Conditions: Myelodysplastic Syndromes
Keywords: Immunosuppression T Cells Hematopoiesis Anti-CD52 Monoclonal Antibody Therapy
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 40
Healthy volunteers: no

Brief summary:
This study will evaluate the safety and effectiveness of a genetically engineered antibody, alemtuzumab (Campath\[R\]) on patients with myelodysplastic syndrome. MDS is made up of malignant stem cell disorders that can mean low levels of red blood cells-that is, anemia-and low counts of white blood cells and platelets. Patients with MDS are at risk for infection, spontaneous bleeding, and possible progression to leukemia, a cancer of bone marrow. Although bone marrow can produce some blood cells, patients with MDS experience a decrease in production of blood cells. Alemtuzumab recognizes specific types of white cells called lymphocytes and destroys them. This study will examine not only the usefulness of the medication but also the side effects in patients with MDS.

Patients ages 18 to 72 who have MDS that requires transfusions and who do not have HIV or a life expectancy of less than 6 months may be eligible for this study. Screening tests include a complete physical examination and medical history. There will be a collection of about 8 tablespoons of blood for analysis of blood counts as well as liver, kidney, and thyroid function; a pregnancy test; an electrocardiogram (EKG) to measure electrical activity of the heartbeat; an echocardiogram (ECHO), which uses sound waves to evaluate heart function; wearing of a Holter monitor for 24 hours while the electrical activity of the heart is recorded; and a bone marrow biopsy. Patients should not receive any vaccines when taking alemtuzumab or for at least 12 months after the last dose. In addition, patients should not take the herbal supplements Echinacea purpurea or Usnea 2 weeks before beginning the study and during it.

For the study, all patients will receive a test dose of 1 mg of alemtuzumab infused into a vein during the course of 1 hour. If the dose is tolerated, the medication will be given at 10 mg doses into the vein for 10 days, as an infusion of 2 hours. Blood samples of 2 tablespoons will be taken daily, and vital signs will be measured daily. The ECHO and 24-hour Holter monitoring will be repeated after patients receive the last dose of the medication. Because suppression of the immune system results from a decrease in white cells that fight infections, patients will take medications to protect them against infections and to treat them if infections occur. If needed, patients will receive blood transfusions for their MDS. Side effects of alemtuzumab involve a temporarily significant lowering of the number of red blood cells, white cells, and platelets. Side effects of the infusion can be rigidity, or stiffness, and fever, as well as risks of infections resulting from the decrease of white blood cells. Blood counts and reactions to all procedures will be carefully monitored throughout the study. After patients receive the last dose of alemtuzumab, they will have follow-up by their referring doctor or at NIH. They must be able to return to NIH after 1 month, 3 months, 6 months, and annually for 5 years after the study. At follow-up visits, there will be blood tests to reevaluate blood counts and test for the presence of viruses. Blood tests will be done weekly for the first 3 months after patients have completed taking alemtuzumab, every other week until 6 months, and then annually for 5 years. There will also be a repeat ECHO at the 3-month visit, and a repeat bone marrow biopsy at the 5-month and 12-month follow-up visits, and as needed after that.

This study may or may not have a direct benefit for participants. For some, the antibody may improve blood counts and decrease the need for transfusions. Knowledge gained in the study may help people in the future.

Detailed description:
Many bone marrow failure syndromes in humans are now recognized to result from immunological mechanisms. These diseases include aplastic anemia, pure red cell aplasia, and some types of myelodysplasia. Patients with these conditions, who may suffer variable degrees of anemia, leukopenia, and thrombocytopenia, alone or in combination, have been shown to respond to a wide variety of immunosuppressive agents, ranging from corticosteroids to cyclosporine (CsA) and horse antithymocyte globulin (h-ATG). However, non-response and relapse continues to be a problem. Why some patients do not respond initially or others respond and then relapse is unclear. Autoreactive T cells may be resistant to the effect of h-ATG/CsA (non-responders), while in others, residual autoreactive T cells expand post-treatment leading to hematopoietic stem cell destruction and recurrent pancytopenia (relapse). Therefore, novel, less toxic immunosuppressive regimens that increase response rates and hematologic recovery and decrease relapse rates are needed.

One such novel therapy, alemtuzumab (Campath\[R\]) is a humanized IgG1 monoclonal antibody directed against the CD52 protein, which is highly expressed on all lymphoid cells and monocytes. Alemtuzumab (Campath\[R\]), produces profound and persistent lymphopenia, affecting predominantly the CD4+ T cell subset. This property has made it attractive in the treatment of a wide range of diseases including rheumatoid arthritis, multiple sclerosis, ocular inflammatory disease, lymphoid malignancies, organ allograft rejection, and in conditioning regimens in stem cell transplantation to prevent graft failure and graft-versus-host disease.

We therefore propose a non-randomized, off label, pilot, Phase I/II study of alemtuzumab (Campath) in MDS patients who are likely to respond to immunosuppression.

Primary endpoints will be changes in peripheral blood counts (platelets, absolute neutrophil count, reticulocyte count, hemoglobin). Secondary endpoints (in transfusion-dependent patients) include improvement in the transfusion requirements (measured as decrease in the number of transfusion administered on as needed basis), duration of response, late effects of treatment, relapse and survival.

Arms:
arm 1: Patients received a 1-mg test dose of alemtuzumab, and the following day, alemtuzumab was administered at 10 mg/dose intravenously for 10 days

Interventions: intervention 1: Alemtuzumab (Campath)
Intervention descriptions: intervention 1: None

Protocol document:
Prot_SAP_000.pdf: 
 
 
05-H-0206 
Christopher Hourigan, M.D.  
April 26, 2018 
1
CLINICAL RESEARCH PROJECT 
 
 
 
 
 
 
 
 
 
 
  
PROTOCOL #05-H-0206 
 
 
 
 
 
 
 
  
Drug Name: Alemtuzumab (Campath®) 
 
 
 
 
 
 
 
  
IND Number: exempt 
 
 
Closed to Accrual: January 27, 2017 
 
 
 
 
 
 
 
 
 
Date: April 26, 2018 
 
 
To: 
Richard Cannon, MD Chair, NHLBI IRB 
 
Title: 
A Pilot Study of Alemtuzumab (Campath®) in Patients with Myelodysplastic Syndrome (MDS) 
 
Other Identifying Words:   
 
Protocol Principal Investigator:  
* Christopher Hourigan, M.D. NHLBI/HB (E)  
 
451-0257 
Bldg 10, CRC 5-5130 
 
Medically Responsible Investigator:  
*Neal S. Young, M.D., Chief, HB, NHLBI (E)  
 
496-5093 
Bldg 10, CRC 3-5142 
 
Associate Investigators:  
 
*Neal S. Young, M.D., Chief, HB, NHLBI (E)  
 
496-5093 
Bldg 10, CRC 3-5142 
Olga Rios, RN, Research Nurse, HB, NHLBI (E) 
 
496-4462 
Bldg 10, CRC 4-5362 
Colin Wu, PhD, Biostatistician, OBR, NHLBI (E) 
 
435-0440 
RKL2, Room 8218 
Xin Tian, PhD, Biostatistician, OBR/NHLBI (E)  
 
435-1298 
RKL2, Room 8217 
Catherine Lai, MD, MPH, Georgetown University Hospital  
3800 Reservoir Road Northwest, 
Washington, DC 20007 
* asterix denotes who can obtain informed consent on this protocol  
 
Medical Monitor: 
John Tisdale, MD, HB, NHLBI (E) 
 
 
 
402-6497 
Bldg. 10, 9N116 
 
 
 
 
Subjects of Study:   
 
Number 
Sex 
 
Age-range 
 
 
 
 
78 
 
Either  
18-72 (inclusive) 
 
 
Project Involves Ionizing Radiation?   
No 
 
Off-Site Project?   
 
