Clinical trial NCT00789776
Conditions: Acute Lymphoblastic Leukemia Acute Myeloid Leukemia Aggressive Non-Hodgkin Lymphoma Diffuse Large B-Cell Lymphoma Previously Treated Myelodysplastic Syndrome Recurrent Chronic Lymphocytic Leukemia Recurrent Chronic Myelogenous Leukemia, BCR-ABL1 Positive Recurrent Indolent Adult Non-Hodgkin Lymphoma Recurrent Mantle Cell Lymphoma Recurrent Plasma Cell Myeloma Recurrent Small Lymphocytic Lymphoma Refractory Chronic Lymphocytic Leukemia Refractory Hodgkin Lymphoma Refractory Plasma Cell Myeloma Refractory Small Lymphocytic Lymphoma Waldenstrom Macroglobulinemia
Sponsor: Fred Hutchinson Cancer Center
Start date: 2008-10-13; Completion date: 2017-05-01
Enrollment: 41 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This phase I/II trial studies the side effects and best dose of donor natural killer (NK) cell therapy and to see how well it works when given together with fludarabine phosphate, cyclophosphamide, total-body irradiation, donor bone marrow transplant, mycophenolate mofetil, and tacrolimus in treating patients with hematologic cancer. Giving chemotherapy, such as fludarabine phosphate and cyclophosphamide, and total-body irradiation before a donor bone marrow transplant helps stop the growth of cancer cells. It may also stop the patient's immune system from rejecting the donor's stem cells. When the healthy stem cells from a donor are infused into the patient they may help the patient's bone marrow make stem cells, red blood cells, white blood cells, and platelets. Giving an infusion of the donor's T cells (donor lymphocyte infusion) may help the patient's immune system see any remaining cancer cells as not belonging in the patient's body and destroy them (called graft-versus-tumor effect). Sometimes the transplanted cells from a donor can make an immune response against the body's normal cells. Giving mycophenolate mofetil and tacrolimus after the transplant may stop this from happening.

Detailed description:
PRIMARY OBJECTIVES:

I. Identification of the maximal feasible dose of NK cells that can be infused one week after nonmyeloablative, human leukocyte antigen (HLA)-haploidentical hematopoietic cell transplant (HCT). (Phase I)

SECONDARY OBJECTIVES:

Once the maximal feasible dose has been identified, accrual will be limited to the cohort containing this cell dose to determine:

I. Incidence of relapse. (Phase II)

II. Incidence of grades III-IV acute graft-versus-host disease (GVHD). (Phase II)

III. Incidence of non-relapse mortality. (Phase II)

OUTLINE: This is a phase I, dose-escalation study of donor NK cell therapy followed by a phase II study.

CONDITIONING: Patients receive fludarabine intravenously (IV) over 30 minutes on days -6 to -2 and cyclophosphamide IV over 1 hour on days -6 and -5. Patients undergo total-body irradiation on day -1.

DONOR BONE MARROW TRANSPLANTATION: Patients undergo donor bone marrow transplantation on day 0.

POST-TRANSPLANTATION IMMUNOSUPPRESSION: Patients receive cyclophosphamide IV over 1 hour on day 3 and mycophenolate mofetil orally (PO) thrice daily (TID) on days 4 to 40, followed by a taper until day 84 in the absence of GVHD. Patients also receive tacrolimus IV continuously or IV once daily (QD) over 1-2 hours or PO twice daily (BID) on days 4 to 84, followed by a taper until day 180 in the absence of GVHD.

DONOR NK CELL INFUSION: Patients undergo donor lymphocyte infusion of NK cells on day 7.

After completion of study treatment, patients are followed up at 6 months, 1 year, 1.5 years, and then every year thereafter.

Arms: arm 1: CONDITIONING: Patients receive fludarabine IV over 30 minutes on days -6 to -2 and cyclophosphamide IV over 1 hour on days -6 and -5. Patients undergo total-body irradiation on day -1.

DONOR BONE MARROW TRANSPLANTATION: Patients undergo donor bone marrow transplantation on day 0.

POST-TRANSPLANTATION IMMUNOSUPPRESSION: Patients receive cyclophosphamide IV over 1 hour on day 3 and mycophenolate mofetil PO TID on days 4 to 40, followed by a taper until day 84 in the absence of GVHD. Patients also receive tacrolimus IV continuously or IV QD over 1-2 hours or PO BID on days 4 to 84, followed by a taper until day 180 in the absence of GVHD.

NK CELL INFUSION: Patients undergo donor lymphocyte infusion of NK cells on day 7.
Interventions: intervention 1: Allogeneic Bone Marrow Transplantation intervention 2: Cyclophosphamide intervention 3: Fludarabine Phosphate intervention 4: Laboratory Biomarker Analysis intervention 5: Mycophenolate Mofetil intervention 6: Natural Killer Cell Therapy intervention 7: Tacrolimus intervention 8: Total-Body Irradiation
Intervention descriptions: intervention 1: Undergo donor bone marrow transplantation intervention 2: Given IV intervention 3: Given IV intervention 4: Correlative studies intervention 5: Given PO intervention 6: Given IV intervention 7: Given IV or PO intervention 8: Undergo total-body irradiation

Protocol documents:
Prot_SAP_001.pdf: 
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FRED HUTCHINSON CANCER RESEARCH CENTER 
UNIVERSITY OF WASHINGTON SCHOOL OF MEDICINE, 
DEPARTMENT OF MEDICINE, DIVISION OF ONCOLOGY 
SEATTLE CHILDREN’S 
 
Previous version: 02/05/2015 
Current version:   02/23/2016 
 
I. Title:  A Phase I/II Study Evaluating the Safety and Efficacy of Adding a Single 
Prophylactic Donor Lymphocyte Infusion (DLI) of Natural Killer Cells Early After 
Nonmyeloablative, HLA-Haploidentical Hematopoietic Cell Transplantation – A Multi 
Center Trial 
 
 
Investigator 
Professional Title 
Phone (206) 
Brenda Sandmaier, MD 
Member, FHCRC; Professor of Medicine, UW 
667-4961 
Shelly Heimfeld, PhD 
Member, FHCRC 
667-4004 
Effie Petersdorf, MD 
Member, FHCRC;  Professor of Medicine, UW 
667-5244 
Ann Woolfrey, MD 
Member, FHCRC; Professor of Pediatrics, UW 
667-4453 
Michael Linenberger, MD 
Associate Member, FHCRC; Professor of 
Medicine, UW 
 
Mohamed Sorror, MD,MSc 
Associate Member, FHCRC, Associate Professor 
of Medicine, UW 
667-2765 
David Maloney, MD, PhD 
Member, FHCRC; Professor of Medicine, UW 
667-5616 
Rainer Storb, MD 
Member/Program Head, Transplantation Biology, 
FHCRC; Professor of Medicine, UW 
667-4407 
 
Statistician: Barry Storer, PhD, Member, FHCRC, 206-667-6151 
 
 
Additional Performance Sites 
Investigators 
Emergency Phone Numbers 
Medical College Wisconsin – 
Children’s Hospital of Wisconsin 
Monica Thakar, MD 
(414) 456-7546    
Medical College of Wisconsin – 
Froedert Hospital 
Parameswaran Hari, 
MD 
(414) 805-4600 
 
Research Nurses: 
 
Michelle Bouvier, RN  (206) 667-6993, pager (206) 995-7658 
 
Emergency 24 Hour Phone:  
FHCRC:    
 
 
Phone:  
(206) 598-8902    FAX: (206) 598-4034 
Seattle Children’s:  Phone: 
(206) 987-2000*  FAX: (206) 987-2764 
 
*Ask Seattle Children’s Operator for “Transplant Provider On-Call” 
 
 
 
 
 
 
 
 


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TABLE OF CONTENTS 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
      page 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
I. 
 
TITLE PAGE…………………………………………………………………………………………...1 
 
II.  
INTRODUCTION………………………………………………………………………………………3 
 
III.  
BACKGROUND………………………………………………………………………………………..3 
 
IV.  
RATIONALE FOR STUDY…………………………………………………………………………..14 
 
V.  
OBJECTIVES…………………………………………………………………………………………14 
 
VI.  
PATIENT SELECTION………………………………………………………………………………14 
 
VII.  
DONOR SELECTION………………………………………………………………………………..16 
 
VIII. 
INFORMED CONSENT……………………………………………………………………………..16 
 
IX.  
PROTOCOL REGISTRATION……………………………………………………………………...17 
 
X.  
PLAN OF TREATMENT……………………………………………………………………………..17 
 
XI.  
DONOR AND RECIPIENT EVALUATIONS……………………………………………………….24 
 
XII.  
DRUGS, IRRADIATION AND MARROW ADMINISTRATION - TOXICITIES AND 
COMPLICATIONS…………………………………………………………………………………...47 
 
XIII.  
INFECTIOUS DISEASE PROPHYLAXIS AND THERAPY……………………………………...50 
 
XIV.  
GRAFT-VERSUS-HOST DISEASE.......................................................................................50 
 
XV.  
EVALUATION AND ENDPOINT DEFINITIONS………………………………………………….50 
 
XVI.  
STATISTICAL ANALYSIS…………………………………………………………………………..52 
 
XVII. 
DATA SAFETY AND MONITORING PLAN AND ADEVERSE EVENT AND SAFETY 
MONITORING………………………………………………………………………………………..54 
 
XVIII. 
RECORDS……………………………………………………………………………………………56 
 
XIX. 
PROJECTED TARGET ACCRUAL………………………………………………………………..56 
 
REFERENCE LIST……………………………………………………………………………………………57 
 
APPENDICES LIST……………………………………………………………………………………………61 
 
 
 
 
 
 
 
 
 


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II. INTRODUCTION 
 
Often the only cure for high-risk hematological malignancies is allogeneic hematopoietic cell 
transplantation (HCT). However, finding suitable HLA-matched donors continues to be markedly 
difficult for certain patients, especially those having small family units, backgrounds of high 
genetic diversity, or those belonging to underrepresented ethnicities. In this regard, HLA-
haploidentical family donors have emerged as easily accessible and viable alternative donor 
sources. In FHCRC Protocol 1667, the use of HLA-haploidentical related donors with 
nonmyeloablative conditioning and potent immunosuppression has proven to be a well-tolerated, 
low-toxicity regimen, with a majority of patients engrafting and having a low incidence of severe 
graft-versus-host disease (GVHD). This is in contrast to many other studies, where graft 
rejection, GVHD, or infections due to delayed immune reconstitution tend to be excessive after 
using HLA-haploidentical donors. In particular, the Perugia group has explored the use of HLA-
haploidentical donors in the myeloablative setting. Using grafts containing a “mega-dose” of 
CD34 enriched, extensively T cell depleted stem cells, they observed an excessive mortality of 
40% due to infectious complications. The lower rates of non-relapse mortality (NRM) seen in 
Protocol 1667 may be credited to using a nonmyeloablative preparative regimen, thus reducing 
toxicity and infectious risks, using  bone marrow over mobilized peripheral blood stem cells 
(PBSC), thus reducing GVHD, and using cyclophosphamide (CY) after HCT, which causes 
bidirectional deletion of alloreactive T cells responsible for GVHD and graft rejection. 
 
However, while we have overcome this strong immunological barrier to provide both engraftment 
and low GVHD, 57% of patients are dying by two years after HCT from either early or late 
relapse despite most having full donor chimerism. This has led to the hypothesis that CY 
treatment after HCT ultimately contributed to the elimination of important donor T as well a 
natural killer (NK) cell clones necessary for immune surveillance and tumor kill. In order to 
preserve the advantage of low GVHD while improving tumor surveillance and control, we now 
propose two separate interventions: 1) continuing to eliminate alloreactive lymphocyte clones 
early after HCT with CY, and later 2) augmenting the graft-versus-tumor (GVT) effect with an 
infusion of purified donor NK cells. Donor lymphocyte infusions (DLI) containing high numbers of 
CD3+ cells can successfully elicit GVT effects after transplantation, but its use is limited in the 
early period after HCT due to the risk of developing myelosuppression, seen particularly in 
marrow-occupying malignancies, and life-threatening GVHD. The add-back of DLI purged of 
alloreactive T cells has also been studied by many groups seeking to improve immune 
reconstitution after T-cell depleted, HLA-haploidentical HCT, although this may not be an 
obvious solution to counteract relapse. Purified NK cell add-back studies have been attempted in 
the clinical setting and have corroborated the feasibility of NK cell enrichment, its ability to 
facilitate GVT effects, and have confirmed its low potential to elicit GVHD and other toxicities in 
the post-transplantation setting. Building upon these promising results, we now propose the use 
of a single, purified infusion of donor NK cells on day +7 after HLA-haploidentical, 
nonmyeloablative HCT as pre-emptive, adoptive immunotherapy to prevent relapse. The effects 
of this donor NK infusion on engraftment kinetics, NK cell phenotype development, killer cell 
immunoglobulin-like receptor (KIR) expression, and T cell reconstitution will also be monitored 
closely after HCT.  
 
III. BACKGROUND 
 
A. A Brief Review of Allogeneic HCT Using HLA-Haploidentical Donors 
 
HLA-haploidentical stem cells have been recognized as an important alternative donor source, 
with multiple groups around the world attempting to improve outcomes using either 


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Table 1. Selected outcomes of interest from HLA- haploidentical related treatment regimens using myeloablative and 
non-myeloablative regimens1 
Institution 
n= 
Preparative 
Regimen 
GVHD ppx 
1˚ Graft 
Failure 
Acute 
GVHD 
NRM 
Outcomes 
MYELOABLATIVE 
FHCRC2 
269 
CY/TBI 
CSP±MTX;MTX only; 
none 
8.5% 
“mod-
severe”: 
67% 
NA 
(17/269: 
aplasia) 
NA 
Royal Marsden 
35 
CY/TBI or CY/Mel 
CSP ±MTX 
29% 
80% 
57% 
OS 6 mo-3 yrs: 
31% 
Milwaukee 
48 
TBI/CY/AraC/ MP 
Ex vivo TCD w/ T10B9 or 
OKT3 
4% 
II-IV: 46% 
42% 
2 yr OS: 21%;        
2 yr DFS: 17% 
JSHCT 
142 
TBI/CY or Bu/CY 
CSP/MTX (87%) or 
others (13%) 
6.3% 
III-IV: 30% 
NA 
NA 
USC 
201 
TBI/VP16/ AraC/CY/ 
ATG 
CSP, partial ex vivo 
TCD (w/ T10B9 or OKT3), 
MP, ATG 
2% 
II-IV: 13% 
III-IV: 15% 
51% 
5 yr OS:19% 
5 yr DFS: 18% 
Beijing, China 
135 
Bu/CY/AraC/ MeCCNU/ 
ATG 
CSP/MTX/ MMF+ in 
vitro TCD w/ ATG 
0% 
II-IV: 40% 
III-IV: 16% 
22% 
2 yr OS: 71% 
2 yr DFS: 64% 
Perugia 
104 
TBI/TT/FLU/ATG 
Ex vivo TCD via CD34 
selection 
7% 
II-IV: 8% 
40% 
5 yr EFS: 47% (for 
pts in remission at 
HCT); 4% (in rel at 
HCT 
Tubingen 
63 
TBI or BU-based with 
FLU/TT/ATG/CY 
Ex vivo TCD via CD34 
or CD133 selection 
17% 
II: 7% 
III-IV: 0% 
29% 
3 yr DFS: 48% 
(NHL/ ALL in 
remission at HCT); 
0% (no remission) 
60%: NMD 
Japan 
Multicenter 
Study 
135 
TBI-based in majority of 
pts 
CSP/FK506/MTX/steroid
s ± ex vivo TCD via 
CD34 selection 
13% 
II-IV: 21% 
47% 
5 yr DFS: 39% (std-
risk pts) 
5% (high-risk pts) 
Canadian 
Multicenter 
Study 
11 
Mel/TT/FLU/ATG 
Ex vivo TCD via CD34 
selection 
0% 
0% 
55% 
9 mo DFS: 9%;   
OS: 9% 
NON-MYELOABLATIVE 
Duke Univ 
49 
FLU/CY/ 
Campath 
MMF±CSP + in vivo 
TCD via Campath 
6% 
II-IV: 16% 
III-IV: 8% 
10.2% 
1 yr OS: 31% 
Tubingen/ 
Dresden 
10 
FLU/TT/Mel/ 
OKT3 
Ex vivo TCD via anti-
CD3/CD19 MAb 
0% 
II: 60%; III: 
0% 
IV: 10% 
30% 
OS: 50% 
Tubingen 
38 
FLU/TT/Mel 
Ex vivo TCD via anti-
CD3/CD19 MAb 
17% 
II-IV: 27% 
2.6% 
EFS: 70% 
(good risk) 
20% (poor risk) 
MGH 
12 
CY/Anti-CD2Mab/ 
Thymic XRT 
CSP±ex vivo TCD via 
CD34 selection and in 
vivo TCD via anti-CD2 
MAb 
0% 
II-IV: 17% 
25% 
15-34 mo DFS: 
17%; OS: 25% 
Osaka Univ 
26 
FLU/BU/ATG 
FK506/MP + in vitro 
TCD via ATG 
4% 
II: 20% 
III-IV: 0% 
15% 
3 yr EFS: 55% 
664day OS: 58% 
Tokyo Univ 
12 
Myelo (n=6): 
TBI/CY/Campath 
RI: FLU/BU/ Campath± 
TBI 4 Gy 
CSP/MTX + in vivo TCD 
via Campath 
0% 
III-IV: 9% 
17% 
1 yr OS: 35% 
JHU 
13 
FLU/CY/TBI 
CSP/MMF + in vivo TCD 
via CY 
31% 
II-IV: 17% 
8% 
6 mo DFS: 38%, 
OS: 46% 
*mechanism not mentioned; Ara-C: cytarabine; ATG: anti-thymocyte globulin; Bu: busulfan; CSP: cyclosporine; 
CY: cyclophosphamide; DFS: disease-free survival; FK506: tacrolimus; FLU: fludarabine; IBMTR: International Bone 
Marrow Transplant Registry; JSHCT: Japan Society for Hematopoietic Cell Transplantation; MeCCNU: methyl-1-(2-
Chloroethyl)-3-Cyclohexyl-1-Nitrosourea; Mel: melphalan; MGH: Massachusetts General Hospital; 
MP: methylprednisolone: MTX: methotrexate; myelo: myeloablative; NA: not available; NMD: non-malignant disease; 
RI: reduced-intensity; TCD: T cell depletion; TT: thiotepa; USC: Univ of South Carolina; XRT: radiation therapy. 


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myeloablative or non-myeloablative regimens. The Perugia group is perhaps the most well-known 
for studying the strategy of using highly T-cell depleted, “mega-dose” CD34-selected   
PBSC preceded by myeloablative conditioning, thus rendering the pt highly lymphopenic, and no 
immune suppression is given after transplantation. Virtually all patients engrafted (95%) and none 
developed GVHD. However, NRM was 40% due to infections, and relapse occurred in 30% of all 
patients, with a more striking effect in those with ALL (50%) compared to AML (10%)3.  
 
Multiple centers have attempted to improve on or replicate the Perugia results with mixed findings. A 
recent review by the group from Duke University1 compiled published results from centers using 
myeloablative and non-myeloablative  regimens with varying degrees of T cell depletion and post-
transplantation immunosuppression for GVHD prophylaxis. The summary of these heterogeneous 
results can be found in Table 1, demonstrating that these institution-dependent treatment plans still 
have considerable room for improvement. Specifically, GVHD, relapse, and infectious complications 
are still having a negative impact on overall survival. 
 
B. HLA-Haploidentical Transplantation for Malignant Diseases Using CY After HCT 
 
1. Using CY after HCT 
 
It has been demonstrated since the 1970’s that high doses of CY are not myeloablative, but are 
immunosuppressive due to its unique pharmacology. Both Jones4 and Kastan5 amongst others have 
studied aldehyde dehydrogenase, correlating high levels (found in hematopoietic progenitor cells) to 
CY resistance and low levels (found in T and B lymphocytes, NK cells) to CY sensitivity. Immature 
erythroid precursors express aldehyde dehydrogenase in intermediate levels. The first study 
demonstrating that CY’s limiting toxicity is not hematopoietic was performed by Storb and 
colleagues6 who showed that Rhesus monkeys receiving up to 200 mg/kg CY will have full 
hematological recovery without need for marrow infusion. Brodsky et al7 have shown that in the 
clinical setting, doses of CY 200 mg/kg can be used to treat severe autoimmune diseases without 
impacting hematological recovery or needing autologous stem cell rescue. In the MHC-mismatched 
murine model, Luznik and colleagues8 confirmed CY’s ability to selectively deplete alloreactive, 
proliferating host and donor lymphocyte clones after using the FLU/TBI-based nonmyeloablative 
conditioning regimen developed at FHCRC. However, it could not be tested in these studies whether 
the beneficial effect of ablating these proliferating T cells to decrease the incidence of GVHD would 
have any undue effects in a malignant disease model. Further, work done at the NIH9 has shown 
that in a fully MHC-mismatched murine transplant model, FLU and CY are synergistic and can 
successfully and preferentially deplete host T cells when compared to myeloablative doses of TBI. 
 
2. Results of phase II nonmyeloablative clinical trials using HLA-haploidentical donors: the Johns 
Hopkins University (JHU) and Seattle collaborative experience 
Based on a successful Phase I study conducted by O’Donnell and colleagues at JHU (n=13)10, a 
Phase II study was developed. Between 1999 and 2006, 68 patients with high-risk hematological 
malignancies requiring allogeneic 
HCT but lacking HLA-matched 
donors were enrolled on 2 
nonmyeloablative, HLA-
haploidentical Phase II clinical 
trials in Seattle and at Johns 
Hopkins University in Baltimore11. 
These studies differed by 
immunosuppression only:  the 
Baltimore regimen utilized 2 
doses while the Seattle regimen 
used 1 dose of CY after HCT 
 
Fig 1. HLA-haploidentical, nonmyeloablative HCT Treatment Plan  
(CY=cycophosphamide, FLU=fludarabine, MMF=mycophenolate mofetil,          
G-CSF= granulcotye colony stimulating factor) 


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(Figure 1). 25% of all patients were from ethnic minorities, in part due to the greater genetic diversity 
seen in African Americans, and highlighting the lower prevalence of underrepresented populations in 
bone marrow registries. Conditioning consisted of CY 14.5 mg/kg/day (days -6 to -5), FLU 30 
mg/kg/day (days -6 to -2), and 2 Gy TBI, followed by unmanipulated, HLA-haploidentical marrow 
allografts, with post-grafting immunosuppression consisting of CY 50 mg/kg (day +3 ± 4), MMF, and 
tacrolimus (both beginning 24 hours after the last dose of CY). Because of the use of HLA-
haploidentical grafts, strong immunological barrier needed to be overcome with this regimen. 
Rejection occurred on average in 9 of 66 (13%) evaluable patients.  Most patients converted to full 
donor chimerism by day +60 (Figure 2). More notable was the cumulative incidence of grade III 
GVHD of 6% at 1 year with no grade IV GVHD (Figure 3).NRM was 4% at day +100 and 15% 
at 1 year with infections being the leading causes of NRM (Table 2). However, relapse was the 
primary contributor to low overall survival, with 58% of patients relapsing by 2 years after 
transplantation, leading to an overall survival of 36% by 2 years. The kinetics of relapse 
demonstrates early and continual disease-related mortality during the first year after HCT, as 
demonstrated by the incidence curve in Figure 4, supporting the need for anti-tumor intervention 
early after HCT.  
In this high-risk population that survives the initial risk of undergoing an HLA-haploidentical HCT, 
relapse continues to be the main cause of mortality. As in other protocols employing in vivo 
lymphocyte depletion to lower GVHD, as is seen with alemtuzumab-based regimens12, relapse is a 
considerable cause of mortality. This supports the need for an early, preemptive intervention to 
facilitate GVT effects after HCT. Figure 5 graphically 
shows the degree of lymphodepletion apparent after 
nonmyeloablative conditioning and the introduction of 
CY early after HCT in 36 patients who have been 
treated in Seattle on Protocol 1667. 
 
 
 
 
 
 
 
 
 
 
Table 2. Causes of Death (Relapse and Non-
Relapse Mortality) Across 2 Studies 
Causes of Death 
Seattle 
(n=28) 
Baltimore 
(n=40) 
Relapse 
12 
19 
GVHD 
1 
1 
Infection 
2 
2 
CNS Hemorrhage 
0 
2 
Unknown 
1 
1 
Secondary AML 
1 
0 
 
Fig 2. Percentage of donor chimerism at days 28-30 and 
56-60 by group. 
 
 
Fig 3. Cumulative incidence of acute 
GVHD in both groups. 


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C. Experiences Using Non-Conventional Forms of DLI as Adoptive Immunotherapy after 
Allogeneic HCT 
 
The infusion of DLI containing high numbers of CD3+ cells can contribute to myelo-suppression, 
especially after marrow-infiltrative diseases, and life-threatening GVHD after HLA-mismatched 
HCT13. To circumvent these issues, a multi-center group from Italy conducted a Phase I study 
testing the safety and feasibility of repeated infusions of allogeneic, ex vivo expanded cytokine-
induced killer cells (CIK), also known as NKT cells, for patients relapsing after 0-1 allelic HLA-
mismatched HCT. Of eleven patients enrolled, none experienced infusional toxicity after CIK infusion, 
and four developed acute GVHD (≤ grade II). However, six showed no clinical response and died of 
rapidly progressive disease. Meyer et al attempted prophylactic DLI, depleted of CD8 cells, on days 
60 and 120 after T-cell depleted, reduced intensity HLA-matched HCT. Of 8 CD8-depleted DLIs 
collected, the median CD3-/CD16+CD56+ dose was 128.4 (range, 82.6-444.2)x 109. Of 23 patients 
enrolled, only 11 met criteria for prophylactic DLI due to the persistence of low-grade acute GVHD. 
Of these 11, 7 developed acute GVHD (I=5, II=1, III=1) and had disease-free survival (DFS) of 
68.2% (95% CI, 37.3-99.1) compared to DFS 52.5% (95%CI, 28.5-76.5) for the whole patient 
population (n=23). Overall, this CD8-depleted DLI was well-tolerated14.  
 
In the HLA-haploidentical setting, the use of DLI purged of alloreactive T cells has been pursued by 
several groups. Brenner’s group from Baylor University co-cultured donor peripheral blood with 
recipient EBV-transformed lymphoblastoid cell lines that were generated six weeks earlier. After 72 
hours of incubation, co-cultures containing alloreactive T-cells were depleted using anti-CD25 
monoclonal antibody, washed, and infused into patients at days 30, 60, and 90 after myeloablative, 
CD34-selected, T-cell depleted HLA-haploidentical HCT. Of 16 patients given T-cell purged DLI, 
GVHD occurred in two (grades II and IV) and higher numbers of effector (rather than memory) T 
cells were generated, thus improving immune reconstitution. However, only five of 16 achieved 
DFSl15, indicating only modest GVL effects in this patient population  This group has further studied 
the ability of expressing a suicide gene, inducible caspase 9, into DLI containing very large numbers 
of allodepleted T cells as a mechanism to allow T cell destruction should adverse events occur, thus 
adding a measure of protection in DLI-induced GVHD16. Additionally, the Perugia group has studied 
the effect of photodynamic purging of alloreactive T cells as a way to improve on DLI containing 
 
 
Fig 4. Incidence of Relapse and NRM 
across both studies 
Fig 5. Absolute Lymphocyte Counts (ALC) in 36 
patients treated on Protocol 1667 in Seattle 
 
1667 ALC Counts
0
50
100
150
200
250
300
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
Days Post-Transplant
Counts (Cells/ul)
Median
Mean


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large numbers of CD3+ cells 17. The latter two studies are in preclinical stages, with no published 
clinical results to date. 
 
D. NK Cells 
 
1.  Biology of NK cells  
 
NK cells are ideal for developing an adoptive immune strategy to decrease relapse after HCT. Due 
to their MHC-unrestricted ability to kill malignant cells, NK cells can provide tumor surveillance when 
T cells cannot. Specifically, malignant cells, which often evade the T cell adaptive immune response 
through down-regulation of MHC class I molecules on their cell surfaces18,19, can still be effectively 
targeted by NK cells. This ability, maintained through the NK cell’s lack of engagement of its MHC-
specific receptors to the target cell, initiates cytolytic activity by suppressing the inhibitory signal that 
normally protect cells from autoimmunity when the receptor binds to its ligand20 21. This phenomenon 
is referred to as “missing self” recognition, allows for NK cell killing of targets when the ligand is not 
expressed. However, NK cells also express receptors that, when in contact with its ligand, can 
generate activating signals based on intracellular signaling through the length of their cytoplasmic 
tails22. This activating signal may be sufficient to engage NK cell activity, despite the presence of 
concurrent inhibitory signals, and as such reduced expression of MHC Class I is not always 
necessary to initiate NK cell function 23. Thus, it is the use of both activating and inhibitory signals 
that influences the overall NK cell immune response. This quality can be exploited for adoptive 
immunotherapy in the HLA-haploidentical setting. Furthermore, it is now understood that NK cells 
can be further split into at least two main categories, those that are CD56bright and those that are 
CD56dim. These different populations perform different functions based on their degree of KIR 
expression, ADCC activity, cytokine production, and graft-versus-tumor effects 24. 
 
NK cell receptors, which recognize antigens at HLA-A, B, or C and are known as killer 
immunoglobulin-like receptors (KIR), are inhibitory receptors and are highly polymorphic; they are 
encoded on chromosome 19 and are clonally distributed within the NK cell repertoire. Those that 
recognize MHC Class I amongst other ligands are the conserved CD94/natural killer group 2 (NKG2) 
receptors, encoded on chromosome 12. The differential expression of these two receptors defines 
the NK cell repertoire. Furthermore, NK cells’ ability to home to sites of infection, produce 
inflammatory cytokines, and kill normal cells infected with parasites, viruses, and bacteria without 
prior sensitization25 further illustrates NK cells’ importance in maintaining a robust system of defense 
against both malignant and infected cells. As such, NK cells play a pivotal role in the innate immune 
response. 
 
2. Effect of immunosuppressive drugs on NK cell function 
 
Although NK cells have been shown to expand quickly after HCT, there is still some concern as to 
the effects of post-HCT immunosuppression on their function. In particular, calcineurin inhibitors, 
which suppress T cell function, are of concern in NK cells due to their shared lymphocyte ancestry. 
In the 1980’s, it was shown that DLA-mismatched dogs receiving CSP alone had higher rejection 
rates than dogs receiving CSP with methotrexate, suggesting that residual host NK cells leading to 
graft rejection were not affected by CSP alone26. Further, dogs undergoing non-myeloablative HCT 
with post-grafting MMF and CSP still have substantially higher lytic activity on in vitro 51Cr release 
NK functional assays compared to untreated controls27. In a rodent model, CSP inhibited T cell but 
not NK cell responses28. Using human NK cells, the Minnesota group has further studied the effect 
of CSP on NK cells using in vitro assays and found that CSP induces a variety of unexpected and 
significant changes in the phenotype and function of cultured NK cells. CSP significantly inhibited the 
expansion of CD56dim cells and preferentially expanded CD56bright cells, which were found to be 
relatively resistant to CSP. However, it was also noted that a minority of these two populations could 
interconvert in vitro after a week of culture in cytokines and growth factors. Although CSP-treated NK 


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cells showed more apoptosis compared to controls, the intracellular content of granzyme B, perforin, 
and surface expression of Fas ligand in NK cells incubated with CSP were not significantly different 
from controls. Additionally, higher percentages of IFN-g producing NK cells were detected in CSP-
treated cultures compared to controls. Mechanistically, CSP works as a calcineurin inhibitor. 
Calcineurin, an integral component of lymphocyte activation, normally becomes triggered through an 
influx of calcium after cell stimulation. Calcineurin then dephosphorylates nuclear factor of activated 
T cells (NFAT), allowing NFAT to enter the nucleus for gene transcription to take place. In this study, 
CSP prevented dephosphorylation of NFAT. Surprisingly, however, NK cells cultured with CSP still 
had higher cytoxicity against K562 and Raji cells, suggesting that the effector function (granule 
release) is not dependent on the nuclear translocation of NFAT29. Thus, it is not expected that the 
donor NK cell infusion will be adversely affected by ongoing post-grafting immunosuppression. 
 
