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Abbreviated Title: RIST using Unrelated Donors NCI Protocol Number: 07-C-0195 X Version Date: September 14, 2018 NCT Number: NCT00520130 PROTOCOL TITLE: Phase II Trial of Targeted Immune-Depleting Chemotherapy and Reduced-Intensity Allogeneic Hematopoietic Stem Cell Transplantation Using 8/8 and 7/8 HLAmatched Unrelated Donors and Utilizing Two Graft-versus-Host Disease Prophylaxis Regimens for the Treatment of Leukemias, Lymphomas, and Pre-malignant Blood Disorders NCI Principal Investigator: Steven Pavletic, MD, MS, ETIB, CCR
10 Center Drive, Room 4-3130 Bethesda, MD 20892 Telephone: 240-760-6174 E-mail: pavletis@mail.nih.gov Investigator Roles:
A. Obtain information by intervening or interacting with living individuals for research purposes B. Obtaining identifiable private information about living individuals C. Obtaining the voluntary informed consent of individuals to be subjects D. Makes decisions about subject eligibility E. Studying, interpreting, or analyzing identifiable private information or data/specimens for research purposes F. Studying, interpreting, or analyzing coded (linked) data or specimens for research purposes G. Some/all research activities performed outside NIH
Commercial Agents: Cyclophosphamide, Mesna, Fludarabine, Etoposide, Vincristine, Doxorubicin, Rituximab, Cytarabine, Methotrexate, Sirolimus, Tacrolimus, Alemtuzumab 1
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precis background

- The major limitations to the broader applicability of allogeneic hematopoietic stem cell transplantation (HSCT) for the treatment of malignancies are lack of suitable donors and therapy-related toxicities which include delayed and incomplete immune reconstitution and graft-versus-host disease (GVHD). Based on the theory that the rapid establishment of donor chimerism was essential for an optimal "graft-versus-tumor" effect, we have employed a strategy of targeted immune-depleting chemotherapy prior to reduced-intensity allogeneic HSCT. It is our intent to investigate this approach in the setting of HLA-matched unrelated donors in a pilot manner.  
- A clearly superior GVHD prophylaxis regimen has not been established in the unrelated donor transplant setting. The best results that have been reported are with the combination of alemtuzumab plus cyclosporine [AC] and the combination of tacrolimus, methotrexate, and sirolimus [TMS]. These two regimens work by mechanisms which are biologically distinct and potentially have markedly different effects upon immune reconstitution that have not been well studied. In addition neither of these regimens has been assessed for their effects on chronic GVHD using the NIH Consensus Conference Criteria. It is our intent to study the effects that these two regimens have on immune reconstitution and chronic GVHD in the setting sequential targeted immune-depleting chemotherapy and reduced-intensity allogeneic HSCT from HLA-matched unrelated donors.  
Objectives:
- Primary objectives:
1) To assess the effects of two biologically distinct GVHD prophylaxis regimens, TMS and AC, on immune reconstitution in patients receiving targeted-immune depletion and reduced-intensity allogeneic HSCT from HLA-matched unrelated donors. As part of a comprehensive assessment of immune reconstitution, the primary immunologic endpoint will be the determination of CD4+ T cell receptor Vβ repertoire by CDR3 spectratyping at 3 months, 6 months, and 12 months post-transplant. Samples will be collected on each of the patients enrolled in the study. However, due to the time and cost involved in the spectratyping analysis, a comprehensive assessment will initially be limited to the first 10 patients on each arm receiving 10 of 10 HLA-matched unrelated donors After completion of this assessment, it will be determined whether or not the analysis should be continued.  
2) To assess overall safety of these two regimens in this setting, as determined by engraftment, acute GVHD, early and late treatment-related mortality, and overall survival.  
3) To determine and monitor incidence, organ severity and overall severity of chronic GVHD prospectively using the newly developed NIH Consensus Conference diagnosis and staging criteria and preliminarily validate those tools for use in clinical practice and trials (Pavletic, Imanguli, and Cowen).  
Eligibility:
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- Adults (18 - 74 years) with advanced or high risk hematologic malignancies including AML, ALL, MDS, CLL, NHL, HL, CML, multiple myeloma, and MPD who lack a suitable HLA-matched sibling.  
- An unrelated donor matched at a minimum of 7 of 8 alleles (HLA-A,-B,-C, and DRB1)
by high-resolution typing, identified through the National Marrow Donor Program.  
- Life expectancy of at least 3 months, ECOG < 2 and relatively normal major organ functions.
Design:
- Patients will receive disease-specific induction chemotherapy (EPOCH-F/R or FLAG)
prior to transplant for disease control and immune depletion. If disease is controlled (≥ SD) and immune depletion objectives have been met, patients may forgo induction chemotherapy and move forward to the transplant conditioning regimen.  
- All patients will receive an identical conditioning regimen consisting of cyclophosphamide 1200 mg/m2/day IV for 4 days and fludarabine 30 mg/m2/day for 4 days.  
- Patients will be stratified according to degree of HLA-match and randomized at the time of enrollment to one of two GHVD prophylaxis regimens:
➢ Arm A (TMS): Tacrolimus, starting day -3 before transplant, given initially at 0.02 mg/kg/day CIV and then an equivalent oral dose (when patient taking po) titrated for a goal level of 5-10 ng/ml; sirolimus given as an initial loading dose of 12 mg p.o. on day -3 pre-transplant, 4 mg starting day -2 pre-transplant titrated for levels 3-12 ng/ml; and methotrexate 5 mg/m2IV on days +1, +3, +6, and +11 post-transplant.  
Tacrolimus and sirolimus will be tapered at day +63, day +119 and day +180 posttransplant as tolerated.  
➢ Arm B (AC): Alemtuzumab at 20 mg/day IV over 8 h on days −8 to −4 pretransplant and cyclosporine, starting day -1 before transplant, given initially at 2 mg/kg IV every 12 hours and then an equivalent oral dose (goal level 150-250 ng/ml) until day 100. Cyclosporine will be tapered by 10% per week between day 100 and six months).
- A maximum of 105 patients will be enrolled and randomly assigned to the two arms in order to yield 44 patients per arm (88 total patients) who are able to be evaluated for development of severe chronic GVHD. This represents an increase of 20 patients over the number permitted prior to this amendment: 25 patients (50 total) randomly assigned to each arm within the 8/8 match group and 13/arm (26 total) within the 7/8 match group (maximum study enrollment = 76 transplanted patients from among up to 85 patients registered).  
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| TABLE OF CONTENTS PRÉCIS 2 TABLE OF CONTENTS 4 1 INTRODUCTION 8 1.1 Study Objectives 8 1.2 Background and Rationale 9 1.3 Primary Aims 21 1.4 Background on Secondary Objectives 23 1.5 Summary 24 1.6 Amendment K Background and Rationale 25 2 ELIGIBILITY ASSESSMENT AND ENROLLMENT 26 2.1 Eligibility Criteria - Recipient on Standard Care Therapy: 26 2.2 Exclusion Criteria - Recipient on Standard Care Therapy: 26 2.3 Inclusion Criteria - Recipient 27 2.4 Exclusion Criteria - Recipient 31 2.5 Inclusion Criteria - Donor 32 2.6 Exclusion Criteria - Donor 33 2.7 Research Eligibility Evaluation - Recipient 33 2.8 Research Eligibility Evaluation - Donor 34 2.9 Patient Registration 35 3 STUDY IMPLEMENTATION 37 3.1 Study Design 37 3.2 Donor Stem Cell Mobilization and Collection (as per NMDP Guidelines) 38 3.3 The following items should be obtained within 48 hours before starting induction  chemotherapy: 39 3.4 The following tests may be performed at any time prior to Treatment Arm enrollment39 3.5 Induction Chemotherapy for the Recipient 39 3.6 Transplant Conditioning Regimen 47 3.7 Allogeneic Peripheral Blood Stem Cell Transplantation 49 3.8 Graft-Versus-Host Disease (GVHD) Prophylaxis 50 3.9 Evaluation During Transplantation 52 3.10 Off Study Criteria 53 4   |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| Version Date: September 14, 2018.      |                                                                                      |
|----------------------------------------|--------------------------------------------------------------------------------------|
| 3.11                                   | Post-Transplantation Evaluation                                                      |
| 4                                      | SUPPORTIVE CARE                                                                      |
| 4.1                                    | Infection Prophylaxis                                                                |
| 4.2                                    | Management of Engraftment Syndrome .                                                 |
| 4.3                                    | Treatment Of Mixed Chimerism Or Persistent/Progressive Disease Post-Transplant... 56 |
| Treatment of Graft-Versus-Host Disease |                                                                                      |
| 4.4                                    |                                                                                      |
| 4.5                                    | Menses Suppression and Contraception                                                 |
| 4.6                                    | Blood Product Support                                                                |
| Nutritional Support                    |                                                                                      |
| 4.7                                    |                                                                                      |
| 4.8                                    | Anti-emetics .                                                                       |
| 4.9                                    | Hematopoietic Growth Factor Support                                                  |
| S                                      | BIOSPECIMEN COLLECTION                                                               |
| 5.1                                    | Correlative Studies for Research                                                     |
| 6                                      | DATA COLLECTION AND EVALUATION .                                                     |
| DATA COLLECTION                        |                                                                                      |
| 6.1                                    |                                                                                      |
| 6.2                                    | Response Criteria                                                                    |
| 6.3                                    | Toxicity Criteria                                                                    |
| 6.4                                    | Statistical Considerations .                                                         |
| 7                                      | HUMAN SUBJECT PROTECTIONS                                                            |
| 7.1                                    | Rationale for Subject Selection                                                      |
| 7.2                                    | Participation of Children                                                            |
| 7.3                                    | Participation of NIH Subjects Unable to Give Consent                                 |
| 7.4                                    | Risks/Benefits Analysis                                                              |
| 7.5                                    | Informed Consent Process and Documentation .                                         |
| 8                                      | SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN                        |
| 77                                     |                                                                                      |
| 8.1                                    | Definitions                                                                          |
| 8.2                                    | NCI-IRB and Clinical Director Reporting                                              |
| 8.3                                    | DATA SAFETY AND MONITORING Plan                                                      |
| 8.4                                    | Record Keeping                                                                       |
| 9                                      | PHARMACEUTICAL INFORMATION                                                           |
| 9.1                                    | CYCLOPHOSPHAMIDE (CTX, Cytoxan, NSC-26271)                                           |  
| 9.2                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | MESNA (Sodium 2-mercaptoethanesulfonate, Mesnum, Mesnex, NSC-113891) 81   |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------|
| 9.3                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | FILGRASTIM (G-CSF, Neupogen) 81                                          |
| 9.4                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | CYCLOSPORINE (Gengraf, Sandimmune, Neoral) 82                             |
| 9.5                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | FLUDARABINE (Fludara, Berlex Laboratories) 83                             |
| 9.6                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | ETOPOSIDE, DOXORUBICIN, and VINCRISTINE (in EPOCH) 84                     |
| 9.7                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | PREDNISONE 85                                                             |
| 9.8                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | RITUXIMAB (Rituxan) 85                                                    |
| 9.9                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | CYTARABINE (Cytosine Arabinoside, Cytosar, ARA-C) 87                      |
| 9.10                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | DIPHENHYDRAMINE 88                                                        |
| 9.11                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | ACETAMINOPHEN 89                                                          |
| 9.12                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | VALACYCLOVIR (Valtrex®) 89                                                |
| 9.13                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | FLUCONAZOLE (Diflucan®) 89                                                |
| 9.14                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | TRIMETHOPRIM/SULFAMETHOXAZOLE (TMP/SMX, Cotrimoxazole, Bactrim,           |
| Septra)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | 90                                                                        |
| 9.15                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | FUROSEMIDE (Lasix) 91                                                     |
| 9.16                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | METHOTREXATE (Methotrexate sodium, MTX, NSC-740) 91                       |
| 9.17                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | SIROLIMUS (Rapamycin) (Rapamune®, Wyeth-Ayerst Laboratories) 91           |
| 9.18                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | Alemtuzumab (CAMPATH-1H, Campath®) 92                                     |
| 9.19                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | Ursodeoxycholic acid (Ursodiol, Actigall®) 93                             |
| 9.20                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | TACROLIMUS (FK-506, Prograf®) 94                                          |
| 10                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           | REFERENCES 96                                                             |
| 11                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           | APPENDICES 107                                                            |
| 11.1                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | Appendix A - PROPHYLAXIS and Treatment of Infectious Complications in     |
| Allogeneic HSCT Recipients 107 11.2 Appendix B: Management of Engraftment Syndrome 114 11.3 Appendix C - Grading and Management of Acute Graft-Versus-Host Disease 116 11.4 Appendix D - NMDP Standards (20th Edition) and NMDP Standard Policies and  Procedures 118 11.5 Appendix E - NMDP Peripheral Blood Stem Cell Collection Protocol 118 11.6 Appendix F - Chronic GVHD Score Sheet [57] 119 11.7 Appendix G - Experimental Transplantation and Immunology Branch Preclinical  Service Policy for Sample Handling 123 11.8 Appendix H: Flow Sheet 125 |                                                                           |  
| 11.9   | Appendix I: Risk of relapse Assessment 128   |
|--------|----------------------------------------------|
| 11.10  | Appendix J: Comobidity Index 132             |
| 11.11  | Appendix K Case Report Forms 141             |  
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1 introduction 1.1 study objectives

1.1.1.1 To assess in a pilot manner the effects of two biologically distinct graft-versushost disease (GVHD) prophylaxis regimens (tacrolimus/methotrexate/sirolimus
[TMS] and alemtuzumab/cyclosporine [AC]) on immune reconstitution in patients receiving targeted-immune depletion and reduced-intensity allogeneic hematopoietic stem cell transplantation (HSCT) from HLA-matched unrelated donors. As part of a comprehensive assessment of immune reconstitution, the primary immunologic endpoint will be the determination of CD4+ T cell receptor Vβ repertoire by CDR3 spectratyping at 3 months and 12 months posttransplant (Friedman and Korngold). This data will be complemented by additional analyses on immune reconstitution as defined by the first five secondary objectives.  
1.1.1.2 To assess in a pilot manner the overall safety, as determined by engraftment, severe (Grade III-IV) acute GVHD, early and late treatment-related mortality, and overall survival, of targeted sequential immune-depleting chemotherapy followed by reduced-intensity allogeneic HSCT from HLA-matched unrelated donors in the treatment of patients with advanced hematologic malignancies.  
1.1.1.3 To determine and monitor incidence, organ severity and overall severity of chronic GVHD prospectively using the NIH Consensus Conference diagnosis and staging criteria and preliminarily validate those tools for use in clinical practice and trials (Pavletic, Imanguli, and Cowen).
1.1.2 Secondary Objectives:  
1.1.2.1 To assess effects of two different GVHD prophylaxis regimens, TMS and AC,
on CD4+ T cell receptor Vβ repertoire by CDR3 spectratyping at 1, 3, 6, and 12 months post-transplant (Hakim Lab).  
1.1.2.2 To assess effects of two different GVHD prophylaxis regimens, TMS and AC,
on CD8+ T cell receptor Vβ repertoire by CDR3 spectratyping at 1, 3, 6, and 12 months post-transplant (Hakim Lab).  
1.1.2.3 To assess effects of two different GVHD prophylaxis regimens, TMS and AC,
on the kinetics of CD4+ and CD8+ T-cells and NK cell depletion and recovery following the use of two different GVHD prophylaxis regimens, TMS and AC, in the setting of reduced-intensity allogeneic HSCT from HLA-matched unrelated donors (Hakim, Gress, and Hardy).  
1.1.2.4 To correlate serum interleukin-7 and interleukin-15 levels during early immune reconstitution after two different GVHD prophylaxis regimens, TMS and AC, in the setting of sequential targeted immune-depleting chemotherapy followed by reduced-intensity allogeneic HSCT from HLA-matched unrelated donors (Mackall and Hakim).  
1.1.2.5 To characterize the pattern of post-transplant CD14+ monocyte production of inflammatory cytokines IL-1-α and TNF-α following the use of two different GVHD prophylaxis regimens, TMS and AC, in the setting of reduced-intensity allogeneic HSCT from HLA-matched unrelated donors (Hakim lab).  
1.1.2.6 To assess the impact of two different GVHD prophylaxis regimens (TMS and AC) on donor/recipient chimerism in the setting of sequential targeted immunedepleting chemotherapy followed by reduced-intensity allogeneic HSCT from HLA-matched unrelated donors.  
1.1.2.7 To determine the incidence and severity of acute GVHD following sequential targeted immune-depleting chemotherapy and reduced-intensity allogeneic HSCT from HLA-matched unrelated donors using two different GVHD prophylaxis regimens, TMS and AC.  
1.1.2.8 To evaluate the feasibility of isolating and expanding clinically relevant numbers of tumor derived lymphocytes from patients' bone marrow or lymph nodes (Hardy).  
1.1.2.9 To prospectively evaluate the pathogenesis of chronic GVHD by examining the immune cell reconstitution in the target tissues and cellular and molecular events preceding and coinciding with the clinical onset of chronic GVHD (Imanguli, Mays, Cowen, Gress, Hakim and Pavletic).  
1.1.2.10 To prospectively assess long and short term cardiac toxicities of EPOCH-F in patients who have received greater than 450mg/m2of anthracyclines prior to study enrollment.
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1.2 background and rationale

Allogeneic hematopoietic stem cell (a.k.a. bone marrow) transplantation (HSCT) is capable of producing sustained remissions in a number of hematologic malignancies. However, therapyrelated toxicities, primarily related to the conditioning regimen and GVHD, and the availability of suitable allogeneic stem cell donors have limited the broader application of allogeneic HSCT.
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1.2.1 reduced-intensity conditioning regimens in

