A Phase I Study of NK Cell Infusion Following Allogeneic Peripheral Blood Stem Cell Transplantation From Related or Matched Unrelated Donors in Pediatric Patients With Hematologic Malignancies
Phase: PHASE1
Conditions: Leukemia, Lymphoma
Sponsor: National Cancer Institute (NCI)

Abbreviated Title: NK cell therapy
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Abbreviated Title: NK Cell Therapy
NCI Protocol #: 11-C-0073 R
Version Date: 07/23/2018
NCT#: NCT01287104
Title: A Phase I Study of NK Cell Infusion Following Allogeneic Peripheral Blood Stem Cell 
Transplantation from Related or Matched Unrelated Donors in Pediatric Patients with 
Hematologic Malignancies
NCI Principal Investigator:
Nirali N. Shah, M.D.
Pediatric Oncology Branch, CCR, NCI
10 Center Drive, CRC Rm 1-1621
Bethesda, MD 20892
Phone: (240) 760-6199
Email: shahnn@mail.nih.gov
Investigational Agents:
Drug Name:
Ex vivo Expanded Allogeneic NK Cells
KT64.4-1BBL Artificial Antigen Presenting Cells
rhIL15
Miltenyi CliniMACS£ System
IND Number:
14402
Sponsor:
Center for Cancer Research, NCI
Manufacturer:
Cell Processing Department, Department of 
Transfusion Medicine, Clinical Center, NIH
Commerical Agents: fludarabine, cyclophosphamide, etoposide, doxorubicin, vincristine, 
prednisone, cytarabine, methotrexate, busulfan, cyclosporine A, tacrolimus

Abbreviated Title: NK cell therapy
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PRECIS
Background
x
Despite progress in pediatric oncology, some patient subsets with hematologic 
malignancies continue to experience extremely poor overall survival. Allogeneic 
Hematopoietic Stem Cell Transplant (HSCT) is effective in some high-risk hematologic 
malignancies.
x
Allogeneic HSCT can be performed safely in these patient populations, but disease 
recurrence is common and new approaches to enhance the antitumor effect of this therapy 
are needed. NK mediated killing appears to confer improved outcomes after HSCT for 
patients with AML and ALL, and NK cell infusions have induced complete remissions in 
patients with AML.
x
Preclinical data demonstrates that activated NK cells readily kill pediatric leukemias, that 
large numbers of activated NK cells can be generated ex vivo using artificial APCs and 
that the post-transplant period may be favorable for expansion and survival of adoptively 
transferred NK cells.
Objectives
x
To assess the feasibility and toxicity of infusing escalating doses of donor-derived 
activated NK cell donor lymphocyte infusions (NK-DLI) on Days 21 ± 3 days and 49 ± 7 
days following HLA-matched, T cell depleted (TCD) PBSCT in patients with high risk 
leukemias who have unrelated donors or related donors
x
To determine if patients treated in this manner experience rapid, sustained donor 
engraftment and acceptable rates of aGVHD (<25% incidence of grade III or grade IV).
Eligibility
x
Patients 4-35 years with hematologic malignancies (e.g., ALL, AML, CML, HD, NHL), with 
a 5/6 or 6/6 HLA-matched related or 9/10 or 10/10 HLA matched unrelated donor.  
Design
x
Pre-transplant disease specific immune depleting chemotherapy and the preparative 
regimen will be the same as that used previously on 02-C-0259 and 01-C-0125, for those 
patients undergoing reduced intensity transplant.
x
For patients with hematologic malignancies, a myeloblative regimen based on current 
Children’s Oncology Group (COG) standard-of-care preparative regimen will also be 
included.
x
Donors will undergo apheresis for filgrastim mobilized PBSC.  This product will be T 
cell and NK cell depleted prior to cryopreservation.  NK cells selected from the product 
will be used for ex vivo activation and expansion using KT64.4-BBL artificial antigen 
presenting cells. 
x
A phase 1 cell dose escalation of donor derived NK-DLI will be performed using 3 dose 
levels (1 x 105, 1 x 106 and 1 x 107 NK cells/kg) infused on days 21±3 post-PBSCT and a 
second infusion on day 49 ± 7 post-PBSCT.
x
Three patients will be enrolled at each dose level, with the cohort expanded to 6 if dose-
limiting toxicity occurs.  An expanded group of 12 patients will be treated at the highest 
dose level tolerated.

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TREATMENT SCHEMA
Donor
Patient 
Stem Cell Harvest and NK Cell Collection
Filgrastim mobilization, for 5-7 days until collection completed
Stem cell collection, target >10 x 106 CD34 cells/kg, minimal 8 x 106 CD34 cells/kg (pre-
selection)
Stem cell cryopreservation and NK cell expansion
Fludarabine-EPOCH Induction Chemotherapy for Lymphoma or Leukemia Patients
1-3 cycles; 21 – 28 day cycles; Cycles 2 and 3 may be dose-modified as outlined in the protocol
Fludarabine, 25 mg/m2 per day IV over 30 minutes daily for 3 days; days 1-3
Etoposide, 50 mg/m2 per day by continuous IV infusion daily for 4 days; days 1-4
Doxorubicin, 10 mg/m2 per day by continuous IV infusion daily for 4 days; days 1-4
Vincristine, 0.4 mg/m2 per day by continuous IV infusion daily for 4 days; days 1-4
Cyclophosphamide, 750 mg/m2 IV over 30 minutes; day 5
Prednisone, 60 mg/m2 per day in 2-4 divided doses PO for 5 days; days 1-5
Filgrastim, 5 Pg/kg per day SQ from day 6 until ANC >1000/Pl x 2 days
Pre-BMT Preparative Regimen (Non-myeloablative)
Transplant Days –5 to -2
Fludarabine, 30 mg/m2 per day IV over 30 minutes daily for 4 days; days –5, -4, -3, -2 of transplant
Cyclophosphamide, 1200 mg/m2 per day IV over 2 hours daily for 4 days; days –5, -4, -3, -2 of 
transplant
Mesna, 1200 mg/m2 per day by continuous IV infusion daily for 4 days; days –5, -4, -3, -2. 
FLAG Induction Chemotherapy for Leukemias (Non-myeloablative)
1-3 cycles; 21 – 28 d cycles; Cycles 2 and 3 may dose-modified as outlined in the protocol
Filgrastim, 5 Pg/kg per day SQ from day 0 until ANC >1000/Pl x 2 days 
Fludarabine, 25 mg/m2 per day IV over 30 minutes daily for 5 days; days 1-5
Cytarabine, 2000 mg/m2 IV over 4 hours daily for 5 days; days 1-5; Begin each dose 3.5 hours after 
completing fludarabine; Conjunctivitis prophylaxis with corticosteroid ophthalmic drops 2 drops to 
each eye every 6 hours starting prior to and continuing until 24 hours after completion of cytarabine.

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Stem Cell Infusion
Transplant Day 0: >4 x 106/kg CD34+ stem cells by IV infusion
NK Cell Infusion
Post-transplant Day 21 (± 3 days): (1 x 105, 1 x 106 or 1 x 107) NK cells/kg by IV infusion.
Followed by a second NK cell infusion of the same cell dose, on Day 49 ± 7 days.
Pre-BMT Preparative Regimen (Myeloablative)
AML Patients:
Day
Treatment
-6
Anti Seizure Prophylaxis*
-5 to -2
Fludarabine 40 mg/m2/dose IV daily (1.3 mg/kg if <10kg)**
-5 to -2
Busulfan 3.2 mg/kg/dose IV daily x 4 doses***
-1
Rest (an additional day may be added)
0
HSCT Infusion
Pre-BMT Preparative Regimen (Myeloablative)
ALL Patients:
Day
Treatment
-6 to -4
TBI 200 cGy BID
-3 to -2
Cyclophosphamide 60 mg/kg/dose IV daily
-3 to -2
Mesna 60 mg/kg/dose IV daily (continuous infusion)
-1
Rest (an additional day may be added)
0
HSCT Infusion

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TABLE OF CONTENTS
PRECIS ......................................................................................................................................2 
TREATMENT SCHEMA ...........................................................................................................3 
TABLE OF CONTENTS............................................................................................................5 
1 
INTRODUCTION.........................................................................................................11 
1.1 
Objectives........................................................................................................11 
1.1.1 Primary............................................................................................................11 
1.1.2 Secondary ........................................................................................................11 
1.2 
Background and Rationale ...............................................................................11 
1.2.1 Allogeneic Stem Cell Transplant for Pediatric Solid Tumors............................11 
1.2.2 Pediatric Leukemias and Lymphoma................................................................ 15 
1.2.3 Current Standard of Care for Eligible Patients.................................................. 16 
1.2.4 Matched Unrelated Donor Transplant in Pediatrics........................................... 23 
1.2.5 T Cell Depletion in Allogeneic Stem Cell Transplantation ............................... 27 
1.2.6 GVHD prophylaxis after T cell depleted AlloHSCT.........................................29 
1.3 
Pre-clinical Models of Natural killer cells: Susceptability of Pediatric Tumors to NK 
Cell Lysis..................................................................................................................30 
1.3.1 Leukemia Models.............................................................................................30 
1.3.2 Neuroblastoma (This study is no longer enrolling patients with solid tumors as of 
Amendment K [see section 1.4.1.3]) ......................................................................... 31 
1.3.3 Ewing sarcoma................................................................................................. 32 
1.3.4 Rhabdomyosarcoma.........................................................................................32 
1.3.5 Natural killer cells and AlloHSCT.................................................................... 32 
1.3.6 Natural killer cells and engraftment.................................................................. 34 
1.3.7 EXAMINING KIR RECEPTORS.................................................................... 34 
1.3.8 Adoptive immunotherapy with NK cells...........................................................34 
1.3.9 Ex-vivo Expansion of NK Cells ....................................................................... 35 
1.4 
Summary: Reduced Intensity Transplantation with T Cell Depleted hematopoetic Stem 
Cell Grafts and Donor NK Cell Infusions .................................................................. 38 
1.4.1 Study Updates..................................................................................................38 
2 
Eligibility Assessment and Enrollment ..........................................................................44 
2.1 
Eligibility Criteria for Patients (Recipient) ....................................................... 44 
2.1.1 Inclusion Criteria: Patient (Recipient) .............................................................. 44 

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2.1.2 Exclusion Criteria: Patient (Recipient) ............................................................. 46 
2.2 
Eligibility Criteria for Donors .......................................................................... 46 
2.2.1 Inclusion Criteria: Donor.................................................................................. 46 
2.2.2 Exclusion Criteria: Donor.................................................................................47 
2.3 
Research Eligibility Evaluation ........................................................................47 
2.3.1 Clinical Evaluation Recipient...........................................................................47 
2.3.2 Clinical Evaluation Donor................................................................................48 
2.3.3 Specialty Consultation......................................................................................48 
2.3.4 Laboratory Serologic Evaluations..................................................................... 48 
2.3.5 Radiologic, Nuclear Medicine, and Specialty Studies (Recipients only) ........... 49 
2.3.6 Pathologic/Tissue Evaluation ...........................................................................49 
2.4 
Registration Procedures.................................................................................... 50 
3 
STUDY IMPLEMENTATION......................................................................................50 
3.1 
Study Design....................................................................................................50 
3.1.1 NK Cell Infusion Definition of Dose Limiting Toxicity (DLT)......................... 51 
3.1.2 Requirements for DLI-NK Cell Infusions......................................................... 51 
3.2 
Allogeneic Peripheral Blood Stem Cell Transplant:..........................................52 
3.2.1 Donor Peripheral Blood Stem Cell Harvest: .....................................................52 
3.2.2 Biopsy of accessible tumor:..............................................................................54 
3.3 
Drug Administration and Treatment Modifications .......................................... 54 
3.3.1 Induction Chemotherapy.................................................................................. 54 
3.3.2 Transplantation: ...............................................................................................57 
3.4 
On Study Protocol Evaluation (Appendix 2 A):................................................ 62 
3.4.1 Baseline ...........................................................................................................62 
3.4.2 Induction Chemotherapy Cycles (EPOCH-F or FLAG cycles) ......................... 62 
3.4.3 Pre-HSCT evaluation .......................................................................................63 
3.4.4 HSCT Hospitalization ......................................................................................63 
3.4.5 Evaluations After HSCT discharge through day 100 (±30 days), as clinically indicated
64 
3.4.6 Evaluations after Day 100 (Appendix 2) (±30 - 90 days): ................................ 64 
3.4.7 Related Donor Evaluations During Treatment (Appendix 2 B) ........................65 
3.5 
NCI/NIH Biologic studies ................................................................................65 
3.5.1 Proposal for Immune Function Studies (Brown)............................................... 65 

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3.5.2 Chimerism Analysis (Kurlander)......................................................................66 
3.5.3 Serum Cytokine Analysis (Lab) .......................................................................66 
3.5.4 TH1/TH2 Cytokine Profile (Frederick Repository)...........................................67 
3.5.5 KIR/HLA genotyping/immunogenetics (Carolyn Hurley – Georgetown Univ.) complete
67 
3.5.6 Viral monitoring for functional Immune Status ................................................67 
3.5.7 Research Testing of Biopsy Samples................................................................67 
3.5.8 NIH Policy on Research Use of Stored Human Samples, Specimens, or Data:..68 
3.6 
Concurrent Therapy ......................................................................................... 68 
3.6.1 Treatment of Graft-Versus-Host Disease:......................................................... 68 
3.6.2 Immunosuppressants........................................................................................ 69 
3.6.3 Infection Prophylaxis ....................................................................................... 69 
3.6.4 GI Protective Agents........................................................................................69 
3.6.5 Hematologic and Blood Product Support..........................................................69 
3.6.6 Concurrent Therapy for Extramedullary Leukemia or CNS Lymphoma: .......... 69 
3.6.7 Treatment of Progressive Disease Post-transplant:............................................70 
3.6.8 Treatment of Mixed Chimerism Post-transplant: .............................................. 71 
3.6.9 Treatment of Graft Failure/Graft Rejection:...................................................... 71 
3.7 
Criteria for Removal from Protocol Therapy and Off Study Criteria.................72 
3.7.1 Criteria for removal from protocol therapy....................................................... 72 
3.7.2 Off-Study Criteria ............................................................................................72 
3.7.3 Off Protocol Therapy and Off-Study Procedure................................................72 
3.8 
End of Study Evaluation (Appendix 2)............................................................. 72 
3.8.1 History and physical examination.....................................................................72 
3.8.2 Laboratory Assessment:  Iron studies (serum iron, ferritin, and transferrin), Complete 
blood count, differential and platelet count. Chem 20 and urinalysis. ........................ 72 
3.8.3 Disease restaging ............................................................................................. 73 
3.8.4 Apheresis. (Optional) ....................................................................................... 73 
3.9 
Duration of study ............................................................................................. 73 
4 
SUPPORTIVE CARE ...................................................................................................73 
4.1 
Infection Prophylaxis ....................................................................................... 73 
4.2 
Mucositis Prophylaxis......................................................................................73 
5 
DATA COLLECTION AND EVALUATION...............................................................73 

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5.1 
Data Collection ................................................................................................73 
5.2 
Response Criteria.............................................................................................74 
5.3 
Toxicity Criteria...............................................................................................74 
5.4 
Data Reporting Exceptions...............................................................................74 
6 
STATISTICAL CONSIDERATIONS ...........................................................................75 
6.1 
Subject Accrual................................................................................................75 
6.2 
Statistics and Feasibility...................................................................................75 
6.3 
Early Stopping Rules........................................................................................ 76 
6.4 
Subject Accrual Goal .......................................................................................76 
7 
HUMAN SUBJECTS PROTECTIONS.........................................................................77 
7.1 
Rationale for subject selection.......................................................................... 77 
7.1.1 Recipients ........................................................................................................77 
7.1.2 Donors............................................................................................................. 77 
7.2 
Participation of Children .................................................................................. 77 
7.3 
Evaluation of benefits and risks/discomforts..................................................... 77 
7.3.1 Recipients ........................................................................................................77 
7.3.2 Donors............................................................................................................. 79 
7.4 
Risk/Benefit Analysis.......................................................................................80 
7.5 
Consent Processes and Documentation.............................................................80 
7.5.1 Recipients ........................................................................................................80 
7.5.2 Related donors ................................................................................................. 81 
7.5.3 Unrelated Donors............................................................................................. 81 
7.5.4 Consent for minors when they reach the age of majority...................................81 
7.5.5 Telephone consent............................................................................................82 
8 
SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING 
PLAN 82 
8.1 
Definitions .......................................................................................................82 
8.1.1 Adverse Events ................................................................................................82 
8.1.2 Suspected adverse reaction...............................................................................82 
8.1.3 Unexpected adverse reaction............................................................................82 
8.1.4 Serious.............................................................................................................82 
8.1.5 Serious adverse events ..................................................................................... 82 
8.1.6 Disability .........................................................................................................83 

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8.1.7 Life-threatening adverse drug experience ......................................................... 83 
8.1.8 Protocol Deviation (NIH Definition) ................................................................ 83 
8.1.9 Non-compliance (NIH Definition).................................................................... 83 
8.1.10 Unanticipated Problem...................................................................................83 
8.2 
NCI-IRB and Clinical Director Reporting ........................................................ 83 
8.2.1 NCI-IRB and NCI CD Expedited Reporting of Unanticipated Problems and Deaths
83 
8.2.2 NCI-IRB Requirements for PI Reporting at Continuing Review....................... 84 
8.2.3 NCI-IRB Reporting of IND Safety Reports......................................................84 
8.3 
IND SPONSOR REPORTING CRITERIA .............................................................84 
8.3.1 Action Plan for Positive Results on Cell Product Safety Testing....................... 85 
8.4 
Data Safety Monitoring Plan ............................................................................ 85 
8.4.1 Principal Investigator/Research Team ..............................................................85 
8.4.2 Sponsor Monitoring Plan.................................................................................. 85 
8.4.3 Safety Monitoring Committee (SMC) .............................................................. 86 
9 
Pharmaceutical Information...........................................................................................86 
9.1 
Fludarabine (Fludara)....................................................................................... 86 
9.2 
Mesna (sodium 2-mercaptoethanesulfonate, Mesnex)....................................... 87 
9.3 
Cyclophosphamide (Cytoxan) ..........................................................................87 
9.4 
Etoposide (VePesid, VP-16)............................................................................. 88 
9.5 
Doxorubicin (Adriamycin) ...............................................................................88 
9.6 
Vincristine .......................................................................................................88 
9.7 
Prednisone .......................................................................................................89 
9.8 
Filgrastim (G-CSF, Neupogen)......................................................................... 89 
9.9 
Pegfilgrastim (recombinant methionyl human G-CSF, Neulasta™).................. 90 
9.10 Cytarabine (Cytosine Arabinoside, Cytosar, ARA-C).......................................90 
9.11 Intrathecal Chemotherapy ................................................................................ 91 
9.11.1 Methotrexate (MTX, amethopterin) NSC #000740......................................... 91 
9.11.2 Hydrocortisone (Cortef, Solu-cortef).............................................................. 92 
9.11.3 Cytarabine (cytosine arabinoside, AraC, Cytosar)........................................... 92 
9.12 Ranitidine (Zantac): ......................................................................................... 93 
9.13 Acetaminophen (Tylenol):................................................................................ 93 
9.14 Diphenhydramine (Benadryl):.......................................................................... 93 

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9.15 Fluconazole (Diflucan):.................................................................................... 93 
9.16 PCP Prophylaxis ..............................................................................................94 
9.16.1 Trimethoprim/Sulfamethoxazole (TMP/SMX) (Bactrim, Septra):...................94 
9.16.2 Pentamadine:..................................................................................................94 
9.17 Intravenous Immunoglobulin (IVIG, Gamimmune, Gammagard, Sandoglobulin):94 
9.18 Palifermin, Kepivance™ (KGF)....................................................................... 94 
9.19 Recombinant Human Interleukin 15 (IL-15).....................................................95 
9.20 Cyclosporine A ................................................................................................95 
9.21 Tacrolimus (FK506, Prograf) ........................................................................... 96 
9.22 Busulfan (Busulfex) ......................................................................................... 96 
9.23 Levetiracetam (Keppra)....................................................................................97 
9.24 Lorezapam (Ativan) .........................................................................................97 
9.25 Radiation Therapy............................................................................................97 
9.25.1 TOTAL BODY IRRADIATION....................................................................97 
9.25.2 CNS Boost .....................................................................................................97 
10 
REFERENCES..............................................................................................................99 
11 
APPENDICES.............................................................................................................117 
11.1 Appendix 1: Performance Status .................................................................... 117 
11.2 Appendix 2: Required Study Evaluations ....................................................... 119 
11.2.1 Appendix 2 A: Patient.................................................................................. 119 
11.2.2 Appendix 2 B: Donor................................................................................... 122 
11.3 Appendix 3: Screening and Baseline Evaluation............................................. 124 
11.4 Appendix 4: Summary of Research Bloods* .................................................. 127 
11.5 Appendix 5: Acute GVHD ............................................................................. 129 
11.6 Appendix 6: Chronic GVHD Exam and grading, Clinician assessment form .. 131 
11.7 Appendix 7: Chronic GVHD, NIH Staging Form........................................... 136 
11.8 Appendix 8: Leukemia/Lymphoma Response Criteria.................................... 139 
11.9 Appendix 9: Solid Tumor Response Criteria (RECIST).................................. 142 
11.10 Appendix 10: Filgrastim Dosing for Donors Prior to Apheresis...................... 143 
11.11 Appendix 12: Engraftment Syndrome (Spitzer, 2001) .................................... 144 
11.12 Appendix 13: TBI Guidelines......................................................................... 145 
11.13 Appendix 14: Calculation of Ideal Body Weight and  Dose Adjustments........ 146 

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1
INTRODUCTION
1.1
OBJECTIVES
1.1.1
Primary
1. Assess the feasibility and toxicity of infusing escalating doses of unrelated or related donor-
derived activated NK cell donor lymphocyte infusions (NK-DLI) on days 21 ± 3 days, and 
49 ± 7 days following allogeneic human leukocyte antigen (HLA)-matched T cell depleted 
(TCD) peripheral blood stem cell transplantation (PBSCT) in patients with high risk 
hematologic malignancies (leukemias and lymphomas).
2. Determine if TCD allogeneic HLA-matched PBSCT + NK-DLI results in rapid, sustained 
donor lymphoid engraftment (>95% at day 100) and acceptable rates of acute graft-versus-
host-disease (aGVHD) (<25% incidence of grade 3 or grade 4).
1.1.2
Secondary
1. Investigate the incidence of cGVHD in patients receiving related or unrelated donor HLA-
matched allogeneic PBSCT followed by NK-DLI. 
2. Investigate the incidence of viral infection and/or reactivation in patients receiving HLA-
matched allogeneic PBSCT followed by NK-DLI. 
3. Compare disease-free (DFS) and overall survival (OS) of patients treated on this protocol 
with our previous experience using T cell-replete matched sibling PBSCT in similar patient 
populations.
4. Correlate post-transplant NK cell, B cells and T cells numbers with select immunologic 
parameters including IL-7 and IL-15 levels.
5. Retrospectively investigate the impact of KIR expression and KIR reactivity on 
progression free survival in patients enrolled on this study. 
1.2
BACKGROUND AND RATIONALE
1.2.1
Allogeneic Stem Cell Transplant for Pediatric Solid Tumors
Advances in multimodal therapies has resulted in an overall survival rate of 75% for all children 
diagnosed with cancer; the greatest success has been seen in patients with ALL, the most 
commonly diagnosed pediatric cancer. However, the management of pediatric patients with 
metastatic and recurrent solid tumors remains a considerable challenge. Depending upon the 
histology, 25-50% of children with newly diagnosed solid tumors have metastatic disease at 
presentation and long-term survival rates using standard therapies for these patients vary from 5-
35%. Furthermore, patients who present with non-metastatic disease but experience poor responses 
to frontline therapy or early recurrence (within 24 months of new diagnosis) have dismal long-
term outcomes.  In addition, patients with desmoplastic small round cell tumor appear to have 
uniquely poor outcomes, even when they present without evidence for disseminated disease.  
Currently, salavage regimens for pediatric solid tumors have response rates in the range of 20-30% 
and rarely lead to durable responses(1). There are several targeted agents (eg. anti-IGFR-1, anti-
VEGF) in phase I and II development that hold promise in pediatric solid tumors, however these 
agents are active in limited diseases and appear not to lead to durable responses or remissions.

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Despite these poor overall outcomes for patients with primary metastatic, early recurrent or poorly 
responsive solid tumors, standard therapeutic multimodality approaches can mediate impressive 
antitumor effects.  We have therefore sought to develop immune based therapies that can be 
incorporated into the standard therapeutic armamentarium for patients with ultra high-risk 
pediatric sarcomas.  Among the approaches under study include the use of allogeneic stem cell 
transplantation.  The rationale for undertaking this approach for ultra high-risk pediatric solid 
tumors is based upon several factors.  First, immune reactivity occurring in the setting of allogeneic 
stem cell transplantation mediates impressive effects in hematologic malignancies, with graft-
versus-leukemia providing substantial antileukemic effects in CML, modest effects in AML and 
lesser but present effects in ALL.  Second, preclinical studies have demonstrated that the allogeneic 
environment may be particularly ripe for administering immune based therapies including tumors 
vaccines, adoptive T cell therapy and NK cell therapy.  Third, an allogeneic GVT effect has been 
seen in both myeloablative and reduced intensity transplantation (RIT) for a variety of solid tumors 
including breast (2, 3), colon (4), renal (4, 5), ovarian, and pancreatic cancers (6).  Fourth, 
improvements in HLA typing and supportive care, and the introduction of reduced-intensity 
conditioning regimens has made allogeneic HSCT safer and may improve outcomes in this heavily 
pre-treated population(7), thus providing new options for incorporating this therapy into a complex 
multimodality regimen for solid tumors.  Indeed, reduced-intensity alloHSCT has been proven to 
be a well-tolerated, feasible approach of therapy for a variety of pediatric tumors; neuroblastoma 
(8 , 9-11) and pediatric sarcoma (12, 13)[POB 02-C-0259 unpublished experience], with the results 
comparable in terms of tumor response and disease control with those observed using conventional 
approaches beyond second-line therapies. 
Fifth, while benefit from allogeneic HSCT for pediatric solid tumors is not proven, several groups 
undertaking this therapy have had encouraging results. A number of case reports have utilized 
allogeneic HSCT in Ewing sarcoma and have suggested a possible GVT effect for this disease(12-
16). Experience with allogeneic HSCT for rhabdomyosarcoma has been limited, with one case 
series reporting a lack of benefit after using matched sibling PBSCs(17), but others reporting more 
success using matched sibling HSCT from PBSCs and bone marrow(18, 19).
Whether GVT 
effects occur in neuroblastoma patients following allogeneic HSCT is not well established.  Early 
studies failed to demonstrate a survival benefit comparing autologous to allogeneic HSCT(8, 20),
however, these studies were not randomized and a larger percentage of high-risk patients were in 
the allogeneic transplant group (20).  More recent case reports have reported some benefit, 
including one using mismatched cord-blood(21) as well as haploidentical bone marrow(10) and 
matched PBSCs(11).  One case series using a reduced-intensity prep followed by haploidentical 
PBSCs showed engraftment in 6/6 patients with neuroblastoma, Ewing sarcoma or 
rhabdomyosarcoma, with NK cell engraftment completed within 4 weeks post-transplant.  The 2 
patients that were still alive had refractory neuroblastoma(22).
The POB has completed a NCI POB 02-C-0259, A Pilot Study of Allogeneic/Syngeneic Blood 
Stem Cell Transplantation in Patients with High-Risk and Recurrent Pediatric Sarcomas, the 
results of which serve as the basis for the backbone of this study. The EPOCH regimen has been 
studied extensively at the NCI and consists of etoposide, vincristine and doxorubicin administered 
as a continuous infusion for four days, cyclophosphamide administered as a bolus on day 5, and 
prednisone given orally for five days. The agents contained in this regimen are known to have 
activity against pediatric sarcomas and other solid tumors. Fludarabine was added the EPOCH 
regimen to enhance the degree of immune depletion.  In this study, as well as several studies in 

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adults in the ETIB, the EPOCH-fludarabine pre-transplant immune depleting regimen was well 
tolerated, achieving effective CD4 depletion and patients who received allogeneic transplant 
following this regimen experienced rapid engraftment (2, 3, 23-25).  Cyclophosphamide and 
melphalan are classical bifunctional alkylators, with a steep dose-response curve, are non-cross-
resistant, differing nonhematologic toxicities, and are potentially synergistic.  The most common 
toxicity of this combination is myelotoxicity and mucositis (26, 27). The use of combination of 
melphalan, cyclophosphamide and fludarabine has been successfully employed in several studies 
(28, 29) including 02-C-259 conducted in the POB. 
To date, a total of 30 donors and 30 recipients have been enrolled on 02-C-0259. Of these, 23 
patients have undergone alloPBSCT while 7 patients were removed from study prior to transplant 
due to progressive disease. To achieve antitumor effects and recipient immune depletion, patients 
received 1-3 cycles of a fludarabine-based induction regimen in combination with EPOCH 
followed by a preparative regimen of cyclophosphamide (1,200 mg/m2/day) and fludarabine (30 
mg/m2/day) for 4 days and one dose of melphalan (100 mg/m2). Unmodified peripheral blood stem 
cell (PBSC) grafts were collected from 5-6/6 HLA-matched first-degree relatives. All donors were 
mobilized with 10 μg/kg/day of filgrastim for 5 days, and apheresis was performed to collect a 
minimum of 4 x 106/kg CD34+ PBSCs. Successful collection was achieved in all donors with a 
single apheresis, except for one due to a technical problem with apheresis and not from poor 
mobilization. Of the 24 patients transplanted, there has been no transplant related mortality (death 
within 100 days due to transplant complications), peri-transplant morbidity has been modest with 
short hospital stays (25 day median), hematopoietic recovery has been rapid (day + 11 is median 
day to ANC >500 and day +12 is median day to platelet > 20K), and full donor engraftment has 
occurred in 100% of patients by day + 28. Donor engraft was independent of CD4 count at entry 
or prior to transplant. Patients with pre-transplant counts as high as 490/mm3 engrafted rapidly 
and there was no mixed chimerism or graft rejection encountered.
In the initial cohort (cohort 1, n=12), GVHD prophylaxis consisted of cyclosporine as a single 
agent. Although high-grade (grade III-IV) aGVHD was not observed, we observed a high 
incidence of low-grade aGVHD and universal occurrence of cGVHD (12/12 evaluable patients). 
The high incidence of GVHD precluded donor lymphocyte infusion (DLI) and required systemic 
immunosuppression in many patients, therefore the GVHD prophylaxis was modified from 
cyclosporine alone in cohort 1 to tacrolimus and sirolimus in cohort 2. The second cohort (n = 11)
has shown similarly low peri-transplant morbidity and full donor engraftment in all patients. 
Further, the rates of aGVHD are significantly reduced, with 3/11 patients developing grade 1, one 
patient with grade 3, and one patient with grade 4 aGVHD. The incidence of cGVHD in the second 
cohort appears to show improvement with the new immunosuppression therapy with 3/10 
evaluable patients having extensive cGVHD and 2/10 with limited cGVHD. Despite the decrease 
in overall GVHD rates and severity, not all patients tolerated the prophylactic regimen consisting 
of tacrolimus and sirolimus and cGVHD occurred almost universally in those who did not receive 
the complete course of immunosuppression with both agents and no patients received DLI.
Thus, the regimen used in cohort 2 of 02-C-0259 accomplished good engraftment, acceptable 
toxicity and acceptable rates of aGVHD and cGVHD.  When evaluating for disease control during 
EPOCH-F therapy, 2 patients had partial responses (PR), 11 patients had stable disease (SD), 5
remained with no evidence of disease (NED), and 12 patients had progressive disease (7 of these 
patients did no go onto transplant. At the day 100 evaluation 11 patients had progressive disease, 

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5 patients had stable disease, 5 patients remained NED and 2 patients previously with disease 
became NED. Graft versus tumor effect was demonstrated in one patient with a skull-based lesion 
that emerged and subsequently regressed after transplant. In addition, several patients who went 
into tranplant with chemotherapy refractory disease were then chemosensitive following 
transplant, including 5 patients that became NED with post-transplant EPOCH therapy. 
Furthermore, median survival of patients on 02-C-0259 was higher than that for a historical control 
group identified a priori with similarly high-risk disease (median survival from diagnosis of 
historical controls 18 mos. vs. 36 month of all patients enrolled on 02-C-0259 vs. 45 months for 
patients receiving BMT on 02-C-0259).  Despite these promising results, disease recurrence 
remains a substantial problem.  Figure 1a shows the survival of all patients enrolled on 02-C-0259 
from the time of enrollment using an intent-to-treat approach.  Figure 1b shows survival of all 
patients receiving transplant.  Five year overall survival was 25% using the intent-to-treat analysis 
and 33% in BMT recipients, which is comparable to historical controls for this ultra high-risk 
patient population.  Moreover, all but one patient experienced disease recurrence, and many 
require chronic therapy with chemotherapy to sustain disease control.  Thus, the antitumor effects 
of this allogeneic stem cell transplant regimen is not adequate to cure these ultra high risk patients. 
Figure 1
Based upon these results, our goal for the current study is to use a similar platform, but to attempt 
to improve disease free survival by augmenting graft-versus-tumor activity.  Graft-versus-tumor 
effects can be T cell mediated or NK cell mediated.  Because of our concerns regarding the high 
rate of GVHD and the requirement for immunosuppression in order to treat this disorder, the 
current study will seek to completely eliminate GVHD using T cell depletion and focus on 
augmentation of NK reactivity.  Evidence from preclinical studies concluded that NK cells do not 
initiate GVHD and therefore it is predicted that NK cell infusions will not induce GVHD.  
Furthermore, activated NK cells can now be readily generated and they show potent killing activity 
of pediatric solid tumors and ALL blasts.  On this basis, we will seek to build upon our previous 
platform by maintaining the previous pre-transplant and peri-transplant preparative regimen, but 
incorporate T cell depletion and escalating NK cell infusions in an attempt to enhance graft-versus-
tumor effects.

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1.2.2
Pediatric Leukemias and Lymphoma
As a group, childhood leukemias represent the most common pediatric malignancy, accounting 
for approximately 32% of cancer in children younger than 15 years and 25% under 20 years of 
age. ALL is by far the most common, comprising approximately 23% of childhood cancer with 
an annual rate of 30 to 40 new cases per million U.S. children. AML accounts for approximately 
4% of pediatric cancer diagnoses and 20% of childhood leukemia with an annual rate in the U.S. 
of 8 per million. Ph+ CML is rare and accounts for approximately 1% of all pediatric cancer, 
although it comprises 10% of leukemia in older adolescents. Although the majority of pediatric 
patients with leukemia are cured, leukemia remains the second most frequent cause of death 
from cancer in children. Similarly, cure rates are excellent for both Hodgkin’s disease and non-
Hodgkin’s lymphomas (NHL) in children, however outcomes are poor for those with refractory 
disease and for those who relapse after standard salvage therapies such as autologous bone 
marrow transplant (ABMT). Conventional alloHSCT appears to offer benefit over ABMT and 
can be used for salvage in those settings.
There have been no large prospective randomized controlled clinical trials to definitively evaluate 
the role of alloHSCT versus chemotherapy in the management of childhood ALL. This is due in 
part to the success of standard front-line chemotherapy and the inherent variability in stem cell 
donor availability. Nonetheless, multiple comparative studies indicate that relapse rates are lower 
after alloHSCT in comparison to chemotherapy, and alloHSCT is standard of care therapy for 
patients in CR#2 or for select very high-risk patients in CR#1(30). Our branch has experience 
delivering a reduced intensity alloHSCT to leukemia and lymphoma patients enrolled on protocol 
01-C0125 using a reduced intensity platform similar to that described above for protocol 02-C-
0259. Lymphoma patients received cycles of EPOCH-F, followed by a non-myeloablative PBSCT 
with a preparative regimen consisting of cytoxan and fludarabine. Leukemia patients received 3 
cycles of FLAG chemotherapy, followed by the same preparative regimen consisting of cytoxan
and fludarabine. A total of 15 recipients have undergone alloHSCT on this protocol. Immune 
depletion with pre-transplant therapy was achieved in all subjects and all patients tolerated 
induction chemotherapy cycles with anticipated side effects. The most common side effects 
included 
nausea, 
vomiting, 
abdominal 
pain, 
elevated 
transaminases, 
neutropenia, 
thrombocytopenia, anemia, neutropenic fever, infection, electrolyte abnormalities, and 
hypertension. Hematopoietic recovery was rapid and complete for all subjects with median times 
to ANC >500/μL of 10 days (range, 7-13) and platelet count >20,000/μL of 11 days (range, 8-16, 
1 not evaluable). There has been rapid lymphoid recovery post-transplant with evidence for early 
recovery of thymic derived T-cells. CD4, CD8, and NK cell counts increased to levels above 
baseline 1 month after transplant.  Similar to the solid tumor study, donor engraft kinetics were 
independent of CD4 count at entry or prior to transplant. All patients engrafted rapidly and there 
was no mixed chimerism or graft rejection encountered. Thirteen of 15 recipients developed 
aGVHD and 12 of 13 with > 100 days of follow-up have developed cGVHD. GVHD has responded 
to therapy in all patients. Although there have been no deaths directly attributable to GVHD on 
this trial, this protocol was modified to use bone marrow rather than PBSCs to reduce cGVHD 
incidence and severity.
Eight of 15 patients (50%) remain alive (median follow-up 34 mo; range 3-81 mo), six (40%) have 
died of relapsed leukemia or lymphoma, and 1 died of toxicity. Six of 15 patients (40%) remain in 
continuous CR (3-60 mo) and 2 (14%) are alive with disease. Of the patients with relapse, only 1 
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in 2 patients. In addition, anti-tumor alloreactivity was suggested by apparent enhanced 
chemotherapy responses in 3 patients with Hodgkin’s.  In summary, this non-myeloablative 
platform can reproducibly induce full donor engraftment with acceptable treatment related 
mortality for patients with high risk hematologic malignancy.  At the same time, disease recurrence 
remains the major cause for treatment failure therefore new approaches are needed to enhance 
antitumor effects of non-myeloablative HSCT for hematologic malignancies.
1.2.3
Current Standard of Care for Eligible Patients 
1.2.3.1
Ewing Sarcoma (This study is no longer enrolling patients with solid tumors as of 
Amendment K [see section 1.4.1.3])
Prognosis of patients with metastatic disease is extremely poor with 6-year event-free survival 
(EFS) of approximately 28% and overall survival (OS) of approximately 30%. Patients with 
bone/bone marrow metastases have a 4-year EFS of approximately 28% and patients with 
combined lung and bone/bone marrow metastases have a 4-year EFS of approximately 14%. The 
overall prognosis for patients with recurrent Ewing sarcoma is equally poor with 5-year survival 
rates following recurrence of 10% to 15%. For recurrent patients, time to recurrence is the most 
significant prognostic factor. Patients who recurred greater than 2 years from initial diagnosis 
(approximately 1 year after completion of therapy) have a 5-year survival of 30% versus 7% for 
patients who recurred within 2 years (31-33).
Standard treatment for patients with metastatic Ewing tumor of bone or bone marrow uses 
alternating vincristine, doxorubicin, cyclophosphamide, and ifosfamide/ etoposide combined with 
local control (surgery and/or radation) with an overall cure rate of approximately 20%. More 
intensive therapies, incorporating high-dose chemotherapy with autologous stem cell support, have 
not shown improvement in EFS (31).
There are no standardized treatments for patients with recurrent Ewing sarcoma. Combinations of 
chemotherapy such as gemcitabine and taxotere, cyclophosphamide, topotecan or irinotecan, and 
temozolomide are active in recurrent Ewing sarcoma family of tumors and can be considered for 
these patients, as well as phase I therapy or palliative care (34).
1.2.3.2
Soft Tissue Sarcomas – Rhabdomyosarcoma (RMS), Desmoplastic Small Round Cell 
Tumor (DSRCT) (This study is no longer enrolling patients with solid tumors as of 
Amendment K [see section 1.4.1.3])
Despite very aggressive therapy, the prognosis for children with metastatic soft tissue sarcomas is 
poor, 37-50% for RMS (35) and 5 year survival rates for DSRCT range from 15% for all patients 
and approach 0% for unresectable patients (36). Standard treatment includes combined treatment 
with chemotherapy, radiation therapy, and surgical resection of pulmonary metastases (34, 35).
There is no standardized care for patients with progressive or recurrent DSRCT or RMS. 
Additional treatment depends on multiple factors, including the sites of recurrence, previous 
treatment, and individual patient concerns. Treatment for local or regional recurrence may include 
wide local excision or aggressive surgical removal of tumor, radiation therapy should be 
considered for patients who have not already received it in the area of recurrence. Previously 
unused, active, single agents or combinations of drugs may also enhance the likelihood of disease 
control and phase I and phase II trials should be considered (34).

