Pilot Study of Prophylactic Dose-Escalation Donor Lymphocyte Infusion After T Cell Depleted Allogeneic Stem Cell Transplant in High Risk Patients With Hematologic Malignancies
Phase: PHASE2
Conditions: Accelerated Phase Chronic Myelogenous Leukemia, Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities, Adult Acute Myeloid Leukemia With Del(5q), Adult Acute Myeloid Leukemia With Inv(16)(p13;q22), Adult Acute Myeloid Leukemia With t(15;17)(q22;q12), Adult Acute Myeloid Leukemia With t(16;16)(p13;q22), Adult Acute Myeloid Leukemia With t(8;21)(q22;q22), Adult Nasal Type Extranodal NK/T-cell Lymphoma, Anaplastic Large Cell Lymphoma, Angioimmunoblastic T-cell Lymphoma, Blastic Phase Chronic Myelogenous Leukemia, Childhood Burkitt Lymphoma, Childhood Chronic Myelogenous Leukemia, Childhood Diffuse Large Cell Lymphoma, Childhood Immunoblastic Large Cell Lymphoma, Childhood Myelodysplastic Syndromes, Childhood Nasal Type Extranodal NK/T-cell Lymphoma, Chronic Phase Chronic Myelogenous Leukemia, Cutaneous B-cell Non-Hodgkin Lymphoma, de Novo Myelodysplastic Syndromes, Extranodal Marginal Zone B-cell Lymphoma of Mucosa-associated Lymphoid Tissue, Hepatosplenic T-cell Lymphoma, Intraocular Lymphoma, Nodal Marginal Zone B-cell Lymphoma, Noncutaneous Extranodal Lymphoma, Peripheral T-cell Lymphoma, Post-transplant Lymphoproliferative Disorder, Previously Treated Myelodysplastic Syndromes, Recurrent Adult Acute Lymphoblastic Leukemia, Recurrent Adult Acute Myeloid Leukemia, Recurrent Adult Burkitt Lymphoma, Recurrent Adult Diffuse Large Cell Lymphoma, Recurrent Adult Diffuse Mixed Cell Lymphoma, Recurrent Adult Diffuse Small Cleaved Cell Lymphoma, Recurrent Adult Grade III Lymphomatoid Granulomatosis, Recurrent Adult Hodgkin Lymphoma, Recurrent Adult Immunoblastic Large Cell Lymphoma, Recurrent Adult Lymphoblastic Lymphoma, Recurrent Adult T-cell Leukemia/Lymphoma, Recurrent Childhood Acute Lymphoblastic Leukemia, Recurrent Childhood Acute Myeloid Leukemia, Recurrent Childhood Anaplastic Large Cell Lymphoma, Recurrent Childhood Grade III Lymphomatoid Granulomatosis, Recurrent Childhood Large Cell Lymphoma, Recurrent Childhood Lymphoblastic Lymphoma, Recurrent Childhood Small Noncleaved Cell Lymphoma, Recurrent Cutaneous T-cell Non-Hodgkin Lymphoma, Recurrent Grade 1 Follicular Lymphoma, Recurrent Grade 2 Follicular Lymphoma, Recurrent Grade 3 Follicular Lymphoma, Recurrent Mantle Cell Lymphoma, Recurrent Marginal Zone Lymphoma, Recurrent Mycosis Fungoides/Sezary Syndrome, Recurrent Small Lymphocytic Lymphoma, Recurrent/Refractory Childhood Hodgkin Lymphoma, Refractory Chronic Lymphocytic Leukemia, Refractory Hairy Cell Leukemia, Relapsing Chronic Myelogenous Leukemia, Secondary Myelodysplastic Syndromes, Small Intestine Lymphoma, Splenic Marginal Zone Lymphoma, T-cell Large Granular Lymphocyte Leukemia, Testicular Lymphoma, Waldenström Macroglobulinemia
Sponsor: University of Chicago

BSD IRB Committee C
The University of Chicago Biological Sciences Division/University of Chicago
Medical Center
5841 S. Maryland Ave., MC7132, I-625, Chicago, IL 60637
FWA00005565
Notification of Continuing Review Approval
Date of Letter: 6/12/2019
Protocol
Number/Submission
Link:
IRB12-1191-CR007
Type of Submission: Continuing Review
Status: Approved
Principal
Investigator:
Hongtao Liu
Protocol Title: Pilot Study of Prophylactic Dose-Escalation Donor Lymphocyte Infusion After T Cell Depleted
Allogeneic Stem Cell Transplant in High Risk Patients With Hematologic Malignancies
Meeting Date: 6/11/2019 12:30 PM
Risk Level: Remaining Activities Minimal Risk
Consent Type: Written Consent Form
Authorization Type: Obtain written consent/authorization
Vulnerable
Population:
Children - Disease/Disorder/Condition
Funding: Externally Funded/Supported
Internally Funded
Protocol Version: 12-1191 Protocol V.7 06252015 CLEAN
Investigator
Brochure(s):
There are no items to display
Stamped Documents: There are no items to display
Approval Date: 6/12/2019
Study Expiration
Date:
6/10/2020
The above-referenced Continuing Review was approved by the IRB.  
Stamped approved documents associated with this study can be found in the study workspace, by following
the Submission Link above.
If you need assistance, please contact the IRB from the submission workspace by clicking the “Send Email to
IRB Team” activity.
Please refer to your IRB’s current policy and procedure manual available
at: http://humansubjects.uchicago.edu/.
https://aurairb6-prd.uchicago.edu/irb/Doc/0/2T67IDLCEKEKDFMFEQ...
1 of 1
7/11/2019, 1:17 PM

PILOT STUDY OF PROPHYLACTIC DOSE-ESCALATION DONOR LYMPHOCYTE 
INFUSION AFTER T CELL DEPLETED ALLOGENEIC STEM CELL TRANSPLANT 
IN HIGH RISK PATIENTS WITH HEMATOLOGIC MALIGNANCIES 
 
 
Coordinating Center:  
The University of Chicago  
5841 S. Maryland Ave., MC2115 
Chicago, IL 60637 USA  
 
Principal Investigators: 
Hongtao Liu, MD, PhD 
Andrew Artz, MD, MS 
 
Co-Investigators:  
 
Michael Bishop, MD. 
  
 
 
 
 
 
 
Wendy Stock, MD 
Richard Larson, MD 
   
 
 
 
 
    Toyosi Odenike, MD  
 
 
 
 
   
 
 
 
 
    Lucy Godley, MD, PhD  
   
 
 
 
 
    Michael Thirman, MD 
   
 
 
 
 
    Jane Churpek, MD 
   
 
 
 
 
    Sonali Smith, MD 
   
 
 
 
 
    Justin Kline, MD 
   
 
 
 
 
    Todd Zimmerman, MD 
   
 
 
 
 
    John Cunningham, MD 
                                             James LaBelle, MD 
                                             Amittha Wickrema, PhD 
   
 
 
 
 
    Yusuke Nakamura, MD, PhD 
 
Statistician:                    
Rangesh Kunnavakkam  
 
Research Nurses:     Paula Del Cerro, R.N., O.C.N. 
  
 
 
 
 
 
 
Linda Schroeder, RN, BSN, MS, AOCNS 
                                             Kelly Kramer, APN 
                                             Caitlin Beaudoin, APN 
                                         
Data Management:     
                                           Lyndsey Schorr, CRA 
   
 
 
 
 
   Alexis Small, CRA 
   
 
 
 
 
   John Tyson, CRA 
   
 
 
 
 
   Ray Robinson, CRA 
   
 
 
 
 
   Lilian Villatuya 
 
Protocol version date:       v. 7.0, 06.25.2015     
 
Institutional Review Board #: 12-1191 
The University of Chicago 
Department of Medicine, 
Section of Hematology/Oncology 
5841 S. Maryland Ave., Chicago, IL 60637 
 

1 
 
 
SCHEMA 
 
 
 
                                                               
 
 
                                                                    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Primary objective: To determine the proportion of patients who receive at least one 
DLI.   
 
Collection of steady-state donor lymphocyte cells and cryopreservation 
in appropriate CD3 enumerated aliquots.  Lymphocytes collected later 
or from mobilized product acceptable. 
Collection of stem cells after G-CSF 
mobilization. BM collection is acceptable
High risk patients with hematologic malignancies undergoing T cell 
depleted Allo-SCT with matched related or unrelated donor consented 
prior to SCT 
Allo-SCT (T cell depleted regimens with Alemtuzumab or ATG). 
Preferably cyto-reduction before conditioning 
Prophylactic DLI: 
Requires no more than Grade I aGVHD and off immune 
suppression for each DLI. First dose ideally will be at day 
75 to day 90 post-SCT, but delayed DLI administration is 
allowed as long as the subject meets pre-DLI criteria. 
Subsequent DLI will be given in 4-8 week intervals.   
 
DLI doses by donor: 
Matched Related Donor (MRD): 
2 x 105/kg ; 5 x 105/kg ; 1 x 106/kg; 2 x 106/kg; 5x106/kg 
 
Matched Unrelated Donor (MUD): 
1 x 105/kg ; 2 x 105/kg ; 5 x 105/kg ; 1 x 106/kg; 2 x 106/kg 
Withdrawal of immunosuppression at day 60 if no more 
than Grade I aGVHD  

2 
 
 
TABLE OF CONTENTS 
SCHEMA ....................................................................................................................................... 1 
 
1. 
OBJECTIVES ....................................................................................................................... 4 
1.1. 
PRIMARY OBJECTIVES .......................................................................................................................... 4 
1.2. 
SECONDARY OBJECTIVES .................................................................................................................... 4 
 
2. 
BACKGROUND ................................................................................................................... 4 
2.1. 
GROWTH IN STEM CELL TRANSPLANT ............................................................................. 4 
2.2. 
TREATMENT OPTIONS FOR RELAPSED AML AFTER SCT ................................................... 5 
2.3. 
DLI FOR RELAPSED AFTER SCT. ....................................................................................... 7 
2.4. 
PROPHYLAXIS DLI AFTER SCT ....................................................................................... 10 
2.5. 
RATIONALES TO CONDUCT PROPHYLACTIC DOSE-ESCALATION DLI TO PREVENT RELAPSE 
IN HEMATOLOGIC MALIGNANCIES AFTER T CELL DEPLETED ALLO-SCT. ........................ 12 
 
3. 
PATIENT SELECTION……………………………………………………………...…..12 
3.1. 
ELIGIBILITY CRITERIA ........................................................................................................................ 13 
3.2. 
EXCLUSION CRITERIA ......................................................................................................................... 14 
3.3. 
ELIGIBILITY TO RECEIVE DLI POST-TRANSPLANT ...................................................................... 14 
3.4. 
HUMAN SUBJECTS AND INCLUSION OF WOMEN AND MINORITIES .......................................... 14 
 
4. 
REGISTRATION PROCEDURES AND RADOMAZATION ...................................... 15 
 
5. 
TREATMENT PLAN ......................................................................................................... 15 
5.1. 
STUDY DESIGN ..................................................................................................................................... 15 
5.2. 
DURATION OF THERAPY ..................................................................................................................... 17 
5.3. 
DURATION OF FOLLOW UP ................................................................................................................ 18 
5.4. 
CRITERIA FOR REMOVAL FROM STUDY .......................................................................................... 18 
5.5. 
PROTOCOL MANAGEMENT PLAN: .................................................................................................... 18 
 
6. 
DOSING DELAYS/DOSE MODIFICATIONS ............................................................... 18 
 
7. 
ADVERSE EVENTS MONITOR AND REPORTING: ................................................. 19 
7.1. 
TOXICITY MONITORING ..................................................................................................................... 19 
7.2. 
TOXICITY REPORTING ......................................................................................................................... 19 
7.3. 
SERIOUS ADVERSE EVENTS .............................................................................................................. 19 
7.4. 
OTHER REPORTING REQUIREMENTS ............................................................................................... 19 
7.5. 
SUPPORTIVE THERAPY ........................................................................................................................ 22 
 
8. 
CORRELATIVE/SPECIAL STUDIES ............................................................................ 23 
 
9. 
CRITERIA FOR STUDY EVALUATION ...................................................................... 24 
 
10. STUDY CALENDAR ......................................................................................................... 22 
 
11. DATA REPORTING / REGULATORY CONSIDERATIONS ..................................... 26 

3 
 
 
12. STATISTICAL CONSIDERATIONS .............................................................................. 27 
13.1. 
STUDY DESIGN/ENDPOINTS .............................................................................................................. 27 
13.2. 
SAMPLE SIZE/ACCRUAL RATE .......................................................................................................... 27 
13.3. 
STATISTICAL CONSIDERATIONS ....................................................................................................... 27 
 
13.  FUTURE DIRECTION ……………………….…………………………..….…………..27 
 
14.  APPENDIX……………………..………………………………….……………………….28 
 
15.  REFERENCES……………………………….……………………………..……………..29 
 
 
 
 

4 
 
1. OBJECTIVES 
 
Primary Objective 
 
 
 
To determine the feasibility of EDR DLI as measured by the proportion of patients who 
receive at least one DLI 
 
 
Secondary Objectives 
 
To assess progression free survival (PFS) at 2 years after stem cell transplant (SCT) for 
high-risk hematologic malignancies receiving T-cell depleted grafts followed by escalating 
dose regimen (EDR) prophylactic DLI compared to historical controls not receiving DLI 
 
To assess the safety of EDR DLI for high-risk hematologic malignancies as measured by 
cumulative incidence of severe grade III-IV acute GVHD 
 
 
To measure outcomes of grade II-IV acute GVHD, non-relapse mortality, overall survival 
and chronic GVHD of EDR DLI 
 
To assess the full donor chimerism rate in the CD3 compartment and immune 
reconstitution after EDR DLI 
 
2. BACKGROUND 
 
2.1  Growth in Stem Cell Transplant: 
There has been tremendous progress in the past couple of decades in allogeneic 
hematopoietic stem cell transplantation (SCT) improving outcomes through incremental 
improvements. Further, access to SCT continues to expand because of larger number of 
stem cell donor options to include HLA-matched unrelated donors (MUD), haploidentical 
related donors, and cord blood units (CBUs), significant improvements in supportive care, 
and introduction of better tolerated reduced intensity conditioning (RIC) regimens (1-4).   
 