Early studies of allogeneic HSCT utilized conditioning regimens containing high doses of alkylating agents often in combination with total body irradiation [1]. The use of such regimens, which are considered "myeloablative", was perceived as essential both for eradication of malignant disease and for suppression of the host-versus-graft response to the donor stem cells (i.e. rejection). These myeloablative conditioning regimens caused severe damage to normal organs within the body resulting in a high degree and incidence of patient morbidity and mortality [2]. This limited the application of allogeneic HSCT to younger (< 60 years) patients, who could withstand and recover from these toxicities. However, subsequent studies and observations challenged assumptions that myeloablative conditioning regimens were necessary to eliminate malignancy and to create the state of immunosuppression to permit the engraftment of the donor hematopoietic and immune systems within the recipient. The high frequency of relapse observed in the setting of T-cell-depleted allogeneic HSCT suggested that a significant proportion of the curative potential of allogeneic HSCT resulted from a T-cell-mediated graftversus-tumor (GVT) [3]. The observation that delayed administration of donor lymphocytes could induce complete remissions remote from the preparative regimen provided further evidence that an immune mechanism primarily mediates an anti-leukemic effects after allogeneic HSCT [4]. With respect to graft rejection, it is also now known that conventional myeloablative regimens do not consistently completely eliminate the host response to the allograft, even in recipients of HLA-matched allografts. For example, 30 to 40% of patients will reject a T-celldepleted, HLA-matched allogeneic stem cell graft after myeloablative conditioning [5]. In addition, there are certain clinical situations where the malignancy is very susceptible to the allogeneic GVT effect (e.g. AML in complete remission, chronic leukemias or low-grade lymphomas). In these clinical situations the use of a myeloablative conditioning regimen may be causing significant toxicity without actually adding any clinical benefit in regard to elimination of the malignancy. These limitations of myeloablative conditioning regimens, combined with the high levels of morbidity and mortality associated with myeloablation, result in a low therapeutic index for this form of allogeneic HSCT. Reducing treatment-related toxicity would improve the therapeutic index of allogeneic HSCT, permitting the broader application and potential benefit of this treatment to more patients. This principle led to the development of less intensive ("nonmyeloablative" and "reduced-intensity") conditioning regimens for allogeneic HSCT, which were designed with the purpose of allowing engraftment of donor cells to exploit the potential allogeneic GVT effect [6-10]. The early reports of studies employing non-myeloablative and reduced-intensity conditioning regimens prior to allogeneic HSCT demonstrated that engraftment occurred in the majority of patients, early (first 100 days after transplantation) transplant-related toxicities were reduced, and that this treatment could be applied to older patients and patients with co-morbidities. However, it was also observed that both nonmyeloablative and reduced-intensity conditioning were associated with high incidences of incomplete donor chimerism, higher incidences of graft rejection/failure, and increased relapse rates, as compared to results with myeloablative conditioning regimens [11,12]. Mixed chimerism and graft rejection has been associated with HLA-disparity, use of unrelated donors, and inversely correlated with amount of therapy prior to transplant [13-15]. It was also observed that a GVT effect was not observed until full donor chimerism was achieved [16-19]. For this reason, a central hypothesis in the design of our transplantation protocols has been that attaining full donor chimerism early after transplantation is central to achieving an enhanced GVT effect, and that accomplishing this goal with a reduced-intensity preparative regimen depends upon the degree of host immunosuppression at the time of HSCT.  
1.2.2 Rationale for Targeted Immune Depletion Induction Prior to Reduced-Intensity Conditioning in the Setting of AlloHSCT from Unrelated Donors; Data from HLAmatched Siblings - CC 99-C-0143, CC 00-C-0119, and CC 03-C-0077 The objective to achieve rapid and complete donor chimerism following allogeneic HSCT led our group to adopt a strategy of sequential targeted immune depletion before transplant conditioning. Murine data from the Fowler Laboratory (NCI) demonstrated that daily administration of a combination of fludarabine and cyclophosphamide resulted in severe reductions in the number of circulating host CD8+and CD4+ T cells to levels comparable to those achieved with myeloablative doses of total body irradiation [20]. This level of T cell depletion was sufficient to permit the engraftment of fully MHC-mismatched (i.e. haploidentical) allografts (F1-into-parent murine model). The degree of myelosuppression with the extended administration of fludarabine and cyclophosphamide was significantly less than with total body irradiation (TBI). Taken together, these studies indicated that extended administration of agents, which were highly immune-depleting yet had nominal myelosuppressive effects, could result in a level of host T cell depletion that would permit the engraftment of haplo-identical stem cells. We used these preclinical murine data as the basis for our clinical trials design. It was our  
 specific intent to design transplantation protocols that would rapidly result in complete donor chimerism, in order to optimize the GVT effect, since these protocols would be used to treat advanced malignancies. For many malignancies, however, the GVT effect may be inadequate by itself, and the cytotoxic effects of the conditioning regimen may therefore play a beneficial, if not necessary, role in the efficacy of allogeneic HSCT [21,22].  
As such, we elected to employ a reducedintensity conditioning regimen, consisting of fludarabine and cyclophosphamide, rather than a TBI-based nonmyeloablative conditioning regimen. This reduced-intensity conditioning regimen has been utilized in all of our transplant protocols for clinical consistency and to permit comparison between protocols. Due to the variability of patients' immune status, we surmised that the level of host T cell depletion required to permit the rapid and complete engraftment of allogeneic stem cells would not be consistently achieved with the reduced-intensity conditioning regimen alone, especially if alternative donor stem cells (e.g. HLA-matched unrelated donors) and/or if T cell depletion of the allograft were to be utilized. To achieve uniform immune depletion, we elected to employ repetitive doses of immunosuppressive agents that had anti-tumor activity, as patients would require disease control prior to receiving a reduced-intensity conditioning regimen. However, we determined that the daily administration of these agents, as given in our murine model, was clinically impractical. As such, we elected to administer these agents at conventional doses in repetitive cycles, similar to how second-line or "salvage" regimens are given prior to autologous HSCT [23]. We refer to these as "induction regimens", as they have the two specific goals of inducing severe host T cell depletion as well as inducing disease remission. The most unique potential advantage is that the induction regimen would permit us to adjust or "target" the level of T cell depletion based upon a patient's pre- existing immune status. Specifically, the number of cycles of the induction regimen is determined by quantification of circulating host T cells after each cycle. In summary the rationale for incorporating "targeted immune-depletion" using induction chemotherapy in our protocol design (Figure 1) was that it: 1) allows us to achieve host T cell depletion in a patient-specific manner, 2) provides disease control, and 3) utilizes chemotherapy at doses with minimal levels of toxicity. The targeted immune-depletion approach has been applied to all of our allogeneic HSCT protocols. We set out to evaluate our targeted immune-depleting approach in a series of sequential trials in which one of the factors that affects engraftment was altered in a step-wise fashion. In each successive study, the barriers to engraftment (e.g. T cell depletion, HLA-disparity) were increased, yet other engraftment variables (e.g. conditioning regimen and stem cell dose) were kept consistent to permit comparisons among protocols. Specifically in our first trial, 99-C-0143, 11 we investigated whether our targeted immune-depletion approach would result in rapid and complete donor chimerism in patients who received T cell replete (TCR; i.e. T cells were not removed from the allograft after collection) allografts from HLA-matched siblings. In 99-C0143, we developed a novel induction chemotherapy regimen consisting of fludarabine in combination with the agents contained in the EPOCH regimen. The main purpose of administering this induction regimen was to achieve a high level of host immunosuppression prior to allogeneic HSCT, in order to attain rapid conversion to full donor chimerism and to facilitate the development of a GVT effect soon after transplantation. As a surrogate marker for the level of immune depletion necessary to achieve this goal, we chose a peripheral blood CD4 count of less than 50 cells/l before administration of the transplant preparative regimen. Our hypothesis was that this level of host CD4+ T cell depletion, would predict for robust engraftment after reduced-intensity allogeneic HSCT and be attainable with up to 3 cycles of induction chemotherapy before transplantation. Twenty patients were enrolled onto pilot arm 99- C-0143. In these 20 patients, the EPOCH-F regimen was very effective at reducing circulating T
cell numbers, as it was observed that the goal of reducing the host circulating CD4+ T cell numbers to less than 50 cells/l was met after one to three cycles of this regimen in the majority of patients. All but one patient had a CD4+ T cell value below 100 cells/l after completing the last cycle of EPOCH-F. Nineteen patients received a TCR allograft from an HLA-matched sibling following the reduced-intensity conditioning regimen consisting of fludarabine and cyclophosphamide. Single agent cyclosporine was given as GVHD prophylaxis. Hematopoietic recovery was brisk with a median time to an absolute neutrophil count greater than 1.0 x 109/L of 10 days. All 19 patients were engrafted and there were no late graft failures. Evaluation of peripheral blood mononuclear cells for chimerism, as determined by a PCR-based typing of variable number tandem repeat (VNTR) polymorphisms for donor and host elements, demonstrated that 18 of 19 patients had greater than 95% donor cells by day +14 post-transplant. Subsequent analyses at days +28 and +100 post-transplant demonstrated that this level of engraftment was sustained. Subset chimerism analysis demonstrated that there was complete donor chimerism in both myeloid (CD15
+or CD33+) and T lymphoid (CD3+) compartments, which were also maintained. Thus, with our approach of targeted immune-depletion we were able to achieve consistent, rapid, complete donor engraftment of TCR, HLA-matched related allogeneic stem cells. However, rapid and complete donor engraftment was also associated with a relatively high incidence (~70%) of clinically significant, defined as Grade II-IV, acute GVHD.  
Overall treatment-related mortality was acceptable at approximately 20% at one year posttransplant. In light of these observations, in a subsequent trial, 03-C-0077, we employed a standard dualagent GVHD prophylaxis regimen, cyclosporine + methotrexate, to determine the impact of this change on hematopoietic recovery, donor/recipient chimerism, and the incidence of acute GVHD. Methotrexate, in addition to its known anti-inflammatory activity, is believed to exert its immunosuppressive effects by inducing apoptosis and clonal deletion of activated T cells [24]. As such, the addition of methotrexate to our prior GVHD prophylaxis of single agent cyclosporine could have theoretically increased the frequency of mixed chimerism after transplantation, as well as the risk of graft rejection. The other transplant variables were otherwise kept the same, as patients again received TCR allografts from HLA-matched siblings. Thirty-one patients with advanced hematologic malignancies, primarily lymphomas were enrolled onto study and underwent allogeneic HSCT after targeted-immune depletion and reduced-intensity conditioning (Bishop - unpublished data). All patients engrafted; there have been no late graft failures with a minimum of two years of follow-up. As had been hypothesized, there was a higher percentage (26%) of patients with early (day +14) mixed donor chimerism, defined as less than 95% donor chimerism; the median donor chimerism at this time point was 90% (range: 35-100%). However, by day+100 post-transplant 94% of patients had achieved full donor chimerism. The incidence of grade II-III acute GVHD was 42%, and there were no cases of Grade IV acute GVHD. The one- and two-year treatment-related mortality rates were 3% and 8%, respectively. At a minimum of two years of follow-up, 19 patients (61%) are alive of which 16 are in a complete remission at the time of this writing. Thus, this study demonstrated that the addition of methotrexate to the sequential targeted immune depletion approach still permitted rapid and complete donor engraftment in the majority of patients and resulted in a lower incidence of acute GVHD, improved treatment-related mortality, and beneficial overall outcomes in patients with high-risk hematologic malignancies. 1.2.3 Donor Availability for Allogeneic Hematopoietic Stem Cell Transplantation Allogeneic HSCT is curative for many hematologic malignancies, but, as previously stated, stem cell donor availability limits the broader application of this treatment. Only 25 to 30% of patients have a sibling donor who is fully matched at the class I and II major histocompatibility complex (MHC) loci, which encode for the human leukocyte antigens (HLA-A, B, C, DRB1, DQB1, and DPB1). This limitation prompted the exploration of alternative stem cell sources for allogeneic HSCT including the use HLA-matched unrelated donors [25]. Enrollment of volunteer donors in national and international registries, such as National Marrow Donor Program (NMDP) in the United States, has facilitated the identification of unrelated stem cell donors for patients without HLA-matched sibling donors. However, transplants from unrelated donors are complicated by higher rates of graft rejection, acute and chronic GVHD, immune dysregulation, and treatment-related mortality. These complications are partially balanced by lower relapse rates attributed to increased histocompatibility differences. These complications have limited the application of myeloablative allogeneic HSCT from HLA-matched unrelated donors to patients less than 55 years of age and without any significant co-morbidities.
untitled h1
1.2.4 effect of hla on

