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1 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
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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
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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.
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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 doselimiting 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 (preselection)
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 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.  
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.  
| 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                                              |
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pre-bmt preparative regimen (myeloablative)

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                                      |  
Stem Cell Infusion Transplant Day 0: >4 x 106/kg CD34+ stem cells by IV infusion
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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.
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5

| 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   |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
6  
| Version Date: 07/23/2018 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   |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
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 7 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   |                                   |
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| 4                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | SUPPORTIVE CARE 73                |
| 4.1                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Infection Prophylaxis 73          |
| 4.2                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Mucositis Prophylaxis 73          |
| 5                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | DATA COLLECTION AND EVALUATION 73 |  
| 8                                                                                                                |                                                                                                                                   |
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| Abbreviated Title: NK cell therapy                                                                               |                                                                                                                                   |
| Version Date: 07/23/2018                                                                                         |                                                                                                                                   |
| 5.1                                                                                                              | DATA COLLECTION                                                                                                                   |
| 5.2                                                                                                              | Response Criteria                                                                                                                 |
| 5.3                                                                                                              | TOXICITY Criteria                                                                                                                 |
| Data Reporting Exceptions                                                                                        |                                                                                                                                   |
| 5.4                                                                                                              |                                                                                                                                   |
| 6                                                                                                                | STATISTICAL CONSIDERATIONS ....................................................................................................75 |
| 6.1                                                                                                              | SUBJECT Accrual                                                                                                                   |
| 6.2                                                                                                              | Statistics and Feasibility .                                                                                                      |
| 6.3                                                                                                              | Early Stopping Rules                                                                                                              |
| 6.4                                                                                                              | Subject Accrual Goal .                                                                                                            |
| 7                                                                                                                | HUMAN SUBJECTS PROTECTIONS .                                                                                                      |
| 7.1                                                                                                              | Rationale for subject selection                                                                                                   |
| 7.1.1 Recipients .                                                                                               |                                                                                                                                   |
| 7.1.2 Donors .                                                                                                   |                                                                                                                                   |
| 7.2                                                                                                              | Participation of Children                                                                                                         |
| 7.3                                                                                                              | Evaluation of benefits and risks/discomforts................................................................ 77                   |
| 7.3.1 Recipients .                                                                                               |                                                                                                                                   |
| 7.3.2 Donors .                                                                                                   |                                                                                                                                   |
| Risk/Benefit Analysis                                                                                            |                                                                                                                                   |
| 7.4                                                                                                              |                                                                                                                                   |
| 7.5                                                                                                              | Consent Processes and Documentation                                                                                               |
| 7.5.1 Recipients .                                                                                               |                                                                                                                                   |
| 7.5.2 Related donors .                                                                                           |                                                                                                                                   |
| 7.5.3 Unrelated Donors .                                                                                         |                                                                                                                                   |
| 7.5.4 Consent for minors when they reach the age of majority................................................. 81 |                                                                                                                                   |
| 7.5.5 Telephone consent                                                                                          |                                                                                                                                   |
| 8                                                                                                                | SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING                                                                          |
| PLAN 82                                                                                                          |                                                                                                                                   |
| 8.1                                                                                                              | Definitions                                                                                                                       |
| 82                                                                                                               |                                                                                                                                   |
| 8.1.2 Suspected adverse reaction                                                                                 |                                                                                                                                   |
| 8.1.3 Unexpected adverse reaction                                                                                |                                                                                                                                   |
| 8.1.4 Serious                                                                                                    |                                                                                                                                   |
| 8.1.5 Serious adverse events .                                                                                   |                                                                                                                                   |
| 8.1.6 Disability .                                                                                               |                                                                                                                                   |  
9  
| 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  
| 83                                                                                                                                                                                                                                                                                                                                                                                               |                                                                 |
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| 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                                                                                                                               |                                                                 |  
| Version Date: 07/23/2018 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 11.10Appendix 10: Filgrastim Dosing for Donors Prior to Apheresis 143 11.11Appendix 12: Engraftment Syndrome (Spitzer, 2001) 144 11.12Appendix 13: TBI Guidelines 145 11.13Appendix 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 donorderived 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-versushost-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 HLAmatched allogeneic PBSCT followed by NK-DLI.  
2. Investigate the incidence of viral infection and/or reactivation in patients receiving HLAmatched 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.
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1.2 background and rationale 1.2.1