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1.2.3.3
Neuroblastoma (This study is no longer enrolling patients with solid tumors as of 
Amendment K [see section 1.4.1.3])
Neuroblastoma is the most common extracranial solid tumor in children, accounting for 6-8% of 
all childhood cancers and over 15% of pediatric cancer deaths (37). For children with high-risk 
neuroblastoma, long-term survival with current treatments is approximately 30%. Children with 
aggressively treated, high-risk neuroblastoma may develop late recurrences, some more than 5 
years after completion of therapy (38, 39). Neuroblastoma risk groups have been defined by the 
Children’s Oncology Group (COG) as follows:
Patients classified as high risk receive treatment with an aggressive regimen of induction 
chemotherapy consisting of high doses of the following agents: cyclophosphamide, ifosfamide, 
cisplatin, carboplatin, vincristine, doxorubicin, etoposide, and topotecan. After a response to 
chemotherapy, resection of the primary tumor should be attempted, followed by myeloablative 
chemotherapy and stem cell rescue. In addition, radiation to the primary tumor site should be 
undertaken whether or not a complete excision was obtained. After recovery, patients are treated 

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with oral 13-cis-retinoic acid for an additional 6 months. For high risk patients in remission 
following HSCT, the chimeric anti-GD2 antibody ch14.18 combined with granulocyte-
macrophage colony stimulating factor (GMCSF), interleukin-2 (IL 2), in conjunction with retinoic 
acid improves has been found to improve EFS and is now standard of care (34, 40).
For patients initially classified as high risk that relapse or have progressive disease the prognosis 
is exceedingly poor and phase I or phase II clinical trials are appropriate and should be considered 
(41).
1.2.3.4
Acute Myelogenous Leukemia (AML)
Non-Transplant Therapy: In most cases, AML treatment consists of intensive induction, 
consolidation, and CNS-directed chemotherapy. Approximately 75 to 90% of children with AML 
will achieve a CR and increasing the treatment intensity of induction improves DFS rates (42-44).
Post-remission consolidation chemotherapy is essential and can be delivered in standard doses or 
as high-dose therapy with autologous stem cell rescue with similar DFS rates (45-48). Despite 
treatment intensification the outcome is guarded for most children with AML, and only about 50% 
are cured with chemotherapy alone (49). Individuals with acute promyelocytic leukemia (FAB 
M3) have a better prognosis, with 80% DFS rates observed when all-trans-retinoic acid is added 
during induction and a maintenance phase (50-53). Young children with trisomy 21 who develop 
AML also have excellent outcomes and require less intensive therapy (54-56).
Transplantation: Given the relative poor outcome for pediatric patients with AML, allogeneic 
HSCT has commonly been used as consolidation in CR1. There have been multiple “genetic 
randomization” studies of matched related allogeneic HSCT in which individuals who have 
matched sibling donors are assigned to transplantation. Allogeneic HSCT confers a lower risk of 
relapse and improves DFS in comparison to chemotherapy with or without autologous rescue (42-
44, 54, 57-62). However, clinical benefits can be offset by transplant-related morbidity and 
mortality, which may eliminate any overall survival advantage in low risk groups (43, 63-66).
Consequently, there is some debate as to whether allogeneic HSCT should be employed in CR1 
or CR2 for AML in childhood (66-68). The ASBMT has published consensus guidelines for the 
use of HSCT in pediatric AML (57) and a suggested approach is presented in Figure 2.
In the U.S., matched related sibling donor HSCT is the most common consolidation therapy 
employed for children with AML in CR1 outside of specific low risk groups. This approach is 
based largely on clinical trials conducted by the Pediatric Oncology Group (POG) and the 
Children’s Cancer Group (CCG) (69, 70). Both groups reported superior outcomes for high and 
intermediate risk patients treated with HSCT in CR1. The 5-year overall survival for patients 
transplanted with a matched sibling donor in CR1 ranges from 52% to 72% (42-44, 46, 57, 62, 69,
70).
Long-term DFS can be achieved in approximately 30% of children with AML who are transplanted 
in CR2 with either matched unrelated or mismatched related donors (62). Consequently, HSCT is 
sometimes reserved for management of patients who relapse after chemotherapy, especially for 
low risk groups (68) or those without sibling donors. 

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Figure 2
1.2.3.5
Acute lymphoblastic leukemia (ALL) 
Clinical and biologic features are used to subtype, risk-stratify and assign therapy at diagnosis. 
Initial risk group assignment is made based on age, peripheral white blood cell count (WBC), 
central nervous system (CNS) involvement, and phenotype (71). Phenotypic classification is 
determined by flow cytometry of lineage-associated cell surface markers. The majority of ALLs 
are of precursor B-cell (pre-B) phenotype (CD10, CD19, HLA-DR, TDT +), 10 to 20% are T-cell 
(CD2, CD3, CD5, and/or CD7 +), and <5% are mature B-cell or Burkitt-type (CD20, surface-
IgM+).
Cytogenetic studies are subsequently used to further define the risk of relapse. The t(12;21) 
translocation, the most frequent recurrent chromosomal translocation associated with childhood 
ALL, is identified in approximately 25% of cases and this is associated with a favorable prognosis 
(72-75). Gene rearrangements of the mixed-lineage leukemia (MLL) gene located at 11q23 is the 
most common cytogenetic finding in infants with ALL, which has an extremely poor prognosis 
(76-79). The so called Philadelphia chromosome (Ph+), which results from a translocation between 
chromosomes 9 and 22, t(9;22), also confers adverse risk (80). The t(1;19) translocation is also 
associated with an increased risk of relapse, but this can be offset by therapy intensification (81,

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82). Hyperdiploidy, which most often includes trisomies of chromosomes 4, 7, and/or 10, carries 
a favorable prognosis (83-86). Hypodiploid cases are at higher risk of relapse (87-90).
The prognosis after relapsed ALL depends on the duration of the first remission (CR1) and the site 
of relapse (91-94). Outcome after short CR1 duration (<12-18 months) is very poor, as is the 
prognosis for individuals who are unable to achieve a second remission. Those with isolated 
extramedullary relapse fair better than those with marrow relapse (95, 96).
There have been no large prospective controlled clinical trials to evaluate the relative efficacy of 
allogeneic HSCT in comparison to chemotherapy for childhood ALL. However, multiple 
comparative studies suggest that relapse rates are lower after HSCT (30). Some of the benefits in 
regard to relapse-free survival are offset by transplant-associated morbidity and mortality (97).
Consequently, HSCT is usually reserved for the management of relapse and it is rarely employed 
for children in CR1 except for those with extremely high-risk features (Figure 3). For those with 
HLA-matched sibling donors, allogeneic HSCT in second remission is considered standard. 
Unrelated donor HSCT is usually reserved for those at high risk of relapse with chemotherapy. 
Importantly, the approach in individual cases will vary based on risk/benefit analysis, donor 
options, and access to transplantation. The American Society for Blood and Marrow 
Transplantation (ASBMT) has published consensus guidelines for the use of HSCT in childhood 
ALL (30) and a suggested algorithms for HSCT in pediatric ALL based on these recommendations 
are presented in Figure 3 and Figure 4.
Figure 3

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Figure 4
A retrospective matched cohort analysis performed by the Children’s Oncology Group (COG) and 
International Bone Marrow Transplant Registry (IBMTR) compared matched related HSCT to 
chemotherapy for children with ALL in CR2. Leukemia-free survival and relapse rates were better 
after HSCT in all patient groups regardless of the CR1 duration (97).
HSCT in first remission has no proven benefits for patients defined as high-risk by WBC count, 
gender, and age. However, transplantation is commonly considered for those at very high risk of 
relapse with standard therapy (e.g., hypodiploidy, induction failure) (Figure 3). Although 
historically HSCT has been considered for children with Ph+ ALL (80, 98), the addition of the 
tyrosine kinase inhibitor imatinib mesylate to chemotherapy appears to have improved non-
transplant outcome (99). Importantly, the use of imatinib in the patient population allows more 
patients to achieve a CR and improves transplant outcomes. Standard of care remains transplant in 
CR1 for these very high risk patients (100).
1.2.3.6
Chronic Myelogenous Leukemia (CML)
CML is characterized by the presence of the Philadelphia chromosome and the associated 
translocation product bcr/abl. CML has three defined clinical phases: chronic, accelerated, and 
blast crisis, with most patients presenting with chronic phase. Response to treatment and survival 
correlate with phase of disease. Blast crisis is clinically indistinguishable from acute leukemia and 
treatment responses are short lived. 
Non-Transplant Therapy: The kinase activity of the bcr/abl fusion protein is inhibited by imatinib 
mesylate (Gleevec) and related kinase inhibitors, and these agents have transformed the approach 
to treatment with CML (101). Imatinib induces complete remissions in most patients with chronic 
phase CML, although continuous treatment appears to be required and resistance may develop 
(102, 103). Thus, there is as of yet no evidence that this new class of kinase inhibitors will be 
curative and it is unclear that TKIs be recommended as a replacement for allogeneic HSCT in 
children who have an HLA-matched donor (104, 105). A number of criteria have been proposed 
for deciding when to proceed from kinase inhibitor therapy to HSCT, including loss of therapeutic 

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response or failure to achieve a complete hematologic response by 3 months or a substantial 
cytogenetic response by 3 to 6 months of treatment (106).
Allogeneic HSCT is the only proven cure for CML and donor availability should be considered 
soon after diagnosis for all children with this disorder. Post-transplant DFS rates are inversely 
related to age and exceed 80% for young children with matched sibling donors in first chronic 
phase. Results are best when HSCT is performed in first chronic phase and with a shorter 
diagnosis-to-transplant interval. Success is substantially diminished for the accelerated phase or 
blast crisis and attempts should be made to induce a second chronic phase prior to transplant  (107,
108). In general, pediatric patients have relatively low risk of transplant-related mortality and 
results are similar with matched unrelated and related donors. Thus, unrelated donor HSCT is 
usually recommended for those who lack sibling donors (101, 104, 106, 108-110).
1.2.3.7
Juvenile Myelocytic Leukemia (JMML)
The myelodysplastic syndromes represent a heterogeneous group of disorders characterized by 
ineffective hematopoiesis, impaired maturation of myeloid progenitors, cytopenias, dysplastic 
changes, and a propensity for the development of AML (111, 112). The major diagnostic groups 
within MDS encountered in pediatric patients include JMML, myeloid leukemia of Down 
syndrome, and MDS occurring de novo and secondary to previous therapy or pre-existing 
disorders (113-115). In general, pediatric MDS carries a poor prognosis and clinical variables have 
little practical utility in guiding therapy (116-119).
JMML is resistant to therapy. Although chemotherapy may reduce disease burden, responses are 
usually short lived and the disease rapidly progresses with a median survival of approximately 1 
year (120). The European Working Group of MDS (EWOG-MDS) in Childhood reported a 
retrospective analysis of 110 cases of JMML. The probability of survival at 10 years was 6% for 
the non-transplant group vs. 39% after transplantation (117).
HSCT is considered the only curative treatment for childhood MDS and JMML. Given the low 
response rates to non-transplant therapies, and because failure rates after HSCT appear lower when 
HSCT is performed soon after diagnosis, strong consideration should be given for early 
transplantation, especially when a matched sibling donor is available. DFS rates of 50 to 64% are 
reported with HSCT. Individuals with JMML who develop GVHD have a lower incidence of 
relapse (111, 121).
Outcome may be improved for individuals transplanted with lower blast percentage and induction 
chemotherapy is commonly employed for patients with elevated bone marrow blasts to induce a 
CR prior to HSCT (119, 122). However, definitive recommendations cannot be made given the 
paucity of data. Given the poor prognosis without transplant and the favorable results of matched 
unrelated donor HSCT in pediatrics, transplantation is usually recommended for children with 
JMML and MDS without regard to the donor type (112, 123-126).
1.2.3.8
Hodgkin’s and Non-Hodgkin’s Lymphoma
Hodgkin’s Lymphoma:
Treatment failure in children and adolescents with Hodgkin lymphoma is typically divided into 
three groups: 1.) Primary progressive disease; 2.) Relapse limited to the site(s) of initial 
involvement (in patients treated with chemotherapy alone); 3.) other relapse. Salvage rates for first 
recurrence range from 10 year OS rates of 55-90% (127-133).

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Initiatial salvage therapy for Hodgkin’s Lymphoma for patients who did not receive prior 
radiotherapy includes further chemotherapy and low-dose involved-field radiation. For the 
remaining patients, treatment of relapse/progression includes an induction chemotherapy, 
followed by high-dose chemotherapy with hematopoietic stem cell transplant (HSCT). Studies 
show better outcomes following autologous versus allogeneic stem cells transplant(134).
Following autologous HSCT, the projected survival rate is 45% to 70% and progression-free 
survival is 30% to 65% (135, 136). For patients who fail following autologous HSCT or for patients 
who cannot mobilize sufficient numbers of autologous stem cells, allogeneic HSCT has been used 
with encouraging results (134, 137-139).
Recurrent Non-Hodgkin’s Lymphoma:
For recurrent or refractory B-lineage non-Hodgkin lymphoma (NHL) or lymphoblastic lymphoma, 
survival is generally 10% to 20% and approximately 60% for recurrent or refractory anaplastic 
large cell lymphoma (140). There is no current standard treatment option for patients with recurrent 
or progressive disease. Disease control can be achieved in 40% of NHL patients using aggressive 
salvage regimens such as dexamethasone, etoposide, cisplatin, cytarabine, and asparaginase, 
maintenance chemotherapy, and transplantation (141). Radiation therapy may have a role in 
treating patients who have not had a complete response to therapy. Rituximab with or without 
cytotoxic chemotherapy has also shown activity in the treatment of B-cell lymphoma 
patients(142). If remission can be achieved, high-dose therapy and stem cell transplantation are 
usually pursued. All patients with primary refractory or relapsed NHL should be considered for 
clinical trials (34).
1.2.4
Matched Unrelated Donor Transplant in Pediatrics
Only 25 to 30% of patients have a sibling donor who is fully matched at the class I and II major 
histocompatibility complex (MHC) loci, which encode for the human leukocyte antigens (HLA-
A, B, C, DR, DQ, and DP). Since 2005, approximately 22 (otherwise eligible) potential candidates 
for the POB sarcoma transplant protocol (02-C-0259) were screened but found to be ineligible 
secondary to the lack of a suitable HLA-compatible sibling donor. During this same time period, 
20 patients were HLA-typed and found to have a matched sibling donor. Of these, only three 
patients did not come to the NIH for further eligibility screening for transplant (one because of 
disease progression). The remaining 17 patients were evaluated and enrolled onto protocol. This 
limitation prompted the exploration of alternative stem cell sources for allogeneic HSCT including 
the use of HLA-matched unrelated donors (MUD). Enrollment of volunteer donors in national and 
international registries, such as the NMDP of the United States, has facilitated the identification of 
unrelated stem cell donors for patients without HLA-matched sibling donors.  Historically, 
transplants from unrelated donors were complicated by higher rates of graft rejection, acute and 
chronic GVHD, immune dysregulation, and treatment-related mortality. However, these 
complications were partially balanced by lower relapse rates (143, 144) and in more recent years, 
complications have been reduced to the levels of matched related donor transplants (144, 145).

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Table 1) Unrelated Donor Bone Marrow Transplantation: Pediatric Series
Patients (n)
Probability 
of myeloid 
engraftment
aGVHD
grades 
2-4
aGVHD
grades 
3-4
cGVHD
Transplant 
Related 
Mortality
Reference
MUD (46)
1 MMUD (42)
100%
83%
98%
37%
62%
60%
69%
24%
51%
Balduzzi 1995
MUD (8) 
1 MMUD (9)
83%
63%
37%
54%
N/A
Davies BJH 1997J
MUD (30)
1 MMUD (20)
93%
49%
67%
23%
30%
50%
55%
Day 100 NRM 
36%
Davies JCO 1997
MUD (28)
MRD (37)
N/A
N/A
64%
38%
32%
14%
57%
26%
11%
19%
Saarinen-Pihkala 
2001
0-1 MMUD (363) 
98%
47%
29%
39%
Day 100 27%
5-year 42%
Bunin 2002
MUD (56)
MMUD (32)
98%
85%
43%
59%
58%
35%
Woolfrey 2002
MUD (33)
1 MMUD (22)
2-3 MMUD(11)
97%
40%
42%
37%
7%
9%
9%
20%
34%
40%
Day 100 10%
Day 180 15%
Overall 22%
Giebel 2003\
MUD (23)
100%
48%
14%
46%
48%
Talano 2006
N/A = not available, n = number, RD = related donor, MUD = matched unrelated donor, MMUD = mismatched unrelated donor, MM = mismatched, mMUD 
= molecular MUD, sMUD = serologic MUD, TRM = transplant related mortality, *high resolution typing (10 allelle), \ 4 received peripheral blood stem 
cells, J data from all patients (0-1 MM) combined, NRM = non-relapse mortality.
Importantly, pediatric patients appear to tolerate alternative donor transplants (MUD and 
mismatched related donors) better than adults. Studies of unrelated donor BMT in the pediatric 
population during the 1980’s through the early 1990’s report high incidences of aGVHD (37-62%) 
and cGVHD (50-69%) (Table 1)(143, 144, 146-151). More recent studies however, suggest a 
decreasing incidence of both aGVHD (21-29%) and cGVHD (39-46%) (152) (153). In 2002, 
investigators in Seattle published results on unrelated-donor marrow transplantation for children 
with ALL. All patients (n=88) received the same conditioning regimen with cyclophosphamide 
and TBI and most received CSA with MTX for GVHD prophylaxis.  Fifty-six patients had an 
HLA-matched donor and 32 had a single antigen mismatch. Acute GVHD was seen in 43% of 
matched recipients and 59% of mismatched recipients. Of those patients surviving past day 80, 
47% developed extensive cGVHD and limited cGVHD was observed in 11% (150).

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1.2.4.1
Direct comparison of related to unrelated donor transplant:
A population-based study of 65 pediatric transplant patients from seven Nordic centers who 
underwent BMT for ALL from either matched sibling donors (n=37) or unrelated donors (n=28) 
was published in 2001. GVHD prophylaxis in the matched sibling group consisted of cyclosporine 
(CSA) in all and methotrexate (MTX) in 67% of patients. GVHD prophylaxis in the unrelated 
donor group consisted of CSA and MTX with 64% receiving anti-thymocyte globulin (ATG) and 
11% also having TCD. Acute GVHD and cGVHD were significantly higher in the unrelated group 
compared to the related group (32% versus 14%; 57% versus 26%). However, the transplant-
related mortality (TRM), event-free survival (EFS) and OS were not significantly different, since 
patients with sibling donors had higher relapse rate and earlier time to relapse (143).
Fifty-seven adult and pediatric hematologic malignancy patients who received MUD BMT were 
matched for disease and stage with 57 recipients of genotypically matched related donor BMT. All 
patients received CSA and MTX for GVHD prophylaxis and MUD patients also received ATG. 
For MUD vs related donor recipients, respectively, incidence of aGVHD grade II–IV was 19% vs 
36%, grade III–IV 10% vs 18%, and cGVHD 44 % vs 51%. Non-relapse mortality was 15% vs 
8% at 100 days, 28% vs 36% at 3 years. Also at 3 years, relapse was 45% vs 42% and disease-free 
survival was 39% vs 37%. None of these differences were significant. The three-year OS was 
identical at 42%. The authors concluded that MUD BMT recipients given pre-transplant ATG have 
similar outcomes to recipients of MRD BMT using conventional drug prophylaxis (152).
In 2006, Uzunel et al. reported on 137 patients who received reduced intensity conditioning for 
alloHSCT in Sweden. Outcomes of 63 patients receiving a sibling donor graft were compared to 
the outcomes of 74 patients receiving a MUD graft. Patients in the two groups were similar in 
terms of diagnosis, age, sex and conditioning regimen. The GVHD prophylaxis was also similar 
in the two groups, with the exception that ATG was given to all of the patients receiving an 
unrelated graft but to only 46% of the patients receiving stem cells from a sibling donor. 
Engraftment was successful in most patients with no significant difference between MUD and 
sibling transplants. No difference in severe aGVHD was found between the groups. However, the 
incidence of cGVHD was higher after sibling transplants. This was probably due to higher donor 
age in this group, since this was the only significant risk factor for cGVHD in multivariate analysis. 
The incidence of TRM was significantly higher after MUD transplantation (40%) than after sibling 
transplantation (16%) (P 0.01). However, because relapse and disease progression were more 
common after sibling transplantation, there was no significant difference in overall survival 
between the two groups (154).
Similarly, a group in Spain reported the results of 58 children with ALL after related (n = 31) or 
MUD (n = 27) HSCT. Characteristics at diagnosis and anti-leukemic treatments were similar in 
both groups. GVHD prophylaxis consisted of CSA alone or CSA and MTX in the related group, 
and the majority of unrelated recipients received CSA, MTX and 2/3 also received ATG. The 5-
year event free survival probability was 43 +/- 9% for the related group and 36 +/- 9% in the MUD 
group (p = .25). The TRM was 16% in the related group and 37% in the MUD group (p = .016). 
The rate of relapse was higher in the related group 36.7% versus 18.6% in the MUD group (p = 
.05). GVHD associated with organ failure or infection caused most of the transplant-related deaths 
in both groups (153).

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1.2.4.1.1 High-resolution typing:
A joint Polish and Italian study prospectively evaluated 63 children transplanted for hematologic 
malignancies from unrelated donors selected by high-resolution typing of both HLA class I and 
class II loci. They evaluated completely matched as well as 1-, 2-, and 3- antigen mismatched 
donors. They observed low incidences of severe aGVHD (7-9%), cGVHD (20-40%) and graft 
failure, with rates comparable to those seen in children transplanted from HLA-identical siblings 
(155).
When comparing unrelated to related stem cell transplants, a group in France analyzed the outcome 
of 89 patients (median age 17 years, range 0-52) with acquired severe aplastic anemia (SAA) 
undergoing HSCT from an unrelated donor between 1989 and 2004 reported to the French Registry 
by 25 centers. They found that patients transplanted during two successive time-periods (1989-
1998 and 1999-2004) had different 5-year survival probabilities of 29% versus 50%, respectively 
(p<0.01). The main difference between the two cohorts was identified as the degree in HLA 
matching between donors and recipients at the allelic level for the ten HLA-A, -B, -C, -DRB1 and 
-DQB1 antigens, which was more frequent in 1999-2004 than in the former period (p=0.0004). In 
multivariate analysis, the only two factors affecting survival were HLA allelic matching (p<0.01) 
and younger age of recipient (p<0.0001). The group concluded that survival after unrelated HSCT 
for SAA has improved significantly over the past 15 years, mainly due to better HLA matching 
and that results for young patients who are fully HLA-matched at the allelic level with their donor 
are comparable to those observed after HSCT from a related donor (156). Additionally, in a recent 
review of 3,857 transplants reported to the NMDP, the authors found that high-resolution typing 
was associated with higher rates of overall survival (157).
Figure 5
Regarding the role of specific allelic level matching, matching at HLA-A, -B, -C and -DRB1 is
often the most important regarding overall outcomes (improved overall survival and lower risk of 
GVHD).  A large retrospective analysis of 334 subjects who underwent a non-T cell depleted 
transplant, found no difference in incidence of acute GVHD whether there was 0 or 1 mismatch in 
the HLA-A,-B, -C, -DRB1 and DQB1 alleles, (Figure 5) though a slight decrease in overall 
survival was seen with each increased degree of mismatch. Notably, most data, including this 
study, are limited to non-T cell depleted transplants, where the risk of GVHD and associated 
complications are inherently higher(158). In another recent review evaluating T-cell replete 
transplantation for adults with AML, amongst 624 subjects who received HLA-identical sibling 
transplants; 1193 who received 8/8 HLA-matched unrelated donor products and 406 who received 
7/8 HLA-matched unrelated donor products, the overall survival and leukemia survival at 3 years 
after transplant did not significantly differ between the groups(159). In a prospective study that 

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evaluated the role of anti-thymocyte globulin (anti-T cell therapy used as one form of T-cell 
depletion) on outcomes between those who did and did not have a single antigen mismatch, showed 
no difference on overall survival or GVHD with a single antigen mismatch(160).
In summary, unrelated volunteer donors utilized in our study will be at least 9/10 allele matched, 
allowing for only a single locus mismatch at HLA-A, B, C, DRB1 and DQB1 loci by high 
resolution typing. The literature supports that despite higher rates of GVHD in the MUD setting, 
overall outcomes are the same due to decrease in relapse in the hematologic malignancy 
population. 9/10 donors who are mismatched at DQB1 will be preferentially chosen, if available,
over mismatches at other loci. 
1.2.5
T Cell Depletion in Allogeneic Stem Cell Transplantation
TCD of the donor graft has been widely established as an effective means of reducing incidence 
and severity of aGVHD as well as cGVHD (161). Current methods of graft manipulation are based 
primarily on removing T-lymphocytes by a variety of antibody-mediated approaches or physical 
methods of separation. CD34 selection as a method to accomplish TCD also eliminates other 
populations of cells (eg, B cells, monocytes, dendritic cells, and others). The loss of the latter may 
potentially weaken engraftment, immune reconstitution, and potentially the GVT effect, but also 
greatly diminishes the risk of EBV-associated lymphoproliferation, and could increase the capacity 
for the host to support NK cell expansion (discussed below). Although TCD has been associated 
with increased relapse rates in CML and AML (162), other studies have not reported increased 
risk of relapse following TCD alloHSCT (163, 164). This may be partly due to disease type or 
other factors. In the proposed study, the use of PBSCs instead of bone marrow, and the use of 
donor NK cells infusions (NK-DLI) following a TCD alloHSCT, may help to ameliorate these 
potential adverse effects, while preserving effective GVHD prevention (165).
Currently, the source of stem cells in transplantation practice is shifting from bone marrow to 
PBSCs. In unrelated alloHSCT, among patients younger than 20 years, marrow was used for 42% 
of unrelated donor transplants in 2003-2006 compared to 59% in 1999-2002. Among adults older 
than 20 years, marrow accounted only for 28% of unrelated donor transplants in 2003-2006 
compared to 66% in 1999-2002 [CIBMTR 2007 summary data]. Neutrophil recovery is more rapid 
following allogeneic PBSCT, as compared with BMT. Moreover, a faster recovery of T cells has 
also been observed following unmanipulated PBSCs (166). Adequate recovery of T-cell and B-
cell function is considered essential for long term event-free survival following alloHSCT, because 
lack of adequate recovery lymphocyte subsets have been associated with infectious morbidity 
beyond the first 100 days post-transplant (165, 167).
Increasingly, more centers are utilizing TCD-PBSCs in both the myeloablative and RIT settings. 
Earlier comparative studies have addressed the recovery of T cells and their functional ability 
following TCD-PBSCT. No significant differences in immune reconstitution
between 
unmanipulated and partial TCD-PBSC in the HLA-matched sibling setting were demonstrated by 
either study. The authors suggested that with partial TCD, GVHD may be prevented, but without 
an apparent increased risk of reduced immune function and opportunistic infections (168, 169).
More recent studies, including one study from the ETIB in the NCI have reported the outcomes 
using TCD-PBSC in a RIT setting (2). Eighteen patients underwent reduced-intensity conditioning 
followed by alloHSCT using a TCD HLA-matched sibling allograft. All evaluable patients (n = 
17) engrafted with no late graft failures. At day +28 post-RIT, 12/17 patients showed complete 
donor chimerism. Full donor chimerism was subsequently achieved in all patients by day 100 after 

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planned DLIs. These data reflect the importance of host immune status prior to RIT and suggest 
that targeted host lymphocyte depletion can facilitate the engraftment of TCD allografts (2).
Similarly, TCD PBSC grafts were shown to accomplish full engraftment after a RIT regimen
following haploidentical alloHSCT at St. Jude’s Children’s Hospital. Notably, patients also 
showed rapid recovery of CD3(+) T-cells, T-cell receptor (TCR) excision circle counts, TCRE
repertoire diversity and NK cells during the first 4 months post-transplantation. These results were 
comparable to those results from a group of patients treated concurrently with a myeloablative 
conditioning regimen. (170). Similarly, in 2008, a German group reported outcomes of 29 patients 
(median age=42) using a RIT and a TCD haploidentical graft following a preparative regimen of 
fludarabine (150-200 mg/m2), thiotepa (10 mg/kg), melphalan (120 mg/m2) and OKT-3 (5 mg/day, 
day -5 to +14) and no posttransplant immunosuppression. Engraftment was rapid with a median 
time to >500 granulocytes/PL of 12 (range, 10-21) days, >20,000 platelets/PL of 11 (range, 7-38) 
days and full donor chimerism after 2-4 weeks in all patients. Incidence of grade II-IV degrees 
GVHD was 48% with the majority having grade II GVHD n=10. TRM in the first 100 days was 
6/29 (20%) with deaths due to idiopathic pneumonia syndrome (n=1), mucormycosis (n=1), 
pneumonia (n=3) or GVHD (n=1). This study shows that in a very high-risk population, 
engraftment was easily achieved and the transplant was fairly well tolerated in a TCD-alloHSCT 
with a reduced intensity regimen using a haploidentical donor (171).
In 2008, Novitzky et al. reported on 15 patients with SAA receiving RIT and TCD PBSC grafts 
from HLA-matched sibling donors. Conditioning consisted of fludarabine 30 mg/m2 daily for 5 
days followed by CY 60 mg/kg on 2 consecutive days. The authors found overall low toxicity and 
rapid engraftment despite TCD, concluding that this approach does not compromise engraftment 
and seems effective for preventing GVHD (172). Another pediatric study, which analyzed 18
consecutive patients aged 5.5-24 years with both malignant and nonmalignant hematologic 
disorders, receiving TCD PBSCT from alternative donors. The preparative regimen included 
single dose total body irradiation (450 cGy), fludarabine (150 mg/m2) and cyclophosphamide (40 
mg/kg). Immunosuppression included ATG and tacrolimus. Grafts were filgrastim, CD34+, TCD
PBSCs in 15 patients and TCD marrows in three patients. All 18 patients engrafted with 100% 
donor chimerism; only one patient developed GVHD. Immune reconstitution was achieved at 
approximately 6 months post-transplant for most patients. These examples illustrated encouraging 
results of TCD transplants from alternative donors using fludarabine-based cytoreduction in 18 
high-risk patients with hematologic disorders, with no evidence of rejection and minimal GVHD
(173).
Table 2) Summary of T Cell Depletion in AlloHSCT
Preparative 
Regimen
Stem Cell 
Source
GVHD
Prophylaxis
CD 34+
Cells/kg
CD 3 +
Cells/kg
Rates of 
aGVHD 
%
Rates of 
aGVHD 
(III-IV)%
Author
RISCT
PBSC
CSA
7.6e6
1e5
59 (II-III)
n/a
Bishop2
Myeloablative
BM
ATG/MP
n/a
n/a
5 (I)
0
Papadopoulos48
Myeloablative
PBSC
CSA
5.8e6
1e5
54 (II-IV)
15
Montero49
RISCT
PBSC
MMF
17.4e6
1.4e5
38 (II-III)
9
Chen55

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RISCT
PBSC
MMF
7.6e6
4.4e4
48 (II-IV)
14
Bethege56
RISCT
PBSC
CSA
4.66e6
n/a
0
0
Novitsky57
RISCT
PBSC/BM
Tacro
10e6
3e3
6 (II-III)
n/a
Chaudhury58
Myeloablative 
PBSC/BM
None
14e6
2.7e4
0
0
Aversa62
Nonmyeloablative
PBSC
None
10.7e6
1e5
0
0
Elhasid64
*total body irradiation (TBI) containing regimen; RISCT – reduced intensity stem cell transplant; BM – bone marrow; 
PBSC – peripheral blood stem cells; ATG – antithymocyte globulin; MP – methylprednisolone; CSA – cyclosporine; MMF 
– mycophenolate mofetil; Tacro – tacrolimus; n/a – not available
1.2.6
GVHD prophylaxis after T cell depleted AlloHSCT
With constantly improving methods of depleting T cells from the stem cell graft in use, the amount 
of residual T cells capable of causing GVHD is diminishing. In recent years, most groups preferred 
positive selection of stem cells as better and more reliable means of TCD as compared to lectin 
agglutination or antibody purging of T cells(174). Given that the drugs used for GVHD 
prophylaxis are immunosuppressive, and potentially deleterious to NK cell function, there is great 
incentive to minimize usage of these agents up front.  Particularly with the use of nonmyeloablative 
preparative regimens, the reduced degree of host inflammation may reduce the chance of GVHD 
developing, making GVHD prophylaxis in the setting of TCD not necessary.  The precedent of 
withholding GVHD prophylaxis after TCD alloHSCT has been realized in multiple studies of 
patients with malignant diseases, including pediatric patients, receiving both HLA-matched or 
haploidentical HSCT.  Due to the heterogeneity of the conditioning regimens between these case 
series, direct comparisons of the optimal regimen between studies are difficult, but are nonetheless 
informative.
In a study by Aversa and colleagues, 17 pediatric and adult patients with end-stage chemoresistant 
leukemia received TCD HSCT of a combination of BM with filgrastim-mobilized PBSCs from 
HLA-haploidentical family members. The conditioning regimen included TBI, ATG, 
cyclophosphamide and thiotepa. No post-transplant GVHD prophylaxis was used.  One patient 
rejected the graft and the other 16 had early and sustained full donor-type engraftment. One patient 
who received a much greater quantity of T lymphocytes than any other patient died from grade IV 
aGVHD. There were no other cases of GVHD greater than or equal to grade II (175).
The same group went on to demonstrate in future studies of haploidentical HSCT with TCD that 
post-transplant GVHD prophylaxis could be withheld without worsening rates of engraftment or 
GVHD (176-178). These studies show that GVHD and graft failure, which limit the use of full-
haplotype mismatched bone marrow transplants, can be overcome in both adult and pediatric 
patients with leukemia with reliable, reproducible CD34+ cell purification, high engraftment rates, 
and prevention of GVHD.
In 2007, Elhasid et al. used TCD through high-dose CD34-cell infusion in 16 pediatric patients 
with matched related donors for nonmalignant diseases. No GVHD prophylaxis was given. All 
patients engrafted, with no graft rejections. All patients survived at a median of 3 years post-
transplantation, and only one developed cGVHD(179).