3. Using highly purified NK cell infusions as adoptive immunotherapy 
 
Further in vitro studies by Aversa et al30 demonstrated that a human AML NOD/SCID mouse model 
infused with human alloreactive NK cells could survive until sacrificed at 120 days compared to 3 
weeks if either left untreated (i.e., no NK cells) or given non-alloreactive human NK cells. In this 
mouse model, NK cells also converted mixed into full donor chimerism and did not elicit GVHD even 
when infused in large numbers. Such observations by the Perugia group suggested that alloreactive 
NK cells in the donor-versus-host direction are responsible for GVL effects, having a stronger impact 
on myeloid malignancies with no contribution to GVHD. 
 
NK cells taken from HLA-haploidentical donors have been used for adoptive immunotherapy both in 
non-transplant and transplant studies. These studies corroborate the ease of NK cell collection and 
the safety of infusing NK cells into patients. One concern for using NK cells has been the ability to 
obtain large enough numbers for infusion to provide adequate immune surveillance. Several groups 
have studied methods of expanding NK cells ex vivo by incubation with either cytokines (i.e., IL-
2),31,32 or Notch ligands33,34. However, in the early period after transplantation when abundant 
cytokines are released35, NK cells will easily expand and thus this issue is of little concern in this 
proposed protocol. 
 
Miller and colleagues36 were the first group to demonstrate the ability of NK cells to expand in vivo 
and used donor NK cell infusions to treat persistent, refractory, or relapsed disease in a non-
transplant setting. NK cells were enriched through the CD3+ depletion of peripheral blood 
mononuclear cell (PBMC) apheresis products from HLA-haploidentical donors. These cells were 
incubated with IL-2 overnight and infused into patients who were then given daily IL-2 injections for 
14 days. All patients (n=43) were primed with one of three different chemotherapeutic regimens to 
prevent immediate rejection of donor cells. These regimens differed in either the degree of 
lymphopenia and/or neutropenia that they elicited. Results demonstrated that those patients 
experiencing the highest degree of chemotherapy-induced lymphopenia (19/43) before the NK cell 
infusion had the most robust in vivo expansion of NK cells (as measured by unique RT-PCR primers 
for donor specific MHC Class I alleles), and of these patients, 5/19 achieved complete remission. IL-
2 stimulated donor NK cells in one case persisted for as long as 138 days. This study suggests that 
the environment into which exogenous NK cells are placed substantially impacts the degree of NK 
cell expansion, function, and persistence. This need for in vivo NK cell expansion, whether by way of 
exogenous IL-2 supplementation as demonstrated in Miller’s study36, or by abundant cytokines 
uniformly released during the early period after HCT35, appears to be crucial in promoting NK cell 
survival and proliferation.  
 
Passweg and colleagues37 developed a pilot study (n=5) evaluating the safety and feasibility of 
collecting and infusing donor NK cells for mixed/declining chimerism or early relapse after 
myeloablative, HLA-haploidentical HCT. The Miltenyi system was used for both CD3 depletion and 
CD56 selection, producing both a highly purified NK cell population (median purity 97.3%) and a 


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high degree of T cell depletion (median log T cell depletion -3.55). After infusing these enriched NK 
cells, no patients on this study experienced infusional toxicities, acute or chronic GVHD, or late 
toxicities. NK cells were infused between 3-26 months after HCT and demonstrated mixed results. 
Specifically, progressive disease (n=1), persistent mixed donor chimerism (n=2), full donor 
chimerism (n=1) and graft failure (n=1) were all observed. Although limited by small numbers, this 
study highlights the need that delayed NK cell infusions given as a last resort in response to clinical 
necessity may not be early enough to prevent undesired outcomes; preemptive therapy may provide 
superior and more durable results.  
 
4.  The effect of KIR ligand mismatches after HCT 
 
Several studies have examined outcomes after KIR-ligand mismatched HCT, reporting both 
encouraging30,38 39 and deleterious40 41 effects. This ongoing controversy may be explained by 
differences in the number of residual host and/or donor T cells present within the host hematopoietic 
microenvironment. Host and donor lymphopenia can be highly variable due to the intensity of 
conditioning or T cell depletion of the donor allograft, respectively. The function of these residual T 
cells on NK cell development is actively being investigated by many groups. A retrospective study 
performed Cooley et al in Minnesota42 examined the role of T cells in HLA-matched donor grafts and 
their effect on NK cell development and subsequent clinical outcomes in patients transplanted for 
CML. Thirty-seven patients received unrelated, unmanipulated marrow while 40 received unrelated, 
T-cell depleted marrow. Recipient samples collected at day +100 (median +98, range +70 to +130) 
were evaluated as a single timepoint and compared to donor pre-transplant samples. Findings 
included: 1) the diminished expression of KIR in patients receiving unmanipulated marrow compared 
to those receiving T cell depleted marrow (p=0.001); 2) no difference in the increased expression of 
CD94/NKG2A between the two types of donor sources; 3) the increased expression of interferon 
gamma-producing NK cells after unmanipulated compared to T cell depleted marrow HCT (p=0.007), 
despite both groups having the same 4-fold increase in NK cells at day +100 (p=0.26), suggesting 
improved NK cell lytic activity in unmanipulated marrow recipients; 4) the increased incidence of 
acute GVHD in those patients having NK cells with higher interferon gamma production; and 5) 
increased KIR expression is independently predictive of increased survival, having a relative risk of 
0.36 (p=0.37). It appears from this study that the interaction between T and NK cells in the early 
period after HCT is complex, and that residual donor T cells may have a detrimental effect on NK 
cell function and development. 
 
A study of NK cell receptor maturation has enabled insight into the lineage-specific commitment of 
CD34+ stem cells into mature, functional NK cells. Cooley et al in Minnesota43 have been able to 
induce differentiation of an immature, poorly-functioning NK cell, defined as lacking inhibitory 
receptors NKG2A-/KIR-/CD56dim, into a productive, effector cell expressing KIR and/or NKG2A 
receptors. The implication of this finding is that our previous understanding of NK cell alloreactivity 
may need to be challenged. Specifically, some NK cells lacking inhibitory receptors may actually be 
developmentally immature cells. After being exposed to a cytokine-rich environment, the cells may 
mature into NK cells expressing inhibitory receptors. As such, these cells, which may have originally 
been considered to be of an alloreactive nature due to their lack of expressing inhibitory receptors, 
may actually promote self-tolerance. Thus, this study provides even more understanding for 
discrepant clinical results seen from multiple centers regarding KIR alloreactivity effects after HCT. 
 
Given the lack of consensus, we will not use KIR criteria for donor selection but will collect samples 
for retrospective evaluation in this study. 
 
 
 


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5.  Effect of NK cells on immune reconstitution after HCT 
 
Other studies have evaluated NK cell reconstitution and found unique patterns in the NK cell 
repertoire after HCT. Shilling et al from Stanford44 identified three unique patterns of NK cell 
reconstitution after patients (related =13; unrelated n=5) underwent HLA-matched HCT for leukemia. 
This group showed that in most cases, it took recipients between 6 months and 3 years to 
recapitulate the donor NK cell phenotype. The first group (n=8) consisted of those whose KIR 
expression became that of donor by 1 year after HCT. None of the recipient/donor pairs had 
coincidental sharing of KIR genotype. The second group (n=5) comprised patients who had reduced 
KIR expression, but whose CD94/NKG2A expression remained high at 1 year after HCT. 
CD94/NKG2A expression is considered an earlier phenomenon of NK cell reconstitution before KIR 
is fully expressed on mature NK cells45. Of this group, one recipient/donor pair had coincidental 
sharing of KIR genotype while another pair, although KIR genotype disparate, had similar NK cell 
repertoires prior to HCT. However, in three patients from whom 3 year post-transplant samples could 
be collected, all eventually converted to KIR repertoires similar to that of their donors. The third 
group (n=5) consisted of patients who developed unique NK cell repertoires which were neither of 
donor nor of host origin. Interestingly, two recipient/donor pairs had identical KIR genotypes. This 
idiosyncratic finding is not fully understandable, although the authors make an observation that 
patients in the third group suffered from major clinical complications after HCT, including acute grade 
IV GVHD, chronic extensive GVHD, and relapse, although this does not fully explain this finding as 
patients in other groups also had relapse and GVHD (although low grade and not extensive). In 
summary, this study showed that in most cases, KIR expression eventually converted to that of 
donor, although it is not well understood why some converted faster, and why others developed 
unique NK cell repertoires neither of host nor donor origin. 
 
Nguyen and colleagues in Paris have specifically looked at NK cell reconstitution after HLA-
haploidentical HCT and also confirmed that residual T cells in the graft affected the function and 
repertoire of NK cells after HLA-haploidentical transplantation. In one of their studies46, 21 patients 
with advanced malignant hematopoietic disease underwent myeloablative conditioning consisting of 
8 Gy TBI (day -5), thiotepa 20 mg/kg/day (day -4), rabbit ATG 5 mg/kg/day (days -3, -2), and 
cyclophosphamide 60 mg/kg/day (days -3, -2), with no post-transplantation GVHD prophylaxis. 
Eleven patients received partial (pTCD) (1.4 - 23 CD3+cells/kg) or extensive (eTCD) (<105 CD3+ 
cells/kg) T cell depleted, GCSF-mobilized PBSC. Findings demonstrated that in pTCD patients, 
CD56dim NK cells were found in similar levels in post-transplant recipients as in their donors. These 
CD56dim cells had higher cytolytic activity than the predominant NK cell found in eTCD patients, 
which were CD56bright NK cells and considered the more primitive subtype. Interestingly, 7 of 10 
patients in the eTCD group died from early relapse (median day +115, range 59-185) despite 8 
having a KIR-ligand mismatch in the graft-versus-host direction, while 1 of 11 patients in the pTCD 
died from relapse with 4 having KIR ligand mismatch in the same direction. This further supports the 
idea that eliminating donor T cells from the graft predisposes patients to relapse, either by direct T 
cell effects or indirectly by way of influencing NK cell reconstitution. In a previous study47, this group 
also suggested that the overexpression of CD94/NKG2A on immature NK cells early after HLA-
haploidentical HCT renders these cells ineffective in killing leukemic blasts.  
 


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E. The Effect of NK Cell Engraftment Kinetics Compared to Other Cell Lineages on Outcomes 
after Allogeneic HCT 
 
Our group has previously 
analyzed lineage-specific 
chimerism 
in 120 patients after non-
myeloablative HCT. A 
statistically-significant 
improvement in relapse-free 
survival was noted only after 
rapid establishment of donor NK chimerism (Table 3)48. A higher statistically significant association 
was seen in a recent update incorporating 282 patients. NK cell chimerism on days 14-42 were 
associated with a lower risk of relapse (p=0.006) and better progression-free survival (p=0.003) in 
time-dependent analyses, and no other lineages emerged as significant. Higher NK cell chimerism 
was not associated with higher rates of 
GVHD (p=0.29), unlike higher T cell 
chimerism (p=0.007) (Table 4). A day-14 
NK cell chimerism level of < 50% was 
associated with increased risks of graft 
rejection (p=0.009). The qualitative 
associations between donor NK cell 
chimerism and graft rejection, GVHD, 
relapse or progression-free survival did 
not change after adjustment for the 
presence of recipient KIR ligands nor 
after adjustment for the number of donor inhibitory or activating KIR genes49.  
 
Savani et al from the NIH looked at the recovery of NK cells in 54 patients receiving T-cell depleted, 
HLA-identical sibling myeloablative HCTs between 1993 and 2005. In a univariate analysis, 
transplants involving donor KIR genes 2DL5A, 2DS1, 3DS1 (found in 46% of patients) and higher 
numbers of inhibitory donor KIR had better transplant outcomes, and by multivariate analysis, day 30 
NK cell counts >150/μL had less relapse (HR 18.3, p=0.039), acute GVHD (HR 3.2, p=0.03), NRM 
(HR 11.4, p=0.03) and improved overall survival (HR 11.4, p=0.03)50. 
 
A group from Korea also examined the effect of non-CD34+ cells and their effect on early 
engraftment. In 69 patients who received either myeloablative (Bu/CY, n=51) or reduced-intensity 
(FLU-based, n=18) regimens, higher numbers of transplanted cytotoxic (CD8+) and NK (CD56+) 
cells predicted earlier engraftment with better HCT outcomes than higher numbers of CD34+ cells51. 
When comparing engraftment kinetics, they found that neutrophil engraftment was highly correlated 
with higher numbers of transplanted NK cells (p<0.001), T cells (p<0.001), MNC (p=0.005), and B 
cell (p=0.032) lineages, but not with numbers of transplanted CD34+ cells (p=0.442). They also 
found that it was higher numbers of transplanted NK and T cells that influenced immune 
reconstitution. Specifically, a higher transplant dose of NK cells was strongly associated with CD3+ T 
cell (p=0.092) and CD56+ NK cell reconstitution between 3 months and 1 year after HCT. This 
further supports the choice of using an enriched and highly purified NK cell infusion rather than 
CD34-selected PBSC as adoptive immunotherapy.  
 
F. Donor Issues 
 
HLA-haploidentical, related marrow donors undergo both a marrow harvest for the initial transplant, 
and then 6-9 days later, a 10-12L leukapheresis (adjusted for donor age/weight and equal to 2-3 
total blood volume (TBV) for both pediatric and adult donors, which is considered Standard Practice 
Table 3. Association between donor chimerism levels and death (any 
cause) or relapse/progression *% donor chimerism modeled as a 
continuous linear variable; hazard ratio presented as increase in 
donor chimerism of 20% 
 
Hazard Ratio* 
95% CI 
P 
Granulocytes 
0.89 
0.72-1.10 
0.28 
T cells 
0.89 
0.72-1.06 
0.19 
Monocytes 
0.89 
0.72-1.08 
0.21 
NK cells 
0.79 
0.64-0.96 
0.02 
Table 4. Increasing T cell rather than NK cell chimerism is 
significantly associated with higher rates of acute GVHD 
 
# Patients with Acute GVHD /  
# Pts at risk (%) 
% donor chimerism on 
day 14 
T-cells 
 
NK cells 
0-50 
21/46 (46%) 
10/20 (50%) 
51-75 
32/62 (52%) 
20/37 (54%) 
76-90 
30/41 (73%) 
31/53 (58%) 
91-100 
9/12 (75%) 
15/23 (65%) 
Trend  
P=.007 
P=0.29 


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for donor apheresis) to collect lymphocytes for NK cell enrichment. If the selected donor is unwilling 
and/or unable to undergo both procedures or storage of autologous blood prior to marrow harvest, 
then another HLA-haploidentical related donor must be found. If the donor is an adult, at least 18 
years old, and peripheral venous access is not adequate then a central venous catheter (CVC) will 
need to be temporarily placed. In such a situation, the risks and benefits of the CVC will need to be 
weighed carefully in order for the donor to make an informed decision. Children less than 12 years of 
age are excluded from being a donor in this study. Children greater than or equal to 12 years of age 
will be considered as a donor for this study if the following two criteria are met: 1) they must be able 
to give assent in the presence of the parent (or legal guardian) and an Attending physician who is 
not a member of the recipient’s care team, 2) they must be able to undergo apheresis using 
peripheral IV access; CVC placement is not an option for children. For donors weighing >40 kg, one 
unit of autologous blood will be collected on two separate occasions, at approximately 3-4 weeks 
and 2-3 weeks prior to the marrow harvest. There must be at least one week between the collection 
of the units, and the second autologous unit must be collected no less than two weeks before the 
marrow harvest.  If the donor weighs <40 kg, only 10 mLs/kg autologous blood will be collected on 
each occasion for storage. Donors will start oral iron supplementation after collection of the first unit 
of autologous blood. For the marrow harvest, no more than 20 mL/kg donor weight of marrow will be 
collected, based on National Marrow Donor Program (NMDP) standards. Donors will likely develop 
anemia due to the marrow harvest. To ensure an adequate hematocrit to safely undergo apheresis, 
the previously-collected autologous blood will be infused into the donor in the recovery room and 
prior to discharge after the marrow harvest. Since autologous blood is not routinely given to an 
otherwise healthy donor in the absence of severe anemia post-operatively, the donors should be 
counseled about the unique indication in this setting and the general risks of blood transfusion. 
There is no expectation that platelet counts will be low following a marrow harvest, thus the patient 
should not need a platelet transfusion prior to the apheresis procedure. One week after the marrow 
harvest, donors will undergo apheresis.  Mononuclear cells (MNC) collected from the apheresis 
procedure will be stored overnight at 1-6 C for Miltenyi CliniMACS processing the following day.  
 
G. Enrichment of NK Cells 
 
Donor MNC will undergo 
both a CD3 depletion 
followed by a CD56 
selection with the Miltenyi 
CliniMACS system using 
Good Tissue Practices. The 
entire volume of processed 
cells, with a safety limit of 
<104/kg of CD3+/CD56- cells, will be infused. This safety limit is based on a dose-finding study 
performed by Lewalle et al where DLI containing various doses of CD3+ cells were given 
prophylactically after T-cell depleted, myeloablative HLA-haploidentical HCT52. After the CD56 
enrichment, the cells will be spun down out of the Miltenyi buffer and resuspended in cell therapy 
laboratory (CTL) standard infusion media (Normosol-R plus 1% Human Serum Albumin) and stored 
at room temperature until ready to be infused.  All reagents and material used during processing and 
final formulation have been verified to be sterile and endotoxin free. Should the CD3+ fraction of the 
product exceed this limit, then the volume of the product will be reduced accordingly while 
maintaining the NK cell dose at ± 20% of the proposed dose level cohort. NK cells will be released 
for infusion into the patient only after the following 3 criteria are met: 1) cell viability must be ≥70%; 
2) Stat gram stain from the microbiology lab, 3) the number of residual CD3+ cells is <104/kg. The 
infusion of the NK cell product will take place on the same day as the processing. If patients 
experience hyperacute GVHD actively requiring systemic therapy (other than prophylactic 
tacrolimus and MMF) or are receiving greater than or equal to 0.25 mg/kg equivalent 
prednisone dose for any reason within one day of the NK cell infusion, we will not infuse the 
Table 5. Results from a non-G-CSF stimulated normal volunteer using 
the Miltenyi CLINIMACS system to perform a CD3 depletion / CD56 
selection   
 
TNC 
CD3+ 
CD3+/CD56+ 
CD56+ 
Start 
9.16 x 109 
4.3 x 109 
3.5 x 108 
9.2 x 108 
CD3 depleted 
2.87 x 109 
5.7 x 106 
3.9 x 106 
7.9 x 108 
CD56 enriched 
4.94 x 108 
1.1 x 105 
1.8 x 106 
4.6 x 108 


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donor NK cells.  If already collected, NK cells may be cryopreserved and may be used off-
study at a later timepoint.  Additionally, steroids are to be avoided within 2 days AFTER receiving 
the NK cell infusion to maximize its effectiveness.  If patients must receive steroids due to medical 
necessity during this time period, document clearly in clinical notes. Patients will remain on study.  
Unused NK cells from which donor identifiers have been removed may also be used for other IRB-
approved research studies, which is described in the Donor Consent. 
 
Three full-scale collections using volunteer donors have been conducted at our Center using CD3 
depletion and CD56 selection via the Miltenyi system. Results of one collection from a non-G-CSF 
stimulated donor are shown (Table 5), demonstrating an enriched, pure NK cell population (94%) 
with a 4.6 log T cell depletion (courtesy of S. Heimfeld). 
 
IV. RATIONALE FOR STUDY 
 
We propose that an infusion of donor NK cells on day +7 after nonmyeloablative, HLA-haploidentical 
HCT will not only provide a temporary boost of mature NK cells to treat low-level disease and hasten 
engraftment, but will also favorably impact early immune reconstitution after HCT. We further 
propose that this infusion of donor NK cells will influence the development of particular NK cell and T 
cell subtypes which will provide both immediate and long-term tumor surveillance, infectious 
monitoring, and sustained engraftment. 
 
V. OBJECTIVES 
 
A. Phase I Objective  
 
Identification of the maximal feasible dose of NK cells that can be infused one week after 
nonmyeloablative, HLA-haploidentical HCT 
 
 
B. Phase II Objectives 
 
Once the maximal feasible dose has been identified, accrual will be limited to the cohort
 
containing this cell dose to determine: 
1.  Incidence of relapse 
2.  Incidence of grades III-IV acute GVHD 
3.  Incidence of non-relapse mortality  
 
VI. PATIENT SELECTION 
 
A.  Inclusion Criteria 
 
1. Patients with the following hematologic malignancies will be permitted although other diagnoses 
can be considered if approved by PCC and the principal investigators: 
 
Aggressive non-Hodgkin lymphomas (NHL) and other histologies such as Diffuse Large B 
cell (DLBC) NHL– a) not eligible for autologous HCT, b) not eligible for high-dose HCT, c) 
after failed autologous HCT, or d) be part of a tandem auto-allo approach for high risk 
patients 
 
Mantle Cell NHL – must be beyond first CR 
 
Low grade NHL– with < 6 month duration of CR between courses of conventional therapy 
 
CLL – must have either 1) failed to meet NCI Working Group criteria for complete or partial 
response after therapy with a regimen containing FLU (or another nucleoside analog, e.g. 2-
CDA, pentostatin) or experience disease relapse within 12 months after completing therapy 
with a regimen containing FLU (or another nucleoside analog); 2) failed FLU-CY-Rituximab 
(FCR) combination chemotherapy at any time point; or 3) have “17p deletion” cytogenetic 
abnormality and relapsed at any time point after any initial chemotherapy. 


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 
Hodgkin Lymphoma – must have received and a) failed frontline therapy, b) not be eligible 
for autologous HCT, or c) or be part of a tandem auto-allo approach for high risk patients. 
 
Multiple Myeloma or Plasma Cell Leukemia – must have received more than one line of prior 
chemotherapy. Consolidation of chemotherapy by autografting prior to nonmyeloablative 
HCT is permitted. 
 
Acute Myeloid Leukemia (AML)–Must have < 5% marrow blasts at the time of HCT  
 
Acute Lymphocytic Leukemia (ALL)-Must have <5% marrow blasts at the time of HCT. 
 
Chronic Myeloid Leukemia (CML) – accepted if they are beyond CP1 and if they have 
received previous myelosuppressive chemotherapy or HCT, and have <5% marrow blasts at 
time of transplant 
 
 Myelodysplasia (MDS)/Myeloproliferative Syndrome (MPS) – (>int-1 per IPSS) after ≥ 1 
prior cycle of induction chemotherapy. Must have <5% marrow blasts at time of transplant. 
 
Waldenstrom’s Macroglobulinemia – must have failed 2 courses of therapy. 
2.  No age restrictions 
3. Patients must be expected to have disease controlled for at least 60 days after HCT 
4.   Patients for whom HLA-matched unrelated donor search could not be initiated or completed due 
to insurance reasons, concerns of rapidly progressive disease, and/or discretion of attending 
physician are eligible for this protocol 
 
B.  Exclusion Criteria 
 
1.  Patients with available HLA-matched related donors 
2. Patients eligible for a curative autologous HCT 
3.  Significant organ dysfunction that would prevent compliance with conditioning, GVHD 
prophylaxis, or would severely limit the probability of survival: 
a. Symptomatic coronary artery disease or ejection fraction <35% or other cardiac failure 
requiring therapy (or, if unable to obtain ejection fraction, shortening fraction of <26%).  If 
shortening fraction is <26% a cardiology consult is required with the PI having final approval 
of eligibility.  
b. DLCO <40% TLC <40%, FEV1 <40% and/or receiving supplementary continuous oxygen. 
 
The FHCRC study PI must approve enrollment of all patients with pulmonary nodules. 
c. Liver function abnormalities: Patient with clinical or laboratory evidence of liver disease will 
be evaluated for the cause of liver disease, its clinical severity in terms of liver function, 
bridging fibrosis, and the degree of portal hypertension. The patient will be excluded if he/she 
is found to have fulminant liver failure, cirrhosis of the liver with evidence of portal 
hypertension, alcoholic hepatitis, esophageal varices, a history of bleeding esophageal 
varices, hepatic encephalopathy, uncorrectable hepatic synthetic dysfunction evinced by 
prolongation of the prothrombin time, ascites related to portal hypertension, bacterial or 
fungal liver abscess, biliary obstruction, chronic viral hepatitis with total serum bilirubin 
>3mg/dL, or symptomatic biliary disease  
4.  HIV seropositive patients 
5. Patients with poorly controlled hypertension despite multiple antihypertensive medications. 
6.  Fertile females who are unwilling to use contraceptive techniques during and for the twelve 
months following treatment, as well as females who are pregnant or actively breast feeding 
7.  Fertile males who are unwilling to use contraceptive techniques during and for the twelve months 
following treatment 
8. Patients with active non-hematologic malignancies (except non-melanoma skin cancers) or 
those with non-hematologic malignancies (except non-melanoma skin cancers) who have been 
rendered with no evidence of disease, but have a greater than 20% chance of having disease 
recurrence within five years 
 
This exclusion does not apply to patients with non-hematologic malignancies that do not require 
therapy 
9. Active infectious disease concerns  


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10. Karnofsky performance score <60 Lansky performance score <60  
11. Life expectancy severely limited by diseases other than malignancy 
12. Patients with a diagnosis of CMML. 
13. CNS involvement with disease refractory to intrathecal chemotherapy.  
14. Patients with AML, MDS, ALL, or CML must not have presence of circulating leukemic blasts 
detected by standard pathology.  
15. Patients with aggressive lymphomas (such as DLBC) must not have bulky, rapidly progressive 
disease immediately prior to HCT. 
16. Patients who have received a prior allogeneic HCT must have no active GVHD requiring 
 
immunosuppressive therapy for at least 21 days prior to start of conditioning 
 
 
VII. DONOR SELECTION 
 
A.  Inclusion Criteria 
 
1. Related, HLA-haploidentical donors who are identical for one HLA haplotype and mismatched for 
any number of HLA-A, -B, -C, DRB1 or DQB1 loci of the unshared haplotype. 
2. Marrow will be the only allowed hematopoietic stem cell source 
3. Haploidentical donor selection will be based on standard institutional criteria, otherwise no 
specific prioritization will be made amongst the suitable available donors. Donors will not be 
selected based on KIR status. 
 
 
B. Exclusion Criteria 
 
1. Children less than 12 years of age. 
2. Children greater than or equal to 12 years of age who have not provided informed assent in  the 
presence of a parent and an Attending physician who is not a member of the recipient’s  
 
care team. 
3. Children greater than or equal to 12 years of age who have inadequate peripheral vein  
 
access to safely undergo apheresis. 
4. Donors unable or unwilling to undergo marrow harvest for the initial HCT, storage of autologous 
blood prior to marrow harvest or apheresis one week after marrow harvest 
5. Donors who are not expected to meet the minimum target dose of marrow cells (1 x 108 
nucleated cells/kg recipient IBW) for the initial HCT. The average nucleated cell content of 
harvested marrow is 22 x 106 nucleated cells/mL or 220 x 108 nucleated cells/Liter. 
6. HIV-positive donors 
7. Donors who are cross-match positive with recipient  
 
VIII. INFORMED CONSENT 
 
Patients will be referred here for consideration of a marrow transplant. Both patient and donor will be 
completely evaluated. The protocol will be discussed thoroughly with patient, donor and family, and 
all known risks to the patient and donor will be described. The goals of the study, requirement for 
data collection, and requirement for release of medical records will be discussed with the patient 
and/or his/her parent/guardian. All potential risks associated with the use of FLU, low dose TBI, CY, 
immunosuppressive drugs, allogeneic HCT with an HLA-haploidentical donor, and NK cell infusion 
from the same HLA-haploidentical donor, will be discussed as objectively as possible with the 
patient. Discussion of potential complications should include graft rejection, GVHD, infections, and 
death. With the donor, discussion about the potential risks of marrow donation, autologous infusion 
of packed red blood cells, and apheresis collection will be discussed.  Donors who are children 
greater than or equal to 12 years of age and less than 18 years of age must willingly sign the assent 


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form in the presence of the parent and an Attending physician independent of the recipient.  If there 
is any question as to the ability of the donor to provide informed assent, another donor must be 
used.  
 
Informed consent from the patient and donor will be obtained using forms approved by FHCRC’s 
Institutional Review Board (IRB). Informed consent will be obtained by the Principal Investigator, Co-
Investigator, or Attending Physician who is familiar with the study but not necessarily an investigator. 
When patients are less than 18 years of age, consent will be obtained from parents or legal 
guardian(s). A summary of the conference will be documented for the medical record detailing what 
was covered. 
 
IX. PROTOCOL REGISTRATION 
 
Eligible patients will be identified by the Clinical Coordinator’s Office, who will register the patients 
with the Registration Office (206-667-4728) between 8:30 am and 4:00 pm, Monday through Friday. 
After hours, the Registration Office can be reached by paging (206) 995-7437. 
 
X. PLAN OF TREATMENT 
 
A. RECIPIENT TREATMENT PLAN 
All pediatric patients will be admitted for the conditioning regimen and will stay inpatient until 
engrafted and other medical issues can be maintained as outpatients.. Adult patients may undergo 
conditioning and post-transplant treatments outpatient as medically appropriate.  If adult patients 
must initiate tacrolimus as a continuous infusion rather than IV bolus, they must be admitted until IV 
bolus/PO formulations can be used.  See Figure 6 for details. 
 
 
Days  
 
−6 
−5 
−4 
−3 
−2 
−1 
Conditioning 
 
 
 
 
 
 
CY 14.5 mg/kg (no MESNA) 
● 
● 
 
 
 
 
FLU 30 mg/m2/day 
● 
● 
● 
● 
● 
 
TBI 200 cGy 
 
 
 
 
 
● 
HCT ( Marrow only acceptable stem cell source) 
0 
Post-Transplantation  +3 
+4 
+7 
+40 
+84 
+180 
CY 50 mg/kg 
● 
 
 
 
 
 
MESNA (dosed 100% CY dose) 
● 
 
 
 
 
 
MMF (refer to XII.F for dosing) 
 
Start 
 
Taper* 
Stop* 
 
Tacrolimus (refer to XII.E for dosing) 
 
Start 
 
 
Taper* 
Stop* 
G-CSF 5g/kg/day   
 
Start^ 
 
 
 
 
Donor NK Cell Infusionº 
 
 
● 
 
 
 
 
Fig 6. HLA-Haploidentical, Nonmyeloablative HCT with Donor NK Cell Infusion Treatment Plan  Conditioning consists of 
CY 29 mg/kg, FLU 150 mg/m2, and 200 cGy TBI. Bone marrow is infused on Day 0. G-CSF begins day+4  after marrow 
infusion and continues until engraftment is seen. Post-transplantation immunosuppression consists of CY (day +3) MMF, and 
tacrolimus. Both MMF and tacrolimus must begin at least 24 hours after the CY infusion in order to prevent blunting of the 
alloreactive response needed for effective CY targeting of proliferating lymphocytes. A single infusion of donor NK cells at a 
dose of either 2.5 or 5 x 106 CD56+/CD3- cells per kg is given on Day +7 (or day of NK cell infusion).  Collections in the 
Phase II portion of the study that do not achieve the NK target dose will be infused and patients will be evaluable 
for the Phase II outcomes 
* Based on GVHD Status                 
^Continue G-CSF until ANC>500 x 3 days 
                                                   
 º Dose Level 1: NK cell dose 2.5 x 106/kg 
   Dose Level 2: NK cell dose 5 x 106/kg 


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  1.  Conditioning Regimen 
 
Menstruating female patients should be placed on an anti-ovulatory agent prior to initiating the 
conditioning regimen.  
 
a. Fludarabine 
 
i. 
Fludarabine dose is based on estimated creatinine clearance 
 
 
 
 
Adults: creatinine clearance may be estimated by the Cockcroft Formula: 
 
CLcr = [(140-age) x weight (kg) x 0.85 (for women only)]/[72 x creat (mg/dl)]. 
 
