Reduced Intensity, Partially HLA Mismatched Allogeneic BMT for Hematologic Malignancies Using Donors Other Than First-degree Relatives
Phase: PHASE1
Conditions: Hematologic Malignancies
Sponsor: Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins

Version date: 
11/04/2020
NCT01203722
 
1 
Reduced intensity, partially HLA mismatched allogeneic BMT for hematologic malignancies using 
donors other than first-degree relatives 
PI: 
Richard Ambinder, MD, PhD 
CRB 389, 1650 Orleans Street 
Baltimore, MD 21287 
Phone 410-955-8839 
Fax 410-955-0960 
Pager 410-283-0311 
ambinri@jhmi.edu 
Co-PI:  Yvette Kasamon, MD 
Coinvestigators: Leo Luznik, Javier Bolaños-Meade, Ephraim Fuchs, Richard Jones, Mary Leffell, Jonathan 
Powell, Christopher Gamper 
Statisticians: Marianna Zahurak, Gary Rosner 
Version date: November 4,, 2020 (Amendment 16) 
Protocol history: 
Original version dated 7/20/2010: IRB approved 8/17/2010 
Protocol version dated 12/28/2010 (Amendment 1): IRB approved 1/18/2011 
 Protocol version dated 2/9/2010 (Amendment 2): IRB approved 3/1/2011  
Protocol version dated 4/13/2011 (Amendment 3): IRB approved 5/3/2011 
 Protocol version dated 5/18/2011 (Amendment 4): IRB approved 6/7/2011 
Protocol version dated 7/19/2011 (Amendment 5): IRB approved 8/9/2011 
 Protocol version dated 8/25/2011 (Amendment 6): IRB approved 9/13/2011 
 Protocol version dated 12/21/2011 (Amendment 7): IRB approval 2/3/2012  
Protocol version dated 11/20/2012 (Amendment 8): IRB approval 12/5/2012 
 Protocol version dated 4/22/2015 (Amendment 9): IRB approval 5/6/2015  
Protocol version dated 11/07/2016 (Amendment 10): IRB approved 12/7/2016  
Protocol version dated 12/20/2016 (Amendment 11): IRB approved 1/11/2017  
Protocol version dated 2/10/2017 (Amendment 12): IRB approved 3/5/2018  
Protocol version dated 7/25/2018 (Amendment 13): IRB approved 8/15/2018 
Protocol version dated 6/23/2020 (Amendment 14): IRB approved 6/29/2020 
Protocol  version dated 7/22/2020 (Amendment 15): IRB approved 10/15/2020 

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INDEX 
Schema 
Post-transplantation immunosuppression: overview 
Regimen B 
Regimen B2 
Regimen C 
1.0 
Introduction 
1.1 
Post-transplantation high-dose cyclophosphamide 
1.2 
Impact of HLA mismatching on outcome 
1.3 
Reduced-intensity conditioning with fludarabine-busulfan 
1.4 
Sirolimus and post-transplantation cyclophosphamide: rationale for study 
1.5 
Special considerations in patients with HIV 
1.6 
Stem Cell Source 
2.0 
Objectives 
2.1 
Primary objectives 
2.2 
Secondary objectives 
3.0 
Selection of Patients and Donors 
3.1 
Patient eligibility 
3.2 
Donor eligibility 
3.3 
Donor prioritization 
3.4 
Donor search criteria 
4.0 
Registration Procedures 
4.1 
Registration requirements 
4.2 
Accrual goal 
5.0 
Treatment Plan 
5.1 
Evaluations and procedures 
5.2 
Overview of study design 
5.3 
Conditioning, transplantation, and post-transplantation cyclophosphamide: 
Regimens B and C 
5.31 
Fludarabine 
5.32 
Pre-transplantation cyclophosphamide 
5.33 
Total body irradiation 
5.34 
Day of rest 
5.35 
Bone marrow transplantation 
5.36 
Post-transplantation cyclophosphamide 
5.4 
Additional post-transplantation therapies 
5.41 
Regimen B: sirolimus and mycophenolate mofetil 
5.42 
Regimen C: tacrolimus and mycophenolate mofetil 
5.43 
Growth factors 
5.44 
Post-transplantation donor lymphocyte infusion (DLI) 
5.5 
Supportive care 
5.51 
Anti-ovulatory treatment 
5.52 
Indwelling central venous catheter 
5.53 
Infection prophylaxis 
5.54 
Antiemetics 

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6.0 
Measurement of Effect and Endpoints 
6.1 
Hematologic parameters 
6.11 
Neutrophil recovery 
6.12 
Platelet recovery 
6.13 
Donor cell engraftment 
6.14 
Graft failure 
6.2 
Graft-versus-host disease 
6.21 
Acute GVHD 
6.22 
Chronic GVHD 
6.3 
Disease and survival endpoints 
6.31 
Progression-free survival 
6.32 
Disease-free survival 
6.33 
Failure-free survival 
6.34 
Overall survival 
6.35 
Non-relapse mortality 
6.36 
Relapse or progression 
6.37 
Minimal residual disease 
6.38 
GVHD-related survival endpoints 
6.4 
Immunologic correlates 
 
7.0 
Study Parameters 
7.1 
Core evaluations 
7.2 
Additional research samples 
 
8.0 
Risks and Reporting Requirements 
8.1 
Drug information 
8.11 
Fludarabine 
8.12 
Cyclophosphamide 
8.13 
Mesna 
8.14 
Mycophenolate mofetil 
8.15 
Sirolimus 
8.16 
Tacrolimus 
8.17 
Concurrent azole therapy 
8.18 
Total body irradiation 
8.2 
Toxicity grading 
8.3 
Toxicity reporting 
8.4 
Monitoring plan 
8.5 
Risks and benefits 
 
9.0 
Statistical Considerations 
9.1 
Primary statistical plan: phase 1 study 
9.11 
First test of a regimen 
9.12 
Continuous safety monitoring of expanded cohorts 
9.13 
Operating characteristics of design 
9.14 
Additional stopping guidelines 
9.2 
Primary statistical plan: phase 2 expansion 
9.21 
Futility monitoring 
9.22 
Continuous safety monitoring 
9.3 
Secondary endpoints 
9.31 
Disease and survival endpoints 
9.32 
Toxicities 

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9.33 
Graft failure and engraftment kinetics 
9.4 
Clinical trial reporting 
 
10.0 
Pathology Review 
 
11.0 
Records to be Kept 
 
12.0 
Patient Consent and Peer Judgment 
 
13.0 
References 
 
14.0 
Appendix 
14.1 
Acute GVHD criteria 

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SCHEMA 
Transplant regimens: overview 
 
Regimen * 
Preparative 
regimen 
High-dose 
cyclophosphamide 
MMF 
Sirolimus 
Tacrolimus 
A 
Flu-Cy-TBI 
Day 3 and 4 
None 
Day 5 - 180 
None 
B 
Flu-Cy-TBI 
Day 3 and 4 
Day 5 - 35 
Day 5 - 180 
None 
B2 
Flu-Cy-TBI 
Day 3 and 4 
Day 5 – 35 
Day 5 – 180 
None 
B3 
Flu-Cy-TBI 
Day 3 and 4 
Day 5 – 35 
Day 5 – 180 
None 
C 
Flu-Cy-TBI 
Day 3 and 4 
Day 5 - 35 
None 
Day 5 - 180 
D 
Flu-Bu 
Day 3 and 4 
Day 5 - 35 
Day 5 - 180 
None 
 
* See Section 5.2 for sequence of study. 

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SCHEMA a 
REGIMEN B 
 
Days –6, –5 
Fludarabine 30 mg/m2  IV QD, adjusted for CrCl 
Cyclophosphamide 14.5 mg/kg IV QD 
 
Days –4, –3, –2 
Fludarabine 30 mg/m2  IV QD, adjusted for CrCl 
 
Day –1 
TBI 400 cGy 
 
Day 0 
 
Infuse T-cell replete bone marrow 
(at least 48 hours after last chemotherapy) 
 
Begin antibiotic prophylaxis (no voriconazole) 
 
Days 3, 4 
Cyclophosphamide 50 mg/kg IV QD 
Mesna 40 mg/kg IV QD in divided doses 
 
Day 5 
Sirolimus loading dose: 6 mg PO once (pts > 18 y) b 
 
Begin MMF 15 mg/kg PO TID (maximum daily dose 3 g/day) 
(Immunosuppression must begin at least 24 hours after Cy completion) 
 
Begin GCSF 5 g/kg SC or IV QD, until ANC > 1000/mm3  over 3 days 
 
Day 6 
Begin sirolimus 2 mg PO QD (pts > 18 y) b, 
target trough 5 – 12 ng/mL 
 
Day 30 (+/- 3 d) 
Assess chimerism in peripheral blood 
 
Day 35 
Discontinue MMF (optional if GVHD is active) 
 
Day 60 (+/- 5 d) 
Assess chimerism in peripheral blood 
Evaluate disease 
 
Day 100 (+/- 5 d) 
GVHD evaluation 
 
Day 180 
Discontinue sirolimus (optional if GVHD is active); 
Day 180 (+/- 21 d) 
 
Assess chimerism in peripheral blood 
Evaluate disease 
 
1 yr  (+/- 30 d) 
Assess chimerism in peripheral blood 
Evaluate disease 
 
 
a. See Sections 5.3 and 5.4 for complete dosing instructions. Up to 2 days of rest may be added after TBI, 
before BMT, per Section 5.34. 
b. See Section 5.41 for sirolimus dosing in younger pts. 

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SCHEMA a 
REGIMEN B2 
 
Days –6, –5 
Fludarabine 30 mg/m2  IV QD, adjusted for CrCl 
Cyclophosphamide 14.5 mg/kg IV QD 
 
Days –4, –3, –2 
Fludarabine 30 mg/m2  IV QD, adjusted for CrCl 
 
Day –1 
TBI 400 cGy 
 
Day 0 
 
Infuse T-cell replete peripheral stem cells 
(at least 48 hours after last chemotherapy) 
 
Begin antibiotic prophylaxis (no voriconazole) 
 
Days 3, 4 
Cyclophosphamide 50 mg/kg IV QD 
Mesna 40 mg/kg IV QD in divided doses 
 
 
Day 5 
Sirolimus loading dose: 6 mg PO once (pts > 18 y) b 
 
MMF 15 mg/kg PO TID (maximum daily dose 3 g/day) 
(Immunosuppression must begin at least 24 hours after Cy completion) 
 
Begin GCSF 5 g/kg SC or IV QD, continue until ANC > 1000/mm3  over 3 days 
 
Day 6 
Begin sirolimus 2 mg PO QD (pts > 18 y) b, 
target trough 5 – 12 ng/mL 
 
Day 30 (+/- 3 d) 
Assess chimerism 
 
Day 35 
Discontinue MMF (optional if GVHD is active) 
 
Day 60 (+/- 5 d) 
Assess chimerism 
Evaluate disease 
 
Day 100 (+/- 5 d) 
GVHD evaluation 
 
Day 180 
Discontinue sirolimus (optional if GVHD is active); 
Day 180 (+/- 21 d) 
 
Assess chimerism 
Evaluate disease 
 
1 yr  (+/- 30 d) 
Assess chimerism 
Evaluate disease 
 
 
a. See Section 5.3 and 5.4 for complete dosing instructions. Up to 2 days of rest may be added after TBI, 
before transplant, per Section 5.34. 
b. See Section 5.41 for sirolimus dosing including in younger pts. 

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SCHEMA a 
REGIMEN C 
 
Days –6, –5 
Fludarabine 30 mg/m2  IV QD, adjusted for CrCl 
Cyclophosphamide 14.5 mg/kg IV QD 
 
Days –4, –3, –2 
Fludarabine 30 mg/m2  IV QD, adjusted for CrCl 
 
Day –1 
TBI 400 cGy 
 
Day 0 
 
Infuse T-cell replete bone marrow 
(at least 48 hours after last chemotherapy) 
 
Begin antibiotic prophylaxis (no voriconazole) 
 
Days 3, 4 
Cyclophosphamide 50 mg/kg IV QD 
Mesna 40 mg/kg IV QD in divided doses 
 
 
Day 5 
Begin tacrolimus 1 mg IV QD (for pts > 18 y) b and 
MMF 15 mg/kg PO TID (maximum daily dose 3 g/day) 
(Immunosuppression must begin at least 24 hours after Cy completion) 
 
Begin GCSF 5 g/kg SC or IV QD, continue until ANC > 1000/mm3  over 3 days 
 
Day 30 (+/- 3 d) 
Assess chimerism 
 
Day 35 
Discontinue MMF (optional if GVHD is active) 
 
Day 60 (+/- 5 d) 
Assess chimerism 
Evaluate disease 
 
Day 100 (+/- 5 d) 
GVHD evaluation 
 
Day 180 
Discontinue tacrolimus (optional if GVHD is active); 
Day 180 (+/- 21 d) 
 
Assess chimerism 
Evaluate disease 
 
1 yr  (+/- 30 d) 
Assess chimerism 
Evaluate disease 
 
 
a. See Section 5.3 and 5.4 for complete dosing instructions. Up to 2 days of rest may be added after TBI, 
before BMT, per Section 5.34. 
b. See Section 5.42 for tacrolimus dosing including in younger pts. 

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1.0 
INTRODUCTION 
Allogeneic blood or marrow transplantation (BMT) is a potentially curative therapy for a variety of 
hematologic malignancies, including the acute and chronic leukemias, myelodysplasia, and lymphomas. 
Recent advances in allogeneic BMT platforms have substantially lowered transplant-related morbidity both 
in the HLA-matched and partially-HLA-mismatched settings. One of these advances is the incorporation of 
high-dose post-transplantation cyclophosphamide (Cy) for prophylaxis of graft-versus-host-disease (GVHD) 
and graft rejection, as developed at Johns Hopkins. This agent, when administered at high doses after 
myeloablative, HLA matched, related or unrelated donor BMT, notably has been found to be effective 
single-agent prophylaxis against GVHD, obviating the need for calcineurin inhibitors (CNI’s) in this setting.1 
In nonmyeloablative, HLA matched or mismatched, related-donor transplantation, the combination of high- 
dose post-transplantation Cy, mycophenolate mofetil (MMF), and tacrolimus has been associated with 
acceptable rates of engraftment and relatively low rates of acute and chronic GVHD.2 
An advantage to nonmyeloablative, partially-HLA mismatched, related-donor BMT is that most 
individuals have multiple potential and readily available donors. However, in some cases patients lack a 
suitable first-degree related donor or an HLA-matched, unrelated donor. In patients infected by the human 
immunodeficiency virus (HIV) it appears that it may be possible to cure patients of HIV by selecting HIV- 
resistant donors as is elaborated below. Transplantation using mismatched, unrelated donors has historically 
been associated with increased rejection rates, severe acute GVHD, extensive chronic GVHD, and increased 
transplant-related mortality.3,4 Due to increases in graft rejection risk and excessive toxicities with increasing 
degrees of mismatch, relatively few transplants using more than 1-locus mismatched, unrelated donors have 
been performed.  Transplantation using partially-HLA mismatched donors who are second-degree as 
opposed to first-degree relatives is also investigational given the greater mismatching at minor 
histocompatibility antigens (mHAg). Thus, if transplantation using mismatched unrelated donors or non- 
first-degree relatives could be performed with an acceptable toxicity profile, an important unmet need would 
be served. Towards this goal, the current study extends our platform of nonmyeloablative, partially HLA- 
mismatched BMT to the use of such donors, investigating postgrafting immunosuppression regimens that 
incorporate high-dose Cy. Of central interest is the incorporation of sirolimus into this postgrafting 
immunosuppression regimen. 
As an alternative to transplantation using bone marrow, there are circumstances in which only 
peripheral blood stem cells are available as a graft source for a variety of different reasons (e.g., the unrelated 
donor doesn’t want to go to the OR) and there are some diseases such as myelodysplasia where peripheral 
blood stem cells may be more effective. While transplants with peripheral blood stem cells (PBSC) have 
higher rates of GVHD than transplants with bone marrow, there are data suggesting that PBSC transplants 
result in lower graft failure and improvements in overall survival (OS) and progressive free survival (PFS) 
71,72,73. Using the methods described above, the PBSC regimen investigates the ability of using mismatched 
unrelated donors or non-first-degree relatives with a PBSC transplant. 
 