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Thus, TCD has been safely performed in both children and adults with leukemia and nonmalignant 
diseases in the setting of no post-transplant prophylaxis.  While the methodology of TCD and 
conditioning regimens vary from study to study, engraftment with low rates of GVHD are 
achievable.
1.3
PRE-CLINICAL MODELS OF NATURAL KILLER CELLS: SUSCEPTABILITY OF PEDIATRIC 
TUMORS TO NK CELL LYSIS 
Our understanding of the biology of NK cells has increased dramatically in the last 20 years.  NK 
cells are lymphoid cells that lyse virally infected cells and tumor cells. They express a variety of 
activating and inhibitory receptors that regulate their function in vivo. The relative contribution of 
activating versus inhibitory signals in controlling NK cell reactivity remain unclear, but likely 
relate to both the status of the NK cell (e.g. activated vs resting) and the status of the target cell 
(presence of ligand for activating receptors and/or presence or MHC or killer immunoglobulin-
like receptors (KIR) ligands).  Activating receptors are not expressed by normal cells but are 
upregulated expressed on stressed, neoplastic and virally infected cells.  The major family of 
inhibitory receptors, called KIR, binds self-MHC class I, which is expressed on almost all 
nucleated cells, and inhibit NK cell function. Both virally infected cells and tumors tend to 
downregulate their MHC class I expression, making them susceptible to NK cell lysis. 
1.3.1
Leukemia Models
Most of the initial studies that elucidated the basic biology of NK cells involved studying their role 
in eliminating leukemic cells in both in vitro and animal models of cancer.  To this day, the gold 
standard for demonstrating a positive control of NK cytotoxicity is to use the well- characterized 
K562 cell line, an erythroleukemia, as a target(1-3).  The following studies highlight some of the 
examples that illustrate how NK cells recognize and destroy hematologic malignancies through a 
variety of effector mechanisms.  
The first NK cell line to make it to clinical trials, NK-92, has features of human activated NK cells 
and shows in vitro and in vivo activity against primary patient-derived leukemic target cells as 
well against leukemia cell lines(4).  In this study, survival times of severe combined 
immunodeficient (SCID) mice bearing the sensitive leukemias were significantly prolonged by 
adoptive cell therapy with NK-92 cells. Based on this selectivity in follow up work, the potential 
of NK-92 cells for adoptive therapy is currently being investigated in phase I/II clinical studies(5).
As mentioned earlier, it is believed that the function of NK cells is governed by summarizing the 
signals by activating and inhibitory receptors(6).  One method that has been examined to tilt the 
balance toward activation has been to block inhibitory receptors with monoclonal antibodies or 
small molecule inhibitors.  Indeed this has been demonstrated to enhance antileukemic activity 
both in vitro and in vivo against AML (7-9), mastocytoma(9) and lymphoma(8, 10).  NK cells can 
also use their activating receptor, NKG2D, to target both leukemic and lymphoma cell lines (11).
Present studies are exploring ways to improve activating signals on NK cells, and one method is 
to engineer chimeric antigen receptors (CARs), which combine the targeting capability of an 
antibody to the cytotoxic potential of an effector cell, onto the surface of NK cells, leading to 
enhanced cytotoxicity against leukemias. For example, modified NK cells carrying a CAR against 
CD20, which is expressed on the surface of mature B cell leukemias and most B-cell lymphomas, 
displays markedly enhanced cytotoxicity in vitro. CD20-specific NK cells also efficiently lysed 
CD20-expressing, but otherwise NK-resistant, established and primary lymphoma and leukemia 

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cells(12).  In addition, genetically modified NK-92 cells expressing a CAR specific for the tumor-
associated ErbB2 (HER2/neu) antigen, which is overexpressed by many tumors of epithelial 
origin, at low effector-to-target ratios specifically and efficiently lysed established and primary 
ErbB2-expressing tumor cells that were completely resistant to cytolytic activity of parental NK-
92 cells(13).
NK cells can utilize a variety of effector mechanisms to eliminate target cells.  Virtually every 
cytotoxic pathway has been verified using a leukemia cell as the target. NK cells can target and 
eliminate ALL cell lines and primary samples coated with human anti-FLT3 monoclonal 
antibodies through ADCC, resulting in prolonged survival and/or reduced engraftment of several 
ALL cell lines and primary ALL samples in nonobese diabetic (NOD)/SCID mice(15). Bortezomib 
is a proteasome inhibitor that has direct antitumor effects, but can also sensitize tumor cells to 
killing via the death ligand, TRAIL, which is expressed on NK cells.  Increases in tumor killing 
were only observed using perforin-deficient NK cells in one study, and thus the increased killing 
was found to be dependent on both TRAIL and FasL(19).  In a murine model of Philadelphia 
chromosome-positive ALL, mice depleted of NK cells were no longer protected when challenged 
with the leukemia(14).
IL-2 has been extensively examined to promote clinical T and NK cell responses.  While in vivo 
administration of IL-2 leads to activation and expansion of NK cells, IL-2 also expands 
immunosuppressive regulatory T cells (Tregs).  Prior depletion of Tregs from IL-2 administration 
has led to improved antitumor effects, and in vitro data correlates with subsequent in vivo survival 
of leukemia-bearing mice, in which co-treatment of IL-2 with anti-CD25 (the high affinity IL-2
receptor expressed on Tregs) led to significantly improved survival compared with mice treated 
with either IL-2 alone or with Treg depletion(16).
Some of the earliest observations of the potential antileukemic activity of NK cells came from 
murine models of allogeneic HSCT.  Allogeneic NK cells given as a DLI after HSCT result in a 
potent GVL effect(17).  Efforts to enhance this effect by giving IL-2 and IL-15 showed that 
recipients of a NK cell infusion had lower clinical GVHD scores and suffered less severe GVHD-
associated weight loss, showed accelerated lymphoid immune reconstitution as well as improved 
survival after leukemia challenge(18).  Clinical trials of allogeneic NK cell infusions will hopefully 
verify the observations of preclinical leukemia and lymphoma models.
1.3.2
Neuroblastoma (This study is no longer enrolling patients with solid tumors as of 
Amendment K [see section 1.4.1.3])
Neuroblastoma is the most common extracranial solid tumor in children, and was one of the first 
pediatric solid tumors explored in preclinical models using NK cell-based immunotherapy. Freshly 
isolated neuroblastoma cells lack surface expression of MHC class I, making them ideal targets 
for circulating NK cells(180, 181).  NK cells isolated from patients have shown cytotoxicity in 
vitro against a variety of neuroblastoma cell lines(182, 183) and in a xenograft model(184). To 
date however, no clinical trial of adoptive NK cell transfer has been performed in neuroblastoma 
patients(7).
The ganglioside GD2 is expressed on neuroblastoma and has been an attractive target for 
immunotherapy(181).  The humanized 14.18 moAb has been conjugated to interleukin-2 (IL-2) 
(hu14.18-IL2) in an attempt to increase NK activation resulting in enhanced antibody-dependent 
cellular cytotoxicity (ADCC)(185).  While NK cells alone do not cure mice with neuroblastoma, 
another group using the chimeric anti-GD2 moAb ch14.18 combined with NK cells cured 70% of 

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mice if the treatment was started 7 days after tumor challenge, whereas waiting 21 days diminished 
the benefit(186).
1.3.3
Ewing sarcoma
Ewing sarcoma is the 2nd most common malignant bone tumor in children.  Preclinical studies 
have demonstrated that Ewing sarcomas express ligands for activating receptors on NK cells, and 
that these receptors are critical for allogeneic NK-mediated lysis of Ewing sarcoma(187).  Notably, 
even chemoresistant Ewing sarcoma cell lines are susceptible to NK-mediated lysis(182).  Yet 
while tumors tested from Ewing sarcoma patients at diagnosis are resistant to NK cell lysis, the 
efficacy of NK cell lysis can be enhanced with IL-15(187, 188).  In addition, Ewing sarcoma cell 
lines express heat shock proteins on their surface whose expression can be upregulated by 
nonlethal heat shock and targeted by NK cells, resulting in tumor lysis(189).
1.3.4
Rhabdomyosarcoma
Rhabdomyosarcoma is the most common soft tissue sarcoma in children.  One of the earliest 
preclinical studies of metastatic rhabdomyosarcoma demonstrated that chemotherapy can 
negatively affect the efficacy of NK cells in controlling pulmonary metastases(190).  NK cells 
were also demonstrated to be active against a patient-derived murine cell line(182).  Further work 
also demonstrated enhanced control of rhabdomyosarcoma in vivo when combining 
cyclophosphamide with IL-15, resulting in NK cell-specific cytotoxicity of the tumor(191).  In 
addition, mice inoculated with doxorubicin-resistant rhabdomyosarcoma show improved 
outcomes following adoptive NK cell therapy(192).  In one clinical trial with IL-2 given to children 
with refractory sarcomas, one patient with rhabdomyosarcoma with lung nodules and a 
retroperitoneal mass had transient (<1 month) evidence of greater than a 50% shrinkage of 
pulmonary lesions(193).
1.3.5
Natural killer cells and AlloHSCT
NK cells can kill their targets through a variety of mechanisms. They express CD16, the Fc 
receptor, which allows the NK cells to participate in ADCC.  They secrete the cytokine gamma 
interferon, which activates macrophages and T cells to destroy target cells.  NK cells also have 
granules that contain perforin, which creates pores in cell membranes, and enzymes called 
granzymes, which enter the pores and induce apoptosis of target cells.  Lastly, they also express 
Fas ligand and TNF-related, apoptosis-inducing ligand (TRAIL), which induces apoptosis in those 
cells that express the appropriate receptor1.   
Allogeneic NK cells administered or reconstituted from MHC mismatched donors can potentially 
induce alloreactivity if the MHC mismatch is such that inhibitory receptors are not ligated and 
therefore do not inactivate the NK cell.  In a landmark study that retrospectively examined HSCT 
from a haploidentical donor to patients with refractory AML, recipients that were missing KIR 
ligands had a lower relapse rate, reduced GVH) and better DFS3. This analysis was based on the 
MHC of the donor and recipient, without investigating the donor KIR genotype or phenotype.   The 
results were consistent with earlier studies in murine models that demonstrated a similar capacity 
for NK cells to control leukemia, as well as a lack of GVHD despite NK cell reactivity and 
enhanced engraftment with NK cell transfer.  These features of NK cell biology:  namely the ability 
to attack malignancy, facilitate engraftment and the inability to induce GVHD has led to great 
interest in utilizing NK cells within the context of alloHSCT.

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Several retrospective studies explored whether the benefits of a KIR mismatch were also observed 
following unrelated donor HSCT. Some large registry studies failed to show a survival benefit 
following unrelated transplantation from KIR ligand mismatched donors4-6, although some studies 
did observe benefit7,8. These contradicting results highlighted the complexity of the issues at play 
when one tries to analyze the effects of NK mediated GVT in the setting of multiple other critical 
variables that influence both clinical outcome and NK biology. For instance, results now suggest 
that TCD of the stem cell graft increases the chance that an NK mediated GVT effect will be 
observed, and several factors likely contribute to this.  First, TCD may allow for enhanced NK cell 
reconstitution and proliferation due to less lymphocyte competition for homeostatic cytokines9,10.
Second, an absence of GVHD (which is reliably prevented by TCD) may facilitate NK cell 
reconstitution and/or the immunosuppressive prophylaxis required for patients who receive TCD 
may limit effective NK reconstitution12. For example, CSA may have a detrimental effect on NK 
cell proliferation in vitro.13 Furthermore, other critical variables, such as stem cell dose,11 and 
leukemia type also influence the chance that a beneficial effect of NK mismatch may be observed 
and are not controlled in such retrospective studies. In adult patients, allogeneic NK cell activity 
seems to produce better DFS for AML than for ALL. However, data from an Italian group suggests 
that ALL may be the better target in pediatric leukemias. Biological support of this observation is 
given by a recent paper documenting that in pediatric recipients of a haploidentical HSCT, donor-
derived alloreactive NK cells are generated and persist for many years(194).
IL-15 has been found to play a major role in NK cell homeostasis. IL-15 is instrumental in NK cell 
differentiation, peripheral expansion and survival (195). Murine models have shown enhanced NK 
cell function after IL-15 administration in tumor-bearing mice and IL-15 transgenic mice resulting 
in increased antitumor activity (195). One approach to improve cell survival, and to optimize the 
cytolytic potential of the NK cells in vivo, is to capitalize on the cytokine milieu in the recipient 
immediately following transplant, when IL-15 levels are increased (Figure 6). In 2008, Boyiadzis 
and Hakim (ETIB/NCI) published results of analysis showing the up-regulation of NK cell 
activating receptors following alloHSCT using a similar lymphodepleting reduced intensity 
regimen (as proposed in this current study) is associated with elevated IL-15 levels. They found 
that NK cells recover rapidly post transplant, reaching a peak at 1 month. This peak was directly 
related to IL-15 levels, which peaked on the day of transplant and remained high within the first 2 
weeks following alloHSCT. The intent of this study is to therefore infuse NK cells early post 
transplant to capitalize on the natural surge of IL-15 in the early days post alloHSCT (195).
Figure 6) Taken from Boyiadzis M. BBMT 
14:290-300 (2008): Correlation of NK cell 
recovery with IL-15 during the post-
transplant period. A) Time courses of NK 
levels (cells/ml) (white squares) and plasma 
IL-15 levels (pg/ml) (black circles). B) Inverse 
relationship overall between NK cell numbers 
and plasma IL-15 levels at all time points in 
the first year. C) Strong inverse correlation 
between NK cell numbers and plasma IL-15
levels at 2 weeks post-transplant, a time when 
NK populations were rapidly recovering and 
IL-15 levels varied over a 10-fold range.

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The optimal cell dose and timing of NK needed to achieve expansion and potentiate tumor kill is 
unknown. On this protocol, we intend to give NK cells on days 1, 7, and 14 in order to capitalize 
on the post-transplant environment, including elevated IL-15 levels, which favors in-vivo NK 
cell expansion. The proposed divided schedule (3 weekly infusions) has been designed to 
maximize cell dose and potentially decrease risk of toxicity. 
1.3.6
Natural killer cells and engraftment
In addition to contributing to anti-tumor responses, the other major potential benefit of adoptively 
transferring NK cells is promotion of engraftment.  Preclinical mouse models have demonstrated 
that NK cells produce IL-1E, IL-6, G-CSF and GM-CSF, leading to enhanced engraftment of 
particularly granulocytic and megakaryocytic lineages (196, 197).  In vitro assays of bone marrow 
cells co-cultured with NK cells, or NK cell supernatants, demonstrate increased hematopoietic 
progenitor colony formation (196).  In vivo models of syngeneic and allogeneic BMT have 
corroborated these findings.  Activated NK cells adoptively transferred to syngeneic mice results 
in an increase in hematopoietic progenitors in the spleen and bone marrow (198).  In vivo depletion 
of NK cells can reverse this effect (199).  Activated NK cells administered with allogeneic bone 
marrow cells also enhances the rate and degree of engraftment, as well as B cell reconstitution, 
likely through the production of cytokines as well as the elimination of the host effector cell’s 
ability to reject the graft (196).  In this study, GVHD was not observed despite using a T cell-
replete graft and a complete MHC-disparate BMT.  Thus, both in vitro and in vivo data suggests 
that NK cells may accelerate the rate and degree of engraftment, and emphasizes the importance 
of having activated NK cells present early during the initial stages after BMT. 
1.3.7
EXAMINING KIR RECEPTORS
While the initial observations of the potency of NK cell alloreactivity examined donor and 
recipient MHC, another approach is to examine the repertoire of donor KIRs and recipient MHC. 
KIR expression by donor NK cells can be tested on the DNA level through genotyping or on the 
protein level by flow cytometry. By HLA typing the recipient, one can determine if the patient 
expresses receptors for the donor expressed KIR molecules expressed by the donor, making them 
susceptible to NK cell lysis. Patients with AML or myelodysplastic syndrome had significantly 
less relapse if they lacked an HLA ligand for the KIR present in the donor(200). Since the 
donor–recipient pairs in this study were HLA-identical siblings, simply examining donor and 
recipient MHC would have predicted no NK alloreactivity. 
1.3.8
Adoptive immunotherapy with NK cells
Donor lymphocyte infusions after allogeneic HSCT can induce durable remissions in CML, and 
is also a treatment for CMV and EBV infections.  However, DLI may exacerbate GVHD after 
allogeneic HSCT. Preclinical and clinical results discussed above suggest that adoptive transfer of 
allogeneic NK cells may be a means of inducing antitumor effects without causing GVHD.  Indeed 
allogeneic NK cells given as a salvage therapy for AML patients can induce remission(201-203).
Miller and colleagues treated 43 patients bearing either advanced solid malignancies or poor 
prognosis AML with immunosuppressive conditioning (high versus low dose) followed by 
haploidentical NK cell infusions along with IL-2 (1.75 x 106 IU/m2) for 14 days in a non-transplant 

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setting. Only the high-dose immunosuppressive regimen (60mg/kg intravenous cyclophosphamide 
and 25mg/m2 intravenous fludarabine) allowed in vivo survival and expansion of donor-derived 
NK cell populations. Briefly, NK cells were isolated from CD3-depleted PBMCs, incubated 
overnight in X-VIVO 15 medium (2 X 106 cells/mL) and 1000 U/mL IL-2 (Proleukin; Chiron, 
Emeryville, CA), and then administered by intravenous infusion. Doses were up to 2 x 107 cells/kg 
dose. Three patients with melanoma and 1 with renal cell carcinoma demonstrated stable disease 
on restaging CT scan performed approximately 6 weeks following NK infusion and subsequently 
received a second course of treatment.
Grade 2 and 3 anemia was observed in 7 and 6 treatment courses, respectively, although 7 of these 
patients had grade 2 anemia at study entry. Two patients experienced grade 4 neutropenia and 2 
others developed grade 3 neutropenia following cyclophosphamide. There were no episodes of 
neutropenic fever and thrombocytopenia was not observed. Hematologic toxicity was transient and 
there was no occurrence of prolonged cytopenia or marrow aplasia. A maximum tolerated dose 
was not reached and patients received the largest cell dose achievable during a single 
lymphapheresis collection. Five out of 19 AML patients achieved complete remission and, 
although only 4/19 AML patients had donors with a KIR-ligand mismatch, 3/4 were among those 
with complete remission. There were no adverse reactions during or after the cell infusion and 
GVHD was not observed(201). Of note the patients who received a second infusion demonstrated 
disease progression 4 to 6 weeks after the second infusion.  Current studies indicate that 
chemotherapy prior to NK cell infusion enhances NK engraftment and in-vivo expansion. Using 
NK cells after HSCT is attractive since the lymphopenic environment generated by the 
conditioning regimen would increases systemic levels of IL-15, which is critical for NK cell 
development. 
The experience by Miller et al was not undertaken following HSCT, but more recently there is 
increasing interest in incorporating NK cell infusions in this setting, and the current approach used 
by Miller is the administration of NK cells following HSCT (Miller, personal communication). As 
noted above, NK cells do not increase the risk of acute GVHD in animal models(204-206). Several 
studies using this approach have been recently presented at the 2008 American Society of 
Hematology (ASH) Annual Meeting. These studies showed that NK cell infusions following 
alloHSCT can be given safely and potentially provide anti-tumor effects (207-210). Furthermore, 
NK cell infusions may promote engraftment, decrease the incidence of aGVHD, and decrease 
infectious complications when given early or co-infused at the time of transplant (207, 210).
Currently there are over 30 clinical trials listed on www.clinicaltrials.gov examining the use of 
NK cell infusions in the autologous, haploidentical and allogeneic settings. Trials are open in both 
pediatrics and adult patient populations, for solid tumors (including neuroblastoma) and 
hematopoetic malignancy (AML, ALL, and lymphoma). Protocols include regimens where NK 
cells are given alone (with or without lymphodepletion) or in combination with chemotherapy or 
monoclonal antibody therapy or following alloHSCT.
1.3.9
Ex-vivo Expansion of NK Cells
Essentially all clinical studies reported thus far with adoptive NK cell therapy have utilized resting 
NK cells purified from peripheral blood with or without incubation in rhIL-2.  Handgretinger et 
al. incorporated ex vivo culture with rhIL15 [Handgretinger, personal communication].  These
approaches incompletely activate NK cells and lead to modest expansion in number approaching 
a maximum of 10-fold expansion.  More recently, several groups have begun utilizing cell-based 

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artificial antigen presenting cells to more efficiently activate and expand NK cells ex vivo.  
Campana and colleagues reported the use of a genetically modified cell line (the leukemia cell line 
K562 transduced with CD137L and membrane-bound IL-15) to activate and expand human NK 
cells.  A median expansion of more than 20-fold was observed after 7 days of culture, with no 
preferential expansion of any NK cell subset(211).  The NK cells generated in this culture system 
express gene expression profiles that are different than those expressed by primary or IL-2-
activated NK cells.  Expanded NK cells showed a high cytotoxicity to myeloid leukemias and even 
some pediatric solid tumors, including Ewing sarcoma, rhabdomyosarcoma and neuroblastoma. 
We have conducted preclinical work with KT.64.41BBL, a CD137L expressing artificial antigen 
presenting cells that were generated using good manufacturing practice (GMP).  This cell line is 
designated KT.64.41BBL and can be used either to expand CD3+ T cells (when incubated with 
either anti-CD3 or HLA-A2 binding peptides) or to expand CD3– NK cells.  For the purpose of 
this protocol, KT.64.41BBL cells will be used to expand CD3– NK cells collected via apheresis 
from the peripheral blood of donors.   The expression of important costimulatory molecules for 
NK cells expansion on KT.64.41BBL as shown below: 
Figure 7
Figure 8
Co-incubation of KT64.4-BBL with PBMC or 
purified NK cells leads to expansion of NK cell 
numbers and activation of the cells.  Using co-
cultures with 1:1 NK cells:APC ratios, expansions 
of 2-3 logs can be accomplished over 14-21 days.  
The expansion is enhanced by the addition of 
rhIL15 to the culture.  In addition to the substantial 
enhancement in NK cell number that is 
accomplished, the expanded NK cells show 
evidence for activation based upon upregulation of 
surface expression of activating NK receptors as 
well as enhanced cytotoxicity of tumors in vitro.

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Figure 9
KT64.4-1BBL expanded NK cells show 
upregulation of NKRs, TRAIL and 4-1BB (left 
panel)  and show enhanced killing of tumor

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1.4
SUMMARY: REDUCED INTENSITY TRANSPLANTATION WITH T CELL DEPLETED 
HEMATOPOETIC STEM CELL GRAFTS AND DONOR NK CELL INFUSIONS
Based on the preceding background information, we intend to employ an induction phase of 
lymphodepleting chemotherapy (EPOCH-F or FLAG), followed by reduced intensity T cell 
depleted alloPBSCT, using a preparative regimen of cyclophosphamide, fludarabine as utilized in 
our previous protocols (02-C-0259 and 01-C-0125). We believe that prevailing evidence predicts 
that we will accomplish rapid engraftment with minimal transplant related morbidity.  In order to 
enhance antitumor effects, we will administer activated NK cell infusions early post transplant.
The protocol will seek to enhance tumor free survival compared to that observed in the previous 
trial.  In addition, we will investigate the biology associated with NK cell reconstitution and in the 
long term, hope to incorporate a multifaceted approach to enhancing immune mediated antitumor 
effects following allogeneic PBSCT for very high risk patients.
1.4.1
Study Updates
1.4.1.1
Enrollment Updates (as of Amendment M, July 2014)
NK 01
The first patient on trial was a pilot patient as required by the FDA, this patient was to receive only 
1 infusion of NK cells after engraftment following transplant. Cohort 1, therefore could not open 
until this pilot patient received EPOCH-F chemotherapy, preparative regimen, transplant, NK-
cells followed by a 28-day observation period. Patient NK01 was enrolled in July 2011. This 
patient received a single cycle of EPOCH-F chemotherapy, but had rapidly progressive disease 
and received urgent radiation therapy for symptomatic lesions. This patient was therefore removed 
from study prior to transplant (no donor was collected) and the patient also never received the IND 
agent (NK-cells). The patient subsequently expired at home due to respiratory failure while in 
hospice. The cause of death was attributed to progression of his disease. An SAE was filed and 
reported to the NCI IRB because the death occurred within 30 days of going off of study.
NK 02: 
Patient NK02 was therefore enrolled as a second pilot patient on September 9, 2011. NK02 was 
the first patient who received transplant (34 year old female with history of recurrent, metastatic 
desmoplastic small round cell tumor) and underwent EPOCH-F followed the preparative regimen 
and stem cell transplant without incidence. She received her peripheral blood stem cells from a 
10/10 matched unrelated donor on October 17, 2011. She engrafted briskly on day +9 (October 
26) and received NK-cell infusion on November 10, 2011. She was discharged on day +12.  She 
subsequently developed rash, fevers and transaminitis, although a skin biopsy was not consistent 
with GVHD.  Despite a small amount of diarrhea, she had no evidence of GVHD and proceeded 
to NK cell infusion on Day 24.  Subsequently patient #1 developed significant diarrhea diagnosed 
as aGVHD with scoring of skin stage 4, GI stage 3, for a Modified Glucksberg score of Grade 4 
acute GVHD. The patient continued to show response at the day 28 restaging but progressed at 
the day 100 restaging.  The patient was taken off treatment on 3/14/12 due to progressive disease 
and died 4/27/12 (7 ½ months post transplant) of progressive disease.
NK03
Based on discussions with the FDA reviewers, patient NK03 was enrolled and treated in a similar 
manner to patient #1.  Patient NK03 was enrolled on October 28, 2011. He received 1 cycle of 

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lymphodepleting chemotherapy with EPOCH-F and subsequently received his stem cell infusion 
on December 7, 2011 from his HLA-identical female sibling donor (NK03 Donor). He briskly 
engrafted and achieved neutrophil recovery > 500/uL on 12/17/11 (day + 10) and platelet recovery 
> 50k/uL on 12/21/11 (day + 14). The NK cell infusion was not given until after engraftment, as 
outlined in the protocol for the first pilot patient on study, with a 28 day follow up delay to assess 
for toxicity, prior to enrolling patients into cohort #1. He received a subsequent NK-Cell infusion 
on 12/21/2011 (day +14). This patient tolerated transplant well with no evidence of acute GVHD 
and no significant infectious complications. His initial re-staging at day +28 showed good disease 
response, however the patient subsequently progressed prior to the day 100 restaging in the CNS 
and chest. The patient was taken off treatment on 2/25/12 after patient withdrew in order to seek 
alternative therapy elsewhere. He died of progressive disease on 5/28/12, 5 ½ months post 
transplant.
NK04
Patient NK04, a 22 year old male with DSRCT, had several abdominal lymph nodes positive on
PET scan prior to enrolling on study. He was enrolled on November 16, 2011 and received 3 cycles 
of EPOCH-F therapy and tolerated chemotherapy very well with continued disease response on 
each cycle. He then received a matched unrelated stem cell transplant on 2/1/12.  He received NK 
DLI infusion #1 on 2/10/12 and NK/ DLI infusion #2 on 3/7/12. He developed a maximum Grade 
2 acute GVHD, which responded to imunosuppression.  Restaging at day 100 showed no evidence 
of disease. Shortly after day 100 evaluation, patient developed septic shock and acute renal failure. 
He was discharged home after a prolonged hospitalization and died in hospice 259 days after 
transplant
NK05
Patient #5, a 15 y/o male with EWS was enrolled with no evidence of disease on December 19, 
2011 and received 3 cycles of EPOCH-F therapy and tolerated chemotherapy very well. He
received his stem cell transplant from a matched unrelated donor on March 7, 2012. He received 
his first dose of NK cells on day +9 following stem cell transplant and subsequently developed 
rash, diarrhea and fevers, categorized as grade 4 aGVHD of the skin and GI tract. He also 
developed transient hypotension at that time. He was started on corticosteroids and his GVHD 
symptoms completely resolved within 48 hours. He was weaned from immunosuppression and 
received a second dose of NK cells on April 10, 2012. He subsequently developed skin rash and 
diarrhea in the setting of fevers and was restarted on corticsteroids, with brisk reversal of symptoms 
again. Patient has undergone restaging every 3 months and remains no evidence of disease, most 
recent visit August 2013. He is currently off all immunosuppression with no signs of GVHD and 
is doing well. He has since developed chronic renal failure, likely multifactorial in etiology, but 
related in part to HCT. Complications included development of thrombotic microangiopathy, 
which may have been related to immunosuppression given for GVHD. Also had a history of 
nephrotoxicity from prior chemotherapy. His current renal status remains stable. Most recent 
disease restaging evaluation at 2 years post transplant shows no definitive evidence for disease, 
but there is a site of concern in the first rib. This will be closely monitored with plans for restaging 
in 6-9 weeks. 
NK06
Patient NK06 (24 y/o male with DSRCT) was enrolled on 2/16/12, and received 2 cycles of 
EPOCH-F chemotherapy and underwent matched related stem cell transplant on April 4, 2012. He 

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subsequently received his first dose of NK cells on April 12, 2012. Restaging on day 28 showed 
no evidence of disease, however his PET scan just prior to day 100 showed progressive disease. 
He received additional NK cell infusions on a single patient exemption trial approved by the FDA 
and NCI IRB. After further disease progression, he was taken off that study and is currently being 
treated at home as per primary oncologist. He continues to tolerate therapy well and has never 
developed evidence of GVHD.Patient has been receiving on-going therapy under the direction of 
his home physician; he remains without evidence of GVHD and tolerating further chemotherapy 
well.
NK07
NK07 was enrolled 5/4/12 and received 1 cycle of EPOCH-F therapy and tolerated chemotherapy 
very well. Patient NK07 had matched related stem cell transplant on 6/20/12. NK DLI infusion 
#1 was on 6/28/12 and NK DLI infusion #2 on 7/26/12.  NK07 developed no acute GVHD nor 
chronic GVHD thus far. Unfortunately patient was found to have relapsed on day +100 restaging.
He was treated at home as per primary oncologis and ultimately passed away from progressive 
disease at day + 321 post-transplant. 
NK08
NK08, was enrolled 5/24/12 and received 3 cycles of EPOCH-F therapy and tolerated 
chemotherapy very well.  Patient received his matched related stem cell transplant on 8/15/12 and 
his NK cell infusions on 8/23/12 and 9/19/12. Patient tolerated cellular infusions well, without 
complications. Furthermore, he developed no acute nor chronic GVHD thus far. Unfortunately 
patient was found to have relapsed on day +100 restaging and died from disease progression 607 
days following HCT.
NK09
NK09 was enrolled on 7/13/12 and received 1 cycle of EPOCH-F therapy. Unfortunately, her 
matched sibling donor experience an anaphylactic reaction to GCSF shot for stem cell 
mobilization, therefore patient was taken off study secondary to no longer having a matched donor 
available. Patient has subsequently relapsed and is receiving alternative chemotherapy.
NK10
NK10 was enrolled on 8/17/12 and received 1 cycle of EPOCH-F therapy. Unfortunately, he had 
rapid progression of disease during this cycle, resulting in a partial cord compression. He was 
therefore taken off study in order to receive emergent radiation therapy. He died in hospice care 
less than 1 month later.
NK11
NK11 was enrolled on 10/25/12 and received 3 cycles of EPOCH-F therapy, which he tolerated 
well. He received his unrelated stem cell transplant on 1/9/13 and the first dose of NK cells on 
1/18/13.  Within 48 hours of NK cell infusion, patient experienced rash, fevers and mild 
hypotension, which responded to fluid bolus. This coincided with engraftment and was thought to 
reflect cytokine effects seen with engraftment, however, patient developed progressive rash and 
diarrhea and was diagnosed with Grade 2 aGVHD on 1/22/13 (stage 3 skin and stage 1 GI), which 
was confirmed by skin and GI biopsy. Patient responded well to steroid therapy, however 
developed reactivation of adenovirus causing a hemorrhagic cystitis and hemorrhagic nephritis. 
Therefore, an aggressive wean of the steroids were attempted and patient had an acute flare of his 

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GVHD resulting in grade 4 GVHD involving his eyes, skin (stage 4), liver (stage 1) and GI tract 
(stage 3). Patient’s course was complicated by GI bleeding and a presumptive Steven Johnson’s 
type reaction (secondary to antibiotics) requiring admission to the ICU. Patient’s aGVHD 
responded to steroid therapy was well controlled with immunosuppression consisting of steroids 
and cyclosporine. He was found to have relapse on day + 156 and was taken off-treatment to 
receive alternative therapy. He died from progressive day at approximately day + 300 post-
transplant. 
NK12 
NK12 is an 18-year-old male with DSRCT who enrolled on study on February 22, 2013. He 
received 2 cycles of EPOCH-F therapy, which he tolerated well. He enrolled on study with no 
evidence of disease and entered transplant as such. He received his stem cell infusion on April 24, 
2013 from an unrelated male donor. He was the first unrelated recipient on this protocol to receive 
tacrolimus immunosuppression for prophylaxis. He received his first NK-Cell infusion on 
5/17/2013 (day +23), and a second infusion on 6/14/2012. He tolerated both infusions well with 
no adverse reactions. He has done fairly well from the transplant standpoint with no evidence of 
aGVHD. He developed disease progression at the 1-year post-transplant evaluation (Day + 383).  
Is alive with disease. 
NK13 
NK 13 is a 13 year-old male with Ewings sarcoma who enrolled on study on March 7, 2013. He 
received 3 cycles of EPOCH-F therapy, which he tolerated well, however following the 3rd cycle 
he was found to have significant, recurrent disease on re-staging scans and he was therefore taken 
off study prior to transplant.
NK14 
NK14 is a 21 year-old female with Ewings sarcoma who enrolled on study on March 21, 2013. 
She received 3 cycles of EPOCH-F therapy, which she tolerated well. She received her stem cell 
transplant from her matched sibling on June 12, 2013 and subsequently her first dose of NK cells 
on Day + 21 (July 3). She tolerated these infusions very well. She subsequently developed 
elevation of her transaminases approximately 10 days following the 1st NK cell infusion. She had 
a liver biopsy for persistent elevation of live enzymes that revealed mild GVHD. She therefore did 
not receive her second NK cell infusion. Her transaminases normalized after several weeks without 
immunosuppressive therapy. At the 1-year evaluation two new lesions (right proximal tibia and 
right anterior 5th rib) were found on PET scan. Subject has since undergone biopsy and results are 
pending. This is worrisome for disease progression.
NK15 
NK 15 is a 26 year-old male with Alveolar Rhabdomyosarcoma who enrolled on study on 
September 18, 2013. He received 1 cycles of EPOCH-F therapy, which he tolerated well.
Unfortunately his pre-transplant restaging revealed progressive disease and he was taken off study 
prior to transplant. 
NK16 
NK16 is a 23 yo M with Acute lymphoblastic leukemia who enrolled on study on November 1, 
2013. He was in a complete remisison and went directly to a myeloablative transplant. He received 
his cells on 11/13/13 and had a rapid engraftment. He received both NK-DLI and remains in an 

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MRD negative complete remission at 6 months post transplant. He also has attained full donor 
chimerism. 
NK17 
NK17 is a 24 yo M with acute lymphoblastic leukemia who enrolled on study on November 8, 
2013. He was in a complete remission and went directly to a myeloablative transplant. He received 
his cells on 11/26/13 and has had a rapid engraftment. He received both NK-DLI and remains in a 
MRD negative complete remission at 6 months post-transplant. He has attained full donor 
chimerism. 
NK18
NK18 is a 25 yo F with acute lymphoblastic leukemia who was in a complete remission and went 
directly to a myeloablative transplant. She received her cells on 1/14/14 from her matched sibling 
brother and had a rapid myeloid engraftment. Unforutnately she never attained donor lymphoid 
engraftment and ultimately rejected her graft requiring a second conventional transplant. She is 
currently 100 days following a second transplant and remains in remission with evidence for grade 
1 GVHD. 
NK19
NK19 is a 18 yo M with acute lymphoblastic leukemia who was in a complete remission and went 
directly to a myeloablative transplant. He received his cells from his matched sibling sister and 
had a rapid myeloid engraftment. He received 1 x 10e4 CD3/kg and was treated on amendment K 
prior to the increased T-cell addback in the new revision. Despite initial robust engraftment, he 
has shown signs for loss of engraftment and is currently being reated with serial DLI. 
1.4.1.2
Rationale for Two Escalation Cohorts
Based on our experience in the first 11 patients enrolled (8 transplanted), the following 
modifications were as per Amendment H:
1.) Separate the matched realted donor recipients and the matched unrelated recipients into 
two cell-dose escalation cohorts. As there has been no acute GVHD seen in the related 
donor recipients (0/4) and 100% aGVHD in the unrelated donor recipients (4/4), we plan 
to separate patients with related and unrelated donors into two cohorts and advance the NK 
cell dose in the related donor cohort and continue at the initial cell dose cohort for the 
unrelated donor recipients. In addition, we will add immunosuppression (cyclosporine) for 
the unrelated cohort in order to decrease the risk of acute GVHD, which although thus far 
is steroid responsive, it has been high grade in several unrelated stem cell recipients and 
has effected the ability to give additional NK cell infusions.
2.) We will delay the first dose of NK cell infusion (from day +7 to day +21), such that it does 
not overlap with the timing of engraftment to help decipher whether the NK cells play a 
role in development or severity of aGVHD.
3.) We will require that sarcoma patients must be in a state of non-measureable disease at the 
time of transplant, as thus far all of the patients with bulky disease have had rapid 
progression on the trial. This has either prevented them from going on to receive a 
transplant, or have had early progression post-transplant, therefore we believe the risk-
benefit ration for these patients to be too great.