2.1.1 T-cell depletion 
T-cell depletion is another approach that enhances tolerability by reducing acute and 
chronic GVHD, complications which result in tremendous transplanted related morbidity 
and mortality. We have employed in vivo T-cell depletion with alemtuzumab for over 10 
years at the University of Chicago and confirmed lower rates of acute and chronic GVHD 
and similar overall survival (5). Recent observational registry studies (6) and now even 
prospective studies have further established that T-cell depletion through either ATG or 
alemtuzumab enables better GVHD free survival (7).  Although relapse in general remains 
highly problematic after transplant (8), particularly for those with active disease at SCT, T-
cell depletion further increases rates of relapse. The relapse rate for patients with AML 
undergoing RIC allo-SCT is in the range of ~25% at one year without T cell depletion and 
in the range of 40-50% at 4 or 5 years with T cell depletion (4, 5, 9, 10).  There has been 
very little progress in reducing the incidence of relapse following allogeneic SCT.  
Therefore, the platform of T-cell depletion enables patients to undergo transplant with low 

5 
 
rates of GVHD but approaches to harness the immune system later after transplant once 
conditioning toxicities have resolved requires novel therapies. 
 
2.1.2 Treatment of high-risk hematologic malignances: 
Despite the high-rates of relapse if not increased treatment related mortality for patients 
entering transplant with poorly controlled hematologic malignancies, some of these patients 
will achieve long-term disease control if not cure after allogeneic SCT. The prognosis for 
such patients absent a transplant remains dismal although better disease control (e.g., lack 
of peripheral blood blasts, longer remission duration), a sibling donor, younger recipient 
age and non-adverse cytogenetics are favorable prognostic factors. Strategies using allo-
SCT as a platform hold appeal to improve long-term outcomes. At the University of 
Chicago, we have developed an interest in improving outcomes for high-risk hematologic 
malignances. One approach we and other have tested with some success is further 
intensification using cyto-reductive chemotherapy immediately before reduced intensity 
conditioning incorporating T-cell depletion with allogeneic SCT Because of the low rate of 
GVHD from T-cell depletion, patients tolerate these regimens surprisingly well and even 
those entering transplant with active disease typically achieve remission (11). Although 
relapse appears to be delayed, most patients eventually develop disease recurrence. This 
protocol assesses maintenance on this established platform. 
 
2.2  Treatment Options for relapsed AML after SCT: 
A general tenant in hematologic malignancies and transplantation specifically is that 
prevention of relapse is more effective than delaying further treatment until the time of 
relapse. Strategies that show efficacy at relapse warrant testing as prophylaxis. The current 
options of management for disease relapse after allo-SCT include withdrawal of immune 
suppression, chemotherapy, second allogeneic transplant, cytokine and adoptive cell 
therapy and DLI.  Withdrawal of immunosuppression can be performed in all patients 
irrespective of hematopoietic stem cell source. But the efficacy is very limited. While 
remission rates approaching 84% were observed in patients with chronic phase CML, they 
were only 10% for AML, and 0% for ALL and advanced phase CML (12) .  
 
2.2.1  
Chemotherapy: For relapsed acute leukemia, both conventional chemotherapy 
and newer biological agents are able to induce significant remission rates, but long-term 
survival is very rare. The use of TKI, including dasatinib and nilotinib in patients with Ph+ 
acute lymphoblastic leukemia (ALL) or 5-azacytidine for relapsed AML may have 
particular benefit. Sorafenib and other FLT3 inhibitors, such as AC220 have demonstrated 
preliminary activity in a small number of patients with relapsed FLT3+ AML (13). 
 
In the AML setting, a retrospective analysis from the Fred Hutchinson Cancer Research 
Center (FHCRC) demonstrated that of those treated for relapse (which is a fraction of the 
actual number of patients relapsing) around 30% of relapsed AML after allo-SCT could 
enter CR after chemotherapy with cytarabine (with and without adriamycin), but the 
median DFS was very short at 9.7 months (14) and the response was highly influenced by 
time to relapse after allo-SCT.  Specifically, 2-year survival estimates for patients relapsing 
less than 100 days, 100-200 days, and greater than 200 days from allo-SCT were 3%, 9%, 
and 19%, respectively. Other studies explored the use of DLI after chemotherapy for 
relapse AML following allo-SCT, and found 1-year survival probability of 10% (95% 

6 
 
confidence interval [CI] 3%-31%) if relapse occurred within 6 months of transplant versus 
44% (95% CI 29%- 68%) if relapse occurred later (15, 16). These data led to the current 
practice standard to offer standard chemotherapy, with and without DLI, only in patients 
who relapse more than 3 to 6 months after allo-SCT, and refer other patients to clinical 
trials or palliative care if no trial available. Our own data showed similar results except 
almost all patients relapsing after T-cell depletion with alemtuzumab were able to receive 
treatment probably in part from low rates of GVHD and high-tolerability of the regimen 
(17). Again, this provides further support for the concept that T-cell depletion is a tolerable 
regimen and platform for subsequent therapy. 
 
Given the relatively futile results of chemotherapy at relapse after allo-SCT, prophylactic 
chemotherapy after allo-SCT for high risk patients has been attempted, especially using 
less toxic hypomethylating agents. A phase 1 trial of azacitidine as post-transplant 
maintenance therapy conducted at the M.D. Anderson Cancer Center demonstrated that 
azacitidine could be given at 32 mg/m2/day for 5 consecutive days every 4 weeks for at 
least 4 cycles with limited side effects in 42 patients who underwent reduced-intensity allo-
SCT for relapsed/refractory AML (18). Low-dose azacitidine was also used by the M.D. 
Anderson group to treat relapsed AML and MDS after allo-SCT, and around 20% long-
term disease control rate was found in patients with early relapses (18). A German group 
also used low dose azacitidine followed with DLI in relapsed AML/MDS patients after 
Allo-SCT with low long-term response rate (19, 20).   
 
 
2.2.2  
Second allogeneic transplant: Patients who have failed an initial Allo-SCT have 
chemo-refractory disease, making additional chemoradiotherapy unlikely to be curative. 
Historically, the role of second allogeneic SCT has been limited by unacceptable relapse 
rates and high mortality rate, depending on previous therapies, age, and time from first 
transplantation. The efficacy of second allogeneic SCT depends on several factors, such as 
underlying malignancy, patient age and performance status, type of conditioning regimen 
employed, and time interval between first and second HSCT. In a large Center for 
International Blood and Marrow Transplant Research (CIBMTR) retrospective study of 
patients with hematologic malignancies undergoing second allogeneic HSCT, transplant-
related mortality was 30% and the relapse rate was 42%, yielding an overall survival rate of 
28% at 5 years post-SCT (21). 
 
Although often suggested, there is no demonstrated benefit to using another donor when 
performing a second transplant after relapse. Generally, outcomes of second transplant are 
better for younger patients and for those with a longer time (6-12 months) from 
transplantation to relapse (i.e, remission duration after SCT). Data from the CIBMTR 
showed a 5-year survival rate of 51% in patients under age 20 years who relapsed more 
than 6 months after transplantation and only 3% in older patients who relapsed within 6 
months after transplantation (21). The European Group for Blood and Marrow 
Transplantation (EBMT) reported the best outcomes for patients with late relapse (292 
days) in remission at time of second transplantation, with 53% survival at 3 years (22).  
High-risk patients relapse earlier post-SCT which also translates into lower chances of 
success with post-SCT therapy.  
 

7 
 
2.2.3  
Novel approaches with immune-therapies  
Innovative and novel immunotherapeutic approaches are currently under investigation. 
Among other approaches, non-specific ex vivo activation and expansion through co-
stimulation of donor T cells have been used safely, with intriguing GVT responses (23). It 
also may be possible to generate leukemia-specific cytotoxic T cells to use in adoptive 
immunotherapy (24). Another strategy of generating CTLs against antigens presented on 
leukemic cells has been attempted by genetically modifying T cells to introduce antigen 
receptors capable of recognizing leukemic cells. Cooper et al. generated T lymphocytes 
engineered to express chimeric antigen receptors (CARs) specific for the CD19 molecule 
that may be able to prevent or treat leukemia relapse in B-ALL patients as these cells 
almost invariably express CD19 (25).  Suppression of negative immune-modulation by 
anti-CTLA4 antibodies, Ipilimumab, has been explored (26). Vaccine strategies with 
tumor-specific antigens or modified tumor cells are other promising approaches to generate 
tumor specific immunity (27, 28). These strategies will likely be most effective in the 
setting of minimal residual disease.  
 
2.3  DLI for relapse after SCT  
Donor lymphocyte infusion (DLI) has been proven to induce remissions post-transplant in 
patients with relapsed hematologic malignancies most consistently observed in patients 
with CML, and to a lesser extent in AML, multiple myeloma and myelodysplasia (29).  
 
2.3.1  
General principles of DLI: source, effective cell dose, timing and toxicity:  
Although G-CSF-stimulated peripheral blood cells for DLI have been used (16),  
typically donor lymphocyte is obtained by leukapheresis of un-stimulated peripheral blood 
(a.k.a., steady-state lymphocytes) which will contain other cell types in addition to CD3+ 
cells, such as dendritic cells, B cells, monocytic cells and natural killer (NK) cells, 
providing a spectrum of allo-reactive and other accessory cells that might play a role in 
graft-versus-tumor effect (30). 
 
The optimal CD3 cell dose in DLI is not established. However, some studies have 
demonstrated that in patients with CML, a dose of 1x107/kg can induce complete donor 
chimerism and a potent graft versus-leukemia (GVL) effect, in some cases in the absence 
of clinical GVHD particularly if given at later time points following transplantation (31). 
On the other hand, Fozza et al demonstrated that the incidence of GVHD was not 
significantly different in patients receiving less than 1 × 107 CD3+/kg compared with the 
patients who received doses greater than 1 × 107 CD3+/kg (32).  
 
The timing of DLI and the interval after DLI required to observe a response from the DLI 
are important factors influencing the effectiveness of this strategy. Patients with relapses 
occurring more than 6 months post-transplantation have higher chances of responding to 
DLI (33). In support of the aforementioned data, Choi et al. found 55% overall survival at 1 
year in patients who were treated for relapse, which occurred greater than 6 months post-
transplantation, as opposed to 0% survival at 1 year in patients treated for relapse that 
occurred within 6 months following SCT (16). Disease response following DLI can be seen 
between 40 days and up to 1 year following DLI. Another predictor of DLI success is the 
tumor burden at the time DLI is administered. Patients with evidence of molecular relapse 
at the time of DLI have better responses even in malignancies not typically viewed as 

8 
 
responsive to DLI, such as ALL, which might support careful screening of patients for 
detection of molecular relapse (33) offer further support for prophylactic approaches 
 
The major complications with DLI are the development of GVHD and cytopenias; marrow 
aplasia has also been appreciated but is quite rare. Acute GVHD develops in up to 40–60% 
of patients who receive DLI. The development of GVHD does not always correlate with 
GVT activity (34). The time interval between SCT and DLI therapy appears to influence 
the likelihood of developing GVHD. A small dose of 1 x 105 T cells/kg can induce GVHD 
if administered on the day of transplant (35), yet a dose of 1 x 107 T cells/kg can be given 
at 12 months post-transplant without GVHD development (31). Other factors which make 
GVHD more likely to occur include donor sex mismatch (female donor to male recipient), 
advanced patient age and mismatch at the mHag level (36).  
 
Aplasia is now a relatively infrequent complication of DLI. It is often transient, but in some 
cases may require hematopoietic stem cell rescue. It was reported historically in 15-20% of 
treated CML patients with an associated mortality rate of ~ 5%. Aplasia is more common 
in hematological relapse of CML, possibly due to poor donor myeloid reserve, and is rarely 
reported in patients with exclusively cytogenetic or molecular relapse (37, 38) or in those 
treated for low levels of recipient mixed chimerism. 
 
Overall, DLI is an effective form of immunotherapy in patients with CML who relapse 
following SCT, with remission rates of approximately 80%. The results in patients with 
acute leukemias and myelodysplasia are disappointing with remission rates in 15–25% of 
patients and often the responses are not durable. 
 
2.3.2  
Strategies to avoid DLI-associated toxicity and improve outcome of DLI 
 
2.3.2.1 Escalated dose regimen (EDR): Administration of DLI as a single bolus of cells 
collected from a single leukapheresis containing variable numbers of CD3+ T cells is 
referred to as a bulk dose regimen (BDR) and this approach is associated with a high 
incidence of GVHD (37-39). The EDR approach is fundamentally different in that the DLI 
product is quantitated for CD3+, CD4+ and CD8+ T-cell numbers and is then administered 
in multiple small aliquots with a dose escalation over time. In this way, the minimum cell 
dose needed to achieve disease remission is administered and with more modest cell doses, 
the likelihood of GVHD may be reduced (31). One study in CML comparing BDR and 
EDR approaches demonstrated equivalent remission rates with both schedules, but a 
significantly lower incidence of GVHD in the EDR cohort (38). It is critical when using the 
EDR schedule to allow an adequate interval between DLI doses to allow for assessment of 
response and toxicity. The optimum interval between doses is yet to be defined, but Dazzi 
et al. report that shorter intervals (rather than total cell dose) leads to a higher incidence of 
GVHD (38).  
 
