Conditions: Graft Versus Host Disease Leukemia Myelodysplastic Syndromes
Keywords: graft versus host disease adult acute lymphoblastic leukemia in remission recurrent adult acute lymphoblastic leukemia childhood acute lymphoblastic leukemia in remission recurrent childhood acute lymphoblastic leukemia adult acute myeloid leukemia in remission recurrent adult acute myeloid leukemia adult acute myeloid leukemia with 11q23 (MLL) abnormalities adult acute myeloid leukemia with inv(16)(p13;q22) adult acute myeloid leukemia with t(15;17)(q22;q12) adult acute myeloid leukemia with t(16;16)(p13;q22) adult acute myeloid leukemia with t(8;21)(q22;q22) childhood acute myeloid leukemia in remission recurrent childhood acute myeloid leukemia childhood myelodysplastic syndromes previously treated myelodysplastic syndromes refractory anemia with excess blasts de novo myelodysplastic syndromes secondary myelodysplastic syndromes
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 41
Healthy volunteers: no

Brief summary:
RATIONALE: Allogeneic hematopoietic stem cell transplant (HSCT) is a treatment that can cure acute leukemia and myelodysplasia. After giving the patient chemotherapy and total body irradiation to stop the growth of cancer and remove the patient's diseased bone marrow, healthy stem cells from a donor are infused into the patient to replace the patient's bone marrow and make red and white blood cells and platelets. Unfortunately HSCT is often complicated by 'graft versus host disease' (GVHD) in which the transplanted cells from a donor can make an immune response against the body's normal cells and cause tissue damage and severe symptoms. Removing a subset of the donor T cells, called 'naive T cells', before transplant may reduce the frequency and intensity of GVHD.

PURPOSE: This phase II trial will determine whether the removal of the naive T cells from donor cells can decrease the rate and severity of graft-vs-host disease while preserving specific immunity against infections in patients with acute leukemia or advanced myelodysplastic syndromes.

Detailed description:
OBJECTIVES:

Primary

* Estimate the probability of grades II-IV acute graft-vs-host disease (GVHD) in patients with acute leukemia or advanced myelodysplastic syndromes treated with CD45RA+ T-cell-depleted allogeneic peripheral blood stem cell transplantation and compare this to relevant historical experience.
* Estimate the probability of graft failure in these patients.

Secondary

* Evaluate immune reconstitution and pathogen-specific T-cell reconstitution in these patients.
* Estimate the probability of transplant-related mortality by day 100 in these patients.
* Estimate the probability of relapse in these patients.
* Estimate the probability and severity of chronic GVHD in these patients.

OUTLINE: This is a multicenter study.

* Myeloablative conditioning regimen: Patients undergo total body irradiation twice daily for 4 days (Days -10 to -7) Patients also receive thiotepa IV over 4 hours for 2 days (Days -6 and -5) and fludarabine phosphate IV over 30 minutes for 5 days (Days -6 to -2.)
* Transplantation: Patients receive a CD34+ enriched allogeneic peripheral blood stem cell (PBSC) product followed by a CD45RA+ T-cell-depleted allogeneic PBSC product on day 0.
* Graft-vs-host disease (GVHD) prophylaxis: Patients will receive Tacrolimus as per cohort 1. If the rate of grade II-IV acute GVHD in the first 35 patients is significantly reduced (compared to historical controls), subsequent patients are enrolled in cohort 2.

  * Cohort 1: Patients receive tacrolimus IV continuously or orally twice daily beginning on day -1 and continuing until day 50, followed by a standard taper in the absence of grade II-IV acute GVHD.
  * Cohort 2: Patients receive tacrolimus IV continuously or orally twice daily beginning on day -1 and continuing until day 30, followed by a rapid taper in the absence of grade II-IV acute GVHD.

Patients are followed actively for at least 1 year post transplant.

Arms:
arm 1: CONDITIONING: Patients undergo total-body irradiation twice daily on days -10 to -7. Patients also receive thiotepa IV over 4 hours on days -6 and -5 and fludarabine IV over 30 minutes on days -6 to -2. TRANSPLANTATION: Patients undergo infusion of CD34+ enriched allogeneic peripheral blood stem cells (PBSC) followed by CD45RA+ T-cell-depleted allogeneic PBSC on day 0.

GRAFT-VS-HOST DISEASE PROPHYLAXIS: Cohort A: Patients receive tacrolimus IV continuously or orally twice daily beginning on day -1 and continuing until day 50 followed by standard taper in the absence of grade II-IV acute GVHD. Cohort B: Patients receive tacrolimus IV continuously or orally twice daily beginning on day -1 and continuing until day 30 followed by rapid taper in the absence of grade II-IV acute GVHD.

Interventions: intervention 1: Fludarabine Phosphate intervention 2: Tacrolimus intervention 3: Thiotepa intervention 4: Total-Body Irradiation (TBI) intervention 5: Magnetic Affinity Cell Sorting intervention 6: Peripheral Blood Stem Cell Transplantation intervention 7: Allogeneic Hematopoietic Stem Cell Transplantation intervention 8: T Cell-Depleted Hematopoietic Stem Cell Transplantation
Intervention descriptions: intervention 1: Fludarabine will be administered in a dose of 25 mg/m2/day IV over approximately 30 minutes for 5 consecutive days (day -6 to -2). The total dose of fludarabine will be 125 mg/m2. intervention 2: Tacrolimus will be administered beginning on day -1 at a dose of 0.03 mg/kg/day by continuous IV infusion.

For the first cohort of 35 patients, 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.

In the second cohort of 25 patients if there is no evidence of grade II GVHD on or prior to day 30, tacrolimus should then be tapered at the rate of approximately 8% of the day 30 dose each week for liquid, and 33% of the day 30 dose per month for capsules. intervention 3: 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). intervention 4: TBI will be given as 165 cGy fractions twice per day x 4 days - total dose 1320cGy (days -10 to -7). intervention 5: Device intervention 6: Patient will undergo a PBSC transplantation intervention 7: Patients who are considered appropriate candidates for allogeneic hematopoietic stem cell transplantation intervention 8: Patients who are eligible will receive a T Cell-Depleted Hematopoietic Stem Cell Transplantation

Protocol document:
Prot_SAP_000.pdf: 
 
 
FRED HUTCHINSON CANCER RESEARCH CENTER 
UNIVERSITY OF WASHINGTON SCHOOL OF MEDICINE 
SEATTLE CHILDREN’S 
 
 
A Multi-center Phase II Study of Selective Depletion of 
CD45RA+ T Cells from Allogeneic Peripheral Blood 
Stem Cell Grafts for the Prevention of GVHD 
 
