An optimal approach would preserve 
the benefit of the rapid engraftment time and low rate of graft failure associated with the 
use of PBSC but substantially reduce the rate of chronic GVHD to lower than that 
observed with either PBSC or BM.  
 
In HCT using HLA-identical donors, GVHD results from recognition of minor 
histocompatibility (H) antigens expressed on recipient tissues by donor T cells.[10] 
Complete depletion of T cells from the donor hematopoietic cell product is a highly 
effective alternative to pharmacologic immunosuppression for preventing GVHD, but is 
complicated by a profound delay in immune reconstitution, which contributes to life 
threatening infections, and has been associated with an increased risk of graft rejection 
and leukemia relapse.[11,12] Laboratory studies have suggested that donor T cells 
specific for recipient minor H antigens are found predominantly within the naïve subset 
of T cells that express the cell surface molecule CD45RA, and that the selective depletion 
of CD45RA+ T cells from PBSC grafts can preserve the subset of pathogen-specific 
memory T cells that lack CD45RA.This protocol will evaluate the selective removal of 
CD45RA+ cells from PBSC as a strategy to reduce chronic GVHD and provide rapid 


Protocol 2660.00 
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recovery of T cell immunity to pathogens in children after HCT from HLA-identical 
unrelated donor transplants.  
 
3.    
Background 
3A. 
GVHD  
The pathogenesis of GVHD involves multiple interacting factors including tissue damage 
resulting from the conditioning regimen, the release of inflammatory cytokines, and the 
activation and proliferation of mature donor T cells that express the αβ T cell receptor 
(TCR) and recognize recipient alloantigens presented as peptides by class I and II major 
histocompatibility complex (MHC or HLA in humans) molecules on antigen presenting 
cells (APCs) [1,13]. The mature T cell repertoire is  “tolerant” to peptides derived from 
self-proteins due to thymic deletion and/or peripheral suppression of autoreactive T cells 
[14]. However, in the setting of allogeneic HCT between HLA-identical individuals, the 
repertoire of peptides displayed on recipient cells will include distinct species that differ 
from those on donor cells as a consequence of polymorphisms in genes outside of the 
MHC, and those peptides can be recognized as minor H antigens by donor T cells.[10] 
The activation of donor T cells to recipient minor H antigens is likely to occur after 
contact with specialized APCs and/or other cell types particularly in the gastrointestinal 
tract and leads to proliferation of T cells and their differentiation to effector cells that 
produce cytokines including IFN-γ and TNFα, and mediate cytotoxicity against recipient 
tissues [10]. The tissues that are most frequently damaged during acute GVHD include 
skin, gastrointestinal tract, and liver, although other organs can be involved, particularly 
when acute GVHD evolves into chronic GVHD.  Chronic GVHD has a characteristic 
clinical presentation which often resembles autoimmune vascular diseases and is distinct 
from acute GVHD. Chronic GVHD can be highly debilitating and prolonged and has a 
20-50% mortality rate due to immune dysregulation and opportunistic infections. [15] 
 
 
The administration of immunosuppressive drugs that interfere with T cell activation or 
proliferation such as methotrexate, cyclosporine, FK506, prednisone, and mycophenolate 
mofetil, alone or in various combinations, are used at most transplant centers to prevent 
GVHD [16]. The development in the early 1980s of the combination of a short course of 
methotrexate with the calcineurin inhibitor cyclosporine represented a significant 
advance in the prophylaxis of GVHD, but major improvements in pharmacologic 
immunosuppression have not been achieved since that time [16,17]. At the FHCRC, a 
short course of methotrexate combined with either cyclosporine or tacrolimus continues 
to be a frequently used immunosuppressive regimen for GVHD prophylaxis after 
allogeneic HCT.  With this regimen of GVHD prophylaxis, following myeloablative 
allogeneic bone marrow transplant from unrelated donors in children at the FHCRC and 
other centers the incidence of grade II – IV acute GVHD is 40-90% and the incidence of 
extensive chronic graft versus host disease is 20-60%.[2-7] Patients who develop GVHD 
typically  require treatment with additional immunosuppressive drugs, which increases 
their risk of post transplant infections and may diminish the graft versus leukemia (GVL) 
effect [18]. Additionally, many patients with acute leukemia will relapse after HCT and 
those with GVHD are less likely to be eligible for, or benefit from, T cell immunotherapy 
designed to augment the GVL effect.   
 
3B. 
T Cell Depletion to Prevent GVHD 
The recognition of recipient minor H antigens by donor T cells is an essential 
requirement for the induction of GVHD after HCT from HLA-identical donors. Thus, 


Protocol 2660.00 
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many centers have used partial or complete depletion of T cells from the donor bone 
marrow or PBSC graft as an alternative approach to pharmacologic immunosuppression 
for preventing GVHD [11,12,19-23]. A variety of methods have been used to remove T 
cells from bone marrow or PBSC grafts including soybean lectin agglutination, 
monoclonal antibodies, and positive selection of CD34+ hematopoietic progenitors 
[11,12,19-22,24]. Depending on the approach, the extent of T cell depletion can vary 
from 2-5 log10.  More complete T cell depletion is generally associated with less GVHD, 
although an absolute minimum threshold dose of donor T cells needed for development 
of GVHD has been difficult to define in humans.  
 
T cell depletion is a highly effective strategy for preventing GVHD in both murine 
models and in humans [11,12,19-23] but recipients of T cell-depleted grafts have poor 
reconstitution of T cell immunity to pathogens, and some studies found T-cell depletion 
to be associated with an increased risk of graft rejection and relapse [11,12,19,25]. The 
Bone Marrow Transplant Clinical Trials Network recently published a multicenter trial of 
complete T cell depletion for AML patients undergoing HCT from HLA-identical sibling 
donors.[22,26]The BMT-CTN study 0303 used TBI (13.75 Gy), Cyclophosphamide, and 
ATG as a conditioning regimen, and the Miltenyi CliniMACS device to select 
CD34+cells from G-CSF mobilized PBSC using anti-CD34-coated immunomagnetic 
beads. CD34 selection with the Miltenyi CliniMACS device provided a 4. 9 log10 
removal of total T cells (3.2-5.9) including both the memory and naïve subsets. All 
patients engrafted with secondary graft failure occurring in only 1 of 44 patients. Low 
rates of acute GVHD (grade II-IV 22.7%, III-IV 4.5%) and extensive chronic GVHD 
(6.8%) were observed. The DFS and OS at three years after CD34+ transplant were 53% 
and 56% respectively for the whole group, 58% and 60% for patients transplanted in 
CR1, and 29 and 38% for patients transplanted in CR2. [22,26]The OS and DFS figures 
are broadly comparable to those seen after T-replete transplant, at least for patients in 
CR1. [27-31] However, as expected the CD34+ selected transplant was associated with a 
high risk of opportunistic viral infections (57% of patients). In particular high-level EBV 
reactivation requiring treatment was observed in 18% of patients and 1 of 44 patients died 
of EBV-PTLD. [31] 
 
Two single center studies performed for MRD and URD recipients with hematologic 
malignancies at Memorial Sloan Kettering Cancer Center employed a similar 
conditioning regimen TBI (13.75 Gy), Thiotepa (10mg/kg over two days), Fludarabine 
(125mg/m2 over five days) and equine ATG (60mg/kg) and CD34+ selection of stem 
cells from G-PBSC or BM from matched (≥ 7/10) unrelated donors with the Isolex 
Device followed by sRBC-rosette depletion of T cells, achieving a 5 log depletion of T 
cells.[20,21] Amongst MUD recipients the rates of aGVHD II-IV and extensive chronic 
GVHD were reported to be 5.8% and 11% respectively. DFS and OS at 4 years were 57 
and 59% respectively, whilst the relapse rate was surprisingly low at 6% at 4 years. 
Opportunistic infections were also frequent in this trial, with several clinically significant 
adenoviral infections (14.7%) including one death, and three (9%) EBV reactivations- 
including one death from EBV PTLD. 
 
In children there is less published experience with CD34+ TCD transplants from 
unrelated donors. The largest study was published by a European group in 2003.[32] 
Purified URD CD34+ stem cells (median 8 x 106/kg, obtained using Miltenyi MACs 
devices) were infused following a range of conditioning regimens each including ATG. 


Protocol 2660.00 
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All patients with an HLA matched URD achieved sustained primary engraftment. Acute 
GVHD was observed in 10% of the patients, and chronic GVHD was <10%. Patients 
with ALL in CR1 (N=14) mostly received TBI containing conditioning regimens and the 
overall survival was 63% amongst this group. Patients with AML were treated without 
TBI (BuMelCy) and had a poor survival (14%). A retrospective analysis of the MSKCC 
pediatric experience with CD34+ TCD using the Isolex device followed by sRBC-rosette 
depletion of T cells was published more recently. Eleven patients with AML or advanced 
MDS received a standard conditioning of Busulphan, Melphalan, Fludarabine and ATG. 
All engrafted and achieved full donor chimerism. The overall survival of the Bu-Mel-Flu 
subgroup is 54.5% with patients dying of relapse (27%), GVHD (9%) or infection 
(9%).[33] 
   
3C. 
Naive T cells (TN) are primarily responsible for GVHD in rodent models  
Transplant approaches that selectively deplete T cells that recognize minor H antigens 
and retain T cells specific for pathogens in the stem cell graft could reduce GVHD and 
improve reconstitution of T cell immunity. Studies in murine models of allogeneic stem 
cell transplantation have provided insights into how such selective manipulation of the T 
cell content of hematopoietic cell grafts might be achieved. Mature CD3+ CD8+ and 
CD3+ CD4+ T cells can be classified into naïve (TN) and memory (TM) subsets that differ 
in cell surface phenotype, prior exposure to cognate antigen, and functional activity 
[34,35]. In mice, the TN subset is CD44- CD62L+ and the TM subset is CD44+ and can be 
further subdivided into a CD62L+ central memory (TCM) population, and a CD62L- 
effector memory (TEM) population [35]. Studies in murine bone marrow transplant 
models have evaluated the potential for T cells derived from TN and TM subsets to cause 
GVHD across both minor and major histocompatibility differences. The first study to 
examine this question employed a multiple minor H antigen mismatched CD4+ dependent 
GVHD model [B10.D2 (H-2d)  BALB/c (H-2d)]. In this model, transplantation of 
irradiated BALB/c mice with T-cell depleted bone marrow combined with unfractionated 
splenocytes or with purified CD4+ CD44- CD62L+ TN from B10.D2 donors caused severe 
GVHD. However, transplantation of T-cell depleted bone marrow with purified CD4+ 
CD44+ CD62L- TM did not cause GVHD and provided for the transfer of T cell immunity 
to a model antigen [36].  
 
The efficacy of removing TN from the stem cell graft for preventing GVHD has been 
confirmed in other murine strain combinations including CD8 dependent minor H 
antigen-mismatched and MHC-mismatched models, and in rats [36-44][Table 1]. It 
should be noted that the intent in these studies was to deplete TN, but the cell selection 
procedure that was utilized in some studies would also remove the TCM subset of TM from 
the graft. In other studies, the selection procedure targeting CD44+ did not allow the 
effects of TCM or TEM to be delineated. In subsequent experiments by the Shlomchik 
group, purified TN or TCM obtained by cell sorting were transplanted with T cell depleted 
bone marrow in a CD8 dependent minor H antigen-mismatched murine model of GVHD 
in the absence of immunosuppression. Mice that received TN developed severe GVHD, 
whereas mice that received TCM developed only mild GVHD [37]. Similar results were 
obtained in an MHC-mismatched model.  These results demonstrate that in murine 
allogeneic minor H antigen and MHC mismatched bone marrow transplantation, the TN 
subset of T cells is primarily responsible for GVHD and bone marrow grafts containing 
only TM exhibit limited capacity to cause GVHD. 


Protocol 2660.00 
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Table 1. Rodent studies of T cell depleted BMT with selective addition of T cell subsets 
 
 
 
 
 
 
 
 
 
 
 
 
 
Transplant Model 
Study 
T Cell Subsets 
Experimental Design 
Outcome 
Multiple Minor H 
Antigen 
Mismatched, CD4 
dependent 
Anderson B et al. 
J Clin Invest, 
112: 101, 
2003[36]  
CD4+ CD44+ CD62L- (TM) 
vs 
CD4+ CD44- CD62L+ (TN) 
B10.D2 (H-2d) 
BALB/c (H-2d) 
No GVHD in mice 
receiving TM,  
severe GVHD in 
mice receiving TN 
Multiple Minor H 
Antigen 
Mismatched, CD8 
dependent 
Zhang Y. et al. 
Blood 103; 3970 
– 3978, 2004[37]  
CD8+ CD44+(TM) 
vs 
CD8+ CD44- (TN) 
CH3.SW (H-2b)  
B6 (H-2b) 
Minimal GVHD in 
mice receiving TM, 
severe GVHD in 
mice receiving TN 
Multiple Minor H 
Antigen 
Mismatched  
CD8 dependent 
Zheng H et al. 
J. Immunology 
182; 5938-5948 
2009 [42] 
CD8+CD44+CD62L+(TCM) 
vs 
CD8+ CD44- CD62L+(TN) 
CH3.SW (H-2b)  
B6 (H-2b) 
Mild GVHD in mice 
receiving TCM, severe 
GVHD in mice 
receiving TN  
MHC Mismatched 
Chen B et al. 
Blood 103: 1534-
1541, 2004[38]  
CD4+ + CD8+ CD62L- (TEM) 
vs 
CD4+ + CD8+ CD62L+ (TN & TCM) 
C57BL/6 (H-2b)  
 BALB/c (H-2d) or 
C3H/HeJ (H-2k) 
No GVHD in mice 
that received CD62L- 
T cells, lethal GVHD 
in recipients of 
CD62L+ T cells 
MHC Mismatched 
Chen B et al 
Blood 109:  
3115-3123 
2007[39] 
CD4+&CD8+CD62L+ CD44-(TN) 
vs. 
CD4+&CD8+ TM (all other), 
CD4+TM (all other), 
CD4+&CD8+ CD62L+ CD44-TCM 
 
C57BL/6 (H-2 b)  
BALB/c (H-2d) 
And  
C57BL/6 (H-2 b)  
to BALB/c (H-2d) 
 
TN Severe GVHD 
TM total No GVHD 
CD4+TMNo GVHD 
TCM No GVHD 
 
MHC Mismatched 
Dutt S et al 
J. Immunology 
179: 6547-6554 
2007 [40] 
CD4+CD62L+ CD44-(TN) 
CD4+CD62L-CD44+(TEM)primed 
CD4+CD62L-CD44+(TEM)unprimed 
 
C57BL/6 (H-2 b)  
BALB/c (H-2d 
TN Severe GVHD 
TEMP Severe GVHD 
TEMUP No/min 
GVHD  
 
MHC Mismatched 
Zheng et al,  
Blood, 111:  
2476-2484.2008 
 [41] 
CD4+CD44+CD62L-(TEM) 
vs 
CD4+CD44-CD62L-(TN) 
B6bm12B6 
No GVHD in TEM 
recipients; GVHD in 
TN recipients. 
MHC Mismatched 
Zheng H et al. 
J. Immunology 
182; 5938-5948 
2009 [42] 
CD8+CD44+CD62L+(TCM) 
vs 
CD8+ CD44- CD62L+(TN) 
C57BL/6 (H-2 b)  
BALB/c (H-2d) 
TN Severe GVHD 
+GVL 
TCM Milder GVHD 
+GVL  
MHC Mismatched 
Dutt S et al, 
Blood 117, 3230-
3239 2011 
[43] 
 
CD8+CD44- (TN) 
vs 
CD8+CD44+(TM)  
C57BL/6 (H-2 b)  
to BALB/c (H-2d) 
 
TN Severe GVHD 
+GVL 
TM total minimum 
GVHD+GVL  
MHC Mismatched 
(Rat Model) 
Xystrakis E et al. 
Eur J Immunol, 
34: 408, 2004 
[44] 
CD4+ CD45RChigh (TN) vs 
CD4+ CD45RClow (TM) 
Parental F1 
(LEWxBN) 
No GVHD in rats 
receiving TM, lethal 
GVHD in rats 
receiving TN  


Protocol 2660.00 
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3D. 
Human T cells specific for minor H antigens are predominantly in the TN subset. 
Inferential data suggests that the results obtained in murine models using selective 
depletion of TN to prevent GVHD might also apply to humans. The TN subset in humans, 
which expresses both CD45RA and CD62L, has been shown to contain the greatest T cell 
receptor (TCR) diversity [45,46]. The TM subset, which contains CD45RO+ CD62L+ TCM 
and CD62L- TEM populations, and a subset of CD62L- cells that re-express CD45RA 
(TEMRA), is comprised of cells that have clonally expanded in response to antigen 
stimulation and contains relatively less TCR diversity compared to TN.  A major fraction 
of the TM subset consists of T cells specific for latent herpes viruses including CMV, 
EBV, herpes simplex virus (HSV) and varicella zoster virus (VZV). Cross reactivity of 
virus-specific TM for major alloantigens has been observed infrequently and in humans 
does not appear to cause GHVD [47,48] , and cross reactivity with minor H antigens has 
not been reported and is likely to be exceedingly rare.  To evaluate the presence of minor 
H antigen-reactive T cells in TN and TM subsets, we purified CD8+ TN (CD45RAbright, 
CD62L+, CD45RO-) and TM (CD45RO+, CD62L+/-, CD45RA-) from leukapheresis 
products obtained from HLA-identical sibling pairs and determined the frequency of 
alloreactive T cells in each subset by limiting dilution analysis. Individuals may be 
exposed to minor H antigens by pregnancy or blood transfusion and develop minor H 
antigen-specific TM responses, therefore donors without a prior history of pregnancy or 
blood transfusion were selected for these experiments. In multiple assays of different 
HLA-matched pairs, the frequency of minor H antigen-reactive T-cells was significantly 
higher in the TN subset than in the TM subset.  Indeed, only very rare cells (~1/2,000,000-
1/6,000,000) in the TM subset exhibited weak recognition of recipient cells in these 
assays, and this weak alloreactivity was not reproducible with repeated testing. Thus, in 
individuals without a history of pregnancy or blood transfusion, minor H antigen-specific 
CD8+ T cells are predominantly in the TN subset [49]. We attempted to evaluate the 
frequency of T cells specific for minor H antigens in CD4+ TN and TM subsets using 
lymphoproliferation assays, but these assays were confounded by high background 
reactivity to autologous cells in both subsets.  
 