2.3.2.2   Manipulation of DLI products: 
In order to decrease the incidence of GVHD, many methods have been attempted including 
depletion of allo-reactive T cells by co-incubation of donor lymphocytes with allogenic 
recipient stimulator cells followed by targeting with immunotoxin-conjugated antibodies 
specific for cell-surface activation markers or antibodies (40, 41). CD8+ T cells are thought 

9 
 
to be the primary mediators of GVHD in humans while CD4+ T cells are reported to 
contribute more to the GVT effect (42). For this reason, a number of groups have explored 
CD8+ T-cell depletion as a strategy to reduce the incidence of GVHD.  CD8+ T-cell 
depletion of the stem cell graft has been reported to reduce the risk of GVHD without a 
parallel increase in relapse rates in several studies (43-45).  
 
Other manipulations under study include engineered tumor-reactive T cells expressing 
either HLA-restricted, heterodimeric TCRs or chimeric antigen receptors (CARs) that 
recognize native cell-surface antigens. Second generation CARs often comprise an 
antibody binding motif and a CD28--CD3 dual signaling receptor which facilitates T-cell 
activation and expansion following stimulation (reviewed in (46)). 
 
On the other hand, Porter et al. demonstrated that infusion of ‘ex vivo’ activated donor 
lymphocytes (using anti-CD3 and anti-CD28 coated beads) in patients with a range of 
hematologic malignancies led to responses where conventional DLI had been 
disappointing.  A total of 17 patients were evaluated and 8 achieved CR. The incidence of 
GVHD in this cohort compared favorably with that of conventional DLI (23). 
 
2.3.2.3  DLI preceded by chemotherapy especially in acute leukemia.  
 
Use of chemotherapy appears to improve the results of DLI. Response rates vary from 10% 
to 60%, with higher response rates than those reported for DLI alone (15, 47). The 
European Group for Blood and Marrow Transplant (EBMT) reported a retrospective 
analysis of 399 patients with AML in first hematologic relapse after transplant and found in 
the DLI subgroup, having less blasts in the BM (<35%), female sex, presence of favorable 
cytogenetics, and CR at the time of DLI were covariates associated with improved survival 
(47). The benefit of chemotherapy prior to DLI is suggested here by the 2-year survival 
>50% for patients that received DLI in CR. 
 
Patients with recurrent AML after allogeneic transplantation have been treated with 
chemotherapy prior to DLI. Chemotherapy was administered because of rapidly 
progressive disease or in an attempt to debulk patients prior to DLI.  Levine et al conducted 
a prospective trial of chemotherapy and G-CSF-stimulated DLI. Patients who emerged 
from the chemotherapy and DLI in complete remission had a 2-year overall survival rate of 
41% (15).  Studies from Japan and Korea treated patients with relapsed acute leukemia 
using chemotherapy followed by DLI reported an overall complete response rate of 33% , 
and 31% estimated overall survival at 24 months respectively (16, 48).   
 
While these results engender some enthusiasm for such a strategy, a number of questions 
remain unanswered. The optimal timing of adoptive immunotherapy, whether administered 
during the nadir or after hematopoietic recovery, is unknown. The contribution of the cell 
therapy to the response rates from chemotherapy alone cannot be determined in the absence 
of a prospective randomized trial.  Our own study in 25 patients with relapsed AML or 
high-risk MDS after transplant did not demonstrate advantage over chemotherapy alone for 
patients who received cellular therapy (second transplant or DLI) after chemotherapy, 
although the sample size was small (17). Our data was consistent with the data from a 
pediatric study on 49 pediatric patients receiving DLI for relapse following transplantation; 

10 
 
there was no advantage of DLI after adjustment for clinical variables compared with a large 
cohort of children with relapsed AML who did not receive DLI after transplant (49). 
 
2.4 Prophylaxis DLI after SCT 
Because DLI seems to be most effective for patients with minimal residual disease, the role 
of prophylactic DLI (pDLI) for high risk patients after stem cell transplant has been 
explored, especially in the setting of T –cell depleted SCT with increased relapse rates. 
Several studies have examined the utility of pDLI to minimize tumor recurrence in 
myeloablative (50-55) and in the RIC setting (56-61).   
 
2.4.1  
Prophylactic DLI in myeloablative T cell depleted SCT 
In the myeloablative setting, the largest series was reported by Montero et al (55).  One 
hundred thirty-eight patients with hematologic malignancies received myeloablative T cell-
depleted peripheral blood stem cell transplant from an HLA-identical sibling donor. 112 
patients with acute graft-versus-host disease (GVHD) grade <2 received 1 or 2 donor 
lymphocyte infusions of 10-50x 106 CD3+ cells/kg between days 45 and 100. Overall 
survival (OS), relapse-free survival, relapse, and transplant-related mortality (TRM) were 
58%, 46%, 40%, and 20%, respectively, after a median follow-up of 4 years. Fifty-three 
(39%) and 21 (15%) patients developed grade 2-4 and 3-4 acute GVHD respectively. 
Forty-two (36%) had limited and 29 (25%) had extensive chronic GVHD. In multivariate 
analysis, disease risk was an independent factor for OS and relapse, day-30 lymphocyte 
count for OS and TRM, and chronic GVHD for OS and relapse. PBSCT with early T cell 
add back leads to comparable rates of chronic GVHD compared with T cell-replete 
PBSCT. However, this chronic GVHD after T cell add back was associated with less 
mortality and retains a protective effect in terms of relapse, at least in the standard-risk 
patients (55).  
 
The patients reported by Schaap received planned DLI at a median of 22 weeks (range: 12–
40) post-SCT, provided the post-SCT immunosuppression was discontinued for at least 2 
months without evidence of active chronic GVHD and no history of acute GVHD above 
grade 1 (53). Similarly, Nakamura reported on patients who had planned non-mobilized 
cryopreserved DLI of 10x106 CD3/kg at day +45 and a second infusion of 50x106 CD3/kg 
at day +100 (52). Alternatively, Lee et al. (54) and Ferra et al. (51) selected DLI dosing 
based on the risk of GVHD and/or relapse risk.  
 
In summary, the risk of relapse ranged from 18 to 69% with TRM occurring in 6–52%. 
This translated into a DFS that exceeded 40% at 2 years (50-55). Schaap’s study (53) 
compared outcomes of patients receiving DLI with those patients not receiving DLI, 
relapse rates were lower resulting in improved LFS. Furthermore, the incidence of acute 
and chronic GVHD and the risk of TRM do not seem to differ from expected outcomes 
after conventional transplants without DLI.  
 
2.4.2  
Prophylactic DLI in non-myeloablative or RIC T cell depleted SCT 
Several studies have analyzed outcomes of pDLI after RIC (56-61) . Barge et al. (56) 
reported on 11 patients, who were given planned DLI at 6 months after RIC MRD SCT 
with in vitro TCD.  The DLI dose, given as unselected mononuclear cells, was based on 
disease status. Patients with relapse or progression at 6 months received 10–100x106 

11 
 
MNC/kg plus IFN-a compared with only 10x106 MNC/kg for patients with stable disease 
or mixed chimerism. For the 11 patients receiving DLI, 5 responded (CR=3; PR=2) and 1 
patient had stable disease. Acute GVHD developed in six patients, chronic GVHD in four 
patients and GVHD accounted for the death of one patient.  
 
In the report from de Lima et al. (57), 12 patients with anticipated life expectancy of less 
than 6 months received fludarabine–melphalan conditioning and sibling PBSC allogeneic 
SCT for active hematologic malignancies, including AML (n=4), myelodysplasia (MDS, 
n=1), ALL (n=3), CML (n=3) and MM (n=1). All patients were scheduled to receive non-
mobilized DLI at days +30, +60 and +90. Six patients received DLI. Of these, four patients 
achieved a CR, only one of whom was in CR at 14 months after SCT. The other three 
patients had either died due to TRM or due to relapse.  
 
Schmid et al (59) studied 75 patients with high-risk AML or MDS who received non-
myeloablative conditioning with fludarabine, cytarabine and low-dose total body 
irradiation. Patients were scheduled to receive prophylactic DLI after 120 days if there was 
no evidence of GVHD and they were off immunosuppression medications. Of the 75 
patients enrolled, only 12 patients were able to receive prophylactic DLI due to early 
relapse, GVHD and other transplant-related complications, demonstrating the challenges of 
this approach. Another study used prophylactic CD8-depleted DLI after reduced-intensity 
transplant (60). In that study, 11 of 23 patients were able to receive DLI. Patients receiving 
CD8-depleted DLI demonstrated accelerated immune reconstitution and minimal GVHD.  
 
Mixed chimerism is common after reduced-intensity regimens and may be associated with 
higher risks of relapse following transplantation. DLI has been used successfully in some 
patients with acute leukemia in order to facilitate conversion to full donor chimerism after 
reduced-intensity transplantation, and seems to lower relapse risk, albeit with a significant 
risk of acute GVHD (58, 61).  
 
2.4.3  
Summary of Feasibility of prophylactic DLI in T-cell depleted SCT. 
In the Schmid study after a non-T-cell depleted regimen, only 12 of 75 (16%) received a 
DLI post-transplant around day 120 because of high rates of aGVHD before planned DLI 
(59). De Lima showed that 6/12 (50%) could receive pDLI planned at days +30, +60 and 
+90 (57).   
 
Minimizing aGVHD after T-cell depletion appears to increase the feasibility of giving 
pDLI. In the setting of TCD myeloablative SCT, Montero et al (55) demonstrated that 112 
out of 138 patients (81%) were able to receive prophylactic DLI between days 45 and 100. 
Several other reports of T-cell depleted regimens demonstrated that 38% to 61% of patients 
received scheduled pDLI (51,53-54,56,60). No studies have evaluated high-risk patients 
alone or followed our unique backbone of cyto-reduction prior to transplant conditioning 
incorporating T-cell depletion.  
 
2.4.4  
Rationale for repetitive DLI. 
A recent study demonstrated that patients with acute myelogenous leukemia or chronic 
myelogenous leukemia in remission following SCT exhibited significant numbers of 
peripheral blood CD8+ T cells that recognized varying combinations of epitopes derived 

12 
 
from leukemia-associated antigens. However, these cells failed to proliferate, release 
cytokines, or de-granulate in response to antigen-specific stimuli.  The use of IL-15 or 
high-dose IL-2, elimination of CD4+ regulatory T cells, and blockade of PD-L all failed to 
rescue responsiveness of these CD8+ T cells in  in vitro assays. Rather, the mechanism for 
CD8+ unresponsiveness after SCT seemed to be replicative senescence (62).  
 
There are multiple sources of chronic stimulation following SCT that may contribute to 
potential T-cell senescence, including GVHD, GVL activity, infection, persistent 
stimulation of T cells by residual leukemia cells, and slow reconstitution of CD4+ T cells 
after SCT during the homeostatic proliferation to repopulate the T-cell pool. On the other 
hand, population dynamics of the T-cell pool after transplant may be influenced by the 
intensity of the conditioning regimen before transplant, T-cell dose within the graft, 
immunosuppressive therapy, and the use of donor lymphocyte infusions (62). 
 
If donor T cells become tolerant or possibly rapidly senescent after SCT as a mechanism 
leading to relapse, in order to preserve and maintain a competent pool of CD8+ T-cell 
precursors after allogeneic HSCT, the use of repetitive DLI once a patient achieves 
remission may be useful.  
 
2.5  Rationale to conduct prophylactic dose-escalation DLI to prevent relapse in 
hematologic malignancies after T cell depleted allo-SCT.  
Since relapse becomes a major issue in non-myeloablative T cell depleted allo-SCT, and 
DLI is a clinically available and established treatment that is most effective for minimal 
residual disease, the role of prophylactic DLI for patients in remission to prevent relapse 
will be assessed in a single arm Phase II study. The study will focus on patients with high 
risk hematologic malignancies that roughly correlate to having leukemia not in remission, 
lymphoma not achieving a partial response, or other disease under poor control undergoing 
allo-SCT. We reviewed the University of Chicago transplant database for patients with 
high-risk AML or MDS who underwent a T-cell depleted (TCD) allogeneic transplant. Of 
the 145 patients with AML or MDS transplanted with active disease, relapse occurred at 
the median of 128 days (24 to 2364 days), with PFS of 137 days (4 to 3272 days) and OS 
of 
214 
days 
(4-3434 
days) 
in 
the 
fludarabine/melphalan/Alentuzumab 
and 
clofarabine/melphalan/Alentuzumab conditioning regimens (unpublished data). From our 
published data, using fludarabine/melphalan/Alentuzumab conditioning regimen, patients 
with high-risk disease had a 39% probability of disease recurrence, a 39% probability of 
treatment-related mortality, and a 25% probability of progression-free survival at 1 year 
after stem cell transplant (63).  In our clofarabine/melphalan/Alentuzumab protocol (11), 
35 out of the total 72 patients accrued had high risk, active disease, and had 1 year PFS of 
31% which is similar to the 25% 1 year PFS using fludarabine/melphalan/Alentuzumab, 
and 1 year relapse rate of 29% for the whole cohort, making the 1 year relapse rate for the 
high 
risk 
patients 
comparable 
to 
1 
year 
relapse 
rate 
of 
39% 
from 
fludarabine/melphalan/Alentuzumab. The cumulative probability of Grade II-IV acute 
GVHD was 33% in flu/mel/campath and 22% in clo/mel/campath study at 1 years, and 1 
year TRM was 33% and 26% respectively (11, 63). Published data using fludarabine+ 
bulsufan+ alentuzumab or ATG had comparable results in high risk hematologic 
malignancies (64, 65).  
 

13 
 
We have opted against a randomized trial for several reasons. This novel approach requires 
feasibility testing of the entire process including enrolling high-risk patients, early 
withdrawal of immune suppression and the ability to escalate DLIs.   
 