NCT00914940 
 
Current Version: 08/25/2014 
 


IRB Approved 
Document Release Date: 09/04/2014 
2222.00 
1R0 REC'D OCT 16 2018 
FRED HUTCHINSON CANCER RESEARCH CENTER 
UNIVERSITY OF WASHINGTON SCHOOL OF MEDICINE 
SEATTLE CHILDREN'S 
Current Version: 08/25/2014 
Previous Version: 07/11/2014 
1. Title of Protocol: A Multi-center Phase II Study of Selective Depletion of CD45RA+ T Cells 
from Allogeneic Peripheral Blood Stem Cell Grafts for the Prevention of GVHD 
Coordinating Site: 
Investigator 
Professional Title 
Telephone number 
Marie Bleakley, MD, 
Assistant Member FHCRC 
(206) 667-6572 
PhD (PI) 
Assistant Professor of Pediatrics, UW 
(206) 469-4487 (pager) 
Stanley Riddell, MD 
Member, FHCRC 
(206) 667-5249 
Professor of Medicine, UW 
Shelly Heimfeld, PhD 
Shelly Heimfeld, PhD 
(206) 667-4004 
Member, FHCRC 
Biostatistician 
Ted Gooley, PhD 
Associate Member, FHCRC 
(206) 667-6533 
Affiliate Associate Professor, UW 
Radiation Oncologist 
Ralph Ermoian, MD 
Assistant Professor of Radiation Oncology, UW 
(206) 598-4100 
Research Staff 
Colette Chaney, RN 
Research Nurse 
(206) 667-5823 
(206) 559-4104 (pager) 
(206) 667-6056 (fax) 
Participating Sites: 
Yale University School of Medicine/Yale New Haven Hospital 
Investigator & IDE Sponsor 
Warren Shlomchik, MD 
Professor of Medicine, Yale University School of 
(203) 737-2478 
Medicine 
Co-Investigators 
Kenneth Roberts, MD 
Associate Professor in Therapeutic Radiology, 
(203) 785-2957 
Yale University School of Medicine 
Stuart Seropian, MD 
Associate Professor of Medicine, Yale University 
(203) 737-5751 
School of Medicine 
Dennis Cooper, MD 
Professor of Medicine, Yale University School of 
(203) 737-5751 
Medicine 
FHCRC IRB Approval 
Emergency (24 hour) Phone: (206) 598-8902; Fax: (206) 598-4034 
NOV 28 2018 
Document Released Date 
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Table of Contents 
2. Introduction 
4 
3. Background 
4 
4. Objectives 
12 
Primary 
12 
Secondary 
12 
5. Patient Selection 
12 
Inclusions 
12 
Exclusions 
12 
6. Donor Selection 
13 
Inclusions 
13 
Exclusions 
13 
7. Evaluation and counseling of patient and donor 
14 
8. Protocol registration 
14 
9. Plan of treatment 
15 
Conditioning regimen 
15 
CNS prophylaxis and testicular irradiation 
16 
Collection of G-CSF mobilized PBSC 
16 
PBSC graft engineering/cell selection 
17 
PBSC infusion 
18 
Post transplant immunosuppression 
18 
Use of hematopoietic growth factors 
20 
10. Evaluation 
20 
Donor evaluation 
20 
Patient pre-transplant evaluation 
21 
Patient post-transplant evaluation guidelines 
22 
11. Irradiation, drugs, and PBSC administration - toxicities and complications 
24 
Total body irradiation 
24 
Thiotepa 
25 
Fludarabine 
25 
PBSC infusion 
25 
G-CSF 
25 
Tacrolimus 
25 
12. Protocol enrollment and special considerations 
27 
Projected target accrual 
27 
Infection prophylaxis 
27 
Ursodeoxycholic acid prophylaxis of hepatic complications 
28 
Management of acute and chronic GVHD 
28 
Management of graft failure 
28 
Management of relapse 
28 
13. Records 
29 
14. Evaluation and statistical considerations 
29 
Type of study 
29 
Definition of endpoints 
29 
Statistical analysis and stopping rules 
31 
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15. Guidelines for reporting and tacking events 
35 
Toxicity Grading 
35 
Definitions 
35 
Tracking of Events 
35 
Reporting to Coordinating Center 
35 
Reporting Requirements to the FHCRC IRB 
36 
Reporting to the Sponsor 
37 
Reporting Requirements to Yale IRB 
37 
Reporting to FDA 
37 
16. Data safety monitoring plan 
38 
Monitoring the progress of trials and the safety of participants 
38 
Plans for assessing data accuracy and protocol compliance 
39 
17. References 
39 
18. Appendices 
Cell selection flow sheet and guidelines 
41 
Product testing 
43 
Positive culture from donor product —action plan 
44 
Infusion of selected cells 
45 
Acute GVHD staging and grading assessment 
47 
Grading of chronic graft versus host disease 
48 
Potential adverse events associated or expected with hematopoietic cell transplantation 
49 
Karnofsky performance status scale 
50 
181. Weight/Adjusted body weight for drug dosing 
51 
I8J. Coordinating center functions 
52 
18K. Radiotherapy treatment plan 
53 
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Introduction 
Graft versus host disease (GVHD) is a frequent complication of allogeneic hematopoietic 
cell transplantation (HCT). 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, the incidence of grade 
II — IV acute GVHD following allogeneic peripheral blood stem cell (PBSC) grafts from 
human leukocyte antigen (HLA) matched sibling donors at the FHCRC and other centers 
is 40-80% [2-4]. In HCT using HLA-identical sibling donors, GVHD results from 
recognition of minor histocompatibility (H) antigens expressed on recipient tissues by 
donor T cells [5]. 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 [6, 7]. 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 peripheral blood stem cell (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 the rate of grades II-IV acute GVHD and provide rapid recovery of T cell 
immunity to pathogens after HCT from HLA-identical sibling donors. 
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 c43 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]. 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 [8]. 
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 these peptides can be recognized as minor H antigens by donor T cells [5]. 
The activation of donor T cells to recipient minor H antigens is likely to occur after 
contact with specialized APCs and leads to proliferation of T cells and their 
differentiation to effector cells that produce cytokines including IFN-y and TNF, and 
mediate cytotoxicity against recipient tissues [5]. 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. 
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, is used at most transplant centers to prevent 
GVHD [9]. The development in the early 1980s of the combination of a short course of 
methotrexate with the calcineurin inhibitor cyclosporine represented a significant 
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advance in the prophylaxis of GVHD, but major improvements in pharmacologic 
immunosuppression have not been achieved since that time [9, 10]. At the FHCRC, a 
short course of methotrexate combined with either cyclosporine or FK506 continues to be 
the most frequently used immtuiosuppressive regimen for GVHD prophylaxis after 
allogeneic HCT. With this regimen of GVHD prophylaxis, the incidence of grade II-IV 
acute GVHD was 61% in patients with acute leukemia that had a HCT from an HLA-
identical sibling donor at the FHCRC since 2003. Patients that develop acute GVHD 
require treatment with additional immunosuppressive drugs, which increases their risk of 
post transplant infections and may diminish the graft versus leukemia (GVL) effect [11]. 
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 sibling donors. 
Thus, 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 [6, 7, 12]. A variety of methods have been 
used to remove T cells from bone marrow or peripheral blood stem cell (PBSC) grafts 
including soybean lectin agglutination, monoclonal antibodies, and positive selection of 
CD34+  hematopoietic progenitors [6, 7, 12]. Depending on the approach, the extent of T 
cell depletion can vary from 2-5 logio More complete T cell depletion is generally 
associated with less GVHD, although an absolute threshold dose of donor T cells for 
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 [6, 7, 12, 13], but recipients of T-cell depleted grafts have poor 
reconstitution of T cell immunity to pathogens, and an increased risk of graft rejection 
and relapse [6, 7, 12-14]. A Phase 2 study at Memorial Sloan Kettering Cancer Center 
(MSKCC) used soybean lectin agglutination and sheep red blood cell rosette depletion to 
remove T cells from donor bone marrow, and administered an intensified pretransplant 
conditioning regimen that included total body irradiation (TBI 
Gy with lung 
shielding to 8 — 9 Gy), cyclophosphamide (Cy), and thiotepa to patients receiving 
allogeneic HLA-identical HCT for AML in remission. ATG was also administered pre 
and post transplant to prevent graft rejection. Graft rejection occurred in 0 of 39 patients 
in the MSKCC study [12]. The toxicity with this conditioning regimen was tolerable with 
2 deaths in the 39 patients due to veno-occlusive disease and interstitial pneumonia, 
respectively. The risk of Epstein-Barr virus lymphoproliferative disease (EBV-LPD) was 
—10% in the MSKCC studies and 6/39 patients died of infectious complications (3 — 
cytomegalovirus (CMV) pneumonia, 1 - toxoplasmosis, 1 — pneumocystis carinii, 1 
fungal pneumonia). The relapse rates for AML patients in V' and 2nd complete remission 
at the time of transplant in the MSKCC study were 3.2% and 12.5% respectively. The 
probability of disease free survival for patients transplanted in 1st  CR was 77.4% at 4 
years, and for patients transplanted in 2nd  CR was 50% at 3 years [12]. The MSKCC 
results are comparable to the results achieved with unmodified PBSC grafts at the 
FHCRC and other centers [2]. Based on these results, the Bone Marrow Transplant 
Clinical Trials Network is currently conducting a multicenter trial of complete T cell 
depletion for AML patients undergoing HCT from HLA-identical sibling donors. The 
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BMT-CTN study is using TBI (13.75 Gy), Cy, and thiotepa as a conditioning regimen, 
and the Miltenyi CliniMACS device to select CD34+cells from G-CSF mobilized PBSC 
using anti CD34 coated imrnunomagnetic beads. CD34 selection with the Miltenyi 
CliniMACS device will provide a 4-5 logo removal of T cells including memory T cells, 
and delayed recovery of T-cell immunity and opportunistic infections are expected to be 
significant obstacles to a successful outcome for these patients. 
Recently, the results of 52 patients (age 19.5 — 62 years) with various hematologic 
malignancies treated at MSKCC with T cell depleted HCT and a conditioning regimen 
consisting of fludarabine (Flu), thiotepa, and TBI (13.75 Gy) without ATG were reported. 
This regimen was well tolerated with a 100 day mortality of —10%. There were no graft 
rejections in this study although two patients received donor lymphocyte infusion for 
mixed chimerism. The rate of acute GVHD was 8%, and the disease free survival was 
72% for patients with standard risk disease and 52% for patients with poor risk disease at 
a median follow-up of 45 months [13]. The cause specific probability of relapse for all 
patients was 17%. Detailed studies of immune reconstitution in these patients were not 
reported, however eight of 52 patients died from infection, and there were two cases of 
EBV-LPD [13]. These findings demonstrate that ATG is not required with this 
conditioning regimen to prevent graft rejection after T cell depleted HCT, and suggest 
that the substitution of Flu for Cy in the regimen may reduce toxicity. 
3C. 
Naive T cells (IN) 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. Recent 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 [15, 
16]. 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 [16]. 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 histocompatiblity differences. The first study to 
examine this question employed a multiple minor H antigen mismatched CD4+ dependent 
GVHD model [B10.D2 (H-2d) 4 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 BI0.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 [17]. 
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 [17-22][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 
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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 [17] 
CD4* CD44+ CD621,- 
(TM) 
vs 
CD4* CD44-  CD62L+ (TN) 
BlO.D2 (H-2 
4BALB/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 [18] 
CD8-' CD44'(Tm) 
VS 
CD8* CD44- (TN) 
CH3.SW (H-2') 
4B6 (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. 
Blood 106 
(abstract 1312), 
2005 [19] 
CD84-CD44+CD621:(Tcm) 
VS 
CD8* CD44- CD62LATO 
CH3.SW (H-2') 
4B6 (H-2b) 
Mild GVHD in 
mice receiving 
Tcm, severe 
GVHD in mice 
receiving TN 
MHC 
Mismatched 
Chen B et al. 
Blood 103: 1534, 
2004 [20] 
CD4+ + CD8+ CD621," 
(TEm) 
VS 
CD4* + CD8+ CD62L+ 
(TN  and Tom) 
C57BL/6 (H-2') 
4 BALB/c (H-2d) 
or C3H/HeJ (H-2k) 
No GVHD in mice 
that received 
CD621; T cells, 
lethal GVHD in 
recipients of 
CD621,* T cells 
MHC 
Mismatched 
Zheng et al, Blood, 
2008.111(4): 2476 
-84.[21] 
CD4a-CD444-CD62L-(TEm) 
VS 
CD4+CD44-CD62L-(TO 
B6
2-4B6 
No GVHD in TEm 
recipients; GVHD 
in TN recipients. 
MHC 
Mismatched 
(Rat Model) 
Xystrakis E et al. 
Eur J Immunol, 
34: 408, 2004 [22] 
CD4' CD45RC 
(TN) vs 
CD4* CD45RCI0w (TM) 
Parental 4F1 
(LEWxBN) 
No GVHD in rats 
receiving TM' 
lethal GVHD in 
rats receiving TN 
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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 [19]. 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. 
Table 1. Rodent studies of T cell depleted BMT with selective addition of T cell subsets 
3D. 
Human T cells s ecific for minor H anti ens are redominantl in the T 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 more than 99% 
of the overall T cell receptor (TCR) diversity [23]. The TM subset, which contains 
CD45R0+ CD621:Tcm and CD62t" TEm populations, and a subset of CD62L- cells that 
re-express CD45RA (TEr4RA), is comprised of cells that have clonally expanded in 
response to antigen stimulation and contains <1% of the overall TCR diversity. 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 only rarely [24] , 
and cross reactivity with minor 1-1 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 
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TM subsets, we purified CD8+ TN (CD45RAbright, CD62L, CD45R0") and TM 
(CD45R0', CD621-+/-, 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 >3 logio higher in the TN subset than in the TM 
subset. Indeed, only very rare cells (-1/10,000,000) 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 [25]. 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 suggest 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 have 
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 [26], 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 
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 
the threshold dose of unselected T cells that causes GVHD after HLA-identical sibling 
HCT [27]. It will therefore allow us to test the hypothesis that CD45RA" T cells can 
safely be transferred with less GVHD. Second, it will 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. 
CD34+ selection consistently yields an enriched progenitor population with an extremely 
low residual T cell content and preserves the CD34+  cells needed for engraftment, 
thereby negating any potential detrimental effects of the T cell manipulation on 
hematopoietic progenitors (S. Heimfeld, personal communication). In 41 CD34+  
selections at the FHCRC the range log T cell depletion was 4.2 to 5.5. Based on a starting 
T cell content of G-CSF mobilized products of 2 x 107 to 1 x 109/kg recipient body 
weight this depletion would result in a residual T cell content of the CD34-positive 
fraction of <= 6 x104 total CDPkg of which 30-70% of the cells will be CD45RA4. The 
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minimum yield of CD34+ cells after CD34 + selection is 40% therefore only G-CSF 
mobilized products where at least 5x106 CD34+ cells/kg of recipient body weight are 
obtained will be processed to ensure that the CD34+ cell dose will be >2x106 CD34+  
cells/kg of recipient body weight. Experience at FHCRC indicates that the products from 
85% of donors will contain >5x106 CD34+ cells/kg of recipient body weight. The CD34 
content of the GCSF mobilized products may potentially be increased further by 
administering GCSF to donors twice daily and a dosing schedule of GCSF 8mcg/kg bid 
will be employed in this protocol. Therefore, it is expected that cell selection will be 
performed for at least 85-90% of the enrolled patients. 
A GMP-grade murine aCD45RA monoclonal antibody that is directly conjugated to 
