Conditions: Lupus Erythematosus, Systemic
Keywords: Refractory SLE Immunoablation Immunological Recovery Efficacy Mechanism of Disease Lupus Systemic Lupus Erythematosus SLE
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 9
Healthy volunteers: no

Brief summary:
This study will examine a new approach to treating patients with severe systemic lupus erythematosus (SLE) that involves collecting stem cells (cells produced by the bone marrow that develop into blood cells) from the patient, completely shutting down the patient's immune system, and then giving back the patient's stem cells. SLE is a chronic, inflammatory disorder of the immune system that can affect many organs. It is called an autoimmune disease because the patient's lymphocytes (white blood cells that normally protect against invading organisms), go out of control and attack the body's own tissues.

Patients between 15 and 40 years of age with severe SLE affecting a major organ that is resistant to standard treatment may be eligible for this study. Candidates are screened with a medical history and physical examination, blood and urine tests, skin tuberculin test, and radiology studies to evaluate the extent of disease. They have endocrinology, nutrition, dental, and social work consultations, ultrasound or MUGA (multi-gated acquisition scan) scan heart imaging, electrocardiogram and lung function tests, bone marrow biopsy, and lymph node aspirate. Depending on which organs are affected, patients may have additional tests, such as lumbar puncture (spinal tap), kidney or lung biopsy, MRI (magnetic resonance imaging) of the brain and spinal cord, and PET (positron emission tomography) scan. They also complete quality of life questionnaires and have disability functional testing and neurocognitive (thinking) assessments.

Participants have a central venous line (plastic tube) inserted into a neck or chest vein for administering stem cells and medicines and for drawing blood. They undergo seven apheresis procedures during the course of the study to collect stem cells for transplant and for research. For apheresis, whole blood is collected through a needle in an arm vein and directed to a cell-separating machine where the white cells are extracted and the rest of the blood is returned to the patient through the same needle.

Patients are primed with three medications (methylprednisolone, rituximab, and cyclophosphamide) through the central line to help control the disease. In addition, a medication called G-CSF (growth colony stimulating factor) is injected under the skin for several days to boost production of stem cells. After enough stem cells have been collected for transplantation (infusion through the central line), patients are admitted to the hospital for an 8-day conditioning regimen followed by transplantation. The conditioning treatment consists of rituximab, fludarabine, and cyclophosphamide to eliminate all the white blood cells from the blood and bone marrow. The stem cells are then infused and the patient is closely monitored by a team of physicians and nurses. When the stem cells have engrafted, the bone marrow has recovered, and the patient feels well enough - usually 2 to 3 weeks after transplant - the patient is discharged from the hospital. Prednisone tapering begins as soon as feasibly possible, but no later then 28 days after transplant.

Patients return to the National Institutes of Health (NIH) Clinical Center for frequent follow-up visits during the first 2 to 3 months following transplant. The time between visits is then extended to once every 3 months the first year, then every 6 months the second year, and then at least yearly for 5 years after the transplant. These visits include a physical examination, blood and urine tests, lumbar puncture (if there is central nervous system involvement), other appropriate biopsies and tests as needed to monitor the patient's health, short apheresis procedures to collect blood for research purposes, and quality of life questionnaires. Some select procedures will be optional. Bone marrow biopsies and lymph node aspirates are done at beginning and at 6, 12, and 24 months after transplant. PET scans are done at 1, 6, 12, and 24 months.

...

Detailed description:
Background:

* Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that can involve almost any organ and can range in severity from mild to life-threatening. In spite of significant improvements in survival of SLE patients over last 20 years, a small but significant portion of patients still develop progressive therapy-refractory disease that impairs organ function and overall survival.
* Since 1996, more than 500 patients have been treated worldwide in pilot trials of autologous hematopoietic stem cell transplantation (autoHSCT) for autoimmune diseases, including about 80 patients with SLE.
* The rationale for autoHSCT in autoimmune disease is to ablate autoreactive immune effectors and allow reconstitution of a new self-tolerant immune system from the

hematopoietic stem cell. Studies have demonstrated acceptable safety and promising short term efficacy of high-dose cyclophosphamide-based (200 mg/kg) autoHSCT for about 60% of patients with advanced refractory SLE and reacquisition of sensitivity to conventional drugs have been demonstrated in many cases. However, these trials were designed to address the primary endpoint of safety and were inadequate for assessing the disease response.

-Numerous questions about the true efficacy of autoHSCT, optimal transplant regimen, patient selection and mechanisms of action remain unaddressed.

Objectives:

* The primary objective is to assess the rate of continuous relapse-free complete clinical responses at 24 months post-transplant, with statistical power of 84% to detect, if greater than 70 percent of patients meet the primary endpoint.
* The long-term goal of this research is to develop a basis for future transplant protocols that would incorporate new cellular or other immunotherapeutic interventions to further improve results of transplants with the ultimate goal to cure SLE.

Eligibility:

-Subjects age 15-40 years who fulfill at least 4 of the 11 criteria for SLE as defined by the

American College of Rheumatology

-Have severe and active lupus, refractory to immunosuppressive therapy. Included are subjects with nephritis, central nervous system (CNS) lupus, pulmonary lupus or hematologic disease

Design:

* Fourteen patients with active and standard dose cyclophosphamide-resistant SLE will be enrolled on this phase II pilot study.
* Study design is intended to improve the efficacy of autoHSCT. A lymphoablative conditioning regimen (rituximab, fludarabine and cyclophosphamide) is explored for the first time in autoimmune disease.
* The treatment schedule consists of two parts; the priming regimen prior to stem cell mobilization and collection, and the conditioning regimen with transplant.
* In contrast to other studies, this study has precisely defined eligibility and disease response criteria with strict schema of tapering immunosuppression that should allow accurate interpretation of the treatment results.
* The study includes a carefully chosen battery of laboratory research studies designed to investigate SLE biology and mechanisms of post-transplant responses.

Arms:
arm 1: Autologous hematopoietic stem cell transplantation (HSCT) in systemic lupus erythematosus (SLE).

SLE is a chronic, inflammatory disease of the immune system. Participants received a priming, conditioning and transplant regimen. Priming regimen consisted of treatment with rituxan, filgrastim, cyclophosphamide, mesna, fludarabine phosphate, and methylprednisolone. Conditioning and transplant regimen consisted of fludarabine, cyclophosphamide, rituxan, filgrastim, mesna, diphenhydramine and stem cell transplant infusion.

Interventions: intervention 1: fludarabine phosphate intervention 2: cyclophosphamide intervention 3: Rituxan (rituximab) intervention 4: filgrastim intervention 5: methylprednisolone intervention 6: immunologic technique intervention 7: laboratory biomarker analysis intervention 8: autologous hematopoietic stem cell transplantation intervention 9: Diphenhydramine intervention 10: Mesna
Intervention descriptions: intervention 1: Conditioning and transplant regimen: 30 mg/m\^2 day intravenous infusion over 30 minutes daily, 4 days (transplant days -6, -5, -4, -3) intervention 2: Priming regimen: 2000 mg/m\^2 intravenous infusion over 2 hours, day 2. Conditioning and transplant regimen: 1200 mg/m\^2 day intravenous infusion daily, 4 days (-6, -5, -4, -3). intervention 3: Priming regimen: 375 mg/m\^2 intravenous day 1, 4. Conditioning and transplant regimen: 750 mg/m\^2 intravenous infusion, day -7. intervention 4: Priming regimen: 10 micrograms/kg/day subcutaneous, starting day 6. Conditioning and transplant regimen: 5 micrograms/kg/day subcutaneous, day +1 until ANC \>500 microliters. intervention 5: Priming regimen:1000 mg intravenous over 30 minutes, day 1. intervention 6: Lymphoablative regimen using cyclophosphamide, rituximab, and fludarabine followed by a CD34 cell selected autologous stem cell transplant. intervention 7: Standard human immunology research laboratory ex vivo studies. intervention 8: Day 0, product will be infused rapidly intravenously after premedication with diphenhydramine 25-60 mg orally or intravenous. intervention 9: Conditioning and transplant regimen 25-50 mg orally or intravenously. intervention 10: Priming regimen: 600 mg/m\^2 intravenous immediately prior to cyclophosphamide and repeat at 4 and 7 hours after the first dose, day 2. Conditioning and transplant regimen:1200 mg/m\^2 per day continuous 24 hour intravenous infusion, daily for 4 days, start concurrently with the start of cyclophosphamide.

Protocol document:
Prot_SAP_000.pdf: 
Page 1 of 115 
 
Abbreviated Title: Autologous transplantation for SLE
CC# 04-C-0095
Amendment G
Version: July 26, 2012  
PROTOCOL TITLE: A Pilot Study of Intensified Lymphodepletion Followed by 
Autologous Hematopoietic Stem Cell Transplantation in Severe Systemic Lupus 
Erythematosus  
Principal Investigator: Steven Z. Pavletic, M.D.
1
 Telephone: 301-402-4899  
 
Scientific Chairpersons:  Frances Hakim, Ph.D.
1  
Telephone: 301-402-3627  Ronald Gress, 
M.D.
1 
Telephone: 301-496-1791  
  
Associated Investigators:  
Juan Gea-Banacloche M.D.
1 
Claude Sportes, M.D.
1 
Daniel Fowler, M.D.
1
Seth Steinberg, Ph.D.
4 
Susan Leitman, M.D.
5 
Diane Arthur, M.D.6 
 
Ann Berger, M.D.13  
Howard Austin, M.D.
8 
  James 
Balow, M.D.
8 
Barbara Mittleman, M.D.
2 
Gabor Illei, M.D.
10 
Sarfaraz Hasni, M.D.
2
 Bradford 
Wood, M.D.
9 
James Shelhamer, M.D.
11 
  Thomas Hughes, Pharm.D.
12 
  
 
 Alicia Armstrong, 
M.D.
7  
Nikolay Nikolov, M.D.
3  
 
1
Experimental Transplantation and Immunology Branch, National Cancer Institute 
 
2
Office of the Clinical Director, National Institute for Arthritis and Musculoskeletal and Skin 
Diseases (NIAMS)  
3
National Institute of Dental and Craniofacial Research
  
4
Biostatistics and Data Management Section, Center for Cancer Research, National Cancer 
Institute  
5
Department of Transfusion Medicine, Clinical Center, National Institutes of Health 
 
6
National Cancer Institute, Center for Clinical Research, Laboratory of Pathology  
7
National Institute of Child Health and Human Development 
8
National Institute for Diabetes, Digestive and Kidney Diseases  
9
Department of Radiology, Clinical Center, National Institutes of Health  
10
 Human Genome Sciences, 14200 Shady Grove Road, Rockville, Maryland 
11
Department of Critical Care, Clinical Center, National Institutes of Health  
12
Pharmacy Department, Clinical Center, National Institutes of Health  
13
Department of Pain and Palliative Care, Clinical Center, National Institutes of Health  


Page 2 of 115 
 
PRECIS  
Background:
•
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that can involve 
almost any organ and can range in severity from mild to life-threatening. In spite of significant 
improvements in survival of SLE patients over last 20 years, a small but significant portion of 
patients still develop progressive therapy-refractory disease that impairs organ function and 
overall survival.   
•
Since 1996, more than 500 patients have been treated worldwide in pilot trials of 
autologous hematopoietic stem cell transplantation (autoHSCT) for autoimmune diseases, 
including about 80 patients with SLE.   
•
The rationale for autoHSCT in autoimmune disease is to ablate autoreactive immune 
effectors and allow reconstitution of a new self-tolerant immune system from the hematopoietic 
stem cell. Studies have demonstrated acceptable safety and promising short-term efficacy of 
high-dose cyclophosphamide-based (200 mg/kg) autoHSCT for about 60% of patients with 
advanced refractory SLE and reacquisition of sensitivity to conventional drugs have been 
demonstrated in many cases. However, these trials were designed to address the primary 
endpoint of safety and were inadequate for assessing the disease-response.  
•
Numerous questions about the true efficacy of autoHSCT, optimal transplant regimen, 
patient selection and mechanisms of action remain unaddressed.   
 
Objectives:  
•
The primary objective is to assess the rate of continuous relapse-free complete clinical 
responses at 24 months post-transplant, with statistical power of 84% to detect, if greater than 
70% of patients meet the primary endpoint.    
•
The long-term goal of this research is to develop a basis for future transplant protocols 
that would incorporate new cellular or other immunotherapeutic interventions to further improve 
results of transplants with the ultimate goal to cure SLE.  
 
Eligibility:  
•
Subjects age 15-40 years who fulfill at least 4 of the 11 criteria for SLE as defined by the 
American College of Rheumatology  
•
Have severe and active lupus, refractory to immunosuppressive therapy.  Included are 
subjects with nephritis, CNS lupus, pulmonary lupus or hematologic disease  
 
Design:
•
Fourteen patients with active and standard dose cyclophosphamide-resistant SLE will be 
enrolled on this phase II pilot study.  
•
Study design is intended to improve the efficacy of autoHSCT.  A lymphoablative 
conditioning regimen (rituximab, fludarabine and cyclophosphamide) is explored for the first 
time in autoimmune disease.  
•
The treatment schedule consists of two parts; the priming regimen prior to stem cell 
mobilization and collection, and the conditioning regimen with transplant.  
•
In contrast to other studies, this study has precisely defined eligibility and disease 
response criteria with strict schema of tapering immunosuppression that should allow accurate 


Page 3 of 115 
 
interpretation of the treatment results.  
•
The study includes a carefully chosen battery of laboratory research studies designed to 
investigate SLE biology and mechanisms of post-transplant responses.   
 
2  
Study Schema
Inclusion criteria  
 
 
Age 15-40 years 
> 4/11 ACR criteria for SLE  
Active and refractory 
lupus:  
-nephritis  
-CNS lupus  
-pulmonary lupus  
-hematologic disease  
Organ function acceptable for 
transplantation  
1. Priming regimen
Dose of administration  
Days 
Methylprednisolone 1000 mg i.v. 
Rituximab 375 mg/m2 i.v. 
Cyclophosphamide 2000 mg/m2 
i.v.  
G-CSF 10 μg/kg/day s.c. starting on 
until the end of the leukapheresis
day 1  
days 1 & 4  
day 2  
day 6
 
 
Leukapheresis and cell processing  
Leukapheresis starts at WBC > 5.0/μL  
CD34 positive selection by Isolex 300i, v 2.5  
Target CD34+ cell infusion dose 3 x 106/kg and at 
minimum 1.5 x 106/kg  
Cryopreserve CD34+ fraction for transplant  
Cryopreserve CD34- fraction for back-up 
Research samples from both fractions
Study design  
Phase II pilot study 
Accrual goal 14 
patients  
Primary efficacy endpoint
Continuous relapse-free complete 
clinical response at 24 mo post- 
2. Conditioning regimen and transplant
Rituximab 750 mg/m2 i.v. 
Fludarabine 30 mg/m2 i.v. 
Mesna 150 mg/m2 i.v. 
Cyclophosphamide 1200 mg/m2 
i.v. 
Mesna 1200 mg/m2 c.i.v. 
Stem cell infusion 
G-CSF 5 μg/kg/day s.c. starting 
day -7  
days -6, -5, -4, -
3 
day -6 only  
days -6, -5, -4, -
3  
days -6 to -3  
day 0  


Page 4 of 115 
 
transplant for at least 3 mo  
Study evaluations
- Clinical response and blood 
immunology studies at: baseline, after 
priming, day 0 prior to stem  cell infusion, 
at 1, 3, 6, 9, 12, 18 & 24 mo  
-Marrow biopsy and aspirate at baseline, 
6, 12 and 24 months lymph node needle 
aspirates or tissue biopsies for disease 
evaluation:  at baseline, 6, 12 and 24 
months.
day 1 until 
ANC> 500/μL 
for one day  
Steroid tapering schedule 
Tapering starts no later than day +28 post-transplant 
Prednisone at least < 10 mg/day at 6 months 
Prednisone at least < 5 mg/day at 12 months 
Indeterminate or minor flare: repeat evaluation in 1- 
2 weeks and may increase prednisone up to 
0.5 mg/kg/day for 2 weeks and taper to 
baseline over 4 weeks (allowed only once)  
Major flare = treatment failure 
   
 
 


Page 5 of 115 
 
TABLE OF CONTENTS  
PRECIS .......................................................................................................................................... 2
Study design ................................................................................................................................... 3
TABLE OF CONTENTS ............................................................................................................. 5
1
INTRODUCTION .................................................................................................................. 8
1.1
Study Objectives .............................................................................................................. 8
1.2
Background and rationale................................................................................................. 8
1.3
SUMMARY ................................................................................................................... 27
2
ELIGIBILITY ASSESSMENT AND ENROLLMENT ....................................................... 28
2.1
Eligibility Criteria .......................................................................................................... 28
2.2
Research Eligibility Evaluation: ..................................................................................... 33
2.3
Patient Registration ........................................................................................................ 36
3
STUDY IMPLEMENTATION ............................................................................................ 36
3.1
Study Design .................................................................................................................. 36
3.2
Drug Administration ...................................................................................................... 37
3.3
Treatment modifications ................................................................................................ 39
3.4
Protocol Evaluation/Monitoring ..................................................................................... 40
3.5
Concurrent therapies ...................................................................................................... 40
3.6
Off Study Criteria ........................................................................................................... 40
3.7
Post SCT Evaluation ...................................................................................................... 41
3.8
Research studies of SLE and transplantation ................................................................. 45
4
Supportive care ..................................................................................................................... 46
4.1
Infection Prophylaxis ..................................................................................................... 46
4.2
Menses Suppression and Contraception ......................................................................... 47
4.3
Blood Product Support ................................................................................................... 47


Page 6 of 115 
 
4.4
Nutritional Support ......................................................................................................... 47
4.5
Anti-emetics ................................................................................................................... 48
4.6
Hematopoietic Growth Factor Support .......................................................................... 48
5
DATA COLLECTION AND EVALUATION ..................................................................... 48
5.1
Data Collection ............................................................................................................... 48
5.2
Response Criteria ........................................................................................................... 48
5.3
Toxicity Criteria ............................................................................................................. 52
5.4
Statistical Section ........................................................................................................... 53
6
HUMAN SUBJECT PROTECTIONS ................................................................................. 55
6.1
Rationale for subject selection ....................................................................................... 55
6.2
Evaluation of Benefits and Risks/Discomforts .............................................................. 55
6.3
Risks/Benefits Analysis.................................................................................................. 56
6.4
Consent and Assent Process and Documentation .......................................................... 56
7
DATA REPORTING ............................................................................................................ 57
7.1
Adverse Event Reporting ............................................................................................... 57
7.2
Record Keeping .............................................................................................................. 59
8
PHARMACEUTICAL AND INVESTIGATIONAL DEVICE INFORMATION .............. 59
8.1
CYCLOPHOSPHAMIDE (CTX, Cytoxan, NSC-26271).............................................. 59
8.2
MESNA (Sodium 2-mercaptoethanesulfonate, Mesnum, Mesnex, NSC113891) ......... 60
8.3
FILGRASTIM (G-CSF, Neupogen®) ........................................................................... 61
8.4
FLUDARABINE (Fludara, Berlex Laboratories) .......................................................... 61
8.5
PREDNISONE ............................................................................................................... 62
8.6
RITUXIMAB (Rituxan) ................................................................................................. 63
8.7
DIPHENHYDRAMINE ................................................................................................. 66
8.8
ACETAMINOPHEN ..................................................................................................... 66


Page 7 of 115 
 
8.9
VALACYCLOVIR (Valtrex®) ...................................................................................... 66
8.10
CASPOFUNGIN (Cancidas
®
) .................................................................................... 67
8.11
TRIMETHOPRIM/SULFAMETHOXAZOLE (TMP/SMX, Cotrimoxazole, Bactrim, 
Septra) 68
8.12
BAXTER ISOLEX 300i Stem Cell Collection System, version 2.5 .......................... 69
8.13
18
FDG-PET ................................................................................................................. 70
9
APPENDICES ...................................................................................................................... 71
9.1
Appendix A .................................................................................................................... 71
9.2
Appendix B: Guidelines for prophylaxis of infectious complications in lymphodepleting 
autologous stem cell transplantation for SLE  Note: ................................................................ 78
9.3
Appendix C: Prednisone taper schema ........................................................................... 82
9.4
Appendix D: Modified SELENA-SLEDAI ................................................................... 83
9.5
APPENDIX E: SLAM (Systemic Lupus Activity Measure) ......................................... 84
9.6
Appendix F ..................................................................................................................... 90
9.7
Appendix G: ANAM ...................................................................................................... 92
9.8
Appendix H: Neurological Rating Scale ........................................................................ 94
9.9
Appendix I: Graceley Scales .......................................................................................... 95
9.10
Appendix J .................................................................................................................. 98
9.11
Appendix K: Ultrasound lymph node surveillance data sheet ................................... 99
9.12
APPENDIX L: Research Specimen Handling.......................................................... 100
9.13
APPENDIX M: Experimental Transplantation and Immunology Branch Preclinical 
Service Policy for Sample Handling ....................................................................................... 105
10 REFERENCES ................................................................................................................... 109


Page 8 of 115 
 
 
6  
1
 INTRODUCTION  
1.1
Study Objectives
1.1.1
Primary objective:  
To evaluate the rate of durable relapse-free complete clinical responses after using a 
lymphodepleting non-myeloablative regimen for autologous hematopoietic stem cell 
transplantation (autoHSCT) in patients with severe systemic lupus erythematosus (SLE).  
1.1.2
Secondary objectives:  
a) To evaluate toxicity of a lymphodepleting autoHSCT regimen in the setting of severe SLE.  
b) To evaluate biological markers of disease after autoHSCT for severe SLE.  
c) To investigate immunological efficacy and mechanisms of responses after 
lymphodepleting autoHSCT for SLE.  
1.2
Background and rationale
1.2.1
Pathogenesis  
Systemic lupus erythematosus (SLE) is the prototypic systemic autoimmune disease that can 
involve almost any organ and can range in severity from mild to life-threatening.  The 
pathogenesis of SLE is complex involving a spectrum of disease promoting mechanisms. A 
general model of pathogenesis is that in a genetically susceptible individual an initial breakdown 
in tolerance creates primary autoreactive effectors, which then propagate the autoimmune 
response by a variety of mechanisms that include positive-feedback amplification loops [1, 3]. 
Autoantibody production, complement activation, immune complex deposition, and leukocyte 
infiltration of target organs are key immunopathogenic events. There is a 24% concordance of 
disease between monozygotic twins, compared to only 2% between dizygotic twins [4] 
suggesting an important effect of the genetic background but at the same time implying an 
important role for environmental or stochastic factors. Although single gene defects can lead to 
lupus-like conditions in animal models, the genetic influence in humans is clearly polygenic and 
may be different from one patient to another. Elegant studies in mice that spontaneously develop 
lupus have demonstrated that different genetic loci are responsible for the initial loss of 


Page 9 of 115 
 
tolerance, the amplification of the autoimmune process and the severity and specific organ 
involvement [5]. Most autoantibodies in human lupus are targeted against conserved nuclear 
antigens, such as DNA, nuclear proteins and nucleosomes [6]. The original inciting event leading 
to loss of tolerance is not known, but in both animal models and humans, central tolerance seems 
to remain intact [1]. In mice immunologic abnormalities can be detected well before the onset of 
clinical symptoms suggesting that the underlying pathogenic mechanisms are different at various 
stages of the disease. At the time of clinical presentation, the immune system of patients with 
systemic lupus is characterized by a myriad of abnormalities. These include abnormalities of T-
cells, B-cells, antigen presenting cells (APC) and the complement and cytokine network. At this 
stage it is difficult to determine what the primary abnormality was, but it is evident that there are 
mechanisms that form positive feedback loops that maintain the auto-inflammatory process and 
may prevent complete remission or cure with the currently used treatments. The T cell/B cell 
interaction is key in the amplification process, therefore targeting these cells is a reasonable 
approach of therapy. Presentation of autoantigens is also abnormal in SLE and modifying the 
way these antigens are presented to the regenerating immune system may lead to tolerance. At 
this point we cannot directly influence antigen presentation, but creating a non-inflammatory 
environment and deleting the potentially “autoreactive” APCs, such as B-cells, may achieve this 
goal.  
1.2.2
B Lymphocytes in the pathogenesis of systemic lupus erythematosus  
Although the number of B cells is low or normal in SLE, they have an increased rate of 
proliferation and increased secretion of immunoglobulins, especially pathogenic IgG 
autoantibodies [7]. The increased B cell activity is T-cell dependent, as demonstrated by isotype 
switching and affinity maturation of B-cells [8] [9] [10] [11]. Additional factors contributing to 
the increased activity of B-cells may involve decreased Fc receptor mediated inhibition or an 
enhanced activity of the mutational machinery [12] [13]. This may either reflect an intrinsic 
defect in the mutational machinery or, more likely, an altered state of B cell activation that 
effects the mutational process and may be associated with an escape of self-reactive B cells from 
the elimination process in the germinal center [13, 14]. Circumstantial evidence based primarily 
on clinical observations suggests that there may be at least two different types of autoantibody 


Page 10 of 115 
 
producing B-cell populations in SLE patients. The first is exemplified by anti-dsDNA antibody 
producing cells. The anti-dsDNA titer exhibits a correlation with disease activity, and responds 
to immunosuppression. It rises before and during disease flares and decreases after 
immunosuppressive therapies. This implies that this population of autoantibody producing cells 
may be proliferating lymphoblasts or early plasmablasts that are continuously replenished in a 
rapid and transient manner that is sensitive to antiproliferative agents. The second type of 
antibody producing cells includes B-cells secreting antinuclear antibody and autoantibodies to 
Ro, La, Sm/RNP and cardiolipin. Titers of these antibodies do not usually correlate with disease 
activity or therapy. These observations suggest that B-cells secreting antinuclear antibodies or 
autoantibodies to the nucleus, Ro, La, Sm/RNP may have fully differentiated to non-
proliferating, long-lived plasma cells that secrete immunoglobulins but do not proliferate and 
thus are not susceptible to antiproliferative agents. The conventional view of the role of B-cells 
in the pathogenesis of lupus has been that B-cells function solely as producers of autoantibodies. 
Emerging data have challenged this theory and indicate that B-cells may also play a role in the 
early events of the pathogenesis of SLE. B-cells are efficient antigen presenting cells for a 
variety of autoantigens [15] and may promote the breakdown of peripheral T-cell tolerance [16] 
[17] possibly by activating populations of T cells with low affinity toward autoantigens [16].  B-
cells from patients with SLE produce higher levels of IL-10 [18] providing a positive autocrine 
feedback loop on B-cells for the production of autoantibodies. B cells may also produce other 
cytokines that could contribute to T-cell activation. The central role of B cells in the 
pathogenesis of lupus was demonstrated in a series of experiments in the MRL/lpr mouse model 
of SLE, that spontaneously develops a lupus-like autoimmune disease in an age-dependent 
manner [19].  First it was shown that B-cell deficient MRL/lpr mice do not develop 
glomerulonephritis (GN) [20] and more surprisingly, had a marked reduction in the number of 
activated memory T cells compared to B cell-intact controls [21]. The significant drop in 
activated memory T cell numbers correlated with the absence of cellular infiltrates in the kidney 
and skin suggesting a connection between T cell amplification and disease. Spontaneous T-cell 
activation was restored in mice reconstituted with B-cells that expressed membrane bound IgM 
but did not secrete soluble immunoglobulins [22]. These mice developed interstitial nephritis and 
vasculitis; histologically renal lesions were similar in appearance to the MRL/lpr animals. The 
presence of end-organ pathology in the absence of circulating antibodies supports the notion that 


Page 11 of 115 
 
the role of B cells in promoting autoimmunity goes beyond the production of autoantibodies. 
This was further corroborated by studies in which B-cell deficient mice were infused with pooled 
serum from diseased MRL/lpr mice. Despite marked glomerular immunoglobulin deposition, no 
renal disease was observed in B-cell deficient mice. Taken together, these data provided 
evidence that the physical presence of B-cells is essential for the clinical manifestations of lupus 
nephritis and T-cells are essential for tissue damage.  
1.2.3
T-cells in SLE  
The pathogenic autoantibodies in SLE show features associated with T cell dependent responses, 
including isotype-switching and somatic mutation. In fact, T-cell clones that can help autologous 
B cells to produce anti-dsDNA antibodies have been isolated from lupus patients. The majority 
of these cells were CD4+, whereas a small fraction were Įȕ TCR+CD4-CD8- and Ȗį TCR+ [23] 
[24] [15]. Similarly, autoreactive T-cell clones that respond to nucleosomes have been isolated 
from lupus patients [25]. Spectratyping of TCR ȕ-chains revealed restricted CDR3 length 
polymorphism and oligoclonal CD4+ T cell activation in peripheral blood from SLE patients. 
These effects were more pronounced in active patients compared patients in remission [26].   
1.2.4
Hypothesis for the central role of T-cell-B-cell interaction in SLE  
Although the primary event in the initiation of autoimmunity is unknown, by the time SLE is 
clinically evident there is an ongoing activation of autoreactive B- and T-cells in the lymphoid 
organs in a self-perpetuating process (Figure 1) [3] [1, 7].  
10  