3. PATIENT SELECTION 
 
3.1. Inclusion Criteria prior to transplant 
1. Age 14 – 75 years 
2.  The clinical trial will be offered to all high risk (defined in 3below) patients with 
hematologic malignancies who require stem cell transplants as part of their standard 
of care using matched related or unrelated donors. 
3. Patients with high risk myeloid or lymphoid malignancies at stem cell transplant 
following 
ASBMT 
criteria  
(http://www.asbmt.org/displaycommon.cfm?an=1&subarticlenbr=35, under disease 
classification), including but not limited to conditions listed. These criteria apply 
BEFORE cyto-reductive therapy given within 28 days of planned conditioning: 
  
 Refractory acute myelogenous or lymphoid leukemia 
 
 
 
Relapsed acute myelogenous or lymphoid leukemia  
 
 
 
Myelodysplastic syndromes with 5% or more blasts  
Chronic myelogenous leukemia in chronic phase 3 or more, blast phase presently, 
or second accelerated phase,  
 Recurrent or refractory malignant lymphoma or Hodgkin’s disease with less than 
a partial response at transplant  
 High risk chronic lymphocytic leukemia defined as no response or stable disease 
to the most recent treatment regimen. 
 
Diseases in response or remission at high risk of relapse at the discretion of the 
attending physician: Some examples include but are not limited to: 
AML in remission with monosomy 5 or 7, deletion of 5q or 7q, 11q23 MLL 
rearrangement or complex karyotype (>= 3 chromosome abnormalities) 
NHL in response that is double hit or triple hit (which are characterized by a 
recurrent chromosome translocation in combination with a MYC/8q24 breakpoint. 
These include but not limited to BCL2+/MYC+; BCL6+/MYC+; CCND1+/MYC+; 
and BCL2+/BCL6+/MYC+) 
bi-phenotypic lineage leukemia 
CLL with 17p deletion 
ALL with t (4,11) et al. 
4. Donors: Matched related or unrelated donor SCT matched at HLA A- B, C, and 
DRB1 by molecular methods. 7 of 8 matched donor acceptable for related donors.  
5. T-cell depletion with ATG (rabbit or horse) or at least 30 mg of Alemtuzumab total in 
the conditioning regimen.  Acceptable conditioning regimens include but not limited 
to fludarabine/melphalan/Alemtuzumab; fludarabine/busulfan/Alemtuzumab; 
fludarabine/melphalan/ATG; fludarabine/busulfan/ATG.    
6. Immune suppression. Planned post-transplant immune suppression should include 
tacrolimus or cyclosporin monotherapy (i.e., calcineurin inhibitor or CN) for 
alemtuzumab regimens and a second immune suppressant for ATG treated patients. 
Other agents may be used if CN intolerance or toxicity occurs post-transplant. 

14 
 
7. Zubrod PS 0-2 or equivalent Karnofsky PS  
8. Eligible for allogeneic transplant in the treating physicians’ judgment and by 
institutional standards.   
 
3.2. Exclusion Criteria prior to Transplant 
1. 
Pregnant or lactating females 
2. 
Hepatitis B with positive viral load prior to transplant conditioning or Hepatitis C 
virus  
3. 
Human immune deficiency virus 
4. 
Psychiatric illness that may make compliance to the clinical protocol unmanageable 
or may compromise the ability of the patient to give informed consent 
5. 
Poor organ function (deviations from the following criteria are allowable only with 
the PIs assent as the risks and benefits must be addressed for patients with incurable 
hematologic malignancies):  
a. Creatinine >/= 2.0 mg/dL 
b. SGOT and SGPT >/= 5 x ULN. Liver biopsy preferred for such patients. 
c. Bilirubin >/= 3 x ULN (unless Gilbert’s syndrome) 
d. DLCO < 50% corrected for hemoglobin 
e. Left ventricular ejection fraction < 40% or equivalent shortening fraction 
<20% in pediatric patients 
6. 
Unlikely to be able to procure additional donor lymphocytes  
 
3.3. Eligibility to receive DLI post-transplant (patients will be followed on protocol even 
if DLI not given) 
  
It is recognized that only some subjects will undergo DLI 
1. 
Donor lymphocytes available or able to be collected 
2. 
No evidence of disease by standard morphology. Minimal residual disease or 
molecular evidence of disease will not exclude.   
3. 
Adequate hematopoietic, renal, and hepatic function at first dose of DLI, defined as: 
 Absolute neutrophil count ≥500/μl 
 Platelet count ≥20,000/μl without transfusion for 7 days. 
 SGOT and SGPT ≤5 x ULN 
 Bilirubin ≤3 x ULN 
4. 
No evidence of Grade II or higher acute GVHD or chronic GVHD at initiation of first 
DLI. 
5. 
No systemic corticosteroids or immunosuppressive drugs (topical acceptable). 
Replacement steroids for adrenal insufficiency are not excluded.  
 
3.4. 
Human Subjects and Inclusion of Women and Minorities 
 
This clinical protocol involves the participation of human patients with hematologic 
malignancies after stem cell transplant to prevent relapse.  Human subjects will be admitted 
to the protocol on a first-come, first-serve basis, provided eligibility criteria are satisfied.  
Subjects will be of either sex and of any race.  Pediatric patients with hematologic 
malignancies undergoing SCT who meets the inclusion criteria could be included in this 
protocol.  

15 
 
4. REGISTRATION PROCEDURES AND ASSIGNMENT 
 
A. The research nurse will ensure that eligibility testing is arranged and the patient is 
registered with the data management office if the patient is eligible for treatment.  
B. A signed informed consent form must be on file before a patient can be registered.  
C. Patient eligibility and the existence of a signed consent form will be checked by data 
management personnel.   
D. Treatment on this protocol must occur at the University of Chicago.    
 
5. TREATMENT PLAN 
 
5.1. Study Design  
 
A. This is an open-label, single arm Phase II study aiming to reduce relapse in extremely 
high risk hematologic malignancies after matched donor stem cell transplant with T cell 
depleted conditioning regimen by early withdrawal of immune suppression followed by 
prophylactic DLI.  Eligible patients will be consented for this study at the time they are 
consented for the allogeneic stem cell transplant. 
 
B. The matched related and unrelated donors will be evaluated and cleared in the routine 
standard fashion and are consented to collection per standard care.  
 
 
Matched related donors (MRD): Matched donors will be evaluated and collected as per 
routine standard procedures. The donor will not be consented to this protocol because 
there is no research procedure performed on donors, and collection of steady state 
lymphocytes before and/or after transplantation is routine.  Clinical consent for collection 
is required. We will request donors to give steady-state donor lymphocytes prior to stem 
cell collection. This involves a short-apheresis procedure usually collecting 10 -12 liters 
of blood. After that, mobilization in standard fashion is started with G-CSF (Neupogen) 
for 4 days with stem cell collection by leukapheresis on day 5 and/or 6 of a large volume 
(e.g., 16-24 liters depending on donor weight). Alternatives to pre-mobilization collection 
include steady-state collection of the donor later closer to the planned DLI infusion or 
using a fraction of the mobilized stem cell product for DLI.  The composition of the DLI 
differs when obtained from a G-CSF mobilized product, although it is not clear if 
outcomes differ. Therefore, steady-state (i.e, non-mobilized) lymphocytes are preferred 
but not mandatory. The steady state donor lymphocytes from the matched donor will be 
collected and cryo-preserved as needed at our Stem cell lab. They will be enumerated by 
CD3 count and stored in appropriate aliquots. Typically, one steady state collection will 
provide an adequate number of CD3 lymphocytes for all the planned DLIs. 
 
Matched unrelated donors (MUD): Steady state collection will likely require approval 
of the National Marrow Donor Program (NMDP), the organization that oversees 
collection of unrelated donor products.  Ultimately, the decision on when to obtain 
lymphocytes will be determined by the NMDP, the donor, and the donor center. We will 
request, steady-state lymphocytes from matched unrelated donor to be collected and 
shipped to the University of Chicago before the first scheduled DLI infusion and the DLI 
will be cryo-preserved as above. As for the related donors, alternative options are using a 

16 
 
portion of the mobilized stem cell product for DLI or steady-state collection of 
lymphocytes later after transplant. These methods do not require approval in advance. 
However, logistical issues may present a barrier to using the mobilized stem cells for DLI 
as this requires that the MUD product has an adequate stem cell dose by CD34 cells/kg 
and enough time to obtain the results to cryo-preserve a portion of the product. This 
protocol will be submitted to the NMDP for approval for the collection of lymphocytes 
from matched unrelated donors. 
 
C. Treatment:  
 
Immune suppression withdrawal: Rapid taper of immune suppression will begin from 
day 60 to 75. It is recommended to reduce the dose 25-50% every 5 days over 10-20 days, 
then discontinue. 
 
Prior to each DLI: 
1. Determine peripheral blood chimerism 
2. Complete blood count and comprehensive metabolic panel 
3. Assess for acute and chronic GVHD. We expect delays especially for people with 
emerging or resolving GVHD to undergo further evaluation. 
4. Three month disease restaging will be done prior to first DLI.  Should the results be 
unavailable, DLI may be given as planned. 
 
DLI after Matched Related Donor (MRD) by recipient weight:   
 
Number 
Dose (CD3/kg) 
1-MRD 
2 x 105 
2-MRD 
5 x 105 
3-MRD 
1 x 106 
4-MRD 
2 x 106 
5-MRD 
5 x 106 
 
 
DLI after Matched Unrelated Donor (MUD) by recipient weight:   
Number 
Dose (CD3/kg) 
1-MUD 
1x 105 
2-MUD 
2 x 105 
3-MUD 
5 x 105 
4-MUD 
1 x 106 
5-MUD 
2 x 106 
 
Patients will get dose escalation DLI at 4 - 8 weeks intervals if there is no grade II-IV 
aGVHD, no chronic GVHD and patient is not receiving system immune suppression. 
Topical GVHD therapy is acceptable, including topical GI therapy (e.g., budesonide) with 
a goal of every 4 weeks. The goal is infuse as close to every 4 weeks as possible. The DLI 
will be conducted in routine fashion. This is typically an outpatient procedure but can be 
done as an inpatient if needed.  
 

17 
 
Timeline of study:  
 
Procedure 
Approximate 
time 
point after transplant  
Interval  before  next 
procedure 
Withdrawal  of  immune  suppression 
(e.g., tacrolimus or cyclosporine) 
60 days 
1 – 3 weeks 
Donor lymphocyte infusion (DLI) 1  
  (Sibling donors 2x 105 CD3 /kg 
  Unrelated donors‐1x 105 CD3 /kg) 
80 days 
4 – 8 weeks 
DLI 2 (Sibling donors‐ 5x 105 CD3 /kg 
  Unrelated donors‐2x 105 CD3 /kg) 
120 days 
4 – 8 weeks 
DLI 3 (Sibling donors‐ 1x 106 CD3 /kg 
  Unrelated donors‐5x 105 CD3 /kg) 
150 days 
4 – 8 weeks 
DLI 4 (Sibling donors‐ 2x 106 CD3 /kg 
  Unrelated donors‐1x 106 CD3 /kg) 
180 days 
4 – 8 weeks 
DLI 5 (Sibling donors‐ 5x 106 CD3 /kg 
  Unrelated donors‐2x 106 CD3 /kg) 
210 days 
4 – 8 weeks 
 
 
After DLI:  
1. Clinic visit the day of or within 3 days of planned DLI to assess as above (prior to each 
DLI).  
2. CBC and comprehensive metabolic panel are recommended every 2 weeks after DLI 
for at least 6 weeks after the last DLI infusion  
3. Long-term follow-up of disease will follow standard clinical practice. Our 
recommended approach has been to repeat disease staging (bone marrow for leukemias 
and imaging for lymphomas) around day 100, day 180, 1 year and as indicated after 
stem cell infusion).   
 
D. Number of Patients: 80 
 
E. Accrual: Target accrual will be 1-2 patients every 4 weeks (12-24 patients/year).   
 
5.2. Duration of Therapy 
 
In the absence of treatment delays due to adverse event(s), treatment may continue for five 
dose-escalating doses in 5-10 months or until one of the following criteria applies: 
 Disease relapse 
 Inter-current illness that prevents further administration of treatment 
 Grade III-IV acute GVHD 
 Grade II acute GVHD lasting more than 8 weeks after last DLI treatment or chronic 
GVHD. Patients could be continued on the study if GVHD resolves within 8 weeks 
from the last DLI infusion off steroid unless only for adrenal insufficiency.  
 Patient decides to withdraw from the study, or 

18 
 
 General or specific changes in the patient’s condition render the patient unacceptable 
for further treatment in the judgment of the investigator. 
 
5.3. Duration of Follow Up 
 
The patients will be followed according to the current post stem cell transplant guidelines.  
For this study, the enrolled patients will be followed for 2 years after stem cell infusion, 
removal from study or until death, whichever occurs first.  Patients removed from study for 
unacceptable adverse events will be followed until resolution or stabilization of the adverse 
event. 
 
5.4. Criteria for Removal from Study 
 
Patients will be removed from study when any of the criteria listed in Section 5.2 applies. 
Patients also may be removed from the study for patient non-compliance, the development 
of a severe medical condition unrelated to their hematologic malignancies or this treatment, 
or a decision from the investigator to discontinue the study. The reason for study removal 
and the date the patient was removed will be documented in the Case Report Form.  
 
5.5. Protocol Management Plan: 
 
For the primary analysis, an intention to treat principle will apply such that all patients 
consenting will be analyzed. Secondary analysis will include only those patients for whom 
immune suppression withdrawal was started and DLI planned.  
 
6. DOSING DELAYS/DOSE MODIFICATIONS 
 
The National Cancer Institute Common Toxicity Criteria Scale (version 4.0) will be used to 
grade toxicities after each DLI infusion.  
All Grade 3 and 4 toxicities, require delaying but not necessarily discontinuing treatment.  
If toxicity occurs, DLI dose can be delayed up to 4 weeks until side effect abates to within 
eligibility criteria. If a patient requires more than 4 weeks delay due to toxicities, then the 
patient will be removed from the study.   
 