3E.  
Depletion of TN from human PBSC grafts as a strategy to reduce GVHD 
The in vivo data in animal models and in vitro analysis of alloreactivity of CD8+ TM and 
TN subsets in HLA-identical siblings suggested that selective depletion of TN from human 
PBSC grafts is a rational strategy to investigate for reducing GVHD and for improving 
the tempo of immune reconstitution after allogeneic HCT. In preclinical studies, we 
examined methods for selectively depleting TN from G-CSF mobilized PBSC products. 
We initially considered using anti-CD62L mAb to deplete TN, based on the data from 
murine models. However, CD62L is subject to proteolytic cleavage by ADAM-17 and 
other proteases released during G-CSF mobilization [50], and we found that cell surface 
CD62L declined on T cells in G-CSF mobilized products. CD45RA is expressed stably 
on all CD8+ and CD4+ TN in G-CSF mobilized PBSC but a minor subset of CD34+ cells 
also express CD45RA. Thus, to remove CD45RA+ T cells without interfering with 
CD34+ progenitors, we used sequential positive selection of CD34+ progenitor cells using 
Miltenyi CD34 immunomagnetic selection, followed by depletion of CD45RA+ cells 
from the CD34-negative fraction. Our goal was to develop a cell selection procedure that 
would result in an engineered stem cell graft that contains >2.0 x 106 CD34+ cells/kg 
recipient body weight, <7.5 x 104 TN/kg and 1-10 x 106 TM/kg. The dose of 1-10 x 106 
CD3+ cells/kg was selected for two reasons. First, this number is nearly equivalent to the 
T cell content of an unmanipulated bone marrow graft and is 10 - 100 fold greater than 


Protocol 2660.00 
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the threshold dose of unselected T cells that causes GVHD after HLA-identical sibling 
HCT [51].  It would therefore allow us to test the hypothesis that CD45RA- T cells can 
safely be transferred with less GVHD.  Second, it would provide a sufficient number of 
memory T cells such that we would reasonably anticipate improved immune 
reconstitution as compared to recipients of only a TCD allograft. 
 
A GMP-grade murine αCD45RA monoclonal antibody that is directly conjugated to 
Miltenyi iron dextran beads was produced by Miltenyi Biotec under contract from the 
NIH RAID program.  Using the new αCD45RA mAb-conjugated bead and the existing 
αCD34-conjugated bead we have now successfully completed more than 30 clinical scale 
cell selection procedures, including preclinical validation runs and procedures for twenty-
four patients who have been treated on our first (adult) Phase II Study of Selective 
Depletion of CD45RA+ T Cells from Allogeneic Peripheral Blood Stem Cell Grafts for 
the Prevention of GVHD using HLA-matched related donors (see section 3H). 
 
3F. 
T cell responses to pathogens are retained after depletion of CD45RA+cells from G-
PBSC 
We have evaluated CD45RA depleted hematopoietic cell products for retention of 
functional memory T cells specific for common pathogens including CMV, EBV, 
influenza, Candida albicans, and VZV. For persistent viruses such as CMV and EBV, a 
major fraction of the CD8+ TM response may reside in the CD45RA+ (TEMRA) subset of 
TM, and the use of αCD45RA for depletion of TN could compromise the transfer of TM 
responses to these pathogens. Therefore, we compared the frequency of CD8+ T-cells 
specific for known epitopes of CMV, EBV and adenovirus in PBSC before and after 
depletion of CD45RA+ cells using an interferon gamma Elispot assay after a single in 
vitro stimulation. Interferon-producing virus-specific CD8+ T-cells were present in both 
the CD45RA-depleted fraction and unselected PBSC at comparable frequencies. CD4+ 
TM cells are uniformly CD45RO+ CD45RA- and should be retained in G-PBSC depleted 
of CD45RA+ T-cells. We analyzed lymphoproliferative responses to CMV, HSV, VZV, 
Adenovirus, Influenza and Parainfluenza and Candida antigens before and after depletion 
of CD45RA+ T-cells from G-PBSC products. To avoid measuring CD8+ T-cells that 
might respond to soluble proteins that are taken up by APC in the culture and enter the 
class I pathway, CD4+ T-cells from the CD45RA depleted product were enriched by 
negative selection prior to plating in the assay. CD4+ TM responses to all the antigen 
preparations were detected in the CD45RA depleted product. These results demonstrate 
that CD8+ and CD4+ memory T cells to common opportunistic pathogens are retained 
after depletion of CD45RA+ cells.  
 
3G.   Preliminary analysis of the first phase II study to evaluate selective depletion of naïve T 
cells from PBSC grafts for preventing acute GVHD in (adult) patients with acute 
leukemia or MDS undergoing allogeneic HLA-identical HCT from a related donor. 
We are conducting a phase II study to evaluate selective depletion of naïve T cells from 
PBSC grafts for preventing acute GVHD in (adult) patients with acute leukemia or MDS 
undergoing allogeneic HLA-identical HCT from a related donor. We employ a 
myeloablative preparative regimen consisting of fludarabine, thiotepa, and TBI that 
provides a high rate of engraftment. The product that is administered to each patient 
consists of an infusion of purified CD34+ cells, obtained by Miltenyi CliniMACS 
selection of G-CSF mobilized cells collected from the donor by apheresis, and an 
infusion of CD45RA- cells obtained by depletion of CD45RA+ cells from the flow-


Protocol 2660.00 
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through remaining after the CD34+ cell selection.  
As discussed above, our goal is to administer a CD34+ cell dose of >2.0 x106/kg of 
recipient body weight and a total T cell dose of 1-10 x 106 CD3+ cells/kg, of which 
<7.5x104/kg are CD45RA+ RO- naïve T cells. The cell selection goals were readily 
achieved in each of the first twenty-four patients treated on the trial. All patients received 
>5.0 x106/CD34+cells/kg, with the median CD34+ dose being 8.1 x106 CD34+cells/kg. 
The median T cell dose administered was 10.00 x 106 CD3+cells/kg (1.59-10.04) with 
most (22/24) patients receiving 9.9-10.04 x 106 CD3+cells/kg. All patients received less 
than 7.5 x104 CD45RA+ RO- naïve T cells per kg with a median of 0.37 x104 CD45RA+ 
RO- naïve T cells per kg (range 0.09-7.46, all but one patient received ≤1 x104 naïve T 
cells/kg). Approximately two thirds of the residual CD45RA+ RO- naïve T cells were 
within the CD34+ cell fraction (median 0.21 x104) and one third were from the CD45RA-
depleted PBSC product (median 0.12 x104). In summary, cell selection process to retain 
CD34+ stem cells and memory T cells whilst depleting naïve T cells is effective and 
reproducible.  
To date we have treated twenty-four patients with acute leukemia or advanced MDS 
(ALL N=16, AML N=6, MDS N=2) with an age range of 19-55 years. Fourteen patients 
were in CR1 without minimal residual disease (MRD) prior to HCT, four patients had 
MRD but were in first morphological remission, and six patients were in CR2 or CR3. 
The median time of follow-up is 545 days (40-1134) (February 2013). The preliminary 
analysis of engraftment and survival is encouraging (Table 2).  
 
Table 2: Engraftment and survival amongst first 24 patients treated with naïve T 
cell-depleted PBSC (FHCRC Protocol 2222) 
Outcome 
 
Neutrophil engraftment (>500/μl) 
24/24 patients 
Time to neutrophil engraftment 
Median 12 days (range 9-18days) 
Platelet engraftment (>20,000/μl) 
24/24 patients 
Time to platelet engraftment 
Median 12 days (range 9-111 days) 
Overall survival  
83% 
Disease free survival  
75% 
Relapse 
3/24 patients 
Death from relapse 
1/24 patients 
Non-relapse mortality all patients 
3/24 patients 
Non-relapse mortality patients <40years 
0/11 
Non-relapse mortality all patients ≥40 years 
3/13 
 
a. Mild acute GVHD grade II-III, universally steroid-responsive, has been observed in 
17/22 (77%) evaluable patients. Fifteen patients developed grade II GVHD and 
responded rapidly to corticosteroids. Two other patients were considered to have 
grade III GVHD on the basis of large volume diarrhea but also responded rapidly to 
prednisone with no subsequent recurrence. In each of the 17 cases GVHD within the 
gastrointestinal tract was observed, predominantly involving the upper GI without 
lower GI involvement (12/17). Skin involvement was also seen in 7/17 patients while 
liver GVHD has not been observed to date. A biopsy of the intestinal tract was 
obtained in all patients diagnosed with gastrointestinal GHVD. The histopathologic 
changes were considered minimal or mild. The high rate of mild upper 
gastrointestinal GVHD is similar to the historical experience for adult patients treated 
with myeloablative TBI containing conditioning with T replete BMT or PBSCT at 
FHCRC where there is a high diagnostic sensitivity and awareness of the potential for 


Protocol 2660.00 
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mild gastrointestinal GVHD without skin involvement [52].   
b. Chronic GVHD: Amongst the 22 patients have completed >100 days of follow-up 
three have been diagnosed with chronic GVHD fulfilling NIH criteria, including one 
mild classic chronic (steroid responsive skin rash and dry eyes) and two late acute 
gastrointestinal with subclinical oral involvement. The frequency of chronic GVHD 
appears to be lower than the historical experience of 45-50% for adult patients 
receiving unmanipulated PBSC transplants from HLA-identical sibling donors.[30]  
c. Discontinuation of immune suppression. The median time to discontinuation of 
prednisone for patients treated on the study was 100 days and the median time to 
discontinuation of all immune suppression was 298 days. Of the 11 patients aged ≤40 
years at the time of HCT the median time to discontinuation of prednisone for 
patients treated on the study was 67 days and the median time to discontinuation of 
all immune suppression was 211 days. All young patients (<40 years) who were alive 
without relapse at one year post HCT completed all immune suppression at or prior to 
that time point. The time to completion of all immunosuppression amongst patients 
treated on this naïve T cell depletion study appears to be considerably faster than in 
concurrent controls who received standard myeloablative PBSC transplants at 
FHCRC, amongst whom 50% of the living patients are still on immune suppression at 
two years after transplant.  
d. Immune reconstitution: The median CD3+ T cell/mm3 of peripheral blood observed in 
recipients of naïve T cell depleted transplant is above 300 at 28 days after transplant 
and remains at this level for the first three months and increases to 700 at 1 year post 
transplant. CD4+ T cells median values are above 100 CD4+ T cells/mm3 of blood for 
the first 3 months and 250 at one year. The numbers of CD4+CD3+ and CD4+CD8+ T 
cells in peripheral blood after naïve T cell depleted HCT exceed those seen after TCD 
HCT over the first 3 months after HCT after which time the levels are similar.[22] 
EBV reactivation has been monitored weekly by quantitative PCR and no EBV 
reactivation has been detected in patients receiving naïve T cell depleted PBSC. In 
comparison a much higher 18% rate of EBV reactivation was seen after TCD 
HCT.[22] In summary, in the first three months after HCT overall immune 
reconstitution appears to be superior in recipients of naïve T cell depleted grafts 
compared to total TCD transplants, with a corresponding reduced level of viral 
reactivation. 
 
3H.  New pediatric clinical trial: A Phase II Study of Selective Depletion of CD45RA+ T 
Cells from Allogeneic Peripheral Blood Stem Cell Grafts for the Prevention of 
GVHD in Children  
The strategy of depleting naïve T cells from stem cell grafts is effective for 
preventing GVHD in murine models. In adult humans naïve T cell depletion of PBSC 
appears to reduce the frequency, severity and duration of chronic GVHD relative to 
unmanipulated PBSC, and to improve immune reconstitution relative to TCD HCT. 
The frequency of acute GVHD is not reduced using naïve T cell depletion and 
tacrolimus monotherapy compared to T cell replete HCT with conventional 
calcineurin inhibitor and methotrexate prophylaxis, but the acute GVHD observed is 
mild and steroid responsive.  
Children are not eligible for the open ongoing trial of naïve T cell depletion as they 
mostly have pediatric donors and bone marrow is preferred to PBSC as a stem cell 
source from younger pediatric donors on research studies due to safety concerns 
primarily related to the insertion of central venous catheters required for the 


Protocol 2660.00 
Page 13 of 57 
collection of apheresis. Selective depletion of naïve T cells from bone marrow grafts 
would not provide a stem cell product with an optimal number of CD34+ cells in 
many patients, as the available number of CD34+ cells in the starting bone marrow 
product is substantially lower than PBSC (median of 2.75 x 106/kg CD34+ cells in 
bone marrow in adults, range1.94-4.53 x 106/kg[9]; median of 6.2 x 106/kg CD34+ 
cells in bone marrow in children Heimfeld personal communication ) and there is typically a 20-
30% loss of CD34+ cells during CD34+ enrichment using current CliniMACs 
technology.  
We plan to study the naïve T cell depletion approach in 20 pediatric recipients of 
PBSC from HLA-identical unrelated donors and to employ tacrolimus and 
methotrexate as pharmacological prophylaxis of GVHD. The primary objective of the 
trial will be to estimate the time to discontinuation of systemic immunosuppression in 
pediatric recipients of CD45RA+ T cell-depleted PBSCT. Unpublished data from our 
center shows that amongst 55 children with a history of leukemia who received a 
myeloablative bone marrow (N=34 ) or PBSC (N=21) transplant from an unrelated 
donor (HLA-A, B, C, DRB1, and DQB1 molecularly matched) following 
conditioning with TBI and cyclophosphamide between July 2002 and December 2010 
the median time to discontinuation of all immunosuppression was 1076 days, with a 
median time to discontinuation of 608 days for bone marrow recipients, and median 
time to discontinuation not reached for recipients of PBSC. The goal is to increase the 
proportion of patients who discontinue all systemic immunosuppression by 608 days 
from the historical value of 50% to 80% in the current trial by depletion of naïve T 
cells from PBSC without observing an increased frequency of graft failure. A 
secondary objective will be to estimate the frequency of acute GVHD in pediatric 
recipients of TN cell-depleted PBSC who receive tacrolimus and methotrexate 
pharmacological prophylaxis.  
 
4. 
Objectives 
4A.  
Primary objectives 
1. Estimate the time to discontinuation of systemic immunosuppression in pediatric 
recipients of CD45RA+ T cell-depleted PBSCT. 
2. Estimate the probability of graft failure in pediatric recipients of CD45RA+ T-cell-
depleted PBSCT. 
 
4B.  
Secondary objectives 
1. Estimate and compare to an appropriate historical cohort the probability of chronic 
GVHD (NIH criteria) requiring treatment with systemic pharmacological 
immunosuppression in pediatric patients who receive CD45RA+ T cell depleted 
PBSC   
2. Estimate the probability of acute GVHD grade II-IV 
3. Estimate the probability of steroid refractory acute GVHD 
4. Evaluate immune reconstitution  
5. Estimate the probability of transplant-related mortality by day 100  
6. Estimate the probability of relapse   
 
5. 
Patient Selection  
5A. 
Inclusions 
1. Patients who are considered appropriate candidates for allogeneic hematopoietic stem 
cell transplantation and have one of the following diagnoses: 


Protocol 2660.00 
Page 14 of 57 
a. Acute lymphocytic leukemia in first or subsequent remission 
b. Acute myeloid leukemia in first or subsequent remission  
c. Acute lymphocytic leukemia in relapse or primary refractory disease with a 
circulating blast count of no more than 10,000/mm3 
d. Acute myeloid leukemia in relapse or primary refractory disease with a 
circulating blast count of no more than 10,000/mm3  
e. Refractory anemia with excess blasts (RAEB-1 and RAEB-2)  
f. Chronic myelogenous leukemia with a history of accelerated phase or blast crisis 
g. Other acute leukemia (including but not limited to ‘biphenotypic’,  
‘undifferentiated’ or ‘ambiguous lineage’ acute leukemia) 
2. Patient age <22 years old  
3. Patient with a HLA-identical (HLA-A, B, C, and DRB1 molecularly matched) 
unrelated donor or related donor capable of donating PBSC. 
 
5B.  
Exclusions 
1. Patients with CNS involvement refractory to intrathecal chemotherapy and/or standard 
cranial-spinal radiation 
2. Patients on other experimental protocols for prevention of acute GVHD 
3. Patients who weigh ≥70kg must be discussed with the principal investigator prior to 
enrolling on the protocol. 
4. Patients who are HIV+  
5. Patients with uncontrolled infections for whom myeloablative HCT is considered 
contraindicated by the consulting infectious disease physician (Upper respiratory tract 
viral infection does not constitute an uncontrolled infection in this context) 
6. Patients with organ dysfunction 
a. Renal insufficiency (creatinine >1.5 mg/dl) 
b. Cardiac ejection fraction < 45% 
c. Patients who can perform pulmonary function tests will be excluded if they have a 
DLCO (corrected for hemoglobin) of <60% predicted. Patients who are unable to 
perform pulmonary function tests (for example, due to young age and/or 
developmental status) will be excluded if the O2 saturation is < 92% on room air. 
d. Liver function abnormality. Patients who have LFTs (including total bilirubin, 
AST and ALT) ≥ twice the upper limit of normal should be evaluated by a GI 
physician unless there is a clear precipitating factor (such as an azole, 
methotrexate, bactrim or another drug) If the GI physician considers that HCT on 
protocol 2660 is contraindicated for that patient the patient will be excluded from 
the protocol.  Patients with Gilbert’s syndrome and no other known liver function 
abnormality and patients with reversible drug-related transaminitis do not 
necessarily require GI consultation and may be included on the protocol.   
7. Patients with a life expectancy <3 months from co-existing disease other than the 
leukemia or RAEB 
8. Patients who are pregnant or breast-feeding  
9. Fertile patients of child bearing age unwilling to use contraception during and for 12 
months post transplant 
10. Patients with a significant other medical conditions that would make them unsuitable 
for transplant 
11. Patients with a known hypersensitivity to tacrolimus  
 
6. 
Donor Selection 


Protocol 2660.00 
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6A.   Inclusions 
1. HLA-matched unrelated donors (HLA-A, B, C, and DRB1 matched based on high-
resolution typing) capable and willing to donate PBSC. 
2. HLA-matched related donors ≥18 years and capable and willing to donate PBSC. 
 