Since its inception, the NMDP has required evaluation of donor-recipient histocompatibility matching (HLA-A, -B, and –DRB1) prior to unrelated donor HSCT [26]. The minimum acceptable match was originally defined by serologic testing at these 3 loci (6 possible antigens) and required at least 5 matches, that is, a "5 of 6 match". This requirement has changed little over the years. Currently, to request a donor for transplantation, the minimal acceptable level of matching remains a 5 of 6 match for HLA-A, -B, and -DRB1. Although only evaluated at antigen level of resolution ("low resolution") for donor release, each of these 3 loci must now be typed at high-resolution by DNA-based methods. High-resolution typing is defined as the identification of alleles based on differences in the antigen recognition site (ARS) domains (Exons 2 and 3 of Class I and exon 2 of Class II genes). In 2005, a requirement for HLA-C typing was added. The most recent studies have clearly shown that transplant outcomes can be improved by matching strategies that increase the overall degree of HLA compatibility above the minimum accepted level by matching also for HLA-C, -DPB1, -DQB1, and haplotypes [27]. As data on high resolution accumulated, the NMDP performed an analysis on the effects of HLA matching (low or high resolution or both) on engraftment, graft-versus-host disease (GVHD), and mortality in 1874 donor-recipient pairs that were retrospectively typed using high resolution techniques for HLA-A, -B, -C, -DRB1, -DQB1, and –DPB1 [28]. There was no effect of any mismatch on engraftment, and only a mismatch at HLA-A affected whether the patients experienced Grade II-IV acute GVHD or chronic GVHD. Single mismatches at HLA-A, -B, -C, and -DRB1 each had similar adverse effects on mortality (relative risk 1.23 - 1.33), as compared to individuals matched at all four loci. The 5-year overall survival rates for a match at all four loci ("8 of 8" as compared to a single mismatch ("7 of 8") was 39% and 31%, respectively. In this analysis, mismatches at HLA-DQ or -DP did not appear to exert any significant effect on survival (5-year overall survival = 39%) or any other outcome. The observed adverse effects on outcome were more evident in transplants with low-resolution versus only high-resolution mismatches. When high-resolution mismatches at HLA-A, -B, -C, and -DRB1 were considered together, adverse effects on survival and GVHD were observed. The authors concluded that matching for HLA-C should be incorporated into algorithms for unrelated donor selection, along with HLA-A, -B, and -DRB1 (i.e. "8 of 8").  
In a subsequent analysis, the NMDP looked at data from 3857 transplantations. Patient-donor pairs were fully typed for HLA-A, -B, -C, -DRB1, -DQB1, -DQA1, -DPB1, and -DPA1 alleles [29]. High-resolution DNA matching for HLA-A, -B, -C, and -DRB1 (8 of 8 match) was the minimum level of matching associated with the highest survival. A single mismatch detected by low- or high-resolution DNA testing at HLA-A, -B, -C or -DRB1 (7/8 match) was associated with higher mortality (relative risk = 1.25; 95% CI, 1.13-1.38; P < 0.001) and 1-year survival of 43% compared with 52% for 8 of 8 matched pairs. Single mismatches at HLA-B or HLA-C appeared to be better tolerated than mismatches at HLA-A or HLA-DRB1. Mismatching at 2 or more loci compounded the risk. Mismatching at HLA-DP or -DQ loci and donor factors other than HLA type were not associated with survival. In multivariate modeling, patient age, race, disease stage, and cytomegalovirus status were as predictive of survival as donor HLA matching. The authors concluded that high-resolution DNA matching for HLA-A, -B, -C, and -DRB1 alleles is associated with higher rates of survival. Based on these results, the NMDP has established guidelines relative to donor selection based on HLA-typing [26]. The optimal donor should be matched at HLA-A, -B, -C, and -DRB1 by highresolution typing. When there are multiple donors with this degree of matching, further matching at HLA-DQ may be utilized; however, the clinical utility does not reach statistical significance. If a mismatch is unavoidable, a single mismatch at HLA-A, -B, -C, and -DRB1 should be sought. In the NMDP data, mismatches at HLA-B and -C may be less detrimental than those at HLA-A and -DRB1. 1.2.5 Experience with Non-myeloablative and Reduced-Intensity Conditioning Regimens from Matched Unrelated Donors:
In light of the high treatment-related morbidity and mortality associated with myeloablative allogeneic HSCT from unrelated donors, clinical studies utilizing non-myeloablative and reduced-intensity conditioning regimens were initiated after encouraging results with these regimens for HLA-matched siblings had been reported. However, because of greater genetic disparity between the donors and recipients, it was hypothesized that non-myeloablative conditioning regimens for unrelated donor transplantation might require more intense immunosuppression than a transplant from an HLA-matched sibling. Since 2001, there have been a number of relatively large single-institution and multi-institutional studies on nonmyeloablative and reduced-intensity allogeneic HSCT utilizing unrelated donors (Tables 2A and 2B) [14,30-34]. It is of significant note that the median ages of recipients have ranged from 44 to 59 years with patients as old as 72 years successfully undergoing this procedure. As had been hypothesized the incidence of graft rejection, ranging from 0 to 21%, was increased. The primary adverse factors related to graft rejection were related to use of non-myeloablative (as opposed to reduced-intensity) conditioning regimens, HLA-mismatching, use of bone marrow rather than mobilized peripheral blood, and a minimal amount of prior therapy prior to transplant. In particular, the incidence of graft rejection was as high as 44% among patients who received unrelated bone marrow after a TBI-based non-myeloablative conditioning regimen in the experience of the Fred Hutchinson Cancer Research Center [14]. Using a variety of GVHD prophylaxis regimens, the reported incidences of Grade II-IV and Grade III-IV acute GVHD have been 21-63% and 6-39%, respectively. The lowest acute GVHD rates have been achieved with regimens employing in vivo T cell depletion with anti-T cell monoclonal antibodies, particularly alemtuzumab [31]. This latter approach has also been associated with increased late rejections and opportunistic infections. The reported incidence of chronic GVHD have varied from 8% to 68% with the lowest rates reported with the use of alemtuzumab. Early (< 100 days post-transplant) treatment-related mortality rates have varied from 10 to 48%; one-year treatment-related mortality rates have varied from 17 to 55%. One-year overall survival rates have been reported to be 32 to 75%.  
| Author/Institute                                                                                             | Patient #                                                    |                               |                                                                             |                                           |                                                   |         |            |      |
|--------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------|-------------------------------|-----------------------------------------------------------------------------|-------------------------------------------|---------------------------------------------------|---------|------------|------|
| Journal/Date                                                                                                 | Median  Age                                                  | HLA                           | Degree                                                                      | Level of HLA Match                        | Conditioning Regimen                              | GVHD    | Stem Cell  | CD34 |
| match                                                                                                        | Dose                                                         |                               |                                                                             |                                           |                                                   |         |            |      |
| Loci                                                                                                         | Prophylaxis                                                  | Source                        | (106/kg)                                                                    | CD3 Dose  ( 108/kg)                       |                                                   |         |            |      |
| Class I                                                                                                      | Class II                                                     |                               |                                                                             |                                           |                                                   |         |            |      |
| Giralt et al. MDACC                                                                                          | N = 86                                                       | 6                             | 6/6                                                                         | Serologic Molecular Flu (25 mg/m2/d) x 5d | FK506 x 6 months                                  | NA      | NA         | NA   |
| Blood 2001                                                                                                   | (URD =  40) 52 years (22-70)                                 | Mel 90–140 mg/m2              | MTX (5 mg/m2)  +1,+3,+6,+11                                                 |                                           |                                                   |         |            |      |
| [Ref #30]                                                                                                    | Clad (12 mg/m2/d) x 5d  Mel 90–140 mg/m2                     |                               |                                                                             |                                           |                                                   |         |            |      |
| Chakraverty et al. UK  multi-institute Blood  2002 [Ref #31]                                                 | N = 47                                                       | 10                            | 9-10/10 (s) serologic  - A,B molecula r - C Molecular Flu (30 mg/m2/d) x 5d | CsA x 3 months                            | BM = 46                                           | NA      | NA         |      |
| [CAMPATH]                                                                                                    | PB = 1                                                       |                               |                                                                             |                                           |                                                   |         |            |      |
| 44 years                                                                                                     | 8-10/10  (m)                                                 | Mel 140 mg/m2                 |                                                                             |                                           |                                                   |         |            |      |
| (18–62)                                                                                                      | CAMPATH (20 mg/kg)  -8,-7,-6,-5,-4,-3                        |                               |                                                                             |                                           |                                                   |         |            |      |
| Kroger et al. German  multi-institute  Blood 2002 [Ref #32] N = 21 (all  multiple  myeloma) 50 years (32-61) | 8                                                            | 7-8/8                         | Serologic Molecular Flu (30 mg/m2/d) x 5d                                   | CsA x 3 months MTX (10 mg/m2) +1,+3,+6    |                                                   |         |            |      |
| ATG (10 mg/kg) -3,-2,-1                                                                                      | 3.9                                                          | NA                            |                                                                             |                                           |                                                   |         |            |      |
| PB = 15 (0.4 - 12.5) BM = 6                                                                                  |                                                              |                               |                                                                             |                                           |                                                   |         |            |      |
| HLA-A,B,  DRB1,DQB1                                                                                          | Mel 100 mg/m2                                                |                               |                                                                             |                                           |                                                   |         |            |      |
| Niederwieser et al.  FHCRC Consortium  Blood 2003 [Ref #33]                                                  | N = 52                                                       | 8 (10)                        | Serologic Molecular Flu (30 mg/m2/d) x 3d                                   |                                           |                                                   |         |            |      |
| 6-8/8 (m)                                                                                                    | TBI 200 cGy                                                  |                               |                                                                             |                                           |                                                   |         |            |      |
| 7-8/8 (s)                                                                                                    | CsA x 2-3 months,  taper until +180) MMF x 40d,taper BM = 13 | 6.0 (1.5 - 21.6)              |                                                                             |                                           |                                                   |         |            |      |
| PB = 39 (BM = 2.7)                                                                                           | 3.4 (0.8 - 10.0) (BM = 0.3)                                  |                               |                                                                             |                                           |                                                   |         |            |      |
| 48 years                                                                                                     | HLA-A,B,C,  DRB1,  (DQB1)                                    |                               |                                                                             |                                           |                                                   |         |            |      |
| (6–65)                                                                                                       |                                                              |                               |                                                                             |                                           |                                                   |         |            |      |
| Maris et al. FHCRC  Consortium  Blood 2003 [Ref #14]                                                         | N = 85                                                       | 10                            | 10/10                                                                       | serologic Molecular Flu (30 mg/m2/d) x 3d | CsA x 3 months,  taper until +180 MMF x 40d,taper | BM = 18 | 6.99 (1.26 | 2.61 |
| -16.4)                                                                                                       |                                                              |                               |                                                                             |                                           |                                                   |         |            |      |
| PB = 71                                                                                                      | (0.8 -37.7)                                                  |                               |                                                                             |                                           |                                                   |         |            |      |
| 53 years                                                                                                     | HLA-A,B,C,  DRB1,DQB1                                        | (81%  molecula r at Class  I) | TBI 200 cGy                                                                 |                                           |                                                   |         |            |      |
| (5–69)                                                                                                       |                                                              |                               |                                                                             |                                           |                                                   |         |            |      |
| Wong et al. MDACC                                                                                            | N = 29                                                       | 6                             | 6/6                                                                         | Serologic Molecular Flu (25 mg/m2/d) x 5d |                                                   |         |            |      |
| 5/6 (n = 2)                                                                                                  | FK506 x 6 - 8  months MTX (5 mg/m2)  +1,+3,+6,+11            | BM = 28                       | 4.46 (0.37                                                                  | NA                                        |                                                   |         |            |      |
| - 8.6)                                                                                                       |                                                              |                               |                                                                             |                                           |                                                   |         |            |      |
| PB = 1                                                                                                       |                                                              |                               |                                                                             |                                           |                                                   |         |            |      |
| Blood 2003                                                                                                   | 59 years                                                     | Mel 90–140 mg/m2              |                                                                             |                                           |                                                   |         |            |      |
| [Ref #34]                                                                                                    | (55-69)                                                      | +/- ATG (n = 17)              |                                                                             |                                           |                                                   |         |            |      |  
Table 2B. Clinical Outcomes of Reported Trials of Non-myeloablative and Reduced-intensity Allogeneic HSCT Utilizing HLA-matched Unrelated Donors  
| Author/Institute                 | Graft     |            |         |        |        |        |          |     |          |
|----------------------------------|-----------|------------|---------|--------|--------|--------|----------|-----|----------|
| Rejection and                    |           |            |         |        |        |        |          |     |          |
| Journal/Date                     | Failure   | Acute GVHD | Chronic | TRM    | OAS    |        |          |     |          |
| GVHD                             |           |            |         |        |        |        |          |     |          |
| II-IV                            | III-IV    | Day 100    | 1 year  | 2 year | 1 year | 2 year |          |     |          |
| Giralt et al.  MDACC Blood  2001 | 5% (n =2) | 62%        | 39%     | 68%    | 37.4%  | NA     | 45% (all | 32% | 28% (all |
| patients)                        | patients) |            |         |        |        |        |          |     |          |
| (11 deaths)                      | (URD)     |            |         |        |        |        |          |     |          |  
| 15%                                                | 20%                             | (~25%)   | 75%               | NA       |     |     |     |     |        |
|----------------------------------------------------|---------------------------------|----------|-------------------|----------|-----|-----|-----|-----|--------|
| 3% (n= 2)                                          | 21.3%                           | 6.4%     | 8% (all  limited) |          |     |     |     |     |        |
| Chakraverty et al.  UK multi-institute  Blood 2002 Kroger et al.  German multiinstitute Blood  2002                                                    | 0                               | 38%      | 19%               | 37%      | 10% | 26% | NA  | 74% | 74%    |
| Limited =  25% Extensive =  12%                    |                                 |          |                   |          |     |     |     |     |        |
| Niederwieser et al.  FHCRC  Consortium Blood  2003 | 12%                             | 63%      | 21%               | 60%      | 11% | 29% | 40% | 44% | 35%    |
| (low CD3;  low CD34)                               | Limited =  30% Extensive =  30% | (19 mo)  |                   |          |     |     |     |     |        |
| Maris et al.  FHCRC  Consortium Blood  2003        | 21% (PB vs.  BM: 15% vs.  44%)  | 52%      | 10%               | 45% (85) | 11% | 17% | NA  | 52% | (~42%) |
| Extensive =  37%                                   |                                 |          |                   |          |     |     |     |     |        |
| Wong et al.  MDACC Blood  2003                     | 14% (n= 4)                      | 41%      | 20%               | 62.5%    | 48% | 55% | NA  | 44% | (~44%) |  
In regard to specific disease outcomes, data (Figure 2A) from the Center for International Blood and Marrow Transplant Research (CIBMTR) demonstrate two-year overall survival rates varying between approximately 25-40% depending of the specific hematologic malignancy. CIBMTR data (Figure 2B) on one-year treatment mortality rates (26-36%) were consistent with reports from single-institution and multi-institutional studies. These data demonstrated that the use of non-myeloablative and reduced-intensity conditioning regimens could successfully be performed in older patient; however, there is a need for significant correlative studies and clinical improvements relative to engraftment, reductions in GVHD, immune reconstitution, treatmentrelated mortality, and overall survival, as a clearly superior approach to this treatment has yet to emerge.  
Figures 2A and 2B: CIBMTR Analysis of Overall Survival and Treatment-related Mortality after NonAblative or Reduced-Intensity HSCT with Unrelated Donors  
  
As described above, the use of a standard dual-agent GVHD prophylaxis regimen, cyclosporine + methotrexate, in 03-C-0077 permitted relative rapid and complete donor engraftment and resulted in an acceptable incidence of clinically significant GVHD and a relatively low overall treatment-related mortality following our approach of targeted immune depletion prior to reduced-intensity conditioning in the setting of allogeneic HSCT from HLA-matched siblings. Similar calcineurin-inhibitor-based GVHD prophylaxis regimens (i.e. FK506)/methotrexate) have been investigated in the setting of reduced-intensity allogeneic stem cell transplantation utilizing unrelated donors [30]. They have been associated with relatively higher rates of GVHD, as compared to other regimens that have been employed, particularly alemtuzumab (Table 2B). It is predicted that the combination of cyclosporine + methotrexate will be inadequately immunosuppressive to prevent clinically severe (grade III-IV) acute GVHD if our targeted immune depletion approach were employed in the setting of reduced-intensity allogeneic HSCT from unrelated donors. The two regimens which have been reported to be most successful at preventing acute GVHD following reduced-intensity allogeneic HSCT from unrelated donors have been the combination of alemtuzumab and cyclosporine [31] and the combination of tacrolimus, methotrexate, and sirolimus [36]. Both regimens have distinct clinical advantages and disadvantages, as well as distinct mechanisms of action. 1.2.7 Tacrolimus/Methotrexate/Sirolimus as Graft-Versus-Host Disease Prophylaxis More recently, the immunosuppressive drug sirolimus, which is effective in the prevention of renal allograft rejection [31], has been evaluated as a third agent for GVHD prophylaxis after allogeneic HSCT from unrelated donors [32]. Sirolimus uniquely inhibits T cell and dendritic cell function through inhibition of mTOR, which in contrast to the mRNA inhibition associated with calcineurin blockade, operates via a post-transcriptional mechanism to reduce cell signaling and modulate protein translation and phosphorylation in T cells [37]. Clinical studies have not evaluated single-agent sirolimus for GVHD prophylaxis, and in fact, murine data indicate that single-agent sirolimus potently abrogates CD8+ T cell mediated GVHD, but not CD4+ T cell mediated GVHD [38]. As such, sirolimus is likely to represent an adjunct to, but not a replacement of, calcineurin inhibitor mediated GVHD prophylaxis. Initial results indicate that addition of sirolimus to GVHD prophylaxis is effective for the prevention of acute GVHD. Investigators at the Dana-Farber Cancer Institute (DFCI) performed a single arm study, which involved unrelated donor or mismatched family member bone marrow transplantation, the GVHD prophylaxis regimen consisted of calcineurin-inhibitor tacrolimus, methotrexate, and sirolimus [36]. Sirolimus was initiated at a loading dose of 12 mg, with a maintenance dose of 4 mg per day throughout the first 100 days post-transplant. The incidence of grade II-IV acute GVHD associated with this regimen was 26% (11/41), which is reduced relative to historical controls previously described above. Sirolimus was also reported to be well-tolerated at this dosing schedule, with hyper-triglyceridemia representing the primary specific drug toxicity. Sirolimus has been of significant interest and investigation in the Fowler Laboratory. In an ongoing clinical protocol, 04-C-0055, short-course sirolimus administration (fourteen day treatment interval) has been evaluated in combination with cyclosporine with and without Th2 cells as GVHD prophylaxis in the setting of non-myeloablative targeted immune depletion and TCR allogeneic HSCT from HLA-matched siblings. The Fowler Laboratory hypothesized that short-course *in vivo* sirolimus would preferentially inhibit unmanipulated donor T cells contained within the allograft, thereby resulting in preferential *in vivo* expansion of sirolimus generated Th2 cells. However, it is also possible that sirolimus alone (i.e. without Th2 cells) would contribute to GVHD prophylaxis, as murine allogeneic bone marrow transplantation experiments in the Fowler Laboratory demonstrated that short-course sirolimus administration in the peritransplant period significantly reduced fully-MHC mismatched graft-versus-host reaction.  
Preliminary results from 04-C-0055 suggest that the arm (III) of this trial receiving only cyclosporine and sirolimus (i.e. no Th2 cells) has resulted in an incidence of acute GVHD similar to that achieved with cyclosporine + methotrexate. In the first 11 evaluable patients on arm III of 04-C-0055, there have only been two case acute GVHD, which were grade I and grade II, respectively (D. Fowler - *personal communication*).  
1.2.8 Alemtuzumab/Cyclosporine as Graft-Versus-Host Disease Prophylaxis: Alemtuzumab is a humanized monoclonal antibody that targets the CD52 antigen, which is highly expressed on both B and T lymphocytes, and has a relatively long half-life that is measured in weeks. Alemtuzumab has been utilized, both ex-vivo and in vivo, to prevent GVHD in individuals undergoing allogeneic HSCT from unrelated donors for over 20 years [39]. More recently has alemtuzumab has been utilized as part of non-myeloablative conditioning regimens. Alemtuzumab has been administered along with non-myeloablative and reduced-intensity conditioning regimens to provide pan-depletion of both host and donor B- and T-cells [31,4042]. These actions may promote engraftment through the depletion of host T cells and prevent GVHD through the in vivo T cell depletion of the allograft. Kottardis and colleagues reported on their use of alemtuzumab in high-risk 44 patients with hematologic malignancies undergoing non-myeloablative allogeneic HSCT [36]. The conditioning regimen consisted of alemtuzumab (20 mg/day on days -8 to -4, fludarabine (30 mg/m2on days -7 to -3), and melphalan (140 mg/m2on day -2). Thirty-six recipients received unmanipulated G-CSF mobilized peripheral blood stem cells from HLA-identical siblings, and 8 received unmanipulated marrow from matched unrelated donors. Additional GVHD prophylaxis consisted of cyclosporine alone for 38 patients and cyclosporine plus methotrexate for 6 sibling recipients. Forty-two of the 43 evaluable patients had sustained engraftment. Results of chimerism analysis demonstrated that 18 of 31 patients studied were full-donor chimeras, while the other patients were mixed chimeras in one or more lineages. There were no cases of grades III-IV acute GVHD. Only 2 patients developed grade II acute GVHD, and only 1 had chronic GVHD. The estimated probability of non-relapse mortality was 11%. Four patients died of regimen-related complications. At a median follow-up of 9 months (range: 3 - 29 months), 33 patients were alive in complete remission or with no evidence of disease progression. Seven patients relapsed or progressed post-transplantation, and four of them subsequently died. Chakraverty reported on 47 patients with hematological malignancy who underwent allogeneic HSCT from HLA-matched, unrelated donors following a non-myeloablative conditioning regimen containing alemtuzumab [31]. Additional GVHD prophylaxis consisted of cyclosporine alone. The median patient age was 44 years. Twenty of the transplants were mismatched for HLA class I and/or class II alleles. Primary graft failure occurred in only 2 of 44 evaluable patients (4.5%). Chimerism studies in 34 patients indicated that the majority (85.3%) attained initial full donor chimerism. Only 3 patients developed grade III to IV acute GVHD, and no patients had developed chronic extensive GVHD at the time of this report. The estimated probability of non-relapse mortality at day +100 post-transplant was 14.9%. At a median followup of 344 days (range: 79-830), the overall and progression-free survival rates at 1 year were 75.5% and 61.5%, respectively. The Spanish and United Kingdom Collaborative Groups for Nonmyeloablative Transplantation investigated the efficacy of alemtuzumab as compared with methotrexate for GVHD prophylaxis in 129 recipients of a non-myeloablative allogeneic HSCT from an HLA-matched sibling enrolled in 2 prospective studies for chronic lymphoproliferative disorders [41]. The conditioning regimens in both studies were based on the same combination of fludarabine and melphalan, but the United Kingdom (UK) study utilized a GVHD regimen consisting of cyclosporine plus alemtuzumab, whereas the Spanish study used cyclosporine plus methotrexate for GVHD prophylaxis. Patients receiving alemtuzumab had a higher incidence of cytomegalovirus (CMV) reactivation (85% vs. 24%, p < 0.001) and a significantly lower incidence of acute GVHD (21.7% vs. 45.1%, p = 0.006) and chronic GVHD (5% vs. 66.7%, p <
0.001). Twenty-one percent of patients in UK study group and 67.5% in Spanish study group had complete or partial responses three months after transplantation (p < 0.001). Eighteen patients in the UK study group received donor lymphocyte infusions to achieve disease control. There was no difference in disease status between the groups with 71% versus 67.5% (p = 0.43) of patients showing complete or partial responses in the UK and Spanish groups, respectively. No significant differences were observed in event-free or overall survival between the two groups. The use of alemtuzumab in the non-myeloablative transplant setting has been complicated with a relative high incidence of cytomegalovirus (CMV) re-activation and infections [42,43]. CMV was monitored by polymerase chain reaction-based assays and treated preemptively in 101 patients who underwent allogeneic HSCT following a non-myeloablative conditioning containing alemtuzumab [39]. Fifty-one patients (50%) were observed to have CMV reactivation at a median of 27 days after transplantation. All three patients who developed clinical CMV disease died of this complication in this series. The median time to CD4+ T-cell count more than 200/microL was 9 months in the 48 patients studied. The probabilities of overall survival and non-relapse mortality at 18 months were 65% and 27.8%, respectively, with no significant difference in survival between patients with and without CMV infection. 1.2.9 Ex vivo T-cell Depletion in Non-myeloablative and Reduced-intensity Allogeneic HSCT Relative to other methods of preventing acute GVHD, there have been reports on the use of ex vivo T cell depletion following myeloablative allogeneic HSCT from unrelated donors, beginning with the first reports on the use unrelated donors [40,41]. Ex vivo T cell depletion resulted in decreased acute GVHD, but it was associated with increased graft failure and opportunistic infections resulting in no clear survival advantage over unmanipulated grafts in the myeloablative setting. This led to a phase III multi-institutional trial in which 405 patients were randomized to either ex-vivo T-cell depletion of the marrow with cyclosporine alone (n = 201) versus an unmanipulated marrow with methotrexate and cyclosporine (n = 204) as GVHD prophylaxis [42]. The primary outcome was 3-year disease-free survival, which was analyzed by intention to treat. Disease-free survival at 3 years was 27% and 34% in recipients of T cell deplete arm and the methotrexate and cyclosporine arm, respectively (p = 0.16). T cell depletion was associated with significantly more rapid neutrophil recovery (15 days vs. 20 days, p < 0.0001), less grade III-IV acute GVHD (18% vs. 37%, p < 0.0001), reduced grade III-IV toxicities (19% vs. 29%, p = 0.017), reduced duration of initial hospitalization, but a higher risk of chronic myelogenous leukemia relapse (20%vs 7%, p=0.009) and CMV infection (28% vs. 17%, p = 0.023) than patients receiving methotrexate and cyclosporine. In a follow-up paper to this trial, the impact of ex vivo T cell depletion on chronic GVHD was analyzed by Pavletic and collaborators [47]. The incidence of chronic GVHD at 2 years was similar between the two arms (T cell depletion = 29% vs. methotrexate/cyclosporine = 34%, p = 0.27). Survival at 3 years from diagnosis of chronic GVHD was also similar (T cell depletion = 51% vs. methotrexate/cyclosporine = 58%; p = 0.29). The incidence of serious infections and leukemia relapse were similar on both treatment arms. In spite of a significant reduction of acute GVHD, T cell depletion did not reduce the incidence of chronic GVHD or improve survival in patients who developed chronic GVHD. There have only been anecdotal reports on the use of ex vivo T cell depletion after either non-myeloablative or reduced-intensity conditioning regimens utilizing either HLA-matched or unrelated donors. One of the larger experiences came from the National Cancer Institute [48]. In this trial, planned, sequential donor lymphocyte infusions given early (day +42) post-transplant resulted in an incidence of acute GHVD similar to that observed with a T cell replete allograft.
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1.3 primary aims

