Conditions: Neoplasm Metastasis
Keywords: Engraftment Peripheral Blood Stem Cells Non-Myeloablative Bone Marrow Transplantation Metastatic Solid Tumors Graft-Versus-Host Disease Cancer Tumor
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 42
Healthy volunteers: no

Brief summary:
The goal of this research study is to identify other types of cancer (malignant neoplasms) that may be treatable with stem cell transplantation (allogenic peripheral blood stem cell transplantation.

Patients with a variety of different types of cancerous tumors that have spread (metastasized) and whose conditions have not improved with stand therapy, will be eligible to participate. Those patients selected to participate in the study will undergo a procedure known as a "mini-transplant". The mini-transplant is a transplantation of stem-cells collected from a sibling (brother or sister) of the patient. Unlike traditional bone marrow transplants, the mini-transplant does not require intense chemotherapy or radiation therapy. Because of this, patients experience fewer and less severe side effects.

This study is open to patients diagnosed with a variety of metastatic solid tumors including esophageal, gastric (stomach), colon, rectal, liver tumors (hepatoma), cancer of the biliary system (cholangiocarcinoma), cancer of the pancreas, lung, breast, prostate, bone (sarcoma), adrenal basal cell, bladder, and adenocarcinomas of unk primary origin.

Detailed description:
The main objective of this study is to identify metastatic neoplasms, which may be susceptible to the GVT effect. We will treat patients with progressive metastatic solid tumors refractory to standard therapy with a non-myeloablative allogeneic PBSC transplant from a family donor. A GVT effect from immunocompetent donor immune cells could extend life expectancy and possibly cure such patients.

Eligible patients will be treated with an allogeneic peripheral blood stem cell transplant from an HLA identical or single HLA antigen-mismatched family donor, using an intensive immunosuppressive regimen without myeloablation ("mini-transplant") in an attempt to decrease the transplant related toxicities while preserving the anti-malignancy and/or anti-host marrow effect of the graft. The low intensity non-myeloablative conditioning regimen should provide adequate immunosuppression to allow stem cell and lymphocyte engraftment. A T-cell replete, donor-derived, granulocyte colony stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSC) will be used to establish hematopoietic and lymphoid reconstitution. We will infuse lymphocytes in patients with \<100% donor T-cell chimerism or with evidence of tumor progression in an attempt to prevent graft rejection and enhance a graft-versus-malignancy effect, respectively.

This trial is open to several different types of metastatic, treatment-refractory, solid neoplasms, breast, cholangiocarcinoma, small intestine/colon/rectal adenocarcinoma, esophageal/gastric, hepatocellular, pancreatic, prostate, and bony/soft tissue sarcomas. The trial design permits up to 10 patients with a specific tumor type to be enrolled to screen for anti-tumor effects. A single complete response in a specific tumor type is an indication to exclude further patients with that diagnosis from the study. Subsequently, a new protocol which focuses on further defining a GVT effect in that disease category will be instituted.

Arms:
arm 1: Cyclosporin beginning day -4 then stem cells given on Day 0 followed by intravenous Methotrexate on days +1, +3, and +6.

Interventions: intervention 1: methotrexate intervention 2: Cyclosporin
Intervention descriptions: intervention 1: Cyclosporin beginning day -4 and intravenous Methotrexate on days +1, +3, and +6 will be given intervention 2: Cyclosporin beginning day -4 and intravenous Methotrexate on days +1, +3, and +6 will be given

Protocol document:
Prot_SAP_000.pdf: 
99-H-0064
Richard W. Childs, M.D.
July 23, 2019
1
CLINICAL RESEARCH PROJECT
Protocol #99-H-0064
Closed to new accrual and follow up care June 2008
Date:
 July 23, 2019
Title: Exploratory study of non-myeloablative allogeneic stem cell transplantation and donor lymphocyte 
infusions for metastatic neoplasms refractory to standard therapy.
Other identifying words: Peripheral blood stem cells, non-myeloablative bone marrow transplantation, 
metastatic solid tumors, engraftment, graft-versus-host disease, graft-versus leukemia, graft-versus-tumor, 
cyclophosphamide, fludarabine, donor apheresis.
Principal Investigator:  Richard Childs, M.D., CMTB, NHLBI (E)
594-8008
Bldg 10, CRC 3-5332
Medically Responsible Investigator:  
*Richard Childs, M.D., CMTB, NHLBI (E)
594-8008
Bldg 10, CRC 3-5332
Associate Investigators:
*Ramaprasad Srinivasan, M.D., UOB, NCI (E)
   
594-8007
Bldg 10, CRC 3-5272 
*Neal S. Young, M.D., HB, NHLBI (E)
   
496-5093
Bldg 10, CRC 3-5142
Lisa Cook, RN, Research Nurse/BMT Coordinator, CMTB, NHLBI/OCD (E) 402-5609 Bldg 10, CRC 3-3485
Nancy Geller, PhD, Director, Office of Biostatistics, NHLBI (E)  
 435-0434 
Rockledge 2, 8210 
Collaborators: 
Li Zhang, MD, Ph.D., Toronto General Research Institute (TGRI) at University Health Network
* Investigators who may obtain informed consent
Subjects of Study:  
Number 
Sex 
Age range 
up to 86
either  
Recipients: 10-80 (both inclusive) Donors: 10-80       
Project involves ionizing radiation? 
No
Off site project? 
No
Multi-institutional project? 
No
DSMB Involvement?
Yes


99-H-0064
Richard W. Childs, M.D.
July 23, 2019
2
PRECIS
The main objective of this study is to identify metastatic neoplasms, which may be susceptible to the 
graft-versus-tumor (GVT) effect. We will treat patients with progressive metastatic solid tumors refractory to 
standard therapy with a non-myeloablative allogeneic peripheral blood stem cell (PBSC) transplant from a family 
donor. A GVT effect from immunocompetent donor immune cells could extend life expectancy and possibly cure 
such patients.
Eligible subjects will be treated with an allogeneic peripheral blood stem cell transplant from an HLA identical or 
single HLA antigen-mismatched family donor, using an intensive immunosuppressive regimen without 
myeloablation ("mini-transplant") in an attempt to decrease the transplant related toxicities while preserving the 
anti-malignancy and/or anti-host marrow effect of the graft. The low intensity non-myeloablative conditioning 
regimen should provide adequate immunosuppression to allow stem cell and lymphocyte engraftment.  A T -cell 
replete, donor-derived, granulocyte colony stimulating factor (G-CSF)-mobilized peripheral blood stem cells 
(PBSC) will be used to establish hematopoietic and lymphoid reconstitution. We will infuse lymphocytes in 
patients with <100% donor T-cell chimerism or with evidence of tumor progression in an attempt to prevent graft 
rejection and enhance a graft-versus-malignancy effect, respectively.
This trial is open to several different types of metastatic, treatment-refractory, solid neoplasms (breast, 
cholangiocarcinoma, small intestine/colon/rectal adenocarcinoma, esophageal/gastric, hepatocellular, pancreatic, 
prostate, and bony/soft tissue sarcomas).  The trial design permits up to 10 subjects with a specific tumor type to 
be enrolled to screen for anti-tumor effects.  A single complete response in a specific tumor type is an indication 
to exclude further subjects with that diagnosis from the study.  Subsequently, a new protocol which focuses on 
further defining a GVT effect in that disease category will be instituted.


99-H-0064
Richard W. Childs, M.D.
July 23, 2019
3
TABLE OF CONTENTS
1.0 
OBJECTIVES ..................................................................................................................................................................4
2.0 
BACKGROUND .............................................................................................................................................................4
3.0 
SCIENTIFIC AND CLINICAL JUSTIFICATION.........................................................................................................6
4.0 
STUDY DESIGN.............................................................................................................................................................7
5.0 
ELIGIBILITY ASSESSMENT........................................................................................................................................7
6.0
CLINICAL EVALUATION OF THE SUBJECT ...........................................................................................................9
7.0
TREATMENT PLAN....................................................................................................................................................11
8.0
DONOR EVALUATION AND PLAN .........................................................................................................................13
9.0 
MANAGEMENT OF SUBJECT COMPLICATIONS .................................................................................................15
10.0 
STUDY MODIFICATIONS..........................................................................................................................................15
11.0
ANCILLARY LABORATORY RESEARCH STUDIES.............................................................................................16
12.0 
RESPONSE CRITERIA ................................................................................................................................................17
13.0  
BIOSTATISTICAL CONSIDERATIONS ...............................................................................................................18
14.0 
DATA  SAFETY AND MONITORING.......................................................................................................................21
15.0 
HUMAN SUBJECT PROTECTION.............................................................................................................................24
16.0 
PHARMACEUTICALS.................................................................................................................................................30
17.0 
REFERENCES...............................................................................................................................................................33
APPENDIX A:  PREPARATIVE REGIMEN............................................................................................................................35
APPENDIX B: ATG DESENSITIZATION...............................................................................................................................36
APPENDIX C: SERIOUS ADVERSE EVENT REPORT FORM ............................................................................................37
APPENDIX D:   APPROVED LIST OF NHLBI LABORATORY STUDIES .........................................................................39
SEE ALSO 
HB PBSCT SUPPORTIVE CARE GUIDELINES (Supportive Care Guidelines)
Marrow stem cell transplant section
Cellular and Molecular Therapeutics Branch, NHLBI
IRB reviewed 10/21/2003 and 6/6/2006


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Richard W. Childs, M.D.
July 23, 2019
4
1.0 
OBJECTIVES
1.1 
To identify an anti-tumor effect of allogeneic PBSC transplantation by induction of a graft-versus-tumor 
(GVT) effect in subjects with a diversity of metastatic solid tumors, which are refractory to standard 
therapy.
1.2 
To evaluate engraftment by bone marrow and blood chimerism analysis.
1.3 
To evaluate the effects of donor lymphocyte infusion (DLI) and cyclosporine A (CSA) withdrawal on 
tumor regression.
2.0 
BACKGROUND
2.1 
Introduction
Despite a number of advances in the treatment of solid tumors, the majority of patients who develop 
metastatic disease die from their underlying cancer. With the exclusion of testicular cancer, germ cell 
tumors, and some lymphomas, solid tumors which have progressed to overt metastatic disease remain 
largely incurable. Although a number of new and novel therapies have been developed to treat patients 
with advanced metastatic diseases, none to date have been shown to be curative and there is no good 
evidence that any of these therapies prolong survival.
There exists overwhelming evidence, both in-vitro and in-vivo, that metastatic solid tumors may be 
susceptible to immune-mediated control (1,2,3). The best support for this immune response comes from 
the demonstration of an autologous anti-tumor response in melanoma and renal cell carcinoma (RCC) 
(11). Tumor-infiltrating lymphocytes (TIL) can be found in a majority of metastatic melanoma lesions 
(27). These TILs can be expanded in-vitro from melanoma tissue and have been shown to consist of a 
predominant population of cytotoxic lymphocytes (CD8+). Expanded TIL populations have potent 
MHC-restricted and non-restricted cytolytic activity against autologous tumor while sparing normal host 
tissue (30).  Transfer of cultured TIL along with IL-2 has resulted in tumor regression in 30%-40% of 
patients with metastatic melanoma (11). A large body of literature exists supporting the in-vitro 
susceptibility of other metastatic solid tumors to T-lymphocytes. TIL, analogous to those seen in 
metastatic melanoma, have been isolated from a diversity of other metastatic solid tumors and have been 
shown in-vitro to be capable of inducing tumor-specific lysis (4,5,6,7,8,9). These findings have led to a 
number of novel clinical trials aimed at generating autologous tumor-specific T -cells as a form of cancer 
immunotherapy. Indeed, a number of cancer centers have shifted away from standard chemotherapeutic 
trials to exploring these new immunotherapeutic approaches utilizing T-cells as the principal effectors.
It is now well established that chronic myelogenous leukemia (CML), myelodysplastic syndromes 
(MDS), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), Hodgkin's and 
non-Hodgkin's lymphomas and multiple myeloma can be cured by allogeneic bone marrow 
transplantation (BMT) (22,23).  This curative effect has been ascribed to the use of myeloablative 
chemo-radiotherapy and the antileukemic effect of the transplant (the graft-versus-leukemia, GVL, effect) 
(24).  The assumption that the intensive myeloablative preparative regimen is essential for the cure of the 
malignancy went unchallenged until the demonstration by Kolb et al, subsequently confirmed by 
numerous investigators, that donor lymphocytes alone exert a powerful antileukemic effect in the context 
of patients relapsing with myeloid leukemias after BMT (20,21,25,26).  This observation has had 
important implications: first, it now appears that some hematological malignancies can be cured with 
preparative regimens of lower intensity, designed to immunosuppress the recipient to allow lymphocyte 
and stem cell engraftment without major cytoreduction of the malignancy by myeloablation (17); second, 