1.1 
Post-transplantation high-dose cyclophosphamide 
The immunologic rationale for administering Cy after transplantation is that recently activated, alloreactive 
T-cells (the cells most responsible for GVHD) are selectively sensitive to the toxic effects of this drug.5 
High-dose Cy, when administered in a narrow window after transplantation, depletes alloreactive T-cells 
from the donor and host and can inhibit both GVHD and graft rejection.5-10 As a form of drug-induced 
immunologic tolerance,11 the strategy of giving high-dose Cy after transplantation takes advantage of the 
heightened cytotoxic sensitivity of proliferating, alloreactive T-cells over non-alloreactive, resting T-cells to 
being killed by a DNA-damaging agent.1 Pre-clinical studies demonstrated that engraftment of major 
histocompatibility complex (MHC)-mismatched bone marrow could be achieved by conditioning mice with 
pre-transplantation fludarabine and low dose (400 cGy) total body irradiation (TBI), with post- 
transplantation Cy.7 Additional studies demonstrated that post-transplantation Cy reduced the incidence and 
severity of GVHD in the setting of MHC-mismatched allogeneic BMT after myeloablative conditioning.6 
 
 
a) Efficacy of single agent post-transplantation cyclophosphamide in GVHD prevention 

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After allogeneic BMT, standard regimens of GVHD prophylaxis consist of a CNI (cyclosporine or tacrolimus) in 
combination with either methotrexate, MMF, or sirolimus. However, acute GVHD still occurs in 35-55% of 
BMT recipients from HLA-matched siblings, and more frequently in unrelated donor BMT recipients.12-16 While 
CNI’s inhibit acute GVHD, they are less effective in preventing chronic GVHD.17  Moreover, they impair 
immune reconstitution by inhibiting T-cell development, potentially increasing the risk of disease relapse.18-20 
Thus, a platform that minimizes the use of CNI’s, minimizes GVHD, and retains the donor graft antitumor 
efficacy would be desirable. 
Toward this end, high-dose Cy on Days 3 and 4 after myeloablative, HLA-matched related or unrelated 
donor BMT recently has been reported to be effective single-agent GVHD prophylaxis in patients with 
hematologic malignancies, obviating the need for CNI’s in this setting.1 Luznik et al studied 117 patients with 
advanced hematologic malignancies received HLA-matched related or HLA-matched unrelated donor allografts 
with a platform of conventional busulfan/cyclophosphamide conditioning, T-cell-replete bone marrow, followed 
by 50 mg/kg/day of Cy on Days 3 and 4 after transplantation as the only GVHD prophylaxis.21 The non-relapse 
mortality (NRM) at Day 100 and 2 years were 9% and 17%, respectively. The 2-year event-free survival (EFS) 
was 39%. The incidences of acute grade II-IV and grade III-IV GVHD were only 43% and 10%, respectively, 
and the incidence of chronic GVHD was only 10%. In addition, this approach was marked by prompt immune 
reconstitution and a low incidence of opportunistic infections including CMV disease; the observed lymphocyte 
reconstitution compared favorably to the levels seen after T-cell-replete allogeneic transplantation with 
cyclosporine and methotrexate for GVHD prophylaxis. 
 
b) Nonmyeloablative, haploidentical BMT: role of postgrafting immunosuppression 
Independent clinical trials have evaluated a nonmyeloablative, partially HLA-mismatched (haploidentical), 
related-donor BMT platform with high-dose post-transplantation Cy, tacrolimus, and MMF for GVHD and 
graft rejection prophylaxis. This approach has been associated with rapid and stable engraftment in most 
patients. Most importantly, this approach has carried acceptable rates of acute GVHD, chronic GVHD, and 
NRM that parallel those seen with nonmyeloablative, HLA-matched transplants.2,22-24 
The postgrafting immunosuppression regimen that underlies recent research efforts at Johns Hopkins 
has been published.2,22,24 Conditioning in these studies has consisted of fludarabine, low-dose Cy, and 400 
cGy TBI. A combined analysis of two independent clinical trials was reported in 2008 (40 patients at Johns 
Hopkins, 28 at Fred Hutchinson Cancer Research Center), evaluating the safety and efficacy of a high-dose 
post-transplantation Cy platform after outpatient nonmyeloablative conditioning and T-cell-replete BMT 
from partially HLA-mismatched related donors (Figure 1).2 Eligible patients were 0.5-70 years of age with 
high-risk myeloid or lymphoid malignancies.  Twenty-one patients (31%) had previously received 
autologous BMT. Conditioning consisted of Cy 14.5 mg/kg/day IV on Days –6 and –5, fludarabine 30 
mg/m2/day IV on Days –6 to –2, and 400 cGy of TBI on Day –1. On Day 0, patients received donor bone 
marrow, which was not T-cell depleted. Following transplantation, high-dose Cy (50 mg/kg) was 
administered on Day 3 (Seattle group), or on Days 3 and 4 (Hopkins). Pharmacologic prophylaxis of GVHD 
was initiated on the day following completion of post-transplantation Cy with tacrolimus and MMF. 
Filgrastim 5 g/kg/day was administered until recovery of neutrophils to >1000/L: 
 
Figure 1: Treatment schema in previous studies 

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Engraftment and chimerism. Median times to recovery of neutrophils and platelets were 15 and 24 days, 
respectively. Graft failure occurred in 9 of 66 evaluable patients (12%); all but one patient with graft failure 
had recovery of autologous hematopoiesis with median times to neutrophil and platelet recovery of 15 days 
(range, 11 – 42) and 28 days (range, 0 – 395 days) respectively. Engrafting patients achieved full donor 
chimerism rapidly; with few exceptions, donor chimerism in patients with sustained engraftment was 
virtually complete (>95%) by 2 months after transplantation. 
 
Hospitalizations and infections. Patients received their initial treatment in the outpatient department. The 
median number of hospitalizations prior to Day 60 was 1 (range 0-4), with a median length of stay of 4 days 
and with neutropenic fever or nonneutropenic infection accounting for 80% of the admissions. Twenty-two 
patients (32%) did not require hospitalization within the first 60 days of transplantation. 
Patients who are seropositive for cytomegalovirus (CMV) are known to be at high-risk for 
reactivating CMV after transplantation, regardless of the serologic status of the donor.25 In this study, CMV 
reactivation occurred in 38% of high-risk patients, without CMV disease or CMV-associated mortality. 
 
Graft-versus-host disease and survival outcomes.  The cumulative incidences of grades II-IV and III-IV 
acute GVHD by Day 200 were <35% and <10%, respectively, on competing-risk analysis (Figure 2). The 
groups did not differ significantly in the incidence of grades II-IV or III-IV acute GVHD, although the risk of 
chronic GVHD appeared to be lower with two doses of Cy. The cumulative incidence of extensive chronic 
GVHD by 1 year was only 5% in the group with two doses of Cy. 
 
Figure 2:  Low incidence of GVHD with post-transplantation Cy 
 
The cumulative incidences of relapse and NRM at 1 year were 51% and 15% respectively, and the EFS 
probability at 1 year was 34%. Similar outcomes were seen in a recent analysis of 185 patients treated on 
these trials and a follow-up phase II trial (J0457).24 
In summary, HLA-haploidentical BMT after nonmyeloablative conditioning and using 2 doses of 
post-transplantation Cy followed by tacrolimus and MMF is a well-tolerated procedure that can be 
administered largely in an outpatient setting. This postgrafting immunosuppression regimen for 
nonmyeloablative, HLA-haploidentical, related-donor BMT has been or is being investigated in several trials 
at Johns Hopkins, including a multicenter phase II trial through the BMT CTN (J0843).26 The toxicity of the 
procedure compares favorably to the toxicity of nonmyeloablative transplantation using unrelated or even 
HLA-identical sibling donors.23 The major cause of treatment failure in this high-risk population is relapse, 
occurring in approximately 50% of patients by 1 year. 
 
1.2 
Impact of HLA mismatching on outcome 

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Historically, HLA typing has been the most important predictor of outcome after allogeneic BMT.4 
Increasing degrees of HLA mismatch between patient and donor at either the antigen or allele level have 
been associated with worse outcomes in numerous series, with respect to GVHD, graft failure, and 
transplant-related mortality.3,27-30  In the setting of myeloablative, unrelated donor transplantation, 1 or 2 
allele mismatching has been associated with increased risk of these complications. Similarly in the setting of 
reduced-intensity transplants, high rates of transplant-related mortality have been observed with the use of 1 
and 2 MHC Class I mismatched donors.31,32 In the Fred Hutchinson experience with a nonmyeloablative 
strategy comprised of fludarabine and 200 cGy TBI followed by cyclosporine and MMF, transplantation 
using HLA Class I mismatched, mostly unrelated donors was associated with a NRM incidence of 22% at 
Day 100 and 36% at 1 year; a grade II-IV acute GVHD incidence of 69%; a grade III-IV acute GVHD 
incidence of 26%; and an incidence of extensive chronic GVHD of 41%.33 On the other hand, supporting the 
possibility of safely performing nonmyeloablative, HLA mismatched transplants from unrelated donors is the 
recent UK experience.34   In this study a regimen of fludarabine, melphalan, and alemtuzumab was followed 
by cyclosporine administration and transplantation from unrelated donors who were either 10/10 matches (n 
= 107) or HLA mismatched (n = 50, with only 3 donors mismatched at 3-4 loci). This approach was 
associated with high rates of durable engraftment and acceptable rates of grade II-IV acute GVHD (20% 
versus 22% respectively) and chronic extensive GVHD (23% versus 24% respectively).34 
The reported effect of HLA disparity on relapse risk varies. However, a lower relapse risk has been 
reported in some series with increasing HLA disparity, suggesting a graft-versus-tumor effect. For example, 
in patients with poor-risk leukemia undergoing related-donor, myeloablative BMT, 2 and 3-locus 
mismatched transplants were associated with a significantly lower relapse than HLA-identical sibling 
transplants.27 Likewise, in patients with high-risk leukemia or myelodysplastic syndrome undergoing 
myeloablative, T-cell replete BMT, significantly lower relapse (p < 0.004) was seen with using 1 antigen 
mismatched, versus no antigen mismatched, donors.35 Following unrelated donor BMT, specific 
combinations of allele mismatches have been linked with lower relapse risk and improved overall survival, 
not necessarily those that lead to severe acute GVHD.36 
However, it is possible that the type of GVHD prophylaxis could influence the balance between 
GVHD toxicity and relapse.  A recent analysis of our nonmyeloablative haploidentical BMT data supports 
this hypothesis and suggests that HLA disparity need no longer be a barrier when selecting amongst potential 
donors.24 We retrospectively analyzed the outcomes of 185 patients with poor-risk hematologic malignancies 
enrolled on three similar clinical trials of related-donor, haploidentical BMT utilizing post-transplantation 
high-dose Cy, MMF, and tacrolimus (J9966, J0457, and the Fred Hutchinson trial).24 Notably, no adverse 
effect of HLA mismatching was found using this approach.24 With increasing degrees of HLA mismatch, no 
deleterious effect was seen on EFS or on the incidence of NRM or acute GVHD (Figure 3). In fact, on 
multivariate analysis, more mismatches were associated with a possibly protective effect on EFS. 
 
 
Figure 3:  A, EFS according to number of antigen mismatches (HLA-A, -B, -Cw, and -DRB1 combined) in 

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either the GVH or HVG direction. B, Cumulative incidences of acute grade II-IV GVHD according to the 
number of antigen mismatches in the GVH direction. 
 
These results suggest an anti-tumor effect of partially HLA-mismatched BMT that is irrespective of 
clinically significant GVHD. Consistent with these observations, a retrospective study of nonmyeloablative 
BMT with post-transplantation high-dose Cy for relapsed/refractory Hodgkin lymphoma (data from Johns 
Hopkins, Seattle, and collaborating sites) found similar overall outcomes with HLA-matched related and 
HLA-haploidentical related donors. Incidences of acute grade III-IV and extensive chronic GVHD were 
similar (11%/35% for HLA-haploidentical, and 16%/50% for HLA matched related transplants, 
respectively).23 The haploidentical transplants actually had significantly lower NRM, a significantly 
decreased risk of relapse, and a significantly improved progression-free survival than HLA-matched related 
transplants. 
Because of the recent progress in prevention of GVHD and graft rejection with high-dose post- 
transplantation Cy, a Johns Hopkins study has been able to examine haploidentical, related-donor BMT for 
poor-risk hematologic malignancies following myeloablative conditioning. There have not been prohibitive 
rates of toxicity or graft rejection on preliminary experience (H. Symons, personal communication). 
 
1.3 
Reduced-intensity conditioning with fludarabine-busulfan 
With reduced-intensity conditioning (RIC), theoretically dose-equivalent regimens have been associated with 
significant differences in outcome, including differences in relapse, toxicities, engraftment kinetics, and 
survival.37 In a CIBMTR retrospective analysis of conditioning intensity, flu-200TBI was associated with 
higher treatment failure rates than flu-bu or flu-melphalan RIC (M. Pasquini, unpublished data). Although 
variability in patient risk and transplant procedure may account for some of these differences, based on such 
concerns flu-200TBI has been omitted from a BMT CTN trial (0901) comparing RIC and myeloablative 
conditioning for these diseases.  Accordingly, and consistent with our programmatic interest at Johns 
Hopkins to extend the experience of nonmyeloablative partially HLA-mismatched BMT with fludarabine 
and TBI (flu-200TBI) to a platform based on fludarabine and busulfan (flu-bu), the current protocol uses the 
latter conditioning strategy. The flu-bu regimen is typically considered to be reduced-intensity or 
nonmyeloablative if it has < 8 mg/kg PO busulfan or IV equivalent, with busulfan dosing in representative 
series ranging from one-quarter to one-half of that used in myeloablative conditioning.37,38 In a Dana Farber 
analysis of RIC transplantation using HLA-matched related or unrelated donors, fludarabine 120 mg/m2 IV + 
busulfan 6.4 mg/kg IV, as compared with fludarabine 120 mg/m2  IV + busulfan 3.2 mg/kg IV, was 
associated with greater progression-free survival (HR 0.6, p = 0.04) without difference in overall survival (V. 
Ho, EBMT 2010 annual meeting).  In the context of nonmyeloablative regimens, one must weigh the 
potential risks of more intensive conditioning against the potentially greater risks of graft rejection and 
relapse with less intensive conditioning. The cumulative doses of fludarabine (150 mg/m2) and busulfan (8 
mg/kg PO or 6.4 mg/kg IV) selected for the current study are standard.37 
With the reduced morbidity of transplantation regimens incorporating high-dose post-transplantation 
Cy for graft rejection and GVHD prophylaxis, relapse has remained the major problem particularly with 
nonmyeloablative transplants. The combination of flu-bu with post-transplantation Cy in the 
nonmyeloablative setting is new. Our group has studied the combination of fludarabine, busulfan, and post- 
transplantation Cy for hematologic malignancies patients undergoing myeloablative, HLA-matched BMT 
(J0844). There has not been an excessive incidence of toxicity on that study to date (L. Luznik, personal 
communication). The toxicities of a reduced intensity, flu-bu conditioning regimen are not expected to differ 
substantially from the flu-low dose Cy-200TBI platform incorporating post-transplantation Cy. This is 
expected to be a more immunosuppressive regimen, however, and the engraftment kinetics and toxicities 
may differ. Given the advances in GVHD prophylaxis with post-transplantation Cy, RIC with flu-bu 
combined with postgrafting immunosuppression that includes high-dose post-transplantation Cy was the 
initial platform for the current study in patients with poor-risk hematologic malignancies. However, based on 
subsequent preliminary engraftment data from a study involving reduced-intensity flu-bu conditioning with 
BMT from first-degree related donors (reduced-intensity flu-bu, followed by high-dose Cy on Days 3 and 4, 
MMF on Days 5-35, and tacrolimus on Days 5-180), it was questioned whether this conditioning regimen is 

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sufficiently immunosuppressive for graft failure prophylaxis.  Given the potentially higher risk of graft 
failure with the use of unrelated and multiply HLA-mismatched donors, the current study has been amended 
(5/18/2011 version date) to change the conditioning regimen to our Johns Hopkins historical standard of flu- 
low dose Cy-200TBI. 
 