1.3.1 Assessment of immune reconstitution following reduced-intensity allogeneic HSCT from unrelated donors.  
Immune reconstitution following allogeneic HSCT is a prolonged process, influenced by a number of factors including, the age and corresponding thymic function of the transplant recipient, and degree histocompatibility between the donor the recipient, as well as the presence or absence of GVHD. However, immune reconstitution has not been well studied in the setting of reduced-intensity allogeneic HSCT from unrelated donors. It is our interpretation of the available medical literature that both of the AC and TMS GVHD prophylaxis regimens are effective in preventing acute GVHD in the setting of reduced-intensity allogeneic HSCT using HLA-matched unrelated donors. However, due to their distinctive biologic differences, it is hypothesized that these two regimens have significantly different effects upon immune reconstitution, which has significant implications to other factors affecting outcomes after transplantation (e.g. infections and relapse). A detailed, prospective analysis on the effects of either of these regimens on immune reconstitution has not been performed, and such an analysis is warranted relative to both transplant biology and clinical outcomes. It is our intent to assess and compare in a pilot manner immune reconstitution following the combination of alemtuzumab and cyclosporine, as employed by the United Kingdom Collaborative Groups for Nonmyeloablative Transplantation group [31] and the combination of tacrolimus, methotrexate, and sirolimus, developed at DFCI [36], in the setting of reduced-intensity allogeneic HSCT from HLA-matched unrelated donors. Immune reconstitution will be assessed in a variety of techniques with which we and our collaborators are recognized for specific expertise. One assessment of immune reconstitution will be an analysis of T-Cell Receptor Vβ repertoire using CDR3 spectratyping. The complementarity-determining region (CDR3) of the T-cell receptor (TCR) is of most importance for the extensive diversity of the TCR [49,50]. The CDR3 plays a key role in defining the specificity of antigen recognition because the region forms the contact site for binding to peptide major histocompatibility complexes (MHCs) expressed by antigen-presenting cells. The CDR3 of both TCRα and TCRβ genes is generated by extensive rearrangement and fusion between the V, D, and J segments and by random insertion and deletion of junctional nucleotides, which yields final products that are quite heterogeneous in size. As a result of these gene rearrangements, each T cell has a unique TCR and the diversity of the T-cell repertoire at any specific time can be characterized by the examination of CDR3 within that population. Healthy individuals demonstrate a diverse and polyclonal TCR repertoire reflected by a Gaussian size distribution of CDR3 regions composed from the TCR Vβ gene [47]. Amplification of the Vβ region expressed in circulating T cells has been used by the technique called CDR3 spectratyping to monitor the Vβ gene expression as a measurement of the level of immune competence in allogeneic HSCT recipients in both the myeloablative and reduced-intensity settings [52,53]. Factors such as T cell depletion, the development of GVHD, high recipient age, and infections are known to impair immune competence after allogeneic HSCT and correlate to oligoclonal or monoclonal CDR3 patterns [54-56]. Different CDR3 sizes and a Gaussian distribution of these peaks should be present in a normal polyclonal expression of a Vβ subfamily [51]. These differences can be quantified and compared. The overall complexity within a Vβ subfamily can be determined by counting the number of discrete peaks per subfamily. Subfamilies can then be graded on a score based on the degree of complexity. Normal complexity is characterized by a Gaussian distribution of transcript sizes, reflecting the presence of polyclonal cDNA species. CDR3 spectratype analysis provides a sensitive method for characterization of the T-cell repertoire as well as for following changes in individual patients over time. CDR3 spectratype analysis is an ideal method to assess differences in immune reconstitution after the administration of these two regimens, which have distinct effects upon T cells over time posttransplant.  
As part of the comprehensive primary objective of assessing the effects of these two GVHD regimens on immune reconstitution, we will specifically assess differences in CD4+ T cell receptor Vβ repertoire by CDR3 spectratyping at 3 months post-transplant. This specific T cell subset was selected as this population generally recovers relative to its effects on other lymphocyte populations (i.e. CD8+ T cells and B lymphocytes). In addition, the homeostatic mechanisms which influence CD4+ T cell reconstitution (i.e. IL-7) are less perturbed by other host environmental factors. We chose three months post-transplant as this is time point at which repertoire diversity previously has been demonstrated to increase. Three months is also the time point at which scheduled tapering of immunosuppression is planned and clinical assessments and decisions are made relative to GVHD and disease status. These data will be complemented by spectratype analysis on both CD4+ and CD8+ T cells at various time points as well as the additional data on immune reconstitution which will be performed as part of the secondary aims.  
This extensive and extremely unique data set on immune reconstitution will be of value to the general transplantation field and in the design of our future protocols utilizing unrelated donors. 1.3.2 Assessment of chronic GVHD in the setting of sequential targeted immune-depleting chemotherapy followed by reduced-intensity allogeneic HSCT from HLA-matched unrelated donors In addition to acute GVHD, chronic GVHD contributes to the morbidity and mortality associated with allogeneic HSCT from unrelated donors [56]. Chronic GVHD has not been well characterized in either the non-myeloablative and reduced-intensity allogeneic HSCT settings, particularly related to the use of unrelated donors. The reported incidences of chronic GVHD following non-myeloablative and reduced-intensity allogeneic HSCT from unrelated donors has varied from 8 - 65% (Table 2B), depending on patient age, degree of HLA-matching, and form of GVHD prophylaxis [14,30-34]. Chronic GVHD has become a specific area of intent research within ETIB. Led by Dr. Steven Pavletic, the Chronic Graft-versus-Host Disease Program is a multi-institutional and extramural collaborative effort which incorporates laboratory and clinical studies including the establishment of one of the world's largest multi-disciplinary clinic for chronic GVHD, utilizing the unique resources and expertise available at the NIH. The efforts by Dr. Pavletic and his NIH collaborators were highlighted by the 2005 NIH-sponsored Chronic GVHD Consensus Project, which has unified the transplant community's approach to chronic GVHD through the activities of focused working groups [61-63]. Patients will be specifically monitored within the Chronic GVHD Clinic, will be offered potential participation on the Chronic GVHD Natural History protocol (04-C-0281), and will potentially be offered participation on therapeutic trials which are currently submitted for review and under development in the NIH. (Please see section 1.6)
1.4 BACKGROUND ON SECONDARY OBJECTIVES  
1.4.1 Assessment of serum Interleukin-7 and Interleukin-15 following Reduced-Intensity Allogeneic HSCT from Unrelated Donors
Immune reconstitution has been a primary research focus of both the Gress and Mackall Laboratories, both of whom are associate investigators on this study [57-61]. Secondary endpoints will continue according to prior studies performed as part of 99-C-0143, 00-C-0119, and 03-C-0077. These studies have included the kinetics of CD4+ and CD8+ T-cells and NK cell depletion and recovery in the setting of sequential immune depletion and reduced-intensity allogeneic HSCT. They have also included, as part of 03-C-0077, the correlation of serum IL-7 and IL-15 levels with lymphocyte subpopulations prior to transplantation and during immune recovery after transplant, and during the development of clinical GVHD, which had never been determined previously in the clinical setting. Preliminary analysis of data from 03-C-0077 demonstrates that there is an association of post-transplant serum IL-7 levels with the development of acute GVHD. The continuation of these studies in the setting of allogeneic HSCT from unrelated donors will potentially provide valuable insights relative to immune reconstitution as it relates to post-transplant toxicities, particularly GVHD and immune dysregulation. 1.4.2 Characterization of post-transplant monocyte production of inflammatory cytokines IL-1α and TNF-α after reduced-intensity allogeneic HSCT
Characterization of the inflammatory cytokine cascade after allogeneic HSCT, aptly termed
"cytokine storm" GVHD [60], has identified IL-1-α and TNF-α as potential targets for GVHD prevention or therapy. Indeed, neutralization of these cytokines can reduce murine gut GVHD [61,62], and importantly, this approach to GVHD prevention can be accomplished without abrogating an allogeneic T cell mediated graft-versus-leukemia (GVL) effect [67,68]. The existence of a similar non-T cell mediated inflammatory cytokine phase of GVHD in human transplantation has been more difficult to prove. Given the role of the macrophage as cell source for inflammatory cytokine-mediated murine GVHD, Fowler and colleagues reasoned that clinical "cytokine storm" might be monitored in a sensitive manner through sequential posttransplant evaluation of circulating monocyte production of IL-1-α and TNF-α using an intracellular flow cytometry assay [65]. Seventeen patients with refractory hematologic malignancy received reduced-intensity conditioning, HLA-matched sibling HSCT, and GVHD prophylaxis consisting of cyclosporine alone. After HSCT, monocyte IL-1-α and TNF-α were measured by intracellular flow cytometry (IC-FCM), and results were correlated with acute GVHD. Post-transplant monocyte IL-1-α increased significantly (p = 0.0065) from week 2 to week 4 post-transplant; however, it was not associated with GVHD severity (p = 1.00). In contrast, increases in monocyte TNF-α were quantitatively reduced and temporally delayed, from week 2 to week 6 (p = 0.076). Elevation of monocyte TNF-α correlated with increased gut GVHD severity (p = 0.0041), with increases in monocyte TNF-α typically preceding the onset of gut GVHD symptoms. Serial measurement of monocyte cytokines, in particular, TNF-α, by ICFCM represents a non-invasive method for GVHD monitoring, potentially allowing identification of patients appropriate for early intervention strategies.
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1.4.3 tumor derived lymphocytes

The prognosis for most patients with hematologic malignancies that have persistent disease or relapse after allogeneic HSCT is poor. Effective therapy for patients who fail withdrawal of immune suppression and administration of donor lymphocyte infusions (DLI) has not been identified. Even when DLI are effective, the response is often not durable and can be accompanied by GVHD. Generation of donor lymphocytes that mediate more potent and specific GVT effects while minimizing GVHD is a major laboratory and clinical effort of the ETIB. We have hypothesized that lymphocytes found in tumor after alloHSCT are of donor origin, and because they are tumor-derived, they may be tumor-specific in their homing and antigen specificity characteristics. In preclinical studies we have developed a method for the activation and expansion of tumor-derived lymphocytes (TDL) through CD3/CD28 co-stimulation and further expansion in IL-2. This approach is being piloted in patients with B cell malignancies who have persistent disease or relapse after AlloHSCT, including recipients of URD allografts. In parallel, ongoing preclinical efforts are focusing on optimization of the TDL cell product development, including cell yields and tumor specificity. Research samples obtained from subjects enrolled on this protocol will provide valuable data that may be of benefit to URD allograft recipients for whom DLI are not always readily available.
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1.5 summary

The major limitations to the broader applicability of allogeneic HSCT for the treatment of malignancies are lack of suitable donor and therapy related toxicities. The use of stem cells from unrelated donors partially alleviates the need for donors, but their use is associated with a higher incidence of graft rejection and mixed donor chimerism, due to histocompatibility difference between the donor cells and the recipient. Based on the theory that the rapid establishment of donor chimerism was essential for an optimal GVT effect, we have employed a strategy of targeted immune-depleting chemotherapy prior to reduced-intensity allogeneic HSCT in a series of sequential trials in which one of the factors that affects engraftment was altered in a step-wise fashion. A recently completed trial, 03-C-0077, demonstrated that targeted immune depletion permitted rapid and complete donor engraftment, a lower incidence of acute GVHD, improved treatment-related mortality, and beneficial overall outcomes in patients with high-risk hematologic malignancies who received a reduced-intensity allogeneic T cell replete HSCT from HLA-matched sibling, using a GVHD prophylaxis regimen consisting of cyclosporine and methotrexate. It is our intent to investigate this approach in the setting of HLA-matched unrelated donors. A clearly superior GVHD prophylaxis regimen has not been established in the unrelated donor setting. The two GVHD prophylaxis regimens with which best results have been reported are alemtuzumab with cyclosporine [31] and the combination of tacrolimus, methotrexate, and sirolimus [36]. Both of these regimens are likely to provide adequate prophylaxis against the development of clinically severe acute GVHD in the setting of targeted immune-depleting chemotherapy prior to reduced-intensity allogeneic HSCT from HLA-matched unrelated donors. However, the two regimens work through biologically distinct mechanisms, and their effects upon immune reconstitution, which have not previously been well studied with either regimen, are predicted to be markedly different, regardless of the presence or absence of acute GVHD. The study design provides a unique opportunity to investigate and compare in a preliminary fashion post-transplant immune reconstitution related to each of these GVHD prophylaxis regimens, which have distinct biologic properties relative to their ability to prevent GVHD. Immune reconstitution will be determined by a variety of techniques with an emphasis on CDR3 spectratyping. There has not been a report on post-transplant immune reconstitution in the available medical literature, as extensive or prospectively performed, as the studies planned in this protocol. These studies are a natural extension of our prior work and take advantage of the unique expertise and resources within the laboratories of the ETIB, intramural programs of the NCI, and the internationally recognized expertise on CDR3 spectratyping at Hackensack University with whom we have formed a formal collaboration. This study will provide preliminary data relative to multiple aspects of immune reconstitution after allogeneic HSCT from unrelated donors, which is important and essential to the understanding of this biology and development of strategies to improve the most important complications after this procedure, specifically GVHD and immune dysregulation. Although both of these GVHD regimens are likely to provide adequate prophylaxis against the development of clinically severe acute GVHD in the setting of targeted immune-depleting chemotherapy prior to reduced-intensity allogeneic HSCT from HLA-matched unrelated donors, this needs to be documented, as well as other clinical endpoints relative to the safety of the procedure. To this end, we will assess, as a primary objective, engraftment, early and late treatment-related mortality, and overall survival and have established stringent stopping rules for each parameter. The results of this pilot trial will be used for the development of subsequent studies related to allogeneic HSCT from unrelated donors incorporating techniques and approaches that are currently under investigation in the ETIB and POB.
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1.6 amendment k background and