99-H-0064
Richard W. Childs, M.D.
July 23, 2019
5
because low-intensity preparative regimens have decreased toxicity, allotransplantation is now being 
formed in patients who were previously not considered candidates for standard "myeloablative" 
procedures due to prohibitive procedure-related mortality (15). Several groups, including our own, have 
begun to investigate this approach to expand the applicability of allotransplantation to other patients 
(14,15,17).  Our own experience at the NIH includes over 100 patients and has also been favorable, with a 
low incidence of treatment-related complications and only one treatment-related death in a cohort at high 
risk for mortality with standard allotransplantation (15).  The decreased toxicity of non-myeloablative 
regimens now makes allogeneic PBSC transplantation appropriate as experimental therapy for patients 
with metastatic solid tumors refractory to standard therapy who have no other potentially curative 
treatment option.
There are a number of logical reasons to evaluate allogeneic PBSC transplantation in patients with 
metastatic solid tumors.  The allograft provides the opportunity to attack the tumor with the donor 
immune system, which is intact and unsuppressed by chemotherapy and has yet to develop tolerance to 
the malignancy. In addition, patients with growing tumors may lack the T-cell repertoire necessary to 
recognize and destroy the tumor, a situation analogous to what is sometimes seen in patients with 
leukemia.  Finally, over and above the presentation of known tumor-specific antigens, the malignant cells 
may present a diversity of allo-antigens to the donor immune system, which have the potential for 
inducing powerful immunodominant responses.  
Recently, we and others explored the possibility of generating anti-tumor effects with allogeneic T-cells 
following HLA matched peripheral blood stem cell transplantation.  Preliminary data indicate that 
reproducible GVT effects can be generated in patients with RCC, malignant melanoma, and breast 
carcinoma (1,2,15).  In addition, anecdotal reports exist of GVT effects in a number of other tumors 
including gastric carcinoma, neuroblastoma, and hepatocellular carcinoma.  
The melanoma and RCC non-myeloablative allogeneic PBSC transplant trials continue to accrue patients 
and show clear evidence that GVT effects can be generated in these tumors.  There is reason, therefore, to 
believe that many other tumors may be susceptible to this approach.
The focus of this study is to identify other solid tumors, which may respond to this treatment approach. 
We now plan to evaluate low-intensity blood stem cell transplants in all patients with incurable metastatic 
solid tumors refractory to standard therapy. The design of the study incorporates the following features:
1)
This is a phase II trial to determine the therapeutic potential of a new transplant approach (GVT 
effect) on incurable metastatic neoplasms which are refractory to conventional therapy.
2)
Patients to be studied:
The original protocol included 15 groups of neoplasms (adrenal, basal cell, breast, transitional cell 
carcinoma of the bladder or uroepithelium, cholangiocarcinoma, small intestine/colon/rectal 
adenocarcinoma, esophageal/gastric, hepatocellular, ovarian, pancreatic, prostate, bony/soft tissue 
sarcomas, small cell lung cancer, non small cell lung cancer, and adenocarcinomas of unknown 
primary origin).
December 2006, the protocol was amended to remove adrenal, basal cell, transitional cell carcinoma of 
the bladder or uroepithelium, ovarian, small cell lung cancer, non small cell lung cancer and 
adenocarcinomas of unknown primary origin due to low accrual issues.  In the  amended protocol 
patients with the following 8 groups of metastatic solid tumors (breast, cholangiocarcinoma, small 
intestine/colon/rectal adenocarcinoma, esophageal/gastric, hepatocellular, pancreatic, prostate, 


99-H-0064
Richard W. Childs, M.D.
July 23, 2019
6
bony/soft tissue sarcomas) refractory to standard therapy, or in which no treatment modality has been 
shown to be curative or to prolong survival will be eligible for study. 
Many of these patients would have received prior dose-intensive chemotherapy and/or radiotherapy and 
would be at increased risk for regimen-related toxicity and mortality with a standard myeloablative 
transplant.  Because the transplant does not incorporate intensive chemotherapy and relies instead on a 
GVT and a graft-versus-marrow effects, which may take months to be effective, only patients with 
expected survivals of 3 months or greater will be eligible for study.  Currently there exists no curative 
option for such patients.
3)
Immunosuppression without myeloablation: Patients will receive chemotherapy sufficient to allow 
donor lympho-hematopoietic engraftment without complete marrow ablation. If the graft is rejected, 
the patient will reconstitute autologous marrow function to pre-transplantation levels.  We will use a 
combination of two or three agents with known immunosuppressive activity in marrow 
transplantation: cyclophosphamide, which produces transient pancytopenia; fludarabine, which 
causes less cytopenia but equivalent immunosuppression; and in patients at increased risk for graft 
rejection (section 7.5.1) antithymocyte globulin (ATG), which is potently immunosuppressive.
4)
Peripheral blood hematopoietic progenitor cell (PBPC) transplant: An unmanipulated peripheral 
blood stem cell collection from a G-CSF stimulated HLA-matched family donor should improve the 
chance of engraftment because of the high stem cell doses infused (>5 x 106/kg CD34+ cells). In 
addition, the allograft will be T -cell replete, which should improve donor engraftment and facilitate 
GVT effects. Patients will receive cyclosporine A (CSA) and methotrexate (MTX) as graft versus 
host disease (GVHD) prophylaxis post-transplant.  Patients with mixed T -cell chimerism at day 30 
will be tapered off CSA over 2 weeks, and patients with 100% donor T -cell chimerism will begin a 
CSA taper at day 60 (through day 100) if no GVHD is present.  Patients with evidence of disease 
progression without grade > II GVHD will have CSA discontinued as early as day 30 regardless of 
chimerism status.
5)
Donor lymphocyte infusion (DLI): Patients who show progressive disease off of CSA and in the absence 
of grade > II GVHD, or who are at risk for graft failure due to incomplete donor T-cell engraftment will 
receive one or more DLI as outlined in section 7.11.
3.0 
SCIENTIFIC AND CLINICAL JUSTIFICATION
This pilot study will evaluate whether allogeneic PBPC transplants have any useful anti-tumor effects in a 
diverse group of metastatic solid tumors, which are progressive and refractory to standard therapy. We 
have in-vitro and in-vivo evidence that GVT effects can be generated using this transplant approach in 
metastatic breast cancer, melanoma and RCC (1,2,15,27). Many other solid tumors have characteristics 
that would make them susceptible to this same GVT effect, including class I MHC expression necessary 
for the presentation of minor histocompatibility antigens and aberrantly expressed mutated peptides 
(29,30).  This experimental approach is ethically justifiable since we are targeting patients with 
progressive metastatic neoplasms in which no known standard therapy is potentially curative or definitely 
capable of extending life expectancy. These patients have no prospect of prolonged survival with any 
standard treatment approach, and are therefore, candidates for this type of investigational therapy.  
There is sufficient experimental evidence and clinical data to indicate that the low dose preparative 
regimen is capable of allowing lympho-hematopoietic engraftment with minimal toxicity. 
Cyclophosphamide alone permits engraftment in patients with severe aplastic anemia (9), and fludarabine 
has been used either alone or in conjunction with Atgam and other agents to achieve engraftment (10).


99-H-0064
Richard W. Childs, M.D.
July 23, 2019
7
Early data indicate that non-myeloablative stem cell transplants are associated with an excellent transplant 
outcome in patients with hematological malignancies.  The presence of high stem cell doses and donor 
lymphocytes is known to facilitate engraftment (18,19). Preliminary studies, including our own 
experience with PBPC transplants and low intensity preparative regimens, indicate that transplant-related 
mortality (TRM) and severe acute GVHD is uncommon when matched family donors are used.
4.0 
STUDY DESIGN
The subject will receive a preparative regimen of cyclophosphamide 60 mg/kg/d IV x 2 days, followed by 
fludarabine 25 mg/m2/d iv over 30 minutes x 5 days (+/- Atgam 40 mg/kg IV x 4 days -5 to -2: section   
7.5.1-7.5.2), followed by a PBPC graft targeted to deliver >5 x 106 CD34+ cells /kg. CSA and MTX for 
GVHD prophylaxis will be used in all subjects. An HLA-matched family donor will receive G-CSF SC 
for 6 days with apheresis collections of PBPC on days 5 and 6 (and day 7 if required).
CSA beginning day -4 and intravenous MTX on days +1, +3, and +6 will be given.  Subjects with mixed 
T-cell chimerism on day 30 will begin a CSA taper. Subjects with 100% donor T-cell chimerism by day 
30 will be tapered off CSA from days 60 through 100 (25% reduction in dose every 10 days-off by day 
100).  CSA will not be tapered in any subjects with grade > II acute GVHD regardless of chimerism 
results.   In addition, subjects with evidence of disease progression without grade > II GVHD will have 
CSA discontinued regardless of chimerism results.
On days, +30, +60 and +100, disease status will be assessed by physical exam and by computerized 
tomography (CT) scans, and subjects with disease progression without grade > II GVHD will have CSA 
discontinued and may receive DLI as outlined in section 7.11.
In addition, on days +30, +60 and +100, the chimeric status of subjects will be assessed by minisatellite 
analysis of the peripheral blood and sometimes bone marrow.  Subjects demonstrating less than 100% 
donor T-cell chimerism on day 30 will be tapered off CSA over a 12 day period and will be re-evaluated.  
If T-cell chimerism remains less than 100% donor, infusions of previously collected and cryopreserved 
donor lymphocytes will be given in an attempt to prevent graft rejection in the absence of grade > II 
GVHD.
5.0 
ELIGIBILITY ASSESSMENT
5.1 
Inclusion criteria -patients
5.1.1
Patients with metastatic solid tumors breast, cholangiocarcinoma, small intestine/colon/rectal 
adenocarcinoma, esophageal/gastric, hepatocellular, pancreatic, prostate, bony/soft tissue 
sarcomas) which are histologically confirmed, progressive and incurable.
Due to low accrual, effective 12/19/2006,  patients with adrenal, basal cell, transitional cell 
carcinoma of the bladder or uroepithelium, ovarian, small cell lung cancer, non small cell lung 
cancer, and adenocarcinomas of unknown primary origin are no longer eligible for the trial (see 
also section 13.1).
5.1.2
Age > 10 to < 80.


99-H-0064
Richard W. Childs, M.D.
July 23, 2019
8
5.1.3 
No known standard therapy for the patient's disease that is potentially curative or definitely 
capable of extending life expectancy. 
5.1.4 
Metastatic disease, which is bi-dimensionally evaluable radiographically.
5.1.5 
No prior treatment for neoplasm within 30 days.
5.1.6
Ability to comprehend the investigational nature of the study and provide informed consent.  
5.1.7 
Availability of HLA identical or single HLA-locus mismatched family donor.
5.1.8 
Willingness and availability to return to the NIH for scheduled follow ups.
5.2
Exclusion criteria for the patient: any of the following
5.2.1 
Pregnant or lactating.
5.2.2 
Age <10 or >80 years.
5.2.3
ECOG performance status of 3 or more, (see Supportive Care Guidelines).
5.2.4
Psychiatric disorder or mental deficiency severe as to make compliance with the BMT treatment 
unlikely, and making informed consent impossible.
5.2.5
Major anticipated illness or organ failure incompatible with survival from PBSC transplant.
5.2.6 
DLCO: < 40% predicted. 
5.2.7 
Left ventricular ejection fraction: < 30%.
5.2.8 
Serum creatinine > 2.5mg/dl or creatinine clearance < 50 cc/min by 24 hr urine collection.
5.2.9
Serum bilirubin >4 mg/dl. 
5.2.10 Transaminases > 5x upper limit of normal.
5.2.11 Oral intake < 1,200 calories/day.
5.2.12 Recent weight loss of >10% of actual body weight.
5.2.13 Life expectancy < 3 months.
5.2.14 Therapy for malignancy within 4 weeks of beginning protocol.
5.2.15 CNS metastatic disease associated with intracranial bleeding, uncontrolled seizure disorder or 
significant intracranial mass effect.
5.2.16 Other malignant diseases liable to relapse or progress within 5 years.
5.2.17 Uncontrolled infection.


99-H-0064
Richard W. Childs, M.D.
July 23, 2019
9
5.3
Inclusion criteria - Donor
5.3.1
HLA identical or single HLA-locus mismatched family donor
5.3.2
Age > 10 up to 80 years old
5.3.3
Ability to comprehend the investigational nature of the study and provide informed consent.
5.4 
Exclusion criteria for the donor: any of the following
5.4.1 
Pregnant or lactating
5.4.2
Donor unfit to receive G-CSF and undergo apheresis. (Uncontrolled hypertension, history of 
congestive heart failure or unstable angina, thrombocytopenia)
5.4.3
Age <10  or >80 years
5.4.4
HIV positive.  Donors who are positive for HBV, HCV or HTLV-I may be used at the discretion 
of the investigator following counseling and approval from the recipient.
6.0
CLINICAL EVALUATION OF THE SUBJECT
Human Biologic materials collected as follows are for the clinical evaluation and management of the 
subject.  The biopsy of the primary neoplasm and metastatic disease, and the bone marrow aspiration and 
biopsy will be read by a pathologist. Samples will be ordered and tracked through the CRIS Screens.  Should 
a CRIS screen not be available, the NIH form 2803-1 will be completed and will accompany the specimen 
and be filed in the medical record.
6.1
Pre-Study Evaluation
6.1.1 
Complete history and physical exam.
6.1.2 
Biopsy confirmation of primary neoplasm and metastatic disease.  Histology slides will be kept 
on file in the pathology department of the clinical center while the subject is being treated on 
protocol.  
6.1.3 
CT scans of the chest, abdomen and pelvis, MRI scan of the brain, as well as any other 
appropriate radiographic or nuclear medicine studies to document evaluable disease within 30 
days of induction of the preparative regimen. Subjects with tumors involving the extremities 
including soft tissue and bony sarcomas will have baseline MRI and/or CT studies of involved 
regions.
6.1.4 
Medium resolution molecular HLA- A, B, DR typing.
6.1.5 
Bone marrow aspiration and biopsy with chromosome analysis, PCR, and flow cytometry as 
appropriate to stage and classify underlying disorder.
6.1.6 
Antibody screen for HBV, HCV, HIV, HTLV-I/II, CMV, EBV, toxoplasma, syphilis. PPD test 
for subjects from areas where tuberculosis is prevalent.


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10
6.1.7 
Coagulation screen, CBC with differential.
6.1.8 
Acute care panel, hepatic panel, and mineral panel.
6.1.9. Chest radiograph, pulmonary function testing: vital capacity FEV-1, DLCO.
6.1.10  Sinus CT scan.
6.1.11  Cardiac function: EKG, MUGA scan, baseline 2-D echocardiogram.
6.1.12  All subjects age 50 or greater, or age 40 or over, with one of the following risk factors: a history of 
high blood pressure, increased cholesterol, smoking, or diabetes or family history of coronary 
disease, will have a baseline cardiac workup, which will include:
1. Stress nuclear perfusion imagery.
2.  Cardiac consultation in subjects age 50 and over.
6.1.13  Nutritional assessment.
6.1.14  Dental review.
6.1.15  Social worker interview.
6.1.16  Ophthalmology consultation.
6.1.17  Interview with members of primary care team and visit to unit.
6.1.18  Consent form signed.
6.1.19  Durable power of attorney form completed.
6.1.20 For subjects with accessible tumors, biopsies will be performed to generate tumor lines for 
in-vitro analysis of donor derived GVT effects
6.2 
In Patient Monitoring
Once daily: CBC with differential, acute care, hepatic, and mineral panels, direct bilirubin, temperature, 
pulse, blood pressure, respiratory rate, weight, caloric intake, abdominal girth.
Twice weekly: reticulocyte, pre-albumin, coagulation screen.
Weekly: CMV surveillance; C-reactive protein; Stool for C difficile when appropriate; Drug levels when 
appropriate (e.g., gentamicin, vancomycin, cyclosporine).
6.3 
Follow Up to day 100: Out-patient
At least weekly: CBC, acute care panel, hepatic and mineral screen, LDH, coagulation screen, 
Temperature, pulse, blood pressure, respiratory rate, weight, C-reactive protein, CMV surveillance. A 
complete physical exam will be repeated weekly. 