 
 
No 
Multi-Institutional Project?   
 
No 
 
 
DSMB Involvement:  
 
 
Yes 
 
 
 
 


 
 
05-H-0206 
Christopher Hourigan, M.D.  
April 26, 2018 
2
PRECIS 
 
 
 
 
 
 
 
 
Many bone marrow failure syndromes in humans are now recognized to result from immunological 
mechanisms. These diseases include aplastic anemia, pure red cell aplasia, and some types of myelodysplasia.  
Patients with these conditions, who may suffer variable degrees of anemia, leukopenia, and 
thrombocytopenia, alone or in combination, have been shown to respond to a wide variety of 
immunosuppressive agents, ranging from corticosteroids to cyclosporine (CsA) and horse antithymocyte 
globulin (h-ATG). However, non-response and relapse continues to be a problem.  Why some patients do not 
respond initially or others respond and then relapse is unclear. Autoreactive T cells may be resistant to the 
effect of h-ATG/CsA (non-responders), while in others, residual autoreactive T cells expand post-treatment 
leading to hematopoietic stem cell destruction and recurrent pancytopenia (relapse). Therefore, novel, less 
toxic immunosuppressive regimens that increase response rates and hematologic recovery and decrease 
relapse rates are needed.  
 
One such novel therapy, alemtuzumab (Campath®) is a humanized IgG1 monoclonal antibody directed 
against the CD52 protein, which is highly expressed on all lymphoid cells and monocytes.  Alemtuzumab 
(Campath®) produces profound and persistent lymphopenia, affecting predominantly the CD4+ T cell subset. 
This property has made it attractive in the treatment of a wide range of diseases including rheumatoid 
arthritis, multiple sclerosis, ocular inflammatory disease, lymphoid malignancies, organ allograft rejection, 
and in conditioning regimens in stem cell transplantation to prevent graft failure and graft-versus-host disease.  
 
We therefore propose a non-randomized, off label, pilot, Phase I/II study of alemtuzumab (Campath®) in 
MDS patients who are likely to respond to immunosuppression.  
 
Primary endpoints will be changes in peripheral blood counts (platelets, absolute neutrophil count, 
reticulocyte count, hemoglobin).  Secondary endpoints (in transfusion-dependent patients) include 
improvement in the transfusion requirements (measured as decrease in the number of transfusion administered 
on ''as needed basis"), duration of response, late effects of treatment, relapse and survival.  
 
 
 
 
 
 
 
 
 
 
 
 


 
 
05-H-0206 
Christopher Hourigan, M.D.  
April 26, 2018 
3
TABLE OF CONTENTS 
1.0   OBJECTIVES 
2.0   BACKGROUND 
3.0  
STUDY DESIGN 
4.0  
ELIGIBILITY ASSESSMENT 
5.0 
 TREATMENT PLAN 
6.0 
   CLINICAL EVALUATION 
7.0 
LABORATORY RESEARCH STUDIES 
8.0 
BIOSTATISTICAL CONSIDERATIONS 
9.0 
DATA AND SAFETY MONITORING 
10.0 HUMAN SUBJECTS PROTECTION 
11.0     PHARMACEUTICALS 
APPENDIX A 
APPENDIX B 
 
 
 
 


 
 
05-H-0206 
Christopher Hourigan, M.D.  
April 26, 2018 
4
1.0   
OBJECTIVES 
The primary objective of this clinical protocol is to assess the safety and efficacy of the genetically 
engineered humanized anti-CD52 monoclonal antibody, alemtuzumab (Campath®) on blood counts 
and transfusion requirements in patients with myelodysplastic syndromes. 
 
2.0   
BACKGROUND 
    
2.1    
Myelodysplastic Syndromes (MDS) 
 
The myelodysplastic syndromes (MDS) are clonal stem cell disorders characterized by cytopenias 
(anemia, neutropenia, thrombocytopenia) due to ineffective hematopoiesis and dysplastic bone 
marrow morphology. As a result, patients with MDS are at risk for symptomatic anemia, infection, 
and bleeding, as well as a variable risk of progression to acute leukemia which is often refractory to 
standard treatment. These disorders may arise de novo or appear years after exposure to potentially 
mutagenic chemotherapy.  
 
Symptoms that derive from low blood counts include:  Anemia which leads to fatigue, weakness, 
lassitude, headaches, and in older patients, dyspnea and chest pain. These manifestations are most 
commonly responsible for the clinical presentation. Thrombocytopenia produces mucosal bleeding:  
petechiae of the skin and mucous membranes, epistaxis, and gum bleeding are frequent and early 
complaints.  Bleeding can be brisk in the presence of accompanying physical lesions, as in gastritis 
and fungal infection of the lungs.  The most feared complication of thrombocytopenia is intracranial 
hemorrhage. Bacterial and fungal infections in the setting of neutropenia are a major cause of 
morbidity and mortality, most often the cause of death in refractory cytopenia associated with MDS. 
 
2.2    Prognostic Indicators 
 
Analysis of previous data demonstrated that certain variables including the patient’s age, whether or not 
they were HLA DR15, and days of red cell transfusion dependence prior to treatment were predictive of 
response. 37  Table 2 shows the relationship between these variables and the predicted probability of 
response in the previous study.   
 
Table 1: Predicted Probability of Response (PPR) to Immunosuppression in MDS   
 
PATIENT’S AGE IN YEARS + DURATION OF RCTD IN MONTHS 
Predicted Probability  
of Response  
DR15 negative patients 
DR15 positive patients 
>58 
>72 
Low                    
50-58 
64-72 
Intermediate       
<50 
<64 
High                   
Key: RCTD = red-cell transfusion dependence  
 
 
 Table 2: The relationship between this status and the response to treatment. 
 
Predicted Probability of Response 
Responders 
Non-Responders 
Low 
1 
14 
Intermediate 
0 
2 
High 
8 
1 
 


 
 
05-H-0206 
Christopher Hourigan, M.D.  
April 26, 2018 
5
When the predicted probability of response criteria were applied to evaluable patients in the current 
study (NHLBI protocol # 00-H-0169), fourteen of 15 (93%) patients with a low probability of 
response were non responders while 7 of 8 (88%) patients with a high probability of response were 
responders. All complete responders were in the high predicted probability of response category. 
None of the patients in the intermediate category responded to treatment.   
 