Pediatrics (Age <17):  Formal measurement such as iothalamate study 
 
 
     (Age <10):  Iothalamate study is recommended 
 
 
 
ii. Fludarabine is administered by IV infusion over 30 minutes on days -6 to -2 
 
 
The dose of fludarabine is based on m2 and will always use Actual Body Weight  
 
Table 6:  Fludarabine dose based on Creatinine Clearance 
Creatinine Clearance ml/min 
Daily Fludarabine Dose (mg/m2) 
> 60 
30 
46-60 
24 
31-45 
22.5 
21-30 
19.5 
<20  
15 
 
b. Cyclophosphamide 
i. 
Cyclophosphamide is administered by IV infusion over 1 hour on days -6 and -5 with IV fluid 
administration  
Refer to standard practice guidelines for administration guidelines. Patients will be monitored 
closely for urine output and hematuria.  MESNA is not required at this dose. 
 
ii. Cyclophosphamide is dosed according to the Adjusted Body Weight if the patient’s Actual 
Body Weight is >100% of the Ideal Body Weight.  
If the Ideal Body Weight (IBW) > Actual Body Weight, then Actual Body Weight will be used.  
 
c. Total Body Irradiation 
200 cGy TBI will be given on day –1 at a rate of 6-15 cGy/min per radiation oncology standard 
guidelines. 
 
2. Marrow Infusion 
 
Donor marrow will be harvested with a target yield of 4 x 108 nucleated cells/kg recipient body 
weight. This volume must not exceed 20 mLs/kg donor weight. The minimum acceptable yield 
should be 1 x 108 nucleated cells/kg recipient IBW. Marrow collection final volume goal should 
be based on the mid-collection total nucleated cell count.  The composition of allografts will be 
determined by flow cytometry.  
See Appendix A for ABO Incompatibility guidelines.  
 
 
 


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3. Post-transplantation Immunosuppression 
 
Immunosuppression to permit engraftment and provide GVHD prophylaxis will be performed with 
Cyclophosphamide (CY), Mycophenolate Mofetil (MMF) and Tacrolimus.  
 
a. Cyclophosphamide 
 
On Day +3, CY will be given as a single dose of 50 mg/kg IV  
 
Use Adjusted Body Weight; if Ideal Body Weight is greater than Actual Body Weight,  use Actual 
Body Weight. Post-transplant CY will be given within 48-72 hours of marrow infusion as a 1 hour 
infusion with MESNA prophylaxis and IV hydration. Refer to standard practice guidelines. Urine 
output and signs of hematuria will be monitored closely.  To maximize the effectiveness of post-
transplant CY, it is critical that immunosuppressive agents are to be avoided FROM THE 
MORNING OF STEM CELL INFUSION until 24 hours AFTER the completion of the post-
transplant CY unless there is medical necessity. This includes corticosteroids as anti-emetics. 
 
b. Mycophenolate Mofetil 
i. 
Starting on Day +4 (24-36 hours after last dose of CY), MMF will be given orally at a dose of 
15 mg/kg based on adjusted body weight every 8 hours.  
 
MMF will be tapered after day +40 (adapted dose-reduction to be discontinued by day+84) if 
there is no evidence of GVHD. 
 
ii. Guidelines for MMF Dose Adjustment and Monitoring  
a)  Initiating MMF therapy: Oral administration of MMF will be at 15 mg/kg orally every 8 
hours (45 mg/kg/day) starting on Day +4. If there is nausea and vomiting at any time 
preventing the oral administration of MMF, MMF should be administered intravenously at 
the appropriate dose.  
b)  Maintaining MMF: Markedly low (<40%) donor T-cell chimerism after HCT may indicate 
impending graft rejection. MMF should be continued at full dose or, if the MMF taper has 
been initiated, reinstitution of full dose MMF should occur. If MMF has been 
discontinued, MMF should be reinitiated at full dose. 
 
 
iii.  Guidelines for MMF Dose Adjustment Based on Toxicity 
a) If in the clinical judgment of the attending physician the observed toxicity is related to 
MMF administration, a dose adjustment may occur. The discontinuation of MMF at any 
point should be discussed with the Study PI and should be documented in the permanent 
medical record and all Case Report Forms (CRF) 
b) Gastrointestinal Toxicity. Severe gastrointestinal toxicities such as gastrointestinal 
hemorrhage have been very rare after nonmyeloablative HCT. In the event of 
gastrointestinal toxicity that requires medical intervention including medication for control 
of persistent vomiting or diarrhea that is considered to be due to MMF after day 28, a 
20% dose reduction will be made or the drug may be given IV. If severe refractory 
diarrhea or overt gastrointestinal bleeding occurs, MMF may be temporarily stopped. The 
MMF should be restarted at 20% reduced dose when the underlying toxicity subsides. 
c)  Neutropenia. Based on previous experience in patients after nonmyeloablative HCT, 
dose adjustments are likely to occur because of hematopoietic adverse effects, in 
particular neutropenia. A thorough evaluation of neutropenia should occur including 
peripheral blood chimerism studies, marrow aspiration and review of marrow suppressive 
medications. If all other potential causes of marrow toxicity are ruled out, dose 
adjustments will only be made for severe, prolonged neutropenia (ANC <500/ul for 5 
days or more) that persists after day +21 post-transplant. Dose reductions should be 
conservative (20%). After day +21, the use of G-CSF will be permitted for severe 
neutropenia. For severe hematological toxicity related to MMF (neutropenia > 5 days 
refractory to G-CSF), MMF may be temporarily stopped. The MMF should be restarted at 


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20% reduced dose when the underlying toxicity subsides. The discontinuation of MMF at 
any point should be discussed with the Study PI and should be documented in the 
permanent medical record and all CRFs. 
 
c. Tacrolimus 
i. 
Starting on Day +4 (24-36 hours after last dose of CY), tacrolimus will be given at a dose of 
0.03 mg/kg/day (for patients <30 kg) continuous infusion over 22-24 hours or 1 mg/day (for 
patients >30kg) IV once a day over 1-2 hr.  
 
Tacrolimus is dosed based on Adjusted Body Weight. Tacrolimus should be changed to an 
oral dosing schedule once oral medications are tolerated and once a therapeutic level (5-15 
ng/ml) is achieved. Oral tacrolimus is dosed twice daily.  Serum levels of tacrolimus should 
be measured on day +8 and then twice weekly thereafter and the dose adjusted accordingly 
to maintain a level of 5-15 ng/ml. Tacrolimus will be tapered after day +84 (adapted dose-
reduction to be discontinued by day +180) if there is no evidence of GVHD. 
 
ii. Guidelines for Tacrolimus Dose Adjustment and Monitoring 
a) If there is nausea and vomiting which prevents oral intake at any time during tacrolimus 
treatment, the drug should be given intravenously at the appropriate dose that was used 
to obtain a therapeutic level. (IV: PO ratio = 1: 4). 
b)  Whole blood trough levels of tacrolimus (i.e., just prior to the next dose) should be 
obtained on Day +8 and then twice weekly until the taper is initiated, unless high  levels 
(>20 ng/ml) are detected or toxicity is suspected, in which case more frequent monitoring 
will be performed as clinically indicated. The dose should be adjusted accordingly to 
maintain a level of 5-15 ng/ml. 
c)  Dose reductions should only be made if tacrolimus toxicity is present or levels exceed 
20ng/ml in the absence of toxicity. Dose reductions of tacrolimus for high levels without 
toxicity should be conservative, e.g. 25%, to avoid inadequate immunosuppression. If 
creatinine is greater or equal to 2 times the baseline level then the tacrolimus dose will 
be reduced by 25%.  
d) Blood pressure, renal function tests (creatinine, BUN), electrolytes and magnesium need 
to be followed at least two to three times per week while receiving tacrolimus to full dose 
and then twice weekly or per attending until tacrolimus is discontinued 
e)  Tacrolimus levels should be performed more frequently when 1) drug is converted from 
oral to IV or IV to oral, 2) dose adjustments are made due to levels outside the 
therapeutic range, or 3) voriconazole (see table below) is initiated or withdrawn 4) if 
toxicity is suspected. Steady state levels will not be achieved for at least 72 hours after 
any change in dosing, i.e. levels determined earlier may not reflect an accurate steady 
state concentration. 
f)  Patients requiring hemodialysis should have tacrolimus levels maintained in the 
therapeutic range (5 to15 ng/ml). 
g) Grapefruit and grapefruit juice affect metabolism of tacrolimus and should be avoided. 
Oral tacrolimus should be taken consistently with or without food.  
 
 
 
 
 


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Table 7:  Medications that Affect Tacrolimus Levels 
Decrease Tacrolimus Levels 
Increase Tacrolimus Levels 
Dilantin 
Steroids 
Phenobarbital 
Fluconazole 
Carbamazepine 
Ketoconazole 
Rifampin 
Itraconazole 
Caspofungin 
Voriconazole* 
 
Cimetidine 
 
Macrolide antibiotics 
 
Calcium channel blockers 
 
Danazol 
 
Metoclopramide 
* When initiating therapy with voriconazole in patients already receiving 
tacrolimus, it is recommended that the tacrolimus dose be reduced to one-
third of the original dose, (a 67% reduction) and followed with frequent 
monitoring of the tacrolimus blood levels. Increased tacrolimus levels have 
been associated with nephrotoxicity. When voriconazole is discontinued, 
tacrolimus levels should be carefully monitored and the dose increased as 
necessary. 
 
4. Growth Factor Support 
Patients will receive G-CSF at 5 g/kg/day IV or SC starting at day +4 and continuing until the ANC 
>500/uL for 3 days. Refer to Section X.A.3.b.iii for information regarding prolonged neutropenia. 
 
5.  Donor NK Cell Infusion 
 
a. Collection of Donor Mononuclear Cells and Enrichment/Purification of Donor NK Cells 
Mononuclear cells (MNCs) will be collected from the COBE apheresis machine on Day +6. If it is 
not feasible to collect cells on Day +6, MNCs can be collected between Days +6 to +9 (although 
the preference is that they be collected on Day +6). For Seattle patients, the apheresis product 
will be transported to the SCCA Cell Therapy Laboratory (Main Lab: 288-1200; Product 
Development Specialist: Lori Jones, 288-8278), where MNCs will be stored overnight at 1-10°C 
for processing the following day.  Other study sites will follow their internal procedures. 
 
 
 
The morning after collection, the MNCs will undergo a CD3 depletion followed by CD56 
 
selection using the Miltenyi CliniMACS system using GTP standard procedures. Details 
 
regarding the enrichment and purification process can be found in Section III.G.  The NK cell 
 
product will be stored at room temperature until ready for infusion. 
 
 
If patients experience hyperacute GVHD actively requiring systemic therapy (other  
 
than prophylactic tacrolimus and MMF) or, for any reason, are receiving greater than   
 
or equal to 0.25 mg/kg equivalent prednisone dose (within one day of the NK cell  
 
 
infusion), we will not infuse the donor NK cells.  If already collected, donor NK cells  
 
may be cryopreserved and may be used off-study at a later timepoint.  Unused NK  
 
 
cells from which donor identifiers have been removed may also be used for other IRB- 
 
approved research studies, which is described in the Donor Consent. 
 
 


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b. Characterization of NK Cell Product and Byproducts 
 
 
 
i. 
NK Cell Product 
 
a)  Cellular Phenotype 
An aliquot of the enriched product will undergo cellular phenotyping.   
 
The number of CD3+ and CD56+ cells from the unmanipulated apheresis product will be 
determined prior to processing to define the degree of NK cell enrichment and log T cell 
depletion. The product will be deemed appropriate for release if the following three criteria 
are met: 1) immediate sterility assessment (i.e., negative gram stain) is clear of foreign 
organisms; 2) cell viability, as ascertained by flow cytometry, is ≥70%; and 3) the number of 
residual CD3+ cells is <104/kg.  It may be necessary to reduce the volume of the NK cell 
product to achieve the appropriate number of CD56+/CD3- cells (within a 20% window) to be 
enrolled at the particular dose level.  Additionally, the volume may need to be reduced to 
achieve a residual CD3 cell number <104/kg. Otherwise the product will not undergo any 
further manipulation to reduce the number of CD3+ cells. 
 
The two dose levels on this study are:  
Dose Level 1 -- NK cell dose of 2.5 x 106 (± 20%) CD56+/CD3- cells per kg.   
Dose Level 2 -- NK cell dose of 5 x 106 (+ 20%) CD56+/CD3- cells per kg. 
 
Once the Phase I study is completed and the maximal feasible dose has been identified, 
subsequent patients will be enrolled on the Phase II study at the maximal feasible dose. 
Collections in the Phase II portion of the study that do not achieve the NK target dose will be 
infused and patients will be evaluable for the Phase II outcomes. 
 
 
b)  The enriched NK cell product may also be phenotyped for expressed NK cell receptors,   
 
or activating and inhibitory KIR.  
 
 
ii. Byproducts 
In addition, the two byproduct fractions containing the CD3+ cells and CD56- cells may also 
be phenotyped for cellular characterization and cryopreserved per batch record worksheet. 
Since these fractions will not be infused into the patient, it may be important to document 
which cell types are being discarded from the final infusion product. KIR analyses will not be 
done on the byproducts as there should be minute numbers of NK cells remaining. 
 
 
c. NK Cell Product Repository 
A cell repository will be created from the NK cell product for use in future studies; this includes 
additional unused NK cells. Cells will be cryopreserved using controlled rate freezing and stored 
in liquid nitrogen freezers. 
 
d. Infusion of Donor NK Cells 
 
Donor NK Cells will be infused into the recipient the same day as the product is processed as 
long as three release criteria are met (refer to X.A.5.b.i).  Due to the length of time it will  take for 
processing, the NK infusion will likely take place in the late afternoon or evening the day after the 
product is collected.  The infusion of these cells will follow established standard procedures for donor 
lymphocyte infusions, including administration of appropriate pre-medications, having anaphylaxis 
medications immediately available, and having close monitoring during and after the infusion.  Adult 
patients may be given the NK cell product as an outpatient based on clinical status.  However, adult 
patients may need to be admitted to the inpatient service in cases of delayed product arrival or need 
for post-infusion monitoring beyond the hours of the SCCA outpatient clinic.  All pediatric patients will 
receive the NK cell product as inpatients. 
 


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Standard guidelines for cellular product infusion currently in place at the SCCA require a 2 hour 
monitoring period to observe any potential adverse events from the end of infusion.   However, in 
order to comply with FDA requirements, the Phase I portion of the study will require a 4 hour window 
of observation after donor NK cell infusion.  All attempts will be made to ensure that the infusion will 
take place as an outpatient.  However, if the 4 hour observation window cannot be completed during 
normal hours of the SCCA outpatient clinic.  Adult patients will need to be admitted for their NK cell 
infusions.  Once the Phase I portion of the study has been completed, we may consider decreasing 
the monitoring period to 2 hours post-infusion for the Phase II portion of the study if no severe 
 
infusional reaction (grade IV toxicity using the Adapted Common Toxicity Criteria) has been 
observed.  
Potential side effects of the NK cell infusion include anaphylaxis, acute GVHD, nausea, vomiting, 
infection, diarrhea, and marrow suppression, in particular prolonged neutropenia.  These side effects 
are considered rare.  One extremely rare potential side effect may be the development of human-
anti-mouse antibodies (HAMA) from the Miltenyi beads.  To maximize effectiveness of the NK cells, 
steroids are to be avoided within 1 day BEFORE until 2 days AFTER receiving the NK infusion 
unless there is medical necessity.  Patients receiving >0.25 mg/kg equivalent prednisone dose within 
1 day prior to the NK cell infusion will not receive NK cells.  Patients not receiving NK cells (for 
any reason) will be taken off-study.  However, patients who must be given steroids for medical 
necessity within 2 days AFTER receiving the NK cell infusion will continue to remain on study. 
 
 
 
DONOR RESEARCH SAMPLES 
Peripheral blood and bone marrow aspirate will be obtained at different timepoints for 
Immune Studies.  Please see Appendix M for details. 
 
 
 
B. DONOR TREATMENT PLAN 
 
Table 8. Donor Treatment Plan 
 
Days 
 
 Week 4 to 3 
Pre 
 Week 3 to 
2 Pre 
Day 0   
+6 
Autologous RBC Collection (1 or 2 Units*) 
● 
● 
 
 
Oral iron supplementation x 7 days 
● 
● 
 
 
Marrow Harvest 
 
 
● 
 
Infusion of Autologous RBC (1-2 Units) 
 
 
● 
● 
COBE Apheresis** 
 
 
 
● 
*If only one unit is needed, the collection can occur 1 to 4 weeks prior to bone marrow harvest 
** COBE Apheresis may take place between days +6 and +9 to allow for scheduling flexibility. Preferred to be 
performed on Day +6. 
 
 
 
1.  Donor Screening Guidelines 
Per FDA guidelines, 21 CFR 1271 will be used for screening and testing the suitability of all donors.  
These guidelines are also reflected within FHCRC Standard Practice Guidelines for Donor Selection.  
Donors will be undergoing both marrow harvest for stem cell collection and apheresis for 
mononuclear cell collection.  Despite being screened for initial donor selection, it is important 
to note that all donors must be re-screened a minimum of 72 hours before, but no more than 
6 days before, apheresis for mononuclear cell collection.  This is to remain compliant with 
FDA regulations 21 CRF 1270 as part of good tissue practice (GTP) guidelines for donor 


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eligibility for cellular product infusions, which mandate donor screening within 7 days of 
cellular product collection.  
 
 
2. Autologous Blood Collection 
Since donors will undergo COBE apheresis approximately 1 week after marrow harvest, 2 Units of 
autologous blood may need to be collected from all adult donors. For pediatric donors greater than 
or equal to 12 years of age who meet other eligibility criteria, this amount will be determined based 
on donor and recipient weights. The first unit will be collected 3-4 weeks prior to the marrow harvest, 
and the second unit collected 2-3 weeks prior to the harvest. There should be at least one week 
between the first and second units. The second unit should be collected no less than 14 days (± 2 
days) prior to the marrow harvest.  If only one auto collection is needed, this may be between 1 and 
4 weeks prior to harvest. After each round of RBC collection, it is recommended that the donor be 
prescribed a 7 day course of oral iron supplementation to help replenish RBC stores.  
 
3. Marrow Harvest and Infusion of Autologous Blood 
It is assumed that 1 liter of aspirated marrow will translate into the equivalent of at least one unit of 
blood loss. Additional blood loss occurs from bleeding into soft tissue during and after the procedure. 
For a harvest of 2 liters, a donor will lose the equivalent of at least 2 units of blood with the product 
plus an additional 1 to 2 units from bleeding and oozing into the soft tissue (personal communication, 
M. Linenberger). Platelet loss is not a problem after marrow harvest. The donor marrow harvest will 
occur on Day 0 per standard procedures.  The total nucleated cell (TNC) goal will be based on the 
recipient’s ideal body weight as specified above. In the absence of ABO or other red cell 
incompatibility, the marrow product will be directly infused into the recipient without manipulation.  If 
major or minor ABO incompatibility exists with significant donor or recipient antibody titers, the 
product will be manipulated according to standard practice procedures (see Appendix A).  Following 
the marrow harvest, donors will be transfused autologous blood during post-anesthesia recovery to 
replenish blood loss from the harvest bleeding into tissues, and to minimize anemia prior to planned 
apheresis at day 6 to 9 post-harvest.   
 
4. Peripheral Blood Mononuclear Cell (MNC) Apheresis 
A CBC will be drawn one day prior to planned MNC apheresis. Apheresis will utilize either large-bore 
peripheral IV access or in adult patients without adequate peripheral veins, a central venous catheter 
placed on the morning of the apheresis procedure.  Donors less than 18 years old must be able to 
collect cells from a peripheral venous access.  The apheresis is preferred to take place on day +6 
(can occur between days +6 and +9 to facilitate scheduling). A total of 10 to 12 liters of donor blood 
volume will be processed by MNC leukapheresis using a Cobe Spectra instrument and following 
Standard Operating Procedures and standard treatment plans of the Apheresis Unit. The apheresis 
product will be transported to the CTL facility for overnight storage and will be processed the 
following day, as described in Section X.A.5.a.. 
 
XI. DONOR AND RECIPIENT EVALUATIONS 
 
A. HLA-Typing of Patient and Potential Donors 
1. As broad a range of potential related donors as possible should be typed. Included are parents, 
siblings, eligible children, and cousins. 
2. Serotyping (HLA-A, B, C) and DNA typing (HLA-A, B, C, DRB1, DQB1) of patient and donor will 
be performed. 
3. Leukocyte and/or florescence activated cell sorter cross match between the patient and donor 
will be done. Recipient must have a negative cytotoxic cross-match to donor lymphocytes, 
otherwise an alternative HLA-haploidentical donor must be identified. 
4. KIR genotype will not be used in determining donor selection. 


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5. Blood samples for HLA-typing should be sent to: Clinical Immunogenetics Lab for HLA-typing 
(heparinized green top tube, 10 cc), 206-667-7700; SCCA G7-200. 
 
 
B.  Donor Evaluations 
 
Per Standard Practice Guidelines, to also include: 
 
1. Complete history and physical examination prior to marrow harvest and interval history and a 
nursing check the day after harvest  
 
2.  Lab tests:  
a) CBC with differential including platelet count: per Institutional Guidelines with marrow 
harvest  
b) ABO Rh blood typing. If the recipient has antibodies against red cell antigens of the donor, 
the titers must be determined. Appropriate RBC depletion of the marrow allograft will need to 
be done per standard procedures. 
c) Infectious disease testing per standard practice, as part of the initial evaluation and within 
1 week of donor apheresis (see X.B.1) 
 
d) Donor Research Tests  
Peripheral blood may be collected from the donor on 1-2 separate occasions during the pre-
transplant work-up in order to perform the Immune Studies (See Appendix M)  
 
3.  Donor Chimerism Reference 
As a pre-transplant reference for subsequent determination of donor chimerism 10 mLs of 
heparinized (green top) peripheral blood from the donor will be drawn. For FHCRC patients: 
Send to Clinical Immunogenetics Lab (206-667-7700; SCCA G7-200) for a DNA-based assay 
that compares the profile of amplified fragment length polymorphisms (ampFLP) of the patient 
and donor. Label the sample with “Protocol 2230.” 
 


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C. Recipient Evaluations 
1.  Pre-transplant Evaluations 
a) See Institutional Standard Practice Guidelines for Standard Pre-Transplant Evaluation 
Guidelines. 
b)   Peripheral blood and bone marrow aspirate may be collected from the recipient during the 
pre-transplant work-up.  If the patient is lymphopenic, an additional peripheral blood draw 
may be required to yield adequate cells for the research test.  See Appendix M for additional 
research specimen requests 
c) See Table 9, 10, 11 and, 12 for disease specific pre-transplant evaluation. 
 
Table 9: Disease-Specific Pre-Transplant Evaluations for Ph (-) ALL, Ph (+) ALL, CML 
 
 
Note: All bone marrow aspirates and biopsies are unilateral and must be collected within 21 days of 
treatment. See Tables 13 and 14 for post-transplant evaluations and table 15 for additional lab instructions. 
 
Specimen / Test / Imaging 
Clinical / 
Research 
Comment 
Bone marrow aspirate 
 
Pathology 
Clinical 
 
Flow Cytometry 
Clinical 
 
Cytogenetics 
Clinical 
 
FISH for clonal abnormalities 
Clinical 
 
PCR for bcr/abl, p.210 breakpoint - *see comment 
Clinical 
*CML only- 
reflective testing for 
FHCRC patients only 
PCR for bcr/abl, p.190 and p.210 breakpoints - *see 
comment  
Clinical 
*Ph (+) ALL only- 
 reflective testing for 
FHCRC patients only 
Bone marrow biopsy 
 
Pathology- *see comment 
Clinical 
*CML only 
Peripheral Blood 
 
Storage for chimerism analysis 
Clinical 
 
PCR for bcr/abl, p.210 breakpoint- *see comment 
Clinical 
*CML only 
 
 
 
 
 
 


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Table 10: Disease-Specific Pre-Transplant Evaluations for AML and MDS/MPD 
 
Note: All bone marrow aspirates and biopsies are unilateral and must be collected within 21 days of 
treatment. See Tables 13 and 14 for post-transplant evaluations and table 15 for additional lab instructions. 
 
Specimen / Test / Imaging 
Clinical / 
Research 
Comment 
Bone marrow aspirate 
 
Pathology 
Clinical 
 
Flow Cytometry 
Clinical 
 
Cytogenetics 
Clinical 
 
FISH for clonal abnormalities 
Clinical 
 
Bone marrow biopsy 
 
Pathology- *see comment 
Clinical 
*MDS/MPD only 
Peripheral Blood 
 
Storage for chimerism analysis 
Clinical 
 
 
 
 
 


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28   
 
 
Table 11: Disease-Specific Pre-Transplant Evaluations for CLL, HL, NHL 
 
Note: All bone marrow aspirates and biopsies are bilateral and must be collected within 30 days of 
treatment. See Tables 13 and 14 for post-transplant evaluations and table 15 for additional lab instructions. 
 
Specimen / Test / Imaging 
Clinical / 
Research 
Comment 
Bone marrow aspirate 
 
Pathology 
Clinical 
 
Flow Cytometry- *see comment 
Clinical 
*No HL 
Cytogenetics 
Clinical 
 
FISH for clonal abnormalities 
Clinical 
 
PCR t(11:14) - *see comment 
Clinical 
*Mantle Cell NHL only 
PCR t(14:18) - *see comment 
Clinical 
*Follicular NHL only 
Bone marrow biopsy 
 
Pathology- *see comment 
Clinical 
*HL – only if history of BM 
involvement 
Peripheral Blood 
 
Storage for chimerism analysis 
Clinical 
 
Quantitative Ig levels 
Clinical 
 
β-2 microglobulin 
Clinical 
 
LDH 
Clinical 
 
ZAP – 70 by flow cytometry- *see comment 
Clinical 
*CLL only– for patients not in CR 
Imaging 
 
CT of chest, abdomen, pelvis (neck if indicated) 
Clinical 
 
 
 
 
 


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29   
Table 12: Disease-Specific Pre-Transplant Evaluations for MM and Waldenstrom’s 
Macroglobulinemia 
 
 
Note: All bone marrow aspirates and biopsies are bilateral *see comment and must be collected within 30 
days of treatment. See Tables 13 and 14 for post-transplant evaluations and table 15 for additional lab 
instructions. 
 
Specimen / Test / Imaging 
Clinical / 
Research 
Comment 
Bone marrow aspirate *see comment                                                *Unilateral bone marrow aspirate 
is acceptable for MM  
 
Pathology 
Clinical 
 
Flow Cytometry 
Clinical 
 
Cytogenetics 
Clinical 
 
FISH for clonal abnormalities 
Clinical 
 
Bone marrow biopsy *see comment                                                   *Unilateral bone marrow  biopsy 
is acceptable for MM 
 
Pathology 
Clinical 
 
Peripheral Blood 
 
Storage for chimerism analysis 
Clinical 
 
SPEP/IFIX 
Clinical 
 
Quantitative Ig levels 
Clinical 
 
β-2 microglobulin 
Clinical 
 
Cryoglobulins, c-reactive protein, serum viscosity - 
*see comment 
Clinical 
*Serum viscosity only for patients with 
>3gm/dL IgM monoclonal protein or 
>4gm/dL IgA or IgG protein 
Urine 
 
UPEP/IFIX 
Clinical 
 
Protein / creatinine clearance 
Clinical 
 
Imaging 
 
MRI –  *see comment 
Clinical 
*MM only. A PET scan is acceptable. 
Skeletal survey –  *see comment 
Clinical 
*MM only 
CT of chest, abdomen, pelvis (neck if indicated) –  
*see comment 
Clinical 
*Waldenstrom’s Macroglobulinemia only 
 
 
 
 


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30   
2. Post-transplant Evaluations 
 
See Table 13 for disease specific post-transplant evaluation on Day +28, 56, 84, etc. This is a 
recommended evaluation schedule. 
 
Patients should be assessed for the need of bisphosphonates and IVIG monitoring and replacement 
therapy per Institutional Guidelines 
 
 
See Standard Practice Guidelines for Evaluation Guidelines Prior to Departure from FHCRC 
System. GVHD evaluation guidelines are as follows: 
 
 
History and physical exam  
 
Skin biopsy 
 
Schirmer's tear test 
 
Pulmonary function test 
 
Oral exam  
 
CXR 
 
Dietician assessment 
 
Gynecological departure assessment (adult female)  
 
See Appendix M for additional research requests 
 


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Table 13: Post-Transplant Evaluation 
This is a recommended evaluation schedule.  
See Tables 9 - 12 for pre-transplant evaluations. Additional lab instructions in Table 15. 
 