 
 
1.4 
Sirolimus and post-transplantation cyclophosphamide: rationale for study 
Mechanistically, immunosuppressive drugs given to control GVHD suppress alloimmunity by nonspecific 
inhibition of alloreactive T-cell activation, proliferation, and differentiation. This is in clear contrast to the 
essential requirement for the induction of stable tolerance, which entails the apoptosis of alloreactive T-cells.39 
Thus, global immunosuppression by blocking T-cell activation and apoptosis precludes and delays the induction 
of transplantation tolerance after allografting. Immunosuppressive drugs can be classified according to their 
action on induction of apoptosis and inhibition of T-cell proliferation.40 Of all the commonly used 
immunosuppressants (steroids, tacrolimus, cyclosporine, MMF, sirolimus, Cy, methotrexate), only methotrexate 
and Cy induce the apoptosis of alloantigen-activated human T-cells, whereas other immunosuppressants mainly 
inhibit their proliferation.40 By promoting tolerance induction, high dose Cy has facilitated the use of alternative 
donor sources, such as HLA-mismatched grafts. Our underlying hypothesis is that high-dose Cy prevents acute 
GVHD by reducing the frequency of alloreactive T effector cells while sparing donor-specific immunity and 
without critically depleting the T regulatory (Treg) cell pool. If the precursor frequency of alloreactive T 
effectors remains high or the Treg pool declines below a critical threshold, then increased differentiation toward 
the pathogenic T effector cells ensues and acute GVHD develops. 
Sirolimus is an immunosuppressive agent that inhibits the mammalian target of rapamycin (mTOR), 
downregulating T-cell proliferation and activation.41 Since it does not inhibit T-cell receptor induced 
signaling, it does not block T-cell receptor-induced tolerance.39 This agent has been used widely to prevent 
graft rejection in solid organ and hematopoietic transplantation, and has been used both to prevent and treat 
acute and chronic GVHD.42 
This study investigates regimens for transplantation that may inhibit graft rejection and GVHD by 
promoting T-cell tolerance. As previously outlined, past regimens have relied heavily on 
immunosuppression with CNI’s.43 However, these agents also inhibit T-cell receptor induced signaling 
required for the generation of T-cell tolerance. On the other hand, activation of Th1 effector cells in the 
setting of mTOR signaling blockade with sirolimus has been shown to induce anergy.44 Therefore, of central 
interest is a postgrafting immunosuppressive approach with mTOR inhibition in combination with other 
agents that promote tolerance induction, such as high-dose post-transplantation Cy. 
 
a) mTOR inhibition promotes anergy and generation of regulatory CD4+ T-cells 
It is thought that cyclosporine and tacrolimus inhibit tolerance induction in vivo by limiting IL-2 production 
and Treg function, while sirolimus does not inhibit tolerance induction biochemically and promotes Treg 
expansion.45,46 In murine models of hematopoietic transplantation, rejection is mediated in part by activation 
of alloreactive CD4+ Th1 cells.47 Activation of Th1 cells in the presence of sirolimus results in anergy upon 
subsequent rechallenge with antigen.44 Critically, this effect of sirolimus depends on the presence of normal 
T-cell receptor signaling during the exposure to sirolimus; thus simultaneous exposure of T-cells to sirolimus 
and a CNI will block the induction of anergy.44 In contrast to committed Th1 cells, activation of naïve T-cells 
in the presence of sirolimus blocks CD4+ T-cell effector differentiation and promotes generation of FoxP3+, 
CD4+ T-cells (Tregs) that can inhibit effector T-cell responses in vitro.48 Laboratory work at Johns Hopkins 
confirmed these findings using a genetic approach based on conditional deletion of mTOR and other 
components of the TORC1 and TORC2 mTOR signaling complexes in murine T-cells.49 These experiments 
have demonstrated that CD4+ T-cell effector differentiation is possible in the absence of either TORC1 or 
TORC2 (Delgoffe and Powell, unpublished observation) but that the absence of both TORC1 and TORC2 
following deletion of mTOR causes naïve T-cells to differentiate into functional Tregs upon activation.49 
 
b) Potential synergy of sirolimus and post-transplantation Cy 

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Sirolimus has the ability to promote T-cell tolerance even in the presence of T-cell costimulation,44 and in 
murine models of haploidentical BMT conditioned with low-dose TBI, can not only prevent graft rejection 
but induce tolerance in the absence of long-term immunosuppression.50 Preclinical data further demonstrate 
that the anti-proliferative effects of sirolimus do not inhibit the effectiveness of post-transplant Cy, and that 
sirolimus and post-transplantation Cy are potentially synergistic in preventing graft rejection and facilitating 
stable mixed chimerism.51 This synergic effect appears to be mediated independently from expression of 
CD25+ Tregs. In murine models of nonmyeloablative, haploidentical BMT involving post-transplantation 
Cy on Day 2, initiation of sirolimus on Day -1 did not block Cy-induced tolerance (Figure 4). Additionally, 
sirolimus administration on either Day -1 through Day 30, or Day +4 through Day 30, in the context of post- 
transplantation Cy was effective in preventing rejection and inducing stable mixed chimerism (Figure 4), 
whereas there was no sustained donor chimerism with either agent alone. 
 
Figure 4: Synergism of sirolimus and Cy in preventing rejection and inducing stable mixed chimerism in 
preclinical models of haploidentical BMT. 
 
The optimal timing of sirolimus initiation in the context of BMT with high-dose post-transplantation 
Cy is not defined. In our clinical trials incorporating high-dose post-transplantation Cy, tacrolimus has been 
initiated on day 5 based on preclinical data. Given the above data, and the efficacy in patients of 
administering tacrolimus on day + 5 though day 180 (together with MMF on Day 5 through Day 35), this 
window has been selected for sirolimus in this study. Following post-transplantation Cy, sirolimus will be 
studied in combination with MMF. Based on the known mechanisms of MMF and sirolimus, MMF is not 
expected to interfere with sirolimus-induced tolerance. 
 
c) Effect of mTOR inhibition on antiviral and antitumor responses 
While these immunosuppressive effects of sirolimus on T-cells would be expected to contribute favorably to 
post-transplantation tolerance, they might also be expected to inhibit desired immune responses against 
pathogens such as CMV and influenza. Despite the theoretically increased risk of such infections while on 
treatment with sirolimus, epidemiologic data from both solid organ transplantation 52 and BMT 53 do not 
seem to bear this concern out. To the contrary, those clinical data suggest a possible anti-CMV effect of 
sirolimus, and data from animal models of LCMV suggest that CD8+ T-cell responses are augmented by 
low-dose sirolimus in vivo.54 Furthermore, sirolimus does not interfere with in vitro function (recognition 
and killing) of anti-mHAg-specific CD8+ T-cell clones, while its delayed in vivo administration does not 
block the graft-versus-tumor effect in murine model of allogeneic BMT. Thus, mTOR inhibition appears to 
be permissive for sustained antiviral and antitumor activity.55,56 
Another rationale for the incorporation of sirolimus into allogeneic transplant regimens is its 
potential anti-lymphoma activity.  mTOR inhibitors (temsirolimus, everolimus) have established clinical 

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activity in relapsed or refractory mantle cell lymphoma.57 Preclinical activity has been demonstrated against 
Hodgkin lymphoma and a variety of non-Hodgkin lymphomas, and phase II trials have suggested single 
agent activity in Hodgkin lymphoma,58 diffuse large B-cell lymphoma,59 and other lymphoid neoplasms. 
In a retrospective analysis of allogeneic transplantation at the Dana Farber Cancer Institute, 
lymphoma patients who received sirolimus following RIC transplantation (mostly with flu-bu) had a similar 
incidence of NRM, but a statistically significantly lower incidence of disease progression, than patients who 
did not receive sirolimus.60 The benefit appeared to be restricted to patients receiving RIC regimens and to 
patients with lymphoma. This effect persisted after adjusting for GVHD and was associated with a 
statistically significant improvement in overall survival. Thus this class of agents may have dual activity 
against GVHD and against selected tumor types. 
 
1.5 
Special considerations in patients with HIV 
A patient with HIV and acute myelocytic leukemia was cured of HIV infection by unrelated allogeneic 
transplantation using a donor who was homozygous for the CCR5delta32 polymorphism that confers 
HIV resistance. Another patient received a cord blood transplant with a CCR5delta32 homozygous donor 
and also appeared to eliminate HIV but the patient relapsed with lymphoma and died. These experiences 
are consistent with our understanding of CCR5 as an HIV coreceptor and suggest that selection of 
appropriate CCR5delta32 homozygous donors may allow additional patients to be cured of HIV. 
 
1.6 
Stem cell source 
There has been a great deal of discussion on the importance of stem cell source on the risk of chronic 
graft-versus-host disease5-9. Several studies have addressed this issue in the related setting. Of the eight 
randomized trials published10-18 only one reported a statistically significant increase in grades II-IV 
acute graft-versus-host disease with the use of peripheral blood stem cells when compared to bone 
marrow (52 vs. 39%)16. Regarding chronic graft-versus-host disease, the results are as follows: 3 studies 
have shown an increase of chronic graft-versus-host disease with peripheral blood stem cells as opposed 
to bone marrow12,16,19. One study showed a trend towards increase in chronic graft-versus-host disease 
with the use of peripheral blood stem cells19. A meta-analysis by Cutler et al. confirmed that both, acute 
and chronic graft-versus-host disease are more common after peripheral blood stem cells than bone 
marrow7. Registry data showed in pediatric patients that chronic graft-versus-host disease was more 
frequent (as well as higher mortality) after peripheral blood stem cells than after bone marrow8. In 
adults, chronic graft-versus-host disease is also more prevalent20. Umbilical-cord stem cells have also 
been a source of grafts in children and young adults. As children tolerate mismatches better than adults, 
interpretation of risk in this group is difficult but it seems that the rate of chronic graft-versus-host 
disease is low for this stem cell sources, especially considering that almost all grafts are 1-3 antigen 
mismatches21,22. In the unrelated setting, a clinical trial by the BMT CTN comparing bone marrow 
versus peripheral blood did not detect significant survival. Peripheral-blood stem cells may reduce the 
risk of graft failure (the overall incidence of graft failure in the peripheral-blood group was 3% [95% CI, 
1 to 5], versus 9% [95% CI, 6 to 13] in the bone marrow group [P=0.002]), whereas bone marrow may 
reduce the risk of chronic GVHD at 2 years (peripheral-blood group was 53% [95% CI, 45 to 61], as 
compared with 41% [95% CI, 34 to 48] in the bone marrow group [P=0.01].17  The proportion of 
patients with extensive chronic GVHD was higher in the peripheral-blood group than in the bone marrow 
group (48% [95% CI, 42 to 54] vs. 32% [95% CI, 26 to 38], P<0.001). Among patients who were alive at 
2 years, 57% of the patients in the peripheral-blood group were receiving immunosuppressive therapy, as 
compared with 37% of those in the bone marrow group (P=0.03). There were no significant between- 
group differences in the incidence of acute GVHD or relapse17. 
 
2.0 
OBJECTIVES 
 
2.1 
Primary objectives 

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1. Phase 1 portion: In reduced-intensity, partially HLA mismatched allogeneic BMT from unrelated 
or non-first-degree related donors, identify a transplant regimen associated with acceptable rates 
of severe acute GVHD (≤ 25%) and transplant-related NRM (≤ 20%) by Day 100. 
2. Phase 2 portion: With the selected transplant regimen, as a measure of immunologic efficacy, 
estimate the 6-month probability of survival without having had acute grade III-IV GVHD or 
evidence of graft failure. 
 
2.2 
Secondary objectives 
1. Estimate the progression-free survival, disease-free survival, overall survival, cumulative 
incidence of progression or relapse, and cumulative incidence of NRM. 
2. Estimate the cumulative incidence of acute grade II-IV GVHD, acute grade III-IV GVHD, and 
chronic GVHD. 
3. Determine the need for systemic immunosuppressive treatment for GVHD beyond the originally 
planned prophylaxis regimen; estimate the cumulative incidence of systemic steroid initiation for 
GVHD, cumulative incidence of non-steroid immunosuppressant use, and cumulative incidence 
of discontinuation of systemic immunosuppression for GVHD treatment; describe the types of 
immunosuppression used for GVHD treatment; and evaluate GVHD composite endpoints 
(GVHD-free relapse-free survival, chronic GVHD-free relapse-free survival). 
4. Describe graft failure frequency, kinetics of T-cell donor chimerism and total leukocyte donor 
chimerism in peripheral blood, and kinetics of neutrophil and platelet recovery. 
5. Characterize immune reconstitution and the immunobiology of sirolimus and post- 
transplantation Cy by analyzing peripheral blood mononuclear cells collected prospectively at 
defined time points. 
 