Miltenyi iron dextran beads has been produced by Miltenyi Biotec under contract from 
the NIH RAID program. Our experience to date both from several mid scale depletions 
of CD45RA + T cells and from full scale depletions on the CliniMACs device indicates 
that the goal of obtaining a total T cell dose of 1-10x 106  
Table 2: Result of Large Scale CD45RA Depletions on GCSF-mobilized Apheresis products 
Unmanipulated PBSC (cells/kg) 
CD45RA depleted PBSC 
(adjusted to 5 x 106 CD3 cells/kg) 
Total TNC 
2.3 x 108  
1.5 x 107 
Total CD3+  T cells 
1.03 x 108  
5 x 106  
CD3' CD45RA + (Naïve) 
5.8 x 107  
9.5 x 103 (8.8 x 102 -2 x 104) 
CD3+  CD45RA- (Memory) 
4.5 x 107  
5 x 106  
CD16+ NK cells 
1.7 x 107  
1.2 x 106  
CD19+  B cells 
1.1 x 107 
1.2 x 104 
CD3+ cells/kg, of which <7.5 x 104/kg will be CD45RA+RO" nave T cells is feasible. 
Residual naïve T cells in the infused product could come from the few contaminating T 
cells in the CD34 + fraction and/or residual nave T cells in the CD34-negative CD45RA 
depleted fraction. We have performed six large-scale CD45RA depletions on GCSF-
mobilized apheresis products and have achieved a nearly 4 log io reduction in 
CD3+CD45RA+ cells (Table 2). If the T cell dose was adjusted to 5 x 106 CD3+ cells/kg 
the infused cells would contain <=2 x104 CD45RA+CD3+ cells/kg after CD45RA 
depletion, even in the event that only a 2.4 logo  depletion was achieved and the donor 
had a particularly high proportion of CD45RA + cells within their original CD3+  
population. Thus, the sum of the cell products after CD34 selection and CD45RA 
depletion is very likely to contain less than 7.5x104 CD45RA+CD3+ cells/kg. CD4+ T 
regulatory cells are CD45R0+ and are retained after CD45RA depletion. NK (CD56+) 
and NK-T (CD56÷, CD3+) are present after CD45RA depletion but are reduced by —1 log, 
while CD19+ and CD20+ B cells are almost completely removed, which should reduce 
the risk of EBV-LPD. 
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 aCD45RA for depletion of TN could compromise the transfer of TM 
responses to these pathogens. Therefore, we compared the frequency of CD8+ T-cells 
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specific for known epitopes of CMV and EBV in PBSC before and after depletion of 
CD45RA + cells using an interferon gamma Elispot assay. Interferon-producing CMV and 
EBV-specific CD8+ T-cells were present in both the CD45RA-depleted fraction and 
unselected PBSC, although there was a reduction in the frequency of these cells in the 
CD45RA-depleted fraction. However, culture of aliquots of the T-cells before and after 
CD45RA depletion with CMV, EBV and influenza peptides for 5 days preferentially 
expanded CMV, EBV, and influenza-specific TM cells in the CD45RA-depleted fraction, 
consistent with the enhanced ability of Tcm to proliferate and differentiate after 
restimulation. CD4+ TM cells are uniformly CD45R0+ CD45RA- and should be retained 
in G-PBSC depleted of CD45RA + T-cells. We analyzed lymphoproliferative responses to 
CMV, VZV, 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. Autologous CD l4 monocytes enriched by immunomagnetic 
selection were used as APCs. 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. It is also anticipated that pathogen specific memory T cell populations 
that are transferred with the stem cell graft will expand in the lymphopenic environment 
post HCT, particularly if CD45RA depletion is effective in preventing GVHD and 
diminishes the need for post transplant corticosteroid therapy. 
Naïve T cells can cause GVHD, but it is uncertain if their removal from a graft could 
compromise immune recovery. The common opportunistic infections in recipients of 
grafts that are depleted of all T cells are consequences of reactivation of latent viruses 
that are controlled by memory T cells. New TN are generated in the thymus from 
precursors within 4-6 months of transplant even following complete TCD in older 
donors. Thus it is anticipated that the capacity of patients to respond to pathogens that 
they have not previously encountered will ultimately be preserved. The newly generated 
TN are likely to be "tolerant" to peptides derived from patient self- proteins due to thymic 
deletion and/or peripheral suppression of autoreactive T cells. 
3G. 
Overview of the Study 
This is a phase 2 study to evaluate selective depletion of naïve T cells from PBSC grafts 
for preventing acute GVHD in patients with acute leukemia or MDS undergoing 
allogeneic HLA-identical HCT from a related donor. We will use a myeloablative 
preparative regimen consisting of fludarabine, thiotepa, and TBI that has resulted in a 
high rate of engraftment when employed with complete T cell depletion without ATG. 
The product that will be administered to each patient will consist of an infusion of 
purified CD34÷ cells/kg of recipient body weight, 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-
through remaining after the CD34+ cell selection. As discussed above, our goal is to 
administer a CD34+ cell dose of >2.0x106/kg of reci?ient body weight and a total T cell 
dose of 1-10 x 106 CD3+ cells/kg, of which <7.5x10 /kg will be CD45RA + RID- naïve T 
cells. 
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The strategy of depleting naïve T cells from stem cell grafts is effective for preventing 
GVHD in murine models without any post grafting immunosuppression but has not been 
evaluated in humans. Thus, for the initial clinical application, we plan to study the safety 
and efficacy of this approach in successive cohorts of patients that will receive 
progressively less prolonged pharmacologic immunosuppression with single agent 
tacrolimus (FK506). The rationale for using single agent tacrolimus as GVHD 
prophylaxis is based on the following considerations. First, randomized studies 
comparing tacrolimus combined with methotrexate to cyclosporine combined with 
methotrexate have suggested tacrolimus is superior to cyclosporine for preventing GVHD 
[4,27]. Second, tacrolimus monotherapy has been shown to result in enhanced neutrophil 
engraftment, shortened hospitalization, and nearly equivalent rates of GVHD compared 
with tacrolimus plus methotrexate [28]. Finally, the administration of standard short 
methotrexate in addition to tacrolimus may cause severe mucosal toxicity due to the 
increased TBI dose employed in this study. Also methotrexate is a cell cycle active agent 
and may interfere with homeostatic and antigen-driven expansion of the memory T cells 
infused with the stem cell graft. 
The first cohort of 35 patients will receive tacrolimus alone as post grafting 
immunosuppression with a planned tacrolimus taper beginning at day 50 in the absence 
of acute GVHD. If the rate of grade II-IV GVHD in this first cohort is reduced 
significantly compared to historical controls, a subsequent group of 25 patients will be 
enrolled and receive a shorter course of tacrolimus for 30 days followed by a rapid taper. 
If the rate of acute GVHD is <10% in both the first and second cohorts combined, future 
protocols may evaluate even shorter or less intense immunosuppressive regimens. The 
risk of graft failure is not expected to be increased given the published results of HCT 
with complete TCD following conditioning with Flu, Thiotepa and TBI. However, graft 
failure will also be a primary safety endpoint in the study and early stopping rules for 
graft failure will be applied. If effective in preventing acute GVHD, this approach to 
allogeneic HCT may reduce chronic GVHD and improve reconstitution of T cell 
immunity to pathogens because of the transfer of memory T cells and the reduced need 
for intensive post transplant immunosuppression. Thus, secondary endpoints will include 
evaluation of the incidence of chronic GVHD and analysis of the tempo of reconstitution 
of both total and pathogen-specific T cells. 
It is not known if the risk of relapse in HCT with selective TN depletion will be increased 
compared with T replete transplantation. Recent studies of complete TCD in acute 
leukemia have not reported higher relapse rates than those reported with =manipulated 
grafts and selective TN depletion will not remove lymphocytes such as NK, NKT and Tcm 
with the potential to induce a GVL effect13. A secondary objective will be to estimate the 
risk of relapse in recipients of CD45RA+ T cell depleted grafts. 
3H. 
Protocol modification (April 2014)  
An interim analysis of protocol 2222 was performed after the first 30 patients were 
treated with a median of 592 days follow-up for surviving patients. In summary, an 
excellent rate of engraftment (100%). a very low (12%) rate of chronic GVHD (a 
secondary endpoint), low rates of relapse (20%) and non-relapse mortality (12%) 
(secondary endpoints), very good immune reconstitution and survival (overall 
survival 80%, disease free survival 68%). In the first 30 patients acute GVHD is not 
lower than the historical benchmark (primary endpoint). However, compared to the 
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first 13 naïve T cell-depleted HCT recipients ( acute GVHD II-IV 85%), acute GVHD 
appears to be lower among 17 subsequent patients (14-30) (53%) who received 
antibiotic prophylaxis directed at gram positive organisms (due to a high rate of 
streptococcus viridans bacteremia amongst the first 13 patients). The original plan 
was to enroll 35 patients with naïve T cell depletion and tacrolimus monotherapy and 
if acute GVHD was reduced in this cohort then to enroll a second cohort of patients 
without tacrolimus. Although the desired reduction in acute GVHD will not be 
achieved in the first 35 patients, the chronic GVHD rates are very low, the general 
outcomes are favorable and it is still possible that acute GVHD will in fact be reduced 
among patients who receive naïve T cell depletion and penicillin. Up to 13 additional 
patients will be enrolled beyond the 35 patients in cohort 1, potentially extending the 
total accrual to 48 patients. Statistical considerations are outlined in section 14C. 
4. 
Objectives 
4A. 
Primary objectives  
I. Estimate and compare to an appropriate historical value the probability of grades II - 
IV acute GVHD in successive cohorts of patients who receive CD45RA+ T cell 
depleted PBSC and: 
tacrolimus for GVHD prophylaxis (day -1 to day 50, then tapered from day 50), 
[Cohort 1]. 
short course tacrolimus for GVHD prophylaxis (day -1 to day 30, then tapered 
from day 30), [Cohort 2]. 
2. Estimate the probability of graft failure in recipients of CD45RA+ T cell depleted 
PBSC. 
4B. 
Secondary objectives  
I. Evaluate immune reconstitution and pathogen-specific T cell reconstitution in 
recipients of CD45RA± T cell depleted PBSC. 
Estimate the probability of transplant-related mortality by day 100 in recipients of 
CD45RA+ T cell depleted PBSC. 
Estimate the probability of relapse in recipients of CD45RA+ T cell depleted PBSC. 
Estimate the probability and severity of chronic GVHD in recipients of CD45RA+ T 
cell depleted PBSC. 
Patient Selection 
5A. Inclusions  
I. Patients who are considered appropriate candidates for allogeneic hematopoietic stem 
cell transplantation and have one of the following diagnoses: 
Acute lymphocytic leukemia in first or subsequent remission 
Acute myeloid leukemia in first or subsequent remission 
Acute lymphocytic leukemia in relapse or primary refractory disease with a 
circulating blast count of no more than 10,000/mm3  
Acute myeloid leukemia in relapse or primary refractory disease with a 
circulating blast count of no more than 10,000/mm3  
Refractory anemia with excess blasts (RAEB-1 and RAEB-2) (if the patient has 
received previous induction chemotherapy within 60 days) 
Chronic myelogenous leukemia with a history of accelerated phase or blast crisis 
(if the patient has received at least one course of induction chemotherapy) 
Other acute leukemia (including but not limited to `biphenotypie, 
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'undifferentiated' or 'ambiguous lineage' acute leukemia) 
Patient age 14 to 55 years 
HLA-identical related donor capable of donating PBSC 
5B. 
Exclusions  
1. Patients < 14 or > 55 years of age 
2. Patients who are pregnant or breast-feeding 
3. Fertile patients unwilling to use contraception during and for 12 months post 
transplant 
4. Patients undergoing second HCT 
5. Patients with CNS involvement refractory to intrathecal chemotherapy and/or standard 
cranial-spinal radiation 
6. Patients on other experimental protocols for prevention of acute GVHD 
7. Patients who are HIV+ 
8. Patients with uncontrolled infections for whom myeloablative HCT is considered 
contraindicated by the consulting infectious disease physician 
9. Patients with organ dysfunction 
Renal insufficiency (creatinine >1.5 mg/di) 
Cardiac ejection fraction <45% 
DLCO corrected (DLCOcorrected) < 60% 
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 2222 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. 
10. Patients with a life expectancy <3 months from co-existing disease other than the 
leukemia or RAEB 
11. Patients with significant other medical conditions that would make them unsuitable 
for transplant 
12. Patients with a known hypersensitivity to tacrolimus 
6. 
Donor Selection 
6A. Inclusions  
Genotypic or phenotypic HLA-identical family members 
Adult donors must have adequate veins for leukapheresis or agree to placement of 
central venous catheter (femoral, subclavian) for collection of PBSC 
Pediatric donors must have adequate veins for leukapheresis (central venous catheter 
is not an option for pediatric donors) 
6B. Exclusions 
Applicable to male patients only: female donors who have previously given birth to 
a male child, or have had a second or third trimester miscarriage or termination of 
pregnancy 
Donors who have received transfusions of red or white blood cells or platelets 
Donors with a CD45 gene mutation with aberrant CD45RA isoform expression 
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Donors <14 years of age 
Donors who for psychological, physiologic, age related or medical reasons are unable 
to undergo PBSC harvest 
Donors who for geographical or social reasons are unable to provide a second 
donation of PBSC in the event of graft failure, EBV lymphoproliferative disease, or 
relapse requiring donor lymphocyte infusion 
Donors who are HIV-1, HIV-2, HTLV-1, HTLV-2 seropositive or with active 
hepatitis B or hepatitis C virus infection 
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)(1). 
Female donors who are pregnant or nursing 
Evaluation and Counseling of Patient and Donor 
Patients will be referred to the Fred Hutchinson Cancer Research Center (FHCRC) or 
Yale University School of Medicine (YUSM) for consideration of a hematopoietic cell 
transplant. Both patient and donor will be evaluated separately. The protocol will be 
discussed thoroughly with patient, donor and other family members if appropriate, and all 
known risks to the patient and donor 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 at FHCRC and YUSM will be obtained using facility 
specific forms approved by the FHCRC Institutional Review Board. A summary of the 
conference detailing what was covered will be dictated for the medical record. 
Protocol Registration 
Overall Procedures: 
Patients enrolled at FHCRC will be assigned to the protocol by the SCCA (Seattle Cancer 
Care Alliance) Clinical Coordinator. Once full consent has been obtained 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. 
Allocation of a study number (unique patient identifier) is required for patients enrolled 
at each site. Each site will keep a log of patients enrolled. 
Once a study number is assigned patients will then be entered into the P2222 (Protocol 
Patient Performance Program (PPPP) database by FHCRC study staff. 
YUSM Procedures: 
When a patient at YUSM is thought to be eligible, pre-screening consent will be obtained 
from the donor and full consent will be obtained from the recipient. When signed 
consents are obtained, YUSM study staff will notify the FHCRC study research nurse by 
email and provide copies of the signed consent forms (consent for patient and pre-
screening consent for the donor) and patient and donor registration forms via fax (206) 
667-6056 or email. 