Page 12 of 115 
 
FIGURE 1. Central role of T-B cell interaction in SLE. Autoantigens are presented to T 
cells in an inflammatory milieu by antigen presenting cells (a) and B cells. Once activated, 
they provide stimulation to hyper-responsive B-cells (b). These autoantigenstimulated B cells 
undergo somatic hypermutation and affinity maturation (c) and produce autoantibodies (d). 
Pathogenic autoantibodies (such as anti-dsDNA ab) deposit in the target organs and elicit an 
inflammatory response (e), leading to organ damage and release of self antigens (f) which are 
taken up by antigen presenting B cells (g) that leads to further T cell activation resulting in a 
positive amplification loop (h). Activated T cells will also migrate to the target organ (i) 
where they contribute to organ damage. Both activated B cells and T cells produce 
inflammatory cytokines that contribute to organ damage and provide an inflammatory milieu 
for abnormal presentation of auto-antigen by APCs (j). Modified from [1, 2].  
Once activated, B cells can form memory cells; differentiate into auto-antibody forming plasma 
cells or plasmablasts; and present auto-antigen to T-cells, inducing proliferation of more 
activated T cells leading to amplification of this process. Deleting B-cells when the disease is 
established may lead to a decrease in autoantigen presentation in the lymphoid organs that may 
in turn decrease the number of autoreactive T-cells and decrease both autoantibody production 
and activation of other effector cells.  Based on the animal data presented above, B-cell depletion 
may also reduce leukocyte infiltration at the sites of inflammation, and ameliorate tissue damage. 
Using agents that delete premature and mature B-cells, but not plasma cells, may decrease the 
 


Page 13 of 115 
 
levels of pathogenic anti-dsDNA antibody levels, without reducing immunoglobulin levels 
globally, which could increase susceptibility to infections. Deleting activated T cells at the same 
time may be necessary to prevent activation of remaining precursors of autoreactive B cells and 
to prevent T cell help to naturally occurring low affinity autoreactive cells that may develop 
during the regeneration of the immune system.  
1.2.5
Epidemiology  
Systemic lupus erythematosus is an autoimmune disorder affecting 24 out of 100,000 individuals 
in the United States [27].  The majority (90%) of patients is female and it is more common in 
minorities, particularly among African-Americans. Clinically, lupus is a heterogeneous disease 
ranging from a relatively mild condition to severe life-threatening disease involving major 
organs, such as the kidney, brain, lung or the hematopoetic system. Lupus nephritis is the most 
common major organ manifestation; about 80 % of patients have microscopic evidence of kidney 
involvement and about half of these develop clinically significant nephritis. Severe, potentially 
life-threatening major organ manifestations occur much less frequently.  
1.2.6
Factors associated with survival  
Overall 10-year survival rates range from 64-93% in North-American series published since 
1990. The standardized mortality rate (SMR), which compares mortality to the general 
population, was found to be four-fold increased in two different lupus cohorts [28], the increased 
mortality risk was higher in younger patients. Regarding the clinical and laboratory features of 
SLE, major organ involvement and persistent overall disease activity are by far the most 
important predictors of poor outcome [reviewed in [29] [30].  
The presence of renal disease was associated with increased risk of mortality in all studies [31] 
[32] [33] [34] [35] [36] [37] [38]. Central nervous system involvement has also been associated 
with decreased survival in several studies [32] [39] [40] [41]. Lung involvement including 
pleuritis, lupus pneumonitis, pulmonary hemorrhage and pulmonary hypertension was found to 
be a predictive factor of mortality [34]. Anemia was an independent predictor of poor survival in 
one study even after adjustment for the presence of kidney disease [32] whereas 
thrombocytopenia was an independent predictor of poor survival in several studies [33] [34] [35] 
[42]. Overall disease activity has been shown to be a risk factor for mortality in a number of 
studies [36] [43] [44] [45] [46]. Greater overall disease activity, as measured by the Systemic 
Lupus Erythematosus Disease Activity Index (SLEDAI) at the time of first visit to a lupus clinic 
and a high weighted SLEDAI score, depicting persistent disease activity were both independent 
predictors of worse survival in a Canadian cohort [34], whereas a high weighted SLEDAI score 
was associated with decreased survival in another study [47]. 
1.2.7
 Causes of Death  
The majority of fatalities among patients with SLE are attributable to active disease with major 
organ involvement, followed by infections  [30]. The infections most commonly occur in the 


Page 14 of 115 
 
setting of active SLE and in patients receiving high dose corticosteroids and/or 
immunosuppressive therapy. Comorbid conditions and other complications of therapy account 
for the remainder of deaths. During the past decade, increasing attention has been directed 
toward a potentially preventable cause of mortality: corticosteroidassociated coronary artery 
disease. In one study the incidence of myocardial infarction was nine-fold higher in SLE patients 
than in controls [48], while another study found that women with lupus in the 35-44 year age 
group were over 50 times more likely to have a myocardial infarction than women of similar age 
in the Framingham Offspring Study [30] [2]. Several studies identified corticosteroid therapy as 
risk factor for cardiovascular disease in SLE patients [29]. Thus, in addition to controlling 
traditional risk factors of atherosclerosis, efforts should be made to minimize the life-long 
cumulative dose of corticosteroids.  
1.2.8
Treatment of major organ manifestations of SLE  
Lupus nephritis is the only major organ manifestation for which effective treatment was 
established in controlled clinical trials, most of which were done at the NIH [49] [50] [51].  
The treatment of other potentially life threatening major organ manifestations, such as cerebritis, 
transverse myelitis, pneumonitis and pulmonary hemorrhage, is based on the experience gained 
in the treatment of severe lupus nephritis combined with organ-specific supportive therapy. 
Immune mediated hemolytic anemia and thrombocytopenia in SLE are treated as their idiopathic 
counterparts, AIHA and ITP [52]. The treatment of severe proliferative lupus nephritis will be 
summarized below. As mentioned above, the same principles apply to the treatment of most 
other major organ manifestations.  
There have been no controlled clinical trials proving the benefit of corticosteroids over 
supportive therapy in lupus nephritis.  Similarly, there have been no studies directly comparing 
conventional prednisone with methylprednisolone pulse therapy.  However, long clinical 
experience amply demonstrates the value of steroids and they remain the cornerstone of the 
management of patients with SLE and lupus nephritis.  
The current standard of treatment for focal and diffuse proliferative lupus nephritis has been 
established in a series of controlled studies of different regimens of cyclophosphamide and 
corticosteroids. The first of a series of controlled trials at the National Institutes of Health has 
shown that cyclophosphamide is superior to corticosteroids and azathioprine in preventing end-
stage renal disease (ESRD) [51]. The next study proved that a 6 month course of monthly bolus 
cyclophosphamide is effective in inducing renal response but prolonged courses (up to over 2 
years) of quarterly pulse cyclophosphamide are needed to maintain response [50]. The most 
recent study showed that renal remission was achieved somewhat more rapidly with the 
combination pulse methylprednisolone and pulse cyclophosphamide therapy [49]. Extended 
follow-up (median 11 years) of this study cohort, demonstrated persistent benefit of 
cyclophosphamide containing regimens compared to methylprednisolone alone. Indeed, the 
majority of the patients who initially received methylprednisolone eventually required 


Page 15 of 115 
 
cyclophosphamide for the control of renal disease. At the end of the extended follow-up, eleven 
of the 82 patients died, 20 patients had doubled their serum creatinine and 15 of these progressed 
to end-stage renal disease [53].   
Approximately one-third to one-half of patients has a relapse of nephritis after achieving partial 
or complete remission of proliferative lupus nephritis. Since each major exacerbation is expected 
to leave residual and cumulative irreversible damage aggressive treatment is recommended for 
patients with moderate or severe nephritic flares. A kidney biopsy is strongly recommended in 
ambiguous situations [54].  
Alternatives to pulse cyclophosphamide induction therapy continue to be used at various centers 
around the world. Azathioprine and mycophenolate mofetil are among the most popular. 
Regarding azathioprine, most studies indicate that this drug adds marginally to the efficacy of 
prednisone alone. Thus, at the present time, azathioprine is used as primary therapy mainly in 
milder forms of lupus nephritis, in patients strongly opposed to use of cyclophosphamide and as 
maintenance therapy after induction of remission with cyclophosphamide. Mycophenolate 
mofetil (MMF) has been claimed to be equivalent to daily oral cyclophosphamide in a small 
study of 42 patients with diffuse proliferative glomerulonephritis. All patients had significant 
improvement and there were no differences in response or early relapse rates between the two 
arms [55]. However, the flare rate in the mycophenolate group was significantly higher at the 2 
and 3 years follow-ups. The comparative efficacy of MMF is being further evaluated in several 
randomized controlled studies.  
1.2.9
Historical background for HSCT in SLE  
Since 1996 a number of investigators worldwide have carried out phase I-II studies of high-dose 
immunosuppression and autoHSCT in approximately 500 patients with autoimmune disease 
[56]. The majority of these transplants have been performed in patients with high-risk therapy-
refractory multiple sclerosis, systemic sclerosis, systemic lupus erythematosus, rheumatoid 
arthritis, juvenile idiopathic arthritis and idiopathic thrombocytopenic purpura. The stage for 
these early studies has been set by demonstrated increase in efficacy and safety of allogeneic and 
autologous HSCT for hematologic malignancies in humans as well as by experimental animal 
models showing efficacy of syngeneic and autologous transplantation in antigen-induced 
autoimmunity [57]. Additional impetus to the field was given by the number of case reports of 
patients with hematologic malignancy or aplastic anemia who had concomitant autoimmune 
disease and were rendered free of both processes after autologous or allogeneic HSCT [58], [59] 
[60]. For the reasons of safety and clarity of initial trial results, international consensus 
guidelines in 1996 recommended that initial studies of HSCT in autoimmune disease should use 
autologous hematopoietic stem cell sources rather than allogeneic [61]. There are several 
theoretical mechanisms for how autoHSCT for autoimmune disease should work. First is the 
high-dose intensity effect by analogy with the treatment of malignant lymphoma; dose-
intensified immunosuppression is supposed to overcome therapeutic resistance and result in 


Page 16 of 115 
 
prolonged anti-inflammatory effect and decrease and delay in target-tissue injury. In that case 
hematopoietic stem cells source serves solely as a vehicle for hematopoietic rescue. The second 
presumed mechanism envisions restoration of self-tolerance by eliminating the bulk of 
autoreactive cells and restoration of the naïve immune system by the process of de-novo immune 
reconstitution from the hematopoietic stem cell. This favorable reconstitution would occur either 
in the absence of the initiating autoantigen or the tolerance would be acquired by the de novo 
process of thymic education in spite of the autoantigen presence. Finally, high-dose therapy 
could be effective in eliminating cell populations which mediate complex and dysregulated 
immune circuits which are associated with the disease (concept of “resetting” the autoimmunity). 
Although there is indirect clinical evidence that at least some of these mechanisms are 
operational in the clinical autoHSCT, their relative contributions are unclear and may be 
different from disease to disease. Studies in monozygotic twins showed only a 15-30% rate of 
concordance for autoimmune disease, indicating that in spite of strong genetic component, 
environmental factors play a significant role in the development of autoimmunity. In fact, around 
70% of patients with autoimmune disease initially respond well to autoHSCT. About 2/3 
demonstrate durable remissions/stabilizations. Those patients who relapse frequently respond to 
salvage agents that were previously ineffective. Reasons for the recurrent autoimmune disease in 
patients after autoHSCT may include insufficient eradication of autoreactive lymphocytes during 
the transplant process or strong genetic component that could not be overcome by the 
immunoablative therapy.  
1.2.10 Summary of preclinical and clinical experience  
a. Animal models of hematopoietic stem cell transplantation Experiments in animal models of 
autoimmune disease provided the rationale and stimulus for clinical studies of autoHSCT in the 
treatment of a variety of severe, refractory autoimmune diseases. There are two types of animal 
models (Table 1): 1. Autoimmune-prone animals that have genetic tendency to spontaneously 
develop either generalized autoimmunity such as the classic model of SLE in NZBxNZW F1 
mice, or organ-specific autoimmunity, such as the model of diabetes mellitus in NOD mice; and 
2. antigen-induced models of autoimmune diseases such as adjuvant arthritis (AA) and 
experimental allergic encephalomyelitis (EAE). In cases of spontaneous autoimmune diseases, 
such as murine models of SLE, syngeneic bone marrow transplantation early in life has not 
prevented autoimmunity; however, allogeneic transplantation using autoimmune resistant strain 
has prevented, or in some instances when given early after the disease onset, ameliorated the 
autoimmune disease. In contrast, studies of antigen-induced autoimmune diseases in EAE and 
AA by van Bekkum and his colleagues have shown both therapeutic and preventive efficacy 
from syngeneic or pseudoautologous transplantation [62] [57].  Application of findings from 
murine lupus-like models to clinical SLE must be done with caution since these mice originate 
from highly inbred strains raised in controlled environments. In outbred human populations, 
environmental exposure and regulatory cells may have a more significant role in modulating 
disease-associated processes. Therefore, antigen-induced models of autoimmunity may be a 


Page 17 of 115 
 
much more realistic tool for translational research. One of the most important messages that 
emerged from the antigen-induced disease models such as adjuvant arthritis and EAE is that, in 
both models, the best results for syngeneic or autologous transplantation have been obtained with 
the strongest lymphomyeloablative regimens [57]. By drawing the analogy to a human situation, 
the rate of cure could be increased by intensifying conditioning regimens but with the cost of 
more toxicity - unless the target cell specificity can be improved. Since most likely target cell 
candidates in overt autoimmune disease are T and B lymphocytes, a high priority is to search for 
agents or regimens that would increase the clinically effective window between lymphotoxicity 
and myelotoxicity. It is reasonable to expect that reinfusing T and B lymphocytes in the 
inoculum would add substantially to the residual population and should be avoided.  
TABLE 1. Animal models of autoimmunity and effect of marrow transplantation [63]  
Disease 
Transplantation effect 
Spontaneous autoimmune disease  
-Models of SLE
(NZBxNZW) F1 mice  
BXSB mice 
(NZWxBXSB) F1 mice   
MRL/lpr mice
 
 
Prevented and treated by allogeneic 
transplantation   
-Model of diabetes 
mellitus NOD mice  
Prevented by allogeneic transplantation 
Antigen-induced autoimmune disease  
 
-Adjuvant arthritis     
Buffalo rat      
Treated by syngeneic and allogeneic 
transplantation  
 
Experimental allergic encephalomyelitis  
Buffalo rat      
SJL mice   
Lewis rat 
Treated by syngeneic and  allogeneic 
transplantation   
 
 
b. Phase I/II studies in SLE  
Since 1996 more than 80 patients have been enrolled in early studies of autoHSCT for severe 
systemic lupus erythematosus worldwide. This experience can be grouped in four ways: a) 
Published literature cases, b) European Bone Marrow Transplant group (EBMT) registry data, c) 


Page 18 of 115 
 
International Bone Marrow Transplant Registry (IBMTR) data, and d) Northwestern University 
experience. Marmont reported in 1997 a first case of autoHSCT where SLE was the only 
indication for transplant (non concomitant disease) in a female patient with recurrent lupus 
nephritis; her SLE relapsed after a three-year steroid-free clinical remission [64]. Since that time 
a total of 29 autoHSCT cases with severe SLE have been reported in the literature by 10 
transplant centers, most patients had severe renal, CNS, pulmonary or hematologic lupus 
manifestations after failing standard treatments (Table 2).  
 
TABLE 2.  Published results on autologous HSCT for non concomitant SLE
Author 
N 
Conditioning regimen 
Outcome
 
Marmont 1997 [64]  1  
thiotepa 15 mg/kg cyclophosphamide 
100 mg/kg  
Relapse after 3 yrs  
Fouillard 1999 [65]  
1  
BEAM  
    Relapse at 9 mo  
Trysberg 2000 [66]  
1  
cyclophosphamide 4940 mg/m2     
TBI 1000 cGy  
CNS lupus still    absent 
at 18 mo but          SLE 
relapsed at         6 mo, 
new AIHA  
Rosen 2000 [67]  
3  
cyclophosphamide 200 mg/kg rabbit 
ATG 270 mg/kg     
methylprednisolone 3000 mg 
In remission at 10, 16 
and 19 mo, on  4-5 mg/d 
PDN  
Marmont 2001 [68]  1  
thiotepa 10 mg/kg     
cyclophosphamide 100 mg/kg  
Relapse at 6 months  
Wulffraat 2001 [69]  2  
cyclophosphamide 200 mg/kg     
rabbit ATG 20 mg/kg     TBI 400 
cGy  
In remission at 12    and 
18 months, off    
immunosuppression  
Musso 2001 [70]  
3  
cyclophosphamide 100 mg/kg rabbit 
ATG 30 mg/kg methylprednisolone 
3000 mg   
2 in remission at 4 and 
30 mo, 1 on PDN 10 
mg/d at 17 mo  
Shaughnessy [71] 
2001  
1  
cyclophosphamide 200 mg/kg horse 
ATG 90 mg/kg     
methylprednisolone 3 mg/kg  
On hemodialysis at 
transplant, died at 2  mo, 
non-engraftment  
Brunner 2001 [72]  
1  
cyclophosphamide 200 mg/kg     
ATG 90 mg/kg  
Pneumonitis resolved    
normal lung function,       
in remission at 21 mo  
Traynor 2002 [73]  
15  
cyclophosphamide 200 mg/kg     
horse ATG 90 mg/kg     
methylprednisolone 3000 mg 
12 in remission 12-66   
months, 2 relapsed, 8 
completely off          
immunosuppression  
TOTAL 
29 
7 (24%) IN REMISSION ON MINIMAL STEROIDS 
13 (45%) IN REMISSION NO IMMUNOSUPPRESSION 
(only 2 remission patients with less than 12 mo follow-up) 
The majority of 29 published SLE cases (87%) responded to high-dose cyclophosphamide-based 
regimens and 20/29 (69%) maintained clinical remissions, 18/20 remissions were durable at 12- 
66 months post-transplant (Table 2). In spite of selection of very poor-risk cases for these early 
trials, only one (3%) transplant-related death was reported in a hemodialysis dependent lupus-
nephritis patient irreversible post-transplant marrow aplasia [71]. Publications of small case 


Page 19 of 115 
 
series are always prone to a possible reporting bias in favor of success. This may be overcome by 
looking at transplant registries. The EBMT international registry seated in Basel is the first 
systematic database to record activities related to BMT in autoimmune diseases and recently 
conducted a preliminary analysis of the reports in transplantation for SLE (David Jayne, personal 
communication). Fifty-tree out of 55 registered patients who received autoHSCT for SLE were 
analyzed. Cases were reported by 23 teams from 12 countries including Peoples Republic of 
China. Median patient age was 28 years (9-52). In 42/53 cases the stem cell source was 
peripheral blood rather than bone marrow and the most common method of stem cell 
mobilization was a combination of cyclophosphamide and G-CSF. Conditioning regimens were 
typically non-myeloablative, high-dose cyclophosphamide (n=32) or BEAM (BCNU, etoposide, 
cytarabine, melphalan) (n=4), with or without anti-thymocyte globulin. Eleven patients received 
TBI-based regimens and one a combination of busulfan and cyclophosphamide. Remission 
(defined as SLEDAI score <3 and prednisone < 10 mg/d) on an intention to treat basis occurred 
in 31 (58%) cases. At the median follow-up of two years, projected survival without evidence of 
disease activity was 59 +/- 17%. Eight out of 31 patients (26%) who achieved remission have 
relapsed and three have died of progressive SLE. There were seven (12%) transplant-related 
deaths in this multi-center series, three from septicemia, one from TTP, one from bleeding, one 
from secondary AML, and one from unknown causes. At the time of last follow-up steroids were 
successfully tapered only in 8 (15%) patients. Fourteen patients were still on steroids for disease 
control, and in additional individuals on post-transplant immunosuppression for disease control 
[(mycophenolate mofetil (n=12), cyclosporine (n=2) or cyclophosphamide (n=1)]. In summary, 
this EBMT experience demonstrated the feasibility of dose-intensification of immunosuppresion 
in patients with severe recurrent SLE many of whom had failed prior cyclophosphamide (70%) 
or other standard therapies. Although some patients are able to stay in remission off steroids, 
durable immunosuppresion-free disease control remains a problem in most and the curative 
potential of this strategy remains uncertain. Transplant-related mortality remains difficult to 
interpret due to the insufficient details about patient clinical status at the time of transplant and 
due to the unknown center-effect typical in such multi-center registry analyses. The International 
Bone Marrow Transplant registry (IBMTR) started tracking activities in the transplants for 
autoimmune diseases more recently, in 2001, and no formal data analysis has been yet 
performed. By November 2002, 27 transplant patients with nonconcomitant SLE have been 
registered with the IBMTR from 8 teams in the United Sates (n=25) and one team from Europe 
(n=2). One patient received an HLA-identical allogeneic sibling transplant and all others 
received autologous transplants. According to the unofficial registry information, survival data 
are available for 16 cases, of which 13 are alive at 12 (3-30) months post-transplant. All deaths 
were within 100 days post-transplant, two due to unspecified infections (one of them after 
allotransplant) and one of idiopathic pneumonia. The only significant single center series of 
autoHSCT for severe SLE is by the Northwestern University group in Chicago. Traynor et al 
published recently a five-year summary analysis of their data [73]. Fifteen patients with 
persistent and severe SLE, 7 of whom were critically ill at the time of transplant, underwent 
autologous HSCT. Stem cells were mobilized with cyclophosphamide 2.0 gm/m
2 
and G-CSF 5 


Page 20 of 115 
 
μg/kg/day. Lymphocytes were depleted from the graft by selection of CD34 positive cells. The 
conditioning regimen used was cyclophosphamide 200 mg/kg, horse antithymocyte globulin 90 
mg/kg, and methylprednisolone 3 gm. Outcome was evaluated by the SLEDAI activity index, 
serum complement levels, serologic features, function of diseased organs and 
immunosuppressive medication requirements. Median follow-up is 36 months (12-66 mo) and no 
deaths occurred post-transplant. All patients demonstrated marked improvement and the 
SLEDAI score declined to < 5 in 12 patients. Only two patients demonstrated recurrent lupus, 
one of whom responded to a course of standard doses of cyclophosphamide that were previously 
ineffective and is currently off immunosuppression. At the time of the last follow-up, 8 patients 
are completely off immunosuppresion, one is receiving i.v. cyclophosphamide and six are on 
tapering prednisone doses of 5-15 mg/day. (After this follow-up was published one patient, who 
is now followed at the NIH and achieved complete clinical remission post-transplant, 
experienced a major lupus flare with pericarditis, rapidly progressing glomerulonephritis, 
pneumonitis and severe hematologic manifestations.) An additional two patients were enrolled in 
to the Northwestern study, and underwent stem cell harvest but died before receiving the 
conditioning regimen. One died of Mucor mycosis two weeks following chemomobilization and 
the other died of lupus cerebritis. Most common toxicities post-transplant included culture 
negative neutropenic fever (n=12), fluid retention necessitating dialysis (n=4), plantar 
dysesthesia or foot drop (n=4) and bacterial infection (n= 4), five patients required transient 
transfer to the intensive care unit. Patients at highest risk for more serious toxicities were those 
who had active glomerulonephritis with concurrent pulmonary or brain manifestations.   
1.2.11 Summary of the current status in transplantation for SLE  
Although dose intensification by high-dose cyclophosphamide-based autoHSCT regimens 
demonstrates major clinical benefit in about 60% of standard-therapy refractory lupus patients, 
there are limitations associated with this procedure at its current stage. The first and major 
limitation is, assessment of the true magnitude of the benefit is obscured due to lack of 
standardization of study entry criteria, clinical response criteria, post-transplant 
immunosuppression schedule, and therapy of relapses. All these early studies were designed 
primarily as toxicity and feasibility trials, with disease response assessments being secondary 
endpoints. Patients enrolled in such studies were frequently too ill and were treated in numerous 
transplant centers, many of which had little experience in applying this treatment to SLE.  The 
second major deficiency of these early studies is the profound lack of the biological studies, so 
the immunological mechanisms that accompany disease responses in patients after autoHSCT for 
SLE are not clear. For example, relative contributions of the dose-intensification versus de-novo
establishment of immune tolerance post-transplant remain to be determined. Current clinical 
research strategies in HSCT for SLE are going in two general directions: One is to test high-dose 
cyclophosphamide-based regimens in the randomized phase III clinical trials as compared to the 
conventional NIH regimen of serial doses of intravenous cyclophosphamide [74]. If successful, 
this strategy may result in the establishment of autoHSCT as a new therapy standard for selected 


Page 21 of 115 
 
patients with severe SLE. However, based on the current clinical experience in animal models 
and humans, high-dose cyclophosphamide alone may not be sufficiently immunodepleting to 
induce cures in the autologous transplant setting [75] [76].  The second clinical research strategy 
implies that the long term goals in autoimmune disease should be achievement of the 
immunological tolerance and cure rather than the delay of disease progression or attenuation of 
symptoms. To achieve these goals, it is critical to explore new approaches to autoHSCT. One 
proposed direction is based on the data from the animal experiments that suggest the use of more 
intense myeloablative preparative regimens to accomplish more effective depletion of the 
memory cells from the host [57]. Although this hypothesis still needs its formal testing in 
humans, the currently available data demonstrate that myeloablative regimens are the major risk 
factor for transplant-related mortality after autologous transplantation in autoimmune disease, 
and this hypothetical benefit may not be without serious increase in risks [57]. An alternative 
strategy to be explored includes the development of more specific immunodepleting regimens 
which spare patient from myeloablation. The main potential disadvantages of autoHSCT are its 
perceived toxicities and costs. On the other hand, in a genetically complex multifactorial disease 
such as SLE, HSCT procedure offers a unique potential of ablation of the disease process that 
could be theoretically followed by the spontaneous or guided reconstruction of a functional 
immune system. Current conventional and novel biological therapies for autoimmune disease 
typically do not eliminate the disease activity but only suppress it with a need for life-long 
pharamacological maintenance. Toxicities of long-term therapies and increasing costs with new 
biologicals are surpassing one-time costs associated with autoHSCT procedure. The situation 
may become even more complicated since biological therapies (to be effective) may need to 
combine multiple agents with additional unpredictable compounding financial and toxic effects. 
On the other hand developing immunotherapeutics could become very valuable tools in future 
transplant regimens if their fundamental efficacy and mechanisms could be established. 
Developing a technology that has a one-time curative potential is a highly attractive therapeutic 
goal for patients with severe autoimmune disease. Study rationale  
1.2.12 Selection of the study population  
A. Disease criteria The general goal is to select patients who have therapy resistant, active severe 
lupus with the potential of long-term response. The specific disease eligibility criteria are defined 
by the following guiding principles:  
•
 Eligible patients must have severe major organ manifestations associated with increased 
mortality or severe disability due to uncontrolled disease activity and/or anticipated toxicity 
associated with conventional immunosuppressive therapy.   
•
 Subjects must have active disease manifestations that are known to respond to 
immunosuppressive therapy. Active disease must be differentiated from permanent damage (eg: 
proteinuria would be only regarded as active if the activity is supported by active sediment or a 
biopsy).  
•
 Treatment-refractory disease is defined as worsening of disease despite adequate 


Page 22 of 115 
 
immunosuppressive therapy or the inability to taper prednisone.  
•
 The minimum length of prior immunosuppressive therapy is defined specifically for each 
organ involved.  
•
 If a patient fulfills criteria on the basis of more than one major organ involvement, the 
primary target organ will be defined based on the degree of assessed risk for morbidity or 
mortality.  
 
B. Age criteria Patients eligible for this protocol will be 15-40 years old. The guiding criteria that 
determined the eligibility age range are the following:  
•
 This age range captures most SLE patients who are potential candidates for this protocol.  
•
 SLE in patients younger than 15 years of age has a different clinical course and there is 
no experience with cyclophosphamide and fludarabine in this age group.   
•
 The upper age limit is based also on the studies from our laboratory on the recovery of 
thymic function after autologous-transplant, showing the lack of the TRECs and CD4
+
 cell 
number recovery in most patients older than 40 years of age. Since certain mechanisms of the 
establishment of the post-transplant immune competence and autoantigen tolerance are 
dependent on functional thymus, patients older than 40 years of age will not be enrolled in this 
pilot study.   
•
 Age above 40 years was the main predictive factor for treatment failure in a large 
registry study of patients transplanted for autoimmune disease [77]. This supports the notion that 
age groups older than 40 should be excluded at least from the pilot studies in this setting.  
 