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Peripheral blood will be drawn weekly until day +60 and in some subjects bone marrow aspirates will be 
taken on days +15, +30, +45, +60 and +100 to assess for donor-host chimerism in the lymphoid and 
myeloid cell lines.  A sample of peripheral blood will also be obtained for chimerism analysis at the time 
of neutrophil recovery (ANC>500).
Radiographic follow up: CT scans and other radiographic studies of areas of evaluable disease will be 
repeated on days +30, +60, +100. CT scans will then be performed every 3 months 
Clinic visits: Day +30 to day +100 up to 2x/week or at subject's request. 
6.4  
Follow Up beyond day 100
At 6, 12, 18, 24, 36, 48 and 60 months (+/- 1 month): CBC, acute care panel, hepatic and mineral screen, 
chest radiograph, bone marrow aspirate (if applicable) and blood sample for chimerism studies and 
karyotype, pulmonary function tests. 
Radiographic follow up: CT scans and other radiographic studies of areas of evaluable disease will be 
repeated every 2 months until 1 year post-transplant. CT scans will then be performed every 3 months 
until 3 years post-transplant; then every 6 months until 5 years post transplant.  
Clinic visits: months 4, 6, 8, 10, 12 then every 3 months thereafter until 3 years then every 6 months until 
5 years. After 5 years follow up visits are not mandatory but yearly communication with the subject and 
the referring physician is continued.
7.0
TREATMENT PLAN
7.1
Pre transplant treatment
Subjects with active disease may receive appropriate chemotherapy or radiotherapy according to standard 
indications to control disease prior to the preparative regimen.
7.2
Apheresis of subject
One collection of 1010 leukocytes by a 10-liter leukapheresis before transplant preparative regimen begins 
and at the time of disease response or acute GVHD for cryopreservation of lymphocytes to be used for 
laboratory investigation of the graft-versus-malignancy or graft-versus-marrow effect or GVHD effectors.
7.3 
Central venous line placement.
A triple lumen Hickman catheter will be placed by an interventional radiologist or a surgeon.
7.4
Infection Prophylaxis and Fever Regimen (see Supportive Care Guidelines)
7.5
Preparative regimen (Appendix A)
7.5.1 
Antithymocyte Globulin (ATgam)
Subjects will receive one of two preparative regimens as described below. Subjects at higher risk 
for graft rejection with this non-myeloablative approach (including heavily transfused subjects or 
subjects who have received donor directed blood products, and single HLA locus mismatched 
subjects) will have Atgam added to the preparative regimen.
7.5.2 
Dose Levels
1 ) 
Cyclophosphamide 60 mg/kg/d IV day -7, -6
Fludarabine 25 mg/m2/d IV day -5, -4, -3, -2, -1


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2) 
Subjects at increased risk for graft rejection-defined above, 7.5.1
Cyclophosphamide 60 mg/kg/d IV day -7, -6
Fludarabine 25 mg/m2/d IV day -5, -4, -3, -2, -1
Antithymocyte globulin 40mg/kg IV days -5,-4,-3,-2
(Methylprednisolone 1 mg/kg day -5 to + 4 then rapid taper)
7.6
Peripheral blood progenitor cell transplant. 
The target for progenitor cells is >5 x 106 CD 34/kg.
Minimum dose for transplant is 2 x 106/kg
7.7 
GVHD  prophylaxis 
All subjects will receive CSA and MTX as GVHD prophylaxis.  CSA (1.5 mg/kg/dose IV BID) will 
begin on day -4.  Dosing will be based on actual body weight unless the subject is “obese” (BMI > 35) 
when practical weight will be used.  Switch to the equivalent oral CsA dose divided bid on day 14 or 
when able to tolerate PO.   MTX will be given intravenously on days +1, +3 and +6.  
Subjects with <100% donor T-cell chimerism on day 30 will begin a 12 day CSA taper (i.e. 25% 
reduction in dose every 3 days). Subjects with 100% donor T-cell chimerism by day 30 will be tapered off 
CSA from days 60 through 100 (25% reduction in dose every 10 days-off by day 100).  CSA will not be 
tapered in any subjects with grade > II acute GVHD regardless of chimerism results.  In addition, subjects 
with evidence of disease progression without grade > II acute GVHD will have CSA tapered regardless of 
chimerism results.
7.8 
Transfusion Support (see Supportive Care Guidelines)
Filtered and irradiated blood products. CMV negative recipients of CMV negative or positive marrow 
will receive CMV negative blood products.
7.9 
Nutrition
Parenteral nutrition will be instituted if daily caloric intake <1000 KCal/day in adults > 18 yrs. Subjects 
10 to 18 years will start parenteral nutrition if they are unable to meet at least half their daily caloric needs 
by mouth (i.e. 50 KCal/kg/day).
7.10 
Hospital Discharge
Subject will be in the hospital for about 4 weeks and will be discharged when the following criteria are 
fulfilled:

Subject afebrile, positive weight balance; no parenteral feeding required.

Platelet transfusion requirement absent or less than 2x weekly.

Subject or family able to care for Hickman line.
7.11 
Donor lymphocyte Infusions (DLI)
Subjects with disease progression or donor T-cell chimerism of <100% following CSA tapering without 
grade > II GVHD will receive donor lymphocyte infusions (previously collected and cyopreserved) based 
on the following algorithm:


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13
Mixed chimerism without disease progression or active GVHD
 
Monthly increments of DLI 5 x 106, 1 x 107, 5 x 107 /CD3/kg.
Disease Progression (independent of chimerism status) without active GVHD 
DLI 1 x 107 and repeat doses depending on response and acute GVHD.
8.0
DONOR EVALUATION AND PLAN
Human Biologic materials collected as follows are for the clinical evaluation and management of the 
donor. Samples will be ordered and tracked through the CRIS Screens.  Should a CRIS screen not be 
available, the NIH form 2803-1 will be completed and will accompany the specimen and be filed in the 
medical record.
8.1 
Pre-study consult and evaluation

HLA-A,-B,-DR typing.

Confirm HLA identity of donor with recipient (subject).

History and physical examination.

Hepatitis B, C, HIV, HTLV-I/II, CMV antibodies, RPR. 

CBC, coagulation screen, acute care, hepatic, mineral screen.

Fit to donate: Orientation -visit to Department of Transfusion Medicine, Inspection of veins to 
determine the need for a central line for apheresis.
8.2 
Pre-consent evaluation and concurrent care of minor donors (donors less than age 18 only) 
For donors less than age 18, a social worker and mental health specialist (psychologist or psychiatrist) 
will meet with the minor donor prior to the assent process to determine willingness to participate.
Donors who are less than 18 years of age will see a pediatric provider (pediatrician, pediatric nurse 
practitioner or pediatric physician’s assistant) who is separate from the transplant team.  This practitioner 
will serve as the donor’s health care provider and advocate during the minor’s participation on the clinical 
trial.
8.3 
Donor lymphocyte collection
Prior to undergoing G-CSF mobilization, the donor may undergo a ten liter apheresis for cryopreservation 
of donor lymphocytes to be infused as necessary after transplantation (section 7.11).  Aliquots of 5 x 106 
CD3/kg, 1 x 107 CD3/kg and 5 x107 CD3/kg will be cryopreserved.  Any remaining cells in excess of that 
required for cryopreservation will be either discarded or used for GVT studies in vitro (section 11.1).
8.4
Donor mobilization with G-CSF      
After medical evaluation and clearance for suitability as an allogeneic donor by the BMT service in 
consultation with DTM, the donor will undergo mobilization with filgrastim (G-CSF) as an outpatient. 
Filgrastim (G-CSF) will be administered based on body weight (see below) for 5-6 days, subcutaneously.  
G-CSF will be administered according to a vial based algorithm to reduce wastage, improve donor 
compliance, and optimize CD34 yields. The doses for days 1-4 may be given at any time of day, but the 
doses for days 5 and 6 must be given very early in the morning, prior to apheresis.  
Donor Wt           Total filgrastim (G-CSF) Dose (range)
 


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14
38 - 48 kg           600 mcg (12.5 to 15.8 mcg/kg)
49 - 56 kg           780 mcg (13.9 to 15.9 mcg/kg)
57 - 60 kg           900 mcg (15.0 to 15.8 mcg/kg)
61 - 67 kg           960 mcg (14.3 to 15.7 mcg/kg)
68 - 108 kg         1080 mcg (10.0 to 15.9 mcg/kg)
> 109 kg             1200 mcg (11.0 or less)
The target for progenitor cells is  >5 x 106 CD 34/kg.
  
The target CD34+ cell dose is 3 x 106/kg
Minimum dose for transplant is 2 x 106/kg
8.5
Peripheral blood stem cell collection & processing   
Leukapheresis procedures will be carried out in the Apheresis Unit, Department of Transfusion Medicine 
(DTM). These procedures will be done via a 2-armed approach or by temporary central venous catheter in 
the femoral position, using the Baxter CS3000Plus instrument or the Cobe Spectra. The volume processed 
per apheresis procedure will be determined by DTM medical staff on the day of apheresis, based on peak 
CD34 cell mobilization response to G-CSF and the CD34 cell dose needed, based on kilogram weight of 
recipient.  This will range from 15 to 35 liters processed per day for 2 to 3 days, not to exceed a total of 
75 liters over 3 days.  In pediatric subjects, defined as less than 40 kg, a maximum of 8 blood volumes 
will be processed per day, for up to 2-3 days. The second day apheresis collection will not be required if 
the CD34 dose is 3 x106/kg or more after the first day of collection. 
Anticoagulation will be accomplished with ACD-A, routinely used in normal donors.  If the donor is 
small or intolerant to ACD-A, and the adverse citrate effects cannot be controlled by usual means 
(slowing flow rate, oral or IV calcium), consideration will be given to using heparin anticoagulation.  
Donors will receive divalent cation prophylaxis to prevent citrate toxicity during apheresis, in accordance 
with standard DTM policies.   
If the minimum CD34+ cell dose (2 x 106 CD34/kg) is not achieved with one mobilization and 2 
apheresis collections, then the PI will consider options of a bone marrow collection, a second course of 
mobilization and apheresis, or no further donor collections and taking the subject off protocol.
The PBSC collections will not be manipulated except for plasma removal or red blood cell depletion as 
needed for ABO or other red blood cell antigen incompatibility.  ABO or other erythrocyte 
incompatibility is not an issue because the resulting number of contaminating erythrocytes is well below 1 
ml using this system. The final cell suspension will be prepared for infusion by transferring the cells into 
a 50-150 ml volume of an infusible solution (Plasmalyte A with 1% human serum albumin), suitable for 
intravenous infusion over 15 minutes to 1 hour, depending on clinical circumstances. PBSC will be either 
cryopreserved for later thawing and infusion, or infused as fresh products within 48 hours of collection.
On rare occasions, subjects may develop marrow failure as a consequence of graft-vs-host hematopoiesis 
or from post transplant drug treatment (i.e. ganciclovir).  When such situations arise, subjects may require 
a stem cell “boost”. Donors will undergo a repeat mobilization with G-CSF (as outlined above) followed 
by CD34+ selection of hematopoietic progenitor cells prior to being infused.
8.5  
Bone marrow harvest
An occasional donor may not mobilize PBPC well. If the subject has already received the conditioning 
regimen, and if analysis of the stem cell content of the first or second apheresis products makes it unlikely 


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Richard W. Childs, M.D.
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15
that additional apheresis will achieve the minimal CD34 dose, the donor will undergo a bone marrow 
harvest under general anesthesia the day following the last apheresis.
9.0 
MANAGEMENT OF SUBJECT COMPLICATIONS
The major complications, are cytomegalovirus (CMV) reactivation, acute and chronic GVHD, 
progression of the original disease, donor-recipient ABO incompatibility and pulmonary engraftment 
syndrome. Subjects with these complications will be treated along the following lines:
9.1 
CMV reactivation (See Supportive Care Guidelines)
9.2 
Acute GVHD (See Supportive Care Guidelines)
9.3
Chronic GVHD (See also Supportive Care Guidelines)
Cyclosporine at standard dose (See Pharmaceuticals, section 16.3).
Prednisone 20-60 mg daily according to severity.
Change to alternate day steroid and cyclosporine therapy when response is established.
Non-responding subjects may be treated with other standard of care therapies such as azathioprine, 
sirolimus, tacrolimus, rituximab, mycophenolate, daclizumab or infliximab PUVA, photopheresis, and/or 
thalidomide at the discretion of the attending physician.
9.4 
Graft rejection
This transplant protocol uses a non-myeloablative preparative regimen.  Therefore, auto-recovery is 
anticipated in subjects who fail to engraft.  Subjects who fail to demonstrate donor T-cell engraftment will 
be taken off study and referred back to their primary physician for further therapy.
9.5 
Progression of metastatic disease
See section 7.7 and 7.11. Subjects who develop progressive disease despite CSA withdrawal and DLI, 
and who remain without grade > II acute GVHD may be treated at the discretion of the principal 
investigator (PI) with standard of care treatment options (i.e. interferon-alpha, low dose IL-2 by 
subcutaneous injection, GM-CSF, and/or chemotherapy) with or without stem cell rescue.  
Subjects with disease progression and grade > II acute GVHD will be taken off study and referred back to 
their referring physician for care.
 