At the time of this submission (October 2010) we have accrued 55 patients to this protocol of which 48 patients have undergone transplant (3 were removed either due to personal request [n =1] or disease progression prior to transplant [n = 2]; the other 4 enrollees are awaiting transplant. As per prior communication, clinical outcomes have either met or exceeded predictions. Relative to safety, all of the criteria for proceeding with the trial (i.e. stopping rules; section **6.4.3**) have been met. Among the 48 transplants there has been one graft failure, which was attributed directly to a poor quality graft, as a second collection from the donor engrafted promptly. The level of acute GVHD has been as expected with grade II-IV acute GVHD being 22% and 33% on the TMS and AC arms, respectively. Grade III-IV acute GVHD has been 11% and 17% on the two arms respectively. Early (< Day +100) treatment-related mortality has been 5% for the entire patient population; the one-year treatment-related mortality has been 17% with no statistical difference in the two arms. Overall survival at one year is greater than 60% on both arms.  
As we had hypothesized engraftment was not only nearly universal but was prompt with median full donor chimerism being 100% at day+28 on both arms. We continue to collect data relative to the primary and secondary aims of immune reconstitution and acute GVHD. In regard to the latter our acute are similar to the results reported in the available medical literature for both the TMS and AC arms. A unique aspect is that we continue to prospectively collect data in regard to chronic GVHD using the NIH Consensus Conference diagnosis and staging criteria [61-63]. There are few if any trials comparing two different GVHD regimens which are prospectively studying chronic GVHD using these criteria, which have become internationally accepted and validated retrospectively. The incidence of chronic GVHD among the first 20 evaluable patients have been 60% and 70% on the TMS and AC arms respectively. The incidence of "severe" chronic GVHD, as defined by NIH Consensus Conference criteria, has been 20% on both arms. As previously stated, chronic GVHD has become a significant research focus in ETIB. These initial chronic results are encouraging, the method of evaluation is unique as the NIH Consensus Conference criteria have been utilized, and a number of highly unique correlative studies have been utilized in this evaluation. Although the data are unique, the number of patients that would potentially be evaluable with the current accrual cap is felt to be insufficient to provide clinically relevant information. Specifically it would be of significant interest to the transplantation community to know if there is (or is not) a difference in severe chronic GVHD between the two GVHD prophylaxis regimens being utilized. In light of the excellent clinical results that have been obtained, we have the unique opportunity to prospectively chronic GVHD and as such have made this a primary objective and have increased accrual number to provide adequate patient numbers to study this clinically important component of allogeneic HSCT.
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2 eligibility assessment and enrollment

2.1 ELIGIBILITY CRITERIA - RECIPIENT ON STANDARD CARE THERAPY:  
2.1.1 The patient is 18 - 74 years of age [14,30,34]. 2.1.2 The patient has a potentially suitable 8/8 donor if they are between the ages of 69-74 years of age or a potentially suitable 8/8 or 7/8 unrelated donor(s) in the National Marrow Registry or Other Available Registry if they are between the ages of 18-74.  
2.1.3 The patient currently does not meet the protocol's eligibility/enrollment criteria for any reason.
2.1.4 There is a high likelihood that the patient, in the opinion of the PI will meet the protocol's eligibility/enrollment criteria to proceed to transplant after standard therapy is completed.  
2.1.5 The patient or legal guardian is able to give informed consent. 2.2 EXCLUSION CRITERIA - RECIPIENT ON STANDARD CARE THERAPY: 2.2.1 HIV infection. There is theoretical concern that the degree of immune suppression associated with the treatment may result in progression of HIV infection.  
2.2.2 Pregnant or lactating. Patients of childbearing potential must use an effective method of contraception (section 4.5). The effects of the chemotherapy, the subsequent transplant
and the medications used after the transplant are highly likely to be harmful to a fetus. The effects upon breast milk are also unknown and may be harmful to the infant.  
| Disease                                                                                                                                                     | Disease Status                                                                                       |                                                                                                       |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------|
| a)                                                                                                                                                          | Relapse/progression after fludarabine and at least one  other salvage regimen or 2 standard regimens |                                                                                                       |
| B-Chronic Lymphocytic Leukemia (CLL) Small Lympho(plasma)cytic Lymphoma                                                                                     | b)                                                                                                   | If 17p deletion present, one standard regimen is  sufficient                                          |
| (B-SLL, B-LPL) [70,71,72]                                                                                                                                   | a)                                                                                                   | T-PLL: Treatment failure after Campath-1H and at  least one other regimen                             |
| Prolymphocytic Leukemia (PLL) T-CLL [73]                                                                                                                    | b)                                                                                                   | B-PLL: Treatment failure after fludarabine and at  least one other salvage regimen                    |
| Hodgkin's Lymphoma [74]                                                                                                                                     | a)                                                                                                   | Primary treatment failure ineligible for autologous  HSCT. Relapse/progression after autologous HSCT. |
| a)                                                                                                                                                          | Chemotherapy-refractory disease                                                                      |                                                                                                       |
| Follicular Lymphoma, Marginal Zone  Lymphomas (splenic, nodal, or  extranodal/MALT type) [72,75]                                                            | b)                                                                                                   | Relapse after > 2 prior regimens                                                                      |
| c)                                                                                                                                                          | Relapse/progression after autologous HSCT                                                            |                                                                                                       |
| a)                                                                                                                                                          | High-risk disease in remission                                                                       |                                                                                                       |
| Burkitt or Acute Lymphoblastic  Lymphomas [76,77]                                                                                                           | b)                                                                                                   | Primary refractory disease                                                                            |
| c)                                                                                                                                                          | Recurrent disease                                                                                    |                                                                                                       |
| d)                                                                                                                                                          | Relapse/progression after autologous HSCT                                                            |                                                                                                       |
| Diffuse Large B-cell Lymphoma,  Follicular Large cell Lymphoma,  Peripheral T-cell Lymphoma, mantle cell  lymphoma, Anaplastic Large Cell  Lymphoma [78-82] | a)                                                                                                   | Primary refractory disease.                                                                           |
| b)                                                                                                                                                          | Relapse/progression after autologous HSCT after  autologous HSCT                                     |                                                                                                       |
| c)                                                                                                                                                          | Stable disease or better response to last therapy                                                    |                                                                                                       |
| a)                                                                                                                                                          | > Stage III                                                                                          |                                                                                                       |
| Cutaneous T-cell Lymphomas (Mycosis  Fungoides, Sezary Syndrome) [83]                                                                                       | b)                                                                                                   | Disease progression > 2 prior regimens, including at  least one systemic therapy.                     |
| a)                                                                                                                                                          | Relapse/progression after autologous HSCT.                                                           |                                                                                                       |
| Multiple Myeloma [32,84-86]                                                                                                                                 | b)                                                                                                   | Plasma cell leukemia                                                                                  |
| c)                                                                                                                                                          | Adverse cytogenetics: del(13q) or 11q translocation.                                                 |                                                                                                       |  
2.3 INCLUSION CRITERIA - RECIPIENT  
| Version Date: September 14, 2018                                                                                                                                                | a)                                                                                           | In first complete Remission with high-risk  cytogenetics.   |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------|-------------------------------------------------------------|
| b)                                                                                                                                                                              | Primary induction failure.                                                                   |                                                             |
| Acute Myelogenous Leukemia [87,88]                                                                                                                                              | c)                                                                                           | In second or greater complete remission.                    |
| d)                                                                                                                                                                              | Secondary AML.                                                                               |                                                             |
| e)                                                                                                                                                                              | In first complete Remission with hyperleukocytosis  at diagnosis                             |                                                             |
| a)                                                                                                                                                                              | First Complete Remission, with high-risk cytogenetics.                                       |                                                             |
| Acute Lymphocytic Leukemia [89,90]                                                                                                                                              | b)                                                                                           | Primary induction failure.                                  |
| c)                                                                                                                                                                              | Second or greater complete remission.                                                        |                                                             |
| a)                                                                                                                                                                              | RAEB I or II                                                                                 |                                                             |
| Myelodysplastic Syndrome [88,91]                                                                                                                                                | b)                                                                                           | High-risk IPSS                                              |
| c)                                                                                                                                                                              | Secondary MDS                                                                                |                                                             |
| Myeloproliferative disorders (Idiopathic  myelofibrosis, polycythemia vera,  essential thrombocytosis, chronic  myelomonocytic leukemia, agnogenic  myeloid metaplasia) [92,93] | a)                                                                                           | Agnogenic myeloid metaplasia with adverse-risk  features    |
| b)                                                                                                                                                                              | Polycythemia vera or essential thrombocythemia in  transformation to secondary AML           |                                                             |
| a)                                                                                                                                                                              | Chronic phase CML                                                                            |                                                             |
| b)                                                                                                                                                                              | Accelerated phase CML                                                                        |                                                             |
| Chronic Myelogenous Leukemia [94]                                                                                                                                               | Not eligible for myeloablative allogeneic HSCT a) First CR for patients with high risk natural killer cell  neoplasms including myeloid/NK cell precursor acute  leukemia, blastic NK-cell lymphoma, aggressive NKcell leukemia and nasal-type extranodal NK-cell  lymphoma in first complete remission.                                                                                              |                                                             |
| NK Cell Neoplasms                                                                                                                                                               | b) Primary induction failure. c) Second or greater CR. a) First CR                           |                                                             |
| T-cell Neoplasm: Adult T-Cell  leukemia/lymphoma, hepatosplenic T-Cell  lymphoma, and enteropathy associated TCell Lymphoma                                                                                                                                                                                 | b) Chemotherapy-refractory disease c) Relapse after greater than or equal to 1 prior regimen |                                                             |
|                                                                                                                                                                                 | 28                                                                                           |                                                             |  
2.3.1 Diagnosis of hematologic malignancy and at least one of the criteria in the "Disease Status" column as specified in the table above.  
2.3.2 Recipients with AML in CR1 must have one of the following:  
2.3.2.1 Adverse cytogenetics with residual disease detectable by flow cytometry, cytogenetic analysis, FISH, or PCR. Adverse cytogenetics in AML are defined as complex karyotype (> 3 abnormalities); inv(3) or t(3;3); t(6;9); t(6;11); monosomy 7; trisomy 8, alone or with an abnormality other than t(8;21), t(9;11), inv(16) or t(16;16); or t(11;19)(q23;p13.1) [95].  
2.3.2.2 Primary induction failure, defined as failure to achieve CR with primary induction chemotherapy [96].  
2.3.2.3 Secondary AML, defined as AML related to antecedent MDS, MPD, or cytotoxic chemotherapy.  
2.3.2.4 Hyperleukocytosis, WBC > 100,000, at diagnosis.  
2.3.2.5 Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene (FLT3-LM; FLTITDs) [125, 126, 127]
2.3.3 Recipients with ALL in CR1 must have one of the following:
2.3.3.1 Adverse cytogenetics or residual disease detectable by flow cytometry, cytogenetic analysis, FISH, or PCR. Adverse cytogenetics in ALL are defined as translocations involving t(4;11), t(1;19), t(8;14), 11q23, t(9;22) or *bcr-abl* rearrangement or complex cytogenetic abnormalities.  
2.3.3.2 Primary induction failure, defined as failure to achieve CR with primary induction chemotherapy [97].
2.3.4 Patients with Cutaneous T-cell Lymphomas (e.g. mycosis fungoides, Sezary syndrome)
must have:
2.3.4.1 Stage III or greater disease 2.3.4.2 Disease which has progressed on or failed to respond to at least 2 therapies including one systemic therapy.  
2.3.5 Recipients with agnogenic myeloid metaplasia must have at least 2 of the following features [98,99]:
2.3.5.1 Hemoglobin < 10 g/dl, or > 10 g/dl with transfusion dependence.  
2.3.5.2 WBC < 4,000 or > 30,000/mm3or requires cytoreductive therapy to maintain.  
2.3.5.3 WBC < 30,000/mm.
2.3.5.4 Abnormal cytogenetics including +8, 12p-.  
2.3.6 Recipients with primary or secondary acute leukemia, refractory anemia with excess blasts (RAEB), CML, or other eligible diagnosis in transformation to acute leukemia must have < 5% blasts in bone marrow and no circulating blasts in peripheral blood at study entry. Recipients who do not meet these criteria may be re-evaluated for study eligibility after receiving standard induction therapy for acute leukemia and determined to be in remission.  
2.3.7 For recipients with CLL or PLL, treatment failure for a specified therapy is defined as relapse within 6 months or failure to achieve remission.  
2.3.7.1 Recipients who are ineligible for a specified therapy (e.g., due to refractory cytopenias) may be considered for enrollment in this protocol.  
2.3.7.2 Recipients who have a 17p/p53 deletion who have never received fludarabine will be eligible. [128, 129, 130]
2.3.7.3 Recipients who have had Richter's transformation who have not received prior chemotherapy for their CLL will be eligible.[131]
2.3.8 For patient with NK cell neoplasms: 1) Patients with high risk natural killer cell neoplasms including myeloid/NK cell precursor acute leukemia, blastic NK-cell lymphoma, aggressive NK-cell leukemia and nasal-type extranodal NK-cell lymphoma in first complete remission. 2) All NK cell neoplasms can be transplanted in: a) Primary induction failure or b) Second or greater complete remission.  
2.3.9 For patients with T-Cell Neoplasms including adult T-cell leukemia/lymphoma, heptosplenic T-Cell lymphoma and enteropathy associated T-cell lymphoma.  
2.3.9.1 First CR. 2.3.9.2 Chemotherapy-refractory disease. 2.3.9.3 Relapse after greater than or equal to 1 prior regimen.  
2.3.10 For patients with non-Hodgkin's lymphoma, they must be determined to have at least stable disease to last therapy [82].  
2.3.11 For patients with chronic phase chronic myelogenous leukemia, patient's disease must have evidence of never responded to or progressed after receiving a tyrosine-kinase inhibitor (e.g. imatinib mesylate).  
2.3.12 Patients with accelerated phase chronic myelogenous leukemia may have < 10% blasts in the peripheral smear or bone marrow at study entry.  
2.3.13 Patients 18 - 74years of age [14,30,34]. 2.3.14 Patient or legal guardian must be able to give informed consent. 2.3.15 All previous cytotoxic chemotherapy must be completed at least 2 weeks prior to study entry, with the exception of the tyrosine kinase inhibitors Imatinib, Nilotinib and Dasatinib which may be continued through induction therapy. Any grade 3 or 4 nonhematologic toxicity of any previous therapy must have resolved to grade 2 or less, unless specified elsewhere in Section 2.1.  
2.3.16 ECOG performance status equal to 0, 1, or 2, and Karnofsky performance status greater than or equal to 60%.  
2.3.17 Life expectancy of at least 3 months.  
2.3.18 Patients with acute leukemia or myelodysplastic syndrome or chronic myelomonocytic leukemia must be in a hematologic remission, defined as <5% blasts present in both blood and marrow to be referred for evaluation. Should a patient have >5% blasts or a donor not be available by the time the patient is ready for enrollment, the patient will be referred back to their primary hematologist-oncologist for treatment. Patients with diseases other than acute leukemia including but not limited to Hodgkin's and NonHodgkin's lymphoma, CLL/SLL, NKTCL, PTCL, must have stable disease to their most recent regimen received within 8 weeks if chemo/radiotherapy or within 12 weeks after prior autologous stem cell transplantation. Should a patient in either of these scenarios have progressive disease or a donor not be available after enrollment, the patient will be referred back to their primary hematologist-oncologist for treatment. If either of these scenarios are not in the best interest of the patient according to the clinical judgment of the PI, then the patient may receive standard treatment for the malignant disease under the current study. If under either of these settings, it becomes apparent that the patient will not be able to proceed to transplant, then he/she must come off study. RecipientSubjects receiving a standard therapy will be told about the therapy, associated risks, benefits alternatives of the proposed therapy, and availability of receiving the same treatment elsewhere, outside of a research protocol.  
2.3.19 Left ventricular ejection fraction > 45% by either MUGA or 2-D echo, obtained within 28 days of enrollment. Patients who have a prior cumulative anthracycline dose greater than 450 mg/m2 will also have a cardiology consult to determine if further anthracycline administration is an absolute contraindication in patients who may require induction chemotherapy with EPOCH-F.  
2.3.20 DLCO > 50% of the expected value when corrected for Hb, obtained within 28 days of enrollment.  
2.3.21 Creatinine < 1.5 mg/dl and creatinine clearance > 50 ml/min/1.73 m2.  
2.3.22 Serum total bilirubin less than 2.5 mg/dl, and serum ALT and AST values less than or equal to 2.5 times the upper limit of normal. Patients with elevations of serum total bilirubin up to 10 mg/dl and/or ALT or AST up to 10 times the upper limit of normal may be considered for participation if such elevations are thought to be due to liver involvement by malignancy. However, in these latter patients, if these values do not decrease to less than or equal to 2.5 times the upper limit of normal during induction chemotherapy, such patients will not be eligible for the transplant phase of the protocol, and will thus be taken off study.  
2.3.23 Minimum absolute neutrophil count of 1,000 cells/μl and minimum platelet count
(without transfusion) of 20,000/mm3. Values below these levels may be accepted at the discretion of the PI if thought to be due to bone marrow involvement by malignancy.  
2.4 EXCLUSION CRITERIA - RECIPIENT 2.4.1 Active infection that is not responding to antimicrobial therapy. 2.4.2 Active CNS involvement by malignancy (patients with known positive CSF cytology or parenchymal lesions visible by CT or MRI).  
2.4.3 Progressive disease within 8 weeks of prior therapy or within 12 weeks after prior autologous stem cell transplantation.  
2.4.4 Active or recent second malignancies unless they have undergone potentially curative therapy for that malignancy and (1) have had no evidence of that disease for 5 years, and/or (2) be deemed at low risk for recurrence (less than or equal to 20% at 5 years).  
2.4.5 HIV infection. There is theoretical concern that the degree of immune suppression associated with the treatment may result in progression of HIV infection.  
2.4.6 Chronic active hepatitis B. Patients may be hepatitis B core antibody positive. For patients with concomitant positive hepatitis B surface antigen, patient will require a hepatology consultation. The risk/benefit profile of transplant and hepatitis B will be discussed with the patient and eligibility determined by the principal investigator.  
2.4.7 Hepatitis C infection. Patient may have a hepatitis C infection. However, each patient will require a hepatology consultation. The risk/benefit profile of transplant and hepatitis C will be discussed with the patient and eligibility determined by the principal investigator and lead associate investigator.  
2.4.8 Pregnant or lactating. Patients of childbearing potential must use an effective method of contraception (section 4.5). The effects of the chemotherapy, the subsequent transplant and the medications used after the transplant are highly likely to be harmful to a fetus. The effects upon breast milk are also unknown and may be harmful to the infant.  
2.4.9 History of psychiatric disorder which may compromise compliance with transplant protocol, or which does not allow for appropriate informed consent (as determined by principal investigator or lead associate investigator).  
2.4.10 No available suitably HLA- matched unrelated donor. 2.4.11 Available suitably HLA- matched unrelated donor is unwilling to donate PBSC, and no alternate donor is found.  
2.5 INCLUSION CRITERIA - DONOR
2.5.1 General donor inclusion criteria specified in the NMDP Standards (20th Edition).  
(Appendix D)
2.5.2 The evaluation of donors shall be in accordance with existing NMDP Standard Policies and Procedures (Appendix D).  
2.5.3 Volunteer unrelated donor matched at a minimum of seven of eight loci (HLA-A, B, C,
DRB1), by high resolution typing (>7/8 allele match) are acceptable donors [14, 26, 28,29].  
2.5.3.1 The preferred donor-recipient pair would be matched at all eight loci (8/8 allele match).  
2.5.3.2 When an 8/8 allele-matched unrelated donor is not available, a single mismatch at HLA-A, -B, -C, or DRB1 will be acceptable in patients who meet all eligility criteria and are 18-69 years of age.  
In the situation where more than one 7/8 match is available donor recipient pairs matched at HLA-DQ will be used. 2.5.4 Ability to give informed consent. 2.5.5 Age 18 years or older. 2.5.6 Donors must be HIV negative, hepatitis B surface antigen negative, and hepatitis C
antibody negative. This is to prevent the possible transmission of these infections to the recipient.
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2.6 exclusion criteria - donor