Evaluation of acute GVHD (aGVHD) on the day of scheduled DLI infusion  
(4 weeks after last DLI) 
No aGVHD 
Proceed to scheduled next level dose of DLI  
Grade I aGVHD 
Proceed to scheduled next level dose of DLI 
Grade II aGVHD 
Delay DLI, re-evaluate after 4 week. 
 
1. GVHD resolves off steroid or systemic immune 
suppression; proceed to next level DLI. 
2.  Still has Grade II aGVHD or higher; off study 
 
Grade III-IV aGVHD 
Off study 
Active Chronic GVHD 
Off study 

19 
 
7. ADVERSE EVENTS MONITORING AND REPORTING:   
 
7.1. Toxicity Monitoring  
 
Patients will be monitored and questioned at every outpatient visit (see test schedule) 
regarding the occurrence and nature of any adverse experiences. An event is defined as any 
change in the physiologic or psychological state other than the primary condition that 
qualifies the patient for the study. More frequent monitoring will be performed in the case 
that an adverse event is noted.  
 
Allogeneic transplant has substantial expected toxicities which are higher for patients 
entering transplant with active disease. The transplant approach prior to immune 
suppression withdrawal and DLI is standard of care and does not require formal 
comparison or stopping rules. The main toxicity related to this approach is acute and 
chronic GVHD.  We will consider terminating the study based on a high-rate of severe (i.e., 
grade III-IV) aGVHD. One must recognize aGVHD takes 4-8 weeks after each DLI to 
develop; one must have approximately 6 months after starting immune withdrawal and DLI 
to determine if severe aGVHD will develop. 
 
7.2. Toxicity Reporting 
 
Toxicities are common following transplant. Therefore, unexpected, grades 3-5 adverse 
events (AEs) will be reported to the UCCCC CCTO Quality Assurance (QA) Coordinator 
by the end of the business day when s/he becomes aware of the event.  Events occurring 
after business hours will be reported to the CCTO by 12pm (noon) the next business day.  
Each event report must indicate where the event meets the IRB’s Unanticipated Problem 
reporting criteria. 
 
The National Cancer Institute Common Toxicity Criteria Scale (version 4.0) will be used to 
grade toxicities. 
  
For this study, early withdrawal of immune suppression (around day 60) and prophylactic 
donor lymphocyte infusions are experimental. The conditioning regimen and transplant 
itself is not experimental.  Adverse event monitoring should occur until 100 days past 
either of these procedures. Deaths will be reported for at least 200 days after transplant or 
100 days past any study procedure, whichever is later. 
 
7.3. Definitions of Adverse Events (AEs) and Serious Adverse Events (SAEs) 
 
Adverse Event - Any untoward medical occurrence in a patient or clinical investigation 
subject administered a pharmaceutical product, medical treatment or procedure and 
which does not necessarily have to have a causal relationship with this treatment. An 
adverse event can therefore be any unfavorable and unintended sign (including an 
abnormal laboratory finding, for example), symptom, or disease temporally associated 
with the use of a medicinal product, medical treatment or procedure whether or not 
considered related to the medicinal product.  
 

20 
 
Life-threatening Adverse Event – Any adverse event that places the patient or subject, 
in view of the investigator, at immediate risk of death from the reaction.  
 
Unexpected Adverse Event – An adverse event is “unexpected” when its nature 
(specificity), severity, or frequency are not consistent with (a) the known or foreseeable 
risk of adverse events associated with the research procedures described in the protocol-
related documents, such as the IRB-approved research protocol, informed consent 
document and other relevant sources of information such as product labeling and 
package inserts; and are also not consistent with (b) the characteristics of the subject 
population being studied including the expected natural progression of any underlying 
disease, disorder or condition any predisposing risk factor profile for the adverse event. 
 
Expected Adverse Event – Any adverse experience, event, incident, interaction or 
outcome that is identified in nature, severity or frequency in the protocol-related 
documents, such as the IRB-approved research protocol, informed consent document 
and other relevant sources of information such as product labeling and package inserts is 
considered an expected adverse event.  Any event that is previously known or 
anticipated to result from the underlying disease, disorder, or condition of the human 
subject or the study population may also be considered an expected adverse event. 
 
Serious Adverse Events – An adverse event that results in any of the following 
outcomes:  
 
• 
Death,  
• 
Life-threatening adverse experience,  
• 
*Inpatient hospitalization or prolongation of existing hospitalization,  
• 
Persistent or significant disability/incapacity or, 
• 
Congenital anomaly/birth defect. 
• 
Is medically significant or requires medical or surgical intervention to 
prevent one of the outcomes listed above 
 
* The initial hospitalization for transplant procedures or planned hospitalizations after 
transplant will not be considered an SAE unless the duration of the hospital stay is 
prolonged beyond what is expected as part of routine care.  During the expected initial 
hospitalization, if toxicities that are not routinely expected after transplant are observed 
(examples listed in the table below), they will be reported as an SAE. Transplant 
procedures are expected to result in hospitalization for 10-20 days after stem cell 
infusion, depending on the stem cell source and patient health.  All other hospitalizations 
unless planned would qualify as an SAE per FDA definitions.   
 
Many toxicities that are grade 3 by CTC are expected and routine for transplant. 
Select Examples 
 
Expected Grade 3 -4 Toxicities for 
Transplant (NOT SAE) 
Not Routinely Expected Grade 3-4 for 
Transplant(SAE) 
Fever after engraftment without a source 
requiring several additional hospital days 
Intensive care unit admission 

21 
 
Fever/infection requiring IV antibiotics 
during neutropenia or related to cathether 
Infection requiring a major surgical 
procedure 
Confusion requiring additional monitoring 
in the room 
Seizure 
Atrial fibrillation or flutter or arrhythmias 
monitoring on the transplant floor 
Arrhythmia requiring monitoring outside 
of the transplant unit, a pacemaker, or 
cardioversion 
Electrolyte disturbances requiring IV 
repletion 
VOD 
Poor nutrition requiring parenteral or 
enteral nutrition (with myeloablative 
regimens) 
 
Acute or chronic GVHD 
 
 
REPORTING GUIDELINES FOR ADVERSE EVENTS . 
 
 
Attribution – The determination of whether an adverse event is related to a medical treatment or 
procedure. Treatment is defined as protocol-defined preparative regimen, GVHD prophylaxis, 
and/or transplant procedure. 
 
Attribution categories: 
Definite – The adverse event is clearly related to the study 
drug/device/procedure/treatment(s). 
 
Probable – The adverse event is likely related to the study          
drug/device/procedure/treatment(s). 
 
Possible – The adverse event may be related to the study          
                   drug/device/procedure/treatment(s). 
 
Unlikely – The adverse event is doubtfully related to the study          
                   drug/device/procedure/treatment(s). 
 
Unrelated – The adverse event is clearly not related to the study          
                       drug/device/procedure/treatment(s). 
Unexpected Event 
Expected Event  
Grades 1 and 2  
Grades 3-5 
Grades 1 - 2 
Grades 3-4 
Grade 5 
Report in Velos 
as AEs.  
Report in Velos 
within 24 hours 
of being notified 
of the event. 
Report to IRB if 
meet reporting 
criteria.  
Not reported 
Reported in 
Velos as an AE.   
No SAE report 
in  Velos or 
IRB is needed.     
Report in 
Velos within 
24 hours of 
being notified 
of event. 

22 
 
Toxicity  
Continuous monitoring for severe and unexpected adverse events will occur and regular 
time-points may be included per protocol. 
 
ALL Serious Adverse Events, whether or not they are considered related to the study 
agent MUST be reported to the sponsor-investigator and to the University of Chicago 
Comprehensive Cancer Center (UCCCC).  Refer to Section 7.4.1 for reporting 
guidelines.  
 
7.4. Other Reporting Requirements 
 
7.4.1. University of Chicago Comprehensive Cancer Center (UCCCC) 
 
 All serious adverse events (as defined in Section 0) and protocol deviations must also be 
reported to the UCCCC Cancer Clinical Trials Office (CCTO) in accordance with the 
UCCCC Data Safety Monitoring Plan.   
 
The Research Nurse or other designated individual should report the SAE/deviation to the 
UCCCC CCTO Quality Assurance (QA) Coordinator by the end of the business day when 
s/he becomes aware of the event.  Events occurring after business hours will be reported to 
the CCTO by 12pm (noon) the next business day.  Each event report must indicate where 
the event meets the IRB’s Unanticipated Problem reporting criteria. 
 
7.4.2. Institutional Review Board (IRB) 
 
Events meeting current IRB reporting criteria must be submitted by the principal 
investigator via the IRB’s electronic submission system within the IRB’s designated 
reporting timeframes.  Details of the IRB’s current reporting policy and timelines can be 
found on their website at: http://bsdirb.bsd.uchicago.edu/forms-guidelines/up.html. 
 
The responsible research nurse and/or clinical research associate/data manager are 
responsible for entering the appropriate information into the IRB’s electronic submission 
system and forwarding the submission to the principal investigator for reporting to the IRB. 
 
7.5. Supportive Therapy 
 
Symptomatic care may be given as required with medications such as anti-emetics and 
analgesics.  
 
GVHD should be clinically suspected when a patient develops skin rash, diarrhea, and liver 
abnormalities at any time point after DLI.  Diagnosis should be confirmed by skin biopsy, 
colonoscopy and biopsy of an involved area to confirm a pathologic diagnosis and to rule 
out other potential causes of the diarrhea; and potential liver biopsy.  Should a diagnosis of 
GVHD be made or suspected, supportive care and non-systemic therapies are favored first. 
This includes topical steroid for localized skin GVHD, symptomatic anti-diarrheal agents, 
i. e., loperamide, for diarrhea. Patients with grade 2 to 4 will have standard GVHD 
treatment according to our protocol with high dose steroids, usually solumedrol at 2mg/kg.   

23 
 
Liver GVHD should be suspected when a patient develops an elevation of transaminases 
with or without an elevation in the bilirubin level. A hepatology consult and liver biopsy 
should be obtained to confirm a pathologic diagnosis whenever possible. 
 
8. CORRELATIVE/SPECIAL STUDIES 
 
In addition to routine clinical tests, we will collect additional samples for possible 
correlative studies.  
 
For the serum Alentuzumab level testing, we will obtain 5-10ml serum/plasma at day 60 at 
the time of immunosuppression withdrawal and at around day 90 or on the day of  first DLI 
to evaluate serum Alemtuzumab levels according to the published methods (66, 67). The 
samples will be 10 cc peripheral blood in Red-top tube, and the serum will be stored in our 
stem cell core facility. 
 
In addition to the standard immune reconstitution studies including lymphocyte subset 
panel 3 and quantitative immunoglobulin levels, an additional 10 cc heparinized (Green 
Top) peripheral blood will be will be collected for detection of regulatory T cell panels and 
potentially 
other 
immune 
markers 
(e.g., 
T-cell 
repertoire) 
at 
the 
time 
of 
immunosuppression withdrawal, prior to first, the third, and the fifth DLI, and 1 year, 18 
months and 2 years after stem cell transplant.  
 
For MRD detection in myeloid leukemia, CLL and lymphoma patients with bone marrow 
involvement, bone marrow aspiration study material will be obtained from the bone 
marrow biopsy prior to stem cell transplant to be used as the baseline sample, since all the 
patients will have active disease before stem cell transplant. For lymphoma patients without 
bone marrow involvement, we will retrospectively obtain lymph node biopsy samples at 
the diagnosis for baseline samples. The follow up MRD samples will be obtained from 
either bone marrow aspiration if available or peripheral blood at the time of 
immunosuppression withdrawal, prior to first, the third, and the fifth DLI, and 1 year, 18 
months and 2 years after stem cell transplant, or at the off study. The samples will be stored 
in our stem cell core facility.  
 
We will store 10-20cc blood at the time of immunosuppression withdrawal, prior to first, 
the third, and the fifth DLI, and 1 year, 18 months and 2 years after stem cell transplant for 
future potential studies.  
 
The following tests are a list of correlative studies most likely to be performed should 
funding and samples permit. 
 
A. Flow cytometry for Treg cells panels. PBMC will be analyzed by flow cytometry 
using combinations of anti-CD4, and CD25, and anti-FoxP3 or CD127 monoclonal 
antibodies to identify the regulatory T cell subpopulation on permeablized cells.  By 
combining the percent-positive cells with the coulter counter data, an absolute number 
of CD4+CD25+ FoxP3+ or CD4+CD25+CD127dim/- cells will be determined. We 
expect these will be done at our Human Immunology Monitoring Facility at University 
of Chicago. 

24 
 
 
B. Measurement of Alemtuzumab level by ELISA. There is no commercially available 
test for the Alemtuzumab level in serum or plasma. A group in MD Anderson 
developed an easy ELISA test (66, 67). Affinity-purified rabbit anti-rat IgG absorbed 
with human IgG will be used to coat flat-button 96-well microtitre plates. After the 
plates are blocked using BSA (2%), patient samples will be added in duplicate and 
incubated. Then, peroxidase-conjugated affinity-purified rabbit anti-human-Fc will be 
added followed by adding substrate to develop color reaction. After 4–8 minutes, the 
reaction will be stopped and plates will be red at 450 nm, and a log reading of samples 
against control will be calculated. 
 
C. Minimal Residual Disease Monitoring: The presence of minimal residual disease in 
myeloid leukemia or MDS will be assessed by monitoring of WT1 transcript levels in 
blood or bone marrow using a quantitative RT-PCR assay. Briefly, total RNA will be 
extracted from blood and bone marrow and cDNA synthesized using standard 
techniques. Amplifications of patient samples, K562 cell line cDNA, and no template 
controls will be performed in triplicate. WT-1 expression levels will be detected using a 
transcript specific primer and probe set. In order to compensate for differences in RNA 
integrity and cDNA synthesis efficiency, the absolute WT1 transcript copy number will 
be normalized to the endogenous control gene Abl. For the lymphoid diseases, MRD of 
CLL will be detected using four color flow cytometry (68), and MRD in NHL will be 
detected using quantitation of IgH or BCL-1/JH copy number using real-time 
polymerase chain reaction (69). 
 