9.6
Donor-recipient ABO incompatibility (see PBSCT Supportive Care Guidelines)
9.7
Pulmonary Engraftment Syndrome:  Subjects who develop pulmonary engraftment syndrome (most 
likely 10-14 days post-transplant) will be treated with steroids.  
10.0 
STUDY MODIFICATIONS


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16
Graft failures will be monitored throughout the study.  Specific need for preparative regimen 
modifications based on the incidence of graft failures will be made as discussed in section 7.5
11.0
ANCILLARY LABORATORY RESEARCH STUDIES
11.1
Sample collection 
During the course of participating on this study, blood and tissue as detailed below will be collected for 
correlative laboratory research studies 

from subject before transplant: One apheresis collection of approximately 1010 leukocytes in a 
volume of  200 ml.

from subject in hospital: Weekly lymphocyte subsets

from subject at planned outpatient visits: cytotoxic T-cell precursor frequency (CTLPf) assays 
and lymphocyte subsets up to day +60, then every 2 weeks until day +100 and then at each 
scheduled follow-up.

from donor at time of leukapheresis:  Donor leukocytes obtained from leukapheresis will be used 
for research studies investigating graft-vs-malignancy effects. The subject's tumor cells and 
normal lymphocytes will serve as targets for all subsequent testing of donor-anti host responses. 

Bone Marrow Samples:  An occasional subject will undergo a pre-transplant bone marrow 
aspirate for disease staging purposes (i.e. extensive small cell lung cancer) or for tumor analysis 
when there is a high likelihood of marrow space involvement with metastatic disease. These 
samples will be read by a pathologist. An extra volume (up to 25 ml) of bone marrow aspirate 
will be collected for research studies at the pre-transplant evaluation and for monitoring minimal 
residual disease.    The cells will be used to investigate lymphocyte interactions with bone 
marrow progenitor cell proliferation. No marrow aspirates solely for research purposes are 
planned
11.2
Intended Use
These specimens will not be read by a pathologist or used for diagnostic purposes.   Studies will not be 
used in assessing the primary endpoint but are undertaken for descriptive or exploratory ancillary research 
as detailed below: 
11.2.1
GVT AND GVHD Studies
Blood and serum from subjects and donors will be obtained by leukapheresis to study genomic and 
proteomic pathways involved in mediating GVHD and in vitro donor T-cell responses to the recipient's 
tumor. The cytotoxic T-cell precursor frequency (CTlPf) assay will be used to evaluate the donor immune 
function at various periods after BMT. 


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17
A fine needle aspirate of accessible tumors will be performed pre-transplant to establish tumor cell lines 
for GVT investigations.  Regressing metastatic lesions will be biopsied if accessible and analyzed for the 
presence of alloreacting TILs. The origin of these cells (donor, host, or mixed chimeric) will be evaluated 
by minisatellite probes.
11.2.2 Chimerism studies
PCR of microsatellites of post-transplant peripheral blood T-lymphocytes, B-cells, NK cells and blood 
myeloid cells will be assessed in serial fashion to determine the chimeric status of the subject as a 
function of time. These studies will be repeated after lymphocyte infusions to assess their effect on host 
chimeric status as well. The relationship between degrees of donor host chimerism, GVHD and tumor 
response will be analyzed.
11.2.3   Other research labs as described on the List of Laboratory Studies Using Human Subject and 
Normal Volunteer Samples in the CMTB Approved by the IRB (Appendix D)
We will share the following de-identified samples with Dr. Li Zhang, Senior Scientist, Toronto General 
Research Institute (TGRI) at University Health Network: peripheral blood stem cells (PBSC), peripheral 
blood mononuclear cells (PBC,) coded as TRA-PBMC, GRE-PBMC and WYN-PBMC, and coded 
Epstein-Bar-Virus (EBV) transformed lymphoblastoid cell line (LCL), coded as TRA-EBV-LCL, GRE-
EBV-LCL and WYN-EBV-LCL,  derived from healthy donors enrolled in protocols 97-H-0196 and 99-
H-0064 and 99-H-0050.
11.3
 Tracking, storage and disposition of Samples
Storage:  Research samples will be stored coded in the secure laboratory of the PI. 
Tracking:  Samples will be ordered and tracked through the CRIS Research Screens.  Should a CRIS 
screen not be available, the NIH form 2803-1 will be completed and will accompany the specimen and be 
filed in the medical record.  Specimens will be entered in the NHLBI Biospecimen Inventory System 
(BSI). Samples will not be sent outside NIH without IRB notification and an executed MTA. 
End of study procedures:  Samples from consenting subjects will be stored until they are no longer of 
scientific value or if a subject withdraws consent for their continued use, at which time they will be 
destroyed. 
Loss or destruction of samples:  Should we become aware that a major breech in our plan for tracking 
and storage of samples has occurred, the IRB will be notified.
12.0 
RESPONSE CRITERIA
12.1
Tumor response criteria
12.1.1 Complete response (CR): disappearance of all signs and symptoms of metastatic disease for a 
period of at least one month.


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18
12.1.2 Partial response (PR): a 50% or greater decrease in the sum of the products of the longest 
perpendicular diameters of all measured lesions lasting for a period of at least one month. No 
new metastatic lesions may appear.
12.1.3 Stable disease (SD): tumor measurements not meeting the criteria of CR, PR, or PD.
12.1.4 Progressive disease (PD): increase of 25% or greater in the sum of the products of the longest 
perpendicular diameters of all measured lesions compared to the smallest previous 
measurements, or the development of any new metastatic disease.
13.0  BIOSTATISTICAL CONSIDERATIONS
13.1
 Design and sample size  
This trial is designed to estimate a novel treatment regimen on inducing disease regression and or 
remission in subjects with a number of different metastatic solid tumors, all of which are refractory to 
standard therapy.  It is designed to screen neoplasms of specific origin and histologic subtype which may 
be sensitive to this approach.  We plan to enroll a maximum of 10 subjects with a particular cancer. If any 
subject achieves a complete response (defined Section 12.0) then we will stop accruing subjects in that 
disease category and plan to write a separate phase II protocol which will evaluate this regimen 
specifically in subjects with this type of cancer.  If we enroll 10 subjects in a specific disease category and 
see no responses, we will be 94% confident that the proportion of complete responses in that category is 
no more than 25% and accrual of these subjects will be terminated (32).  
In November 2002, a retrospective analysis for all subjects on HB protocols undergoing non-
myeloablative allogeneic stem cell transplantation following cyclophosphamide/fludarabine based 
conditioning was conducted to determine the impact of adding mycophenolic acid mofetil (MMF) to CSA 
as GVHD prophylaxis.  In this analysis we evaluated the incidence and severity of acute GVHD in our 
first 66 consecutive subjects who received CSA alone (Group I) versus the next 78 consecutive subjects 
who received CSA + MMF (Group II).  We saw no beneficial impact of adding MMF to CSA on the 
incidence of acute grade II-IV, acute grade III-IV GVHD or chronic GVHD.   As a consequence, we 
amended the protocol to replace MMF with the more conventional GVHD prophylactic agent MTX as 
follows:
1) Discontinued the use of MMF as it has clearly failed to reduce the incidence of acute or 
chronic GVHD. 
2) Continued CSA as GVHD prophylaxis using the same dose and scheduling regimen that was 
used for subjects in Group I and Group II. 
3)Added MTX to CSA as GVHD prophylaxis in the same fashion and doses as are currently 
given on protocol 97-H-0196. CSA and MTX have been used together for over a decade and are 
considered "standard agents" for GVHD prophylaxis
In December 2006, in response to low accrual and following recommendation of the DSMB and approval 
of the IRB the types of solid tumors to be included was reduced from 15  (adrenal, basal cell, breast, 
transitional cell carcinoma of the bladder or uroepithelium, cholangiocarcinoma, small 
intestine/colon/rectal adenocarcinoma,  esophageal/gastric, hepatocellular, ovarian, pancreatic, prostate, 
bony/soft tissue sarcomas, small cell lung cancer, non small cell lung cancer, and adenocarcinomas of  


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Richard W. Childs, M.D.
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19
unknown primary origin) to 8 (breast, cholangiocarcinoma, small intestine/colon/rectal adenocarcinoma,  
esophageal/gastric, hepatocellular, pancreatic, prostate, bony/soft tissue sarcomas). Therefore,  the revised 
sample size will be a maximum of 86 subjects (8 disease groups x 10 subjects/group) + 6 subjects in no 
longer accruing disease categories (1 adenocarcinoma, 1 adrenal, 1 basal cell, 1 lung cancer and 2 ovarian 
cancer subjects).
13.2
Parameters to be monitored 
1)
Regression of metastatic disease.
2) 
Degrees of donor-recipient lymphoid and myeloid chimerism by minisatellite probe analysis of 
peripheral blood and marrow cells (marrow will be collected on days +15, +30, +45, +60, & +100).
3) 
Neutrophil recovery (days to neutrophil count of 0.5 x 109/1 and 1.0 x 109/1).
4) 
Platelet recovery (days to platelet count of 50 x 109, days to transfusion independence).
5) 
Red cell recovery (days to transfusion independence).
6) 
Effect of CSA and MMF withdrawal and DLI on donor-host lymphoid and myeloid chimerism 
measured by mini satellite DNA analysis of T-lymphocytes and myeloid cells.
7) 
Incidence and severity of acute and chronic GVHD.
8) 
Non-hematological effects attributable to the preparative regimen.
9) 
Allograft cell dose (CD34+ cell dose, CD3+ cell dose) infused. 
13.3 
Other endpoints
13.3.1 The proportion of graft failures: The proportion of graft failures will continue to be monitored.  If 
there are 4 graft failures among the first 14 subjects, or 5 among the first 28 subjects, or 6 among 
the first 42 subjects, we will reassess our preparative regimen because such results are 
inconsistent with prior beliefs that the graft failure rate is most likely to be 5% and unlikely to be 
greater than 15%.  A beta distribution with parameters 2 and 38 was used as the prior distribution 
for this calculation.
13.3.2 The proportion of subjects with Grade 3 or higher acute (day 100 or less) and chronic GVHD will 
also be estimated with 95% confidence intervals.
13.4
Stopping rule for safety 
We will monitor 200 day transplant related mortality (TRM) in all of the neoplasms combined and wish 
to stop this trial if the 200 day TRM proportion is higher than 30-40%.   We take a beta distribution with 
parameters 4 and 6 and for our prior distribution, consistent with the belief that the proportion of TRM at 
200 days is centered at .4 and is not likely to be greater than .6 (prior probability .05) or less than .2 
(prior probability .03).  We stop if the probability that the 200 day TRM is greater than .4 is at least .9.  
The stopping rule below was assessed by simulation based on the first 80 subjects and results in .7 
overall probability of stopping early if the true 200 day TRM proportion is .5, .2 overall probability of 


99-H-0064
Richard W. Childs, M.D.
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20
stopping early if the true 200 day TRM proportion is .4, and .02 overall probability of stopping early if 
the true 200 day TRM proportion is .3.  The stopping rule is: 
It should be noted that stopping is permitted prior to accrual of a complete cohort of 10 subjects. 
  
13.5 
Withdrawal from study
   
13.5.1 Withdrawal by the subject from the transplant procedure
Subjects and their donors will be given ample opportunity to withdrawal from the study prior to 
admission for transplant. Thereafter, the nature of the procedure does not permit safe withdrawal 
from the protocol.  
The subject and donor have the right at any time to elect not to participate in the research aspects 
of the protocol (donation of blood and bone marrow for non-routine tests).
13.5.2 Withdrawal by the physician from experimental protocol
Subjects who fail to achieve allogeneic marrow engraftment (donor chimerism or mixed 
chimerism) will be removed from study after full autologous marrow recovery is achieved.
Subjects with disease progression after the day +160 restaging who have active grade >II GVHD 
or who have failed to respond to a previous lymphocyte transfusion and/or to interferon-alpha or 
IL-2 may be removed from study.
Subjects with disease progression associated with a significant decline in performance status 
which negates further treatment on protocol (i.e. DLI) will be removed from study.
13.6
Data management
No. subjects
No. TRM
7-10
7
11-20
12
21-30
17
31-40
21
41-50
25
51-60
30
61-70
34


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21
The PI will be responsible for overseeing entry of data into an in-house password protected electronic 
system and ensuring data accuracy, consistency and timeliness. The PI, associate investigators (AI), 
CMTB fellows, research nurses and/or a contracted data manager will assist with the data management 
efforts.   
All human subjects personally identifiable information (PII) as defined in accordance to the Health 
Insurance Portability and Accountability Act,  eligibility and consent verification will  be recorded in 
DIR’s Clinical Data System (CDS) or the Laboratory of Cardiac Energetics (LCE) database.  Primary 
data obtained during the conduct of the protocol will be kept in secure network drives or in approved 
alternative sites that comply with NIH security standards.  Primary and final analyzed data will have 
identifiers so that research data can be attributed to an individual human subject participant, e.g., study-
specific identifying number (SSPIN) generated by CDS or other unique code or minimum PII required for 
subject identification.
End of study procedures:  Data will be stored in locked cabinets and in a password protected database 
until it is no longer of scientific value. 
Loss or destruction of data:  Should we become aware that a major breech in the our plan to protect 
patient confidentiality and trial data has occurred, the IRB will be notified
Data will not be sent outside NIH without IRB notification and an executed MTA or CTA.  An MTA has 
been executed and a copy is on file with the Office of Clinical Affairs for the transfer of sample to Pierre 
Ronco,  MD, PhD, Head, Nephrology Department,  Unit 702 At Tenon Hospital, Paris, France  tel : + 33 
1 56 01 66 39/38 pierre.ronco@tnn.aphp.fr. 
Publication Policy: Given the research mandate of the NIH, patient data including the results of testing 
and responses to treatment will be entered into an NIH-authorized and controlled research database. Any 
future research use will occur only after appropriate human subject protection institutional approval such 
as prospective NIH Intramural IRB review and approval or an exemption from the NIH Office of Human 
Subjects Research (OHSR).
14.0 
DATA SAFETY AND MONITORING
14.1
Safety Monitoring  
Principal Investigator: Accrual, efficacy, and safety will be monitored by the PI. The study will be 
continuously evaluated for any unusual or unpredicted complications with the aim of detecting and 
preventing unacceptable increase in morbidity and mortality over that anticipated from unmanipulated 
bone marrow transplantation.
NIH Intramural IRB. Accrual and safety data will be reviewed annually by the Institutional Review Board 
(IRB). Prior to implementation of this study, the protocol and the proposed patient consent and assent 
forms will be reviewed and approved by the properly constituted Institutional Review Board (IRB) 
operating according to 45 CFR 46 Code of Federal regulations.  This committee will also approve all 
amendments to the protocol or informed consent, and conduct continuing annual review so long as the 
protocol is open to accrual or follow up of subjects. 