2.6.1 Donor exclusion will be in accordance with existing NMDP Standards (20th Edition).  
(Appendix D)  
2.6.2 In addition to NMDP donor exclusion criteria, for the purposes of this protocol, donors who are unwilling to donate PBSC and only wish to pursue a bone marrow donation will be excluded. An alternate donor will be selected if possible, but in the event that no alternate donor is available, the patient will be removed from the trial.  
2.6.3 Current treatment with lithium.
2.6.4 Presence of sickle hemoglobin as demonstrated by appropriate testing such as hemoglobin electrophoresis.  
2.7 RESEARCH ELIGIBILITY EVALUATION - RECIPIENT 2.7.1 Typing for HLA-A, -B, -C, -DRB1, and –DQB1. 2.7.2 The following clinical, laboratory and radiologic assessments must be performed in the patient (recipient) within 28 days before the initiation of induction or conditioning chemotherapy (Section 3.3) to determine disease status:
2.7.2.1 Complete medical history and physical examination. 2.7.2.2 Nutritional assessment (initial consult). 2.7.2.3 Dental consultation to assess need for teeth cleaning or removal. 2.7.2.4 Social work consultation. 2.7.2.5 Antibody screen for hepatitis A, B, and C; HIV, HTLV-I/II, CMV, adenovirus, EBV, HSV, Toxoplasma, and syphilis.  
2.7.2.6 PPD test (if considered to be in a high-risk group). 2.7.2.7 PT, PTT, and ABO typing. 2.7.2.8 Urinalysis. 2.7.2.9 24-hour urine collection for determination of creatinine clearance. 2.7.2.10 Unilateral bone marrow aspirate and biopsy. Flow cytometry, cytogenetics, and molecular testing via polymerase chain reaction (PCR) should be performed on marrow aspirates as deemed clinically appropriate for specific diseases.  
2.7.2.11 Chest radiograph. 2.7.2.12 Electrocardiogram and 2D ECHO or MUGA scan. 2.7.2.13 Pulmonary Function Test including DLCO 2.7.2.14 CT scans of chest, abdomen, and pelvis (neck included if measurable disease is present).  
2.7.2.15 CT or MRI of the head. 2.7.2.16 Examination of CSF cytology, cell counts and routine chemistries (i.e. glucose and protein) for patients at risk for CNS involvement.  
2.7.2.17 PET scan for recipients with non-Hodgkin's lymphoma or Hodgkin's lymphoma.  
2.7.2.18 Skeletal survey (for multiple myeloma patients only). 2.7.2.19 Other specific serum or urine diagnostic studies clinically appropriate for the evaluation of recipient's malignancy (e.g., SPEP and UPEP for multiple myeloma).  
2.7.2.20 All biopsy specimens will be reviewed by the Laboratory of Pathology/CCR/NCI, for confirmation of the histologic diagnosis prior to enrollment on study.  
2.7.2.21 Biopsy material or blood samples from patients with B-cell malignancies will be tested for CD20 expression, to confirm eligibility for rituximab with induction chemotherapy. CD20 expression will be determined by immunohistochemistry or flow cytometry in the Laboratory of Pathology/CCR/NCI.
2.7.2.22 Flow cytometric analysis of peripheral blood for baseline measurement of CD3, CD4, CD8, and CD56 positive lymphocyte populations.  
2.7.2.23 Baseline determination of serum IL-7 and IL-15 levels, for correlation with NK
and T-cell subpopulations.  
2.8 RESEARCH ELIGIBILITY EVALUATION - DONOR 2.8.1 Donor eligibility will be determined by the NMDP-affiliated Donor Center Physician according to the most recent and stringent FDA, NMDP, and AABB regulations.  
2.8.2 Exceptions to donor eligibility (e.g. foreign travel, tattoos) which do not automatically exclude the donor will be reviewed by the Principal Investigator. The recipient will be informed and consented to any exceptions which could potentially increase the risk of their transplant.  
2.8.3 The following clinical, laboratory and radiologic assessments will be performed by the NMDP donor center. Documentation of donor clearance and consent will be delivered to the transplant center through the NMDP search coordinating unit.  
2.8.3.1 Complete medical history and physical examination.  
2.8.3.2 Antibody screen for hepatitis B, and C; HIV, HTLV-I/II, CMV, HSV, and syphilis. Hepatitis A, Adenovirus, EBV, and Toxoplasma screens will be performed upon receipt of the PBSC product if additional donor blood is available at the time of collection. The results of these screens do not affect the eligibility of the donor for collection or inclusion on this protocol.  
.
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2.8.3.3 cbc with differential pt

2.8.3.4 Acute care panel, hepatic panel, and mineral panel. 2.8.3.5 Urinalysis.  
2.8.3.6 Urine HCG in women of childbearing potential.  
2.8.3.7 Chest radiograph.  
2.8.3.8 Electrocardiogram.
2.8.3.9 PCR test of DNA mini-satellite regions for future determination of chimerism will be performed at NIH  
2.8.3.10 Data on donor characteristics will be entered in the C3D database for later assessment
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2.9 patient registration 2.9.1 protocol

The patient's entry date on protocol is considered to be the day that consent form has been signed by the recipient and confirmation from the NMDP that the requested donor is available, meets the inclusion criteria, and had provided informed consent relative to stem cell donation. The treatment start date is considered to be the day the recipient begins his/her initial induction cycle (Section 3.3).
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2.9.2 registration

Authorized staff must register an eligible candidate with the NCI Central Registration Office (CRO) within 24 hours of the patient (recipient) signing consent. The CRO will provide the randomized treatment assignment to the PI based on random assignments determined by the study statistician. Following amendment F, patients will be stratified for 7/8 or 8/8 HLA allele matches, and will be randomized separately according to this stratification. The patient may not sign consent before the patient has been determined to be eligible and confirmation of donor clearance has been received from the NMDP. The patient will not be registered before she/he has signed consent and confirmation from the NMDP that the donor has signed the NMDP "Statement of Intent to Donate" form at the respective institution at which the donor stem cells will be collected. A registration Eligibility Checklist from the web site (http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) must be completed and sent via encrypted email to: NCI Central Registration Office (HOIS) ncicentralregistrationl@mail.nih.gov. After confirmation of eligibility at the CRO, the CRO staff will call Pharmacy to advise them of the acceptance of the patient on the protocol prior to the release of any investigational agents and their randomized treatment assignment. Verification of Registration will be forwarded electronically via e-mail. Please note, it is very important for all registrars to acquire encrypted e-mail from NIH Help Desk, since the verification of registration includes patient's information. A recorder is available during non-working hours.
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2.9.3 off protocol therapy and

Authorized staff must notify Central Registration Office (CRO) when a patient is taken off protocol therapy and when a subject is taken offstudy. A Participant Status Updates form from the web site (http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) main page must be completed and sent via encrypted email to: NCI Central Registration Office (HOIS) ncicentralregistration-l@mail.nih.gov.
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3 study implementation 3.1 study

GVHD then taper at day, +63,  
  
  
b) Alemtuzumab (Campath®) at 20 mg/day IV over 8 h on days −8 to −4 pre-transplant.
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allogeneic hematopoietic stem cell transplantation