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Additional modifications to the protocol are based on the poor accrual for patients with 
acute leukemias. We believe this is due to the fact that myeloablative transplant is the 
standard of care for these patients and reduced intensity transplant is therefore a deterant 
for referrals. We will keep a reduced intensity option available for those patients who are 
unlikely to tolerate ablation, such as some previously transplanted patients. Therefore:
4.) We will add a myeloablative preparative option for the patients with hematologic 
malignancies in order to increase accrual of this population – the preparative regimens are 
derived from current Children’s Oncology Group (COG) ALL (ASCT0431) and AML 
(AAML1031) frontline transplant protocols.
1.4.1.3
Rationale for Closing Accrual to Solid Tumors
As of Amendment K, 15 subjects have been treated on this study (see table below). Ten of these 
subjects have received HSCT, and all but three have experienced early progressive disease.  At 
this time, since most of our experience has been with solid tumors, we will focus future efforts 
on accruing subjects with hematologic malignancies to determine the outcomes of this regimen 
in this patient population. 
1.4.1.4
Rationale for Increasing T-cell dose (“add-back”) in recipients of related donor products
Due to a recent occurrence of secondary graft rejection and based on an analysis of all subjects 
transplanted to date, we have identified that there is a discrepancy in CD3 donor chimerism 
between related and unrelated donor recipients. The median donor CD3 chimerism at day 14 in 
recipients of related and unrelated donors was 11% (range, 2-73%) and 93% (range, 64-100%) 
respectively (p=0.01). At day 28, at the median donor CD3 chimerism was 45.5% (range, 8-92%) 
and 100% (range, 92-100%) in related and unrelated donor recipients, respectively (p=0.006).
Importantly no high grade GVHD has been observed in recipients of products from related donors.  
Because of the risk of graft rejection and the increased relapse risk associated with sustained low 
donor lymphoid chimerism, subsequent recipients of matched related donor products will receive 

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a higher T-cell add back (1-2 x 105 CD3/kg) in the initial stem cell product. This represents a one-
log increase from 1-2 x 104 CD3/kg.Since this increased T-cell dose will increase the risk of 
GVHD, recipients of related donor products will also receive calcinurin inhibitor for GVHD 
prophylaxis.  This will also make the related and unrelated donor cohorts more consistent as the 
same GVHD prophylaxis was already instituted in the unrelated donors due to GVHD (outlined in 
sections 1.4.1.1 and 1.4.1.2).
2
ELIGIBILITY ASSESSMENT AND ENROLLMENT
2.1
ELIGIBILITY CRITERIA FOR PATIENTS (RECIPIENT)
2.1.1
Inclusion Criteria: Patient (Recipient)
2.1.1.1
Hematologic Malignancies Diagnoses:
a) Acute lymphoblastic leukemia (ALL) with a history of bone marrow relapse in clinical 
remission (CR) #2 or greater, or in CR#1 if prior induction failure; or with an M1 marrow 
if unable to achieve CR.
b) Philadelphia chromosome positive ALL patients who;
1) Have progressed through or relapsed following TKI therapy or conventional 
myeloablative therapy
OR
2) Are ineligible to receive tyrosine kinase inhibitor (TKI) therapy AND myeloablative 
HSCT
c) Acute Myelogenous Leukemia (AML) with a history of bone marrow relapse in 
remission CR #2 or greater; or with an M1 marrow if unable to achieve CR; or in CR#1 if
prior induction failure; or any of the following High-Risk categories:
1) FLT3/ITD+ with high allelic ratio > 0.4 (HR FLT3/ITD+) regardless of low risk 
features.
2) Presence of monosomy 7, monosomy 5, or del5q, without inv(16)/t(16;16) or t(8;21) 
cytogenetics or NPM or CEBPĮ mutations.
3) AML without inv(16)/t(16;16), t(8;21), NPM, CEPBĮ mutations, monosomy 7, 
monosomy GHOTRU+5)/7,7'EXWZLWKHYLGHQFHRIUHVLGXDO$0/
at end of Induction I.
d) Hodgkin’s and Non-Hodgkin’s Lymphoma with refractory disease or relapse after at least 
one salvage regimen, or after autologous stem cell transplant 
e) Juvenile Myelocytic Leukemia (JMML) with <10% blasts in marrow and blood, who are 
not eligible for effective standard therapies.
f)
Chronic Myelogenous leukemia (CML) with history of blast crisis (ALL/AML) or 
progressive disease failing Tyrosine-kinase inhibitor (TKI).
2.1.1.2
Age:  4 to < 35 years old at the time of enrollment. 
2.1.1.3
All previous cytotoxic chemotherapy must be completed at least 2 weeks prior to study 
entry.  Any prior non-hematologic vital organ toxicity (cardiac, pulmonary, hepatic, 
renal) of any previous therapy must have resolved to grade 1 or less, unless specified 
elsewhere in Section 2.1. 
Exceptions:

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45
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x
There is no time restriction in regard to prior intrathecal chemotherapy provided there is 
complete recovery from any acute toxic effects; or
x
Subjects receiving standard ALL maintenance chemotherapy will not require washout. 
2.1.1.4
All previous immunologic or molecularly targeted therapy must be completed at least 2
weeks prior to study entry. Any prior non-hematologic toxicity of any previous therapy 
must have resolved to grade 1 or less, unless specified elsewhere in Section 2.1, or 
except BCR-ABL tyrosine kinase in patients who have Ph+ ALL, where there will be 
no washout period.
2.1.1.5
Prior investigational therapy must be completed at least 30 days prior to study entry.
2.1.1.6
Patients with prior autologous or allogeneic transplant are eligible. Patients must be > 
100 days post transplant and have no evidence of active GVHD.
2.1.1.7
Performance status: ECOG 0, 1 or 2; or Karnofsky of t 60, or for children d10 years of 
age, Lansky t 60 (Appendix 1). Life expectancy >3 months. 
2.1.1.8
Availability of HLA-matched related or unrelated donors. Related donors must be 5 or 
6/6 antigen matched. Unrelated donors must be at least 9/10 allele matched.
2.1.1.9
&DUGLDFIXQFWLRQ/HIWYHQWULFXODUHMHFWLRQIUDFWLRQE\08*$ or ECHO,
IUDFWLRQDOVKRUWHQLQJE\(&+2.
2.1.1.10 3XOPRQDU\IXQFWLRQ'/&2RIWKHH[SHFWHGYDOXHFRUUHFWHGIRUDOYHolar volume 
and hgb for reduced intensity transplant and '/&255% for myeloablative regimen.
For children who are unable to cooperate for PFTs, the criterion is: No evidence of 
dyspnea at rest, no exercise intolerance, and no requirement for supplemental oxygen 
therapy.
2.1.1.11 /LYHUIXQFWLRQ6HUXPWRWDOELOLUXELQPJGOVHUXP$67DQG$/7[XSSHU
limit of normal. Patients with Gilbert syndrome are excluded from the requirement of a 
normal bilirubin. (Gilbert syndrome is found in 3-10% of the general population, and is 
characterized by mild, chronic unconjugated hyperbilirubinemia in the absence of liver 
disease or overt hemolysis).
2.1.1.12 Renal function:  Age-adjusted normal serum creatinine according to the following table 
RUDFUHDWLQLQHFOHDUDQFHPOPLQ/1.73 m2.
2.1.1.13 Marrow 
function: 
ANC must be > 750/mm3 (unless due to underlying disease in which case there is no 
grade restriction), platelet count must be t 75,000/mm3 (not achieved by transfusion) 
Age (years)
Maximum serum creatinine (mg/dl)
d5
0.8
>5 d 10
1.0
>10 d 15
1.2
> 15
1.5

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unless due to underlying disease in which case there is no grade restriction). 
Lymphopenia, CD4 lymphopenia, leukopenia, and anemia will not render patients 
ineligible.
2.1.1.14 Ability to give informed consent.   For patients <18 years of age their legal guardian 
must give informed consent.  Pediatric patients will be included in age-appropriate 
discussion in order to obtain verbal assent.
a. Durable power of attorney form completed (patients t18 years of age only).
b. Female patients (and when relevant their male partners) must be willing to practice 
birth control (including abstinence) during and for two months after treatment, if of 
childbearing potential.  
2.1.2
Exclusion Criteria: Patient (Recipient)
2.1.2.1
Uncontrolled infection.  
2.1.2.2
Active CNS malignancy as defined by: 
a.
Lymphoma: tumor mass on CT scan or leptomeningeal disease
b. Leukemia: CNS 2 or CNS 3 classification (Appendix 8).
2.1.2.3
Lactating or pregnant females (due to risk to fetus or newborn).
2.1.2.4
HIV positive (due to unacceptable risk associated with severe immune suppression).
2.1.2.5
Hepatitis B surface antigen (HBsAg) positive or hepatitis C antibody positive with 
elevated liver transaminases.  All patients with chronic active hepatitis (including those 
on treatment) are ineligible.
2.1.2.6
Patients who require systemic corticosteroid or other immunosuppressive therapy for
GVHD.  Immunosuppressive therapy must be stopped at least 28 days prior to protocol 
C1D1. Steroids for physiologic replacement, for ALL maintenance or topical agents 
and/or inhaled corticosteroids are permitted.
2.1.2.7
High risk of inability to comply with transplant protocol, or inability to give appropriate 
informed consent in the estimation of the PI, social work, psychiatry, or the stem cell 
transplant team.
2.1.2.8
Fanconi Anemia
2.1.2.9
Clinically significant systemic illness (e.g. serious active infections or significant
cardiac, pulmonary, hepatic or other organ dysfunction), that in the judgment of the PI 
would likely compromise the patient’s ability to tolerate protocol therapy or 
significantly increase the risk of complications.
2.2
ELIGIBILITY CRITERIA FOR DONORS
2.2.1
Inclusion Criteria: Donor

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2.2.1.1
:HLJKWNLORJUDPVDQGIRUXQUHODWHGGRQRUV> 18 years.
2.2.1.2
HLA-matched related or unrelated allogeneic donors.  Genotypically HLA identical 
twins may serve as stem cell donors. Related donors must be 5 or 6/6 antigen-matched.
Unrelated donors must be at least 9/10 allele matched.
2.2.1.3
For donors <18 years of age, he/she must be the oldest suitable donor, their legal 
guardian must give informed consent, the donor must give verbal assent, and he/she 
must be cleared by social work and a mental health specialist to participate.
2.2.1.4
For donors t 18 years of age, ability to give informed consent.
2.2.1.5
Adequate peripheral venous access for apheresis or consent to use a temporary central 
venous catheter for apheresis.
Donor selection will be in accordance with NIH/CC Department of Transfusion 
Medicine (DTM) criteria and, in the case of an unrelated donor, the National Marrow 
Donor Program (NMDP) standards and FDA 21 CFR 1271. 
2.2.2
Exclusion Criteria: Donor
2.2.2.1
History of medical illness that in the estimation of the PI or DTM/NMDP physician 
poses prohibitive risk to donation including, but not limited to, stroke, hypertension that 
is not controlled with medication, or heart disease.  Individuals with symptomatic 
angina or a history of coronary bypass grafting or angioplasty will not be eligible. 
2.2.2.2
Anemia (Hb < 11 gm/dl) or thrombocytopenia (<100,000/Pl).
2.2.2.3
Identical twins will be excluded; the lack of MHC incompatibility will alter the toxicity 
profile in such a way as to make the results uninterpretable.
2.2.2.4
Breast feeding or pregnant females. Donors of childbearing potential must use an 
effective method of contraception during the time they are receiving filgrastim.  The 
effects of cytokine administration on a fetus are unknown and may be potentially 
harmful. The effects upon breast milk are also unknown and may potentially be harmful 
to the infant.
2.2.2.5
High risk of inability to comply with protocol requirements as determined by the 
principal investigator and donor center team.
2.2.2.6
Positive screening test for transfusion-transmissible infection in accordance with DTM 
or NMDP donation standards, including HIV-positive, hepatitis B surface antigen 
(HBsAg) positive or hepatitis C antibody positive.
2.3
RESEARCH ELIGIBILITY EVALUATION
2.3.1
Clinical Evaluation Recipient

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2.3.1.1
All patients will be screened by complete medical history and physical examination. 
Pre-transplant history and physical documentation templates for the recipient can be 
found in CRIS. 
2.3.1.2
Eligibility evaluation for recipient participation in this trial is outlined in Appendices 2 
& 3 and must be completed within 30 days of study entry unless otherwise specified.  
2.3.2
Clinical Evaluation Donor
2.3.2.1
All donors will be screened by complete medical history and physical examination. The 
evaluation of minor donors will be performed by a practitioner with pediatric expertise. 
2.3.2.2
Donor eligibility is outlined in Appendix 3 and must be completed within 30 days of 
study entry unless otherwise required by CC DTM or NMDP donation standards.
2.3.2.3
Donors at NIH will complete a Blood Bank screening questionnaire in the Department 
of Transfusion Medicine (DTM).  NIH Donors will have an interim health assessment 
performed by the DTM staff immediately before each collection procedure.
2.3.2.4
For unrelated donors, all evaluations will be performed at an NMDP-approved donor 
center, with the exception of specified blood samples to be sent to the NIH for analysis.
2.3.3
Specialty Consultation
2.3.3.1
Dental consultation to assess need for teeth cleaning, caries correction or extraction 
(patients only).
2.3.3.2
Social work consultation (patient and related donor).
2.3.3.3
'XUDEOHSRZHURIDWWRUQH\IRUPFRPSOHWHGSDWLHQWV\HDUVRIDJH
Related donors (at NIH) who are minors will be evaluated by a mental health specialist 
with pediatric expertise (psychologist or psychiatrist) prior to the assent process to 
determine willingness to participate (donors < 18 years of age only).
2.3.4
Laboratory Serologic Evaluations 
***In attempt to preserve adequacy of peripheral access for apheresis, antecubital 
phlebotomy will be avoided when possible during donor screening
2.3.4.1
Typing for HLA-A, -B, C and –DRB1 performed in at the NIH DTM (related donor and 
patient).  HLA typing may be performed at any time prior to entry without time 
limitation. The resolution of the HLA typing must distinguish among similar alleles 
being inherited in the family. If an unrelated search is required, the patient will be HLA 
typed at allele resolution for HLA-A, B, C, DRB1, DQB1 by the C. W. Bill Young 
Marrow Program/ Georgetown University, 3rd Floor, 11333 Woodglen Drive, 
Rockville, MD 20852, 301-998-8900.  Buccal swabs will provide sufficient material for 
this testing.  Typing for unrelated donors will be performed through the NMDP 
Customized Typing Program according to NMDP standards (allele resolution for HLA-
A, B, C, DRB1, DQB1), with source documentation at NIH.
2.3.4.2
Blood type, screen, and, if blood type identical, RBC phenotyping, must be performed 
at the NIH DTM laboratory (related donor and patient) or at a NMDP.  In cases of 
major ABO incompatibility isohemagglutinin titers must be performed. Typing for 
unrelated donors may be performed at an NMDP Donor Center according to NMDP 

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49
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standards, with source document verification at NIH. Typing may be performed at any 
time prior to entry without time limitation.
2.3.4.3
Leukemia Patients: Unilateral bone marrow aspirate and biopsy. Cytogenetics and flow 
cytometry analysis (through the NCI Laboratories of Pathology) should be performed 
on bone marrow aspirate if disease can be followed by those modalities. Aspiration 
should be repeated within 14 days prior to the first cycle of induction chemotherapy and 
within 14 days prior to starting transplant preparative regimen.
OR
Lymphoma Patients: Bilateral bone marrow aspirates and biopsies for lymphoma patients 
with a prior history of bone marrow involvement, if clinically indicated.
2.3.4.4
Infectious disease testing per Blood Bank/NMDP standards to include HIV, HTLV-I/II, 
HBV, HCV, CMV, and syphilis (donor).  Donor may also have HIV/HCV nucleic acid 
test and West Nile virus (WNV) antigen and VZV serology screening (according to the 
standards of DTM Blood Bank/NMDP). Donor testing will be performed no more than 
30 days prior to each collection.
2.3.4.5
CBC with differential (patient and donor).  Must be repeated within 72 hours of first 
collection and within 24 hours of subsequent collections (donor).
2.3.4.6
Chemistry 20 (Chem 20): electrolytes, glucose, BUN, creatinine, AST, ALT, alkaline 
phosphatase, bilirubin (total and direct), albumin, calcium, magnesium, phosphorus, 
uric acid, lactate dehydrogenase (LDH), total protein, CK.
2.3.4.7
Protime (PT), partial thromboplastin time (PTT), (patient and donor) 
2.3.4.8
Urine or blood EHCG in post-pubertal females (patient and donor).
2.3.4.9
24-hour urine for creatinine clearance (patient only, if creatinine is abnormal).
2.3.5
Radiologic, Nuclear Medicine, and Specialty Studies (Recipients only)
2.3.5.1
CT scans of the head, chest, abdomen and pelvis and primary tumor.
2.3.5.2
MRI scans of the head and primary tumor, if clinically indicated.
2.3.5.3
FDG-PET scan on all patients as clinically indicated. Technetium99m bone imaging if 
clinically indicated.
2.3.5.4
MIBG Scan for patients with MIBG avid neuroblastoma.
2.3.5.5
Pulmonary function tests (vital capacity, FEV-1, DLCO).
2.3.5.6
Electrocardiogram and MUGA scan or ECHO.
2.3.6
Pathologic/Tissue Evaluation
2.3.6.1
Tissue will be reviewed when available, by the NCI Department of Pathology, and the 
diagnosis must be confirmed prior to enrollment. When tissue is not available, reports 
from a CLIA certified laboratory is sufficient

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2.4
REGISTRATION PROCEDURES
Authorized staff must register an eligible candidate and related donor with NCI Central 
Registration Office (CRO) within 24 hours of signing consent.  A registration Eligibility 
Checklist from the website (http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) must be 
completed and sent via encrypted email to:  NCI Central Registration Office
ncicentralregistration-l@mail.nih.gov.  After confirmation of eligibility at Central Registration 
Office, CRO staff will call pharmacy to advise them of the acceptance of the patient and donor 
on the protocol prior to the release of any investigational agents.  Verification of Registration 
will be forwarded electronically via e-mail to the research team. A recorder is available during 
non-working hours. 
NIH UID numbers will be generated on unrelated donors while maintaining the anonymity 
required by the NMDP, and a completed eligibility checklist must be emailed to the CRO for 
registration of unrelated donors. The CRO will also be notified for removal of patients and 
donors from study.
3
STUDY IMPLEMENTATION
3.1
STUDY DESIGN 
This will be a dose escalation study of NK-DLI infusions following alloHSCT. All patients will 
receive the exact same disease-specific treatment regimen from either a related or unrelated 
matched allogeneic PBSC and NK cell donor.
All lymphoma recipients may receive 1 to 3 cycles of EPOCH-F to achieve immune depletion 
and for concurrent disease control. This will be followed by a myeloablative or non-
myeloablative PBSCT, as clinically indicated.
Leukemia patients with M1 marrow, but detectable minimal residual disease (MRD) by flow 
may receive 1 to 3 cycles of FLAG or EPOCH-F, followed by either a myeloablative or non-
myeloablative PBSCT. Those patients in a CR with no evidence of MRD may proceed directly to 
transplant preparative regimen, however, if there is a delay secondary to donor availability, they 
may receive 1 to 3 cycles of FLAG or EPOCH-F chemotherapy. Those patients with poor lung 
function, a history of prior total body irradiation, or who under the discretion of the PI are felt to 
be at high risk of death from a myeloablative preparative regimen will receive a non-myeloablive 
transplant, regardless of disease status.
Patients will be enrolled to 3 dose levels in 2 cohorts as defined below.  The first patient in each 
GRVHFRKRUWZLOOEH\HDUVRIDJH,IWKe first patient does not experience DLT by 28 days 
after the first NK cell infusion, subsequent patients < 18 years of age may be treated with NK-
'/,,IWKHILUVWSDWLHQWDGXOWLQDFRKRUWH[SHULHQFHV'/7WKHDGGLWLRQDOSDWLHQWV\HDUV
of age in that dose cohort must complete 28 days post NK infusion without further DLT, before 
treating pediatric patients.  
Prior to enrolling on the first cohort, a single patient 18 years of age or older will receive DLI-
NK cells once ANC reaches at least 1000 cells/μL or within 42 days following transplant, 
whichever comes first.  This will allow analysis of the potential risk for NK induced graft loss. 
This first patient must be monitored for DLT for at least 21 days following the last NK dose to 
evaluate for late toxicity prior to infusing NK cells to patients enrolled in cohort 1.  In the 

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51
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absence of DLT, the first patient may proceed to a second infusion of DLI-NK within 3 months of 
the initial NK infusion.  
Subsequently cohorts of patients entered onto the study will receive DLI-NK on day 21 (± 3
days). A second infusion of DLI-NK will be given on Day 49 (approximately 28 days after the 
first infusion) (± 7 days) if toxicity attributable to NK cells has resolved. At the completion of 
each dose level, the toxicities of all patients in that level will be analyzed for trends and reported 
to the FDA, and in the absence of DLT, accrual will proceed to the next dose level. In the 
absence of dose limiting toxicity, the dose of DLI-NK will be escalated by 1 log per level, as 
defined in the table below.  Given the differences in toxicities experienced by patients receiving 
PBMC and DLI-NK cells from related donors versus unrelated donors, the dose levels will be 
escalated in two cohorts: Cohort 1: Patients receiving cells from related donors versus Cohort 2: 
Patients receiving cells from unrelated donors.
Dose Level
Cohort 1: Patients with Related 
Donors
Cohort 2: Patients with Unrelated 
Donors
Dose Level 1
1 X 105 NK Cells/kg infused on day 
7 (± 3d), 35 (± 7d) COMPLETED
1 X 105 NK Cells/kg infused on day 21
(± 3d), 49 (± 7d)
Dose Level 2
1 X106 NK Cells/kg infused on day 
21 (± 3d), 49 (± 7d) 
1 X106 NK Cells/kg infused on day 21
(± 3d), 49 (± 7d) 
Dose Level 3
1 X 107 NK Cells/kg infused on day 
21 (± 3d), 49 (± 7d)
1 X 107 NK Cells/kg infused on day 21
(± 3d), 49 (± 7d)
Patient #1:
1 X 105 NK Cells/kg infused on day 42 or following ANC recover to at least 1000 
cells/μL (whichever comes first). 
If a patient is enrolled in a dose cohort but cell processing does not yield a sufficient number of 
cells to meet the cohort dose target, if the product otherwise fulfills the release criteria, the 
patient will be treated as planned but analyzed as additional data in the lower dose cohort 
according to the number of cells available.  That patient will be replaced in the cohort enrollment 
numbers to ensure sufficient data for toxicity analysis. 
3.1.1
NK Cell Infusion Definition of Dose Limiting Toxicity (DLT)
Dose-limiting toxicity is defined as follows: 
x
All grade 3 – 5 allergic reactions at infusion of NK-DLI
x
Grade 4 neutropenia lasting > 28 days
x
Grade 4 or 5 organ toxicity within 30 days of the last dose of NK-DLI, at least possibly 
related to the NK-DLI
x
Steroid refractory Grade 3 or 4 acute GVHD within 45 days of the last dose of NK-DLI
3.1.2
Requirements for DLI-NK Cell Infusions

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52
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3.1.2.1
Requirements for the first DLI-NK infusion
x
Equal to or < Grade 1 aGVHD
x
Off corticosteroids for aGVHD treatment
x
No uncontrolled infection is present
x
No oxygen requirement is present 
x
No serious condition that in the opinion of the PI places the patient at increased risk from 
NK cell infusion
3.1.2.2
Requirements for second DLI-NK Cell Infusion
The second DLI-NK cell infusion can be given if the following criteria are met:
x
Equal to or < Grade 1 aGVHD
x
Off corticosteroids for aGVHD treatment
x
ANC > 500
x
Platelets > 20 K without transfusion support
x
No uncontrolled infection is present
x
No oxygen requirement is present
x
Baseline or < Grade 3 ALT, AST and serum bilirubin.
x
No serious condition that in the opinion of the PI places the patient at increased risk from 
NK cell infusion
The dose of the second DLI-NK cell infusion may be reduced to the next lower dose, at the PI’s 
discretion if grade 1 GVHD is present. The infusion of the second DLI-NK may be delayed up to 
100 days post-transplant to allow for resolution of any active issues that may have placed the 
patient at increased risk from NK cell infusion at the assigned time-point of the second infusion. 
3.2
ALLOGENEIC PERIPHERAL BLOOD STEM CELL TRANSPLANT:
3.2.1
Donor Peripheral Blood Stem Cell Harvest: 
3.2.1.1
Donor Mobilization and Collection of CD34+ PBSCs: 
Related Donor stem cell and NK cell mobilization with filgrastim 
After medical evaluation and clearance for suitability as an allogeneic donor, donors will 
undergo mobilization with filgrastim (G-CSF, Neupogen, Amgen). The first dose of filgrastim 
will be given in the clinic or doctor’s office where the donor will be observed for approximately 
90 minutes to rule out evidence of allergic reaction.
Filgrastim administration:
Minor donors ZLOOUHFHLYHILOJUDVWLPDVDQRXWSDWLHQWDWDGRVHRIȝJNJGD\HDFKPRUQLQJE\
subcutaneous (SQ) injection beginning 4 days prior to scheduled collection. Adult donors will be 
dosed according to DTM “higher dose algorithm” as components will undergo further 
processing; and a high transplant cell dose is required (CD34 > 8 x106/kg).  GSCF dosing 
instructions can be found in Appendix 10.
3.2.1.2
Donor stem cell collection by apheresis 
For related donors, PBSC collection will be performed in the Dowling Apheresis Clinic of the 
Department of Transfusion Medicine (DTM). Bilateral peripheral venous access will be used 

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whenever possible. Alternatively, a temporary central venous catheter (CVC) will be placed for 
collection. If this is necessary, the donor will be admitted to the Clinical Center for CVC 
placement prior to starting apheresis. The CVC will be inserted by Critical Care or Interventional 
Radiology staff with the assistance of anesthesiology as indicated. The donor will remain 
hospitalized until collections are completed and the CVC is removed. 
A DTM physician is within the immediate vicinity of the procedure or available within one 
minute by pager. Donors will receive divalent cation prophylaxis (Ca) to prevent citrate toxicity 
during apheresis, in accordance with standard DTM policies.  The target (or “optimum”) CD34 
cell number harvested in the DTM Cell Processing Laboratory is > 10 x 106/kg, with a minimum 
of 8 x 106/kg. The minimum post-selection CD34 dose that must be recovered in order to 
proceed with further therapy is 4 x 106/kg.  Since approximately 60% of the CD34 cells in the 
apheresis product are recovered following CD34 cell selection, the targeted CD34 dose to be 
collected by apheresis is >8 x .6 = 4.8 x 106/kg recovered after CD34 selection of the apheresis 
component.
The volume processed per apheresis procedure will be determined by DTM medical staff on the 
day of apheresis, based on peak CD34 cell mobilization response to filgrastim, optimum and 
minimum CD34 cell dose needed, and kilogram weight of recipient.  Volume processed will 
range from 15 to 35 liters per procedure for 1 to 3 consecutive daily procedures, not to exceed a 
total of 75 liters over 3 days.  In pediatric subjects, defined as less than 40 kg, a maximum of 5 
total blood volumes will be processed per procedure, for up to 2-3 consecutive daily procedures. 
Collection will be conducted using the Cobe Spectra, or an equivalent instrument. This 
procedure typically takes 4 to 6 hours.
If > 10 x 106 CD34+ cells per kg (recipient weight) are harvested after the first cycle of 
mobilization and apheresis, no further mobilization or collection will be performed, and the 
patient will be eligible to receive the stem cell transplant with that stem cell dose. In the event 
that < 10 x 106 CD34+ cells per kg are harvested after the first cycle of mobilization and 
apheresis, the donor may need to undergo additional mobilization and collection, depending on 
the final cell dose. 
Unrelated donors will be collected according to similar procedures as described above, but at 
NMDP Centers in accordance with approved NMDP policies and procedures.  Unrelated donor 
apheresis products will be transported immediately after donation to the NIH DTM Cell 
Processing Section for processing.
3.2.1.3
Stem Cell Product Processing
The PBSC from all donors’ apheresis product will undergo targeted T-cell depletion by CD 34 
positive selection (anti-CD34 beads) over a CliniMACS device (Miltenyi Biotec). Product will 
be adjusted post-selection for a cell count of 1 x 104 CD3+ cells/kg, but will allow up to a
maximum of 2 x 104 CD3+ cells/kg for unrelated donor products. In recipients of matched
related donor products, the CD3+ count will range from 1-2 x 105 CD3+ cells/kg. Any 
additional CD3+ T-cells will be cryopreserved for potential future DLI. DLI will be stored in up 
to three aliquots at the following doses: 1 x 106 CD3/kg; 1 x 107 CD3/kg and 1 x 108 CD3/kg, as 
feasible. If these doses cannot be attained, decisions regarding the appropriate dose and possible 
aliquots will be based on standard CPS, DTM procedures. PBSCs will typically be infused fresh, 

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but if needed, may be cryopreserved by methods currently in use and detailed in the Cell 
Processing Laboratory (CPS), DTM standard operating procedures, and stored in either the vapor 
or liquid phase of a liquid nitrogen storage tank according to standard CPS, DTM procedures. 
The concentration of CD34+ cells in the apheresis product will be determined by flow cytometry, 
and the number of CD34+ cells in each bag will be calculated. For collections from related 
donors ONLY, where the pre-collection CD34 count predicts a yield of greater than 15 x 106
CD34+ cells/kg of recipient body weight, 10% of the total collection will be cryopreserved as 
separate aliquots and stored for research. 
3.2.1.4
NK Cell Product Processing
NK Cells will be immunomagnetically selected from the CD34+ cell depleted apheresis product 
using the CliniMacs device. Cells will be cryopreserved using standard techniques.  
Approximately 7-10 days prior to first infusion, the NK cell product will be thawed and placed in 
culture with irradiated KT64.4-1BBL artificial antigen presenting cells and rhIL15.  Co-culture 
may be maintained for 8-10 days until the target dose is achieved according to standard operating 
procedures in DTM.  If target cell dose is achieved, cells may be frozen and subsequent infusions 
given from the thawed product. The culture will be maintained in a closed system using bags.  
On the day of infusion, cells will be given fresh and infused into the patient as outlined in section 
3.3.2.9. Cryopreserved cells may be used as clinically indicated. 
3.2.2
Biopsy of accessible tumor:  
Where possible, biopsies will be obtained from adult patients with accessible tumor in order to
establish tumor cell lines. Since the ability to generate immune responses against tumor cell 
lines from these patients represents an experimental question from which there is no benefit to 
individual patients, risks associated with acquisition of additional tumor tissue from patients with 
an established diagnosis must be minimized.  If a biopsy is being obtained solely for 
experimental purposes, procedures used to obtain the additional tissue should be limited to fine 
needle aspiration, core biopsy, or open biopsy of readily accessible lesions.  Patients should not 
be subjected to extensive surgeries such as thoracotomy or laparotomy.  In addition, this 
procedure is entirely optional and the patient refusal of a biopsy will not prevent enrollment on 
this trial.
3.3
DRUG ADMINISTRATION AND TREATMENT MODIFICATIONS
3.3.1
Induction Chemotherapy
Unless the patient already has a functioning permanent CVC, one will be placed prior to 
initiation of chemotherapy.  Patients may receive up to three cycles of induction chemotherapy 
regardless of CD4 count at the time of study entry.
The following tables are guidelines for schedules, rates and volumes, which, since these are 
standard induction chemotherapy regimens may vary slightly based on the clinical situation and 
guidance from personnel in the Pharmacy Department and clinical staff.  
3.3.1.1
Induction Chemotherapy for lymphoma patients and some patients with leukemia:
EPOCH- Fludarabine may be administered as outpatient:

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Drug
Dose
Days
Fludarabine
25 mg/m2 per day IV
infusion over 30 minutes ± 10 
minutes,
daily for 3 days
Days 1,2,3
Etoposide
50 mg/m2 per day continuous IV
infusion over 24 hours ± 60 
minutes, daily for 4 days
Days 1,2,3,4
Doxorubicin
10 mg/m2 per day continuous IV
infusion over 24 hours ± 60 
minutes, daily for 4 days
Days 1,2,3,4
Vincristine
0.4 mg/m2 per day continuous IV
infusion over 24 hours ± 60 
minutes, daily for 4 days
Days 1,2,3,4
Cyclophosphamide
750 mg/m2 IV Infusion over 30 
minutes ± 10 minutes x 1 dose
Day 5
Prednisone
60 mg/m2 per day in 2-4 divided 
doses PO daily for 5 days
Days 1,2,3,4,5
Filgrastim or 
pegfilgrastim
Filgrastim:  5 Pg/kg per day SQ
Pegfilgrastim:  6 mg SQ, one 
dose. 
Filgrastim:  Daily from day 6 until ANC 
>1000/Pl x 2 consecutive CBC’s
Pegfilgrastim: for patients t 40 kg, given 24-36
hours post chemotherapy (next cycle of chemo 
therapy must be >14 days).
3.3.1.2
Induction chemotherapy for patients with leukemias (Non-myeloablative regimen):
Drug
Dose
Supportive Care
Days
Fludarabine
25 mg/m2 per day 
IV infusion over 
30 minutes ± 10 
minutes, daily for 
5 days.
IV pre-hydration 1 hour prior to fludarabine daily 
for 5 days with 0.9% NaCl and KCl 10 meq/liter 
at a rate of 90 ml/m2/hour to a maximum rate of 
100 ml/hour. Following fludarabine infusion, 
restart fluid at a rate of 90 ml/m2/hour to a 
maximum rate of 100 ml/hour continue until 
cytarabine infusion begins.
Days 1,2,3,4,5
Cytarabine*
2000 mg/m2 IV 
infusion over 4 
hours ± 30 
a. Corticosteroid ophthalmic drops 2 drops to 
each eye every 6 hours starting prior to first 
dose and until 24 hours after the last dose of 
cytarabine completed.
Days 1,2,3,4,5

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minutes, daily for 
5 days.
b. Begin cytarabine doses 3.5 hours after 
completion of the preceding fludarabine. 
b. Infuse in 250 ml of D5W. 
c. IV post-hydration x 4 hours daily for 5 days 
with 0.9% NaCl and KCl 10 meq/liter at a rate 
of 90 ml/m2/hour to a maximum rate of 100 
ml/hour.
d. In the event of signs of CNS toxicity, 
cytarabine infusion will be interrupted and the 
M.D. notified. No further cytarabine will be 
administered if there is CNS toxicity (any 
grade) deemed related to cytarabine.
Filgrastim
5Pg/kg per day 
SQ beginning the 
day PRIOR to 
initiation of 
chemotherapy.
Daily from day 
0 until ANC 
>1000/Pl x 2 
consecutive 
days
3.3.1.3
Subsequent Cycles: Patients may receive subsequent cycles of chemotherapy as early 
as day 22 and up to 28 days after the previous cycle was initiated. Toxicities must 
resolve to < grade 2 in order to receive the next cycle with the following exceptions: 
liver function test elevations (if believed due to malignancy, manage as outlined in 
section 2.1.1.11), neutropenia (ANC must be > 750/mm3 unless due to underlying 
disease in which case there is no grade restriction), lymphopenia, CD4 lymphopenia, 
leukopenia, and anemia (no grade restriction). An additional 14 days of recovery time is 
allowed before administration of subsequent cycles for resolution of toxicity or as 
medically indicated (e.g., documented infection).
3.3.1.4
Dose Modification for Cycle 2 and Cycle 3 
1.) Dose Reduction: If the prior cycle of induction chemotherapy is associated with 
reversible grade 4 non-hematologic toxicity, dose reduction for that patient’s subsequent 
cycles will be performed. In addition, if 2 patients experience the same grade 4 non-
hematologic toxicity, dose reduction will be performed for all subsequent cycles for all 
patients. Dose reduction will be as follows:
a) EPOCH-F: Etoposide, doxorubicin, and vincristine will be reduced from 4 days to 3 
days of administration and prednisone will be reduced from 5 days to 4 days. In the 
event of this change, cyclophosphamide will be given on day 4 and filgrastim will be 
started on day 5. The doses of all medications will remain unchanged. 
b) FLAG: Fludarabine and cytarabine will be reduced from 5 days to 4 days of 
administration. The doses of all medications will remain unchanged. 
3.3.1.5
Anti-emetics: 
Routine anti-emetic prophylaxis and treatment should be employed. Corticosteroids may be used 
if clinically indicated.

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3.3.1.6
Maximum dose of doxorubicin:  
As long as it has been determined that patients have adequate cardiac function as outlined in 
section 2.1.1.9, doxorubicin will be administered until a maximum cumulative total dose of 500 
mg/m2.  Once this dose level has been reached, induction chemotherapy will not include 
doxorubicin.  All other agents will be given according to the parameters outlined elsewhere in 
this section.
3.3.2
Transplantation: 
On day 22 after the final cycle of induction chemotherapy, patients will be eligible to start pre-
HSCT preparative regimen.  However, transplant may be postponed for up to an additional 14 
days in order to facilitate cell processing, patient recovery from illness or other unanticipated 
delays.  The following clinical parameters must be met in order to proceed to the transplant 
portion of this protocol:
3.3.2.1
Clinical Parameters Required for BMT:  
3.3.2.1.1 Non-hematologic toxicities of induction chemotherapy must resolve to < grade 2 in 
order to proceed. If more than 14 days of additional recovery time is necessary for 
resolution of toxicity or as medically indicated (e.g., documented infection), PI 
approval is required prior to proceeding to HSCT. 
3.3.2.1.2 ECOG performance status of 0,1 or 2RU.DUQRIV\RUIRUFKLOGUHQ\HDUVRI
DJH/DQVN\Appendix 1).
3.3.2.1.3 Life expectancy > 3 months.
3.3.2.1.4 &DUGLDFIXQFWLRQ/HIWYHQWULFXODUHMHFWLRQIUDFWLRQE\08*$RU(&+2
IUDFWLRQDOVKRUWHQLQJE\(&+2
3.3.2.1.5 Renal function:  age adjusted normal serum creatinine according to the table shown in 
2.1.10 or a creatinine clearance of >60 ml/min/1.73 m2.
3.3.2.1.6 /LYHUIXQFWLRQVHUXPGLUHFWELOLUXELQPJGOVHUXP$67DQG$/7[XSSHU
limit of normal.
3.3.2.1.7 Disease Status: Patients must meet same disease criteria as enrollment as defined in 
section 2.1.1.1
3.3.2.2
Inpatient care: 
Patients will be admitted for peri-transplant care to NIH in accordance with CC and nursing 
policies.  Patients will be hospitalized in single positive pressure isolation rooms.
3.3.2.3
Pre-HSCT Non- Myeloablative Preparative Regimen: 
The following table is a guideline for schedules, rates and volumes, which, since these are standard 
pre-transplant chemotherapy regimens may vary slightly based on the clinical situation and 
guidance from personnel in the Pharmacy Department and clinical staff.  
Pre-transplant chemotherapy will be administered as follows:
Drug
Dose
Days
Fludarabine
30 mg/m2 per day IV infusion
Transplant Days –5, -4, -3, -2

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over 30 minutes ± 30 minutes, daily for 
4 days
Cyclophosphamide
1200 mg/m2 per day IV infusion
Over 2 hours ± 30 minutes, daily for 4 
days
Transplant Days –5, -4, -3, -2
Mesna*
1200 mg/m2 per day by continuous IV 
infusion, daily for 4 days.
Transplant Days –5, -4, -3, -2
*Mesna will be administered at a dose of 1200 mg/m2 per day by continuous IV infusion on days 
–5, -4, -3, and –2. The first 100 mg/m2 will be mixed with the cyclophosphamide in an 
appropriate amount of fluid based on the hydration rate. Immediately upon completion of this 
initial infusion, mesna at a dose of 1100 mg/m2 will be infused over 22 hours until the next 
cyclophosphamide dose or until 22 hours after completion of the final dose of 
cyclophosphamide.
3.3.2.4
Pre-HSCT Myeloablative Preparative Regimen for AML or subjects who have received 
prior TBI, as clinically indicated:
The following table is a guideline for schedules, rates and volumes, which, since these are standard 
pre-transplant chemotherapy regimens may vary slightly based on the clinical situation and 
guidance from personnel in the Pharmacy Department and clinical staff.
Day
Treatment
-6
Anti Seizure Prophylaxis*
-5 to -2
Fludarabine 40 mg/m2/dose IV daily (1.3 mg/kg if <10kg)**
-5 to -2
Busulfan 3.2 mg/kg/dose IV daily x 4 doses***
-1
Rest (may be extended an additional day if needed)
0
HSCT Infusion
Note: Dose adjustments will be made for obesity, defined as > 125% of IBW: 
Adjusted weight = 1.25*IBW (Please see Appendix 13 for IBW and dose adjustment 
calculations.)
* Seizure prophylaxis is mandatory. The preferred regimen is lorazepam (0.02-0.05 mg/kg/dose, 
maximum dose: 2 mg) given 30 minutes prior to each busulfan dose and then continuing for at 
least 24 hours after last busulfan dose. Alternative acceptable regimen is levetiracetam beginning 
12 hours prior to busulfan and continue at least 24 hours after completion of busulfan.  The dose 
of levetiracetam is 10 mg/kg/dose PO BID (maximum dose: 1000 mg). Loading dose and 
therapeutic monitoring are not necessary. For patients with a known seizure disorder, consider IV 
dosing with loading and therapeutic monitoring. 
NJ)OXGDUDELQHPJP,9GDLO\LQ1D&ORU':RYHUPLQXWHVRQ'D\V-5, -
4, -3, -2 for a total of 4 doses. 