Disease 
Specimen/ Test/ Imaging 
Clinical/ 
Research 
Comment 
Days 
Years 
Annual 
x 5 years 
**28 
56 
84 
180 
1 
1.5 
Ph (-) ALL 
BM aspirate* If CR documented at one year, and there is recovery of normal blood counts, bone marrow after one year may be obtained based on clinical  
                        judgment 
Chimerism  
Clinical 
 
 
 
x 
 
x 
 
 
Pathology 
Clinical 
 
x 
x 
x 
x 
x 
x 
x 
Flow cytometry 
Clinical 
 
x 
x 
x 
x 
x 
x 
x 
Cytogenetics 
Clinical 
*If abnormal pre-transplant 
*See 
commen
t 
*See 
comment 
*See 
commen
t 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
FISH for clonal 
abnormalities 
Clinical 
*If abnormal pre-transplant 
*See 
commen
t 
*See 
comment 
*See 
commen
t 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
Peripheral blood 
Chimerism 
Clinical 
**Needed on Day 7, 14, 28, 
56, 84, 180, and 1 year 
**See table 14 for peripheral blood chimerisms 
T and B cell subsets (CD3, 
CD4, CD8, B and NK 
cells) 
Clinical 
 
 
 
x 
 
x 
 
 
GVHD evaluation 
Clinical 
See text for details 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Disease 
Specimen/ Test/ Imaging 
Clinical/ 
Research 
Comment 
Days 
Years 
Annual 
x 5 years 
**28 
56 
84 
180 
1 
1.5 
Ph (+) ALL 
 
BM aspirate* If CR documented at one year, and there is recovery of normal blood counts, bone marrow after one year may be obtained based on clinical judgment 
Chimerism 
Clinical 
 
 
 
x 
 
x 
 
 
Pathology 
Clinical 
 
x 
x 
x 
x 
x 
x 
x 
Flow cytometry 
Clinical 
 
x 
x 
x 
x 
x 
x 
x 
Cytogenetics 
Clinical 
 
x 
x 
x 
x 
x 
x 
x 
FISH for  bcr/abl and other 
clonal abnormalities 
Clinical 
 
x 
x 
x 
x 
x 
x 
x 
PCR for bcr/abl, p.190 and 
p.210 breakpoints 
Clinical 
 
x 
x 
x 
x 
x 
x 
x 
Peripheral blood 
Chimerism 
Clinical 
**Needed on Day 7, 14, 28, 
56, 84, 180, and 1 year 
**See table 14 for peripheral blood chimerisms 
T and B cell subsets(CD3, 
CD4, CD8, B and NK 
cells) 
Clinical 
 
 
 
x 
 
x 
 
 
PCR for bcr-abl, p.190 and 
p.210 breakpoints 
Clinical 
*If Bone Marrow not 
obtained, reflexive testing for 
FHCRC patients only  
*See 
commen
t 
*See 
comment 
*See 
commen
t 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
GVHD evaluation 
Clinical 
See text for details 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Disease 
Specimen/ Test/ Imaging 
Clinical/ 
Research 
Comment 
Days 
Years 
Annual 
x 5 years 
**28 
56 
84 
180 
1 
1.5 
AML 
BM aspirate* If CR documented at one year, and there is recovery of normal blood counts, bone marrow after one year may be obtained based on clinical judgment 
Chimerism 
Clinical 
 
 
 
x 
 
x 
 
 
Pathology 
Clinical 
 
x 
x 
x 
x 
x 
x 
x 
Flow cytometry 
Clinical 
 
x 
x 
x 
x 
x 
x 
x 
Cytogenetics 
Clinical 
*If abnormal pre-transplant 
*See 
commen
t 
*See 
comment 
*See 
commen
t 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
FISH for clonal 
abnormalities 
Clinical 
*If abnormal pre-transplant 
*See 
commen
t 
*See 
comment 
*See 
commen
t 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
Peripheral blood 
Chimerism 
Clinical 
**Needed on Day 7, 14, 28, 
56, 84, 180, and 1 year 
**See table 14 for peripheral blood chimerisms 
T and B cell subsets(CD3, 
CD4, CD8, B and NK 
cells) 
Clinical 
 
 
 
x 
 
x 
 
 
GVHD evaluation 
Clinical 
See text for details 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Disease 
Specimen/ Test/ Imaging 
Clinical/ 
Research 
Comment 
Days 
Years 
Annual 
x 5 years 
**28 
56 
84 
180 
1 
1.5 
MDS/ MPD 
 
 
BM aspirate *see biopsy 
                       ** If CR documented at one year, and there is recovery of normal blood counts, bone marrow after one year may be obtained based on clinical judgment 
Chimerism 
Clinical 
 
 
 
x 
 
x 
 
 
Pathology 
Clinical 
 
x 
x 
x 
x 
x 
x 
x 
Flow cytometry 
Clinical 
 
x 
x 
x 
x 
x 
x 
x 
Cytogenetics 
Clinical 
*If abnormal  pre-transplant 
*See 
commen
t 
*See 
comment 
*See 
commen
t 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
FISH for clonal 
abnormalities 
Clinical 
*If abnormal  pre-transplant 
*See 
commen
t 
*See 
comment 
*See 
commen
t 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
BM biopsy 
Pathology 
Clinical 
*For pts. with evidence or 
history of myelofibrosis 
*See 
commen
t 
*See 
comment 
*See 
commen
t 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
Peripheral blood 
Chimerism 
Clinical 
**Needed on Day 7, 14, 28, 
56, 84, 180, and 1 year 
**See table 14 for peripheral blood chimerisms 
T and B cell subsets(CD3, 
CD4, CD8, B and NK 
cells) 
Clinical 
 
 
 
x 
 
x 
 
 
GVHD evaluation 
Clinical 
See text for details 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Disease 
Specimen/ Test/ Imaging 
Clinical/ 
Research 
Comment 
Days 
Years 
Annual 
x 5 years 
**28 
56 
84 
180 
1 
1.5 
CML 
BM aspirate *see biopsy 
                        ** If CR documented at one year, and there is recovery of normal blood counts, bone marrow after one year may be obtained based on clinical judgment 
 
Chimerism  
Clinical 
 
 
 
x 
 
x 
 
 
Pathology 
Clinical 
 
x 
x 
x 
x 
x 
x 
x 
Flow cytometry 
Clinical 
 
x 
x 
x 
x 
x 
x 
x 
Cytogenetics 
Clinical 
 
x 
x 
x 
x 
x 
x 
x 
FISH for bcr-abl and other 
clonal abnormalities 
Clinical 
 
x 
x 
x 
x 
x 
x 
x 
PCR for bcr-abl, and p.210 
breakpoint 
Clinical 
*If abnormal pre-transplant, 
Reflexive testing for FHCRC 
patients only  
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
BM biopsy 
 
Pathology 
Clinical 
*If abnormal pre-transplant 
 
 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
Peripheral blood 
 
Chimerism 
Clinical 
**Needed on Day 7, 14, 28, 
56, 84, 180, and 1 year 
**See table 14 for peripheral blood chimerisms 
T and B cell subsets(CD3, 
CD4, CD8, B and NK cells) 
Clinical 
 
 
 
x 
 
x 
 
 
PCR for bcr-abl, and p.210 
breakpoint 
Clinical 
*If abnormal pre-transplant 
AND bone marrow not 
obtained, Reflexive testing for 
FHCRC patients only 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
GVHD evaluation 
Clinical 
See text for details 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Disease 
Specimen/ Test/ Imaging 
Clinical/ 
Research 
Comment 
Days 
Years 
Annual 
x 5 years 
**28 
56 
84 
180 
1 
1.5 
CLL 
BM aspirate *see biopsy 
                       ** If CR documented at one year, and there is recovery of normal blood counts, bone marrow after one year may be obtained based on clinical judgment 
 
Chimerism  
Clinical 
 
 
 
x 
 
x 
 
 
Pathology 
Clinical 
 
x 
x 
x 
x 
x 
x 
x 
Flow cytometry 
Clinical 
 
x 
x 
x 
x 
x 
x 
x 
Cytogenetics 
Clinical 
*If abnormal pre-transplant 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
FISH for clonal 
abnormalities 
Clinical 
*If abnormal pre-transplant 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
BM biopsy 
 
Pathology 
Clinical 
 
 
 
x 
x 
x 
x 
x 
Peripheral blood 
 
Chimerism 
Clinical 
**Needed on Day 7, 14, 28, 
56, 84, 180, and 1 year 
**See table 14 for peripheral blood chimerisms 
T and B cell subsets(CD3, 
CD4, CD8, B and NK cells) 
Clinical 
 
 
 
x 
 
x 
 
 
Flow cytometry 
Clinical 
*If peripheral blood 
involvement pre-transplant 
AND bone marrow not done 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
Quantitative Ig levels 
Clinical 
*If abnormal pre-transplant 
*See 
comment  
*See 
comment  
*See 
comment  
*See 
comment  
*See 
comment  
*See 
comment  
*See comment  
β-2 microglobulin 
Clinical 
 
 
 
x 
 
x 
 
 
LDH 
Clinical 
 
 
x 
x 
x 
x 
x 
x 
Imaging 
 
CT chest, abdomen, pelvis 
(neck if indicated) 
Clinical 
*Day 56 only if abnormal  pre-
transplant 
 
*See 
comment 
x 
x 
x 
x 
x 
GVHD evaluation 
Clinical 
See text for details 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 


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Disease 
Specimen/ Test/ Imaging 
Clinical/ 
Research 
Comment 
Days 
Years 
Annual 
x 5 years 
**28 
56 
84 
180 
1 
1.5 
HL - 
No history of 
BM 
involvement 
BM aspirate* If CR documented at one year, and there is recovery of normal blood counts, bone marrow after one year may be obtained based on clinical judgment 
 
Chimerism 
Clinical 
 
 
 
x 
 
x 
 
 
Pathology 
Clinical 
 
 
 
x 
 
x 
 
 
Cytogenetics 
Clinical 
*If abnormal pre-transplant  
 
 
*See 
comment 
 
*See 
comment 
 
 
FISH for clonal 
abnormalities 
Clinical 
*If abnormal pre-transplant 
 
 
*See 
comment 
 
*See 
comment 
 
 
Peripheral blood 
 
Chimerism 
Clinical 
**Needed on Day 7, 14, 28, 
56, 84, 180, and 1 year 
**See table 14 for peripheral blood chimerisms 
T and B cell subsets(CD3, 
CD4, CD8, B and NK cells) 
Clinical 
 
 
 
x 
 
x 
 
 
Quantitative Ig levels 
Clinical 
*If abnormal pre-transplant 
*See 
comment  
*See 
comment  
*See 
comment  
*See 
comment  
*See 
comment  
*See 
comment  
*See comment  
β-2 microglobulin 
Clinical 
 
 
 
x 
 
x 
 
 
LDH 
Clinical 
 
 
x 
x 
x 
x 
x 
x 
Imaging 
 
CT chest, abdomen, pelvis 
(neck if indicated) 
Clinical 
*Day 56 only if abnormal  pre-
transplant 
 
*See 
comment  
x 
x 
x 
x 
x 
GVHD evaluation 
Clinical 
See text for details 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Disease 
Specimen/ Test/ Imaging 
Clinical/ 
Research 
Comment 
Days 
Years 
Annual 
x 5 years 
**28 
56 
84 
180 
1 
1.5 
HL - 
History of BM 
involvement 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
BM aspirate *see biopsy 
                       **  If CR documented at one year, and there is recovery of normal blood counts, bone marrow after one year may be obtained based on clinical judgment 
 
Chimerism  
Clinical 
 
 
 
x 
 
x 
 
 
 
Pathology 
Clinical 
 
x 
x 
x 
x 
x 
x 
x 
Cytogenetics 
Clinical 
*If abnormal pre-transplant  
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
FISH for clonal 
abnormalities 
Clinical 
*If abnormal pre-transplant  
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
BM biopsy 
 
Pathology 
Clinical 
 
 
 
x 
x 
x 
x 
x 
Peripheral blood 
 
Chimerism 
Clinical 
**Needed on Day 7, 14, 28, 
56, 84, 180, and 1 year 
**See table 14 for peripheral blood chimerisms 
T and B cell subsets(CD3, 
CD4, CD8, B and NK cells) 
Clinical 
 
 
 
x 
 
x 
 
 
Quantitative Ig levels 
Clinical 
*If abnormal pre-transplant 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
β-2 microglobulin 
Clinical 
 
 
 
x 
 
x 
 
 
LDH 
Clinical 
 
 
x 
x 
x 
x 
x 
x 
Imaging 
 
 
 
 
 
 
 
 
 
CT chest, abdomen, pelvis 
(neck if indicated) 
Clinical 
*Day 56 only if abnormal  pre-
transplant 
 
*See 
comment  
x 
x 
x 
x 
x 
GVHD evaluation 
Clinical 
See text for details 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Disease 
Specimen/ Test/ Imaging 
Clinical/ 
Research 
Comment 
Days 
Years 
Annual 
x 5 years 
**28 
56 
84 
180 
1 
1.5 
NHL –  
No history of 
BM 
involvement 
 
*see separate 
section for 
additional PCR 
on Mantle Cell 
and Follicular 
NHLs in 
suspected CR 
BM aspirate* If CR documented at one year, and there is recovery of normal blood counts, bone marrow after one year may be obtained based on clinical judgment 
   Chimerism 
Clinical 
 
 
 
x 
 
x 
 
 
 
Pathology 
Clinical 
 
 
 
x 
 
x 
 
 
 
Flow cytometry 
Clinical 
 
 
 
x 
 
x 
 
 
 
Cytogenetics 
Clinical 
*If abnormal pre-transplant 
 
 
*See 
comment 
 
*See 
comment 
 
 
Peripheral blood 
 
Chimerism (CD3+) 
Clinical 
*Days 56 and 180 only if <50% 
on day 28 
x 
*See 
comment 
x 
*See 
comment 
x 
 
 
 
Chimerism (CD33+) 
Clinical 
 
 
 
x 
 
 
 
 
 
Chimerism  (NK CD56+) 
Clinical 
Optional for outside institutions 
x 
 
 
 
 
 
 
 
Flow cytometry 
Clinical 
* If peripheral blood 
involvement pre-transplant 
AND bone marrow not done 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
 
β-2 Microglobulin 
Clinical 
 
 
 
x 
 
 
 
 
 
LDH 
Clinical 
 
 
 
x 
x 
x 
x 
x 
Imaging 
 
CT of chest, abdomen, pelvis 
(neck if indicated) 
Clinical 
*If abnormal pre-transplant 
 
*See 
comment 
x 
x 
x 
x 
x 
GVHD evaluation 
Clinical 
See text for details 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Disease 
Specimen/ Test/ Imaging 
Clinical/ 
Research 
Comment 
Days 
Years 
Annual 
x 5 years 
**28 
56 
84 
180 
1 
1.5 
NHL -- 
History of BM 
involvement  
 
*see separate 
section for 
additional PCR 
on Mantle Cell 
and Follicular 
NHLs in 
suspected CR 
BM aspirate *see biopsy 
                        ** If CR documented at one year, and there is recovery of normal blood counts, bone marrow after one year may be obtained based on clinical judgment 
 
Chimerism  
Clinical 
 
 
 
x 
 
x 
 
 
Pathology 
Clinical 
 
x 
x 
x 
x 
x 
x 
x 
Flow cytometry 
Clinical 
 
x 
x 
x 
x 
x 
x 
x 
Cytogenetics 
Clinical 
*If abnormal pre-transplant  
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
FISH for clonal 
abnormalities 
Clinical 
*If abnormal pre-transplant  
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
BM biopsy 
 
Pathology 
Clinical 
 
 
 
x 
x 
x 
x 
x 
Peripheral blood 
 
Chimerism 
Clinical 
**Needed on Day 7, 14, 28, 
56, 84, 180, and 1 year 
**See table 14 for peripheral blood chimerisms 
Flow cytometry 
Clinical 
*If peripheral blood 
involvement pre-transplant 
AND bone marrow not done 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
T and B cell subsets(CD3, 
CD4, CD8, B and NK cells) 
Clinical 
 
 
 
x 
 
x 
 
 
Quantitative Ig levels 
Clinical 
*If abnormal pre-transplant 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
β-2 microglobulin 
Clinical 
 
 
 
x 
 
x 
 
 
LDH 
Clinical 
 
 
x 
x 
x 
x 
x 
x 
Imaging 
 
CT chest, abdomen, pelvis 
(neck if indicated) 
Clinical 
*Day 56 only if abnormal  pre-
transplant 
 
*See 
comment  
x 
x 
x 
x 
x 
GVHD evaluation 
Clinical 
See text for details 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 


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Disease 
Specimen/ Test/ Imaging 
Clinical/ 
Research 
Comment 
Days 
Years 
Annual 
x 5 years 
**28 
56 
84 
180 
1 
1.5 
Mantle Cell 
NHL in 
suspected CR 
BM aspirate *in addition to complete NHL restaging 
                        ** If CR documented at one year, and there is recovery of normal blood counts, bone marrow after one year may be obtained based on clinical judgment 
 
PCR for t(11:14) 
Clinical 
*If abnormal pre-transplant  
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
Peripheral blood  *in addition to complete NHL restaging 
 
PCR for t(11:14) 
Clinical 
*If abnormal pre-transplant 
AND bone marrow not done 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
Follicular Cell 
NHL in 
suspected CR 
BM aspirate *in addition to complete NHL restaging 
                        ** If CR documented at one year, and there is recovery of normal blood counts, bone marrow after one year may be obtained based on clinical judgment 
 
PCR for t(14:18) 
Clinical  
*If abnormal pre-transplant  
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
Peripheral blood  *in addition to complete NHL restaging 
 
PCR for t(14:18) 
Clinical  
*If abnormal pre-transplant 
AND bone marrow not done 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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42   
Disease 
Specimen/ Test/ Imaging 
Clinical/ 
Research 
Comment 
Days 
Years 
Annual 
x 5 years 
**28 
56 
84 
180 
1 
1.5 
MM 
 
 
Omit 
SPEP/IFIX and 
UPEP/IFIX for 
non-secretory 
MM 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
BM aspirate   * If CR documented at one year, and there is recovery of normal blood counts, bone marrow after one year may be obtained based on clinical judgment 
 
Chimerism 
Clinical 
 
 
 
x 
 
x 
 
 
Pathology 
Clinical  
 
x 
x 
x 
x 
x 
x 
x 
Flow cytometry 
Clinical   
 
x 
x 
x 
x 
x 
x 
x 
Cytogenetics 
Clinical  
*If abnormal pre-transplant  
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
FISH for chrom. 13 (and 
other clonal) abnormalities  
Clinical 
*If abnormal pre-transplant  
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
Peripheral blood 
 
Chimerism 
Clinical 
**Needed on Day 7, 14, 28, 
56, 84, 180, and 1 year 
**See table 14 for peripheral blood chimerisms 
T and B cell subsets(CD3, 
CD4, CD8, B and NK cells) 
Clinical 
 
 
 
x 
 
x 
 
 
SPEP and IFIX 
Clinical 
 
 
 
x 
x 
x 
x 
x 
Quantitative Ig levels 
Clinical 
*If abnormal pre-transplant 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
β-2 microglobulin 
Clinical 
 
 
 
x 
x 
x 
x 
x 
Cryoglobulins, C-reactive 
protein, viscosity 
Clinical 
*If abnormal pre-transplant 
 
 
*See 
comment 
*See 
comment 
*See 
comment 
 
*See comment 
Urine 
 
Protein/creatinine clearance 
Clinical 
 
 
 
x 
x 
x 
x 
x 
UPEP and IFIX 
Clinical 
*If abnormal pre-transplant  
 
 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
Imaging 
 
Complete skeletal survey 
Clinical 
 
 
 
 
 
x 
 
x 
Skeletal MRI 
Clinical 
 
 
 
 
 
x 
 
x 
GVHD evaluation 
Clinical 
See text for details 
 
 
x 
 
 
 
 
 
 
 
 
 


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Disease 
Specimen/ Test/ Imaging 
Clinical/ 
Research 
Comment 
Days 
Years 
Annual 
x 5 years 
**28 
56 
84 
180 
1 
1.5 
Waldenstrom’s 
Macro-
globulinemia 
 
Omit SPEP/IFIX 
and UPEP/IFIX 
for non-secretory 
Waldenstrom’s 
Macro-
globulinemia 
 
BM aspirate   * If CR documented at one year, and there is recovery of normal blood counts, bone marrow after one year may be obtained based on clinical judgment 
 
Chimerism 
Clinical 
 
 
 
x 
 
x 
 
 
Pathology 
Clinical  
 
x 
x 
x 
x 
x 
x 
x 
Flow cytometry 
Clinical  
 
x 
x 
x 
x 
x 
x 
x 
Cytogenetics 
Clinical  
*If abnormal pre-transplant  
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
FISH for chrom. 13 (and 
other clonal) abnormalities  
Clinical 
*If abnormal pre-transplant  
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
Peripheral blood 
 
 
Chimerism 
Clinical 
**Needed on Day 7, 14, 28, 
56, 84, 180, and 1 year 
**See table 14 for peripheral blood chimerisms 
T and B cell subsets(CD3, 
CD4, CD8, B and NK cells) 
Clinical 
 
 
 
x 
 
x 
 
 
SPEP and IFIX 
Clinical 
 
 
 
x 
x 
x 
x 
x 
Quantitative Ig levels 
Clinical 
*If abnormal pre-transplant 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
β-2 microglobulin 
Clinical 
 
 
 
 
x 
x 
x 
x 
Cryoglobulins, C-reactive 
protein, viscosity 
Clinical 
*If abnormal pre-transplant 
 
 
*See 
comment 
*See 
comment 
*See 
comment 
 
*See comment 
Urine 
 
 
Protein/ creatinine 
clearance 
Clinical 
 
 
 
x 
x 
x 
x 
x 
UPEP and IFIX 
Clinical 
*If abnormal pre-transplant  
 
 
*See 
comment 
*See 
comment 
*See 
comment 
*See 
comment 
*See comment 
Imaging 
 
 
 
 
 
 
 
 
 
 
CT chest, abdomen, pelvis 
(neck if indicated) 
Clinical 
*Day 56 only if abnormal  pre-
transplant 
 
*See 
comment 
x 
x 
x 
x 
x 
GVHD  evaluation 
Clinical 
See text for details 
 
 
x 
 
 
 
 
 
 
 
 
 
 


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Table 14: Post-Transplant Peripheral Blood Chimerism Evaluation 
This is a recommended evaluation schedule. See Tables 9 - 12 for pre-transplant evaluations, table 13 for post-transplant evaluations.  
Additional lab instructions in Table 15. 
 
Disease 
Test 
Clinical/ 
Researc
h 
Comment 
Days 
1 
Year 
7 
14 
28 
56 
84 
180 
All 
Diseas
es 
Chimerism (CD3+) 
Clinical 
*If donor NK cell infusion is occurring 
on Day +7, obtain chimerism sample pre-
NK cell infusion 
*See 
comme
nt 
x 
x 
x 
x 
x 
x 
Chimerism 
(CD33+) 
Clinical 
*If donor NK cell infusion is occurring 
on Day +7, obtain chimerism sample pre-
NK cell infusion 
*See 
comme
nt 
x 
x 
x 
x 
x 
x 
Chimerism (NK 
CD56+) 
Clinical 
*If donor NK cell infusion is occurring 
on Day +7, obtain chimerism sample pre-
NK cell infusion 
*See 
comme
nt 
x 
x 
x 
x 
x 
x 
 
 
 
 
 
 
 


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Table 15: Additional Lab Instructions 
Note: All bone marrow tests are done on aspirate unless specifically identified as biopsy. All instructions apply to both pre- and post-transplant 
evaluations unless identified otherwise.  
Off-site providers may use local facilities for the tests.  
 
Volumes represent desired amounts. 
 
 
Specimen / 
Test 
Type 
Instructions 
Lab Name 
Contact Information 
Bone marrow 
 
Chimerism  
Clinical 
1-3mL bone marrow in 
green-top tube 
Clinical Immunogenetics 
Lab 
Seattle Cancer Care Alliance 
 (206) 288-7700 
Pathology 
(aspirate) 
Clinical 
2mL bone marrow in 
EDTA/ formalin 
SCCA Pathology Lab 
Seattle Cancer Care Alliance 
(206) 288-1355 
Pathology (biopsy) 
Clinical 
1cm bone marrow in 
formalin OR mounted in 
paraffin 
SCCA Pathology Lab 
Seattle Cancer Care Alliance 
 (206) 288-1355 
Flow Cytometry 
Clinical 
2mL bone marrow in green-
top tube 
UW Hematopathology Lab 
Seattle Cancer Care Alliance 
 (206) 288-7060 
Cytogenetics 
Clinical 
3mL bone marrow in green-
top tube 
SCCA Cytogenetics Lab 
Seattle Cancer Care Alliance 
 (206) 288-1390 
FISH 
Clinical 
2mL bone marrow in green-
top tube 
SCCA Cytogenetics Lab 
Seattle Cancer Care Alliance 
 (206) 288-1390 
PCR  for bcr-abl 
and p190 and/or 
p210 
Clinical 
3mL bone marrow in 
lavender-top tube 
Label “protocol 2230” 
UW Molecular 
Hematopathology Lab  
Mailstop G7-800 825 Eastlake Ave, 
East Seattle, WA 98109 
(206) 288-7060 
PCR for t(11:14) or 
t(14:18) 
Clinical 
2mL bone marrow in 
lavender-top tube 
UW Molecular 
Hematopathology Lab  
Mailstop G7-800 825 Eastlake Ave, 
East Seattle, WA 98109 
(206) 288-7060 
Peripheral blood 
 
Chimerism  
(CD3+), (CD33+) 
NK(CD56+) 
Clinical 
10mL blood in green-top 
tube for Flow sorting, then 
to CIL 
UW Hematopathology Lab, 
routed to Clinical 
Immunogenetics Lab 
Mailstop G7-800 825 Eastlake Ave, 
East Seattle, WA 98109 
(206) 288-7060 
Flow Cytometry 
Clinical 
10mL blood in green-top 
tube 
UW Hematopathology Lab 
Seattle Cancer Care Alliance 
 (206) 288-7060 
T and B cell 
Clinical 
1mL blood in lavender-top 
UW Hematopathology Lab 
Seattle Cancer Care Alliance 


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Specimen / 
Test 
Type 
Instructions 
Lab Name 
Contact Information 
subsets(CD3, 
CD4, CD8, B and 
NK cells) 
 (206) 288-7060 
SPEP/IFIX 
Clinical 
3mL blood in red-top tube 
UW Department of 
Laboratory Medicine 
University of Washington 
(800) 713-5198 
Quantitative Ig 
Levels 
Clinical 
3mL blood in red-top tube 
SCCA Alliance Lab 
Seattle Cancer Care Alliance 
(206) 288-2057 
β-2 Microglobulin 
Clinical 
3mL blood in red-top tube 
UW Department of 
Laboratory Medicine 
University of Washington 
(800) 713-5198 
LDH 
Clinical 
3mL blood in red-top tube 
SCCA Alliance Lab 
Seattle Cancer Care Alliance 
(206) 288-2057 
PCR for bcr-abl 
and p190 and/or 
p210 
Clinical 
7mL blood in lavender-top 
tube 
Label “protocol 2230” 
UW Molecular 
Hematopathology Lab  
Mailstop G7-800 825 Eastlake Ave, 
East Seattle, WA 98109 
(206) 288-7060 
PCR for t(11:14) or 
t(14:18) 
Clinical 
5mL blood in lavender-top 
tube 
UW Molecular 
Hematopathology Lab  
Mailstop G7-800 825 Eastlake Ave, 
East Seattle, WA 98109 
(206) 288-7060 
 
ZAP – 70 by Flow 
cytometry 
(pre-transplant only) 
Clinical 
5mL blood in green-top 
tube 
UW Hematopathology Lab 
Mailstop G7-800 825 Eastlake Ave, 
East Seattle, WA 98109 
(206) 288-7060 
 
See Standard Practice Guidelines for T and B cell subsets 
 
Outside institutions may use VNTR analysis (sex- matched transplants) or sex chromosome FISH-analysis (sex-mismatched 
transplants) for chimerism analysis. 
 
 


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XII. Drugs, Irradiation and Marrow Administration - Toxicities and Complications 
 
A. Cyclophosphamide 
 
1. Description 
 
Cyclophosphamide is an alkylating agent which prevents cell division primarily by cross 
linking DNA strands. Cyclophosphamide is cell cycle non-specific. Cyclophosphamide is  not 
stem cell toxic. 
2.  Storage and Administration 
Cyclophosphamide for injection is commercially available in 2000 mg vials, which are 
reconstituted with 100 ml sterile water for injection. The concentration of the reconstituted 
product is 20 mg/ml. The calculated dose will be diluted further in 250-500 ml of Dextrose 5% 
in water. Each dose will be infused over 1-2 hr (depending on the  total volume). 
3. Side Effects and Toxicity 
Clinical toxicities of cyclophosphamide include alopecia, nausea and vomiting, headache and 
dizziness, hemorrhagic cystitis, cardiotoxicity, immunosuppression, myelosuppression, 
pulmonary fibrosis, increased hepatic enzymes and syndrome of appropriate anti-diuretic 
hormone (SIADH). 
 
B. MESNA 
 
1.  Description 
Mesna is a prophylactic agent used to prevent hemorrhagic cystitis induced by the 
oxazaphosphorine (cyclophosphamide and iphosphamide). It has no intrinsic cytotoxicity  and 
no antagonistic effects on chemotherapy. Mesna binds with acrolein, the urotoxic metabolite 
produced by the oxazaphosphorine, to produce a non-toxic thioether and  slows the rate of 
acrolein formation by combining with 4-hydroxy metabolites of oxazaphosphorine. 
 
2. Storage and Administration 
Mesna is commercially available in 200 mg, 400 mg and 1000 mg vials containing a 
100mg/ml solution. Each dose of Mesna will be diluted further in 50 ml of normal saline to be 
infused over 15 min. Mesna dose will be based on the CY dose being given but can be 
omitted when the CY dose is less than 1000 mg/m2 (or 33.3 mg/kg). The total daily dose of 
Mesna is equal to 100% of the total daily dose of cyclophosphamide. 
 
3.  Side Effects and Toxicity 
At the doses used for uroprotection Mesna is virtually non-toxic. However, adverse effects, 
which may be attributable to Mesna, include nausea and vomiting, diarrhea, abdominal pain, 
altered taste, rash, urticaria, headache, joint or limb pain, hypotension and fatigue. 
 
C. Fludarabine 
 
 
1. Description  
Fludarabine’s active metabolite 2-fluoro-ara-A is an antimetabolite that inhibits DNA primase, 
DNA polymerase alpha and Ribonucleotide nuclease.  
 
2. Dosage and Administration 
Fludarabine monophosphate is commercially available as a 50 mg/vial, which is 
reconstituted with 2 ml of sterile water, resulting in a 25mg/ml solution. The desired dose is 
further diluted to concentrations of 0.04-1 mg/ml in normal saline or 5% dextrose (50-100ml) 
for injection and will be administered by IV infusion over 30 minutes or longer.  
 
Fludarabine will be administered by IV infusion over 30 minutes in a dose of 30 mg/m2/day 
on days -6 to -2.  
 
 


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3. Side Effects and Toxicities 
The dose of fludarabine used in this protocol is nonmyeloablative; however, it can cause 
severe immunosuppression particularly in the CD4+ T cell compartment. 
Immunosuppression increases the risk of infection, which can be life threatening. In addition, 
clinical toxicities of fludarabine monophosphate include: myelosuppression, primarily 
lymphopenia and granulocytopenia, alopecia, rash, dermatitis, nausea, vomiting, anorexia, 
stomatitis, diarrhea, somnolence, fatigue, peripheral neuropathy, mental status changes, 
cortical blindness, hepatocellular toxicity with elevation in serum transaminases, 
neurotoxicity and interstitial pneumonitis. These effects are reversible when the drug is 
discontinued. Immunosuppression observed with the use of fludarabine increases the risk of 
infection which can be life-threatening. 
 
D.  Total Body Irradiation 
TBI given at high doses in conventional transplants may cause nausea, vomiting, diarrhea, 
temporary hair loss, and painful swelling of the salivary glands for a few days. TBI may destroy 
normal marrow cells in addition to the cancer cells. The doses of TBI (200-300 cGy) used in this 
protocol is about one-sixth of that used in conventional transplant protocols, and severe acute 
side effects have so far not been observed. TBI has been associated with causing sterility and 
there is a risk of major genetic damage to any children conceived after transplantation. There is 
a risk that a small percentage of patients may develop a secondary cancer resulting from this 
treatment.  
 
E.  Tacrolimus 
 
1.  Description 
Tacrolimus, also known as FK-506, is a macrolide immunosuppressive agent. Tacrolimus 
inhibits lymphocytes by forming a complex with FKBP-12, calcium, and calmodulin, leading 
to the decrease in the phosphatase activity of calcineurin. Calcineurin mediates the first 
intracellular signal required for T-cell activation after antigen recognition by the T-cell 
receptor. This drug is used with corticosteroids for prophylaxis of organ rejection in patients 
receiving allogeneic liver transplants and for  prophylaxis of GVHD in the setting of HCT. It 
is also used for immunosuppression after kidney, cardiac, pancreas, pancreatic islet cell and 
small bowel transplantation. This drug is well-absorbed orally. It is metabolized in the liver by 
unknown mechanisms, but demethylation and hydroxylation have been proposed based on 
in vitro studies. The metabolized products are excreted in the urine. 
 