 
3.0 
SELECTION OF PATIENTS AND DONORS 
 
3.1 
Patient eligibility 
1. Patient age 0.5-75 years old. 
2. Absence of a suitable related or unrelated bone marrow or peripheral stem cell donor who is 
molecularly matched at HLA-A, -B, -Cw, -DRB1, and -DQB1. 
3. Absence of a suitable partially HLA-mismatched (haploidentical), first-degree related donor. 
Note: Determination of matching is based on allele or allele group level typing. To be 
considered haploidentical, the donor and recipient must be identical at at least one allele of 
each of the following genetic loci: HLA-A, -B, -Cw, -DRB1, and -DQB1.  A minimum 
match of 5/10 is therefore required, and will be considered sufficient evidence that the donor 
and recipient share one HLA haplotype. Donors who are homozygous for the CCR5delta32 
polymorphism are given preference. 
4. Eligible diagnoses: 
a. Relapsed or refractory acute leukemia (acute myeloid leukemia or acute lymphoblastic 
leukemia or lymphoma) in second or subsequent remission, with remission defined as 
<5% bone marrow blasts morphologically 
b. Poor-risk acute leukemia in first remission, with remission defined as <5% bone marrow 
blasts morphologically: 
• 
AML with at least one of the following: 
• 
AML arising from MDS or a myeloproliferative disorder, or secondary 
AML 
• 
Presence of Flt3 internal tandem duplications 
• 
Poor-risk cytogenetics: Complex karyotype [> 3 abnormalities], inv(3), 
t(3;3), t(6;9), MLL rearrangement with the exception of t(9;11), or 
abnormalities of chromosome 5 or 7 
• 
Primary refractory disease 

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• 
ALL (leukemia and/or lymphoma) with at least one of the following: 
• 
Adverse cytogenetics such as t(9;22), t(1;19), t(4;11), or MLL 
rearrangement 
• 
Clear evidence of hypodiploidy 
• 
Primary refractory disease 
• 
Biphenotypic leukemia 
c. MDS with at least one of the following poor-risk features: 
• 
Poor-risk cytogenetics (7/7q minus or complex cytogenetics) 
• 
IPSS score of INT-2 or greater 
• 
Treatment-related MDS 
• 
MDS diagnosed before age 21 years 
• 
Progression on or lack of response to standard DNA-methyltransferase 
inhibitor therapy 
• 
Life-threatening cytopenias, including those generally requiring greater than 
weekly transfusions 
d. Interferon- or imatinib-refractory CML in first chronic phase, or non-blast crisis CML 
beyond first chronic phase. 
e. Philadelphia chromosome negative myeloproliferative disease. 
f. Chronic myelomonocytic leukemia. 
g. Juvenile myelomonocytic leukemia. 
h. Low-grade non-Hodgkin lymphoma (including SLL and CLL) or plasma cell neoplasm 
that has progressed after at least two prior therapies (excluding single agent rituximab 
and single agent steroids), or in the case of lymphoma undergone histologic conversion; 
patients with transformed lymphomas must have stable disease or better. 
i. 
Poor-risk CLL or SLL as follows: 11q deletion disease that has progressed after a 
combination chemotherapy regimen, 17p deletion disease, or histologic conversion; 
patients with transformed lymphomas must have stable disease or better. 
j. 
Aggressive non-Hodgkin lymphoma as follows, provided there is stable disease or better 
to last therapy: 
• 
NK or NK-T-cell lymphoma, peripheral T-cell lymphoma (including 
angioimmunoblastic T-cell lymphoma, hepatosplenic T-cell lymphoma, 
subcutaneous panniculitic T-cell lymphoma, and other variants), T-cell 
prolymphocytic leukemia, or blastic/blastoid variant of mantle cell lymphoma; 
or 
• 
Hodgkin or aggressive non Hodgkin lymphoma that has failed at least one 
multiagent regimen, and the patient is either ineligible for autologous BMT or 
autologous BMT is not recommended. 
Eligible subtypes of aggressive non-Hodgkin lymphoma include: 
• 
mantle cell lymphoma 
• 
follicular grade 3 lymphoma 
• 
diffuse large B-cell lymphoma or its subtypes, excluding primary CNS 
lymphoma 
• 
primary mediastinal large B-cell lymphoma 
• 
large B-cell lymphoma, unspecified 
• 
anaplastic large cell lymphoma, excluding skin-only disease 
• 
Burkitt’s lymphoma or atypical Burkitt’s lymphoma (high-grade B-cell 
lymphoma, unclassifiable, with features intermediate between diffuse 
large B-cell lymphoma and Burkitt’s), in complete remission 
5. Patients with CLL, SLL, or prolymphocytic leukemia must have < 20% bone marrow 
involvement by malignancy (to lower risk of graft rejection). 
6. One of the following, in order to lower risk of graft rejection: 

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a. Cytotoxic chemotherapy, an adequate course of 5-azacitidine or decitabine, or 
alemtuzumab within 3 months prior to start of conditioning; or 
b. Previous BMT within 6 months prior to start of conditioning. 
Note: Patients who have received treatment outside of these windows may be eligible if it is 
deemed sufficient to reduce graft rejection risk; this will be decided on a case-by-case basis 
by the PI or co-PI. 
7. Any previous BMT must have occurred at least 3 months prior to start of conditioning. 
8. No active extramedullary leukemia or known active CNS involvement by malignancy. Such 
disease treated into remission is permitted. 
9. Adequate end-organ function as measured by: 
a. Left ventricular ejection fraction  35%, or shortening fraction > 25%, unless cleared by 
a cardiologist 
b. Bilirubin ≤ 3.0 mg/dL (unless due to Gilbert’s syndrome or hemolysis), and ALT and 
AST < 5 x ULN 
c. FEV1 and FVC > 40% of predicted; or in pediatric patients, if unable to perform 
pulmonary function tests due to young age, oxygen saturation >92% on room air 
10. ECOG performance status < 2 or Karnofsky or Lansky score > 60. 
11. Not pregnant or breast-feeding. 
12. No uncontrolled bacterial, viral, or fungal infection (infection is permitted if there is evidence of 
response to medication). 
Note: HIV-infected patients are potentially eligible. Eligibility of HIV-infected patients 
will be determined on a case-by-case basis. 
 
3.2 
Donor eligibility 
1. Potential donors consist of: 
b. Unrelated donors 
c. Second-degree relatives 
d. First cousins 
2. Donor must not be HLA identical to the recipient. 
3. The donor and recipient must be identical at at least 5 HLA alleles based on high resolution 
typing of HLA-A, -B, -Cw, -DRB1, and -DQB1, with at least one allele matched for a HLA class 
I gene (HLA-A, -B, or -Cw) and at least one allele matched for a class II gene (HLA-DRB1 or - 
DQB1). 
4. Meets institutional selection criteria and medically fit to donate. 
5. Lack of recipient anti-donor HLA antibody. 
Note: In some instances, low level, non-cytotoxic HLA specific antibodies may be 
permissible if they are found to be at a level well below that detectable by flow cytometry. 
This will be decided on a case-by-case basis by the PI and one of the immunogenetics 
directors. Pheresis to reduce anti-HLA antibodies is permissible; however eligibility to 
proceed with the transplant regimen would be contingent upon the result. 
6. Has not donated blood products to recipient. 
 
Donor prioritization criteria are designated in Section 3.3. 
 
3.3 
Donor prioritization 
Eligible donors will be prioritized in the following order: 
1. Donor matched with the recipient for at least one allele each at HLA-A, -B, -Cw, -DRB1, and - 
DQB1. 
2. Fewest number of HLA-mismatched loci between donor and recipient. Preference will be given 
for donors who are matched for at least one allele each of HLA-A, -B, and -DRB1. 

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3. If multiple donors are available and the total number of mismatches is the same, donors who are 
mismatched at the allele level are prioritized over those who have antigen level mismatches; e.g., 
a 1 allele mismatch has priority over a 1 antigen mismatch. 
4. Major ABO compatibility. 
5. CMV serostatus: CMV negative donor preferred, if the recipient is CMV negative; CMV 
positive donor preferred, if the recipient is CMV positive. 
6. ABO compatibility preferred over minor incompatibility. 
 
 
Other considerations, such as donor age, health history and anti-donor HLA antibody status, may be 
prioritized over the above criteria. For patients with HIV infection, donors who are CCR5delta32 
homozygous may be prioritized over other donors so long as they meet the donor eligibility criteria 
in Section 3.2. 
 
4.0 
REGISTRATION PROCEDURES 
 
4.1 
Registration requirements 
Patients will be registered in the CRMS.  The following are additionally required: 
• 
Signed and dated informed consent 
• 
Patient eligibility checklist 
 
A registration may be cancelled, provided that protocol treatment has not been begun. 
 
4.2 
Accrual goal 
The accrual goal is to transplant up to 99 patients such that at least 20 patients will have mismatched 
unrelated peripheral blood donors using Regimen B2, per Sections 5.2 and 9.0. Up to 10 HIV+ 
patients with partially matched unrelated CCRd32 homozygous donors will have transplants using 
Regimen B2 as part of Cohort B3.  Up to 3 additional patients per regimen may be transplanted to 
replace inevaluable patients (maximum number of transplants, 114). Additional patients may be 
screened and registered, in order to identify the target number of patients who meet all eligibility 
criteria and receive the transplant. 
 
Every effort will be made to recruit women and minorities to this study. 
 
5.0 
TREATMENT PLAN 
 
5.1 
Evaluations and procedures 
Required evaluations are designated in Section 7.0. 
 
5.2 
Overview of study design 
Up to 3 regimens involving flu-Cy-200TBI conditioning (Regimens A though C) were initially 
planned (2 containing sirolimus and 1 containing tacrolimus), summarized in Table 1 below. Criteria 
for moving from one regimen to another are based upon the number of patients developing severe 
acute GVHD, transplant-related NRM, or graft failure, as detailed in Section 9.0 (Statistical 
Considerations). Whether a regimen is deemed prohibitive or not prohibitive is based specifically on 
the safety and stopping criteria in Section 9.0. 
The first cohort of patients enrolled prior to the 5/18/11 version date (receiving flu-bu 
followed by post-transplantation Cy, MMF, and sirolimus) is now designated as having received 
Regimen D. Study of this regimen was stopped, per the Background section. The protocol was 
amended 12-21-2011 to remove Regimen A containing sirolimus without MMF. The other regimens 
will be studied in the following order: 

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• 
Begin with Regimen B (MMF + sirolimus), and expand if appropriate. 
• 
If Regimen B is prohibitive at any point, move to Regimen C (MMF + tacrolimus) and 
expand if appropriate. 
• 
If Regimen C is prohibitive, accrual will stop pending external review. 
 
Table 1: Transplant regimens 
Regimen 
Preparative 
regimen 
High-dose 
cyclophosphamide 
MMF 
Sirolimus 
Tacrolimus 
A 
Flu-Cy-TBI 
Day 3 and 4 
None 
Day 5 - 180 
None 
B 
Flu-Cy-TBI 
Day 3 and 4 
Day 5 - 35 
Day 5 - 180 
None 
B2 
Flu-Cy-TBI 
Day 3 and 4 
Day 5 – 35 
Day 5 – 180 
None 
B3 
Flu-Cy-TBI 
Day 3 and 4 
Day 5 – 35 
Day 5 – 180 
None 
C 
Flu-Cy-TBI 
Day 3 and 4 
Day 5 - 35 
None 
Day 5 - 180 
D 
Flu-Bu 
Day 3 and 4 
Day 5 - 35 
Day 5 - 180 
None 
 
Additional cohorts “B2” and “B3” will be added using peripheral blood donor cells according to 
the “B” Prep regimen. 
 
Accrual need not pause while the first 5 patients on a given regimen complete monitoring to 
Day 100 (specified in Section 9.0). In the event that more than 5 patients are enrolled during this 
time, accrual to that regimen will continue. This is justifiable based on preliminary data with MMF + 
sirolimus and our historical data in nonmyeloablative, related-donor BMT with MMF + tacrolimus. 
 
5.3 
Conditioning, transplantation, and post-transplantation cyclophosphamide: Regimens B, B2, 
B3 and C 
The preparative regimen for Regimens B and C consists of fludarabine, Cy, and TBI, with post 
grafting immunosuppression consisting of high-dose Cy with two other immunosuppressants 
(MMF and sirolimus, or MMF and tacrolimus respectively). Post grafting immunosuppression 
other than high-dose Cy is specified in Section 5.4. 
5.31 
Fludarabine 
Fludarabine 30 mg/m2/day (adjusted for renal function) is administered over a 30-60 minute IV 
infusion on Days –6 through –2 (maximum cumulative dose, 150 mg/m2), if no days of rest before 
transplantation is planned. 
 
The body surface area (BSA) for fludarabine dosing is based on actual body weight. 
 
For decreased creatinine clearance (CrCl), fludarabine dosage is reduced as follows: 
CrCl ≥ 70 ml/min – fludarabine 30 mg/m2 
CrCl 40-69 ml/min - fludarabine 24 mg/m2 
CrCl 20-39 ml/min – fludarabine 20 mg/m2 
CrCl < 20 ml/min – fludarabine 15 mg/m2 
 
Alternatively, dose-adjustments of fludarabine for decreased CrCl can follow current institutional 
standard. 
 
For patients > 18 years old, CrCl will be estimated by the Cockcroft Formula, based on body weight: 
CrCl = (140 – age) x weight (kg)   x   0.85 for females 
PCr  x 72 
 
When calculating CrCl: if Actual Body Weight is less than Ideal Body Weight, Actual Body Weight 
will be used; if Actual Body Weight is between 100-120% of Ideal Body Weight, Ideal Body Weight 

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will be used; and if Actual Body Weight is > 120% of Ideal Body Weight, 25% Adjusted Body 
Weight 
For patients <18 years old, CrCl will be estimated by the Schwartz equation: 
Schwartz equation: CrCl (mL/min/1.73m2)= [length (cm) x k] / serum creatinine 
k = 0.45 for infants 1 to 52 weeks old 
k = 0.55 for children 1 to 13 years old 
k = 0.55 for adolescent females 13-18 years old 
k = 0.7 for adolescent males 13-18 years old 
 
CrCl may change during the days fludarabine is given. Adjustment in fludarabine dose due to 
creatinine changes during conditioning is permitted per institutional standard. 
 
5.32 
Pre-transplantation cyclophosphamide 
Cy 14.5 mg/kg/day is administered as a 1-2 hour IV infusion on Days –6 and –5 after hydration. 
Mesna 11.6 mg/kg IV daily on Days –6 and –5 is not required, but may be given. 
 
Cy and mesna are dosed according to IBW, unless the patient weighs less than IBW, in which case 
dose these drugs according to actual weight. 
 
5.33 
Total body irradiation 
400 cGy TBI is administered in a single fraction on Day –1. Radiation sources, dose rates, and 
shielding follow institutional practice. 
 
5.34 
Day of rest 
A day of rest, i.e. after preparative regimen completion and prior to bone marrow infusion, is not 
routinely scheduled for Regimens B and C. Up to two days of rest may be added in this window 
based on logistical considerations or clinically as indicated. For one day of rest, fludarabine would 
be administered on Days –7 through –3, pretransplantation Cy on Day –7 and Day –6, and TBI on 
Day –2. For two days of rest, fludarabine would be administered on Days –8 through –4, 
pretransplantation Cy on Day –8 and Day –7, and TBI on Day –3. Should logistical issues preclude 
one of these schedules, TBI may be given on Day 0, prior to bone marrow infusion, with PI or co-PI 
permission. 
 
5.35 
Hematopoietic cell transplantation 
On Day 0, the harvested bone marrow or peripheral blood stem cells are infused. 
 
The graft will not be manipulated to deplete T-cells. Processing for ABO incompatibility follows 
institutional practices. Guidelines for cellular infusion are established and outlined in the allogeneic 
BMT standing orders. 
 
 
5.37 
Post-transplantation cyclophosphamide 
Hydration with Cy, management of volume status, and monitoring for hemorrhagic cystitis will 
follow institutional standards. 
 
Mesna is given in divided doses IV 30 minutes pre- and at 3, 6, and 8 hours post-Cy, unless patients 
are treated in the Children’s Center in which case mesna is dosed per pediatric oncology standard 

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(e.g., divided doses IV 30 minutes pre- and at 3, 6, and 9 hours post-Cy). The total daily dose of 
mesna is equal to 80% of the total daily dose of Cy. 
 
Cy and mesna are dosed according to IBW, unless the patient weighs less than IBW, in which case 
dose these drugs according to actual body weight. 
 
Cy 50mg/kg IV, over 1-2 hours (depending on volume), is given on Day 3 post-transplantation 
(ideally between 60 and 72 hours after marrow infusion) and on Day 4 (approximately 24 hours after 
Day 3 cyclophosphamide). 
 
It is crucial that no systemic immunosuppressive agents are given until at least 24 hours after 
the completion of the post-transplantation Cy.  This includes corticosteroids as anti-emetics. 
 