The FHCRC study nurse will coordinate with the FHCRC regulatory coordinator to 
ensure that the completed patient and donor registration forms and consent documents are 
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faxed to the FHCRC Registration Office. The FHCRC Registration Office will generate a 
unique patient identifier. 
The unique patient identifier will be provided by email to the designated YUSM research 
nurse within 48 business hours after receipt of the signed patient consent and donor pre-
screening consent forms. 
If pre-screening indicates that the donor is eligible, which includes primarily 
documentation of normal CD45RA expression, a signed donor consent form for 
continued participation in the study will be obtained and provided via fax to FHCRC 
(206) 667-6056 and the FHCRC research staff will fax the donor consent to the FHCRC 
Registration Office. The source documents for pre-screening testing of CD45RA 
expression will be submitted to the FHCRC research nurse when available or may be held 
until the full eligibility package is assembled for both patient and donor. 
The FHCRC study nurse and FHCRC PI will review all source documents supporting 
eligibility criteria for the patient and donor and will verify if the patient and donor are 
eligible. 
An email will be sent to Dr. Warren Shlomchik (warren.shlomchik(4ale.edu) containing 
the FHCRC Pl's determination of eligibility within 48 hours (during the Monday-Friday 
workweek) of receiving all required source documentation. 
Informed consent must be signed by the transplant recipient or his/her legal guardian and 
confirmation of eligibility must be verified by the coordinating center PI prior to the 
performance of any study-specific treatment. 
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 schematically in 
Table 3. 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). 
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). 
Patients will receive fludarabine 25 mg/m2/day ( m2 always to be 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). 
Day -1 will be a day of rest. 
GCSF-mobilized CD34 enriched PBSC and CD45RA depleted cells will be infused on 
day 0. 
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Table 3. Conditioning regimen 
Day relative to infusion of GCSF mobilized CD34 enriched PBSC and CD45RA det,leted cells 
Treatment 
-10 
-9 
-8 
-7 
-6 
-5 
-4 
-3 
-2 
-1 
0 
1 
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 G-PBSC 
CD34 + and CD45RA- cells 
X 
+1- 
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. No dose of intrathecal therapy will be given within 72 
hours of CD34 enriched PBSC infusion. Male patients with ALL will receive a 400 cGy 
testicular irradiation boost pre-transplant during the conditioning regimen. 
Collection of G-CSF mobilized PBSC  
I. G-CSF administration to donors: All donors will receive G-CSF 16 µg/kg/day 
administered as 8 µg/kg two times per day by subcutaneous injection for 5 
consecutive days on day -5 thru day-1, and in some cases in the morning on Day 0. 
Donors who fail to mobilize adequately and require a day +1 collection may also 
receive GCSF 8 µg/kg in the pm on Day 0. G-CSF doses will be administered at 
approximately 8:00 a.m. and between approximately 4:30 pm and 8 pm each day in 
the Outpatient Department. For Yale patients, the weekend p.m. dose may be 
administered in the in-patient BMT unit. 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 must be confirmed with the personnel in the Apheresis 
Facility. Day 0 should be fixed on a Tuesday-Thursday. 
2. PBSC collection by apheresis: Donors may undergo vein to vein apheresis 
collections. If apheresis cannot be performed by this technique, a percutaneous 
Mahurkar catheter may be inserted for adult donors. PBSCs will be collected using 
standard apheresis procedures. We will aim to process up to 18L liters of whole blood 
per collection for all donors. The first apheresis will be performed on day -1, and 
stored in the refrigerator at 4° C overnight according to the Standard Operating 
Procedures for overnight storage of PBSC products at each institution. A second 
PBSC collection will be performed on the morning of day 0. 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). Cell selection will be performed on day 0 (9D & Appendix A) and the final 
product will be tested for quality control (9D.3) and infused on day 0 (9E). In a small 
minority of cases CD34 + cell target numbers may not be achieved by day 0 and a third 
PBSC collection will be required on day I. This PBSC product will not undergo any 
cell selection process and will be infused on day 1 (9D and 9E). 
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Table 4. Treatment schema-donor 
Day relative to infusion of GCSF mobilized CD34 enriched PBSC and CD45RA depleted cells 
Treatment 
-5 
-4 
-3 
-2 
-1 
0 
1 
G-CSF 8 µg/kg s.c. bid. 
X 
X 
X 
X 
X 
+/- 
X 
X 
X 
X 
X 
+/- 
Apheresis 
X 
X 
+/- 
9D. 
PBSC graft engineering - CD34 + cell selection and depletion of CD45RA + cells  
We will perform cell selections only when the PBSC product from days -1 and 0 
combined contains >5x106 CD34 + cells/kg of recipient body weight to ensure that the 
CD34+ cell dose will be >2x106 CD34' cells/kg of recipient body weight. 
If the PBSC product from days -1 and 0 combined contains <5x106 CD344-  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. These 
patients will receive GVHD prophylaxis with tacrolimus and methotrexate (see 
section 9F). Up to 10% of patients enrolled on protocol 2222 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 7.5 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 >90% of patients. 
The cell selection will be performed as outlined below and in the flow chart provided as 
Appendix A. 
I. CD34' cell selection (Also see Appendix A) 
The apheresis products obtained on day —1 and day 0 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 Procedure. The goal is to obtain a target CD34 cell dose of 
>2.0x106/kg of recipient body weight to administer with the engineered graft and this 
goal is expected to be achieved for all products that undergo cell selection. The 
CliniMACS system of CD34 selection results in a 4-5 logio depletion of CD3+ cells 
which will result in a median CD3 cell dose of <6x I04/kg of which 30-70% may be 
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). The goal is to administer <7.5 x104/kg of 
CD45RA+RO- cells and a total CD3+ T cell dose of 1-10 x106/kg to the patient 
including both the CD34 + enriched fraction and the CD45RA depleted fraction. To 
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achieve these total T cell numbers, a proportion of the CD45RA depleted cells will be 
administered to the patient following administration of the positively selected CD34+  
cell product. 
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"): 
Viability testing 
Sterility testing 
Total nucleated cell count 
Immunophenotyping by flow cytometry will be performed on the initial apheresis 
product and cells that have completed the selection process: 
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 
Additional cell markers will be evaluated and may include 
CD3+ CD45RA + CD45R0- CCR7+ (TN) 
CD8+CD45RA+ CD45R0- CCR7+ (CD8 TN) and CD4+CD45RA+  
CD45R0- CCR7+ (CD4 TN) 
CD3' CD45R0+ (TM), CD3+CD8+CD45R0+ (CD8 TM)  and CD3+ CD4+  
CD45R0+ (CD4 TM) 
CDT CD45R0+ CCR-7+ (Tcm) and CD3+  CD45R0+ CCR-T (TEm) 
CD8 + CD45R0+ CCR-7+ (CD8 Tcm) and CD8+ CD45R0+ CCR-7- (CD8 
TEm), and CD4 + CD45R0+ CCR-7+ (CD4 Tcm) and CD4 + CD45R0+  
CCR-T (CD4 TEN) 
CD3+/- CD56+, CD3+/- CD16+, CD14+, CD19+ and CD20+  
CD4 + CD25+FoxP3+. 
a. 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 cytolcine staining and/or 
tetramer analysis. 
PBSC Infusion  
The CD34+ selected and CD45RA-depleted products will be infused through a central 
venous catheter on day 0 of the transplant. The CD34+ product will be infused first, 
followed as soon as possible thereafter, by the CD45RA-depleted product (see Appendix 
D). If additional CD34+ cells are required (see section 9D.1 and Appendix A and D) 
unselected PBSC will be administered through the central venous catheter on day 0 or 1. 
Post-transplant immunosuppression  
In the first cohort of 35 patients, GVHD prophylaxis will consist of tacrolimus (FK506) 
alone for 50 days followed by a standard taper if there is no GVHD (see section 9F. 1 b). If 
in the first cohort, the rate of acute GVHD grades II or higher is significantly lower than 
historical controls (see section 14C Statistical Analysis) and the rate of acute GVHD 
grades III-1V does not exceed 15%, a subsequent cohort of 25 patients will receive a 
shorter course of tacrolimus alone for 30 days post transplant followed by a more rapid 
taper if they do not develop GVHD (see section 9F. 1 c). 
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In the event that the target cell doses cannot be achieved for a patient using the cell 
selection procedure described in section 9D.1 and 2 and Appendix A and the patient 
requires the infusion of unmanipulated G-PBSC methotrexate will be administered in 
addition to tacrolimus as immunosuppression. 
I. Tacrolimus administration  
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. 
For the first cohort of 35 patients, 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. 
In the second cohort of 25 patients if there is no evidence of grade II GVHD on or 
prior to day 30, tacrolimus should then be tapered at the rate of approximately 8% 
of the day 30 dose each week for liquid, and 33% of the day 30 dose per month 
for capsules. 
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 practices. Patients with GVHD may be enrolled in 
investigational protocols for GVHD treatment. 
If there is evidence of disease progression and no evidence of GVHD prior to day 
28, patients in either cohort should taper tacrolimus and all other 
inununosuppressive 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  
Tacrolimus levels should be maintained in the range of 5-15 ng/ml. Whole blood 
trough levels should be obtained on approximately day 2, then approximately 
once each week or more often if clinically indicated, such as when IV to PO 
dosing is initiated. Dose adjustments are made if the levels are outside the 
therapeutic range, or if there is evidence of toxicity that may be related to 
tacrolimus. 
When initiating therapy with voriconazole in patients receiving tacrolimus, it is 
recommended that the tacrolimus dose be reduced to one-third of the original 
dose, (a 67% reduction) and followed with frequent monitoring of the tacrolimus 
blood levels. When voriconazole is discontinued, tacrolimus levels should be 
carefully monitored and the dose increased as necessary. 
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. 
Blood pressure, renal function tests (creatinine, BUN), electrolytes and 
magnesium should be monitored regularly as per institutional standard practice 
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guidelines. 
3. Methotrexate administration 
Most patients treated on this protocol will not receive any methotrexate. If patients do 
require the infusion of unmanipulated G-PBSC because of inadequate CD34+ cell 
content before or after cell processing they will receive methotrexate in addition to 
tacrolimus as immunosuppression. Methotrexate should be administered to this 
subgroup of patients on days +1, +3, +6 and +11 at a dose of 5 mg/m2 by IV push. 
Calculation of m2 should be according to institutional standard practice guidelines. 
The first dose of methotrexate should be given approximately 24 hours after 
completion of all cell infusions, and no sooner than 24 hours after completion of cell 
infusions. If clinically necessary methotrexate levels may be obtained and/or 
methotrexate dose adjusted according to institutional standard practice guidelines. 
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 as indicated in Section 12 E. 
Evaluation 
10A. Donor evaluation 
Pre-donation evaluation should be conducted as per standard institutional practice. The 
following should be obtained for all donors: 
I. Female donors should have a birth history obtained. Male patients with female donors 
who are pregnant, have given birth to male children or who have had a second or 
third trimester miscarriage (or termination of pregnancy) are not eligible for inclusion 
in this protocol as they may have been immunized to minor H antigens. 
All donors should have a blood transfusion history obtained. Donors who have 
received transfusions of red or white blood cells or platelets are not eligible for 
inclusion in this protocol as they may have been immunized to minor H antigens. 
A full vaccination history. 
Testing by flow cytometry for the CD45 gene mutation with aberrant CD45RA 
isoform expression should have been completed prior to enrollment on protocol 2222. 
Donors with aberrant CD45RA isoform expression are not eligible for inclusion on 
this protocol. 
Confirmatory HLA typing between recipient and donor. 
Leulcocytotoxic and/or Fluorescence Activated Cell Sorter cross match between 
recipient and donor. For Yale patients, One Lambda Luminex mixed screen kit as 
well as single antigen kit will be used for HLA antibody screening, and anti-human 
globulin enhanced complement-dependent lymphocytotoxicity method will be 
employed for crossmatching. 
ABO and Rh typing and two way red cell cross match between recipient and donor. 
DNA for storage for chimerism studies. For FHCRC patients, samples will be sent to 
the Clinical Immunogenetics Laboratory (206) 288-1128. For Yale patients, samples 
will be sent to the Molecular Diagnostics Laboratory (203) 688-6985. 
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 
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according to institutional standard practice. Tests are performed using FDA licensed, 
cleared, and approved test kits in a CLIA-certified laboratory. 
CMV and EBV serologies performed within 30 days of donation. 
Serum pregnancy qualitative within 2 weeks of the start of conditioning 
G-CSF and monitoring blood draws (including CBC and Hepatic Function Panel with 
LDH) should be conducted per institutional standard practice guidelines. 
If collection of donor lymphocytes is required post transplant for the treatment of 
relapse or EBV post transplant lymphoproliferative disorder, the PBSC donor should 
be re-evaluated as per institutional standard practice. 
Research Tests: A 90 cc sample of donor blood should be collected (preferably before 
the donor starts GCSF) in heparinized or ACD tubes for immunologic studies, 
specifically TM responses to pathogens by functional assays which may include 
ELISPOT, lymphoproliferation assays, intracellular cytokine staining and/or tetramer 
analysis. SCCA patients send the sample to the Riddell lab D3-313 and contact the 
protocol 2222 research technician at (206) 667-4804 or Marie Blealdey at (206) 469-
4487. For Yale patientssend the sample to the FHCRC Riddell lab. Some assays on 
these cells may also be performed at Yale. 
10B. Patient pre-transplant evaluation 
Patient pre-transplant evaluation should be conducted as per standard institutional 
practice. The following should be obtained for all patients: 
1. History 
Possible antecedent causes for the development of leukemia or MDS including 
prior cytotoxic therapy. 
Hematologic, cytogenetic and flow cytometric findings at diagnosis and at the 
time of enrollment. 
Prior therapies and response to therapy 
2. Laboratory evaluation 
Bone marrow aspirate for morphology with standard cytogenetics and where 
appropriate, molecular cytogenetics, within 30 days of the start of conditioning. 
Bone marrow flow cytometry for determination of blast counts within 30 days of 
the start of conditioning. 
Lumbar puncture with CSF evaluation within 30 days of the start of conditioning. 
Confirmatory HLA typing between recipient and donor. 
Leukocytotoxic and/or Fluorescence Activated Cell Sorter cross match between 
recipient and donor. For Yale patients, One Lambda Luminex mixed screen kit as 
well as single antigen kit will be used for HLA antibody screening, and anti-
human globulin enhanced complement-dependent lymphocytotoxicity method 
will be employed for crossmatching. 
ABO and Rh typing and two way red cell cross match between recipient and 