6B.  
Exclusions  
1. Donors who are HIV-1, HIV-2, HTLV-1, HTLV-2 seropositive or with active 
hepatitis B or hepatitis C virus infection  
2. Donors who fail eligibility requirements for donation of cells or tissue per section 21 
CFR 1271 for donation of a HCT/P will be excluded unless use of the cells complies 
with 21 CFR 1271.65(b)(iii) (urgent medical need) or with 21 CFR 1271.65(b)(i) 
(allogeneic use in a first-degree or second-degree relative) 
3. Unrelated donors donating outside of the USA or Germany. 
 
7. 
Evaluation and Counseling of Patient  
Patients will be referred to the Fred Hutchinson Cancer Research Center (FHCRC) for 
consideration of a hematopoietic cell transplant. The protocol will be discussed 
thoroughly with patient, and other family members if appropriate, and all known risks to 
the patient will be described. The procedure and alternative forms of therapy will be 
presented as objectively as possible, and the risks and hazards of the procedure explained 
to the patient or, in the case of minors, to the patient's responsible legal guardian. Consent 
will be obtained using forms approved by the FHCRC Institutional Review Board.  A 
conference summary detailing what was covered will be dictated for the medical record. 
Related donors will be evaluated separately at the FHCRC. The protocol will be 
discussed thoroughly with donor and other family members if appropriate. The procedure 
and alternative forms of therapy will be presented as objectively as possible, and the 
known risks and hazards of the procedure explained to the donor and in the case of 
minors, to the donor's responsible legal guardian. Consent will be obtained using forms 
approved by the FHCRC Institutional Review Board.   
Unrelated donors will be managed by the NMDP (US donors) or DKMS (German 
donors). 
 
 
8. 
Protocol Registration  
Patients enrolled will be assigned to the protocol by the SCCA (Seattle Cancer Center 
Alliance) Clinical Coordinator.  Once full consent has been obtained from patients and if 
applicable from related donor the SCCA Consent Coordinator will register the patient 
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.  The FHCRC regulatory coordinator will then complete a patient registration form 
and fax it to the FHCRC Registration Office who will generate a unique patient identifier. 
 This patient will then be entered into the Protocol Patient Performance Program 
(PPPP) database by FHCRC study staff. Informed consent must be signed by the subject 
or their legal guardian prior to the performance of any study related procedures or 
assessments. 
 
9. 
Plan of Treatment  
9A. 
Conditioning Regimen  
The conditioning regimen will consist of fludarabine, thiotepa, and fractionated total 
body irradiation (TBI) and will be administered as outlined below and shown 


Protocol 2660.00 
Page 16 of 57 
schematically in Table 2. Note that for chemotherapy administered during conditioning 
and other drugs listed in this protocol, in keeping with institutional practice  it is 
acceptable to administer a dose within 10% of the protocol specified dose.  
TBI will be given as 165 cGy fractions twice per day x 4 days – total dose 1320cGy (days 
-10 to -7).  
1. After completing the TBI, patients will be treated with thiotepa 5 mg/kg/day (adjusted 
body weight –see appendix I) administered intravenously over approximately 4 hours 
on each of two consecutive days (days -6 and -5). If actual weight is less than ideal 
body weight, actual body weight will be used. The total dose is 10 mg/kg (adjusted 
body weight - see appendix I).  
2. Patients will receive fludarabine 25 mg/m2/day (m2 based on actual weight see 
appendix I) administered intravenously over approximately 30 minutes for 5 days 
beginning on the first day of thiotepa (days -6 to -2).  
3.  Day -1 will be a day of rest. 
4.  GCSF-mobilized CD34 enriched PBSC and CD45RA depleted cells will be infused 
on day 0. 
 
Table 3a. Treatment Plan –patients with unrelated donors 
Day of the week 
(example only) 
M 
T 
W 
Th 
F 
S 
S 
M 
T 
W 
Th 
F 
S 
S 
Day relative to transplant 
of CD34+ cells 
-10 
-9 
-8 
-7 
-6 
-5 
-4 
-3 
-2 
-1 
0 
0/1 
2 
3 
TBI  
165 cGy BID x 4 days  
X 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
Thiotepa  
5 mg/kg/day x 2 days 
 
 
 
 
X 
X 
 
 
 
 
 
 
 
 
Fludarabine  
25 mg/m2/day x5 days 
 
 
 
 
X 
X 
X 
X 
X 
 
 
 
 
 
Infusion of  
CD34+ stem cells and  
CD45RA+-depleted cells 
 
 
 
 
 
 
 
 
 
 
X 
+/-2 
+/-2 
+/-2 
Donor GCSF 
 
 
 
 
 
 
X 
X 
X 
X 
X 
+/-2 
+/-2 
 
 
Donor apheresis 
 
 
 
 
 
 
 
 
 
 
X 
 
+/-2 
+/-2 
 
Cell selection 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
1 A single apheresis will be collected on day -1, and transported as soon as possible on day-1. The selected product 
will be infused on day 0.2  On rare occasions a second apheresis could be required to make the cell selection goals 
and would be collected on day+1 or +2 and infused without cell selection on day+1, +2 or +3. (Also see section 9C, 
9D &9E and Appendix A). 
Table 3b. Treatment Plan –patients with related donors 
Day of the week 
(example only) 
M 
T 
W 
Th 
F 
S 
S 
M 
T 
W 
Th 
F 
S 
S 
Day relative to transplant 
of CD34+ cells 
-10 
-9 
-8 
-7 
-6 
-5 
-4 
-3 
-2 
-1 
0 
0/1 
2 
3 
TBI  
165 cGy BID x 4 days  
X 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
Thiotepa  
5 mg/kg/day x 2 days 
 
 
 
 
X 
X 
 
 
 
 
 
 
 
 
Fludarabine  
25 mg/m2/day x5 days 
 
 
 
 
X 
X 
X 
X 
X 
 
 
 
 
 
Infusion of  
CD34+ stem cells and  
 
 
 
 
 
 
 
 
 
 
X 
+/-2 
+/-2 
+/-2 


Protocol 2660.00 
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CD45RA+-depleted cells 
Donor GCSF 
 
 
 
 
 
 
X 
X 
X 
X 
X 
+/-2 
 
 
 
Donor apheresis 
 
 
 
 
 
 
 
 
 
 
X1 
+/-1 
+/-2 
+/-2 
 
Cell selection 
 
 
 
 
 
 
 
 
 
 
 
X 
+/- 
 
 
1 An apheresis collection will be performed on day -1, and may also be performed on day 0. The selected product 
will be infused on day 0. (Also see section 9C, 9D &9E and Appendix A). 2On rare occasions an additional 
collection of PBSC, or bone marrow could be required. 
 
9B.  
Central Nervous System Prophylaxis and Testicular Irradiation 
Patients will have a diagnostic lumbar puncture prior to the preparative regimen. 
Intrathecal chemotherapy will be given according to institutional standard practice if a. 
the CSF is positive for malignant cells; b. the patient has a prior history of CNS disease; 
c. the patient has a history of ALL; or d. the patient is otherwise deemed to be at 
significant risk of CNS disease. Intrathecal chemotherapy may be omitted if the attending 
physician considers that it is contraindicated for an individual patient based on prior 
toxicities or comorbidities. No dose of intrathecal therapy will be given within 72 hours 
of CD34+ stem cell infusion. Male patients with ALL will receive a 400 cGy testicular 
irradiation boost pre-transplant during the conditioning regimen.  For patients with a 
history of CNS leukemia a CNS irradiation boost may also performed if it is clinically 
indicated in the opinion of the pediatric BMT physician and radiation oncologist. 
 
9C. 
Collection of GCSF mobilized PBSC (see also appendix A) 
All donors will receive GCSF administered by subcutaneous injection for 5 consecutive 
days on days -5 thru day-1. Unrelated donors will receive a dose of 10 µg/kg/day of 
GCSF according to standard NMDP and DKMS practice for unrelated donors whilst 
related donors will receive a dose of 16 µg/kg/day of GCSF according to FHCRC 
standard practice. On infrequent occasions 1-2 additional doses of GCSF may be required 
for further PBSC collections if the cell infusion goals are not met initially. The schedule 
of G-CSF administration and PBSC collections will be determined when the schedule for 
the conditioning regimen and day 0 is established and should be confirmed with the 
personnel in the Cellular Therapy Laboratory. Day 0 should usually be fixed on a 
Monday-Friday. Typically, for URD transplant recipients the first apheresis would be 
performed on a Wednesday and transported to the SCCA Cellular Therapy Laboratory for 
cell selection on a Thursday. PBSC collection by apheresis: Unrelated donors will 
undergo vein to vein apheresis collections. Vein to vein apheresis is also preferred for 
related donors but insertion of a central venous catheter for apheresis collection is 
permitted for related donors if necessary. PBSCs will be collected on day -1 (unrelated 
donors and related donors) and sometimes also day 0 (some related donors).  
On rare occasions CD34+ cell target numbers may not be achieved according to the 
specified plans, and an additional PBSC collection would be requested and infused 
without cell selections.  
  
9D.  
PBSC graft engineering - CD34+ cell selection and depletion of CD45RA+ cells (see also 
appendix A) 
• Prior to cell selection, a sample of the PBSC products will be analyzed for the content 
of total nucleated cells, CD34+ cells, CD3+, CD3+ CD45RA+ RO- and total CD45RA+ 
cells and an aliquot of cells will be obtained for subsequent analysis of other cell 
subsets (see section 9D.3 below).   


Protocol 2660.00 
Page 18 of 57 
• Cell selections will only be performed when the PBSC product from day -1 or a 
pooled collection from day-1 and day 0 contains ≥5x106 CD34+ cells/kg of recipient 
body weight to ensure that the final CD34+ cell dose will be ≥2x106 CD34+ cells/kg 
of recipient body weight. In most cases we expect that cell selections will be 
performed on a PBSC product containing ≥10x106 CD34+ cells/kg of recipient body 
weight which should provide a final CD34+ cell dose of ≥5x106 CD34+ cells/kg of 
recipient body weight. 
•  If the available PBSC product contains <5x106 CD34+ cells/kg of recipient body 
weight the PBSCs will be infused without any cell selection and the patients will not 
be included in the analysis of study efficacy end points. We expect that ≤5% of 
patients enrolled on protocol 2660 may receive a G-PBSC product that does not 
undergo any cell selection. 
• For the patients in whom cell selection is performed we expect to achieve:  
 The CD34 goal  of a minimum of 2.0 x 106 CD34+ cells/kg of recipient body 
weight in all patients 
 A maximum of 5 x 104 CD45RA+RO-CD3+ T cells/kg recipient body weight and 
a range of 1-10 x 106/kg total CD3+ T cells in >95% of patients. 
• Cell processing would commence ideally within 24 hours of the end of an apheresis 
collection, but may be delayed for up to 48 hours if necessary. 
• Timing (for additional details please see Appendix A): 
• Unrelated donors  
 The apheresis will be performed on day -1, and transported as soon as 
possible on day -1.  
 Cell selections will be performed on day 0 using the day -1 apheresis  
 The cells will be infused as soon as possible after cell selection and release 
testing are completed. 
• Related donors 
 The first apheresis will be performed on day -1. A second apheresis may 
be performed on day 0, depending on the size of the recipient and the 
CD34+ content of the day -1 apheresis product (Appendix A). 
 Cell selections will be performed on day 0 using the day -1 apheresis, or a 
pooled product from the combined day-1 and day 0 apheresis depending 
on the size of the recipient and the CD34+ content of the day -1 apheresis 
product. 
 The cells will be infused as soon as possible after cell selection and release 
testing are completed. 
• On rare occasions CD34+ cell target numbers may not be achieved according to 
the specified plans, and an additional PBSC collection would be requested and 
infused without cell selections.  
 
The cell selection will be performed as outlined below and in the flow chart provided as 
Appendix A.  
 
1. CD34+ cell selection (Also see Appendix A) 
The apheresis products will be processed by CD34+ cell selection using the clinical-
grade Miltenyi anti CD34+ conjugated iron dextran microbeads and magnetic 
selection with the CliniMACs device using the institution’s Standard Operating 
Procedures. The goal is to obtain a target CD34 cell dose of >5.0x106/kg (minimum 
of >2.0x106/kg) of recipient body weight and this goal is expected to be achieved for 


Protocol 2660.00 
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all products that undergo cell selection. The CliniMACS system of CD34 selection 
results in a 4-5 log10 depletion of CD3+ cells and typically results in a median CD3+ 
cell dose of 2 x104/kg of which ≤ 30% are CD45RA+RO-. 
2.  CD45RA+ cell depletion to remove TN cells (See also Appendix A) 
The CD34-depleted flow through fraction(s) will be collected and processed to 
deplete CD45RA+ cells using clinical grade anti-CD45RA-conjugated iron dextran 
beads and magnetic selection with the CliniMACS device according to the 
institution’s standard operating procedures. The phenotype and content of T cells that 
remain in the product after removal of CD45RA+ cells will be determined by flow 
cytometry (see section 9D3, below). We will plan to administer a total CD3+ T cell 
dose of 10 x106/kg to the patient including both the CD34+ enriched fraction and the 
CD45RA depleted fraction. 10 x106/kg total T cells is highly likely to contain < 5 
x104/kg of CD3+CD45RA+RO- cells/kg on the basis of our experience to date. If 
necessary the total T cell dose may be reduced to maintain a maximum infusion of 5 
x104/kg of CD3+CD45RA+RO- cells/kg.  
3.  Analysis of the engineered cell products.  Samples will be taken from the apheresis 
products before and after the CD34 selection and depletion of CD45RA+ cells, and 
analyzed as follows (also see Appendix B “Product Testing”): 
a. Viability testing 
b. Sterility testing  
c. Total nucleated cell count 
d. Immunophenotyping by flow cytometry will be performed on the initial apheresis 
product(s) and cells that have completed the selection process:  
i. The following cell subsets will be enumerated to guide cell selection and to 
determine whether the goals for the composition of the  product are achieved:  
- 
CD34+ cells  
- 
CD3+ cells  
- 
CD3+ CD45RA+ RO- cells  
ii. Additional cell markers may be evaluated such as 
- 
CD3+ CD45RA+ CD45RO- CCR7+ (TN)  
- 
CD8+CD45RA+ CD45RO- CCR7+ (CD8 TN) and CD4+CD45RA+ 
CD45RO- CCR7+ (CD4 TN) 
- 
CD3+ CD45RO+ (TM), CD3+CD8+ CD45RO+ (CD8 TM) and CD3+ CD4+ 
CD45RO+ (CD4 TM) 
- 
CD3+ CD45RO+ CCR-7+ (TCM) and CD3+ CD45RO+ CCR-7- (TEM) 
- 
CD8+ CD45RO+ CCR-7+ (CD8 TCM) and CD8+ CD45RO+ CCR-7- (CD8 
TEM), and CD4+ CD45RO+ CCR-7+ (CD4 TCM) and CD4+ CD45RO+ 
CCR-7- (CD4 TEM) 
- 
CD3+/- CD56+, CD3+/- CD16+, CD14+, CD19+ and CD20+  
- 
CD4+ CD25+FoxP3+. 
e. Specific T cell responses to pathogen-derived antigens may be performed on 
aliquots of the CD45RA depleted cell product and PBSC prior to CD45RA 
depletion using functional assays which may include ELISPOT, intracellular 
cytokine staining and/or tetramer analysis. 
 
 
9E. 
PBSC Infusion  
The CD34+ selected product and CD45RA-depleted product will be infused through a 
central venous catheter on day 0 or + 1 (‘second day 0’) of the transplant (see Appendix 
D).  The CD34+ product will be infused first followed as soon as possible by the 


Protocol 2660.00 
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CD45RA-depleted product. In the very unlikely event that additional CD34+ cells are 
required (see section 9D.1 and Appendix A and D) unselected PBSC will be 
administered. 
 
9F. 
Post-transplant immunosuppression 
GVHD prophylaxis will consist of tacrolimus and methotrexate. Tacrolimus (FK506) will 
be started on day -1 and should be continued for 50 days followed by a standard taper if 
there is no GVHD (see section 9F.1b). Methotrexate should be administered on day +1, 
+3, +6, +11 (see section 9F.3). 
 
1. Tacrolimus administration  
a. Tacrolimus will be administered beginning on day -1 at a dose of 0.03 mg/kg/day 
by continuous IV infusion per institutional standard practice guidelines. 
Tacrolimus doses are based on actual body weight. If actual weight is more than 
ideal body weight, it is recommended to use adjusted body weight. Conversion to 
the oral formulation of tacrolimus (IV: PO ratio of 1:4) may be made when oral 
feeding is established. Oral tacrolimus is recommended to be given in two divided 
daily doses every 12 hours on an empty stomach. Children < 6 years old may 
require oral dosing every eight hours to maintain target serum trough levels. 
b. If there is no evidence of grade II-IV acute GVHD on or prior to day 50, 
tacrolimus should then be tapered at the rate of approximately 5% of the day 50 
dose each week for liquid, and 20% of the day 50 dose per month for capsules.  
c. If there is evidence of acute GVHD, then the standard recommendations for 
treatment of acute GVHD and tapering of immunosuppression should be followed 
according to institutional practice. Patients with GVHD may be considered for 
enrollment in investigational protocols for GVHD treatment after discussion with 
the principal investigator.   
d. If there is evidence of disease progression and no evidence of GVHD prior to day 
50, patients should taper tacrolimus and all other immunosuppressive agents 
within 2 weeks and be observed for the development of GVHD. The taper may be 
accelerated or tacrolimus may be discontinued, as clinically indicated.  
 