1.2.13 Selection of response criteria  
The ultimate goal of the study is to evaluate the sustained remission-inducing potential of 
intensive immunoablation followed by autoHSCT. The response criteria are designed to reflect 
major favorable responses of the underlying autoimmune process in the target organ with a 
concomitant improvement of the general disease activity. An important aspect of evaluating 
response is the differentiation between disease activity and irreversible damage that is already 
present at the time of autoHSCT. Since concomitant corticosteroid therapy is a significant 
confounder of outcome, the protocol includes a standard regimen of steroid taper and successful 
tapering of corticosteroids a necessary element of response. Different grades of responses 
(complete, partial or no response) are defined for every target organ. All definitions will 
incorporate three aspects of response:  
1. Response in the primary target organ.  
2. Improvement in general disease activity.  
3. Ability to taper prednisone according to a predetermined schedule. Response will be 
evaluated at various time-points. Flares will be defined for all target organs and will be 
classified as severe and mild. At the end of the study patients will be classified as complete 
responders, partial responders or non-responders based on the degree and duration of 
response as defined for the primary target organ. The occurrence or absence of flares will be 
incorporated in the final classification of patients.  


Page 23 of 115 
 
1.2.14 Fludarabine and cyclophosphamide  
It is now evident, that even if given in highest possible doses (200 mg/kg), cyclophosphamide 
does not sufficiently eliminate T-cells from the host and the recurrences of autoimmunity in the 
autologous setting or graft-rejections in the allogeneic setting are frequent problems. One 
strategy to address the insufficient lymphodepleting potential of cyclophosphamide is to use it in 
conjunction with total-body irradiation or high-doses of busulfan at myeloablative doses. 
Unfortunately, high-intensity myeloablative regimens correlate with a significant increase in the 
regimen-related mortality. An alternative strategy is to search for new regimens with better 
lymphotoxicity versus myelotoxicity ratio. In a separate line of investigation in B-cell chronic 
lymphocytic leukemia, in vitro studies demonstrated a synergistic antitumor effects of 
fludarabine and cyclophosphamide and were first to support the use of this combination in 
clinical trials [78]. These studies in BCLL showed that patients resistant to either drug respond to 
the combination of the two with good tolerance, most common side-effects being 
immunosuppression and increased incidence of herpes simplex and varicella-zoster infections.  
In studies at the NCI in a murine F1-into-parent marrow transplant rejection model, a preparative 
regimen using the combination of daily fludarabine and cyclophosphamide showed much better 
selectivity in lymphodepletion and better efficacy in preventing the graft rejection than lethal 
total-body irradiation [79]. We took these experimental results into the clinic and incorporated 
this regimen in two NCI transplant protocols, 99-C-0143 (immunoablative allogeneic 
transplantation for hematologic malignancies) and CC 00-C0119 (immunoablative allogeneic 
transplantation and graft T-cell depletion for metastatic breast cancer). Sixty patients have been 
treated on these studies and pertinent findings demonstrated profound lymphodepletion after 
fludarabine/cyclophosophamide, excellent full engraftment rate and only 3% preparative 
regimen-related mortality (one sepsis and one cardiac arrhythmia) in a very high-risk group of 
allogeneic transplant patients, many of whom had advanced age or decreased organ function. 
This daily concurrent fludarabine plus cyclophosphamide regimen has been adopted as a current 
standard preparative regimen for allogeneic stem cell transplantation at the NCI for its excellent 
immunodepleting properties and good clinical tolerance [80]. The same will be used in the 
current autoHSCT protocol for SLE. Various other schedules and combinations of fludarabine 
and cyclophosphamide are the mainstay of preparative regimens for nonmyeloablative allogeneic 
stem cell transplantation worldwide; however, no study to date addressed its role as 
lymphodepletion in transplantation for autoimmune disease.   
1.2.15 Phase I/II lupus nephritis study of cyclophosphamide plus fludarabine at NIAMS  
For their immunosuppressive effects nucleoside analogs fludarabine and cladribine have been 
explored and shown activity in systemic lupus, rheumatoid arthritis, dermatomyositis and 
polymyositis [81] [82] [83] [84, 85].  Based on these experiences, we conducted a Phase I/II 
study to evaluate a short course of low-dose cyclophosphamide combined with fludarabine in 
patients with lupus nephritis (LN) (Illei et al, manuscript in preparation). Patients with active 
proliferative LN were treated with monthly oral boluses of low-dose cyclophosphamide (0.5 


Page 24 of 115 
 
gm/m
2
 on day 1) and subcutaneous fludarabine (30 mg/m
2
 on days 1-3). Prednisone was 
aggressively tapered to a low-dose, alternate day schedule. Complete response (CR) was defined 
as stable creatinine, proteinuria < 1 gm/day and inactive urine sediment (<10 RBC/hpf and no 
cellular casts). Partial response was defined as > 50% reduction in 24 h proteinuria, if baseline > 
2 g/day, with persistently active urine sediment; or inactive urine sediment (if active at baseline) 
with proteinuria < 2 g/day if nephrotic at baseline or < 50% of baseline if non-nephrotic. The 
primary and secondary outcomes were determined at 12 months. Patients were regularly 
followed for at least 24 months to establish long-term effects of therapy. Thirteen patients were 
enrolled in the study; 11 received three or more cycles. The study was terminated early because 
of the higher than expected rate of bone marrow suppression. One patient had severe aplastic 
reaction and died from a subsequent transfusion-associated graft versus host disease (TA-
GVHD); two other patients had self-limited grade 4 neutropenia, not associated with any clinical 
sequelae. Three patients had mild leukopenia. In terms of efficacy, 8/10 patients with at least 24 
months post-treatment follow-up had sustained significant response: 5 achieved and maintained 
CR, 3 had significant response but continued to have persistent stable mild hematuria (n=2) or 
low grade proteinuria (n=1). Additional treatment was started in the 2 patients who had no 
significant response and in 1 patient with partial response who had a kidney biopsy at 24 months 
showing mild focal proliferative nephritis – much improved from her baseline. Prolonged CD4 
lymphocytopenia was noted in most patients (mean CD4: 98/uL at 7 months and 251/uL at 12 
months); this was not associated with an increased rate of infection.  
1.2.16 Rationale for Rituximab  
Rituximab is a chimeric IgG1 human/mouse anti-CD20 monoclonal antibody with human 
constant regions and mouse variable regions [86]. It is the first monoclonal antibody approved 
for clinical use in cancer and has become part of the standard therapy for patients with B-cell 
non-Hodgkin’s lymphoma. To date more than 300,000 patients have been treated with rituximab 
worldwide where this antibody has been used alone or in combinations with cytotoxic agents or 
hematopoietic stem cell transplantation. The monoclonal antibody is generally well tolerated up 
to single-infusion doses of 2,250 mg/m
2
 [87]. Adverse events are typically infusion-associated, 
mostly occur with the first dose and most commonly include chills, fever and rigor related to the 
release of cytokines. Rituximab treatment in lymphoma patients results in impaired secondary 
immune responses to recall antigens but does not result in decrease in immunoglobulin levels or 
increase in the number of infections [88]. Rituximab binds avidly to the CD20 antigen which is 
expressed on 95% of B-cell lymphoma cells and on normal B-cells and in much lower levels or 
not at all on plasma cells. Importantly, CD20 is not present on precursor B-cells or hematopoietic 
stem cells. The CD20 antigen is an appealing target for a selective immunotherapy for several 
reasons: CD20 does not circulate in the plasma, it is not shed from the surface of CD20
+
 cells 
after antibody binding, and it is not internalized or down regulated [86]. Although the function of 
CD20 is not fully understood, factors such as complement-mediated lysis, effector cell-mediated 


Page 25 of 115 
 
lysis, induction of apoptosis and interference with calcium influx into cell contribute to the 
cytotoxic activity. Infusion of rituximab to lymphoma patients results in selective depletion of 
CD20
+ 
lymphocytes lasting 6-12 months. Terminal half-life of rituximab in lymphoma patients is 
variable and ranges between 76.3 h after the first infusion to 205.8 h after the fourth infusion 
[89]. Many autoimmune disorders and especially SLE are mediated in large part by B-cells and 
the elimination of autoreactive B-cell clones provides a rationale for the increasing use of 
rituximab in these diseases. Currently, the most promising information about the efficacy of 
rituximab is available from the treatment of idiopathic thrombocytopenic purpura, autoimmune 
hemolytic anemia, rheumatoid arthritis, dermatomyositis, and type II mixed cryoglobulinemia, as 
well as in systemic lupus erythematosus [90]. For example, in rheumatoid arthritis a short 
induction dose of rituximab was well tolerated and resulted in durable responses in majority of 
patients as well as a significant additive effect if used in combination with a short course of 
relatively low-doses of cyclophosphamide or methotrexate [91].  In an ongoing study in the 
United States, Anolik et al reported on 18 patients with active but non-organ threatening SLE 
who were treated with three dose levels of rituximab, a single infusion of 100 mg/m2 (n=6), a 
single infusion of 375 mg/m2 (n=6) and a full course of four weekly doses of 375 mg/m2.  
Rituximab was well tolerated and it induced a dose-dependent depletion of CD19+ lymphocytes. 
Furthermore, the efficacy of B-cell depletion was influenced by the polymorphism of the Fc 
receptor genotype on immune effector cells but this effect seemed to be abrogated by the higher 
levels of rituximab in the higher-dose cohorts. The degree of B-cell depletion correlated with the 
clinical improvement in the symptoms of SLE, 7/12 patients who had good depletion of B-
lymphocytes (<1% CD19+ lymphocytes) had a statistically significant decrease in disease 
activity (SLAM) scores at 2-3 months post-rituximab. In contrast, those patients who did not 
achieve a good CD19+ cell depletion experienced a continuous increase in their SLAM scores 
during the same period of time [92]. Overall, the early data indicate promising safety and 
efficacy of rituximab in the treatment of patients with SLE warranting future larger trials.  
Another report from England by Leandro et al described six patients with active SLE resistant to 
standard immunosuppressive therapy who were treated on an open-label basis. During the two-
week period each patient received two 500 mg infusions of rituximab, two 750 mg infusions of 
cyclophosphamide, and high-doses of oral corticosteroids. No significant side-effects were 
observed during the follow-up. Five out of six patients had improved as evidenced by British 
Isles Lupus Assessment Group global scores from a median of 14 (range 9-27) at baseline to a 
median of 6 (range 3-8) at 6 months. Manifestations of SLE such as fatigue, arthralgia/arthritis, 
and serositis responded particularly well to this protocol. Disease parameters improved after 
rituximab in most patients as assessed by the increase in hemoglobin, decrease in the erythrocyte 
sedimentation rate, increase in complement level, and in two cases by the improvement in renal 
function and proteinuria. At the follow-up of 6-18 months, two patients are still well enough 
without additional immunosuppression despite fluctuating evidence of disease activity, two 
patients relapsed at 7 and 8 months [93]. In another report from Europe, Perrotta et al reported a 
resolution of SLE related autoimmune hemolytic anemia after two doses of 375 mg/m2 of 


Page 26 of 115 
 
rituximab that remained durable at 7 months after therapy [94].  These preliminary data on 
efficacy and safety of rituximab in SLE and its potent B-cell depleting activity provide the 
rationale for its use in combination with other cytotoxic and immunodepleting drugs for 
autologous stem cell transplantation. Immunodepleting regimens with high-dose 
cyclophosphamide or combinations of cyclophosphamide and fludarabine are more biased 
towards T-cell depletion than B-cell depletion and their use in combination with a potent anti-B-
cell agent is rational given the role of both cell populations in the pathogenesis of the disease.  
Rituximab sensitizes to and significantly enhances the efficacy of cytotoxic drugs on neoplastic 
lymphocytes in vitro [95]. Combinations of rituximab with fludarabine and cyclophosphamide 
have been shown in clinical trials to be very effective and safe in non-transplant therapy of B-
CLL, resulting in a dramatic increase of profound molecular remissions and clinical responses 
providing the rationale for further use of this combination for lymphodepletion. Khouri used with 
success a combination of rituximab, cyclophosphamide, and fludarabine in a conditioning 
regimen for non-myeloablative allogeneic stem cell transplantation in low-grade lymphoma [96]. 
The combination was extremely well tolerated with promising clinical anti-lymphoma efficacy 
and low-incidence of acute graft-versus-host disease, suggesting a potential clinical benefit of 
added depletion of malignant and non-malignant B-lymphocytes. Finally, a number of trials used 
rituximab in conjunction with autologous stem cell transplantation for patients with lowgrade 
non-Hodgkin’s lymphoma, showing promising safety and efficacy in this setting [86].  
1.2.17 Rational for priming regimen and stem cell support  
The priming regimen to be used in this study has several purposes: a) mobilization of 
hematopoietic stem cells, b) control of disease activity, c) in vivo graft-purging, and d) control of 
infusional side-effects. The addition of cyclophosphamide to G-CSF for hematopoietic stem cell 
mobilization in autoimmune disease results in better CD34+ cell yields, better control of the 
autoimmune disease and the elimination of G-CSF associated disease flares [97, 98]. The 2 
gm/m2 cyclophosphamide dose has been shown to be as effective in obtaining good CD34+ cell 
doses but is less toxic than 4 gm/m2 cyclophosphamide. The one-time bolus dose of high-dose 
methylprednisolone at the beginning of mobilization has been commonly used in autoimmune 
disease protocols to accomplish a rapid disease control, attenuate antibody-induced infusional 
side effects, and prevent flares. Several studies of autologous stem cell transplantation for B-
CLL and mantle cell lymphoma showed the efficacy and safety of rituximab when used for in 
vivo graft purging of malignant cells from the stem cell product [99-103]. Administration of 
rituximab resulted in dramatic reduction of B-cells from the graft, no interference with the 
engraftment and high-rate of long-term molecular remissions post-transplant in patients with 
advanced low-grade B-cell malignancies. In vivo administration of rituximab for B-cell depletion 
does not interfere with the procedures for the CD34 cell selection [100, 104]. There is an 
additional possibility that administration of G-CSF may enhance the effector cell function and 
improve killing of the target cells by the anti-CD20 monoclonal antibody [105-107]. To 
minimize the reinfusion of memory T-cells, hematopoietic stem cell product will be further 


Page 27 of 115 
 
depleted ex vivo of T-cells by using CD34 cell positive selection. It is expected that such 
manipulation will provide about 4 log T-cell depletion from the graft with >90% CD34 cell 
purity. The hematopoietic stem cell support in the current protocol is given to minimize the 
myelotoxicity of the regimen and to build a foundation for the future graft engineering protocols.  
1.2.18 Rationale for the proposed therapy  
The goal of the proposed treatment is to delete the majority of auto-reactive immune cells and 
create a non-inflammatory environment for the regenerating immune system in which the 
potential for the development of tolerance against nuclear antigens is maximal. 
Cyclophosphamide is standard therapy for major organ manifestations in lupus and the 
combination of low dose cyclophosphamide and fludarabine was shown to be effective in lupus 
nephritis in a pilot study. The inclusion of rituximab will ensure profound depletion of B cells. 
This regimen is expected to lead to profound and durable responses in most patients. We doubled 
the single dose of rituximab in the conditioning regimen. The rationale for this higher rituximab 
dosing is for the sake of simplicity and also to be consistent with the intention of dose-
intensification during conditioning [108]. This is based also on the evidence from the dose 
escalation trial conducted by O’Brien et al where rituximab was given safely up to the six times 
higher single doses than prescribed by the drug label [87]. Since toxicities of rituximab are most 
common during the first infusion, we still decided to adhere to the standard dose during the 
initial administration in the priming regimen. Available information supports also a clear dose-
response effect of rituximab. The ultimate goal of this study is to evaluate if reeducation of the 
immune system and cure can be achieved with intensive immunoablative therapy approaches.    
1.3
SUMMARY
This proposal is a phase II pilot study of intensive lymphoablative preparative regimen using the 
combination of fludarabine, cyclophosphamide and rituximab followed by autoHSCT for 
patients with severe SLE. The primary goal is to assess the rate of durable complete clinical 
responses. It is expected that such transplant regimen with attenuated doses of cyclophosphamide 
would result in improved efficacy/toxicity ratio in treating patients with severe SLE. The study 
also incorporates tightly defined patient selection and clinical response criteria and precise 
definitions for the use of post-transplant immunosuppression and therapy of flares. Such strictly 
defined criteria should improve the interpretation of the results of the study. Major components 
of this research are the in-depth studies of SLE biology and transplantation immunology. The 
long-term goal is to develop a basis for future transplant protocols that would incorporate new 
cellular or other immunotherapeutic interventions to further improve results of transplants with 
the ultimate goal of cure for SLE. 


Page 28 of 115 
 
2
 ELIGIBILITY ASSESSMENT AND ENROLLMENT  
2.1
Eligibility Criteria  
2.1.1
Inclusion criteria  
1. Age 15-40 years  
2. Must fulfill at least 4 of the following 11 criteria for SLE as defined by the American College 
of Rheumatology [109, 110]:  
 
•
Malar rash.  Fixed erythema, flat or raised, over the malar eminences, tending to spare 
the nasolabial folds.  
•
Discoid rash.  Erythematous raised patches with adherent keratotic scaling and follicular 
plugging; atrophic scarring may occur in older lesions.  
•
Photosensitivity.  Skin rash as a result of unusual reaction to sunlight, by patient history 
or physician observation.  
•
Oral ulcers.  Oral or nasopharyngeal ulcerations, usually painless, observed by a 
physician.  
•
Arthritis.  Nonerosive arthritis involving two or more peripheral joints, characterized by 
tenderness, swelling, or effusion.  
•
Serositis.  a.) Pleuritis - convincing history of pleuritic pain or rub heard by a physician or 
evidence of pleural effusion OR b.) Pericarditis - documented by ECG or rub or evidence of 
pericardial effusion  
•
Renal disorder.  a.) Persistent proteinuria greater than 0.5 grams per day or greater than 
3+ if quantitation not performed OR b.) Cellular casts – may be red cell, hemoglobin, granular, 
tubular, or mixed.  
•
• Neurologic disorder.  a.) Seizures – in the absence of offending drugs or known 
metabolic derangements; eg, uremia, ketoacidosis, or electrolyte imbalance  
•
OR b.) Psychosis - in the absence of offending drugs or known metabolic derangements; 
eg, uremia, ketoacidosis, or electrolyte imbalance  
•
Hematologic disorder. a.) Hemolytic anemia – with reticulocytosis OR b.) Leukopenia – 
less than 4000/μL total on two or more occasions OR c.) Lymphopenia – less than 1500/μL on 
two or more occasions OR d.) Thrombocytopenia – less than 100,000/μL in the absence of 
offending drugs  
•
Immunologic disorder.  a.) Anti-DNA: antibody to native DNA in abnormal titer OR b.) 
Anti-SM: presence of antibody to SM nuclear antigen OR c.) Positive finding of 
antiphospholipid antibodies based on (1) an abnormal serum level of IgG or IgM anti-cardiolipin 
antibodies, (2) a positive test result for lupus anticoagulant using a standard method, or (3) false 
positive serologic test for syphilis known to be positive for at least 6 months and confirmed by 
Treponema Pallidum immobilization or fluorescent treponemal antibody absorption test  
•
Antinuclear antibodies.  An abnormal titer of ANAs by immunofluorescence or an 
equivalent assay at any point in time in the absence of drugs known to be associated with “drug-
induced lupus” syndrome.  
 


Page 29 of 115 
 
3. Have severe and active lupus, refractory to immunosuppressive therapy, defined as one of 
the following (a-d):  
a. Nephritis: Biopsy proven Diffuse Proliferative Glomerulonephritis (WHO Class IV) with or 
without superimposed membranous changes  
i. Active disease:  
1. A kidney biopsy within three months of enrollment showing active WHO Class IV disease. 
Activity will be determined based on the presence of endocapillary cellular proliferation 
compromising the capillary loops or cellular crescents or necrosis on light microscopy or 
subendothelial deposits on electron microscopy.  
2. If a biopsy is contraindicated patients can be enrolled if they had a previous biopsy showing 
Diffuse Proliferative Glomerulonephritis (WHO Class IV) and at the time of enrollment have 
all of the following:  
a. Proteinuria > 1gm/day  
b. Active urine sediment defined as hematuria (> 10 RBC/hpf on a nephrology urinalysis of a 50 
mL urine sample) with dysmorphic RBC and/or cellular casts  on a nephrology urinalysis of a 50 
mL urine sample  
c. Low C3 (<69 mg/dL) and/or elevated dsDNA antibodies (>25EU) 3. Need for prednisone >20 
mg/day due to increased renal activity after at least 6 months of cyclophosphamide.  
ii. Treatment resistant: Patients with active disease after at least 6 months of mycophenolate 
mofetil or intravenous pulse cyclophosphamide ± iv methylprednisolone and daily oral 
prednisone, or  
Early flare: those who have reactivation of their nephritis during or within 6 months of 
completing cyclophosphamide therapy  
Recalcitrant disease: two or more recurrences of lupus nephritis within five years of enrollment. 
All flares must have received adequate therapy and at least one of the episodes must have been 
treated with minimum 6 months of intravenous pulse cyclophosphamide ± iv methylprednisolone 
and maintenance oral prednisone. 
 b. CNS lupus: Lupus CNS manifestations indicative of encephalitis or myelitis or vasculitis. 
Concomitant CNS diseases should be excluded. (e.g. infections, multiple sclerosis; patients 
fulfilling MS and SLE criteria will be excluded). Clinical signs and symptoms must be supported 
by objective findings of CNS inflammation.  
i. Active disease:  
Signs/symptoms that are accepted for disease activity:  


Page 30 of 115 
 
x
Clinical signs and symptoms compatible with focal CNS damage   
x
Severe global neurocognitive/psychiatric impairment (eg: psychosis, organic brain syndrome, 
severe depression)  
x
Intractable seizures       
Clinical findings must be supported by at least one of the following: 
a) MRI findings consistent with transverse myelitis or CNS vasculitis   
-Signs of inflammation on MRI are either the presence of Gadolinium (Gd)-
enhancing lesions, or the increase of the number and/or volume of T2-
weighted lesions (or lesions showing up on FLAIR imaging). We will use the 
standard MS protocol sequences, which are routinely used in the Clinical 
Center to evaluate inflammatory CNS lesions.  
b) If patient has seizures/psychiatric signs and symptoms in the absence of clear signs 
of vasculitis or cerebritis by MRI, the CSF should show protein elevation 
above normal levels and abnormal number of WBCs or intrathecal IgG 
synthesis/or oligoclonal bands.  
c) Need for prednisone >20 mg/day due to increased CNS activity (see above) after at 
least three monhs of cyclophosphamide therapy.  
ii. Treatment resistant:  
a) Active disease after a minimum of three months of oral or intravenous cyclophosphamide, or  
b) Early flare: reactivation of CNS lupus within 6 months of completing cyclophosphamide 
therapy  
c) Recalcitrant disease: two or more recurrences of CNS lupus within five years of enrollment. 
All flares must have received adequate therapy and at least one of the episodes must have 
been treated with minimum 3 months of oral or intravenous cyclophosphamide  
c. Pulmonary lupus  


Page 31 of 115 
 
 Recalcitrant disease: two or more recurrences of pulmonary as described above within five years 
of enrollment. All flares must have received adequate therapy and at least one of the episodes 
must have been treated with minimum 3 months of oral or intravenous cyclophosphamide  
d) Hematologic disease
i) Active disease:  
a) Severe immune-mediated thrombocytopenia (platelet count < 20,000/mm
3
 or < 50,000/mm
3
 
with history of bleeding), or b) Severe immune-mediated anemia (requiring transfusions to 
maintain Hb> 8.0 g/dL or to treat symptoms of anemia) c) Need for prednisone >20 mg/day due 
to increased hematologic lupus activity after therapy as described in section ii.a).  
ii) Treatment resistant:  
a) Active disease as defined above after a minimum of three months of high dose oral or pulse 
corticosteroids ± IVIg (or WinRho) and splenectomy, or  
b) Early flare: reactivation of hematologic lupus (as defined above) within 6 months of 
completing above therapy.  
i.  
Active disease:  
a)  
Lung biopsy  showing active pneumonitis, alveolitis  or pulmonary vasculitis 
after the minimally required therapy within one month of enrollment or  
b)  
If a biopsy is contraindicated within one month of enrollment, patients may be 
included if they had a biopsy at the start or during cyclophosphamide treatment 
showing active pneumonitis, alveolitis or pulmonary vasculitis (as above) and 
have abnormal or worsening pulmonary function tests with a chest CT 
consistent with active pneumonitis, alveolitis or vasculitis within 2 weeks of 
enrollment and at the time of enrollment have a CT consistent with active 
disease. 
c)  
Need for prednisone >20 mg/day due to increased pulmonary lupus activity 
after minimum of three months of cyclophosphamide. Treatment resistant:  
d)  
Ongoing or recurrent active pulmonary lupus after a minimum of three months 
of oral or intravenous cylophosphamide, or Early flare: reactivation of 
pulmonary lupus (as defined above) within 6 months of completing 
cyclophosphamide therapy.  
  


Page 32 of 115 
 
c) Recalcitrant disease: two or more recurrences of immune-mediated thrombocytopenia or 
anemia, as described above, within five years of enrollment. All flares must have received 
adequate therapy and at least one of the episodes must have been treated by splenectomy.  
2.1.2
Exclusion Criteria  
1. Inability to provide written informed consent prior to entry in the protocol  
2. Pregnant or lactating women. Women of childbearing potential are required to have a 
negative pregnancy test at screening  
3. Women of childbearing potential who are not practicing or who are unwilling to practice 
birth control during the entire study   
4. Men who are unwilling to practice birth control for the first 6 months after the transplant  
5. Evidence of infection with hepatitis B, hepatitis C, or HIV  
6. History of malignancy other than basal cell carcinoma of the skin  
7. DLCO corrected <45%  
8. LVEF <45%, determined by ECHO cardiogram or MUGA   
9. SGOT or SGPT >2x upper limit of normal (unless active myopathy is proven by elevation of 
serum aldolase levels and the patient has no obvious hepatic disease) and/or bilirubin >2.0 
(unless due to isolated hemolysis).   
 


