Clinical trial NCT00741455
Conditions: Hematologic Malignancies
Keywords: Non-myeloablative transplant Chronic Myelogenous Leukemia Acute Myelogenous Leukemia Acute Lymphoblastic Leukemia Multiple Myeloma Non-Hodgkin's Lymphoma Myelodysplastic syndrome Chronic Lymphocytic Leukemia Hodgkin's Disease
Sponsor: Dartmouth-Hitchcock Medical Center
Start date: 2004-06-01; Completion date: 2020-05-01
Enrollment: 18 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The purpose of this study is to determine disease-free survival, overall survival, time to progression, regimen-related toxicity and/or treatment-related mortality in patients with hematologic malignancies treated with non-myeloablative chemotherapy followed by allogeneic stem cell transplant.

Detailed description:
Allogeneic bone marrow transplantation (BMT) became feasible in the 1960s after elucidation of the Human Leukocyte Antigen (HLA) complex. Since then, the therapy has evolved into an effective treatment for many hematologic disorders. Otherwise incurable malignancies are frequently cured by this approach, with the likelihood of cure ranging from 10% to 85%, depending on the disease and the disease status. The treatment strategy incorporates very large doses of chemotherapy and often radiation to eliminate cancer cells and to immunosuppress the recipient to allow the engraftment of donor cells. Donor cells give rise to hematopoiesis within two to three weeks, rescuing the patient from the effects of high dose therapy. In the ideal situation, immune recovery and recipient-specific tolerance occurs over the following 6-18 months, and the patient is cured of their underlying malignancy, off immunosuppression, with a functionally intact donor-derived immune system. However, complications are common and include fatal organ damage from the effects of high dose chemotherapy, infection, hemorrhage, and, in particular, graft-versus-host disease (GvHD). A realistic estimate of transplant-related mortality in the standard HLA-matched sibling setting is approximately 25%. The risk of treatment-related mortality limits the success and certainly precludes its use in older patients. Thus, new strategies in transplantation are needed.

With the growing understanding that much of the curative potential of allogeneic bone marrow or stem cell transplant (SCT) is from an immune anti-tumor effect of donor cells, known as graft-versus-leukemia (GvL) or graft-versus-tumor (GvT), a new strategy is being employed that shifts the emphasis from high-dose chemo-radiotherapy to donor-derived, immune-mediated anti-tumor therapy. In this approach, patients receive preparative regimens that, while having some anti-tumor activity, are mainly designed to be immunosuppressive enough to allow engraftment of donor stem cells and lymphocytes. Engrafted lymphocytes then mediate a GvL effect; if the GvL effect of the initial transplant is not sufficient, then additional lymphocytes may be infused (achievement of engraftment allows additional lymphocytes to "take" in the recipient without requiring any additional conditioning of the recipient). The lower intensity of the preparative regimen lessens the overall toxicity by minimizing the doses of chemo-radiotherapy. In addition, less intensive preparative regimens may be associated with less GvHD, as much evidence suggests that high-dose therapy contributes to the syndrome of GvHD by causing tissue damage, leading to a cytokine milieu which enhances activation of graft-versus-host (GvH) effector cells. Thus, such an approach may allow the safer use of allogeneic transplants in standard populations and may allow extension of allogeneic transplantation to patients who could not receive standard (myeloablative) transplants because of age or co-morbidities. This protocol investigates a non-myeloablative transplant approach, using fludarabine and cyclophosphamide, to allow engraftment of allogeneic cells, which may then mediate anti-tumor effects.

Arms: arm 1: Chemotherapy, stem cell transplantation, HLA-Matched related allogeneic stem cell transplantation, leukapheresis, G-CSF, peripheral blood stem cell transplant, fludarabine, cyclophosphamide, donor lymphocyte infusion, cyclosporine, methotrexate
Interventions: intervention 1: Stem Cell Transplant intervention 2: G-CSF intervention 3: Fludarabine intervention 4: cyclophosphamide intervention 5: Cyclosporine intervention 6: Methotrexate
Intervention descriptions: intervention 1: Donor: Prior to mobilization, leukapheresis to collect CD3+ cells. The donor will then receive G-CSF (10 mcg/kg/day) with leukapheresis collection of peripheral blood stem cells on days 5, 6 and 7 as needed. Goal of leukapheresis will be \> 5 x 106 CD34+cells/kg of recipient.

Patient: Peripheral Blood Stem Cell (PBSC) Transplant. Fludarabine 25mg/m2/d IV over 30 minutes on days -6 to -2, followed by cyclophosphamide 1g/m2/d IV on days -3 and -2. This will be followed by allogeneic stem cell infusion 48 hours later.

Donor Lymphocyte Infusion (DLI) and Adjustment of Immunosuppression: Cyclosporine (CSA) and methotrexate (MTX) will be used for GvHD prophylaxis with target CSA levels of 200-400 ng/ml. intervention 2: 10 mcg/kg/day on days 5, 6, and 7 intervention 3: 25 mg/m2/d IV over 30 minutes on days -6 to -2 intervention 4: 1 g/m2/d IV on days -3 and -2 intervention 5: used for GvHD prophylaxis with target CSA levels of 200-400 ng/ml intervention 6: used for GvHD prophylaxis with target CSA levels of 200-400 ng/ml

Protocol documents:
Prot_SAP_000.pdf: 
 
 
NON-MYELOABLATIVE CHEMOTHERAPY FOLLOWED BY 
HLA-MATCHED RELATED ALLOGENEIC STEM CELL 
TRANSPLANTATION FOR HEMATOLOGIC MALIGNANCIES 
 
 
Protocol Number: 
 
D0345 
 
Coordinating Center: 
Dartmouth-Hitchcock Medical Center 
 
 
 
 
One Medical Center Drive 
 
 
 
 
Lebanon, NH 03756 
 
Principal Investigator: 
John M. Hill, Jr., M.D. 
 
 
 
 
Bone Marrow Transplant Program 
 
 
 
 
Dartmouth-Hitchcock Medical Center 
 
 
 
 
One Medical Center Drive 
 
 
 
 
Lebanon, NH 03756 
 
 
 
 
Phone: 603-650-4628 
 
 
 
 
Fax: 603-650-7791 
 
 
 
 
e-mail: John.M.Hill@Hitchcock.org  
 
 
Sub-Investigators: 
 
Kenneth R. Meehan, M.D. 
Elizabeth M. Bengtson, M.D. 
Marc Gautier, M.D. 
Christopher Lowrey, M.D. 
 
 
Cell Processing: 
 
Zbigniew M. Szczepiorkowski, M.D., Ph.D. 
 
Biostatistician: 
 
Tor D.Tosteson, Sc.D. 
Zhong-Ze Li, MS 
 
 
Protocol History: 
Original 
 
 
21 January 2004 
Amendment 1  
 
1 December 2004 
 
 
 
Amendment 2  
 
12 November 2007 
 
 
Amendment 3  
 
26 November 2008 
Amendment 4  
 
18 October 2010 
 
 
 
 
 
 
 


 
TABLE OF CONTENTS 
 
TREAMENT SCHEMA ................................................................................................................. i 
1.0 
OBJECTIVES .................................................................................................................... 1 
2.0  
INTRODUCTION ............................................................................................................. 1 
3.0   
STUDY DESIGN............................................................................................................... 6 
4.0  
ELIGIBILITY CRITERIA................................................................................................. 6 
5.0  
STAGING WORKUP AND PATIENT CLINICAL EVALUATION .............................. 8 
6.0 
DONOR EVALUATION ................................................................................................ 10 
7.0 
TREATMENT PLAN ...................................................................................................... 11 
8.0 
MANAGEMENT OF POST-TRANSPLANT COMPLICATIONS ............................... 15 
9.0 
POTENTIAL TOXICITIES, HAZARDS AND DISCOMFORTS ................................. 15 
10.0 
WITHDRAWAL FROM THE STUDY .......................................................................... 16 
11.0 
RESPONSE CRITERIA AND EVALUATION OF RESULTS ..................................... 16 
12.0 
STATISTICS ................................................................................................................... 17 
13.0 
CHIMERISM STUDIES ................................................................................................. 18 
14.0 
REGISTRATION GUIDELINES .................................................................................... 18 
15.0 
DATA COLLECTION AND DATA AND SAFETY MONITORING .......................... 18 
16.0 
ADVERSE DRUG REACTION (ADR) REPORTING .................................................. 19 
17.0 
REFERENCES ................................................................................................................ 20 
Appendix I:  
Disease Staging and Response Definitions ...................................................... 23 
Appendix II: 
Prophylaxis And Treatment Of Infection ........................................................ 25 
Appendix III: 
GvHD Grading Scale ....................................................................................... 26 
Appendix IV: 
Toxicity Scale .................................................................................................. 28 
 Appendix V: 
 DHMC BMT Standard Operating Procedures (SOPs)………………………32 
 


i 
SCHEMA 
 
Informed Consent and Pre-study Evaluations 
 
 
 