5.4 
Additional post-transplantation therapies 
 
5.41 
Regimen B and B2 and B3: Sirolimus and Mycophenolate Mofetil 
 
5.411 Sirolimus 
For patients > 18 years old: A one-time sirolimus loading dose, 6 mg PO, is given on Day 5, 
at least 24 hours after Cy completion. Sirolimus is then continued at a maintenance dose 
(start 2 mg PO QD), with dose adjustments to maintain a trough of 5 – 12 ng/mL as 
measured by HPLC or immunoassay. There is no planned taper. Sirolimus prophylaxis is 
discontinued after the last dose on Day 180, or may be continued if there is GVHD. 
Sirolimus troughs should be checked at minimum weekly. 
 
For patients < 18 years old: Sirolimus dosing is based on actual body weight; however an 
adjusted body weight may be used if the actual weight is > 50% greater than IBW. A one- 
time sirolimus loading dose, 3 mg/m2 PO with the dose not to exceed 6 mg, is given on Day 
5, at least 24 hours after Cy completion. Sirolimus is then continued at a maintenance dose 
(start 1 mg/m2 PO QD, maximum 2 mg PO QD), with dose adjustments to maintain a trough 
of 5 – 12 ng/mL as measured by HPLC or immunoassay.  There is no planned taper. 
Sirolimus prophylaxis is discontinued after the last dose on Day 180, or may be continued if 
there is GVHD.  Sirolimus troughs should be checked at minimum weekly. 
 
Sirolimus may be discontinued earlier than Day 180 in the context of relapse, progression, 
graft failure, or prohibitive toxicity. It is suggested that patients with suspected graft failure 
remain on sirolimus until at least the ~Day 60 chimerism assessment. Decisions regarding 
early discontinuation of immunosuppression will be made on a case-by-case basis in 
consultation with the PI or co-PI. 
 
5.412 Mycophenolate Mofetil 
MMF begins on Day 5, at least 24 hours after completion of post-transplantation Cy. The 
MMF dose is 15 mg/kg PO TID (actual body weight) with total daily dose not to exceed 3 
grams (i.e. maximum 1 g PO TID). An equivalent intravenous dose (1:1 conversion) may 
instead be given. Dose modification guidelines are provided in Section 8.15. MMF 
prophylaxis is discontinued after the last dose on Day 35, or may be continued if there is 
GVHD. 

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5.42 
Regimen C: Tacrolimus and Mycophenolate Mofetil 
 
5.421 Tacrolimus 
Tacrolimus begins on Day 5, at least 24 hours after completion of post-transplantation Cy. 
 
For patients > 18 years old, the tacrolimus starting dose is 1 mg IV QD. The starting dose of 
tacrolimus may be increased with PI or co-PI permission should institutional practice 
guidelines change. Tacrolimus can be changed to a PO BID dosing schedule once a stable 
therapeutic level is achieved and the patient can tolerate PO medications.  Dose is adjusted 
to maintain a serum trough level of 10 – 15 ng/mL. 
 
For patients < 18 years old, the starting dose of FK-506 is 0.015mg/kg IV Q12 hours, based 
on ideal body weight, unless actual body weight is less. Tacrolimus can be changed to a PO 
BID dosing schedule once a stable therapeutic level is achieved and the patient can tolerate 
PO medications.  Dose is adjusted to maintain a serum trough level of 10 – 15 ng/mL. 
 
Tacrolimus is discontinued after the last dose on Day 180, or may be continued if GVHD is 
present. At PI or co-PI discretion, cyclosporine (target concentration 200-400 ng/mL) may 
be substituted for tacrolimus if the patient is significantly intolerant of tacrolimus. 
 
Tacrolimus may be discontinued earlier than Day 180 in the context of relapse, progression, 
graft failure, or prohibitive toxicity. It is suggested that patients with suspected graft failure 
remain on tacrolimus until at least the ~Day 60 chimerism assessment. Decisions regarding 
early discontinuation of immunosuppression will be made on a case-by-case basis in 
consultation with the PI or co-PI. 
 
5.422 Mycophenolate Mofetil 
MMF begins on Day 5, at least 24 hours after completion of post-transplantation Cy. The 
MMF dose is 15 mg/kg PO TID (actual body weight) with total daily dose not to exceed 3 
grams (i.e. maximum 1 g PO TID). An equivalent intravenous dose (1:1 conversion) may 
instead be given. Guidelines for dose modification are provided in Section 8.15. MMF 
prophylaxis is discontinued after the last dose on Day 35, or may be continued if there is 
GVHD. 
 
5.43 
Growth factors 
GCSF (filgrastim) begins on Day 5 at a dose of 5 mcg/kg/day (actual body weight) IV or 
subcutaneously (rounding to the nearest vial dose is allowed), until the absolute neutrophil count 
(ANC) is ≥ 1,000/mm3 over the course of three days. Additional GCSF may be administered as 
warranted.  Pegfilgrastim (Neulasta®) and GM-CSF are not permitted. 
 
5.44 
Post-transplantation donor lymphocyte infusion (DLI) 
Prophylactic post-transplantation DLI (for persistent detectable malignancy, prophylaxis in the 
absence of detectable malignancy, or mixed donor chimerism) is not permitted before Day 100, as 
this carries a high risk of GVHD. The use of DLI will be recorded and such patients will be 
censored for analysis of disease and graft failure outcomes, GVHD, and related transplant-related 
toxicity outcomes.  Analysis of outcomes without such censoring is also planned. 
 
5.45 
Other post-transplantation therapy 
Preemptive systemic cancer therapy is permitted post-transplantation (e.g., DNA-methyltransferase 
inhibitor, tyrosine kinase inhibitor, rituximab for CD20+ malignancy). Intrathecal chemotherapy and 
consolidative radiation therapy are permitted. The use of such posttransplantation therapies other 
than intrathecal chemotherapy will be tracked. 

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5.5 
Supportive care 
Patients will receive transfusions, nutritional support, infection prophylaxis and treatment, and other 
supportive care according to standard of care and institutional guidelines. 
 
5.51 
Anti-ovulatory treatment 
Menstruating females should begin an anti-ovulatory agent before starting the preparative regimen. 
 
5.52 
Indwelling central venous catheter 
A double lumen central venous catheter is required for administration of IV medications and blood 
products. 
 
5.53 
Infection prophylaxis 
Patients will receive infection prophylaxis and treatment according to institutional guidelines. 
Infection prophylaxis should include agents or strategies to prevent herpes simplex, CMV (e.g., 
CMV PCR screening and preemptive therapy), Pneumocystis jirovecii, fungal infections, and 
infections from oral flora secondary to mucositis. Post-transplantation immunizations will be given 
per institutional standard. 
 
Because of the extreme interaction between sirolimus and voriconazole or posaconazole, 
prophylactic voriconazole or posaconazole is not permitted while on sirolimus. All azole 
antifungals with the exception of fluconazole should be discontinued at least 1 week prior to 
sirolimus initiation. 
 
5.54 
Antiemetics 
Note that dexamethasone should not be used as an anti-emetic agent after the graft is infused, in the 
absence of relapsed/progressive disease.  Such use will not constitute a protocol deviation. 
 
6.0 
MEASUREMENT OF EFFECT AND ENDPOINTS 
 
6.1 
Hematologic parameters 
 
6.11 
Neutrophil recovery: Post-nadir ANC > 500/mm3 for three consecutive measurements on 
different days. The first of the three days will be designated as the day of neutrophil 
recovery. 
 
6.12 
Platelet recovery: Platelet count > 20,000/mm3 or > 50,000/mm3 with no platelet 
transfusions in the preceding seven days, and maintained on at least three consecutive 
measurements on different days. The first day of those three consecutive measurements will 
be designated as the day of initial platelet recovery. 
 
6.13 
Donor cell engraftment: Mixed donor chimerism is defined as > 5%, but < 95%, donor. 
Full donor chimerism is defined as > 95% donor. 
 
Prior to transplantation, a sample of peripheral blood from the patient, and either harvested 
bone marrow or blood from the donor, are collected for genetic studies to establish a 
baseline for subsequent chimerism assays. 
 
Donor chimerism from T-cells (CD3+ sorted) and whole blood (total nucleated cells) from 
the peripheral blood will be serially determined per Section 7.0, and more frequently as 
indicated. Methods may include (i) PCR analysis of variable number of tandem repeats 
(VNTR) in PBMC if informative, (ii) restriction fragment length polymorphism (RFLP) if 

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the donor and recipient RFLPs are informative, (iii) fluorescence in-situ hybridization 
(FISH) for Y-chromosome markers on PBMC if the donor is male and patient is female, (iv) 
cytogenetic analysis, (v) flow cytometric analysis of HLA-A, B or DR on lymphocytes in the 
peripheral blood if haploidentical and suitable reagents exist. Chimerism may also be 
determined from the bone marrow. 
 
6.14 
Graft failure: < 5% donor chimerism in blood and/or bone marrow on ~Day 30 or after and 
on all subsequent measurements, in the absence of documented bone marrow involvement 
by malignancy. . 
▪ 
Primary graft failure: < 5% donor chimerism in blood and/or bone marrow by ~ 
Day 60 
▪ 
Secondary graft failure: Achievement of > 5% donor chimerism, followed by 
sustained < 5% donor chimerism in blood and/or bone marrow. 
 
Less than 5% donor T-cell chimerism, but with > 5 % donor chimerism in total leukocytes, 
is not considered graft failure. 
 
6.2 
Graft-versus-host disease 
 
6.21 
Acute GVHD: Acute GVHD is graded by standard clinical criteria (Appendix).61 All 
suspected cases of acute GVHD must be confirmed histologically by biopsy of an affected 
organ (skin, liver, or gastrointestinal tract). Date of symptom onset, date of biopsy 
confirmation of GVHD, maximum clinical grade, and dates and types of treatment will be 
recorded. Dates of symptom onset of grade II or higher GVHD and grade III-IV GVHD will 
be recorded. 
 
The cumulative incidence of grade II-IV and grade III-IV acute GVHD will be determined 
through competing risk analysis. Relapse/progression, graft failure, and death are 
considered competing risks for GVHD for study purposes, including stopping rules. In 
addition, GVHD will be reported with only graft failure and death regarded as competing 
risks. 
 
6.22 
Chronic GVHD: Chronic GVHD is graded by NIH consensus criteria62 and Seattle 
criteria.63 Date of onset, date of biopsy proof (if any), dates and types of treatment, and 
extent will be recorded. The cumulative incidence of chronic GVHD (overall, and according 
to extent) will be determined through competing risk analysis. 
 
6.3 
Disease and survival endpoints 
 
6.31 
Progression-free survival: Interval from Day 0 to date of first objective disease progression 
or relapse, death from any cause, unplanned treatment of disease persistence, or last patient 
evaluation.  Patients without such failures will be censored at the last date they were 
assessed and deemed free of relapse or progression. Disease persistence in the absence of 
progression is not considered a PFS failure unless it leads to treatment. 
 
6.32 
Disease-free survival: Interval from Day 0 to date of first objective detection of disease 
persistence, progression or relapse, or last patient evaluation. Patients without such failures 
will be censored at the last date they were assessed and deemed disease-free. Disease 
persistence posttransplantation, followed by disappearance of detectable disease in the 
absence of treatment, is not considered a DFS failure. 
 
6.33 
Failure-free survival: In the phase 2 expansion, failure-free survival, as it pertains to 

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“immunologic success” of the transplant regimen, is the interval from Day 0 to the date of 
severe acute (grade III-IV) GVHD, graft failure, non-relapse mortality, or last patient 
evaluation. Patients without these events will be censored at the last date they were assessed 
and deemed failure-free. Patients who relapse, progress, or receive unplanned treatment for 
disease persistence will be censored on that date of failure. 
 
6.34 
Overall survival: Interval from Day 0 to date of death from any cause or last patient contact. 
 
6.35 
Non-relapse mortality: Death without evidence of disease progression or relapse. 
Relapse/progression and unplanned treatment of disease persistence are competing risks for 
non-relapse mortality. 
 
6.36 
Relapse or progression: Defined per the following response criteria: 
• 
Lymphoma: 2007 International Working Group (IWG) criteria for lymphoma 64 
• 
Acute leukemia: 2010 European LeukemiaNet criteria,65  based on 2003 IWG criteria 66 
• 
MDS: 2006 IWG criteria 67 
 
Designation of disease status in other histologies will also follow standard criteria. Non- 
relapse mortality is a competing risk for relapse/progression. 
 
6.37 
Minimal residual disease (MRD): MRD is defined by the sole evidence of malignant cells 
by flow cytometry, FISH, PCR or other techniques, in absence of morphological or 
cytogenetic evidence of disease in blood or marrow. Since the frequency and sensitivity of 
testing for MRD are variable, evidence of MRD will not be sufficient to meet the definition 
of relapse or progression in this study, but will be captured in the case report forms along 
with data on changing management in response to MRD detection. 
 
6.38 
GVHD-related survival endpoints 
 
• 
GVHD-free relapse-free survival (GFRFS): Interval from Day 0 to acute grade 
III-IV GVHD, systemic treatment of chronic GVHD, or PFS failure, whichever 
occurs first. Patients without these failures will be censored at the last date they 
were assessed and deemed failure-free. 
 
Chronic GVHD-free relapse-free survival (cGFRFS): Interval from Day 0 to a chronic GVHD event 
(variably defined as either moderate or severe chronic GVHD, or systemic treatment of any chronic GVHD) 
or PFS failure, whichever occurs first. Patients without these failures will be censored at the last date they 
were assessed and deemed failure-free. 
 
7.0 
STUDY PARAMETERS 
 
7.1 
Core evaluations 
The following table summarizes the minimum testing and clinical assessments required for study 
purposes. This is in addition to other testing and assessments indicated as standard of care, which 
may be collected for study purposes. Telemedicine visits are an acceptable alternative if deemed 
appropriate by the investigator, and will be conducted in accordance with established Johns 
Hopkins policies. 