D. Additional studies. There may be additional studies geared toward gaining a better 
understanding and predictors of disease relapse, GVHD, and immunologic activity of 
this strategy. 
 
9. CRITERIA FOR STUDY EVALUATION 
 
The proportion of patients receiving one pDLI, proportion receiving all pDLIs, PFS at 2 
year after stem cell transplant, highest Grade acute GVHD, relapse rate, and non-relapse 
mortality at 2 years after stem cell transplant are the relevant endpoints.   
 
Acute GVHD will be scored according to the criteria proposed by Przepiorka et al (70). 
Limited Chronic GVHD is defined as GVHD with limited skin involvement only or 
presenting with liver function abnormalities only. All other presentations of chronic GVHD 
are defined as extensive and will require treatment. The diagnosis and staging of chronic 
GVHD will be done according to the published NIH Consensus (71).  
 
Relapse will be recorded by the day of initial detection of malignant cells, if these cells 
were on subsequent testing confirmed to be increasing in number. The molecular detection 
of MRD will not be taken into account for the definition of clinical recurrence. The 
diagnosis of disease recurrence will be based on clinical and pathological criteria. 
 
 

25 
 
10. STUDY CALENDAR: 
The following calendar summarizes the required samples specifically for this study. 
Tests and Procedures 
Baseline
&  
 
IS 
withdrawal 
(~Day 60 
post SCT) 
+/- 7 days 
Before 
first DLI 
 (-10 to -1 
day) 
Day 0 
of 1st 
DLI 1 
(-7 to 0 
days) 
Day 0 
of 2rd 
DLI 2  
(-7 to 0 
days) 
Day 0 
of 3th 
DLI 3 
(-7 to 0 
days) 
Day 0 
of 4th 
DLI 4 
(-7 to 0 
days) 
Day 0 
of 5th 
DLI 5 
(-7 to 0 
days) 
One 
month 
after all 
DLI;  12, 
and 24 
months 
post-SCT 
or  At 
time off 
study 
(+/-7 
days) 
 
Standard tests 
 
 
 
 
 
 
 
 
 
History 
and 
exam 
(including vital signs 
and PS) 
X 
X 
 
X 
X 
X 
X 
X 
 
CBC, Plt, diff  
 
X 
 
X 
X 
X 
X 
X 
 
BMP, LDH, LFT 
 
X 
 
X 
X 
X 
X 
X 
 
GVHD Assessment 
 
X 
 
X 
X 
X 
X 
X 
 
Toxicities Assessment 
 
X 
 
X 
X 
X 
X 
X 
 
Disease restaging: Bone 
marrow biopsy and/or 
CT imaging with or 
without 
PET 
for 
lymphoma 
X 
 
X* 
 
 
X1 
 
X1 
X 
Cytogenetic analysis 
X 
 
X* 
 
 
X1 
 
X1 
X 
Chimerism (blood or 
Bone Marrow )  
 
 
X* 
Xb 
Xb 
Xb 
Xb 
Xb 
Xa,b 
Immune reconstitution  
 (lymphocyte 
subset 
panel  and Quantitative 
Igs levels)   
 
 
X 
 
X 
 
X 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
Research 
samples 
(optional but strongly 
recommended) 
 
 
 
 
 
 
 
 
 
Serum/plasma 
sample 
(10cc blood in Red top 
tube) 
X 
X 
 
X 
 
 
 
 
 
Regulatory T cell panel 
1/2#   
(10cc in Green top 
tube)3 
X 
X 
 
X 
 
X 
 
 
X2 
MRD testing  ( BM 
aspiration  or peripheral 
blood or 10cc) 3  
(Green top tube) $ 
Xb 
Xb 
X* 
Xb 
 
Xb 
 
 
X2,a,b 
Extra research sample 
for proteomics analysis 
(10cc blood in Green 
top tube) 
X 
X 
 
X 
X 
X 
X 
X 
X2 

26 
 
 
 
1  Only clinically indicated. No need to do if the date falls within 2 weeks window of the scheduled 
bone marrow aspiration and biopsy. 
2
 Only need one month after  DLI5, and at 1 year post-SCT or at the time of off study.  
3 Samples will be stored and tests will be done in the batch fashion at the end of the study. 
& Baseline samples will be done prior to cyto-reduction chemotherapy or prior to conditioning 
chemotherapy. 
* This will be done -1 to 10 days before the infusion of first DLI. This bone marrow biopsy is 
required. a Using samples from bone marrow biopsy. 
# Regulatory T cell panel 1: CD4+ CD25+ FoxP3+ 
  Regulatory T cell panel 2: CD4+ CD25+ CD127 dim/- 
$ 10cc bone marrow aspiration will be obtained from the Bone marrow biopsy prior to the cyto-
reduction chemotherapy or prior to conditioning chemotherapy. For lymphoma patients without 
bone marrow involvement, the original lymph node biopsy samples will be retrospectively retrieved 
for baseline sample for MRD detection. 
b.  From Peripheral blood. 
 
All the patients will get the standard follow-up after the stem cell transplant according to 
our established transplant guidelines, which includes biweekly clinical visit in first month 
after discharge from the hospital after SCT, weekly visit in first 3 months.  
 
Transplant patients will have bone marrow aspiration and biopsy at the following time 
points for cytogenetics analysis, chimerism per standard care. Peripheral blood samples 
will be obtained at the same time for immune re-constitution by checking lymphocyte 
subsets and quantitative immunoglobin levels. This is a recommended schedule. Variations 
to this schedule will not be considered a protocol deviation.  
 
Day 28 (± 1 wk) post-SCT 
Day 80-90 (± 1 wk) post-SCT before the first DLI infusion, if first DLI infusion will be 
delayed, a bone marrow biopsy is strongly recommended prior to the first DLI infusion. 
Day 180 (± 1 wk) post-SCT 
Day 365 (± 1 wk) post-SCT 
Day 730 (± 1 wk) post-SCT 
At relapse  
 
11. DATA REPORTING / REGULATORY CONSIDERATIONS 
 
A. The University of Chicago medical records will be utilized for all patients.  Data will be 
entered into a data management file within 3 weeks after each evaluation of the patient.  
After the patient goes off treatment, follow-up information will be collected and entered 
into the data management file every 3 months by telephone contact.  
 
B. Pathologic diagnosis and HLA typing will be recorded in a conventional way with a 
record being placed in the patient’s permanent record and data management file.  
However, investigational correlative assay results will not be made part of the medical 
record.  
 

27 
 
C. Data and safety monitoring for this trial will be carried out in accordance with the 
University of Chicago Comprehensive Cancer Center Data and Safety Monitoring (DSM) 
Plan.  Briefly, accrual, toxicity, and response data will be reviewed weekly at the 
transplant patient care conference. Adverse events will be reported to the principal 
investigator, IRB, as described in section 7.2.  Decisions will be made regarding study 
continuation, amendment, or closure at the weekly meeting and a note will be signed by 
the principal investigator documenting this decision.  External monitoring of accrual is 
performed by the Accrual Monitoring Committee.  The study will be independently 
audited annually by the Cancer Clinical Trials Office of the University of Chicago 
Comprehensive Cancer Center in accordance with DSM Plan.  
 
12. STATISTICAL CONSIDERATIONS 
 
12.1. Study Design 
Patients will be assigned to Matched related donor SCT group or Matched unrelated donor 
SCT group according to the type of donor for their stem cell transplant.  
 
12.2. Sample Size/Accrual Rate 
  
A total of 80 patients will be accrued with an accrual rate at 10-15 patients per year. 
 
12.3. Statistical Considerations  
 
Statistical analysis: 
We expect that this protocol will enroll approximately equal numbers of patients 
undergoing MRD or MUD SCT with more than 50% of patients from both groups 
combined will be able to receive at least one DLI treatment. We also intend to analyze the 
2 year PFS, OS, and rate of aGVHD among the patients who receive at least one DLI 
treatment. All the enrolled patients will be followed up at least 2 years from stem cell 
infusion as long as they are still alive. Baseline characteristics will be summarized using 
descriptive statistics.  Intention to treat analysis will be used to determine the percentage of 
patients who receive at least one DLI treatment. Progression-free survival (time to relapse 
or death as a result of any cause) and overall survival will be computed using the Kaplan-
Meier product-limit estimate and expressed as probabilities with a 95% CI.  Acute and 
chronic GVHD, and treatment-related mortality will be estimated by cumulative incidence 
method. Cumulative incidence of treatment-related mortality with relapse of the original 
disease as the competing risk will be calculated.  
Sample size: 
We will determine the number of subjects who undergo withdrawal of immune suppression 
and receive at least one DLI.  For this estimate, subjects who consent but do not proceed to 
transplant will be considered non-evaluable. Based on prior studies outlined in the 
background with heterogeneous disease risk and regimens, around 40-60% received pDLI 
after T-cell depleted regimens. We expect to increase the proportion receiving at least one 
pDLI to around 70% with around 15% having progressive disease after transplant and 
another 15% having GVHD or other clinical concerns that preclude DLI (e.g., infection, 
unwillingness to proceed, or lack of available lymphocytes). Thus we will test the null 

28 
 
hypothesis of a 50% pDLI rate against a 70% alternative.  To achieve 85% power with a 
two sided test with alpha set at 0.05 requires n= minimum 56 patients.   
 
Should feasibility be established, this regimen will also need to demonstrate adequate disease 
activity to be pursued further.  We will collect preliminary data on  the efficacy of this 
treatment strategy for very high risk disease as measured by improved progression-free 
survival (PFS) compared  to historical data in very similar patients (i.e, similar disease risk, 
similar regimens but no pDLI)  at our institution.  PFS was calculated for two major disease 
categories of AML and Non-Hodgkin Lymphoma treated on Alemtuzumab-based protocols 
who entered transplant with high-risk disease (not in CR for AML and not in CR or PR for 
NHL) from our historical institutional experience.   There are two populations in which one 
can calculate PFS. We can evaluate the entire cohort by intention to treat or an enriched 
cohort of only those who are eligible to begin early immune suppression. We prefer the 
latter as this tests the efficacy (or biologic activity) of early immune suppression 
withdrawal and DLI for high-risk diseases. It is unlikely that there will be bias in our 
comparison as the major criteria for removal--significant aGVHD and disease relapse are 
unlikely to be affected by the investigators or subjects. Alternative approaches are required 
to salvage patients with very early relapse or early significant GVHD.  
 
We determined outcomes for AML patients without GVHD and relapse at 80 days post 
stem cell transplant as these are the patients who would be eligible for early withdrawal of 
immune suppression and DLI. The 2 year PFS was 32.3% (95% CI 15.4% to 50.5%) for 
AML/MDS patients and 37.9% (95% CI 10.8% -65.4 %) for NHL patients. These are 
better than the outcomes of the entire cohort because of censoring as above. Outcomes for 
higher risk hematologic malignancies tend to be similar to each other (as expected as that is 
the inherent purpose of “high-risk” classification) thus justifying combining NHL, AML 
and other high risk diseases. Otherwise too few patients exist to generate a reliable 
historical control rate. Collapsing AML and NHL into one category, we have a null, two-
year PFS rate of 35% based on our historical controls.  
 
Stopping rules: 
We will monitor aGVHD regularly and have derived a formal stopping rule. After every 10 
patients have passed 6 months of follow up from immune suppression withdrawal, the 
incidence of aGVHD will be calculated.  Patients who relapse or develop significant 
aGVHD before tapering will not be counted. Overall rates in historical controls after T-cell 
depleted regimens with alemtuzumab of aGVHD have been very low, at around 15%, with 
even fewer cases of severe aGVHD.   As the regimen tested in this trial may reduce relapse 
at the expense of higher risks of aGVHD, we will allow a somewhat higher rate to occur. 
Specifically, if the lower limit of the 95% confidence interval exceeds 25% for severe 
(grade III-IV) GVHD, the study will halt accrual. Either protocol modification will occur or 
the study will be terminated. These modifications may include reduced DLI dose or 
number of infusions, delay of stopping immune suppression, or possibly continued immune 
suppression even after DLI.  
 
Numbers of severe aGVHD events for every 10 subjects that warrant protocol stopping or 
revision are: 
 

29 
 
Severe 
aGVHD 
events 
Enrolled 
Subjects 
 
Observed aGVHD 
(%) 
95% CI 
6 
10 
60% 
30.3-78.3 
9 
20 
45% 
26.0-65.3 
13 
30 
43% 
27.9-58.9 
16 
40 
40% 
26.9-54.22 
19 
50 
38% 
26.5-50.6 
However, high rates of aGVHD that do not meet the stopping rules may still warrant 
protocol modification at the discretion of the investigators. 
 
The pre-defined stopping rule was never met during the enrollment of the first 56 subjects 
defined by the original protocol. Thus, severe GVHD has been demonstrated to not be a 
concern for this study. The amended protocol will allow us to enroll more patients to the 
study for better efficacy evaluation. We expect to close the study after the requested 
number of 80 subjects has been reached. 
 
While it is acceptable to have significant and severe aGVHD for curative intent 
approaches, the major problem with high rates of severe aGVHD is that this would make 
this a poor platform to integrate additional therapies later as severe aGVHD often prevents 
further immune modulation. 
 
Chronic GVHD also remains of interest but can occur years after aGVHD and thus 
stopping rules based on this outcome cannot be formulated. We will further monitor the 
proportion undergoing early immune withdrawal with or without DLI continuously.  
 