donor. 
DNA for storage for chimerism studies. For FHCRC patients, samples will be sent 
to the Clinical Immunogenetics Laboratory (206) 288-1128. For Yale patients, 
samples will be sent to the Molecular Diagnostics Laboratory (203) 688-6985. 
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 within 30 days of the start of conditioning. 
CMV, EBV, EISV, VZV serologies within 30 days of the start of conditioning. 
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CMV and EBV plasma PCR and aspergillus galactomannan EIA obtained and run 
within 2 weeks prior to starting conditioning. 
Quantitative immunoglobulins should be obtained within 30 days of the start of 
conditioning. 
I. 
CBC within 2 weeks of the start of conditioning. 
LFTs, to include ALT, AST, and Bili T/D, within 2 weeks of the start of 
conditioning. 
Serum chemistry, to include Na, K, Cl, CO2, BUN, Cr, Ca, Mg, and Phos, with in 
2 weeks of the start of conditioning. 
Serum pregnancy qualitative within 2 weeks of the start of conditioning. 
Research Tests: For FHCRC patients only: Approximately 90 cc of peripheral 
blood, in heparinized or ACD tubes should be sent to the FHCRC Riddell lab for 
immunological studies. 
3. Other evaluations: 
a. MUGA scan or echocardiogram within one month of the start of conditioning. 
IOC. 
Patient post transplant evaluation guidelines  
See Institutional standard practice Policy Guidelines for standard evaluation procedures 
during the first 100 days post transplant, evaluation prior to departure and long-term 
follow-up. In addition: 
Bone marrow aspiration should be performed at baseline and on approximately days 
+28, day +56 and between days +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. 
Donor and recipient chimerism of CD3+ and CD33+ subsets in the peripheral blood 
should be evaluated at approximately day +28 +56, and +80 by STR polymorphism. 
Peripheral blood chimerism studies may also be performed at approximately 6 and 12 
months post transplant. 
Quantitative immunoglobulins should be assessed at baseline and on approximately 
days +30, +60 and + 90 and if possible on approximately day +180 and +360. 
Antibody responses to recall antigens (tetanus toxoid, diphtheria toxoid and measles 
virus) should be assessed at one year post transplant if possible. 
CMV PCR should be performed once every week until day 100 or departure, and 
after departure according to institutional guidelines. Preemptive antiviral therapy 
should be instituted for a positive PCR according to institutional guidelines. 
Surveillance for EBV should be performed using a quantitative EBV DNA PCR assay 
approximately weekly until day 100 or departure. Infectious disease consultation 
should be obtained for patients who develop EBV DNA levels of >1000 copies/ml 
plasma on any test. For Yale patients, EBV DNA is reported as number of 
genomes/100,000 WBC's. Infectious disease consultation will be obtained if EBV 
DNA levels are > 1000 genomes/100,000 WBC's. 
Galactomannan EIA (GMEIA) should be performed approximately once per week 
between day 28 and day 100 or departure. Infectious disease consultation should be 
obtained for patients who develop a positive GMEIA. 
Research Tests: 
a. Immune reconstitution should be assessed on approximately day +28, +56, and 
between approximately day +80 and +100. Where possible immune reconstitution 
should also be assessed at approximately day +180 and +360. SCCA patients send 
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approximately 90 cc of heparinized (or ACD) peripheral blood to the Riddell lab 
D3-313 contact the protocol 2222 research technician on (206) 667-4804 or Marie 
Bleakley (206) 469-4487. For Yale patients, 90 cc of heparinized (or ACD) 
peripheral blood will be sent to the FHCRC Riddell lab. 
i. Lymphocyte subsets will be enumerated using flow cytometry. Cell markers 
may include: 
CD3+ CD45RA+ CD45R0- CCR7+ (TN) cells 
CD8+CD45RA+ CD45R0- CCR7+ (CD8 TN) and CD4+CD45RA+  
CD45R0- CCR7+ (CD4 TN) cells 
CD3+ CD45R0+ (TM), CD3+CD8+ CD45R0+ (CD8 TM)  and CD3+ CD4+  
CD45R0+ (CD4 TM)  cells 
CD3+ CD45R0+ CCR-7 (Tcm) and CD3+  CD45R0+ CCR-T (Tt.m) cells 
CD8+ CD45R0+ CCR-7+ (CD8 Tcm) and CD8+  CD45R0+ CCR-T (CD8 
TEm), cell number and CD4 + CD45R0+ CCR-7+ (CD4 Tcm) and CD4+  
CD45R0+ CCR-T (CD4 TEm) cells. 
CD3+/- CD56+, CD3+/- CD16+, CD14+, CD19+ and CD20+ cells 
CD4 + CD25+FoxP3+ cells. 
Specific T cell responses to pathogens (such as CMV, EBV, Candida, and 
VZV) will be performed using functional assays which may include 
ELISPOT, lymphoproliferation assays, intracellular cytokine staining, and/or 
tetramer analysis. 
Specific T cell responses to leukemia associated antigens may be performed in 
a subset of patients using functional assays including intracellular cytokine 
staining and/or tetramer analysis. 
T cell receptor excision circle (TREC) analysis may be performed on a subset 
of patients. 
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A summary of patient evaluations post-transplant is provided in Table 5. 
Table 5. Patient evaluations over the course of the study 
Anproximate days nost trans lant 
Study Assessments/ 
Testing 
Baseline 
0 
7 
14 
21 
28 
35 
42 
49 
56 
63 
70 
77 
Between 80-100 
180 
360 
History, physical 
exam, height' and 
weight 
X 
X 
X 
X 
X 
Kamofsky 
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 
Serum chemistry 2 
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 
X 
EBV PCR 
X 
X 
X 
X 
X 
X 
X 
X 
X 
x 
x 
x 
x 
Aspergillus GM-EIA 
X 
X 
X 
X 
X 
X 
x 
x 
x 
X 
Quantitative 
Immunoglobulins 
for IgG, IgA and IgM 
x 
x 
x 
x 
x 
x 
Quantitative blood 
lymphocyte subset 
evaluation 
X 
X 
X 
X 
X 
Antibody response to 
recall antigens 
X 
Peripheral blood T 
cell & myeloid 
chimerism (CD3-  & 
CD33') 
X 
Baseline 
DNA 
X 
X 
X 
X 
X 
Bone marrow 
aspirate/biopsy' 
X 
X 
x 
x 
x 
Morbidity 
assessments' 
X 
X 
X 
X 
X 
X 
X 
X 
x 
x 
x 
x 
x 
x 
Acute GVHD 
X 
X 
X 
X 
X 
X 
X 
X 
x 
x 
x 
x 
Chronic GVHD6 
x 
x 
Skin biopsy 
x 
Oral medicine 
consult 
x 
Pulmonary function 
test including DLCO 
x 
x 
x 
Testa 
_Research 
Blood lymphocyte 
flow cytometry 
analysis and specific 
T cell responses 
X 
(FHCRC 
patients 
only) 
X 
X 
X 
X 
X 
I . 
Height on baseline only 
Serum Chemistry should include the following: Na, K, CI, CO2, BUN, Cr, Ca, Mg, Phos, ALT, AST, and BILIT/D 
CMV PCR or antigen monitoring is suggested to continue on a weekly basis for at least one year beyond day 100 for patients who have 
had CMV reactivation in the first 100 days and/or have active GVHD requiring steroids or other agents. 
Bone marrow aspiration; evaluation with a. histopathology b. flow cytometry c. cytogenetics (FISH and PCR informative for disease 
status eg BCR-ABL for Ph+ ALL) 
Morbidity assessments refer to evaluation for adverse events including standard clinical and laboratory evaluation. 
Chronic GVHD evaluation should be performed between approximately day 80 and 100, and on day 360 where possible. 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 be 
asked to return for a LTFU chronic GVHD consult. 
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 +1-7 days of time points < day 100; +/-30 days for time points > day 100). 
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. 
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11A. Total Body Irradiation (TBI)  
I. 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: 
Myelosuppression is the major dose limiting toxicity. 
Erythema may occur in the first 24 hours. 
Hyperpigmentation may occur in the first month following TBI. 
Oral ulceration, anorexia, nausea, vomiting and diarrhea, fatigue and alopecia 
occur frequently. 
Parotitis 
Decreased production of saliva and tears 
Hepatic dysfunction and rarely liver failure. 
Late effects include cataracts, growth failure, gonadal failure and sterility, 
hypothyroidism, pulmonary dysfunction and secondary malignancies. 
11B. Thiotepa 
I. 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: 
The major dose limiting toxicity of thiotepa is myelosuppression. 
Oral ulceration, anorexia, nausea, vomiting and diarrhea, fatigue and alopecia 
occur frequently. 
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. 
Dizziness and headache 
Hepatic toxicity including elevation in bilirubin and transaminases, and hepatic 
damage can occur. 
Rarely reported toxicities include CNS toxicity with somnolence, confusion, 
seizures, forgetfulness and inappropriate behavior. 
Allergic reactions during infusion occur rarely. 
Late effects include sterility and secondary malignancies. 
11C. Fludarabine 
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. 
Toxicity: 
Immunosuppression is the major toxicity of fludarabine. 
Oral ulceration, anorexia, nausea, vomiting and diarrhea, fatigue and alopecia 
occur frequently. 
Myelosuppression (lymphopenia, granulocytopenia, and anemia) is common. 
Numbness and tingling in hands or feet and visual changes occur and rarely 
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somnolence, mental state changes, cortical blindness, coma and other 
neurotoxicity 
e. Other reported toxicities include rash, hepatocellular toxicity, hemolytic anemia 
and interstitial pneumonitis. 
PBSC infusion 
Refer to Appendix D for infusion of selected cells. 
G-CSF  
For donor stem cell mobilization- see section 9C. 
Tacrolimus administration  
1. Also see section 9F for information about tacrolimus administration and dosage 
adjustments. 
2. Administration and dosage: 
Intravenous dosing - The IV formulation of tacrolimus is supplied as a sterile 
solution of 5 mg/ml ampules in polyoxyethylated castor oil (Cremophor FCL). 
The drug is diluted in D5W in glass or other non-PVC container. 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. 
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). For better absorption, it is 
recommended that Tacrolimus capsules be taken on an empty stomach. 
Tacrolimus should not be taken with grapefruit juice or other beverages 
containing bergamottin as it may increase blood levels. If patient vomits within 
one hour of oral administration, repeat dosing is recommended and if vomiting 
persists, switch to IV administration is recommended. 
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. 
3. Toxicity 
Side effects are generally reversible and may include: 
Rise in serum creatinine, electrolyte wasting, hemolytic uremic syndrome, and 
renal failure. 
Nausea and vomiting and hepatic dysfunction. 
Hypertension, 
Increases in cholesterol and triglycerides 
Paresthesia, tremors, seizures, headache, insomnia, dizziness, depression, 
confusion, hallucinations, psychosis, myoclonus, neuropathy, agitation. 
Blurred vision, photophobia. 
Hirsutism 
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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 
1 
1 
2 
Not Hispanic or Latino 
24 
34 
58 
Ethnic Category Total of All Subjects* 
25 
35 
60 
Racial Categories 
American Indian / Alaska Native 
0 
0 
0 
Asian 
0 
1 
1 
Native Hawaiian or Other Pacific 
Islander 
0 
0 
0 
Black or African American 
1 
1 
2 
White 
24 
33 
57 
Racial Categories: Total of All 
Subjects* 
25 
35 
60 
12B. Infection Prophylaxis  
1) Antibiotics 
(a) Prophylactic antibacterial antibiotics should be used for all patients that develop 
neutropenia (ANC <500/m1) 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 according to institutional practice. The 
recommended standard regimens are: 
FHCRC Standard: Penicillin VK 1 gram PO BID starting at day -2 until not 
able to take oral therapy and then IV Penicillin G one million units IV every 
six hours (4 million units total) until day +10. 
Yale Standard: Ceftriaxone Igm IV Q24 beginning day -2. 
Penicillin 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 
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practice guidelines (e.g. Cefazidime). Prophylactic penicillin or ceftriaxone may 
be discontinued when vancomycin or an alternative anti-streptococcus viridans 
agent is initiated. 
Prophylactic fluconazole (400 mg/day) should be used from the first day of 
conditioning or sooner until day +75 post transplant unless patients are participating 
in a study of antifungal prophylaxis. 
Preemptive therapy for CMV reactivation should be administered according to 
institutional standard practice guidelines. 
Infectious disease consultation should be obtained for patients who develop EBV 
DNA levels of >1000 copies/ml plasma on any test. For Yale patients, EBV DNA is 
reported as number of genomes/100,000 WBC's. Infectious disease consultation will 
be obtained if EBV DNA levels are > 1000 genomes/100,000 WBC's. 
Infectious disease consultation should be obtained for patients who develop a positive 
Galactomannan EIA test only after D+28. 
Patients should receive acyclovir or valacyclovir through day +365 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, foscamet or other effective antiviral drug for management of CMV. 
After engraftment, patients should receive prophylaxis for pneumocystis carinii 
according to institutional standard practice guidelines. 
12C. Ursodeoxycholic acid (UDCA) prophylaxis of hepatic complications of transplant 
1. UDCA should be given to prevent hepatic complications according to standard 
practice guidelines. Specifically UDCA should be given from approximately 2 weeks 
prior to the start of conditioning until day +90 post transplant. If a patient develops 
GVHD continuation of UDCA beyond day +90 may be warranted at the discretion of 
the attending physician. The recommended dose of urosodeoxycholic acid (UDCA) is 
12 mg/kg/day po divided in 2-3 daily doses with food. 
12D. Management of acute and chronic GVHD  
I. Patients who develop > grade II acute GVHD should be treated with systemic 
corticosteroids and 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 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 
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I. Patients who have persistent leukemia or who relapse after transplant may be treated 
according to institutional practice which can include but are not limited to 
chemotherapy and/or donor lymphocyte infusions (DLI), or may be eligible for 
research protocols evaluating immunotherapy with antigen-specific T cells. 
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, Yale University School of Medicine and the 
Yale-New Haven Hospital. 
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. 
For Yale patients, primary research data may also be collected and maintained by the data 
coordinator and stored at the Yale Cancer Center Clinical Trials Office (CTO) in a locked 
room. Research records will only be accessible to research personnel involved in the 
study or those performing auditing functions. 
Information gathered from this study regarding patient outcomes and adverse events will 
be made available to the sponsor and the Federal Drug Administration. All precautions to 
maintain confidentiality of medical records will be taken. 
Evaluation and statistical considerations 
Type of study 
This is a prospective phase II study of allogeneic stem cell transplantation using PBSC 
that are selectively depleted of CD45RA+ T cells. 
Definition of endpoints  
1. Acute GVHD 
Definition: 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. 
Evaluation: During the inpatient stay, clinical evaluations will occur daily. 
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During the outpatient stay, each patient should be evaluated at least weekly until 
day +30 and then at least every other week 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. An attending pathologist will interpret the biopsy material. The 
decision tcl initiate GVHD therapy will be made by the attending 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, one 
from each study site and one from a transplant center not involved in the study. 
c. Endpoints for acute GVHD that will be collected include: 
Presence of acute GVHD grades II-IV 
Time to onset of GVHD 
Requirement for methylprednisolone-equivalent dose of >=0.75mg/kg at day 
28 
Cumulative dose of methylprednisolone equivalent during the first eight 
weeks 
Requirement for secondary systemic therapy for acute GVHD 
2. Graft failure 
a. Definition: Graft failure is defined operationally as: 