2.3 
FAB Classification  
 
No single morphologic finding is diagnostic of myelodysplastic syndrome; rather, the combination of 
dysplastic features in the peripheral blood and/or bone marrow is necessary.  The standard stains 
(hematoxylin and eosin, Romanowsky) will be done on the bone marrow sample as well as the 
Prussian blue stain for iron and the reticulin stain for fibrosis. 
 
The common thread between the five subgroups below is dyspoiesis.  Quantitative abnormalities of 
myelodysplastic syndromes 
 
Table 3. The World Health Organization (WHO) classification of myelodysplastic syndromes (MDS).  
 Disease 
Blood Findings 
Bone Marrow Findings 
Refractory anemia (RA) 
Anemia  
No or rare blasts  
< 1 x 109/L monocytes 
Erythroid dysplasia only  
< 10% grans or megas dysplastic  
< 5% blasts  
< 15% ringed sideroblasts 
Refractory anemia with  
ringed sideroblasts (RARS) 
Anemia  
No blasts 
Erythroid dysplasia only  
< 10% grans or megas dysplastic  
15% ringed sideroblasts  
< 5% blasts 
Refractory cytopenia with  
multilineage dysplasia (RCMD) 
Cytopenias (bicytopenia or 
pancytopenia)  
No or rare blasts  
No Auer rods  
< 1 x 109/L monocytes 
Dysplasia in 
10% of cells in two or 
more  
myeloid cell lines  
< 5% blasts in marrow  
No Auer rods  
< 15% ringed sideroblasts 
Refractory cytopenia with 
multilineage  
dysplasia and ringed sideroblasts  
(RCMD-RS) 
Cytopenias (bicytopenia or 
pancytopenia)  
No or rare blasts  
No Auer rods  
< 1 x 109/L monocytes 
Dysplasia in 
10% of cells in two or 
more  
myeloid cell lines  
15% ringed sideroblasts  
< 5% blasts  
No Auer rods 
Refractory anemia with excess blasts  
– 1 (RAEB-1) 
Cytopenias  
< 5% blasts  
No Auer rods  
< 1 x 109/L monocytes 
Unilineage or multilineage dysplasia  
5%–9% blasts  
No Auer rods 
Refractory anemia with excess blasts  
– 2 (RAEB-2) 
Cytopenias  
5%–19% blasts  
Auer rods +/–  
< 1 x 109/L monocytes 
Unilineage or multilineage dysplasia  
10%–19% blasts  
Auer rods +/– 
Myelodysplastic syndrome,  
unclassified (MDS-U) 
Cytopenias  
No or rare blasts  
No Auer rods 
Unilineage gran or mega dysplasia  
< 5% blasts  
No Auer rods 


 
 
05-H-0206 
Christopher Hourigan, M.D.  
April 26, 2018 
6
MDS associated with isolated del (5q) 
Anemia  
< 5% blasts  
Platelets normal or increased 
Normal to increased megakaryocytes  
with hypolobulated nuclei  
< 5% blasts  
No Auer rods  
Isolated del (5q) 
2.4 
Evidence for Immune Mechanisms Causing Cytopenia of MDS 
 
There is a growing body of clinical and experimental evidence suggesting that the cytopenia(s) in 
MDS is T-cell mediated in a proportion of cases. 
 
2.4.1 Clinical evidence of immune mechanisms 
 
• 
Twenty-one of sixty-one patients (33%) with MDS treated at the NIH with h-ATG 
immunosuppression (protocol 95-H-0189) became transfusion-independent. 
• 
Three of 18 patients (17%) with MDS treated with CsA immunosuppression (96-H-0142, 99-
CC-0021) became transfusion independent   
• 
In a separate report, two patients with hypoplastic MDS responded to h-ATG and CsA either 
alone or in combination with durable responses. 1   
• 
In various other case reports, 8 of 13 patients with hypoplastic MDS have responded to h-ATG 
treatment 2,3   
• 
h-ATG treatment improved the cytopenia of MDS that had evolved from aplastic anemia. 4   
• 
In 17 cytopenic patients with MDS of the refractory anemia subtype, 82% responded to 
treatment with CsA alone 5.   
• 
Four patients with autoimmune diseases and myelodyplasia showed hematological responses to 
CsA.6  
• 
In 30 patients with autoimmune diseases and MDS, 6 showed a hematological response to 
steroid treatment including one complete cytogenetic response.7  
• 
CsA induced a remission in an infant with myelodysplastic syndrome8  
• 
Our experience with 133 patients treated over a 10 year period with ATG or ATG/CsA 
demonstrating response rates of over 70% in patients in high or intermediate risk groups (see 
section 2.4) 
 
 
2.4.2 
Experimental evidence of immune mechanisms 
 
• 
Patients with MDS have a high suppressor: helper lymphocyte ratio similar to that found in 
aplastic anemia, where the presence of cytotoxic CD8 cells producing interferon-gamma has 
been linked to the suppression of hematopoiesis. 9,10,11,12   
• 
In MDS, CD8 T cells suppress granulocyte progenitor cell growth, and, in patients responding 
to h-ATG, T cell mediated colony inhibition is lost after treatment 13  
• 
Immunohistochemical examination of the marrow of MDS patients showed increased staining 
for TNF-alpha and TGF-beta compared to controls 14,15    
• 
The percentage of T helper cells and T suppressor cells demonstrating an activated phenotype 
is increased in MDS 
• 
Depletion of lymphocytes increased the in vitro hemopoiesis in long-term bone marrow 
cultures (LTBMC) from patients with myelodysplastic syndrome (MDS). 16    
 
In aplastic anemia, where a similar T-cell mediated marrow suppression is believed to occur, the 
response rate to immunosuppression with h-ATG alone or CsA alone is 40-50%.  This response rate 


 
 
05-H-0206 
Christopher Hourigan, M.D.  
April 26, 2018 
7
was increased to 70-80% by combining the 2 agents. 17,18  Responders to immunosuppression have a 
major survival advantage over non-responders.  As in aplastic anemia, more intensive 
immunosuppression may improve the response rate in MDS compared to h-ATG or CsA alone. 
 
  
2.5 
Treatment Options for MDS 
 
2.5.1  Bone marrow transplantation 
 
The only definitive treatment is bone marrow transplantation; unfortunately, treatment-related 
mortality precludes the application of the procedure for patients older than 60 years and those lacking 
a suitable matched sibling donor. Our institution reported on 43 patients who received either a full 
myeloablative or reduced intensity (age >55) T-cell depleted peripheral blood stem cell for MDS.  
Actuarial 3 year overall survival was 64%, relapse 26% and treatment related mortality 23%80.   
While this is feasible option for younger patients with HLA matched siblings, treatment related 
mortality increases with age and degree of HLA mismatch.  Recent data from the SEER database 
reports the median age of diagnosis of MDS to be 76 years old 81.  
 