a) Mobilized PBSCT, day 0 (requested dose > 7 x 106 CD34+ cells/kg)
b) Filgrastim, 10 mcg/kg/day SC, starting day 0, Continue until ANC > 5000 x 1 day and 1000 x 3 days Study accrual: number transplanted = 44arm (88 total) for 8/8 and 13/arm (26 total) for 7/8.  
Study total = 76 transplanted patients 3.2 DONOR STEM CELL MOBILIZATION AND COLLECTION (AS PER NMDP GUIDELINES)  
3.2.1 Donors will receive filgrastim for stem cell mobilization at a dose of 10 to 12 μg/kg/day by subcutaneous injection, according to the NMDP PBSC Protocol (Appendix E). The filgrastim dose will not exceed 1200μg/day. Per NMDP standards, filgrastim will be administered at approximately the same time each day for the first four days. The fifth dose will be given at least one hour prior to apheresis. There will be an additional, sixth dose of filgrastim in cases where a second PBSC is performed on day 6.  
3.2.2 Apheresis will start on the fifth day of filgrastim administration. 12-24 liters will be processed daily based on recipient weight (Table 3). The actual volume processed may be adjusted by the NMDP Donor Center or Transplant Center Medical Director based on the pre-collection CD34+ count in order to meet the desired cell dose.  
3.2.3 The target cell dose will be greater than or equal to 7 x 106 CD34+ cells/kg-recipient weight.  
3.2.4 In the event that the donor collection is terminated early for donor-related medical concerns, the donor will be offered the opportunity to consent to a bone marrow donation, and this protocol will allow the infusion of a bone marrow graft. This is considered only in the unlikely circumstance where the recipient has already received conditioning and the product is being collected simultaneously. The recipient will be removed from the study, but will be continued to be managed for all transplant-related care issues and complications.  
| Recipient Weight (kg)   | Volume Processed (L)   | Procedure                  |
|-------------------------|------------------------|----------------------------|
| < 35                    | 12                     | Single 12 liter apheresis. |
| 36 - 45                 | 15                     | Single 15 liter apheresis. |  
| 46 - 55   | 18   | Single 18 liter apheresis or two 12 L aphereses.     |
|-----------|------|------------------------------------------------------|
| 56 - 65   | 22   | Single 22 liter apheresis or two 12 liter aphereses. |
| > 65      | 24   | Single 24 liter apheresis or two 12 liter aphereses. |  
3.2.5 Cells may be processed, cryopreserved, and stored in liquid nitrogen until the day of transplant upon delivery to the NIH Department of Transfusion Medicine (DTM). In the event that cryopreservation is required, special permission will be obtained from NMDP.  
3.2.6 If an ABO incompatibility exists between the donor and patient, the graft will be processed in the cell processing laboratory, according to standard DTM operating procedures.  
3.3 THE FOLLOWING ITEMS SHOULD BE OBTAINED WITHIN 48 HOURS BEFORE STARTING
INDUCTION CHEMOTHERAPY:  
3.3.1 Adequate central venous access is required for all patients undergoing transplantation on this protocol.  
3.3.2 CBC with differential.
3.3.3 Acute care panel, hepatic panel, and mineral panel.  
3.3.4 Urine HCG in women of childbearing potential.  
3.4 THE FOLLOWING TESTS MAY BE PERFORMED AT ANY TIME PRIOR TO TREATMENT ARM
ENROLLMENT
3.4.1 Typing for HLA-A, -B, -C, -DRB1, and –DQB1 3.4.2 Risk of relapse assessment (see Appendix I)
3.4.3 Comorbidity Index assessment (see **Appendix** J)  
3.5 INDUCTION CHEMOTHERAPY FOR THE RECIPIENT
3.5.1 A major goal of the induction chemotherapy is to establish severe host immune T cell depletion prior to the allogeneic HSCT. However, there may be individuals who have already experienced severe host T cell depletion with their most recent therapy and additional chemotherapy prior to the transplant conditioning regimen may be placing additional, unnecessary toxicities upon these individuals. As such, patients meeting all of the following criteria will not receive induction chemotherapy and proceed directly to transplant:
3.5.1.1 Fewer than 100 CD4+ cells/ μl and/or within the statistical margin of error of the CD4+ T-lymphocyte depletion goal ≤ 115 CD4 cells/l of blood at study enrollment, which must be at least 28 days beyond the start of last cycle of therapy prior to enrollment.  
3.5.1.2 Demonstration of response that is at least 28 days beyond the start of cycle of therapy prior to enrollment.
3.5.2 Induction Chemotherapy - After enrollment, recipients will receive either EPOCH-F/R or FLAG induction chemotherapy according to their underlying diagnosis, as indicated in the following table [96,97]:  
| Diagnosis                                                                                                                                                                                                                    | Induction Chemotherapy Regimen   |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------|
| Acute myelogenous leukemia (AML) Secondary AML Acute lymphocytic leukemia (ALL) Lymphoblastic lymphoma                                                                                                                       | FLAG                             |
| Myelodysplastic syndrome (RAEB) Myeloproliferative disorder (MPD) Chronic myelomonocytic leukemia (CMML) Chronic myelogenous leukemia (CML) Hodgkin's lymphoma Non-Hodgkin's lymphoma (NHL)  (except lymphoblastic lymphoma) | EPOCH-F/R                        |
| Chronic lymphocytic leukemia (CLL) Prolymphocytic leukemia (PLL) Multiple myeloma                                                                                                                                            |                                  |  
3.5.3 EPOCH-Fludarabine/Rituximab Induction Chemotherapy (EPOCH-F/R):  
| Drug                      | Dose                                                                                          | Days            |
|---------------------------|-----------------------------------------------------------------------------------------------|-----------------|
| Rituximab                 | 375 mg/m2 IV infusion via specified  rate titration. (Patients with CD20+  malignancies only) | Day 1 only      |
| Fludarabine               | 25 mg/m2 per day IV infusion over 30                                                          | Days 1, 2, 3, 4 |
| minutes, daily for 4 days |                                                                                               |                 |  
| Etoposide                               | 50 mg/m2 per day continuous IV        | Days 1, 2, 3, 4                                             |
|-----------------------------------------|---------------------------------------|-------------------------------------------------------------|
| infusion over 24 hours daily for 4 days |                                       |                                                             |
| Doxorubicin                             | 10 mg/m2 per day continuous IV        | Days 1, 2, 3, 4                                             |
| infusion over 24 hours daily for 4 days |                                       |                                                             |
| Vincristine                             | 0.4 mg/m2 per day continuous IV       | Days 1, 2, 3, 4                                             |
| infusion over 24 hours daily for 4 days |                                       |                                                             |
| Cyclophosphamide                        | 750 mg/m2 IV infusion over 30 minutes | Day 5 only                                                  |
| Prednisone                              | 60 mg/m2 per day PO daily for 5 days  | Days 1, 2, 3, 4, 5                                          |
| Filgrastim                              | 5 g/kg per day SC                    | Daily from day 6 until ANC  > 1000/l x 2 following  nadir. |  
3.5.3.1 Only patients with CD20+ B-cell malignancies will receive rituximab as part of the induction chemotherapy regimen, as per Section 2.5.2.20. The initial infusion should begin at a rate of 50 mg/hr; if no hypersensitivity reaction is observed after the first 30 minutes, the infusion rate may be escalated in 50 mg/hr increments every 30 minutes, to a maximum rate of 400 mg/hr. If a hypersensitivity reaction occurs, the infusion should be temporarily slowed or halted, with resumption at half the previous rate upon improvement in patient symptoms. If the first infusion is tolerated well, infusions during subsequent cycles of EPOCH-F/R can begin at 100 mg/hr and be increased by 100 mg/hr increments at 30-minute intervals to a maximum rate of 400 mg/hr. However, if the first infusion is not tolerated well, then the guidelines for the initial infusion should be followed for subsequent administration. Pre-medication with acetaminophen 650 mg PO and diphenhydramine 25 to 50 mg I.V./p.o. will be administered 30 to 60 minutes prior to the beginning of each rituximab infusion. Rituximab should be infused prior to the administration of fludarabine and the start of infusional EPOCH chemotherapy on the first day of each cycle.  
3.5.3.2 Fludarabine will be given immediately after the rituximab infusion is completed on day 1. If venous access permits, fludarabine may be given concurrently with the start of infusional EPOCH chemotherapy on days 1 through 4 of each cycle (preferred); otherwise, fludarabine will be given before the start of EPOCH infusional agents on days 1-4.  
3.5.3.3 Etoposide, doxorubicin, and vincristine will be prepared and administered according to NIH Clinical Center Pharmacy standard guidelines.  
3.5.3.4 The total vincristine dose per cycle will not be capped, even if it exceeds 2.0 mg over four days.  
3.5.3.5 Fluconazole should not be taken during the period of EPOCH-F/R
administration.  
3.5.3.6 Cyclophosphamide will be administered on day 5 of each cycle immediately after the prior day's infusion is completed. Hydration with 1 liter of 0.9% sodium chloride is recommended to begin one hour prior to cyclophosphamide, infused over two hours.  
3.5.3.7 Filgrastim will be initiated on day 6 at a dose of 5 μg/kg/day; it will be continued until the ANC is greater than 1000 cells/μl on two consecutive biweekly measurements following the ANC nadir, or greater than 5000 cells/μl on 1 biweekly measurement. Biweekly measurements are obtained at least 3 days apart (e.g., Monday and Thursday). Only biweekly CBC values will be used to determine the duration of filgrastim administration during each cycle, even if additional CBC's are obtained.
3.5.3.8 EPOCH-F/R Dose Modification for Cycles 2 and 3:  
- Dose adjustments above starting dose level (level 1) consist of a 20% escalation in the daily doses of etoposide, doxorubicin, and cyclophosphamide, relative to the last cycle administered on study.  
- Dose adjustments below starting dose level (level 1) consist of a 20% reduction in the cyclophosphamide dose only, relative to the last cycle administered on study.  
- Dose adjustments are based on non-hematologic toxicities and the duration of ANC nadir during the previous cycle, using consecutive biweekly CBC/differential (> 3 days apart). Only biweekly CBC values will be used to determine dose adjustment, even if additional CBC's are obtained.  
3.5.3.9 Fludarabine will be reduced by 20% for patients with creatinine clearance less than 70 ml/minute/1.73 m2.
3.5.3.10 The number of cycles of EPOCH-F/R are specified in section **3.5.5**.  
Rules for dose adjustment are summarized in the following table:  
| -   | ANC < 500/l on 1 or fewer measurements     |    | Increase 1 dose level above  previous cycle.   |
|-----|---------------------------------------------|-----|------------------------------------------------|
| -   | No grade III or IV non-hematologic toxicity |     |                                                |
| -   | ANC < 500/l on 2 measurements, or transient (< 7 days) grade III nonhematologic toxicity                                             |    | Same dose level as previous cycle.             |
| -   | No persistent (> 7days) grade III nonhematologic toxicity                                             |     |                                                |
| -   | No grade IV non-hematologic toxicity        |     |                                                |  
| -                                           | ANC < 500/l on 3 measurements, or persistent grade III non-hematologic   |                                              |                                                                     |
|---------------------------------------------|---------------------------------------------------------------------------|----------------------------------------------|---------------------------------------------------------------------|
| toxicity, or                                |                                                                          | Decrease 1 dose level below  previous cycle. |                                                                     |
| transient grade IV non-hematologic toxicity |                                                                           |                                              |                                                                     |
| -                                           | ANC < 500/l on 4 or more measurements,  at dose level 1 or lower; or     |                                             | Discontinue EPOCH-F/R; proceed  to transplant conditioning regimen. |
| -                                           | Persistent grade IV non-hematologic  toxicity                             |                                              |                                                                     |  
3.5.4 Fludarabine/Cytarabine/Filgrastim Induction Chemotherapy (FLAG):  
| Drug                                                    | Dose                              | Days               |
|---------------------------------------------------------|-----------------------------------|--------------------|
| Fludarabine                                             | 25 mg/m2 per day IV               | Days 1, 2, 3, 4, 5 |
| infusion over 30 minutes, Daily for 5 days              |                                   |                    |
| Cytarabine                                              | 2,000 mg/m2 IV                    | Days 1, 2, 3, 4, 5 |
| infusion over 4 hours, Daily for 5 days                 |                                   |                    |
| Filgrastim                                              | 5 g/kg per day SC                | Daily from day 0   |
| until ANC >1000/l x 2                                  |                                   |                    |
| beginning 24 hours PRIOR to  initiation of chemotherapy | following nadir (Section 3.3.4.1) |                    |  
3.5.4.1 Filgrastim will be initiated within 24 hours prior to the initiation of cytarabine and fludarabine (the day prior to the first day of chemotherapy or Day 0). Filgrastim will be administered at a dose of 5 μg/kg/day will be continued until the ANC is greater than 1000 cells/μl on two consecutive days following ANC nadir.  
3.5.4.2 Begin each cytarabine dose 3.5 hours after completion of the preceding fludarabine dose.  
3.5.4.3 Corticosteroid ophthalmic drops will be administered 2 drops to each eye every 6 hours, starting prior to first dose of cytarabine and continuing until 24 hours after the last dose of cytarabine has been completed.  
3.5.4.4 In the event of signs of CNS toxicity, the cytarabine infusion will be interrupted and the M.D. notified. No further cytarabine will be administered if there is CNS toxicity (any grade) deemed related to cytarabine.
3.5.4.5 FLAG Dose Modification for Cycle 2 (individual recipients)
- If the prior cycle of induction chemotherapy is associated with reversible grade 4 non-hematologic toxicity, dose reduction for that patient's subsequent cycles will be performed. Dose reduction will be as follows:
- Fludarabine and cytarabine will be reduced from 5 days to 4 days of administration. The doses of all other medications will remain unchanged.  
3.5.4.6 FLAG Dose Reduction (study population):
- If any 2 of 4 consecutive recipients experience the same grade 4 non-hematologic toxicity, dose reduction will be performed for all subsequent cycles for all patients. Dose reduction will be as follows:
- Fludarabine and cytarabine will be reduced from 5 days to 4 days of administration. The doses of all other medications will remain unchanged.  
3.5.4.7 Fludarabine will be reduced by 20% for patients with creatinine clearance less than 70 ml/minute/1.73 m2.
3.5.4.8 The number of cycles of FLAG are specified in 3.3.5.  
3.5.5 Determination of Number of Cycles of Induction (FLAG or EPOCH-F/R) Chemotherapy:
3.5.5.1 Because a major goal of the induction chemotherapy is to establish severe host immune T cell depletion prior to the allogeneic HSCT, the number of induction chemotherapy cycles administered will be determined principally by the level of host T cell depletion achieved.  
3.5.5.2 The CD4 count will be measured by FACS analysis at least 48 hours after the cycle's last dose of filgrastim and within three days before the next scheduled cycle (i.e. day 22). FACS analysis will be performed by a CLIA certified laboratory using a flow cytometry methodology to determine the percentage of circulating CD4+ cells. A simultaneous CBC will be drawn in order to calculate the absolute CD4 number.  
3.5.5.3 If a patient has fewer than 100 CD4 cells/l and/or within the statistical margin of error of the CD4+ T-lymphocyte depletion goal ≤ 115 CD4 cells/l of blood on the final CD4 determination of the induction chemotherapy cycle, then that patient will receive the transplant preparative regimen. If there are ≥ 100 CD4 cells/l, an additional cycle of induction chemotherapy will be administered.  
These criteria may be used for each induction chemotherapy cycle. A minimum of one cycle of induction chemotherapy will be administered. However, a maximum of three cycles of induction chemotherapy can be administered.  
3.5.5.4 Patients will receive the second cycle of chemotherapy as specified:
- EPOCH-F/R will begin the second cycle of chemotherapy on day 22 after the first cycle was initiated. Patients must have a post-nadir ANC >500/µL and post-nadir platelet count >50,000/µL in order to proceed to next cycle. However, up to two weeks of additional recovery time may be provided if medically indicated before administration of the second cycle (e.g., for delay in neutrophil or platelet recovery, documented infection, or grade III or higher III non-hematologic toxicity resulting from the induction chemotherapy regimen).  
- FLAG will begin on day 28 after the first cycle was initiated. However, up to two weeks of additional recovery time may be provided if medically indicated before administration of the second cycle (e.g., for delay in neutrophil recovery to 1000 cells/l, documented infection or other complication resulting from the induction chemotherapy regimen).  
3.5.5.5 Patients who develop excessive neutropenia during chemotherapy will go straight to transplantation
- If a patient receiving EPOCH-F/R develops neutropenia of less than 500 cells/l on 4 or more consecutive biweekly CBC's during any cycle of induction chemotherapy administered at dose level 1 or lower, the patient will receive no further induction chemotherapy. At that point, the patient will receive the transplant preparative regimen, even if the CD4 count is > 100 cells/l. Only biweekly CBC values (at least 3 days apart, e.g. Monday and Thursday) will be used to define the duration of neutropenia during each cycle, even if additional CBC's are obtained.  
- If a patient receiving FLAG chemotherapy continues to have neutropenia defined as ANC<500 which continues until ≥ day 28 of treatment, the patient will receive no further induction chemotherapy even if the CD4 count at the time of recovery is ≥ 100 cells/l.  
3.5.5.6 In patients who previously have received a total anthracycline dose >
450mg/m2a MUGA or 2-D echo will be repeated after each cycle of EPOCHFR. If the ejection fraction is less than 45% and/or new wall motion abnormalities have occurred the next planned cycle will be held for 2 weeks and a repeat MUGA or 2-D echo will be performed. If the abnormalities persist the patient will be removed from protocol. If they have resolved the patient may proceed with the next planned course of EPOCH-FR however the Adriamycin will be removed.  
3.5.5.7 If the maximum cycles (3 for EPOCH-F/R and 2 for FLAG) of induction chemotherapy has been administered on the study or prior to the study enrollment, then the patients may proceed to the transplant preparative regimen, even if the CD4 count is still ≥ 100 cells/l.  
3.5.5.8 Patients who develop progressive disease or relapse during or after the completion of the induction chemotherapy will not be eligible for the transplant phase of the protocol, and will be removed from the study (Section **3.10**).
3.5.6 Concurrent Therapy for Extramedullary Leukemia or CNS Lymphoma:  
3.5.6.1 Concurrent therapy or prophylaxis for testicular leukemia, CNS leukemia, and CNS lymphoma including standard intrathecal chemotherapy and/or radiation therapy will be allowed as clinically indicated after approval by the PI. Such treatment may continue until the planned course is completed. Subjects must be in CNS remission at the time of protocol enrollment.
3.5.7 Concurrent Therapy for bcr-abl (+) CML, AML or ALL  
3.5.7.1 Patients receiving tyrosine kinase inhibitors (e.g. imatinib, nilotinib, and dasatinib) may continue through induction therapy. Such treatments must be discontinued one week prior to initiating the conditioning regimen.
There has been an increasing amount of data over the past three years showing that the administration of TKI's with induction and consolidative chemotherapy provides clinical benefit relative to remission. The proposed amendment would allow for the continued use of tyrosine kinase inhibitors though induction therapy. The concomitant use of TKI with conventional chemotherapy is becoming the standard of care for patients with bcr-abl(+) leukemias. The Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL) treated 45 patients with newly diagnosed Ph+ ALL and initiated imatinib with consolidative chemotherapy (HAM:
mitoxantrone with intermediate-dose cytarabine) consolidation in good early responders (corticosensitive and chemo-sensitive ALL) or earlier during the induction course in combination with dexamethasone and vincristine in poor early responders (cortico-resistant and/or chemo-resistant ALL). Imatinib was then continuously administered until stem cell transplantation. Overall, complete remission (CR) and BCR-ABL real-time quantitative polymerase chain reaction (RQPCR) negativity rates were 96% and 29%, respectively. All of the 22 CR patients (100%) with a donor actually received allogeneic SCT in first CR. At 18 months, the estimated cumulative incidence of relapse, disease-free survival, and overall survival were 30%, 51%, and 65%,
respectively. These 3 end points compared very favorably with results obtained in the preimatinib LALA-94 trial. The authors of the paper concluded that their study confirmed the value of the combined approach and encourages prospective trials to define the optimal chemotherapy that has to be combined with imatinib. In addition, it appears that the use of TKI prior to transplant is not associated with increased transplant-related morbidity (TRM) or poorer outcomes [122, 123]. 3.5.8 Interim Evaluation During Induction Chemotherapy  
3.5.8.1 The following studies will be obtained in all EPOCH-F/R or FLAG recipients during each cycle of induction chemotherapy on a biweekly basis, at least 3 days apart:
3.5.8.1.1 Complete blood count with differential
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3.5.8.1.2 acute care mineral and