Page 33 of 115 
 
10.Calculated glomerular filtration rate < 30 ml/min using the MDRD equation estimate  
[111] :  
GFR (ml/min/173 m
2
) =186.3 x (Pcr) 
-1.154
 x (age) 
-0.203
 x 1.212 (if black) x 0.742 (if female)  
 Late flare (patients who have a single episode of target organ flare, that is not within the time 
frame defined as early flare), will not be considered as treatment failures until they receive the 
minimally required therapy for this flare episode and fail to respond to it  
 Abnormal bone marrow cytogenetics  
 Significant concurrent medical condition or any significant circumstance that could affect the 
patient's ability to tolerate or complete the study  
 Live vaccines within 4 weeks of starting the priming regimen  
 
2.1.3
Concurrent therapy  
It is anticipated that all subjects prior to enrollment in this protocol will be on 
immunosuppressive therapy and moderate-to high doses of corticosteroids. It is also anticipated 
that the priming regimen will provide short-term control of lupus. Therefore, all 
immunosuppressive and disease modifying treatments, with the exception of steroids, will be 
discontinued before the beginning of the priming regimen. Analgesics (except NSAIDs) 
including narcotics will be allowed if needed. Flares that occur before HSCT will be treated with 
corticosteroids. Prednisone will be tapered according to a predetermined schedule based on the 
dose at the time of transplant. The treatment of flares after HSCT is specified later in the protocol 
(section 5.2.5). The following treatments must be discontinued before mobilization;  
a. Pulse cyclophosphamide at least 3 weeks before mobilization  
b. Antimalarials at least 2 weeks before mobilization  
c. Methotrexate, mycophenolate mofetil, azathioprine, oral daily cyclophosphamide, 
leflunomide, cyclosporine or any other medication for lupus at least 1 week before mobilization  
 
2.2
Research Eligibility Evaluation:  
2.2.1
To confirm the eligibility status the following clinical, laboratory and radiologic 
screening assessments must be performed at the time of the evaluation at NIAMS:  
a) Complete medical history and physical examination  
b) CBC with differential, PT, PTT, ESR  
c) Chem 20 panel  
d) ANA, ENA, dsDNA, RF, C3, C4, lupus anticoagulant, anticardiolipin antibody, 
direct and indirect Coombs test   


Page 34 of 115 
 
e) ABO typing, and infection disease markers: Anti-HIV-1/2, Anti-HTLV-I/II, Anti-
HCV, Anti-HBc, HBSAg, Anti-HA, Anti-CMV, NAT (nucleic acid testing) for 
HIV, HCV and WNW, EBV, adenovirus, HSV and BK virus serology, RPR, 
Toxo-titers  
f)
 Nephrology and routine urine analysis  
g) Urinalysis  
h) 24 hrs urine collection x2 for renal SLE pts only  
i)
TSH, free T3, free T4  
j)
Pregnancy test  
k) Lumbar puncture if CNS SLE  (unless contraindicated)  
l)
Kidney biopsy for renal SLE (unless contraindicated) and lung biopsy for 
pulmonary SLE (unless contraindicated). The Laboratory of Pathology will 
review all biopsy specimens of SLE involved organs. CCR; kidney biopsies will 
be sent to AFIP as per routine CC policy  
m) Bronhoalveolar lavage to exclude active infection if pulmonary SLE  
n) Chest CT for pulmonary SLE 
o) MRI scans for CNS SLE 
p) Neurocognitive testing, if necessary for evaluation for NPSLE 
q) Other diagnostic studies as clinically indicated to evaluate SLE disease activity  
r)
STATCLOT  
Additional testing to be performed as part of the eligibility screening at the NCI:  
s) History and physical  
t)
Female reproductive endocrinology consultation  
u) Chest x-ray  
v) Social work consultation  
w) Cardiac ECHO or MUGA for ejection fraction, ECG  
x) Pulmonary function tests with DLCO measurements  
y) Unilateral bone marrow aspirate and biopsy (CBC/Diff + Lym Pheno TBNK 
panel at the same day with bone marrow biopsy) for flow cytometry, conventional 
and FISH cytogenetics and research studies  
z) Other specific serum or urine diagnostic studies clinically appropriate for the 
health status evaluation  


Page 35 of 115 
 
2.2.2
Following studies should be completed at the NCI prior to starting the priming regimen 
(duplicate testing will not be performed, unless medically indicated, if the previous test 
was done within two weeks): 
a) History and physical  
b) CBC, differential, acute care panel, hepatic panel, mineral panel  
c) ANA, ENA, dsDNA, RF, C3, C4, lupus anticoagulant, anticardiolipin antibody  
d)  Nephrology and routine urine analyses  
e) Urinalysis  
f)
Pneumococcal and tetanus serum titers  
g) FSH, LH, Estradiol (female) and Testosterone (male), Inhibin A and B  
h) Nutritional assessments consult  
i)
Social work consults  
j)
Dental consult  
k)  HLA-A, -B, and –DR typing  
l)
PPD  
m) Placement of central venous line  
n) Lymph node ultrasound survey and fine needle aspirates for conventional 
cytology and research studies  
o) FDG-PET scan focused on the lymphoid organs  
p) Leukapheresis for the peripheral blood mononuclear cells for baseline research 
studies  
q) Storage of mononuclear cells, RNA and sera for future research  
r)
Quality of life assessments  
s) Neurocognitive testing: every patient able to cooperate will undergo 
neurocognitive testing with ANAM, a computerized neurocognitive test that is not 
language dependent. Cooperative patients with good English will also have a set 
of traditional neurocognitive tests. We will apply a panel that is used in a study 
evaluating neurocognitive function in SLE  
t)
SLEDAI, SLAM, SLICC-ACR damage index.  


Page 36 of 115 
 
2.3
Patient Registration  
2.3.1
The patient's entry date on protocol is considered to be the day that treatment consent 
forms have been signed by the patient. The treatment start date is considered to be the 
day the recipient begins her/his priming regimen.  
2.3.2
Authorized staff must register an eligible candidate with Central Registration no later 
than 24 hours after patient has signed consent forms. A registration checklist must be 
completed confirming eligibility and faxed to the NCI Central Registration Office at 301-
480-0757. After confirmation of eligibility at the NCI Central Registration Office, the 
NCI Central Registration Office staff will call Pharmacy to advise them of the acceptance 
of the patient on the protocol.  For registration, authorized staff should call 301-402-1732 
between the hours of 8:30 a.m. and 5:00 p.m., Monday through Friday.  A recorder is 
available during non-working hours.  
2.3.3
Principal investigator will hold a formal meeting of the coinvestigators that will 
summarize and document the eligibility criteria for each patient prior to entry onto study. 
Meeting participants will include at minimum: principal investigator, clinical 
chairperson, one study physician with marrow transplant expertise, one study physician 
with SLE expertise, one physician with primary lupus-affected organ expertise; study 
research nurse, and an external physician with SLE expertise who is not a co-investigator 
on this study (Sandy Via, MD, of University of Maryland agreed to fulfil this function for 
the purpose of this protocol as a primary external SLE expert). In case the primary 
external expert is not available or in an unusual situation where the external expert is at 
the same time a referring physician, an alternate independent SLE expert will be 
designated by the PI or the clinical chairperson. The study eligibility will be properly 
documented and signed by all meeting participants. If necessary, the meeting can also 
include communication over the phone in the form of a conference call – as long as all 
necessary documentation was provided to the participating investigators and experts.  
3
STUDY IMPLEMENTATION  
3.1
Study Design
This is a phase II pilot study of autologous hematopoietic stem cell transplantation with the goal 
to enroll 14 patients. The treatment schedule consists of two parts, the priming regimen and the 
conditioning regimen with transplant.  


Page 37 of 115 
 
3.2
Drug Administration
3.2.1
Priming regimen:  
Drug 
Dose 
Days
 
Methylprednisolone* 
 
1000 mg i.v. over 30 minutes 
on day 1 
Rituximab 
375 mg/m2 i.v 
on days 1 and 4 
Cyclophosphamide 
2000 mg/m2 i.v. infusion/2 
hours 
on day 2 
Mesna 
600 mg/m2 i.v. immediately 
prior to cyclophosphamide and 
repeat at 4 and 7 hours after 
the first dose 
on day 2 
 
Filgrastim (G-CSF) 
10 ȝg/kg/day s.c. 
starting day 6** 
*An alternative parenteral glucocorticoid may be used at equivalent doses depending on 
the availability. **G-CSF is given daily approximately at 7.00 AM until the last day of 
leukapheresis, G-CSF can be stopped earlier if total WBC exceeds 100,000/μL.   
3.2.2
 Hydration with 0.9% NaCl i.v. will begin at 500 cc/hr and run for one hour  before 
starting cyclophosphamide, then will be continued at 200 cc/hr during cyclophosphamide 
and then at 250 cc/hr for additional 5 hours after  finishing cyclophosphamide infusion. 
Mesna will be given by i.v. route over 15-30 minutes, 600 mg/m
2
 immediately prior 
cyclophosphamide (time 0),  and then 4 and 7 hours after the start of the 
cyclophosphamide.   
- Chem 20 including serum potassium level will be checked before and 8 hours in 
to hydration. Serum potassium will be supplemented as clinically necessary. 
During hydration, 20 mg of furosemide can be administered once by i.v. route to 
maintain diuresis, with additional doses of furosemide to be given as needed for 
weight gain due to fluid retention. In general, furosemide doses should be 
separated by at least a four-hour observation interval. Routine hematologic 
monitoring, cardiac monitoring, and urinalysis will be performed as deemed 
clinically necessary.  


Page 38 of 115 
 
3.2.3
Patients will visit the DTM Dowling Apheresis Clinic during the week prior to apheresis 
for a venous assessment.  Prior to starting the priming regimen all patients will be 
scheduled for placement of an apheresis compatible central venous catheter by the 
Vascular Access Device (VAD) service of the Critical Care Medicine Dept (10D).  
3.2.4
Apheresis will start on the day the WBC first exceeds >5,000/μL. 15-25 liters will be 
processed daily. At least one hour will elapse between G-CSF administration and the 
beginning of leukapheresis. Apheresis and filgrastim administration will continue daily 
until a target cell dose of at least 3 x 10
6
 CD34+ cells/kg-recipient weight is obtained 
after completion of cell processing and allocation of research samples, or until 3 
consecutive procedures are performed, whichever comes first. Starting at latest on day 10 
after the priming dose of cyclophosphamide patients will be started on early morning 
monitoring of CBC and CD34 counts so that samples arrive to the laboratory by 6.30-
7.00 AM and the apheresis can start by 7.30-8.00 AM. Unless the WBC is rising very 
rapidly from the prior day, leukapheresis for stem cell collection will not start unless the 
circulating CD34 cell count is >8/μL. Maximal total number of peripheral blood stem cell 
collection procedures will be 3.  
3.2.5
T-cell depletion will be performed in the cell processing laboratory of the  Department of 
Transfusion Medicine using the Baxter Isolex 300i Stem Cell   Collection System version 
2.5, for CD34 positive selection.  The infused progenitor cell target dose will be > 3 x 10
6
 
CD34+cells/kg, it  will be attempted not to infuse >8 x 10
6
 CD34+cells/kg. 
Transplantable  minimal progenitor cell dose to be infused will be 1.5 x 10
6
 CD34+ 
cells/kg, patients who do not collect minimal number of stem cells will not be evaluable 
for the primary endpoint and will go off study. Samples of the  stem cell product will be 
obtained for extended immunophenotyping  including CD3, CD4, CD8, CD19, CD20, 
CD56, CD14, CD33, and CD34  surface markers and for the FISH cytogenetics. Cells 
will be cryopreserved and stored according to DTM policy and procedure until needed 
for infusion.  
3.2.6
Conditioning Regimen and Transplant: Three weeks after administration date of 
cyclophosphamide patients will become eligible to start the transplant conditioning 
regimen. Any non-hematologic toxicities of mobilization must resolve to at least < grade 
2 in order to proceed. If more than 14 days of additional recovery time is required for 
resolution of toxicity or other medical complications (for example documented infection), 
PI approval is required prior to initiation.  
Transplant Conditioning Regimen
Drug
Dose and administration 
Days* 


Page 39 of 115 
 
Rituximab 
750 mg/m2 i.v. infusion
Transplant Day -7 
Fludarabine
30 mg/m2 per day i.v. infusion 
over 30 minutes, daily for 4 days
Transplant Days -6, -5, -4, -3 
Cyclophosphamide
1200 mg/m2 per day i.v. infusion 
over 2 hours, daily for 4 days
Transplant Days –6, -5, -4, -3 
Mesna 
1200 mg/m2 per day continuous 
24 hr i.v. infusion, daily for 4 
days, start concurrently with the 
start of cyclophosphamide
Transplant Days –6, -5, -4, -3 
Stem cell infusion 
Product will be infused rapidly 
i.v. after premedication with 
diphenhydramine 25-50 mg orally 
or i.v.
Day 0 
Filgrastim (G-CSF) 
5 ȝg/kg/d s.c.
Day +1 until ANC >500/ȝL
3.2.7
Hydration during the conditioning regimen:  
a) Hydration will be initiated 12 hours prior to cyclophosphamide infusion (on day –7 of the 
transplant), consisting of 0.9% sodium chloride supplemented with 10 mEq/liter 
potassium chloride (KCl) at an initial rate of 100 ml/hour.  For patients with poor oral 
intake, the rate of hydration may be increased as clinically indicated to meet fluid 
requirements. Hydration will continue until 24 hours after the last cyclophosphamide 
dose has been completed.  
b) During hydration, serum potassium level will be monitored every 12 hours. If serum 
potassium is > 4.5 mEq/l, KCl will be removed from the saline infusion. If serum 
potassium is < 3.0, KCl concentration in the saline will be increased to 20 mEq/L.  
c) During hydration, 20 mg of furosemide will be administered daily by IV route to 
maintain diuresis, with additional doses of furosemide to be given as needed for weight 
gain due to fluid retention. In general, furosemide doses should be separated by at least a 
four-hour observation interval.  
d) During hydration, if fluid intake exceeds urine output by greater than 500 ml during an 8-
hour period, an additional 20 mg of furosemide will be administered.  
e) Routine hematologic monitoring, cardiac monitoring, and urinalysis will be performed as 
deemed clinically necessary.  
3.3
Treatment modifications 
 Patients with GFR 30-70 ml/min/1.73m
2 
will have fludarabine dose reduction of 20%.  


Page 40 of 115 
 
3.4
Protocol Evaluation/Monitoring
3.4.1
The following studies will be obtained in the patient during hospitalization for 
transplantation:  
3.4.1.1  CBC and differential count twice daily.  
3.4.1.2  Chem 20 twice daily during the conditioning, then daily.  
3.5
 Concurrent therapies  
3.5.1
 Corticosteroids:  
3.5.1.1  Pre-HSCT: all immunosuppressive and disease modifying drugs will be discontinued 
before mobilization. Flares in disease activity before HSCT will be controlled with 
corticosteroids as clinically indicated. The daily dose of prednisone will be maximized 
at 1mg/kg with 80 mg/day being the maximal dose; if a patient requires higher doses, 
they will be treated with pulse methylprednisolone (1 gm methylprednisolone i.v. for 1-
3 days).  
3.5.1.2   Post-HSCT: corticosteroids will be tapered according to Appendix C. Tapering can be 
started as early as clinically feasible as but not later than Day 28 post transplant. The 
dose of prednisone will be decreased every two weeks by the equivalent of 10 mg/day 
for doses > 35mg/day and by the equivalent of 5mg/day for doses 35 mg/day. After 
reaching 20 mg/day a taper to alternate day dosing will be attempted. If patients show 
no clinical sign/symptoms of lupus activity, a more rapid taper can be attempted; if this 
results in reappearance of disease activity the dose can be increased to the dose shown 
in Appendix C Table for that time point. This will not be considered a flare. The taper 
can be suspended in case of signs and symptoms of adrenal insufficiency or severe 
steroid withdrawal. Stress doses of steroids can be used for intercurrent infections or 
other stressful situations, when clinically indicated.  The goal is to discontinue 
prednisone unless signs and symptoms of adrenal insufficiency or severe steroid 
withdrawal develop.   
3.6
 Off Study Criteria
3.6.1
Patients will be removed from protocol for any of the following reasons:  
a) Inadequate stem cell collection (Section 3.2.4).   
b) Irreversible non-hematologic toxicity (> grade 3) during the priming.  
c) The patient refuses to continue therapy or withdraws consent.  


Page 41 of 115 
 
d) No response to therapy (see 5.2.6). Patient can still be followed every 6 months up to 
five years for survival and the SLE treatment history data.  
e) In addition, the patient may at any time be removed from protocol at the principal 
investigator’s discretion, if the PI deems the patient to be at unacceptable risk to 
remain on study.  Reasons for this action may include (but are not limited to) disease 
progression with declining organ function/performance status before transplantation; 
inadequate family/caregiver support; noncompliance.  
3.6.2
Authorized physicians must notify Central Registration office when a patient is taken off 
study.  
3.7
Post SCT Evaluation
3.7.1
After completion of therapy the patient will be followed for potential complications 
related to HSCT. After discharge from the transplant inpatient service, the patient will be 
followed at least twice weekly in the NCI outpatient setting until Day +28 and then at 
least weekly until discharge home. The patient will be seen thereafter in follow-up at the 
Clinical Center to evaluate disease status, immunological status and late problems related 
to HSCT, at minimum monthly for the first six months, and then at 9, 12, 18, and 24 
months post-transplant, then yearly up to five years post-transplant, or at the time of the 
disease flare or progression. Evaluation of immunological status will include assessments 
of peripheral blood lymphocyte and mononuclear cell subpopulations by flow cytometry 
to determine the efficacy of lymphocyte depletion and the time course of lymphocyte 
reconstitution. Enclosed is the flow-chart summarizing the standard pre and post-
transplant evaluations: 


Page 42 of 115 
 
 
3.7.2
Study Calendar 
 
Study Evaluations  
Screening 
NIAMS NCI  
Baseline
* 
 After 
Primin
g  
Conditio
ning Day 
-7  
Conditioni
ng Day -1  
Scheduled Follow-up**  
History and physical  
x x  
x  
 
x  
x  
x  
CBC and differential   
x x  
x  
 
x  
x  
x  
ESR  
x  
x  
 
x  
x  
x  
PT/PTT  
x  
 
 
 
 
 
Chem 20 panel  
x  
x  
 
x  
x  
x  
ANA, ENA, dsDNA, RF, C3, 
x  
x  
 
x  
x  
x  
ABO typing, TTV viral 
screen  
x  
 
 
 
 
 
Lym Pheno TBNK panel  
x  
 
 
 
 
At 4, 5, 7, 8 mo  
Lupus anticoagulant  
x  
x  
 
x  
 
x  
Anticardiolipin antibody  
x  
x  
 
x  
 
x  
STATCLOT  
x  
 
 
 
 
 
Direct and indirect Coombs 
test  
x  
 
 
 
 
x  
Urinalysis  
x  
x  
 
x  
x  
x  
24 Hrs URINE if renal SLE   
x (Twice)  
 
 
 
 
At 3, 6, 9, 12, 18, 24 mo  
TSH, free T3, free T4  
x  
 
 
 
 
 
Pneumococcal and tetanus 
Abs  
 
x  
 
 
 
At 12 mo & before boost  
Fertility consult  
x  
 
 
 
 
 
Pregnancy test  
x  
x  
 
 
 
 
FSH, LH, Estradiol (f), 
Testosterone (m) ( l )  
 
x  
 
x  
 
x  
Inhibin A&B  
 
x  
 
 
 
At 24 months  
Chest x-ray  
x  
 
 
 
 
Q month 1st 3 mo  
Nutritional assessment  
 
x  
 
 
 
x  


Page 43 of 115 
 
Social work consult  
x  
x  
 
 
 
x  
Dental consult  
 
x  
 
 
 
 
HLA typing  
 
x  
 
 
 
 
PPD test  
 
x  
 
 
 
 
Cardiac ECHO/MUGA & 
ECG  
x  
 
 
 
 
 
Pulmonary function & DLCO 
 x  
 
 
 
 
x if lung SLE  
Lumbar puncture for CNS 
SLE  
x  
 
 
 
 
At 1, 3, 6, 9, 12, 18 and 24 
mo  
Marrow biopsy  
x  
 
 
 
x  
At 6,12, 24 mo  
Marrow cytogenetics  
x  
 
 
 
 
At 6,12, 24 mo  
Lymph node FNA/US guided   
x  
 
 
 
At 6, 12, 24 mo  
PET scans  
 
x  
 
 
x  
At 1, 6, 12 and 24 mo  
Kidney biopsy if renal SLE  
x  
 
 
 
 
At 12 mo  
Lung biopsy if lung SLE  
x  
 
 
 
 
 
BA lavage if lung SLE  
x  
 
 
 
 
At 12 mo  
Chest CT for lung SLE  
x  
 
 
 
 
At 1, 3, 6, 9, 12, 18 and 24 
mo  
MRI scans for CNS SLE  
x  
 
 
x  
 
At 1, 3, 6, 9, 12, 18 and 24 
mo  
Store serum  
 
x  
 
x  
x  
“  
Store PBMNC  
 
x  
 
x  
x  
“  
Blood research studies  
x  
x  
x  
x  
x  
“ (please see footnote#)  
Stem Cell Collection  
 
 
x  
 
 
 
Quality of life questionnaires  
 
x  
 
 
 
“  
SLEDAI and SLAM 
 
 x  
 
x  
x  
At 1, 3, 6, 9,12,18 and 24 mo  
SLICC-ACR Damage Index  
 
x  
 
 
 
At 6, 12, 18, and 24 mo  
HAQ, SF-36, KFFS  
 
x  
 
x  
x  
At 3, 6, 9, 12, 18 and 24 mo  
Neurocognitive testing 
ANAM  
 
x  
 
 
 
At 12 and 24 mo  
Leukapheresis for 
research***  
 
x  
 
 
 
At 1, 3, 6, 9, 12, 18, 24 mo 
and relapse  
*Duplicate testing will not be performed unless medically indicated if the previous test was done within two weeks. Please note: 
None of the optional invasive tests done solely for research indication will be offered to patients younger than 18 years of age 


Page 44 of 115 
 
(day-1 marow biopsy, PET scans, lymph node aspirates, 12 mo kidney biopsy and 12 mo BAL) 
**Post-transplant follow-up evaluations are monthly during first six months, then at 9, 12, 18, 24 months then yearly for five 
years, or at the time of documented flare or progression.    
*** For leukapheresis to collect cells for in vitro research five liters will be processed by continuous leukapheresis (two needle, 
two arm device) in patients with adequate bi-lateral peripheral venous access. In patients who do not have adequate peripheral 
venous access, two liters of blood will be processed by discontinuous flow apheresis (single-needle device). The goal is to 
obtain approximately 2 x 109 MNCs per procedure.
#
An additional peripheral blood draw for research will take place at 2 weeks, 
7, 8 months post-transplant. Also, if at monthly evaluations during first nine months absolute B-cell count in peripheral blood 
gets for the first time >25/μL, an extra research blood draw will be repeated for studies of early B-cell recovery.  
 


Page 45 of 115 
 
3.7.3
Patient flow coordination between two institutes Candidates for this study will be 
identified through the NIAMS Lupus clinic. The eligibility will be assessed by the 
primary rheumatology team in consultation with relevant subspecialists and thereafter by 
the NCI transplant team. After confirmation of eligibility informed consent signature will 
be obtained and patients’ care will be transferred to the NCI primary team until the 
discharge from the transplant inpatient and day-hospital service, which is anticipated to 
occur by the day 100 post-transplant. While patient is under the care of the NCI primary 
team, a rheumatology team member will attend transplant inpatient rounds at least 
2x/weeks on the inpatient service and at least 1x/week on the outpatient service. All SLE-
specific clinical response evaluations will be conducted by the NIAMS team. At the 
points when patients return to the NIH for the routine post-transplant long-term follow-up 
(beyond 100 days), evaluations will be scheduled in both NIAMS and NCI facilities. The 
NCI research team will hold the ultimate responsibility that all protocol-related data are 
collected, recorded on case report forms and stored as planned.  
3.8
Research studies of SLE and transplantation
Research studies will focus on the degree and nature of the immune cell depletion and the 
regeneration of the immune system following immunoablation and autoHSCT. Special emphasis 
will be put on the evaluating B- and T-lymphocyte and dendritic cell (DC) subsets and their 
functional status. Clinical correlations with lupus activity will be made. We will attempt to 
obtain a detailed and comprehensive picture of the degree and nature of the immune cell 
depletion and immune reconstitution following HSCT by analyzing not only the changes in 
peripheral blood but also the bone marrow and secondary lymphoid organs, such as lymph nodes 
and thymus either directly or indirectly. To achieve these goals, we will collect routine CBC at 
each time point TBNK assay and phenotyping is done in addition to the other research blood 
samples throughout the protocol and collect PBMCs for research by apheresis at pre-specified 
time-points.  Bone marrow biopsy will be done at baseline and at 6,12 and 24 months. To 
evaluate the role of lymph nodes, we will perform ultrasound surveillance and fine needle 
aspiration at baseline and at 6, 12 and 24 months. In addition, FDG-PET studies using GE 
Advance Scanners will be done to evaluate the metabolic and immunologic activity of peripheral 
lymph nodes and the thymus before priming, before conditioning and 1, 6, 12 and 24 months 
post transplant. This is based on the results of a previous study that suggested that activity in the 
lymph nodes is increased in all lupus patients but only patients with active disease have 
increased FDG uptake in the thymus. PBMCs, bone marrow samples and lymph node aspirates 
will be characterized by multiparameter FACS analysis, functional studies and mRNA 
microarrays. Cytogenetic studies using FISH will be done at baseline, 6, 12 and 24 months after 
the transplant to detect small clones of abnormal myeloid progenitor cells.  
Specific research studies:  
1
T- and B-lymphocyte, NK and DC subsets and their activation status will be determined 


Page 46 of 115 
 
by multiparameter FACS analysis using a panel of B- and T-cell and myeloid-cell 
specific antibodies from PBMCS, bone marrow and fine needle aspirates of lymph nodes.  
2
Detection of TRECs from PBMCs to assess the number of recent thymic emmigrants  
3
Assays of T- cell, B-cell and NK function.  
4
B-cells: ELISPOT assays to detect anti-dsDNA producing plasma cells.  
5
RNA isolation: mRNA from whole blood and B- and T-cell subsets will be isolated and 
tested with mRNA micro-arrays for change in expression of inflammatory molecules.  
6
Serum cytokine levels using multiplex cytokine assays.  
7
Urine cytokine levels.  
 
Serum, urine, CSF , cells and RNA will be stored and may be used for future studies evaluating 
markers of lupus activity and/or imunnoregeneration.  A more detailed description of research 
studies is given in Appendix A. Please refer to Appendix M for a description of the processes 
used in this study for the tracking and storage of human specimens.  The PI will report any loss 
or destruction of samples to the IRB.  
4
SUPPORTIVE CARE  
4.1
Infection Prophylaxis
 See Appendix B for detailed description. All patients will receive:  
4.1.1
Prophylaxis against Pneumocystis carinii pneumonia, prior to priming and continuing 
until transplantation, and resuming at the time of neutrophil and platelet recovery. PCP 
prophylaxis will continue until 12 months, steroids are tapered and CD4 counts >200/μL, 
whichever comes later. Trimethoprim/sulfamethoxazole is the preferred regimen, but 
alternative regimens are available for patients with a history of adverse reactions to 
Bactrim.  See Appendix B, Section 9.1.1 for details.  
4.1.2
 Caspofungin during the period of neutropenia post-transplant. See Appendix B, Section 
9.1.2 for details.  
4.1.3
 Valacyclovir for prophylaxis against herpes simplex virus and varicella zoster virus 
infection/reactivation.  This therapy will start at the time of chemomoblization, 
continuing through transplantation until 12 months, steroids are tapered, and 
CD4>200/μL.  See Appendix B, Section 9.1.3.1 for details.  
4.1.4
 If pre-transplant serology for Cytomegalovirus (CMV) is positive, monitoring for CMV 
reactivation by weekly testing for CMV antigenemia and clinical evaluation will be done. 
Positive antigenemia will be treated according to the schema in the Appendix B, Section 


Page 47 of 115 
 
9.1.3.2. Weekly monitoring will continue through day +100 and until CD4 count 
>200/μL – see Appendix B).  
4.1.5
Monitoring for Epstein-Barr virus reactivation by polymerase-chain reaction testing, and 
treated preemptively with a single dose of rituximab as outlined in the appendix B, 
section 9.1.4.   
4.1.6
 Intravenous antibiotics while neutropenic post-transplant. Ceftazidime is preferred as 
described in Appendix B, Section 9.1.5.  
4.1.7
 PPD testing prior to starting the chemomobilization and if positive will be treated with 
INH and pyridoxine as outlined in the Appendix B, section 9.1.6.  
4.1.8
 Standard vaccinations beginning 12 months post-transplant.  See Appendix B, Section 
9.1.7 for details.  
4.2
 Menses Suppression and Contraception
Menstruating pre-menopausal women who have not undergone hysterectomy will be placed on 
adequate suppression regimen as recommended by the gynecology consultants. Female 
transplant recipients will be advised to use contraception for at least 2 years after transplantation 
and to additionally have their male partners use condoms. Male transplant recipients will be 
advised to use contraception, preferably condoms, for 6 months after transplantation.  
4.3
 Blood Product Support
Patients will receive packed red blood cells and platelets as needed to maintain Hb > 8.0 gm/dl, 
and plts > 10,000/mm
3
 (or higher, if clinically indicated).  All blood products with the exception 
of the stem cell product will be irradiated.  
4.3.1
Leukocyte-reduction filters will be utilized for all blood and platelet transfusions to 
decrease sensitization to transfused leukocytes and decrease the risk of CMV infection.  
4.4
Nutritional Support
When mucositis or other patient condition prevents adequate PO intake, parenteral 
hyperalimentation will be instituted and discontinued under the direction of the dietary service. 
Oral intake will resume when clinically appropriate under the supervision of the dietary service 
of the Clinical Center.  