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Richard W. Childs, M.D.
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22
DSMB: The NHLBI Data Safety and Monitoring Board will review the protocol at six or twelve month 
intervals.  A progress report will be forwarded to the DSMB at these times and their recommendations will 
be expeditiously implemented. The DSMB may recommend early termination of the study for 
considerations of safety and efficacy.  Since this study was closed to accrual and follow up care in June 
2008, a final report was submitted to the DSMB and this study is no longer reviewed by the DSMB.
14.2 
Adverse Events 
The definitions of the Reportable Events will be followed per Policy 801.
NIH IRB and CD reporting
Expedited Reporting
Events requiring expedited reporting will be submitted to the IRB per Policy 801 “Reporting Research 
Events”.
Reports to the IRB at the time of Continuing Review:
The PI or designee will refer to HRPP Policy 801 “Reporting Research Events” to determine IRB 
reporting requirements.
Reports to the CD:
The PI or designee will refer to NHLBI DIR Policy to determine CD reporting requirements and 
timelines. 
DSMB:  All SAEs will be included for review by the DSMB at six to twelve month intervals.  Reports of 
serious adverse events that are unexpected and thought to be related to the experimental will also be 
forwarded immediately to the Data and Safety Monitoring Board (DSMB).  All SAEs will be 
included for review semiannually by the DSMB. 
15.0 
HUMAN SUBJECT PROTECTION
15.1
Rationale for subject selection
No subjects will be excluded from participation based on gender, race or ethnicity. All patients with 
eligible diseases itemized in section 5.1 will be considered for the protocol. Because the transplant does 
not incorporate intensive chemotherapy and relies instead on a GVT and a graft-versus-marrow effects, 
which may take months to be effective, only patients with expected survivals of 3 months or greater will 
be eligible for study.  
Strategies for patient recruitment: Oncologists throughout the country will be informed of the protocol by 
letter. Information about the protocol will be posted on Clinicaltrials.gov, Clinical Center Studies, and the 
NHLBI patient recruitment websites.  The protocol will also be listed in the PDQ directory.  A 
recruitment flyer for the protocol will also be submitted to several oncology journals.
15.2
Participation of children
Participation of children as stem cell transplant recipients: In principle, age is not a consideration.  In 
practice, we are limiting the protocol to patients over the age of 10 years as the diseases under study on 
this protocol (metastatic, treatment-refractory, solid neoplasms (breast, cholangiocarcinoma, small 


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23
intestine/colon/rectum, esophageal/gastric, hepatocellular, pancreatic, prostate, bony/soft tissue sarcomas) 
would be relatively rare in children as compared to adults and an extraordinary effort would be needed to 
accrue sufficient number of children to access safety and/or efficacy.  
Participation as stem cell donors: :As the stem cell transplant on this protocol is not an accepted standard 
clinical intervention for the disease under investigation and the donor would not be donating the stem 
cells outside the context of this research, the donors become subjects because the investigators are 
intervening with them through the stem cell harvest procedure. The stem cell collection procedure is 
considered part of the research for the donors and the risks and benefits of the harvest procedure are 
considered as risks and benefits of the research for the donors.
As participants in research studies: Pediatric donors may participate only in those laboratory studies that 
the IRB finds involves no greater than minimal risk to children provided that adequate provisions are 
made for soliciting the assent of the children and the permission of their parents or guardians (see section 
15.4.5)
15.3 
Hazards and Discomforts
15.3.1 The transplant recipient
Related to the transplant:  The mortality from conventional BMT may be as high as 40%.  Although our 
data as well as that of others suggest a significant reduction in transplant related mortality with 
non-myeloablative PBSC transplantation, the procedure nevertheless carries risk.  It is therefore only 
appropriate to carry out this experimental procedure in the context of life-threatening metastatic cancer, 
which is incurable with conventional therapy and with full informed consent from the patient and donor. 
The specific hazards of this study using a non-myeloablative preparative regimen and high PBPC content 
graft are graft rejection, graft-versus-host disease and progression of metastatic disease. The major 
discomforts are those of nausea, mucositis, anorexia, diarrhea, fever and malaise, and intolerance of the 
isolation period. 
Related to the common drugs used in BMT:
Antithymocyte Globulin: Febrile reactions, allergic symptoms including rash, wheezing, anaphylaxis, 
cytopenias, serum sickness, nausea, vomiting, diarrhea, arthralgias, hypotension, tachycardia, 
bradycardia, fever, chills, myelosuppression. Several cases of a severe lung injury related to ATgam 
treatment have been reported. Although this side effect appears extremely rare, it is serious and can be 
fatal. There is no information about the mechanism or specific treatment for this condition. A few patients 
recovered after intensive medical support including use of a breathing machine. 
Cyclosporine: CSA is metabolized primarily in liver but the major toxicity is renal. Side effects include 
renal impairment, reversible renal insufficiency, hemolytic uremic syndrome, elevated bilirubin and 
transaminases that normalize with continued administration or reduced dose, hypertrichosis, headaches, 
nausea, gingival hypertrophy, parasthesias (painful hands and feet), hypertension, hypomagnesium, 
bilirubinemia, hypertrichosis, nausea, tremor, and seizure.  An extremely rare complication of 
cyclosporine is blindness, which may be irreversible
Cyclophosphamide: Immediate: tingling and metallic taste, nausea and vomiting, ADH-like effect, 
cardiotoxic at high doses (>70 mg/kg). Rare - pulmonary toxicity, urticaria and flushing, mucositis 
Delayed: Marrow suppression, nausea, mucositis, rash, hemorrhagic cystitis, myocardial damage, 
alopecia, infertility


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24
  
Fludarabine: Myelosuppression, fever and chills, nausea and vomiting, malaise, fatigue, anorexia, 
weakness, and rarely hemolysis and pulmonary toxicity, hemolytic anemia and interstitial pneumonitis. 
Serious opportunistic infections have occurred in CLL patients treated with fludarabine.  
Methotrexate:  Mucositis, nausea, dizziness, neutropenia, thrombocytopenia, malaise, fatigue, fever, 
melena, headaches, blurred vision, rashes, alopecia, elevated liver functions, 
Hematologic: Myelosupression [leukopenia (nadir 7 days) thrombocytopenia, anemia] 
Hepatic:  Acute (elevated transaminases) and chronic (fibrosis and cirrhosis) hepatic toxicity. Chronic 
toxicity has generally occurred after prolonged use (generally 2 years or more) and after a total dose of 
at least 1.5 grams.
Urogenital:  Severe nephropathy or renal failure, azotemia, cystitis, hematuria; defective oogenesis or 
spermatogenesis, transient oligospermia, menstrual dysfunction and vaginal discharge; infertility, 
abortion, fetal defects. With high doses of methotrexate, close attention to renal function including 
adequate hydration, and urine alkanization are essential for safe administration. 
Gastrointestinal:  Gingivitis, pharyngitis, stomatitis, anorexia, nausea, vomiting, diarrhea, hematemesis, 
melena, gastrointestinal ulceration and bleeding, enteritis. Should be used with extreme caution in the 
presence of peptic ulcer disease or ulcerative colitis. Therapy may be discontinued if ulcerative 
stomatitis or other severe GI adverse reactions occur.
Pulmonary:  Interstitial pneumonitis deaths have been reported, and chronic interstitial obstructive 
pulmonary disease has occasionally occurred. Pulmonary symptoms or a nonspecific pneumonitis may 
be indicative of a potentially dangerous lesion and require interruption of treatment and careful 
investigation; infection needs to be excluded. This lesion can occur at all dosages.
Skin: Erythematous rashes, pruritis, urticaria, photosensistivty, pigmentary changes, alopecia, ecchymosis, 
telangiectasia, acne, furunculosis.
Central Nervous System: Headaches, drowsiness, blurred vision. There have been reports of 
leukoencephalopathy following intravenous administration of methotrexate to patients who have 
craniospinal irradiation. Aphasia, hemiparesis, paresis, and convulsions have also occurred following 
administration of methotrexate. Following low doses, occasional patients reported transient subtle 
cognitive dysfunction, mood alteration, or unusual cranial sensations.
Other:  Opportunistic infections, arthralgia/myalgia, loss of libido/impotence, diabetes, and osteoporosis. 
A few cases of anaphylactoid reactions have been reported.  
Antimicrobials in general: Allergic reactions, renal impairment (gentamicin, vancomycin, amphotericin, 
acyclovir), "red man" syndrome -vancomycin, hepatic damage (acyclovir, rifampicin)
15.3.2 The donor
Related to filgrastim (G-CSF): G-CSF has been given to large numbers of normal donors without major 
side effects or long term consequences. The immediate side effects of G-CSF are bone pain, fatigue, 
insomnia, myalgia, headache, chills, low grade fever, rash, injection site irritation, exacerbation of pre 
existing inflammatory conditions, splenomegaly, reversible elevation in uric acid, LDH, and leucocyte 
alkaline phosphates. These are usually mild and are self limiting.  Reversible thrombocytopenia, with 
platelet counts falling to the region of 100,000/cu mm is frequent.   G-CSF administration could result in 
splenic rupture, a potentially life-threatening complication. Up to now five cases of splenic rupture have 
been reported in healthy donors (Becker et al. 1997, Falzetti et al. 1999, Balaguer et al. 2004, Dincer et al. 
2004, Nuamah et al. 2006). Donors will be asked to avoid vigorous activities and to report any left upper 
abdominal and/or shoulder pain to the research team or the on-call physician for the NIH Department of 
Transfusion Medicine at 301-496-1211. 


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25
Patients with ongoing ischemic heart disease have been reported to have angina seemingly temporally 
related to G-CSF administration and apheresis. The discomfort from G-CSF mobilization and 
leukapheresis for collection of blood stem cells are probably lower than those associated with marrow 
harvesting.  
Related to Central line placement:  It is estimated that about 50% of the donors will require intravenous 
central line placement to successfully complete apheresis. Intravenous line placement in the femoral vein 
using a temporary double-lumen Arrow catheter carries a small risk of bleeding, bruising or pain and a 
very low risk of accidental injury to the adjacent artery and nerve. Some patients may experience a 
vasovageal reaction (lightheadedness, or rarely, fainting due to temporary lowering of blood pressure). 
These risks are minimized by using only trained experienced MICU staff for the procedure.
Related to Leukapheresis:  Adverse reactions related to leukapheresis include hypotension resulting from 
transient blood volume loss and cutaneous paresthesia from the use of anticoagulant.  The former toxicity 
can be corrected by postural changes and volume replacement.  The latter is manageable with slowing the 
rate of anticoagulant infusion and/or providing calcium supplement.  In exceptional instances the donor 
may be required to donate PBPC a third time or to give bone marrow. Donation of PBPC on three 
successive days significantly increases the risk of thrombocytopenia ( < 1 00,000/ul).  However, 
thrombocytopenia is transient and unlikely to cause clinical sequelae.  There is no additional risk to the 
donor giving marrow after PBPC donation (than would normally be associated with bone marrow 
harvesting).
15.4
Risks in relation to Benefit
This protocol was closed to accrual and follow up in June 2008; all subjects are off study and this 
protocol is in data analysis phase only therefore, the research currently does not involve greater than 
minimal risk. 
15.4.1 For Adult Transplant Subjects 
Clinically, the approach is ethically acceptable because we are targeting a patient group with incurable 
neoplasms, which have become refractory to conventional therapy. The protocol also aims to decrease the 
risk of transplant-related mortality, thus making patients with advanced metastatic disease candidates for 
this therapy. The risk of death due to complication from the allogeneic BMT procedure is justified by the 
anticipated benefit of potentially eradicating the subject’s underlying metastatic disease and the relation 
of the anticipated benefit to the risk is at least as favorable to the subjects as presented by available non-
transplantation therapies.
Therefore, for adult transplant recipients on this protocol, the research involves greater than minimal risk 
to subjects with the prospect of direct benefit (45 CFR 46.102).
15.4.2 For Pediatric Transplant Subjects  
The inclusion of children satisfies the criteria set forth in 45 Code of Federal Regulations 46, Subpart D: 
46.405 as follows:
(a) the risk is justified by the anticipated benefit to the subjects: Clinically the approach is ethically 
acceptable because we are offering children >10 years of age with incurable metastatic neoplasms, 
which have become refractory to conventional therapy an alternative to palliative care.
 


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(b)  the relation of the anticipated benefit to the risk is at least as favorable to the subjects as that presented 
by available alternative approaches. The protocol aims to decrease the risk of transplant-related 
mortality, thus making more patients candidates for potentially curative therapy and
 (c) adequate provisions are made for soliciting the assent of the children and permission of their parents or 
guardians, as set forth in 46.408.
Therefore for pediatric transplant recipients on this protocol, the research involves greater than minimal 
risk but presents the prospect of direct benefit to the individual subjects.
15.4.3  For Adult Donors
Normal identical or single HLA locus mismatched family members will be co-enrolled into this study as 
stem cell donors. The stem cell collection aspect of this protocol is not investigational.  Despite the risks 
associated with this procedure (section 15.3), potential benefit does exist for family donors. The donor 
derives psychosocial benefit from donating stem cells both at the time of donation and possibly into the 
future, especially in view of the reduced life expectancy due to this disease in a family member.  Other 
potential benefits include detection of illnesses, determination of blood cell counts, and evaluation of 
kidney and liver function in the potential donor at the time of screening.   
 