Failure to reach an ANC of >500/p.1 for 3 consecutive days by day 28 
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 be 
considered graft failure. 
b. Evaluation: Engraftment endpoints will include: 
Time to ANC of >500/uL on the first of three consecutive days. 
Time to ANC of >1,000/uL on the first of three consecutive test results 
Time to RBC transfusion independence. 
Time to platelet count >20,000/µL for 3 days without transfusion. 
Time to platelet count >50,000/µL for 3 days without transfusion. 
Chimerism analysis of CD3 and CD33 cells in peripheral blood performed on 
or around day 28, 56, 80-100, 180 and 360. Additional peripheral blood or 
marrow chimerism studies may be performed as part of the evaluation of cases 
of graft failure. 
3. Relapse 
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. 
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 5. 
Suspected extramedullary sites of recurrent disease should be evaluated by biopsy 
and/or lumbar puncture if clinically indicated. 
4. Chronic GVHD 
a. Definition: Chronic GVHD will be diagnosed using NIH criteria outlined in 
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Appendix F. 
b. Chronic GVHD will be defined operationally as the occurrence of compatible 
symptoms. Evaluation: The primary chronic GVHD endpoint will be the 
occurrence of chronic GHVD requiring systemic immunosuppression. Additional 
chronic GVHD endpoints may include: 
Severity of GVHD — Mild versus Moderate versus Severe. 
Duration of immune suppression (other than the standard tacrolimus taper) in 
months after diagnosis. 
Number of cycles of immune suppressant therapy. A cycle is defined as either 
a nine-month course of a calcineurin inhibitor and prednisone given according 
the Long Term Follow-Up standard policy at the FHCRC or a course of any 
second-line immunosuppressant therapy given on or off protocol. 
Use of additional immune suppressive agents other than first line therapy 
(prednisone and tacrolimus/CSP). 
5. 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. Acute GVHD 
The primary objective of this single-arm Phase II study is to estimate the probability 
of grades II-IV acute GVHD and compare this to relevant historical experience. The 
goal is to observe a statistically significant reduction in the probability of GVHD 
compared to this historical experience without an accompanying increase in the 
probability of graft failure. Primary endpoints are grades II-IV acute GVHD and 
graft failure. Secondary endpoints include grades III-IV acute GVHD, relapse, 
transplant-related mortality, rate of engraftment, and chronic GVHD. Based on the 
historical experience at the FHCRC with unmanipulated PBSC grafts for 74 patients 
who received a first transplant from a matched related donor on protocols 179, 739, 
and 1106 since 2001, the probability of grade II-IV acute GVHD is 61%, and the 
probability of grade III-IV acute GVHD is 19%. The rates of grade II-IV and grade 
III-IV acute GVHD were 62% and 17% respectively, for 29 AML and ALL patients 
who received unmanipulated PBSC grafts from HLA-matched related donors and 
received post transplant irrununosuppression with tacrolimus and methotrexate on 
Protocol 1932. Based on these data, we shall use a rate of grade II-IV acute GVHD 
of 60% as our benchmark for comparison in the current trial. Table 6 summarizes the 
number of evaluable patients needed in the current trial to achieve 90% power to 
observe a statistically significantly (at the one-sided level of .05; power estimated 
from chi-square test) reduced probability of grades II-IV acute GVHD compared to 
this fixed value for various assumed-true probabilities. 
Table 6 
Fixed Historical Probability 
Assumed-true Probability, 
CD45RA depleted 
Number Patients 
.60 
.40 
52 
.60 
.35 
33 
.55 
.30 
22 
Based on these numbers, we propose to enroll 35 evaluable patients in the first cohort 
of patients on the current trial (which will provide 92% power if the assumed-true 
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GVHD rate CD45RA-depleted patients is 35%). If 15 or fewer of the 35 patients 
(43% or less) develop grades II-IV acute GVHD, then the probability of GVHD will 
be considered to be sufficiently low to proceed with the second cohort of patients. 
The probability of this occurring if the true probability of grades II-IV GVHD is .60 
is .03. Such an occurrence would therefore give us high confidence that the true 
GVHD rate is less than the benchmark of 60%. GVHD will be treated as a binary 
endpoint, and as such, patients who do not develop GVHD will be regarded as non-
failures for purposes of evaluating GVHD, regardless of whether they died or 
survived to day 100 without GVHD. If 15 or fewer patients develop grades II-IV 
acute GVHD and the limits for graft failure (14.C.2) are not met, a second cohort of 
25 patients will be treated with a shorter course of tacrolimus in an effort to see if the 
reduced GVHD rate can be maintained with a further reduction in 
immunosuppression. This study will not be capable of proving statistically that the 
GVHD rate with reduced immunosuppression is no worse than it is with the 
immunosuppression regimen utilized in the initial cohort of 35 patients. We propose 
to enroll 25 patients with short course tacrolimus, and if 10 or fewer (40% or less) 
experience grades II-IV acute GVHD the reduced immunosuppression will be 
considered promising and worthy of further study. If the true probability of grades II-
IV acute GVHD is 0.50, then the probability of observing 10 or fewer cases of grades 
II-IV GVHD among 25 patients is 0.21. 
Modification April 2014 
An interim analysis of protocol 2222 was performed after the first 30 patients were 
treated with a median of 592 days follow-up for surviving patients. In summary, an 
excellent rate of engraftment (a primary endpoint), a very low (12%) rate of chronic 
GVHD (a secondary endpoint), low rates of relapse and non-relapse mortality 
(secondary endpoints), very good immune reconstitution and survival were observed. 
In the first 30 patients acute GVHD is not lower than the historical benchmark 
(primary endpoint). However, compared to the first 13 naïve T cell-depleted HCT 
recipients (85%), acute GVHD appears to be lower among 17 subsequent patients 
(14-30) (53%) who received antibiotic prophylaxis directed at gram positive 
organisms (due to a high rate of streptococcus viridans bacteremia amongst the first 
13 patients). The original plan was to enroll 35 patients with naïve T cell depletion 
and tacrolimus monotherapy and if acute GVHD was reduced in this cohort then to 
enroll a second cohort of patients without tacrolimus. Although the desired reduction 
in acute GVHD will not be achieved in the first 35 patients, the chronic GVHD rates 
are very low, the general outcomes are favorable and it is still possible that acute 
GVHD will in fact be reduced among patients who receive naïve T cell depletion and 
penicillin. 
We acknowledge that through the first 30 patients enrolled to this trial attainment of 
our primary endpoint is not possible (15 or fewer cases of grades 2-4 acute GVHD 
among 35 patients). We further acknowledge that the original version of the protocol 
did not have stated as a primary or secondary endpoint the probability of grades 2-4 
acute GVHD among patients who received penicillin for infection prophylaxis. 
Given the observation that patients who received penicillin might have a lower rate of 
grades 2-4 acute GVHD (the primary endpoint of this Phase II trial), we are extending 
accrual to 2222 to an intended sample size of n=35 patients who will receive 
penicillin. The same design considerations will hold for the "penicillin patients" as 
were in place in the original protocol. We acknowledge that this will lead to a bias in 
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the estimate of our primary endpoint, as we plan to include the 17 patients already 
enrolled and treated with penicillin in our analysis since their outcomes have already 
been observed (9/17 (53%) patients with grades 2-4 acute GVHD). However, this 
observed rate is closer to the null hypothesis (GVHD probability of 60%) than the 
alternative, so under the alternative hypothesis that the probability of GVHD is 35%, 
the bias will be positive relative to 35% (in fact, based on a simulation study, the bias 
under alternative hypothesis is roughly 8%; under the null hypothesis, the bias is 
approximately 2%). And while it was not intended as such, and we fully 
acknowledge this, one might loosely think of the first 17 patients who received 
penicillin as the first stage in at two-stage design, after which the trial could be 
terminated if "too many" cases of GVHD are observed. Towards that end, the 
probability of 9 or more cases of GVHD among 17 patients under the null hypothesis 
of 35% is .10; under the assumption that the true probability of GVHD is 40%, the 
probability of 9 or more cases of GVHD among 17 patients is .20. Therefore, if this 
truly were the first stage of a two-stage trial, we would not have enough evidence to 
reasonably terminate the trial. While the power to detect a statistically significant 
difference relative to the fixed benchmark of 60% is certainly lower with outcomes 
already observed for roughly half the patients, the conditional power (given 9/17 
already observed) under the alternative hypothesis of 35% GVHD is 55% (at the one-
sided significance level of .05; at the one-sided significance level of .10, the 
conditional power is 73%. Given the observed outcome of 9/17, the probability of 
"success" after 35 patients (15 or fewer cases of GVHD, or 6 or fewer among the next 
18) under then null hypothesis is p=.02 (i.e., the type I error rate). The protocol will 
be terminated if it becomes apparent that the primary endpoint will not be met among 
the penicillin patients. In other words, if a 16th case of grades 2-4 acute GVHD 
occurs among the 35 patients treated with penicillin (or a 7th case among the 
additional 18 patients post-modification), then success for our primary endpoint will 
not be attainable and the study will be terminated. 
It is theoretically plausible, although unlikely, that severe, steroid refractory GVHD 
could be increased even though T-cells are being removed. Because of this, we will 
have in place stopping rules for this outcome. These rules will be separately applied 
for each cohort. If there is sufficient evidence to suggest that the true probability of 
steroid refractory GVHD exceeds 5%, the study will be stopped. Sufficient evidence 
will be taken to be an observed rate whose lower one-sided 90% confidence limit 
exceeds 5%. Operationally this will occur if any of the following observed ratios 
occur: 2 of the first 10 or fewer, 3 of the first 22 or fewer, or 4 of the first 35 or fewer 
patients develop steroid refractory GVHD. If the true probability of GVHD is 0.03, 
the probability of stopping after 20 or 35 patients is approximately 0.05 and 0.06, 
respectively. If the true probability is 0.23, then the probability of stopping is 
approximately 0.90 and 0.98, respectively. The presence of 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. 
2. Graft Failure 
Graft failure will be closely monitored throughout this study. A true probability of 
graft failure over 5% will be considered excessive. As with GVHD, graft failure will 
be treated as a binary outcome. If two graft failures are seen among the first 16 
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patients, 3 among the first 30 patients, or 4 among the first 35 patients enrolled the 
trial will be stopped due to excessive graft failure. These limits correspond to 
observed rates of graft failure whose lower one-sided 80% confidence limits exceed 
5%. Demanding higher confidence that the estimated graft failure rate exceeds 5% 
before stopping means that the chances the trial would stop when the true graft failure 
rate is over 5% are unacceptably low. If the true failure rate is as low as 1%, then the 
probability that the trial stops after 20 or 35 patients is approximately .01, and .01, 
respectively. If the true failure rate is 16%, these probabilities are .78 and .91, 
respectively. 
These stopping rules for the modification will be applied to the group of patients 
receiving penicillin. 
Graft failure will continue to be closely monitored in the second cohort of 25 patients. 
Since shortening the tacrolimus post transplant should not increase the probability of 
graft failure, the experience of the initial group of 35 patients will be considered when 
assessing graft failure. Extending the stopping rules for graft failure listed above, 4 
failures among the first 46 patients or 5 among the first 60 patients will be considered 
excessive. If the true probability of graft failure is .01, the probability of stopping 
after 45 or 60 patients is approximately .02 and .02, and if the true probability of 
failure is .16 the probability of stopping after 45 and 60 patients is approximately .96 
and .99, respectively. 
Relapse 
Relapse will also be carefully monitored. While formal stopping rules will not be put 
in place for relapse due to the relatively long period of time post-transplant that this 
complication is of concern, a Data Safety and Monitoring Board (DSMB) will be in 
place to meet roughly every 6 months (or whenever the last patient in each group of 
five patients reaches day 100, whichever is sooner) to review the data. This DSMB 
will be composed of Drs' Eneida Nemecek, Elihu Estey, Soheil Meshinchi, Blythe 
Thomson and Barry Storer. Rough guidelines for acceptable rates of relapse will be 
50% for patients transplanted in relapse and 25% for patients transplanted in 
remission. 
Failure to deliver therapy 
If CD45RA-depleted PBSC cannot be delivered as planned for the first 2 patients 
enrolled, 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. This rule will be reinstated for the subsequent enrollment. 
Patient withdrawal from treatment or study 
All patients who receive infusion of PBSC with or without cell selection will be 
considered evaluable for study endpoints. Patients in whom tacrolimus is 
discontinued, or switched to cyclosporin, due to concerns for toxicity will still be 
considered on study. 
When a patient withdraws consent from P2222 they will no longer have research 
samples taken and the research team will no longer have access to clinical records to 
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follow them for study endpoints. 
The only exception is for FHCRC patients who have signed the general consent and 
authorization form allowing their leftover specimens and medical records to be used 
for research. If the FHCRC patient has 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 
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/ftpl/CTCAE/About.html  
Definitions  
Definitions associated with reportable events can be found on the FHCRC's Institutional 
Review Office (IRO) extranet website (Table 7 FHCRC IRB Forms for Reporting). 
Unanticipated adverse device effect means any serious adverse effect on health or 
safety or any life-threatening problem or death caused by, or associated with, a device, if 
that effect, problem, or death was not previously identified in nature, severity, or degree 
of incidence in the investigational plan or application (including a supplementary plan or 
application), or any other unanticipated serious problem associated with a device that 
relates to the rights, safety, or welfare of subjects. (21 CFR 812.3) 
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: 
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. 
Grade 3-5 Blood/Bone Marrow adverse events occurring between day 43 and day 100 
will be collected. 
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, I year, and then yearly through 5 years after day 0 whenever 
possible. 
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. All treatment related mortality occurring in the first 100 days will be 
reported to all parties in an expedited fashion. 
Grade 3-5 infusion reactions and grade 4-5 thrombotic microangiopathy (TMA) 
occurring in the first 100 days will be reported to all parties in an expedited fashion. 
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Reporting to Coordinating Center 