Page 48 of 115 
 
4.5
Anti-emetics   
Anti-emetic usage will follow Clinical Center Guidelines as well as recommendations from the 
Pharmacy service.  
4.6
 Hematopoietic Growth Factor Support   
All patients will start filgrastim (GCSF) 5μg/kg/day on day +1. The dose will be given 
subcutaneously (SC). G-CSF will be continued until the absolute neutrophil count (ANC) is > 
500/μL, it can be resumed if ANC drops or stays below 1000/μL per discretion of the attending 
physician if clinically indicated.  
5
DATA COLLECTION AND EVALUATION 
5.1
 Data Collection  
Data will be prospectively collected and entered into the Center for Clinical Research database. 
All laboratory adverse events will be collected from grade 2. Protocol-specific case report 
forms will be developed and filled out by the NIAMS or NCI teams depending on the phase of 
the protocol and the type of evaluations (all lupus-specific evaluations such as SLEDAI will be 
performed exclusively by the rheumatologists). Working relationships between the teams are 
described in section 3.7.2. NCI research team is ultimately responsible for the appropriate data 
collection and storage as defined by the protocol.  
5.2
 Response Criteria
5.2.1
 Primary efficacy endpoint: Continuous relapse-free complete clinical response at 24 
months post-transplant. Patients will be considered to achieve this endpoint if they fulfill 
the criteria for complete clinical response at anytime and maintain it for at least three 
months.  
5.2.2
 Definitions:  
I. Complete clinical response:  
•
Complete response in the target organ and no clinical signs of active lupus as determined 
by a SLEDAI score of  3  
•
 Prednisone 10mg/day at 6 months and 5mg/day at 12 months or later  
 
II. Partial response:  
•
At least partial response in the target organ (defined on an organ basis)  
•
At least 50 % improvement in general disease activity as measured by SLEDAI  
•
At least 50% decrease in prednisone dose at 6 months and 10mg/day at 12 months or 


Page 49 of 115 
 
later  
 
III. No response:  
•
No response in target organ  
•
Inability to taper prednisone due to increased disease activity  
•
Need for additional immunosuppressive therapy  
 
5.2.3
 Secondary efficacy endpoints:  
•
Time to flare-free complete response   
•
Time to complete clinical response (in months)  
•
Time to partial response (in months)  
•
Time to relapse (in months)  
•
Improvement in general disease activity measured by SLAM and SLEDAI  
•
Time to normalization of dsDNA and complement levels  
•
Pain scale, quality of life  
•
Functional assessment by HAQ score  
•
Neurocognitive testing and depression scale  
 
5.2.4
 Organ-specific efficacy definitions:  
Renal:  
Complete response  
a.
Stable or improved renal function: <30% increase in serum creatinine or <20% decrease 
in GFR and  
b. Inactive urine sediment (< 10 RBC/hpf and no cellular casts) and   
c.
Improvement in proteinuria  
 
i.
If nephrotic range proteinuria at baseline, decrease in proteinuria to < 1.0 g/day or  
ii.
If non-nephrotic range proteinuria at baseline: proteinuria < 0.5 gm/day  
Partial Response  
a.
Stable or improved renal function, as above and  
b. Improvement in proteinuria (as above) with a persistently active urine sediment  
or  


Page 50 of 115 
 
Inactive urine sediment and non-nephrotic proteinuria and >50% decrease from 
baseline  
 
No response  
a.
Not fulfilling complete or partial response   
Relapse 
a.
 Minor: increase in hematuria and/or > 50% increase in proteinuria and stable 
renal function (as defined above)  
b.  Major: reappearance of sustained (repeatedly present at least 1 month apart) 
nephritic sediment with biopsy proven proliferative nephritis or for patients where 
a biopsy is contraindicated: reappearance of sustained (repeatedly present at least 
1month apart) nephritic sediment with sustained > 30% increase in serum 
creatinine or > 20% decrease in GFR  
 
5.2.5
CNS Lupus  
Complete response:  
Stable or improved clinical exam defined by the Scripps Neurological Ration Scale (NRS), 
(Appendix H) and no objective signs of active CNS inflammation.  
a.
Stable or improved clinical exam: no more than a 5 point worsening on the NRS 
required on two consecutive exams (3 months apart). The presence and frequency 
of seizures before HSCT will be documented. If the seizures disappear, this will 
be considered a response. If seizures persist, but do not increase in frequency, it 
will be assumed that this is a response to treatment assuming that other objectives 
measure of CNS inflammation, such as MRI and/or CSF normalize. If the seizure 
frequency and/or severity increase, we will consider this a treatment failure. De 
novo occurrence of seizures will similarly be considered a failure.  
b. No objective signs of CNS inflammation: No signs of inflammation on MRI 
and/or CSF  
 
(Normal CSF protein or cell count within normal range with the protein/IgG ratio 
equal or improved compared to pretreatment).   
Partial response: Not meeting the criteria for either complete or no response  
No response/failure:  


Page 51 of 115 
 
a) Persistence or re-appearance of any of the signs/symptoms that  
define active CNS lupus b) NRS worsening of >15 points c) Any requirement for specific 
treatment of CNS lupus-associated signs/symptoms, e.g. seizures Relapse  
a. Minor: Anything less than major relapse  
b. Major: Longer than 24 hours appearance/re-appearance of signs/symptoms compatible with 
CNS lupus. These findings should not be otherwise attributable, i.e. to interfering infections, 
compromise of pulmonary, renal function, or other Pulmonary:  
Complete response:  
Ƈ Improvement in DLCO by >50% or normal DLCO and no evidence of alveolitis on high 
resolution CT Partial response: Ƈ stable DLCO ( <150% and > 85% of baseline) and no evidence 
of alveolitis on high resolution CT  
No response:  
Ƈ 15% worsening in DLCO or evidence of alveolitis on high resolution CTRelapse a. Minor: 
any of the following: new onset or worsening dyspnea on exertion, <20% decrease in DLCO, 
new infiltrate of unknown significance on chest CT   
b. Major: any of the following: pulmonary hemorrhage, >20% decline in DLCO, dyspnea at rest, 
need for oxygen, respiratory failure, new or worsening alveolitis on high-resolution CT  
 
For both minor and major relapse other causes of pulmonary disease such as pulmonary emboli 
and infections have to be excluded Hematologic:  
Complete response:  
Ƈ Platelet count > 100,000/mm
3
 and normalization of Hb with no signs of hemolysis and 
independent of transfusions or any other therapy for at least three months  
Partial response:  
Ƈ self-sustained (platelet counts >50,000/mm
3
 without signs of bleeding and Hb > 10g/dL No 
response:  
Ƈ not meeting criteria for complete or partial response  Relapse:  
a. Minor: platelets >20,000-100,000 without signs of bleeding or Hb > 8 g/dL – normal  
b. Major: platelets <20,000 or <100,000 with bleeding or requiring transfusions to maintain Hb > 
8.0g/dL or to treat symptoms of anemia  


Page 52 of 115 
 
 
5.2.6
Treatment of relapses (flares)  
1. Indeterminate or minor:  
a. Repeat evaluation in 1-2 weeks  
b. Increase prednisone up to 0.5 mg/kg/day for 2 weeks, taper prednisone to baseline over 4 
weeks  
 
2. Major: treatment failure Treat as clinically indicated  
5.2.7
 Assessment of response  
Response to therapy will be assessed every 3 months following transplant. At each time-point 
patients will be classified as   
x
Complete clinical responders or  
x
Partial responders or  
x
Non-responders  
based on the criteria defined previously. The final outcome of the patients will be determined at 
24 months. They will be classified as  
x
Responders, if they fulfill criteria for relapse-free complete clinical response  
x
Partial responders: if they fulfill criteria for partial response and have maintained it for at 
least 3 months or they had one (1) episode of indeterminate/minor flare and maintained 
partial or complete clinical response for at least 3 months (as above)  
x
Non-responders: if they never achieved partial or complete response criteria or they had a 
major flare or they had more than one indeterminate/minor flare that required increase in 
their prednisone dose.  
 
5.3
Toxicity Criteria
This study will use the CTC version 3.0 for toxicity and adverse event reporting. A copy of the 
CTC version 3.0 can be downloaded from the CTEP home page (http://ctep.cancer.gov). Known 
toxicities for each agent used in this protocol are listed in section 8.0 of this protocol.  


Page 53 of 115 
 
5.4
Statistical Section 
The primary objective of this study is to determine if the strategy identified in section 3.1 is 
associated, in a pilot fashion, with a sufficiently high success-rate to warrant further study in 
subsequent trials. The primary efficacy endpoint will be continuous relapse-free complete 
clinical response at 24 months post transplant, which will be evaluated as a dichotomous 
variable. Using an exact binomial test from nQuery Advisor, if 14 subjects are enrolled, there is 
84% power to be able to detect the difference between a null proportion of 70% with success and 
a more desirable alternative as high as 95%, using a one-sided 0.05 alpha level test.  In other 
words, if we conducted this study repeatedly, and if the true major response rate were 95%, then 
with 14 patients, 84% of the time, we would obtain the result that the lower bound of a one-sided 
95% confidence interval exceeded 70%. Since we are also concerned about the possible toxicity 
associated with the proposed therapy, this will also be closely monitored and used to determine 
the suitability of continuing to enroll patients onto the trial. It would be expected that there is no 
more than a 5% probability of any individual patient experiencing an early non-lupus related 
death on the study. As such, the probability of observing 0 such deaths in 14 patients would be 
49% and 1 such death in 14 would be 36%. Thus, there is approximately 85% probability that no 
more than one such death would be observed in 14 patients if the true probability of this 
happening to any individual patient were 5%. If one such death occurs prior to accrual of the 14 
patients, then this would result in a re-evaluation of protocol procedures, but not termination of 
accrual. If there are two early non-lupus related deaths on the study, the trial will cease 
enrollment. This more likely indicates that the true probability of these events is perhaps 20% 
rather than 5%, since the probability of 2 or more events in 14 is 80% if the true probability were 
20%.   
The evaluation for continuous relapse-free complete clinical response at 24 months post 
transplant will be based on 14 patients who are able to mobilize sufficient numbers of stem cells. 
An occasional patient who is not able to mobilize sufficient stem cells to infuse at least 1.5 x 10
6
 
selected CD34+ cells/kg will be taken off study and considered inevaluable for response. 
However, the trial will terminate early due to toxicity if there are two early non-lupus related 
deaths in any enrolled patients, regardless of whether they are evaluable for response or not.  In 
order to assure 14 evaluable patients, a total of up to 16 patients may be enrolled.  
All patients will be monitored closely by the principal investigator and other clinical personnel 
associated with the trial. Should any severe toxic events arise other than that specifically referred 
to above, the investigators will carefully decide how to proceed with the treatment of any 
subsequent patients on the protocol in order to protect patient safety.  
All secondary evaluations of biologic markers and other similar laboratory determinations will 
be done with exploratory intent. In view of the limited number of subjects to be included in this 
pilot trial, non-parametric evaluation of changes from baseline will be performed, as well as 
descriptive analyses. In any cases in which p-values are reported from these analyses, they will 
be reported as being unadjusted for multiple comparisons, and will be carefully noted to be the 


Page 54 of 115 
 
result of hypothesis generating studies, requiring replication in larger populations in order to 
confirm any effects noted.  
As noted in section 5.2.3, a number of secondary efficacy endpoints have been proposed. All 
endpoints relating to time to an event will be analyzed via the Kaplan-Meier method. Other 
measures which involve baseline and then follow-up evaluations will be done by subtracting the 
baseline evaluation from each follow-up evaluation (see section 3.7.1 for details of the follow-up 
points), and using paired non-parametric tests to compare the changes. Since these evaluations 
will be considered secondary, the results will not be adjusted for multiple comparisons, and will 
be carefully stated as being the result of exploratory evaluations.  
It is expected that approximately one such patient per month may be enrolled onto this trial; thus 
accrual should be completed in 1.5 years, depending on the referral of such patients to the NCI.  
5.4.1
 Data and Safety Monitoring Plan  
5.4.2
In accordance with NCI IRB policies, this pilot study does not require independent 
monitors or review by a Data Safety and Monitoring Board. However, an independent 3-
person review committee has been established to review all deaths to determine if those 
are related to the underlying disease or to the experimental treatment. The committee 
consists of 2 lupus experts (Mark Gourley NIEHS, Peggy Crow, Hospital for Special 
Surgery, NY) and 1 bone marrow transplant expert (Harry Malech, NIAID). In 
exceptional special circumstances (for example but not limited to - inability to serve on 
the panel, or in an unusual situation where the SLE expert is at the same time a referring 
physician), PI in consultation with the clinical chairperson or one of the scientific 
chairpersons can designate a substitute for a member of this committee and inform 
immediately the IRB about this change. Also it is anticipated that deliberations of this 
committe will be done by a conference call under provision that all necessary and 
requested documentation on the respective patient is provided in advance. The committee 
will provide its assessment to the PI who will forward it to the IRB and FDA, as 
appropriate. If the death is considered to be at least probably related to the experimental 
therapy the protocol procedures will be re-evaluated but enrollment will not be 
terminated. If there are two early non-lupus related deaths on the study, the trial will 
cease enrollment.  
5.4.3
The PI and Clinical Chairperson will provide continuous, close monitoring, with prompt 
reporting of unexpected or serious adverse events to the IRB and FDA (Section 7.1).  On 
a weekly basis, the PI, Clinical Chairperson, Protocol Research Nurse, and Transplant 
Coordinator will review clinical and laboratory data from each of the transplant 
recipients.  A complete summary of patient outcomes will be provided annually to the 
IRB and FDA. Protocol Support Office of the Medical Oncology Clinical Research Unit, 
CCR, NCI will assist with annual internal auditing of subject case records. After accrual 


Page 55 of 115 
 
of 3 subjects, the Protocol Support Office will also perform a protocol activation audit to 
review adherence to the informed consent procedures, eligibility documentation, and 
initial treatment.  
5.4.4
The expected heterogeneity of study participants with respect to risk factors for toxic 
events renders these parameters impractical to evaluate in a statistical manner; 
accordingly, formal stopping rules will not incorporate toxic events but will instead be 
monitored as described in statistical section.  
6
 HUMAN SUBJECT PROTECTIONS  
6.1
 Rationale for subject selection  
Subjects with severe treatment refractory SLE will be enrolled. The gender distribution (F:M) for 
lupus is 9:1 and the peak age of onset is in the third and fourth decade. SLE is ~3 times more 
common in African-Americans therefore women and minorities are expected to be well 
represented in this study.  If the referral base cannot provide a population that is representative of 
the population of the US, then alternative methods for recruitment will be considered such as 
contacting our referring physicians and request the appropriate study subjects and/or contacting 
health professionals in urban tertiary referral centers for African-, Hispanic-, or Asian-American 
subject recruitment.  
6.1.1
 Participation of children  
This is a study that involves greater than a minor increment over minimal risk with the prospect 
of direct benefit to subjects. Minors over the age of 15 will be enrolled in the study. The clinical 
course of patients older than 15 years is similar to adult patients and we have experience with 
this age group. SLE in patients younger than 15 years of age has a different course and there is 
no experience with fludarabine and cyclophosphamide or rituximab in lupus patients in this age 
group, therefore patients under 15 years of age will not be included in the study. If the safety of 
this treatment is established, younger patients may be considered for future studies. 
6.2
Evaluation of Benefits and Risks/Discomforts 
The patient population eligible may benefit from this treatment by achieving the remission of 
their disease or prolonged disease control. It is expected that this benefit may occur in at least 
half of the cases, in this population of patients who failed all available standard therapies. 
Achievement of durable responses is expected to result in improved survival, less disability, and 
better quality of life. This is however a more than standard risk procedure with estimated 
procedure-related mortality risk of up to 5%, there is also a theoretical but probably small risk of 
irreversible procedure-related injury to vital organs due to transplant-related complications such 
as unusual drug-induced side effects or infections or, in theory, a risk for bone marrow injury 
that would result in secondary myelodysplastic syndromes. Other more common risks would be 


Page 56 of 115 
 
reversible side-effects of chemotherapy, antibodies, growth factors, mobilization, apheresis, or 
central-line placement. Transient negative psychological impacts of the transplant process on the 
patient and caregiver are possible. Adverse legal, social or financial impacts are not expected but 
all attempts will be made to anticipate them, prevent them and attenuate them from the very 
beginning and through the transplant process. Alternative treatments for these patients exist but 
none of them are likely to result in disease remissions, including continuation of pulse-
cyclophosphamide with or without additional other immunosuppression. Another alternative are 
other investigational therapies as available. Confidentiality of the subjects will be protected by 
safe-record keeping and the avoidance of the usage of patient identifiers. In case of the adverse 
effects of the study related therapy all medical intervention will be provided at the site by the 
study team. 
6.3
Risks/Benefits Analysis 
The anticipated risks of this therapy are considered justifiable given the fact that for this patient 
population, no effective standard therapies exist. These patients are at a much higher risk for 
mortality and disability from their disease or its conventional treatments than of this therapy if 
the therapy provides the anticipated results. To ensure the anticipated risk/benefit ratio, the 
protocol has implemented careful monitoring of toxicities, stopping rules, as well as a data safety 
monitoring plan. In addition to the potential benefit to the patients enrolled in this study, there is 
a strong potential for a broader benefit to future patients by acquiring a better knowledge of the 
systemic lupus disease process and ways to cure it. Some patients younger than 18 years of age 
(15-18 years) are anticipated to be enrolled in this study. Although the overall risk is considered 
to be greater than minimal, there is a strong potential for a therapeutic benefit and intent built in 
this protocol that is in accordance with federal guidelines for approvable research in children. In 
fact, due to the better prospects for complete immune reconstitution in younger individuals, 
children are those who are the most likely to have successful outcome with transplant therapy. 
6.4
Consent and Assent Process and Documentation 
The procedures and treatments involved in this protocol, with their attendant risks and 
discomforts, potential benefits, and potential alternative therapies will be carefully explained to 
the patient by the study team and the principal investigator personally. Particular emphasis will 
be placed on the subject’s understanding that this treatment is investigational, that it may not 
result in the direct benefit, and that there is a short term risk form the procedure-related mortality 
of 5%. An informed consent document, and assent for individuals under age 18, will be obtained 
and signed from the patients by the PI (or one of his clinical associates), in the presence of a 
NIAMS and an NCI team representative and an independent monitor who is familiar with the 
ethical and regulatory requirements for informed consent. The independent monitor will 
ascertain that the subject understood that this is an experimental approach, which may not lead to 
direct benefit and may be associated with significant risks, including death. The independent 
monitors who agreed to observe and document the process of obtaining the consent are Ms 


Page 57 of 115 
 
Mildred Wilson, RN and Beverly Barham RN. Both are research nurses at NIAMS with all 
necessary training and both are currently doing the observed consent for the neurocognitive 
lupus study at NIAMS. Thereafter, the Principal Investigator (or his designee) and the subject 
(and/or his/her legal representative) will sign the consent document in the presence of an 
independent witness. The independent monitor will also assist the investigators to identify 
subjects for whom additional assessment of decisional capacity would be appropriate. Such 
participants will then be evaluated for their decisional capacity to participate in this protocol by 
the consultation psychiatry liaison service. The original signed consent and assent documents 
will be kept with the patient’s chart. The Data Management Section will also retain a copy of the 
informed consent and assent documents. A copy of the informed consent and assent documents 
will also be given to the patient.  
7
 DATA REPORTING  
7.1
 Adverse Event Reporting
 This protocol is registered with the FDA under IND number 66,867 (Holder: Dr Steven 
Pavletic). The IND was withdrawn from the FDA in May 2012.  Reports of adverse events will 
be made using the Common Terminology Criteria for Adverse Events version 3.0 for reference 
according to the guidelines published by the DCTD, NCI on June 10, 2003. A copy of the 
CTCAE v3.0 can be obtained from the CTEP home page (http://ctep.cancer.gov). Guidelines for 
AE reporting are summarized as follows:  
7.1.1
 The protocol PI will report to the NCI-IRB:  
All serious adverse events (SAEs) that are not in the consent form, but are possibly, probably or 
definitely related to the research.  An SAE is defined as an untoward medical occurrence 
that:resulted in a death;
 was life-threatening; required or prolonged hospitalization; caused persistent or significant 
disability/incapacity; resulted in congenital anomalies or birth defects; or required intervention to 
prevent permanent impairment or death.  All other deaths not included in the SAE category 
above.  
 All grade 3 and 4 (CTCAE) events that are not in the consent and that are possibly, probably or 
definitely related to the research, but not included in the SAE category above. All life-
threatening or disabling events (i.e., grade 4, except for myelosuppression), which may be due to 
administration of the study drug(s). Since hospitalization or re-hospitalization are more a rule 
rather than exception in HSCT trials, hospitalization by itself will not automatically qualify as an 
adverse event [112].  Failure to recover neutrophil count >500/μL by day +21 post-transplant.  
Cases of secondary AML/MDS Reports must be received by the NCI-IRB within 7 days of 
notification of the event.  Severe adverse events due to FDG-PET will be also reported to Dr. 
Peter Herscovitch, Building 10, Room 1 C 495 (301-451-4248).  As of 2012, this protocol was 
removed from the FDG IND. 


Page 58 of 115 
 
7.1.2
 FDA reporting requirements include:  
(1) Reporting any unexpected fatal or life-threatening adverse events associated with the use of 
the drugs by telephone (301-827-2090) or fax (301-827-2531) no later than 7 calendar days after 
initial receipt of the information (21 CFR 312.32(c)(2);  
(2) Reporting any adverse experience associated  with use of drugs that is both serious and 
unexpected in writing no later than 15 calendar days after initial receipt of information (21 CFR 
312.32(c)(1); and  
(3) Submitting annual progress reports (21 CFR 312.33).  
 
7.1.3
 The principal investigator will review each report and determine whether an adverse 
event is expected or unexpected and its possible relationship to the research study based 
on the information available in the protocol consent document, drug label, status of the 
underlying SLE disease and preexisting conditions.  
7.1.4
 All fatal events will be reported to an independent review committee within 10 days. The 
committee will determine if these events are related to the experimental treatment.  
7.1.5
Information on adverse events that do not qualify for immediate reporting will be 
collected at 3-month intervals from the case report forms, C3D database and medical 
records. The following information, specifically, will be collected and reported:  
•
 All Grade 2 events that are not in the consent form, but are possibly, probably or 
definitely related to the research;  
•
 All Grade 3 and 4 events that are possibly, probably or definitely related to the research;  
•
 All Grade 5 events regardless of attribution;  
•
 All Serious Events regardless of attribution.  
 
The study personal will compile this information for the PI who will notify IRB if action is 
warranted.  Beginning at the one year post transplant follow up, only those adverse events that 
meet the reporting criteria above will be recorded.  


Page 59 of 115 
 
7.1.6
 Annual progress reports will be submitted in parallel to the IRB and the FDA. Such 
annual reports will also include the summary of any adverse events on this protocol.  
7.2
Record Keeping
7.2.1
 All patients must have signed an Informed Consent, and an on-study confirmation of 
eligibility form will be filled out before entering on the study. Study data will be reported 
as case-report forms and study summaries.  
8
 PHARMACEUTICAL AND INVESTIGATIONAL DEVICE INFORMATION  
 
8.1
 CYCLOPHOSPHAMIDE (CTX, Cytoxan, NSC-26271)
8.1.1
 Supply – Cyclophosphamide will be purchased by the NIH Clinical Center Pharmacy 
Department from commercial sources and is supplied as a lyophilized powder in various 
vial sizes.  
8.1.2
 Preparation - will be reconstituted with sterile water for injection to yield a final 
concentration of 20 mg/ml as described in the package insert.  
8.1.3
 Storage and Stability - The vials are stored at room temperature. Following 
reconstitution as directed, solutions of cyclophosphamide are stable for 24 hours at room 
temperature, or 6 days when refrigerated at 2-8° C.  
8.1.4
 Route of Administration - The cyclophosphamide used in this regimen will be mixed in 
100 ml normal saline and given as an IVPB over 2 hours.  
8.1.5
 Toxicities:  
a) Nausea and vomiting - variable; symptomatically improved with standard anti-emetics and/or 
benzodiazepines [e.g., lorazepam].  
b) Water retention – cyclophosphamide may rarely provoke the syndrome of inappropriate 
antidiuretic hormone secretion and resultant hyponatremia, usually manifested 12-48 hrs after IV 
administration, necessitating frequent accurate assessment [q 1-2 hrs] of intake, urine output and 
urine specific gravity.  This effect can be counteracted by furosemide. Fluid restriction is not 
feasible during administration of high dose cyclophosphamide.  
c) Cardiomyopathy -cyclophosphamide may cause severe, sometimes lethal, hemorrhagic 
myocardial necrosis or congestive cardiomyopathy. Patients may present with congestive 
cardiomyopathy as late as 2 weeks after the last dose of cyclophosphamide. The clinical 
syndrome has been observed in patients receiving the dose of cyclophosphamide in this regimen 


Page 60 of 115 
 
in allogeneic transplantation patients. In an attempt to minimize this complication, patients with 
significant cardiac dysfunction are excluded from this protocol [see patient eligibility]. 
Congestive failure is managed according to standard medical therapeutics.  
d) Hemorrhagic cystitis – this is a serious, potentially life-threatening complication related to 
injury of the bladder epithelium by cyclophosphamide metabolites.  Although subclinical 
hematuria is not uncommon at this dose level, clinically significant hematuria or serious 
hemorrhage can usually be avoided by maintaining a high urine volume and frequent voiding and 
the administration of Mesna. Diuresis is maintained for 24 hrs after completion of last dose by 
parenteral infusions of 0.9% Sodium Chloride with potassium chloride, as specified in section 
3.2. Careful monitoring of serum and urine electrolytes is mandated.  Furosemide may be 
required to ensure this diuresis. Continuous bladder irrigation may be used for control of 
significant hematuria.  
e) Sterility after cyclophosphamide is a possibility and more commonly occurs in female patients 
above age 26-30 years.  
f) Less common but serious complications include pulmonary fibrosis and secondary 
malignancies. Less common but reversible toxicities include alopecia and skin rash.  
8.2
 MESNA (Sodium 2-mercaptoethanesulfonate, Mesnum, Mesnex, NSC113891)
8.2.1
 Supply – Mesna will be will be purchased by the NIH Clinical Center Pharmacy 
Department from commercial sources and is supplied as a 100mg/ml solution.  
8.2.2
 Preparation  
 Priming regimen: The Mesna should be prepared in 100 ml of either 5% Dextrose in Water, 
USP or 0.9% sodium chloride injection, USP and infused over 15-30 minutes.  
 Conditioning regimen: Dilute up to 20 mg Mesna/ml fluid in D5W or 0.9% Sodium Chloride. 
Mesna should be started concurrently with the the start of cyclophosphamide at 1200 mg/m
2
 in 
500 ml by continuous IV infusion over 24 hour infusion for four doses (days -6, 5, -4, and -3).  
8.2.3
 Storage and Stability – Intact ampules are stored at room temperature. Diluted solutions 
(1 to 20 mg/dl) are physically and chemically stable for at least 24 hours under 
refrigeration.  Mesna is chemically stable at room temperature for 48-72 hours in D5W, 
48-72 hour in D5W/0.45% sodium chloride, or 24 hours in 0.9% sodium chloride.  
8.2.4
 Administration - To be administered intravenously as continuous infusion.  
8.2.5
 Toxicities - Nausea, vomiting, diarrhea.  


Page 61 of 115 
 
8.3
 FILGRASTIM (G-CSF, Neupogen®)  
8.3.1
 Supply – Commercially available as filgrastim injection in a concentration of 300μg/ml 
in 1ml (300μg) and 1.6ml (480μg) vials.  
8.3.2
 Preparation – For subcutaneous administration, the appropriate prescribed dose is drawn 
up from the vial with no further dilution prior to administration. For intravenous 
administration, the commercial solution for injection should be diluted prior to 
administration.  It is recommended that the prescribed dose be diluted with dextrose 5% 
in water to a concentration greater than 5μg/ml. Dilution of filgrastim to a final 
concentration of less than 5μg/ml is not recommended at any time. Do not dilute with 
saline at any time; product may precipitate. Filgrastim diluted to concentrations between 
5 and 15μg/ml should be protected from absorption to plastic materials by the addition of 
Albumin (Human) to a final concentration of 2mg/ml. When diluted in 5% dextrose or 
5% dextrose plus Albumin (Human), filgrastim is compatible with glass bottles, PVC and 
polyolefin IV bags, and polypropylene syringes.  The dose may be “rounded down” to 
within 10% of patient’s calculated dose to use the drug cost-effectively.  
8.3.3
 Storage and Stability – Filgrastim for injection should be stored in the refrigerator at 2
o
 
to 8
o
C (36
o
 to 46
o
F). Avoid shaking.  
8.3.4
 Administration – Subcutaneous injection is preferred.  If clinically indicated, filgrastim 
may be administered as an intravenous infusion over 4 or 24 hours.  
8.3.5
 Toxicities – Medullary bone or skeletal pain is the most commonly reported toxicity.  In 
addition, reversible elevations in uric acid, lactate dehydrogenase, and alkaline 
phosphatase are common laboratory abnormalities. Four cases of splenic rupture have 
been reported in healthy donors when given filgrastim or other myeloid growth factors 
for peripheral blood stem cell mobilization; 1 of these cases resulted in fatality. Five 
additional cases of splenic rupture have been reported in cancer patients undergoing 
chemotherapy or peripheral blood stem cell mobilization; splenic rupture may have 
contributed to deaths in 2 of these cases. One additional death due to splenic rupture after 
filgrastim therapy was reported to the manufacturer without additional information. 
According to the manufacturer, the reporting rate for splenic rupture with filgrastim is 
less than 1 in 486,000.  
8.4
 FLUDARABINE (Fludara, Berlex Laboratories)  
8.4.1
Supply - Fludarabine monophosphate will be purchased by the NIH Clinical Center 
Pharmacy Department from commercial sources and is supplied as a white, lyophilized 


Page 62 of 115 
 
powder.  Each vial contains 50 mg of fludarabine phosphate, 50 mg of mannitol, and 
sodium hydroxide to adjust pH. Fludara is stored at room temperature.  
8.4.2
8.4.2 Preparation - FLUDARA IV should be prepared for parenteral use by aseptically 
adding Sterile Water for Injection, USP. When reconstituted with 2 ml of Sterile Water 
for Injection, each ml of the resulting solution will contain 25 mg of Fludarabine 
Phosphate, 25 mg of mannitol, and sodium hydroxide to adjust the pH to 7–8.5. 
Fludarabine will be diluted in 100 to 125ml of either 5% dextrose in water or 0.9% 
sodium chloride, and infused IV over 30 minutes.  
8.4.3
Storage and Stability - Reconstituted FLUDARA IV is chemically and physically stable 
for 24 hours at room temperature or for 48 hours if refrigerated. Because reconstituted 
FLUDARA IV contains no antimicrobial preservative, care must be taken to assure the 
sterility of the prepared solution; for this reason, reconstituted FLUDARA IV should be 
used or discarded within 8 hours.  
8.4.4
8.4.4 Administration - Parenteral drug products should be inspected visually for 
particulate matter and discoloration prior to administration.   
8.4.5
 Toxicities – Fludarabine toxicities include myelosuppression (dose limiting toxicity), 
fever, nausea, vomiting, stomatitis, diarrhea, gastrointestinal bleeding, anorexia, edema, 
skin rashes, myalgia, headache, agitation, hearing loss, transient episodes of somnolence 
and fatigue, auto-immune hemolytic anemia, auto-immune thrombocytopenia, 
paresthesias, peripheral neuropathy, renal, and pulmonary toxicity (intersititial 
pneumonitis).  Severe fatal CNS toxicity presenting with loss of vision and progressive 
deterioration of mental status were encountered almost exclusively after very high doses 
of fludarabine monophosphate.  Such toxicity has only rarely been demonstrated at the 
25-30 mg/m
2
/day dosage of fludarabine. Very rarely described complications include 
transfusion-associated graft-versus-host disease, thrombotic thrombocytopenic purpura, 
and liver failure. Tumor lysis syndrome following fludarabine administration has been 
observed, especially in patients with advanced bulky malignant disease. Opportunistic 
infections (protozoan, viral, fungal, and bacterial) have been observed postfludarabine, 
especially in heavily pre-treated individuals, and in individuals receiving fludarabine 
combined with other agents.  
8.5
PREDNISONE  
8.5.1
Supply – Prednisone will be purchased by the NIH Clinical Center Pharmacy Department 
from commercial sources. Prednisone is commercially available as tablets, in strengths of 
1, 2.5, 5, 20, and 50 mg.  