 
Pre-Transplant Treatment Regimen 
 
 
 
Transplant (Day 0) 
 
 
 
Post-Transplant Treatment Regimen 
 
 
 
Post Transplant Follow-Up  
Days +30 & +60 (both +3 days), Days +100, +180, +365 (all +15 days) 
 
 
 
Short Term Follow-up  
(Every three months for two years following 1 year visit, then every year for 5 years or until death or relapse, whichever occurs first) 
 
 
Long Term Follow-up (Yearly survival check) 
 
Fludarabine 
25mg/m2 
IV 
Once daily over 30 
minutes on days -6 
through -2 
Cyclophosphamide 
1mg/m2 
IV 
Once daily over 2 
hours on days -3 and  
-2 
Mesna 
20% of 
cyclophosphamide 
dose 
IV 
15 minutes prior to 
cyclophosphamide, 4 
and 8 hours following 
cyclophosphamide 
Cyclosporine 
3mg/kg if IV 
5mg/kg if PO 
IV/PO 
Once daily starting on 
day -4 if IV, Twice 
daily starting on day -
4 if PO 
Methotrexate 
10mg/m2 
IV 
Days +1, +3 and +6 


i 
1.0 
OBJECTIVES 
Primary Objectives 
 To evaluate engraftment by measuring donor-recipient chimerism in lymphoid and 
myeloid lineages 
 To investigate the effect of donor lymphocyte infusion on donor-host chimerism 
 To determine the incidence and severity of acute and chronic graft-versus-host disease  
 To assess the following: 
 treatment-related mortality 
 time to progression 
 disease-free survival 
 overall survival 
 regimen-related toxicity  
Secondary Objective       
 To evaluate disease response rate as defined in section 12.3    
 
2.0  
INTRODUCTION 
 
2.1 
Allogeneic Bone Marrow Transplantation  Background 
Allogeneic bone marrow transplantation (BMT) became feasible in the 1960s after 
elucidation of the Human Leukocyte Antigen (HLA) complex.  Since then, the therapy has 
evolved into an effective treatment for many hematologic disorders [1]. Otherwise 
incurable malignancies are frequently cured by this approach, with the likelihood of cure 
ranging from 10% to 85%, depending on the disease and the disease status. The treatment 
strategy incorporates very large doses of chemotherapy and often radiation to eliminate 
cancer cells and to immunosuppress the recipient to allow the engraftment of donor cells.  
Donor cells give rise to hematopoiesis within two to three weeks, rescuing the patient from 
the effects of high dose therapy.  In the ideal situation, immune recovery and recipient-
specific tolerance occurs over the following 6-18 months, and the patient is cured of their 
underlying malignancy, off immunosuppression, with a functionally intact donor-derived 
immune system.  However, complications are common and include fatal organ damage 
from the effects of high dose chemotherapy, infection, hemorrhage, and, in particular, 
graft-versus-host disease (GvHD).  A realistic estimate of transplant-related mortality in the 
standard HLA-matched sibling setting is approximately 25%.  The risk of treatment–related 
mortality limits the success and certainly precludes its use in older patients.  Thus, new 
strategies in transplantation are needed. 
 


2 
 
2.2 
Shifting emphasis in BMT toward adoptive immunotherapy 
With the growing understanding that much of the curative potential of allogeneic bone 
marrow or stem cell transplant (SCT) is from an immune anti-tumor effect of donor cells, 
known as graft-versus-leukemia (GvL) or graft-versus-tumor (GvT) [2-5], a new strategy is 
being employed that shifts the emphasis from high-dose chemo-radiotherapy to donor-
derived, immune-mediated anti-tumor therapy.  In this approach, patients receive 
preparative regimens that, while having some anti-tumor activity, are mainly designed to be 
immunosuppressive enough to allow engraftment of donor stem cells and lymphocytes [6-
11].  Engrafted lymphocytes then mediate a GvL effect; if the GvL effect of the initial 
transplant is not sufficient, then additional lymphocytes may be infused (achievement of 
engraftment allows additional lymphocytes to “take” in the recipient without requiring any 
additional conditioning of the recipient).  The lower intensity of the preparative regimen 
lessens the overall toxicity by minimizing the doses of chemo-radiotherapy.  In addition, 
less intensive preparative regimens may be associated with less GvHD, as much evidence 
suggests that high-dose therapy contributes to the syndrome of GvHD by causing tissue 
damage, leading to a cytokine milieu which enhances activation of graft-versus-host (GvH) 
effector cells [12]. Thus, such an approach may allow the safer use of allogeneic 
transplants in standard populations and may allow extension of allogeneic transplantation 
to patients who could not receive standard (myeloablative) transplants because of age or 
co-morbidities. This protocol investigates a non-myeloablative transplant approach, using 
fludarabine and cyclophosphamide, to allow engraftment of allogeneic cells, which may 
then mediate anti-tumor effects. 
 
2.3 
Graft-vs-Leukemia 
In the past, it was thought that allogeneic transplant cured malignancies simply by allowing 
the delivery of high doses of chemotherapy and radiation. However, it has become 
increasingly clear over the past few years that much of the curative potential of allogeneic 
SCT derives from an anti-tumor effect mediated by donor-immune cells. This GvL 
phenomenon may account for as much as half of the curative potential of the allogeneic 
SCT process.  
 
Numerous animal studies since the mid-1950s have documented an anti-leukemic effect of 
allogeneic bone marrow transplantation [2,3]. Observations in humans have strongly 
suggested the existence of a GvL effect in human allogeneic BMT [4,5]: relapse rates of 
syngeneic BMT recipients are higher than in allogeneic BMT recipients; allogeneic 
recipients who do not develop GvHD relapse more frequently than allogeneic recipients 
who do develop GvHD; and recipients of T-cell depleted BMT relapse more frequently 
than recipients of non-T-cell depleted BMT. In addition, GvL has been purposefully 
induced simply by withdrawal of immune suppression (e.g., cyclosporine, steroids), which 
presumably allows the expansion of allogeneic donor lymphocytes with anti-tumor 
reactivity; remissions following this immunologic maneuver have been reported in AML, 
CML and NHL. 
 
Based on the potential power of the GvL effect and its presumed mediation by donor 
leukocytes (T cells and/or natural killer [NK] cells), several groups have infused leukocytes 
obtained from the original bone marrow donor into patients with relapsed malignancy after 


3 
 
allogeneic BMT [16-26]. Most patients treated with donor leukocyte infusions (DLI) have 
had chronic myelogenous leukemia; durable complete remissions have been reported in 50-
70% of these patients. Ongoing remissions have also been reported in patients with AML, 
ALL, multiple myeloma, NHL and CLL. 
 
DLIs are often associated with GvHD.  In one series, acute and chronic GvHD occurred in 
60% of patients [23]; however, GvHD was closely associated with disease response 
(p<.00001). Several observations have suggested that it may be possible to separate GvL 
from GvHD in some cases. Occasional patients receiving typical doses of DLI (108 T 
cells/kg) have been observed who had complete responses but did not develop GvHD [22, 
23].  A higher percentage of patients receiving lower doses of donor leukocytes (107 T 
cells/kg) had complete responses but no GvHD [27, 28].  The incidence and severity of 
GvHD (both acute and chronic) also appears to be significantly lower when escalating 
doses of DLI are used, with similar or improved GvL effect [27, 28].  Two groups have 
administered DLI depleted of CD8+ cells and observed GvL without GvHD in the majority 
of patients [29, 30]. 
 
Response to DLI in patients with the following diseases provides definitive evidence for 
GvL in a percentage of patients. 
 
2.4 
Chronic Myelogenous Leukemia 
The GvL effect is most pronounced in CML. An allogeneic GvL effect in CML is 
supported by the following evidence: 
 
a. A lower risk of relapse in recipients who develop GvHD after allogeneic BMT 
(relative risk 0.24 for patients with both acute and chronic GvHD, p= .03) [5]. 
b. A trend towards increased risk of relapse in syngeneic transplant recipients (relative 
risk 2.95, p=.08) 
c. An increased risk of relapse in recipients of T cell depleted allografts (relative risk 
5.14, p= .0001); 
d. Cyclosporine withdrawal-induced remission in CML patients in relapse after 
allogeneic BMT [14];  
e. Complete remission following treatment with DLI in 50-75% of patients with CML 
in relapse after allogeneic BMT [22, 23]. 
 