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 535%. 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 longterm 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, antiVEGF) 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. 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 graftversus-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(1216). 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 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-crossresistant, 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, 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 1**a shows the survival of all patients enrolled on 02-C-0259 from the time of enrollment using an intent-to-treat approach. **Figure 1**b 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-versustumor effects.  
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 nonHodgkin'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-C0259. 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 patient relapsed prior to day 100 (day 95). A graft-vs.-leukemia effect was clinically demonstrated 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
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1.2.3.1 ewing sarcoma (this study

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).  
1.2.3.3
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neuroblastoma (this study is no

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:  
| Group Assignment Schema Used for COG-9641 and COG-A3961 Studies a                                                                               |              |              |                     |              |              |
|-------------------------------------------------------------------------------------------------------------------------------------------------|--------------|--------------|---------------------|--------------|--------------|
| INSS Stage                                                                                                                                      | Age          | MYCN Status  | INPC Classification | DNA Ploidy b | Risk Group   |
| 0–21 y                                                                                                                                          | Any          | Any          | Any                 | Low          |              |
| 2A/2B                                                                                                                                           | <365 d       | Any          | Any                 | Any          | LOW          |
| 2365 d–21 y                                                                                                                                     | Nonamplified | Any          | Low                 |              |              |
| 2365 d-21 y                                                                                                                                     | Amplified    | Favorable    | Low                 |              |              |
| 2365 d–21 y                                                                                                                                     | Amplified    | Unfavorable  | High                |              |              |
| 3                                                                                                                                               | <365 d       | Nonamplified | Any                 | Any          | Intermediate |
| < 365 d                                                                                                                                         | Amplified    | Any          | Any                 | High         |              |
| 2365 d-21 y                                                                                                                                     | Nonamplified | Favorable    | Intermediate        |              |              |
| 2365 d–21 y                                                                                                                                     | Nonamplified | Unfavorable  | High                |              |              |
| 2365 d-21 y                                                                                                                                     | Amplified    | Any          | High                |              |              |
| <548 d [13-15]                                                                                                                                  | Nonamplified | Any          | Intermediate        |              |              |
| a                                                                                                                                               | Any          |              |                     |              |              |
| < 365 d                                                                                                                                         | Amplified    | Any          | Any                 | High         |              |
| 2548 d–21 y                                                                                                                                     | Any          | Any          | High                |              |              |
| 4S°                                                                                                                                             | <365 d       | Nonamplified | Favorable           | >            | Low          |
| <365 d                                                                                                                                          | Nonamplified | AN           | =1                  | Intermediate |              |
| <365 d                                                                                                                                          | Nonamplified | Unfavorable  | Any                 | Intermediate |              |
| <365 d                                                                                                                                          | Amplified    | Any          | Any                 | High         |              |
| The COG-9641 and COG-A3961 trials established the current standard of care for neuroblastoma patients in terms of risk group                    |              |              |                     |              |              |
| ent and treatment                                                                                                                               |              |              |                     |              |              |
| ° DNA Pioidy: DNA Index (DN > 1 is favorable, = 1 is unfavorable; hypodiploid tumors (with DI < 1) will be treated as a tumor with a DI > 1 (DI |              |              |                     |              |              |
| < 1 [hypodiploid] to be considered favorable ploidy;                                                                                            |              |              |                     |              |              |
| immediate chemotherapy for four cycl                                                                                                            |              |              |                     |              |              |
| 6 INSS stage 3 or stage 4 patients with clinical symptoms as listed above should receive immediate chemotherapy                                 |              |              |                     |              |              |
| ° INSS stage 4S infants with favorable biology and clinical symptoms should be treated with immediate chemotherapy until asymptomatic           |              |              |                     |              |              |
| (2–4 cycles). Clinical symptoms include: respiratory distress with or without hepatomegaly or cord compression and neurologic deficit or        |              |              |                     |              |              |
| inferior vena cava compression and renal ischemia; or genitourinary obstruction; or gastrointestinal obstruction and vomiting; or               |              |              |                     |              |              |
| coagulopathy with significant clinical hemorrhage unresponsiveeplacement therapy                                                                |              |              |                     |              |              |  
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 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 granulocytemacrophage 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).
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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 (4244, 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.  
  