Page 63 of 115 
 
8.5.2
Storage and Stability - Prednisone tablets should be stored in the container provided away 
from heat. The product labeling bears the manufacturer's expiration dating for stability.  
8.5.3
Administration - Prednisone will be administered at a maximum dose of 1 mg/kg/day or 
at a maximum total daily dose of 80 mg orally and according the enclosed post-transplant 
tapering guidelines (appendix). In patients unable to tolerate oral medication, 
methylprednisolone can be substituted at an equivalent dosage, diluted in a small volume 
(e.g. 25-50ml) of 5% dextrose in water or 0.9% sodium chloride and infused over 15 
minutes. To reduce gastrointestinal side effects, prednisone should be taken with food.  
8.5.4
Toxicities -Prednisone frequently causes gastritis, immunosuppression, muscle wasting, 
fluid retention, and hyperglycemia.  
8.6
RITUXIMAB (Rituxan)
8.6.1
Supply – Rituximab will be obtained commercially and is supplied as a 10 mg/ml sterile, 
preservative-free solution for injection, in vials of 100 mg and 500 mg.  
8.6.2
Preparation – The desired dose will be diluted to a final concentration of 1 to 4 mg/mL in 
either 0.9% Sodium Chloride or D5W.  
8.6.3
 Storage and Stability – Rituximab vials should be stored at 2–8°C (36–46°F) and should 
be protected from direct sunlight.  
8.6.4
Administration – First Infusion: The rituximab solution for infusion should be 
administered intravenously at an initial rate of 50 mg/hr. Rituximab should not be mixed 
or diluted with other drugs.  If hypersensitivity or infusion reactions do not occur, 
escalate the infusion rate in 50 mg/hr increments every 30 minutes, to a maximum of 400 
mg/hr. If hypersensitivity (non-IgE-mediated) or an infusion reaction develops, the 
infusion should be temporarily slowed or interrupted. The infusion can continue at one-
half the previous rate upon improvement of patient symptoms. If the first infusion is 
tolerated well, subsequent infusions can begin at a rate of 100 mg/hr and will be 
increased by 100 mg/hr increments at 30-minute intervals.  However, if the first infusion 
is not tolerated well, then the guidelines for the initial infusion should be followed for 
subsequent administration. Premedication will routinely be administered 30 to 60 minutes 
prior to the beginning of each rituximab infusion, consisting of acetaminophen 650 mg 
PO and diphenhydramine 50 mg IV.  
8.6.5
Toxicities – The most serious adverse reactions caused by rituximab include infusion 
reactions, tumor lysis syndrome, mucocutaneous reactions, hypersensitivity reactions, 
cardiac arrhythmias and angina, and renal failure.  


Page 64 of 115 
 
a) Fatal and Severe Infusion Reactions:  Deaths within 24 hours of rituximab infusion have been 
reported. Approximately 80% of fatal infusion reactions occurred in association with the first 
infusion.  Severe reactions typically occurred during the first infusion with time to onset of 30 to 
120 minutes.  Signs and symptoms of severe infusion reactions may include hypotension, 
angioedema, hypoxia or bronchospasm, and may require interruption of rituximab 
administration. The most severe manifestations and sequelae include pulmonary infiltrates, acute 
respiratory distress syndrome, myocardial infarction, ventricular fibrillation, and cardiogenic 
shock.  In the reported cases, the following factors were more frequently associated with fatal 
outcomes: female gender, pulmonary infiltrates, and chronic lymphocytic leukemia or mantle 
cell lymphoma.  Management of severe infusion reactions:  The rituximab infusion should be 
interrupted for severe reactions and supportive care measures instituted as medically indicated 
(e.g., intravenous fluids, vasopressors, oxygen, bronchodilators, diphenhydramine, and 
acetaminophen).  In most cases, the infusion can be resumed at a 50% reduction in rate (e.g., 
from 100 mg/hr to 50 mg/hr) when symptoms have completely resolved. Patients requiring close 
monitoring during first and all subsequent infusions include those with pre-existing cardiac and 
pulmonary conditions, those with prior clinically significant cardiopulmonary adverse events and 
those with high numbers of circulating malignant cells (= 25,000/mm
3
) with or without evidence 
of high tumor burden.  
b) Severe Mucocutaneous Reactions: Mucocutaneous reactions, some with fatal outcome, have 
been reported in patients treated with rituximab.  These reports include paraneoplastic 
pemphigus (an uncommon disorder which is a manifestation of the patient’s underlying 
malignancy), Stevens-Johnson syndrome, lichenoid dermatitis, vesiculobullous dermatitis, and 
toxic epidermal necrolysis.  The onset of the reaction in the reported cases has varied from 1 to 
13 weeks following rituximab exposure. Patients experiencing a severe mucocutaneous reaction 
should not receive any further infusions and seek prompt medical evaluation.  Skin biopsy may 
help to distinguish among different mucocutaneous reactions and guide subsequent treatment. 
The safety of readministration of RITUXAN to patients with any of these mucocutaneous 
reactions has not been determined.  
c) Infusion reactions and lymphopenia are the most commonly occurring adverse reactions. Mild 
to moderate infusion reactions consisting of fever and chills/rigors occur in the majority of 
patients during the first rituximab infusion.  Other frequent infusion reaction symptoms included 
nausea, pruritus, angioedema, asthenia, hypotension, headache, bronchospasm, throat irritation, 
rhinitis, urticaria, rash, vomiting, myalgia, dizziness, and hypertension.  These reactions 
generally occur within 30 to 120 minutes of beginning the first infusion, and resolved with 
slowing or interruption of the rituximab infusion and with supportive care (diphenhydramine, 
acetaminophen, IV saline, and vasopressors).  In an analysis of data from 356 patients with 
relapsed or refractory, low-grade NHL who received 4 (N = 319) or 8 (N = 37) weekly infusions 
of rituximab, the incidence of infusion reactions was highest during the first infusion (77%) and 


Page 65 of 115 
 
decreased with each subsequent infusion (30% with fourth infusion and 14% with eighth 
infusion).  


Page 66 of 115 
 
8.7
DIPHENHYDRAMINE
8.7.1
Supply – Commercially available.  Diphenhydramine HCl injection is available in an 
injectable solution at a 50mg/ml concentration in single dose ampules, syringes and vials 
as well as multi-dose vials from multiple manufacturers.  
8.7.2
Preparation – Diphenhydramine HCl may be given by direct intravenous injection 
without additional dilution.  Alternatively the prescribed dose may be diluted in a small 
volume (e.g. 25-50ml) of 5% dextrose in water (D5W) or 0.9% sodium chloride (NS) and 
infused over 10-15 minutes.  
8.7.3
Storage and Stability – Store commercially available injectable product at controlled 
room temperature.  
8.7.4
Administration – Diphenhydramine HCl injection may be administered by direct IV 
injection (IV push) at a rate generally not exceeding 25mg/min. Alternatively, 
diphenhydramine HCl injection may be diluted and given over 10-15 minutes (see 
Preparation).  
8.7.5
Toxicities – Sedation, sleepiness, dizziness, disturbed coordination, epigastric distress, 
thickening of bronchial secretions. Diphenhydramine can provide additive effects with 
alcohol or other CNS depressants. Diphenhydramine can cause anticholinergic side 
effects (e.g. dry mouth, fixed or dilated pupils, flushing, urinary retention). 
Diphenhydramine should be used with caution in patients with a history of bronchial 
asthma, increased intraocular pressure, hyperthyroidism, cardiovascular disease or 
hypertension.  
8.8
 ACETAMINOPHEN  
8.8.1
 Supply – Commercially available as 325 mg or 500 mg tablets for oral administration 
from multiple manufacturers.  
8.8.2
 Storage – Store at controlled room temperature.  
8.8.3
Administration – Oral. For analgesia and antipyresis, the usual dose is 650 to 1000 
milligrams every 4 to 6 hours, to a maximum of 4 grams/day.  
8.8.4
Toxicities – No toxicities are anticipated to result from single doses of acetaminophen 
administered as premedication for rituximab infusions.  
8.9
 VALACYCLOVIR (Valtrex®)  


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8.9.1
 Supply – Commercially available as 500mg tablets and 1gm tablets. Dose adjustment is 
necessary in patients with significant renal impairment (refer to the manufacturer's 
labeling for dose adjustment guidelines.)  
8.9.2
Pharmacology – Valacyclovir is the hydrochloride salt of L-valyl ester of the antiviral 
drug acyclovir. After oral administration, valacyclovir is rapidly absorbed from the GI 
tract and nearly completely converted to acyclovir and L-valine by first-pass intestinal or 
hepatic metabolism.  
8.9.3
Storage and Stability – Oral tablets should be stored at 15
o
 to 25
o
C (59
o
 to 77
o
F).  
8.9.4
Administration – Oral.  
8.9.5
Toxicities – Nausea and/or vomiting, headache, dizziness, abdominal pain, 
dysmenorrhea, arthralgia, acute hypersensitivity reactions, elevations in liver enzyme 
laboratory values (e.g. AST).  Renal failure and CNS symptoms have been reported in 
patients with renal impairment who received valacyclovir or acyclovir at greater than the 
recommended dose. Dose reduction is recommended in this patient population (refer to 
the manufacturer's labeling for dose adjustment guidelines).  
8.10 CASPOFUNGIN (Cancidas
®
)
8.10.1  Supply: Commercially available.  
8.10.2  Product description: Caspofungin is available in 70mg and 50mg single-use lyophilized 
vials that require reconstitution with appropriate diluent and final dilution prior to 
administration.     
8.10.3  Preparation: For reconstitution, aseptically add 10.5ml of an appropriate diluent (see 
manufacturer’s package insert for suggested diluents) to either the 70mg or 50mg 
lyophilized vial. Then aseptically transfer 10ml of the reconstituted caspofungin to an IV 
bag (or bottle) containing 250ml of 0.9% sodium chloride injection or another suitable 
parenteral solution (see manufacturer’s package insert for suggested solutions). NOTE: 
DO NOT USE DILUENTS CONTAINING DEXTROSE AS CASPOFUNGIN IS NOT 
STABLE IN DILUENTS CONTAINING DEXTROSE. In fluid restricted patients 
receiving the 50mg dose, a volume of 100ml (instead of 250ml) of an appropriate 
parenteral solution can be utilized.  
8.10.4  Storage and Stability: The lyophilized vials should be stored refrigerated at 2
o
 to 8
o
 C 
(36
o
 to 46
o
 F). Reconstituted caspofungin may be stored at < 25
o
C (< 77
o
F) for one hour 


Page 68 of 115 
 
prior to the preparation of the patient infusion solution. The diluted product (final patient 
infusion solution) in the IV bag or bottle should be stored at < 25
o
 C (< 77
o
F) for 24 
hours or at 2
o
 to 8
o
C (36
o
 to 46
o
 F) for 48 hours.  
8.10.5  Route of administration:  Parenteral. Parenteral doses should be administered by slow IV 
infusion over approximately one hour.  
8.10.6  Toxicities:  
Fever, nausea, vomiting, flushing, infused-vein complications (e.g. phlebitis), and headache have 
generally been reported in clinical trials at an incidence > 2%. Possible histamine-mediated 
symptoms have been reported including isolated reports of rash, facial swelling, pruritis, 
sensation of warmth, or bronchospasm. Anaphylaxis has been reported during administration of 
caspofungin.  In post marketing experience, rare cases of clinically significant hepatic 
dysfunction has been reported. Elevations in liver transaminases (AST, ALT), total serum 
bilirubin, and serum alkaline phosphatase have been reported in clinical trials at an incidence 
usually > 2%. Concomitant use of caspofungin with cyclosporine has been associated with a 
higher risk of liver tranasaminase elevations in healthy subject volunteers and concomitant use is 
not recommended by the manufacturer.  
8.11 TRIMETHOPRIM/SULFAMETHOXAZOLE (TMP/SMX, Cotrimoxazole, Bactrim, 
Septra)
8.11.1  Supply – Commercially available as a single strength tablets containing trimethoprim 
80mg and sulfamethoxazole 400mg and a double strength (DS) tablet containing 
trimethoprim 160mg and sulfamethoxaole 800mg. It is also available in a oral suspension 
at a concentration of 40mg of trimethoprim and 200mg sulfamethoxazole per 5ml. 
Parenteral TMP/SMX is available in a solution for injection at a concentration of 80mg of 
trimethoprim and 400mg of sulfamethoxazole per 5ml.  
8.11.2  Preparation – For parenteral administration, the commercial solution for injection must 
be diluted prior to administration. It is recommended that each 5ml of the solution for 
injection be diluted with 100-125 ml or, if fluid restriction is required, in 75ml of 
dextrose 5% in water.  Normal saline (0.9% sodium chloride) may be substituted as a 
diluent but the resulting solutions have reduced stability. Consult with pharmacy for 
questions regarding diluent, volume, and expiration.  
8.11.3 Storage and Stability – Oral tablets and oral suspension should be stored at 15
o
 to 30
o
C 
(59
o
 to 86
o
F) in a dry place and protected from light. TMP/SMX for injection should be 
stored at room temperature between 15
o
 to 30
o
C (59
o
 to 86
o
F) and should not be 


Page 69 of 115 
 
refrigerated. Stability of intravenous doses after final dilution is dependent on 
concentration and diluent. Consult with pharmacy for questions regarding stability and 
expiration dating.  
8.11.4 . Administration – Oral and parenteral.  Parenteral doses should be administered by an 
intravenous infusion over 60 to 90 minutes.  
8.11.5 Toxicities – The most common adverse effects from TMP/SMX are gastrointestinal 
disturbances (nausea, vomiting, anorexia) and allergic skin reactions (such as rash and 
urticaria). Fatalities associated with the administration of sulfonamides, although rare, 
have occurred due to severe reactions, including Stevens-Johnson syndrome, toxic 
epidermal necrolysis, fulminant hepatic necrosis, agranulocytosis, aplastic anemia and 
other blood dyscrasias.  For TMP/SMX injection, local reaction, pain and slight 
irritationon IV administration are infrequent. Thrombophlebitis has rarely been observed.  
8.12 BAXTER ISOLEX 300i Stem Cell Collection System, version 2.5
8.12.1 Availability - The Isolex 300i Stem Cell Separation System, version 2.5, is an automated 
cell separation system based on immunomagnetic methods to purify CD34+ 
hematopoietic progenitor cells.  It is manufactured by Baxter Healthcare Corporation and 
has been approved by the FDA; however, use of this system in this protocol is considered 
an “off label” use. CD34+ cell selection is performed according to the manufacturer’s 
instructions and the DTM standard operating procedure based on those instructions.  
8.12.2 Preparation and Administration – A leukapheresis concentrate of mobilized peripheral 
blood stem cells is incubated with human IVIgG, filtered through a standard blood 
recipient filter, and attached to the disposable set of the Isolex system. Using the Isolex 
closed system, the cells are washed to remove platelets and plasma, and concentrated for 
incubation with a murine anti-human CD34 monoclonal antibody. After washing, the 
sensitized cells are rosetted with paramagnetic microspheres coated with sheep anti-
murine antibody.  The rosetted cells are separated from the remaining cells by exposure 
to the primary magnet.  A release agent is added to release the CD34+ cells from the 
paramagnetic microspheres coated with sheep anti-murine antibody.  The released 
CD34+ cells are washed, concentrated, and transferred to a plastic bag for infusion or 
cryopreservation.  
8.12.3 Toxicities – There are no significant toxicities associated with the infusion of autologous 
CD34+ cells that have been purified by the Isolex method.  The cells will be 
cryopreserved and theoretically there may be adverse effects associated with the use of 
dimethylsulfoxide (DMSO) as a cryoprotectant such as hives, flushing, chest tightness, or 
cardiovascular instability. However, these effects are unlikely to occur or would be 


Page 70 of 115 
 
relatively minor because the dose of DMSO is very low with elected CD34+ cells. The 
stem cell product will be additionally infused after premedication with diphenhydramine 
(Benadryl) 25-50 mg p.o. or i.v.  
8.13
18
FDG-PET
8.13.1 Supply  
 Fluorine-18-fluorodeoxyglucose (FDG) is an investigational new drug approved by the FDA for 
use in a limited number of patients.  The current holder of this IND#23195 is Dr. Peter 
Herscovitch, Chief, PET Department. The FDG is produced as outlined in the IND. FDG is an 
analog of glucose labeled with F-18 (half-life is109 minutes). The FDG is taken up by the same 
mechanism as glucose and then is phosphorylated.  Once intracellular and  phosphorylated, it is 
not subject to the breakdown by glucose phosphatase and therefore accumulates intracellularly.    
8.13.2 Preparation  
The FDG in this protocol will be provided by the PET Department, CC, NIH or from a 
commercially available source in compliance with the FDG IND.  The formulations will be 
prepared on the day of use and injected within 9 hours of formulation. The FDG is typically 
prepared at a specific activity of > 1 Ci/mmol.  Radionuclide purity is analyzed periodically and 
to date no radionuclide impurities have been detected.  Preparation  
The FDG for human use is in sterile isotonic phosphate buffer and is passed over terminal 
millipore filter (0.22μ). It has high specific activity and is isomerically pure. A sample of each 
FDG preparation will be tested for pyrogenicity by the limulus amebocyte lysate assay; sterility 
and radiochemical purity by thin layer chromatography by the Quality Control Group of the PET 
department. Quality control will be performed as outlined in the IND. Alternatively the FDG will 
be purchased from a commercial vendor who provides FDG for clinical use.  
8.13.3 Administration  
The product is in liquid form and will be administered intravenously.  
8.13.4 Dosimetry  
FDG dosimetry has been calculated using methodology of the Medical Internal Radiation Dose 
(MIRD) Committee of the Society of Nuclear Medicine. The cumulative radiation dosimetries 
with bladder voiding 1.5 hours after injection (for a total of 15 mCi) are summarized in the RSC 
form 88-23(A).  All radioactive drugs will be administered under the supervision of an 
authorized user. The dosimetry for FDG and transmission scans performed under this protocol is 
given in the NIH 88-23 form.  


Page 71 of 115 
 
8.13.5 Toxicity  
FDG has been used extensively at the NIH since 1983 and at many other centers throughout the 
world and it is well tolerated. Because we use microgram amounts there are no clinically 
significant side-effects.   
9
APPENDICES  
9.1
Appendix A 
. Summary of laboratory research studies  
A-1Examination of B cell precursor and mature B cells in SLE before and after auto-HSCT (By 
the laboratories of Amrie Grammer and Peter Lipsky)  
SLE-prone individuals may have an abnormality in the generation and/or    maintenance of their 
functional repertoire of B cells expressing specific Ig  genes and may have been subjected to 
conditions that bias specific  immunoglobulin variable region usage, thereby predisposing to the  
development of autoAb [113]. Intrinsic B cell over-reactivity may provide the drive for 
pathogenic autoAb formation by overwhelming the normal mechanisms that protect against 
autoimmunity and thereby permit  the escape of pathogenic autoantibody producing progeny. 
AutoHSCT of  SLE patients will determine whether deleting a large percentage of memory B-
cells and effector plasma cells and replacing them with autologous stem cell-derived naïve cells 
in a non-inflamed environment will lead to long  lasting remission in systemic autoimmune 
disease. The goal of this research is to examine the extent of depletion of  memory B-cells and 
effector plasma cells as a result of the conditioning  regimen and the nature of the surviving cells 
and the cells reconstituted    from the autologous stem cells and to relate these parameters to the 
extent of clinical response and the length and quality of remission.  
The human B cell repertoire is generated following differentiation of pluripotent hematopoietic 
stem cells in the bone marrow to immature B cells that migrate through the blood to finish their 
differentiation to mature B cells in the spleen. The techniques that can be used to monitor the 
nature and completeness of B cell depletion before transplantation as well as reconstitution of B 
cell subsets include multiparameter flow cytometry, single cell PCR of sorted B cell populations 
for Ig rearrangement, repertoire and mutational analysis, as well as microarray and the proteomic 
technique immunoaffinity capillary electrophoresis (ICE) for analysis of genes and proteins that 
are not able to be detected by multiparameter flow cytometery.  
A-2. Reconstitution of regulatory and effector T cell populations
following autologous HSCT in systemic lupus (By the laboratory of Fran  
Hakim and Ronald Gress)


Page 72 of 115 
 
Systemic lupus patients display multiple defects in T cell function, resulting in problems 
distinguishing which defects may be primary and which secondary effects of the disorder or of 
therapies applied. Immunoablation provides a unique opportunity to attempt to dissect primary 
and secondary defects in T lymphocytes of lupus patients and to correlate them with disease 
activity. Durable remissions in SLE following cyclophosphamide treatment and autologous 
transplant have been associated with a normalization of T cell populations, repertoire and 
function in a few patients studied [114]. Certainly the depletion of activated T cells associated 
with the disease process is a key element in remission. Recovery of naïve T cell populations 
through a renewal of thymopoiesis post transplant would also contribute to the recovery of a 
broad T cell receptor repertoire. T cell regeneration will be evaluated by studying the peripheral 
T cell receptor repertoire and subsets of T cells by multicolor FACS analysis. The role of thymus 
will be evaluated by studying the presence of recent thymic emigrants determined by the 
presence of TRECs.  
Special emphasis will be placed on studies of regulatory T lymphocytes (some of these studies 
will be done in Dr. Lipsky’s laboratory). Little is known of the role of TR in the pathogenesis of 
systemic autoimmune disorders in man. The complete lack of TR in the scurfy mouse results in 
an aggressive lethal autoimmune disorder that can be corrected by early transplant of 
CD4+CD25+ TR cells [115]. On the other hand, depletion of TR by neonatal thymectomy in 
lupus-prone (SWR x NZB)F1 mice increased levels of autoantibodies and organ specific 
autoimmunity, but also shifted the immunoglobulin isotype of the predominant autoantibodies 
and reduced glomerulonephritis [116]. Some of the immunological abnormalities present could 
be consistent with a reduction in TR, such as the common finding of an inability to make TGFȕ, 
a key cytokine in generation and function of TR. We have recently shown that levels of 
CD4+CD25+ TR cells are abnormal in active lupus patients in contrast to those with inactive 
lupus [117]. Furthermore, the TR cells in lupus patients lack suppressive activity in vitro.  
One potential explanation for durable remission with auto-HSCT is the recovery of regulatory T 
cell populations. Immune reconstitution studies post transplant have focused on expansion of 
residual memory cells and generation of new naïve T cells. The ability of the adult thymus to 
reconstitute TR populations following transplant has never been examined. The severe reduction 
in lymphocyte populations produced by the fludarabine and cyclophosphamide regimen and the 
CD34 selected graft should provide optimal conditions for assessing the recovery of TR cells.  
We will assess the levels of Tr cells in at the pretreatment time point and  establish a time course 
of recovery of TR cells in the peripheral blood by the use of multiparameter flow cytometry, 
functional analysis and quantitative  RT-PCR.  
 A-3. Feasibility of Th2-cell type differentiation (By the laboratory of Daniel Fowler)  
As previously referenced, SLE is an autoimmune disease with a complex disease pathogenesis. 
With respect to the role of T cells in SLE, there is considerable debate whether the Th1/Th2 
paradigm of T cell cytokine biology applies to this disease. As such, we have generated the 


Page 73 of 115 
 
hypotheses that, like most inflammatory disorders, Th1-type immunity plays a role in SLE 
pathogenesis, and that Th2-type immunity present in SLE patients may represent an unsuccessful 
attempt at immune auto-regulation. Significantly, recent murine data would lend support to this 
hypothesis [118]. This biology would be analogous to that of GVHD, which is initiated by Th1-
type cells, yet after disease establishment, both Th1- and Th2-type patterns of T cells can 
contribute to disease pathogenesis.  However, in the allogeneic transplant setting, if Th2-type 
cells are predominant at initiation of the transplant, the initial Th1-type response is abrogated, 
and acute GVHD is prevented. By extension of these principles, one would predict that in the 
setting of immune ablation and autologous transplantation for the therapy of SLE, that re-
establishment of an anti-inflammatory immune response that is devoid of Th1-type elements and 
enriched for Th2-type elements might prevent the re-development of an SLE autoimmune 
predisposition. Given this possibility, using established methods of T cell expansion we have 
developed, we will evaluate the feasibility of whether CD4
+
 T cells from SLE patients (at the 
time of study entry) can be induced into a state of Th2-type differentiation. In the event that this 
is possible, such an autologous Th2-type product might be utilized in future studies to promote 
the reconstituting immune system to a Th2 phenotype after transplantation for SLE. 40 ml of 
peripheral blood will be harvested from SLE patients at the time of study entry. CD4
+
 T cells 
will be enriched by negative selection, and an initial CD4 cell cytokine phenotype will be 
determined by ELISA assay and Miltenyi cytokine secretion assay. Such CD4 cells will then be 
expanded under in vitro culture conditions in an attempt to polarize towards Th1- or Th2-
phenotypes.  In addition to this Th2 cell therapy feasibility evaluation, post-autoHSCT time 
points will also be evaluated by the same methods. These evaluations will be aimed at 
determining whether autologous transplantation resulted in a shift in the capacity of patient CD4 
cells to develop towards Th1 or Th2 phenotypes.      
B . Cytogenetics
(By the laboratory of Diane Arthur)  
A relatively rare but serious potential complication of autoHSCT is the  development of 
secondary, therapy-related myelodysplastic syndrome (t-MDS) or acute myelogenous leukemia 
(t-AML). Although documented most extensively inpatients transplanted for non-Hodgkin's 
lymphomas, t-MDS and t-AML have alsobeen reported following autoHSCT for non-
hematologic solid tumors and nonmalignant diseases [119] (D. Jayne personal communication). 
Both pretransplantation and transplant-related factors likely play a role in the occurrence of 
secondary malignancy, but the relative contributions of these various factors remain a matter of 
some controversy. Factors that have been implicated include dose, type, and duration of prior 
cytotoxic therapy; total body irradiation; number and source of hematopoietic stem cells infused; 
chemotherapy priming of peripheral blood stem cells; and older age. The preparative regimen for 
patients entered on this study has been carefully chosen to decrease myelotoxicity and optimize 
immunodepletion.  Nonetheless, we anticipate these patients will be at some risk for developing 