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Table 2: Core evaluations 
Baseline a,b 
D30 
+/- 10d 
D60 
+/- 15 d 
If question 
of graft 
failure 
D100 
+/- 15 d 
D180 
+/- 30 d 
D365 
+/- 45 d c 
Standard pre/post 
transplant evaluations a, b 
History and physical exam 
X 
X 
X 
X 
X 
X 
ECOG performance status 
X 
Karnofsky or Lansky score 
X 
CBC / differential d 
X 
X 
X 
X 
X 
X 
X 
Comprehensive metabolic 
panel e 
X 
X 
X 
X 
X 
X 
Infectious disease titers f 
X 
Fasting cholesterol and serum 
triglycerides (sirolimus arms 
only) 
X 
X 
Xo 
Serum HCG (if applicable) 
X 
EKG 
X 
LV ejection fraction or 
shortening fraction 
X 
Pulmonary function tests m 
X 
Bone marrow biopsy and 
aspirate with flow cytometry 
and relevant cytogenetic and 
molecular studies g 
X 
X, with 
chimerism 
studies h, n 
X, with 
chimerism 
studies 
X h 
X h 
CT of sinuses 
X 
CT, PET/CT, or MRI of chest, 
abdomen, and pelvis 
(lymphoma and CLL only) 
X 
X 
X 
Response assessment to last 
therapy i 
X 
HLA typing 
X 
Lymphocytotoxic antibody 
screen 
X 
Donor marrow or blood for 
VNTR or RFLP analysis j 
X 
Patient blood for baseline 
VNTR or RFLP analysis j 
X 
Peripheral blood chimerism, 
both total leukocyte (unsorted) 
and T-cell sorted j 
X 
X 
X 
X 
X 
GVHD and other morbidity 
assessments k 
X 
X 
X 
X 
X 
 
 
a Baseline evaluations should occur < 1 month before initiation of conditioning therapy, with the exception of 
the following: cardiac and pulmonary evaluations may occur < 8 weeks prior, and the HLA typing and 
baseline studies for chimerism determinations may occur at any point prior. Results of evaluations performed 
before study entry as standard of care may be used for research purposes and to fulfill study requirements. 
b Demographics and baseline characteristics will be captured.  Characteristics to be recorded include: age, 

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gender, race/ethnicity, performance status, disease type, remission status, prior treatments including prior 
autologous transplantation, donor age, donor relationship to patient, donor gender, CMV serostatus of patient 
and donor, ABO compatibility. 
c   All patients will be considered off study with regard to monitoring for adverse effects at 1 year after 
transplantation. Patients should continue to follow-up at Johns Hopkins at least yearly on study. Follow-up 
data may be captured more frequently for study purposes. Data that will continue to be recorded beyond 1 
year include disease status, vital status, and GVHD. Patients who relapse or progress will also be 
considered off study. .  
d At minimum, CBC/differential should also be performed twice a week from start of preparative regimen, 
until ANC is >1000/µL over course of 3 days, then weekly until 12 weeks post-transplantation, and 
periodically thereafter; those need not be captured in the CRF. 
e Chemistries include: BUN, creatinine, sodium, potassium, chloride, AST, ALT, total bilirubin, alkaline 
phosphatase. At minimum, these should be performed weekly until 12 weeks post-transplantation, then periodically 
until off immunosuppression; those need not be captured in the CRF. 
f Standard infectious disease evaluations include: CMV IgG, HSV IgG, VZV IgG, Hepatitis panel (Hep B surface Ag, 
Hep B core antibody, Hep C antibody), RPR, HIV antibody, and HTLV I/II antibody. 
g Flow cytometry in diseases other than Hodgkin’s lymphoma. Follow-up studies should include relevant cytogenetics 
and molecular markers to detect residual disease, i.e. repeat of studies found to be positive at baseline. 
h Bone marrow aspiration and biopsy will be done at the discretion of the physician caring for the patient 
i  Include comparison of pre- and post-treatment scans with bidimensional measurements where relevant. 
j  Collect 10 cc lavender top. 
k GVHD and other morbidity assessments are also standardly performed weekly until Day 100 and may be collected 
via telemedicine. Results of these and subsequent assessments may be collected for research purposes. Patients may 
be asked to complete GVHD questionnaires. 
l m For pediatric patients unable to perform PFT’s, document oxygen saturation on room air. 
n If an adequate bone marrow biopsy is performed for suspected graft failure before but in close proximity to 
Day 60 evaluations, the Day 60 bone marrow biopsy may be omitted at PI or co-PI discretion. 
o Fasting cholesterol and serum triglycerides (sirolimus arms only) day 100 assessments to be done between 
day 90 – day 130. 
 
 
8.0 
RISKS AND REPORTING REQUIREMENTS 
 
8.1 
Drug information 
 
8.11 
Fludarabine (Fludara®) 
Fludarabine is a fluorinated nucleoside analog. After phosphorylation to fluoro-ara-ATP the drug 
appears to incorporate into DNA and inhibit DNA polymerase alpha, ribonucleotide reductase and 
DNA primase, thus inhibiting DNA synthesis. Excretion of fludarabine is impaired in patients with 
impaired renal function. 
 
Fludarabine toxicities include: 
a. Neurotoxicity: Agitation or confusion, blurred vision, loss of hearing, peripheral neuropathy or 
weakness have been reported. Severe neurologic effects, including blindness, coma, and death 
may occur; severe CNS toxicity is rarely seen with doses in the recommended range for 
nontransplant therapy. The dose used in this study is approximately 1.5 times the usual one- 
course dose given in non-transplant settings. Doses and schedules similar to those used in this 
study have been used in adult and pediatric patients without observed increase in neurotoxicity. 
b. Anemia: Life-threatening and sometimes fatal autoimmune hemolytic anemia has been reported 
after one or more cycles of therapy in patients with or without a previous history of autoimmune 
hemolytic anemia or a positive Coombs’ test and who may or may not be in remission.  
Corticosteroids may or may not be effective in controlling these episodes. The majority of patients 
re-challenged developed a recurrence of the hemolytic process. 

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c. Cardiovascular: Deep venous thrombosis, phlebitis, transient ischemic attack, and aneurysm (1%) 
are reported. 
d. Fever: 60% develop fever. 
e. Rash: 15% develop a rash, which may be pruritic. 
f. Digestive: Gastrointestinal side effects include: nausea/vomiting (36%), diarrhea (15%), stomatitis (9%), 
anorexia (7%), GI bleeding and esophagitis (3%), mucositis (2%), liver failure, abnormal liver function 
test, constipation, dysphagia (1%) and mouth sores. 
g. Some other effects include: Chills (11%), peripheral edema (8%), myalgias (4%), osteoporosis (2%), 
pancytopenia, arthralgias (1%), dysuria (4%), urinary tract infection and hematuria(2%); renal failure, 
abnormal renal function test, and proteinuria (1%); and, very rarely, hemorrhagic cystitis and pulmonary 
toxicity. 
 
Dose adjustments of fludarabine are required for renal insufficiency if the estimated CrCl is < 60 mL/min (per 
Sections 5.31 and 5.41). Fludarabine is dosed in this study according to actual body weight. 

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8.12 
Cyclophosphamide (Cytoxan®) 
Cyclophosphamide is an alkylating agent. It is activated by the liver cytochrome P450 system to 
cytotoxic metabolites, which form cross-links with DNA.  It is cell cycle-nonspecific. 
 
Toxicities include: 
Nausea, vomiting, diarrhea, headache, dizziness, hemorrhagic cystitis, fluid weight gain/edema, 
SIADH, transaminitis, cardiomyopathy, pericarditis, rash, mucositis, alopecia, cytopenias, sterility, 
and rarely, secondary myelodysplastic syndrome and anaphylaxis. 
 
Dose adjustments for cyclophosphamide will not be made. Cyclophosphamide is dosed in this study 
according to IBW, unless actual body weight is less. 
 
8.13 
Mesna 
Mesna (sodium-2-mercaptoethanesulphonate) is a prophylactic agent used to prevent hemorrhagic 
cystitis induced by the oxasophosphorines (cyclophosphamide and ifosphamide). It has no intrinsic 
cytotoxicity and no antagonistic effects on chemotherapy. Mesna binds with acrolein, the urotoxic 
metabolite produced by the oxasophosphorines, to produce a non-toxic thioether and slows the rate 
of acrolein formation by combining with 4-hydroxy metabolites of oxasophosphorines. 
 
Toxicities: At the doses used for uroprotection, mesna is virtually non-toxic. However, potential 
adverse effects include nausea and vomiting, diarrhea, abdominal pain, altered taste, rash, urticaria, 
headache, joint or limb pain, hypotension, and fatigue. 
 
Dose adjustments for mesna will not be made. The total daily dose of mesna is equal to 80% of the 
total daily dose of cyclophosphamide in this study. Mesna is dosed in this study according to IBW, 
unless actual body weight is less. 
 
8.14 
Mycophenolate Mofetil (MMF; Cellcept®) 
MMF is an ester prodrug of the active immunosuppressant mycophenolic acid (MPA). 
 
Toxicities include: 
Pancytopenia, infection, nausea, vomiting, diarrhea, allergic reactions, hypertension, headache, 
dizziness, insomnia, hyperglycemia, electrolyte imbalances, rash, and leg cramps/bone pain. 

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Drug interactions: MMF activity is decreased with oral antacids and cholestyramine. There are no 
pharmacokinetic interactions with cotrimoxazole, oral contraceptives, or cyclosporine. Acyclovir or 
ganciclovir blood levels may increase due to competition for tubular secretion. High doses of 
salicylates or other highly protein-bound drugs may increase the free fraction of MPA and 
exaggerate the potential for myelosuppression. 
 
Dose adjustments: No dose adjustments are required for liver dysfunction. For renal insufficiency, 
MMF dosing should not be modified unless dialysis is needed, in which case MMF can be reduced 
to 25-50% of the starting dose. 
 
8.15 
Sirolimus (rapamycin, Rapamune®) 
Sirolimus is an immunosuppressant that inhibits cytokine-stimulated T-cell activation and 
proliferation, and also inhibits antibody formation. 
 
Drug formulations: The mean bioavailability of sirolimus after administration of the tablet is ~27% 
higher than the oral solution. Sirolimus oral tablets are not bioequivalent to the oral solution. Clinical 
equivalence has been demonstrated at the 2-mg dose level; however, it is not known if higher doses 
are clinically equivalent on a mg to mg basis. 
a) Sirolimus oral solution: Sirolimus oral solution (1 mg/mL) should be stored protected from 
light and refrigerated at 2°C to 8°C (36°F to 46°F). For dilution, the appropriate dose should 
be measured using an amber oral syringe, then added to a glass or plastic container that holds 
at least 60 mL. Before taking the dose, it should be diluted with water or orange juice then 
taken immediately; it should not be diluted with grapefruit juice. The syringe should be 
discarded after one use. Sirolimus oral solution provided in bottles may develop a slight haze 
when refrigerated, which does not affect product quality; allow the product to stand at room 
temperature and shake gently until the haze disappears. 
b) Sirolimus tablets: Sirolimus tablets are available in 1 mg and 2 mg tablets that cannot be 
crushed or broken. Sirolimus tablets should be stored at 20° to 25° C (68°–77°F), protected 
from light. 
 
Toxicities: The most common adverse reactions of sirolimus are: peripheral edema, 
hypertriglyceridemia, hypercholesterolemia, hypertension, increased creatinine, constipation, 
abdominal pain, nausea, diarrhea, headache, fever, urinary tract infection, anemia, 
thrombocytopenia, arthralgia, pain. Adverse reactions that have resulted in rates of sirolimus 
discontinuation >5% were increased creatinine, hypertriglyceridemia, and thrombotic 
thrombocytopenic purpura (TTP) / thrombotic microangiopathy (TMA). Sirolimus toxicities are 
summarized in Table 4 below: 

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Table 4: Sirolimus toxicities 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
(L): Toxicity may also occur later. 
a Significant transaminitis, generally without sequellae, may occur. Sirolimus has been associated 
with higher rates of venoocclusive disease after myeloablative conditioning. 
b Incidence 3% to < 20% in a trial of kidney transplantation. In allogeneic BMT, increase in TMA 
from 4.2% with tacrolimus or cyclosporine alone, versus 10.8% with tacrolimus/sirolimus 
combination was noted.68 
c Lipid-lowering agent may be required; consider if fasting serum triglycerides are > 2.5 x ULN, and 
recommend starting if > 800 mg/dL. 
 
Drug interactions: Sirolimus is known to be a substrate for both cytochrome CYP3A4 and P- 
glycoprotein. Agents that may increase sirolimus levels include tri-azole drugs (especially 
voriconazole and posaconazole*), amiodarone, calcium channel blockers, macrolide antibiotics (but 
not azithromycin), micafungin, gastrointestinal prokinetic agents (cisapride, metoclopramide), 
cimetidine, cyclosporine, grapefruit juice, and HIV protease inhibitors. Agents that may decrease 
sirolimus levels include anticonvulsants (carbamezepine, phenobarbital, phenytoin), rifamycins, St. 
John’s Wort. 
Common (>20%) 
Occasional (5-20%) 
Rare (<5%) 
Immediate (within 1-
2 days) 
Headache (L), 
hypertension (L), 
immunosuppression 
(L), fever, nausea, 
diarrhea, constipation 
Chest pain, 
insomnia, 
dyspepsia, 
vomiting, dyspnea 
Hypotension, 
asthma, cough, flu-
like syndrome, 
tachycardia, 
anorexia, 
hypersensitivity 
Prompt (within 2-3 
Tremor (L), renal 
Elevated LFT’s (with 
Pleural and pericardial 
weeks) 
dysfunction, pain 
elevated sirolimus 
effusions, pulmonary 
(abdominal, back, 
a, stomatitis, infections 
toxicity (non-infectious 
arthralgias), 
hyperlipidemia c 
(including UTI, URI), 
mild 
pneumonitis, 
BOOP, pulmonary 
(hypercholesterolemia, 
leukopenia, electrolyte 
thombosis, myalgias 
hypertriglyceridemia), 
disturbances 
hyperglycemia, edema 
(hyper/hypokalemia [L], 
including peripheral 
hypophosphatemia, 
edema, anemia 
hypomagnesemia [L]), 
rash, hives, pruritus, 
delayed wound healing 
or dehiscence (L), 
proteinuria, 
TTP/HUS/TMA b 
especially with 
concurrent CNI 
Delayed (any time later 
Acne 
Kidney disease, CHF, 
during therapy, 
ascites, arthrosis, bone 
excluding above 
necrosis, osteoporosis 
conditions) 
Late (any time after 
completion of 
treatment) 
Lymphoproliferati
ve disorders, skin 
malignancies 
Unknown frequency 
and timing 
Embryo/fetotoxic; unknown whether excreted in human milk 

Version date: 
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34 
 
 
 
Dose adjustments: The sirolimus dose is adjusted to maintain a serum trough level of 5-12 ng/mL. 
Changes in levels due to altered bioavailability should be apparent within 24-48 hours. For sirolimus 
without CNI as in this study, a 20-25% reduction of sirolimus dose is recommended for trough levels 
>12 – 18 ng/mL, and a 20-25% increase is recommended for trough levels < 5 ng/mL. 
Renal failure does not affect the excretion of sirolimus. Excretion is reduced in liver failure; 
impaired hepatic function should prompt consideration of reduction in sirolimus maintenance doses 
but no dose adjustment of the loading dose is necessary. 
Due to extreme interactions with voriconazole and posaconazole, these drugs are 
relatively contraindicated during sirolimus therapy. Sirolimus dose is to be reduced by 90% 
when voriconazole is initiated and should also be significantly reduced with posaconazole. 
Dosing guidelines are provided in Section 8.18. 
 
8.16 
Tacrolimus (FK-506, Prograf®) 
Tacrolimus is a macrolide immunosuppressant that inhibits lymphocytes through calcineurin 
inhibition. 
 
Drug formulation: Tacrolimus injection must diluted with 0.9% Sodium Chloride or 5% Dextrose to 
a concentration between 0.004 mg/mL and 0.02 mg/mL prior to use. Diluted infusion solution should 
be stored in glass or polyethylene containers and discarded after 24 hours. PVC-free tubing is 
preferable for more dilute solutions. Due to chemical instability in alkaline media, tacrolimus 
injection should not be mixed or co-infused with solutions of pH 9 or greater (e.g., ganciclovir or 
acyclovir). Supplied as a 5 mg/mL solution, to be stored between 5° - 25°C, and as capsules (0.5 mg, 
1 and 5 mg) to be stored at room temperature, 15°- 30° C. 
 
Toxicities: There is a spectrum of well-described toxicities of tacrolimus. Toxicities include renal 
insufficiency, hypertension, hyperglycemia, hypomagnesemia, hypokalemia, nausea, diarrhea, 
headache, neurologic toxicity including tremor and leukoencephalopathy, infection, and rarely 
thrombotic thrombocytopenic purpura (TTP). 
 