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 ,
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**.
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figure 3

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 nontransplant 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).
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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 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).
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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
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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). 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).
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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).
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1.2.4 matched unrelated donor transplant

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 (HLAA, 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).  
| Table 1) Unrelated Donor Bone Marrow Transplantation: Pediatric Series Patients (n) Probability  of myeloid  engraftment aGVHD aGVHD cGVHD Transplant  Related  Mortality   | Reference   |     |     |             |             |                        |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------|-----|-----|-------------|-------------|------------------------|
| grades                                                                                                                                                                      | grades      |     |     |             |             |                        |
| 2-4                                                                                                                                                                         | 3-4         |     |     |             |             |                        |
| MUD (46)                                                                                                                                                                    | 100%        | 83% | 37% | 60%         | 24%         | Balduzzi 1995          |
| 1 MMUD (42)                                                                                                                                                                 | 98%         | 62% | 69% | 51%         |             |                        |
| MUD (8)                                                                                                                                                                     | 83%         | 63% | 37% | 54%         | N/A         | Davies BJH 1997J       |
| 1 MMUD (9) MUD (30)                                                                                                                                                         | 93%         | 49% | 23% | 50%         | Day 100 NRM | Davies JCO 1997        |
| 36%                                                                                                                                                                         |             |     |     |             |             |                        |
| 1 MMUD (20)                                                                                                                                                                 | 67%         | 30% | 55% |             |             |                        |
| MUD (28)                                                                                                                                                                    | N/A         | 64% | 32% | 57%         | 11%         | Saarinen-Pihkala  2001 |
| MRD (37)                                                                                                                                                                    | N/A         | 38% | 14% | 26%         | 19%         |                        |
| 0-1 MMUD (363)                                                                                                                                                              | 98%         | 47% | 29% | 39%         | Day 100 27% | Bunin 2002             |
| 5-year 42%                                                                                                                                                                  |             |     |     |             |             |                        |
| MUD (56)                                                                                                                                                                    | 98%         | 85% | 43% | 58%         | 35%         | Woolfrey 2002          |
| MMUD (32)                                                                                                                                                                   | 59%         |     |     |             |             |                        |
| MUD (33)                                                                                                                                                                    | 97%         | 40% | 7%  | 20%         | Day 100 10% | Giebel 2003\           |
| 1 MMUD (22)                                                                                                                                                                 | 42%         | 9%  | 34% | Day 180 15% |             |                        |
| 2-3 MMUD(11)                                                                                                                                                                | 37%         | 9%  | 40% | Overall 22% |             |                        |
| 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                                |             |     |     |             |             |                        |  
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). 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 transplantrelated 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 5year 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 (19891998 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).  
  
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 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 Bcell 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 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).  
RISCT PBSC CSA 7.6e6 1e5 59 (II-III) n/a Bishop2  
| GVHD          |          |        |                    |                            |        |    |                |
|---------------|----------|--------|--------------------|----------------------------|--------|----|----------------|
| Prophylaxis   | CD 34+   | CD 3 + | Rates of  aGVHD  % | Rates of  aGVHD  (III-IV)% | Author |    |                |
| Cells/kg      | Cells/kg |        |                    |                            |        |    |                |
| Myeloablative | BM       | ATG/MP | n/a                | n/a                        | 5 (I)  | 0  | Papadopoulos48 |  
| Preparative   | Stem Cell   |
|---------------|-------------|
| Regimen       | Source      |  
Myeloablative PBSC CSA 5.8e6 1e5 54 (II-IV) 15 Montero49 RISCT PBSC MMF 17.4e6 1.4e5 38 (II-III) 9 Chen55  
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
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1.2.6 gvhd prophylaxis after t