Therefore, for adults participating as stem cell donors, the research involves a greater than minimal risk 
but presents the prospect of direct benefit to the individual subject   (45 CFR 46.102).
15.4.4 For Pediatric Donors
The inclusion of children as donors satisfies the criteria set forth in 45 Code of Federal Regulations 46, 
Subpart D: 46.405 as follows:
 (a) the risk is justified by the anticipated benefit to the subjects:  Despite the risks associated with this 
procedure, it is standard clinical practice to include children as donors for allogeneic peripheral 
blood stem cell transplantation. Increased risk is due to G-CSF administration and central line 
placement.  Leukapheresis in children, as discussed under 15.2, is safe and effective both in our 
experience and in others’, and there have never been any leukapheresis-related deaths in the history 
of the Transfusion Medicine department at NIH.
(b) the relation of the anticipated benefit to the risk is at least as favorable to the subjects as that presented 
by available alternative approaches. As with adults the pediatric donor derives psychosocial benefit 
from donating stem cells especially in view of the reduced life expectancy due to this disease in a 
family member.  Other potential benefits include detection of illnesses, determination of blood cell 
count and evaluation of kidney and liver function in the potential donor at the time of screening. 
(c)
adequate provisions are made for soliciting the assent of the children and permission of their parents 
or guardians, as set forth in 46.408.
Therefore for children participating as stem cell donors the research involves greater than minimal risk 
but presents the prospect of direct benefit to the individual subjects.
15.4.5 For Pediatric donors – healthy volunteers- involved in laboratory research studies.  
The inclusion of children satisfies the criteria set forth in 45 Code of Federal Regulations 46, Subpart D: 
46.404 as follows:


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(a) The research does not involve greater than minimal risk.  Blood and bone marrow specimens for 
research are obtained concurrently with clinically indicated sampling.  Therefore, there is no risk 
associated with sample collection for research because research will only performed on material 
obtained during standard clinical intervention.
Research specimens will be stored in the Dr. Childs’s laboratory. Samples will never be labeled with 
the child’s name. Samples will be assigned a unique code known only to the PI, which will serve as a 
link to the child’s clinical information collected as part of this research protocol.  No samples will be 
provided to investigators outside the branch. Therefore confidentiality is protected.
Only those laboratory tests approved by the IRB and involving not greater than minimal risk will be 
conducted (See Appendix D laboratory studies D1-D6 are determined to be greater than minimal 
risk).  Research will not include genetic testing.  Therefore, there is no genetic testing associated 
risks. 
(b) adequate provisions is made for soliciting the assent of the children and permission of their parents or 
guardians, as set forth in 46.408.
Therefore, for Pediatric donors – healthy volunteers- participating in laboratory research studies on this 
protocol, the research does not involve greater than minimal risk  (45 CFR 46.404).
15.5
Informed Consent Process and Procedures
The investigational nature and research objectives of this trial, the procedures and attendant risks and 
discomforts will be carefully explained to the subject and a signed informed consent document will be 
obtained prior to entry onto this study.  Drs. Childs, Srinivasan, Barrett, Young will lead this discussion. 
If the subject is a minor, the parent who signs the consent for the minor must be a legally recognized 
parent or guardian.  Where deemed appropriate by the clinician, and the child's parent or guardian, the 
child will also be included in all discussions about the trial and a minor's assent will be obtained.   The 
parent or guardian will sign on the designated line on the informed consent attesting to the fact that the 
child had given assent.
If the donor is a minor, assent will not be sought until an evaluation by a social worker and mental health 
specialist (psychologist or psychiatrist) is completed to determine the minor donor’s willingness to 
participate.   As detailed above, the parent who signs the consent for the minor must be a legally 
recognized parent or guardian.  Where deemed appropriate by the clinician, and the child's parent or 
guardian, the child will also be included in all discussions about the trial and a minor donor’s assent will 
be obtained.   The parent or guardian will sign on the designated line on the informed consent attesting to 
the fact that the child had given assent
When the assent is not age appropriate, the study will be explained to the child and the assent will be 
obtained verbally from the child.
Re-Consent for Minors when they reach the age of majority:  When a pediatric subject reaches age 
18, continued participation will require re-consenting of the now adult with the standard protocol consent 
document to ensure legally effective informed consent has been obtained. Should sample or data analysis 
continue following completion of active participation and the subject has reached 18 years of age, we will 
attempt to contact the subject using the last known contact information to obtain consent for continued 


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28
use of data or samples  collected during their prior visit. Given the length of time that may have transpired 
for some of the subjects since their last visit for this study, we request waiver of informed consent for 
those individuals who after good faith efforts to contact them, we are unable to contact.
Requirements for Waiver of Consent consistent with 45 CFR 46.116 (d):
(1) The research involves no more than minimal risk to the subjects.
a.
Analysis of samples and data from this study involves no additional risks to subjects.
(2) The waiver or alteration will not adversely affect the rights and welfare of the subjects.
a.
Retention of these samples or data does not affect the welfare of subjects. 
(3) The research could not practicably be carried out without the waiver or alteration.
a.
Considering the length of time between a minor’s enrollment and their age of majority, it is 
possible that more than a few subjects may be lost to follow up.  A significant reduction in 
the number of samples analyzed could impact the quality of the research. 
(4) Whenever appropriate, the subjects will be provided with additional pertinent information after 
participation.
a.
We only plan to request a waiver of reconsent for those subjects who have been lost to 
follow-up.  
At any time during participation in the protocol that new information becomes available relating to risks, 
adverse events, or toxicities, this information will be provided orally or in writing to all enrolled or 
prospective patient participants.  Documentation will be provided to the IRB and if necessary the 
informed consent amended to reflect relevant information.
15.6
CONFLICT OF INTEREST
The PI assured that each AI listed on the protocol title page received a copy of the NIH’s Guide to 
preventing conflict of interest.  Investigators added subsequent to the initial circulation were provided a 
copy of the document when they were added. Copies of the Conflict of Interest Statement were forwarded 
to the Clinical Director.  No initial or subsequent members of the research team reported a potential 
conflict of interest.  
This protocol has no associated patents, CRADAs, or CTAs.  An MTA has been executed and a copy is 
on file with the Office of Clinical Affairs for the transfer of sample to Pierre Ronco,  MD, PhD, Head, 
Nephrology Department,  Unit 702 At Tenon Hospital, Paris, France  tel : + 33 1 56 01 66 39/38 
pierre.ronco@tnn.aphp.fr 
 
16.0 
PHARMACEUTICALS
16.1
ANTITHYMOCYTE GLOBULIN (Atgam)
Other: Antithymocyte Gammaglobulin, Antithymocyte Globulin, Atgam, Antithymocyte 
Immunoglobulin.
Compound: Principally monomeric IgG, prepared from plasma or serum of healthy horses hyper-
immunized with human thymus lymphocytes.
Action: Immunosuppressive agent. Exact mechanism of immunosuppression of ATgam has not been fully 
elucidated but may involve elimination of antigen-reactive T-cells in peripheral blood and/or 
alteration of T-cell function.
Side effects: see section 15.3, Risks and discomforts. 


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16.2
CYCLOPHOSPHAMIDE
Generic: cyclophosphamide
other: Endoxan, Cytoxan, Neosar,  NSC#: 26271
Chemical: 2-bix (2-chloroethyl) amino tetrahydro-2H-1,3,2-oxazaphosphorine-2-oxide monohydrate.
Classification: Alkylating agent
Action:  Prevents cell division by altering DNA 
Metabolism: Converted in the liver by microsomal enzymes to active  nitrogen mustard like 
compounds.Excreted by the kidney as metabolites and unconverted cyclophosphamide.
Availability: 100, 200, and 500 mg vials. Commercial: Meade Johnson and Adria.
Storage: Stable at room temperature. After reconstitution use within 24 hours. Administration: Dissolved 
in 250 ml D5W and administered over one hour. 
Precautions: Institute  antiemetic therapy with a serotonin antagonist before administration and continue 
with anti-emesis for 24 hours following administration.
To prevent hemorrhagic cystitis: give mesna prior to cyclophosphamide. Continue with mesna following 
cyclophosphamide; IV hydration as per Appendix A.
Side effects: See section 15.3, Risks and discomforts. 
16.3
CYCLOSPORINE (CSA)
Dose:1.5 mg/kg/dose BID IV will begin on day -4 through day 100. Dosing will be based on actual body 
weight unless the patient is “obese” (BMI > 35) when practical weight will be used.  Switch to the 
equivalent oral CsA dose divided bid on day 14 or when able to tolerate PO.  
Levels: Serum levels monitored weekly
Mechanism of Action: Blocks T cell activation by inhibiting transport of cytoplasmic component of 
Nuclear factor of activated T-cells (NFAT) into nucleus, thereby preventing IL-2 transcription.
Metabolism: CSA is metabolized primarily in liver but predominate toxicity is renal. 
Dose Modifications 
Renal insufficiency
Creatinine >2.0 mg/dl reduce dose 25%
Creatinine >3.0 mg/dl reduce dose 75%
Creatinine >4.0 mg/dl hold
Hepatic insufficiency
If increase in bilirubin or transaminases >100% over baseline decrease by 
2mg/kg/d
Serum levels
Adjust dose for therapeutic range (200-400ug/ml)
Side effects: See section 15.3, Risks and discomforts. 
16.4
FLUDARABINE PHOSPHATE
Generic: fludarabine phosphate 
Other: Fludara, Berlex
Chemical: 2-fluoro-ara-monophosphate
Classification: Fluorinated nucleotide analog
Action: Inhibits DNA synthesis
Metabolism: Converted intracellularly to the active metabolite 2-fluoro-ara-ATP.
Availability: 50 mg for iv use only. Commercial: Biospherics
Side effects: See section 15.3, Risks and discomforts. 
16.5
G-CSF (Human recombinant granulocyte colony stimulating factor)


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G-CSF (Amgen), Filgrastim
Classification: glucoprotein
Action: Regulates the production of neutrophils from bone marrow progenitor cells and mobilizes 
primitive hematopoietic stem cells from the bone marrow into the circulation.   
Metabolism: Absorption and clearance of G-CSF follow first order kinetics.  A positive linear correlation 
occurs between the parenteral dose and both the serum concentration and area under the 
concentration-time curves.  The elimination T1/2 is 3.5 hours.
Administration:   G-CSF may be given sc a single daily dose or iv over 30 min.  
G-CSF is a protein that is produced by normal human bone marrow cells.  G-CSF used in this study is 
purified from the product of E. coli with recombinant DNA technology. The only differences between 
this product and natural G-CSF are an additional N-terminal methionine necessary for expression in 
E. coli and the fact that this product is unglycosylated.  G-CSF is approved by the US Food and Drug 
Administration to be used in preventing chemotherapy-induced leukopenia. This drug has been used 
in cancer patients for prolonged periods of time without major toxicities. The extensive use of this 
drug in many patients also proved its safety at a dose of 10 g/kg/day. The most frequent side effect is 
bone pain originating from expansion of cells inside the bone marrow. It can affect up to 80% of the 
subjects at this dosage range.  The pain is usually mild and can be alleviated most of the times by 
acetaminophen or other non-steroidal analgesics and will cease altogether once G-CSF is 
discontinued. G-CSF is associated with increased inflammation in patients with autoimmune diseases 
or subjects with infection, however these patients are not eligible for this study.  These are readily 
reversible after cessation of the drug. Leukocytosis is expected after administration of G-CSF.  Other 
adverse reactions associated with the use of G-CSF as cited in the literature include: fatigue (50%), 
headache (30%), muscle aches (25%), insomnia (rare), fever (rare), transient but reversible increases 
of alkaline phosphatase, lactate dehydrogenase, and uric acid levels, exacerbation of preexisting skin 
rashes, and thrombocytopenia (mild, reversible).
Availability: Recombinant G-CSF is available from the pharmacy at the National Heart, Lung and Blood 
Institute in colorless glass, single-use vials containing either 300g in 1.0 ml vials or 480g in 1.6 ml 
vials (300 g/ml).  It is formulated as a sterile, clear, colorless liquid in a 10 mm sodium acetate 
buffer at pH 4.0.  The quantitative composition (per ml) is:
Recombinant G-CSF 
300 g
Acetate 
0.59 mg
Mannitol 
50 mg
Tween 80TM
0.004%
Sodium 
0.035 mg
Water for injection (qs ad) to 1.0 ml
Storage: The intact vials of G-CSF should be stored under refrigeration (2 - 8C).
Stability: G-CSF in the intact vial is stable for 36 months when stored in a refrigerator at 2 - 8C.  A 
single brief exposure (up to 7 days) to elevated  temperatures (< 37C) does not affect the stability.  
G-CSF should not be frozen, and vials, which have been frozen should not be used.
Side effects: See section 15.3 Risks and Discomforts.
16.6
METHOTREXATE 
Generic:  Methotrexate sodium injection
Other: Rheumatrex® (Methotrexate)
Classification: Antimetabolite:Immunosuppressive agent