2. Monitoring of tacrolimus levels and dose adjustment  
a. It is recommended that tacrolimus levels are maintained in the range of 5-15 
ng/ml. It is recommended that whole blood trough levels are obtained on 
approximately day 2, then approximately once each week or more often if 
clinically indicated. Dose adjustments are recommended if the levels are outside 
the therapeutic range, or if there is evidence of toxicity that may be related to 
tacrolimus.  
b. When initiating therapy with voriconazole or posaconazole in patients receiving 
tacrolimus, it is recommended that the tacrolimus dose be reduced according to 
institutional practice, and followed with frequent monitoring of the tacrolimus 
blood levels.  
c. Weekly tacrolimus levels can be discontinued during a tacrolimus taper when the 
dose has been reduced by 25% if the patient has an adequate oral intake, volume 
status, renal function and the absence of toxicities that might be attributed to 
tacrolimus.  
d. Blood pressure, renal function tests (creatinine, BUN), electrolytes and 
magnesium should be monitored regularly as per institutional standard practice 


Protocol 2660.00 
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guidelines. 
 
 
3. Methotrexate administration 
In patients weighing >10kg methotrexate should be administered by IV push on days 
+1, +3, +6 and +11 at a dose of 15 mg/m2 (on day +1) and a dose of 10 mg/m2 (on day 
+3, +6, +11). Calculation of m2 should be according to institutional standard practice 
guidelines. Children weighing 10kg or less will have methotrexate dosed per kg rather 
than per /m2  and should receive 0.5mg/kg on day +1 and 0.33mg/kg on days +3,+6, 
and +11. The first dose of methotrexate should be given at approximately 24 hours, 
but no sooner than 24 hours after completion of the cell infusion. The day 11 
methotrexate dose may be omitted if the patient develops severe mucositis, a pleural 
effusion, ascites, or another relative contraindication to methotrexate administration. 
 
9.G.  Use of hematopoietic growth factors 
There will not be routine post-transplant use of growth factors in patients enrolled on this 
protocol. Growth factors may be recommended by the Attending Physician to manage 
slow engraftment or in the event of infectious complications as indicated in Section 12 E. 
 
10. 
Evaluation 
10A. Donor evaluation 
Pre-donation evaluation for unrelated US donors should be conducted as per NMDP 
(National Marrow Donor Program) standard procedure. Pre-donation evaluation for 
unrelated German donors should also be conducted according to the same FDA required 
screening and eligibility tests. Infectious disease markers (IDM) testing for both NMDP 
and DKMS donors is performed in CLIA (Clinical Laboratory Improvement 
Amendments) certified laboratories using FDA licensed, cleared and approved test kits. 
IDM testing for DKMS donors is sent to the central NMDP contracted laboratory 
(currently Labs Inc, Centennial, Colarado).    
Related donors evaluation: The following should be obtained for all related donors:  
1. Screening for high-risk behavior and HepBsAg, antiHepB core antibody, HBV NAT, 
antiHep C antibody, HCV NAT, HTLV-1 and HTLV-2 antibodies, a serologic test for 
syphilis, and HIV (1 & 2) antibodies and HIV NAT, all performed within 30 days of 
donation and West Nile Virus NAT testing and Trypanosoma antibody testing 
according to institutional standard practice. These tests are performed using FDA 
licensed, cleared, and approved test kits in a CLIA-certified laboratory. 
2. CMV and EBV serologies performed within 30 days of donation. 
3. Serum pregnancy qualitative within 2 weeks of the start of conditioning 
4. G-CSF and monitoring blood draws (including CBC and Hepatic Function Panel with 
LDH) should be conducted per institutional standard practice guidelines.  
5. Research Tests: A 90 cc sample of related donor blood should be collected 
(preferably before the donor starts GCSF) in sodium heparin or ACD tubes for 
immunologic studies, specifically T cell responses to pathogens by functional assays 
which may include ELISPOT, lymphoproliferation assays, intracellular cytokine 
staining and/or tetramer analysis (SCCA patients send to the Bleakley lab and contact 
the protocol 2660 research technician at (206) 667-4804 or Marie Bleakley at (206) 
469-4487).  
 
10B. 
Patient pre-transplant evaluation 


Protocol 2660.00 
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Patient pre-transplant evaluation should be conducted as per standard institutional 
practice. Results of tests and/or procedures conducted as part of that evalution may be 
used for eligibility determination. It is recommended that the following information is 
obtained for all patients:  
1. History  
a. Possible antecedent causes for the development of leukemia or MDS including 
prior cytotoxic therapy. 
b. Hematologic, cytogenetic and flow cytometric findings at diagnosis and at the 
time of enrollment. 
c. Prior therapies and response to therapy 
 
2. Laboratory evaluation 
a. Bone marrow aspirate for morphology with standard cytogenetics and where 
appropriate, molecular cytogenetics, within 30 days of the start of conditioning. 
b. Bone marrow flow cytometry for determination of blast counts within 30 days of 
the start of conditioning. 
c. Lumbar puncture with CSF evaluation within 30 days of the start of conditioning. 
d. Panel reactive antibody testing and, if indicated (e.g. if PRA>10% activity or as 
determined by clinical immunogenetics laboratory specialist recommendations) 
leukocytotoxic and/or fluorescence activated cell sorter cross match between 
recipient and donor.  
e. ABO and Rh typing and two way red cell cross match between recipient and 
donor as per standard practice.  
f. HepBsAg, antiHepB core antibody, antiHep C antibody, HTLV-1, and HTLV-2 
antibodies, a serologic test for syphilis, and HIV (1 & 2) and HIV p24 antigen or 
HIV PCR quantitation within 30 days of the start of conditioning. 
g. CMV, EBV, HSV, VZV serologies within 30 days of the start of conditioning.  
h. CMV, EBV and adenovirus plasma PCR obtained and run within 2 weeks prior to 
starting conditioning.  
i. CBC within 2 weeks of the start of conditioning. 
j. LFTs, to include ALT, AST, and Bili T/D, within 2 weeks of the start of 
conditioning. 
k. Serum chemistry, to include Na, K, Cl, CO2, BUN, Cr, Ca, Mg, and Phos, within 
2 weeks of the start of conditioning 
l. Serum pregnancy qualitative testing within 2 weeks of the start of conditioning in 
females of childbearing age. 
3. Other evaluations: 
a. Echocardiogram within one month of the start of conditioning. 
b. Pulmonary Function test (patients > 6 years if considered developmentally 
capable of PFTs by attending physician and PFT lab technician) or documented 
O2 saturation on room air.  
 
10C. 
Patient post transplant evaluation guidelines 
See Standard Practice Manual for standard evaluation procedures during the first 100 
days post transplant, evaluation prior to departure and long-term follow-up. Post 
transplant evaluation prior to day 80 will in most cases be performed whilst the patient is 
under the care of the SCCA pediatric BMT team. After day 80 evaluations may be 
performed under the supervision of the SCCA pediatric BMT team and/or the patient’s 


Protocol 2660.00 
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local physician. Specific recommendations (which may be modified by the clinical team 
as deemed clinically appropriate) include: 
1. It is recommended that bone marrow aspiration is performed at baseline and on 
approximately days +28 and between approximately day +80 and + 100. Bone 
marrow aspiration may also be performed at approximately one year after transplant. 
Additional bone marrow aspirations and/or biopsies may be performed at the 
discretion of the treating physicians. 
2. It is recommended that donor and recipient chimerism of CD3+ and CD33+ subsets in 
the peripheral blood is evaluated at approximately day +28  +56, and +80 by STR 
polymorphism. Peripheral blood chimerism studies are also recommended to be 
performed at approximately 6, 9 and 12 months post transplant.  
3. It is recommended that quantitative immunoglobulins (to include IgG, IgA and IgM) 
are assessed at approximately days +28, +56 and +80 and if possible on 
approximately day +180, +270 and +360.  
4. CMV, EBV and Adenovirus monitoring is recommended for all patients. Specifically 
it is recommended that: 
a. CMV PCR should be performed at least once every week until day 180 according 
to institutional standard practice. Preemptive antiviral therapy should be instituted 
for a positive PCR according to institutional guidelines for recipients of T cell 
depleted allogeneic HCT (or an IRB approved research protocol for CMV 
management). CMV PCR monitoring is suggested to continue for at least one 
year beyond transplant for patients who have had CMV reactivation in the first 
100 days and/or have active GVHD requiring steroids or other agents. 
b. Surveillance for EBV reactivation should be performed using a quantitative EBV 
DNA PCR assay approximately weekly until day 180. Infectious disease (ID) 
consultation should be obtained for patients who develop EBV DNA levels of 
>1000 copies/ml plasma on any test.  
c. Surveillance for Adenovirus reactivation should be performed using a quantitative 
PCR assay (including adenovirus AF, and BCDE serogroups) approximately 
weekly until day 180. ID consultation should be obtained for patients who 
develop Adenoviral DNA levels of >300 copies/ml plasma on any test.  
d.  HHV6 monitoring by PCR may also be performed in the first 1-3 months after 
transplant at a frequency and for a duration determined by the patients attending 
physician as clinically indicated.  
5. Research Tests  (Also see appendix K for pediatric research blood draw guidelines)  
a. It is recommended that a 4ml peripheral blood sample is obtained (sent to 
SCCA/UWMC Hematopathology lab) for research  flow cytometry for lymphocyte 
subset evaluation on approximately days +28, +56 and + 80 and if possible on 
approximately day +180, +270 and +360. Specifically, it is recommended that the 
following subsets are enumerated: CD3+, CD3+CD4+, CD3+CD8+, CD4+TN 
(CD3+/CD4/CD45RA+/ CD45RO-/CD62L+), CD8+TN 
(CD3+/CD8+/CD45RA+/CD45RO-/CD62L+), NK (CD3-CD56+CD16+), B cells 
(CD19+), Tregs (CD4+/CD25+/CD127-).  
b. It is recommended that peripheral blood (≤1ml/kg, maximum 50ml for a single 
draw) is obtained on days +28, +56, +80-100, +180, +270 and +360 for research tests 
that may include 
i. T cell responses to EBV and possibly to other pathogens performed using 
functional assays which may include ELISPOT, lymphoproliferation assays, 
intracellular cytokine staining, and/or tetramer analysis.  


Protocol 2660.00 
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ii. T cell receptor excision circle (TREC) analysis 
iii.  TCR Vβ spectratyping or deep sequencing 
iv. Additional lymphocyte subset analysis which may include detailed analysis of 
T regulatory cell subsets. 
In smaller patients it may not be possible to perform all of the tests specified above 
due to the smaller recipient blood volume and the need to avoid contributing to 
anemia or hypovolemia. Blood samples for research testing will not exceed the 
volumes specified in Appendix K. Research samples may also be deferred or declined 
by the patient, their guardian or attending physician depending on the patient’s 
clinical status and preferences. The research tests performed will be prioritized by the 
PI depending on the number of available cells and the patient’s clinical course. 


Protocol 2660.00 
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A summary of recommended patient evaluations post-transplant is provided in Table 4. 
 
Table 4. Patient evaluations over the course of the study 
Approximate days post transplant 
Study Assessments/ 
Testing  
Baseline 
7 
14 
21 
28 
35 
42 
49 
56 
63 
70 
77 
Between 
80-100 
180 
270 
360 
 
History, physical 
exam, height1 and 
weight 
X 
 
 
 
X 
 
 
 
X 
 
 
 
X 
X 
X 
X 
Karnofsky/Lansky 
performance status 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
X 
Automated CBC with 
differential, platelet 
count 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serum chemistry 2   
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
CMV PCR  
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Weekly to day 180 
 
 
EBV PCR 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Weekly to day 180 
 
 
Adenoviral PCR 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Weekly to day 180 
 
 
Quantitative 
Immunoglobulins 
for IgG, IgA and IgM  
 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
X 
X 
X 
Quantitative blood 
lymphocyte subset 
research evaluation 
 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
X 
X 
X 
Peripheral blood T 
cell & myeloid 
chimerism (CD3+ & 
CD33+) 
X 
Baseline 
DNA 
 
 
 
X 
 
 
 
X 
 
 
 
X 
X 
X 
X 
Bone marrow 
aspirate/biopsy3 
X 
 
 
 
X 
 
 
 
 
 
 
 
X 
 
 
X 
Acute GVHD 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Chronic GVHD4 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
X 
X 
Skin biopsy5 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
Oral medicine5 
consult 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
Pulmonary function 
test including DLCO5 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
X 
Research Tests6 
 
 
 
 
X 
 
 
 
X 
 
 
 
X 
X 
X 
X 
Post transplant time points represent guidelines for performance of required evaluations. Due to numerous factors influencing 
scheduling (pt and provider availability, testing services limitations etc), variation in evaluation performance dates is anticipated and 
acceptable to the protocol (e.g., within +/- 7 days of time points < day 100; +/- 30 days for time points > day 100). There may also be 
variation in the content of the evaluation performed particularly beyond day 100 depending on the patient’s health status, size, 
geographical location, physician and/or PI recommendations and unanticipated variables. In particular the timing of blood draws may 
be spaced out or some tests not obtained to ensure that excessive blood is not drawn from small children at any given time, or over a 
given period. 
1. 
Height on baseline only 
2. 
Serum Chemistry should include the following: Na, K, Cl, CO2, BUN, Cr, Ca, Mg, Phos, ALT, AST, and BILIT/D 
3. 
Bone marrow aspiration; evaluation with a. histopathology b. flow cytometry c. cytogenetics and/or molecular testing where clinically 
relevant (FISH and PCR informative for disease status eg BCR-ABL for Ph+ ALL)  
4. 
Chronic GVHD evaluation should be performed between approximately day 80 and 100, and on day 180, 270, 360 where possible. Day 
180, 270, 360 evaluations should be performed at the SCCA if possible, or patients may have a ‘home town evaluation’ conducted by 
their regular medical provider with guidance by SCCA Pediatric long term follow-up (LTFU) as appropriate. Patients with symptoms or 
signs compatible with chronic GVHD between day 100 and day 360, based on report by the PMD and/or patient, should evaluated by the 
pediatric LTFU team at the SCCA or using telemedicine and/or medical photography in consultation with the patients regular medical 
provider as appropriate. 
5. 
Optional test: May or may not be performed depending on the age and developmental stage of the child and the recommendation of the 
pediatric attending physician. 
6. 
Research tests: In smaller patients it may not be possible to perform all of the tests specified in section 10C.6.due to the smaller recipient 
blood volume and the need to avoid contributing to anemia or hypovolemia. Blood samples for research testing will not exceed the 
volumes specified in Appendix K. Research samples may also be deferred or declined by the patient, their guardian or attending physician 
depending on the patient’s clinical status and preferences. The research tests will be prioritized by the PI depending on the number of 
available cells and the patient’s clinical course. 
 
 
 


Protocol 2660.00 
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11. 
Drugs, Irradiation and PBSC Administration - Toxicities and Complications. 
Note that for chemotherapy administered during conditioning and other drugs listed in 
this protocol, in keeping with institutional practice, it is acceptable to administer a dose 
within 10% of the protocol specified dose.  
 
11A.   Total Body Irradiation (TBI) 
1. TBI will be given as 165 cGy fractions twice per day x 4 days (days -10 to -7) to all 
patients using the linear accelerator at a rate of 6-7 cGy/min. Dosimetry calculations 
will be performed by the radiation oncologist.   
2. Toxicity: 
a. Myelosuppression is the major dose limiting toxicity.  
b. Erythema may occur in the first 24 hours. 
c. Hyperpigmentation may occur in the first month following TBI.  
d. Oral ulceration, anorexia, nausea, vomiting and diarrhea, fatigue and alopecia 
occur frequently.  
e. Parotitis  
f. Decreased production of saliva and tears 
g. Hepatic dysfunction and rarely liver failure. 
h. Late effects include cataracts, growth failure, gonadal failure and sterility, 
hypothyroidism, pulmonary dysfunction and secondary malignancies.  
 
11B.   Thiotepa  
1. Dosage. Thiotepa will be administered in a dose of 5 mg/kg/day (adjusted body 
weight) IV over approximately 4 hours for 2 consecutive days (day -6 and day -5). 
The total dose is 10 mg/kg (adjusted body weight). If actual weight is less than ideal 
body weight, actual body weight will be used.  Thiotepa is available in 15 mg vials 
and is reconstituted with sterile water resulting in an isotonic solution with 10 mg/ml 
of thiotepa.  
2. Toxicity  
a. The major dose limiting toxicity of thiotepa is myelosuppression.  
b. Oral ulceration, anorexia, nausea, vomiting and diarrhea, fatigue and alopecia 
occur frequently. 
c. Occasionally patients develop a skin rash that involves darkening of the skin and 
peeling, particularly in the axillary and inguinal folds. Thiotepa is secreted in 
sweat, therefore the axillary and inguinal areas should be washed twice daily 
during administration and for 2 days after administration.  
d. Dizziness and headache  
e. Hepatic toxicity including elevation in bilirubin and transaminases, and hepatic 
damage can occur. 
f. Rarely reported toxicities include CNS toxicity with somnolence, confusion, 
seizures, forgetfulness and inappropriate behavior.  
g. Allergic reactions during infusion occur rarely. 
h. Late effects include sterility and secondary malignancies.  
 
11C.  Fludarabine 
1. Dosage: Fludarabine will be administered in a dose of 25 mg/m2/day ( m2 always 
based on actual body weight)  IV over approximately 30 minutes for 5 consecutive 
days (day -6 to -2). The total dose of fludarabine will be 125 mg/m2. 
2. Toxicity 


Protocol 2660.00 
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a. Immunosuppression is the major toxicity of fludarabine. 
b. Oral ulceration, anorexia, nausea, vomiting and diarrhea, fatigue and alopecia 
occur frequently. 
c. Myelosuppression (lymphopenia, granulocytopenia, and anemia) is common. 
d. Numbness and tingling in hands or feet and visual changes occur and rarely 
somnolence, mental state changes, cortical blindness, coma and other 
neurotoxicity 
e. Other reported toxicities include rash, hepatocellular toxicity, hemolytic anemia 
and interstitial pneumonitis. 
 