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 fullhaplotype 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 posttransplantation, and only one developed cGVHD(179).  
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.
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1.3 pre-clinical models of natural

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 immunoglobulinlike 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.
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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 cells(12). In addition, genetically modified NK-92 cells expressing a CAR specific for the tumorassociated 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 NK92 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 GVHDassociated 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 mice if the treatment was started 7 days after tumor challenge, whereas waiting 21 days diminished the benefit(186).
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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).
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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).
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1.3.5 natural killer cells and

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. 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, donorderived 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 posttransplant 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.  
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.
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1.3.6 natural killer cells and

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 cellreplete 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.
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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.
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1.3.8 adoptive immunotherapy with nk

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 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.
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1.3.9 ex-vivo expansion of nk

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 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-2activated 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 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 cocultures 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 1.4 summary reduced

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
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1.4.1.1 enrollment updates (as of

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, NKcells 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.
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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.  
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 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.  
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  
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.  
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 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 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, 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 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 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 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 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 posttransplant.  
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.  
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 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.  
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 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 MRD negative complete remission at 6 months post transplant. He also has attained full donor chimerism.  
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 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 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 riskbenefit ration for these patients to be too great.
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 ulikely 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.
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1.4.1.3 rationale for closing accrual

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.  
| Best Response   |                 |             |           |               |             |               |                |               |              |                      |                 |
|-----------------|-----------------|-------------|-----------|---------------|-------------|---------------|----------------|---------------|--------------|----------------------|-----------------|
| wing transplan  |                 |             |           |               |             |               |                |               |              |                      |                 |
| Patient         | Gender          | Age         | Race      | Disease       | Donor       | NK Cell Dose  | acute GVHD     | hronic GVHD   | +NK cells    | Relapse              | Status          |
| 2 y             | Ewing's         | N/A         | N/A       | N/A           | N/A         | N/A           | N/A            | N/A           |              |                      |                 |
| 1 x 10e5/kg     |                 |             |           |               |             |               |                |               |              |                      |                 |
| 34 y            | c               | DSRCT       | MUD       | Grade IV GVHD | Non         | PR            | Dy 100         | DoD day + 190 |              |                      |                 |
| 3 y             | MSD             | 1 x 10eS/kg | PR        | DoD day + 171 |             |               |                |               |              |                      |                 |
| M               | c               | Ewing's     | Non       | Day 70        |             |               |                |               |              |                      |                 |
| M               | 2 y             | C           | DSRCT     | MUD           | 1 x 10e5/kg | Grade II GVHD | None           | CR            | NED          | DoSepsis - day + 191 |                 |
| 5               | M               | 16 y        | c         | Ewing s       | MUD         | 1 x 10e5/kg   | Grade III GVHD | None          | Remained NED | NED                  | NED             |
| 1 x 10e5/kg     | On chemotherapy |             |           |               |             |               |                |               |              |                      |                 |
| 6               | M               | 24 y        | s         | DSRCT         | MSD         | None          | None           | CR/NED by PET | Day 100      |                      |                 |
| M               | 18 y            | c           | ARMS      | MSD           | 1 x 10c5/kg | None          | None           | Remained NED  | Day 85       | On chemotherapy      |                 |
| 8               | M               | 14 y        | c         | Ewing s       | MSD         | 1 x 10eS/kg   | None           | None          | Remained NED | Day 93               | On chemotherapy |
| 9               | F               | 15 y        |           | RMS           | N/A         | N/A           | N/A            | N/A           | N/A          | N/A                  | N/A             |
| 10              | M               | 9 y         | C         | Ewing's       | N/A         | N/A           | N/A            | N/A           | N/A          | N/A                  | N/A             |
| 11              | M               | 15 y        | c         | Ewing's       | MUD         | 1 x 10e5/kg   | Grade IV GVHD  | None          | Remained NED | Day 156              | On chemotherapy |
| 12              | M               | 18 y        | c         | DSRCT         | MUD         | 1 x 10eS/kg   | None           | Remained NED  | NED          | NED                  |                 |
| Non             |                 |             |           |               |             |               |                |               |              |                      |                 |
| 13              | M               | 13 y        | c         | Ewing S       | N/A         | N/A           | N/A            | N/A           | N/A          | N/A                  | N/A             |
| 14              | F               | 21 y        | c         | Ewing s       | MSD         | 1 x 10e6/kg   | iver GVHD®     | TO EARLY      | ained NED    | NED                  | NED             |
| N/A             | TOO EARLY       | N/A         | TOO EARLY | N/A           | n/a         |               |                |               |              |                      |                 |
| 15              | M               | 26 y        | c         | ARMS          | N/A         |               |                |               |              |                      |                 |
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1.4.1.4 rationale for increasing t-cell