Drug interactions: Tacrolimus is well absorbed orally. Tacrolimus is extensively metabolized by the 
cytochrome P-450 (CYP3A4) system and metabolized products are excreted in the urine. Drugs that 
may increase tacrolimus levels include tri-azole drugs (especially voriconazole and posaconazole), 
nephrotoxic drugs, calcium channel blockers, cimetidine and omeprazole, metoclopramide, 
macrolide antibiotics, quinupristin/dalfopristin, danazol, ethinyl estradiol, methylprednisolone, and 
HIV protease inhibitors. Drugs that may decrease tacrolimus levels include some anticonvulsants 
(phenobarbital, phenytoin, carbamezepine), caspofungin, rifamycins, and St. John’s wort. 
 
Dose adjustments: The tacrolimus dose is adjusted to maintain a serum trough level of 10-15 ng/mL. 
Patients with hepatic or renal insufficiency should receive doses at the lower end of therapeutic 
concentrations.  No dose adjustments are required in patients undergoing hemodialysis. 
Due to extreme interactions with voriconazole and posaconazole, the tacrolimus dose 
should be empirically lowered when these azoles are initiated. Dose adjustments for therapy with 
other azoles may be indicated (see Section 8.18). The tacrolimus loading dose in this study takes 
fluconazole prophylaxis into account. 
 
8.16 
Concurrent azole therapy 
Triazole antifungal medications are expected to increase serum CNI and sirolimus levels; therefore 
dosages of CNIs and sirolimus should be adjusted accordingly. Guidelines are provided in Table 5 
below. In the event of suspected or documented fungal infection, alternative antifungal therapy 
should be considered. 

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35 
 
 
 
 
Table 5: Suggested pre-emptive dose reduction of tacrolimus or sirolimus when azoles are 
initiated at steady state levels of tacrolimus or sirolimus 
Antifungal 
Tacrolimus 
Sirolimus 
Dose ↓ 
Comment 
Dose ↓ 
Comment 
Voriconazole 
67% 
Strongly advised 
90% 
Essential 
Posaconazole 
67% 
Advised 
67% 
Advised 
Itraconazole 
50% 
Advised 
No data 
No data 
Fluconazole 
25% 
Consider 
50% 
If > 400 mg qd 
 
Notes: 
a. If voriconazole is given IV or if voriconazole and sirolimus are not given together, the effect on 
sirolimus bioavailability will be weaker. Empiric dose reduction is advised; however, guidelines 
are not well established. 
b. Regarding dose increases of CNI’s or sirolimus when azoles are stopped: Reversal of azole- 
mediated inhibition of cytochrome CYP3A4 (and others) and P-glycoprotein is gradual. 
Therefore, immediate significant dose increases are not advised. Rather, tacrolimus and 
sirolimus dose increases should be cautious and based on more frequent monitoring of levels as 
appropriate. 
 
8.17 
Total Body Irradiation (TBI) 
TBI can cause: nausea and vomiting, diarrhea, parotitis (rapid onset within 24-48 hours, usually self- 
limited), generalized mild erythema (usually within 24 hours, resolving in 48-72 hours), 
hyperpigmentation, fever, mucositis, alopecia, and pancytopenia. Late effects include: cataracts (10- 
20%), hypothyroidism, nephropathy, interstitial pneumonitis, veno-occlusive disease, carcinogenesis, 
and sterility. 
 
8.2 
Toxicity grading 
Toxicities are graded using NCI’s Common Terminology Criteria for Adverse Events (CTCAE), 
Version 4.0. 
 
8.3 
Toxicity reporting 
The agents being used in the study are used extensively in the BMT setting, have well-defined 
toxicity profiles, and are FDA approved. In addition, there are many expected toxicities of 
allogeneic BMT. The following are examples of toxicities that are serious but not unexpected: 
Grade 4 cytopenias; neutropenic fever and sepsis; bacterial, fungal, or viral (CMV, BK virus) 
infection; severe mucositis; severe GVHD; hepatic veno-occlusive disease; pulmonary toxicities; 
hemorrhagic cystitis; bleeding without hemodynamic compromise. 
For study purposes, the following will be recorded and reported in accordance with IRB 
requirements: 
• 
Any hospitalization and its reason in the first year of transplant. 
• 
Neutropenic fever is an expected, common complication; as such, hospitalizations 
for grade 4 neutropenic fever will be reported in real-time to the IRB with 
hospitalizations for lesser grade neutropenic fever routinely reported on a yearly 
basis. 
• 
Any death before Day 200, and any later death which is potentially transplant-related. 
• 
Any unexpected, serious events deemed significant by the PI 
 
In addition, the following toxicities will be tracked for study purposes and reported on a yearly basis 
to the IRB, or earlier if warranted: 

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• 
Clinically significant infections during the first year of transplant, with the exception of 
uncomplicated, culture-negative neutropenic fever. This includes CMV disease, bacterial 
infections, and documented or suspected fungal infections. 
• 
CMV reactivation (including asymptomatic reactivation) 
• 
Hepatic veno-occlusive disease 
• 
Grade 3 or greater pulmonary toxicity during the first year of transplant that is 
potentially transplant-related 
 
Additional toxicities may be tracked. This is in addition to evaluating hematologic parameters, 
GVHD, and disease and survival endpoints outlined in Section 6.0. 
 
All patients will be considered off study with regard to monitoring for adverse effects at 1 
year after transplantation. Further, all patients who relapse will be considered off study. 
 
8.4 
Monitoring plan 
The SKCCC Compliance Monitoring Program will provide external monitoring for JHU affiliated 
sites in accordance with SKCCC DSMP (Version 6.0, 02/21/2019). The SMC Subcommittee will 
determine the level of patient safety risk and level/frequency of monitoring. All trial monitoring and 
reporting will also be reviewed annually by the SKCCC Safety Monitoring Committee. The PI will 
review data to assure the validity of data, as well as the safety of the subjects. The PI will also 
monitor the progress of the trial. The PI will review safety reports and clinical trial efficacy 
endpoints and to confirm that the safety outcomes favor continuation of the study. 
 
8.5 
Risks and benefits 
Allogeneic BMT carries risk for major morbidity and mortality, the risk of which is expected to 
increase, potentially substantially, with the use of HLA-mismatched unrelated donors or second- 
degree relatives. Major toxicities and risks of the transplant procedure include acute and chronic 
GVHD, severe infection, immunosuppression which may be prolonged, graft failure, end-organ 
damage, thrombotic microangiopathy, and death. High-dose post-transplantation cyclophosphamide 
appears to significantly lower the risk of GVHD in other settings. 
The potential benefits of this trial are palliation of disease-related symptoms and 
prolongation of overall or event-free survival, including the possibility of long-term disease-free 
survival and cure. 
 
9.0 
STATISTICAL CONSIDERATIONS 
 
9.1 
Primary statistical plan: phase 1 study 
The primary objective is to identify a reduced-intensity transplant regimen incorporating high-dose 
post-transplantation Cy that carries acceptable rates of severe (grade III-IV) acute GVHD and 
transplant-related NRM by Day 100. Up to two immunosuppressive regimens with flu-Cy-TBI 
conditioning (sirolimus + MMF, or tacrolimus + MMF, all with post-transplantation 
cyclophosphamide) will be studied. As described in the Background, sirolimus has been prioritized 
over tacrolimus for initial study, and study of a regimen with flu-bu conditioning was stopped. 
 
A Bayesian monitoring rule for two adverse events, grade III-IV acute GVHD by Day 100 and 
transplant-related NRM by Day 100, will be used to examine the safety of each regimen along a 
decision tree. The study will be continuously monitored (for each occurrence of any such adverse 
event) for safety. A regimen will be considered prohibitive if the probability of severe acute GVHD 
is convincingly >25% or the probability of transplant-related NRM is >20% by this time point. This 
design is based on the Bayesian posterior probability derived for the bivariate case.69 The likelihood 
assumes that occurrence of one adverse event precludes that of the other. Although GVHD and 
NRM are not mutually exclusive, the Day 100 mortality from acute GVHD is expected to be low. 
Therefore, a patient who experiences grade III-IV acute GVHD then NRM will be counted once as 

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37 
 
 
having the GVHD adverse event.  In the absence of previous experience with these types of

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38 
 
 
 
transplant regimens, a bivariate uniform prior distribution is assumed. The posterior distribution is a 
product of piecewise beta densities and the calculation has been programmed using Mathematica.70 
 
Each time a new regimen is tested, it will first be tested in a cohort of 5 patients. If a patient begins 
the preparative regimen but does not receive the transplant (e.g., because of a donor issue), that 
patient will be replaced. When a final regimen has been selected, phase 1 study of this regimen will 
be expanded to a planned total of 20 patients, including the initial 5 patients. Per Section 5.2, phase 
1 expansion may begin while evaluation of the first cohort of 5 patients is in progress. If a regimen 
is tested with an expanded sample size and a stopping boundary is met, the study of that regimen 
will stop. The stopping rule will hold enrollment to the expansion cohort if, by Day 100, the 
posterior probability is 80% or higher that the severe acute GVHD risk exceeds 25% or transplant- 
related NRM risk exceeds 20%. A regimen will additionally be considered prohibitive if, during its 
initial or expanded phase 1 testing, the frequency of graft failure by ~Day 60 convincingly exceeds 
35%, per Section 9.14. 
 
The sequence of regimens is designated in Section 5.2. 
 
The B2 cohort will include up to 20 patients with safety monitoring as outlined below. 
 
Cohort B3: HIV patients with CCRd32 homozygous donors (N=10): 
We would like to accrue additional HIV patients with CCRd32 homozygous donors (who are resistant to 
HIV infection). You may be aware of the “Berlin patient” reported in 2009 and the “London patient” 
reported 10 years later in 2019 who had fully matched unrelated marrow transplants from HIV resistant 
donors and were cured of HIV74-75. There have been so few because it is difficult to find HLA-matched 
HIV-resistant donors76.  
 
The J1055 protocol has demonstrated that unrelated bone marrow donors needn’t be HLA matched77, that 
related donors who are haploidentical need not be first degree relatives78, and most recently that the 
approach is successful with a peripheral blood stem cell graft as well as bone marrow (in preparation). This 
work has led to a national trial based on ours that confirmed the bone marrow results, and a follow-up 
national trial not yet activated to confirm our peripheral blood stem cell results. The J1055 has allowed 
enrollment of HIV patients. Because of the more relaxed requirements for HLA matching, it has been 
possible to identify unrelated partially mismatched HIV resistant donors in most instances. We have 
transplanted two in a little over a year on J1055. The first died in the second month after transplant. He had 
been hospitalized with severe CMV and adenovirus infections (not uncommon after allo transplant).  A 
second patient is early after transplant and is alive and fully engrafted and being followed closely in our 
outpatient clinic. With this amendment, we add an additional cohort of 10 patients with HIV needing 
transplant for standard indications but having partially matched unrelated HIV-resistant donors identified. 
The stopping rule and operating characteristics of the rule for this 10 patient cohort are given in the sections 
9.12 and 9.13. 
 
 
Upon completion of the above, a phase 2 expansion cohort is planned for this study. 
 
9.11 
First test of a regimen 
Table 6 below shows the stopping rule for the initial test of a regimen after the first 5 patients, when the 
allowed probabilities of severe acute GVHD and transplant-related NRM are 25% and 20%, respectively, 
by Day 100. The bolded cells are combinations of severe acute GVHD and NRM events by this time point 
that would prevent further study of the regimen, with a threshold for the Bayesian posterior probability of 
80%. As an example, if we were to observe k1 = 2 patients with severe acute GVHD and k2 = 2 patients 
with transplant-related NRM by Day 100, the posterior probability that the frequency of either event 
exceeds that allowed would be 92% and study of the regimen should be stopped. Similar tables have been 
calculated for sample sizes of 6 to 20, and stopping boundaries for continuous monitoring are summarized 
in Table 7. 

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39 
 
 
 
Table 6: Posterior probabilities that the severe acute GVHD risk exceeds 25% or NRM risk exceeds 
20% by Day 100, with a sample size of 5 
 
NRM (k2) 
0 
1 
2 
3 
4 
5 
aGVHD (k1) 
0 
0.03 
0.12 
0.37 
0.79 
0.98 
1 
1 
0.10 
0.27 
0.65 
0.94 
1 
1 
2 
0.32 
0.62 
0.92 
0.99 
1 
1 
3 
0.74 
0.93 
0.99 
1 
1 
1 
4 
0.97 
1 
1 
1 
1 
1 
5 
1 
1 
1 
1 
1 
1 
 
 
9.12 
Continuous safety monitoring 
As long as a stopping boundary is not met, we expect to accrue a total of 20 patients in the phase 1 portion 
using the chosen regimen.  Tables similar to Table 6 have been calculated to monitor the study 
continuously as each patient in the cohort reaches the Day 100 benchmark. The stopping boundaries from 
these tables, using a posterior probability threshold of 80%, have been consolidated in Table 7. The 
combinations of severe acute GVHD and transplant-related NRM events by Day 100 that lead to stopping 
are shown as row and column headers with the range of sample sizes in the body of the table. As an 
example, when 7 patients have completed the 100 day observation period, the combinations of adverse 
events (severe acute GVHD, transplant-related NRM) by that time point that would stop the trial are: (5,0), 
(4,1), (3,2), (2,3), (1,4) and (0,4). 
 
Table 7:  Stopping boundaries based on the number of severe acute GVHD and NRM events 
by Day 100 using a posterior probability threshold of 80%. The table entries are the sample sizes 
for which the row and column numbers of severe acute GVHD cases and transplant-related NRM 
cases, respectively, constitute the stopping rule. 
 
NRM 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
aGVHD 
0 
5-7 
8-10 
11-13 
14-16 
17-19 
20-23 
24- 
1 
5-6 
7-8 
9-11 
12-14 
15-17 
18-20 
21-24 
25 
2 
5 
6-7 
9-10 
11-12 
13-15 
16-18 
19-20 
22-25 
3 
5-6 
6-7 
8-9 
10-11 
12-14 
15-17 
18-20 
21-23 
24-25 
4 
5-6 
7-8 
8-9 
10-11 
12-13 
14-16 
17-18 
19-20 
22-24 
25 
5 
7-9 
9-10 
10-11 
12-13 
14-16 
17 
18-20 
21-22 
23-25 
6 
10-11 
11-12 
12-13 
14-15 
16-17 
18-19 
20-22 
23-24 
25 
7 
12-13 
13-14 
14-16 
16-17 
18-19 
20-21 
22-23 
24-25 
8 
14-16 
15-17 
17-18 
18-19 
20-21 
22-23 
24-25 
9 
17-19 
18-19 
19-20 
20-22 
22-23 
24-25 
10 
20-21 
20-22 
21-23 
23-24 
24-25 
11 
22-24 
23-25 
24-25 
25 
12 
25 
 
 
 
 
 
Continuous safety monitoring for HIV cohort (N=10): 
Because there was an event in one of the two HIV patients with a resistant donor in the previous cohort, the 

Version date: 
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40 
 
 
rule has been modified to start after the second patient.  The thresholds for allowed probabilities of severe 
acute GVHD and transplant-related NRM remain the same, 25% and 20% by Day 100, respectively, as well 
as the 80% threshold for the posterior probability, 
 
 
Table showing when to stop based on the number of acute GVHD (aGVHD) cases and non-relapse 
mortalities (NRMs).  Table entries are the sample sizes for which the row and column numbers of 
acute GVHD cases and non-relapse mortalities constitute the stopping rule.  The rule is based on 
allowed probabilities of severe acute GVHD and transplant-related NRM of 25% and 20%, 
respectively, by Day 100. 
 