2.5.2 Growth factors 
 
Until recently, most patients are treated with transfusions and growth factors to improve blood counts.  
The disease transforms into acute myeloid leukemia (AML) in up to 40% of patients with MDS but 
an equal proportion of patients die from bleeding or infection resulting from cytopenias. The 
prognosis for patients with MDS/AML is very poor with high chemotherapy complication rates and a 
short duration of remission in responders. Additionally, symptoms of anemia, and frequent trips to the 
treating physician’s office for blood and/or platelet transfusions significantly impact quality of life.  
In low risk MDS patients, erythropoietin provides a modest benefit for the treatment of anemia and 
many studies have shown a superior benefit when granulocyte stimulating factor (GCSF) is added to 
erythropoietic 78;79.  Additionally, GCSF alone can stimulate the production of neutrophils to prevent 
infectious complications from long-term neutropenia79.  Unfortunately, growth factors work only for a 
finite period of time and patients eventually succumb to anemia requiring transfusion support and 
prolonged neutropenia.  The prognosis for patients with MDS/AML is very poor with high 
chemotherapy complication rates and a short duration of remission in responders. 
 
2.5.3  Azacitidine (AzA-CR, 5-AZC) and Decitabine 
 
Azacitidine has been designated an orphan product for the use in the treatment of myelodysplastic 
syndromes but has shown only a 10-30% response rate. 19 Its use requires close monitoring as 
cytopenias and central nervous system toxicities (lethargy, somnolence, confusion) are a frequent 
complications. The drug was recently licensed and is widely available to those who wish to use it off 
study.   5-azacytidine, can reduce transfusion requirements, delay time to leukemic transformation, an 
improve quality of life  when compared to supportive care 9;10 This trial will not exclude patients who 
have tried and failed Azacitidine nor does it preclude its use in patients who have failed treatment.   
 
Decitabine, another hypomethelating agent, has shown to improve hematologic parameters (improve 
cytopenias and decrease blast counts) in some patients with MDS. 83    A study of 170 average and 
high risk MDS patients showed an overall response of 17% with a 9% complete response (Normal 
peripheral counts and ≤ 5% marrow blasts) and 13% hematologic improvement (improvement in 
peripheral cell counts).  In this population there was a trend (although statistically non-significant) 
towards increased leukemia free progression time and death.  While overall survival is improved with 
both drugs, some patients are limited in their ability to tolerate these medications secondary to 


 
 
05-H-0206 
Christopher Hourigan, M.D.  
April 26, 2018 
8
worsening cytopenias.  Additionally, all patients eventually progress or die of complications of their 
disease despite treatment.82;83 
 
2.5.4  Revlimid® (lenolidomide) 
 
Revlimid® was approved by the United States Food and Drug Administration on December 27, 2005 
for the treatment of low-or-intermediate risk MDS associated with deletion of 5q, a chromosomal 
(cytogenetic) abnormality, with or without other cytogenetic abnormalities. Revlimid is structurally 
similar to thalidomide, a drug known to cause severe birth defects. Additional studies are ongoing in 
animals to address whether there is a risk that Revlimid will also cause birth defects when taken 
during pregnancy. While these studies are under way, the company is marketing Revlimid under a 
risk management plan called RevAssist, designed to prevent fetal exposure.  
Under RevAssist, only pharmacists and prescribers registered with the program will prescribe and 
dispense Revlimid. The program requires patients, including female patients undergoing mandatory 
pregnancy testing, to give informed consent before starting Revlimid. Physicians are to check 
pregnancy tests, limit prescriptions to a one-month mail supply, and report any pregnancies to FDA. 
FDA and the manufacturer will re-evaluate the risk management plan when results of further animal 
testing for birth defects are completed. 
The labeling for Revlimid will include a Black Box Warning and a Medication Guide regarding the 
prevention of fetal exposure. Additional Black Box Warnings include the potential need to lower the 
dose due to suppressed blood counts and increased risk of blood clots. Common side effects reported 
with Revlimid include thrombocytopenia (low platelet count), neutropenia (low white blood cell 
count), diarrhea, pruritis (itch), rash, and fatigue.  
2.5.5  Antithymocyte globulin (h-ATG)  
 
h-ATG is currently approved for the treatment of aplastic anemia by the Food and Drug 
Administration.  The mechanism by which h-ATG improves bone marrow failure in aplastic anemia 
and MDS is not fully known.  Several modes of action are possible.  After treatment with h-ATG, 
circulating levels of lymphocytes drop to 10% pretreatment level, through a variety of mechanisms; 
these include Fc receptor complement-dependent lysis, opsonization and phagocytosis by 
macrophages, and immunomodulation leading to long-term depletion via antibody dependent cell-
mediated cytotoxicity and activation induced apoptosis.   
 
While it is generally believed that h-ATG administration leads to depletion of immune competent cells, 
its exact mechanism of action remains unclear. 20  h-ATG preparations contain a variety of antibodies 
recognizing human T-cell epitopes, which are directed against activated T-cells or activation antigens. 
21,22 After treatment with h-ATG, circulating levels of lymphocytes decline only transiently, but the 
number of activated T -cells is decreased for prolonged periods of time. 23 This effect is also reflected in 
the decrease of lFN-γ and possibly TNF production after therapy with h-ATG. 24 
 
Susceptibility to h-ATG-induced apoptosis is restricted to activated cells, dependent on IL-2 and 
prevented by cyclosporine A, FK506 and rapamycin. 25 h-ATG includes antibodies against T-cell 
markers such as CD2, CD3, CD4, CD8, CD11a, CD18, CD25, CD44, CD45, HLA-DR, HLA Class I 
heavy chains, and beta-2-microglobulin.  While absolute numbers of lymphocytes return to baseline 
after h-ATG, patients who respond to therapy show a decrease in the number of activated T cells at 
three months post treatment.  Tissue culture preparations of peripheral blood lymphocytes treated 
with h-ATG produce hematopoietic colony stimulating factors, suggesting a possible stimulatory role 


 
 
05-H-0206 
Christopher Hourigan, M.D.  
April 26, 2018 
9
in vivo.  h-ATG binds to numerous other cell types in addition to lymphocytes, including cells of the 
bone marrow. 26,27,28  The response to h-ATG may be mediated by circulating factors produced in this 
immunologically activated state, although response to h-ATG has not correlated with severity or 
presence of clinical serum sickness.  
 
 
 
2.5.6 
Cyclosporine (CsA)  
 
Cyclosporine (CsA) binds to intracellular receptors termed immunophilins, inhibiting in turn the 
activity of calcineurin, which results in the blocking of interleukin-2 production and T cell activation 
and proliferation. 
In vivo, CsA inhibits the release of IL-2 from activated T-cells and consequently decreases T cell 
proliferation.29,30,31 Production of gamma interferon is also reduced while growth factor production is 
unaffected. 32  In vitro, CsA inhibits transcription of IL-2 mRNA. 33  Cell culture studies have shown 
a predominant effect on helper/inducer lymphocytes at low dose, with loss of subtype specificity at 
higher doses.34 B cells are also affected with an early direct inhibition of activation and a decrease in 
antibody production.   In addition to inhibition of T-cell proliferation, CsA induces apoptosis of 
immune-competent cells.35  
 
CsA is not cytotoxic.  It has no direct stimulatory or suppressive effect on bone marrow cells in 
culture.  To inhibit T cell activation, it must be present at the time of antigen stimulation.  This is 
consistent with studies that show no improvement in colony formation in bone marrow cultures of 
patients with aplastic anemia after addition of CsA, even though improvement is shown in the same 
cultures with T cell depletion.36 
 
2.5.7 
Combination therapy with h-ATG and/or CsA   
 
The goal of immunosuppressive regimens applied to the treatment of aplastic anemia is inhibition of 
immune-mediated destruction of hematopoietic progenitor and stem cells. Although activated 
lymphocytes may be more susceptible to the action of CsA and h-ATG, these agents lack specificity 
for autoreactive T-cell clones. 
 