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< 10 kg: Fludarabine 1.33 mg/kg IV daily in 0.9% NaCl or D5W over 30 minutes on Days -5, -4, 
-3, -2 for a total of 4 doses. 
*** Busulfan administered intravenously via a central venous catheter as a  3-hour infusion once 
daily for 4 consecutive days for a total of 4 doses. Doses will be adjusted to achieve an overall 
exposure target AUC of 900-1500 (micromole/liter)*minute. 
3.3.2.5
Pre-HSCT Myeloablative Preparative Regimen for ALL:
The following table is a guideline for schedules, rates and volumes, which, since these are standard 
pre-transplant chemotherapy regimens may vary slightly based on the clinical situation and 
guidance from personnel in the Pharmacy Department and clinical staff.
Prep should be 
continuous.
Day
Treatment*, **
-6 to -4
TBI 200 cGy BID (see Appendix 12 for TBI guidelines)
-3 to -2
Cyclophosphamide 60 mg/kg/dose IV daily
-3 to -2
MESNA 60 mg/kg/dose IV daily
-1
Rest (may be extended an additional day if needed)
0
HSCT Infusion
Note: Dose adjustments will be made for obesity, defined as > 125% of IBW: 
Adjusted weight = 1.25*IBW (Please see Appendix 13 for for IBW and dose adjustment 
calculations.)
*The order of the cyclophosphamide/TBI may be switched to accommodate scheduling needs
**A CNS boost, if indicated, will precede the start of the conditioning regimen.
Mesna infusion should start simultaneously with the cyclophosphamide and be administered by 
continuous IV infusion on days -3 and -2.  The Mesna infusion should continue for a minimum 
of 22 – 24 hours after the last dose of cyclophosphamide.  The entire Mesna dose prescribed 
should be administered each day of cyclophosphamide therapy.
3.3.2.6
Hydration During Pre-HSCT Preparative Chemotherapy: 
IV hydration will be initiated 12 hours prior to cyclophosphamide (on day –7 of the non 
myeloablative transplant, day -4 of the myeloablative ALL transplant) using 0.45% NaCl with 
5% dextrose supplemented with KCl 10 meq/liter at a rate of 100 ml/m2/hour continuing until 24 
hours after the last cyclophosphamide dose has been completed.   During hydration, IV 
furosemide (0.5 mg/kg/dose to a maximum of 20 mg) may be administered as needed to maintain 
normal urine output and fluid balance. During hydration, serum electrolyte levels will be 
monitored every 12 hours and IV fluid content and KCl supplementation will be adjusted to 
maintain normal serum electrolyte levels. 

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Hydration volume, composition and/or rate may be modified based on patient condition and 
investigator assessment, but may not go below the minimum of 65 ml/m2/hour.
3.3.2.7
GVHD Prophylaxis with calcineurin inhibitor:
x
Tacrolimus will be initiated on the day -1 (at least 24 hours before the stem cell infusion)
typically starting at a dose of 0.02 - 0.03 mg/kg/day as a continuous infusion. Once oral 
feedings are tolerated and there is resolution of mucositis, parenteral tacrolimus will be 
discontinued.  Twelve hours later, oral tacrolimus will be initiated at a dose of 0.1-0.15 
mg/kg/day in two divided doses every 12 hours.  
x
The tacrolimus dose will be titrated to maintain a trough level of 5-10 ng/ml. Tacrolimus 
levels should NOT be drawn from the CVL lumen used for tacrolimus infusion.
OR
x
Cyclosporine-A will be initiated on the day before the transplant (day –1) starting at a dose 
of 2 mg/kg/dose IV over 2-hours q 12 hours.
x
The dose will be titrated to maintain an approximately trough level of 200-250 ng/ml 
(monoclonal RIA methodology). CSA levels should not be drawn from the CVL lumen 
used for CSA infusion. 
x
Once the patient is able to tolerate oral feedings, CSA will be converted to an equivalent 
oral dose. The starting oral dose should be approximately 2-times the IV dose given in a q 
12 hour schedule. The dose should be titrated to a trough level of 200-250 ng/ml 
(monoclonal RIA methodology).
x
Tacrolimus is preferred over cyclosporine-A, however if tacrolimus is unavailable, or the 
laboratory reagents needed to monitor tacrolimus serum levels are unavailable, 
cyclosporine-A may be substituted,
x
Calcineurin inhibitor will continue until day 100 post-transplant, at which point it will be 
gradually tapered as long as the level of acute-GVHD is < grade 2 and there is no chronic-
GVHD. Taper will consist of a 5 to 10% dose reduction each week and will be discontinued 
by around day 180 post-transplant.
x
The decision to taper Caclineurin inhibitor before day 100 will only be permitted if 
clinically indicated and after PI approval. Specifically, taper before day 100 may be 
permitted for the treatment of progressive disease and/or low levels of donor chimerism.
3.3.2.8
Anti-emetics: 
Routine anti-emetic prophylaxis and treatment should be employed. Corticosteroids may be used 
if clinically indicated.
3.3.2.9
Stem Cell Infusion: 
On day 0, the patient will receive fresh donor CD34+ PBSCs via intravenous infusion. 
(Cryopreserved cells may also be used in clinically indicated.)
If the stem cells are cryopreserved, the product will be thawed and administered IV immediately 
at a rate of approximately 10-15 ml/min or as tolerated based on volume status and/or DMSO 
toxicity.  Do not exceed 20 ml of product per kg body weight (if 10% DMSO) or 40 ml of 
product per kg body weight (if 5% DMSO) so that per 24-hour period so that total DMSO 
administered is less that 1 mL/kg/day.  

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The target dose of the PBSC is > 5 x 106 CD34+ cells per kg recipient body weight.  
a.
Pre-medications: Patients will receive the following medications approximately 30-60
minutes prior to stem cell infusion: 
i.
Diphenhydramine 0.5 - 1 mg/kg/dose (maximum 50 mg/dose) IV 
ii.
Acetaminophen 15 mg/kg/dose (maximum 650 mg/dose) PO 
iii.
If the patient has been premedicated earlier in the day for blood product or other 
infusion, pre-medication is not warranted if given within 4 hours of stem cell 
infusion.
b. Hydration During Stem Cell Infusion: IV hydration will be initiated at a minimum of 12
hours prior to stem cell infusion (on day –1) using 0.9% NaCl supplemented with KCl 10 
meq/liter.  Patients will receive hydration at a rate of 90 ml/m2/hour to a maximum rate of 
100 ml/hour. Hydration will continue at a minimum of 12 hours after the stem cell 
infusion has been completed. 
c.
Monitoring During Stem Cell Infusion (Inclusive of T-cell Addback as a separate 
infusion): General monitoring guidelines will include vital signs and oxygen saturation q 
15 minutes (+/- 10 minutes) until one hour after completion of infusion (s) and will 
follow NIH Clnical Center Nursing and Patient Care Services Procedure “Administration 
of products for Cellular Therapy.” 
(http://intranet.cc.nih.gov/nursing/practicedocs/procedures_pdf/PRO_Cellular_Therapy_I
nfusion.pdf)
d. Supplemental oxygen will be available at the bedside.
e.
If an allergic or other acute reaction occurs, studies appropriate for investigation of a 
transfusion reaction will be performed (urinalysis, CBC, Coomb’s test).
3.3.2.10 Growth Factor Administration Post-transplant: 
After stem cell infusion on day 0, filgrastim will be started at a dose of 5Pg/kg/day SQ, unless 
contraindicated. Filgrastim will continue until the ANC is greater than 5000 cells/Pl (post nadir) 
on two consecutive blood draws.  If the patient has impaired renal function, pegfilgrastim may be 
administered in place of filgrastim at 6 mg RQHWLPHGRVHIRUSDWLHQWVNJRQGD\
3.3.2.11 NK Cell Infusion: 
On day 21 (+/- 3 days) after stem cell infusion, the dose of NK cells will be infused 
intravenously over approximately 10 -20 minutes.  In the absence of DLT, the NK cell infusion 
will be repeated 28 (+/- 7 days) days following the first infusion for a total of 2 infusions, as per 
the dose descriptions in Section 3.1.  Any adverse reaction should prompt interruption of the 
infusion and should be reported to the principal investigator.
Patients will receive the following medications approximately 30-60 minutes prior to NK cell 
infusion: 
1.) Diphenhydramine 0.5 - 1 mg/kg/dose (maximum 50 mg/dose) IV; 
2.) Acetaminophen 15 mg/kg/dose (maximum 650 mg/dose) PO
3.) If the patient has been premedicated earlier in the day for blood product or other infusion, 
pre-medication is not warranted if given within 4 hours of stem cell infusion
Safety monitoring during and after infusion will follow NIH clnical center guidelines 
(http://intranet.cc.nih.gov/nursing/practicedocs/procedures_pdf/PRO_Cellular_Therapy_Infusion
.pdf) and will include vital signs (prior to infusion, every 15 minutes (+/- 10 minutes) for 1 hour 

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after the start of infusion, every 30 minutes (+/- 15 minutes) for the second hour and then hourly 
until stable.
An NK-DLI infusion may be delayed in patients with significant complications (eg. severe 
infection, engraftment syndrome, GVHD flare) that according to the protocol PI or AI, may be in 
the best interest of the patient, and later administered when the patient has sufficiently recovered.  
Cells must meet the same release criteria as the originally scheduled dose regimen.  For 
statistical analysis, patients receiving infusions outside of the planned ± 3 day or 7 day time 
frame will be analyzed separately and reported descriptively.
NK cells may remain in culture for up to an additional 7-14 days. If both doses of NK cells can 
be delivered within the prescribed time, this will be considered feasible delivery of NK-DLI.
If a DLT occurs, no further NK cells will be given to that patient.
3.4
ON STUDY PROTOCOL EVALUATION (APPENDIX 2 A):
3.4.1
Baseline
3.4.1.1
Spinal fluid for cell count and cytology (for patients with acute leukemia or NHL).
3.4.1.2
PCR of mini-satellite regions (STR profile) for future determination of chimerism 
(donor and patient).  These studies may be performed at any time prior to entry without 
time limitation.
3.4.1.3
Antibody screen for varicella, HSV, and toxoplasma (patient only) and CMV, EBV 
(patient and donor).
3.4.1.4
PPD with appropriate control (for patients considered to be at high risk).
3.4.1.5
CBC with differential (patient and donor).  Must be repeated within 72 hours of first 
collection and within 24 hours of subsequent collections (donor).
3.4.1.6
TBNK (patient only)
3.4.1.7
Urinalysis, (patient and donor).
3.4.1.8
Lupus anti-coagulant (patient only), as indicated.
3.4.1.9
Serum iron, ferritin and transferrin (patient only).
3.4.1.10 24-hour urine for creatinine clearance, if indicated.
3.4.1.11 Spot urine for calcium/creatinine ratio (patient only).
3.4.1.12 Radiologic studies that measure identifiable disease may be repeated prior to each cycle 
of immune depleting chemotherapy and prior to the conditioning regimen, at the PI’s 
discretion.
3.4.2
Induction Chemotherapy Cycles (EPOCH-F or FLAG cycles)

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3.4.2.1
H&P prior to each cycle and weekly during induction.
3.4.2.2
CBC, differential, platelets and prior to each cycle and two times per week during 
induction.
3.4.2.3
Laboratory evaluations: Sodium (Na), Potassium (K), Chloride (Cl), Total CO2 
(bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total, 
Magnesium total (Mg), Inorganic Phosphorus, Alkaline Phosphatase, ALT/GPT, 
AST/GOT, Total Bilirubin, Direct Bilirubin, LDH, Total Protein, Total CK, Uric Acid,-
prior to each cycle and one time per week during induction.
3.4.2.4
Urinalysis prior to each cycle.
3.4.2.5
Urine or Serum Pregnancy test prior to each cycle, for all females of childbearing 
potential.
3.4.2.6
Disease staging evaluation - as clinically indicated based on disease and sites of disease 
(see section 2.2).
3.4.2.7
TBNK or NK panel (CD4 count) within 7 days prior to each cycle.
3.4.3
Pre-HSCT evaluation
3.4.3.1
H&P 
3.4.3.2
CBC, differential, platelets 
3.4.3.3
Laboratory evaluations: Sodium (Na), Potassium (K), Chloride (Cl), Total CO2 
(bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total, 
Magnesium total (Mg), Inorganic Phosphorus, Alkaline Phosphatase, ALT/GPT, 
AST/GOT, Total Bilirubin, Direct Bilirubin, LDH, Total Protein, Total CK, Uric Acid
3.4.3.4
Urinalysis
3.4.3.5
Complete disease staging evaluation – staging based on disease and sites of disease
within 17 days prior to start of transplant preparative regimen.
3.4.3.6
EKG and MUGA or ECHO
3.4.3.7
Research studies (Section 3.5, Appendix 4) (When ALC > 200 K/uL ONLY)
a.
Lymphocyte Phenotyping:
-
TBNK (CD4 count) or 
-
NK Subset (except on Fridays) within 7 days prior to each cycle (See Appendix 4).
3.4.4
HSCT Hospitalization
3.4.4.1
H&P daily
a.
Twice daily weights (from transplant through date of engraftment)
b.
DAT daily on days 6-10 (for patients with ABO histo-incompatabilities ONLY)
3.4.4.2
CBC, differential, platelets daily.
3.4.4.3
Laboratory evaluations: Sodium (Na), Potassium (K), Chloride (Cl), Total CO2 
(bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total, 
Magnesium total (Mg), Inorganic Phosphorus, Alkaline Phosphatase, ALT/GPT, 

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AST/GOT, Total Bilirubin, Direct Bilirubin, LDH, Total Protein, Total CK, Uric Acid
daily.
3.4.4.4
Type and screen every 4 days (as clinically indicated)
3.4.4.5
Reticulocyte count, haptoglobin, IGG level, EBV, CMV, HHV6, adenovirus monitoring 
weekly (during hospitalization).
3.4.4.6
Urinalysis daily 
3.4.4.7
Research studies (Section 3.5, Appendix 4)
3.4.4.8
Disease staging evaluation (as clinically indicated based on disease sites)
3.4.5
Evaluations After HSCT discharge through day 100 (±30 days), as clinically indicated
3.4.5.1
H&P twice weekly
3.4.5.2
CBC, differential, platelets weekly.
3.4.5.3
EBV, CMV, HHV6, adenovirus monitoring weekly.
3.4.5.4
Type and screen and IGG level, weekly as clinically indicated.
3.4.5.5
Laboratory evaluations: Sodium (Na), Potassium (K), Chloride (Cl), Total CO2 
(bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total, 
Magnesium total (Mg), Inorganic Phosphorus, Alkaline Phosphatase, ALT/GPT, 
AST/GOT, Total Bilirubin, Direct Bilirubin, LDH, Total Protein, Total CK, Uric Acid
weekly. 
3.4.5.6
Iron studies - serum iron, transferrin, ferritin, reticulocyte count, monthly as clinically 
indicated.
3.4.5.7
Bone marrow biopsy and aspirate day 28 (as indicated).
3.4.5.8
Research studies (Section 3.5, Appendix 4).
3.4.5.9
Blood for bcr-abl PCR 28-35 days post final NK cell infusion.
3.4.6
Evaluations after Day 100 (Appendix 2) (±30 - 90 days):
Disease evaluations may occur before Day 100 or at any time after day 100 based on signs and 
symptoms or PI’s suspicion of disease recurrence.  Follow-up visits will be clinically determined 
after day 100, but at least once monthly. NIH scheduled visits will occur around day 100, day 
150, day 180, day 270, and day 365 post-transplant. These timepoints are used a guidelines but 
may be altered based on each patient’s clinical condition. Patients will then be followed
approximately every 3 months for the second post-transplant year, and then every 6 months for 
the third post-transplant year, and then yearly until at least 3 years post-transplant.  Evaluations 
will include the following as clinically indicated:
3.4.6.1
H&P including symptoms of GVHD (see Appendices 6-8), infection, and respiratory 
dysfunction. 
3.4.6.2
CBC and reticulocyte count
3.4.6.3
Laboratory evaluations: Sodium (Na), Potassium (K), Chloride (Cl), Total CO2 
(bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total, 

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Magnesium total (Mg), Inorganic Phosphorus, Alkaline Phosphatase, ALT/GPT, 
AST/GOT, Total Bilirubin, Direct Bilirubin, LDH, Total Protein, Total CK, Uric Acid
3.4.6.4
IgG levels monthly until IVIG supplementation discontinued.
3.4.6.5
Blood type, red cell phenotype, and ABO isohemagglutinin titers (in cases of ABO 
incompatibility only) as clinically indicated.
3.4.6.6
Antibody titers to previous vaccinations or infections (e.g., diphtheria, HIB, HBSAb, 
pneumococcus, tetanus, varicella) at or beyond 12 months post-HSCT and off IVIG for 
at least 3 months.
3.4.6.7
Disease staging evaluation (as clinically indicated based on disease sites): All studies 
that identify disease will be performed around day 100, every 3 months for the first 
year, every 6 months for the second and third year, and yearly for years 4-5, or as 
clinically indicated.
3.4.6.8
Organ function evaluation:
a.
Pulmonary function tests with DLCO q 3 months if GVHD, otherwise every 6 – 12 
months and as clinically indicated.
b. Cardiac tests: EKG and MUGA or ECHO at around 6 months, 1 year, and then yearly 
until year 5 ± 3-4 months.
c.
Endocrine studies as clinically indicated.
3.4.6.9
Research studies (Section 3.5, Appendix 4) including KIR/HLA 
genotyping/immunogenetics at or around Day 100±30 days.
3.4.7
Related Donor Evaluations During Treatment (Appendix 2 B)
3.4.7.1
Donors will have an interim health assessment performed by DTM staff immediately 
before each collection procedure.
3.4.7.2
Infectious disease testing will be performed no more than 30 days prior to each 
collection.
3.4.7.3
If a CVC is required, CBC, PT, PTT, and history and physical must be performed 
within 72 hours prior to placement.
3.4.7.4
Donors will have a physical examination, including a CBC, in the ETIB or POB 
clinic or by their primary physician within one week of each collection.
3.5
NCI/NIH BIOLOGIC STUDIES 
Blood sample volume for the research purposes of this study will be restricted. The amount of 
blood drawn from adults (those 18 years of age or older) for research purposes shall not exceed 
10.5 mL/kg or 550 mL, whichever is smaller, in an 8 week period. The amount of blood drawn 
for research purposes from pediatric patient subjects (those under 18 years of age) will not 
exceed 5 mL/kg in a single day, and no more than 9.5 mL/kg over an 8 week period.
In the event that blood draws are limited due to these restrictions, research studies will be 
performed in order of priority as listed below and in Appendix 4.
3.5.1
Proposal for Immune Function Studies (Brown)

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3.5.1.1
Background: to evaluate the effects of the chemotherapy regimen on host immune 
depletion/reconstitution.
3.5.1.2
Rationale: Analysis will consist of flow cytometry to detect depletion of lymphoid sub-
populations during therapy.
3.5.1.3
Objectives / Specific Aims: 
3.5.1.3.1 Immunophenotyping
Blood samples (5ml lavender top tube) will be drawn 
Samples should be delivered to the Clinical Pathology Flow Cytometry Laboratory, Attn. 
Margaret Brown, Building 10, Room 2C410, 301-496-4879.  Test must be scheduled prior to 
sending. 
Time points for Lymphocyte Phenotyping–NK Subset samples will be: 1) On-study; post-HSCT
at 24 hours (+/- 24 hours) post NK#1 and #2 infusion (+/- 48 hours) and on day 28 (+/- 7), day 
60 (+/- 21), and day 100 (+/- 30), and then every 3 months (+/- 1 mo) for the first two years post-
transplant. Samples will not be drawn if ALC is < 200 K/uL.
Time points for Lymphocyte Phenotyping TBNK samples will be: 1) Prior to cycle 2 and 3 of 
induction chemotherapy (within 7 days of each cycle);  2) Prior to pre-HSCT preparative 
chemotherapy, (day –7), and 3) Prior to HSCT (day 0) if ALC is > 200 K/uL. Samples will not 
be drawn if ALC is < 200 K/uLSamples should be delivered to the Clinical Pathology Flow 
Cytometry Laboratory, Attn. Margaret Brown, Building 10, Room 2C410, 301-496-4879.
3.5.2
Chimerism Analysis (Kurlander)
Blood samples (10cc and, if doing cell sorting, an additional 20* cc in yellow top ACD tubes) 
will be drawn to evaluate the relative percentage of donor and host chimerism post-transplant. 
Samples should be delivered to the clinical laboratory of Dr. Roger Kurlander in Building 10, 
hematology laboratory (301-435-6399) 
* Cell sorting will be done on day 14 and repeated on day 28 if mixed chimerism (<98% donor) 
is found on day 14.
Timepoints for samples will be: 1) Eligibility; 2) Day 14 (+/- 5), day 28(+/- 7), day 60(+/- 21),
day 100(+/- 30) ; and 2) After day 100 as clinically indicated.
3.5.3
Serum Cytokine Analysis (Lab)  
Blood samples (5 cc in red-top serum collection tubes) will be drawn to evaluate circulating 
cytokine levels (including IL7 and IL15) and possible correlation with the degree of CD4 
lymphopenia, immune reconstitution, engraftment, and clinical complications such as graft 
versus host disease and infections. The serum will be separated and frozen for subsequent 
analysis by high sensitivity colorimetric enzyme linked immunosorbant assay. 
Timepoints for samples will be: 1) On-Study; 2) Prior to pre-HSCT preparative chemotherapy 
(day –7); 3) Prior to HSCT (day 0); and 4) day prior to or morning of each NK infusion (before 
infusion) then day 1, 2, 7 (+/- 48 hr) and 14 (+/- 48 hr) days post each NK infusion, then days 60
(+/- 21) and 100(+/- 30), and then every 3 months (+/- 1 mo) for the first two years post-
transplant.

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Samples should be picked up by courier (301-846-5893) and delivered to Frederick Repository.
3.5.4
TH1/TH2 Cytokine Profile (Frederick Repository)
Blood samples (1 cc in red-top serum collection tubes) will be drawn to evaluate the types of 
cytokines that are present in the allogeneic environment, and correlate their levels with 
occurrence of potential GVHD and infectious complications. Blood samples will be analyzed by 
multiplex cytokine bead array technology.  
Timepoints for samples will be: 1) On-Study; 2) Prior to pre-HSCT preparative chemotherapy 
(day –7); 3) Prior to HSCT (day 0); and 4) and post-HSCT on day 1, day prior to or morning of 
each NK infusion (before infusion) then day 1, 2, 7 (+/- 48 hr) and 14 days (+/- 48 hr) post each
NK infusion, then days 60 (+/- 21) and 100 (+/- 30), and then every 3 months (+/- 1 mo) for the 
first two years post-transplant.
Samples should be picked up by courier (301-846-5893) and delivered to Frederick Repository.
3.5.5
KIR/HLA genotyping/immunogenetics (Carolyn Hurley – Georgetown Univ.) complete
3.5.5.1
Objective: Retrospectively evaluate for the presence or absence of specific KIR genes to 
determine if the presence of a specific KIR in the absence of its HLA ligand (ie KIR 
"mismatch") correlates with enhanced engraftment, increased anti-tumor activity and 
less infectious complications.
3.5.5.2
Rationale: Patients receiving KIR mismatched NK-DLI may have better NK activity due
to the absence of inhibitory signals, but it is not clear if KIR mismatch is needed in the 
setting of multiple activating signals provided during the NK cell expansion process.
3.5.5.3
Methods:  Presence or absence of specific KIR genes will be determined by locus specific 
PCR amplification followed by gel electrophoresis.  The panel of primer pairs will detect 
KIR2DL1-2DL5, 2DS1-2DS5, 3DL1-3DL3, 3DS1 and two pseudogenes.  Followup 
sequencing of KIR alleles may be performed if required to identify expressed alleles.  
Timepoint for samples will be prior to SCT (day 0) and day 100.  Samples (1-2 ml 
blood in ACD or 3 buccal swabs will be kept at room temperature and will picked up by 
the research nurse. They will then be shipped deidentified to Lihua Hou / Carolyn 
Hurley at C. W. Bill Young Marrow Program/Georgetown University, 3rd Floor, 11333 
Woodglen Drive, Rockville, MD 20852, 301-998-8900.
3.5.6
Viral monitoring for functional Immune Status 
3.5.6.1
Objective: Serially monitor viral reactivation in transplant patients.  Compare viral 
reactivation to historic controls on protocol 02-C-0259 and 01-C-0125. Monitor for a 
meaningful correlation between circulating NK cell levels and viral reactivation.
3.5.6.2
Rationale: Patients receiving TCD grafts carry a higher risk for viral infections and 
reactivation. Viral reactivation is a marker for T-cell immune dysregulation. In 
particular, HHV6 is a marker of cell mediated viral response and a marker of 
inflammation.
3.5.6.3
Methods: Weekly PCR surveillance of EBV, CMV, HHV6, and adenovirus through the 
Clinical Center central laboratory, Section of   Microbiology.
3.5.7
Research Testing of Biopsy Samples

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3.5.7.1
Xenograft Models: Human tumors can be transferred to immunodeficient mice. These 
xenografts models can be used to study tumor biology and treatment. Samples from 
consenting adult patients with solid tumor malignancies will be used for pre-clinical 
testing and development of immunotherapy strategies. Recent studies exemplify the 
important role for xenograft models in the pre-clinical development of IGFR-1
antibodies. Informed consent will be required for this purpose, and subjects will be 
given the option to decline having xenograft models established from their samples.
Sample requirements: Fresh tumor samples in DMEN + 10% FBS, (optimally 0.5 cm in 
one dimension). Samples will be minced into 1mm fragments and injected into 
immunodeficient mice in a location appropriate for specific tumor growth (adrenal 
gland for neuroblastoma, bone for osteosarcoma). Must schedule with lab in advance.
3.5.7.2
Cell line establishment: Attempts will be made to establish solid tumor cell lines lines 
with the aim to identify potential molecular targets for new therapy development. Cell 
lines may also be used to perform in vitro testing of new agents. Informed consent will 
be required for this purpose, and subjects will be given the option to decline having cell 
lines established from their samples.
Sample requirements: Fresh tumor samples in DMEN + 10% FBS, (optimally 0.5 cm in 
one dimension). Samples will be minced into 1mm fragments and cells will be placed in 
flasks with complete growth media and incubated in humidified tissue culture incubator.
3.5.8
NIH Policy on Research Use of Stored Human Samples, Specimens, or Data: 
Blood and tissue collected during the course of this study will follow NIH guidelines for the 
research use of human samples. Most samples will initially be sent to the Central Repository for 
NCI/Frederick using established procedures. All samples will be stored in monitored 
freezers/refrigerators either in the Central Repository for NCI/Frederick or in the investigator’s 
laboratory at specified temperatures with alarm systems in place. All samples will be tracked by 
unique sample identifiers. A secure computer database system will be used to track all samples 
collected on this protocol; the system will contain data that includes but is not limited to the 
unique sample identifiers, storage locations and conditions, biologic study results, information 
about the sample origins (as appropriate), and corresponding records of all derivatives generated 
from samples collected on this protocol. The system will employ mechanisms for restricting 
users to viewing only the level of data appropriate for each individual user, will provide the 
capability to audit any data modification, and will be maintained and backed up according to 
established standards. 
Specimens will be disposed of in accordance with the environmental protection laws, regulations 
and guidelines of the Federal Government and the State of Maryland. Any new uses of human 
subject samples collected during the course of this trial must be reviewed and approved by the 
NCI IRB. Any loss or unintentional destruction of the samples will be reported to the IRB.
3.6
CONCURRENT THERAPY 
3.6.1
Treatment of Graft-Versus-Host Disease:
In the event a patient develops GVHD, the subsequent procedures will be followed. Standard 
clinical and/or biopsy criteria will be used to establish the diagnosis of GVHD, attempts will be
made to obtain histiologic confirmation. Acute GVHD (aGVHD) will be graded by the CIBMTR 

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criteria (Appendix 5) and Chronic GVHD (cGVHD) by the NIH cGVHD Consortium 
Consensus Criteria (Appendix 6 and Appendix 7). Patients will be monitored for GVHD over 
the entire 5-year post-HSCT observational period.  Patients with grade 1 acute GVHD will not 
receive systemic therapy.  In general, patients with > grade 2 acute GVHD will be treated with 
corticosteroids. Patients who fail to respond satisfactorily to corticosteroids will be considered 
for other standard anti-GVHD therapies, and/or investigational GVHD treatment protocols if 
available. Simiarly, patients with cGVHD will be treated with corticosteroids and/or calcineurin 
inhibitors. Patients will also be considered for other standard anti-GVHD therapies, and/or 
investigational GVHD treatment protocols if available. Established guidelines, which are 
regularly reviewed and updated for the treatment of GVHD as outlined by the NIH BMT 
consortium will be followed.  These guidelines can be found at 
http://intranet.cc.nih.gov/bmt/clinicalcare/guidelines.shtml.
3.6.2
Immunosuppressants
Calcineurin inhibitor as described in section 3.3.2.6.
3.6.3
Infection Prophylaxis
Infectious prophylaxis as outlined in section 4.1.
3.6.4
GI Protective Agents
Patients on corticosteroids must be on H2 blockade or other gastrointestinal protective agents.
3.6.5
Hematologic and Blood Product Support
3.6.5.1
Platelet transfusion: In attempt to prevent bleeding, platelet counts should be maintained 
> 20,000/ul (or as clinically indicated) and >50,000 prior to apheresis. Single donor 
platelets should be employed whenever possible in attempt to decrease donor exposure 
and the risk of alloimmunization.
3.6.5.2
Red blood cell transfusion: Packed RBCs should be used to maintain a hemoglobin > 8
gm/dL for ABO compatible transplants and >10 gm/dL in ABO minor mismatch 
transplants (or as clinically indicated).
3.6.5.3
Irradiation: To reduce the risk of transfusion associated graft-vs-host disease, all cellular 
blood products must be irradiated.
3.6.5.4
Leukodepletion: To decrease the risk of febrile reactions and alloimmunization, 
platelets and red cells should be leukoreduced.
3.6.6
Concurrent Therapy for Extramedullary Leukemia or CNS Lymphoma: 
Concurrent therapy or prophylaxis for testicular leukemia, CNS leukemia, and CNS lymphoma 
including standard intrathecal chemotherapy and/or radiation therapy will be allowed as 
clinically indicated after approval by the PI. Such treatment may continue until the planned 
course is completed. Patients must be in CNS remission at the time of protocol enrollment.
3.6.6.1
CNS Prophylaxis for Patients with ALL: Patients with ALL may receive intrathecal (IT) 
methotrexate for 5 doses after transplant.
a. Dose: By age as follows:

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Age (years)
Methotrexate (mg)*
Volume (ml)
3-8
12
8
>9
15
10
b. Administration: To facilitate CNS delivery, the volume of CSF removed should 
be approximate the volume administered and patients should remain prone for 30 
minutes after IT administration.
c. Schedule: Approximately weekly x 5 doses to begin after myeloid recovery, not 
before post-transplant day 28.
d. Antiemesis: Routine (e.g., ondansetron) should be considered prior to each 
dose of methotrexate in patients with a history of nausea associated with 
intrathecal chemotherapy.
*Methotrexate may be substituted with hydrocortisone/cytarabine, or Triple IT 
(MTX/AraC/HC) may be given, if clinically indicated, and dosing will be 
performed as per standard Children’s Oncology Group (COG) doses (example 
below):
Age (years)
Cytarabine (mg)
Hydrocortisone (mg)
3-8
24
12
>9
30
15
3.6.7
Treatment of Progressive Disease Post-transplant: 
Patients who have progressive disease after day +100 post-transplant may receive donor 
lymphocyte infusions (DLI) since this has been effective in the management of post-BMT 
relapse. Patients who have been started on immunosuppression for GVHD treatment will 
undergo sequential withdrawal of immunosuppression prior to DLI. As long as there is no 
evidence for active GVHD, patients may be eligible to receive unmodified DLI. If deemed 
necessary to combat rapidly progressive disease, adjunctive chemotherapy using standard 
disease-specific agents may be employed in conjunction with this approach with PI approval. 
The specific chemotherapy to be used should be decided by the patient’s oncology and BMT 
teams in consultation with the PI. Lympholytic agents (e.g., corticosteroids) should be 
avoided. Alternatively, in the event of progressive disease after day +100, the patient may be 
taken off-study and referred for conventional therapy or participation on other NCI 
investigational trials for which they might be eligible.
3.6.7.1
LEUKAPHERESIS PROCEDURE TO COLLECT CELLS FOR DLI: Donation of DLI shall be 
by standard lymphapheresis, during which 2 to 5 blood volumes will be processed as 
estimated by recipient weight and target cell harvest dose (maximum 5 total blood 
volumes) in children (defined as less than 40 kg), or 7 to 10 liters of blood in adults will 
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DTM staff in consultation with PI). Non-mobilized donor lymphocytes will be collected 
by apheresis and processed as per standard NIH DTM procedures. The lymphapheresis 
will undergo red cell or plasma depletion for major or minor ABO incompatibility, 
respectively, according to CPS, DTM policies and procedures. In the event that some or 
all of the donor lymphocytes will be administered in the future, the lymphapheresis 
product will be cryopreserved and stored according to CPS, DTM standard operating 
procedures.  10% of the total lymphapheresis product of related donor ONLY will be 
cryopreserved as a separate aliquot and stored for research. Use of cryopreserved DLI 
collected as part of the original PBSCT collection may be used if clinically indicated.
3.6.7.2
DLI ADMINISTRATION: DLI will generally be administered at a starting dose of 1 x 106
CD3+ T-cells/kg recipient body weight by IV infusion. A higher starting may be used as 
clinicaly indicated. Additional DLI may be administered in escalating doses in increments 
of 0.5-1 log10 to a maximum dose of 1 x 108 CD3+ T-cells/kg every 2-4 weeks provided
there has been no improvement in malignant disease and there remains no evidence of 
GVHD. 
Patients will receive the following medications approximately 30-60 minutes prior to DLI
cell infusion: 
1.) Diphenhydramine 0.5 - 1 mg/kg/dose (maximum 50 mg/dose) IV or PO over 
10-15 minutes; 
2.) Acetaminophen 15 mg/kg/dose (maximum 650 mg/dose) PO
3.) If the patient has been premedicated earlier in the day for blood product or other 
infusion, pre-medication is not warranted if given within 4 hours of stem cell 
infusion
Safety monitoring during and after infusion will include vital signs (blood pressure, heart 
rate, respiratory rate) prior to infusion, and 15 minutes post infusion and more frequently 
if clinically indicated.  
3.6.8
Treatment of Mixed Chimerism Post-transplant: 
If a patient has < 95% donor lymphoid chimerism on day +28 post-transplant, repeat analysis 
will be performed until complete chimerism is achieved or a pattern of stable mixed chimerism 
or graft rejection is established. For patients with significant reductions in donor chimerism 
as determined by the PI, DLI (as per Section 3.6.7) will be considered. If patient is on 
immunosuppression for GVHD) sequential withdrawal of immunosuppression will precede 
DLI administration. This approach has been successfully employed in pediatric patients with 
ALL, AML, and MDS who had progressive mixed chimerism after conventional 
myeloablative allogeneic BMT(212). If at any time a patient has < 5% donor lymphoid 
chimerism, they will be taken off treatment and consideration will be given for conventional 
HSCT.
3.6.9
Treatment of Graft Failure/Graft Rejection:
If a patient develops graft rejection (as evident by decreasing hematopoiesis and decreasing 
CD3 donor chimerism) or has graft failure (lack of engraftment) they will be taken off 
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treatment of graft rejection will be determined as clinically indicated but may include 
serotherapy such as anti-thymocyte globulin, alemtuzumab and/or second transplant.
3.7
CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY CRITERIA
3.7.1
Criteria for removal from protocol therapy
x
Unacceptable toxicity as defined in section 3.1.1.
x
Inability to proceed with transplantation (failure to meet criteria).
x
Progressive disease during induction therapy.
x
Irreversible non-hematologic grade 4 toxicity during induction chemotherapy. Any other 
non-hematologic toxicity during induction chemotherapy of sufficient severity or 
duration that in the opinion of the PI poses prohibitive risks to further chemotherapy.
x
Progressive disease post tranplant, which requires anti-neoplastic therapy.
x
Patients unable to comply with study requirements will come off treatment.
x
< 5% donor lymphoid chimerism at or beyond day +28, or graft rejection/failure
x
Enrollment on another clinical trial for treatment of progressive disease or graft rejection.
x
Recipients will come off treatment upon completion of study follow-up period (5 year 
post PBSCT). These patients with be followed for survival and development of chronic 
GVHD.
3.7.2
Off-Study Criteria
x
Recipient death.
x
Recipient withdrawal of consent
x
Lost to follow-up
x
Investigator discretion
x
Completed study follow-up period
x
Donors will come off-study at the time of recipient off-study date
Note: all minors who sign the treatment consent will be kept on study and followed until the age 
of 18 to allow for consent.
3.7.3
Off Protocol Therapy and Off-Study Procedure
Authorized staff must notify Central Registration Office (CRO) when a subject is taken off 
protocol therapy and when a subject is taken off-study. A Participant Status Updates Form from 
the website (http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) main page must be 
completed and sent via encrypted email to:  NCI Central Registration Office 
ncicentralregistration-l@mail.nih.gov.
3.8
END OF STUDY EVALUATION (APPENDIX 2)
The following tests and procedures should be performed, if possible, at the time a patient comes 
off study regardless of the reason for coming off study, unless the test or procedure has been 
performed in the last 4 weeks (2 weeks for physical examination and laboratory assessment). 
3.8.1
History and physical examination. 
3.8.2
Laboratory Assessment:  Iron studies (serum iron, ferritin, and transferrin), Complete blood 
count, differential and platelet count. Chem 20 and urinalysis. 