As FHCRC is the coordinating center reportable events occurring at the FHCRC and 
participating sites will be collected by the FHCRC PI and/or study nurse and reported to 
FHCRC IRB. Reportable events occurring in YUSM patients will be reported by the 
YUSM investigator or representative to the FHCRC PI and/or study nurse. The trial 
coordinators at collaborating centers or the local PIs will verbally report all events 
requiring expedited reporting (including, but not limited to, all patient deaths regardless 
of cause, occurring days 0-200 post-transplant procedure) within 72 hours of learning of 
the event and an official report of the event is faxed to the coordinating center (FHCRC) 
at 206-667-6056 within seven days utilizing the FHCRC Adverse Event Reporting Form 
and the FHCRC Expedited Reporting Form for Unanticipated Problems or 
Noncompliance. Follow-up information to a reportable event report must be submitted as 
soon as the relevant information is available. 
The FHCRC PI and research nurse will meet regularly to review all reported events. If 
the event meets FHCRC IRB current reporting obligations it will be sent to them. 
After the FHCRC IRB has reviewed the event report it will be disseminated to all 
participating sites/investigators, the DSMB, and the study sponsor. 
Reporting Requirement to FHCRC IRB  
The Principal Investigator, study nurse, or coordinator shall submit to the FHCRC IRB 
reportable events occurring in Cancer Consortium patients and multi-center sites 
according to current reporting policies as outlined in Table 7 FHCRC IRB Policies for 
Reportable Events. 
As FHCRC is the coordinating center reportable events occurring at the FHCRC and 
participating sites will be collected by the FHCRC P1 and/or study nurse and reported to 
FHCRC IRB. 
All reportable events should be submitted on the relevant FHCRC Forms (URLs linking 
to the FHCRC IRO webs ite are found in Table 8 FHCRC IRB Forms for Reporting). 
Table 7 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.thcrc.org/EN/sections/iro/irb/ae.html  
IRB Policy 1.1 
Reporting Obligations for 
Principal Investigators 
http://extranet.fhcrc.orv./EN/sections/iro/irb/policv/index.html 
IRB Policy 2.2 
Continuing Review 
11.4•://extranet.fhcrc.org/EN/sections/iro/irb/policy/index.html  
IRB Policy 1.13 
Investigational New Drugs 
(END), Biologics and 
Investigational Device 
http://extranet.fherc.org/EN/sections/iro/irb/policy/index.html  
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Exemptions (IDE) 
Table 8 FHCRC IRB Forms for Re ortin 
Adverse Event Reporting Form 
htip
t ://extranet. fhc rc . org/EN/sectio ns/i ro/i rb/forms/i ndex.htm I 
Expedited Reporting Form for Unanticipated 
Problems or Noncompliance 
http://extranet.fhcrc.org/EN/sections/iro/irb/forms/index.html  
Reporting to the Sponsor 
Unanticipated adverse device events, grade 3-5 infusion reactions and grade 4-5 
thrombotic microangiopathy (TMA) occurring in the first 100 days post-transplant will 
be reported and sent via email to the sponsor, Dr. Warren Shlomchik 
(warren.shlomchik@yale.edu) and the IDE coordinator, as soon as possible but no later 
than 10 working days of learning of the event. A confirmatory email communication 
must be received from at least one of these individuals. If a confirmation email is not 
received within 2 working days, Dr. Shlomchik and the IDE coordinator should be 
contacted again by email and additionally by telephone or FAX. 
In addition, graft failure, death or grade 4 non-hematologic events not resolving to grade 
2 or less within 96 hours occurring in the first 200 days post-transplant, will be reported 
to the sponsor of the IDE, Dr. Warren Shlomchik, as soon as possible but in no event 
later than 10 working days after the investigator first learns of the event. 
Reporting Requirement to Yale IRB  
All reportable events whether originating at Yale or a collaborating center will be 
reported by Yale study staff to the Yale University Human Investigation Committee 
(HIC) using HIC Form 6A or the Protocol Deviation or Unanticipated Problem Involving 
Risks to Subjects or Others Report Form within 48 hours of Yale learning of the event. A 
copy of the HIC Adverse Event Policy is available at: 
http://www.yale.edu/hrpp/policies/index.html#  
A copy of the HIC Adverse Event and Deviation or Unanticipated Problem report form is 
available at: 
http://www.yale.edu/hrpp/forms-templates/biomedical.html  
Reporting to the FDA 
For studies conducted under an IDE (Investigation Device Exemption) the FDA 
regulations outline the following sponsor obligations regarding unanticipated adverse 
device effects: 
A sponsor shall immediately evaluate any unanticipated adverse device effect. 
21CFR812.46 (b) (1) 
A sponsor who conducts an evaluation of a unanticipated adverse device effect under 21 
CFR 812.46(b) shall report the results to FDA within 10 working days after the sponsor 
first receives notice of the effect. Thereafter the sponsor shall submit such additional 
reports concerning the effect as FDA requests. 21CFR812.150 (b) (1) 
A Med Watch 3500A form will be completed and sent to the FDA 
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A sponsor who determines that an unanticipated adverse device effect presents an 
unreasonable risk to subjects shall terminate all investigations or parts of investigations 
presenting that risk as soon as possible. Termination shall occur not later than 5 working 
days after the sponsor makes this determination and not later than 15 working days after 
the sponsor first received notice of the effect. 21CFR812.46 (b) (2) 
16. 
Data safety monitoring plan 
16A. Monitoring the progress of trials and the safety of participants 
The FHCRC PI and IDE sponsor are responsible for monitoring this multi-institutional 
clinical trial, with oversight by a Data and Safety and Monitoring Board (DSMB), the 
Protocol and Data Monitoring Committee (PDMC) at the FHCRC, the Yale Cancer 
Center Office of Protocol Review and Monitoring and the IRBs at the respective 
institutions. 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 
months after HCT. 
A DSMB will be in place to meet approximately every 6 months (or whenever the last 
patient in each group of five patients reaches day 100, whichever is sooner) to review the 
data particularly as it relates to engraftment, grades III-IV GVHD and relapse. The 
DSMB will also review all adverse events reported to the coordinating center (FHCRC). 
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 Coordinating Center IRB (FHCRC) and the PI. The FHCRC study staff 
will send a copy of the reports to the IDE sponsor, as well as to the trial 
coordinators/local Investigators' of this protocol. Each institution will then distribute this 
report per their internal policies. The DSMB will discontinue the review of outcomes 
when all subjects on this trial have completed all protocol-specified follow-up. The 
DSMB for protocol 2222 will be composed of Drs' Eneida Nemecek, Elihu Estey, Soheil 
Meshinchi, Rebecca Gardner, and Barry Storer. 
P2222 DSMB Members 
Name 
Affiliation 
Position 
Dr. Eneida Nemecek Oregon Health & Science University 
DSMB Chair 
Dr. Barry Storer 
FHCRC 
DSMB Biostatistician 
Dr Soheil Meshinchi 
FHCRC 
DSMB Clinical Investigator 
Dr. Elihu Estey 
SCCA 
DSMB Clinical Investigator 
Dr. Rebecca 
Gardner 
Seattle Children's Research Institute 
DSMB Clinical Investigator 
All members have experience in the management of patients with leukemia and 
myelodysplasia and in the conduct and monitoring of clinical trials. 
The PDMC at FHCRC and the Quality Assurance Compliance and Safety Committee 
(QUACS) at Yale 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. YUSM also has an IRB that will review and 
monitor the protocol. Each IRB will be contacted by the local Investigator in accordance 
with their IRB reporting guidelines. 
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An external monitor will be retained to monitor study progress at both the FHCRC and at 
Yale. The scope of monitoring will be based on the FHCRC/UW Data and Safety 
Monitoring Plan: http://www.cancerconsortium.org/rto/prr/DSMPlan.pdf . 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, the IDE sponsor and the PI at 
FHCRC. Each institution will then distribute this report per their internal policies. 
Flow of information concerning clinical trial participants originates with the clinicians 
and nurses in the clinic or referring clinicians at other institutions and is transmitted to the 
FHCRC Research Nurse. At the FHCRC and YUSM, 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. 
168. Plans for assuring data accuracy and protocol compliance  
The study has a research nurse at each site that follows patients to confirm eligibility, 
reporting of adverse events, reporting of events which are part of the safety-monitoring 
plan, and protocol adherence. Each site's investigator 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 +180) and again approximately 
yearly for 5 years after HCT via chart review and entered into electronic/paper based 
protocol specific Case Report Forms (CRFs). The principal investigator at each site will 
review the CRFs and the primary source documents verifying by signature 
(paper/electronic) their data accuracy. 
The Coordinating Center PI will review and send queries to the collaborating investigator 
if CRFs are incomplete or inaccurate. 
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. 
17. 
References 
Ferrara JL, Reddy P. Pathophysiology of graft-versus-host disease. Semin Hematol 43:3-10, 2006. 
Bensinger WI, Martin PJ, Storer B, et al. Transplantation of bone marrow as compared with peripheral-
blood cells from HLA-identical relatives in patients with hematologic cancers. N Engl J Med 344:175-81, 
2001. 
Oehler VG, Radich JP, Storer B, et al. Randomized trial of allogeneic related bone marrow transplantation 
versus peripheral blood stem cell transplantation for chronic myeloid leukemia. Biol Blood Marrow 
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Transplant 11:85-92, 2005. 
Ratanatharathorn V, Nash RA, Przepiorka D, et al. Phase III study comparing methotrexate and tacrolimus 
(prograf, FK506) with methotrexate and cyclosporine for graft-versus-host disease prophylaxis after HLA-
identical sibling bone marrow transplantation. Blood 92:2303-14, 1998. 
Chao NJ. Minors come of age: Minor histocompatibility antigens and graft-versus-host disease. Biol Blood 
Marrow Transplant 10:215-23, 2004. 
Champlin RE, Passweg JR, Zhang MJ, et al. 1-cell depletion of bone marrow transplants for leukemia from 
donors other than HLA-identical siblings: advantage of T-cell antibodies with narrow specificities. Blood 
95:3996-4003, 2000. 
Ho VT, Soiffer RJ. The history and future of 1-cell depletion as graft-versus-host disease prophylaxis for 
allogeneic hematopoietic stem cell transplantation. Blood 98:3192-204, 2001. 
Goodnow CC, Sprent J, Fazekas de St Groth B, Vinuesa CG. Cellular and genetic mechanisms of self 
tolerance and autoimmunity. Nature 435:590-7, 2005. 
Chao NJ, Chen BJ. Prophylaxis and treatment of acute graft-versus-host disease. Seznin Hematol 43:32-41, 
2006. 
Storb R, Deeg HJ, Whitehead J, et al. Methotrexate and cyclosporine compared with cyclosporine alone for 
prophylaxis of acute graft versus host disease after marrow transplantation for leukemia. N Engl J Med 
314:729-35, 1986. 
Brown JM. Exogenous administration of immunomodulatory therapies in hematopoietic cell 
transplantation: an infectious diseases perspective. Curr Opin Infect Dis 18:352-8, 2005. 
Papadopoulos EB, Carabasi MH, Castro-Malaspina H, et al. T-cell-depleted allogeneic bone marrow 
transplantation as postremission therapy for acute myelogenous leukemia: freedom from relapse in the 
absence of graft-versus-host disease. Blood 91:1083-90, 1998. 
Jakubowski AA, Small TN, Young JW, et al. 1-cell depleted transplantation for adults with hematologic 
malignancies: sustained engraftment of HLA-matched related donor grafts without the use of anti-
thymocyte globulin. Blood 110:4552-9, 2007. 
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 
73:1340-50, 1989. 
Lanzavecchia A, Sallusto F. Dynamics of T lymphocyte responses: intermediates, effectors, and memory 
cells. Science 290:92-7, 2000. 
Kaech SM, Wherry EJ, Ahmed R. Effector and memory T-cell differentiation: implications for vaccine 
development Nat Rev Immunol 2:251-62, 2002. 
Anderson BE, McNiffJ, Yan J, et al. Memory CD4+ T cells do not induce graft-versus-host disease. J Clin 
Invest 112:101-8, 2003. 
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 103:3970-8, 2004. 
Zheng H, Matte C, Anderson BE, et al. CD8+ central memory T cells mediate graft-versus-host disease 
although retain graft-versus-leukemia effect Blood 106:2005. 
Chen BJ, Cui X, Sempowski GD, Liu C and Chao NJ. Transfer of allogeneic CD62L- memory T cells 
without graft-versus-host disease. Blood 103:1534-41, 2004. 
Zheng H, Matte-Martone, Hongmei L, et al. Effector memory CD4-- T cells mediate graft-versus-leukemia 
without inducing graft versus host disease. Blood 111: 2476-84, 2008. 
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 CD45RC1ow subset Eur J Immunol 34:408-17, 2004. 
Arstila TP, Casrouge A, Baron V, Even J, Kanellopoulos J, Kourilsky P. A direct estimate of the human 
alphabeta T cell receptor diversity. Science 286:958-61, 1999. 
Burrows SR, Khanna R, Silins SL, Moss DJ. The influence of antiviral T-cell responses on the alloreactive 
repertoire Immunol Today 20:203-7, 1999. 
Bleakley M, Mollerup A, Chaney C, Brown M, Riddell S. Human minor histocompatibility antigen-
specific CD8+ T cells are found predominantly in the CD45RA+ CD62L+ Naive T cell subset. Blood 
106:2005. 
Smalley DM, Ley K. L-selectin: mechanisms and physiological significance of ectodomain cleavage. J Cell 
Mol Med 9:255-66, 2005. 
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 96:2062-8, 2000. 
Uberti JP, Ayash L, Braun T, Reynolds C, Silver S, Ratanatharathorn V. Tacrolimus as monotherapy or 
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combined with minidose methotrexate for graft-versus-host disease prophylaxis after allogeneic peripheral 
blood stem cell transplantation: long-term outcomes. Bone Marrow Transplant 34:425-31, 2004. 
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Product has >2.0 x 
No — 106/kg CD34 + cells in 
CD34 + fraction 
CD45RA depletion 
f  
Determine number of CD3+ T 
cells and CD45RA + RO-CD3+  
T cells in CD34+ and CD34 
depleted CD45RA depleted 
fractions. Remove aliquot of 
CD45RA depleted fraction for 
infusion to deliver a total of 
<7.5 x 104 CD45RA+RO-CD3+  
T cells/Icg and 1-10 x 106  
Yes 
Infuse CD34 + and CD34 depleted 
CD45RA depleted fractions on day 0 
If? 7.5 x 10 CD45RA 
RO-  CD3+ T cells/kg in 
cell volume required to 
-• deliver 1-10 x 106 CD3+ T 
cells/kg refer to the cell 
selection guidelines 
below (3b) 
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Day - I 
Day 0 
APPENDIX A 
Cell Selection Flow Sheet 
Apheresis #1 
Stored overnight 
Always collect apheresis #2 
Process apheresis products #1 and #2 if CD34' 
count is  .5.0 x 106 cells/k2  
CD34 selection 
CD34 positive fraction 
CD34 depleted fraction 
If <2.0 x 106 /kg CD34+ 
cells in CD34 + fraction 
refer to the cell selection 
guidelines below (3a) 
Based on prior experience with cell selection of PBSC, we expect the following: 
A total CD34 cell dose of >5x106 cells/kg of recipient body weight will be available from two donor 12-
16L leukapheresis collections for 85% of donors. A total CD34' cell dose of >2.0x106 cells/kg of 
recipient body weight will be obtained from CD34 positive selection from an apheresis product 
containing 5x106  CD34+cells/Icg of recipient body weight. 
The number of residual donor CD45R.A 4RO- CD3+ T cells in the CD34 selected product that will 
contribute to the overall dose of CD45RA+RO- CD3-  T cells will be <4.2.x 1 04/kg of recipient body 
weight. 
Depletion of CD45RA+ cells from the CD34 negative fraction will yield sufficient T cells to administer 1-
10 x106 CD3+ cells/kg and <3x104 CD45RA+  R0- CD3+ cells/kg of recipient body weight for >90% of 
patients. 
The maximum number of CD45RA+ R0 CD3+  cells anticipated in the sum of the CD34 selected product 