Between 1998 and 2003, 108 patients with MDS and FAB classification RA, RARS or RAEB were 
enrolled in Immunosuppressive therapy (IST) treatment protocols at our institution: 11 received 
cyclosporine (CsA) to maintain levels above 100 ng/ml for up to 6 months; 66 received h-ATG 
(Pharmacia, 40mg/kg x 4 days); and 31 received a combination of h-ATG/CsA.  Previously, we 
reported that responses to h-ATG alone could be predicted in individuals who were HLA DR15, of 
younger age and had a shorter duration of red cell transfusion dependence. 37  We used these factors to 
develop a score segregating patients into high, intermediate and low probabilities of response. We found 
that 90% high probability patients and 1% of low probability patients responded to h-ATG+CsA 
(P<0.001). The mean time to response to h-ATG/CsA was 6 weeks.  The mean time to response to h-
ATG alone was 3 months. 
 
To analyze effectiveness of treatments of different immunosuppressive intensity, prognostic scores 
were assigned to 95 patients receiving CsA, h-ATG, or h-ATG+CsA. In patients with a low 
probability of response, no differences in responsiveness were seen between therapies (5% h-ATG 
alone, 5% h-ATG/CsA and 10% CsA alone responded to therapy). 37    However, in patients with a 
high probability of response, there was a statistically significant increase in the response rate of 
patients receiving h-ATG regimens compared to patients receiving  CsA + h-ATG (p=0.02).  Patients 
with cytogenetic abnormalities were more likely to be in the group of patients with a low probability 
of response with the exception of patients with trisomy 8; these patients were just as likely to respond 


 
 
05-H-0206 
Christopher Hourigan, M.D.  
April 26, 2018 
10
to treatment with immunosuppressive therapy as were patients with normal cytogenetics. These 
results confirm the predictive value of the immunosuppression-response score and suggest that h-
ATG rather than CsA is the more effective agent inducing hematological responses in susceptible 
MDS patients (see Table 2, section 2.2) 
2.6 Alemtuzumab (Campath®) 
 
2.6.1. Description  
 
Alemtuzumab (Campath®) is a humanized IgG1 monoclonal antibody directed against the CD52 
protein, which is highly expressed on all lymphoid cells and monocytes. 38  Alemtuzumab 
(Campath®) is a recombinant DNA derived humanized monoclonal antibody that is directed against 
the 21-28 kD cell surface glycoprotein, CD52.  CD52 is expressed on the surface of normal and 
malignant B and T lymphocytes, NK cells, monocytes, macrophages and tissues of the male 
reproductive system.  Alemtuzumab (Campath®) is an IgG1 kappa with human variable framework 
and consant regions and complementarity-determining regions from a murine (rat) monoclonal 
antibody (Campath-1G).  The alemtuzumab (Campath®) antibody has an approximate molecular 
weight of 150 kD.  Alemtuzumab (Campath®) is produced in a mammalian cell (Chinese hamster 
ovary) suspension culture in a medium containing neomycin.  Neomycin is not detectable in the final 
product.  The rationale for using the humanized form of the monoclonal antibody is the decreased 
formation of antibodies against alemtuzumab (Campath®), which limit efficacy and the potential for 
re-administration of the drug. 
 
2.6.2 
Clinical pharmacology   
 
 
Alemtuzumab (Campath®) binds to CD52, a non-modulating antigen that is present o the surface of 
essentially all B and T lymphocytes, a majority of monocytes, macrophages and NK cells.  Analysis 
of samples collected from multiple volunteers has not identified CD52 expression on erythrocytes or 
hematopoetic stem cells.  The proposed mechanism of action is antibody-dependent lysis of cells 
following cell surface binding.  Alemtuzumab (Campath®) Fab binding was observed in lymphoid 
tissues and the mononuclear phagocyte system.  Significant binding was also observed in the skin and 
male reproductive tract (epididymis, sperm, seminal vesicle).  Mature spermatozoa stain for CD52, 
but neither spermatogenic cells nor immature spermatozoa show evidence of staining.  
 
 
Alemtuzumab (Campath®) pharmacokinetics displayed nonlinear elimination kinetics.  In CLL 
patients treated with Campath®, mean half-life was 11 hours (range 2 to 32 hours) after the first 30 
mg dose and 6 days (range 1 to 14 days) after the last 30 mg dose. Systemic clearance decreased with 
repeated administration due to decreased receptor-mediated clearance (i.e. loss of CD52 receptors in 
the periphery).   
 
 
2.6.3 
The CD52 antigen  
 
The CD52 antigen is a heavily glycosylated lymphocyte glycoprotein with a small protein sequence 
of only 12 amino acids, being attached to the cell membrane by a glycosylphosphatidylinositol (GPI) 
anchored protein. Other proteins on T and B lymphocytes like CD48, CD55 and CD59 also are 
expressed as GPI-anchored proteins. Mutations in the phosphatidylinositolglycan (PIG-A) gene in 
hematopoietic stem cell results in an acquired deficiency of the GPI anchor and is etiologic for PNH. 
39,40 In patients treated with Campath-1H, emergence of CD52-, GPI-deficient lymphocytes have 
given rise to a transient PNH-like clone. 41  However, a classical PNH mutation in the PIG-A gene has 
not been identified in most studies, suggesting an alternate pathway for the presence of GPI-deficient 


 
 
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April 26, 2018 
11
cells. 42 One study using ultra sensitive techniques detected mutations in the PIG-A gene prior to 
Campath-1H treatment in patients who develop CD52 deficient lymphocytes after therapy. 43 Despite 
the persistence of these cells, there have been no adverse reactions associated to their emergence with 
no clinical findings suggestive of PNH. 44 Emergence of a PNH cell clone population among 
lymphocytes will be monitored in our study, but we do not expect it to be of clinical significance. 
CD52 is not present on hematopoietic cells and therefore, Campath-1H does not influence 
proliferation and development of hematopoietic progenitor cells and should not impact on established 
PNH or the emergence of an expanded PNH clone.  
 