In addition, there is evidence that supports a graft-vs.-tumor effect in other hematologic 
diseases as noted below:  
 
2.5  
Acute Myelogenous Leukemia and Acute Lymphoblastic Leukemia 
a. A lower risk of relapse in recipients who develop GvHD after allogeneic BMT 
(relative risk 0.34 for patients with both acute and chronic GvHD, p= .0003) [5]; 
b. An increased risk of relapse in syngeneic transplant recipients (relative risk 2.58, p= 
.008) [5]; 
c. Cyclosporine withdrawal-induced remission in AML (>ALL) patients in relapse 
after allogeneic BMT [5]; 


4 
 
d. Complete remission to treatment with DLI alone in patients in cytogenetic-only 
relapse after allogeneic BMT (4 of 9 patients in one series) (Collins, RH et al. 
unpublished observations); 
e. Ongoing disease-free survival in 20% of patients in hematologic relapse after 
allogeneic BMT who are treated with combined DLI and chemotherapy (Collins, 
RH et al. manuscript in preparation) 
 
2.6 
Multiple Myeloma   
a. A lower risk of relapse in patients treated with allogeneic as opposed to autologous 
transplantation (an alternative explanation is re-infusion of tumor cells in 
autologous transplants) [26]; 
b. Clear-cut complete remissions to DLI in patients in relapse after allogeneic BMT 
[25, 26]. 
 
2.7 
Non-Hodgkin’s Lymphoma 
a. A lower risk of relapse in patients treated with allogeneic as opposed to autologous 
transplantation.  In two large studies, patients with lymphoma were assigned to 
allogeneic versus autologous transplants based on the availability of a matched 
sibling donor [31, 32].  Relapse rates were significantly reduced in recipients of 
allografts; 
b. Withdrawal of immunosuppression-induced remission (associated with GvHD in 
relapsed NHL after allogeneic BMT) [16]. 
c. Clear-cut disease response to DLI [16, 23]. Overall, the number of NHL patients 
treated with DLI is low, making an estimate of response rate difficult                   
at this time. 
 
2.8 
Myelodysplastic syndrome (MDS) 
A graft-vs.-tumor effect in MDS is supported by complete responses to DLI in patients 
with relapsed disease after allogeneic BMT (4 of 10 patients; Collins, RH et al. 
unpublished observations). However, more patients need to be treated to allow definitive 
assessment of response to this maneuver. 
 
 
 
2.9 
Chronic Lymphocytic Leukemia (CLL)    
 
a. Relapse rates are significantly reduced in CLL allografts versus autografts [33] 
(again, reinfusion of tumor cells with autografts is an alternative explanation); 
b. Clear-cut tumor responses have been observed in patients with residual or relapsed 
disease treated with DLI [24].  Not enough patients have yet been reported to allow 
estimation of response rate. 
 
2.10 
Hodgkin’s Disease 
A graft-versus-tumor effect is supported mainly by the reduced relapse rate in allogeneic 
versus autologous or syngeneic transplant recipients [34]. To date, very few DLIs have 
been reported in Hodgkin’s disease;  1 of 5 patients in a series has responded (Collins et al, 
unpublished data). In addition, Porter et al. have reported two patients with relapsed 
Hodgkin’s disease after allogeneic BMT who achieved responses, including a complete 
response after DLI [35]. 


5 
 
 
2.11 
Allogeneic Peripheral Blood Stem Cell (PBSC) Transplants 
Stem cells for allogeneic transplantation may be obtained from either the bone marrow or 
the blood. Peripheral blood stem cells are obtained by apheresis after the donor has 
received granulocyte colony stimulating factor (G-CSF) for several days to mobilize stem 
cells from the marrow to the blood [36].  The more immunologically mature peripheral 
blood stem cells and higher number of T cells in this pheresis collection increases the rate 
and overall success of engraftment relative to bone marrow-derived stem cells, particularly 
with regard to the platelet cell line.  Despite these immunologic features, peripheral blood 
stem cell transplants do not result in a higher incidence of acute GvHD, though 
retrospective case-control studies do suggest that the incidence of chronic GvHD may be 
higher. 
 
2.12 
Non-Myeloablative Preparative Regimens 
As described in detail above, allogeneic BMT has curative potential in several hematologic 
malignancies. Due to the toxicity of standard BMT procedures, however, older patients or 
younger patients with co-morbid illnesses or organ dysfunction cannot be offered this 
potentially curative therapy.  Since the curative potential of allogeneic BMT may be 
derived from the anti-tumor effect of donor immune cells, it is possible to harness this 
effect by discontinuing immunosuppression after donor cell engraftment, or by infusing 
additional immune cells without immunosuppression. The non-myeloablative therapy is 
immunosuppressive enough to allow donor cell engraftment [37] [6-11]. Allogeneic 
peripheral blood stem cells are infused following therapy, and once engrafted these donor 
immune cells may eliminate residual tumor cells via the GVL effect. If the malignancy 
persists, then the GvT effect may be enhanced by stopping immunosuppression or by 
infusing additional donor immune cells (DLI).  Attainment of mixed (donor/recipient) 
chimerism after engraftment serves as the necessary platform for subsequent DLI. 
 
We will use this non-myeloablative regimen to treat hematologic malignancies. The design 
of the study incorporates some of the following key features: 
 
1. Immunosuppression without myeloablation: Patients will receive chemotherapy 
sufficient to allow donor lymphohematopoietic engraftment without complete 
marrow ablation.  If the graft is rejected, the patient will reconstitute autologous 
marrow and survive.  We will use a combination of two agents with known 
immunosuppressive activity in BMT: Cyclophosphamide, which produces 
transient pancytopenia, and Fludarabine, which causes less cytopenia but 
equivalent immunosuppression. 
2. PBSC transplant:  An unmanipulated peripheral blood stem cell collection from 
a G-CSF stimulated HLA-matched related donor should improve the chance of 
engraftment because of the high stem cell dose (>5 x 106/kg CD34+ cells) and 
the presence of donor lymphocytes.  Low intensity preparative regimens have a 
low incidence of severe GvHD.  To further reduce the risk of GvHD, patients 
will receive cyclosporine (CSA) for the first 100 days after the transplant. 


6 
 
3. Use of donor lymphocyte infusions (DLI) for patients with less than full donor 
chimerism and/or residual disease to optimize post-transplant engraftment and 
anti-tumor effect. 
3.0   
STUDY DESIGN 
3.1 
Collection of Donor Cells  
Collection of donor cells will be performed per the institution’s standard practice.  
The goal of leukapheresis will be > 5 x 106 CD34+cells/kg.  In the unlikely event of 
a suboptimal PBSC collection, the donor will undergo a bone marrow harvest (non-
mobilized) following a delay of at least 3 days.  
 
3.2 
Patient Step I - Peripheral Blood Stem Cell (PBSC) Transplant   
(Refer to Section 7.2 for details) 
Participants will receive fludarabine 25mg/m2/d IV over 30 minutes on days -6 to    
-2, followed by cyclophosphamide 1g/m2/d IV on days -3 and -2. This will be 
followed by allogeneic stem cell infusion 48 hours later. The allogeneic HSC graft 
will aim to deliver greater than 3.5 x 106 CD34+ cells/kg of recipient weight, with a 
minimum of  2 x 106 CD34+ cells/kg of recipient weight.   
 
3.3 
Patient Step II - Donor Lymphocyte Infusion (DLI) and Adjustment of 
Immunosuppression (Refer to Sections 7.4 and 7.5 for details) 
Cyclosporine (CSA) and methotrexate (MTX) will be used for GvHD prophylaxis 
with target serum CSA levels of 200-400 ng/ml.  
Refer to the Treatment Section (Section 7.5) for adjustment and tapering of 
immunosuppression and for Donor Lymphocyte Infusions. 
 