3.5.8.2 The following studies will be obtained in recipients with lymphoma, CLL,
PLL, or multiple myeloma at the end of each cycle of EPOCH-F/R induction chemotherapy:
3.5.8.2.1 Routine chemistry hematology panels and 24 hour Creatinine Clearance (if clinically indicated).
3.5.8.2.2 Urine or serum HCG in women of childbearing potential  
3.5.8.2.3 For recipients with lymphoma: computed tomography scans of chest/abdomen/pelvis (and neck, if clinically indicated).  
3.5.8.2.4 For recipients with multiple myeloma: serum protein electrophoresis; serum Ig level (free light chains); 24h collection of urine for urine protein electrophoresis, immunofixation, albumin, and creatinine clearance; β2microglobulin; immunofixation if M protein is undetectable; CRP
3.5.8.3 The following studies will be obtained in recipients with acute leukemia, lymphoblastic lymphoma, MDS, MPD, CMML, or CML at the end of each cycle of FLAG induction chemotherapy:
3.5.8.3.1 Routine chemistry hematology panels and 24 hour Creatinine Clearance (if clinically indicated).
3.5.8.3.2 Urine or serum HCG in women of childbearing potential  
3.5.8.3.3 For recipients with lymphoblastic lymphoma: computed tomography scans of chest/abdomen/pelvis (and neck, if clinically indicated).  
3.5.8.3.4 Bone marrow aspirate and biopsy, with flow cytometry, cytogenetics, FISH
and/or PCR as clinically appropriate to monitor disease status.  
3.6 TRANSPLANT CONDITIONING REGIMEN 3.6.1 The following studies will be obtained in the recipient within 28 days before the initiation of the conditioning regimen:
3.6.1.1 Bone marrow aspiration and biopsy; flow cytometry, cytogenetics, and molecular studies as clinically appropriate 3.6.1.2 For recipients with lymphoma, CLL, or PLL: computed tomography scans of chest/abdomen/pelvis (and neck, if clinically indicated); FDG-PET scan, if evaluable disease was present on baseline PET.  
3.6.1.3 For recipients with multiple myeloma: serum protein electrophoresis; serum Ig level; 24h collection of urine for urine protein electrophoresis; B2microglobulin; immunofixation if M protein is undetectable; bone marrow biopsy; skeletal survey.
3.6.1.4 Pulmonary function testing including DLCO 3.6.1.5 2-D echo or MUGA scan 3.6.2 The following studies will be obtained in the recipient within 48 hours of the initiation of the transplantation conditioning regimen:
3.6.2.1 CBC with differential. 3.6.2.2 PT, PTT. 3.6.2.3 Acute care panel, hepatic panel, and mineral panel.  
3.6.2.4 Urinalysis and 24 hour Creatinine Clearance.
3.6.2.5 Urine or serum HCG in women of childbearing potential.  
6.2.1.6 PCR test of DNA mini-satellite regions for future determination of chimerism 3.6.3 On day 22 after the start of the last cycle of EPOCH-F/R or day 28 after the start of the last cycle of FLAG chemotherapy, patients will be eligible to start the transplant conditioning regimen and should meet the following criteria (performed after completion of induction therapy) Patients who already met all these criteria at the study enrollment may be considered for proceeding with transplant conditioning per PI decision and in consultation with relevant sub-specialist services when pertinent:  
3.6.3.1 ECOG performance status equal to 0, 1, or 2, and Karnofsky performance status greater than or equal to 60%.  
3.6.3.2 Left ventricular ejection fraction > 45% by either MUGA or 2-D echo.
3.6.3.3 DLCO > 50% of the expected value when corrected for Hb.  
3.6.3.4 Creatinine < 1.5 mg/dl and creatinine clearance > 50 ml/min/1.73 m2.  
3.6.3.5 Serum total bilirubin less than 2.5 mg/dl, and serum ALT and AST values less than or equal to 2.5 times the upper limit of normal.  
3.6.3.6 No evidence of active infection that is not responding to antimicrobial therapy. 3.6.3.7 Verification of donor eligibility received from NMDP.
3.6.3.8 Verification of disease status:  
a) Recipients with primary or secondary acute leukemia, myelodysplastic syndrome, blastic phase CML, myeloproliferative disorders, or other eligible diagnoses in transformation to acute leukemia must have < 5% blasts in bone marrow and no circulating blasts after induction therapy.  
b) Recipients with chronic or accelerated phase CML must have < 10% blasts in the peripheral smear or bone marrow after induction therapy.  
c) For patients with non-Hodgkin's lymphoma, Hodgkin's disease, multiple myeloma, CLL, PLL, or other lymphoid malignancy must be determined to have at least stable disease to induction therapy.
3.6.4 Non-hematologic toxicities of induction chemotherapy must resolve to <grade 2 in order to proceed. If more than 14 days of additional recovery time is required for resolution of toxicity or other medical complications (for example, prolonged neutropenia or documented infection), PI approval is required prior to initiation of the transplant conditioning regimen.  
3.6.5 Fludarabine/Cyclophosphamide/Mesna:  
Drug Dose Days  
| Drug                                          | Dose                                                                      | Days                           |
|-----------------------------------------------|---------------------------------------------------------------------------|--------------------------------|
| Fludarabine                                   | 30 mg/m2 per day                                                          | Transplant Days –6, -5, -4, -3 |
| IV infusion over 30 minutes, daily for 4 days |                                                                           |                                |
| Cyclophosphamide                              | 1200 mg/m2 per day                                                        | Transplant Days –6, -5, -4, -3 |
| IV infusion over 2 hours, daily for 4 days    |                                                                           |                                |
| Mesna                                         | 1200 mg/m2 per day                                                        | Transplant Days –6, -5, -4, -3 |
| Continuous IV infusion, Daily for 4 days      |                                                                           |                                |
| 3.6.5.1                                       | Mesna infusion should start concurrently with cyclophosphamide on day –6. |                                |  
3.6.5.2 Fludarabine will be reduced by 20% for patients with creatinine clearance less than 70 ml/min/1.73 m2.  
3.6.6 Hydration during the conditioning regimen:  
3.6.6.1 Hydration will be initiated 12 hours prior to cyclophosphamide infusion (on day –7 of the transplant), consisting of 0.9% sodium chloride supplemented with 10 mEq/liter potassium chloride (KCl) at an initial rate of 100 ml/hour. For recipients with poor oral intake, the rate of hydration may be increased as clinically indicated to meet fluid requirements. Hydration will continue until 24 hours after the last cyclophosphamide dose has been completed.  
3.6.6.2 During hydration, serum potassium level will be monitored every 12 hours. If serum potassium is > 4.5 mEq/l, KCl will be removed from the saline infusion. If serum potassium is < 3.0, KCl concentration in the saline will be increased to 20 mEq/l.  
3.6.6.3 During hydration, 20 mg of furosemide will be administered daily by IV route to maintain diuresis, with additional doses of furosemide to be given as needed for weight gain due to fluid retention. In general, furosemide doses should be separated by at least a four-hour observation interval.  
3.6.6.4 During hydration, if fluid intake exceeds urine output by greater than 500 ml during an 8-hour period, an additional 20 mg of furosemide will be administered.  
3.6.6.5 Routine hematologic monitoring, cardiac monitoring, and urinalysis will be performed as deemed clinically necessary.  
3.7 ALLOGENEIC PERIPHERAL BLOOD STEM CELL TRANSPLANTATION
3.7.1 On day 0, the recipient will receive PBSC. If the PBSC product is cryopreserved, it will be administered intravenously immediately after thawing. Infused CD3 cell dose per kg recipient weight will be recorded 3.7.2 Diphenhydramine and/or meperidine are preferred for the treatment of acute DMSO
toxicities (e.g. chills, myalgias); steroids are not permitted.  
3.8 GRAFT-VERSUS-HOST DISEASE (GVHD) PROPHYLAXIS 3.8.1 GVHD Prophylaxis Arm Assignment - At the time of study enrollment, the CRO will inform the investigators of the patient's assignment to their respective GVHD prophylaxis arm. Patients will be randomized to receive one of two specific GVHD prophlaxis regimens at the time of enrollment: Tacrolimus + Methotrexate + Sirolimus (TMS) versus Alemtuzumab + Cyclosporine (AC).  
3.8.1.1 Tacrolimus + Methotrexate + Sirolimus (TMS Arm) [36]:
a) Tacrolimus will be initiated on day -3 before the transplant. Tacrolimus will be administered continuous intravenous infusion at 0.02 mg/kg/day. Subsequently doses will be adjusted according to trough levels monitored at least biweekly and/or upon symptoms or alteration in renal function. The target serum level for tacrolimus will be 5 to 10 ng/mL.  
b) When the recipient is able to take oral medications (typically 10 to 21 days after transplantation), tacrolimus will be converted to an equivalent oral dose. The total daily dose will be divided into two equal doses, one dose given approximately every 12 hours.  
c) This dose of tacrolimus will continue until day +63 (+/- 2 days), and then reduced by one-third as long as the severity of GVHD is less than grade 2 and patient is not requiring systemic steroids. Tacrolimus will be subsequently reduced by one third on day +119 (+/- 2 days). Tacrolimus will then be completely discontinued by day +180 (i.e. 6 months) discontinued if there are no signs of GVHD.  
d) Methotrexate will be given at 5 mg/m2IV over 15 minutes on days +1, +3, +6, and +11.  
Each day's dose of methotrexate will not be administered until approved that day by the transplant attending after the transplant team has evaluated the patient. Doses will be withheld for the development of grade III or IV mucositis or clinical evidence of venoocclusive disease. Administration of the combination of trimethoprim and sulfamethoxazole (i.e., Bactrim) or non-steroidal anti-inflammatory drugs is contraindicated during methotrexate administration. Methotrexate administration will be assessed on each planned day of administration, and dose adjustments will be based upon the following criteria:  
| Creatinine      | Total Bilirubin   | Methotrexate Dose   |
|-----------------|-------------------|---------------------|
| < 2.0 mg/dL     | <5.0 mg/dL        | 100%                |
| 2.0 - 3.0 mg/dL | ----              | 50%                 |
| > 3.0 mg/dL     | > 5.0 mg/dL       | Hold Dose           |  
e) Methotrexate may also be held or dose adjusted at the discretion of attending physician or P.I. for severe mucositis or third space fluid collection (e.g. pleural effusion, ascites, or edema. In addition, leucovorin (folinic acid) rescue may be considered under the direction of the attending physician or PI. The recommended dose regimen of leucovorin is 10mg IV or PO every 6 hours starting 24 hours after methotrexate administration and continued until undetectable methotrexate levels or upon the discretion of the attending physician or P.I.. Leucovorin should be held 12 hours prior to the any subsequent methotrexate dose and not resumed (if indicated) for 24 hours after a methotrexate dose.  
f) Sirolimus will be initiated on day –3 before the transplant procedure. Sirolimus will be administered by mouth as sirolimus tablets at an initial "loading dose" of 12 mg, p.o., on day –3 of transplantation.  
g) On day –3 pre-transplant and subsequently, sirolimus dosing will be 4 mg, p.o., each day.  
Sirolimus dose should be adjusted to maintain therapeutic concentrations in the range of 3 to 12 ng/ml, with sirolimus trough levels drawn on a Monday, Wednesday, Friday schedule (if inpatient) or a Monday, Thursday schedule (if outpatient).
h) This dose of sirolimus will continue until day +63 (+/- 2 days), and then reduced by onethird as long as the severity of GVHD is less than grade 2 and patient is not requiring systemic steroids. Sirolimus will be subsequently reduced by one third on day +119 (+/- 2 days). Sirolimus will then be completely discontinued by day +180 (i.e. 6 months) if there are no signs of GVHD.  
i) Because voriconazole greatly reduces sirolimus clearance via cytochrome p450 inhibition, the use of voriconazole should be avoided in recipients of sirolimus. However, if voriconazole is deemed necessary while the patient is receiving sirolimus, the dose of sirolimus will be reduced by 90% [98].
j) Fluconazole has less of an effect on sirolimus clearance, and therefore will be used for fungal prophylaxis in all patients.  
k) For sirolimus levels, 2.5 cc of blood will be collected into EDTA tubes and analyzed at the NIH Clinical Center.  
3.8.1.2 Alemtuzumab and Cyclosporine (AC Arm) [31]:
3.8.1.2.1 Alemtuzumab (CAMPATH-1H, Campath®) at 20 mg/day by intravenous
(IV) infusion over 8 h on days −8 to −4 (total alemtuzumab = 100 mg).  
3.8.1.2.1.1 Patients are to be pre-medicated with diphenhydramine 50 mg p.o.  
and acetaminophen 650 mg p.o. 30 minutes prior to each alemtuzumab infusion.  
3.8.1.2.1.2 In light of the relatively high risk of infusion-related toxicities, the initial infusion rate should be 0.5 mg/hour, which may increased every 15 minutes as tolerated by increments of 0.5 mg/hour until the target rate of 2.5 mg/hour is achieved.  
3.8.1.2.1.3 In cases where severe infusion-related events occur, the rate should be slowed or discontinued and patients should receive hydrocortisone 200 mg intravenously after approval by treating physician. Once determined by the treating responsible physician that the patient is stable, the infusion can be re-initiated or advanced as per guidelines in 3.6.1.2.1.2.  
3.8.1.2.1.4 Other drug substances should not be added or simultaneously infused through the same intravenous line that alemtuzumab is administered.  
3.8.1.2.2 Cyclosporine will be initiated on the day –1 before the transplant.  
Cyclosporine will be administered by IV infusion at 2 mg/kg/dose every 12 hours; infusion will be over a 2-hour period. Subsequently doses will be adjusted according to trough levels monitored at least biweekly and/or upon symptoms or alteration in renal function. The target range for serum cyclosporine levels will be 150–250 g/ml.  
3.8.1.2.3 When the recipient is able to take oral medications (typically 10 to 21 days after transplantation), cyclosporine will be converted to an equivalent oral dose. The total daily dose will be divided into two equal doses, one dose given approximately every 12 hours.  
3.8.1.2.4 This dose of cyclosporine will continue until day +100, and then will be tapered as long as the severity of GVHD is less than grade 2. Cyclosporine will be tapered by reducing the dose by approximately 10% from the last dose administered each week to a dose of 25 mg/day. Cyclosporine will then be completely discontinued by six months if there are no signs of GVHD.
3.9 EVALUATION DURING TRANSPLANTATION 3.9.1 The following studies will be obtained in the recipient during hospitalization for transplantation:
3.9.1.1 CBC twice daily; differential count daily 3.9.1.2 Acute care and mineral panels twice daily; hepatic panel and LDH daily 3.9.1.3 Type and screen every 4 days  
3.9.1.4 GVHD prophylaxis (cyclosporine, tacrolimus, sirolimus) trough level twice weekly, starting day +1 or day +2.
3.9.2 Donor/recipient chimerism studies:  
3.9.2.1 Baseline determination in donor and recipient (prior to start of respective induction chemotherapy).
3.9.2.2 After transplantation in recipient (+/- 3 days):  
a) Peripheral blood lymphoid, myeloid, and total chimerism on day +14, +28, +42, +60, if lymphoid and myeloid are ≥ 95% then will check again at day +100. It will then be
obtained monthly until day +180, and every three months thereafter until day +365(+/- 3 days at all timepoint intervals). .  
b) Bone marrow chimerism on days +28, +100, and 12 months. Lymphoid & myeloid subset chimerism will also be measured at these time-points if donor chimerism was < 95% on the previous study.  
3.9.2.3 Chimerism may be measured at other time-points if clinically indicated (e.g., to determine effect of manipulating immune suppression in order to increase donor chimerism).
3.9.3 The patient will be evaluated at least twice a week during the first 100 days after transplantation for the development of acute GVHD.
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3.10 off study criteria

Prior to removal from study, effort must be made to have all subjects complete a safety visit approximately 30 days following the last dose of study therapy. 3.10.1 The recipient will be removed from protocol for any of the following reasons:
3.10.1.1 Inadequate stem cell collection from the selected donor defined as less than or equal to 2 x 106 CD34+ cells/kg. If this rare event should occur, another eligible donor may be enrolled and may undergo filgrastim mobilization and PBSC collection as outlined in Section 0. If no other eligible donor is available or if there is insufficient time to arrange donor cell collection, then the recipient will be removed from the trial; however, the available cells will be infused and the patient will receive standard post-transplant care.  
3.10.1.2 Recipients with acute leukemia (primary or secondary), MDS, MPD, CMML-2, or blastic phase CML must be in remission after FLAG induction chemotherapy (section **3.3.4**), defined as < 5% blasts on bone marrow aspirate and the absence of circulating blasts in peripheral blood.  
3.10.1.3 Progressive disease during or after completion of EPOCH-F/R induction chemotherapy (section **3.3.3**).
3.10.1.4 Non-hematologic toxicity of > grade 3 for the recipient while receiving induction chemotherapy (EPOCH-F/R or FLAG), that does not resolve within 14 days after the next cycle of induction chemotherapy or the transplant conditioning regimen is due to be administered. However, recipients with grade 3 or higher infections may remain on study and continue with protocol treatment, if the infection or infections are responding to antimicrobial therapy and are not felt to pose an excessive risk in the opinion of an infectious diseases consultant.  
3.10.1.5 Failure to meet pre-transplant eligibility criteria defined in section**2.7.**
3.10.1.6 The donor or recipient refuses to continue therapy. 3.10.1.7 Lost to Follow-Up 3.10.1.8 Death 3.10.2 In all cases, the reason(s) for withdrawal will be documented. 3.10.3 In addition, a patient may at any time be removed from protocol at the principal investigator's discretion, if the P.I. deems the subject to be at unacceptable risk to remain on study. Reasons for this action may include (but are not limited to) disease progression with declining organ function/performance status before transplantation; inadequate family/caregiver support; noncompliance.  
3.11 POST-TRANSPLANTATION EVALUATION 3.11.1 After completion of therapy the recipient will be followed for potential complications related to allogeneic SCT. The recipient will be followed at least twice weekly in the outpatient setting until at least Day +100 after discharge from the hospital.  
3.11.2 The recipient will be seen in follow-up at the Clinical Center to evaluate disease status and late complications of allogeneic HSCT, at days +28 and +100; and at 6, 9, 12, 18, 24 months and then annually post-transplant thereafter up until the 5 year timepoint unless earlier evaluation is clinically indicated. At these times recipients will undergo the following tests to determine clinical response:
3.11.2.1 Routine chemistry and hematologic panels. 3.11.2.2 Bone marrow aspiration and biopsy with flow cytometry, cytogenetics, and molecular studies as clinically appropriate.  
3.11.2.3 For recipients with lymphoma: computed tomography scans of chest/abdomen/pelvis (and neck, if clinically indicated). FDG-PET scan will be obtained 28 and 100 days post transplant in all patients with lymphoma (if indicated). For recipients who enter into CR or CRu with negative PET, follow-up evaluation will include a PET scan at 6 and 12 months post transplant. Other time points may be included if clinically indicated.  
3.11.2.4 For recipients with multiple myeloma: serum protein electrophoresis with M
protein; serum Ig level; 24h collection of urine for urinary protein excretion, protein electrophoresis and M protein; B2-microglobulin; immunofixation if M
protein is undetectable; skeletal survey will be performed as clinically indicated..  
3.11.2.5 Pulmonary Function Test (PFT): (exception: D28 evaluation is only if clinically indicated).  
3.11.2.6 Patients who have previously received a total anthracycline dose > 450mg/m2 will also have a MUGA or 2-D echo will at these timepoints.  
3.11.3 Any patient who develops chronic GVHD (before or after day 100) and all patients with acute GVHD manifestations at 6, 9, 12, 15, 18, 21 and 24 months will be referred in the Chronic GVHD Clinic as part of their scheduled routine follow-up.  
3.11.4 Any patient who experiences post-transplant relapse will be referred to the Natural History of Disease Outcomes post Allogeneic Hematopoietic Stem Cell Transplantation protocol 11-C-0125. Patients will not be taken off study in this case but can be enrolled on both studies concurrently.  
3.11.5 After 5 year follow up all key protocol research endpoints are achieved there would be no protocol scheduled follow up visits and all further follow up would be based solely on clinical decisions by the PI or LAI.
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4 supportive care 4.1 infection

(For a full description of infection prophylaxis please refer to the NIH BMT Consortium Supportive Care Guidelines at http://intranet.cc.nih.gov/bmt/clinicalcare/guidelines.shtml)
4.1.1 All recipients will receive prophylaxis against Pneumocystis jiroveci (previously known as *Pneumocystis carinii*) pneumonia, beginning with the first cycle of the induction therapy, continuing until transplantation, and resuming at the time of platelet recovery. Pneumocystis prophylaxis will continue until immunosuppression is discontinued and the CD4 count is > 200 for 3 months. Trimethoprim/sulfamethoxazole is the preferred regimen.  
4.1.2 All recipients will receive fluconazole for prophylaxis against yeast infections. Because of its interaction with vincristine, fluconazole should not be taken concurrently with EPOCH-F/R chemotherapy. Therefore, fluconazole will start on day 6 of the first cycle of pre-transplant induction chemotherapy and will be held on days 1 through 5 of each subsequent cycle. Fluconazole will then continue through transplantation until day +100 and immunosuppression is discontinued. Fluconazole may be stopped during therapy with an alternative antifungal agent.  
4.1.2.1 Although there is evidence that ketoconazole can raise the AUC of imatinib, as well as cyclosporine, there are no specific recommendations within the medical literature that any of these agents require a specific dose modification. However, it is advised to monitor more closely for the known adverse effects of these agents and to adjust or discontinue these agents as appropriate if an adverse event occur.  
4.1.3 During the peri-transplant period, recipients will receive prophylactic broad-spectrum antibiotics with ceftazidime from the onset of neutropenia (ANC < 500/μl) or first fever (T > 38.3°C) until neutropenia resolves. Recipients with allergy to cephalosporin antibiotics will receive an alternative regimen for prophylaxis as recommended by the infectious diseases consultant. This practice is adopted in recognition of the historically high infectious morbidity and mortality associated with allogeneic HSCT from unrelated donors. At other times, antibiotic therapy for neutropenic recipients will be managed in accordance with CDC guidelines. Recipients with neutropenia and fever that persists for longer than 4 days despite broad-spectrum antibiotics will receive empiric antifungal therapy with caspofungin, liposomal amphotericin B, poseconazole, or voriconazole, in accordance with standard practices. Empiric antifungal therapy may be started at other times, if clinically indicated. Voriconazole should not be administered during the time of sirolimus in patients randomized to that arm (section **3.8.1.1**).  
4.1.4 All recipients will receive valacyclovir for prophylaxis against herpes simplex virus and Varicella zoster virus infection/reactivation. This therapy will start on day 1 of pretransplant induction chemotherapy, continuing through transplantation until day +100 and immunosuppression is discontinued. Valacyclovir will be discontinued while patients are receiving IV acyclovir, valganciclovir, ganciclovir, or foscarnet.  
4.1.5 Recipients with positive pre-transplant serology for Cytomegalovirus (CMV) and/or CMV-sero-positive donors will be monitored for CMV reactivation by clinical evaluation and weekly testing with CMV antigenemia or PCR assays as clinically appropriate. CMV reactivation or disease will be treated according to the schema in Appendix A. Weekly monitoring will continue through 6 months post transplant (longer for recipients on continued immunosuppression).  
4.1.6 All recipients will undergo monitoring for reactivation of Epstein-Barr virus (EBV) by weekly quantitative PCR assays. Pre-emptive treatment with rituximab and/or donor lymphocyte infusion (DLI) will be considered for recipients with EBV reactivation, in consultation with infectious diseases staff. Monitoring will continue through day +180 post transplantation (longer for recipients deemed at continued high risk by infectious diseases staff).  
4.1.7 All patients will receive ursodeoxycholic acid (Ursodiol) for the prevention of hepatic complications after allogeneic stem cell transplantation [99,100]. Ursodiol will start day -6 prior to the transplant and will continue until day +98 post-transplant. Patients weighing less than 90kg will receive 300mg orally twice daily (600mg total dose each day). Those patients weighing more than 90kg will receive 300mg orally each morning and 600mg orally each evening (900mg total dose each day).  
4.1.8 Recipients receiving immunosuppression for chronic GVHD will receive penicillin V for prophylaxis against bacterial infections. All recipients will undergo vaccinations as indicated in Appendix A, beginning 6 months after transplantation. Administration of live vaccines will be avoided during the first 2 years after transplantation or in patients with ongoing chronic GVHD or immunosuppression.