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31
Dose: 5 mg/m2 intravenously to be given on post-transplant days +1, +3, and +6. Methotrexate will be 
held if the serum creatinine is > 3mg/dl.  Patients with elevated liver function tests (i.e. 
transaminases) greater than 3 times the upper limit of normal may have methotrexate held at the 
discretion of the principal investigator.
General:  Rheumatrex® (Methotrexate) is an antimetabolite which binds to dihydrofolic acid reductase, 
thereby preventing the reduction of folic acid to tetrahydrofolate. It interferes with DNA synthesis, 
repair, and cellular replication. Actively proliferating tissues are in general more sensitive to this effect.
Administration:  Methotrexate Sodium for Injection, freeze dried, preservative free, low sodium, for single 
use only, is available in 20 mg, 50 mg, and 1 gram vials, containing approximately 0.14, 0.33, and 7 
meq of sodium respectively. Reconstitute immediately prior to use with an appropriate sterile, 
preservative-free medium.
Side effects: See section 15.3, Risks and Discomforts
16.7
MYCOPHENOLATE MOFETIL (MMF)
Generic:  Mycophenolate mofetil
Other:  CellCept, Roche Laboratories
Classification:  Immunosuppressive agent
Dose: 1 gram p.o. twice daily given to some patients with steroid resistant acute GVHD or steroid 
dependent chronic GVHD.
Action: Mycophenolic acid produces potent, noncompetitive inhibition of inosine monophosphate 
dehydrogenase, thus blocking de novo synthesis of guanosine nucleotides; as lymphocytes depend 
upon the de novo pathway for purine synthesis, lymphocyte proliferation is inhibited. In vitro and in 
vivo studies have demonstrated the ability of mycophenolic acid to block proliferative responses of T 
and B lymphocytes, and inhibit antibody formation and the generation of cytotoxic T-cells.
Metabolism: Mycophenolate mofetil is rapidly converted to it’s active metabolite, mycophenolic acid in 
the liver.  Mycophenolic acid is subsequently metabolized to mycophenolic acid glucuronide which is 
inactive and is excreted via the urine and bile.  Enterohepatic recirculation of mycophenolic acid may 
occur.  The elimination half-life of mycophenolic acid is approximately 11 to 18 hours.  
Side effects: See section 15.3, Risks and Discomforts.
17.0 
REFERENCES
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Eibl B, Schwaighofer H, Nachbaur D et al. Evidence for a graft-versus-tumor effect in a patient treated with marrow 
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Or R; Ackerstein A; Nagler A; Kapelushnik J; Naparstek E; Samuel S; AmarA; Bruatbar C; Slavin S. Allogeneic 
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Ueno NT; Rondon G; Mirza NQ et al. Allogeneic peripheral-blood progenitor-cell transplantation for poor-risk patients 
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Rabinowich H., Cohen R., Bruderman I., et al. Functional analysis of mononuclear cells infiltrating into tumors: Lysis of 
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Heo D., Whiteside L., Johnson J., et al. Long-term interleukin 2 dependent growth and cytotxic activity of tumor 
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6 
Takagi S., Chen K., Schwarz R., et al Functional and phenotypic analysis of tumor infiltrating lymphocytes isolated from 
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July 23, 2019
32
7 
Heo D., Whiteside L., Kanbour A., et al. Lymphocytes infiltrating human ovarian tumors. Journal of Immunology 1988, 
140: 4042-4049
8
 Rabinowich H., Cohen R., 8ruderman I., et al. Functional analysis of mononuclear cells infiltrating into tumors: Lysis of 
autologous human cells by cultured infiltrating lymphocytes. Cancer Research 1987, 47: 173-177
9 
Saito T., Tanaka R., Yoshida S., et al. Immunohistochemical analysis of tumor infiltrating lymphocytes and major 
histocompatibility antigens in human gliomas and metastatic brain tumors. Surg Neurol 1988, 29:435-442
10 Parkhurst M., Salgaller M., Southwood S., et al. Improved induction of melanoma reactive CTL with peptides from the 
melanoma antigen gp100 modified at HLA-A*0201-binding residues. The J of Immunology 1996, 157: 2539-2548
11 Rosenberg S.A., Yang J.C., Topalian S.L., et al. Treatment of 283 consecutive patients with metastatic melanoma or 
renal cell cancer using high dose IL-2. JAMA 1994,271:12:907-913
12 Khouri I, Keating MJ, Przepioka et el. Engraftment and induction of GVL with fludarabine (FAMP)-based 
non-myeloablative preparative regimen in patients with CLL and lymphoma. Blood 88;10:suppl,310a,1996.
13 Giralt S, Estey E, Albitar M, van Besienk K: Engraftment of allogeneic hematopoietic progenitor cells with purine 
analog containing chemotherapy. Blood 1997, Jun 15;89 (12)4531-6 -
14 Khouri IF, Keating M, Korbling Met al. Transplant lite: induction of graft-versus-malignancy using fludarabine-based 
nonablative chemotherapy and aHogeneic blood progenitor-cell transplantation as treatment for lymphoid malignancies. 
J Clin Oncol, 1998: 16;8, 2817 -24
15 Childs R., Bahceci E., Barrett J., et al. Non-myeloablative allogeneic peripheral blood stem cell transplants for malignant 
diseases reduces transplant related mortality. Blood 1998, 92:10:Abstract # 552-137a
16 Childs R., Clave E., Barrett J., Et al. Kinetics of engraftment in non-myeloablative allogeneic peripheral blood stem cell 
transplants: An analysis of hematopoetic lineage specific chimerism. Blood 1998, 92:10: Abstract # 2137-520a
17 Slavin S, Nagler A, Naparstek E et al. Non-myeloablative stem cell transplantation and cell therapy as an alternative to 
conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and non-malignant 
hematologic diseases. Rapid Communication Blood 1998;91 :756-763
18  Schmitz N, Greger P, Suttorp Met al. Primary transplantation of allogeneic peripheral blood progenitor cells mobilized 
by Filgastrim (G-CSF). Blood 1995, 85: 1666-1674.
19 Goodman JM. Peripheral blood stem cells for allografting. Blood 1995, 85: 1413-1415. 
20 Kolb HJ, Schattenberg A, Goldman JM, et al. Graft-versus-Ieukemia effect of donor lymphocyte transfusion in marrow 
grafted patients. Blood 86: 2041-2050, 1995.
21 Kolb HJ, Mittermuller J, Clemm C et al. Donor leukocyte transfusions for treatment of recurrent chronic myelogenous 
leukemia in marrow transplant recipients. Blood 76:2462-2465, 1990.
22 The clinical practice of stem cell transplantation. Barrett AJ and Treleavan JG (Eds) Isis Medical Media Oxford 1998.
23 Armitage JO. Bone marrow transplantation. N Eng J Med 330: 827-38, 1994
24 Weiden PL, Sullivan KM, Flournoy N, Storb R, Thomas ED, The Seattle Marrow Transplant Team: Antileukemic effect 
of chronic graft-versus-host disease. Contribution to improved survival after allogeneic marrow transplantation. N Engl.J 
Med 304: 1529-1533, 1981


99-H-0064
Richard W. Childs, M.D.
July 23, 2019
33
25 Porter DL, Roth MS, McGarigle C, Ferrara JLM, Antin JH. Induction of graft-versus-host disease as immunotherapy for 
relapsed chronic myeloid leukemia. N Engl J Med 330: 100-106, 1994
26 Khouri I, Keating MJ, Przepiorka a, et al. Stem cell transplantation (SCT) for chronic lymphocytic leukemia (CLL): 
graft-versus-Ieukemia (GVL) without acute graft-versus-host disease (GVHD). Blood 86: 457a, 1995.
27 Rivoltini L, Barracchini K, Viggiano V, et al. Quantitative correlation between HLA class I expression and recognition 
of melanoma cells by antigen-specific T lymphocytes. Cancer Res 1995, 55: 3149-3157
28 Childs R., Clave E., Barrett J., et al: Complete regression of metastatic renal cell cancer following non-myeloablative 
allogeneic PBSC transplantation; Evidence for the graft-versus-tumor effect. In press- Journal of Clinical Oncology 1999
29 Ruiz-Cabello F, Perz-Ayala M, Gomez 0, et al. Molecular analysis of MHC-Class-l-alterations in human tumor cell 
lines. Cancer (Suppl) 1991, 6: 123-130
30 De Bueger M, Bakker A, Van Rood JJ, et al. Tissue distribution of human minor histocompatibility antigens: Ubiquitous 
versus restricted tissue distribution indicates heterogeneity among human cytotoxic T Iymphocyte-defined non-MHC 
antigens. J Immunol 1992, 149: 1788-1796
31 Rosenburg S.A, Spiess P., Lafreniere R. Anew approach to the adoptive immunotherapy of cancer with tumor infiltrating 
lymphocytes. Science 1986,233:1318-1321
32 Gehan EA. The determination of the number of patients required in a preliminary and follow-up trial of a new 
chemotherapeutic agent. J Chron Dis 1961; 13:346-351


99-H-0064
Richard W. Childs, M.D.
July 23, 2019
34
APPENDIX A:  PREPARATIVE REGIMEN
DOSE LEVEL I
Cyclophosphamide regimen
Hour
Day -7
0
At midnight
Hydrate: NS 2.6ml/kg/hr 10 mEq/l KCl (starting 12 hours
pre-cyclophosphamide and continue hydration until 24 hours after last 
cyclophosphamide infusion)
 
11
Ondansetron 8 mg IV q 12 hrs or an equivalent serotonin antagonist + decadron 8 
mg PO q12 hrs x 1 day.
Mesna (per PBSCT Supportive Care Guidelines)
Furosemide 20 mg iv.
12
Cyclophosphamide 60 mg/kg IV in 250 ml D5 in 1 hr.
13
Mesna (per Supportive Care Guidelines) 
Day - 6
11
Ondansetron 8 mg IV q 12 hrs or an equivalent serotonin antagonist +  decadron 
8 mg PO q12 hrs x 1 day.
Mesna (per Supportive Care Guidelines)
Furosemide 20 mg iv
12
Cyclophosphamide 60 mg/kg IV in 250 ml D5 IV over 1 hr 
13
Mesna (per Supportive Care Guidelines) 
Day -5
Stop IV hydration (24 hours after last cyclophosphamide dose)
Monitor electrolytes and fluid balance while receiving hydration. 
Day -5 to Day -1
Hour 
9
Fludarabine 25 mg/m2 IVBP over 30 minutes for 5 days
DOSE LEVEL II
Same as dose level I except patients will receive ATgam at a dose of 40 
mg/kg/d for 4 consecutive days on day -5 to day -2.


99-H-0064
Richard W. Childs, M.D.
July 23, 2019
35
 APPENDIX B: ATG DESENSITIZATION
An intravenous line must be in place, and emergency medications (diphenhydramine 50 mg, epinephrine 1:1000, 
hydrocortisone 100mg) should be at the bedside prior to initiating desensitization. Desensitization should be 
performed in a limb where a tourniquet can be proximally placed if a reaction occurs.  Injections are given 
intravenously every 15 minutes according to the following timetable.  If an injection produces a large local 
reaction, the same dose is repeated before progressing to the next larger dose.  Vital signs should be recorded 
every 15 minutes. 
Dose #
Concentration of 
ATG (mg/ml)
Volume/Dose
Rate
Cumulative 
Dose (microg)
          1
0.0001
1 ml/0.1 mcg
Push
0.1
2
0.0005
1 ml/0.5 mcg
Push
0.6
3
0.002
1 ml/2 mcg
Push
2.6
4
0.005
1 ml/5 mcg
Push
7.6
5
0.01
1 ml/10 mcg
Push
17.6
6
0.01
3 ml/30 mcg
Push
47.6
7
0.01
10 ml/100 mcg
Push
147.6
8
0.01
160 ml/1600 mcg
16 ml/min for 
10 min (total 
160ml) 
1747.6
9
Therapeutic 
Infusion  
 


99-H-0064
Richard W. Childs, M.D.
July 23, 2019
36
 APPENDIX C: SERIOUS ADVERSE EVENT REPORT FORM (As July 1, 2019 this form no 
longer being used)
Definition of a serious adverse event: For the purposes of this form, a serious adverse event is any 
untoward medical occurrence that results in death, is life-threatening, requires or prolongs hospitalization, 
causes persistent or significant disability/incapacity, results in congenital anomalies/birth defects, or in the 
opinion of the investigator  represents other significant hazards or potentially serious harm to research 
subjects or others. A serious adverse event is considered unexpected if it is not described in the Package 
Insert or in the Investigator's Brochure (for FOA investigational agents), in the protocol, or in the informed 
consent document. 
Please complete the information requested below and forward it to the NHLBI IRB via the 
NHLBI Office of Clinical Affairs (OCA), Melissa Bryant, CRC 4-1581, Room  4-1580 with a 
copy to Dr. Richard Cannon, Clinical Director, Clinical Research (CRC-4-1581, as soon as 
possible, but no later than seven (7) days in the case of death or life-threatening serious 
adverse events or within fifteen (15) days after the occurrence of all other forms of serious 
adverse events.  In addition, continue to follow FDA and the NIH Office of Biotechnology 
Activities (OBA) reporting requirements if your research involves an IND / IDE or gene transfer.
1. Protocol number:   99-H-0064
2. Protocol title:   Exploratory study of non-myeloablative allogeneic stem cell transplantation and donor 
lymphocyte infusions for metastatic neoplasms refractory to standard therapy
3. Principal Investigator:  Richard W. Childs, M.D.
    Institute:
         NHLBI/HB
    Office:
                      Bldg 10, Room CRC 3-5332
    Phone:                           301-594-8008
    FAX:
    E-mail:                          childsr@NHLBI.NIH.gov
4. a. Date of serious adverse event: 
b. Date the Sponsor/Principal Investigator were first made aware of this event. ( please note that it is the 
investigators responsibility to report this event within the stipulated time frame once they are made aware 
of the event) 
c. If there is significant lag time from the date the event occurred to the date the event was reported, please 
provide a brief explanation.
5. Location of serious adverse event (e.g., at NIH or elsewhere):
6. Was this an unexpected adverse event? Yes [ ]     No [ ]
7. Brief description of subject(s) 
Sex:
 Age: 
    (do not include identifiers)
            Diagnosis:
8. Brief description of the nature of the serious adverse event (attach description separately if           more 
space needed):


99-H-0064
Richard W. Childs, M.D.
July 23, 2019
37
9. Category (outcome) of the serious adverse event:
1. [ ] death 
2. [ ] life-threatening
3. [ ] hospitalization-initial or prolonged
          