4.0  
ELIGIBILITY CRITERIA 
4.1 
Inclusion Criteria 
4.1.1 Age:  18-75 years 
4.1.2 Diseases 
a. Chronic myelogenous leukemia (CML) 
 First chronic phase or later 
 Accelerated phase 
b. Acute myelogenous or lymphoblastic leukemia (AML or ALL) 
 Second or subsequent remission 
 First remission with poor risk features, including, but not limited to:   
For AML- complex chromosome karyotype, abnormalities of 
chromosome 5 or 7, 12p-, 13+, 8+, t(9;22), t(11;23) 
For ALL- t(9;22), t(4;11), t(1;19), myeloid antigen coexpression   
 Patients who have failed an autologous PBSC transplant 
c. Myelodysplastic syndrome (MDS) 
d. Multiple myeloma  - high risk myeloma (poor responders, relapse after 
autologous PBSCT, chromosome 13 abnormalities)  
e. Hodgkin’s disease 


7 
 
 Primary refractory disease 
 Relapsed disease (first relapse or later) 
 Patients who have failed an autologous PBSC transplant 
f. Non-Hodgkin’s lymphoma 
Low grade (by Working Formulation) 
 Relapsed, progressive disease after initial chemotherapy 
 Primary refractory disease or failure to respond (>PR) to initial 
chemotherapy 
 Patients who have failed an autologous PBSC transplant 
Intermediate grade (by Working Formulation) 
 Relapsed disease 
 Primary refractory disease or failure to respond (>PR) to initial chemo  
 Mantle cell lymphoma  
 Patients who have failed an autologous PBSC transplant 
g. Chronic lymphocytic leukemia (CLL) 
 Patients newly diagnosed with poor prognostic factors,  including CD38 
expression, Chromosome 11 or 17 abn  
 T-CLL/PLL  
 Relapsed or progressive disase, or refractory after Fludarabine 
 Patients who have failed an autologous PBSC transplant 
4.1.3 Donor Availability: Six of six matched HLA A, B and DR identical sibling 
(or parent or child) or 5/6 related donor with single mismatch at Class I 
antigen (A or B) 
4.1.4 Karnofsky performance status of  >70%  
4.1.5 Serum bilirubin <2x upper limit of normal; transaminases <3x normal 
(unless due to disease) 
4.1.6 24 hr urine creat clearance of > 40 ml/min. 
4.1.7 DLCO 50% predicted 
4.1.8 Left ventricular ejection fraction 35% 
4.1.9 Voluntary written informed consent must be given before performance of 
any study-related procedure, with the understanding that consent may be 
withdrawn by the participant at any time without prejudice to future medical 
care. The participant must have the ability to understand and the willingness 
to sign a written informed consent document. 
 
4.2 
Exclusion Criteria 
4.2.1 Major organ dysfunction 
4.2.2 Pregnant or lactating female 
4.2.3 Active infection 
4.2.4 Psychological problems that preclude compliance and completion of the 
clinical trial 
4.2.5 Any other condition, that in the judgement of the investigator, affects 
participant safety or overall participation 
 
 


8 
 
5.0  
STAGING WORKUP AND PATIENT CLINICAL EVALUATION 
5.1 
Pre-study evaluation  
            *Based on clinical considerations, each patient will begin treatment on this    
              protocol within a clinically relevant timeframe following completion of the    
              pre-transplant evaluation 
5.1.1 HLA typing (molecular) 
5.1.2 Patholologic evaluation confirming the diagnosis.  
5.1.3 Disease staging (varies by disease- see appendix) 
5.1.4 Serologic testing (IgM and IgG):  CMV; HSV; VZV; toxoplasmosis  
5.1.5 Antibody screen for Hep A, HBV, HCV, HIV, HTLV I/II, VDRL (or RPR) 
5.1.6 CBC with differential, PT/PTT, CMP 
5.1.7 ABO Rh testing 
5.1.8 PSA (males over age 40 or with 1st degree family history of prostate cancer) 
5.1.9 Serum pregnancy test (female with child-bearing potential; if clinically 
appropriate) 
5.1.10 Mammogram and PAP smear (females) – within the past 12 months or as 
clinically indicated 
5.1.11 Pulmonary function tests with DLCO 
5.1.12 Cardiac function: EKG, MUGA scan (or ECHO) 
5.1.13 24 hr urine for creatinine clearance as clinically indicated 
5.1.14 Dental evaluation (within the past 6 months; panorex as clinically indicated) 
5.1.15 Ophthalmology assessment (if clinically indicated) 
5.1.16 Psychiatric and Social Work assessment 
5.1.17 Refer to Appendix I for disease-specific tests 
 
5.2 
Patient Follow Up Evaluation (Post Transplant) 
5.2.1 Post-transplant monitoring   
a. DHMC BMT SOPs for monitoring and evaluation of allogeneic HSCT 
recipients will be followed. 
b. Re-evaluation: Disease re-evaluation will be performed on days +30, +60, 
+100, +180, +365 (all visits +/- 15 days) and as otherwise clinically 
indicated.  Disease assessment testing will vary with disease (see appendix 
IV for appropriate testing). 
c. Chimerism assay (VNTR or STR analysis); FISH analysis for y- and x- 
specific microsatellite markers in the setting of sex mismatched transplants. 
5.2.2 Lineage-specific Chimerism Analysis 
a. Peripheral blood and/or bone marrow will be obtained on days +30, +60, 
+100, +180 and +365 (all visits +/- 15 days) (and as otherwise clinically 
indicated) to determine the degree of donor-host chimerism in lymphoid 
and myeloid lines using PCR analysis of microsatellite variable number of 
tandem repeat (VNTR) or short tandem repeat (STR) markers. 
b. Serial lineage-specific post-transplant chimerism analysis will be performed 
on peripheral blood (preferred) or bone marrow (if WBC inadequate) using 
PCR of informative minisatellite regions (VNTR or STR) to identify 
differences between the donor and recipient.  Complete donor chimerism 
(CDC) will be defined as the presence of at least 95% donor DNA in the T-
lymphoid (or unfractionated) sample analyzed.         


9 
 
 
This lineage-specific chimerism analysis, allowing direct characterization of 
asynchronous lymphoid and myeloid engraftment, will require the specific 
enrichment of myeloid (CD33+) and T-lymphoid (CD3+) cells prior to 
performing chimerism testing.  Cell enrichment kits (RosetteSep: Stem Cell 
Technologies, Vancouver, BC), utilizing antibody-mediated cell separation 
and density centrifugation, will be used to select the cells of interest. 
 
5.2.3 Graft-versus-Host Disease Evaluation 
a. Post-transplant acute GvHD assessment (diagnosis and grading) will be  
undertaken as outlined in the DHMC BMT SOP for the Diagnosis, Grading 
and Treatment of Acute Graft-versus-Host Disease.  
b. Day 100 screening  for chronic GvHD (tests below) will be performed at the 
discretion of the treating physician, based on clinical indication, per DHMC 
BMT SOP for Chronic GvHD Diagnosis and Treatment. Recommended 
evaluation for GvHD is listed below:  
1. Skin Biopsy : 4mm punch biopsy- iliac crest/forearm/back  
2. Oral Biopsy from lower lip if clinically indicated, based on 
symptoms or history of acute GvHD.  
3. Schirmer test if clinically indicated, based on symptoms or history of 
acute GvHD. 
4. CBC and liver associated enzymes: AST, bilirubin, alk. Phos. 
5. Pulmonary function tests (ABG, if clinically indicated) 
6. Gynecologic evaluation if clinically indicated 
7. Karnofsky score 
c. Screening and monitoring beyond day 100 will include the following 
workup, performed at minimum every 3 months for 2 yrs, then every year x 
5 yrs or relapse, whichever occurs first: 
1. Physical exam  
2. CBC/differential, reticulocyte count  
3. Liver, renal and comprehensive metabolic panel 
4. Additional tests and disease staging studies will be performed as 
clinically indicated by the treating physician.  
 
 
 
 
 
 
 
 
 
 
 
 
 


10 
 
6.0 
STUDY CALENDAR  
 
   
 
Pre- 
Studya 
Day +30 
(+/- 15) 
 
Day +60 
(+/- 15) 
 
Day +100 
(+/- 15) 
 
Day 
+180 
(+/- 15) 
 
Day 
+365 
(+/- 15) 
 
Short Term 
Follow-up j 
(Until Relapse) 
Long Term Follow-
Up k  
(Yearly for 
Survival) 
 
Informed consent 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HLA typing (molecular) 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pathologic Evaluationb 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical Exam 
X 
X 
X 
X 
X 
X 
X 
 
Disease Specific Stagingc 
X 
 
 
 
 
 
 
 
Bone Marrow Biopsy 
X 
X 
X 
X 
X 
X 
 
 
 
EKG 
 
X 
 
 
 
 
 
 
 
 
ECHO or MUGA 
 
X 
 
 
 
 
 
 
 
 
Pulmonary Function Test with DLCO 
 
X 
 
 
 
 
 
 
 
 
 
 
 
Dental Evaluationd 
 
X 
 
 
 
 
 
 
 
 
Mammograme 
 
X 
 
 
 
 
 
 
 
 
PAP smeare 
 
X 
 
 
 
 
 
 
 
 
PSAf  
 
X 
 
 
 
 
 
 
 
 
Psychiatric and Social Work Assessment 
 
X 
 
 
 
 
 
 
 
 
  
 
 
Opthalmology Assesmente 
X 
 
 
 
 
 
 
 
  
 
 
 
CBC w/Diff 
X 
X 
X 
X 
X 
X 
X 
 
 
PT and PTT 
X 
 
 
 
 
 
 
 
 
 
CMP 
X 
X 
X 
X 
X 
X 
X 
 
LDH 
X 
X 
X 
X 
X 
X 
X 
 
B-HCG (serum)g 
X 
 
 
 