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3.8.3
Disease restaging 
3.8.4
Apheresis. (Optional)
3.9
DURATION OF STUDY
Patients will be followed annually for up to year 3 after completing treatment on study, and 
annually by phone for survival status.
4
SUPPORTIVE CARE
4.1
INFECTION PROPHYLAXIS 
Routine measures to prevent infection in accordance with standard of care practice. For patients 
enrolled at the NIH, the recommendations following the NIH Allogeneic Stem Cell 
Transplantation guidelines should be employed 
(http://intranet.cc.nih.gov/bmt/education/infectious-mgmt-guidelines.shtml). 
4.2
MUCOSITIS PROPHYLAXIS
Kepivance™ (palifermin), a human keratinocyte growth factor (KGF), may be administered to 
reduce the incidence or severity of mucositis.  Palifermin will be administered at a dose of 60 
mcg/kg/day IV X 3 doses, given twice.  The first 3 daily doses should be administered prior to 
the preparative chemotherapy with the 3rd dose completed at least 24 hours prior to starting the 
preparative regimen.  The last 3 daily doses should be administered starting on day +1.  
5
DATA COLLECTION AND EVALUATION
5.1
DATA COLLECTION
The PI will be responsible for overseeing entry of data into an in-house password protected 
electronic system and ensuring data accuracy, consistency and timeliness. The principal 
investigator, associate investigators/research nurses and/or a contracted data manager will assist 
with the data management efforts.  All data obtained during the conduct of the protocol will be 
kept in secure network drives or in approved alternative sites that comply with NIH security 
standards. Primary and final analyzed data will have identifiers so that research data can be 
attributed to an individual human subject participant.
All AEs, including clinically significant abnormal findings on laboratory evaluations, regardless 
of severity, will be followed until satisfactory resolution. AEs should be reported up to 30 days 
following the last dose of study drug.  AEs that are considered treatment related, expected, 
continuing, but not resolvable by 30 days after treatment completion (e.g., alopecia) will not be 
followed after the 30-day period. 
Donor AE data will be collected through 1 week following collection. 
An abnormal laboratory value will be considered an AE if the laboratory abnormality is 
characterized by any of the following:
x
Results in discontinuation from the study
x
Is associated with clinical signs or symptoms 
x
Requires treatment or any other therapeutic intervention
x
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x
Is judged by the Investigator to be of significant clinical impact
If any abnormal laboratory result is considered clinically significant, the investigator will provide 
details about the action taken with respect to the test drug and about the patient’s outcome.
Recipient and Donor data will also be reported to the Center for International Blood and Marrow 
Transplant Research (CIBMTR) registry as required by law.  Recipients and donors will be 
offered the opportunity to consent to the use of this information, and additional baseline and 
outcome data submission, through one of two NIH protocols, depending on their donor source.  
Consent to these protocols is optional and does not affect the recipient’s access to transplant or to 
unrelated donor grafts.  For unrelated donor graft recipients, this data submission element 
ensures compliance with standards described in the Transplant Center Agreement between the 
NIH Clinical Center and the NMDP.
End of study procedures: Data will be stored according to HHS, FDA regulations, and NIH 
Intramural Records Retention Schedule as applicable. 
Loss or destruction of data: Should we become aware that a major breach in our plan to protect 
subject confidentiality and trial data has occurred, the IRB will be notified.
5.2
RESPONSE CRITERIA
Standard disease specific clinical and laboratory response criteria will be employed as detailed in 
Appendix 9 and Appendix 10.
5.3
TOXICITY CRITERIA 
The following adverse event management guidelines are intended to ensure the safety of each 
patient while on the study.  The descriptions and grading scales found in the revised NCI 
Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for AE 
reporting. All appropriate treatment areas should have access to a copy of the CTCAE version 
4.0. A copy of the CTCAE version 4.0 can be downloaded from the CTEP web site 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm#ctc_40).
5.4
DATA REPORTING EXCEPTIONS
For patients who meet off-treatment criteria, but remain on-study and who are undergoing 
treatment of toxicity related to the investigational therapy (e.g., second transplant for 
treatment of graft rejection), only adverse events will be recorded and reported for those events 
which are > grade 3 or serious, are previously unexpected AND are deemed possibly, probably 
or definitely related to a procedure, test, or treatment, or require intervention. Adverse events 
resulting from other disease-related treatments not associated with this investigational therapy
(i.e. treatment for progression of disease), or from events not related to the patient’s disease will 
NOT be recorded or reported.
Expedited reporting will not apply to events arising from participation in another clinical trial or 
alternative treatment for disease progression.

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6
STATISTICAL CONSIDERATIONS
6.1
SUBJECT ACCRUAL
Subjects of both genders, from all racial and ethnic groups are eligible for this trial if they meet 
the criteria outlined in Section 2.1. To date, there is no information that suggests differences in 
disease response among racial or ethnic groups or between the genders, indicating that results of 
the trial will be applicable to all groups. Efforts will be made to extend the accrual to a 
representative population, but in a pilot study with limited accrual, a balance must be struck 
between patient safety considerations and limitations on the number of individuals exposed to 
potentially toxic or ineffective treatments on the one hand and the need to explore gender, racial, 
and ethnic aspects of clinical research on the other. If differences in outcome that correlate to 
gender, age, racial, or ethnic identity are noted, accrual may be expanded or additional studies 
may be performed to investigate those differences more fully. 
6.2
STATISTICS AND FEASIBILITY
The first primary objective of this study is to assess the feasibility and toxicity associated with 
infusion of escalating doses of donor derived natural killer (NK) cells (NK-DLI) on Days 21 (± 3
days) and 49 days (± 7 days) following infusion 1, following allogeneic HLA-matched T cell 
depleted (TCD) peripheral blood stem cell transplantation (PBSCT) in patients with metastatic or 
recurrent pediatric solid tumors and leukemias. Because of the experience in the first 8 patients 
transplanted, recipients will now be divided into 2 cohorts: 1) matched related donor recipients 
and 2) unrelated donor recipients. Patients will be enrolled onto this trial using a conventional 
Phase I dose escalation design in which 3 patients per dose level, in each cohort are entered, with 
3 additional patients enrolled at the dose level, following the usual guidelines for a phase I trial, 
based on the development of DLTs. After enrollment of the first six patients on study without 
DLT, cohort 1 was expanded by a maximum of 6 patients to study the pathophysiology of 
symptoms observed to date.  Because no GVHD was seen in related donor recipients (n = 4),
dose level 1 for related donor recipients will be closed and patients with related donors will start 
accrual onto dose level 2. Since the expansion of dose level 1, one additional unrelated donor 
recipient had been transplanted and developed grade 4 GVHD. Although the GVHD was steroid 
responsive and therefore did not meet definition of DLT, we find the incidence and severity of 
the GVHD in these patients unacceptable. Therefore, all subsequent recipients will be treated 
with GVHD prohylaxis and patients with unrelated donors will continue to enroll on cell dose 
level 1. In addition to assessment for DLT, if >1 of the additional 3 patients with unrelated 
donors, enrolled at the cohort 1 cell dose level experience Grade 3 or any experience grade 4
acute GVHD, the NK cell dose level will not be escalated without further consultation with the 
FDA and IRB regarding a protocol modification.
Consistent with a phase I design, the highest dose level at which 0/6 or 1/6 patients has a DLT 
will be expanded. If at any dose level, there are 2 or more patients who experience a DLT, then 
no further dose escalations will occur. At the expanded dose level, if 3 or more of 9, or 4 or more 
of 12 patients experience a DLT, consideration for de-escalation and an expanded cohort at a 
lower dose level will be given through a protocol amendment.  The feasibility objective would be 
met if >=80% of patients receive 2 doses of NK cell infusions within 56 days of HSCT.  As 
described in the protocol, patients are scheduled to receive NK-DLI on Days 21 (± 3 days) and 
49 (± 7 days) but the doses may be delayed based upon clinical judgment of the treating team or 

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concern for toxicity related to previous infusions. Moreover, it is possible that patients may not 
receive adequate cells because of toxicity, product failure, or product contamination.
A second primary objective of this trial is to determine if full donor lymphoid engraftment in T 
cell depleted, MHC matched sibling or unrelated donor transplants with NK-DLI can be 
accomplished using this regimen. Donor engraftment is defined as >95% donor lymphoid 
chimerism (CD3+ T-cells in peripheral blood) on post-BMT day +100. The highest dose 
explored will enroll 12 total patients in order to allow adequate numbers of patients to receive the 
regimen for more accurate estimates of engraftment, aGVHD and toxicity at that dose level.  
Because NK cells are presumed to enhance engraftment rates, only patients enrolled on the 
highest dose level will be evaluable for the engraftment endpoint.  If however, at any dose level, 
engraftment is problematic and primary graft failures occur in 2 patients, the protocol stopping 
rule for insufficient engraftment described below will be triggered.  It would be desirable to have 
greater than 75% of patients at the highest NK dose level show sustained donor lymphoid 
engraftment (>95% at day 100). Thus, if 8 or more of 12 patients engraft, this will be considered 
adequate since the upper one sided 90% CI about 7/12 is 0.781.  
A third primary objective of this trial is to determine rates of aGVHD in TCD, MHC matched 
sibling or unrelated donor transplants given NK-DLI. Upon completion of the trial, the incidence 
of all aGVHD will be reported with appropriate confidence intervals at all dose levels. All 
histologies will be analyzed together for this end point, since the incidence of aGVHD is not 
likely to be influenced by this variable.  In addition, however, we will evaluate the incidence of 
aGVHD as it relates to histology for exploratory purposes in an attempt to identify any 
unexpected relationships that might exist.  A rate of <25% grade III or grade IV acute GVHD per 
cohort would be required to consider this approach of interest for further development.  All 
patients enrolled, regardless of NK dose level, will be evaluable for the aGVHD endpoint.  If 
however, excessive aGVHD is observed, this will trigger the stopping rules described below.
6.3
EARLY STOPPING RULES
If the number of patients who have ever been treated on this trial at any dose level 1) develop 
steroid refractory grade 3 or grade 4 aGVHD at any time point, 2) show primary graft failure -
defined as failure to achieve independence from platelet transfusion or ANC never reaching > 
500 cells/mcl by day 35 post-BMT, or 3) experience treatment related mortality within 100 days 
(defined as toxicity from the preparative regimen, infection, aGVHD or therapy for aGVHD),
exceeds 1/5 (upper 90% confidence interval of 58%), 3/10 (upper 90% CI of 55%), or 4/15 
(upper 90% CI of 46%) or 6/20 (upper 90% CI of 47%), then accrual to the trial will terminate as 
soon as this can be evaluated, regardless of how successful the clinical outcomes have been, as 
this will be considered excessive toxicity.
In addition, if a single patient (exception: patients with bcr-abl+ leukemia) develops a positive 
bcr-abl PCR confirmed by repeat testing, there will be a temporary suspension to accrual and no 
products infusions will be given to patients currently on study.
6.4
SUBJECT ACCRUAL GOAL
As stated in section 3.1, three to 6 recipients of unrelated donors will be treated on cell dose level 
1, and three to 6 recipients will be treated on cell dose 2 per cohort, with an additional six 
recipients enrolled on cohort 3, or the highest cohort explored. Thus, a total of up to 38 recipients 
(and 38 donors) may be enrolled on this trial. Prior to enrolling on the first cohort, a single 

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patient will receive DLI-NK cells once ANC reaches at least 1000 cells/μL or within 42 days 
following transplant. Due to toxicities observed in the first patient, a second patient received this 
regimen.  Once these first two patients had undergone transplant and accomplished 
hematopoietic recovery, cohort 1 was opened 28 days after the last NK cell infusion. With a rare 
possibility of not achieving the targeted cell numbers per cohort, an additional 3 recipient/donor 
pairs may be enrolled if needed to replace patients who do not meet the targeted cell dose for the 
assigned cohort, for a maximum of 43 recipients and 43 donors.  With an approximately 5-7
patients enrolling per year, anticipated accrual is 3-4 years with 3 year post-transplant follow up. 
It is therefore likely this trial will remain open for approximately 6 - 8 years.
7
HUMAN SUBJECTS PROTECTIONS
7.1
RATIONALE FOR SUBJECT SELECTION
7.1.1
Recipients
Allogeneic hematopoietic stem cell transplants are being performed at increasing rates each year 
for patients with malignant diseases. Patients with hematologic malignancies will be the subjects 
for this study. This patient group was chosen because current treatment options offer little to no 
hope for survival and new approaches to therapy are desperately needed.  While the diseases 
targeted in this trial affect all races, and genders and subjects of both genders, efforts will be made 
to extend accrual to a representative population, but in a small pilot trial, it may be difficult if not 
impossible to achieve complete balance in this regard. The trial will be listed on the NCI and NIH 
websites and clinical trials search sites. Individuals with HIV disease will not be candidates for 
this protocol, due to the high risk of post-transplant complications in this population. Individuals 
who are pregnant or lactating will not be candidates for this protocol, due to risk to the fetus or 
newborn.
7.1.2
Donors
Related donors will be first-degree HLA-matched family members of the transplant recipient. In 
most cases the best donor is a sibling. All donors will be selected in accordance with NIH CC 
Department of Transfusion Medicine standards. Minors will only be selected when they represent 
the most suitable stem cell donor for the individual recipient. In such cases, the oldest suitable 
donor will be selected and approval to participate will require medical review by a pediatric 
practitioner, evaluation by a social worker, and psychological assessment by a mental health 
specialist with pediatric expertise. For donors who are minors, informed consent from their legal 
guardian and donor verbal assent will be obtained.
Unrelated donors will be HLA-matched (at least 9/10 allele) and will be selected in accordance 
with National Marrow Donor Program (NMDP) standards.
7.2
PARTICIPATION OF CHILDREN
The age range of patients eligible for this trial is 4 to 35 years of age. Physicians, nurses, and 
multidisciplinary support teams have expertise in the management of children undergoing 
PBSCT.  All children enrolled on this protocol will be followed and cared for in the Pediatric 
Oncology Branch clinic. 
7.3
EVALUATION OF BENEFITS AND RISKS/DISCOMFORTS 
7.3.1
Recipients

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Survival for the population of patients eligible for this trial is limited.  This population has a very 
high risk of dying from their disease, and other therapeutic options are primarily experimental. 
Although the overall treatment plan is experimental, the PBSCT portion of this study is an 
acceptable therapeutic option for patients in this population, and pre-clinical immunologic studies 
indicate there may be some benefit to the addition of NK cell administration.  The goal of this 
study is to determine the safety and feasibility of this approach and to determine if transplantation 
of allogeneic (related or unrelated) HLA-matched NK cells post PBSCT can result in full donor 
engraftment (>95% by day 100), while reducing the incidence of Graft-versus-Host Disease 
(GVHD), and reducing the risks associated with infection.  Patients participating in this trial will 
be treated with therapeutic intent, and the response to therapy will be monitored.  The potential 
benefits from PBSCT alone are prolonged disease remission and/or reduction in cancer-related 
symptoms, while the potential additional benefits of the NK cell infusion are unknown at this time.  
This protocol involves greater than minimal risk, but does present the potential for direct benefit 
to individual subjects according to 21CFR50.52, 46.405.
7.3.1.1
Risks associated with PBSCT
The primary risks to patients participating in this research study include toxicity of the 
chemotherapy and immunosuppressive drugs and allogeneic transplantation. The chemotherapy 
utilized in this study has the risk of direct drug toxicity. In addition, the immune suppression 
resulting from this therapy is associated with an increased risk of infections. The primary risks 
associated with allogeneic stem cell transplantation are graft rejection, GVHD and regimen 
related toxicity. This protocol utilizes a profoundly immunosuppressive induction and 
preparative regimen designed to facilitate rapid hematologic engraftment.  Indeed, patients 
treated with a similar regimen and T cell depleted graft in the ETIB have shown >90% myeloid 
engraftment in the majority of patients by day 28 and in all patients by day 98. In addition, the 
POB and the ETIB have extensive experience with this induction and preparative regimen with T 
cell replete grafts and have seen universally brisk engraftment, therefore graft rejection is not 
anticipated to be limiting.  In this regimen, melphalan has been added for additional 
cytoreductive effects.  While this may increase the probability that the preparative regimen is 
myeloablative, it lessens the chance for graft rejection even further and thus we believe that graft 
rejection is very unlikely in this protocol.
Thus, the evidence suggests that GVHD and tumor recurrence/progression will represent the 
primary cause of morbidity and mortality post-transplant.  Because of the history of significant 
rates of GVHD in previous POB trials, the graft will be T cell depleted and the risk for GVHD is 
likely to be far lower.  The protocol provides for detailed and careful monitoring of all patients to 
assess for toxicity and response to treatment.  All patients entered on the trial will have high risk 
pediatric malignancy for which standard therapy is ineffective.  Further, the chemotherapy drugs 
used on this protocol are known to be effective for the types of cancer that these patients will 
have. 
7.3.1.2
Risks associated with allogeneic NK cell administration
In previous studies, grade 2 and 3 anemias have been reported though most patients had anemia 
at study entry. Neutropenia has also been reported, but this was following chemotherapy agents. 
There were no episodes of neutropenic fever and thrombocytopenia was not observed. 
Hematologic toxicity was transient and there was no occurrence of prolonged cytopenia or 
marrow aplasia. Reports suggest that engraftment is improved, rates of GVHD and infections are 

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lower with NK cell infusions, however, a negative impact on engraftment and the development 
of GVHD are theoretic risks.
Based on experiences on this trial, however, there is an apparent increase in GVHD in recipients 
of unrelated donor products in comparison to recipients of related donor products, which is 
thought to be at least possibly related to the allogeneic NK cell administration. Because of this 
disparity, several changes have been made to the protocol to ameliorate the potential risk of NK 
related GVHD. 1) NK cells are now given no earlier than day 21 (+/- 3) days post PBSCT to 
avoid concurrent engraftment syndrome which may be exacerbated by NK cell administration; 2) 
Recipients of unrelated donor products will receive immunosuppression to ameliorate the 
potential risk of GVHD. 
7.3.1.3
Tumor biopsy
In the event a patient has a readily accessible tumor, adult patients may be asked to undergo 
biopsy in order to obtain tissue for research evaluation (see section 3.2.2).  Standard techniques 
will be used for percutaneous biopsies and may include CT and / or ultrasound guidance. 
Although direct benefit from research conducted on this tumor biopsy is unlikely, participation in 
this research may allow patients some benefit in knowing their contribution may lead to a greater 
understanding of their cancer and potential benefit to others in the future. Patients enrolled on 
this protocol suffer from extremely rare malignancies with very low survival rates and often have 
a strong desire to participate in research that may lead to a better understanding of their disease.
7.3.2
Donors
The donor stem cell collection procedure on this trial is conducted per standard clinical routine.
There are a number of potential benefits to donors who participate on this trial. The most 
probable is psychological benefit from contributing to medical care designed to improve the 
health of another. Donors appreciate that their participation represents an essential component to 
a transplant, which is performed with curative intent. This psychological benefit may be derived 
both at the time of donation, as well as in the future, since it is hoped that the transplant will 
result in prolongation and/or improvement in the quality of the recipient’s life. Donor 
participation may also help advance scientific knowledge about stem cell transplantation and 
lead to improvements in the treatment of cancer. (213)
The main risks to donors are the possible side effects of stem cell harvesting.  Stem cell donation 
is a safe procedure that is routinely performed in healthy children and adults. The potential side 
effects of filgrastim used for mobilization are outlined in Section 9.8. The most common side 
effects of apheresis are pain and bruising at IV sites. Side effects of a temporary central venous 
catheter, required for young children, include pain, bleeding, bruising, infection, thrombosis, and 
vascular perforation. Mild side effects from citrate anticoagulant are common and include chills, 
numbness and tingling sensations ("pins and needles"), anxiety, muscle cramps, and nausea. 
More serious side effects due to citrate-induced low calcium levels are uncommon and include 
low blood pressure, seizures, weakness, and tetany. Citrate reactions rapidly resolve away when 
the collection is slowed down or stopped. Transient mild thrombocytopenia is common after 
donation. To prevent dilutional anemia, the extracorporeal circuit must be primed with 1 unit of 
red blood cells for small children. Side effects of blood draws include pain and bruising, 
lightheadedness, and rarely, fainting. There is the possibility that the donor will have negative 
psychological effects (e.g., guilt, sadness) if the transplant is unsuccessful or if complications 

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(e.g. GVHD) contribute to recipient morbidity or mortality. Donors will be closely monitored 
and procedures to minimize risks and prevent side effects are incorporated into all aspects of the 
protocol. The POB, DTM, and NIH CC and NMDP Donor Sites have broad expertise to 
adequately manage side effects, including in pediatric donors (related donors only).
7.4
RISK/BENEFIT ANALYSIS
All patients enrolled on this study are at very high risk of disease recurrence and from dying of 
their disease.  Patients entered on the trial will be treated with therapeutic intent and response to 
the therapy and possible toxicity will be closely monitored. Patients on this study may directly 
benefit from the chemotherapy administered as it may provide anti-tumor activity.  Successful 
allogeneic PBSCT may offer patients a curative therapy for their cancer.  It is hypothesized that 
the addition of NK cell infusions may improve engraftment, reduce the incidence of GVHD and 
lower infection risks; although these results are not known at this time.  
Therefore, this protocol involves greater than minimal risk to patients, but presents the potential 
for direct benefit to individual subjects and therefore meets Federal requirements for patients that 
are minors.  (45CFR46.405)
7.5
CONSENT PROCESSES AND DOCUMENTATION 
7.5.1
Recipients
A signed informed consent document will be obtained prior to entry onto the study. The PI or an 
associate investigator on the trial will obtain consent. The PI, an Associate Investigator, or their 
designee will be available to answer all questions from patients and their parents or guardians. 
The investigational nature and research objectives of this trial, the procedures and treatments 
involved and their attendant risks and discomforts and potential benefits, and alternative 
therapies will be carefully explained to the patient and/or the patient’s parents or guardian if 
he/she is of minor age. 
The investigators are requesting a waiver from the IRB to allow only one parent to sign the 
informed consent to enter a child on the protocol. Because many patients must travel to the NIH 
from long distances at substantial expense, requiring both parents to be present for the consent 
process could be a financial hardship for many families. When guardianship status of the child is 
uncertain, documentation of custody status must be obtained.
Where deemed appropriate by the clinician and the child’s parent(s) or guardian, the child will 
also be included in all discussions about the trial and age-appropriate language will be used to 
describe the procedures and tests involved in this study, along with the risks, discomforts and 
benefits of participation.  Written assent will not be obtained from children as the study holds out 
the prospect of direct benefit that is important to the health and well-being of the child and is 
available only in the context of the research. Verbal assent will be obtained as appropriate for 
children ages > 7 and the parent or guardian will sign the designated line on the informed 
consent attesting to the fact that the child has given assent.  Children under the age of 7 will not 
be required to provide assent as they typically do not have the cognitive ability to fully 
understand the nature of research. The consent/assent process will be documented in the child’s 
medical record, including the assessment of the child’s ability to provide assent (verbal versus 
written) as applicable. All children will be contacted after they have reached the age of 18 to 

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determine whether they wish to continue on the trial and informed consent will be obtained from 
them at that time.
7.5.2
Related donors
Informed consent will also be obtained from all donors entered on this trial. Donors < 18 years of 
age will be evaluated by a social worker and mental health specialist (psychologist or 
psychiatrist) prior to the assent process to determine willingness to participate. If willingness to 
participate has been confirmed, verbal assent, as described above, will also be obtained. The 
attached informed consent documents contain all elements required for consent. In addition, the 
Principal Investigator, Associate Investigator, or their designee will be available to answer all 
questions from patients, donors, and their parents or guardians.
7.5.3
Unrelated Donors 
Informed consent will be obtained from all unrelated donors entered on this trial. This process 
will be conducted at the NMDP Donor Center using the Protocol Informed Consent Document 
for Unrelated Donors, which will be approved by both the NCI and NMDP IRBs. Per NMDP 
regulations, and to maintain donor confidentiality, the signed informed consent document will 
not be sent to the NIH. Instead, the NMDP will issue a certification notice to the NIH confirming 
donor consent.
7.5.4
Consent for minors when they reach the age of majority 
When a pediatric subject reaches age 18, continued participation (including ongoing interactions 
with the subject or continued analysis of identifiable data) will require consenting of the now 
adult with the standard protocol consent document to ensure legally effective informed consent 
has been obtained. Given the length of time that has transpired for some of the subjects since 
their last visit for this study, we request waiver of informed consent for those individuals who 
have completed their participation in the research study.
Requirements for Waiver of Consent consistent with 45 CFR 46.116 (d):
(1) The research involves no more than minimal risk to the subjects.
a.
Analysis of samples and data from this study involves no additional risks to 
subjects.
(2) The waiver or alteration will not adversely affect the rights and welfare of the 
subjects.
a.
Retention of these samples or data does not affect the welfare of subjects. 
(3) The research could not practicably be carried out without the waiver or alteration.
a.
Considering the length of time between the minor’s last contact with the 
research team and their age of majority, it will likely be very difficult to locate 
them again. A significant reduction in the number of samples analyzed is 
likely to impact the quality of the research. 
(4) Whenever appropriate, the subjects will be provided with additional pertinent 
information after participation.
a.
We only plan to request a waiver of reconsent for those subjects who have 
been lost to follow-up or who, prior to the approval of Amendment Q, have 
been taken off study prior to reaching the age of majority.

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7.5.5
Telephone consent 
The informed consent document will be sent to the subject.  An explanation of the study will be 
provided over the telephone after the subject has had the opportunity to read the consent form.  
The subject will sign and date the informed consent. A witness to the subject’s signature will 
sign and date the consent.
The original informed consent document will be sent back to the consenting investigator who 
will sign and date the consent form with the date the consent was obtained via telephone.  
A fully executed copy will be returned via mail for the subject’s records.
The informed consent process will be documented on a progress note by the consenting 
investigator and a copy of the informed consent document and note will be kept in the subject’s 
research record.
8
SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING
PLAN
8.1
DEFINITIONS
8.1.1
Adverse Events
Any untoward medical occurrence in a human subject, including any abnormal sign (for example, 
abnormal physical exam or laboratory finding), symptom, or disease, temporally associated with 
the subject’s participation in research, whether or not considered related to the subject’s 
participation in the research.
x
8.1.2
Suspected adverse reaction
Suspected adverse reaction means any adverse event for which there is a reasonable possibility 
that the drug caused the adverse event. For the purposes of IND safety reporting, ‘reasonable 
possibility’ means there is evidence to suggest a causal relationship between the drug and the 
adverse event. A suspected adverse reaction implies a lesser degree of certainty about causality 
than adverse reaction, which means any adverse event caused by a drug.
8.1.3
Unexpected adverse reaction
An adverse event or suspected adverse reaction is considered “unexpected” if it is not listed in the 
investigator brochure or is not listed at the specificity or severity that has been observed; or, if an 
investigator brochure is not required or available, is not consistent with the risk information 
described in the general investigational plan or elsewhere in the current application. "Unexpected”, 
also refers to adverse events or suspected adverse reactions that are mentioned in the investigator 
brochure as occurring with a class of drugs or as anticipated from the pharmacological properties 
of the drug, but are not specifically mentioned as occurring with the particular drug under 
investigation.
8.1.4
Serious 
An Unanticipated Problem or Protocol Deviation is serious if it meets the definition of a Serious 
Adverse Event or if it compromises the safety, welfare or rights of subjects or others.
8.1.5
Serious adverse events

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An adverse event or suspected adverse reaction is considered serious if in the view of the 
investigator or the sponsor, it results in any of the following:
x
Death,
x
A life-threatening adverse drug experience
x
Inpatient hospitalization or prolongation of existing hospitalization
x
A persistent or significant disability/incapacity or substantial disruption of the ability to 
conduct normal life functions
x
A congenital anomaly/birth defect.
x
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered a serious adverse drug experience when, based upon 
appropriate medical judgment, they may jeopardize the patient or subject and may require 
medical or surgical intervention to prevent one of the outcomes listed in this definition.
8.1.6
Disability
A substantial disruption of a person’s ability to conduct normal life functions.
8.1.7
Life-threatening adverse drug experience
Any adverse event or suspected adverse reaction that places the patient or subject, in the view of 
the investigator or sponsor, at immediate risk of death from the reaction as it occurred, i.e., it does 
not include a reaction that had it occurred in a more severe form, might have caused death.
8.1.8
Protocol Deviation (NIH Definition)
Any change, divergence, or departure from the IRB approved research protocol.
8.1.9
Non-compliance (NIH Definition)
The failure to comply with applicable NIH Human Research Protections Program (HRPP)
policies, IRB requirements, or regulatory requirements for the protection of human research 
subjects.
8.1.10 Unanticipated Problem
Any incident, experience, or outcome that:
x
Is unexpected in terms of nature, severity, or frequency in relation to 
(a) the research risks that are described in the IRB-approved research protocol and 
informed consent document; Investigator’s Brochure or other study documents, and
(b) the characteristics of the subject population being studied; AND
x
Is related or possibly related to participation in the research; AND
x
Suggests that the research places subjects or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or 
recognized.
8.2
NCI-IRB AND CLINICAL DIRECTOR REPORTING
8.2.1
NCI-IRB and NCI CD Expedited Reporting of Unanticipated Problems and Deaths

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The PI will report in the NIH Problem Form to the NCI-IRB and NCI Clinical Director:
x
All deaths, except deaths due to progressive disease
x
All Protocol Deviations
x
All Unanticipated Problems
x
All non-compliance
Reports must be received within 7 days of PI awareness via iRIS.
8.2.2
NCI-IRB Requirements for PI Reporting at Continuing Review
The protocol PI will report to the NCI-IRB:
1. A summary of all protocol deviations in a tabular format to include the date the deviation 
occurred, a brief description of the deviation and any corrective action.
2. A summary of any instances of non-compliance
3. A tabular summary of the following adverse events:
a) All Grade 2 unexpected events that are possibly, probably or definitely related to the 
research;
b) All Grade 3 and 4 events that are possibly, probably or definitely related to the research;
c) All Grade 5 events regardless of attribution;
d) All Serious Events regardless of attribution;
NOTE: Grade 1 events are not required to be reported.
8.2.3
NCI-IRB Reporting of IND Safety Reports
Only IND Safety Reports that meet the definition of an unanticipated problem will need to be 
reported to the NCI IRB.
8.3 IND SPONSOR REPORTING CRITERIA 
During the first 30 days after the subject receives investigational agent/intervention, the
investigator must immediately report to the sponsor, using the mandatory MedWatch form 3500a 
or equivalent, any serious adverse event, whether or not considered drug related, including those 
listed in the protocol or investigator brochure and must include an assessment of whether there is 
a reasonable possibility that the drug caused the event. For serious adverse events that occur 
more than 30 days after the last administration of investigational agent/intervention, only report 
those that have an attribution of at least possibly related to the agent/intervention.
Required timing for reporting per the above guideline:
x
Deaths (except death due to progressive disease) must be reported via email within 24 
hours.  A complete report must be submitted within one business day. 
x
Other serious adverse events including deaths due to progressive disease must be reported 
within one business day
Events will be submitted to the Center for Cancer Research (CCR) at: CCRsafety@mail.nih.gov
and to the CCR PI and study coordinator.

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8.3.1
Action Plan for Positive Results on Cell Product Safety Testing
8.3.1.1
In the unlikely event that a positive sterility test or mycoplasma test result is obtained 
after distribution of the cell product, or after administration of the product to the patient, 
the following steps will be initiated IMMEDIATELY:
a.
CPS, DTM personnel will notify the principal investigator, Dr. Nirali Shah, MD at 
(240) 760-6199 or by pager 301-496-1211.  As soon as the identification and 
sensitivity report from positive sterility is available, a copy of the final report will be 
sent to the PI.  The DTM QS officer and CPS Supervisor will determine the need for
quality improvement based on the nature and extent of the incident.
b. If CPS is unable to reach Dr. Shah within 15 minutes, contact the page operator (301-
496-1211) to page the Pediatric Oncology Branch Fellow on-call.  NOTE: Dr. Shah 
or the POB Fellow on-call will contact the attending physician for the POB Service, 
who will determine the extent of the work-up of a positive culture in consultation 
with staff from the Microbiology Service.
c.
Dr. Shah /attending physician will discuss the positive results with the patient/parents, 
and specify the clinical therapy, antibiotic regimen and/or monitoring plan in 
consultation with staff from the Microbiology Service.
d. A contaminated sample of a product that has been administered to a patient will be 
handled in the same fashion as a Grade 4 toxicity (CTC version 4.0) and the Sponsor 
will be responsible for notifying the NCI IRB and the FDA.  A full written report 
with description of events, laboratory findings, clinical evaluation and treatments 
would be submitted to the NCI IRB within 7 working days.  The Sponsor will be 
responsible for filing this report with the FDA within 15 calendar days. 
8.4
DATA SAFETY MONITORING PLAN 
8.4.1
Principal Investigator/Research Team 
The Principal Investigator will review all adverse events. The Principal Investigator will meet 
with the research team at frequent and regularly scheduled intervals, to determine treatment 
modifications and treatment based toxicities, initiating enrollment to the next lower weight based 
group, to monitor trends in adverse events, and determine if trends are noted.
Adverse events will be tracked by the PI. If trends are noted and/or risks warrant it, accrual will 
be interrupted and/or the protocol and/or consent document will be amended accordingly. If 
trends in toxicities are noted or stopping rules are met, the PI will temporarily suspend 
enrollment while reviewing the episodes with the NCI IRB and FDA to determine if any changes 
to the protocol or the informed consent are warranted.  At the completion of each cohort, a 
toxicity summary with both acute and delayed toxicities will be formally reported to the FDA 
prior to opening the subsequent cohort. In addition, if a toxicity occurs after D 28 in any patient, 
that meets DLT criteria, is serious or is unexpected, it will be reported to the FDA immediately, 
and when applicable, discussions with the FDA and IRB regarding dose adjustments will occur.
The principal investigator will personally conduct or supervise the investigation and provide 
appropriate delegation of responsibilities to other members of the research staff. 
8.4.2
Sponsor Monitoring Plan

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As a sponsor for clinical trials, FDA regulations require the CCR to maintain a monitoring 
program. The CCR’s program allows for confirmation of: study data, specifically data that could 
affect the interpretation of primary study endpoints; adherence to the protocol, regulations, and 
SOPs; and human subjects’ protection. This is done through independent verification of study 
data with source documentation focusing on:
x
Informed consent process
x
Eligibility confirmation
x
Drug administration and accountability
x
Adverse events monitoring
x
Response assessment.
The monitoring program also extends to multi-site research when the CCR is the coordinating 
center.
This trial will be monitored by personnel employed by a CCR contractor. Monitors are qualified 
by training and experience to monitor the progress of clinical trials. Personnel monitoring this 
study will not be affiliated in any way with the trial conduct. 
8.4.3
Safety Monitoring Committee (SMC)
This protocol will require oversight from the Safety Monitoring Committee (SMC).  Initial 
review will occur as soon as possible after the annual NCI-IRB continuing review date.   
Subsequently, each protocol will be reviewed as close to annually as the quarterly meeting 
schedule permits or more frequently as may be required by the SMC.   For initial and subsequent 
reviews, protocols will not be reviewed if there is no accrual within the review period. Written 
outcome letters will be generated in response to the monitoring activities and submitted to the 
Principal investigator and Clinical Director or Deputy Clinical Director, CCR, NCI.
9
PHARMACEUTICAL INFORMATION
9.1
FLUDARABINE (FLUDARA)
Availability: Fludarabine monophosphate is commercially available and will be supplied by the 
Clinical Center Pharmacy. FLUDARA IV, is supplied as a white, lyophilized powder. Each vial 
contains 50 mg of fludarabine phosphate, 50 mg of mannitol, and sodium hydroxide to adjust 
pH. Fludara is stored at room temperature.
Storage and Stability: Reconstituted FLUDARA IV is chemically and physically stable for 24 
hours at room temperature, or for 48 hours if refrigerated. Because reconstituted FLUDARA IV 
contains no antimicrobial preservative, care must be taken to assure the sterility of the prepared 
solution; for this reason, reconstituted FLUDARA IV should be used or discarded within 8 
hours. 
Preparation: FLUDARA IV should be prepared for parenteral use by aseptically adding Sterile 
Water for Injection, USP. When reconstituted with 2 ml of Sterile Water for Injection, each ml of 
the resulting solution will contain 25 mg of Fludarabine Phosphate, 25 mg of mannitol, and 
sodium hydroxide to adjust the pH to 7-8.5. The product may be further diluted for intravenous 
administration in 50 ml of 5% Dextrose for Injection USP, or in 0.9% Sodium Chloride, USP.