11D. PBSC infusions  
Refer to Appendix D for infusion of selected cells. 
 
11E.  Tacrolimus administration 
1. Also see section 9F for information about tacrolimus administration and dosage 
adjustments. 
2. Administration and dosage 
a. Intravenous dosing - The standard mode of IV administration is by continuous 
infusion over 22-24 hours. Tacrolimus should be initiated as an IV continuous 
infusion on day –1 at a dose of 0.03 mg/kg/day based on actual body weight. If 
actual weight is greater than ideal body weight, it is recommended to use adjusted 
body weight.  
b. Oral dosing – The oral formulation of tacrolimus is supplied as 0.5 mg, 1 mg or 5 
mg capsules or as oral syrup (0.5 mg/ml).  
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 (Q 12 hour) doses. 
Children < 6 years old may require oral dosing every eight hours to maintain 
target serum trough levels. 
3. Toxicity  
Side effects are generally reversible and may include: 
a. Rise in serum creatinine, electrolyte wasting, hemolytic uremic syndrome, and 
renal failure. 
b. Nausea and vomiting and hepatic dysfunction. 
c. Hypertension, 
d. Increases in cholesterol and triglycerides 
e. Paresthesia, tremors, seizures, headache, insomnia, dizziness, depression, 
confusion, hallucinations, psychosis, myoclonus, neuropathy, agitation. 
f. Blurred vision, photophobia. 
g. Hirsutism 
 
11F.  Methotrexate administration 
1. Also see section 9F for information about methotrexate administration and dosage 
adjustments. 
2. Administration and dosage  
a. In patients weighing >10kg methotrexate should be administered by IV push on 
days +1, +3, +6 and +11 at a dose of 15 mg/m2 (on day +1) and a dose of 10 
mg/m2 (on day +3, +6, +11). Calculation of m2 should be according to 
institutional standard practice guidelines.  
b. Children weighing 10kg or less will have methotrexate dosed per kg rather than 


Protocol 2660.00 
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per /m2  and should receive 0.5mg/kg on day +1 and 0.33mg/kg on days +3,+6, 
and +11.  
c. The first dose of methotrexate should be given at approximately 24 hours, but no 
sooner than 24 hours after completion of the cell infusion.  
d. If clinically indicated methotrexate levels may be obtained and/or methotrexate 
dose adjusted according to institutional standard practice guidelines and/or 
leucovorin rescue may be considered. In the case of severe mucositis or in the 
presence of effusions, ascites, or another relative contraindication to methotrexate 
administration, the day 11 methotrexate may be withheld at the discretion of the 
attending physician.  
3. Toxicity 
a. In this setting of HCT, mucositis is the primary toxicity related to methotrexate. 
b. If there is also renal dysfunction methotrexate clearance may be delayed. 
c. Methotrexate may cause an elevation in serum transaminases. 
d. Severe skin reactions after single or multiple doses of methotrexate have 
occasionally been reported. 
 
 
12. 
Protocol Enrollment and Special Considerations 
12A. 
Projected Target Accrual 
ETHNIC AND GENDER DISTRIBUTION CHART 
 
TARGETED / PLANNED ENROLLMENT:  Number of Subjects 
 
Ethnic Category 
Sex / Gender 
Females 
Males 
Total 
Hispanic or Latino
0
0
0
Not Hispanic or Latino
8
12
20
Ethnic Category Total of All Subjects*
8
12
20
Racial Categories 
American Indian / Alaska Native
0
0
0
Asian
0
0
0
Native Hawaiian or Other Pacific Islander
0
0
0
Black or African American
0
0
0
White
8
12
20
Racial Categories: Total of All Subjects*
8
12
20
 


Protocol 2660.00 
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12B. 
Infection Prophylaxis  
1) Antibiotics 
(a) Prophylactic antibacterial antibiotics should be used for all patients that develop 
neutropenia (ANC <500/ml) according to institutional standard practice 
guidelines or according to currently active protocols. 
(b) In order to prevent early Streptococcus viridians bacteremia all patients should 
receive additional prophylactic therapy.  Patients should be started on oral 
penicillin daily beginning at day -2 until they are unable to take PO, when they 
should be moved to penicillin IV through day +10.  Patients with a known allergy 
to penicillin should receive Vancomycin IV from day -2 through day +10. The 
recommended standard regimens are: 
(i) Standard: Penicillin VK 25mg/kg PO BID (maximum 1 gram PO BID) 
starting at day -2 until not able to take oral therapy and then IV Penicillin G 
25,000U/kg IV every 6 hours (maximum one million units IV every six hours, 
4 million units total/day) until day +10.  
(ii) If Penicllin Allergic:  IV Vancomycin 15 mg/kg IV every 12 hours from day -
2 through day +10 
(c) Neutropenic fever: In patients with any active mucositis and neutropenia, IV 
Vancomycin (15 mg/kg IV at least every 12 hours) or an alternative antibiotic 
regimen covering streptococcus viridans should be given in addition to the other 
empiric neutropenic fever antibiotic therapy specified by institutional standard 
practice guidelines (e.g. Cefazidime).  Prophylactic penicillin may be 
discontinued when vancomycin or an alternative anti-streptococcus viridans agent 
is initiated. 
2) Prophylactic fluconazole or an alternative antifungal medication should be used from 
the first day of conditioning or sooner until day +75 post transplant in accordance 
with standard practice (or according to an IRB approved anti-fungal research 
protocol) 
3) Preemptive therapy for CMV reactivation should be administered according to 
institutional standard practice guidelines for a T cell depleted allogeneic HCT (or 
according to an IRB approved CMV research protocol). 
4) Infectious disease consultation should be obtained for patients who develop EBV 
DNA levels of >1000 copies/ml plasma on any test.   
5)  Infectious disease consultation should be obtained for patients who develop 
Adenovirus DNA levels of >300 copies/ml plasma on any test.   
6) Patients should receive acyclovir or valacyclovir according to institutional standard 
practice guidelines as prophylaxis for HSV and VZV. Acyclovir or valacyclovir can 
be withheld during periods that the patient is receiving ganciclovir, foscarnet or other 
effective antiviral drug for management of CMV. 
7) After engraftment and ≥20-30 days after HCT, patients may receive prophylaxis for 
pneumocystis carinii according to institutional standard practice guidelines.  
 
12C.  Ursodeoxycholic acid (UDCA) prophylaxis of hepatic complications of transplant 
1. UDCA is recommended to prevent hepatic complications according to standard 
practice guidelines.  
 
12D.  Management of acute and chronic GVHD 
1. Patients who develop > grade II acute GVHD should be treated with systemic 


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corticosteroids and/or with beclomethasone or budesonide if clinically indicated. 
Patients failing initial therapy will be eligible for second line therapy. Second line 
immunosuppressant therapy includes but is not limited to sirolimus, mycophenolate 
mofetil, monoclonal antibodies, pentostatin, denileukin difitox, thalidomide and 
extracorporeal photopheresis, given on or off study protocols.  
 
2. Patients who develop chronic GVHD and require systemic immunosuppressant 
therapy should be treated with immunosuppressive therapy according to institutional 
guidelines or may be treated on research protocols. 
 
12E.  Management of Delayed Engraftment 
1. If the ANC has not reached 100 by Day 21, a bone marrow examination may be 
performed to ascertain cellularity. If the ANC is <100 on Day 21, G-CSF, GM-CSF 
or other appropriate cytokines may be utilized and chimerism studies should be 
performed. Care of patients that have continued poor graft function after cytokine 
administration or who reject their grafts after initial engraftment is at the discretion of 
the attending physician.  
 
12F. 
Management of Relapse 
1. Patients who are at high risk of relapse, have persistent leukemia or who relapse after 
transplant may be treated according to institutional practice which can include but are 
not limited to chemotherapy, small molecule inhibitors and/or donor lymphocyte 
infusions (DLI), or may be eligible for research protocols including immunotherapy 
with antigen-specific T cells.  
 
13. 
Records 
The medical record containing information regarding treatment of the patient will be 
maintained as a confidential document, within the guidelines of the Fred Hutchinson 
Cancer Research Center, Seattle Children’s, the University of Washington Medical 
Center, the Seattle Cancer Care Alliance.  
Each patient is assigned a unique patient number to assure patient confidentiality.  
Patients should not be referred to by this number, by name, or by any other individual 
identifier in any publication or external presentation. The Clinical Statistics Department 
maintains a patient database at FHCRC to allow storage and retrieval of patient data 
collected from a wide variety of sources. The licensed medical records departments, 
affiliated with the institution where the patient receives medical care, maintains all 
original inpatient and outpatient chart documents.  
 
The primary research records will be contained and accessed through CORE, an 
encrypted, password-protected web site maintained by the FHCRC Clinical Research 
Data Systems division. Access is restricted to personnel authorized in writing by 
the FHCRC principal investigator.  
 
Information gathered from this study regarding patient outcomes and adverse events will 
be made available to the Federal Drug Administration. All precautions to maintain 
confidentiality of medical records will be taken. 
 
14.   Evaluation and statistical considerations 
14A. Type of study 


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This is a prospective phase II study of allogeneic stem cell transplantation in pediatric 
patients using PBSC that are selectively depleted of CD45RA+ T cells. 
 
14B. 
Definition of endpoints 
1. Estimate the time to discontinuation of systemic immunosuppression in pediatric 
recipients of CD45RA+ T cell-depleted PBSCT (a. including and b. excluding 
calcineurin inhibitors) 
a. Definitions:  
i. Time from transplant to the final discontinuation of all systemic immune 
suppression. 
ii. Time from transplant to the discontinuation of all systemic immune 
suppression with the exception of calcineurin inhibitors.  
b. Evaluation: The research team will follow the study participants via the database 
of the FHCRC long term follow-up program (LTFU) and the patient’s primary 
care physician and/or oncologist as required to determine the final date of 
completion of immunosuppression as defined.  
2. Graft failure 
a. Definition: Graft failure is defined operationally as:  
i. Failure to reach an ANC of >500/µl for 3 consecutive days by day 28 
ii. Irreversible decrease in ANC to <100 after an established donor graft:  If the 
reduction in ANC is the result of relapse, as determined by histopathology, 
flow cytometry or molecular studies, this will not be considered graft failure.  
If there is a reasonable explanation, such as viral infection or drug effect that 
may be responsible for a reversible decrease in ANC, this will be not 
necessarily be considered graft failure. 
b. Evaluation: Engraftment endpoints will include: 
i. Time to ANC of >500/uL on the first of three consecutive days. 
ii. Time to ANC of >1,000/uL on the first of three consecutive test results 
iii. Time to platelet count >20,000/µL for 3 days without transfusion. 
iv. Time to platelet count >50,000/µL for 3 days without transfusion.  
v. It is recommended that chimerism analysis of CD3 and CD33 cells in 
peripheral blood be performed on or around day 28, 56, 80-100, 180, 270 and 
360. Additional peripheral blood or marrow chimerism studies may be 
performed as clinically indicated.   
3. Chronic GVHD 
a. Definition: Chronic GVHD will be diagnosed using NIH criteria outlined in 
Appendix F. Chronic GVHD will be defined operationally as the occurrence of 
compatible symptoms fulfilling NIH criteria and requiring treatment with 
systemic immunosuppression.  
b. Evaluation will be conducted by the FHCRC pediatric LTFU team at day 80-100 
and where possible on approximately day 180, 270, 360 and yearly up to 5 years 
after HCT. The patient’s primary care physician and/or pediatric oncologist will 
conduct clinical assessments between these time points as required. 
i.  The primary chronic GVHD endpoint will be the occurrence of chronic GHVD 
meeting NIH criteria and requiring systemic pharmacological immunosuppression 
(systemic corticosteroids and/or other systemic agents, excluding calcineurin 
inhibitors which are generally not considered adequate as sole agents for the 
treatment of chronic GVHD)  
ii. A secondary chronic GVHD endpoint will be use of additional immune 


Protocol 2660.00 
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suppressive agents other than first line therapy (first line therapy is considered 
prednisone and tacrolimus/cyclosporin).  
c. If patients do not develop chronic GVHD after transplant but do relapse and 
then receive donor lymphocyte infusion or antigen specific T cells as treatment 
of relapse they will not be considered evaluable for the chronic GVHD 
endpoint. 
4. Acute GVHD 
a. Definition:  
i. Acute GVHD will be diagnosed and graded using the clinical and laboratory 
criteria in Appendix E. Acute GVHD is defined operationally as the occurrence of 
compatible symptoms or signs in the skin, gastrointestinal tract, or liver prior to 
Day +100. In most cases histology of biopsy material of at least one involved 
organ will be used to confirm acute GVHD, but biopsy is not absolutely required 
for the diagnosis. 
ii. Steroid refractory GVHD will be defined by the decision of the attending 
physician to initiate secondary systemic therapy for acute GVHD due to concerns 
that corticosteroids are providing inadequate control of acute GVHD 
manifestations. 
b. Evaluation: During the inpatient stay, clinical evaluations will occur daily.  
During the outpatient stay, each patient should be evaluated at least weekly until 
departure.  Clinical evaluations will be performed by an attending physician. 
Cutaneous, hepatic and gastrointestinal GVHD should be confirmed by biopsy 
except where medically contraindicated or considered medically unnecessary. An 
attending pathologist will interpret the biopsy material. The decision to initiate 
GVHD therapy will be made by the attending pediatric BMT physician.  
At the conclusion of the study, two pathologists with experience in GVHD will 
independently review the histology of biopsy specimens. An acute GVHD grade 
will be assigned for each patient by three experienced transplant physicians.  
c. Endpoints for acute GVHD that will be collected include: 
i. Presence of acute GVHD grades II-IV 
ii. Presence of acute GVHD grade III-IV 
iii. Presence of steroid refractory acute GVHD 
5. Relapse 
a. Definition: Relapse is defined by the presence of malignant cells in marrow, 
peripheral blood, or extramedullary sites by histopathology. Minimal residual 
disease is defined as the presence of malignant cells in the marrow, peripheral 
blood, or extramedullary sites detectable only by molecular methods, 
cytogenetics, or flow cytometry, but not observed by histopathology. 
b. Evaluation: Testing for recurrent malignancy in the blood and bone marrow will 
be performed by monitoring the CBC and bone marrow as outlined in Table 3.  
Suspected extramedullary sites of recurrent disease may be evaluated by biopsy 
and/or lumbar puncture if clinically indicated.  
 
6. Transplant related mortality (TRM). TRM is defined as mortality in any patient for 
whom there has not been a diagnosis of relapse. 
 
14C. 
Statistical Analysis and Stopping Rules 
1. Duration of immunosuppression 
The first primary objective is to estimate the time-to-discontinuation of systemic 
immunosuppression in pediatric recipients of CD45RA+ T cell-depleted PBSCT.  The 


Protocol 2660.00 
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goal is to observe a statistically significant reduction in the time-to-discontinuation of 
systemic immunosuppression compared to the historical experience without an 
accompanying increase in the probability of graft failure.  
Unpublished data from FHCRC shows that amongst 55 children with a history of 
leukemia who received a myeloablative bone marrow (N=34 ) or PBSC (N=21) 
transplant from an unrelated donor (HLA-A, B, C, DRB1, and DQB1 molecularly 
matched) following conditioning with TBI and cyclophosphamide between July 2002 
and December 2010 the median time to discontinuation of all immunosuppression 
was 1076 days, with a median time to discontinuation of 608 days for bone marrow 
recipients, and median time to discontinuation not reached for recipients of PBSC. 
Based on these data, we shall use a median time to discontinuation of systemic 
immunosuppression of 608 days as our benchmark for comparison in the current trial. 
The goal is to increase the proportion of patients who discontinue all systemic 
immunosuppression from the historical value of 50% to 80% in the current trial.  
Evaluation of 20 patients will provide 90% power to observe a statistically significant 
difference between (the fixed probability of) 50% and 80% of patients discontinuing 
by 608 days (with a 1-sided significance level of 0.05).  
Pediatric patients with HLA-identical related donors ≥18 years will also be eligible 
for enrollment on the protocol but only a few such patients are likely to be enrolled 
and GVHD may be less frequent and less prolonged in these patients. Therefore the 
time to discontinuation of immunosuppression data for the subgroup of pediatric 
patients with related donors will be analyzed separately.  
 
2. Graft Failure 
Graft failure will be closely monitored throughout the study. A true probability of 
graft failure of 10% will be considered excessive. If there is sufficient evidence to 
suggest that the true probability of graft failure exceeds 10%, the study will be 
stopped.  Sufficient evidence will be taken to be an observed rate whose lower one-
sided 90% confidence limit exceeds 10%.  Operationally this will occur if any of the 
following observed ratios occur: 2 of the first 5 or fewer, 3 of the first 11 or fewer, 4 
of the first 18 or fewer, or 5 of the first 20 of fewer patients develop graft failure.  If 
the true probability of graft failure is 0.05, the probability of stopping after 10 or 20 
patients is approximately 0.03 and 0.04, respectively.  If the true probability is 0.3, 
then the probability of stopping is approximately 0.67 and 0.88, respectively 
(probabilities estimated from 5,000 simulations).  
 
3. Failure to deliver therapy 
If the CD34+ stem cells cannot be delivered as planned for the first 2 patients 
enrolled, or for any other two consecutive patients, the study will be suspended for a 
period of time in order to optimize the procedure. This is defined as failure to deliver 
a minimum of 2.0x 106 CD34+ cells/kg recipient body weight. If an improved 
procedure is achieved, the study may be resumed.  
 
4. Patient withdrawal from treatment or study 
All patients who receive infusion of PBSC with or without cell selection will be 
considered evaluable for study safety endpoints. If they received PBSC with cell 
selection they will be evaluable for study efficacy endpoints. 
 