 
 
 
 
Creatine Clearance from 24 hour urine 
X 
 
 
 
 
 
 
 
Serologic Testingh 
X 
 
 
 
 
 
 
 
Antibody Testingi 
X 
 
 
 
 
 
 
 
 
 
 
ABO Rh 
X 
 
 
 
 
 
 
 
Reticulocyte Count 
 
X 
X 
X 
X 
X 
 
 
Chimersim Testing (from peripheral 
blood or bone marrow) 
 
X 
X 
X 
X 
X 
 
 
Survival Check 
 
 
 
 
 
 
 
X 
 
a. Based on clinical considerations, each patient will begin treatment on this protocol within a clinically relevant timeframe following 
completion of the pre-transplant evaluation 
b. Pathologic evaluation confirming the diagnosis will be based on institutional diagnostic evaluations per disease 
c. Disease staging varies by disease, refer to appendix I 
d. Dental evaluation must be within past 6 months; panorex as clinically indicated 
e. AS CLINICALLY INDICATED 
f. For males 40 years or older with first degree family history of prostate cancer 
g. In females of child-bearing potential 
h. Serologic Testing includes the following: IgM, IgG, CMV, HSV, VZV, toxoplosmosis 
i. Antibody Testing includes the following: Hep A, HBV, HCV, HIV, HTLV I/II, VDRL (or RPR) 
j. Short term follow-up will occur every three months for two years following 1 year visit, then every year for 5 years or until death or relapse, 
whichever occurs first. 
k. Long term follow-upwill consist of yearly survival checks 


11 
 
 
 
7.0 
TREATMENT PLAN  
 
7.1 
Patient Treatment Plan 
The patient will be admitted to the hospital as clinically indicated during 
administration of the nonmyeloablative conditioning regimen, stem cell infusion 
and immediate post-transplant period.  A central venous catheter will be placed 
prior to the start of therapy (triple lumen Hickman catheter preferred). 
7.1.1 Supportive Measures - Infectious Disease 
a. Infectious Disease SOP:  DHMC BMT SOP for infection prophylaxis and 
empiric selective gut decontamination in allogeneic HSCT recipients will be 
followed. 
b. Fungal cultures: Surveillance fungal blood cultures will be obtained at 
admission in the following high-risk patients: 
 
 Steroid treatment for > 3 months duration  
 History of prolonged neutropenia (ANC < 500 for > 21 days)  
 Neutropenia of uncertain duration  
c. VRE: Stools will be tested for VRE if clinically indicated. 
d. Transfusion of Blood Products: Leukocyte reduced and irradiated blood 
products will be used.  CMV sero-negative recipients of CMV sero-negative 
stem cells will receive CMV negative blood products, if available. 
                   7.1.2   Preparative regimen  
Fludarabine:  Fludarabine will be administered at 25mg/m2/day IV on days 
–6, -5, -4, -3 and -2 as an IV infusion over 30 minutes each day for 5 days.     
For potential toxicities, see Section 9.0. 
 
Cyclophosphamide:  Cyclophosphamide will be administered at 1g/m2/day 
IV over 2 hours on days -3 and -2.  All patients will receive hydration as 
outlined below (section 7.3.2.1).  Mesna will be given for hemorrhagic 
cystitis prophylaxis (outlined in section 7.3.2.2).  For potential toxicities, see 
Section 
9.0. 
Recommended 
supportive 
anti-emetic 
therapy 
with 
Cyclophosphamide will be as follows: 
 Dexamethasone 10mg IV/po 30 min. prior to Cyclophosphamide  
 Dolasetron 100mg IV/po 30 min. prior to Cyclophosphamide  
 Lorazepam 0.5-2mg IV/po 30 min. prior to Cyclophosphamide, then 
q4-6h prn for breakthrough nausea or anxiety 
 Prochlorperazine 5-10mg po OR Promethazine 12.5-25mg IV q6h 
prn for breakthrough nausea/vomiting   
7.1.3 Transplant:  The allogeneic peripheral blood stem cell product will be 
infused on Day 0.  Stem cell infusion preparation and administration will be 
performed as outlined in the DHMC SOP for PBSC Reinfusion.  
 
 
 
 
 


12 
 
7.2 
Treatment Schema 
7.2.1 Day –6 through day -2 
7.3.1.1 Fludarabine 25 mg/m2 IV daily over 30 minutes. (If serum creatinine 
is 1.6-2.5 mg/dl, fludarabine is reduced to 20 mg/m2 daily x 5 days) 
7.2.3 Day –3 and day -2 
 
7.3.1.2 Recommended hydration:  One liter of 0.9% NaCl to be infused over 
1-2 hours prior to initiation of Cyclophosphamide, followed by 0.9% 
NaCl infused at 250 ml/hr for 4 hours post-Cyclophosphamide. 
7.3.1.3 Mesna at 20% of Cyclophosphamide dose (200 mg/m2) as an IV 
bolus infusion 15 minutes prior to Cyclophosphamide.  Repeat same 
intravenous 
dosage 
four 
and 
eight 
hours 
following 
Cyclophosphamide.  If oral, use Mesna tablets at 40% of 
Cyclophosphamide dose (400mg/m2) 2 hours and 6 hours after 
Cyclophosphamide.  If vomiting occurs within 2 hours of oral Mesna 
intake, repeat dosing is necessary.        
7.3.1.4 Cyclophosphamide (cytoxan) 1g/m2 IV over 2 hours. 
 
7.3 
GvHD prophylaxis  
7.3.1 Cyclosporine (CSA) 3 mg/kg per day, in divided doses, will be administered 
IV beginning on day – 4. When feasible, oral CSA at 5mg/kg twice daily 
will be substituted, as tolerated, with dosage adjustments made to maintain 
serum levels in the 200-400 mg/ml range. CSA levels will be checked on 
day -1 and every Monday, Wednesday and Friday, thereafter.  A taper will 
be started between days +60-100 as clinically indicated (see below). The 
precise cyclosporine schedule will depend on chimerism status and on the 
presence and severity of acute GvHD encountered (see below). 
7.3.2 Methotrexate (MTX) will be administered at a dose of 10mg/m2 
intravenously on days +1, +3, and +6.   
7.4 
Post-transplant Intervention 
7.4.1 The disease status and chimeric status will be assessed in all patients on 
days +30, +60, +100, +180, +365 (all visits +/- 15 days) for  and as 
otherwise clinically indicated.  Post-transplant decisions pertaining to 
withdrawal of immunosuppression and donor lymphocyte infusions 
(DLI) will be based both on the rapidity and extent of donor cell 
engraftment and the patient’s overall disease status.   
 Disease:  Analysis of disease status will be undertaken using standard 
morphologic, cytogenetic and molecular methods, along with flow 
cytometry, where applicable, to assess marrow/blood for persistent or 
progressive disease. 
 Chimerism:  Donor/recipient chimerism status will be interpreted by 
lineage-specific VNTR or STR PCR analysis of bone marrow, peripheral 
blood T cells and granulocytes as outlined in Section 5.3. 
7.4.2 Day #30: No DLI will be undertaken at day +30. Withdrawal of CSA 
immunosuppression will be done in the following situations:  
 Clear disease progression in the absence of significant graft-versus-host 
disease (> grade II), warranting a rapid taper of cyclosporine therapy (over 2 
weeks). 