and the CD45RA depleted product will be 7.5 x 104 CD45RA+RO-CD3+ T cells/kg. 
Cell Selection Guidelines  
1. We will perform cell selections only when the PBSC product from days -1 and 0 combined 
contains >5x106 CD34+ cells/kg of recipient body weight. If the PBSC product from days -1 and 
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0 combined 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. The patients that receive G-PBSC that does not undergo cell selection will receive 
GVHD prophylaxis with tacrolimus and methotrexate (see section 9F). 
Priority will be given to administering adequate numbers of CD34 ÷ cells to achieve reliable 
engraftment. In most cases we expect to infuse 5-10 million CD34 + cells/kg. The infused CD34+  
cell number may be limited to 10 million CD34 + cells/kg at the PI's discretion or less (within the 
range of 2-10 million CD34 + cells/kg) if necessary to achieve the limit of <7.5 x 104  
CD45RA+CD45RCYCD3+ cells/kg. 
Situations may arise where the goals for cell selection are not achieved with processing of the two 
leukapheresis collections. These unusual situations will be discussed with the protocol PI and a 
recommendation made. General guidelines are as follows: 
If a total CD34 cell dose of 
x 106 cells/kg is not achieved after processing of two 
leukapheresis collections all available CD34+ cells will be administered to the patient on 
day 0 and a third apheresis collection will be performed on day 1 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 and will 
receive GVHD prophylaxis with tacrolimus and methotrexate (see section 9F). 
If < 7.5 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-lox 106/kg total CD3+ T cells) as described above, the following steps may be 
required: 
Repeat the CD45RA depletion of the CD34 depleted fraction using a second magnetic 
column. 
If there is >5 x 104  CD3+ CD45RA+ RO- cells in the CD34 + fraction and >7.5 x 106  
CD34 + cells/kg in the CD34 + fraction, a second column pass of an aliquot of the CD34 + 
cells may be performed but a minimum of 2.0 x 106  CD34 cells/kg MUST be 
maintained. 
If 1 x 106 CD3+ cells/kg and <7.5 x 104CD3+CD45RA+ RO" cell/kg cannot be achieved 
by i. or ii. 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 <7.5 x 104CD3+CD45RA+ RC/ 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 with tacrolimus 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. 
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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. 
Safety: Samples from the final products 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 affiliated with each cell processing center, according to validated procedures. Samples 
will be inoculated into thioglycollate broth and/or agar media. Media will be incubated at appropriate 
temperatures, and inspected for bacterial and/or fungal growth over a period of 14 days. Subculture onto 
agar plates will be performed to investigate any turbidity noted in broth cultures, and if positive cultures are 
found organism species identification will take place. Each institution 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. After final product processing a sample will be pulled and a rapid gram stain 
will be performed with an acceptance criterion of "No Organisms Detected". If there is a positive gram 
stain, the attending physician will be notified. The attending physician will decide whether to administer the 
product, and if infused whether to begin prophylactic antibiotics. If the product is administered, we would 
await product culture results and if positive, modify antibiotic therapy accordingly and proceed as per 
Appendix C. 
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+CD45R0-) 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. 
CD4514A" 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+CD45R0-), and total T cells (CD3+) will also be performed by multi-color flow cytometry, 
The aim is to infuse a maximum of 7.5 x 104  CD3*CD45RA+ ROlkg 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,. 
All fractions will in addition be phenotyped by multi-color flow cytometry for the presence of other cell 
lineages and T-cell subpopulations (see section 9D3) 
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. 
Stability: The first leukapheresis collection will be stored overnight at 4C prior to CD34 selection and 
CD45RA depletion, with infusion to take place that same day. 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 
will take place within 6 hours after completion and release of the processed 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, 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 will notify the FHCRC IRB, the sponsor, Warren Shlomchik, as soon as possible, but no 
later than 10 calendar days of finding out of the event. The sponsor will notify the FDA as outlined in 
section 15. 
4. After notification of the attending physician, the following actions will take place. 
The attending physician will notify the patient (recipient) of the positive culture. 
The attending physician or primary care provider will perform a thorough examination of the 
patient (recipient). 
Blood samples will be obtained from the patient (recipient) for cultures (bacterial, fungal, and 
viral cultures). 
Samples of other fluids will be obtained for cultures if clinically indicated. 
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: 
Assays for identification and sensitivity of the organism have been completed. 
Results of the patient (recipient) blood cultures are available. 
The absolute neutrophil count exceeds 500 cells/gl. 
Unexplained clinical signs or symptoms of systemic infection have resolved. 
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 fraction) 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. 
For FHCRC patients: 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. 
For YNHH patients: Questions regarding ABO mismatches should be directed to the Blood Bank 
Transfusion Medicine Service or the 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, unless specified differently in the protocol or standard treatment plan. 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 selected cell volume is generally less than 300 ml total 
Pre-medication 
At FHCRC 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 platelet transfusion guidelines for allergic or febrile 
reactions. At YNHH diphenhydramine may be given as a premedication as per YNHH standard practice. 
Filtration 
Product should be filtered through a blood administration set with 150-260 micron mesh size. 
Product Infusion Rate 
Adults and Pediatrics > 20 kg: 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 
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100m1 each so volume overload is not expected. Volume reduction may be required if the cell volume of either 
individual infusion exceeds 20 mUlcg adult recipient weight, 15 mUkg pediatric recipient weight, or if the patient 
is volume overloaded by clinical criteria. Request volume reduction by contacting the Cellular Therapy 
Laboratory. 
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 or YNHH 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. For YNH patients, the YUSM Transfusion Services Office may be consulted. 
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 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 fat is highly unlikely in the selected cell products. 
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 DISEASE' 
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/100m1) 
+2 
bilirubin (3-5.9mg/100m1) 
+3 
bilirubin (6-14.9mg/100m1) 
+4 
bilirubin > 15mg/100m1 
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/lcg/day)*  
+2 
>1,000 ml of stool/day (>15m1 of stool/kg/day)+ 
+3 
>1,500 ml of stooUday.  (>20m1 of stool/kg/day)t  
+4 
2,000 ml of stool/day. 25m1 of stool/kg/day)t  
In the absence of infectious/medical cause 
t For pediatric patients 
Severity of GVHD 
Grade! 
+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. Hematopoletie Cell Transplantation Ed: D. Thomas, K. Blume, S. Forman, Blackwell 
Sciences; 1999, pages 518-519. 
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APPENDIX F 
GRADING OF CHRONIC GRAFT-VERSUS-HOST DISEASE 
It
' 0,1 
Chronic GVHD 
Guidelines 
11:1:1
10m4  
Chronic GVHD 
Appendix D 
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APPENDIX G 
Potential Adverse Events Associated or Expected with Hematopoietic Cell Transplantation 
Opportunistic infections, including viral and fungal infections, can results in severe pulmonary, neurologic, 
hepatic and other organ dysfunction, and possible death. 
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. 
Cardiac toxicity. Cardiotoxicity (for example congestive heart failure, pericardial effusion, EKG changes) is 
uncommonly associated with chemotherapy agents and TBI and these sequelae may prove lethal. 
Pulmonary toxicity. Diffuse interstitial pneumonitis of unknown etiology occurs with some regularity after 
BMT. Interstitial fibrosis and diffuse alveolar hemorrhage occur less frequently. Each are well-described 
complications of intensive chemotherapy and TBI regimens and may prove lethal. 
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. 
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. 
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. 
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. 
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. 
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 TB!. 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 Performance Status Scale 
1CARNOFSKY PERFORMANCE STATUS SCALE 
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 
REFERENCE 
Karnofsky DA: Meaningful clinical classification of therapeutic responses to anti-cancer drugs. Editorial: Clin 
Pharmacol Ther 2:709-712, 1961. 
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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. 
BSA is calculated in M2. The formula by definition adjusts for both under and over weight individuals. 
The formula for this calculation is: 
Niactual weight in kg x height in cm 
60 
Body weight is measured in kg. The ideal body weight (IBW) will be calculated in the following ways 
For Adult Patients: 
SWOG/CTN Formulas will be used to calculate ideal weights 
Males: 50 kg + (2.3 kg/inch over 5 feet) 
Females: 45.5 kg + (2.3 kg/inch over 5 feet) 
Patients less than 5 feet: subtract 2.3 kg/inch 
Height in inches will be rounded to the nearest whole number. 
Height with inches < 1/2  inch will be rounded down to the nearest whole inch 
Example: 5 feet 5 1/4  inches will be rounded to 5 feet 5 inches 
Height with inches > 1/2  inch will be rounded up to the nearest whole inch 
Example: 5 feet 6 1/2  inches will be rounded to 5 feet 7 inches 
(Note: The dietitians will document the conversion from height in centimeters to height in inches as well 
as the rounded height used to calculate the ideal weight on the green Nutrition Demographic / 
Anthropometric nutrition cardex form.) 
For Pediatric Patients: 
Post pubertal adolescents (females > 12 years old and males > 14 years old): 
Ideal weight will be assessed using the BMI. If the child's normal BMI [weight in kg/(height in meters)2 ] 
is between the 25-75th percentile, the child may be considered at IBW. 
Children whose BMI exceeds the 75th percentile: Ideal weight will be the 75th percentile BMI weight. 
Children 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 
For both Adult and Pediatric Patients: 
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 
COORDINATING CENTER FUNCTIONS 
Outside Center — PI Communication 
Study Management, data analysis, and Data and Safety Monitoring 
a. Study Management: 
i. 
Each local PI is responsible for selection, training and oversight of local study coordinators 
The Coordinating Center registers subjects on the study and assigns study IDs 
One copy of the research data is retained by the site. Another data set (identified only by 
study IDs) is transmitted to the Coordinating Center to create the master data file. All data are 
kept in locked areas and password protected databases accessible only to study staff 
iv. 
The quality of data is monitored in an ongoing fashion with the study team and corrective 
action plans instituted as necessary 
b. Data Analysis: 
Study staff review data for completeness as it is submitted by the sites 
The study statistician is responsible for data cleaning and the conduct of analyses as outlined 
in the protocol and grant 
c. Data Safety and Monitoring: 
i. 
The collaborating centers will report all adverse events, unanticipated problems and non- 
compliance as described in section 15. 
Protocol and informed consent document management 
A master protocol is maintained by the Coordinating Center and distributed to the sites for 
customization and local IRB review 
All protocol and consent modifications initiated by the Coordinating Center are sent to the 
Collaborating Sites following approval by the Coordinating Center IRB, for review and approval by 
the local IRB 
Changes required by local IRBs are reviewed by the Coordinating Center and approved prior to 
implementation at local sites 
III. 
Assurance of local IRB OHRP-approved assurance 
Each site provides their OHRP assurance number and evidence of IRB certification 
Study staff monitor maintenance of institutional assurance and IRB certification 
IV. 
Assurance of local IRB approvals 
The Coordinating Center maintains copies of the most current collaborating site Consent Forms and 
IRB approval documentation 
No site may enroll subjects until the Coordinating Center has received confirmation of local IRB 
approval 
Each site is responsible for preparation and submission of their continuing reviews. Any changes to 
the protocol or consent form will be communicated to the Coordinating Center 
Sites are required to have active IRB approvals to participate in any study related activities 
V. 
Any substantive modification by the Collaborating Institution related to risks or alternative 
procedures is appropriately justified 
a. The Coordinating Center reviews any modifications to consent forms to ensure that site consents do 
not delete or change the basic or additional elements or alternatives required in the sample consent 
form 
VI. 
Informed consent is obtained from each subject in compliance with HHS regulations 
Subjects must provide written informed consent prior to study participation 
The Coordinating Center verifies eligibility and signed consent prior to assigning a study ID number 
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APPENDIX K 
Radiotherapy Treatment Guidelines (FHCRC Protocol 2222) 
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. 
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: 
I. 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. 
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. 
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. 
Lower Mediastinum (Point 4): this reference point is defined along the patient's longitudinal axis at the level 
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of the xiphisternal notch. The reference point is midway between the entrance and exit points of the opposed 
beams 
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. 
Knee (Point 6): this reference point is defined along the midline in the midplane of the knee at the level of the 
patella. 
Ankle (Point 7): this reference point is defined along the midline at the midplane of the ankle at the level of 
the lateral malleolus. 
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. 
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 1-20%, -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 radiation oncology study 
coordinators, Dr. Kelly or Dr. Roberts: 
Ralph Ermoian, MD 
Department of Radiation Oncology 
1959 NE Pacific St. 
Box 356043 
Seattle, WA, 98195 
Work: 206 598 4100 
Email: ralphpe@uw.edu  
Kenneth Roberts, MD 
Yale University School of Medicine 
Department of Therapeutic Radiology 
Hunter Radiation Therapy Center 
P.O. Box 208040 
New Haven, CT 06520-8040 
Phone : (203) 785-2957 
Fax : (203) 785-4622 
Email : kenneth.roberts@yale.edu  
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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