2.  Dosage and Administration 
a. Oral 
 
Tacrolimus capsules – 0.5 mg, 1 mg or 5 mg capsules. (There is currently no liquid 
solution available.)  
 
For better absorption, it is recommended that Tacrolimus capsules be taken on an 
empty stomach. 
 
Tacrolimus should not be taken with grapefruit juice as it may increase blood levels. 
 
If patient vomits within one hour of oral administration, repeat dose. 
 
If vomiting persists, switch to IV administration. 
 
b. Intravenous 
 
Sterile solution of 5 mg/ml ampules in polyoxyethylated castor oil (Cremophor FCL). 
 
Diluted in D5W in glass or other non-PVC container. 
 
Final dilution volume: 50-250 ml dependent upon patient size and Tacrolimus dose. 
 
Infusion time:  
o 
Pediatrics (<30 kg) – 22-24 hours (continuous infusion 0.03 mg/kg/day). Convert 
to twice daily oral dosing once therapeutic levels achieved. 
o 
Adults and Pediatrics (>30 kg) – single daily IV dose of 1 mg over 1-2 hours.  
Convert to twice daily oral dosing once therapeutic levels achieved. 


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c.  Conversion from IV to PO dosing of tacrolimus. 
Patients should be converted to an oral dose at 4 times the IV dose to be given in divided 
(Q 12 hour) doses. For children aged < 6 years old who have sub-therapeutic levels, the 
dose interval may need to be reduced to every 8 hours.  
 
3. Side effects and Toxicities  
 
Renal – Rise in serum creatinine, hemolytic uremic syndrome. 
 
Neurological – Peripheral: paresthesia, tremor. Central: seizures, headache, 
 
insomnia, dizziness, depression, confusion, hallucinations, psychosis, 
 
myoclonus, neuropathy, agitation. 
 
Gastrointestinal – Nausea, vomiting, anorexia, constipation, diarrhea. 
 
Cardiovascular – Hypertension, myocardial hypertrophy. 
 
Endocrine – Hyperglycemia, hyper/hypokalemia, hypophosphatemia, 
 
hypomagnesemia. 
 
Integument – Itching, rash. 
 
Hematologic – Leukocytosis, thrombocytopenia, leukopenia, anemia, PTLD, 
 
thrombotic microangiopathy. 
 
Liver – Abnormal liver function tests. 
 
Ocular – Blurred vision, photophobia. 
 
Respiratory – Pleural effusion, atelectasis, cough, dyspnea. 
 
Musculoskeletal – Arthralgia. 
 
 
See Section X.A.3.c for information about administration and dosage adjustments. 
 
F.  Mycophenolate Mofetil 
 
1.  Description 
MMF is the morpholinylethylester of mycophenolic acid (MPA) and reversibly inhibits  inosine 
monophosphate dehydrogenase, particularly the type II isoform that is more  prominent in 
activated lymphocytes. As a result of the inhibition of de novo purine  synthesis, proliferation 
of B and T lymphocytes is blocked and antibody production is  inhibited. 
 
2.  Storage and Administration 
MMF is available in an oral and an intravenous formulation. The oral formulation is supplied 
in 250mg hard gelatin capsules and can be stored at room temperature. MMF for IV 
administration is supplied as a lyophilized powder in a glass vial containing the equivalent of 
500mg.  
 
3.  Side Effects and Toxicity 
1. Precautions: MMF has been studied extensively among patients after nonmyeloablative 
HCT. Previous clinical studies in patients after allografting suggest that the principal 
adverse reactions associated with the administration of MMF include nausea, vomiting, 
neutropenia, diarrhea, and on one occasion bloody diarrhea. In the setting of marrow 
transplantation, several etiologic factors may contribute to alterations in gastrointestinal 
and hematologic parameters.  MMF has an increased incidence of digestive system 
adverse events, including GI tract ulceration, and hemorrhage (3% of patients receiving 
MMF). GI tract perforations have rarely been observed. Most patients in these studies 
were also on other drugs known to be associated with these complications. Up to 2% of 
patients receiving MMF for prevention of rejection developed severe neutropenia (ANC 
<500). The development of neutropenia may be related to MMF itself, concomitant 
medications, viral infections or some combination of these causes. MMF dose adjustments 
will be made if clinically indicated if in the opinion of the attending physician, no other 
cause is thought to be responsible for the abnormality.  These adjustments should be 


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discussed with the principal investigator and documented in the medical records and the 
clinical reporting form (CRF). Dose adjustments are described in Section X.A.3.b. 
 
 
 
XIII. 
INFECTIOUS DISEASE PROPHYLAXIS AND THERAPY 
 
Patients will receive prophylaxis for pneumocystis carinii pneumonia (PCP), varicella zoster virus 
(VZV), herpes simplex virus (HSV), and encapsulated bacterial and fungal organisms per 
Standard Practice Guidelines. Recommendations are listed in Appendix B of the protocol.  
 
Highlights include: 
1. Sulfamethoxazole/trimethoprim should be started pre-transplant, discontinued on day -2, and 
reinstituted at Day +30 only if the ANC >500 for three consecutive days; use alternative 
prophylaxis if ANC <500 by Day+30. 
2. Acyclovir should be provided to all transplant patients except those having both negative 
HSV and VZV serostatus. For duration, refer to chart in Appendix B  
3.  Fluconazole should be given until Day +75 
 
XIV. 
GRAFT-VERSUS-HOST DISEASE 
 
A. Diagnosis and Treatment 
1. Refer to Appendices C and D for diagnosis and to Standard Practice Guidelines for latest 
treatment recommendations for acute and chronic GVHD, respectively 
2. Prophylaxis with daily Bactrim and acyclovir must continue while receiving 
immunosuppression for Acute GVHD >Grade II or Chronic Extensive GVHD 
 
B. Stopping Rules for GVHD  
A stopping rule is provided that will stop the study if there is evidence that the incidence of grade 
III/IV acute GVHD is greater than 25% within the first 100 days after HCT.  Details of the rule are 
described in Section XVI. 
 
XV. EVALUATION AND ENDPOINT DEFINITIONS 
 
A. Study Description 
This is a Phase I/II study using nonmyeloablative conditioning (FLU, CY, TBI), HLA-
haploidentical marrow as the stem cell source, potent post-transplantation immunosuppression 
(CY, MMF, tacrolimus), followed by a single NK cell infusion early after HCT. 
 
B.  Definition of endpoints 
1. Non-relapse Mortality (NRM):   
a. Definition:  Death in any patient for whom there has not been a diagnosis of relapse or 
disease progression. 
2.  Relapse: 
a.  Definition: Presence of malignant cells in marrow (>5% blasts by morphology), peripheral 
blood (circulating blasts), or extramedullary sites (enlarging lymphadenopathy, soft tissue 
masses) not evident at the time of HCT. Presence of new or recurrent cytogenetic 
abnormalities in patients who have previously cleared this abnormality. 
b.  Evaluation: Testing for recurrent malignancy will be performed using disease-specific, 
established testing methods, including but not limited to:  marrow and peripheral blood 
morphology, immunophenotyping, cytogenetics, and molecular assays; peripheral blood 
counts and tumor markers; radiological evaluation (CT, MRI, PET scans); and biopsy-
proven disease.  
 
 


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3. Progression: 
 
Definition:  Advancement of malignancy after HCT despite previous achievement of stable 
 
disease (Table 16).  
 
4.  Acute GVHD: 
a.  Definition: Acute GVHD will be defined by the criteria outlined in Appendix C. 
b.  Evaluation: GVHD will be assessed by a by a single clinician using the criteria in 
Appendix C. Biopsy for confirmation of diagnosis of skin, liver and GI tract GVHD will be 
performed as indicated. 
c.  The time to onset of GVHD and the number and types of immune suppressant therapies 
will be determined. 
5.  Chronic GVHD: 
a.  Definition: Chronic GVHD is defined by criteria outlined in Appendix D. 
b.  Evaluation: Chronic GVHD endpoints will include: 
i. Extent (limited or clinical extensive) of GVHD 
ii. Duration of immune suppression in months after diagnosis 
iii. Number of cycles of immune suppressant therapy 
iv. Need for immune suppressive agents other than first line therapy  
 
 
6. Engraftment: 
a. Time to ANC >500/uL on the first of three consecutive days. 
b. Mixed chimerism:  5-95% donor T-cells (CD3+) in peripheral blood. 
c. Full donor chimerism: > 95% donor CD3+ T-cells in peripheral blood. 
d. Donor engraftment: Having mixed or full donor chimerism. 
e. Increasing donor chimerism: a 20% absolute increase in the CD3+ T-cell chimerism 
compared to the previous months’ chimerism evaluation.  
f. 
increasing donor chimerism: a 20% absolute decrease in the CD3+ T-cell chimerism 
compared to the previous months’ chimerism evaluation. 
g. Low donor chimerism: <40% CD3+ T-cells after HCT on two consecutive evaluations 
within a 4 week period. The two evaluations must be at least 14 days apart. Low donor 
Table 16:  Definitions of Disease Progression  
 
Multiple 
Myeloma  
or Plasma 
Cell 
Leukemia 
 
Increasing bone pain or 
 
Increase in serum/urine monoclonal protein by 25% or 
 
Increase in circulating or bone marrow blasts by 25% 
CLL, NHL, 
Hodgkin 
Lymphoma 
 
New sites of lymphadenopathy or 
 
Increase of ≥ 25% in lymph node size (assessed by CT scan) 
 
Blood or  marrow involvement with clonal B cells (lymphoma) 
 
Increase of ≥ 25%  marrow involvement or 
 
Increase of ≥ 25% blood involvement of lymphocytes > 50x103/μl) with 
clonal B-cells (CLL) 
CML 
 
Inability to control platelet or granulocyte counts 
 
Increase in baseline number of metaphases demonstrating the Ph+ 
chromosome by 25% 
 
Any other new cytogenetic abnormality 
 
Transformation to accelerated phase or blast crisis 
MDS, AML 
 
Aberrant myeloblasts may clear only slowly from the marrow after non-
myeloablative HCT. Thus, the presence of residual (<5%) MDS or AMLcells 
early post-HCT cannot in and of itself be considered evidence for relapse. 
However, if subsequent marrows show an increase in abnormal cells >5%, 
this will be considered progressive disease. 
 


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chimerism should always be confirmed with repeat blood T-cell and granulocyte 
chimerism analysis. VNTR analyses or FISH studies (in sex mismatched patients) of 
sorted peripheral blood CD3+ T-cells will be used to quantify chimerism. The same 
assay should be used for a given patient for repeated studies of chimerism. VNTR and 
FISH analyses will also be performed on marrow aspirates. Therapeutic decisions (i.e. 
donor lymphocyte infusion) will be made based  
on the results of sorted T-cell studies 
of peripheral blood. 
h. Rejection: the inability to detect, or less than 5% donor T-cells (CD3) as a proportion of 
the total T-cell population after nonmyeloablative HCT.  
i. 
Graft failure: grade IV thrombocytopenia and neutropenia after Day +21 that lasts > 2 
weeks and is refractory to growth factor support. 
 
XVI. STATISTICAL ANALYSIS 
 
This is a phase I/II study with an initial dose escalation for the targeted NK cell dose, followed by 
a single cohort phase II at the identified safe dose. 
 
If a patient initially enrolls on this study but is unable to receive a donor NK cell infusion for any 
reason, the patient will not be included in the statistical analyses for DLT criteria, graft failure, 
GVHD, relapse, or NRM and they will be considered off-study.   
 
Reasons for not receiving a donor NK cell infusion include, but are not limited to:   
1)  Patient being actively treated for hyperacute GVHD requiring additional systemic 
immunosuppressive therapy (other than prophylactic tacrolimus and MMF) 
2)  Patient receiving >0.25 mg/kg equivalent prednisone dose prior within one day of donor NK 
cell infusion;  
3)  Any patient medical concerns or issues that prohibit infusion of the NK cell product 
4)  Any donor medical concerns or issues that prohibit collection of the NK cell product 
 
Phase I   
 
Two possible targeted doses of NK cells are defined: 2.5x106 cells/kg and 5x106 cells/kg.  Three 
patients will be initially entered at the lower dose level.  If none of the 3 experience DLT (as 
defined below) within 28 days of the donor NK cell infusion, then escalation to the higher dose 
level will occur.  If 1 of the 3 patients experiences DLT then an additional 3 patients will be 
enrolled at the lower dose level, and if no additional DLTs are observed then escalation to the 
higher dose may occur.   If a 2nd DLT is observed at the lower dose level in any number of 
patients, then the study will be suspended pending review by our DSMB and consideration of a 
protocol amendment to establish a lower dose level. 
 
At the higher dose level, the first 6 patients enrolled will be considered part of ‘phase I’.  If no 
more than 1 DLT is observed among these 6, then ‘phase II’ enrollment will commence at this 
dose level and continue for an additional 29 patients.   If a 2nd DLT is observed in the first 6 
patients, then the lower dose level will be considered the phase II dose and the additional phase 
II enrollment of 29 patients will commence at that dose level, provided that at least 6 patients 
have been enrolled with no more than 1 DLT. 
 
During the phase I enrollment period, patients may be enrolled at the current dose level pending 
completion of the 28 day observation period for the final patient required to permit dose 
escalation, provided that at least 2 patients at the current dose level have completed the 28 day 
observation period without DLT. 
 
DLT is defined as having at least one of the following adverse events, independent of the 
attribution to the NK cell infusion, within the first 28 days after the NK cell infusion:  
 
1) Grade IV infusional toxicity (based on the “Adapted Common Toxicity Criteria” or CTC)  


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2) Grade IV regimen-related toxicity (based on Adapted CTC); 
 
3) Grade IV acute GVHD  
 
4) Non-relapse mortality 
 
Phase II 
 
The phase II efficacy evaluation will target a total enrollment of 35 patients: the 6 patients 
enrolled during phase I plus the additional 29 patients enrolled during phase II.  All patients 
receiving NK cell infusions during phase II will be counted, even if the infused cell dose does not 
meet the targeted dose established in phase I. The sample size is chosen to provide adequate 
power to evaluate a possible reduction in the 1-year relapse rate from the historical reference 
rate of 55% observed in previous studies. With 35 patients, we have approximately 80% power 
to detect a 20% reduction in the 1-year relapse rate, at a 1-sided 0.10 level of significance.  This 
power calculation is intended only as an approximate point of reference.  Because of the 
heterogeneity of the patient populations, relapse rates are not likely to be directly comparable, 
and further analysis taking this heterogeneity into account will be required in order to determine 
the potential effectiveness of the regimen. 
 
Stopping rules will be imposed for: 
 
 
Day 200 NRM with a threshold rate of 25%  
 
Day 100 grade III/IV acute GVHD, with a threshold rate of 25%, or   
 
Day 100 graft failure, with a threshold rate of 10%.   
 
The limits for NRM and GVHD are somewhat higher than the rates observed in the previous 
haploidentical donor studies, but we are allowing for the possibility that a decrease in early 
relapse might lead to a modest increase in these adverse endpoints yet still yield a better overall 
outcome.  The study will stop at any time that there is reasonable evidence that the true rate of 
these events exceeds the threshold rate.  Reasonable evidence will be taken to mean that the 
lower bound of a 1-sided 80% confidence interval exceeds the threshold.  Operationally, the 
stopping rules would be triggered by the number of patients experiencing the event in question 
as outlined below: 
 
 
Day 200 NRM (25% threshold): 5/10, 8/20, or10/30 patients  
 
Day 100 grade III/IV acute GVHD (25% threshold): 5/10, 8/20, or10/30 patients    
 
Day 100 graft failure (10% threshold): 3/10, 4/20, or 5/30 patients 
 
These stopping rules will be evaluated at least every 10 patients.  Both the patients and events 
observed among the 6 patients enrolled during phase I will ‘count’ for purposes of evaluating the 
stopping rules.  The operating characteristics of these rules are in Table 17 below. 
 
 
 
True rate of 
day 200 NRM 
or day 100 GVHD 
 
Probability 
of 
stopping1 
 
Average N 
at 
stopping1 
 
True rate of 
graft failure 
 
Probability 
of 
stopping1 
 
Average N 
at  
stopping1 
0.3 
55% 
25 
.15 
61% 
23 
0.35 
74% 
20 
.20 
83% 
18 
0.4 
89% 
16 
.25 
94% 
14 
0.45 
96% 
13 
.30 
99% 
11 
 
    1 estimated from 10,000 Monte Carlo simulations 
 
 


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Enrollment may continue pending evaluation of these endpoints at the 10 patient benchmarks; 
however, the outcome of subsequently enrolled patients may not override the stopping rule if 
triggered by patients currently enrolled. 
Other endpoints will include evaluation of rejection, overall survival, chronic extensive GVHD, the 
contribution of KIR ligand alloreactivity to relapse risk, and the effects of a donor NK cell infusion 
on post-transplant immune reconstitution. 
 
 
XVII. DATA SAFETY AND MONITORING PLAN AND ADVERSE EVENT AND SAFETY 
MONITORING 
 
A. Monitoring the progress of trials and the safety of participants 
 
The principal investigators (PI) will monitor this study, with oversight by a Data Safety and 
Monitoring Board (DSMB), the Protocol and Data Monitoring Committee (PDMC) and the 
Institutional Review Board (IRB). The PI reviews toxicity data weekly for the first 100 days after 
HCT, and outcome data for each individual patient at a minimum of 3 months after HCT.  
 
Serious adverse events are reported to the trial coordinator, the study nurse or directly to the PI 
within 72 hours of the event during the initial 28 days after NK infusion. An official report of an 
adverse event is faxed to the coordinating center within seven days during the initial 28 days and 
up to day 180 post HCT. The adverse event report is reviewed by PI. If the adverse event meets 
the FHCRC criteria for reporting, then an official signed report is submitted to the FHCRC 
Institutional Review Office (IRO). All deaths, regardless of the cause, are reported to the IRB. 
This protocol has a DSMB responsible for monitoring patient safety on this clinical trial. The 
DSMB meets twice a year and all outcome data is reviewed including all adverse events 
reported to the FHCRC IRO. The DSMB confirms that the trial has not met any stopping rules 
and reviews any patient safety problems necessitating discontinuation of the trial. A report from 
the DSMB is submitted to the FHCRC IRB as well as the trial coordinators/local PIs of this 
protocol. Furthermore, the FHCRC also has a PDMC that reviews the progress of the protocol 
with respect to the monitoring plan at the time of each annual renewal. As with initial review, 
annual IRB review and approval is also required.  
 
With respect to safety, patients are monitored for the development of GVHD, myelosuppression, 
infections and rejection. All patients, regardless of diagnosis, will be considered in the safety 
analysis. GVHD events will be closely monitored and severity of GVHD graded. Formal stopping 
rules for GVHD grades III-IV and graft failure before day 100, and NRM before day 200 are 
provided. 
 
Flow of information concerning clinical trial participants originates with the clinicians and nurses 
in the clinic or referring clinicians at other institutions and is transmitted to the FHCRC Trial 
Coordinator. At the FHCRC, health care providers and rotating attending physicians assess 
patients and record their observations regarding toxicity and response outcomes in the medical 
record.  This documentation is extracted by the study nurse within 140 days +/- after HCT via 
chart review and collection of copies of source documents and entered into a hard copy or 
electronic Case Report Form (CRF).  The PI reviews the official CRF and primary source 
documents. After the CRFs are verified, they are signed by the PI. Thus, multiple health care 
providers provide independent observations and participate in monitoring this trial. The PI may 
be a clinician for some patients entered on this trial. However, assessments are the sum total of 
the primary health care provider (fellow or physician assistant), floor or outpatient nurse and the 
PI or other attending clinician involved with the patient averting possible conflict of interest 
having the PI as the attending clinician for protocol patients. If determination of adverse events is 
controversial, co-investigators will convene on an ad hoc basis as necessary to review the 
primary data and render a decision. 
 


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B. Plans for assuring compliance with requirements regarding the reporting of adverse 
events  
 
The adverse event reporting will follow the FHCRC Guidelines for serious adverse event (SAE) 
reporting (Appendix E). These guidelines detail the expedited reporting requirements and 
definitions of particular events. All SAEs that meet expedited reporting criteria are reported to the 
IRO within 10 days by the investigator, trial coordinator, or research nurse upon learning of the 
event. A completed SAE report form, signed by the PI, must be received by the IRO within 10 
calendar days. The PI reviews all SAEs and annual reports at the time of submission. For 
patients being cared for at the FHCRC, health care providers communicate with the PI, trial 
coordinator or research nurses as events occur triggering subsequent reporting. All serious 
adverse events are reported to the IRB at the time of annual renewal and at the Mixed 
Chimerism Meeting. The PI for a study is responsible for this reporting and the IRO assures 
adverse event reporting on an annual basis. The PI in the annual application for grant 
continuation will summarize reports of toxicities. Furthermore, stopping rules and interim analysis 
provides an additional safeguard for adverse event analysis and reporting in this protocol. All 
collaborating PIs have fulfilled all NIH requirements for training in human subjects’ protection. 
 
In regards to monitoring for the FDA, all acute adverse event monitoring will occur until 28 days 
after the NK cell infusion.  We will plan to report all unexpected SAEs and grades 3-4 infusional 
reactions in an expedited fashion.  We plan to expedite reporting on any patient with an ANC 
< 500 by day 28 after NK cell infusion.  In addition, we will plan to include a separate listing of 
infusional toxicities, in addition to other required elements, in our annual reports to the FDA. 
Duration for monitoring GVHD, relapse, and survival for purposes of reporting to the FDA will be 
for 1 year after transplant.  At FHCRC, we will continue collecting long-term follow-up data for 
many years for our internal use. 
  
C. Plans for assuring that any action resulting in a temporary or permanent suspension of 
an NCI-funded clinical trial is reported to the NCI grant program director responsible for 
the grant  
 
The procedure of CD3+ depletion and CD56+ selection will be performed under an investigator-
sponsored IND, with FDA oversight. Serious and unexpected adverse experiences must be 
reported to the IND holder and the Principal Investigator. The IND holder and the principal 
investigator must report all serious and unexpected adverse events to the IRB, the FDA and the 
Miltenyi Biotec, Inc. Any temporary or permanent suspension, as determined by the PI, IRB, or 
PDMC, of this clinical research trial will be reported to the NCI grant program director by the PI.  
1. Unexpected and related fatal or life-threatening events are reported to FDA by phone or FAX 
within 7 calendar days. 
2. A written report will be sent to FDA within15 calendar days for any serious unexpected 
adverse events for which is a reasonable possibility that the event may have been caused by 
study device. 
3. All serious adverse events occurring during this study, whether or not attributed to study 
drug, will be included in the IND holder’s annual IND report to FDA. 
 
D. Plans for assuring data accuracy and protocol compliance 
 
This protocol has a DSMB that is responsible for reviewing protocol data and safety endpoints. 
The DSMB meets on a twice-yearly basis, reviews a report of appropriate endpoint data, and 
compiles a report that is submitted to the IRB, PDMC Chairman, and Protocol Office. 


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At the FHCRC, health care providers and rotating attending physicians assess patients and 
record their observations in the medical record. This documentation is extracted by one of the 
study nurses at 100 days after HCT via chart review and entered into an electronic Case Report 
Form (CRF). The study nurses also continue to follow patients after day 100, review source 
documentation, and complete CRFs at 6 month and then yearly intervals per protocol. The CRFs 
are printed directly from the database, and the PI reviews the CRFs and the primary source 
documents for data accuracy. When the CRFs are verified, the PI signs them, and the data are 
incorporated into a central database by the data manager. Thus, multiple health care providers 
provide independent observations and participate in assessments on this trial. The study has 
dedicated nurses who at a minimum follow patients to confirm eligibility; reporting of adverse 
events; reporting of events, which are part of the safety-monitoring plan, and protocol 
adherence. The PI, data coordinators, and research nurses are responsible for review and 
maintenance of all patient records to ensure data integrity and protocol adherence. 
 
 
XVIII. RECORDS 
 
Clinical records will be maintained as confidentially as possible. Data will be collected maintained 
by Clinical Statistics. The investigator will ensure that data collected conform to all established 
guidelines for coding, collection, key-entry and verification. Each patient is assigned a unique 
patient number to assure patient confidentiality. Any publication or presentation will refer to 
patients by this number and not by name. The licensed medical records department, affiliated 
with the institution where the patient receives medical care, maintains all original inpatient and 
outpatient chart documents. Patient research files are kept in a locked room. They are 
maintained by the FHCRC data collection staff, which is supervised by an A.R.T. Access is 
restricted to personnel authorized by the Division of Clinical Research. Information gathered from 
this study regarding patient outcomes and adverse events will be made available to the Federal 
Drug Administration and to Miltenyi Biotec, Inc. All precautions to maintain confidentiality of 
medical records will be taken. 
 
  
XIX. PROJECTED TARGET ACCRUAL 
 
 
Table 18. Projected Accrual based on ethnicity and racial category 
TARGETED / PLANNED ENROLLMENT                            Number of Subjects 
 
Ethnic Category 
Sex / Gender 
Females 
Males 
Total 
Hispanic or Latino 
0 
1 
1 
Not Hispanic or Latino 
14 
20 
34 
Ethnic Category Total of All Subjects* 
14 
21 
35 
 
RACIAL CATEGORIES 
American Indian / Alaska Native 
1 
0 
0 
Asian 
0 
2 
1 
Native Hawaiian or Other Pacific Islander 
0 
0 
0 
Black or African American 
1 
2 
1 
White 
12 
17 
33 
Racial Categories:  Total of All Subjects* 
14 
21 
35 


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APPENDICES 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
      page 
 
 
A 
ABO Incompatibility Guidelines………………………………………………………………………… 61 
 
B 
Infectious Disease Guidelines………………………………………………………………………….. 63 
 
C 
Grading of Acute GVHD………………………………………………………………………………… 64 
 
D 
Evaluation of Chronic GVHD…………………………………………………………………………… 65 
 
E 
Study Coordinator’s Manual……………………………………………………………………………. 67 
 
F 
Core Case Report Form………………………………………………………………………………… 71 
 
G Patient Demographics and Registration Form……………………………………………………….. 72 
 
H 
Notice of Death…………………………………………………………………………………………... 76 
 
I 
Serious Adverse Event Report Form…………………………………………………………………...77 
 
J 
The Hematopoietic Cell Transplant-Comorbidity Index (HCT-CI) 9/07/10………………………….79 
K  Adapted Common Toxicity Criteria ……………………………………………………………………..81 
L  Coordinating Center Functions ………………………………………………………………………... 89 
M  Research Specimens.…………………………………………………………………………………... 91 
N  Intrathecal Therapy Administration.…………………………………………………………………... 96 
 
 
 
 
 
 


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APPENDIX A 
 
ABO INCOMPATIBILITY GUIDELINES 
 
Red Blood Cell - Incompatibility (Major): 
Occasional patients may have antibodies directed against red blood cell antigens found on the 
donor's cells. These are generally ABO or Rh antigens, although incompatibility with other red cell 
antigens identified by donor-recipient crossmatch may occur. If the recipient shows an anti-donor 
RBC antibody titer of  1:32, the RBC content of the marrow product must be reduced to  20mL in 
order to avoid a hemolytic transfusion reaction when the product is infused. Incompatible marrow 
products  600 mL will be RBC depleted by processing through an apheresis instrument and 
recovering marrow mononuclear cells (MNC) containing  20mL of residual RBC. Incompatible 
marrow products < 600 mL will undergo RBC depletion by the starch sedimentation method. Refer to 
the Clinical Coordinator's Patient Information Sheet for instructions regarding management of a 
specific patient.  
Post transplant blood component support will be according to Standard Practice Guidelines. 
 
Timing: Every attempt should be made to infuse red cell depleted marrow products within 2 hours of 
depletion. 
Expected Results: Red blood cell depleted marrow products will contain < 20ml residual red blood 
cells and  80% recovered marrow MNC. 
 
Red Blood Cell - Incompatibility (Minor): 
Occasional donors may have antibodies directed against red blood cell antigens (ABO, Rh, or other 
antigen system) found on the recipient's cells. The risk of hemolysis of recipient red cells 
immediately after transplant may occur if the donor antibody titer is  1:256 and the volume of 
infused plasma (for recipient  20 kg) is > 200mL. If the donor antibody titer is  1:256 the marrow 
product must be plasma reduced to a final volume of  200 mL (for recipient  20 kg). For recipients 
< 20 kg, see the Standard Practice Manual for guidelines regarding final plasma-reduced marrow 
volumes (see flowsheet below). Due to the high number of lymphocytes in the marrow inoculum, 
recipients of minor ABO mismatched products may also be at risk for a delayed type of hemolysis 
that can be severe. Refer to the Clinical Coordinator's Patient Information Sheet for instructions 
regarding management of a specific patient.  
Post transplant blood component support will be according to Standard Practice Guidelines. 
 
Timing: Every attempt should be made to infuse plasma-depleted PBMC within 2 hours of depletion. 
Expected Results: The plasma depletion should not affect the nucleated cell recovery. 
 
Red Blood Cell – Bidirectional Incompatibility: 
Patients undergoing transplants for bidirectional RBC incompatibility should be managed according 
to both algorithms shown below. Most red cell depletion techniques also deplete plasma from the 
marrow component with no additional cell loss. Refer to the Clinical Coordinator's Patient Information 
Sheet for instructions regarding management of a specific patient.  
 
 
 
 


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Post transplant blood component support will be according to Standard Practice Guidelines. 
 