Page 74 of 115 
 
t-MDS or t-AML due, in part, to previous cytotoxic chemotherapy, particularly the alkylating 
agent cyclophosphamide [120], and perhaps to cyclophosphamide and fludarabine in the priming 
and preparative regimens.  Recent studies have shown that small clones of abnormal myeloid 
progenitor cells, not evident by routine G-banding, can be detected prior to transplant using 
interphase fluorescence in-situ hybridization (FISH) techniques in patients who go on to develop 
t-MDS or t-AML after transplant [121, 122]. The investigators suggest that pre-transplant FISH 
studies can potentially identify patients at increased risk of developing secondary malignancy 
and, therefore, permit more informed clinical management.To test the hypothesis that 
prospective interphase FISH analyses can detect small clones of abnormal myeloid progenitor 
cells prior to transplant, we propose to perform interphase FISH analyses using a carefully 
chosen panel of probes on bone marrow collected at baseline and 6, 12, and 24 months after 
transplant, as well as purified CD34+ stem cells collected for re-infusion on Day 0.  Based upon 
prior studies done in our laboratory and others, we anticipate the most common chromosomal 
abnormalities following alkylating agent chemotherapy will be loss or deletion of the long arms 
of chromosomes 5 and 7 (-5/5q-, -7/7q-).  Other common abnormalities include deletions of band 
13q14, the short arm of chromosome 17, and the long arm of chromosome 20; and trisomy of 
chromosome 8. We will therefore employ a panel of six probes to detect these abnormalities.   
Routine G-banded metaphase chromosome analysis will be performed on all bone marrow 
aspirates in the CAP-accredited NCI/CCR/LP Clinical Cytogenetics Laboratory under the 
direction of Dr. Diane Arthur. Processing and analysis will be performed according to 
standardized laboratory protocols. Final reports will be issued to the patients' medical records, 
with copies to Dr. Pavletic. Interphase FISH analyses will be done on all bone marrow aspirates, 
and also on purified CD34+ stem cells. These analyses will also be performed in Dr. Arthur's 
laboratory according to procedures established and quality-controlled for each probe in her 
laboratory.  A panel of six probes purchased from Vysis, Inc. will be applied to each sample 
including LSI EGR1/D5S23, D5S721 Dual Color Probe, LSI D7S486/CEP 7 Dual Color Probe, 
LSI 13 (RB1) 13q14 SpectrumOrange Probe, LSI p53 (17p13.1) SpectrumOrange Probe, LSI 
D20S108 (20q12) SpectrumOrange Probe, and CEP 8 DNA Probe Kit.  The FISH tests will be 
performed on a research basis, with results to Dr. Pavletic only.  
C . F-18 FDG Positron Emission Tomography to Assess the Distribution of Activated 
Lymphocytes in Systemic Lupus Erythematosus
(By Peter Herscovitch, M.D. Participating investigator: M. Whatley).   
Positron emission tomography (PET) is a physiology-based method of imaging based on the 
uptake and metabolism of radiopharmaceutical by the tissues. PET with FDG is a functional 
imaging technique that provides information about glucose metabolism. FDG has been used 
successfully to image a variety of tumors [123]. FDG accumulation in tissue is by no means 
tumor specific and accumulation has been reported in a variety of infectious and inflammatory 
processes [124, 125]. Uptake in sarcoidosis has been reported and patients with AIDS often have 
hypermetabolic nodes.  Similar findings have been found in our study of patients with AIDS and 


Page 75 of 115 
 
high viral loads at the clinical center (protocol 00-I-0109). Furthermore patients with 
autoimmune lymphoproliferative disorder (ALPS) who have a defect in apoptosis and who 
typically exhibit enlarged lymph nodes will often have increased uptake of FDG in their 
peripheral nodes (based on our experience here at NIH (02-I-0308).  
Patients who are receiving G-CSF typically will change their FDG biodistribution exhibiting a 
marked increase in bone marrow uptake and increase in splenic uptake.  
In patients with active SLE both T and B-lymphocytes express higher levels of activation 
markers compared to inactive patients and effective treatment leads to a decrease in activated 
lymphocytes [84]. The anatomic site of lymphocyte activation is unknown in humans.  
In a recently completed pilot study, we compared the pattern of PET images in lymphoid organs 
between patients with active and inactive disease. There were two important findings of this 
study. First, both active (n=10) and inactive (n=9) lupus patients had increased FDG uptake in 
lymph nodes when compared to healthy volunteers, with no statistical difference between the 
two groups. This suggests that the metabolic and probably immunologic activity of lymphoid 
organs is enhanced, not only in active, but also in clinically quiescent SLE patients. It would be 
of interest to see whether achieving clinical remission after the autoHSCT results in a more 
normal PET pattern. Second, thymic uptake, was demonstrated in 5/10 active patients compared 
to 0/9 of inactive patients. This suggests a qualitative difference in the immune response between 
different stages of clinical activity and points toward an important role for the thymus during 
active disease. Age might be a confounding factor but thymic uptake was seen in active patients 
even in their fourth decade, when the thymus is usually involuted. In fact, 3/5 patients with 
thymic uptake were 29 years or older. Thymic uptake was seen in only 1/10 healthy controls of 
similar age. Imaging the thymus will be of interest for several reasons. The correlation between 
thymic activity and regeneration of the T-cell repertoire following immunoablation is of general 
interest, whereas the correlation between thymic uptake, changes in T lymphocyte subsets and 
disease activity will provide important information about the pathogenesis of lupus.  
     Eligibility Assessment and Enrollment for PET studies Exclusion Criteria:  
i)  Patients who weigh  136 kg (weight limit for the PET scanner table).  
ii)  Patients with uncontrolled glucose due to diabetes may be studied,  
 but will be evaluated separately.  
iii)  If logistical difficulties arise patients may not be scanned and this will  
 not be considered a protocol violation.  
iv)  Pregnancy test for women of childbearing potential prior to imaging  
 within 24 h of imaging.  
v)  Patients under the age of 18.  
 
 FDG-PET scanning  


Page 76 of 115 
 
FDG-PET scans of the whole body will be performed at baseline. On day -1 before transplant 
and at 1, 6, 12, and 24 months post-transplant. The patients will be fasting for at least 6 hours 
except for water.  They will be encouraged to drink fluids prior to the FDG injection.  An 
intravenous line with a 0.9% saline solution will be inserted into the subject’s arm for isotope 
injection. Lines should be inserted prior to arrival for their PET scan. The patient will be 
positioned on the FDG-PET scanning bed.  A transmission scan using Ga-68/Ge-68 rotating pin 
source of positron emitting radioactivity will be performed to measure the attenuation.  These 
measurements will be used to correct the emission data. Typically, each area imaged will require 
a 3 minute transmission scan (total transmission scan < 8 min per study).  This will be followed 
by an emission scan of the same area.  Transmission scans will be used for attenuation 
correction. In total there will be a 1 h uptake period and less than 90 min imaging time  
One blood sample for glucose and insulin determination will be obtained during the scanning 
sequence.  The patients will be instructed to void frequently for the next 6 hours. Patients will be 
allowed to void after their emission scan.  These scans will be reconstructed and images will be 
reviewed visually. The PET scan images will be read by a physician experienced in the 
interpretation of whole body PET imaging. The region of interest will be performed over areas in 
the baseline and follow up scans that will include the liver, spleen, bone marrow and any lymph 
node regions or areas that are felt to warrant evaluation by the nuclear physican. We will use 
quantitative or semi-quantitative parameters to assess lesion activity.   
For FDG-PET scans the following data will be collected at baseline and follow up:  
i. SUV max and mean  
ii. Metabolic volume  
iii. Comparison with CT (if available)  
iv. Comparison with fine needle aspirates (if available)  
-As many patients as possible that are being accrued into the protocol will be imaged with FDG.  
Patient’s baseline images will compare to the follow up images. Human Subject Protections  
-FDG-PET is an approved procedure for the evaluation of patients with metastatic cancer. In this 
study the main purpose of the FDG scan is to try evaluate if we can see metabolic changes 
related to lymphocyte homing and trafficking, thus no direct benefit is expected from serial 
FDG-PET imaging.  
D . Ultrasound surveillance and fine needle aspiration of lymph nodes
(By Bradford J. Wood, Ziv Neeman and Andrea Abati) For the lymph node specimen research 
sampling patients will undergo a limited superficial ultrasound survey at regular intervals during 
the protocol, including pre-transplantation (not to exceed 6 weeks prior to transplantation), and 6, 


Page 77 of 115 
 
12, and 24 months following transplantation. This limited ultrasound will include a survey of 
bilateral axillae, groins (inguinal and femoral nodes), and the neck (cervical chains). Grossly 
abnormal nodes will be measured and recorded on the imaging datasheet (Appendix K). The 
largest node in each region (6 regions: left and right neck, groin, and axilla) will be measured in 
3 dimensions, and the exact location will be recorded in relation to nearby anatomy and vessels 
for future localization. Depth below skin surface will be recorded. Note will be made if no nodes 
are visible on ultrasound in a region.  
Fine needle aspiration: Fine needle aspirations will be performed with 23 or 22 gauge needles 
(like Chiba cutting needle) using standard image guided technique with ultrasound. Clinical 
Center guidelines will be adhered to for all stages of the procedure following the special 
procedures policies. If conscious sedation is required, Clinical Center sedation guidelines will be 
adhered to. Standard laboratory studies will be performed within 7 days of the procedures to 
include platelets and PT/PTT. History and physical will be on chart within 30 days of procedure. 
Research specimen paperwork will be used. Cytology staff will be available at the time of the 
aspirations for rapid bedside interpretation to estimate the quantity and quality of lymphoid 
material sampled.   
Patients will be prepped and draped in the usual sterile fashion, and the subcutaneous tissues will 
be anesthetized with 1 – 2 % lidocaine along the chosen pathway. This should render the 
remainder of the procedure relatively pain-free following the mild burning sensation induced by 
lidocaine anesthetic. 22 or 23 gauge needles will be inserted into the nodal target with real-time 
ultrasound visualization to ensure accurate sampling. The first pass will be a “capillary” pass, 
with no suction applied, to minimize red blood cell contaminant in the specimen. Subsequent 
passes will have suction applied, usually with an automatic syringe. All specimens will be split 
between slide and test tube with normal saline (or fixative such as RPMI).  
Up to three nodes may be sampled in a given patient, at a given time period. Fine needle 
aspirates may be performed at baseline (pre-transplantation), as well as at 6, 12 and 24 months 
following transplantation.   
Ultrasound guided fine needle aspiration of superficial nodes is a very low-risk procedure. The 
location of the nodes superficially makes bleeding less likely since pressure can be held to 
control any theoretical bleeding. Such small needles have a much lower risk of causing bleeding 
or other complications that larger needles typically used for image-guided biopsy procedures. 
Measurements will be made of nodes before and after aspiration, and the size pre-aspiration may 
be used for the timeline measurement.  


Page 78 of 115 
 
 
9.2
Appendix B: Guidelines for prophylaxis of infectious complications in 
lymphodepleting autologous stem cell transplantation for SLE  Note:
The practice guidelines included in this Appendix are based upon the best available clinical and 
circumstantial evidence and may change as additional data become available. Although the 
current protocol is in the autologous transplantation setting, this patient population of lupus 
patients is considered to be at an infection risk that is potentially higher than in a non-
lymphodepleting autologous transplantation protocol, therefore the whole infection prevention 
philosophy here is to err on the side of safety and at minimum matches the allogeneic stem cell 
transplantation protocol prophylaxis standards.   
Pneumocystis carinii pneumonia
PCP prophylaxis will start prior to priming. After finishing leukapheresis and prior to starting the 
conditioning regimen, all patients, except those with lupus nephritis, will complete (as latest by 
day -1) a 7 day course of trimethoprim 160 mg/sulfamethoxazole 800 mg (TMP/SMX 160/800), 
one tablet PO BID. Lupus nephritis patients will continut the usual 3x/week prophyliactic 
schedule until day -1. The treatment will resume post transplant when ANC is >500/μL and 
unsupported platelets >50,000/mm
3
 at one tablet a day dosing three days per week (e.g., on each 
Monday, Wednesday and Friday). This will continue for 12 months post transplant and until 
CD4 count >200/μL and prednisone dose < 5 mg/day, whichever comes later.  
9.2.1.1 The alternative regimens for PCP prophylaxis are less effective than TMP/SMX and 
include:  
-Pentamidine inhalation 300 mg every 4 weeks  
-Dapsone PO 100 mg/day  
-Atovaquone PO 1500 mg/day  
9.2.2
Fungal Infections (including Aspergillus)  
Since majority of patients will be on long-term high doses of steroids, caspofungin usually at 50 
mg i.v./day will be used starting on day 2 until post-transplant ANC is >1000/μL.  
9.2.3
Viral pathogens  
9.2.3.1 Herpes Simplex Virus (HSV) and Varicella Zoster Virus (VZV)  
a) All patients will receive valacyclovir 500 mg PO QD for suppression of HSV and VZV 
infection/reactivation.  This prophylaxis will begin when patients start the priming regimen and 


Page 79 of 115 
 
continue throughout transplantation. Valacyclovir will continue for 12 months after 
transplantation, until CD4 count >200/μL and prednisone <5mg/day, whichever comes later. If a 
patient cannot take oral medications, acyclovir 250 mg/m
2
 IV q12h will be substituted for 
valacyclovir.  
9.2.3.2 Cytomegalovirus (CMV) Preemptive Therapy:  Patients with positive pre-transplant 
serologies for CMV will undergo weekly monitoring of CMV antigenemia. 
Antigenemia monitoring will begin at the time of starting priming regimen and continue 
through day +100 or until CD4 count is > 200/μL. Antigenemia will be assessed if 
clinically indicated independently of the pre-transplant CMV serology status, for 
example if fever of unknown origin, decreasing counts or pulmonary symptoms occur. 
If a patient has an ANC < 1000/μL or the specimen is obtained from a long distance 
CMV monitoring will be performed via polymerase chain reaction (PCR) instead of 
antigenemia. If antigenemia becomes positive in two consecutive samples (expressed as 
the number of positive cells per 400,000 white blood cells) before day +100, then 
patients will be treated with oral valganciclovir, IV ganciclovir, or IV foscarnet 
according to the following schema:  
# Of 
(+) cells 
 
Rx: 
VALGANCICLOVIR
 
Rx: 
IV 
GANCICLOVIR 
Monitoring 
Reinduction 
Any 
Valganciclovir 
induction: 900 mg PO 
q12h for 7 days, then 
maintenance: 900 
mg/24h until 
antigenemia is 
negative x 2 weeks* 
Ganciclovir 
induction: 5 
mg/kg IV q12h 
for 7 days, then 
maintenance: 5 
mg/kg/24h 5 
days/week until 
antigenemia is 
negative x 2 
weeks* 
 
Weekly 
antigenemia 
 
1.
If 
antigenemia 
becomes 
positive 
after being 
negative 
2.
For 
doubling of 
antigenemia 
 
 
*I.e., on two consecutive weekly antigenemia tests.  
NOTES:  
 
x
Valganciclovir and ganciclovir may cause bone marrow suppression.  If ANC < 1000: 
Start filgrastim (G-CSF) 5-10 μg/kg/d  


Page 80 of 115 
 
x
Weekly IVIG infusion has no proven benefit in the setting of CMV reactivation without 
established disease.  Therefore, its use will be limited to patients with CMV pneumonitis 
(or other target organ infection, at the investigators’ discretion).  
 
Indications for IV ganciclovir:   
x
clinical suspicion of malabsorption  
Indications for foscarnet:  
x
ANC < 500/μl despite G-CSF  
x
Platelets < 20,000/mm
3 
 
x
Resistance to ganciclovir: rising antigenemia after 3 weeks with no response to 
ganciclovir reinduction  
Dose Adjustments for Renal Insufficiency  
 
 
 
Valganciclovir 
Calculat 
ed CrCl  
Induction  Maintenance  
>60  
900 mg 
q12h  
900 mg q24h  
40-59  
450 mg q 
12h  
450 mg q 24h 
25-39  
450 mg q 
24h  
450 mg q 48h 
10-24  
450 mg q 
48h  
450 mg 
2x/week  
Ganciclovir 
Calculated 
CrCl  
Induction  
Maintenance  
>70  
5 mg/kg 
q12h  
5 mg/kg q24h  
>50-69  
2.5 mg/kg 
q12h  
2.5 mg/kg q24h  
25-49  
2.5 
mg/kg/24h  
1.25 mg/kg/24h  
10-24  
1.25 
mg/kg/24h  
0.625 mg/kg/24h  
<10  
1.25 mg/kg 
3x/wk  
0.625 mg/kg 3x/wk 
 
 
9.2.4
Epstein-Barr virus  
All patients will be monitored following the completion of the conditioning regimen at weekly 
intervals for EBV-DNA by a quantitative assay at least until the day 100 post-transplant and until 
CD4 count is >200/μL, whichever comes later. Patients experiencing an EBV reactivation more 
than or equal to 1000 geg/mL will be candidates for preemptive therapy using a single 375 


Page 81 of 115 
 
mg/m
2
 infusion of rituximab [126]. Prior to such preemptive rituximab infusion patients will be 
carefully examined for signs and symptoms to exclude EBV post-transplant lymphoproliferative 
disorder (PTLD), including if needed, CT scans and bone marrow biopsy. EBV load will be 
monitored daily during the first 72 hours then twice weekly until 2 negative (<50 geg/mL) tests 
were obtained and thereafter at the each outpatient visit.EBV-PTLD is extremely rare after 
autologous stem cell transplantation but is more commonly described in transplants for both 
malignant and/or autoimmune disease in situations of profound T-cell depletion by graft 
manipulations or anti-T cell antibody administration.   
9.2.5
Bacterial Pathogens  
Since most of these patients will be on steroids, ceftazidime at doses of 2 g IV Q 8 hrs will be 
given prophylactically during the period of post-transplant neutropenia (defined as ANC 
<500/μL).   
9.2.6
Tuberculosis  
All patients will undergo PPD testing prior to starting chemomobilization, those with positive 
skin tests would receive prophylactic INH 300 mg QD with pyridoxine 50 mg QD.  
9.2.7
Vaccination  
All patients will undergo standard HSCT immunizations that include: 1. seasonal flu vaccine that 
may start at 6 months post-transplant, 2. Tetanus-Diphteria toxoid, Haemophilus influenza type 
B conjugate, inactivated Polio at 12, 18, and 24 months. 3. Pneumococcal  7-valent conjugate 
will be given at 12 months only  


Page 82 of 115 
 
9.3
Appendix C: Prednisone taper schema 
 
Week  Maximally allowed daily dose of prednisone mg/day (or equivalent)#  
 
0 – 2  
80  
75  
70  
65  
60
55
50
45
40
35  
30  
25
20
3 – 4  
80  
75  
70  
65  
60  
55  
50  
45  
40  
35  
30  
25  
20  
5 – 6  
70  
65  
60  
55  
50  
45  
40  
35  
30  
30  
25  
20  
15  
7- 8  
60  
55  
50  
45  
40  
35  
30  
30  
25  
25  
20  
15  
10  
9 – 10  
50  
45  
40  
35  
30  
30  
25  
25  
20  
20  
15  
10  
10  
11 –12  
40  
35  
30  
30  
25  
25  
20  
20  
15  
15  
10  
10  
*  
13 – 14  
30  
30  
25  
25  
20  
20  
15  
15  
10  
10  
10  
*  
*  
15 –16  
25  
25  
20  
20  
15  
15  
10  
10  
10  
10  
*  
*  
*  
17 – 18  
20  
20  
15  
15  
10  
10  
10  
10  
*  
*  
*  
*  
*  
19 – 20  
15  
15  
10  
10  
10  
10  
*  
*  
*  
*  
*  
*  
*  
21 – 22  
10  
10  
10  
10  
*  
*  
*  
*  
*  
*  
*  
*  
*  
23 -24  
10  
10  
*  
*  
*  
*  
*  
*  
*  
*  
*  
*  
*  
25 - 26  
*  
*  
*  
*  
*  
*  
*  
*  
*  
*  
*  
*  
*  
48  
<5mg/day  
 
 
* A taper from the equivalent of 10 mg/day can start. Decrease dose every 4 weeks by 2.5 
mg/day. Taper to 0 mg/day, if tolerated.  
#: Taper to alternate day regimen, if tolerated, for doses <20 mg/day  


Page 83 of 115 
 
9.4
Appendix D: Modified SELENA-SLEDAI 
Descriptor  
Definition  
Wt
Present 
Seizure  
Recent onset (last 10 days). Exclude metabolic, infections or drug cause, or 
seizure due to past irreversible  
8  
 
Psychosis  
Altered ability to function in normal activity due to severe disturbance in the 
perception of reality. Include hallucinations, incoherence, marked loose 
associations, impoverished thought content, marked illogical thinking, 
bizarre, disorganized, or catatonic behavior. Exclude uremia and drug 
causes.  
8  
 
Organic Brain 
Syndrome  
Altered mental function with impaired orientation, memory or other 
intellectual function, with rapid onset and Syndrome fluctuating clinical 
features. Include clouding of consciousness with reduced capacity to focus, 
and inability to sustain attention to environment, plus at least two of the 
following: perceptual disturbance, incoherent speech, insomnia or daytime 
drowsiness, or increased or decreased psychomotor activity. Exclude 
metabolic, infectious or drug causes.  
8  
 
Visual 
Disturbance  
Retinal and eye changes of SLE. Include cytoid bodies, retinal hemorrhages, 
serous exudate or hemorrhages in the choroid, optic neuritis, scleritis or 
episcleritis. Exclude hypertension, infection, or drug causes.  
8  
 
Cranial Nerve 
Disorder  
New onset of sensory or motor neuropathy involving cranial nerves. Include 
vertigo due to lupus.  
8  
 
Lupus 
Headache  
Severe persistent headache: may be migrainous, but must be non-responsive 
to narcotic analgesia.  
8  
 
CVA  
New onset of cerebrovascular accident(s). Exclude arteriosclerosis or 
hypertensive causes  
8  
 
Vasculitis  
Ulceration, gangrene, tender finger nodules, periungual infarction, splinter 
hemorrhages, or biopsy or an angiogram proof of vasculitis.  
8  
 
Arthritis  
More than 2 joints with pain and signs of inflammation (i.e., tenderness, 
swelling, or effusion).  
4  
 
Myositis  
Proximal muscle aching weakness, associated with elevated creatine 
phosphokinase/adolase or electromyogram changes or a biopsy showing 
myositis.  
4  
 
Urinary Casts  
Heme-granular or red blood cell casts.   
4  
 
Hematuria  
>10 red blood cells/high power field. Exclude stone, infection or other cause. 4  
 
Proteinuria  
New onset or recent increase of more than 0.5 g/24 hours.  
4  
 
Pyuria  
>5 white blood cells/high power field. Exclude infection.  
4  
 
Rash  
Ongoing inflammatory lupus rash.  
2  
 
Alopecia  
Ongoing abnormal, patchy or diffuse loss of hair due to active lupus.  
2  
 
Mucosal Ulcers  
Ongoing oral or nasal ulcerations due to active lupus.  
2  
 
Pleurisy  
Classic and severe pleuritic chest pain or pleural rub or effusion or new 
pleural thickening due to lupus.  
2  
 
Pericarditis  
Classic and severe pericardial pain or rub or effusion, or electrocardiogram 
confirmation.  
2  
 
Low 
Complement  
(Will not be rated in this study.)  
N/
A  
N/A  
Increased DNA 
Binding  
>25% binding by Farr assay or above normal range for testing laboratory.  
2  
 
Fever  
>38 C. Excluding infectious causes.  
1  
 
Thrombocytope
<100,000-platelets/ mm3.  
1  
 


Page 84 of 115 
 
nia  
Leukopenia  
<3,000 white blood cells/mm3.  Excluding drug causes.  
1  
 
TOTAL SCORE (Sum of weights for conditions present)  
 
 
 
MD Global Rating of SLE Activity
(over last month, use most active rating during the month)  
(assume that “NONE” and “MOST ACTIVE” refer to what patient has experienced) 
 
0 (NONE)  
Mild   
Moderate  
 
Severe  
Patient Global Rating of Their Disease Activity
0 (NONE)           
10 (MOST)  
9.5
APPENDIX E: SLAM (Systemic Lupus Activity Measure)
 
Date last seen at NIH (mm/dd/yyyy): ____/____/_______  
Today’s date (mm/dd/yyyy): ____/____/_______  
Rater:_________________________________________________  
Constitutional
Absent or normal 
Mild 
Moderate 
Severe 
Not recorded 
Weight Loss 
Physical Examination, VITALS, Weight 
 
 
 
< 10% body weight 
 
 
> 10% 
 
 
Fatigue 
Source Document for Physician, Review of systems 
 
 
 
 
Fever 
Source Document for Physician, Review of systems 
Physical Examination, VITALS, Temperature 
 
 
 
37.5 – 38.5ºC 
 
 
> 38.5ºC 
 
 
 
 
  
Integument
Absent or 
normal 
Mild 
Moderate 
Severe 
Not recorded 
Oral/nasal ulcers, 
or 
Periungal 
erythema, or 
3 
2
1
0 
3 
1
0 
0
3 
1
0 


Page 85 of 115 
 
Malar rash, or 
Photosensitive 
rash, or 
Nail fold infarct 
Physical Examination, Skin 
 
 
 
Present 
 
 
 
 
Alopecia 
Physical Examination, Skin 
 
 
 
 
 
 
 
 
 
 
 
 
 
Integument
(Continued)
Absent or 
normal 
Mild 
Moderate 
Severe 
Not recorded 
Erythematous, 
maculopapular 
rash, or 
Discoid lupus, or 
Lupus profundus, 
or 
Bullous lesions 
Physical Examination, Skin 
 
 
 
Present 
 
 
 
 
Vasculitis 
Leukocytoclastic 
vasculitis, or 
Urticaria, or 
Palpable purpura, or 
Livedo reticularis, or 
Ulcer, or 
Panniculitis 
 
 
 
 
 
Physical Examination, Skin 
 
 
 
< 20% TBA 
 
20-50% 
TBA 
 
> 50% TBA 
or necrosis 
 
3 
3 
2
2
1
0 
1
0 
2
1
0 
0
1
0 


Page 86 of 115 
 
Eye
 
 
 
 
 
Cytoid bodies 
Physical Examination, HEENT 
 
 
 
Present 
 
 
Visual acuity 
< 20/200 
 
 
Hemorrhages (retinal 
or choroidal), or 
Episcleritis 
 
 
Physical Examination, HEENT 
 
 
 
Present 
 
 
Visual acuity 
< 20/200 
 
 
Papillitis, or 
Pseudotumor cerebri 
Physical Examination, HEENT 
 
 
 
Present 
 
 
Visual acuity 
< 20/200 or 
field cut
 
 
Reticuloendothelial 
Absent or 
normal 
Mild 
Moderate 
Severe 
Not recorded 
Diffuse 
lymphadenopathy 
(cervical, axillary, 
epitrochlear) 
Physical Examination, Lymph nodes 
 
 
 
Shotty 
 
> 1cm × 1.5cm 
 
 
 
Hepato- or 
splenomegaly 
 
Physical Examination, Abdomen 
 
 
 
Palpable only with 
inspiration 
  
Palpable only 
without 
inspiration
 
 
Pulmonary
Absent or 
normal 
Mild 
Moderate 
Severe 
Not recorded 
Pleural effusion 
Pleurisy 
Source Document for Physician, Review of systems 
 
 
 
Shortness of breath or 
pain only with 
 
Shortness of breath 
or pain only with 
 
Shortness of breath or 
pain at rest, decreased 
 
 
3
2
1
0 
2
2
3 
1
0 
1
0 
0 
1
3 
3 
1
0 
3 
1
0 


Page 87 of 115 
 
prompting. Exam 
normal or nearnormal 
exercise decreased 
breath sounds and 
dull lower lobe (s) 
breath sounds and dull 
middle and lower lobes 
Pneumonitis 
 
Source Document for Physician, Review of systems 
 
 
 
X –ray infiltrate only 
  
Shortness of 
breath with 
exercise at rest 
  
Shortness of breath 
 
Cardiovascular
Absent or 
normal 
Mild 
Moderate 
Severe 
Not recorded 
Raynaud’s 
Source Document for Physician, Review of systems 
 
 
 
Present 
 
 
 
 
 
Hypertension 
 
Physical Examination, VITALS, BP 
 
 
 
Diast. 90-105 
  
 
 
Diast. 105-115 
 
Diast. > 115 
Carditis 
 
Source Document for Physician, Review of systems 
 
 
 
Pericarditis by 
EKG&/or RUB 
&/oreffusion byEcho; 
no symptom 
  
Chest pain or 
arrhythmia 
 
Myocarditis 
withhemodynamic 
compromise&/or 
arrhythmia 
 
Gastrointestinal 
Absent or 
normal 
Mild 
Moderate 
Severe 
Not recorded 
Abdominal pain 
(Serositis, pancreatitis, 
ischemic bowel, etc) 
Source Document for Physician, Review of systems 
 
 
 
Complaint Limiting 
pain 
 
 
 