When a patient withdraws consent from P2660 they will no longer have research 


Protocol 2660.00 
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samples taken and the research team will no longer have access to clinical records to 
follow them for study endpoints unless they have signed the general FHCRC consent 
and authorization form allowing their leftover specimens and medical records to be 
used for research.  If they have signed that consent then they will still be evaluable for 
clinical endpoints although research blood draws would be discontinued. If the patient 
refused to sign this general consent or withdrew general consent to the use of clinical 
data for research purposes then no further follow-up would be done. 
 
15. 
Guidelines for Reporting and Tracking Events 
15A. Toxicity Grading  
Toxicities will be graded according to the current version of the NCI Common 
Terminology Criteria for Adverse Events (CTCAE) Version 4.  The full text of the NCI 
CTCAE is available online at: 
 
http://evs.nci.nih.gov/ftp1/CTCAE/About.html 
 
15B. 
Definitions 
Definitions associated with reportable events can be found on the FHCRC’s Institutional 
Review Office (IRO) extranet website.  
 
According to ICH guidelines (Federal Register. 1997; 62(90):25691-25709) and 21 CFR 
312.32, IND Safety Reports, and ICH E2A, Definitions and Standards for Expedited 
Reporting, an adverse event is defined as follows: 
 
An adverse event is any untoward medical occurrence in a clinical investigation subject 
administered a medicinal product and which does not necessarily have a causal 
relationship with this treatment. An AE can therefore be any unfavorable and unintended 
sign (including an abnormal laboratory finding), symptom, or disease temporally 
associated with the use of a medicinal product, whether or not considered related to the 
medicinal product. 
 
Abnormal laboratory values for laboratory parameters specified in the study should not 
be recorded as an adverse event unless an intervention is required (repeat testing to 
confirm the abnormality is not considered intervention), the laboratory abnormality 
results in a serious adverse event or the adverse event results in study termination or 
interruption/discontinuation of study treatment. 
 
Medical conditions present at screening (i.e., before the study treatment is administered) 
are not adverse events and should not be recorded on adverse event pages of the CRFs.  
These medical conditions should be adequately documented on the subject chart.  
However, medical conditions present at baseline that worsen in intensity or frequency 
during the treatment or post-treatment periods should be reported and recorded as adverse 
events. 
 
15C.  Tracking and reporting of events 
 
Patients enrolled in this study are receiving treatments that are generally associated with 
high rates of “expected” adverse events (outlined in Appendix G as well as Section 11 of 
the protocol). The following events will be tracked and reported:   


Protocol 2660.00 
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1. Non-hematologic adverse events assessed as Grade 3-5 per NCI CTCAE, expected or 
unexpected, from the start of study treatment (pre-transplant conditioning) through 
day 100 will be collected, with the exception of some abnormal lab values and 
medical conditions present at screening (as noted in section 15B). 
2. Grade 3-5 Blood/Bone Marrow adverse events occurring between day 60 and day 100 
will be collected.  
3. Graft versus host disease assessment done as part of routine care will be reviewed 
approximately once weekly through day 100. Details about GVHD symptoms, 
diagnosis, treatment, and outcome will be collected. After day +100, GVHD data will 
be captured at day +180, +270, 1 year, and then yearly through 5 years after day 0 
whenever possible. 
4. Relapse, graft failure, and death data will be captured as they occur in the first 200 
days post transplant for all patients, and from 200 days up to 5 years whenever 
possible.  
5. All treatment related mortality attributed to the investigational cell product occurring 
in the first 100 days will be reported to the IRB and the FDA as stated in sections 
15.D and E. 
6. Grade 3-5 infusion reactions will be reported to the IRB and FDA as stated in 
sections 15.D and E. 
 
15D. Reporting Requirement to FHCRC IRB 
The Principal Investigator, study nurse, or coordinator shall submit to the FHCRC IRB 
reportable events according to current reporting policies as outlined in FHCRC IRB 
Policies for Reportable Events.   
 
The FHCRC PI and research nurse will meet regularly and will together review all 
reported events that could potentially meeting reporting requirements.  If the event meets 
FHCRC IRB current reporting obligations it will be sent to them. 
 
All reportable events should be submitted on the relevant FHCRC Forms (URLs linking 
to the FHCRC IRO website are found in Table 5 FHCRC IRB Forms for Reporting).   
 
Table 5. FHCRC IRB Policies for Reportable Events.  
(Relevant FHCRC policies include, but are not limited to the following documents. Please also 
refer to the FHCRC IRO website. ) 
IRB Policy 2.6 
Adverse Events and Other 
Unanticipated Problems 
Involving Risks to Subjects or 
Others 
http://extranet.fhcrc.org/EN/sections/iro/irb/ae.html 
IRB Policy 1.9 
Noncompliance with the Office 
of the Director’s Human 
Research Protection Program 
Policy 
http://extranet.fhcrc.org/EN/sections/iro/irb/ae.html 
IRB Policy 1.1 
Reporting Obligations for 
Principal Investigators 
http://extranet.fhcrc.org/EN/sections/iro/irb/policy/index.html 
IRB Policy 2.2 
Continuing Review 
http://extranet.fhcrc.org/EN/sections/iro/irb/policy/index.html 
IRB Policy 1.13 
Investigational New Drugs 
(IND), Biologics and 
Investigational Device 
Exemptions (IDE) 
http://extranet.fhcrc.org/EN/sections/iro/irb/policy/index.html 
 


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Table 6 FHCRC IRB Forms for Reporting 
Adverse Event Reporting Form 
http://extranet.fhcrc.org/EN/sections/iro/irb/forms/index.html  
Expedited Reporting Form for Unanticipated 
Problems or Noncompliance 
http://extranet.fhcrc.org/EN/sections/iro/irb/forms/index.html  
 
15E.  Reporting to the FDA 
As a study conducted under IND (Investigational New Drug) regulations we will comply 
with the FDA regulations regarding safety reporting 21CFR312.32 including the 
following requirements: 
1. 
A sponsor must promptly review all information relevant to the safety of the drug 
21CFR312.32 (b). 
2. 
A sponsor must notify FDA in an IND safety report of potential serious risks, as 
soon as possible but in no case later than 15 calendar days after the sponsor determines 
that the information qualifies for reporting under 21 CFR312.32 (c)(1). Information that 
is required to be reported includes, but is not limited to, a. Serious and unexpected 
adverse reactions and b. An increased rate of occurrence of serious suspected adverse 
reactions.  
3. 
The IND safety report must be completed and sent to the FDA in a narrative format, 
on FDA Form 3500A, or an electronic format. 
4. 
A sponsor must also notify FDA of any unexpected fatal or life-threatening 
suspected adverse reaction as soon as possible but in no case later than 7 calendar days 
after the sponsor’s initial receipt of the information 21CFR312.32 (c)(2). 
 
16. 
Data safety monitoring plan 
16A. Monitoring the progress of trials and the safety of participants 
The FHCRC PI/Sponsor is responsible for monitoring this clinical trial, with oversight by 
a Data and Safety and Monitoring Board (DSMB), the Data Safety Monitoring 
Committee (DSMC) and the IRB at the FHCRC. This is a Phase II study and the 
assessment of risk is considered above minimal. The PI reviews outcome data for each 
individual patient at approximately 3 and 12 months after HCT, at a minimum. 
 
A DSMB will be in place to meet approximately every 6 months to review the data 
particularly as it relates to engraftment, grades III-IV GVHD and relapse. 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 and the PI. The DSMB will discontinue the review of outcomes when 
all subjects on this trial have completed all protocol-specified follow-up.  
 
P2660 DSMB Members 
Name 
Affiliation 
Position 
Eneida Nemecek, MD Oregon Health & Sciences University DSMB Chair 
Mary Elizabeth 
Percival, MD, MS 
Fred Hutch 
DSMB Clinical Investigator 
Rebecca Gardner, 
MD 
Seattle Children’s Research Institute 
DSMB Clinical Investigator 
Soheil Mesenchi, MD Fred Hutch 
DSMB Clinical Investigator 


Protocol 2660.00 
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Vicky Wu, PhD 
Fred Hutch 
DSMB Biostatistician 
 
All members will have experience in the management of patients with leukemia and 
myelodysplasia and in the conduct and monitoring of clinical trials. At least some of the 
members of the DSMB will have experience in the management of pediatric patients with 
leukemia and myelodysplasia. 
 
The DSMC at FHCRC will review the progress of the protocol with respect to the 
monitoring plan at the time of each annual renewal. As with initial review, annual 
FHCRC IRB review and approval is also required.  
 
A protocol monitor (from the FHCRC Clinical Research Support Office or an external 
monitor) will be retained to monitor study progress. The scope of monitoring will be 
based on the FHCRC/UW Data and Safety Monitoring Plan: 
http://centernet.fhcrc.org/CN/depts/iro/irb/dsm/ Per the DSMP subjects will be randomly 
selected for verification.  An initial monitoring visit is expected within six months of 
enrollment of the first subject and preferred prior to enrollment exceeding 4 subjects as 
100% verification is expected during an initial visit.  Monitoring reports will be 
forwarded to the DSMB, and the Principal Investigator/Sponsor at FHCRC.  
 
Flow of information concerning clinical trial participants originates with the clinicians 
and nurses in the clinic and is transmitted to the FHCRC Research Nurse. At the FHCRC 
health care providers and rotating attending physicians assess patients and record their 
observations regarding toxicity and response outcomes in the medical record. Thus, 
multiple health care providers provide independent observations and participate in 
monitoring this trial. The PI may be a clinician for some patients entered on this trial. 
However, assessments are the sum total of the multiple clinicians 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. 
 
 16B. Plans for assuring data accuracy and protocol compliance 
The study has a research nurse that follows patients to confirm eligibility, reporting of 
adverse events, reporting of events which are part of the safety-monitoring plan, and 
protocol adherence. The PI and research nurse are responsible for review and 
maintenance of all patient research records to ensure data integrity and protocol 
adherence. 
 
Health care providers and rotating attending physicians assess patients and record their 
observations in the medical record. This documentation is extracted by the site’s research 
staff by approximately day 100 (and no later than day +130) and again at approximately 1 
year (no later than 15 months) after HCT via chart review and entered into electronic 
protocol specific Case Report Forms (CRFs). The principal investigator will review the 
CRFs and the primary source documents verifying by signature (electronic) their data 
accuracy.  
 
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.  


Protocol 2660.00 
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Enriched T Cell-Depleted Grafts in a Multicenter Trial for Acute Myeloid Leukemia-Blood and Marrow 
Transplant Clinical Trials Network (BMT CTN) Protocol 0303. Biol Blood Marrow Transplant (2011). 
25. 
Keever CA, Small TN, Flomenberg N et al. Immune reconstitution following bone marrow transplantation: 
comparison of recipients of T-cell depleted marrow with recipients of conventional marrow grafts. Blood 
73(5), 1340-1350 (1989). 
26. 
Keever-Taylor CA, Devine SM, Soiffer RJ et al. Characteristics of CliniMACS(R) System CD34-enriched 
T cell-depleted grafts in a multicenter trial for acute myeloid leukemia-Blood and Marrow Transplant 
Clinical Trials Network (BMT CTN) protocol 0303. Biol Blood Marrow Transplant 18(5), 690-697 (2012). 
27. 
Walter RB, Pagel JM, Gooley TA et al. Comparison of matched unrelated and matched related donor 
myeloablative hematopoietic cell transplantation for adults with acute myeloid leukemia in first remission. 
Leukemia 24(7), 1276-1282 (2010). 
28. 
Ringden O, Pavletic SZ, Anasetti C et al. The graft-versus-leukemia effect using matched unrelated donors 
is not superior to HLA-identical siblings for hematopoietic stem cell transplantation. Blood 113(13), 3110-
3118 (2009). 
29. 
Doney K, Gooley TA, Deeg HJ, Flowers ME, Storb R, Appelbaum FR. Allogeneic hematopoietic cell 
transplantation with full-intensity conditioning for adult acute lymphoblastic leukemia: results from a 
single center, 1998-2006. Biol Blood Marrow Transplant 17(8), 1187-1195 (2011). 
30. 
Mielcarek M, Storer B, Martin PJ et al. Long-term outcomes after transplantation of HLA-identical related 
G-CSF-mobilized peripheral blood mononuclear cells versus bone marrow. Blood 119(11), 2675-2678 
(2012). 
31. 
Pasquini MC, Devine S, Mendizabal A et al. Comparative Outcomes of Donor Graft CD34+ Selection and 
Immune Suppressive Therapy As Graft-Versus-Host Disease Prophylaxis for Patients With Acute Myeloid 
Leukemia in Complete Remission Undergoing HLA-Matched Sibling Allogeneic Hematopoietic Cell 
Transplantation. J Clin Oncol 30(26), 3194-3201 (2012). 
32. 
Lang P, Handgretinger R, Niethammer D et al. Transplantation of highly purified CD34+ progenitor cells 
from unrelated donors in pediatric leukemia. Blood 101(4), 1630-1636 (2003). 
33. 
Kobos R, Steinherz PG, Kernan NA et al. Allogeneic hematopoietic stem cell transplantation for pediatric 
patients with treatment-related myelodysplastic syndrome or acute myelogenous leukemia. Biol Blood 
Marrow Transplant 18(3), 473-480 (2012). 
34. 
Lanzavecchia A, Sallusto F. Dynamics of T lymphocyte responses: intermediates, effectors, and memory 
cells. Science 290(5489), 92-97 (2000). 
35. 
Kaech SM, Wherry EJ, Ahmed R. Effector and memory T-cell differentiation: implications for vaccine 
development. Nat Rev Immunol 2(4), 251-262 (2002). 
36. 
Anderson BE, Mcniff J, Yan J et al. Memory CD4+ T cells do not induce graft-versus-host disease. J Clin 
Invest 112(1), 101-108 (2003). 
37. 
Zhang Y, Joe G, Zhu J et al. Dendritic cell-activated CD44hiCD8+ T cells are defective in mediating acute 
graft-versus-host disease but retain graft-versus-leukemia activity. Blood 103(10), 3970-3978 (2004). 
38. 
Chen BJ, Cui X, Sempowski GD, Liu C, Chao NJ. Transfer of allogeneic CD62L- memory T cells without 
graft-versus-host disease. Blood 103(4), 1534-1541 (2004). 
39. 
Chen BJ, Deoliveira D, Cui X et al. Inability of memory T cells to induce graft-versus-host disease is a 
result of an abortive alloresponse. Blood 109(7), 3115-3123 (2007). 
40. 
Dutt S, Tseng D, Ermann J et al. Naive and memory T cells induce different types of graft-versus-host 
disease. J Immunol 179(10), 6547-6554 (2007). 
41. 
Zheng H, Matte-Martone C, Li H et al. Effector memory CD4+ T cells mediate graft-versus-leukemia 
without inducing graft-versus-host disease. Blood 111(4), 2476-2484 (2008). 
42. 
Zheng H, Matte-Martone C, Jain D, Mcniff J, Shlomchik WD. Central memory CD8+ T cells induce graft-
versus-host disease and mediate graft-versus-leukemia. J Immunol 182(10), 5938-5948 (2009). 
43. 
Dutt S, Baker J, Kohrt HE et al. CD8+CD44(hi) but not CD4+CD44(hi) memory T cells mediate potent 
graft antilymphoma activity without GVHD. Blood 117(11), 3230-3239 (2011). 
44. 
Xystrakis E, Bernard I, Dejean AS, Alsaati T, Druet P, Saoudi A. Alloreactive CD4 T lymphocytes 
responsible for acute and chronic graft-versus-host disease are contained within the CD45RChigh but not 
the CD45RClow subset. Eur J Immunol 34(2), 408-417 (2004). 


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45. 
Arstila TP, Casrouge A, Baron V, Even J, Kanellopoulos J, Kourilsky P. A direct estimate of the human 
alphabeta T cell receptor diversity. Science 286(5441), 958-961 (1999). 
46. 
Robins HS, Campregher PV, Srivastava SK et al. Comprehensive assessment of T cell receptor {beta} 
chain diversity in {alpha}{beta} T cells. Blood (2009). 
47. 
Burrows SR, Khanna R, Silins SL, Moss DJ. The influence of antiviral T-cell responses on the alloreactive 
repertoire. Immunol Today 20(5), 203-207 (1999). 
48. 
Melenhorst JJ, Leen AM, Bollard CM et al. Allogeneic virus-specific T cells with HLA alloreactivity do 
not produce GVHD in human subjects. Blood 116(22), 4700-4702 (2010). 
49. 
Bleakley M, Otterud BE, Richardt JL et al. Leukemia-associated minor histocompatibility antigen 
discovery using T-cell clones isolated by in vitro stimulation of naive CD8+ T cells. Blood 115(23), 4923-
4933 (2010). 
50. 
Smalley DM, Ley K. L-selectin: mechanisms and physiological significance of ectodomain cleavage. J Cell 
Mol Med 9(2), 255-266 (2005). 
51. 
Nash RA, Antin JH, Karanes C et al. Phase 3 study comparing methotrexate and tacrolimus with 
methotrexate and cyclosporine for prophylaxis of acute graft-versus-host disease after marrow 
transplantation from unrelated donors. Blood 96(6), 2062-2068 (2000). 
52. 
Martin PJ, Mcdonald GB, Sanders JE et al. Increasingly frequent diagnosis of acute gastrointestinal graft-
versus-host disease after allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant 
10(5), 320-327 (2004). 
 