 2.7 
Clinical Experience Alemtuzumab (Campath®) 
 
Alemtuzumab produces profound and persistent lymphopenia, affecting predominantly the CD4+ T 
cell subset. This property has made it attractive in the treatment of a wide range of diseases including 
rheumatoid arthritis, multiple sclerosis, ocular inflammatory disease, lymphoid malignancies, and in 
conditioning regimens in stem cell transplantation to prevent graft failure and graft-versus-host 
disease.45,46,47,48,49,50,51,52,53,54,55,56  
 
 
 
2.7.1 
Rheumatoid arthritis     
 
 
The mechanisms by which Campath-1H induces its lymphocytotoxic effect appear to be complement-
mediated cytotoxicity, antibody-dependent cellular cytotoxicity and induction of apoptosis. Most data 
regarding the degree and duration of lymphopenia have been derived from patients with rheumatoid 
arthritis. 57,58 A decrease in lymphocyte and monocyte counts are seen immediately after 
administration. Initial cell recovery is noticed with CD16+ NK cells and CD14+ monocytes returning 
to normal levels within 1-2 months, and CD 19+ B cells within 3-6 months. Cytotoxic CD8+ T cell 
begin to recover at about 3 months after treatment and helper CD4+ T cells may remain suppressed 
for years. Metteson et al demonstrated that 22% of patients remained lymphopenic at 6 months after 
treatment, with CD4+ and CD8+ T cells being 14% and 13% pretreatment levels, respectively. 59 
Others have shown that T cell subsets can remain suppressed for an even longer period, not rising 
above 20% of pretreatment levels at any time after 18 months of therapy. 60  
Despite the prolonged and profound lymphopenia, infectious complications in practice have been 
minor, most occurring in the first 8 weeks and the majority being well controlled and self-limiting. 
Fatal opportunistic infections have been infrequent.   
 
Campath-1H has been administered in rheumatoid arthritis by subcutaneous and intravenous routes. 
With the subcutaneous injection, clinical improvement was short- lived, with a high incidence of anti-
Campath-1H antibodies. 61  In an intravenous dose-ranging study, 41 patients with RA who had failed 
at least one disease-modifying anti-rheumatic drug received a total of 100, 250 or 400 mg over 5 or 
10 days. 62 Profound lymphopenia developed in all patients with clinical responses appearing more 
sustained in patients receiving 250 or 400 mg.  Approximately 50% of patients achieved a 50% 
Paulus response at 31 days and 20% maintained a 50% Paulus response at least 6 months. There were 
2 fatal infections.  One patient died from infection with Coccidioides immitis (methotrexate was 
added after Campath-1H) and another of suspected hemolytic-uremic syndrome.  Overall, infectious 
complications were minor (mainly herpetic) and all occurred within 8 weeks of therapy.  
 
In rheumatoid arthritis and autoimmune diseases the half-life of Campath-1H has been estimated to 
be 5 - 9 days. It is likely that the half-life in aplastic anemia will be similar, even though no studies to 
date have evaluated the pharmacokinetics of Campath-1H in these patients.  
 


 
 
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Toxicities seen with Campath-1H are short-term infusion-related and long-term due to profound, 
sustained lymphopenia. Early symptoms include fever, rash, rigors, diarrhea, hypotension and nausea, 
probably related to cytokine release of TNF-α, interferon-γ, and IL-6. 63 Treatment is mainly 
symptomatic with discontinuation of the infusion, restarting it as tolerated at a slower rate.  
 
Opportunistic infections are seen most occurring within the first 8 weeks of treatment and have 
generally been mild. As expected, viral infections with herpes simplex and cytomegalovirus occur at 
a high frequency.   Despite the long duration of CD4+ T cell lymphopenia after treatment with 
Campath-1H, few fatalities have been reported secondary to opportunistic infections, suggesting that 
the recovery of B cells and NK cells along with low but existing levels of CD4+ and CD8+ cells are 
adequate to avoid infectious catastrophes. However, serious, sometimes fatal, opportunistic infections 
do occur following treatment with Campath-1H.   
 
2.7.2 
Chronic lymphocytic leukemia (CLL) 
 
The efficacy and safety of Campath-1H in CLL was evaluated in a prospective, non-comparative 
phase II trial conducted at 21 centers in the United States and Europe. 64 Ninety-three patients with 
high-risk CLL refractory to alkylating agents and fludarabine received Campath-1H 30mg , 3 times a 
week, up to a maximum of 12 weeks. Overall response was 33%, with 2% complete and 31% partial 
responses. Median time to progression was 4.7 months, 9.5 months for responders and overall median 
survival was 16 months, 32 months for responders.  
 
The most common adverse event was infusion-related and was generally grade 1 or 2 in severity. 
They included rigors, fever,  nausea, vomiting and rash.  Infections occurred in 51 patients (55%) 
during the study, being mild to moderate in 26, and more severe (grade 3 or 4) in 25 patients. 
Septicemia occurred in 14 patients (15%), with grade 3 or 4 in 10 patients.  Superficial candidiasis 
occurred in 9 patients; and viral reactivation with cytomegalovirus (n=7) or Herpes simplex (n=6) in 
13 patients. A total of 18 patients developed opportunistic infections, 11 during treatment and 7 in the 
follow-up period. Opportunistic infections included Pneumocystis carinii (n=1); Aspergillus 
pneumonia (n=1); rhinocerebral mucormycosis (n=1); systemic candidiasis (n=1); cryptococcal 
pneumonia (n=1, fatal); herpes zoster (n=4, follow-up); pulmonary aspergillosis (n=1, follow-up, 
fatal); Listeria meningitis (n=1, follow-up). The most common opportunistic infection was CMV 
reactivation (n=7). Nine deaths occurred during treatment or within 30 days of the last administration 
of Campath-1H (3 progressive disease, 3 pneumonia, 1 pulmonary embolism, 1 sepsis, 1 
rhinocerebral mucormycosis) and 19 deaths occurred between 30 and 180 days (11 progressive 
disease, 6 infections, 1 respiratory distress, 1 inanition). Patients who died during treatment were 
more likely to have advanced disease at study entry (Rai III or IV) and have failed to respond to 
Campath-1H.  
 
Most patients experienced transient cytopenias during treatment, with neutropenia most commonly 
seen during weeks 5 and 6 (30% of patients) and thrombocytopenia during the first 2 weeks of 
therapy. Neutrophil and platelet counts recovered by 2 months follow-up.  
 
Two smaller phase II trials in patients with chemotherapy and fludarabine refractory CLL (24 and 29 
patients each) also showed similar response rates of about 30%. 65,66 Antimicrobial prophylaxis was 
not routinely recommended in these trials with infection rates somewhat higher compared to the 
larger pivotal study.  Based on the data from these studies, Campath-1H received FDA approval in 
May 2001 for patients with B-cell chronic lymphocytic leukemia who have been treated with 
alkylating agents and have failed fludarabine therapy. 
 


 
 
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2.7.3 Transplant setting to prevent graft rejection and GVHD 
 
Alemtuzumab has also been used as part of conditioning regimens in stem cell transplantation to 
prevent graft rejection and graft-versus-host disease. 67,68 In solid organ transplantation Campath-1H 
has been used with cyclosporine in renal allograft recipients to help prevent rejection and reduce 
further immunosuppressive therapy. 69 In bone marrow transplantation for aplastic anemia, Campath-
1G (rat anti-human IgG2b monoclonal antibody directed against the same CD52 antigen) has been 
used in HLA-identical sibling donors as well as matched unrelated donors with low incidence of 
graft-versus host disease and graft failures. 70,71 
 
2.7.4 
Immune cytopenias 
 
There are no published studies of Campath-1H in patients with aplastic anemia, but a recent case 
series used Campath-1H in a diverse population of 21 patients with severe autoimmune cytopenias 
resistant to standard therapy. 72 The diseases included were: 4 with autoimmune neutropenia (AIN), 4 
with autoimmune hemolytic anemia (AIHA), 4 with immune thrombocytopenia purpura (ITP), 3 with 
Evan’s syndrome, 3 with autoimmune cytopenias, 1 with ITP and AIN and 1 with ITP and pure red 
cell aplasia (PRCA). Campath-1H was given as a daily dose of 10 mg intravenously for 10 days. CsA 
was given to 7 patients and was introduced following Campath-1H. Apart from first dose reactions, 
Campath-1H was well tolerated in all patients. 
 