13 
 
 Donor T-cell chimerism status of <40%, as this predicts for loss of the graft 
despite DLI [38].  Patients with this level of donor chimerism and no 
evidence of disease response or significant GvHD will begin a CSA taper at 
a rate of 25% every 10 days with intended plateau of the taper if day +60 
reassessment demonstrates a >95% level of donor T-cell status. 
 Patients with 40-94% donor chimerism on day +30 after transplant and 
stable yet persistent disease with <grade II GvHD will begin a slow CSA 
taper (10% per week).  (Patients with >95% chimerism status on day +30 
and no disease progression will continue to receive CSA until day +60, with 
reassessment at that time.) 
7.4.3 Day #60: 
 Patients demonstrating >95% donor chimerism at day +60 with a complete 
disease response and < grade II GvHD will begin a CSA taper by 5% every 
week, with the goal to discontinue CSA by 6-7 months post-transplant, 
unless a change in disease or chimerism status is noted on subsequent 
evaluation. 
 Patients demonstrating 40-94% donor chimerism at day +60 without 
evidence of progressive disease or significant GvHD will begin a CSA taper 
by 10% per week with discontinuation at day +130 in an attempt to prevent 
graft rejection.  Absence of a clear trend toward complete donor T-cell 
chimerism at 2 week follow-up (ie., <95% donor chimerism) and no GvHD 
progression will prompt consideration for DLI at 1 x 10e7 cells per kg 
recipient weight, depending on disease and immunosuppressive status.  
 Patients demonstrating <40% donor T-cell chimerism status at day +60 
without evidence of disease response or significant GvHD will begin a CSA 
taper of 25% every 10 days.  Subsequent improvement in chimerism status 
by <20% at 1-2 week follow-up (or ongoing total donor chimerism of < 
40%) or the development of clear disease progression will prompt 
administration of DLI of 1 x 10e7 cells/kg.           
 Patients demonstrating imminent graft rejection and/or clear disease 
progression at day +60 in the absence of > grade II GvHD will undergo 
rapid tapering of CSA over a 2 week period.  Failure to demonstrate a clear 
response at 1-2 week follow-up (and no GvHD progression) will prompt 
immediate DLI of 1 x 10e7 cells/kg with subsequent dose escalation as 
indicated (see DLI dose escalation section below).      
 Patients who have undergone withdrawal of immunosuppression at the time 
of their day +30 assessment (above) who demonstrate <20% improvement 
in donor T-cell chimerism status or persistent/progressive disease at day +60 
despite that intervention will be administered DLI of 1 x 10e7 cells/kg.  
7.4.4 Day #60–100:  Patients demonstrating >95% donor chimerism with disease 
progression between days +60 and +100 (without grade > II GvHD) will 
have their CSA  tapered (either by 25% every 10 days or 100% over a 2 
week period, depending on the extent of disease and tempo of progression). 
If these patients demonstrate ongoing disease progression at 1-2 week 
follow-up and no evidence of grade > II GvHD, DLI of 1 x 10e7 cells/kg 
will be administered.   


14 
 
7.4.5 DLI / Dose Escalation:  All patients not converting to >95% donor 
chimerism 
upon 
follow-up 
assessment 
after 
withdrawal 
of 
immunosuppression will be considered for donor lymphocyte infusion. 
Patients undergoing DLI will be considered for up to 3 escalating doses of 
donor T-cells at 1 x 10e7, 5 x 10e7, and 1 x 10e8 CD3+ cells/kg recipient 
weight at 1-6 month intervals, depending on their overall disease state, 
chimerism status and extent of graft-versus-host disease, based on 
subsequent monthly or more frequent follow-up.   
Mandatory prerequisites for DLI will include the following: 
 Minimal persistence of donor T-cell (at least 1-5% donor CD3 cells) 
chimerism.  Supportive stem cell infusion and/or immune suppression 
may be utilized to supplement DLI, as clinically indicated.      
 Discontinuation of all immunosuppressive therapy for at least 2 weeks 
without significant GvHD flare (ie., not ≥ Grade II) 
 Control of significant leukocytosis as indicated (e.g., with hydroxyurea 
and/or leukapheresis) for patients in hematologic relapse prior to DLI.  
Such intervention should be discontinued at least one day prior to 
initiation of DLI.  Patients with rapidly progressive malignancies 
requiring salvage chemotherapy should have this discontinued at least 3 
weeks prior to DLI. 
 Karnofsky score ≥ 50%. 
 
Consideration for post-DLI dose escalation will be required until one or 
more of the following is confirmed: 
 Attainment of complete donor (>95-100% T-cell) chimerism                  
 Presence of ≥ grade II acute GvHD or clinical extensive chronic GvHD  
 Regression of malignant disease 
 
      Patients with chronic myelogenous leukemia (CML) who have been 
managed with the tyrosine kinase inhibitor imatinib mesylate (Gleevec; STI-
571) at the time of referral will discontinue this agent prior to transplant.  In the 
setting of persistent or progressive disease on post-transplant evaluation, this 
agent may be reinitiated, if clinically indicated, either alone or prior to DLI.    
 
7.5 
Use of donor lymphocyte infusions (DLI) following development of aGvHD: 
Patients developing grade II or greater GvHD after transplant or after DLI will not 
receive further lymphocyte add-back (DLI) unless GvHD has been successfully 
treated (i.e. off steroids) or tumor progression is documented. 
 
7.6 
Acute GvHD following DLI:  See DHMC BMT SOP on the Treatment of Acute 
GvHD. 
 
7.7 
Hospital Discharge 
Recommended guidelines for discharge include the following:  
1. 
Afebrile with ANC > 0.5 x 109/l and no active, untreated infection. 
2. 
Patient can perform daily activities at home, with caregiver assistance. 


15 
 
3. 
Patient able to tolerate oral fluids and medications sufficiently to maintain 
adequate hydration (goal > 2 liters po/day). 
4. 
Patient and caregiver’s understanding of outpatient care is adequate. 
 
8.0 
MANAGEMENT OF POST-TRANSPLANT COMPLICATIONS 
8.1 
The major complications of allogeneic transplantation include CMV reactivation, 
veno-occlusive disease (VOD), acute and chronic GvHD, disease progression, graft 
failure and opportunistic infection (bacterial/fungal). Patients with these 
complications will be treated according to DHMC Stem Cell Transplant Standard 
Operating Procedures (SOPs). 
 
8.2 
Treatment of Acute and Chronic GvHD: 
Patients who develop grade >II GvHD will be treated initially with steroids, 
followed by other immunosuppressive agents, as clinically indicated.  Unless the 
GvHD has been successfully treated, these patients will not receive additional donor 
lymphocyte infusions.  (See DHMC BMT SOP on the Treatment of Acute and 
Chronic GvHD.)   
 
9.0 
POTENTIAL TOXICITIES, HAZARDS AND DISCOMFORTS 
9.1 
The patient: The mortality from conventional allogeneic BMT may be as high as 
40% or greater. Although we anticipate that this protocol is relatively safe, the 
procedure, nevertheless, carries some risk. The major hazards (GvHD, VOD, 
infection, graft failure, and disease progression) will be reviewed in detail by the 
BMT Attending Physician during discussions with all prospective patients and their 
families. The major discomforts are those of nausea, mucositis, anorexia, diarrhea, 
fever and malaise. Side effects of the common drugs used in this regimen include: 
Cyclophosphamide 
Marrow suppression, nausea, mucositis, rash, hemorrhagic cystitis, 
myocardial damage, alopecia, infertility, nausea and vomiting. 
Fludarabine 
Myelosuppression, fever and chills, nausea and vomiting, malaise, fatigue, 
anorexia, weakness, neurologic toxicity, and interstitial pneumonitis. 
Serious opportunistic infections have occurred in CLL and other patients 
treated with fludarabine. 
Ganciclovir 
Nausea, anorexia, abdominal discomfort, pancytopenia, neutropenia, 
peripheral neuropathy, renal damage (reversible on drug discontinuation). 
Cyclosporine 
Renal impairment, hypertension, painful hands and feet, elevated bilirubin, 
hypertrichosis, nausea, tremor, seizure, hypomagnesemia, thrombotic 
thrombocytopenic purpura (TTP). 
Methotrexate 
Mucositis, myelosuppression, renal impairment, hepatic damage, rash, 
alopecia, fever, nausea, vomiting, diarrhea, abdominal pain, GI bleeding, 
intestinal perforation. 


16 
 
Antimicrobials (in general) 
Allergic reactions, renal impairment, nausea, vomiting, hepatic damage, 
marrow suppression 
Antithymocyte globulin 
Allergic reactions, anaphylaxis, serum sickness, rash, fever, chills,   
myelosuppression 
 
10.0 
WITHDRAWAL FROM THE STUDY 
10.1 
Withdrawal from the transplant procedure 
Participants may withdraw from the trial prior to the recipient’s admission to the 
hospital for the transplant. Thereafter, the nature of the procedure does not permit 
safe withdrawal from the protocol.   
10.2 
Withdrawal from experimental protocol 
10.2.1 The participant has the right at any time not to participate in the trial.  All 
participants who sign an informed consent will be followed, regardless of 
their clinical status unless the participant withdraws consent for using their 
health information for research purposes. 
10.2.2 Although all patients that sign an informed consent will be followed, 
patients will not be treated according to the trial if the following occurs: 
 The recipient fails to achieve allogeneic marrow engraftment (donor 
chimerism or  mixed chimerism)  
 Patients with disease relapse after the day +180 restaging who have 
active grade > II GvHD or who have failed to respond to a previous 
donor lymphocyte transfusion  
 Patients with disease progression associated with a significant decline in 
performance status, which negates further treatment on the trial (e.g., 
lymphocyte add-back) 
 
11.0 
RESPONSE CRITERIA AND EVALUATION OF RESULTS  
11.1 
OBJECTIVES 
 To evaluate disease response rate as defined in section 12.3. 
 To evaluate engraftment by measuring donor-recipient chimerism in lymphoid and 
myeloid lineages 
 To investigate the effect of donor lymphocyte infusion on donor-host chimerism 
 To determine the incidence and severity of acute and chronic graft-versus-host 
disease (GvHD) 
 To assess the following: 
 treatment-related mortality 
 time to progression 
 disease-free survival 
 overall survival 
 regimen-related toxicity 
 


17 
 
12.0 
STATISTICS  
 
12.1 
Endpoints – The endpoints of this trial include rates of successful engraftment, 
complete donor chimerism, graft-versus-host disease (acute and chronic), mortality, 
toxicity, and tumor response rate.  The trial will be amended or closed if excessive 
toxicity or mortality is determined. The specific toxicity stopping criteria are as  
follows:   
o Greater than 33% incidence of severe (Grade III/IV) graft-versus-host 
disease at day +30 among the first 6 patients enrolled in this study. 
o Greater than 33% mortality at day +60 among the first 6 patients enrolled in 
this study. 
                         