 
 
 
 
NRM 
 
 
 
 
 
 
0 
1 
2 
3 
4 
5 
aGVHD 
 
 
 
 
 
 
0 
 
 
2 
3-4 
5-7 
8-10 
1 
 
2 
3-4 
5-6 
7-8 
9-10 
2 
2 
3-4 
5 
6-7 
9-10 
 
3 
3-4 
5-6 
6-7 
8-9 
10 
 
4 
5-6 
7-8 
8-9 
10 
 
 
5 
7-9 
9-10 
10 
 
 
 
6 
10 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
9.13 
Operating characteristics of design 
The probability of stopping the phase 1 study early under different scenarios is shown in Table 8. By 
Day 100 the allowed probability of severe acute GVHD (A1) is 25% and the allowed probability of 
transplant-related NRM (A2) is 20%, while the simulated adverse events (k1 or k2) were either equal 
to or greater than the allowed amount. The probability of stopping early was calculated from 500 
simulated trials for a minimum sample size of 5 or maximum sample sizes of 20 per a given regimen 
with 3 different posterior probability thresholds: 0.60, 0.70 and 0.80. The stopping boundaries in 
Table 7 use a posterior probability threshold of 80%. 

Version date: 
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41 
 
 
 
 
Table 8: Probability of stopping early under three adverse event scenarios with 
continuous monitoring and a minimum sample size of 5 or a maximum sample size of 
20 per given regimen 
Posterior 
Cohort size 
Equivalence: 
Increase both: 
More NRM: 
Threshold 
A1=.25, A2=.20 
A1=.25, A2=.20 
A1=.25, A2=.20 
k1=.25, k2=.20 
k1=.35, k2=.35 
k1=.25, k2=.45 
0.60 
5 
31.0 
64.4 
61.6 
0.70 
5 
17.4 
44.0 
39.4 
0.80 
5 
11 
30.6 
31.8 
0.60 
20 
51.8 
89.8 
92.6 
0.70 
20 
37.8 
84.4 
90.0 
0.80 
20 
29.8 
76.8 
82.8 
 
 
Operating characteristics HIV cohort (N=10): 
Probability of stopping early under three adverse event scenarios with continuous 
monitoring starting after the second patient, and posterior threshold of 80%. 
 
Posterior 
Cohort size 
Equivalence: 
Increase both: 
More NRM: 
Threshold 
A1=.25, A2=.20 A1=.25, A2=.20 
A1=.25, A2=.20 
k1=.25, k2=.20 
k1=.35, k2=.35 
k1=.25, k2=.45 
0.80 
10 
29.4 
61.8 
70.4 
 
 
 
 
9.14 
Additional stopping guidelines 
Early stopping guideline for graft failure: Phase 1 study of a given regimen will stop if the 
frequency of graft failure by Day 60 evaluations convincingly exceeds 35% in evaluable 
patients. For this stopping guideline, each regimen will be separately assessed using a one-sided 
exact binomial 90% confidence bound. We will stop study of a given regimen if 4 out of the 
first 5, 6 out of first 10, or 9 out of first 15 evaluable patients experience graft failure by this 
time point (corresponding lower one-sided 90% confidence limits: 0.416, 0.354, 0.404, and 
0.385). Patients who die before Day 60 evaluations without evidence or documentation of > 5% 
donor chimerism, who have < 5% donor chimerism in the context of any bone marrow 
involvement by persistent or progressive malignancy, or who have < 5% donor chimerism 
without assessment of bone marrow disease status if the bone marrow was involved 
pretransplantation may be considered inevaluable for this stopping guideline. However, 
chimerism data in these and the other patients may be considered in the overall evaluation of 
engraftment, and a go/no-go decision rendered on this basis. 
 
9.2 
Primary statistical plan: phase 2 expansion 
The study will accrue an additional 45 patients to a phase 2 expansion cohort, using the 
regimen chosen in the initial phase (effective with protocol version 1/30/2017). The 
primary objective of this portion of the study will be to evaluate the “immunologic 
efficacy” of the chosen regimen. 
Immunologic efficacy is herein defined as surviving to 6 months posttransplant without 
having had severe acute GVHD or evidence of graft failure. Accrual to the phase 2 portion 
may begin while full evaluation of the phase 1 portion is in progress. The 20 patients in 
the safety/ regimen finding portion of the study will be included in the analysis of this 
expansion cohort. 
 

Version date: 
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42 
 
 
9.21 
Futility monitoring 
The non-parametric Kaplan-Meier estimate will be used to monitor the failure-free survival 
(FFS) function at 6 months wherein, from an immunologic standpoint, failure is defined as 
transplant- related non-relapse mortality, severe acute GVHD, or graft failure. Patients will 
be censored at the time of relapse. Futility monitoring will start after the 5th patient has been 
enrolled in the expansion cohort. There will be two interim analyses for futility, after the 5th 
and 15th patient have been enrolled in the expansion cohort. The study is designed to stop for 
futility if there is 80% certainty that the 6-month FFS is below 40%. 
 
The study design operating characteristics assume a total sample size of 45 patients, a 5-year 
recruitment period, and additional follow-up of 6 months. The following table summarizes the 
operating characteristics of the futility stopping rule under various scenarios for the 
underlying exponential FFS, based on 1000 simulations. For futility monitoring we 
optimistically characterize the uncertainty of the 6 month FFS estimate with the prior: 
beta(3,2). This implies that our prior guess at the 6 month FFS in this study is 60% and there 
is 90% certainty that it is between 25% and 90%. Because patients will be censored at the time 
of relapse for the primary endpoint, the simulations assume an exponential hazard rate (per 
month) for relapse of 0.02. The resulting overall proportion of censored observations in the 
simulations, Table 9, is close to what we might see if the true 6-month FFS is similar to that 
for nonmyeloablative haplo transplants from first-degree related donors, i.e. approximately 
23% censoring when the true 6-month FFS is 0.60. 
Table 9. Sample size of 45 with a null 6-month FFS of 0.40, prior beta(3,2) and an alternative 
of 0.60 
6-month 
FFS 
Prob Stop for 
Futility 
Avg N 
Estimated 
6-month FFS 
lo 95% 
Post Int'l 
hi 95% 
Post Int'l 
Overall % 
censored 
0.30 
1.00 
25.2 
0.30 
0.13 
0.50 
9.6 
0.35 
0.99 
25.5 
0.34 
0.16 
0.52 
11.2 
0.40 
0.97 
26.2 
0.39 
0.21 
0.55 
12.9 
0.45 
0.91 
27.9 
0.43 
0.23 
0.59 
14.8 
0.50 
0.77 
31.1 
0.48 
0.27 
0.64 
17.0 
0.55 
0.56 
35.2 
0.53 
0.35 
0.70 
20.0 
0.60 
0.33 
39.3 
0.60 
0.40 
0.75 
23.1 
0.65 
0.17 
42.0 
0.64 
0.46 
0.79 
26.9 
0.70 
0.06 
44.0 
0.69 
0.50 
0.83 
31.9 
 
9.22 
Continuous safety monitoring 
Table 10 provides expanded stopping boundaries to monitor the study continuously for safety 
as patients 21 through 45 in the phase 2 cohort reach the Day 100 benchmark.  As in the phase 
1 portion, the allowed probabilities of severe acute GVHD and transplant-related NRM are 
25% and 20% respectively by Day 100. The stopping boundaries use a posterior probability 
threshold of 80%. The combinations of severe acute GVHD and transplant-related NRM events 
by Day 100 that lead to stopping are shown as row and column headers, with the range of 
sample sizes in the body of the table. As an example, when 26 patients have completed the 
100-day observation period, the combinations of adverse events (severe acute GVHD, 
transplant-related NRM) by that time point that would stop the trial are: (12,2), (12,1), (10,5), 
(10,4), (9,6), (8,7), (5,9), (3,9), (2,10) and (0,10). 
 
If Regimen B (with post-transplant Cy, MMF, and sirolimus) is selected for phase 2 
expansion, but its study then stops due to futility or safety, Regimen C (with post-transplant 
Cy, MMF, and tacrolimus) will be evaluated and expanded to phase 2 study if appropriate. 

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Table 10: Phase 2 expansion stopping boundaries, based on the number of severe acute GVHD 
and transplant-related NRM events by Day 100, using a posterior probability threshold of 
80%. The table entries are the sample sizes for which the row and column numbers of severe acute 
GVHD cases and transplant-related NRM cases, respectively, constitute the stopping rule. 
 
NRM 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
aGVHD 
0 
5 -7 
8 - 
10 
11- 
13 
14- 
16 
17- 
19 
20- 
23 
24- 
26 
27- 
30 
31- 
33 
34- 
37 
38- 
41 
42- 
44 
45 
1 
5 -6 
7 -8 
9 - 
11 
12 - 
14 
15- 
17 
18- 
20 
21- 
24 
25- 
27 
28- 
31 
32- 
34 
35- 
38 
39- 
42 
43- 
45 
2 
5 
6 -7 
9 - 
10 
11 - 
12 
13- 
15 
16- 
18 
19- 
20 
22- 
25 
26- 
28 
29- 
32 
34- 
35 
36- 
39 
40- 
43 
44- 
45 
3 
5 -6 
6 -7 
8 -9 
10 - 
11 
12 - 
14 
15- 
17 
18- 
20 
21- 
23 
24- 
26 
28- 
29 
30- 
33 
34- 
36 
37- 
40 
41- 
44 
45 
4 
5 -6 
7 -8 
8 -9 
10 - 
11 
12- 
13 
14- 
16 
17- 
18 
19- 
20 
22- 
24 
25- 
27 
28- 
30 
31- 
34 
35- 
37 
38- 
41 
42- 
45 
5 
7 -9 
9- 
10 
10 - 
11 
12 - 
13 
14- 
16 
17 
18- 
20 
21- 
22 
23- 
25 
26- 
28 
29- 
32 
33- 
35 
36- 
38 
39- 
42 
43- 
45 
6 
10-11 
11- 
12 
12- 
13 
14- 
15 
16- 
17 
18- 
19 
20- 
22 
23- 
24 
25- 
27 
28- 
30 
31- 
33 
34- 
36 
37- 
40 
41- 
43 
44- 
45 
7 
12-13 
13- 
14 
14- 
16 
16- 
17 
18- 
19 
20- 
21 
22- 
23 
24- 
27 
27- 
29 
30 
31- 
34 
35- 
38 
39- 
41 
42- 
44 
45 
8 
14-16 
15- 
17 
17- 
18 
18- 
19 
20- 
21 
22- 
23 
24- 
25 
26- 
28 
29- 
30 
31- 
33 
34- 
36 
37- 
39 
40- 
42 
43- 
45 
9 
17-19 
18- 
19 
19- 
20 
20- 
22 
22- 
23 
24- 
25 
26- 
27 
28- 
30 
31- 
32 
33- 
35 
36- 
37 
38- 
40 
41- 
44 
45 
10 
20-21 
20- 
22 
21- 
23 
23- 
24 
24- 
26 
26- 
27 
28- 
29 
30- 
32 
33- 
34 
35- 
36 
37- 
39 
40- 
42 
43- 
45 
11 
22-24 
23- 
25 
24- 
25 
25- 
27 
27- 
28 
28- 
30 
30- 
32 
33- 
34 
35- 
36 
37- 
38 
39- 
41 
42- 
44 
45 
12 
25-27 
26- 
27 
26- 
28 
28- 
29 
29- 
30 
31- 
32 
33- 
34 
35- 
36 
37- 
38 
39- 
40 
41- 
43 
44- 
45 
13 
28-30 
28- 
30 
29- 
31 
30- 
32 
31- 
33 
33- 
34 
35- 
36 
37- 
38 
39- 
40 
41- 
42 
43- 
45 
14 
31-33 
31- 
33 
32- 
34 
33- 
34 
34- 
35 
35- 
37 
37- 
38 
39- 
40 
41- 
42 
43- 
44 
45 
15 
34-36 
34- 
36 
35- 
36 
35- 
37 
36- 
38 
38- 
39 
39- 
41 
41- 
43 
43- 
44 
45 
16 
37-39 
37- 
39 
37- 
39 
38- 
40 
39- 
41 
40- 
42 
42- 
43 
44- 
45 
45 
17 
40-42 
40- 
42 
40- 
42 
41- 
43 
42- 
44 
43- 
45 
44- 
45 
18 
43-45 
43- 
45 
43- 
45 
44- 
45 
45 
 
9.3 
Secondary endpoints 
 
9.31 
Disease and survival endpoints 
The probabilities of 1-year and longer-term progression-free survival, disease-free survival, overall 
survival, GFRFS, and cGFRFS after transplantation will be estimated and reported with 90% 
confidence intervals using the Kaplan-Meier method. In addition, the proportion of patients who are 
progression-free and who are alive will be reported at 1 year with 90% exact binomial confidence 
intervals, in patients who have been followed for that minimum time. 
Cumulative incidences of progression/relapse and NRM will be estimated with competing 
risk analyses using Gray’s method.  The disease and survival endpoints will be described for the 

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44 
 
 
 
group as a whole, for the final selected regimen, and if numbers permit, for myeloid versus lymphoid 
histologies without formal statistical comparison. 
 
9.32 
Toxicities 
The cumulative incidence of acute GVHD (grade II-IV, grade III-IV) and chronic GVHD (overall, 
and according to extent) will be estimated through competing-risk analysis, wherein 
relapse/progression, graft failure, and death are competing risks for GVHD. We also plan to report 
the cumulative incidence of GVHD with only graft failure and death regarded as competing risks. 
The cumulative incidences of systemic steroid initiation for GVHD treatment, of non-steroid 
immunosuppression use, and of discontinuation of immunosuppression for GVHD treatment will be 
similarly estimated through competing-risk analysis, wherein graft failure and death, or graft failure, 
death and relapse/progression, are considered competing risks. The frequency of steroid use for 
GVHD will be reported. The number and types of systemic immunosuppression used for GVHD 
treatment will be reported descriptively. 
Other selected toxicities will be reported descriptively. 
 
9.33 
Graft failure and engraftment kinetics 
Times to neutrophil and platelet recovery will be described with medians and ranges, and with 
cumulative incidence functions with death before count recovery as a competing risk. 
The amount of donor chimerism in T cells and total leukocytes at ~Day 30, ~Day 60, and 
beyond will be described. The graft failure frequency will be described with exact 90% binomial 
confidence intervals. 
 
9.4 
Clinical trial reporting 
Up to two reports of preliminary outcomes are planned, including publication of the phase 1 results, 
in order to describe the experience with feasibility, toxicity, overall clinical outcomes, and laboratory 
correlative studies. These analyses will be descriptive in nature rather than formal interim analyses. 
 
10.0      PATHOLOGY REVIEW 
In accordance with standard institutional practice, at least one specimen diagnostic of the malignancy (from 
the original diagnosis and/or relapse) must be reviewed by the Johns Hopkins department of pathology prior 
to starting protocol therapy. In cases diagnosed solely by peripheral blood flow cytometry, the diagnostic flow 
cytometry report must be reviewed. 
 
11.0 
RECORDS TO BE KEPT 
Records to be filed include the following: 
1. Patient consent form 
2. Registration form 
3. Case report forms, including patient-donor HLA reports 
4. Adverse event report form(s) 
5. Follow-up assessments 
 
The principal investigator will review case report forms on a regular basis. Case report forms will be 
supported by primary source documents. 
 
12.0      PATIENT CONSENT AND PEER JUDGMENT 
Current federal, NCI, state, and institutional regulations regarding informed consent will be followed. 
 
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APPENDIX 
Clinical grading of acute GVHD 
 
 
From Przepiorka D et al. 1994 Consensus Conference on Acute GVHD Grading. BMT 1995; 15: 825-828.