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Administration: Parenteral drug products should be inspected visually for particulate matter and 
discoloration prior to administration. Fludarabine will be mixed in 50 ml of 0.9% NaCl, and 
infused i.v. over 30 minutes.
Toxicity: Fludarabine toxicities include myelosuppression (dose limiting toxicity), fever, nausea, 
vomiting, stomatitis, diarrhea, gastrointestinal bleeding, anorexia, edema, skin rashes, myalgias, 
headache, agitation, hearing loss, transient episodes of somnolence and fatigue, autoimmune 
hemolytic anemia, autoimmune thrombocytopenia, paresthesias, peripheral neuropathy, renal, 
and pulmonary toxicity (interstitial pneumonitis). Severe fatal CNS toxicity presenting with loss 
of vision and progressive deterioration of mental status has occurred after very high doses 
(approximately 4 times higher than the standard doses employed in this protocol). Severe 
neurologic toxicity has only rarely been demonstrated at the 25-30 mg/m2/day dosage of 
fludarabine. Rarely describe complications include transfusion-associated graft-versus-host 
disease, thrombotic thrombocytopenic purpura, and liver failure. Tumor lysis syndrome 
following fludarabine administration has been observed, especially in patients with advanced 
bulky disease. Opportunistic infections (protozoan, viral, fungal, and bacterial) have been 
observed post-fludarabine, especially in heavily pre-treated individuals, and in individuals 
receiving fludarabine combined with other agents. 
9.2
MESNA (SODIUM 2-MERCAPTOETHANESULFONATE, MESNEX)
Availability: Commercially available and will supplied by the Clinical Center Pharmacy.
Stability: Mesna may be diluted in D5W, 0.9%NaCl, or D5/0.45NaCl to concentrations between 
0.6 mg/ml and 20 mg/ml for intravenous infusions.  These concentrations are stable at room 
temperature for 24 hours.
Administration: Will be administered by continuous i.v. infusion on days –6, -5, -4, and –3 of the 
transplant during high dose cyclophosphamide administration. Dose of mesna will be 1200 
mg/m2 per day by continuous i.v. infusion for four consecutive days (days –6, -5, -4, and –3). 
Toxicity May be associated with nausea, vomiting, diarrhea, or headaches.
9.3
CYCLOPHOSPHAMIDE (CYTOXAN)
Availability: Commercially available as a lyophilized powder in 2 gram vials and will supplied 
by the Clinical Center Pharmacy.
Storage and Stability: Following reconstitution as directed, cyclophosphamide is stable for 24 
hours at room temperature or 6 days when kept at 2-8C. 
Preparation: Reconstitution with 100 ml of sterile water for injection to a final concentration of 
20 mg/ml.
Administration: Cyclophosphamide will be diluted in 0.9% NaCl and infused over 30 minutes 
during induction and 2-hours during pre-HSCT preparative therapy. 
Toxicity: Hematologic toxicity occurring with cyclophosphamide usually includes leukopenia 
and thrombocytopenia. Anorexia, nausea, and vomiting occur, especially after high-dose 
cyclophosphamide; diarrhea, hemorrhagic colitis, and mucosal and oral ulceration have been 
reported. Sterile hemorrhagic cystitis occurs in about 20% of patients; severity can range from
microscopic hematuria to extensive cystitis with bladder fibrosis. Mesna has been used 
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mesna is not effective in preventing hemorrhagic cystitis in all patients. Patients who receive 
high dose cyclophosphamide may develop interstitial pulmonary fibrosis, which can be fatal. 
Hyperuricemia due to rapid cellular destruction may occur, particularly in patients with 
hematologic malignancy. Hyperuricemia may be minimized by adequate hydration, 
alkalinization of the urine, and/or administration of allopurinol. If allopurinol is administered, 
patients should be watched closely for cyclophosphamide toxicity (due to allopurinol induction 
of hepatic microsomal enzymes). At high doses, cyclophosphamide can result in a syndrome of 
inappropriate antidiuretic hormone secretion; hyponatremia with progressive weight gain without 
edema occurs. At high doses, cyclophosphamide can result in cardiotoxicity. Deaths have 
occurred from diffuse hemorrhagic myocardial necrosis and from a syndrome of acute 
myopericarditis; in such cases, congestive heart failure may occur within a few days of the first 
dose. Other consequences of cyclophosphamide cardiotoxicity include arrhythmias, potentially 
irreversible cardiomyopathy, and pericarditis. Other reported adverse effects of 
cyclophosphamide include headache, dizziness, and myxedema; faintness, facial flushing, and 
diaphoresis have occurred following IV administration. 
9.4
ETOPOSIDE (VEPESID, VP-16)
Availability: Commercially available and will be supplied by the Clinical Center Pharmacy.
Etoposide Toxicity: Includes nausea, vomiting, stomatitis, diarrhea, neutropenia, 
thrombocytopenia, and alopecia. Secondary AML has been associated with etoposide. 
Bradycardia and hypotension are sometimes observed with etoposide administration.
9.5
DOXORUBICIN (ADRIAMYCIN)
Availability: Commercially available and will be supplied by the Clinical Center Pharmacy.
Doxorubicin Toxicity: Includes nausea, vomiting, stomatitis, myelosuppression, and 
cardiotoxicity. Cardiotoxicity is particularly noted after cumulative doses of greater than 550 
mg/m2. Other toxicities include nausea, vomiting, stomatitis, diarrhea, and alopecia. Skin 
infiltration of doxorubicin causes tissue necrosis.
9.6
VINCRISTINE 
Availability: Commercially available and will be supplied by the Clinical Center Pharmacy.
Vincristine Toxicity Vincristine causes neurological toxicities such as paresthesias, jaw pain, 
ataxia, foot-drop, cranial nerve palsies, paralytic ileus, constipation, abdominal pain, and loss of 
deep tendon reflexes. It is also a vesicant, and occasionally causes alopecia and 
myelosuppression. Because fluconazole can delay the clearance of vincristine, fluconazole will 
be discontinued during days 1, 2, 3, and 4 of induction chemotherapy.
Storage and Stability: Stability studies conducted by the Pharmaceutical Development Service, 
Pharmacy Department, NIH Clinical Center, have demonstrated that admixtures of vincristine, 
doxorubicin, and etoposide in 0.9% Sodium Chloride for Injection (at concentrations, 
respectively, of 1, 25, and 125 mcg/ml; 2, 50, and 250 mcg/ml; and 2.8, 70, and 350 mcg/ml) are 
stable for at least 36 hours at room temperature when protected from light. 
Administration of these Agents as Continuous Infusion: In this study, the daily dose of 
vincristine, doxorubicin, and etoposide (i.e. the 24 hour supply) will be admixed together in 500 
ml of 0,9% Sodium Chloride Injection and delivered with a suitable infusion pump through a 

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central venous access device. All 3-in-1 admixtures dispensed from the Pharmacy will contain a 
24-hour supply of etoposide, doxorubicin, and vincristine PLUS an overfill (excess) fluid and a 
proportional amount of drug to compensate for volume lost in administration set tubing to ensure 
delivery of the entire 24 hour dose.  Before dispensing 3-in-1 admixtures, Pharmacy staff will 1) 
purge all air from the drug product container, 2) attach an administration set appropriate for use 
with a portable pump and the set will be 3) primed close to its distal tip and 4) capped with a 
Luer-locking cap. Bags will be exchanged daily for 4 consecutive days to complete a 96-hour 
drug infusion. Portable pumps used to administer etoposide + doxorubicin + vincristine 
admixtures will be programmed to deliver 528 ml per day at 22 ml/hr. At the end of an infusion, 
some residual fluid is expected because overfill fluid and drug were added; however, nurses are 
asked to return to the Pharmacy for measurement any drug containers that appear to contain a 
greater amount of residual drug than expected.
9.7
PREDNISONE
Availability: Commercially available and will be supplied by the Clinical Center Pharmacy; 
available in solid or liquid dosage forms. Tablet strengths include 1, 2.5, 5, 20, and 50 mg forms, 
and the oral solution is at 1 mg/ml. 
Administration: Prednisone will be administered at a dose of 60 mg/m2 orally in 2-4 divided 
doses on days 1, 2, 3, 4, and 5 of fludarabine-EPOCH induction chemotherapy regimen. In 
patients unable to tolerate oral medication, methylprednisolone can be substituted at the same 
dosage, diluted in 25 ml of D5W, and infused over 15 minutes. To reduce gastrointestinal side 
effects, prednisone should be taken with food. 
Toxicity: Prednisone frequently causes gastritis or ulcers, hypertension, immunosuppression, 
muscle wasting, fluid retention, increased appetite, psychiatric disturbances, hyperglycemia, and 
skin changes.  In addition, steroids can also cause pancreatitis, impaired growth, menstrual 
abnormalities, osteoporosis, osteonecrosis, cataracts, pseudotumor cerebri, glaucoma, and 
seizures. 
9.8
FILGRASTIM (G-CSF, NEUPOGEN)
Availability: Filgrastim is a commercially available and will be supplied by the Clinical Center 
Pharmacy.  This product differs from the natural form due to its absence of N-terminal o-
glycosylation. Filgrastim has undergone tests for USP pyrogen, limulus amebocyte assay, 
sterility and the general safety test. The nucleic acid content can be no greater than 10 pg/unit 
dose. The final product is clear, colorless sterile solution that is free of particulates; the product is 
not less than 95% pure. It is provided in 1 ml vials of 300 mcg or 1.6 ml vials of 480 mcg. 
Storage and Stability: Intact vials should be stored in the refrigerator; do not freeze. Product is 
stable for at least 1 year when refrigerated.
Preparation: Do not dilute with sodium chloride solutions.
Administration: Inject subcutaneously at 5-10 mcg/kg/day as per treatment plan.
Toxicity: Filgrastim may cause bone pain, constitutional symptoms such as fever, fatigue and 
myalgias, splenomegaly, thinning hair, pain at the injection site, bone pain, worsening of pre-
existing inflammatory conditions, and elevations in uric acid, LDH, and alkaline phosphatase.

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9.9
PEGFILGRASTIM (RECOMBINANT METHIONYL HUMAN G-CSF, NEULASTA™)
Availability: Pegfilgrastim is a commercially available and will be supplied by the Clinical 
Center Pharmacy.  Pegfilgrastim is a covalent conjugate of recombinant methionyl human G-
CSF (filgrastim) and monomethoxypolyethylene glycol. To produce pegfilgrastim, a 20 kD 
monomethoxypolyethylene glycol molecule is covalently bound to the N-terminal methionyl 
residue of filgrastim. The average molecular weight of pegfilgrastim is approximately 39 kD.  
Pegfilgrastim is supplied in 0.6 mL prefilled syringes for subcutaneous injection, containing 6 
mg pegfilgrastim (based on protein weight), in a sterile, clear, colorless, preservative-free 
solution (pH 4.0) containing acetate (0.35 mg), sorbitol (30.0 mg), polysorbate 20 (0.02 mg), and 
sodium (0.02 mg) in water for injection, USP.
Storage and Stability: Pegfilgrastim should be stored refrigerated and protected from light until 
time of use. Shaking or freezing should be avoided. Before injection, Pegfilgrastim may be 
allowed to reach room temperature for a maximum of 48 hours but should be protected from 
light.
Administration:  Inject subcutaneously as per treatment plan.
Toxicity:  See filgrastim above.
9.10 CYTARABINE (CYTOSINE ARABINOSIDE, CYTOSAR, ARA-C)
Availability: Commercially available.
Storage and Stability: The reconstituted drug is stable for 48 hours at room temperature. The 
drug will be further diluted in an age appropriate amount of 5% Dextrose Injection or 0.9% 
Sodium Chloride Injection prior to administration. 
Administration: Cytarabine is available for intravenous and intrathecal administration.  The 
manufacturer supplies the drug as unreconstituted lyophilized powder in vials. With each vial, an 
ampule of bacteriostatic water for injection containing 0.945% benzyl alcohol is provided.  To 
prepare high dose (2 gm/m2) cytarabine intravenous infusions, each vial of drug should be 
reconstituted with 2 cc (100 mg) or 10 cc (500 mg) of sterile water for injection USP (i.e. not the 
manufacturer-provided diluent) which contains no preservative. Begin each cytarabine dose 3.5 
hours after completion of the preceding fludarabine dose.
Toxicity: IV Administration: Acute DLT consists of severe leukopenia and thrombocytopenia.  
Nausea and vomiting may be dose limiting at higher doses.  Other adverse reactions include 
diarrhea, immunosuppression, anorexia, stomatitis, oral ulceration, flu-like syndrome, fever, 
hepatic dysfunction, and alopecia. At high doses, as in this protocol, keratoconjunctivitis, 
dermatitis, and central nervous system toxicity (e.g., ataxia, somnolence, coma, dysarthria) may 
occur. Occasionally, the CNS impairment is not fully reversible. Renal impairment will enhance 
toxicity.  In the event signs of CNS toxicity, the cytarabine will be interrupted and the M.D. 
notified. In the event of signs of CNS toxicity, the cytarabine infusion will be interrupted and the 
M.D. notified. No further cytarabine will be administered if there is CNS toxicity (any grade) 
deemed related to cytarabine.
Conjunctivitis Prophylaxis: Corticosteroid ophthalmic drops will be administered 2 drops to each 
eye every 6 hours starting prior to first dose and continuing until 24 hours after the last dose of 
cytarabine has been completed.

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9.11 INTRATHECAL CHEMOTHERAPY
9.11.1 Methotrexate (MTX, amethopterin) NSC #000740
Route: Intrathecal therapy (methotrexate single agent)
Source and Pharmacology: A folate analogue that inhibits the enzyme dihydrofolate reductase, 
halting DNA, RNA, and protein synthesis. Initial IV half-life is about 1.2 hours, with a second 
phase of 10.4 hours. About 50% is bound to protein. Transport into the cell is carrier-mediated. 
Once in the cell, MTX (Glu)n are formed, the number of which are related to the cytocidal effect. 
Once MTX (Glu)n are formed, they do not pass back out of the cell unless converted back to 
MTX. After oral administration, about 60% of a 30 mg/m2 dose is rapidly absorbed from the GI 
tract, with peak blood levels at 1 hour. Above this dose, absorption decreases significantly. 
Absorption can be very erratic, varying between 23% to 95%. A 20-fold difference between peak 
levels of drug has been reported (0.1 to 2mM). There is significant enterohepatic circulation of 
MTX: 9% of MTX is excreted in feces. MTX is excreted unchanged in the urine, except at high 
doses when it is partially metabolized to hydroxy-MTX and excreted.
Formulation and Stability: Intact vials may be stored at room temperature (22°-25°C) and are 
stable for at least 2 years or until date of expiration. INTRATHECAL MTX: Available in various 
dosages in preservative-free liquid, 25mg/ml in a 2ml vial, or as a lyophilized powder. 
Reconstitute the powder with buffered saline solution. The Methotrexate solutions may be 
further diluted with buffered saline or the patient's own CSF. After mixing it should be used 
within 24 hours, since MTX contains no antibacterial preservative. 
Guidelines for Administration: The drug will be filtered and mixed in sterile preservative free 
saline less than 4 hours prior to administration. Intrathecal delivery should be approximately 
isovolumetric (ml CSF out = ml drug in) via lumbar puncture (LP) in the lateral decubitus 
position. The patient should remain in prone or Trendelburg position for approximately 30 
minutes post LP to facilitate drug circulation throughout the CNS. Leucovorin rescue should be 
administered 5 mg PO/IV q 6 hours x 4 doses, starting 24 hours after methotrexate.
Supplier: Commercially available. See package insert for further information.
Toxicities: The following toxicities may occur when methotrexate is given alone into the spinal 
fluid:
Common
Happens to 21-100 
children out of every 100
Occasional
Happens to 5-20 children 
out of every 100
Rare
Happens to < 5 children 
out of every 100
Immediate:
Within 1-2 days of
receiving drug
Headache, abnormally high 
number of cells in the spinal 
fluid
Vomiting, fever, rash, 
drowsiness, stiff neck, 
irritation of tissues in the 
brain/spinal cord, 
seizures (L), partial 
paralysis
Prompt:
Decrease in the number 
of red and white blood 
cells and platelets made 
in the bone marrow, 

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Within 2-3 weeks, 
prior to the next 
course
drowsiness, unsteady 
walk
Delayed:
Any time later 
during therapy
Learning disability
Thinning of nerve fiber 
insulation resulting in 
difficulty with loss of 
memory, concentration, 
balance and walking, 
which may get worse 
with time1 (L) 
Late:
Any time after the 
completion of 
treatment
Increasingly  poor  
nervous system function1
1 May be enhanced by HDMTX and/or cranial irradiation.
(L) Toxicity may also occur later.
Methotrexate crosses the placenta to the fetus. Fetal toxicities and teratogenic effects of 
methotrexate (either alone or in combination with other antineoplastic agents) have been noted in 
humans. The toxicities include: congenital defects, chromosome abnormalities, malformation, 
severe newborn myelosuppression, pancytopenia, and low birth weight.
Dose Modification: Hold for any CNS toxicity.
9.11.2 Hydrocortisone (Cortef, Solu-cortef)
Route: Intrathecal therapy (as clinically indicated)
Source and Pharmacology: Commercially available. See package insert for further information.
Synthetic steroid akin to natural adrenal hormone, cortisol.  It binds with steroid receptors on 
nuclear membrane, impairs cellular mitosis and inhibits protein synthesis.  It is phase specific, 
killing cells primarily during S phase.  It has a catabolic effect on proteins and alters the kinetics 
of peripheral blood leukocytes.  It is excreted in the urine and catabolized in the liver.
Toxicity: The following toxicities may occur when hydrocortisone is given intrathecally: nausea 
and vomiting, headache, pleocytosis, fever, somnolence, meningismus, learning disability, 
leukoencephalopathy.
Formulation and Stability: Available as 100 mg, 250 mg, 500 mg, and 1000 mg vials for 
aqueous injection.  In powder form, the drug is stable for 2 years at room temperature.  After 
reconstitution, it is stored at room temperature, and should be discarded after 3 days.
Intrathecal Administration: IT hydrocortisone should be reconstituted with physiologic buffered 
diluents (lactated Ringer’s, 0.9% sodium chloride, Elliott’s B solution).  Do not use 
Bacteriostatic Water to reconstitute for IT use; use only preservative free solutions.
Guidelines for Administration:  Hydrocortisone will be given by IT administration in an age-
specified dose and will be mixed with cytarabine.
9.11.3 Cytarabine (cytosine arabinoside, AraC, Cytosar)

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Route: Intrathecal therapy (as clinically indicated)
Source and Pharmacology: Commercially available. See package insert for further information.  
Deoxycytidine analogue which is metabolized to ARA-CTP, a substance which inhibits DNA 
polymerase.  It is S phase specific, and thus affects DNA synthesis. 
Toxicity: The following toxicities may occur when cytarabine is given intrathecally: Nausea, 
vomiting, headache, pleocytosis, arachnoiditis, rash, fever, somnolence, meningismus, 
convulsions, paresis, myelosuppression, ataxia, learning disability. CNS impairment may not be 
fully reversible.  No further cytarabine will be administered if there is CNS toxicity (any grade) 
deemed related to cytarabine.
Formulation and Stability: A freeze-dried powder available in 100 mg, 500 mg, 1 G and 2 G 
vials.  The unreconstituted form of the drug is stable at room termperature for at least 2 years. 
Intrathecal Administration: IT cytarabine should be reconstituted with physiologic buffered 
diluents (lactated Ringer’s, 0.9% sodium chloride, Elliott’s B solution).  Do not use 
Bacteriostatic Water to reconstitute for IT use; use only preservative free solutions.
Guidelines for Administration:  Cytarabine will be given by IT administration in an age-specified 
dose and will be mixed with hydrocortisone.
9.12 RANITIDINE (ZANTAC):
Will be given as a pre-medication.  It will be supplied by the Clinical Center Pharmacy.  The 
most common side effects are fatigue, dizziness, headache, and diarrhea. See package insert for 
additional information.
9.13 ACETAMINOPHEN (TYLENOL):
Will be given as a pre-medication.  It will be supplied by the Clinical Center Pharmacy.  The 
most common side effect seen with high doses or chronic usage is hepatotoxicity. Rarely, 
sensitivity reactions can occur. See package insert for additional information.
9.14 DIPHENHYDRAMINE (BENADRYL):
Will be given as a pre-medication IV over 10-15 minutes.  It will be supplied by the Clinical 
Center Pharmacy.  The most common side effects are sleepiness, dizziness, restlessness, and 
irritability. See package insert for additional information.
9.15 FLUCONAZOLE (DIFLUCAN):
Will be given as prophylaxis against susceptible yeast and fungi.  It will be supplied by the 
Clinical Center Pharmacy.  Fluconazole increases the AUC and serum concentration of 
tacrolimus and sirolimus.  Thus, careful monitoring of levels is important when these 
medications are administered concurrently.  Significant interactions with oral hypoglycemics, 
coumarin-type anticoagulants, phenytoin, rifampin, theophylline, cisapride, astemizole, rifabutin 
and tacrolimus have been observed. As described in section 3.3.2.7, interactions with vincristine 
also occur. Rare mild side effects including headache, nausea and abdominal pain have been 
reported.  Elevations in transaminases can occur. Hepatotoxicity is usually, but not always, 
reversible. Fluconazole has been associated with rare cases of severe hepatic toxicity, including 
fatalities.  Other adverse effects include anaphylaxis, and rash (occasionally severe). See package 
insert for additional information.

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9.16 PCP PROPHYLAXIS
9.16.1 Trimethoprim/Sulfamethoxazole (TMP/SMX) (Bactrim, Septra):  
Will be given as prophylaxis against pneumocystis.  It will be provided by the Clinical Center 
Pharmacy.  Side effects include a reduction in blood counts, gastrointestinal disturbances 
(nausea, vomiting and anorexia) and allergic reactions. Interactions with thiazide diuretics, 
phenytoin, and methotrexate occur and require caution when used concurrently.  Due to 
photosensitivity, patients should avoid prolonged exposure to the sun. Fatalities associated with 
the administration of sulfonamides, although rare, have occurred due to Stevens-Johnson 
syndrome, toxic epidermal necrolysis, fulminant hepatic necrosis and blood dyscrasias.  See 
package insert for additional information.
9.16.2 Pentamadine:
Will be given as prophylaxis against pneumocystis, as a substitute for TMP/SMX.  It will be 
provided by the Clinical Center Pharmacy.  It is supplied in 300 mg per nebulizer.  Dosing will 
be as per the CC BMT Guidelines (http://intranet.cc.nih.gov/bmt/clinicalcare/pdf/Table_VII.pdf).  
Side effects reported with the use of Pentamidine include metallic taste, coughing, bronchospasm 
in heavy smokers and asthmatics; increased incidence of spontaneous pneumothorax in patients 
with previous PCP infection or pneumatoceles, or hypoglycemia. IV formulation may be used if 
there is a contraindication to inhaled delivery. 
9.17 INTRAVENOUS IMMUNOGLOBULIN (IVIG, GAMIMMUNE, GAMMAGARD,
SANDOGLOBULIN):
Will be used for infection prophylaxis in the post-transplant period.  It will be provided by the 
Clinical Center Pharmacy and will be administered by standard guidelines.  Side effects vary 
depending on the preparation but can included anaphylactic reactions, hypotension, fever, chills, 
headache, urticaria, and aseptic meningitis.  In general, these effects are infusion-related and can 
be treated by decreasing the rate of infusion. Rarely, acute renal failure has been reported, 
predominantly in individuals with pre-existing renal dysfunction. Although donor screening and 
inactivation procedures substantially reduce the risk of viral transmission, immune globulin is 
made from human plasma and, therefore, carries a risk of transmission of infectious agents. See 
package insert for the individual preparations for additional information.
9.18 PALIFERMIN, KEPIVANCE™(KGF)
Kepivance™ (palifermin) is a human keratinocyte growth factor (KGF) produced by 
recombinant DNA technology in Escherichia coli (E coli). It will be supplied by the Clinical 
Center Pharmacy. See the package insert for complete details.
Palifermin is supplied as a sterile, white, preservative-free, lyophilized power for IV injection 
after reconstitution with 1.2 mL of sterile water for injection, USP.  Palifermin will be 
administered to decrease the incidence and duration of oral mucositis.  It may be administered at 
60 mcg/kg/day IV bolus injection for 3 days, twice:
1. The first 3 daily doses should be administered prior to the preparative chemotherapy,
with the 3rd dose at least 24 hours prior to starting the preparative regimen, 
2. The last 3 daily doses should be administered starting on day -1.

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Toxicities associated with use of palifermin include: rash, erythema, edema, pruritus, 
dysesthesia, tongue discoloration, tongue thickening, alteration of taste, pain arthralgias and 
dysesthesia.  
9.19 RECOMBINANT HUMAN INTERLEUKIN 15 (IL-15)
Source and Pharmacology:
rhIL-15 will be purchased by the POB Branch from CellGenix™ for use in cell preparation.  
rhIL-15 is produced under GMP conditions for investigational use; the source is a DNA 
sequence encoding the mature IL-15 protein expressed in E. coli.  For clinical ex vivo cell 
culture use only.
Formulation and Stability:
Lyophilized from a 250 mcg/mL, 0.2 mcm-sterile filtered solution in PBS (300 mM NaCl) + 
0.1% Tween 20.  Each vial contains 50 mcg (+/- 10%).  Product is stable for 2 years from date of 
production when stored as directed.  This study will require approximately 2 vials per patient for 
cell development.
Storage: Store vials at -20°C to -80°C; avoid repeated freeze-thaw cycles.
9.20 CYCLOSPORINE A
Availability: Cyclosporine A is commercially available as either an injectable concentrate or as a 
microemulsion in capsules or liquid. Capsules are available in 25 and 100 mg strengths. Liquid 
solution is available as 100 mg/ml.
Storage and Stability: Stable at room temperature.
Administration: Cyclosporine will be started by IV infusion at day –1 before the transplant at a 
dose of 2 mg/kg/dose (over a two hour period) for two doses per day. Once the patient is able to 
tolerate oral feedings, CSA will be converted to an equivalent oral dose. The starting oral dose 
should be approximately 2-times the IV dose given in a q 12 hour schedule. The dose should be 
titrated to a trough level of 150-300 ng/ml (monoclonal RIA methodology). 
Drug Interactions Cyclosporine has important interactions with multiple medications. Most 
commonly, CSA levels are altered due to induction or inhibition of hepatic metabolism. Azole 
antifungal agents (e.g., fluconazole), macrolides (e.g., erthyromycin), ciprofloxacin, 
metronidazole, grapefruit juice, cimetidine, metoclopramide, oral contraceptive pills, and 
corticosteroids may result in elevated cyclosporin A levels. Agents that decrease CSA levels 
include nafcillin, anticonvulsants (e.g., phenytoins, barbituates), and octreotide. CSA levels 
should be monitored more closely when any such agents are co-administered, in the setting of 
impaired renal or hepatic function, and when other nephrotoxic or hepatoxic drugs are used. 
Cisapride is contraindicated due to risk of QT prolongation and arrhythmias. 
Toxicity: Includes nephrotoxic effects, which are manifested as increases in BUN and creatinine, 
hypertension, hyperkalemia, and hyperuricemia; nephrotoxicity is increased by co-administration 
of acyclovir, aminoglycoside antibiotics, and amphotericin B. Nervous system effects include a 
fine hand tremor, seizures, headache, paresthesis, flushing, and confusion. Cyclosporine-induced 
seizures may be associated with concurrent high-dose corticosteroid therapy, hypertension, and 
hypomagnesemia. Rarely, cyclosporine can induce a syndrome of severe neurotoxicity 
characterized by cortical blindness, quadriplegia, seizures, and coma. Adverse dermatologic 

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effects include hirsutism and gingival hyperplasia. Elevated liver transaminases may occur. 
Adverse GI effects include diarrhea, nausea and vomiting, anorexia and abdominal discomfort. 
9.21 TACROLIMUS (FK506, PROGRAF)
Supply: Tacrolimus will be obtained by the NIH Clinical Center Pharmacy Department from 
commercial sources and is available in capsules (0.5 mg, 1mg, and 5mg), and as a parenteral 
concentrate for injection (5 mg/ml, 1 ml ampules).
Preparation: For parenteral doses, tacrolimus injection concentrate (5 mg/ml) should be diluted 
to a final concentration of 0.004 to 0.02 mg/ml in dextrose 5% in water or sodium chloride 0.9%. 
Parenteral doses of tacrolimus will be prepared in non-PVC containers and infused with non-
PVC administration sets/tubing.
Storage and Stability: Capsules and ampules of parenteral concentrate bear expiration dates and 
are stored at room temperature. Tacrolimus concentrate for injection that has been diluted to a 
final concentration of 0.004 to 0.02 mg/ml is stable for 24 hours in 5% dextrose or 0.9% sodium 
chloride injection in glass, PVC or non-PVC plastic containers. To minimize the potential for 
sorption to PVC plastic bags and tubing as well the leaching of phthalate plasticizer (DEHP) into 
the solution, only non-PVC plastic bags and intravenous administration sets should be utilized.
Administration: Tacrolimus may be given intravenously over 24 hours or orally.
Toxicities: Acute tacrolimus nephrotoxicity is usually manifested by a moderate decline in renal 
excretory function, which is readily reversible by a decrease in drug dosage. Although some 
degree of transient renal dysfunction may occur in patients with therapeutic levels of tacrolimus, 
significant renal toxicity is associated with elevated trough or steady state levels. In addition to 
an increase in BUN and creatinine, hyperkalemic hyperchloremic acidosis, low fractional 
excretion of sodium and the onset of hypertension with hypomagnesemia are seen with 
tacrolimus nephrotoxicity. Insulin dependent post transplant diabetes mellitus was reported in 
20% kidney transplant patients, which was reversible in 50% of the patients at 2 years post 
transplant. Hypertension occurs in up to 60% of patients. Hypomagnesaemia can be associated 
with neurological symptoms, including seizures, cerebellar ataxia and depression. Dose-related 
hepatotoxicity, manifested by elevation of serum transaminases and bilirubin, has been reported. 
The drug carries a warning for increased susceptibility to infection and the possible development 
of lymphoma may result from resulting immunosuppression.
9.22 BUSULFAN (BUSULFEX)
General: Busulfex is a bifunctional alkylating agent approved for use as a conditioning agent 
prior to allogeneic hematopoieitc stem cell transplantation. The IV formulation is administered 
as Busulfex (Busulfex, Otsuka America Phamraceutical, Inc.)
Supply: For patient administration, IV Busulfex is purchased by the NIH Clinical Center 
Pharmacy Department from commercial sources. The drug is supplied as a clear, colorless sterile 
solution in 10ml single use vials. Each vial of BUSULFEX contains 60 mg (6mg/ml) of 
busulfan.
Storage and Stability: Unopened vials of BUSUILFEX are stable until the date indicated on the 
package when stored under refrigeration at 2-6 degrees C (36-46 degrees F).

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Administration: BUSULFEX must be diluted prior to use with either 0.9% Sodium chloride or 
5% Dextrose Injection. The diluent quantity should be 10 times the volume of BUSULFEX so 
that the final concentration of busulfan is approximately 0.5 mg/ml. BUSULFEX should be 
administered intravenously via a central venous catheter as a two hour infusion every 24 hours 
for a total of 4 doses over four days.
Toxicities: 1) At the indicated dose and schedule BUSULFEX induces profound 
myelosuppression; 2) All patients should be pre-medicated with phenytoin or Clonazepam plus 
Levetiracetam (Keppra) since busulfan is known to cross the blood brain barrier and induce 
seizures; 3) Nausea, vomiting, and stomatitis are common side-effects of busulfan.
Drug Interactions: Itraconazole increases the clearance of busulfan by up to 25%. Phenytoin 
increases the clearance of busulfan by 15%.
9.23 LEVETIRACETAM (KEPPRA)
Supply: 500 mg tablets
Preparation: None
Storage and Stability: Tablets are stored at room temperature.
Route of Administration: Orally. Children: the dose is 10 mg/kg/dose PO BID (maximum dose: 
1000 mg). Adults: drug will be given as 500mg po every 12 hours. Dosing starts the night before 
BUSULFEX and continues until 24 hours after last Busulfan dose.
Toxicities: Leukopenia, hepatic abnormalities.
9.24 LOREZAPAM (ATIVAN)
General: benzodiazepine
Administration: Oral or IV, 0.02-0.05 mg/kg/dose, maximum dose: 2 mg) given 30 minutes prior 
to each busulfan dose and then continuing for at least 24 hours after last busulfan dose.
Toxicity: CNS depression, confusion, paroxysimal agitation.
9.25 RADIATION THERAPY
9.25.1 TOTAL BODY IRRADIATION
Chemical name: Not applicable
Known toxicities: The side effects of radiation have been well described. The most common 
include nausea and mucositis. There also exists a risk of hypothyroidism, cataracts, interstitial 
pneumonitis, nephropathy, and an unspecified long-term risk of developing secondary 
malignancies. Importantly, the majority of the non-neoplastic effects were subclinical and/or 
reversible. Studies attempting to evaluate the risk attributed to radiation within a preparative 
regimen have implicated secondary malignancies, however this is difficult to ascribe since 
secondary malignancies are also increased with cGVHD and other post-transplant complications. 
Pediatric patients who require sedation in order to undergo TBI will be asked to sign a separate 
consent outlining the risk of the selected anesthesia.
9.25.2 CNS Boost

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Chemical name: Not applicable
Known toxicities: For the CNS boost, the common short-term side effects include skin redness 
and irritation in the area treated, hair loss in the area treated, fatigue, and uncommonly 
headaches, weakness, numbness, or other neurologic deficit which may require steroid treatment, 
nausea, vomiting, weight loss, and rarely decrease blood cell count and excessive need of sleep. 
Long-term side effects of whole-brain radiation therapy, which may occur months to years after, 
are mild decrease in memory or ability to think (which may be exacerbated in those who have 
received prior radiation therapy to the brain), scalp discoloration and thickening, permanent 
thinning of the hair in the treated area. Uncommonly, severe memory loss, cataracts, permanent 
loss of brain cells, seizures, hearing loss. Rarely, death of brain cells can cause swelling in the 
brain and require long-term steroid treatment, hospitalization and/or surgery, loss or decrease in 
vision. Extremely rare is development of new tumors in the scalp, skull or brain. 

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10
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178.
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Society of Clinical Oncology. 2005;23(15):3447-54.
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Elhasid R, Arush MB, Zaidman I, Leiba R, Barak AB, Postovsky S, et al. Safe and 
Efficacious Allogeneic Bone Marrow Transplantation for Nonmalignant Disorders Using Partial 
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Blood and Marrow Transplantation. 2007;13(3):329-38.
180.
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cell-mediated killing of freshly isolated neuroblastoma cells: critical role of DNAX accessory 
molecule-1-poliovirus receptor interaction. Cancer research. 2004;64(24):9180-4.
181.
Prigione I, Corrias M, Airoldi I, Raffaghello L, Morandi F, Bocca P, et al. 
Immunogenicity of Human Neuroblastoma. Annals of the New York Academy of Sciences. 
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Chin T, Toy C, Vandeven C, Cairo MS. Lymphokine-activated killer cytotoxicity in 
neonatal mononuclear cells: in vitro responses to tumor cell lines from pediatric solid tumors. 
Pediatric research. 1989;25(2):156-60.
183.
Klob S, Bochennek K, Huenecke S, Zimmermann S-Y, KuÁi S, M¸ller T, et al. A novel 
five-colour flow cytometric assay to determine NK cell cytotoxicity against neuroblastoma and 
other adherent tumour cells. Journal of Immunological Methods. 2007;325(1-2):140-7.
184.
Castriconi R, Dondero A, Cilli M, Ognio E, Pezzolo A, De Giovanni B, et al. Human NK 
cell infusions prolong survival of metastatic human neuroblastoma-bearing NOD/scid mice. 
Cancer immunology, immunotherapy. 2007;56(11):1733-42.
185.
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Clinical Trial of the hu14.18-IL2 (EMD 273063) as a Treatment for Children with Refractory or 
Recurrent Neuroblastoma and Melanoma: a Study of the Children's Oncology Group. Clinical 
cancer research : an official journal of the American Association for Cancer Research. 
2006;12(6):1750-9.
186.
Capitini CM, Cooper LJ, Egeler RM, Handgretinger R, Locatelli F, Sondel PM, et al. 
Highlights of the First International "Immunotherapy in Pediatric Oncology: Progress and 
Challenges" Meeting. J Pediatr Hematol Oncol. 2009;31(4):227-44.
187.
Verhoeven DHJ, de Hooge ASK, Mooiman ECK, Santos SJ, ten Dam MM, Gelderblom 
H, et al. NK cells recognize and lyse Ewing sarcoma cells through NKG2D and DNAM-1
receptor dependent pathways. Molecular immunology. 2008;45(15):3917-25.
188.
Wu A, Wiesner S, Xiao J, Ericson K, Chen W, Hall W, et al. Expression of MHC I and 
NK ligands on human CD133+ glioma cells: possible targets of immunotherapy. Journal of 
Neuro-Oncology. 2007;83(2):121-31.
189.
Multhoff G, Botzler C, Wiesnet M, Eissner G, Issels R. CD3- large granular lymphocytes 
recognize a heat-inducible immunogenic determinant associated with the 72-kD heat shock 
protein on human sarcoma cells. Blood. 1995;86(4):1374-82.
190.
Nolibé D, Poupon MF. Enhancement of pulmonary metastases induced by decreased lung 
natural killer cell activity. Journal of the National Cancer Institute. 1986;77(1):99-103.
191.
Evans R, Fuller JA, Christianson G, Krupke DM, Troutt AB. IL-15 Mediates Anti-tumor 
Effects after Cyclophosphamide Injection of Tumor-Bearing Mice and Enhances Adoptive 
Immunotherapy: The Potential Role of NK Cell Subpopulations. Cellular Immunology. 
1997;179(1):66-73.
192.
Belova OB, Vinnichuk UD, Shlakhovenko VA, Berezhnaya NM. Efficacy of different 
immunotherapy approaches toward treatment of doxorubicin-resistant and doxorubicin-sensitive 
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193.
Bauer M, Reaman GH, Hank JA, Cairo MS, Anderson P, Blazar BR, et al. A phase II 
trial of human recombinant interleukin-2 administered as a 4-day continuous infusion for 
children with refractory neuroblastoma, non-Hodgkin's lymphoma, sarcoma, renal cell 
carcinoma, and malignant melanoma. A Childrens Cancer Group study. Cancer. 
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194.
Pende D, Marcenaro S, Falco M, Martini S, Bernardo ME, Montagna D, et al. Anti-
leukemia activity of alloreactive NK cells in KIR ligand-mismatched haploidentical HSCT for 
pediatric patients: evaluation of the functional role of activating KIR and re-definition of 
inhibitory KIR specificity. Blood. 2008:blood-2008-06-164103.
195.
Boyiadzis M, Memon S, Carson J, Allen K, Szczepanski MJ, Vance BA, et al. Up-
regulation of NK cell activating receptors following allogeneic hematopoietic stem cell 
transplantation under a lymphodepleting reduced intensity regimen is associated with elevated 
IL-15 levels. Biology of blood and marrow transplantation : journal of the American Society for 
Blood and Marrow Transplantation. 2008;14(3):290-300.
196.
Murphy WJ, Keller JR, Harrison CL, Young HA, Longo DL. Interleukin-2-activated 
natural killer cells can support hematopoiesis in vitro and promote marrow engraftment in vivo. 
Blood. 1992;80(3):670-7.
197.
Gewirtz AM, Xu WY, Mangan KF. Role of natural killer cells, in comparison with T 
lymphocytes and monocytes, in the regulation of normal human megakaryocytopoiesis in vitro. 
Journal of immunology (Baltimore, Md : 1950). 1987;139(9):2915-24.
198.
Murphy WJ, Longo DL. Hematopoietic growth-promoting effects of natural killer cells in 
mice in response to in vivo administration of human interleukin-2. J Natl Cancer Inst. 
1992;84(15):1195-200.
199.
Murphy WJ, Raziuddin A, Mason L, Kumar V, Bennett M, Longo DL. NK cell subsets in 
the regulation of murine hematopoiesis. I. 5E6+ NK cells promote hematopoietic growth in H-2d
strain mice. Journal of immunology (Baltimore, Md : 1950). 1995;155(6):2911-7.
200.
Hsu KC, Keever-Taylor CA, Wilton A, Pinto C, Heller G, Arkun K, et al. Improved 
outcome in HLA-identical sibling hematopoietic stem-cell transplantation for acute myelogenous 
leukemia predicted by KIR and HLA genotypes. Blood. 2005;105(12):4878-84.
201.
Miller JS, Soignier Y, Panoskaltsis-Mortari A, McNearney SA, Yun GH, Fautsch SK, et 
al. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in 
patients with cancer. Blood. 2005;105(8):3051-7.
202.
Passweg JR, Stern M, Koehl U, Uharek L, Tichelli A. Use of natural killer cells in 
hematopoetic stem cell transplantation. Bone Marrow Transplant. 2005;35(7):637-43.
203.
Passweg JR, Tichelli A, Meyer-Monard S, Heim D, Stern M, Kuhne T, et al. Purified 
donor NK-lymphocyte infusion to consolidate engraftment after haploidentical stem cell 
transplantation. Leukemia. 2004;18(11):1835-8.
204.
Ruggeri L, Capanni M, Urbani E, Perruccio K, Shlomchik WD, Tosti A, et al. 
Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. 
Science (New York, NY). 2002;295(5562):2097-100.
205.
Murphy W, Koh C, Raziuddin A, Bennett M, Longo D. Immunobiology of natural killer 
cells and bone marrow transplantation: merging of basic and preclinical studies. Immunological 
reviews. 2001;181(1):279-89.
206.
Asai O, Longo DL, Tian ZG, Hornung RL, Taub DD, Ruscetti FW, et al. Suppression of 
graft-versus-host disease and amplification of graft-versus-tumor effects by activated natural 

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killer cells after allogeneic bone marrow transplantation. The Journal of clinical investigation. 
1998;101(9):1835-42.
207.
Lang P. Co-Transfusion of Donor NK Cells in Haploidentical Stem Cell Transplantation. 
Blood. 2008;112(ASH Annual Meeting Abstracts ):Abstract 2907.
208.
Rizzieri D. Safety Trial of NK Cell Enhanced Donor Lymphocyte Infusions from a 3-5/6 
HLA Matched Family Member Following Nonmyeloablative Allogeneic Stem Cell 
Transplantation. Blood. 2008;112(ASH Annual Meeting Abstracts):Abstract 342.
209.
Siegler U, Kalberer CP, Nowbakht P, Sendelov S, Meyer-Monard S, Wodnar-Filipowicz 
A. Activated natural killer cells from patients with acute myeloid leukemia are cytotoxic against 
autologous leukemic blasts in NOD//SCID mice. Leukemia. 2005;19(12):2215-22.
210.
Isgro A. Donor’s NK Cells May Influence the Engraftment in Pediatrics Patients after T-
Cell Depleted Haploidentical Stem Cell Transplant for Thalassemia. Blood. 2008;112(ASH 
Annual Meeting Abstracts):Abstract 4595.
211.
Imai C, Iwamoto S, Campana D. Genetic modification of primary natural killer cells 
overcomes inhibitory signals and induces specific killing of leukemic cells. Blood. 
2005;106(1):376-83.
212.
Sanders JE, Thomas ED, Buckner CD, Doney K. Marrow transplantation for children 
with acute lymphoblastic leukemia in second remission. Blood. 1987;70(1):324-6.
213.
Wiener LS, Steffen-Smith E, Fry T, Wayne AS. Hematopoietic stem cell donation in 
children: a review of the sibling donor experience. J Psychosoc Oncol. 2007;25(1):45-66.