 
MAJOR ABO INCOMPATIBLE 
 
 
 
 
 
< 20ml RBC total 
      
Infuse without modification 
 
 
 
 
> 20ml RBC total 
      
RBC depletion of component to  20 mL 
Recipient anti- 
Donor titer 
 
 
 
 
 
Infuse without modification 
 
MINOR ABO INCOMPATIBLE 
 
 
 
 
 
Plasma depletion of component to  200 mL (recipient  20 kg). See  
 
 
           Standard Practice Manual for volume guidelines for recipient < 20 kg)  
 
Donor anti- 
Recipient titer 
 
 
 
 
 
Infuse without modification or plasma volume restriction  
 
 
 
 
 
 
 
  
  
 
 
 
  
  
 
 
 
  
  
 
 
 
  
  
 
 
 
 
 
 
 
 
 
 
 
 1:128 
 1:256 
 1:16 
 1:32 


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APPENDIX B 
 
INFECTIOUS DISEASE GUIDELINES 
 
 
Herpes Simplex and Varicella Zoster Virus Prevention and Treatment 
Herpes Simplex and 
Varicella Zoster Virus  
CMV Prevention: Surveillance and Preemptive Therapy 
cmvprevention.pdf
 
CMV Disease: Diagnosis and Treatment 
cmvdiseasetreatmen
t.pdf
 
 
Antifungal Therapy Guidelines 
antifungal_therapy.p
df
 
 
Pneumonia / Pneumocystis Jiroveci Prophylaxis 
pneumocystisjiroveci
 
 
Antibiotic Prophylaxis for Encapsulated Bacteria in Allogeneic Patients with Chronic GvHD 
Requiring Immunosuppressive Therapy 
Antibiotic Prophylaxis
for Encap Bacteria  
 
Vaccinations  
 
Foscarnet 
foscarnet.pdf
 


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APPENDIX C 
GRADING OF ACUTE GRAFT-VERSUS-HOST DISEASEa 
 
Severity of Individual Organ Involvement (stage) 
Skin 
 
 +1 
 
a maculopapular eruption involving less than 25% of the body 
surface 
  
 
 
+2 
a maculopapular eruption involving 25-50% of the body surface 
  
 
 
+3 
generalized erythroderma 
  
 
+4 
generalized erythroderma with bullous formation and often with 
desquamation 
 
Liver 
 
+1 
 
bilirubin (2.0-3.0 mg/100 ml) 
  
 
 
+2 
bilirubin (3-5.9 mg/100 ml)  
  
 
 
+3 
bilirubin (6-14.9 mg/100 ml)  
  
 
 
+4 
bilirubin > 15 mg/100 ml  
 
Gut 
 
Diarrhea is graded +1 to +4 in severity. Nausea and vomiting and/or 
anorexia caused by GVHD is assigned as +1 in severity 
  
 
 
The severity of gut involvement is assigned to the most severe involvement  
                           noted. Patients with visible bloody diarrhea are at least stage +2 gut and 
grade  
                           +3 overall 
 
Diarrhea 
 
  
 
 
+1 
 1000 ml of liquid stool/day* ( 15ml of stool/kg/day)† 
  
 
 
+2 
>1,000 ml of stool/day* (> 15ml of stool/kg/day)† 
  
 
 
+3 
>1,500 ml of stool/day* (> 20ml of stool/kg/day)† 
  
 
 
+4 
2,000 ml of stool/day* ( 25ml of stool/kg/day)† 
 
*In the absence of infectious/medical cause 
†For pediatric patients 
 
Severity of GVHD (grade) 
Grade I  
+1 to +2 skin rash 
  
 
 
No gut or liver involvement 
 
 
Grade II 
+1 to +3 skin rash  
 
 
 
+1 gastrointestinal involvement and/or +1 liver involvement  
 
Grade III 
+2 to +4 gastrointestinal involvement and/or 
  
 
 
+2 to +4 liver involvement 
  
 
 
with or without a rash 
 
Grade IV 
Pattern and severity of GVHD similar to grade 3 with extreme constitutional 
symptoms or death 
_______________ 
a From “Graft-vs-host disease” Sullivan, Keith M. Hematopoietic Cell Transplantation Ed: D. Thomas, K. Blume, S. 
Forman, Blackwell Sciences; 1999, pages 518-519 
 
 
 
 


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APPENDIX D 
EVALUATION OF CHRONIC GRAFT-VERSUS-HOST DISEASEa 
 
In all cases, concomitant processes (i.e. infections or drug reactions) must be ruled out. Karnofsky or 
Lansky Clinical Performance scores, 60%, > 15% weight loss, and recurrent infections are usually 
signs of clinical extensive chronic GVHD. Abnormalities that could indicate chronic GVHD are 
categorized by organ systems as listed below. 
 
Skin 
Erythema, dryness, pruritus, pigmentary changes (i.e. hyperpigmentation, vitiligo), 
mottling, papulosquamous plaques, nodules, exfoliation, macular-papular or 
urticarial rash, scleroderma, morphea (one or several circumscribed, indurated 
and shiny lesions) 
 
Nails                    Ridging, onychodystrophy, onycholysis 
 
Hair 
Premature graying, (scalp hair, eyelashes, eyebrows), thinning scalp hair, 
alopecia, decreased body hair 
 
Mouth 
Dryness, burning, gingivitis, mucositis, striae, atrophy, erythema, lichenoid 
changes, ulcers, labial atrophy or pigmentary changes, tooth decay, tightness 
around the mouth 
 
Eyes                     Dryness, burning, blurring, gritty eyes, photophobia, pain 
 
Vagina/vulva   
Dryness, dyspareunia, stricture or stenosis, erythema, atrophy or lichenoid 
changes not included 
 
Liver 
Elevated liver function tests not due to other causes (alkaline phosphatase  3x 
upper limit of normal, AST or ALT 4x upper limit of normal or total serum bilirubin 
 2.5; in the absence of chronic GVHD involving other organs, liver biopsy is 
required to confirm diagnosis) 
 
Lung 
Bronchiolitis obliterans (see diagnostic indicators), cough, wheezing, dyspnea on 
exertion, history of recurrent bronchitis or sinusitis 
 
GI                         Anorexia, nausea, vomiting, weight loss, dysphasia, odynophagia, 
                             malabsorption 
 
Fasciitis 
Stiffness and tightness with restriction of movement, occasionally with swelling 
pain, cramping, erythema and induration, most commonly affecting forearms, 
wrists and hands, ankles, legs, and feet, inability to extend wrists without flexing 
the fingers or the elbows, contractures 
 
Serositis  
  
Chest pain or cardiopulmonary comprise due to pericarditis or pleuritis 
 
Muscle                   Proximal muscle weakness, cramping 
 
Skeletal 
Arthralgia of large proximal girdle joints and sometimes smaller joints 
 
 
 
 
 


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Laboratory testing and diagnostic indicators of chronic GVHDa 
 
Eye 
Schirmer’s test with a mean value  5mm at 5 minutes, or symptomatic with 
values of 6-10mm or keratitis detected by slit lamp examination 
 
Liver 
Elevated liver function tests not due to other causes (see definition of clinical 
limited and extensive chronic GVHD) 
 
Lung 
New obstructive lung defect defined as FEV1 < 80% of predicted with either an 
FEF 25-75 <65% of predicted or RV >120% of predicted, or a decrease of 
FEV1/FVC by > 12% within a period of less than 1 year. A diagnosis of 
bronchiolitis obliterans requires negative microbiological tests from 
bronchoalveolar lavage and evidence of air trapping by high resolution end-
expiratory and end-inspiratory CAT scans o the chest. A thoracoscopic lung 
biopsy may be necessary in order to confirm the diagnosis of bronchiolitis 
obliterans in patients who have obstructive lung disease without air trapping when 
chronic GVHD involving other organs is absent 
 
Esophagus 
Esophageal web formation, stricture or dysmotility demonstrated by barium 
swallow, endoscopy or manometry 
 
Muscle              
Elevated CPK or aldolase, EMG findings consistent with myositis 
 
Blood 
Thrombocytopenia (usually 20,000-100,000/l), eosinophilia, 
hypogammaglobulinemia, hypergammaglobulinemia, and autoantibodies occur in 
some cases 
 
_________________ 
a From Standard Practice Guidelines for “Chronic Graft-versus-Host Disease Classification at 
the time of presentation” developed by Long Term Follow-Up at the FHCRC 
 
 
 
 
 


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APPENDIX E 
 
STUDY COORDINATOR’S MANUAL 
I. Introduction 
 
The mixed chimerism protocols have been opened to multiple sites to increase the referral base and 
accrual. Because of this expansion of collaborators, the data collection procedures are being revised. 
The procedure manual was created to assure consistency of data reporting across the centers and 
to assure compliance with regulations. General expectations of collaborators are that they will 
comply with appropriate regulatory requirements, specified protocol requirements, and provide 
outcome data. 
 
The manual translates working procedures for study coordination. Its goal is to describe the 
procedures with sufficient clarity to ensure that all study centers will use the same procedures and 
follow-up schedules for participant data management and reporting. Changes to the manual and 
relevant forms will be made as soon as practical and will become effective on receipt of the revised 
procedures at the study centers, unless otherwise noticed. 
 
II. Institutional Review Board Review of Protocols and Modifications 
 
All research protocols proposed for use that involves human subjects must be reviewed and 
approved by the Institutional Review Board (IRB) prior to implementation. New protocols will 
undergo review at the FHCRC IRB and then will be distributed to sites that wish to participate for 
their IRB’s review. For Centers that have a Federal Wide Assurance (FWA), formal collaboration 
includes submission of a form 310 and a copy of the IRB approved protocol and consent forms to 
the FHCRC.  For sites without a FWA, an FWA form needs to be filed. Once the paperwork is 
submitted to the Office for Human Research Protection, the approval process can take up to a 
couple of months, and must be completed before collaboration on a protocol can begin.  
 
In addition, all amendments and/or revisions to on-going, approved activities must be submitted for 
review and approved prior to implementation at an institution.  No revisions may be implemented at 
outside institutions without the prior approval of the FHCRC Principal Investigator.  The FHCRC and 
the local site’s IRB must review all protocol activities at least once annually. This must be done 
within 365 days of the last review regardless of the policies of the institution. A copy of annual 
renewal approvals must be received for collaboration to continue for the next year. 
 
III. Registrations 
 
Collaborating Institutions: The principal investigator of the collaborating institution who will register 
the patient with the FHCRC will identify eligible patients. Registration will include completion of the 
eligibility checklist/demographic form. This form will be faxed (206-667-5378) prior to treatment 
initiation. Patients should be registered prior to treatment initiation for valid registration 
 
IV. Reporting Adverse Events 
 
The following guidelines are the minimum serious adverse event (SAE) reporting guidelines for 
Category 1 and 2 studies conducted at the Fred Hutchinson Cancer Research Center.   


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Expedited Reporting Requirements  
All unexpected and serious adverse events which may be due to study treatment or intervention 
must be reported to the FHCRC Institutional Review Office as soon as possible but within at least 10 
calendar days of the investigator learning of the event. 
Definitions 
 
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.  
 
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.  Study toxicities are graded using the 
adapted NCI Common Toxicity Criteria v.4.0 (where appropriate use the criteria for transplant 
patients.)  All Grade 4 (life-threatening) toxicities occurring between the start of any protocol 
intervention and day 180 that meet expedited reporting requirements must be reported as soon as 
possible but within at least 10 calendar days of the investigator learning of the event to the IRO and 
72 hours to the IND holder.  
 
Unexpected Adverse Event – An adverse event, the nature or severity of which is not consistent 
with the applicable product information (e.g., Investigator’s Brochure for an unapproved 
investigational product or package insert/summary of product characteristics for an approved 
product).  If applicable product information is not available, such as for studies that do not involve 
pharmaceutical products or devices, an unexpected adverse event is an adverse event that was not 
described in the study protocol or informed consent. 
 
Serious Adverse Event (SAE) – Any adverse event occurring that results in any of the following 
outcomes:   
 Death – start of any protocol intervention to day 180, regardless of cause,  
 a life-threatening adverse event (see above)  
 a persistent or significant disability/incapacity 
 a congenital anomaly  
 requires intervention to prevent permanent impairment or damage.  
 
Hospitalization, in general, will not be considered a serious adverse event as approximately half of 
evaluable MRD patients AND the majority of evaluable URD patients receiving nonmyeloablative 
transplants were hospitalized.  Hospitalization will be considered a serious adverse event if it fulfills 
the criteria for a serious and unexpected adverse event as described above. 
 
To ensure no confusion or misunderstanding exist of the differences between the terms “serious” 
and “severe,” which are not synonymous the following note of clarification is provided: 
 
The term “severe” is often used to describe the intensity (severity) or a specific event 
(as in mild, moderate or severe myocardial infarction); the event itself, however, may 
be of relatively minor medical significance (such as severe headache).  This is not the 
same as “serious,” which is based on patient/event outcome or action criteria usually 
associated with events that pose a threat to a patient’s life or functioning.  
Seriousness (not severity) serves as a guide for defining regulatory obligations. 
 
Attribution - The FHCRC designation for the determination of whether an adverse event is related 
to a medical product, treatment or procedure will be as follows: 
 Related – includes adverse events that are definitely, probably, or possibly related to  
 
 
  
the medical treatment or procedure. 


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 Not Related – includes adverse events are doubtfully related or clearly not related to  
 
 
  
the medical treatment or procedure. 
 
The FHCRC Serious Adverse Event (SAE) Report Form should be completed for all adverse events 
that meet the expedited reporting requirements.   All available information should be submitted but it 
is acceptable to fax an incomplete report form at the initial report.  A completed report should be 
faxed as soon as possible but must be received within 10 calendar days. 
 
It is the responsibility of the FHCRC Principal Investigator to notify NIH and the responsibility of the 
IND holder to notify the FDA or other agencies of serious adverse events as required in the protocol. 
 
Serious adverse events that do not meet the requirement for expedited reporting (not related to 
study treatment or expected) will be reported to the IRB as part of the annual renewal of the protocol. 
 
FHCRC is acting as the Coordinating Center for this multi-institutional study, and it is the 
responsibility of the FHCRC Principal Investigator (or designee) to complete the FHCRC Serious 
Adverse Event Report for all serious adverse events that meet the expedited reporting requirements 
that are received from the participating sites.   
 
 
 
Procedure for Reporting Serious and Unexpected Adverse Events from Participating Sites 
 
Regulations defining the responsibilities for reporting serious and unexpected adverse reactions are 
defined above.  Serious and unexpected adverse events must be reported to the FHCRC 
Investigator and the IND holder within 72 hours of learning of the event. This includes patient deaths, 
regardless of cause, occurring at the start of any protocol intervention and day 180 post-transplant 
procedure.  The immediate telephone report must be followed by faxed comments to the FHCRC 
Trial Coordinator at (206) 667-5378. This will be followed by detailed written report (See Appendix I) 
within 3 working days.  The report must include the date and time of onset, severity and duration of 
the event, the relationship to the study, the treatment given and eventual outcome.  Follow-up 
information to a SAE report must be submitted as soon as the relevant information is available. 
 
Obligation of Investigators 
 
All grade 3 or 4 adverse events, or highly unusual grade 2 adverse events, using the modified (for 
HSCT) NCI Common Toxicity Criteria v.4.0 which occur between the start of any protocol 
intervention and day 100 during the study will be recorded on the Case Report Form (Appendix F).  
These adverse events which are observed by the Investigator or reported by the patient whether or 
not attributed to the study, will be recorded on the Case Report Form. Attributes will include a 
description, date of onset, maximum severity, and assessment of relationship to the study agent or 
other suspect agent(s). 
 
Adverse events will be graded accordingly: 0 = none, 1 = mild, 2 = moderate, 3 = severe, 4 = life 
threatening or debilitating, and 5 = fatal.   All Grade 4 (life-threatening) or Grade 5 (fatal) events on 
the adapted HSCT NCI scale meet expedited reporting requirements. 
 
Association or relatedness to the study agent will be graded as follows: 1 = unrelated, 2 = unlikely, 3 
= possibly, 4 = probably, and 5 = definitely related. 
 
V. Case Report Forms 
 
Case report forms must be completed for all patients registered onto the protocols and submitted to 
the FHCRC data coordinating center. The first case report form (day 28) is due on day 50.  For 


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outside centers a Staging Form must accompany the form with the patient staging at registration, 
day 28, day 56, day 84 and day 100. Staging forms should also be completed with each Follow Up 
Form completed on day 180, 1 year, 1.5 years, 2 years, 3 years, and yearly thereafter. For Outside 
Centers, case report forms are expected to be submitted no later than 30 days following the 
scheduled follow up date.  
 
VI. Protocol Monitoring 
 
As the coordinating center, FHCRC will monitor accrual at the outside institutions. The guidelines 
below are intended to guide the reviewers in their assessment of items that significantly alter the 
clinical effectiveness of the treatment or the evaluation of its toxicity. 
 
A. Registration/Randomization 
1. Patient was registered prior to treatment and approval by FHCRC PI occurs prior to 
randomization.  
2. Information given at registration represents actual data in medical records (stage, diagnosis, 
cell type, etc.) 
 
B. Informed Consent/IRB Approval Dates 
1. The consent was signed prior to registration. 
2. The consent is in language was approved by the institution’s IRB. IRB approval and 
reapproval are documented including appropriate use of full-board review and proper review 
of appropriate amendments or revisions 
3. Consent was dated and has written witness signature. IRB approval was obtained prior to 
the patient signing the consent form and start of treatment. 
 
C. Patient Eligibility 
1. Eligibility criteria and exclusion criteria were met. 
2. Treatment/Intervention Administration 
3. Doses were modified according to protocol 
4. Accurate documentation of drug administration 
 
D. Study Tests/Evaluation 
1. Protocol specified laboratory tests or diagnostic studies are available 
 
2. Appropriate record of protocol intervention is documented. 
 
E. Study Events/Adverse Drug Experience 
1. Serious Adverse Evens reported according to protocol specifications 
 
F. 
Follow-Up 
1. Disease status assessed according to the required protocol guidelines documenting 
response to treatment. 
2. Accurate determination of cancer progression 
 
 
 


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APPENDIX F 
 
CORE CASE REPORT FORM 
 
 
 
 
 
 
 


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APPENDIX G 
 
PROTOCOL 2230 PATIENT DEMOGRAPHICS AND REGISTRATION FORM 
Please Fax this completed form to (206)-667-5378 for patient registration. 
Questions regarding eligibility should go to Brenda Sandmaier, M.D., 206 667 4961. 
UPN: _____________ 
Patient Name: 
_______________________________ 
(Last) 
________________________ 
(First) 
_____  
(MI) 
Date of Birth: _____ / _____ / __________  Age:________ 
 (Mo)    (Day)         (Year) 
Gender (choose one): 
 Male    
 Female   
 Unknown 
Patient Diagnosis: 
_______________________________________________   
Planned Day 0: _____/______/_____ 
 
(Mo) (Day)  (Year) 
Ethnicity (choose one):  Instruct the patient to select one of the following. 
 Hispanic (A person of Cuban, Mexican, Puerto Rican, South or Central American, or other 
Spanish culture or origin, regardless of race.  Term “Spanish Origin” can also be used in 
addition to “Hispanic” or “Latino”.) 
 Not Hispanic or Latino 
 
 Declined to Report 
Race (check all that apply):  Instruct the patient to select one or more of the following. 
  American Indian/Alaska Native (A person having origins in any of the original peoples of North, 
Central, or South America, and who maintains tribal affiliations or community attachment). 
  Asian (A person having origins in any of the original peoples of the Far East, Southeast, Asia, 
       or the Indian subcontinent including, for example, Cambodia, China, India, Japan, Korea, 
      Malaysia, Pakistan, the Philippine Islands, Thailand and Vietnam). 
  Black/African American (A person having origins in any of the black racial groups of Africa). 
  Native Hawaiian/Pacific Islander (A person having origins in any of the original peoples of 
Hawaii, Guam, Samoa or other Pacific Islands). 
  White (A person having origins in any of the original peoples of Europe, the Middle East or North 
Africa). 
  Research subject does not know race 
  Declined to report 
 
NK CELL DOSE LEVEL 
 Dose Level 1 -- NK cell dose of 2.5 x 106 (± 20%)   
 
 
 
 
 
 
 
CD56+/CD3- cells per kg.    
                      
 
                      
 Dose Level 2 -- NK cell dose of 5 x 106 (± 20%)  
 
 
 
 
 
 
 
CD56+/CD3- cells per kg. 
 
                   
Creatinine Clearance:                                                        date: 
 
 
Fludarabine Dose: 
 
 
Signature of Principal Investigator:                                                               date: 
 
 


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Protocol 2230 Eligibility   
 
One of the following criteria questions (1-6) must be marked “Yes” for the patient to enter on 
2230. (The following diseases will be permitted although other diagnoses can be considered if 
approved by PCC and the principal investigator.) 
  
*Patients must be not eligible for conventional autologous HCT.  Patients for whom HLA-matched 
unrelated donor search could not be initiated or completed due to insurance reasons, concerns 
of rapidly progressive disease, and/or discretion of attending physician are eligible for this protocol.  
Patients must be expected to have disease controlled for at least 60 days after HCT.   
 
1) Yes 
 No 
 
Aggressive NHLs and Other Histologies Such as Diffuse large B cell 
(DLBC) NHL– a) not eligible for autologous HCT, b)  not eligible for high-dose 
HCT, c) after failed autologous HCT, or d) be part of a tandem auto-allo 
approach for high risk patients 
Low grade NHL– with < 6 month duration of CR between courses of 
conventional therapy. Mantle Cell NHL -must be beyond first CR. CLL – must 
have either 1) failed to meet NCI Working Group criteria for complete or partial 
response after therapy with a regimen containing FLU (or another nucleoside 
analog, e.g. 2-CDA, pentostatin) or experience disease relapse within 12 months 
after completing therapy with a regimen containing FLU (or another nucleoside 
analog); 2) failed FLU-CY-Rituximab (FCR) combination chemotherapy at any 
time point; or 3) have “17p deletion” cytogenetic abnormality and relapsed at any 
time point after any initial chemotherapy. HL – Must have received and a) failed 
frontline therapy, b) not be eligible for autologous HCT, or c) or be part of a 
tandem auto-allo approach for high risk patients. 
 
 
2) Yes 
 No 
 
Multiple myeloma or Plasma Cell Leukemia – must have received more than 
one line of prior chemotherapy. Consolidation of chemotherapy by autografting 
prior to nonmyeloablative HCT is permitted. 
 
3) Yes 
 No 
 
AML/ALL – must have < 5% marrow blasts at the time of HCT  
 
4) Yes 
 No 
 
CML – Patients will be accepted if they are beyond CP1 if they have received 
previous myelosuppressive therapy or HCT, < 5% marrow blasts at the time of 
transplant 
 
5) Yes 
 No 
 
MDS/MPD - (>int-1 per IPSS) after ≥ 1 prior cycle of induction chemotherapy. 
Must have <5% marrow blasts at time of transplant. 
 
6) Yes 
 No 
 
Waldenstrom’s Macroglobulinemia – must have failed 2 courses of therapy. 
 
Each of the following questions (7-20) must be marked “No” Or “NA” for the patient to enroll on 2230. 
 
7)  Yes 
 No 
  Patient is eligible for a curative autologous transplant. 
 
8)  Yes 
 No 
  NA 
  Patient received a prior allogeneic HCT and has active GVHD  
                             requiring immunosuppressive therapy for at least 21 days prior to start of  
                             conditioning 
 
9)  Yes 
 No 
 NA 
    Patient has available HLA-matched related donors                      
 
10)  Yes 
 No 
  Patient has significant organ dysfunction that would prevent compliance 
with conditioning, GVHD prophylaxis, or would severely limit the 
probability of survival: 


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a. Symptomatic coronary artery disease or ejection fraction <35% or other 
cardiac failure requiring therapy (or, if unable to obtain ejection fraction, 
shortening fraction of <26%).  If shortening fraction is <26% a cardiology 
consult is required with the PI having final approval of eligibility.  
PI Signature:                                                     Date:           /           /          .                    
b. DLCO <40% TLC <40%, FEV1 <40% and/or receiving supplementary 
continuous oxygen.  The FHCRC study PI must approve enrollment of all 
patients with pulmonary nodules. 
PI Signature:                                                     Date:           /           /          .                    
c. Liver function abnormalities: Patient with clinical or laboratory evidence of 
liver disease will be evaluated for the cause of liver disease, its clinical 
severity in terms of liver function, bridging fibrosis, and the degree of 
portal hypertension. The patient will be excluded if he/she is found to 
have fulminant liver failure, cirrhosis of the liver with evidence of portal 
hypertension, alcoholic hepatitis, esophageal varices, a history of 
bleeding esophageal varices, hepatic encephalopathy, uncorrectable 
hepatic synthetic dysfunction evinced by prolongation of the prothrombin 
time, ascites related to portal hypertension, bacterial or fungal liver 
abscess, biliary obstruction, chronic viral hepatitis with total serum 
bilirubin >3mg/dL, or symptomatic biliary disease.  
                   
11) Yes 
 No 
   Patient is HIV seropositive 
 
12) Yes 
 No 
  Patient has poorly controlled hypertension despite multiple antihypertensive  
medications. 
  
13) Yes 
 No 
 NA 
  Fertile female who is unwilling to use contraceptive techniques during and 
for the twelve months following treatment, and/or a female who is pregnant or 
actively breast feeding. 
 
14) Yes 
 No 
 NA 
  Fertile male who is unwilling to use contraceptive techniques during and 
for the twelve months following treatment.  
 
15) Yes 
 No 
  Patients with active non-hematologic malignancies (except non-melanoma     skin 
cancers) or those with non-hematologic malignancies (except non-melanoma 
skin cancers) who have been rendered with no evidence of disease, but have a 
greater than 20% chance of having disease recurrence within five years 
This exclusion does not apply to patients with non-hematologic malignancies that 
do not require therapy 
 
16) Yes 
 No 
  Patient has active CNS involvement with disease refractory to intrathecal 
chemotherapy.   
 
17) Yes 
 No 
 Patient has a Karnofsky score <60 / Lansky score <60 
 
18) Yes 
 No 
  Patient has active infectious disease concerns. 
 
 
19) Yes 
 No 
 Patient’s life expectancy severely limited by diseases other than malignancy.  
 
20) Yes 
 No 
  NA 
  Patient has AML, MDS, ALL, or CML and has presence of circulating 
                               leukemic blasts detected by standard pathology.           
 
21) Yes 
 No 
  NA 
  Patient has aggressive lymphomas (such as DLBC) and has bulky,  
rapidly progressive disease immediately prior to HCT. 
 
22) Yes 
 No 
  NA 
 Patients with a diagnosis of CMML. 
 


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Note: HCT-Comorbidity Score is: _________ 
 
Signature of person completing form:    
 
 
 
 
 
 
 
 
 
Date:  
 
 
 
 
FHCRC Patients:  
 
Patient signed IRB approved consent form.  Date:   
 
 
 
 
 
 
 
 
 
IRB file number:    
 
 
 
 
 
      
 
Date of IRB approval:   
              
 
 
 
Signature of Principal Investigator:  
 
 
 
                                     Date:               
 
 
(post signing of consent)     
 
Outside Center Patients:  
 
Signature of person completing form:    
 
 
 
 
 
 
 
 
Date:  
 
 
 
 
 
Patient signed IRB approved consent form.    Date:    
 
 
 
 
 
 
 
 
IRB file number:    
 
 
 
 
 
     Date of IRB approval:                 
 
              
            
 
Signature of Local Principal Investigator    
 
 
 
                         Date:  
        
 
 
 
Signature of FHCRC Principal Investigator   
 
 
 
                         Date:                
 
 
 
 


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APPENDIX H 
 
NOTICE OF DEATH 
 
 
Patient ID:   
 
 
 
 
 
 
 
 
 
 
Date of Death:   
 
 
 
 
 
 
 
 
 
Place of Event:   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
    
 
 
Apparent cause of death (Please be specific.  Attach hospital summary or death summary when 
possible): 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Form completed by:  ________________________________  
 
 
Date:   ______________ 
 


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APPENDIX I 
SERIOUS ADVERSE EVENT REPORT (SAE) Form IRO-08 
Fred Hutchinson Cancer Research Center 
Clinical Research Division 
Institutional Review Office 
 
FHCRC IR File Number: 
 
FHCRC Protocol Number: 
 
FHCRC Unique Patient #  
 FHCRC/SCCA   
  Other 
Gender:  
 Male  
 Female 
Age:   
FHCRC Principal Investigator:  
 
 
Phone Number: 
Mailstop: 
 
Date of Report:              
 
 
 Initial Report 
 Follow-Up Report #  
 Other 
 
Date study staff became aware of event: __________ 
 
Date Serious Adverse Event Started: 
Date Ended:      
Or 
 Ongoing (if ongoing – must submit follow up report) 
Adverse Event: 
 
 
Describe the Serious Adverse Event including a summary of all relevant clinical information. 
(Or attach a MedWatch Form or other SAE reporting form if one has been completed.)  Use Page 2, if necessary: 
 
Outcomes Attributed to adverse event:  (Check all that apply) 
 Death            /           /     
 Life-Threatening 
 Hospitalization (initial or prolonged) 
 
 Disability 
 Congenital Anomaly 
 Required intervention to prevent permanent                      
impairment/damage 
Specify Agent(s) and/or Procedure(s) involved in this protocol: 
 
#1 
Pharmaceutical product/medical 
treatment/procedure 
 
#2 
Pharmaceutical product/medical 
treatment/procedure 
 Not Related (Unrelated, Unlikely) 
 Not Related (Unrelated, Unlikely) 
 Related (Possible, Probable, Definite) 
 Related (Possible, Probable, Definite) 
 
 
 Follow-up Report Required 
 Final Report (PI must sign final report) 
 
Report Completed by: 
 
Date: 
 
The PI has determined that the consent form must be revised: 
 Yes      
 No 
 
Does this study involve the deliberate transfer of recombinant DNA or DNA or RNA derived from recombinant 
DNA, into human subjects (human gene transfer)? 
 yes  
 no  If yes and the activity involves the SCCA 
outpatient clinic, a copy of this Protocol Modification Form and any supporting documents to be reviewed and 
approved, will be forwarded to the FHCRC’s Institutional Biosafety Committee (IBC) by the Protocol Office 
(Mailstop:  LM-230). 
 
 
Signature of Principal Investigator 
 
 
Date: 


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FHCRC IR File Number: 
 
FHCRC Protocol Number: 
 
FHCRC Unique Patient #  
Date of Report:              
Describe the Serious Adverse Event including a summary of all relevant clinical information. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Appendix J 
The Hematopoietic Cell Transplant-Comorbidity Index (HCT-CI) 9/7/10 
Assign scores appropriately if the patient has any of these comorbidities 
Patient ___________________________________ (name), UPN______________ Date___________ 
Instructions: Circle applicable scores and provide actual value or cause of co-morbidity. Fax to FHCRC w/registration. 
Comorbidities 
Definitions 
HCT-CI 
scores 
Actual Lab 
Values/Comments 
Arrhythmia 
Atrial fibrillation or flutter, sick sinus syndrome, and 
ventricular arrhythmias requiring treatment in the 
patient’s past history 
1 
 
Cardiac 
Coronary artery disease†, congestive heart failure, 
myocardial infarction in patient’s past history or EF 
of 50% at time of HCT 
1 
 
Inflammatory bowel 
disease 
Crohn’s disease or ulcerative colitis requiring 
treatment in the patient’s past history 
1 
 
Diabetes 
Requiring treatment with insulin or oral 
hypoglycemic, but not diet alone, at time of HCT 
1 
 
Cerebro-vascular disease 
Transient ischemic attack or cerebro-vascular 
accident in patient’s past history 
1 
 
Psychiatric disturbance 
Depression/anxiety requiring psychiatric consult or 
treatment at time of HCT 
1 
 
Hepatic – mild 
Chronic hepatitis, Bilirubin >ULN- 1.5 X ULN, or 
AST/ALT >ULN-2.5XULN at time of HCT 
1 
 
Obesity 
Patients with a BMI of 
>35 for adults or with BMI-for-age percentile of 
≥ 95th percentile for children at time of HCT 
1 
 
Infection 
Documented infection or fever of unknown etiology 
requiring anti-microbial treatment before, during and 
after the start of conditioning regimen 
1 
 
Rheumatologic 
SLE, RA, polymyositis, mixed CTD, polymyalgia 
rheumatica in patient’s past history  
2 
 
Peptic ulcer 
Requiring treatment in patient’s past history 
2 
 
Renal 
Serum creatinine >2 mg/dl, on dialysis, or prior renal 
transplantation at time of HCT 
2 
 
Moderate pulmonary 
DLco and/or FEV1 >65%-80% or 
Dyspnea on slight activity at time of HCT 
2 
 
Prior solid tumor 
Treated at any time point in the patient’s past history, 
excluding non-melanoma skin cancer 
3 
 
Heart valve disease 
At time of HCT excluding mitral valve prolapse 
3 
 
Severe pulmonary 
DLco and/or FEV1 65% or 
Dyspnea at rest or requiring oxygen at time of HCT 
3 
 
Moderate/severe hepatic 
Liver cirrhosis, Bilirubin >1.5 X ULN, or AST/ALT 
>2.5XULN at time of HCT 
3 
 
Please provide (KPS): 
 
Karnofsky Performance Score = _______% 
Total Score 
=________ 
Signature of Provider: 
________________ 
 
 


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†One or more vessel-coronary artery stenosis, requiring medical treatment, stent, or bypass graft. 
EF indicates ejection fraction; ULN, upper limit of normal; SLE, systemic lupus erythmatosis; RA, rheumatoid arthritis; CTD, connective 
tissue disease; DLco, diffusion capacity of carbon monoxide; FEV1, forced expiratory volume in one second; AST, aspartate aminotransferase; 
ALT, alanine aminotransferase. 