Peritoneal 
signs/ascites 
 
 
Neuromotor 
 
 
Stroke syndrome 
Mononeuritis multiplex, 
 
 
Single TIA 
  
 
 
2
3 
1
0 
3 
2
1
0 
2
3 
1
0 
2
3 
1
0 
1
0 
2
3 
1
0 


Page 88 of 115 
 
Transient ischemic attack 
(TIA), 
Reversible ischemic 
neurologic deficit 
(RIND), 
Cerebrovascular accident 
(CVA), 
Retinal vascular 
thrombosis 
Multiple 
TIA/RIND, or 
mononeuritis 
multiplex, or 
Cranial 
neuropathy 
or Chorea 
CVA/myelitis, 
retinalvascular 
occlusion 
 
Seizure 
 
Source Document for Physician, Review of systems 
 
 
 
1-2/month > 2/month 
  
 
 
Status epilepticus 
 
Cortical dysfunction 
 
Source Document for Physician, Review of systems 
 
 
 
Mild 
depression/personality 
disorder or cognitive 
deficit 
  
Change in sensorium 
or severe depression 
orlimiting cognitive 
impairment 
 
Psychosis or 
dementia or coma 
 
 
Headache 
(including migraine 
equivalents) 
 
Source Document for Physician, Review of systems 
 
 
 
Symptoms or 
transient neuro deficit 
with normal activities 
  
Interferes 
somewhat 
meningitis 
 
Incapacitating/aseptic 
 
 
Myalgia/myositis 
 
Source Document for Physician, Review of systems 
 
 
 
       Complaint Limits 
some activity  
  
 
 
Incapacitating 
 
 
Joints
Absent or 
normal 
Mild 
Moderate 
Severe 
Not recorded 
Joint pain from 
synovitis, and/or 
tenosynovitis 
Physical Examination, Musculoskeletal 
 
 
 
Arthralgia only 
 
Objective 
inflammation 
 
Limited function 
 
 
Other 
Absent or 
normal 
Mild 
Moderate 
Severe 
Not recorded 
3 
2
1
0 
2
3 
1
0 
2
3
1
0 
2
3
1
0 
2
3 
1
0 


Page 89 of 115 
 
 
(Write rules for ascertainment and ad hoc scale)
 
 
 
 
 
 
 
 
 
 
Laboratory 
 
 
Hematocrit 
 
> 35 
 
30-35 
 
  
25-29.9 
 
< 25 
 
 
WBC 
 
> 3500 
 
3500-2000 
 
  
2000-1000 
 
< 1000 
 
 
Lymphocyte count 
 
1500-4000 
 
1499-1000 
  
999-500 
 
< 499 
 
 
Platelet count 
 
>150T 
 
100-150T 
  
99-50T 
 
< 50T 
 
 
ESR (Westergren) 
 
>25 
 
25-50 
  
51-75 
 
>75 
 
 
Serum creatinine, or 
creatinine clearance 
 
0.5-1.3 
mg/dLor 
80-100% 
CrCl 
 
1.4-2 mg/dL or 79-
60% CrCl 
  
2.1-4 mg/dL or 
30-60% CrCl 
 
> 4 mg/dL or < 
30% CrCl 
 
 
Urine sediment 
 
 
 
> 5 RBC &/or WBC/hpf 
or 0 to 1-3 granular &/or 
cellular casts/hpf or  1-
2+ proteinuria or < 500 
mg/L 24° urine protein 
  
> 10 RBC &/or 
WBC/hpf or > 3 
granular &/or cellular 
casts/hpf or 3 or 4+ 
proteinuria or 500-
3500 mg/L 24° urine 
protein 
 
> 25 RBC or 
WBC/hpf or red 
cell cast or > 4+ 
proteinuria or > 
3500 mg/L 24° 
urine protein 
 
 
2
3
1
0 
2
3
1
0 
2
3
1
0 
2
3
1
0 
2
3
1
0 
2
3
1
0 
2
3
1
0 
3 
2
1
0 


Page 90 of 115 
 
9.6
Appendix F 
 


Page 91 of 115 
 
 


Page 92 of 115 
 
9.7
Appendix G: ANAM
The ANAM test will be administered by trained NIAMS investigators in a room both quiet and 
confidential. The ANAM test is on a CD-ROM, available on a computer in the clinic. Each 
participant’s results will be stored on the NIAMS’s hard drive and will be labeled only with 
his/her unique identification number that will be assigned at his/her entrance to the study and 
will be kept in the participant’s research chart. Coded data will be exported to a CDROM and 
will be shipped to Dr Joseph Bleiberg at the National Rehabilitation Hospital for interpretation. 
The following ANAM subtests will be conducted;  
x
Simple reaction time (SRT)  
x
Sternberg memory (STN)  
x
Mathematical processing (MTH)  
x
Matching to sample (MSP)  
x
Spatial processing (SPD)  
x
Code substitution (CDD)  
Conventional neurocognitive tests 
The whole test will take approximately 25 minutes. The following battery of neuropsychological 
tests will be administered to each participant by trained NIAMS investigators in the same order 
using standardized instructions and pre-recorded audio tapes in a quiet room.  
x
California Verbal Learning Test (memory)  
x
Rey-Osterrieth Complex Figure Test Copy (Visual-Spatial Processing)  
x
Stroop Color-Word Test (interference) (Complex Attention/Executive Functions)  
x
Wechsler Adult Intelligence Scale (WAIS-III) Digit Symbol Substitution Test 
(Psychomotor Speed)  
x
Trail Making Test (Part B) (Complex Attention/Executive Functions)  
x
Controlled Oral Word Association Test (Language)  
x
Rey-Osterrieth Complex Figure Test Recall (Spatial Memory)  
x
Grooved Pegboard (Motor function)  
The whole procedure will take approximately 1.5 hours. Participants will be allowed to drink 
water and to take a break as necessary.  
Self-report instrument for depression  
Depression will be evaluated by Beck Depression Inventory – Second Edition (BDI-II), which 
will be administered to each participant by trained NIAMS investigators using standardized 
instructions. The BDI-II constitutes the latest revision of the original Beck Depression Inventory 
(BDI), an instrument that was extensively used over a long period of time for measuring the 


Page 93 of 115 
 
severity of depression. The BDI-II consists of 21 self-administered items that measure affective, 
cognitive, and somatic symptoms of depression. The BDI-II requires approximately 15 minutes 
to complete. Each item evaluates a category according to a scale of four possible responses of 
increasing severity, and the total score ranges from 0 to 63. In analyzing data, we will follow the 
cut score guidelines suggested for patients diagnosed with major depression. Thus, participants 
will be categorized into four levels based on the total scores: minimal (0-13); mild (14-19); 
moderate (20-28); severe (29-63).   


Page 94 of 115 
 
9.8
Appendix H: Neurological Rating Scale
 
From Sipe et al, Neurology (Cleveland) 34:1368-1372, 1984  


Page 95 of 115 
 
9.9
Appendix I: Graceley Scales 
9.9.1
Intensity  


Page 96 of 115 
 
 


Page 97 of 115 
 
 
9.9.2
Unpleasantness 
 


Page 98 of 115 
 
9.10 Appendix J 
FACIT-Sp (version 4)  
Below is a list of statements that other people with your illness have said are important. By
circling one (1) number per line, please indicate how true each statement has been for you 
during the past 7 days.  
 
PYSICAL WELL-BEING 
Not 
at all 
A 
little 
bit 
Some 
Quite 
a bit 
Very 
much 
GP1  
 
I have a lack of energy  
0 
1  
2 
3 
4 
GP2  
 
I have nausea  
0 
1  
2 
3 
4 
GP3  
Because of my physical condition, I have trouble 
meeting the needs of my family 
0 
1  
2 
3 
4 
GP4  
 
I have pain.  
0 
1  
2 
3 
4 
GP5  
 
I am bothered by side effects of treatment  
0 
1  
2 
3 
4 
GP6  
 
I feel ill 
0 
1  
2 
3 
4 
GP7  
 
I am forced to spend time in bed 
0 
1  
2 
3 
4 
 
SOCIAL/FAMILY WELL-BEING 
Not 
at all 
A 
little 
bit 
Some 
Quite 
a bit 
Very 
much 
GP1  
 
I have a lack of energy 0  
0 
1  
2 
3 
4 
GP2  
 
I have nausea  
0 
1  
2 
3 
4 
GP3  
Because of my physical condition, I have trouble 
meeting the needs of my family 
0 
1  
2 
3 
4 
GP4  
 
I have pain.  
0 
1  
2 
3 
4 
GP5  
 
I am bothered by side effects of treatment  
0 
1  
2 
3 
4 
GP6  
 
I feel ill 
0 
1  
2 
3 
4 
GP7  
 
I am forced to spend time in bed 
0 
1  
2 
3 
4 
Sp1  


Page 99 of 115 
 
Sp2  
9.11 Appendix K: Ultrasound lymph node surveillance data sheet
Name sticker  
Date  
Circle one:  
0,  
6,  
12,  
24 months  
 
Region /  
location/  
depth /  
size /   aspirated? /   notes  
L groin  
R groin  
L axilla  
R axilla  
L neck  
R neck  


Page 100 of 115 
 
9.12 APPENDIX L: Research Specimen Handling 
    TIME POINTS
PRODUCTS
SEND TO  
2° recipient
PRE TREATMENT- Baseline 
PBL/HAKIM  
1GTT/1CPTred/green  
Fran Hakim 
5A07  
(10ml hep/10ml red&green 
heparized cell prep.tub  
PBL/FOWLER  
4GTT  
Dan Fowler 
12C210  
(10ml hep tube)  
SERUM  
1RTT (10 ml red top, 
serum tub  
Fran Hakim 
5A07 
 (10 ml red top, serum tube)  
LEUKAPHERESIS (1 - 
2 x109 )  
entire bag  
Fran Hakim 
5A07  
200 E6 Amrie Grammer  
MARROW  
3-5ml, heparized,  
Diane Arthur, cytogenetics  
MARROW  
Remainder of 10ML 
ASPIRATE  
Amrie Grammer 
6D50  
 
PBL/GRAMMER(the 
same day w/B  
1CPT red/green  
Amrie Grammer 
6D50  
 
HAKIM  
TBNK (Lym Pheno 
TBNK panel  
Clinical 
Immunology  
 
KIDNEY  
BIOPSY  
Clinical lab; 3rd 
pass to AG  
AG & FH request laser 
scanning cytometer  
PBL/Illei  
1CPT red/green  
Gabor Illei 9S210 8ml CPT  
RNA/ILLEI  
1 Special red top tube  
Gabor Illei 9S210 2.5 ml  
Plasma/ILLEI  
6ml, purple tube  
Gabor Illei 9S210 6ml  
LYMPH NODE  
FNA (B.Wood, 
Z.Neeman)  
Andrea Abati  
AG 6D50 (Fran will 
collaborate in T cell data)  
URINE  
FIRST IN MORNING  
Gabor Illei 9S228  
PBSC APHERESIS  
 
 
 
PBL/HAKIM  
1GTT, before apheresis  
Fran Hakim 
5A07  
 
BAG FACS  
1x107  
Fran Hakim 
5A07  
 
CD34  
1x107  
Diane Arthur, cytogenetics  
CD34  
1x107  
Amrie Grammer 
6D50  
(Fran will collaborate in T cell 
data)  
CD34  
any yield ? 8 x106/kg  
Amrie Grammer 
6D50  
Sarah for mice  
non-CD34 ELUATE  
200 x106  
Amrie Grammer 
6D50  
(Fran will collaborate in T cell 
data)  
 
 
 
 
CONDITIONING 
(DAY -7)  
 
 
 
FACS/HAKIM  
1GTT  
Fran Hakim 
5A07  
 
PBL/Illei  
1CPT red/green  
Gabor Illei 9S210 8ml CPT  
RNA/ILLEI  
1 Special red top tube  
Gabor Illei 9S210 2.5 ml  
Plasma/ILLEI  
6ml, purple tube  
Gabor Illei 9S210 6ml  


Page 101 of 115 
 
SERUM/HAKIM  
1RTT  
Fran Hakim 
5A07  
 
 
 
 
 
CONDITIONING 
(DAY -1)  
 
 
 
PBL/HAKIM  
1GTT  
Fran Hakim 
5A07  
 
SERUM/HAKIM  
1RTT  
Fran Hakim 
5A07  
 
URINE  
FIRST IN MORNING  
Gabor Illei 9S228  
MARROW  
10ML ASPIRATE  
Amrie Grammer 
6D50  
Fran will collaborate  
PBL/GRAMMER(the 
same day w/B  
1CPT red/green  
Amrie Grammer 
6D50  
 
 
 
 
 
2 WEEKS  
 
 
 
PBL/HAKIM  
1GTT/6CPTred/green  
Fran Hakim 
5A07  
 
SERUM/HAKIM  
1RTT  
Fran Hakim 
5A07  
 
1 MONTH  
 
 
 
PBL/HAKIM  
1GTT/6CPTred/green  
Fran Hakim 
5A07  
 
Plasma/ILLEI  
6ml, purple tube  
Gabor Illei 9S210 6ml  
SERUM/HAKIM  
1RTT  
Fran Hakim 
5A07  
 
URINE 
 
 
 
2 MONTHS 
 
 
 
Plasma/ILLEI 
 
 
 
URINE 
 
 
 
3 MONTHS 
 
 
 
PBL/HAKIM  
1GTT/1CPTred/green  
Fran Hakim 
5A07  
 
PBL/FOWLER  
4GTT  
Dan Fowler 
12C210  
 
PBL/Illei  
1CPT red/green  
Gabor Illei 9S210 8ml CPT  
RNA/ILLEI  
1 Special red top tube  
Gabor Illei 9S210 2.5 ml  
Plasma/ILLEI  
6ml, purple tube  
Gabor Illei 9S210 6ml  
SERUM  
1RTT  
Fran Hakim 
5A07  
 
LEUKAPHERESIS (2-3 
x108)  
entire bag  
Fran Hakim 
5A07  
Sort/phenotype with Randy  
URINE  
FIRST IN MORNING  
Gabor Illei 9S228  
4 MONTHS  
 
Hakim 
 6 CPT  
Fran Hakim 
5A07  
 
Hakim  
TBNK (Lym Pheno 
TBNK panel)  
Clinical 
Immunology  
 


Page 102 of 115 
 
Plasma/ILLEI  
6ml, purple tube  
Gabor Illei 9S210 6ml  
URINE  
FIRST IN MORNING  
Gabor Illei 9S228  
 
 
 
 
5 MONTHS  
 
Hakim 
 6 CPT  
Fran Hakim 
5A07  
 
Hakim  
TBNK (Lym Pheno 
TBNK panel)  
Clinical 
Immunology  
 
Plasma/ILLEI  
6ml, purple tube  
Gabor Illei 9S228 6ml  
URINE  
FIRST IN MORNING  
Gabor Illei 9S210  
 
 
 
 
TIME POINTS  
PRODUCTS  
SEND TO  
2° recipient  
6 MONTHS  
 
 
 
PBL/HAKIM  
1GTT/1CPTred/green  
Fran Hakim 
5A07  
 
SERUM  
1RTT  
Fran Hakim 
5A07  
 
PBL/Illei  
1CPT red/green  
Gabor Illei 9S210 8ml CPT  
RNA/ILLEI  
1 Special red top tube  
Gabor Illei 9S210 2.5 ml  
Plasma/ILLEI  
6ml, purple tube  
Gabor Illei 9S210 6ml  
PBL/GRAMMER(the 
same day w/B  
1CPT red/green  
Amrie Grammer 
6D50  
 
LEUKAPHERESIS (3-4 
x 108 )  
entire bag  
Fran Hakim 
5A07  
Sort/phenotype with Randy  
LYMPH NODE  
FNA (B.Wood, 
Z.Neeman)  
Andrea Abati  
AG 6D50 (Fran will 
collaborate in T cell data)  
URINE  
FIRST IN MORNING  
Gabor Illei 9S228  
MARROW  
3-5ml, heparized,  
Diane Arthur, 
cytogenetics  
 
MARROW  
Remainder of 10ML 
ASPIRATE  
Amrie Grammer 
6D50  
Fran will collaborate  
 
 
 
 
7 MONTHS  
 
 
 
Hakim 
 6 CPT  
Fran Hakim 
5A07  
 
Hakim  
TBNK (Lym Pheno 
TBNK panel)  
Clinical 
Immunology  
 
 
 
 
 
8 MONTHS  
 
 
 
Hakim 
 6 CPT  
Fran Hakim 
5A07  
 
Hakim  
TBNK (Lym Pheno 
TBNK panel)  
Clinical 
Immunology  
 
 
 
 
 
9 MONTHS  
 
 
 
PBL/HAKIM  
1GTT/1CPTred/green  
Fran Hakim 
5A07  
 
PBL/Illei  
1CPT red/green  
Gabor Illei 9S210 8ml CPT  


Page 103 of 115 
 
RNA/ILLEI  
1 Special red top tube  
Gabor Illei 9S210 2.5 ml  
Plasma/ILLEI  
6ml, purple tube  
Gabor Illei 9S210 6ml  
SERUM  
1RTT  
Fran Hakim 
5A07  
 
LEUKAPHERESIS (3-4 
x 108 )  
entire bag  
Fran Hakim 
5A07  
Sort/phenotype with Randy  
URINE  
FIRST IN MORNING  
Gabor Illei 9S228  
 
 
 
 
12 MONTHS  
 
 
 
PBL/HAKIM  
1GTT/1CPTred/green  
Fran Hakim 
5A07  
 
PBL/FOWLER  
4GTT  
Dan Fowler 
12C210  
 
PBL/Illei  
1CPT red/green  
Gabor Illei 9S210 8ml CPT  
RNA/ILLEI  
1 Special red top tube  
Gabor Illei 9S210 2.5 ml  
Plasma/ILLEI  
6ml, purple tube  
Gabor Illei 9S210 6ml  
SERUM  
1RTT  
Fran Hakim 
5A07  
 
PBL/GRAMMER(the 
same day w/B  
1CPT red/green  
Amrie Grammer 
6D50  
 
LEUKAPHERESIS (1 x 
109 ) 
Entire bag 
Fran Hakim 
5A07 
Sort/ phenotype with Randy 
MARROW 
3 – 5 mL, heparinized 
Diane Arthur, 
cytogenetics 
 
MARROW 
Remainder of 10 mL 
aspirate 
Amrie Grammer 
6D50 
 
KIDNEY 
Biopsy 
Clinical lab; 3rd 
pass to A 
AG & FH request laser 
scanning cytometer 
LYMPH NODE 
FNA ( B. Wood, Z. 
Neeman) 
Andrea Abati 
AG 6D50 (Fran will 
collaborate in T cell data) 
URINE 
FIRST IN MORNING 
Gabor Illei 9S228
 
18 MONTHS 
 
 
 
PBL/HAKIM  
1GTT/1CPTred/green  
Fran Hakim 
5A07  
 
PBL/Illei  
1CPT red/green  
Gabor Illei 9S210 8ml CPT  
RNA/ILLEI  
1 Special red top tube  
Gabor Illei 9S210 2.5 ml  
Plasma/ILLEI  
6ml, purple tube  
Gabor Illei 9S210 6ml  
SERUM  
1RTT  
Fran Hakim 
5A07  
 
LEUKAPHERESIS (1 
x109 )  
entire bag  
Fran Hakim 
5A07  
Sort/phenotype with Randy  
URINE  
FIRST IN MORNING  
Gabor Illei 9S228  
 
 
 
 
24 MONTHS  
 
 
 
PBL/HAKIM  
1GTT/1CPTred/green  
Fran Hakim 
5A07  
 
PBL/FOWLER  
4GTT  
Dan Fowler 
 


Page 104 of 115 
 
12C210  
PBL/Illei  
1CPT red/green  
Gabor Illei 9S210 8ml CPT  
RNA/ILLEI  
1 Special red top tube  
Gabor Illei 9S210 2.5 ml  
Plasma/ILLEI  
6ml, purple tube  
Gabor Illei 9S210 6ml  
SERUM  
1RTT  
Fran Hakim 
5A07  
 
PBL/GRAMMER(the 
same day w/BM  
1CPT red/green  
Amrie Grammer 
6D50  
 
LEUKAPHERESIS (1 -
2 x109 )  
entire bag  
Fran Hakim 
5A07  
Sort/phenotype with Randy  
MARROW  
3-5ml, heparized,  
Diane Arthur, 
cytogenetics  
 
MARROW  
Remainder of 10ML 
ASPIRAT  
Amrie Grammer 
6D50  
 
LYMPH NODE  
FNA (B.Wood, 
Z.Neeman)  
Andrea Abati  
AG 6D50 (Fran will 
collaborate in T cell data)  
URINE  
FIRST IN MORNING  
Gabor Illei 9S228  
 
 
 
 
RELAPSE  
 
 
 
PBL/HAKIM  
1GTT/1CPT; if no 
leukapheresis, get 10  
Fran Hakim 
5A07  
 
PBL/Illei  
1CPT red/green  
Gabor Illei 9S210 8ml CPT  
RNA/ILLEI  
1 Special red top tube  
Gabor Illei 9S210 2.5 ml  
Plasma/ILLEI  
6ml, purple tube  
Gabor Illei 9S210 6ml  
SERUM  
1RTT  
Fran Hakim 
5A07  
 
PBL/GRAMMER  
1CPT red/green  
Amrie Grammer 
6D50  
 
LEUKAPHERESIS (1 -
2 x109 )  
entire bag  
Fran Hakim 
5A07  
Sort/phenotype with Randy  
MARROW  
3-5ml, heparized,  
Diane Arthur, 
cytogenetics  
 
MARROW  
Remainder of 10ML 
ASPIRAT  
Amrie Grammer 
6D50  
 
KIDNEY  
BIOPSY  
Clinical lab; 3rd 
pass to A  
AG & FH request laser 
scanning cytometer  
LYMPH NODE  
FNA (B.Wood, 
Z.Neeman)  
Andrea Abati  
AG 6D50 (Fran will 
collaborate in T cell data)  
URINE  
FIRST IN MORNING  
Gabor Illei 9S228  
 


Page 105 of 115 
 
 
9.13 APPENDIX M: Experimental Transplantation and Immunology Branch Preclinical 
Service Policy for Sample Handling  
9.13.1 Storage/Tracking  
Normal donor and patient blood and tissue samples, collected for the purpose of research under 
IRB approved protocols of the Experimental Transplantation and Immunology Branch, may be 
archived by the ETIB Preclinical Service. All data associated with archived clinical research 
samples is entered into the ETIB Preclinical Service’s Microsoft Excel databases on frozen cells 
and plasma. These databases are stored on the NCI group drive in the ETIB Preclinical Service 
folder. Access to this folder is limited to ETIB clinical staff, requiring individual login and 
password. All staff in the Preclinical Service laboratory have received annually updated 
NIH/CIT training and maintain standards of computer security.  
The data recorded for each sample includes the patient ID, name, trial name/protocol number, 
date drawn, treatment cycle/post transplant time point, cell source (e. g. peripheral blood, 
lymphapheresis, mobilized peripheral blood stem cells, marrow, pleural fluid) as well as box and 
freezer location. Patient demographics that correlate treatment outcomes and therapies with the 
samples can be obtained only through the NCI/ETIB clinical records. As of January 2007, all 
newly received samples will receive a unique bar code number, which will be added to the 
sample Preclinical Service database. Only this bar code will be recorded on the sample vial and 
the vials will not be traceable back to patients without authorized access to the Preclinical 
Service database. All non-coded samples previously archived will be stripped of identifiers prior 
to distribution for any use other than as a primary objective of the protocol under which they 
were collected.  
Samples are stored in locked freezers at -85°C (sera and plasma) or under liquid nitrogen (cells), 
according to stability requirements.  These freezers are located onsite at the Preclinical Service 
laboratory (12C216) (-85° freezer) or in ETIB common equipment space (CRC/3-3273). Access 
to samples from a protocol for research purposes will be by permission of the Principal 
Investigator of that protocol or through his/her submission and IRB approval of the NCI IRB 
Authorization Form (appended) stipulating whether IRB review is not necessary or IRB approval 
is granted for the pursuit of this new research activity. All researchers are required to sign a form 
(attached) stating that the samples are only to be used for research purposes associated with 
objectives of the original protocol for which the samples were collected, or (using only unlinked 
or coded samples) for an IRB approved protocol as stipulated on the IRB Authorization Form, 
and that any unused samples must be returned to the Preclinical Service laboratory.  
9.13.2 Protocol Completion/Sample Destruction  
Once primary research objectives for the protocol are achieved, researchers can request access 
to remaining samples, providing they have both approval of the Principal Investigator of the 
original protocol under which the samples or data were collected and either an IRB approved 
protocol and patient consent or the IRB Authorization Form stipulating that the activity is 
exempt from IRB review (see attached authorization form from the NCI IRB).  


Page 106 of 115 
 
Samples, and associated data, will be stored permanently unless the patient withdraws consent. 
If researchers have samples remaining once they have completed all studies associated with the 
protocol, they must be returned to the Preclinical Service laboratory.  
The Preclinical Service staff will report to the Principal Investigators any destroyed samples, if 
samples become unsalvageable because of environmental factors (ex. broken freezer or lack of 
dry ice in a shipping container), lost in transit between facilities or misplaced by a researcher. 
The Principal Investigators will annually report this information to the IRB.   


Page 107 of 115 
 
9.13.3 ETIB Preclinical Service Form for Requesting Samples  
Please fill in the relevant information and initial and date the form below. Note that all unused 
samples or portions of samples are to be returned to the ETIB Preclinical Service laboratory.  
I am requesting the following samples from transplant protocols of the Experimental 
Transplantation and Immunology Branch for analysis as a primary objective of the protocol 
under which the samples were obtained. I am a principle/associate investigator under Protocol 
_____________.  
Investigator Name ______________________  Initials____ Date __________  
I am requesting coded samples of cells/plasma/sera of patients obtained from transplant protocols 
of the Experimental Transplantation and Immunology Branch for analysis under IRB Protocol 
__________. Relevant demographic data may be made available, but patient identifiers have 
been stripped from these samples.  
Investigator Name ________________________ Initials____ Date __________  
Attached is the IRB authorization Form indicating that no IRB review is necessary for the 
stipulated research activity.  
Investigator Name ________________________ Initials____ Date __________ 
************************************************************************ 
******  
Protocol Number  
Cell Type/Plasma  
Transplant Stage   
Request to Conduct Research for Same Use of Stored Human Samples, Specimens,or Data 
Collected in a Terminated NCI-IRB Protocol  Protocol Number of Terminated Protocol:  Please 
Check One: [ ] Exempt from IRB Review:  
If the NIH investigator CANNOT identify the subjects, then the research activity may be exempt 
from IRB review and approval (Under 45CFR46.101(b). Use form I on this OHSR Website 
http://ohsr.od.nih.gov/info/info.html .  
[ ] Same Use as Described in the Terminated Protocol  
For this submission, complete the 1195 for as with any other initial protocol, respond to the 
required new protocol information, and provide a copy of the Consent Form/s from the 
Terminated Protocol:  
I. Brief Description of Data/Specimens (how many, types, storage):  
II. Research/Analysis being Conducted:  
III. Timeframe for Analysis of Data/Specimens: (Please note – if longer than one year the 


Page 108 of 115 
 
requirement for Continuing Review of the Research applies.)  
IV. Indicate how the Rights and Welfare of Human Subjects will be protected and Risks 
to Human Subjects will be minimized: (This section must include a description of 
the processes for maintaining confidentiality of identifiable 
data/specimens/samples and what will happen to identifiable 
data/specimens/samples at the end or completion of the protocol. Where human 
samples or specimens are involved - describe how they will be stored; how they 
will be tracked; and under what circumstances would the PI report to the IRB loss 
or destruction of the samples/specimens.   
9.13.4 NIAMS Laboratory Policy for Research Use, Storage and Disposition of Human 
Subject’s Samples and Data.  
Research records will be stored in locked files in our offices and in password-protected 
databases on a secure server. Clinical data will be stored indefinitely in the Medical Records 
department of the NIH Clinical Center where they will be secured and confidentiality will be 
protected under the standard procedures of these departments.   
Serum, plasma, and DNA samples will be stored in locked freezers in Building 10.  All stored 
biological samples and specimens will be coded with a study identification number only. Only 
NIH investigators associated with this study will have access to the secure database linking the 
study identification number with individual patients.  Research data and samples may be shared 
with additional investigators in the future.  Any data or samples sent to these investigators will 
be coded and will not include any patient identifying information.  Codes need to be retained to 
allow the possibility of adding new patient-derived information in the future.   
We do not plan to destroy research-related data or specimens at the completion of the 
protocol, as these data may be useful in the future care of subjects.  The IRB will be notified 
at each annual review of any data or biological specimens lost or destroyed.  


Page 109 of 115 
 
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