4. [ ] disability / incapacity
5. [ ] congenital anomaly / birth defect
6. [ ] required intervention to prevent permanent impairment
7. [ ] other
10. Relationship of Serious Adverse Event to research:
[ ] 1 = Unrelated (clearly not related to the research)
[ ] 2 = Unlikely (doubtfully related to the research)
[ ] 3 = Possible (may be related to the research)
[ ] 4 = Probable (likely related to the research)
[ ] 5 = Definite (clearly related to the research) .
11. Have similar adverse events occurred on this protocol? Yes [ ] No [ ]
If "Yes", how many? _______   Please describe.
12. What steps do you plan to take as a result of the adverse event reported above? Provide
      documentation to the IRB for review and approval of any of the steps checked below. 
[ ] no action required 
            
[ ] amend protocol
[ ] amend consent document                          
[ ] inform current subjects
[ ] terminate or suspend protocol                   
[ ] other (describe)
Signature of Principal Investigator:______________________________ Date:___________


99-H-0064
Richard W. Childs, M.D.
July 23, 2019
38
 APPENDIX D:   APPROVED LIST OF NHLBI LABORATORY STUDIES
IRB APPROVED NHLBI CMTB LABORATORY RESEARCH STUDIES v. 2.5.2013
DESCRIPTION OF LABORATORY STUDY BY BRANCH SECTION
Does this test 
pose a greater 
than minimal 
risk to 
pediatric 
subjects per 
45 CFR 
46.404?
Does this 
test pose a 
greater than 
minimal 
risk to 
healthy 
pediatric 
donors per 
45 CFR 
46.404?
A
Stem Cell Allotransplantation Section (Dr. A. John Barrett) – no longer active
A.1
Measurement of lymphocyte function and immune responses directed toward 
allogeneic tissues, malignant cells, and infectious agents.  Assay of a variety of 
antigens, including standard proliferation, cytotoxicity, and intracellular cytokine  
detection including GVHD predictive markers. Measurement of antigen-specific 
responses including employment of tetramers, ELISPOT technique, gene 
amplification-based assays, and flow cytometry.  Selection of cells using 
immunomagnetic beads or flow cytometry.  Culture, expansion, and selection of 
cells. Surface marker analysis of PB MC using flow cytometry. Cytokine/chemokine 
analysis of plasma/serum samples using ELISA and/or Luminex techniques.
No
No
A.2
Generation of cell lines for the study of immune cell interactions with other cells.  
Transformation of B-lymphocytes using Epstein-Barr virus.  Derivation of malignant 
cell lines from patient leukemic or solid tumor samples.
No
No
A.3
Infection of cells and cell lines with recombinant genes to ascertain the effects of 
expressed molecules on immune responses and on growth and development. 
Transfection of cell lines with specific molecules to study antigen-specific responses.
No
No
A.4
Assays of peripheral blood and bone marrow progenitor cells including primitive and 
late erythroid progenitor-derived colonies, myelomonocytic colonies, and primitive 
multi- potential progenitor-derived colonies.
No
No
A.5
Injection of human cells into experimental animals to study the immune system and 
the growth of normal and malignant cells under varying conditions.
No
No
A.6
Testing of selection methods, cell isolation, and cell expansion leading to the 
development of new cell-based therapies requiring scale-up for clinical application.
No
No
A.7
Identification of individual T cell clones by their T cell receptor sequence.
No
No
A.8
Measurement of tumor and tissue specific antigens in cells of subjects and donors by 
mRNA,protein, or peptide expression in cells or fluids.
No
No
A.9
Laser capture micro dissection of cells from biopsies for GVHD to determine 
clonotypes.
No
No
A.10
DNA and RNA typing of genes that control immune responses in lymphocytes.
No
No
A.11
Microassay studies utilizing cellular DNA, cDNA, and RNA for neoplasia and host-
tumor interactions.
No
No
B
Molecular Hematopoiesis  Section  (Dr. Cynthia Dunbar) 
B.1
Flow cytometric analysis of cell surface and cytoplasmic proteins, including cell 
adhesion molecules, putative retroviral receptors, and markers of differentiation, 
using bone marrow and mobilized peripheral blood cells.
No
No


99-H-0064
Richard W. Childs, M.D.
July 23, 2019
39
B.2
Hematopoietic progenitor-derived colony ascertainment in vitro (as described above), 
and engraftment of immunodeficient mice for detection of human stem cell number 
and function.
No
No
B.3
Testing ability of hematopoietic progenitor cells to be transduced with retroviral, 
lentiviral, and novel gene transfer vectors in vitro.
No
No
B.4
Reprogramming of adult mature cells, including skin fibroblasts and blood cells, into 
induced pluripotent stem cells in vitro.
No
No
C
Cell Biology Section (Dr. Neal Young) 
C.1
Studies of blood and bone marrow hematopoietic progenitor numbers, including early 
and late erythroid progenitors, myelomonocytic progenitors, and multi-potential 
progenitor cells.  In addition, bone marrow may be placed in long-term bone marrow 
culture to assess the function of stroma and stem cells and to assay more primitive 
progenitors, as well as organelle culture.  Whole or selected bone marrow 
populations are cultured short-term for CD34 cell expansion.
No
No
C.2
Assays of apoptosis in hematopoietic cells and their progeny, using flow cytometric 
methods such as annexin and caspase-3 staining, propidium iodide uptake, and 
mitochondrial permeability tests.
No
No
C.3
Separation and functional study of cell populations characteristic of paroxysmal 
nocturnal hemoglobinuria, identified by absence of glycosylphosphatidylinositol 
anchored proteins.
No
No
C.4
Studies of mutation rates in hematopoietic cells and in buccal mucosa cells, using 
conventional hypoxanthine phosphoribosyltransferase activity functional assays, 
sequencing of mitochondrial DNA after specific gene amplification, and 
measurement of GPI-anchored deficient cells in blood and bone marrow.
No
No
C.5
Assays of immune function of T-cells, including intracellular cytokine staining, 
ELISPOT, semiquantitative gene amplification for gamma-interferon, tumor necrosis 
factor, interleukin-2, and other cytokines, and functional assessment in co-culture 
using specific neutralizing monoclonal antibodies.  In addition, peripheral blood 
lymphocytes are subjected to spectratyping for CDR3 size distribution as well as 
nucleotide sequence of CDR3 peaks obtained.
No
No
C.6
Studies of engraftment of human normal and diseased bone marrow and peripheral 
blood in immunodeficient mice in order to determine the presence of hematopoietic 
repopulating stem cells as well as functional differences among selected populations.
No
No
C.7
Flow cytometric analysis of blood and bone marrow for lymphocyte phenotype, 
especially for evidence of activation of lymphocytes, for markers of apoptosis, and 
for antigens associated with primitive and mature hematopoietic cell populations.
No
No
C.8
Flow cytometric analysis of blood and bone marrow for hematopoietic stem cell 
progenitors and CD34 positive cells.
No
No
C.9
Studies of chromosomal instability in myelopdysplastic syndromes including BM cell 
and CD34 cell response to PAS crosslinking and examination of the cytotoxic effect 
of lymphocytes to the abnormal clone of cells.
No
No
C.10
Surface Enhanced Laser/Desorption Ionization (SELDI)  time-of-flight mass 
spectrometry (Ciphergen)  (proteomics methodology).
No
No
C.11
Mitochondrial DNA (mtDNA) sequence heterogeneity.
No
No
C.12
Measurement of EBV viral load.
No
No
C.13
Measurement of EBV LMP-1 via RT-PCR for LMP-1 RNA or flow cytometry for 
LMP-1.
No
No
C.14
Outgrowth assay of EBV transformed B cells.
No
No
C.15
Quantification of serumchemokines and cytokines (e.g. SDF-1, IL-10, IL-6, CXCR4, 
CXCL12).
No
No
C.16
Quantification of EBV cytotoxic T cells (tetramerstaining).
No
No


99-H-0064
Richard W. Childs, M.D.
July 23, 2019
40
C.17
Telomere length measurement by Southern blot, Q-PCR, flow-fish, in situ 
hybridization and STELA
No
No
C.18
Telomere repair complex gene mutations by nucleotide sequencing of some or all of 
the following:  DKC1 ,TERC, TERT, SBDS, NOp10 , NHP2.
No
No
C.19
Analysis of  inflammatory markers and/or bacterial, viral, fungal or protozoal 
elements in plasma or serum using molecular, colorimetric, enzymatic, flow 
cytometric or other assays in subjects receiving immunosuppressive therapy, 
chemotherapy and/or bone marrow transplantation.
No
No
C.20
Confocal microscopic imaging of bone marrow.
No
No
C.21
Characterization of intracellular signaling proteins by cell permeabilization and flow 
cytometry, and quantitative immunoblots.
No
No
C.22
Assays for chromosomal aneuploidy by florescence in situ hybridization (FISH) and 
other molecular techniques.
No
No
C.23
Conversion of human dermal fibroblasts into hematopoietic progenitors using Oct4 
transfection.
No
No
D
Virus Discovery Section (Dr. Neal Young) THESE ASSAYS WILL NOT BE 
PERFORMED ON SAMPLES FROM HEALTHY PEDIATRIC DONORS
D.1
Assays of serum, blood cells, and bone marrow cells for B19 parvovirus and possible 
B19  variants using gene amplification, cell culture, and hematopoietic colony 
inhibition assays.
No
N/A
D.2
Assays of blood, bone marrow, liver, and other tissues for potentially novel viruses, 
using a variety of techniques including RNA and DNA assays, differential display, 
gene amplification with conserved and random primers, cell culture assays, 
immunohistochemical methods, and inocculation of mice, rabbits, and monkeys, as 
well as antibody measurements.
No
N/A
D.3
Assays of blood, bone marrow, and liver for known viruses, including herpesviruses 
such as cytomegalovirus, human herpesviruses 6, 7, and 8, enteric viruses such as A-
6, circiviruses, and parvoviruses, using assays as in (2).
No
N/A
D.4
Spectra-typing of blood cells to determine response to known or putative viral 
infections.
No
N/A
D.5
HLA typing or subtyping to determine risk factors/determinants for hepatitis-AA 
studies.
No
N/A
D.6
Cytotoxic lymphocyte assays with intracellular cytokine measurement for 
determining anti-viral response and lymphocyte cloning to obtain clones with 
specific antiviral activity.
No
N/A
E
Solid Tumor Section (Dr. Richard Childs)
E.1
Cr51 cytotoxicity assay to evaluating killing of patient tumor cells by patient NK cell 
clones and T-cells.
No
No
E.2
ELISA for IL-12  maturity of DC's made from subjects monocytes.
No
No
E.3
ELISA for IFN ã to evaluate specificity of CTL clones.
No
No
E.4
H thymidine uptake to evaluate proliferation potential of antigen specific T-cells.
No
No
E.5
PCR of STR to assess chimerism status of cellular subsets grown in-vitro or retrieved 
from subjects post-transplant.
No
No
E.6
Flow sorting of PBL and/or tissue samples to evaluate chimerism of different subsets.
No
No
E.7
Surface marker analysis of peripheral blood mononuclear cells using flow cytometry.
No
No
E.8
cDNA expression arrays to evaluate T-cells expression/gene patterns in subjects with 
GVHD and a GVT effect.
No
No
E.9
Geno typing of tumor or tissue samples by high density cDNA arrays.
No
No
E.10
VHL mutation analysis on kidney cancer tissue.
No
No


99-H-0064
Richard W. Childs, M.D.
July 23, 2019
41
E.11
Transduction of dendritic and tissue cells with tumor antigens using plasmids, viral 
vectors and hybrid fusions.
No
No
E.12
Lasar capture microdisection of cells from tumor biopsies and tissue samples  to 
determine origin (donor vs patient).
No
No
E.13
Quantification of polyoma virus BK exposure by serology and PCR in stem cell 
transplant donors and recipients from blood and urine samples.
No
No
E.14
Quantification of polyoma virus BK specific T cells in stem cell transplant donors 
and recipients from peripheral blood samples.
No
No
E.15
Determination of origin of neovasculature endothelial cells in tumor and tissue 
samples obtained from subjects post transplant.
No
No
E.16
Quantification of lymphocyte subsets CD34 progenitors and endovasculator 
progenitors in G-CSF mobilized peripheral cell allografts.
No
No
E.17
Testing for polyoma virus BK latency in CD34 progenitors, B cells and T cells in the 
G-CSF mobilized peripheral cell allografts.
No
No
E.18
Determination of etiology of membraneous nephropathy using serum from subjects.
No
No
E.19
Serum Proteomic patterns analysis to diagnose complications related to allogeneic 
transplantation.
No
No
E.20
Determine cell origin (donor vs patient) of tissue samples using IHC, IF, sorting, and 
FISH.
No
No
F
Lymphoid Malignancies Section (Dr. Adrian Wiestner)
F.1
Culture of cells from research subjects to investigate molecular disease mechanisms, 
model host tumor interactions, and to test effect of drugs on cell survival and cellular 
functions.
No
No
F.2
Generation of stable cell lines for the study of hematologic malignancies.
No
No
F.3
Modifications of cells using standard expression systems or biologic molecules, e.g. 
interfering RNA, to investigate the effects of candidate genes on cellular functions.
F.4
Identification and monitoring of B or T cell populations as identified by flow 
cytometry and by their B cell or T cell receptor expression.
No
No
F.5
Measurement of gene expression in cells or tissues. Techniques frequently used 
include gene expression profiling on microarrays, quantitative RT-PCR, Western 
blotting, flow cytometry and ELISA assays.
No
No
F.6
Analysis of chromosomal abnormalities or mutations in malignant cells and non-
malignant cells including FISH technology and DNA sequencing.
No
No
F.7
Assays of immune function of B-cells and T-cells, including intracellular cytokine 
staining, ELISPOT, quantitative RT-PCR for cytokines or other immune regulatory 
genes.
No
No
F.8
Analysis of antibody specificities in serum and antigen specificity of the B-cell 
receptor on cells. Techniques may include expression of antibodies in phage display 
systems, generation of antibodies in cell culture systems and use of such antibodies to 
screen for cognate antigens.
No
No
F.9
Transplantation of human cells into mice (xenograft model) to study disease biology 
and to investigate the effect of experimental therapy.
No
No
F.10
Measurements of drug concentrations, biologic molecules and disease markers in 
blood, serum, and plasma.
No
No