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APPENDIX K 
 
Adapted from 
COMMON TOXICITY CRITERIA (CTC) 
Version 4.0 
 
Grade 
Adverse Event 
3 
4 
5 
 
BLOOD AND LYMPHATIC SYSTEM DISORDERS 
 
Disseminated 
intravascular 
coagulation 
Laboratory findings and 
bleeding 
Life-threatening 
consequences; urgent 
intervention indicated 
Death 
Febrile neutropenia 
ANC <1000/mm3 with a 
single temperature of 
>38.3 degrees C (101 
degrees F) or a sustained 
temperature of >=38 
degrees C (100.4 degrees 
F) for more than one hour 
Life-threatening 
consequences; urgent 
intervention indicated 
Death 
Hemolysis 
Transfusion or medical 
intervention indicated 
(e.g., steroids) 
Life-threatening 
consequences; urgent 
intervention indicated 
Death 
Hemolytic uremic 
syndrome 
Laboratory findings with 
clinical consequences 
(e.g., renal insufficiency, 
petechiae) 
Life-threatening 
consequences, (e.g., CNS 
hemorrhage or 
thrombosis/embolism or 
renal failure) 
Death 
Grade 
Adverse Event 
3 
4 
5 
 
CARDIAC DISORDERS 
 
Atrial fibrillation 
Symptomatic and 
incompletely controlled 
medically, or controlled 
with device (e.g., 
pacemaker), or ablation 
Life-threatening 
consequences; urgent 
intervention indicated 
Death 
Atrial flutter 
Symptomatic and 
incompletely controlled 
medically, or controlled 
with device (e.g., 
pacemaker), or ablation 
Life-threatening 
consequences; urgent 
intervention indicated 
Death 
Atrioventricular block 
complete 
Symptomatic and 
incompletely controlled 
medically, or controlled 
with device (e.g., 
pacemaker) 
Life-threatening 
consequences; urgent 
intervention indicated 
Death 
Constrictive pericarditis 
Symptomatic heart failure 
or other cardiac 
symptoms, responsive to 
intervention 
Refractory heart failure or 
other poorly controlled 
cardiac symptoms 
Death 


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APPENDIX K (cont’d) 
 
Heart failure 
Severe with symptoms at 
rest or with minimal activity 
or exertion; intervention 
indicated 
Life-threatening 
consequences; urgent 
intervention indicated 
(e.g., continuous IV 
therapy or mechanical 
hemodynamic support) 
Death 
Left ventricular systolic 
dysfunction 
Symptomatic due to drop 
in ejection fraction 
responsive to intervention 
Refractory or poorly 
controlled heart failure 
due to drop in ejection 
fraction; intervention such 
as ventricular assist 
device, intravenous 
vasopressor support, or 
heart transplant indicated 
Death 
Myocardial infarction 
Severe symptoms; cardiac 
enzymes abnormal; 
hemodynamically stable; 
ECG changes consistent 
with infarction 
Life-threatening 
consequences; 
hemodynamically 
unstable 
Death 
Myocarditis 
Severe with symptoms at 
rest or with minimal activity 
or exertion; intervention 
indicated 
Life-threatening 
consequences; urgent 
intervention indicated 
(e.g., continuous IV 
therapy or mechanical 
hemodynamic support) 
Death 
Pericardial effusion 
Effusion with physiologic 
consequences 
Life-threatening 
consequences; urgent 
intervention indicated 
Death 
Pericardial tamponade 
 - 
Life-threatening 
consequences; urgent 
intervention indicated 
Death 
Ventricular arrhythmia 
Medical intervention 
indicated 
Life-threatening 
consequences; 
hemodynamic 
compromise; urgent 
intervention indicated 
Death 
Grade 
Adverse Event 
3 
4 
5 
GASTROINTESTICAL  
DISORDERS 
 
Ascites 
Severe symptoms; 
invasive intervention 
indicated 
Life-threatening 
consequences; urgent 
operative intervention 
indicated 
Death 
Diarrhea 
Increase of >=7 stools per 
day over baseline; 
incontinence; 
hospitalization indicated; 
severe increase in ostomy 
output compared to 
baseline; limiting self care 
ADL 
Life-threatening 
consequences; urgent 
intervention indicated 
Death 
 


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APPENDIX K (cont’d) 
 
Duodenal ulcer 
Severely altered GI 
function; TPN indicated; 
elective operative or 
endoscopic intervention 
indicated; limiting self care 
ADL; disabling 
Life-threatening 
consequences; urgent 
operative intervention 
indicated 
Death 
Gastric ulcer 
Severely altered GI 
function; TPN indicated; 
elective operative or 
endoscopic intervention 
indicated; limiting self care 
ADL; disabling 
Life-threatening 
consequences; urgent 
operative intervention 
indicated 
Death 
Gastritis 
Severely altered eating or 
gastric function; TPN or 
hospitalization indicated 
Life-threatening 
consequences; urgent 
operative intervention 
indicated 
Death 
Lower gastrointestinal 
hemorrhage 
Transfusion, radiologic, 
endoscopic, or elective 
operative intervention 
indicated 
Life-threatening 
consequences; urgent 
intervention indicated 
Death 
Mucositis oral 
Severe pain; interfering 
with oral intake 
Life-threatening 
consequences; urgent 
intervention indicated 
Death 
Oral hemorrhage 
Transfusion, radiologic, 
endoscopic, or elective 
operative intervention 
indicated 
Life-threatening 
consequences; urgent 
intervention indicated 
Death 
Pancreatitis 
Severe pain; vomiting; 
medical intervention 
indicated (e.g., analgesia, 
nutritional support) 
Life-threatening 
consequences; urgent 
intervention indicated 
Death 
Typhlitis 
Symptomatic (e.g., 
abdominal pain, fever, 
change in bowel habits 
with ileus); peritoneal signs 
Life-threatening 
consequences; urgent 
operative intervention 
indicated 
Death 
Grade 
Adverse Event 
3 
4 
5 
 
GENERAL DISORDERS AND ADMINISTARTION SITE CONDITIONS 
 
Multi-organ failure 
Shock with azotemia and 
acid-base disturbances; 
significant coagulation 
abnormalities 
Life-threatening 
consequences (e.g., 
vasopressor dependent 
and oliguric or anuric or 
ischemic colitis or lactic 
acidosis) 
Death 


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APPENDIX K (cont’d) 
 
Grade 
Adverse Event 
3 
4 
5 
 
HEPATOBILIARY DISORDERS 
 
 
Cholecystitis 
Severe symptoms; 
radiologic, endoscopic or 
elective operative 
intervention indicated 
Life-threatening 
consequences; urgent 
operative intervention 
indicated 
Death 
Grade 
Adverse Event 
3 
4 
5 
 
IMMUNE SYSTEM DISORDERS 
 
Allergic reaction 
Prolonged (e.g., not rapidly 
responsive to symptomatic 
medication and/or brief 
interruption of infusion); 
recurrence of symptoms 
following initial 
improvement; 
hospitalization indicated for 
clinical sequelae (e.g., 
renal impairment, 
pulmonary infiltrates) 
Life-threatening 
consequences; urgent 
intervention indicated 
Death 
Immune system 
disorders - Other, 
specify 
Severe or medically 
significant but not 
immediately life-
threatening; hospitalization 
or prolongation of existing 
hospitalization indicated; 
disabling;  limiting self care 
ADL 
Life-threatening 
consequences; urgent 
intervention indicated 
Death 
Grade 
Adverse Event 
3 
4 
5 
 
INFECTIONS AND INFESTATIONS 
 
Enterocolitis infectious 
IV antibiotic, antifungal, or 
antiviral intervention 
indicated; radiologic, 
endoscopic, or operative 
intervention indicated; 
profuse watery diarrhea 
with signs of hypovolemia;  
bloody diarrhea; fever; 
severe abdominal pain; 
hospitalization indicated 
Life-threatening 
consequences; urgent 
intervention indicated 
Death 


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Infections and 
infestations - Other, 
specify 
Severe or medically 
significant but not 
immediately life-
threatening; hospitalization 
or prolongation of existing 
hospitalization indicated; 
disabling; limiting self care 
ADL 
Life-threatening 
consequences; urgent 
intervention indicated 
Death 
Grade 
Adverse Event 
3 
4 
5 
 
INVESTIGATIONS 
 
Alanine 
aminotransferase 
increased 
>5.0 - 20.0 x ULN 
>20.0 x ULN 
 - 
Aspartate 
aminotransferase 
increased 
>5.0 - 20.0 x ULN 
>20.0 x ULN 
 - 
Blood bilirubin 
increased 
>3.0 - 10.0 x ULN 
>10.0 x ULN 
 - 
Carbon monoxide 
diffusing capacity 
decreased 
Asymptomatic decrease  of 
>8 units drop;  >5 units 
drop along with the 
presence of pulmonary 
symptoms (e.g. , >Grade 2  
hypoxia or >Grade 2 or 
higher dyspnea) 
 - 
 - 
Cardiac troponin I 
increased 
Levels consistent with 
myocardial infarction as 
defined by the 
manufacturer 
 - 
 - 
Cardiac troponin T 
increased 
Levels consistent with 
myocardial infarction as 
defined by the 
manufacturer 
 - 
 - 
Creatinine increased 
>3.0 baseline; >3.0 - 6.0 x 
ULN 
>6.0 x ULN 
 - 
Weight gain 
>=20% from baseline 
 - 
 - 
Grade 
Adverse Event 
3 
4 
5 
 
METABOLISM AND NUTRITIONAL DISORDERS 
 
Hypercalcemia 
Corrected serum calcium 
of >12.5 - 13.5 mg/dL;>3.1 
- 3.4 mmol/L;  Ionized 
calcium  >1.6 - 1.8 mmol/L; 
hospitalization indicated 
Corrected serum calcium 
of >13.5 mg/dL; >3.4 
mmol/L;  Ionized calcium  
>1.8 mmol/L; life-
threatening 
consequences 
Death 
Hypertriglyceridemia 
>500 mg/dL - 1000 mg/dL;  
>5.7 mmol/L - 11.4 mmol/L 
>1000 mg/dL; >11.4 
mmol/L;  life-threatening 
consequences 
Death 
Hyperuricemia 
>ULN - 10 mg/dL (0.59 
mmol/L) with physiologic 
consequences 
>10 mg/dL; >0.59 mmol/L; 
life-threatening 
consequences 
Death 


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APPENDIX K (cont’d) 
 
Tumor lysis syndrome 
Present 
Life-threatening 
consequences; urgent 
intervention indicated 
Death 
Grade 
Adverse Event 
3 
4 
5 
 
NEOPLASMS BENIGN, MALIGNANT, AND UNSPECIFIED (INC CYSTS AND POLYPS) 
 
 
Treatment related 
secondary malignancy 
Non life-threatening 
secondary malignancy 
Acute life-threatening 
secondary malignancy; 
blast crisis in leukemia 
Death 
Grade 
Adverse Event 
3 
4 
5 
 
NERVOUS SYSTEM DISORDERS 
 
Dysarthria 
Severe impairment of 
articulation or slurred 
speech 
 - 
 - 
Intracranial 
hemorrhage 
Ventriculostomy, ICP 
monitoring, intraventricular 
thrombolysis, or operative 
intervention indicated 
Life-threatening 
consequences; urgent 
intervention indicated 
Death 
Ischemia 
cerebrovascular 
 - 
 - 
 - 
Leukoencephalopathy 
Severe symptoms; 
extensive T2/FLAIR 
hyperintensities, involving 
periventricular white matter 
involving 2/3 or more of 
susceptible areas of 
cerebrum +/- moderate to 
severe increase in SAS 
and/or moderate to severe 
ventriculomegaly 
Life-threatening 
consequences; extensive 
T2/FLAIR 
hyperintensities, involving 
periventricular white 
matter involving most of 
susceptible areas of 
cerebrum +/- moderate to 
severe increase in SAS 
and/or moderate to 
severe ventriculomegaly 
Death 
Seizure 
Multiple seizures despite 
medical intervention 
Life-threatening; 
prolonged repetitive 
seizures 
Death 
Syncope 
Fainting; orthostatic 
collapse 
 - 
 - 
Nervous system 
disorders - Other, 
specify 
Severe or medically 
significant but not 
immediately life-
threatening; hospitalization 
or prolongation of existing 
hospitalization indicated; 
disabling;  limiting self care 
ADL 
Life-threatening 
consequences; urgent 
intervention indicated 
Death 


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APPENDIX K (cont’d) 
 
Grade 
Adverse Event 
3 
4 
5 
 
RENAL AND URINARY DISORDERS 
 
Chronic kidney disease 
eGFR or CrCl 29 - 15 
ml/min/1.73 m2 
eGFR or CrCl <15 
ml/min/1.73 m2; dialysis 
or renal transplant 
indicated 
Death 
 
Renal and urinary 
disorders - Other, 
specify 
Severe or medically 
significant but not 
immediately life-
threatening; hospitalization 
or prolongation of existing 
hospitalization indicated; 
disabling;  limiting self care 
ADL 
Life-threatening 
consequences; urgent 
intervention indicated 
Death 
Grade 
Adverse Event 
3 
4 
5 
 
RESPIRATORY, THORACIC, AND MEDIASTINAL DISORDERS 
 
Adult respiratory 
distress syndrome 
Present with radiologic 
findings; intubation not 
indicated 
Life-threatening 
respiratory or 
hemodynamic 
compromise; intubation or 
urgent intervention 
indicated 
Death 
Apnea 
Present; medical 
intervention indicated 
Life-threatening 
respiratory or 
hemodynamic 
compromise; intubation or 
urgent intervention 
indicated 
Death 
Bronchopulmonary 
hemorrhage 
Transfusion, radiologic, 
endoscopic, or operative 
intervention indicated (e.g., 
hemostasis of bleeding 
site) 
Life-threatening 
respiratory or 
hemodynamic 
compromise; intubation or 
urgent intervention 
indicated 
Death 
Hypoxia 
Decreased oxygen 
saturation at rest (e.g., 
pulse oximeter <88% or 
PaO2 <=55 mm Hg) 
Life-threatening airway 
compromise; urgent 
intervention indicated 
(e.g., tracheotomy or 
intubation) 
Death 
Pleural effusion 
Symptomatic with 
respiratory distress and 
hypoxia; surgical 
intervention including chest 
tube or pleurodesis 
indicated 
Life-threatening 
respiratory or 
hemodynamic 
compromise; intubation or 
urgent intervention 
indicated 
Death 


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Pneumonitis 
Severe symptoms; limiting 
self care ADL; oxygen 
indicated 
Life-threatening 
respiratory compromise; 
urgent intervention 
indicated (e.g., 
tracheotomy or intubation) 
Death 
Pulmonary edema 
Severe dyspnea or 
dyspnea at rest; oxygen 
indicated; limiting self care 
ADL 
Life-threatening 
respiratory compromise; 
urgent intervention or 
intubation with ventilatory 
support indicated 
Death 
Respiratory failure 
 - 
Life-threatening 
consequences; urgent 
intervention, intubation, or 
ventilatory support 
indicated 
Death 
Grade 
Adverse Event 
3 
4 
5 
 
SKIN AND SUBCUTANEOUS TISSUE DISORDERS 
 
Erythema multiforme 
Target lesions covering 
>30% BSA and associated 
with oral or genital 
erosions 
Target lesions covering 
>30% BSA; associated 
with fluid or electrolyte 
abnormalities; ICU care or 
burn unit indicated 
Death 
Grade 
Adverse Event 
3 
4 
5 
 
VASCULAR DISORDERS 
 
Capillary leak 
syndrome 
Severe symptoms; 
intervention indicated 
Life-threatening 
consequences; urgent 
intervention indicated 
Death 
Hypotension 
Medical intervention or 
hospitalization indicated 
Life-threatening and 
urgent intervention 
indicated 
Death 
Thromboembolic event 
Thrombosis (e.g., 
uncomplicated pulmonary 
embolism [venous], non-
embolic cardiac mural 
[arterial] thrombus), 
medical intervention 
indicated 
Life-threatening (e.g., 
pulmonary embolism, 
cerebrovascular event, 
arterial insufficiency); 
hemodynamic or 
neurologic instability; 
urgent intervention 
indicated 
Death 
Vasculitis 
Severe symptoms, medical 
intervention indicated (e.g., 
steroids) 
Life-threatening; evidence 
of peripheral or visceral 
ischemia; urgent 
intervention indicated 
Death 
 
 
 
 
 
 


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Appendix L 
COORDINATING CENTER FUNCTIONS 
 
Outside Center – PI Communication in Hematologic Malignancies 
 
I. 
Study Management, data analysis, and Data and Safety Monitoring 
 
a. Study Management: 
i. 
Each local PI is responsible for selection, training and oversight of local study 
coordinators 
ii. 
The Coordinating Center registers subjects on the study and assigns study IDs 
iii. 
One copy of the research data is retained by the site. Another data set (identified 
only by study IDs) is transmitted to the Coordinating Center to create the master 
data file. All data are kept in locked areas and password protected databases 
accessible only to study staff 
iv. 
The quality of data is monitored in an ongoing fashion with the study team and 
corrective action plans instituted as necessary  
b. Data Analysis: 
i. 
Study staff review data for completeness as it is submitted by the sites 
ii. 
The study statistician is responsible for data cleaning and the conduct of 
analyses as outlined in the protocol and grant 
c. Data Safety and Monitoring: 
i. 
The trial coordinators at collaborating centers or the local PIs will fax an official 
report of an SAE to the Coordinating Center (FHCRC) within 72 hours if the 
event occurred during the initial 28 days post NK infusion, otherwise within seven 
days 
ii. 
The SAE report is reviewed by the Overall PI. If the SAE meets the FHCRC 
criteria for reporting then an official signed report is submitted to the IRB 
iii. 
An independent DSMB will meet at six-month intervals and all outcome data is 
reviewed including all adverse events and SAEs reported to the Coordinating 
Center along with those officially reported to the IRB 
iv. 
      A report from the DSMB is submitted to the IRB as well as the trial 
coordinators/local PIs participating in the protocol 
 
II.  
Protocol and informed consent document management 
a. A master protocol is maintained by the Coordinating Center and distributed to the sites 
for customization and local IRB review 
b. All protocol and consent modifications initiated by the Coordinating Center are sent to 
the Collaborating Sites following approval by the Coordinating Center IRB, for review 
and approval by the local IRB 
c. Changes required by local IRBs are reviewed by the Coordinating Center and 
approved prior to implementation at local sites 
 
III. Assurance of local IRB OHRP-approved assurance 
a. Each site provides their OHRP assurance number and evidence of IRB certification 
b. Study staff monitor maintenance of institutional assurance and IRB certification 
 
IV. Assurance of local IRB approvals 
a. The Coordinating Center maintains copies of the most current collaborating site 
Consent Forms and IRB approval documentation 
b. No site may enroll subjects until the Coordinating Center has received confirmation of 
local IRB approval 
c. Each site is responsible for preparation and submission of their continuing reviews. 
Any changes to the protocol or consent form will be communicated to the Coordinating 
Center 
d. Sites are required to have active IRB approvals to participate in any study related 
activities 
 


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V. 
Any substantive modification by the Collaborating Institution related to risks or 
alternative procedures is appropriately justified 
a.   The Coordinating Center reviews any modifications to consent forms to ensure that site 
consents do not delete or change the basic or additional elements or alternatives 
required in the sample consent form 
 
VI. 
Informed consent is obtained from each subject in compliance with HHS regulations 
a. Subjects must provide written informed consent prior to study participation 
b. The Coordinating Center verifies eligibility and signed consent prior to assigning a 
study ID number 
 
 
 
 


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Appendix M 
Research Specimens  
 
 
 
Pre-Transplant 
 
Recipient: 
 
 
1-2 mL bone marrow aspirate in lavender-top EDTA tube for Immunophenotyping 
 
10 mL blood in green-top heparin tube for Immunophenotyping (if lymphopenic, up to an 
additional 40 ml may be requested) 
 
10 mL blood in green-top heparin tube for NK Cytotoxicity Assay 
 
10 mL blood in green-top heparin tube for Cell Repository 
 
10 mL blood in lavender-top EDTA tube for KIR Genotyping 
 
5 mL blood in lavender-top EDTA tube for T Cell Spectratyping and TREC analysis 
 
Donor: 
 
 
10 mL blood in green-top heparinized tube for Immunophenotyping 
 
10 mL blood in green-top heparinized tube for Cell repository 
 
10 mL blood in green-top heparinized tube for NK cytotoxicity 
 
10 mL blood in lavender-top EDTA tube for KIR genotyping 
 
5 mL blood in lavender-top EDTA tube for T cell spectratyping and TREC analysis 
 
 
Day +7: 
 
Recipient: 
 
30-60 minutes PRE-NK infusion 
 
10 mL blood in green-top heparin tube for Immunophenotyping  
15 minutes from end of NK infusion 
 
10 mL blood in green-top heparin tube for Immunophenotyping  
 
 
Donor (from NK cell final product): 
 
 
0.25 aliquot for immunophenotyping, cryopreservation, and KIR phenotyping 
 
 
Day +14: 
 
Recipient: 
 
 
10 mL blood in a green top heparin tube for Immunophenotyping 
 
 
Day +28: 
 
Recipient: 
 
 
10 mL blood in green-top heparin tube for Immunophenotyping 
 
10 mL blood in green-top heparin tube for NK cytotoxicity 
 
5 mL blood in lavender-top EDTA tube for TREC analysis 


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Day +56: 
 
Recipient: 
 
 
10 mL blood in a green top heparin tube for Immunophenotyping 
 
 
Day +84: 
 
Recipient: 
 
 
10 mL blood in green-top heparin tube for Immunophenotyping 
 
10 mL blood in green-top heparin tube for NK cytotoxicity 
 
5 mL blood in lavender-top EDTA tube for T cell spectratyping and TREC analysis 
 
 
One Year: 
 
Recipient: 
 
 
10 mL blood in green-top heparin tube for Immunophenotyping 
 
5 mL blood in lavender-top EDTA tube for T cell spectratyping and TREC analysis 
 
 
INSTRUCTION FOR RESEARCH LABS 
 
SAMPLES OBTAINED IN OR SENT TO SEATTLE: 
 
Distribution of Samples for Processing:  
 
Heimfeld Lab: David Yaddock, DE 554, Fred Hutchinson Cancer Research Center, 1100 
Fairview Ave N, Seattle, WA  98109 
 
Sample Processing at HEIMFELD LAB: 
 
Bone marrow aspirate:  
o 
Immunophenotyping:  
 
Arrives: EDTA tube, fresh, room-temperature 
 
Action: Perform Immunophenotyping 
 
Peripheral blood: 
o 
Immunophenotyping:  
 
Arrives: Heparin tube, fresh, room-temperature.  
 
Action: Perform immunophenotyping. 
o 
KIR genotyping:  
 
Arrives: EDTA tube, fresh, room-temperature.  
 
Action: Sample to be ficolled, PBMC collected, cryopreserved, stored. 
 
See section “Every 3-6 months” for shipping instructions 
o 
T cell spectratyping and TREC analysis:  
 
Arrives: EDTA tube, fresh, room-temperature.  
 
Action: Prepare TREC card (5 spots) and store in ziplock bag at room 
temperature. Remainder of sample to be ficolled, PBMC collected, 
cryopreserved, stored. 
o 
NK Cytotoxicity Assay:  
 
Arrives: Heparin tube, fresh, room-temperature. 


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 
Action: Ship fresh sample at room temperature via overnight mail to 
Milwaukee, WI (M-Th only). Email or call lab the day before sample 
arrival: 
 
Shipping Address: 
Clinical Research Coordinator – Protocol 2230 
MACC Fund Research Center, Suite 3018 
8701 Watertown Plank Road 
Milwaukee, WI 53226 
 
Phone/Email: 
o 
crconc@mcw.edu 
o 
Cell Repository: 
 
Arrives: Heparin tube, fresh, room-temperature 
 
Action: Store sample 
 
Every 3-6 Months: 
o 
Send batched, TREC cards and cryopreserved PBMC (from ficolled blood 
samples) to Milwaukee, WI for KIR genotyping, TREC, and T cell spectratyping.  
Cryopreserved PBMC samples must be sent on dry ice.  TREC cards must be 
doubled bagged (Ziploc-to ensure they stay dry) and placed on TOP of the 
sealed Styrofoam container (must NOT be packaged in with the dry ice)  Email or 
call lab the day before sample arrival, ship M-Th only. 
o 
Send with samples:  KIR Genotype Invoice that contains Client #3513 and 
Subject ID#.  Put on outside of specimen bag: Completed Sticker for” Diagnostic 
Laboratories, Blood Center of Wisconsin…” 
 
 
Shipping Address: 
Clinical Research Coordinator – Protocol 2230 
MACC Fund Research Center, Suite 3018 
8701 Watertown Plank Road 
Milwaukee, WI 53226 
 
Phone/Email: 
o 
crconc@mcw.edu 
 
SAMPLES OBTAINED IN OR SENT TO MILWAUKEE: 
 
Distribution of samples for processing: 
Malarkannan’s Lab 
 Cell Repository 
 NK Cytotoxicity Assay 
Ellis Lab 
 KIR genotyping 
Verbsky Lab 
 T cell spectratyping and 
TREC analysis 
 
Sample Processing in HEIMFELD Lab 
 
Bone marrow aspirate: 
o 
Immunophenotyping:  
 
Arrives: EDTA tube, fresh, room-temperature 
 
Action: Ship fresh sample at room temperature via overnight mail to 
Heimfeld Lab in Seattle (M-Th only). Email or call lab the day before 
sample arrival. 
 
Shipping Address: 
David Yaddock (Heimfeld Lab Manager)  
DE-554 
Fred Hutchinson Cancer Research Center  
1100 Fairview Ave N  
Seattle, WA 98109 
 
Phone/Email: 
o 
David Yaddock: 206-667-4609; dyadock@fhcrc.org 


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 
Peripheral blood: 
o 
Immunophenotyping: 
 
Arrives: Heparin tube, fresh, room-temperature.  
 
Action: Ship fresh sample at room temperature via overnight mail to 
Heimfeld Lab in Seattle (M-Th only). Email or call lab the day before 
sample arrival. 
 
Shipping Address: 
David Yaddock (Heimfeld Lab Manager)  
DE-554 
Fred Hutchinson Cancer Research Center  
1100 Fairview Ave N  
Seattle, WA 98109 
 
Phone/Email: 
o 
David Yaddock: 206-667-4609; dyadock@fhcrc.org 
 
Sample Processing in MALARKANNAN  Lab: 
 
o 
NK Cytotoxicity Assay:  
 
Arrives: Heparin tube, fresh, room-temperature. 
 
Action: Perform NK Cytotoxicity Assay 
o 
Cell Repository: 
 
Arrives: Heparin tube, fresh, room-temperature 
 
Action: Store sample 
 
Sample Processing at ELLIS LAB: 
 
Peripheral blood: 
o 
KIR Genotyping: 
 
Arrives: EDTA tube, fresh, room-temperature (if drawn in Milwaukee) or 
cryopreserved if shipped from Seattle. 
 
Action: Perform KIR Genotyping 
 
 
Sample Processing at VERBSKY/GORSKI LAB: 
 
Peripheral blood: 
o 
T cell spectratyping and TREC analysis:  
 
Arrives:  
 
Milwaukee sample: EDTA tube, fresh, room-temperature  
 
Seattle sample:  Cryopreserved +TREC card in ziplock bag 
 
Action:  
 
Milwaukee sample: (To Verbsky Lab) Prepare TREC card (5 spots) 
and store in ziplock bag at room temperature in Verbsky Lab for 
future analysis. 
 
Remaining blood sample to be ficolled, PBMC collected, 
cryopreserved and stored in Verbsky Lab for future T-Cell 
Spectratyping. 
 
Verbsky Lab will deliver cryopreserved blood sample to Gorski Lab 
for T-Cell Spectratyping. 
 
 
 
 
 
 


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FHCRC Version 02/23/2016 
96   
Appendix N 
Intrathecal Therapy Administration 
 
 
intrathecaltherapy-c
ombined.pdf
 

ICF_000.pdf: 
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Page 1 of 16 
FRED HUTCHINSON CANCER RESEARCH CENTER 
UNIVERSITY OF WASHINGTON SCHOOL OF MEDICINE, 
DEPARTMENT OF MEDICINE, DIVISION OF ONCOLOGY 
SEATTLE CHILDREN’S 
 
Consent to Participate in a Research Study called: 
A Phase I/II Study Evaluating the Safety and Efficacy of Adding a Single Prophylactic 
Donor Lymphocyte Infusion (DLI) of Natural Killer Cells Early After Nonmyeloablative, 
HLA-Haploidentical Hematopoietic Cell Transplantation – A Multi Center Trial 
 
 
Note: If you are a parent or guardian of a patient younger than 18 years old and have been asked 
to sign this form, the 'you' in this document refers to the patient. 
 
Investigators (doctors in charge of this study):  
 
  
 
 
Brenda Sandmaier, M.D., 
Member, FHCRC, Professor of Medicine, UW 
Shelly Heimfeld, PhD 
 Member, FHCRC 
Effie Petersdorf, MD 
Member, FHCRC,  Professor of Medicine, UW 
Ann Woolfrey, M.D. 
Member, FHCRC, Professor of Pediatrics, UW 
Michael Linenberger M.D. 
Associate Member, FHCRC, Professor of Medicine, UW 
Mohamed Sorror, M.D., MSc 
Associate Member, FHCRC, Associate Professor of Medicine, UW 
David Maloney, MD, PhD 
Member, FHCRC, Professor of Medicine, UW 
Rainer Storb, M.D. 
Member/Program Head, Transplantation Biology, FHCRC, 
Professor of Medicine, UW 
 
 
Statistician: Barry Storer, PhD, Member, FHCRC 
 
Additional Performance Sites 
Investigators 
Emergency Phone Numbers 
Medical College Wisconsin - 
Children’s Hospital of Wisconsin 
Monica Thakar, MD 
(414) 456-7546    
Medical College of Wisconsin– 
Froedert Hospital 
Parameswaran Hari, 
MD 
(414) 805-4600 
 
 
Research Nurse: Michelle Bouvier, RN (206) 667-6993, pager (206) 995-7658 
 
 
 
 
 
 
 
Emergency 24 Hour Phone:  
FHCRC:   
 
 
Phone:  
(206) 598-8902    
FAX: (206) 598-4034 
Seattle Children’s:  
Phone:  
(206) 987-2000*  
FAX: (206) 987-2764 
 
*Ask Seattle Children’s Operator for “Transplant Provider On-Call” 
 
 
The purpose of this informed consent form is to let you know about a clinical trial (a type of 
research study). This form tells you about the purpose, risks and benefits, and describes what is 
involved in the study. It also tells you what other choices you have. 
Up to 40 people will be in the phase I and phase II portions of the study. Your study doctor will 
explain the clinical trial to you. Clinical trials include only people who choose to take part. Please 
take your time to make your decision about taking part. You may discuss your decision with 
your friends and family. You can also discuss it with your health care team. If you have any 
questions, you can ask your study doctor for further information. There is no guarantee that this 
procedure will be successful. 


2230.00A 
 
Page 2 of 16 
Even if you join this study, you may stop at any time. There is no penalty for stopping. If you 
decide not to be part of this study, you will continue receiving medical care. If you would like to 
be part of this study, we will ask you to sign this form. You will get a copy to keep. 
Why is this study being done? 
Sometimes it is difficult to find a “matched donor” in the family (related donor) or in the Bone 
Marrow Registry (unrelated donor).  When this happens, we may choose a donor who is only 
partially matched to you.  This can sometimes cause problems, such as graft rejection (when 
the body rejects the new cells), graft-versus-host disease (GVHD) (when the new cells attack 
the body), or even death.  Therefore, there is a great need to develop safer and more effective 
protocols when using mismatched donors. 
 