13.  
FUTURE DIRECTIONS 
Depending on the results of this study, we will modify or design the future studies. The 
first possible modification may be to include leukemia patients with high-risk cytogenetics 
that are under disease control (remission) as outcomes are similar to those with active 
disease. We have excluded these patients initially as historically cytoreduction is not 
typically given prior to transplant since they do not have morphologic disease. If high early 
aGVHD is observed, we will continue immune suppression with DLI, since some data 
indicate DLI maintains a benefit even if infused with immune suppression. If high rate of 
acute GVHD occurs with escalated DLI doses, we will not escalate DLI or we will stop 
DLI after full donor chimerism for patients starting with mixed chimerism. If high rates of 
chronic GVHD are found, we will continue immune suppression during DLI or give 
cyclophosphamide after DLI. If there is no change in relapse rates after prophylactic DLI, 
we will either further escalate DLI doses or add disease specific agents or immune 
stimulation, such as basiliximab, or ipiluzumab.  
 
 
 
 
 
 
 

30 
 
 
Reference: 
1. 
Copelan, E.A. 2006. Hematopoietic stem-cell transplantation. N Engl J Med 354:1813-
1826. 
2. 
Gooley, T.A., Chien, J.W., Pergam, S.A., Hingorani, S., Sorror, M.L., Boeckh, M., 
Martin, P.J., Sandmaier, B.M., Marr, K.A., Appelbaum, F.R., et al. Reduced mortality 
after allogeneic hematopoietic-cell transplantation. N Engl J Med 363:2091-2101. 
3. 
Diaconescu, R., Flowers, C.R., Storer, B., Sorror, M.L., Maris, M.B., Maloney, D.G., 
Sandmaier, B.M., and Storb, R. 2004. Morbidity and mortality with nonmyeloablative 
compared with myeloablative conditioning before hematopoietic cell transplantation from 
HLA-matched related donors. Blood 104:1550-1558. 
4. 
Sorror, M.L., Sandmaier, B.M., Storer, B.E., Franke, G.N., Laport, G.G., Chauncey, T.R., 
Agura, E., Maziarz, R.T., Langston, A., Hari, P., et al. 2011. Long-term outcomes among 
older patients following nonmyeloablative conditioning and allogeneic hematopoietic cell 
transplantation for advanced hematologic malignancies. JAMA 306:1874-1883. 
5. 
van Besien, K., Kunavakkam, R., Rondon, G., De Lima, M., Artz, A., Oran, B., and 
Giralt, S. 2009. Fludarabine-melphalan conditioning for AML and MDS: alemtuzumab 
reduces acute and chronic GVHD without affecting long-term outcomes. Biol Blood 
Marrow Transplant 15:610-617. 
6. 
Soiffer, R.J., Lerademacher, J., Ho, V., Kan, F., Artz, A., Champlin, R.E., Devine, S., 
Isola, L., Lazarus, H.M., Marks, D.I., et al. 2011. Impact of immune modulation with 
anti-T-cell antibodies on the outcome of reduced-intensity allogeneic hematopoietic stem 
cell transplantation for hematologic malignancies. Blood 117:6963-6970. 
7. 
Finke, J., Bethge, W.A., Schmoor, C., Ottinger, H.D., Stelljes, M., Zander, A.R., Volin, 
L., Ruutu, T., Heim, D.A., Schwerdtfeger, R., et al. 2009. Standard graft-versus-host 
disease prophylaxis with or without anti-T-cell globulin in haematopoietic cell 
transplantation from matched unrelated donors: a randomised, open-label, multicentre 
phase 3 trial. Lancet Oncol 10:855-864. 
8. 
van den Brink, M.R., Porter, D.L., Giralt, S., Lu, S.X., Jenq, R.R., Hanash, A., and 
Bishop, M.R. Relapse after allogeneic hematopoietic cell therapy. Biol Blood Marrow 
Transplant 16:S138-145. 
9. 
Wingard, J.R., Majhail, N.S., Brazauskas, R., Wang, Z., Sobocinski, K.A., Jacobsohn, D., 
Sorror, M.L., Horowitz, M.M., Bolwell, B., Rizzo, J.D., et al. 2011. Long-term survival 
and late deaths after allogeneic hematopoietic cell transplantation. J Clin Oncol 29:2230-
2239. 
10. 
Kahl, C., Storer, B.E., Sandmaier, B.M., Mielcarek, M., Maris, M.B., Blume, K.G., 
Niederwieser, D., Chauncey, T.R., Forman, S.J., Agura, E., et al. 2007. Relapse risk in 
patients with malignant diseases given allogeneic hematopoietic cell transplantation after 
nonmyeloablative conditioning. Blood 110:2744-2748. 
11. 
van Besien, K., Stock, W., Rich, E., Odenike, O., Godley, L.A., O'Donnell, P.H., Kline, J., 
Nguyen, V., Cerro, P.D., Larson, R.A., et al. 2012. Phase I-II Study of Clofarabine-
Melphalan-Alemtuzumab 
Conditioning 
for 
Allogeneic 
Hematopoietic 
Cell 
Transplantation. Biol Blood Marrow Transplant. 
12. 
Elmaagacli, A.H., Beelen, D.W., Trenn, G., Schmidt, O., Nahler, M., and Schaefer, U.W. 
1999. Induction of a graft-versus-leukemia reaction by cyclosporin A withdrawal as 
immunotherapy for leukemia relapsing after allogeneic bone marrow transplantation. 
Bone Marrow Transplant 23:771-777. 

31 
 
13. 
Metzelder, S., Wang, Y., Wollmer, E., Wanzel, M., Teichler, S., Chaturvedi, A., Eilers, 
M., Enghofer, E., Neubauer, A., and Burchert, A. 2009. Compassionate use of sorafenib 
in FLT3-ITD-positive acute myeloid leukemia: sustained regression before and after 
allogeneic stem cell transplantation. Blood 113:6567-6571. 
14. 
Mielcarek, M., Storer, B.E., Flowers, M.E., Storb, R., Sandmaier, B.M., and Martin, P.J. 
2007. Outcomes among patients with recurrent high-risk hematologic malignancies after 
allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant 13:1160-
1168. 
15. 
Levine, J.E., Braun, T., Penza, S.L., Beatty, P., Cornetta, K., Martino, R., Drobyski, W.R., 
Barrett, A.J., Porter, D.L., Giralt, S., et al. 2002. Prospective trial of chemotherapy and 
donor leukocyte infusions for relapse of advanced myeloid malignancies after allogeneic 
stem-cell transplantation. J Clin Oncol 20:405-412. 
16. 
Choi, S.J., Lee, J.H., Kim, S., Seol, M., Lee, Y.S., Lee, J.S., Kim, W.K., Chi, H.S., and 
Lee, K.H. 2004. Treatment of relapsed acute myeloid leukemia after allogeneic bone 
marrow transplantation with chemotherapy followed by G-CSF-primed donor leukocyte 
infusion: a high incidence of isolated extramedullary relapse. Leukemia 18:1789-1797. 
17. 
Pollyea, D.A., Artz, A.S., Stock, W., Daugherty, C., Godley, L., Odenike, O.M., Rich, E., 
Smith, S.M., Zimmerman, T., Zhang, Y., et al. 2007. Outcomes of patients with AML 
and MDS who relapse or progress after reduced intensity allogeneic hematopoietic cell 
transplantation. Bone Marrow Transplant 40:1027-1032. 
18. 
Jabbour, E., Giralt, S., Kantarjian, H., Garcia-Manero, G., Jagasia, M., Kebriaei, P., de 
Padua, L., Shpall, E.J., Champlin, R., and de Lima, M. 2009. Low-dose azacitidine after 
allogeneic stem cell transplantation for acute leukemia. Cancer 115:1899-1905. 
19. 
Lubbert, M., Bertz, H., Wasch, R., Marks, R., Ruter, B., Claus, R., and Finke, J. 2009. 
Efficacy of a 3-day, low-dose treatment with 5-azacytidine followed by donor 
lymphocyte infusions in older patients with acute myeloid leukemia or chronic 
myelomonocytic leukemia relapsed after allografting. Bone Marrow Transplant 45:627-
632. 
20. 
Lubbert, M., Bertz, H., Ruter, B., Marks, R., Claus, R., Wasch, R., and Finke, J. 2009. 
Non-intensive treatment with low-dose 5-aza-2'-deoxycytidine (DAC) prior to allogeneic 
blood SCT of older MDS/AML patients. Bone Marrow Transplant 44:585-588. 
21. 
Eapen, M., Giralt, S.A., Horowitz, M.M., Klein, J.P., Wagner, J.E., Zhang, M.J., Tallman, 
M.S., Marks, D.I., Camitta, B.M., Champlin, R.E., et al. 2004. Second transplant for 
acute and chronic leukemia relapsing after first HLA-identical sibling transplant. Bone 
Marrow Transplant 34:721-727. 
22. 
Bosi, A., Laszlo, D., Labopin, M., Reffeirs, J., Michallet, M., Gluckman, E., Alessandrino, 
P.E., Locatelli, F., Vernant, J.P., Sierra, J., et al. 2001. Second allogeneic bone marrow 
transplantation in acute leukemia: results of a survey by the European Cooperative Group 
for Blood and Marrow Transplantation. J Clin Oncol 19:3675-3684. 
23. 
Porter, D.L., Levine, B.L., Bunin, N., Stadtmauer, E.A., Luger, S.M., Goldstein, S., Loren, 
A., Phillips, J., Nasta, S., Perl, A., et al. 2006. A phase 1 trial of donor lymphocyte 
infusions expanded and activated ex vivo via CD3/CD28 costimulation. Blood 107:1325-
1331. 
24. 
Falkenburg, J.H., Wafelman, A.R., Joosten, P., Smit, W.M., van Bergen, C.A., Bongaerts, 
R., Lurvink, E., van der Hoorn, M., Kluck, P., Landegent, J.E., et al. 1999. Complete 
remission of accelerated phase chronic myeloid leukemia by treatment with leukemia-
reactive cytotoxic T lymphocytes. Blood 94:1201-1208. 

32 
 
25. 
Cooper, L.J., Topp, M.S., Serrano, L.M., Gonzalez, S., Chang, W.C., Naranjo, A., Wright, 
C., Popplewell, L., Raubitschek, A., Forman, S.J., et al. 2003. T-cell clones can be 
rendered specific for CD19: toward the selective augmentation of the graft-versus-B-
lineage leukemia effect. Blood 101:1637-1644. 
26. 
Bashey, A., Medina, B., Corringham, S., Pasek, M., Carrier, E., Vrooman, L., Lowy, I., 
Solomon, S.R., Morris, L.E., Holland, H.K., et al. 2009. CTLA4 blockade with 
ipilimumab to treat relapse of malignancy after allogeneic hematopoietic cell 
transplantation. Blood 113:1581-1588. 
27. 
Rezvani, K., Yong, A.S., Mielke, S., Savani, B.N., Musse, L., Superata, J., Jafarpour, B., 
Boss, C., and Barrett, A.J. 2008. Leukemia-associated antigen-specific T-cell responses 
following combined PR1 and WT1 peptide vaccination in patients with myeloid 
malignancies. Blood 111:236-242. 
28. 
Van Tendeloo, V.F., Van de Velde, A., Van Driessche, A., Cools, N., Anguille, S., Ladell, 
K., Gostick, E., Vermeulen, K., Pieters, K., Nijs, G., et al. Induction of complete and 
molecular remissions in acute myeloid leukemia by Wilms' tumor 1 antigen-targeted 
dendritic cell vaccination. Proc Natl Acad Sci U S A 107:13824-13829. 
29. 
Deol, A., and Lum, L.G. 2010. Role of donor lymphocyte infusions in relapsed 
hematological malignancies after stem cell transplantation revisited. Cancer Treat Rev 
36:528-538. 
30. 
Loren, A.W., and Porter, D.L. 2006. Donor leukocyte infusions after unrelated donor 
hematopoietic stem cell transplantation. Curr Opin Oncol 18:107-114. 
31. 
Mackinnon, S., Papadopoulos, E.B., Carabasi, M.H., Reich, L., Collins, N.H., Boulad, F., 
Castro-Malaspina, H., Childs, B.H., Gillio, A.P., Kernan, N.A., et al. 1995. Adoptive 
immunotherapy evaluating escalating doses of donor leukocytes for relapse of chronic 
myeloid leukemia after bone marrow transplantation: separation of graft-versus-leukemia 
responses from graft-versus-host disease. Blood 86:1261-1268. 
32. 
Fozza, C., Szydlo, R.M., Abdel-Rehim, M.M., Nadal, E., Goldman, J.M., Apperley, J.F., 
and Dazzi, F. 2007. Factors for graft-versus-host disease after donor lymphocyte 
infusions with an escalating dose regimen: lack of association with cell dose. Br J 
Haematol 136:833-836. 
33. 
Raiola, A.M., Van Lint, M.T., Valbonesi, M., Lamparelli, T., Gualandi, F., Occhini, D., 
Bregante, S., di Grazia, C., Dominietto, A., Soracco, M., et al. 2003. Factors predicting 
response and graft-versus-host disease after donor lymphocyte infusions: a study on 593 
infusions. Bone Marrow Transplant 31:687-693. 
34. 
Tomblyn, M., and Lazarus, H.M. 2008. Donor lymphocyte infusions: the long and 
winding road: how should it be traveled? Bone Marrow Transplant 42:569-579. 
35. 
Kernan, N.A., Collins, N.H., Juliano, L., Cartagena, T., Dupont, B., and O'Reilly, R.J. 
1986. Clonable T lymphocytes in T cell-depleted bone marrow transplants correlate with 
development of graft-v-host disease. Blood 68:770-773. 
36. 
Dazzi, F., and Fozza, C. 2007. Disease relapse after haematopoietic stem cell 
transplantation: risk factors and treatment. Best Pract Res Clin Haematol 20:311-327. 
37. 
Collins, R.H., Jr., Shpilberg, O., Drobyski, W.R., Porter, D.L., Giralt, S., Champlin, R., 
Goodman, S.A., Wolff, S.N., Hu, W., Verfaillie, C., et al. 1997. Donor leukocyte 
infusions in 140 patients with relapsed malignancy after allogeneic bone marrow 
transplantation. J Clin Oncol 15:433-444. 
38. 
Dazzi, F., Szydlo, R.M., Craddock, C., Cross, N.C., Kaeda, J., Chase, A., Olavarria, E., 
van Rhee, F., Kanfer, E., Apperley, J.F., et al. 2000. Comparison of single-dose and 