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11
APPENDICES
11.1 APPENDIX 1: PERFORMANCE STATUS 
PERFORMANCE STATUS CRITERIA Karnofsky and Lansky performance scores are intended to be multiples of 10.
ECOG (Zubrod)
Karnofsky
Lansky
Score
Description
Score
Description
Score
Description
0
Fully active, able to carry on all pre-disease 
performance without restriction.
100%
Normal, no complaints, no 
evidence of disease. 
100%
Fully active, normal.
90%
Able to carry on normal activity; 
minor sighs of symptoms of 
disease.
90%
Minor restrictions in physically 
strenuous activity.
1
Restricted in physically strenuous activity but 
ambulatory, able to carry out light or 
sedentary work, e.g., light housework, office 
work.
80%
Able to carry on normal activity 
with effort; some signs or 
symptoms of disease.
80%
Active, but tires more quickly.
70%
Cares for self, unable to carry on 
normal activity or do active 
work.
70%
Both greater restriction of, and less 
time spent in, play activities.
2
Ambulatory and capable of all self-care but 
unable to carry out any work activities. Up and 
about more than 50% of waking hours. 
60%
Requires occasional assistance 
but is able to care for most of 
own needs.
60%
Up and around, but minimal active 
play; keeps busy with quieter 
activities.
50%
Requires considerable assistance 
and frequent medical care.
50%
Gets dressed, but lies around much 
of the day; no active play; able to 
participate in quiet play and 
activities. 
3
Capable of only limited self-care, confined to 
bed or chair more than 50% of waking hours
40%
Disabled; requires special care 
and assistance. 
40%
Mostly in bed; participates in quiet 
activities.

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30%
Severely disabled; 
hospitalization indicated, 
although death not imminent.
30%
In bed; needs assistance even for 
quiet play.
4
Completely disabled.  Cannot carry on any 
self-care. Totally confined to a bed or chair
20%
Very ill; hospitalization 
necessary;  active supportive 
treatment required.
20%
Often sleeping; play entirely limited 
to very passive activities.
10%
Moribund, fatal process 
progressing rapidly
10%
No play; does not get out of bed
5
Dead
0%
Patient expired
0%
Unresponsive; Dead

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11.2 APPENDIX 2: REQUIRED STUDY EVALUATIONS
11.2.1 Appendix 2 A: Patient
Required Study Evaluations
PATIENT
Treatment Phase
Eligibility
On-
Study
Induction
Pre-
Pre-
BMT prep
Day
D 1 - 21
Induction 
#2&3
BMT
-6 to -1
0
14
28±7
days
60±21
days
100±30
days
180±30
days
6-12
MO±3 
MO
12-60
MO±3 
MO
Off-
Treatme
nt
History & Physical - vital signs
X
X
weekly
X
X
daily
daily until discharge
2/week
2/week
2/week
monthly
monthly
monthly
X
Pathology Review
X
HLA Typing
X
Type/Screen
X
q 4 days
q 4 days until d/c
X*
X*
X*
X*
X*
DAT*
daily on days 6-10*
RBC Phenotype/Isohem Titer*
X
X*
X*
X*
X*
X*
Urinalysis
X
X each cycle
X
daily
daily 
until d/c
CBC
X
X
2x/week
X
X
daily
daily until d/c
weekly
weekly
weekly
monthly
monthly
X*
X
Reticulocyte count
weekly
weekly until d/c
weekly
weekly
weekly
X*
X*
X*
PT, PTT, Lupus anti-coagulant
X
Chemistry
X
X
2x/week
X
X
QD
daily until d/c
X*
X*
X*
X*
X*
X*
X
Iron, Transferrin, Ferritin
X
monthly
monthly
monthly
Urine CrCl, Ca/Cr*
X
Urine Pregnancy*
X
X
X
EKG
X
X
X
*
*

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Echo or MUGA
X
X
X
*
*
PFT/DLCO
X
GVHD
X
*
*
Disease and GVHD Staging Evaluation
X
X
X
X
X
X
q 3mos
q 3mos
Bone Marrow*
X
<14 days
X*
X*
X*
X*
X*
X*
X*
CSF*
X
DPA (>18 years old only)
X
Endocrine function tests*
*
*
*
Bcr-abl PCR blood (28-35d post NK)
Consults
Social Work
X
Dental
X
ID Monitoring & Prophylaxis
Infectious Disease Markers
X
Viral Serology (HSV, VZV, EBV, Toxo)
X
PPD/Control*
X
Fluconazole
Start, hold during vincristine
PCP Prophylaxis
Start
Viral monitoring and prophylaxis
Start
Viral Monitoring (CMV, EBV, HHV6, adeno virus)
weekly
weekly
weekly
weekly
weekly
X*
X*
X*
X*
IgG Level
X
weekly
weekly
weekly
weekly
weekly
X*
X*
X*
Post-vaccine antibody titers
X*
X*
X*

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Research Studies
Serum Storage
X
Immune surveillance (TBNK or flow)
X
X
X
X
Chimerism - Peripheral Blood
X
X
X
X
X
*
*
*
*
Chimerism - Bone Marrow
*
*
*
*
*
KIR/HLA Genotyping
Anytime prior to  HSCT
X
Immune Reconstitution
X
X
X
X
X
q 3 mo
Lymphocyte Proliferation
X
X
X
X
q 6 mo
* as clinically indicated

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11.2.2 Appendix 2 B: Donor
Treatment Phase
Eligibility
On-
Study
Pre-
Pre-
Post-
1-2
week
Day
CVL
Collection
Collection
Collection
F/U
History & Physical
X
<72 
hrs
X
DTM Screen
X
X
HLA Typing
X
Type/Screen
X
Red Cell Phenotype*
X*
Isohemmaglutinin Titer*
X*
Urinalysis
X
CBC
X
<72 
hrs
X
X
PT, PTT
X
<72 hrs
Chemistry
X
Infectious Disease 
Markers
X
<30 days
X
Viral Serology (EBV)
X
CD34 Count
X

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Urine Pregnancy*
X*
Consults
Social Work
X
Child 
Psychology/Psychiatry
X*
Research Studies
Serum Storage
X
Chimerism (STR profile)
X
KIR testing
X
^ Avoid antecubital phlebotomy if possible.
* as clinically indicated

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11.3 APPENDIX 3: SCREENING AND BASELINE EVALUATION
RECIPIENT INFORMATION
DONOR INFORMATION
Name
MR#
Diagnosis
Stem Cell Collection Dose
DOB (Age)
Wt(kg)/Ht(cm)/BSA(m2)
Sex
Test
Date
Results
Comments/Inititals
Date
Results
Comments/Inititals
History & Physical
Immunization Records
Donor Questionairre
Diagnostic Pathology
Blood Type
Antibody Screen
RBC Phenotype*
Isohemmaglutinin Titer*
Urinalysis
CBC
PT, PTT, Lupus anticoagulant
Chemistry
Urine CrCl
Urine Ca/Cr
Iron, Transferrin, Ferritin
HLA: initial and  confirmatory
Chimerism (STR Profile)
HIV-1/2
NAT
HTLV-I/II

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RPR
HB S Ag
HB core Ab
HCV
CMV IgG/IgM
Toxo IgG
HSV IgG
Varicella IgG
EBV Panel
PPD/Control*
Fanconi Anemia test (MDS only)
Urine Pregnancy*
EKG
Echo (SF) or MUGA (EF)
PFT/DLCO-VAcorr
Staging: 
PET/CT
BM
CSF
Consults
Social Work
Dental
Child Psych (minors only)
CVL / Consent
Informed Consent
CIBMTR Consent
IDMs-BMT <30 days of collection
Workup reviewed and confirmed complete by:

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Attending Physician
date
Form date: 08/15/05

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11.4 APPENDIX 4: SUMMARY OF RESEARCH BLOODS*
SAMPLE ACQUISITION TIME POINTS
Study
Laboratory 
Location
Pre-Study
Induction
Pre-BMT 
Prep
Post-BMT
Volume
Tube
Notes
Donor & 
Recipient 
Serum 
Storage
NCI-Frederick
Donor and 
Recipient
10 ml
Red top
Sample refrigerator on patient unit
^Courier to NCI-Frederick repository 301-
846-5893 to schedule pick-up
Immuno-
Phenotyping
(Lymphocyte 
Phenotyping-
TBNK panel 
or NK 
Subset)**
Clinical Path Flow 
Cytometry 
10/2C410     
6-4879
On-Study
(Lymphocyte 
Phenotyping-
NK Subset)
Prior to 
Each 
Cycle
Day –7, 0 if 
ALC >200
1st day ALC >200; 24 hours 
+/- 24 hours post NK#1 and 
#2 infusion. Day 28( ± 7), 60(
± 21), 100( ± 30), and every 3 
months ( ± 1 mo) until 2 yrs
5 ml
Purple top 
(EDTA)
Schedule before sending
X 64879
Chimerism 
(STR Profile)
Dr. Kurlander 
Hematology Lab 
435-6399
Eligibility
Day 14^, 28^+, 60, 100+, then 
PRN; both whole blood and 
WBC STR chimerism.                
10 ml ^20 
ml +1-2
ml 
marrow
Blood: yellow top 
(ACD) Marrow: 
heparinized 
syringe
^20 ml for cell sorting (until 100% donor);  
+ blood and marrow. Schedule with lab X 
56399
Cytokine  
Levels
(IL-7/IL-15)
10/ C210     
2-1298
On-Study
Day –7, Day 
0
Day prior to or morning of 
EACH NK infusion, the Day 
1, 2, 7, &14 ( ± 48 hr each 
)after each NK cell infusion, 
then D 60( ± 21), D100( ±
30), and then q 3 mos ( ± 1
mo) for 2 yrs
4 ml
red top
Sample refrigerator on patient unit
^Courier to NCI-Frederick repository
TH1/TH2 
cytokine 
profile
SAIC-Frederick
(301) 846-1707
On-Study
Day –7, Day 
0
Day prior to or morning of 
EACH NK infusion, the Day 
1, 2, 7, &14 ( ± 48 hr each )
after each NK cell infusion, 
then D 60 ( ± 21), D100 ( ± 
30), and then q 3 mos ( ± 1
mo) for 2 yrs
4 ml
red top
Sample refrigerator on patient unit
^Courier to NCI-Frederick repository

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KIR 
genotyping
Delbrook RN
1W-3750
Recipient 
-Prior to 
prep
Donor –
prior to
PBSC 
collection
Day 100 ± 30d
1 -2 mL 
Blood: yellow top 
(ACD) tube OR
3 buccal swabs 
Sample stored and sent at room temp.   
Deidentified prior to sending and label 11-
C-0073–subject ID#. Send to Lihua 
Hou/Carolyn Hurley at C. W. Bill Young 
Marrow Program, Georgetown University, 
3rd Floor, 11333 Woodglen Drive, 
Rockville, MD 20852, 301-998-8900.
Lymphocyte 
Proliferation
NCI-Frederick
Day 28 ( ± 7), 60 ( ± 21), 100(
± 30), 180 ( ± 30), and every 6 
months ( ± 30d) until 2 yrs 
20 ml
green top (sodium 
heparin)
Sample refrigerator on patient unit
^Courier to NCI-Frederick repository
Blasts (Bone 
Marrow or 
Blood)
NCI-Frederick
Relapse
10ml 
marrow;
20ml 
blood
Preservative-free 
heparin
Sample refrigerator on patient unit
^Courier to NCI-Frederick repository
Single-Pass 
Apheresis
DTM
Relapse: post-subsequent 
remission
^Courier to NCI-Frederick repository 
Donor 
Lymphocytes
DTM
Only if DLI collection
^Courier to NCI-Frederick repository
BCR abl 
PCR; p210
Kurlander Lab 
435-6399
28-35 days post final NK 
infusion
10 ml 
Blood: yellow 
ACD tube-
Sent as clinical lab (must be arranged with 
Dr. Roger Kurlander lab).
*In the event that blood draws are limited due to patient size, research studies are listed in order of priority.
^Courier to NCI-Frederick repository 301-846-5893 to schedule pick-up
**May be substituted for TBNK panel if lymphocyte phenotyping cannot be scheduled. 

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11.5
APPENDIX 5: ACUTE GVHD
Does the patient have Acute GVHD?  No  / Yes  o Date of the first diagnosis of aGVHD? __/__/____ 
Did the patient have an aGVHD flare since last exam? No    Yes o Date:  __/__/____
Select the appropriate stage, certainty of the system involved and indicate whether a biopsy was completed. 
Skin
Certainty
Comments
Biopsy
Rash
Stage
Y/N
 Rash not attributable to GVHD
N/A
 No rash
0
Unlikely Possible  Probable  Certain
No  Yes  Pending
 <25% BSA
1
 25-50% BSA 
2
 >50%
3
 bullae and desquamation
4
Liver
Certainty
Comments
Biopsy
Bilirubin
Stage
Y/N
 Bilirubin not attributable to GVHD
N/A
 <2.0 mg/dl
0
Unlikely Possible  Probable  Certain
No  Yes  Pendin
 2-3.0 mg/dl
1
 3.1-6.0 mg/dl (3.1-8.0 Glucksberg)
2
 6.1-15 mg/dl (8.1-15.0 Glucksberg)
3
 >15 mg/dl
4
Upper GI 
GI Tract
Certainty
Comments
Biopsy
Persistent 
N/V
Diarrhea ml/day 
(peds ml/m2/day)
Stage
Y/N
 Diarrhea not attributable to 
GVHD
N/A
 No (0)
 No diarrhea
0
Unlikely Possible 
Probable 
Certain
No Yes Pending

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 Yes (1)
 500-1,000 (< 280)
1
 1,000-1,500 (280-555)
2
 > 1,500 (555-833)
3
 severe abdominal pain (w/, 
w/o ileus)
4
Grade* Modified Glucksberg Criteria
Grade* IBMTR
Skin
Liver
GI
Skin 
(max 
stage)
Liver 
(max 
stage)
GI 
(max 
stage)
1
1-2
0
0
A
1
0
0
2
1-3
1
1
B
2                or
1-2                or
1-2
2o
0
1
1
C
3                or
3                  or
3
2s
4
0
0
D
4                or
4                   or
4
3
2-4
2-4
&/or  
2-4  (1 only >2)
4
3-4
2-4
2-4
Modified 
Glucksberg 
Criteria
CIBMTR Criteria
Date
Current aGVHD Grade:
Maximum aGVHD Grade
What was the max stage in the 1st 100 days per organ: 
Skin:____(0-4), Lower GI:____(0-4), Upper GI (i.e. nausea/vomiting):_____(0-1), Liver_____(0-4)
Other organ system involvement: No / Yes o  Lung o biopsy proven: No /Yes 
 Other ____________

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11.6 APPENDIX 6: CHRONIC GVHD EXAM AND GRADING, CLINICIAN ASSESSMENT FORM
Were Functional Performance tests completed? No    Yes  (Must be greater than 4 years old)
Select the tests performed:
 Walk Time: Number of laps: _______ (x 50 feet) + final partial lap: _____ feet  = ______ feet walked in 2 minutes  
Total Distance Walked in 2 Minutes: 
 Grip Strength:
Grip Strength (Dominant Hand)  _______________________
 Range of Motion: 
 Not performed See Physical Therapy Report 
Skin: Does the patient have skin changes? No    Yes o   Check and score all findings that apply.
Skin Component
Findings
Scoring 
 Erythematous rash of any sort
            ____% BSA (max 100%)
 Moveable sclerosis 
            ____ % BSA (max 100%)
 Non-moveable sclerosis (hidebound/non-
pinchable) or subcutaneous sclerosis/fasciitis
             ____% BSA (max 100%)
 Ulcer(s): select the largest ulcerative lesion, 
and measure its largest dimension in cm and 
mark location of ulcer
Location: ________________
Largest dimension: _______cm
Eyes: Does the patient have decreased tear production? No    Yes
Schirmer’s Tear Test completed? No    Yes   o  With anesthesia    Without anesthesia
Bilateral Schirmer’s Tear Test
(without anesthesia) > 9 years or older
Right Eye:  ____________ mm of wetting
Left Eye:  _____________ mm of 
wetting
Lungs: Were Pulmonary Function Tests completed? No    Yes

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Lungs
x
Bronchiolitis Obliterans
x
Pulmonary 
Function 
Tests 
with 
Diffusing Capacity 
FEV-1
% Predicted
Single Breath DLCO (adjusted for 
Hg)
                                         
                               % Predicted
Gastrointestinal:
Upper GI 
x Early satiety  OR
x Anorexia     OR
x Nausea/Vomiting
Score:
0= no symptoms
1=mild, occasional symptoms, with little reduction in oral intake during the past week
2=moderate, intermittent symptoms, with some reduction in oral intake during the past week
3=more severe or persistent symptoms throughout the day, with marked reduction in oral intake, on almost every 
day of the past week
GI/Esophageal
x Dysphagia     OR
x Odynophagia
Score:
0= no esophageal symptoms
1=Occasional dysphagia or odynophagia with solid food or pills during the past week
2=Intermittent dysphagia or odynophagia with solid foods or pills, but not for liquids or soft foods, during the past 
week
3=Dysphagia or odynophagia for almost all oral intake, on almost every day of the past week
Lower GI
x Diarrhea
Score:
0= no loose or liquid stools during the past week
1= occasional  loose or liquid stools, on some days during the past week
2=intermittent loose or liquid stools throughout the day, on almost every day of the past week, without requiring
intervention to prevent or correct volume depletion
3=voluminous diarrhea on almost every day of the past week, requiring intervention to prevent or correct volume 
depletion
Mucous Membrane: Does the patient have mucosal changes? No    Yes
Mucosal 
change
No cGvHD
Mild
Moderate
Severe
Erythema
None
0
Mild or moderate 
erythema (<25%)
1
0RGHUDWH

RU
Severe erythema (<25%)
2
Severe 
erythema 

3
Lichenoid
None
0
Hyperkeratotic 
changes(<25%)
1
Hyperkeratotic 
changes(25-50%)
2
Hyperkeratotic 
changes (>50%)
3
Ulcers
None
0
None
0
8OFHUVLQYROYLQJ
3
Severe 
ulcerations 
(>20%)
6
Mucoceles*
None
0
1-5 mucoceles
1
6-10 scattered mucoceles
2
Over 10 mucoceles
3

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*Mucoceles scored for lower labial and soft palate only
Total 
mucosal 
changes
Genital: Does the patient have involvement of the genital involvement?   No    Yes
 Erythema  Lichen planus-like features  Erosions/fissures  Ulcers 
Female patients only:  Agglutination of the clitoral hood  Vulvar sclerosis  Vestibular tenderness  Vaginal 
scarring/stenosis 
Health Care Provider Global Ratings:
In your opinion, do 
you think that this 
patient’s 
chronic 
GvHD 
is 
mild, 
moderate or severe?
 0=none
 1= mild
 2=moderate
 3=severe
Where would you rate the severity of this patient’s chronic GvHD 
symptoms on the following scale, where 0 is cGVHD symptoms that are not 
at all severe and 10 is the most severe cGVHD symptoms possible:
0       1         2         3         4         5         6         7         8          9         10
Not at all                                                                                   Most severe
                                                                                                   Symptoms
                                                                                                     Possible
Provider rating:  ____________                                                                             
Over the past month would you say 
that this patient’s cGvHD is
 +3= Very much better 
 +2= Moderately better
 +1= A little better
 0= About the same
 -1=A little worse
 -2=Moderately worse 
 -3=Very much worse

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Appendix 6 (continued): Chronic GVHD Exam and Grading
Does the patient have cGVHD? o
No    Yes
First diagnosis of cGVHD? Yes No o Date of first cGVHD diagnosis __/__/__
Onset of 1st diagnosis of chronic GVHD was:
   Progressive (acute GVHD progressed directly to chronic GVHD)
   Interrupted (acute GVHD resolved, then chronic GVHD developed)
   De novo (acute GVHD never developed)
Diagnosis was based on:  histologic evidence / biopsy proven   clinical evidence  
 both   unknown
Did cGVHD flare since last visit?  N/A  No  Yes  o Date:  __/__/__
Maximum grade of chronic GVHD (Seattle Criteria):

Limited - localized skin involvement and/or hepatic dysfunction due to cGVHD
   Extensive - one or more of the following:
– generalized skin involvement; or,
– liver histology showing chronic aggressive hepatitis, bridging necrosis or cirrhosis; or,
– involvement of eye: Schirmer’s test with < 5 mm wetting; or
– involvement of minor salivary glands or oral mucosa demonstrated on labial biopsy; or
– involvement of any other target organ
Overall severity of chronic GVHD (NIH Consensus Criteria):
 Mild – signs and symptoms do not interfere substantially with function and do not progress 
once appropriately treated with local therapy or standard systemic therapy (corticosteroids and/or 
cyclosporine or FK 506)

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 Moderate– signs and symptoms interfere somewhat with function despite appropriate therapy 
or are progressive through first line systemic therapy (corticosteroids and/or cyclosporine or FK 
506)
 Severe – signs and symptoms limit function substantially despite appropriate therapy or are 
progressive through second line therapy

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11.7 APPENDIX 7: CHRONIC GVHD, NIH STAGING FORM
NIHCGVHD SCORE
SCORE 0
SCORE 1
SCORE 2
SCORE 3
SKIN
Clinical features:
Maculopapular rash
Lichen planus-like 
features
Papulosquamous 
lesions or ichthyosis
Hyperpigmentation
Hypopigmentation
Keratosis pilaris
Erythema
Erythroderma 
Poikiloderma
Sclerotic features 
Pruritus
Hair involvement
Nail involvement
% BSA 
involved
No 
Symptoms 
<18% BSA with 
disease signs but 
NOsclerotic 
features
19-50% BSA OR 
involvement with 
superficial sclerotic 
features “not 
hidebound” (able to 
pinch)
>50% BSA OR
deep sclerotic features 
“hidebound” (unable 
to pinch) ORimpaired 
mobility, ulceration or 
severe pruritus 
MOUTH
No 
symptoms 
Mild symptoms 
with disease signs 
but not limiting oral 
intake significantly
Moderate 
symptoms with 
disease signs with
partial limitation of 
oral intake
Severe symptoms 
with disease signs on 
examination with
major limitation of 
oral intake
EYES
Mean tear test (mm):
>10
6-10
<5
Not done
No 
symptoms 
Mild dry eye 
symptoms not 
affecting ADL 
(requiring eyedrops 
< 3 x per day) OR
asymptomatic 
keratoconjunctivitis 
sicca
Moderate dry eye 
symptoms partially 
affecting ADL 
(requiring drops > 3 
x per day or punctal 
plugs), WITHOUT
vision impairment
Severe dry eye 
symptoms 
significantly affecting 
ADL (eyeware to 
relieve pain) OR
unable to work 
because of ocular 
symptoms ORloss of 
vision caused by 
keratoconjunctivitis 
sicca

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GITRACT
No 
symptoms
Symptoms such 
as dysphagia, 
anorexia, nausea, 
vomiting, 
abdominal pain or 
diarrhea without 
significant weight 
loss (<5%)
Symptoms 
associated with mild 
to moderate weight 
loss (5-15%)
Symptoms 
associated with 
significant weight loss 
>15%, requires 
nutritional supplement 
for most calorie needs 
OResophageal 
dilation
LIVER
Normal 
LFT
Elevated 
Bilirubin, AP*, 
AST or ALT <2 x 
ULN
Bilirubin >3 mg/dl 
or Bilirubin, 
enzymes 2-5 x ULN 
Bilirubin or 
enzymes > 5 x ULN

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NIHCGVHD SCORE
SCORE 0
SCORE 1
SCORE 2
SCORE 3
LUNGS*
FEV1
DLCO
No 
symptoms
FEV1 > 
80% OR 
LFS=2
Mild symptoms 
(shortness of breath 
after climbing one 
flight of steps)
FEV1 60-79% 
OR LFS 3-5
Moderate 
symptoms (shortness 
of breath after 
walking on flat 
ground)
FEV1 40-59% 
OR LFS 6-9
Severe symptoms 
(shortness of breath  at 
rest; requiring 02)
FEV1 <39% OR 
LFS 10-12
JOINTS AND FASCIA
No 
symptoms
Mild tightness of 
arms or legs, 
normal or mild 
decreased range of 
motion (ROM) 
AND not affecting 
ADL
Tightness of arms 
or legs OR joint 
contractures, 
erythema due to 
fasciitis, moderate 
decrease ROM AND 
mild to moderate 
limitation of ADL
Contractures WITH 
significant decrease of 
ROM AND significant 
limitation of ADL 
(unable to tie shoes, 
button shirts, dress 
self etc.) 
GENITAL TRACT
No 
symptoms 
Symptomatic 
with mild signs on 
exam ANDno 
effect on coitus and 
minimal discomfort 
with gynecologic 
exam
Symptomatic with 
moderate signs on 
exam ANDwith 
mild dyspareunia or 
discomfort with 
gynecologic exam
Symptomatic 
WITH advanced signs 
(stricture, labial 
agglutination or severe 
ulceration) AND
severe pain with 
coitus or inability to 
insert vaginal 
speculum

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11.8 APPENDIX 8: LEUKEMIA/LYMPHOMA RESPONSE CRITERIA
Bone Marrow Classification
 
% blasts (at least 200 cells counted) 
M1 
<5% 
M2 
5 - 25% 
M3 
> 25% 
CNS Classification
 
CSF Cytospin 
CNS 1 
0 blasts on cytopsin 
CNS 2 
White cell count < 5/μl, blasts on cytopsin 
CNS 3 
White cell count > 5/μl; blasts on cytospin 
Acute Leukemia (Adapted from Cheson BD, et al. Revised recommendations of the International Working Group 
for Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for therapeutic 
trials in acute myeloid leukemia. J Clin Oncol 2003;21:4642-4649)
Complete Response (CR)

M1 marrow, absence of peripheral blasts, absence of extramedullary sites of disease, peripheral 
blood neutrophil count > 1,000/PL and platelet count > 100,000/PL.

Morphologic CR with incomplete blood count recovery (CRi): Above CR criteria without 
specified blood counts.

Cytogenetic CR (CRc): In addition to above CR criteria, reversion to normal karyotype for those 
with previously detected cytogenetic abnormality.

Molecular CR (CRm): In addition to above CRc criteria, normalization of previously detected 
molecular cytogenetic abnormality.
Partial Response (PR)

M2 marrow and a decrease in the percentage of marrow blasts and absolute peripheral blast count 
by at least 50%, absence of extramedullary sites of disease, peripheral blood neutrophil count >
1,000/PL and platelet count > 100,000/PL.

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Stable Disease (SD)

No change in marrow classification (i.e., M status) and less than a 50% decrease to less than a 
50% increase in absolute peripheral blast count or extent of extramedullary disease
Progressive Disease (PD)

Worse marrow classification (i.e., M status) with at least a 50% increase in the percentage of 
marrow blasts.
Or

No change in marrow classification (i.e., M status), but a 50% or greater increase in absolute 
peripheral blast count or extent of extramedullary disease
Chronic Myelogenous Leukemia
Hematologic
Cytogenetic
Molecular
CR
Normal CBC, no 
splenomegaly
0% Ph+ cells
Undetectable bcr/abl by 
PCR
PR
> 50% decrease in 
peripheral WBC and WBC 
< 20,000; persistent 
splenomegaly
> 50% decrease in 
Ph+ cells
Detectable bcr/abl by 
PCR
SD
< 50% decrease in 
peripheral WBC 
No new cytogenetic 
abnormalities
Detectable bcr/abl by 
PCR
PD
Worse CBC or 
splenomegaly
New cytogenetic 
abnormalities
Detectable bcr/abl by 
PCR
Lymphoma (Adapted from: Cheson BD, Horning SH, Coiffier B, et al: Report of an international workshop to 
standardize response criteria for non-Hodgkin's lymphomas. J Clin Oncol 1999;17: 1244-53)
Response
Physical Exam
Lymph Nodes
Lymph Node Masses
Bone Marrow
CR
Normal 
Normal
Normal
Normal
CRu
Normal
Normal
Normal
Normal
Normal
> 75% decrease x 3 mos
Indeterminate
Normal or indeterminate
PR
Normal
Normal
Decrease in liver/spleen
Normal
> 50% decrease
> 50% decrease
Normal
> 50% decrease
> 50% decrease
Positive
Irrelevant
Irrelevant

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PD
Enlarging liver/spleen, 
new sites
New or increased
New or increased
Reappearance
Complete Remission (CR): Disappearance or reduction to normal size of all measurable and 
evaluable disease (lymph nodes <1.5 cm or nodal masses decreased by > 75% in the sum of the 
products of the greatest diameters lasting for > 3 months consistent with scar),  negative bone 
marrow, non-palpable spleen.
Partial Response (PR): Reduction in tumor burden by at least 50%. The sum of the products of 
perpendicular measurements of all (or a representative number of) masses judged to be malignant 
must decrease by at least 50%. None of these masses may show an increase >25% in size and no 
new malignant lesions should appear. Sites of disease which cannot be quantitated, such as bone 
marrow aspirates or lesions showing wide fluctuations prior to treatment, cannot be used to 
determine a PR. While defined splenic nodules can be considered measurable for the purpose of 
determining a PR, total spleen size cannot, although a PR is ruled out if the spleen size increases 
> 25%.
Stable Disease: A <50% decrease to a <50% increase in the sum of the diameter product of all 
measurable lesions.
Progressive Disease: A > 50% increase in the diameter product of any existing single lesion or the 
appearance of any new lesions.

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11.9 APPENDIX 9: SOLID TUMOR RESPONSE CRITERIA (RECIST)
1. The CTEP response criteria for solid tumors (RECIST) outlined below will be used to assess 
response:
2. Only patients with measurable disease at baseline can have objective tumor response evaluated 
as an endpoint. 
x Measurable disease: The presence of at least one measurable lesion.  If the 
measurable disease is restricted to a solitary lesion, its neoplastic nature should be 
confirmed by cytology/histology, if possible.
x Measurable lesions: Lesions that can be accurately measured in at least one 
GLPHQVLRQZLWKORQJHVWGLDPHWHUPPXVLQJFRQYHQWLRQDOWHFKQLTXHVRU
mm with spiral CT scan.
x Non-measurable lesions: All other lesions, including small lesions (longest 
diameter < 20 mm with conventional techniques or < 10 mm with spiral CT scan), 
bone lesions, leptomeningeal disease, ascites, pleural/pericardial effusion, 
inflammatory breast disease, lymphangitis cutis/pulmonis, cystic lesions, and also 
abdominal masses that are not confirmed and followed by imaging techniques.
3. All measurable lesions up to a maximum of five lesions per organ and ten lesions in total, 
representative of all involved organs should be identified as target lesions and recorded and 
measured at baseline.  Target lesions should be selected on the basis of their size (those with the 
longest diameter) and their suitability for accurate repeated measurements.
4. All measurements should be taken and recorded in metric notation, using a ruler or calipers.  
All baseline evaluations should be performed as closely as possible to the beginning of treatment 
and never more than 4 weeks before the beginning of the treatment.
5. The same method of assessment and the same technique should be used to characterize each 
identified and reported lesion at baseline and during follow-up.
6. Clinical lesions will only be considered measurable when they are superficial (e.g., skin 
nodules and palpable lymph nodes).  For the case of skin lesions, documentation by color 
photography, including a ruler to estimate the size of the lesion, is recommended. 
7. Tumor Response:  
x Complete Response: the disappearance of all target lesions
x Partial Response: at least a 30% decrease in the sum of the longest diameter of all 
target lesions 
x Progressive disease: at least a 20% increase in the sum of the longest diameter of 
all target lesions 
x Stable disease: neither sufficient shrinkage to qualify for partial response nor 
sufficient increase to qualify for progressive disease
From: Therasse, P., et al., New guidelines to evaluate the response to treatment in solid tumors. European 
Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National 
Cancer Institute of Canada. J Natl Cancer Inst, 2000. 92(3): p. 205-16

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11.10APPENDIX 10: FILGRASTIM DOSING FOR DONORS PRIOR TO APHERESIS
Minor Donors: 
7KHGRQRUZLOOUHFHLYHILOJUDVWLPDVDQRXWSDWLHQWDWDGRVHRIȝJNJGD\HDFKPRUQLQJ
by subcutaneous (SQ) injection beginning 4 days prior to scheduled collection. The donor 
and/or a family member will be taught to administer the SQ injections when possible. 
Otherwise, injections will be given in the POB outpatient clinic. The filgrastim should 
be given as early as possible after awakening in the morning. This is especially important 
on days 5, 6, and 7 of the injections. Filgrastim will be continued for 5, 6, or 7 days as 
required to complete collection of the target dose. The donor will continue filgrastim for 
the complete 7-day period, unless notified by the transplant team that the target dose was 
reached before day 7.
Adult Donors (>18 years) :
Timing of dose: Doses for days 1-4 should be given as early as possible after awakening 
in the morning, but the dose for day 5 and if necessary, days 6 and 7 must be given early 
in the morning, at least one hour and preferably two hours prior to starting apheresis. 
Filgrastim is administered according to a vial-based algorithm to reduce wastage. Two 
dosing algorithms are recommended for use in the Clinical Center. The standard dose 
algorithm uses doses in the range of 10 to 12 mcg/kg /day, with a higher dose given to
lighter weight donors to improve CD34 yields.  The higher dose algorithm will be utilized 
in this protocol as it is intended for use in (1) autologous donors who have received prior 
myelotoxic agents; (2) donors whose components will undergo further processing; 
(3) protocols in which a high transplant cell dose is required (CD34 > 8 x106); and
(4) situations with large weight discrepancy between donor and recipient (recipient > 
donor).  The total dose is capped at 1200 mcg/kg/day.  
Higher-Dose Filgrastim Algorithm:
Donor Wt
Total Daily Filgrastim Dose (range)
38 - 48 kg            
600 mcg 
(12.5 to 15.8 mcg/kg)
49 - 56 kg            
780 mcg 
(13.9 to 15.9 mcg/kg) 
57 - 60 kg            
900 mcg 
(15.0 to 15.8 mcg/kg)
61 - 67 kg            
960 mcg 
(14.3 to 15.7 mcg/kg)
68 - 108 kg          
1080 mcg 
(10.0 to 15.9 mcg/kg)
> 109 kg              
1200 mcg 
(11.0 or less)
Predictable side effects of filgrastim, including headache, bone pain, and myalgia, may be 
treated with acetaminophen or ibuprofen.

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11.11APPENDIX 11: ENGRAFTMENT SYNDROME (SPITZER, 2001)
Diagnosis of Engraftment Syndrome (ES) is made based on the presence of all three (#) 
major criteria, or two (2) major criteria with one or more minor criteria.
Major Criteria
x
7HPSHUDWXUHƕC with no identifiable infectious etiology
x
Erythrodermatous rash involving more than 25% of body surface area and not 
attributable to a medication
x
Noncardiogenic pulmonary edema, manifested by diffuse pulmonary infiltrates
consistent with this diagnosis, and hypoxia.
Minor Criteria
x
+HSDWLFG\VIXQFWLRQZLWKHLWKHUWRWDOELOLUXELQPJGORUWUDQVDPLQDVHOHYHOV
two times ULN
x
5HQDOLQVXIILFLHQF\VHUXPFUHDWLQLQHWZRWLPHEDVHOLQH
x
:HLJKWJDLQRIEDVHOLQHERG\weight
x
Transient encephalopathy unexplainable by other causes.
,QDGGLWLRQ(6VKRXOGRFFXUZLWKLQKRXUVRIHQJUDIWPHQWGHILQHGDVDQHXWURSKLOFRXQWRI
500/μL for 2 consecutive days). 

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11.12APPENDIX 12: TBI GUIDELINES
x
TBI will be delivered to a total midplane dose of 12Gy and median lung dose of 6Gy. 
Radiation will be fractionated twice daily over 3 days (days -6, -5, -4). Opposed lateral 
fields with lung compensation will be treated to 2 Gy for each fraction to a total cumulative 
dose of 12Gy. 
x
Mediastinal fields will be treated AP-PA concurrently with the opposed lateral fields at a 
dose of 100cGy twice daily to bring the mediastinal dose to 12Gy with a median lung dose 
of 6 Gy.
x
Fractions will be delivered with at least a 6 hour interfraction interval. Gonadal shielding 
will not be used. Head and neck compensation and other technical modifications may be 
made at the discretion of the treating radiation oncologist.
x
For patients with CNS positive disease at any time after diagnosis, CNS boost may be 
delivered unless contraindicated.  The final decision of appropriateness of delivering the 
boost and boost dose will be made by the PI in consultation with the treating radiation 
oncologist. The CNS boost will be delivered as a 6 Gy boost in once daily 2 Gy fractions. 
The boost will be delivered prior to TBI. 
x
Patients with sites identified by nuclear imaging or other clinically accepted diagnostic 
tests may receive a boost to these areas as clinically indicated at any time point pre- or post 
transplantation.

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11.13APPENDIX 13: CALCULATION OF IDEAL BODY WEIGHT AND DOSE ADJUSTMENTS
Formulation for deriving the weight to be used in dose calculations in morbidly obese 
candidates.
1.
Definition
Obesity is defined as a > 125% of IBW.
2.
Calculation of ideal body weight is performed using the standard, published formula:
Male: 50 + 2.3(Hgt - 60) where Hgt is in inches, and the result is expressed in kg. 
ex. The ideal weight of a 5'10" male = 50 + 2.3(10) = 73 kg. 
Female: 45.5 + 2.3(Hgt - 60), where height is in inches, and the result is in kg.
3. Dose adjustments for pre-transplant myeloablative chemotherapy will be based on an adjusted 
weight. 
Adjusted weight =1.25 x IBW
4.  Calculation of the “practical weight.”
Calculate the midway point, halfway between the actual and ideal body weights (ie the 
average of the two numbers). This is the "practical weight" which may be used for
calculating the targeted cell dose.
Example:
Patient’s actual weight = 143 kg.
Patient’ s actual height 173 cm = 69 in
IBW formula = 50 + 2.3(9) = 70.7 kg
Midway point between 70.0 and 143 = 107 kg.
The weight we would use in targeting cell dose is 107 kg.