                        The 7th patient will not be enrolled until the results from the first 6 patients are  
                        analyzed and it has been determined that there is no excessive toxicity.  If at any  
                        subsequent time these toxicity criteria are exceeded, accrual to the trial will be  
                        suspended and the DSMB will be notified and asked to make a recommendation for  
                        continuing, stopping or modifying the trial.   
12.1.1 Engraftment will be defined as neutrophil recovery to > 0.5 x 109/L for 3 
consecutive days and an untransfused platelet count > 20 x 109/L. 
12.1.2 Chimeric studies will be undertaken on post-transplant days +30, +60, +100, 
+180 and +365 (all visits +/- 15 days) and as otherwise clinically indicated, 
according to the method of lineage-specific chimerism analysis described in 
detail in Section 5.3.2.  Complete donor chimerism will be defined as the 
presence of at least 95% donor DNA in the T-lymphoid (or unfractionated) 
sample analysis. 
12.1.3 Acute and chronic GvHD will be assessed according to the criteria and 
grading scales of the International Bone Marrow Transplant Registry, as 
depicted in Appendix III. 
12.1.4 Statistical analysis for the primary endpoints – Straightforward descriptive 
statistical methods will be used to summarize the primary endpoints.  Rates 
of successful engraftment, complete donor chimerism, graft-versus-host 
disease (acute and chronic), and toxicity will be computed with 95% 
confidence intervals. Toxicity will be summarized according to type and 
grade.  In addition, rates of any grade 3 or worse toxicity will be computed.  
Mortality, measured from the time of transplant, will be assessed using the 
product-limit method, and the survival curve will be graphed.  
12.2 
One of the endpoints of this study is tumor response rate as defined in section 12.3.  
It is expected that a response rate between 40% and 60% would represent a 
successful outcome and would suggest that further studies be conducted to more 
completely evaluate efficacy of the experimental treatment (i.e., the treatment will 
be accepted for further study).  The target response rate for this trial is 45%, and a 
response rate of 20% or less would indicate that the experimental treatment should 
not be recommended for further study (i.e., the treatment would be rejected).   
12.3 
Statistical analysis for assessment of tumor response rate will utilize an optimal 2-
stage design.  In the first stage, 10 patients evaluable for response will be accrued. 
If 3 or more patients experience tumor response (complete or partial) in the first 
stage, the study will progress to the second stage; otherwise, the study will be 


18 
 
terminated and the treatment will be rejected.  In the second stage, 12 additional 
patients evaluable for response will be accrued, for a total of 22 patients.  If 8 or 
more patients experience tumor response in the full patient sample, then the 
treatment will be accepted for further study; otherwise, it will be rejected.  Tumor 
response criteria will vary by disease, as outlined in Appendix I.  (Note: patients 
being transplanted with high-risk disease in first or subsequent remission will not be 
evaluable for response by standard criteria. In this patient subset, determination of 
treatment efficacy will be based empirically on the absence or development of 
relapse by morphologic, immunophenotypic, cytogenetic and/or molecular criteria).  
Tumor response rate will be determined as the ratio of number of complete and 
partial responders to the total number of patients evaluable for response.  The 
complete response rate will be determined as the ratio of complete responders to the 
total number of patients evaluable for response.  A 95% confidence interval for the 
response rate will be computed using exact binomial methods. 
12.4 
Sample size justification – The sample size is based on Simon’s optimal 2-stage 
design (Simon R. Optimal two-stage designs for phase II clinical trials. Controlled 
Clinical Trials 10:1-10; 1989.). The probability of early stopping is 0.68 and 0.10 
for true remission rates of 20% and 45%, respectively.  If the true response rate is 
20%, the probability of accepting the treatment for further study is 0.05 (statistical 
significance level).  For a true response rate of 45%, this probability is 0.81 
(statistical power). With 22 patients, the 95% confidence interval for the response 
rate will have a half width not exceeding +/- 21%.   
 
13.0 
CHIMERISM STUDIES  
13.1 
VNTR and STR analysis of post-transplant peripheral blood lymphocytes and bone 
marrow will be assessed in serial fashion to determine the chimeric status of the 
patient as a function of time. These studies will be repeated after lymphocyte add 
back to assess the effect of DLI on host chimeric status as well. The relationship 
between degrees of donor-host chimerism, GvHD and tumor response will be 
analyzed.   
See Section 5.3  (Lineage-specific Chimerism Analysis).  
 
14.0 
REGISTRATION GUIDELINES  
 
14.1 
This is a non-randomized study. 
14.2 
To enter eligible patients or discuss a patient’s eligibility, please contact Dr. John 
Hill or any of the BMT physicians or the BMT Clinical Research Associate (Cynara 
Nayar, MPH. at 603-650-6240.  Eligibility will be documented as part of the admit 
packet. No patients shall be entered on study without consultation with the Principal 
Investigator and BMT Research team. 
 
15.0 
DATA COLLECTION AND DATA AND SAFETY MONITORING 
The Safety and Data Monitoring Committee of the Norris Cotton Cancer Center will 
review this protocol on a quarterly basis.  The Dartmouth College IRB will review this 
protocol on an annual basis.  All data will be maintained in a closed data base with each 


19 
 
subject identified by a unique patient number (UPN), with access to patient names only for 
those involved in clinical care.  All patients will provide informed consent prior to study 
entry.  Necessary information will be forwarded to the North American Bone Marrow 
Transplant Registry (NABMTR). 
 
16.0 
ADVERSE DRUG REACTION (ADR) REPORTING 
Investigators are required by law to notify the IRB of adverse drug reactions.  In addition, 
the study investigators should be notified.  All investigators are required to report 
secondary malignancies occurring on or following treatment on NCI-sponsored protocols 
using commercial drugs. Reporting is to be performed in the same manner as reporting 
Adverse Drug Reactions, including (within ten working days) completion of the FDA Form 
1639.  
 
Reporting requirements and procedures depend upon: 1) whether investigational agents are 
suspected of causing toxicity, 2) whether the possibility of such a toxicity was reported in 
the protocol, consent form, or manufacturer’s literature [Expected Toxicity], 3) the severity 
or grade of the toxicity.   
 
All grade 4 and 5 toxicities should be reported to the study investigators immediately.  
 
The FDA form 1639 is used for reporting commercial drug toxicities. 
 
All adverse reactions should also be reported to the local Institutional Review Board.   
 
Each patient must be fully informed concerning this study, including pertinent adverse 
reactions.  All institutional or other Federal regulations and guidelines concerning informed 
consent will be fulfilled. 


20 
 
 
17.0 
REFERENCES 
 
1. Thomas E, Blume K, Forman S, editors. Hematopoietic Cell Transplantation. 2nd ed. Malden, 
ME: Blackwell Science, Inc.; 1999 
2. Barnes D, Loulit L, Neal F. Treatment of murine leukemia with X-rays and homologous bone 
marrow. British Medical Journal 1956;2:626-30. 
3. Truitt RL, Johnson BD. Principles of graft-vs. leukemia reactivity. Biol Blood Marrow 
Transplant 1995;1(20:61-68 
4. Gale RP, Champlin RE. How does bone marrow transplantation cure leukemia? Lancet 
1984;2(?8393):28-30 
5. Horowitz MM, Gale RP, Sondel PM, Goldman JM, Kersey J, Kolb HJ, et al. Graft-versus-
leukemia reactions after bone marrow transplantation. Blood 1990; 75(3):555-62. 
6. Giralt S, Estey E, Albitar M, van Besien K, Rondon G, Anderlini, et al. Engraftment of 
allogeneic hematopoietic progenitor cells with purine analog-containing chemotherapy: 
harnessing graft-vs.-leukemia effect without myeloablative therapy. Blood 1997;89(12):4531-
36 
7. Khouri IF, Keating M, Korbling M, Przepiorka D, Anderlini, O’Brien S, et al. Transplant-lite: 
induction of graft-versus-malignancy using fludarabine-based nonablative chemotherapy and 
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21 
 