Of the 4 patients with AIN, a sustained response was seen in three, with the fourth patient requiring 
fewer G-CSF injections. Of the 4 patients with AIHA, 2 with warm type autoimmune hemolytic 
anemia responded and one with cold type hemolytic anemia had a partial response. In the 4 patients 
with PRCA, 2 responded with one having a relapse after the CsA level was found to be sub-
therapeutic. Of the 3 patients with Evan’s syndrome, 2 had an initial response but later one relapsed, 
and the third had a transient response only.  A response was seen in 2/3 patients with autoimmune 
cytopenia, although one relapsed having responded again to a second course of Campath-1H. The one 
patient with ITP and AIN had a sustained response from the ITP, although he remained neutropenic.  
 
Of the 7 patients who died, 2 had autoimmune cytopenia (1-Guillain Barre syndrome, 1-TTP), 2 had 
Evan’s syndrome (1 – cerebral hemorrhage, 1 – recurrent bronchial carcinoma), 1 had ITP (cerebral 
hemorrhage), 1 with AIHA (intractable intravascular hemolysis and systemic venous thrombosis), 1 
had PRCA (non-Hodgkin’s lymphoma transformation). A profound and predictable lymphopenia 
developed in all patients. B lymphocyte recovery occurred before 3 months, CD8+ T cells recovered 
at variable times ranging from 1-36 months and the median CD4 T cell at last follow up visit was 140 
(range 15-560). Despite the lymphopenia there was only one patient who developed a mild viral 
infection with scattered vesicular skin lesions treated with acyclovir. There was no correlation 
between CD4 counts and response. 
 
In a case report, a 51 year old female with a 5 year history of autoimmune neutropenia failed 
treatment with G-CSF, azathioprine, cyclosporine, one course of horse ATG, one course of rabbit 
ATG and prednisolone. 73 She was then treated with Campath-1H 10 mg/d for 10 days. Two weeks 
later, the neutrophil count began to rise and normalized after 2.5 months, which was maintained eight 
months after treatment.  
 
2.8 
Use of Serial Immunosuppressive Therapies  
 
Repeated courses of immunosuppression are commonly administered in human autoimmune diseases, 
including aplastic anemia and myelodysplastic syndromes.   In patients who are refractory to initial 


 
 
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14
horse ATG/CsA, repeated courses of immunosuppression often are employed in order to improve 
marrow function.  
 
2.9 
CsA Therapy Following Alemtuzumab (Campath®)  
 
Alemtuzumab (Campath®) is more immunosuppressive than ATG, but because of concern for 
potential relapse in the months after the effects of alemtuzumab wane, we will allow the institution of 
CsA in patients responding to alemtuzumab but relapsing following initial response at 3 months (see 
section 5.2.2). As the half-life of alemtuzumab is 12 days, the potential for increased toxicity should 
be minimal.  The addition of CsA may rescue relapsed patients from worsening pancytopenia and all 
its complications. The rationale for using CsA in the relapse setting comes from the experience of 
improvement in the blood counts with addition of this agent in patients who relapse after h-ATG. 74   
 
2.10  Scientific and Clinical Justification of the Protocol 
 
Selected MDS patients generally respond very well to h-ATG or CsA. However, there is substantial 
morbidity associated with h-ATG administration in this group of patients. In a previous trial using h-
ATG/CsA to treat MDS, 7/38 patients died within a three month period of time following treatment; 
three deaths were directly attributable to h-ATG.  In addition, 6/38 had a life threatening toxicity 
directly attributable to the h-ATG. 
 
Our initial experience with alemtuzumab (Campath®) in severe aplastic anemia as salvage treatment for 
patients who have failed a first course of h-ATG have been encouraging. Infusion- related toxicities have 
been modest and immunosuppression-related toxicities, including infections, compare favorably to what 
is observed with h-ATG or r-ATG.  In our preliminary experience of 12 patients, hematologic response 
rates have been as at least equivalent to r-ATG.  Should alemtuzumab (Campath®) produce comparable 
responses to h-ATG but without the toxicity, alemtuzumab (Campath®) would be an attractive treatment, 
particularly in this group of patients who have substantial co-morbidities.   
 
In our previous experience treating 133 patients with MDS with immunosuppression, we demonstrated the 
superiority of combination ATG and CsA over either drug alone (p=0.02) (unpublished data). Furthermore, 
we noted that many patients relapsed following  discontinuation of CsA, and required long-term 
continuation of the medication to maintain normal counts.  Campath-1H is more immunosuppressive than 
ATG, but we are concerned about potential for relapse in the months after the effects of Campath-1H wane.  
Therefore we will allow institution of CsA in patients responding to Campath-1H but relapsing 3 months 
after initial response to Campath-1H measured at 3 months.  As the half-life of Campath-1H is 12 days this 
would minimize the potential for increased toxicity. We will collect data on relapse rates and recovery of 
relapsing patients after institution of CsA. 
 
We will treat patients who relapsed following Campath-1H with CsA regardless of prior response to 
CsA treatment. In patients who did not respond to CsA prior to Campath-1H, it is possible that CsA 
may have activity in the post Campath-1H setting due to a Campath-related alteration of the 
underlying disease.   
 
 
We therefore propose to evaluate this new immunosuppressive therapy, alemtuzumab, to treat the 
cytopenias of MDS and to have CsA added to the treatment regimen in those subjects who relapse 
after initial response to Campath-1H.  
 
 


 
 
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15
3.0  
STUDY DESIGN  
 
 
We propose a non randomized, off label, Phase I/II study of Alemtuzumab (Campath®) in MDS 
subjects who are likely to respond to immunosuppression based in our previous model (see section 
2.2 prognostic indicators)  
 
 
 
 
 
 
 
4.0  
ELIGIBILITY ASSESSMENT 
 
All patients 18 years old or over with MDS who lack a suitable matched sibling marrow donor will be 
considered for enrollment.  Patients who have a suitable matched sibling donor will be referred for 
consideration of allogeneic bone marrow transplantation.  Patients not willing to undergo 
transplantation will be considered for protocol participation. Relimid treatment will be discussed 
particularly with patients diagnosed with 5q minus. 
 
Patients who have responded to prior treatment with h-ATG or h-ATG/CsA will be eligible.  Patients 
who have failed to respond to prior treatment with h-ATG or h-ATG/CsA will not be eligible for 
protocol participation.  
 
4.1 
Inclusion Criteria 
 
4.1.1    MDS with WHO classification of RA, RARS, RCMD-RS, and RCMD and RAEB-1  
 
4.1.2 
Anemia requiring transfusion support with at least one unit of packed red blood cells per 
month for greater than or equal to 2 months 
 
 
 
 
 
OR 
   
Anemia (hemoglobin <9 or a reticulocyte count <60,000) 
  
 
 
OR  
  
thrombocytopenia (platelet count less than 50000/µL) 
  
 
 
 OR  
  
neutropenia (absolute neutrophil count less than 500/µL). 