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 a higher T-cell add back (1-2 x 105 CD3/kg) in the initial stem cell product. This represents a onelog 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**).
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2 eligibility assessment and enrollment

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 GHOTRU+5)/7,7'EXWZLWKHYLGHQFHRIUHVLGXDO$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.
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exceptions

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 &DUGLDFIXQFWLRQ/HIWYHQWULFXODUHMHFWLRQIUDFWLRQE\08*$ or ECHO,
IUDFWLRQDOVKRUWHQLQJE\(&+2.
2.1.1.10 3XOPRQDU\IXQFWLRQ'/&2RIWKHH[SHFWHGYDOXHFRUUHFWHGIRUDOYHolar volume and hgb for reduced intensity transplant and '/&255% 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 /LYHUIXQFWLRQ6HUXPWRWDOELOLUXELQPJGOVHUXP$67DQG$/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 RUDFUHDWLQLQHFOHDUDQFHPOPLQ/1.73 m2.  
| Age (years)   | Maximum serum creatinine (mg/dl)   |
|---------------|------------------------------------|
| d5            | 0.8                                |
| >5 d 10       | 1.0                                |
| >10 d 15      | 1.2                                |
| > 15          | 1.5                                |  
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)
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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.
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2.2 eligibility criteria for donors

2.2.1 Inclusion Criteria: Donor 2.2.1.1 :HLJKWNLORJUDPVDQGIRUXQUHODWHGGRQRUV> 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.
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2.3 research eligibility evaluation

2.3.1 Clinical Evaluation Recipient  
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 'XUDEOHSRZHURIDWWRUQH\IRUPFRPSOHWHGSDWLHQWV\HDUVRIDJH
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 HLAA, 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 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.
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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.
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3 study implementation 3.1 study

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 nonmyeloablative 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 nonmyeloablative 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 GRVHFRKRUWZLOOEH\HDUVRIDJH,IWKe 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- '/,,IWKHILUVWSDWLHQWDGXOWLQDFRKRUWH[SHULHQFHV'/7WKHDGGLWLRQDOSDWLHQWV\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 DLINK 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

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    | Cohort 2: Patients with Unrelated     |
|-------------------------------|------------------------------------|---------------------------------------|
| Donors                        | Donors                             |                                       |
| Dose Level 1                  | 1 X 105 NK Cells/kg infused on day | 1 X 105 NK Cells/kg infused on day 21 |
| 7 (± 3d), 35 (± 7d) COMPLETED | (± 3d), 49 (± 7d)                  |                                       |
| Dose Level 2                  | 1 X106 NK Cells/kg infused on day  | 1 X106 NK Cells/kg infused on day 21  |
| 21 (± 3d), 49 (± 7d)          | (± 3d), 49 (± 7d)                  |                                       |
| Dose Level 3                  | 1 X 107 NK Cells/kg infused on day | 1 X 107 NK Cells/kg infused on day 21 |
| 21 (± 3d), 49 (± 7d)          | (± 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
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.
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related donor stem cell and

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.
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filgrastim administration

Minor donors ZLOOUHFHLYHILOJUDVWLPDVDQRXWSDWLHQWDWDGRVHRIȝJNJGD\HDFKPRUQLQJE\ 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 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, 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.
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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.
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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.