Protocol 2660.00 
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2. We will also perform cell selection only when the T cells in the PBSC product include a distinct CD45RO+, 
CD45RA- population observed by flow cytometry. ≤5% of donor products are expected to have a CD45 variant in 
which all the T cells express CD45RA and there is therefore no CD45RO+, CD45RA- population. If there is no 
CD45RO+, CD45RA- population the PBSCs will be infused without any cell selection and the patients will not be 
included in the analysis of study efficacy end points.  
3. Situations may arise where the goals for cell selection are not achieved with cell processing. These very unusual 
situations will be discussed with the protocol PI and a recommendation made. General guidelines are as follows: 
a) If a total CD34 cell dose of ≥2.0 x 106 cells/kg is not achieved after the processing the apheresis collection(s) all 
available CD34+ cells will be administered to the patient as soon as possible and an additional apheresis collection 
will be requested and the un-manipulated G-PBSC product will be administered to the patient without any cell 
selection procedure. These patients will not be included in the analysis of study efficacy end points.   
b) If < 5 x 104CD3+ CD45RA+ RO- cells/kg in the sum of the CD34+ fraction and CD45RA depleted fraction 
cannot be achieved by simply reducing the number of CD3+ and/or CD34+ cells within the target range (minimum of 
2.0 x 106/kg CD34+ cells and a range of 1-10 x 106/kg total CD3+ T cells) as described above, the following steps 
may be required:   
i. 
Repeat the CD45RA depletion of the CD34 depleted fraction using a second magnetic column. 
ii. 
If 1 x 106 CD3+ cells/kg and <5 x 104CD3+CD45RA+ RO- cell/kg cannot be achieved by i. then the patients 
should receive the CD34 selected product (minimum of 2.0 x 106 CD34+ cells/kg) and may receive an 
aliquot of the CD3+ cells (i.e. less than 1 x 106 CD3+ cells/kg) containing <5 x 104CD3+CD45RA+ RO- 
cell/kg. These patients who do not meet the goals for cell selection will not be included in the analysis of 
study end points. Patients will receive immunosuppression as described in the protocol unless they receive 
only the CD34+ selected product and < 1 x 105 CD3+ cells/kg in which case they will receive no 
immunosuppression.  
3. In a typical case we expect to a. perform cell selections on the day -1 apheresis collection (unrelated donors, 
some related donors), or a product pooled from the day -1 and day 0 collection (some related donors), b. to use a 
portion of the apheresis collection(s) containing at least 8-10 x 106 CD34+ cells/kg for the cell selections, c. to 
reserve at least 3-5 x 106 CD34+ cells /kg as a back-up for infusion of unselected PBSC into the patient if 
unanticipated problems arise in the cell selection procedure, and d. (for URD recipients) to cryopreserve at least 
1 x 107 CD3+ cells/kg for potential future donor lymphocyte infusion (DLI). We also plan to cryopreserve any 
available additional unmanipulated PBSC left over from the cell processing requirements as a back-up in the 
unlikely event of graft failure. In the unlikely event that there are insufficient unmanipulated PBSC to 
cryopreserve as DLI for URD recipients we may instead cryopreserve aliquots of the CD34-depleted fraction to 
be used as DLI.  
Apheresis Collection Guidelines 
Collection of unrelated donor GCSF mobilized apheresis products will be managed by the NMDP (US donors) or 
DKMS (German donors). Collection of related donor GCSF mobilized apheresis products will be managed by the 
SCCA apheresis center. The following represents a general guideline 
Unrelated donors 
1. A large volume apheresis collection (processing approximately 24L of blood) will be performed on day -1 and 
transported as soon as possible on day -1.  
Related donors 
1. A large volume apheresis collection (processing approximately 18L of blood) will be performed on day -1. The 
CD34 content will be evaluated on day-1.  
a) If  ≥ 15 million CD34 cells/kg have been obtained from the day -1 no further apheresis is required. Cell 
processing can commence on day 0 using 10 million CD34 cells/kg with 5 million CD34 cells/kg held in 
the event of cell processing problems, and cryopreserved if not required. 
b) If  <15 million CD34 cells/kg have been obtained from the day -1 collection a second collection will be 
obtained on day 0 to a maximum of 18L.  
i. 
The second collection may be totaled with the day -1 apheresis for a pooled or two separate cell 
selection(s) on day 0 and/or day +1 ( e.g. if the day -1 apheresis was ≤ 8 x 106 CD34/kg and the total of 
the day-1 and 0 aphereses is ≥ 5 x 106/kg )  
ii. 
The second collection may also be held as a back-up in case of unanticipated problems in the cell 
selection procedure of the first apheresis and subsequently cryopreserved if not required for cell 
selection.   
On very rare occasions the minimum CD34+ cell target number of 2 x 106/kg may not be achieved before or after 
cell processing and an additional donor apheresis collection may be requested and infused without cell selections. 


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APPENDIX B 
Product Testing 
 
The CD34-enriched and CD45RA-depleted cells, because they are derived solely as the result of a cell 
selection process with no culturing required, will be considered as having undergone “minimal 
manipulation”. Each of the CD34-enriched and CD45RA-depleted cell batches will be tested for safety, 
purity and potency, identity and stability as indicated below. In general, once myeloablative therapy has 
been initiated, the patient must receive the CD34-selected PBSC product, to reconstitute his/her 
hematopoietic system.  
 
a) Safety: Samples from the final products (CD34+ and CD45RA-) will be sent for sterility testing for 
bacterial and/or fungal contamination of products according to methods specified in 21 CFR 610.12. The 
tests will be performed in clinical laboratories. If positive cultures are found organism species identification 
will take place.  FHCRC has defined procedures and action plans in place to notify appropriate personnel 
and take appropriate measures if positive cultures are detected after infusion has taken place.  
 
b) Purity and potency:   
CD34+ product:  Viability will be determined by flow cytometry using exclusion of propidium iodide or 7-
AAD with a notification specification set of <70% viable cells (i.e. If there are <70% viable cells in the 
CD34+ product the PI will be notified). Quantitation of CD34+ cells will be performed by flow cytometry, 
with a notification specification of <70% final purity and CD34+ cell dose of <= 2.0 x 106 CD34+ cells/kg. 
Quantitation of residual naïve T-cells (CD3+CD45RA+CD45RO-) and total T cells (CD3+) will be 
performed by multi-color flow cytometry, however no specification will be set  for these cell subsets other 
than the total dose indicated below.  
 
CD45RA- product:  Viability will be determined by flow cytometry using exclusion of propidium iodide or 
7-AAD, with a notification specification set of <70% viable cells. Quantitation of residual naïve T-cells 
(CD3+CD45RA+CD45RO-), and total T cells (CD3+) will also be performed by multi-color flow cytometry, 
The aim is to infuse a maximum of 5 x 104 CD3+CD45RA+ RO-/kg and a range of 1-10 x 106 CD3+ cells/kg 
in the sum of the CD45RA-depleted and CD34+ cell products and a minimum of 2.0 x 106 CD34+ cells/kg. 
 
c) Identity:  Proper identification of all the intermediate and final cell products will be assured by defined 
process and label controls as specified in standard operating procedures within the cell processing facilities 
to ensure the recipient is receiving the correct cell products. 
 
d) Stability: The first leukapheresis collection will be transported and stored for a total of up to 48 hours 
from the end of apheresis collection at 4C prior to CD34 selection and CD45RA depletion. Unmanipulated 
leukapheresis products are routinely stored this duration and longer, particularly in matched unrelated donor 
transplantation, and we therefore do not anticipate that stability will be a major concern for this study.  
Viability, which is an important measure of stability, will be assessed immediately after all processing is 
completed, and infusion of the CD34+ cells and CD45RA-depleted cells will take place within 6 hours after 
completion and release of the cell products.  


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APPENDIX C 
Positive Culture from Donor Product Action Plan 
 
In the event that a sterility-testing culture sample turns positive (after the infusion has taken place), the following 
actions will take place immediately: 
 
1. The director or designee of the Cell Therapeutics Laboratory will notify the recipient’s attending 
physician and the Principal Investigator or the designated study nurse of the positive culture result. 
2. Identification of the organism and sensitivity testing will be completed. 
3. The FHCRC PI /Sponsor will notify the FHCRC IRB, as soon as possible, but no later than 10 calendar 
days of finding out of the event and will notify the FDA as outlined in section 15.  
4. After notification of the attending physician, the following actions will take place. 
a. The attending physician will notify the patient (recipient) of the positive culture. 
b. The attending physician or primary care provider will perform a thorough examination of the 
patient (recipient). 
c. Blood samples will be obtained from the patient (recipient) for cultures (bacterial, fungal, and 
viral cultures). 
d. Samples of other fluids will be obtained for cultures if clinically indicated. 
e. Assays for identification of the organism and sensitivity testing will be completed and results 
reported to the attending physician. 
5. The patient will be treated with empiric antimicrobial agents until the following endpoints are reached: 
a. Assays for identification and sensitivity of the organism have been completed. 
b. Results of the patient (recipient) blood cultures are available. 
c. The absolute neutrophil count exceeds 500 cells/µl. 
d. Unexplained clinical signs or symptoms of systemic infection have resolved. 
 
e. Exceptions to the administration of antibiotics must be based upon lack of criteria 5 a-d. These 
exceptions must be discussed by the attending physician with the PI 
6. The patient will be monitored daily for signs of systemic infection.  
7. Quality Assurance measures will be enacted. 
  


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APPENDIX D 
Infusion of Selected Cells 
 
General Guidelines 
The selected cells (CD34+ and CD34 depleted CD45RA depleted fractions) will be infused through a blood 
administration set filter. The cells will be suspended in clinical grade Normosol-R (Hospira) plus HSA (Baxter) 
for infusion. Central venous access is preferred for infusion. A peripheral IV line may be used only if central 
access is not available. Under no circumstances will these cells be irradiated. The staff caring for the patient must 
be familiar with the practices and complications of HPC infusion. Staff should be prepared to treat the recipient 
for an acute hemolytic transfusion reaction. 
 
Questions regarding ABO mismatches should be directed to the SCCA Transfusion Services Office or the 
SCCA Transfusion Medicine attending on call. Other questions regarding handling of selected cells should 
be directed to the Medical Director of the Cellular Therapy Laboratory. 
 
 
Timing 
After completion of the conditioning regimen cells should not be infused earlier than 36 hrs after the last dose of 
chemotherapy. Selected cells will be infused as soon as they are available. The CD34+ fraction will be given first 
followed as soon as possible, by the CD34-depleted CD45RA-depleted fraction. If a delay is anticipated because 
of timing of conditioning regimen or patient medical status as determined by the attending physician, cells may be 
stored in the refrigerator (4°C). It is aimed to commence administration of each fraction within 6 hours from the 
end of processing of that fraction. 
 
Volume 
The CD34+ cell volume will be approximately 50ml or less, and the CD34-depleted, CD45RA-depleted cells will 
also be approximately 50ml or less. 
 
Pre-medication 
No pre-medication will be given unless patient has previously reacted to blood or platelet transfusions, then 
premedication will be administered as per patient’s transfusion guidelines for allergic or febrile reactions.  
 
Filtration 
Products should be filtered through a blood administration set.  
 
Product Infusion Rate 
Begin at 0.5 times maintenance rate for 15 minutes, then increase to 1.5 times maintenance rate as tolerated.  
 
Monitoring 
Vital signs before infusion, after 15 minutes, then hourly during infusion, and at completion of infusion. If any 
reaction occurs, notify primary care provider. 
 
Concomitant Infusions: 
No medications or fluids may be given "piggy-back" with the selected cells, although they may be given through 
the other lumen of a double lumen catheter. Amphotericin, antibodies, investigational medications or blood 
products should not be given concomitantly because of difficulty in evaluating reactions. Cells must not be 
infused during plasmapheresis or dialysis. 
 
Reactions: 
Volume Overload: Generally, the CD34+ fraction and CD34 depleted CD45RA depleted cell volumes will be 50 
ml each so volume overload is not expected. Volume reduction may be required if the cell volume of either 
individual infusion exceeds 15 ml/kg recipient weight (highly unlikely), or if the patient is volume overloaded by 
clinical criteria. Request volume reduction by contacting the Cellular Therapy Laboratory. 
 


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Transfusion Reaction: Recipients who have clinically significant antibodies to AB or other antigens found on 
donor red cells may be at risk for an acute hemolytic transfusion reaction (i.e. major mismatch). Red cell 
depletion (or recipient plasma exchange) may be indicated in some cases. Patients also may experience 
hemoglobinuria from damaged cells in the cell inoculum. Delayed hemolysis may occur in the setting of major 
mismatch and rebound of recipient antibodies after plasmapheresis (if performed). Refer to the Clinical 
Coordinator's Patient Information Sheet for instructions regarding red cell reduction of product (or 
plasma exchange of recipient). The SCCA Transfusion Services Offices may also be consulted as a resource 
for management of specific patients. 
 
Patients with minor mismatch (i.e. donor antibodies against patient red cells) may experience hemolytic 
transfusion reactions. Circulating donor antibodies will be removed during processing of the manipulated cells by 
the Cellular Therapy Laboratory so early hemolytic transfusion reactions are unlikely. Delayed reactions can 
occur about 5-14 days after marrow infusion. Delayed reactions may occur from formation of antibodies by donor 
lymphocytes against either recipient or incompatible transfused red cells (see Red Blood Cell Infusion 
Guidelines).The SCCA Transfusion Services Office may be consulted as a resource for management of 
specific patients.  
 
Allergic Reaction: Recipients may have allergic reactions (chills, fever, hives) to the selected cell product. Please 
note, these products have been manipulated in the laboratory and may contain foreign proteins or reagents. 
Treatment is the same (diphenhydramine, meperidine, hydrocortisone) as for reactions to platelet transfusions. For 
anaphylaxis, treat per Institutional standard practice Guidelines, “Anaphylaxis Emergency / Drug Chart 
Reference”. 
 
Pulmonary Micro-Embolism: Fat and particulates may result in micro-emboli. Patients may complain of chest 
pain, dyspnea, or coughing. Excessive fat is highly unlikely in the selected cell products but can be removed by 
centrifugation by the Cellular Therapy Laboratory after discussion with the Medical Director. Slowing of infusion 
and administration of oxygen may alleviate mild dyspnea during infusion.  
 
Excessive Anti-coagulation: Selected cells will not be anticoagulated with heparin and/or citrate solutions. When 
heparin is used, rapid or large volume infusions may result in transient anti-coagulation of the recipient. 


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APPENDIX E 
Acute GVHD Staging and Grading Assessment 
GRADING OF ACUTE GRAFT-VERSUS-HOST DISEASEa 
Severity of Individual Organ Involvement 
Skin 
+1 
a maculopapular eruption involving less than 25% of the body surface 
+2 
a maculopapular eruption involving 25-50% of the body surface 
+3 
generalized erythroderma involving >50% of the body surface 
+4 
generalized erythroderma with bullous formation and often with 
desquamation 
Liver 
+1 
bilirubin (2.0-2.9 mg/100ml) 
+2 
bilirubin (3-5.9mg/100ml) 
+3 
bilirubin (6-14.9mg/100ml) 
+4 
bilirubin > 15mg/100ml 
Gut 
Diarrhea is graded +1 to +4 in severity. Nausea and vomiting and/or anorexia 
caused by GVHD is assigned as +1 in severity. 
The severity of gut involvement is assigned to the most severe involvement noted.  
Patients with visible bloody diarrhea are at least stage +2 gut and grade +3 overall 
Diarrhea 
+1 
≤ 1000 ml of liquid stool/day* (≤ 15ml of stool/kg/day)† 
+2 
>1,000 ml of stool/day* (> 15ml of stool/kg/day)† 
+3 
>1,500 ml of stool/day* (> 20ml of stool/kg/day)† 
+4 
2,000 ml of stool/day* (≥ 25ml of stool/kg/day)† 
*In the absence of infectious/medical cause 
†For pediatric patients 
Severity of GVHD 
Grade I 
+1 to +2 skin rash 
No gut or liver involvement 
Grade II 
+1 to +3 skin rash and/or 
+1 gastrointestinal involvement and/or +1 liver involvement 
Grade III 
+4 skin involvement and/or 
+2 to +4 gastrointestinal involvement and/or 
+2 to +4 liver involvement with or without a rash 
Grade IV 
Pattern and severity of GVHD similar to grade 3 with extreme constitutional 
symptoms or death 
a. From “Graft-vs-host disease” Sullivan, Keith M. Hematopoietic Cell Transplantation Ed: D. Thomas, K. Blume, S. Forman, Blackwell 
Sciences; 1999, pages 518-519. 




Protocol 2660.00 
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APPENDIX G 
Potential Adverse Events Associated or Expected with Hematopoietic Cell Transplantation 
 
1. Opportunistic infections, including viral and fungal infections, can results in severe pulmonary, neurologic, 
hepatic and other organ dysfunction, and possible death. 
 
2. Gastrointestinal toxicity. Nausea and vomiting can be anticipated during the entire course of ablative therapy. 
Mucositis and diarrhea should be expected. Prednisone can cause GI bleeding. 
 
3. Cardiac toxicity. Cardiotoxicity (congestive heart failure, pericardial effusion, EKG changes) is uncommonly 
associated with chemotherapy agents and TBI and these sequelae may prove lethal. 
 
4. Pulmonary toxicity. Diffuse interstitial pneumonitis and diffuse alveolar hemorrhage of unknown etiology 
occurs with some regularity after BMT. Interstitial fibrosis occurs less frequently. Each are well-described 
complications of intensive chemotherapy and TBI regimens and may prove lethal. 
 
5. Hepatic toxicity. Veno-occlusive disease of the liver is a common toxicity of high-dose chemoradiotherapy and 
may result in death. Tacrolimus may cause elevation of ALT/AST. 
 
6. Renal dysfunction. Chemoradiotherapy may uncommonly cause renal dysfunction. More commonly, 
nephrotoxicity results from tacrolimus and generally responds to dose reduction. Rarely, idiopathic or calcineurin 
inhibitor-associated hemolytic-uremic syndrome may occur and may be progressive and fatal. A syndrome of 
moderate renal insufficiency and hemolysis has been seen 5-7 months post HSCT after intensive conditioning 
plus TBI. 
 
7. Hemorrhagic cystitis, manifested either as gross or microscopic hematuria, is a common toxicity after high-
dose chemoradiotherapy, but usually associated with regimens that include cyclophosphamide. Hemorrhagic 
cystitis may predispose to a long-term increased risk of bladder cancer. 
 