To address these problems, we will test a nonmyeloablative transplant from a haploidentical 
donor. 
 
Unlike a standard transplant, a nonmyeloablative transplant uses milder or lower 
doses of drugs or radiation to leave some of the immune system intact. Drugs help 
your body accept the new donor cells. 
 
A haploidentical donor is related to the patient, but has a different tissue type.  This 
tissue type is matched by at least 50% to the patient.  A haploidentical transplant is 
considered an “alternative” transplant to traditional methods. 
 
As compared to conventional transplants with matched related and unrelated donors, 
haploidentical  transplants have been associated with a higher risk of:  
 
GVHD 
 
Infections. 
 
Relapse after transplant. 
 
Rejection of the new bone marrow. 
 
This trial is asking whether the addition of extra cells from the same haploidentical donor one 
week after transplant can help prevent these problems.  These cells are known as natural killer 
cells or NK cells.  The addition of NK cells is considered investigational therapy. 
 
NK cells are a type of white blood cell. 
 
They have been shown to fight infections and fight cancer cells. 
 
They have been shown to help new bone marrow stem cells engraft. 
 
When we do the transplant followed by an infusion of donor NK cells, we will want to find out: 
 
Are adding NK cells after non-myeloablative, haploidentical transplant safe? 
 
Will these NK cells reduce the rates of relapse and graft rejection? 
 
Will these NK cells help fight infections after transplant? 


2230.00A 
 
Page 3 of 16 
 
Hospitalization 
Pediatric patients (those under 18 years of age) may require hospitalization for this treatment. 
All of the research studies will be done in the hospital.  You will be discharged to the outpatient 
department when the inpatient doctor decides you are ready. You may need hospitalization at 
other times if you get sick. 
Adult patients (those 18 years of age and older) the treatment, including infusion of donor bone 
marrow cells and NK cells, can be done as an outpatient.  You may get hospitalized at other 
times if you get sick. 
Procedures that will be done as part of the transplantation 
Conditioning: 
You will get drugs and radiation to suppress the immune system and destroy the abnormal 
marrow, so the donor’s healthy cells can grow and produce new blood cells. This is called 
“conditioning.” You will get these drugs through the central venous catheter: 
 
Cyclophosphamide: 2 doses total, on days −6 and −5.   
 
Fludarabine: 1 dose per day for 5 days (day −6 through day −2.).  
 
Total Body Irradiation: The day before the transplant, you will get 1 dose of radiation. 
(If the patient is too young to be still, anesthesia will be provided.) 
You will also get medications to help prevent nausea and vomiting. 
Bone marrow transplant: 
The donor’s bone marrow stem cells will be given through the central venous catheter, similar to 
a blood transfusion. From these stem cells will grow the components found in blood: red blood 
cells that carry oxygen, white blood cells that fight infections, and platelets that make clots. 
Post-transplant immunosuppression: 
Starting on day 3 after the transplant, we will give you strong immune suppressing drugs. The 
purpose of these drugs is to allow the new bone marrow cells to be accepted and to try to 
reduce the risk of GVHD. 
The drugs are: 
 
Cyclophosphamide: One dose on day 3 after transplant through the central venous 
catheter. 
 
Mesna:  4 doses with each dose (1 just before the cyclophosphamide and 3 just 
after). Mesna is not chemotherapy. It is a medicine that helps protect your bladder 
from the side effects of high-dose cyclophosphamide. 
 
Tacrolimus twice a day beginning on day 4 until 84 days after the transplant, first by 
central venous catheter, then by mouth.  The dose will then be gradually reduced 
over a period of 13 more weeks, until day 180.  
 
Mycophenolate mofetil (or MMF): Three times a day beginning on day 4 until day 40, 
first by central venous catheter, then by mouth.  The dose will then be gradually 
reduced over a period of 6 more weeks, until day 84.  
If you develop GVHD or we think your body may reject the donor cells, you may need to take 
the drugs for a longer time or we may need to add other drugs. 


2230.00A 
 
Page 4 of 16 
Post-transplant bone marrow recovery 
Starting on day 4 after transplant, we will start granulocyte colony stimulating factor (GCSF) to 
help the new white blood cells grow. This will be given either as an injection under the skin, or 
through an IV or central venous catheter. GCSF will continue until enough new white blood cells 
grow. 
 
Post-transplant donor NK cell infusion 
The same haploidentical family member who gave you cells for your bone marrow transplant will 
also give you cells for your NK cell infusion.  One week after transplant, you will receive the NK 
cells as a single infusion through your central venous catheter. 
 
 
You will also have blood tests at least three times a week to check kidney function 
and blood cell counts early after the transplant 
 
Other blood tests routinely done include liver function and electrolytes (blood salts). 
 
In addition, a bone marrow biopsy will be needed at various time points. The bone 
marrow and blood samples will be evaluated for the presence of donor cells to 
determine how well you have accepted the new stem cells. 
 
You will undergo scheduled screening tests to make sure that your cancer has not 
come back.  This screening is specific for your type of cancer.  Your doctor can 
provide more details regarding your screening tests. 
Here is the schedule of conditioning and post-transplant procedures: 
 
 
Days before transplant 
 
 
−6 
−5 
−4 
−3 
−2 
−1 
Conditioning 
 
 
 
 
 
 
Cyclophosphamide 
● 
● 
 
 
 
 
 
 
 
 
 
 
 
Fludarabine 
● 
● 
● 
● 
● 
 
TBI 
 
 
 
 
 
● 
 
Days after transplant 
+3 
+4 
+7 
+40 
+84 
+180 
Post-Transplantation  
 
 
 
 
 
 
Cyclophosphamide 
● 
 
 
 
 
 
Mesna  
● 
 
 
 
 
 
MMF  
 
Start 
 
Taper* 
Stop* 
 
Tacrolimus 
 
Start 
 
 
Taper* 
Stop* 
G-CSF**  
 
Start 
 
 
 
 
Donor NK Cell Infusion 
 
 
● 
 
 
 
* We will reduce the dose around this time, depending on your GVHD status. 
** Continues until enough new white blood cells grow. 
 
 
 
 
 
 
 
 
 


2230.00A 
 
Page 5 of 16 
Blood and Bone Marrow Tests for Research 
 
 
 
We will use your blood and tissue samples for research to study your immune system recovery.  
This may involve sending your research samples to outside research labs. Samples will be 
stored indefinitely.  
 
If we want to use your tissue in the future for a purpose not described in this consent, we must 
first send a request to the Institutional Review Board (IRB) for review and approval for all 
proposed new research. 
How long will I be in the study? 
Your treatment at the SCCA will last about 3½ months, but could be longer. You may be asked 
to return for follow up at 6 months and every year thereafter, to help manage some of the 
complications of the transplant. 
We would like to keep track of your medical condition for the rest of your life, to understand the 
long-term effects of the transplant. But you may be taken off the study and followed less often if: 
 
The study treatment does not work for your disease; 
 
You develop a serious side effect that you cannot tolerate or that cannot be 
controlled with other medications; 
 
Your health gets worse; 
 
You are unable to meet the requirements of the study (for example, you cannot take 
the medicine as prescribed or you refuse follow-up); 
 
You start other treatments for your disease; 
 
Other treatments for your disease become available; 
 
By your request. 
What are the side effects (risks)? 
Transplant 
We expect that the transplant itself will have relatively minor side effects (like a blood 
transfusion). However, after the transplant you will be at very high risk for complications, 
including death. The side effects of transplant are related to the chemotherapy, radiation 
therapy, and immunosuppression that you will be receiving.  You may need red blood cell and 
platelet transfusions and antibiotic therapy. 
 
Sample 
Sample Size 
Timing 
Bone marrow 
aspiration  
Up to 2 ml (about ½ 
teaspoon) 
 
 
Before transplant, We will try to collect this sample 
at the same time as your regularly scheduled doctor 
visits. 
Blood  
Up to 45 ml (about 3 
tablespoons) 
Before and after transplant, and after you return 
home at approximately Day +7, +14, +28, +56, +84, 
and one year . We will try to collect these samples 
at the same time as your regularly scheduled doctor 
visits. 


2230.00A 
 
Page 6 of 16 
Side effects that we know about now are described in the table. Side effects are categorized 
into either: 
 
Likely side effects: Side effects that may occur in 10% or more of patients (this means 
that 10 or more patients out of 100 might get this). Certain side-effects in this category 
could occur in virtually all patients. 
 
Less likely side effects: Side effects that may occur in 3-9% of patients (this means 
that 3 to 9 patients out of 100 might get this). 
 
Rare side effects: Side effects that do not occur very often, but may occur in less than 
3% of patients (this means that 1 or 2 patients out of 100 might get this).    
 
With any drug or combination of drugs, there may be complications or side effects that we do 
not know about. 
Likely Side Effects  
Less Likely Side Effects  
Rare Side Effects  
Graft-versus-host disease 
Allergic reaction with infusion of 
marrow and/or NK cells (including 
itching, hives, flushing, 
hypersensitivity, shortness of 
breath, wheezing, chest tightness, 
skin rashes, fever, chills, muscle 
stiffening, severe breathing 
problems) 
Sores in mouth and/or 
throat 
Nausea 
Jaundice (yellowish discoloration of 
sclera) 
Hair loss 
Vomiting 
Rejection/graft failure 
Skin or nail discoloration 
Diarrhea 
Fluid retention (bloating or swelling) 
Nail changes 
Loss of appetite 
Weakness 
Painful burning on the skin 
of the hands and feet 
Fever 
Fatigue 
Irregular menses or 
stopping of menses 
Lowered white blood cell 
counts (may lead to 
infection) 
Seizure 
Infertility (inability to have 
children) in women or 
Sterility for men 
Lowered platelet counts 
(may lead to bleeding) 
Tremor 
Failure of heart function 
Lowered red blood cell 
counts (may lead to 
anemia, fatigue, shortness 
of breath) 
Muscle or joint pain 
Failure of liver function 
Infection 
Red Blood Cell Destruction 
Failure of brain function 
Time away from work 
 
Impairment or failure of 
kidney function 
 
 
Failure of lung function 
 
 
Bleeding or dysfunction of 
the central nervous system 
 
 
 


2230.00A 
 
Page 7 of 16 
NK Cell Infusion 
We expect that the NK cell infusion itself will have relatively minor side effects.  Potential side 
effects are listed in the table and all are considered rare (less than 3% of patients, or 1-2 out of 
100 patients or less).  There may be complications or side effects that we do not know about. 
 
Graft-versus-host disease 
Nausea 
Vomiting 
Infection  
Prolonged neutropenia (low white count) 
Diarrhea 
Possible allergic reaction (including itching, hives, 
flushing [red face], shortness of breath, wheezing, 
chest tightness, skin rash, fever, chills, stiff muscles, or 
trouble breathing) 
Marrow suppression 
 
There is a very small risk (less than 1%, or 1 out of 200 patients or less) of developing 
antibodies against proteins that are leftover on the NK cells.  This leftover protein is needed to 
select the NK cells from the donor’s blood and is a critical part of the NK cell collection process. 
 
Side effects may occur at the time of transplant or after. They may require hospitalization, 
medication, or therapy. Risks and side effects vary from person to person. Your doctor may be 
able to change or give medications to make some of the side effects less bothersome. 
Side effects such as GVHD and infections can be very serious, long lasting, and/or life 
threatening. Please talk with your study doctor about these side effects. If you want to read 
more about the side effects from study drugs, please ask your doctor or pharmacist for more 
information. 
In addition to the above risks, there is the risk of organ failure, including heart, kidney, lung, 
brain, liver or other body parts. This risk is increased in those patients who have pre-existing 
damage to any organ system. Although the aim of this study is to reduce the risks of having 
bone marrow transplants, the side effects of treatment could be severe and include a risk 
of death. 
There is a risk that your disease will come back, even if the transplant works at first. Also, the 
transplant may be less successful at treating your disease than a standard stem cell transplant 
from a matched related donor, and the rate of disease recurrence may be higher. 
Rejection of donor cells: There is a risk that you will reject the donor blood stem cells and that 
the donor cells will not be detected after transplant. Transplants from haploidentical donors are 
at a higher risk of rejection. If this occurs, we expect your own blood counts to recover.  When 
this happens, you are also at higher risk of relapse if you have a history of leukemia.  Rarely, 
rejection may occur without recovery of cells. This type of rejection can be fatal. It can only be 
treated by a second stem cell transplant. 


2230.00A 
 
Page 8 of 16 
 
Cyclophosphamide is a type of chemotherapy used in stem cell transplants to try and reduce 
the risk of rejection and/or GVHD.  
 
Likely side effects 
Less likely 
Rare 
Low white blood cell 
count  
Low red blood cell count 
Diarrhea 
Vomiting 
Liver damage 
Lower sperm production 
in men 
Hair loss 
Nausea 
Loss of appetite 
Missing or stopping 
menstrual cycle in 
women 
Sores in mouth or on lips 
Blood in urine 
Fatigue 
Lower platelet count (mild) with 
increased risk of bleeding 
Darkening of nail beds 
Fetal damage (if pregnancy occurs 
while taking cyclophosphamide) 
Secondary cancers 
Infertility 
Lung scarring (fibrosis) with 
cough and shortness of 
breath 
Heart failure (with high 
doses) 
Decrease in sodium level in 
the blood with high doses 
 
Cyclophosphamide can cause bleeding in your bladder. Getting more fluid through a vein or 
your catheter and drinking extra liquids may prevent this. If the bleeding becomes severe, the 
bladder may need to be washed out with a salt-water solution, using a bladder catheter (thin 
plastic tube). 
DO NOT take any aspirin or nonsteroid anti-inflammatory medications (e.g., Ibuprofen, Motrin, 
Advil). Report easy bruising or bleeding such as a nose bleed, bleeding of gums when you 
brush your teeth or have black/bloody stools.  
Mesna is used to prevent bleeding from the bladder, a possible side effect of 
cyclophosphamide. It is not chemotherapy. 
 
Likely side effects  
Less likely 
Nausea 
Vomiting 
Diarrhea 
 
Abdominal pain 
Altered taste 
Rash 
Hives  
Headache 
Joint or limb pain 
Low blood pressure 
Fatigue 
 


2230.00A 
 
Page 9 of 16 
Fludarabine is a chemotherapy used to try and reduce the risk of rejection. 
 
Likely side effects 
Less 
likely 
Rare 
Low white blood cell count with an increased risk of 
infection (from bacteria, fungi or viruses) 
Low platelet count with an increased risk of bleeding 
Anemia 
Nausea 
Diarrhea 
Fatigue 
Vomiting 
Trouble seeing or problems 
with your eyes 
Numbness or tingling in your 
fingers or toes 
Confusion or coma 
Pneumonia 
Secondary cancers 
 
Total body irradiation (TBI) is “full body” radiation used to help weaken or destroy your abnormal 
bone marrow cells. The dose used in this protocol will be much lower than that used in standard 
transplant protocols. TBI may destroy normal bone marrow cells. 
 
Likely side effects  
Less likely 
Nausea 
Fatigue 
Temporary hair loss  
Vomiting 
Diarrhea 
Painful swelling of the parotid gland (a gland 
under the chin) for a few days 
Cataracts (an opacity or whitening of the lens) 
in the eye 
Secondary cancers 
Sterility 
Major genetic damage to children conceived 
after transplant 
Damage to the lungs 
 
 
Patients receiving radiation have a risk of developing a secondary cancer later in life.  You will 
also be receiving diagnostic exams (CT scans, chest x-rays, bone scan, etc) to help follow your 
progress. These exams will result in a radiation dose to you, but these doses are very small in 
comparison to the therapy dose you will receive. They are not expected to increase your health 
risk. 
TBI may also cause damage to the lining of the mouth, called “mucositis” that requires 
pain medication and temporary administration of fluids and nutrition by vein.  TBI may 
cause diarrhea that lasts for a few days.  In addition TBI can cause damage to the lungs 
and often causes the formation of cataracts many years after its administration.     
 
 


2230.00A 
 
Page 10 of 16 
Tacrolimus is a drug used to treat GVHD. During treatment, we will monitor the levels of 
tacrolimus in your blood, to see if we need to adjust the dose. 
 
Likely side effects 
Less likely 
Rare 
High blood pressure 
(hypertension) 
Shaking of hands (tremor) 
Changes in liver or kidney 
function 
Altered levels of 
magnesium, calcium, 
potassium, and sugars in 
the blood 
Headache 
Pain in the hands or 
feet. 
Increases in 
cholesterol and 
triglyceride levels 
Nausea/vomiting 
Changes in how clearly 
one can think 
Trouble sleeping 
Patients have had seizures, but it is 
unclear whether tacrolimus, other 
drugs, or a combination of drugs were 
responsible. 
Renal failure from damage to the blood 
vessel walls and destruction of red 
blood cells by a condition called 
hemolytic uremic syndrome (HUS) 
 
 
Mycophenolate Mofetil (MMF) is a drug used for suppressing the immune system. It is 
reasonably well tolerated by patients who have had nonmyeloablative transplants. There are a 
small number of patients who have received solid organ transplants and had reversible fall in 
their red cell or white cell count while receiving MMF. Additionally, cases of Pure Red Cell 
Aplasia (PRCA) have been reported in some patients receiving MMF. PRCA is a condition in 
which the bone marrow stops producing red blood cells. In some instances, PRCA can be 
reversed by reducing or stopping MMF. Your blood counts will be monitored closely and if 
significant decrease is noted, dose adjustments or stopping your MMF may be indicated. 
 
Cases of progressive multifocal leukoencephalopathy (PML) have occurred in some patients 
receiving MMF. PML is a rare disorder that affects the central nervous system, and is most often 
found in patients with suppressed immune systems. It occurs when the polyomavirus (or JC 
virus) is activated, and can cause neurologic symptoms including weakness on one side of the 
body, lack of emotion, confusion, cognitive difficulties, and loss of coordination. It can cause 
permanent disability and is sometimes fatal. You should notify your doctor immediately if you 
develop any of the above symptoms. 
 
MMF has caused birth defects in humans. The United States Food and Drug Administration 
(FDA) requires that women who take part in this study must use two forms of contraception if 
they are fertile and not abstinent. 
 
FOR WOMEN WHO COULD BECOME PREGNANT: Birth defects could occur if you take MMF 
while you are pregnant. As discussed above, you must use 2 effective forms of contraception if 
you are fertile and sexually active. You should talk to your doctor to find out which methods of 
birth control would be most effective for you. You must notify your doctor and the coordinating 
center for the study immediately if you become pregnant while you are taking MMF. You should 
not breast feed while you are taking MMF. 
Likely side effects 
Less likely 
Rare 
Nausea  
Birth defects 
Vomiting  
Diarrhea and abdominal discomfort 
Lower red blood cell count that is reversible 
Lower white blood cell count with increased risk of 
infection 
Stomach bleeding 
Blood in stools 
Secondary cancers 


2230.00A 
 
Page 11 of 16 
 
Granulocyte Colony Stimulating Factor (known as GCSF, Neupogen, or growth factor) is a drug 
used to help increase your white blood cell count after transplant.  
 
 
 
Graft-versus-host-disease (GVHD) is a known complication of bone marrow transplantation. It 
has occurred in 50 - 70% of patients on similar studies. When you receive stem cells from a 
donor with a different tissue type than yours, the risk of GVHD is higher.  However, we are 
hoping to reduce this complication to less than 30% by using cyclophosphamide on day +3 after 
transplant. 
GVHD can occur early after the transplant (called acute GVHD) or late after transplant (called 
chronic GVHD). In GVHD, donor cells react against cells of your body. GVHD may cause skin 
damage (rash, discoloration, and tightness); gastrointestinal damage (nausea, vomiting, and 
diarrhea); liver damage (abnormal liver function tests and jaundice); lung damage, dry mouth or 
sores of the mouth, dry eyes, weight loss, and hair loss. GVHD may be mild or severe. It may 
require prolonged treatment (sometimes for years) with immune suppressing drugs to reduce 
inflammation. Severe GVHD and complications of its treatment can result in death. 
Side effects of GVHD include: 
 
Acute 
Chronic 
Skin rash 
Lack of appetite, stomach cramps, diarrhea or “full” feeling 
in stomach  
Intestinal bleeding 
Problems of the liver  
Problems of the stomach 
Nausea 
Vomiting 
Death 
Eye problems 
Skin problems 
Liver problems 
Problems of the mouth, lips, and 
throat 
Lung problems 
And all the risks listed under 
“Acute” 
Early (acute) or late (chronic) GVHD may become so bad that it results in death. GVHD is 
treated with drugs that weaken the immune system. This makes you more likely to get 
infections. 
A very effective drug for treating GVHD is prednisone, which is a type of steroid. However, 
prednisone is often required at high doses for 9 months to several years. Depending on the 
dose and duration of treatment, side effects may include weight gain, depression or psychosis 
(mood swings), cataracts, diabetes, muscle loss, bone loss or fractures, avascular necrosis 
(break down) of hip or other joints that may require joint replacement, high blood pressure and 
others. 
Reproductive risks 
Risk to the Unborn:  You should not become pregnant or father a child while on this study. The 
treatments in this study have NOT been proven to be safe at any stage of pregnancy. 
Therefore, if you are pregnant or nursing, you are not eligible for this study. Women who have 
the potential of becoming pregnant or men who have the potential of fathering a child must use 
two forms of effective birth control or abstinence for one year after transplant. If you are still on 
Likely side effects 
Less likely 
Muscle aches or pain  
Bone pain 
Itching  
Skin rash  
Headache 
Blood vessel inflammation (vasculitis) 
Ruptured spleen 


2230.00A 
 
Page 12 of 16 
MMF for more than one year after transplant, you must continue using two forms of birth control 
until you have been off MMF for at least 2 months.  Effective birth control would be defined as 
the following: 1) refraining from all acts of vaginal intercourse (ABSTINENCE); 2) consistent use 
of birth control pills; 3) injectable birth control methods (Depo-Provera); 4) tubal sterilization or 
male partner who has undergone a vasectomy; 5) placement of an IUD (intrauterine device); 
and, 6) use, with every act of intercourse, of a diaphragm with contraceptive jelly and/or 
condoms with contraceptive foam. 
Sterility and Future Childbearing Potential for Men and Women: Chemotherapy and/or 
irradiation may affect fertility. In males who have reached puberty and have received 300 cGy 
radiation, most men will have return of sperm counts by 30 months.  But some men will not 
recover sperm counts, and sterility can still occur. Female patients may find that their menstrual 
cycle becomes irregular or stops permanently. However, this DOES NOT MEAN THAT YOU 
CANNOT BECOME PREGNANT OR FATHER A CHILD, and you must use some effective 
method of birth control for at least one year after the transplant. Damage to reproductive tissue 
may result in birth defects or permanent inability to have a child or become pregnant. You 
should discuss these risks and options in detail with your doctor before entering this study. 
Cyclophosphamide can sometimes make it harder to have a baby (infertility). The doses in this 
study are much lower than in a standard transplant, so your risk of infertility is also lower.  Since 
you will be receiving a combination of both radiation and cyclophosphamide, the risk of infertility 
or sterility may be higher than receiving each alone.   
If you are a male and have reached puberty, you can talk to your doctor about ways of storing 
sperm. If you are a woman and have reached puberty, egg storage is much more difficult. Talk 
to your doctor if you are interested in learning more about this.  
Other risks 
If you have severe side effects, which can include low blood counts, infections, bleeding, and 
failure of the donor stem cells to grow, you may need red blood cell and platelet transfusions. 
Red blood cell and platelet transfusions have a very small risk of transmitting serious infections. 
You may need antibiotics or other medications to treat the side effects. 
There is a risk that you might develop a fatal lymphoma, called Post-Transplant 
Lymphoproliferative Disease (PTLD). The risk of PTLD is small; it has occurred in 5% of 
patients who were given standard (high-dose) transplant regimens. All these patients had either 
a diagnosed immune deficiency before the transplant, or severe GVHD after transplant that 
required intensive immune suppressive therapy. If you have either of these risk factors you may 
be at risk for development of PTLD. 
Are there benefits to taking part in this study? 
There may or may not be direct medical benefits to patients in this study. We hope that this 
treatment will result in a cure of your bone marrow failure or leukemia and help your immune 
system function better after transplant. Some patients may not directly benefit, but may be 
encouraged by knowing that being in this study may help future patients with the same disease.  
What other treatment options are there? 
Instead of being in this study, you can decide to have: 
 
Treatments that help with the symptoms of your disease. These may include 
antibiotics, blood transfusions, enzyme replacement, special diets, as well as other 
standard medical treatments. 
 
Treatments that are considered experimental. For example, some patients may be 
eligible for gene therapy or other experimental forms of stem cell transplant. 


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 
A conventional stem cell transplant, which uses higher doses of chemotherapy and 
radiation therapy. 
 
Waiting for a bone marrow donor who has a better tissue match to you. 
 
No treatment. 
 
Comfort care. 
Before you agree to this study, please talk with your doctor about these and other options that 
may become available. 
Will my medical information be kept private?  
The individuals responsible for this study will try to keep your personal information as private as 
possible and ensure that it is disclosed only in accordance with state and federal law and the 
terms of this consent. However, it is not possible to guarantee absolute privacy. In addition to 
disclosures in accordance with state and federal law, the organizations that are listed below 
may inspect or copy your research records for quality assurance and data analysis. Your 
research records will identify you by a unique patient number (UPN) that may be linked to your 
name and will include things such as your medical record, medical history, results of your blood 
tests and exams, reports from your treatment, and reports of your office visits.  For any 
published reports, a different coding system not linked to your name will be used. 
Who has access to my research record information? 
You will receive some services, tests, or procedures at University of Washington clinical facilities 
(“UW Medicine”) and/or at the Seattle Cancer Care Alliance (“SCCA”) as part of this study. This 
means that information about this study will become part of your medical record.   If you do not 
already have a UW Medicine or SCCA medical record for clinical purposes, one will be created for 
you. Basic information such as the name and number of the study, the study sponsor, the names 
and contact information of the study staff will be included in your medical record.    
 
Research procedures and test results may also be put in your medical record.  This will include 
things such as your medical history, results of your blood tests and exams, reports from your surgery 
and treatment, reports of your office visits. 
 
If you have already given or in the future decide to give permission to any person or group (such as 
an insurance company or employer) to have access to your medical record, the information about 
this study and your participation in it will be included.  This could affect your ability to get life 
insurance or a new job.   
 
It is important for you to consider the possible risks of including study information in your medical 
record before you sign this consent form. 
 
We will try to keep your personal information as private as we can.  We cannot guarantee absolute 
privacy.  Your personal information may be disclosed if required by law.  Some people may see or 
copy your medical records. They would do this as part of their research, to make sure this study is 
being done correctly and safely, or to evaluate the results. Organizations that may inspect and/or 
copy your research records for quality assurance and data analysis include the groups listed below.   
 
Researchers involved with this study 
 
Fred Hutchinson Cancer Research Center (FHCRC), University of Washington (UW), 
Seattle Children’s, and Seattle Cancer Care Alliance (SCCA) 
 
Medical College of Wisconsin 
 
US Food and Drug Administration (FDA), National Institutes of Health (NIH), and Office 
for Human Research Protections (OHRP) 


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 
Institutional Review Boards (IRB) (groups who review the study to protect your rights as 
a research participant) 
 
Miltenyi Biotec 
What are the costs of taking part in this study? 
Miltenyi Biotec will cover the cost of the reagents used to select the NK cells from your donor’s 
cells collected post transplant on day 7. Research funding will provide the remainder of the 
laboratory costs of the NK cell selection.  You or your insurance company will pay the rest of the  
medical expenses relating to, or arising from, this study.  If you are injured or become ill from 
taking part in this study, emergency medical treatment is available but will be provided at the 
usual charge. No funds have been set aside to pay you in the event of injury. You or your 
insurance company will be charged for continuing medical care and/or hospitalization. 
Taking part in this study may lead to added costs for you or for your insurance company due to 
the frequency of blood tests required. If you have any questions concerning your costs, financial 
responsibilities, and or medical insurance coverage for this activity, please ask your physician or 
contact the SCCA Patient Financial Services Department at (206) 288-1113. 
Will I be paid to take part in this research study?  
You will not be paid for taking part in this study. 
Do I have to be part of the study? 
Joining this study is up to you. You are free to say yes or no, or to drop out after joining. 
However, stopping treatment during conditioning or after the transplant could have very serious 
health consequences, even death. Stopping MMF and tacrolimus after the transplant could lead 
to rejection of the donor stem cells, or to life-threatening GVHD. If you are thinking about 
dropping out of this study, please tell your doctor and the study doctor. 
What are my rights as a study participant? 
 
You do not have to join this study. You are free to say yes or no. Your regular 
medical care will not change. 
 
If you join this study, you do not have to stay in it. You may stop at any time (even 
before you start). There is no penalty for stopping. Your regular medical care will not 
change. 
 
If you get sick or hurt in this study, you do not lose any of your legal rights to seek 
payment by signing this form. 
 
During the study, we may learn new information you need to know. For example, 
some information may affect your health or well-being. Other information may make 
you change your mind about being in this study. If we learn these kinds of 
information, we will tell you. 


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For more information 
If you have questions or concerns about this study, you may talk to your doctor anytime. Other 
people you can talk to are listed below. 
 
For Questions About 
Please Contact 
This study and what it involves 
Your doctor (attending physician) or one of the investigators 
listed at the beginning of the consent 
Your rights as a participant in a 
research study 
Karen Hansen, in the Institutional Review Office, FHCRC at 
206-667-4867 
Your bills and health insurance 
coverage 
Seattle Cancer Care Alliance, Patient Financial Services at 
206-288-1113 
Research use of your blood or 
tissue sample, or research files 
Clinical Research Division, Data Management Office, 
FHCRC/Clinical Research Division at 206-667-4728 
Research related injury 
Your physician or one of the investigators listed at the 
beginning of this consent 
Emergency care 
Emergency (24 hour) phone: UWMC(206) 598-8902; Seattle 
Children’s(206) 987-2032 
Medical records? 
Contact the Seattle Cancer Care Alliance's Director of    
Health Information Management at 206-288-2174. 


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Signature 
Before you sign this consent form, make sure of the following: 
 
You have read this consent form, or someone has read it to you. 
 
This study has been explained to you. 
 
You had the chance to ask as many questions as you wanted. 
 
You understand you can ask more questions anytime. 
 
You understand you (or your insurer) will have to pay the costs of being in this study, 
including treatment for side effects. 
 
You understand your medical records will be available to the doctors, staff, and other 
groups working on this study. 
 
You agree to join this study. 
Participant (age 14+) / date 
 
 
Parent or legal guardian (for participants 
under 18 years) / date 
 
 
Other parent or legal guardian (if 
reasonably available) / date 
 
Medical Staff Person’s Statement 
I have discussed the above research study, including the study procedures and possible 
alternatives and risks, with the person signing above. I encouraged questions and have 
answered them to the best of my ability. A copy of the signed consent form will be given to the 
participant. 
  
 
 
 
 
 
Medical Staff Person’s Signature 
 
 
 Date 
 
 
 
Printed Name and Title of Medical Staff Person 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Signature of Any Additional Staff Person Present During Consent Process (if present) 
 
 
2230.00A – Current version: 04/14/2016 
Previous version: 12/09/2014 
Copies: Patient, Medical Records, Research File 
 
 
Signed Consent MUST be sent to Data Management – 
LF-229FHCRC, 1100 Fairview Avenue North, Seattle, WA 98109-1024