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discontinuation 
of 
immunosuppression. 
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23 
 
Appendix I:  Disease Staging and Response Definitions  
 
Staging  
 
1. CML  
a. 
Physical Exam 
b. 
Bone marrow aspiration and biopsy  
c. 
Cytogenetics  
d. 
BCR/ABL gene rearrangement by PCR  
 
2. AML, MDS  
a. 
Physical Exam 
b. 
Bone marrow aspiration and biopsy  
c. 
Cytogenetics  
d. 
Disease-specific translocation by FISH (as indicated) 
 
3. Multiple myeloma  
a. 
Physical Exam 
b. 
Bone marrow aspiration and biopsy  
c. 
M-protein measurement (SPEP or 24 hour urinary light chain excretion light chain 
disease)  
d. 
SIEP, UIEP  
e. 
Cytogenetics 
 
4. NHL, HD, CLL (only tests that were abnormal initially, unless otherwise indicated) 
a. 
Physical exam  
b. 
CT scans  
c. 
Bone marrow aspiration and biopsy  
d. 
Cytogenetic analysis  (as indicated) 
e. 
Disease-specific translocation by PCR (as indicated)  


24 
 
Appendix I (continued):  
Response Definitions  
 
1.  CML  
a. Complete response (CR): Resolution of bone marrow and blood morphologic abnormalities 
and absence of Philadelphia chromosome by standard cytogenetic analysis.  
b. Molecular complete remission: In addition to criteria above for complete remission, 
BCR/ABL rearrangement by RT-PCR is absent.  
2.  AML/ALL  
a. Complete response: Resolution of abnormal blood and bone marrow morphology with less 
than 5% blasts and resolution of disease-specific chromosome abnormality (if present) by 
standard cytogenetic analysis.  
b. Molecular complete remission: In addition to criteria above for a complete response, 
disease-specific rearrangement is absent by PCR analysis.  
3.  MDS 
a. Complete response: Resolution of abnormal blood and bone marrow morphology with less 
than 5% blasts and resolution of disease-specific chromosome abnormality (if present) by 
standard cytogenetic analysis.  
b. Partial response: Decrease in bone marrow and peripheral blood blast count by > 50%.  
c. Molecular complete remission: In addition to criteria above for a complete response, 
disease-specific rearrangement is absent by PCR analysis.  
4.  Multiple myeloma  
a. Complete response: Bone marrow less than 5% plasma cells, polytypic by 
immunohistology, 
and 
absence 
of 
M-protein 
on 
electrophoresis 
and 
immunoelectrophoresis.  Resolution of chromosome abnormality (e.g., chromosome 13 
deletion), if present, by standard cytogenetic analysis.  Absence of skeletal lesion 
progression by bone survey. 
b. Partial response: Reduction in M-protein by > 50% (and absence of skeletal lesion 
progression by bone survey). 
5. NHL, HD, CLL  
a. Complete response: Resolution of disease by physical findings and normalization of all 
previously abnormal clinical tests such as bone marrow examination and computed 
tomographic scans (CT). In addition, resolution of disease-specific chromosome 
abnormality (if present) by standard cytogenetics.  
b. Partial response: Reduction in disease volume in all measurable sites by > 50%, using 
product of diameters.  
c. Molecular complete remission: In addition to criteria for a complete response, disease-
specific rearrangement is absent by PCR analysis.  
 


25 
 
Appendix II: Prophylaxis and Treatment of Infection 
 
 
Antibacterial Prophylaxis: 
 
Ciprofloxacin 750 mg PO BID beginning day 0 until neutrophil recovery  
Ciprofloxacin 400 mg IV q12h if unable to take ciprofloxacin by mouth 
 
Bactrim DS one tablet PO every Monday, Wednesday, and Friday, from count recovery until 6 
months following BMT (longer if ongoing immunosuppressive therapy and/or chronic GvHD). 
 
Antifungal Prophylaxis: 
 
Fluconazole 400 mg PO/IV QD beginning day 0 until engraftment (if day +30 chimerism status 
suboptimal, continue until day +75). Fluconazole will be discontinued if started on IV 
amphotericin. 
 
Antiviral Monitoring and Prophylaxis: 
 
1. 
CMV antigenemia testing will be undertaken between day +10 and day +100 for 
pre-transplant CMV seropositive patients or patients receiving CMV seropositive 
stem cells at transplant.  If CMV antigenemia is detected, Ganciclovir will be 
initiated.  
2. 
HSV positive recipient or donor:  Acyclovir 400 mg PO TID (or 250 mg/m2 IV 
q12h if unable to take PO) from first day of preparative regimen until discharge; at 
discharge, change to valacyclovir 500 mg PO QD until day +100.  
3. 
HSV negative recipient and donor:  no prophylaxis 
 
Immunoglobulin:  
 
Weekly IgG level surveillance starting day +1 until day +100.  When levels fall below 400mg/dL, 
give IVIG 125mg/kg/wk IV.  If IgG is still <400 mg/dL on subsequent checks, increase to IVIG 
250 mg/kg (or 500 mg/kg, if necessary) to ensure IgG > 400mg/dL prior to day +100 post-
transplant.  After day +100 post-transplant, IVIG will only be continued on a monthly basis and in 
the setting of persistent IgG <400mg/dL .  This will not be given beyond day +360 post-transplant.   
 
Fever Regimen: 
 
Fever above 38.2 C or sustained fever with two or more readings above 38.0 C. Any temperature 
with clinical suspicion of bacteremia:  see DHMC SOP for Antimicrobial Infections and Protocol. 
 


26 
 
 
Appendix III: GvHD Grading Scale 
 
1.  Acute GvHD  (see DHMC SOP for the Diagnosis and Treatment of Acute GvHD).  
 
 
a. If patient develops acute GvHD at any time after BMT, acute GvHD is graded as outlined 
below. 
b. If patient dies before day 30 and has not developed acute GvHD, then the patient is 
considered not evaluable for GvHD. 
 
Clinical Stage of Acute GvHD according to Organ System 
Stage 
Skin 
Liver 
Intestine 
+ 
Maculopapular rash < 25% of 
body surface 
Bilirubin 2-3 mg/dl 
>500-1000 ml diarrhea /day or 
nausea, anorexia or vomiting 
with biopsy (EGD) confirmation 
of upper GI GvHD 
++ 
Maculopapular rash  25-50% if 
body surface 
Bilirubin 3-6 mg/dl 
>1000-1500 ml diarrhea/day 
+++ 
Maculopapular rash . 50% of 
body surface area or generalized 
erythroderma 
Bilirubin 6-15 mg/dl 
>1500 ml diarrhea/day 
++++ 
Generalized erythroderma with 
bullous formation and 
desquamation  
Bilirubin > 15mg/dl 
>1500 ml diarrhea/day plus 
severe abdominal pain with or 
without ileus 
 
 
 
Overall Clinical Grading of Severity of Acute GvHD 
Grade 
Skin 
Liver 
Gut 
I 
1-2 
0 
0 
II 
0 
0 
1-3 
1-3 
1-3 
3 
0-1 
1 
0-1 
1 
0 
X 
1 
0-1 
1 
0-1 
0 
X 
 
III 
0-3 
0-3 
0-3 
2-3 
0-3 
4 
0-2 
2-3 
0-3 
IV 
0-3 
4 
0-4 
0-4 
4 
0-4 


27 
 
 
Appendix III (Continued): 
 
2.  Chronic Graft-vs.-Host Disease (See DHMC SOP for the Diagnosis and Treatment of 
Chronic GvHD). 
l. 
If patient develops chronic GvHD at any time after BMT, chronic GvHD is graded as 
outlined below.  
m. 
If the patient dies before day 100 and has not developed chronic GvHD, then the patient is 
considered not evaluable for chronic GvHD.  
•  Limited chronic GvHD 
Either or both  
 Localized skin involvement  
 Hepatic dysfunction due to chronic GvHD  
•   Extensive chronic GvHD  
Either  
 Generalized skin involvement, or  
 Localized skin involvement and/or hepatic dysfunction 
Plus  
 Liver histology showing chronic aggressive hepatitis, or  
 Involvement of eye (Schirmers test with less than 5mm wetting), or  
 Involvement of minor salivary glands or oral mucosa demonstrated on labial 
biopsy, or  
 Involvement of any other target organ  
 
3.  Pancytopenia  
Occurrence of an absolute neutrophil count less than 500/l or platelet count less than 20,000/l at 
any time after DLI, with the pancytopenia not deemed to be due to underlying hematologic disease 
or chemotherapy.  
  
 
 


28 
 
 
Appendix IV: Toxicity Scale 
 
 
See NCI Common Toxicity Criteria (version 2).