8. Central nervous system toxicity. Radiation and chemotherapy can cause CNS toxicity, including seizures, 
depressed mental status, or leukoencephalopathy. Calcineurin inhibitors can cause seizures or other CNS toxicity. 
 
9. Marrow aplasia. Severe neutropenia, thrombocytopenia, and anemia, is expected to occur for a period of 7 to 42 
days following infusion of marrow. Transfusion of platelets and red blood cells is expected as supportive care. 
Transfusion of blood products may be associated with acquisition of HIV or a hepatitis virus. Neutropenia may 
increase the risk for acquiring serious infection. Thrombocytopenia may increase the risk of life-threatening 
hemorrhage. Hemorrhagic or infectious complications during the expected period of aplasia may result in death. 
Primary and secondary graft failure occur uncommonly. 
 
10. Miscellaneous. Alopecia and sterility are expected complications of the program as a whole. Cataract 
development is possible after TBI and/or steroids. Deficiencies of growth hormone, thyroid hormone, and sex 
hormones are possible after TBI. Calcineurin inhibitors can cause transient gingival hyperplasia, tremor, seizure, 
hypertension, headache, dysesthesia, metabolic complications and hirsutism. Steroid therapy can also contribute 
to fluid retention, easy bruising, hypertension, aseptic necrosis of bone, metabolic complications including 
diabetes mellitus and increased susceptibility to infection. Hospitalization during conditioning and recovery 
period is expected to be 5-9 weeks in duration. 


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APPENDIX H 
Karnofsky/Lansky Performance Status Scale 
 
 
  
KARNOFSKY PERFORMANCE STATUS SCALE (RECIPIENT ≥16 YEARS) 
Percentage 
 
100 
Normal, no complaints, no evidence of disease 
90 
Able to carry on normal activity; minor signs or symptoms of disease 
80 
Normal activity with effort; some signs or symptoms of disease 
70 
Cares for self; unable to carry on normal activity or do active work 
60 
Requires occasional assistance, but is able to care for most of his/her needs 
50 
Requires considerable assistance and frequent medical care 
40 
Disabled; requires special care and assistance 
30 
Severely disabled, hospitalization indicated.  Death not imminent 
20 
Very sick, hospitalization necessary, active supportive treatment necessary 
10 
Moribund, fatal processes, progressing rapidly 
0 
Dead 
 
LANSKY PERFORMANCE STATUS SCALE (RECIPIENT <16 YEARS) 
Percentage 
 
100 
Fully active 
90 
Minor restriction in physically strenuous play 
80 
Restricted in strenuous play, tires more easily, otherwise active 
70 
Both restrictions of and less time spent in active play 
60 
Ambulatory up to 50% of the time, limited active play with assistance/supervision 
50 
Considerable assistance required for any active play, fully able to engage in quiet play 
40 
Able to initiate quiet play 
30 
Needs considerable assistance for quiet activity 
20 
Limited to very passive activity initiated by others (e.g. TV) 
10 
Completely disabled, not even passive play 
0 
Dead 
 


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APPENDIX I 
Weight / Adjusted Body Weight for Drug Dosing 
 
Drug Dosing By Body Size: 
Drug dosing will be based on either body surface area (BSA) or body weight. 
1. BSA is calculated in M2. The formula by definition adjusts for both under and over-weight individuals. 
The formula for this calculation is: 
  __________________________ 
√actual* weight in kg x height in cm 
60 
BSA calculations used in this protocol are based on actual body weight for all individuals, not on adjusted weight. 
 
2. Body weight is measured in kg. Body weight may be adjusted in patients who are above ideal body weight. 
 
Calculating adjusted weight. Calculating adjusted weight requires determining an ideal body weight first. 
The ideal body weight (IBW) will be calculated in the following ways 
For Pediatric Patients: 
Pre-pubertal children (females < 12 years old and males < 14 years old): 
Ideal weight will be calculated by matching weight at the 50th percentile for length-for-age or 
height-for-age at the 50th percentile. 
Post pubertal adolescents (females > 12 years old and males > 14 years old): 
Ideal weight will be assessed using the body mass index (BMI) [weight in kg/height in 
meters squared)]. If the child’s normal BMI is between the 25-75th percentile, the child may 
be considered at IBW. 
Adolescents whose BMI exceeds the 75th percentile: Ideal weight will be the 75th percentile BMI 
weight. 
Adolescents whose BMI is below the 25th percentile: Ideal weight will be the 25th percentile BMI 
weight 
Note: When deviating from these age ranges (based on early or late maturity), the dietitian will 
document the rationale in the nutrition assessment. 
 
Weight / Adjusted Body Weight for Drug Dosing 
 
Adjusted Body Weight will be calculated as follows: 
Ideal Weight + 0.25 (actual weight – ideal weight) 
 
Weight Shifts After Initial Evaluation: 
Individuals with significant weight shifts after the initial evaluation will have the adjusted body weight reassessed 
by the Clinical Nutrition Staff as appropriate. 
 


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APPENDIX J 
Radiotherapy Treatment Guidelines  
 
1.1 Total Body Irradiation  
 
1.1.1 Patients 
Every patient will receive total body irradiation as part of the preparatory regimen for stem cell transplantation.  
The treatment dose regimen will be 165 cGy BID over 4 treatment days to a total dose of 1320 cGy. 
Lung shielding will be used as described below. 
All male patients having ALL will receive a testicular boost as part of the transplant regimen. 400 cGy for one 
fraction will be delivered by an en fosse electron field. For patients with a history of CNS leukemia a CNS 
irradiation boost may also performed if it is clinically indicated in the opinion of the pediatric BMT physician and 
radiation oncologist. 
 
1.1.2 Equipment 
 
1.1.2.1 Modality: 
High-energy photons with energy ≥ 6MV photons should be utilized. Although there is no upper limit on the 
energy as long as the skin dose requirements can be met, it is recommended that 18MV or lower be used. 
The selection of energy is determined by the dose uniformity criterion. 
 
1.1.3 Target Volume 
The total body will be treated, including the head and feet, in one field (except in certain circumstances).  Care 
should be taken to insure that the patient is entirely within the 90% isodose decrement line of the beam (i.e., not in 
the penumbra region of the beam).   
 
1.1.4 Target Dose 
The prescription point is defined as the point along the longitudinal axis of the patient at the midline at the level of 
the umbilicus (see Point 5, section 1.5.4.1). No tissue inhomogeneity correction will be made in the calculation of 
dose to the prescription point.  The absorbed dose along the patient’s head to toe axis (line formed by the 
intersection of the midsagittal plane and the midcoronal plane) shall be within 10% of the prescribed dose.  The 
dose at selected anatomical points shall be calculated and these calculations are to be submitted as part of the 
quality assurance.  Measurements of patient dimensions needed for the calculation of the prescription dose will be 
made at the time of the simulation for lung blocks.  Measurement and calculations of required monitor units 
necessary for each treatment will be performed in the patient treatment position for AP-PA fields: either upright 
position or the reclining, lateral decubitus position . In the event the patient is intended to be treated in an upright 
position, but proves too ill for treatment, dose calculation will have been pre-calculated to permit treatment in the 
lateral decubitus position). 
 
1.1.4.1 Prescription Point: 
The following reference points will be determined: 
 
1. Head (Point 1): this reference point is defined along the longitudinal axis of the skull at the greatest mid-
separation (immediately superior to the nasal bridge).  The depth should be taken as midway between the entrance 
and exit points of the opposed radiation beams. 
2.  Neck (Point 2): this reference point is defined along the patient’s longitudinal axis at the level of C3/C4 
(approximate mid-neck, but chosen for the thinnest mid-separation of the neck).  The point is taken to be midway 
between the entrance and exit point of the beam. 
3.  Upper Mediastinum (Point 3):  this reference point is defined along the patient’s longitudinal axis at the level 
of the angle of Louis.  The reference point is midway between the entrance and the exit points of the opposed 
beams. 
4. Lower Mediastinum (Point 4):  this reference point is defined along the patient’s longitudinal axis at the level 
of the xiphisternal notch.  The reference point is midway between the entrance and exit points of the opposed 


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beams 
5. Umbilicus (Point 5): THE PRESCRIPTION POINT is defined along the patient’s longitudinal axis at the level 
of the umbilicus.  The prescription point is midway between the entrance and exit points of the opposed beams. 
6. Knee (Point 6):  this reference point is defined along the midline in the midplane of the knee at the level of the 
patella. 
7.  Ankle (Point 7):  this reference point is defined along the midline at the midplane of the ankle at the level of 
the lateral malleolus. 
8.  Shielded Lung Dose (Point 8):  this reference point is located on the right chest wall under the lung block.  It 
is centered (both medial/lateral and cephalocaudad) under the lung block as projected on the patient’s skin.  The 
depth should be taken as midway between the entrance and exit points of the opposed radiation beams.  Dose 
measurements at this location will be taken during a fraction with lung shielding in place. 
9.  Unshielded Lung Dose (Point 9): This reference point is the same as point 8.  Dose measurements at this 
location will be taken during a fraction without lung shielding in place.  The depth should be taken as midway 
between the entrance and exit points of the opposed radiation beams. 
 
1.1.4.2 Dose definition: 
The absorbed dose is specified as centigray (cGy)-to-muscle. 
 
1.1.4.3 Prescribed Dose, and Fractionation, and Timing:  
165 cGy will be delivered in one fraction. 
 
1.1.4.4 Dose Rate: 
A mid-plane dose rate of between 6 and 15 cGy per minute is required. 
 
1.1.4.5 Dose Uniformity: 
The objective is to keep the dose throughout the body, defined to extend to within 2 mm of the skin surface, to at 
least 90% of the prescription dose. In addition, the brain dose shall not exceed 107% of the prescription dose.  
 
For AP/PA treatments, partial transmission lung blocks will be used to limit the overall total lung dose. The dose 
at the midpoint of the thickest part of the body, while in the treatment position, should be determined and if 
necessary, modifications made to the treatment to raise the dose in this region to at least 90% of the prescription 
dose. 
 
In order to satisfy the requirement that the skin dose at a depth of 2 mm is within at least 90% of the prescription 
dose, beam spoilers or other equally effective devices should be used. The field size shall be such that no part of 
the patient extends into the portion of the penumbra region where the dose is less than 90% of the central axis 
dose. 
 
1.1.5 Treatment Technique 
Patients will be treated using AP/PA fields in an upright seated or standing position in a TBI positioning device.  
Treatment will be delivered with equally weighted parallel opposed portals, with each treatment including both 
AP and PA fields.  An acceptable alternate arrangement will include equally weighted AP-PA parallel opposed 
fields delivered to the patient in a lateral decubitus position on a treatment couch or gurney. 
 
Young patients requiring anesthesia will be treated in an AP/PA configuration at extended distance.  If more than 
a single field is needed to accomplish treatment, the field junction should be at the level of the thighs. 
 
1.1.5.1 Dose calculation for the Prescription Point 
The calculation of the treatment time or the monitor units for the prescribed dose can be carried out using standard 
techniques. However, TBI presents special problems relative to the routine treatment situation in that the field 
sizes are much larger and the treatment distances much longer.  The TBI percent depth dose (PDD) or Tissue 
Maximum Ratio (TMR) and output factors should be measured under TBI treatment conditions for a range of 
phantom sizes to establish the database for TBI beam-on time calculations or to validate the calculation 
methodology. 
 


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Typically, a calculation methodology will be adopted which uses PDD or TMR and output factors measured 
under standard conditions but then modified to account for the larger treatment distance. For example, modified 
values for inverse square corrected percentage depth dose or tissue-air ratios and tissue-phantom ratios are 
necessary for some treatment units when the patient is positioned at a long distance from the photon source and 
near the floor or one wall of the room. Also, some deviation from an exact inverse square decrease with distance 
has been demonstrated for certain geometries. 
 
Measurements of dose at the center of a phantom about the size of the typical patient should be performed and 
compared to the calculated dose. If differences are found, additional correction factors should be introduced to the 
calculation method. 
 
1.1.5.2 Critical Organ Dose Points 
The required dose calculations should be performed for the 9 points referenced above (1.5.4.1). The midline dose 
at these locations should be recorded on the TBI Summary Form.  
 
1)  The dose can be calculated based on the thickness at each location and factors appropriate to the TBI treatment 
conditions. 
 
It is recommended that entrance and exit TLDs or diodes be placed on the patient at each required dose 
assessment location.  The midline dose can be calculated from these measurements making the appropriate 
corrections to the readings and then averaging the corrected values. 
 
In younger patients it is also recommended that TLDs or diodes be placed underneath the lung blocks to 
document the transmission dose and scatter dose. 
 
1.2 Lung Shielding 
Lung shielding shall be used in all patients 
 
1.2.1 Lung Block design 
Lung blocks will confirm to the following guidelines:  The lateral edges will be 1.0 – 1.5 cm from the inner 
border of the ribs; the inferior edges will be 1.0 – 1.5 cm from the dome of the apex of the diaphragm; the 
superior borders will be 1.0 – 1.5 cm below the clavicles; the medial border 2.0 – 2.5 cm from the lateral edges of 
the thoracic vertebral bodies.  No contouring of the lung shields will be done around the hilum unless there is a 
residual abnormal hilar adenopathy, in which case the margins around the hilar mass will be 1.0 – 1.5 cm. 
 
1.2.2 Timing 
Lung blocks will be employed for sequential treatments starting with the first treatment.  Should patient infirmity 
preclude upright positioning during a fraction when lung shielding is prescribed, that patient may be treated in the 
lateral decubitus position without lung shielding, and lung shielding can be deferred until the next treatment 
fraction. Alternatively, lung blocks may be used in the lateral decubitus position for patients being treated 
exclusively in that position. 
 
For children receiving TBI under anesthesia, treatments will be performed in a modified supine and prone 
position, with the appropriate lung shielding as specified in the protocol. 
 
1.2.2.1 Fractionation Schema 
For a fractionation scheme of 165 cGy bid x 4 days; lung shielding will be used for the first 4 fractions.  This 
schema is calculated to deliver a nominal dose of approximately 825 cGy to both lungs, without correction for 
lung homogeneity (660 cGy x 0.25 = 165 cGy + 660 cGy = 825 cGy). 
 
1.2.3 Electron boost 
No compensatory electron boost of that portion of the chest wall shielded by the lung blocks is required. 
 
The lung dose will be reported on the TBI Summary form. 
 


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1.3 Testes Boost 
 
1.3.1 Patients 
All patients with biopsy-proven testicular leukemia at relapse will receive a testicular radiation boost (400 cGy in 
1 fraction) prior to TBI.  It is recognized that there will some patients who previously received radiotherapy prior 
to relapse.  These patients will be treated similarly. 
 
1.3.2 Equipment 
1.3.2.1 Modality 
Electron beams. The selection of energy is determined by the dose uniformity criterion, to cover the testes with no 
less than the 80% isodose curve. 
 
1.3.3 Target Volume 
The target volume consists of the testes in the scrotal sac. (Note: Cremasteric reflex may move testes high up in 
the inguinal canal.) 
 
1.3.4 Target Dose 
400 cGy 
 
1.3.4.1 Prescription Point 
The prescription point is to Dmax 
 
1.3.4.2 Dose definition 
The absorbed dose is specified as centigray (cGy)-to-muscle. 
 
1.3.4.3 Prescribed Dose and Fractionation 
The total dose to the prescription point shall be 400 cGy in 1 fraction. 
 
1.3.4.4 Timing 
Testicular irradiation should start within 3 treatment days of the beginning of the TBI preparative regimen if they 
have not already received testicular irradiation as part of their induction therapy. 
 
1.3.4.5 Dose Uniformity: 
The variations of dose within the planning target volume shall be within +20%, -20% of the dose to the 
prescription point. The uniformity requirement can in general be met with an electron beam of appropriate energy 
provided bolus is used, which is the simplest technique.  
 
1.3.5 Treatment Technique 
 
1.3.5.1 Patient Position 
The patient shall be treated in the supine position. 
 
1.3.5.2 Field-shaping 
Field shaping can be done with electron cutout blocks of the appropriate thickness. 
 
 
 
1.3.6 Normal Tissue Sparing 
 
1.3.6.1 Perineum: 
The testes shall be supported posteriorly and, if possible, extended caudally in order to minimize perineal 
irradiation. The field shall not be angled towards the perineum. 
 
1.3.6.2 Penis: 
The penis shall be excluded from the field by fixing it to the skin over the symphysis pubis. 


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1.4 Questions 
Questions regarding the radiotherapy section of this protocol should be directed to the study radiation oncologist 
coordinators, Dr. Ralph Ermoian (206)598-4100. 
 
 
 
1.5 Definitions of Deviation in Protocol Performance 
Prescription Dose 
Minor Deviation: 
The dose to the prescription point differs from that in the protocol by between 6% and 10%. 
Major Deviation: 
The dose to the prescription point differs from that in the protocol by more than 10%. 
 
Dose Uniformity 
Minor Deviation: 
The dose to any of the reference points in Section 16.5.5.1 differs from the protocol dose by more 
than 10% but less than 20%. 
Major Deviation: 
The dose to any of the reference points in Section 16.5.5.1 differs from the protocol dose by more 
than 20%. 


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Appendix K:  Guidelines for Pediatric Research Blood Draws  
 
Maximum recommended blood draws 
A single research blood draw will be ≤1ml/kg to a maximum of 50ml. 
 The research blood draw volume will not exceed the lesser of 100 ml or 3 ml per kg in an 8-week period.   
 
 
 
Weight in 
kilograms 
Maximum total amount at a single blood draw 
(ml) 
(≤1ml/kg or <50ml whichever is less) 
Maximum total amount in 8-week period (ml)  
(≤3ml/kg or <100ml whichever is less) 
5-6.9 
5 
15 
7-8.9 
7 
20 
9-9.9 
9 
25 
10-14.9 
10 
30 
15-19.9 
15 
45 
20-24.9 
20 
60 
25-29.9 
25 
75 
30-34.9 
30 
90 
35-39.9 
35 
100 
40-44.9 
40 
100 
45-49.5 
45 
100 
≥50 
50 
100