33 
 
escalating-dose regimens of donor lymphocyte infusion for relapse after allografting for 
chronic myeloid leukemia. Blood 95:67-71. 
39. 
Porter, D.L., Collins, R.H., Jr., Hardy, C., Kernan, N.A., Drobyski, W.R., Giralt, S., 
Flowers, M.E., Casper, J., Leahey, A., Parker, P., et al. 2000. Treatment of relapsed 
leukemia after unrelated donor marrow transplantation with unrelated donor leukocyte 
infusions. Blood 95:1214-1221. 
40. 
Hartwig, U.F., Nonn, M., Khan, S., Link, I., Huber, C., and Herr, W. 2008. Depletion of 
alloreactive donor T lymphocytes by CD95-mediated activation-induced cell death 
retains antileukemic, antiviral, and immunoregulatory T cell immunity. Biol Blood 
Marrow Transplant 14:99-109. 
41. 
Wehler, T.C., Nonn, M., Brandt, B., Britten, C.M., Grone, M., Todorova, M., Link, I., 
Khan, S.A., Meyer, R.G., Huber, C., et al. 2007. Targeting the activation-induced antigen 
CD137 can selectively deplete alloreactive T cells from antileukemic and antitumor 
donor T-cell lines. Blood 109:365-373. 
42. 
Kamiryo, Y., Eto, M., Yamada, H., Yajima, T., Harano, M., Takeuchi, A., Tatsugami, K., 
Hamaguchi, M., Naito, S., and Yoshikai, Y. 2009. Donor CD4 T cells are critical in 
allogeneic stem cell transplantation against murine solid tumor. Cancer Res 69:5151-
5158. 
43. 
Champlin, R., Jansen, J., Ho, W., Gajewski, J., Nimer, S., Lee, K., Territo, M., Winston, 
D., Tricot, G., and Reichert, T. 1991. Retention of graft-versus-leukemia using selective 
depletion of CD8-positive T lymphocytes for prevention of graft-versus-host disease 
following bone marrow transplantation for chronic myelogenous leukemia. Transplant 
Proc 23:1695-1696. 
44. 
Giralt, S., Hester, J., Huh, Y., Hirsch-Ginsberg, C., Rondon, G., Seong, D., Lee, M., 
Gajewski, J., Van Besien, K., Khouri, I., et al. 1995. CD8-depleted donor lymphocyte 
infusion as treatment for relapsed chronic myelogenous leukemia after allogeneic bone 
marrow transplantation. Blood 86:4337-4343. 
45. 
Alyea, E.P., Soiffer, R.J., Canning, C., Neuberg, D., Schlossman, R., Pickett, C., Collins, 
H., Wang, Y., Anderson, K.C., and Ritz, J. 1998. Toxicity and efficacy of defined doses 
of CD4(+) donor lymphocytes for treatment of relapse after allogeneic bone marrow 
transplant. Blood 91:3671-3680. 
46. 
Roddie, C., and Peggs, K.S. 2011. Donor lymphocyte infusion following allogeneic 
hematopoietic stem cell transplantation. Expert Opin Biol Ther 11:473-487. 
47. 
Schmid, C., Labopin, M., Nagler, A., Bornhauser, M., Finke, J., Fassas, A., Volin, L., 
Gurman, G., Maertens, J., Bordigoni, P., et al. 2007. Donor lymphocyte infusion in the 
treatment of first hematological relapse after allogeneic stem-cell transplantation in adults 
with acute myeloid leukemia: a retrospective risk factors analysis and comparison with 
other strategies by the EBMT Acute Leukemia Working Party. J Clin Oncol 25:4938-
4945. 
48. 
Takami, A., Okumura, H., Yamazaki, H., Kami, M., Kim, S.W., Asakura, H., Endo, T., 
Nishio, M., Minauchi, K., Kumano, K., et al. 2005. Prospective trial of high-dose 
chemotherapy followed by infusions of peripheral blood stem cells and dose-escalated 
donor lymphocytes for relapsed leukemia after allogeneic stem cell transplantation. Int J 
Hematol 82:449-455. 
49. 
Levine, J.E., Barrett, A.J., Zhang, M.J., Arora, M., Pulsipher, M.A., Bunin, N., Fort, J., 
Loberiza, F., Porter, D., Giralt, S., et al. 2008. Donor leukocyte infusions to treat 

34 
 
hematologic malignancy relapse following allo-SCT in a pediatric population. Bone 
Marrow Transplant 42:201-205. 
50. 
Barrett, A.J., Mavroudis, D., Tisdale, J., Molldrem, J., Clave, E., Dunbar, C., Cottler-Fox, 
M., Phang, S., Carter, C., Okunnieff, P., et al. 1998. T cell-depleted bone marrow 
transplantation and delayed T cell add-back to control acute GVHD and conserve a graft-
versus-leukemia effect. Bone Marrow Transplant 21:543-551. 
51. 
Ferra, C., Rodriguez-Luaces, M., Gallardo, D., Encuentra, M., Martin-Henao, G.A., Peris, 
J., Ancin, I., Sarra, J., Berlanga, J.J., Garcia, J., et al. 2001. Individually adjusted 
prophylactic donor lymphocyte infusions after CD34-selected allogeneic peripheral blood 
stem cell transplantation. Bone Marrow Transplant 28:963-968. 
52. 
Nakamura, R., Bahceci, E., Read, E.J., Leitman, S.F., Carter, C.S., Childs, R., Dunbar, 
C.E., Gress, R., Altemus, R., Young, N.S., et al. 2001. Transplant dose of CD34(+) and 
CD3(+) cells predicts outcome in patients with haematological malignancies undergoing 
T cell-depleted peripheral blood stem cell transplants with delayed donor lymphocyte 
add-back. Br J Haematol 115:95-104. 
53. 
Schaap, N., Schattenberg, A., Bar, B., Preijers, F., van de Wiel van Kemenade, E., and de 
Witte, T. 2001. Induction of graft-versus-leukemia to prevent relapse after partially 
lymphocyte-depleted allogeneic bone marrow transplantation by pre-emptive donor 
leukocyte infusions. Leukemia 15:1339-1346. 
54. 
Lee, C.K., de Magalhaes-Silverman, M., Hohl, R.J., Hayashi, M., Buatti, J., Wen, B.C., 
Schlueter, A., Strauss, R.G., and Gingrich, R.D. 2002. Prophylactic T cell infusion after T 
cell-depleted bone marrow transplantation in patients with refractory lymphoma. Bone 
Marrow Transplant 29:615-620. 
55. 
Montero, A., Savani, B.N., Shenoy, A., Read, E.J., Carter, C.S., Leitman, S.F., Mielke, S., 
Rezvani, K., Childs, R., and Barrett, A.J. 2006. T-cell depleted peripheral blood stem cell 
allotransplantation with T-cell add-back for patients with hematological malignancies: 
effect of chronic GVHD on outcome. Biol Blood Marrow Transplant 12:1318-1325. 
56. 
Barge, R.M., Osanto, S., Marijt, W.A., Starrenburg, C.W., Fibbe, W.E., Nortier, J.W., 
Falkenburg, J.H., and Willemze, R. 2003. Minimal GVHD following in-vitro T cell-
depleted allogeneic stem cell transplantation with reduced-intensity conditioning 
allowing subsequent infusions of donor lymphocytes in patients with hematological 
malignancies and solid tumors. Exp Hematol 31:865-872. 
57. 
de Lima, M., Bonamino, M., Vasconcelos, Z., Colares, M., Diamond, H., Zalcberg, I., 
Tavares, R., Lerner, D., Byington, R., Bouzas, L., et al. 2001. Prophylactic donor 
lymphocyte infusions after moderately ablative chemotherapy and stem cell 
transplantation for hematological malignancies: high remission rate among poor 
prognosis patients at the expense of graft-versus-host disease. Bone Marrow Transplant 
27:73-78. 
58. 
Massenkeil, G., Nagy, M., Lawang, M., Rosen, O., Genvresse, I., Geserick, G., Dorken, 
B., and Arnold, R. 2003. Reduced intensity conditioning and prophylactic DLI can cure 
patients with high-risk acute leukaemias if complete donor chimerism can be achieved. 
Bone Marrow Transplant 31:339-345. 
59. 
Schmid, C., Schleuning, M., Ledderose, G., Tischer, J., and Kolb, H.J. 2005. Sequential 
regimen of chemotherapy, reduced-intensity conditioning for allogeneic stem-cell 
transplantation, and prophylactic donor lymphocyte transfusion in high-risk acute 
myeloid leukemia and myelodysplastic syndrome. J Clin Oncol 23:5675-5687. 

35 
 
60. 
Meyer, R.G., Britten, C.M., Wehler, D., Bender, K., Hess, G., Konur, A., Hartwig, U.F., 
Wehler, T.C., Ullmann, A.J., Gentilini, C., et al. 2007. Prophylactic transfer of CD8-
depleted donor lymphocytes after T-cell-depleted reduced-intensity transplantation. 
Blood 109:374-382. 
61. 
Dey, B.R., McAfee, S., Colby, C., Sackstein, R., Saidman, S., Tarbell, N., Sachs, D.H., 
Sykes, M., and Spitzer, T.R. 2003. Impact of prophylactic donor leukocyte infusions on 
mixed chimerism, graft-versus-host disease, and antitumor response in patients with 
advanced hematologic malignancies treated with nonmyeloablative conditioning and 
allogeneic bone marrow transplantation. Biol Blood Marrow Transplant 9:320-329. 
62. 
Beatty, G.L., Smith, J.S., Reshef, R., Patel, K.P., Colligon, T.A., Vance, B.A., Frey, N.V., 
Johnson, F.B., Porter, D.L., and Vonderheide, R.H. 2009. Functional unresponsiveness 
and replicative senescence of myeloid leukemia antigen-specific CD8+ T cells after 
allogeneic stem cell transplantation. Clin Cancer Res 15:4944-4953. 
63. 
van Besien, K., Artz, A., Smith, S., Cao, D., Rich, S., Godley, L., Jones, D., Del Cerro, P., 
Bennett, D., Casey, B., et al. 2005. Fludarabine, melphalan, and alemtuzumab 
conditioning in adults with standard-risk advanced acute myeloid leukemia and 
myelodysplastic syndrome. J Clin Oncol 23:5728-5738. 
64. 
Kroger, N., Bornhauser, M., Ehninger, G., Schwerdtfeger, R., Biersack, H., Sayer, H.G., 
Wandt, H., Schafer-Eckardt, K., Beyer, J., Kiehl, M., et al. 2003. Allogeneic stem cell 
transplantation after a fludarabine/busulfan-based reduced-intensity conditioning in 
patients with myelodysplastic syndrome or secondary acute myeloid leukemia. Ann 
Hematol 82:336-342. 
65. 
Lim, Z.Y., Ingram, W., Brand, R., Akthari, M., Milojkovic, D., Ho, A.Y., Devereux, S., 
Pagliuca, A., Duarte, R.F., and Mufti, G.J. 2007. Clonal gammopathies following 
alemtuzumab-based 
reduced 
intensity 
conditioning 
haematopoietic 
stem 
cell 
transplantation: association with chronic graft-versus-host disease and improved overall 
survival. Bone Marrow Transplant 40:747-752. 
66. 
Jilani, I., Keating, M., Giles, F.J., O'Brien, S., Kantarjian, H.M., and Albitar, M. 2004. 
Alemtuzumab: validation of a sensitive and simple enzyme-linked immunosorbent assay. 
Leuk Res 28:1255-1262. 
67. 
Chakraverty, R., Orti, G., Roughton, M., Shen, J., Fielding, A., Kottaridis, P., Milligan, 
D., Collin, M., Crawley, C., Johnson, P., et al. Impact of in vivo alemtuzumab dose 
before 
reduced 
intensity 
conditioning 
and 
HLA-identical 
sibling 
stem 
cell 
transplantation: pharmacokinetics, GVHD, and immune reconstitution. Blood 116:3080-
3088. 
68. 
Bottcher, S., Ritgen, M., Fischer, K., Stilgenbauer, S., Busch, R.M., Fingerle-Rowson, G., 
Fink, A.M., Buhler, A., Zenz, T., Wenger, M.K., et al. Minimal residual disease 
quantification is an independent predictor of progression-free and overall survival in 
chronic lymphocytic leukemia: a multivariate analysis from the randomized GCLLSG 
CLL8 trial. J Clin Oncol 30:980-988. 
69. 
Liu, H., Johnson, J.L., Koval, G., Malnassy, G., Sher, D., Damon, L.E., Hsi, E.D., Bucci, 
D.M., Linker, C.A., Cheson, B.D., et al. 2012. Detection of minimal residual disease 
following induction immunochemotherapy predicts progression free survival in mantle 
cell lymphoma: final results of CALGB 59909. Haematologica 97:579-585. 
70. 
Przepiorka, D., Weisdorf, D., Martin, P., Klingemann, H.G., Beatty, P., Hows, J., and 
Thomas, E.D. 1995. 1994 Consensus Conference on Acute GVHD Grading. Bone 
Marrow Transplant 15:825-828. 

36 
 
71. 
Filipovich, A.H., Weisdorf, D., Pavletic, S., Socie, G., Wingard, J.R., Lee, S.J., Martin, P., 
Chien, J., Przepiorka, D., Couriel, D., et al. 2005. National Institutes of Health consensus 
development project on criteria for clinical trials in chronic graft-versus-host disease: I. 
Diagnosis and staging working group report. Biol Blood Marrow Transplant 11:945-956. 
 
 

37 
 
APPENDIX 
 
PRZEPIORKA CRITERIA 
FOR ACUTE GVHD 
 
Consensus Criteria for Grading of Acute GVHD