 
 
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4.1.3 
Off all other treatments for MDS (except filgrastim [G-CSF]), erythropoietin, and transfusion 
support and related medications) for at least four weeks.   Filgrastim (G-CSF) can be used 
before, during and after the protocol treatment for patients with documented neutropenia 
(<500/Ul) as long as they meet the criteria for anemia and/or thrombocytopenia as stated 
above.  
 
4.1.4 
Ages 18-72 (inclusive) 
 
 
 4.2 
Exclusion Criteria 
 
4.2.1 
Chronic myelomonocytic leukemia (CMML) , WHO RAEB-2 (see section 2.2) 
 
4.2.2 
Secondary MDS 
 
4.2.3 
Failure to respond to prior therapy with ATG or ATG/CsA 
 
4.2.4 
Prior therapy with combination chemotherapy 
 
4.2.5 
Transformation to acute leukemia (FAB sub-group RAEB-T, i.e., greater than 20% blasts 
in marrow aspirate) 
 
4.2.6 
Failure to discontinue the herbal supplements Echinacea purpurea or Usnea barbata (Old 
Man’s Beard) within 2 weeks of enrollment  
 
4.2.7 
Active infection not adequately responding to appropriate therapy 
 
4.2.8 
HIV positive patients 
 
4.2.9 
Active malignant disease (excluding non-melanoma skin carcinoma) 
 
4.2.10 
Moribund status or concurrent hepatic, renal, cardiac, neurologic, pulmonary, infectious, or 
metabolic disease of such severity that it would preclude the patient’s ability to tolerate 
protocol therapy or that death within 7-10 days is likely  
 
4.2.11 
Life expectancy less than 6 months 
 
4.2.12 
Low predicted probability of response (see section 2. 2) 
 
4.2.13 
Previous hypersensitivity to alemtuzumab (Campath®) or its components 
 
4.2.14 
Current pregnancy, or unwilling to take oral contraceptives or refrain from pregnancy if of 
childbearing potential 
 
4.2.15 
Not able to understand the investigational nature of the study or give informed consent 
 
 
5.0 
 
TREATMENT PLAN   
 


 
 
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5.1 
Alemtuzumab (Campath®) Administration 
All subjects will initially receive a test dose of 3 mg by subcutaneous injection the day before the first 
full dose.  
If tolerated, alemtuzumab (Campath®) will be administered at 10 mg/dose for 10 days 
subcutaneously.  
Subjects will be admitted to the Clinical Center hospital for study drug initiation only if necessary 
based on the adverse reactions they experience. If the following study drug injection is tolerated well 
(toxicity <  grade 2) the subject may be discharged and receive the remainder of the treatment course 
as an outpatient.  
Due to the subcutaneous administration of alemtuzumab subjects will be treated as outpatients 
without increasing the risk of side-effects 85-88 
5.2 
.Cyclosporine (CsA) Administration 
5.2.1  Subjects who fail to respond to Campath-1H 
Subjects who fail to respond to Campath-1H are not eligible to have CsA added to their treatment 
regimen, rather, will be taken off study per section 8.6. 
5.2.2 
Subjects who have responded to Campath-1H and then relapse 
Subjects who relapse after initial response to Campath-1H measured at 3 months (see section 6.4) will 
have CsA added to the protocol therapy regimen unless otherwise contraindicated. CsA will be started 
at 10 mg/kg/d by mouth in divided doses q12 hr.  Dosing will be based on ideal body weight and will 
be adjusted to maintain a target level of 200 – 400 ng/ml. If the blood counts improve, the CsA will 
be continued and tapered off gradually and the subject will continue to be followed on study. The 
CsA may be continued for 2-3 months or longer at the discretion of the PI to determine if 
immunosuppression with CsA will further improve the blood counts.  
• 
 If there is no improvement, the CsA will be discontinued and the subject will go off study.   
• 
If further relapses occur while the subject in on therapeutic CsA (blood level of 200-400 
ng.ml), then the subject will go off study. 
5.3 
Pre-medications and Management of -Adverse Reactions 
Subjects will receive pre-medication with diphenhydramine 25 to 50 mg, orally or by IV, and 
acetaminophen 650 mg 30 minutes prior to the injection.  
 
Adverse reactions will be treated symptomatically (e.g., anti-emetics, IV fluid hydration, 
acetaminophen, antihistamines, inhaled bronchodilators).  
 
5.4 
Supportive Treatment  


 
 
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• 
Transfusional supportive care (e.g., blood and platelets) as clinically indicated. 
 
• 
Growth factors if deemed necessary by the investigator 
  
5.5 Infection Prophylaxis and Monitoring 
 
• 
Pneumocystis prophylaxis:  Aerosolized pentamidine will be used as prophylaxis against 
Pneumocystis carinii, 300 mg every 4 weeks by inhalation beginning the first month of therapy and 
to continue for at least 6 months. If at 6 months CD4+ cells are < 200 /µL, Pneumocystic carinii 
prophylaxis will be continued until CD4+ cells > 200 /µL.  Trimethoprim/ sulfamethoxazole, 
Dapsone or other prophylactic regimen against Pneumocystic carinii may be substituted at the 
discretion of the PI. This prophylaxis may be continued at the discretion of the PI. 
 
• 
Antiviral prophylaxis:  Valacyclovir 500 mg once daily will be given for at least 2 months to 
subjects regardless of HSV serology status.  If at 2 months CD4+ cells are < 200 /µL, antiviral 
prophylaxis will be continued until CD4+ cells > 200 /µL.  This drug may be continued at the 
discretion of the PI. 
 
• 
Antibacterial prophylaxis:  Ciprofloxacin 500 mg BID until ANC > 500/µL.  This drug may be 
continued at the discretion of the PI.  Potential drug interactions will be reviewed with the subject 
prior to initiating prophylatic therapy.  In subjects who cannot tolerate ciprofloxacin, another drug 
may be substituted at the discretion of the PI.  
 
• 
EBV and CMV monitoring:  Subjects will be monitored for EBV and CMV PCR in the blood 
prior to treatment, then weekly for the first month, every other week in the second month, 
monthly for another 6 months and at 12 months.   In case of a positive PCR for CMV, treatment 
will be instituted when clinical symptoms are attributed to CMV.  EBV positivity by PCR will 
also be placed in context of clinical signs and symptoms.   
 
5.6 
Fever Management of Neutropenic Subjects (All subjects) 
 
Subjects with a single temperature of 38.5 °C or two readings of 38.0 °C or greater will be evaluated 
for infection including cultures of blood and urine and any other suspicious sites prior to beginning 
empiric therapy. Antibiotics will be initiated following current infectious disease guidelines.   
 
5.7 
Instructions to Subjects 
 
Regarding immunizations and Campath: Subjects who have recently received alemtuzumab 
(Campath®), should not be immunized with live viral vaccines due to the immunosuppression.  In 
addition, other persons living in the household should not take oral polio vaccine since there is a 
chance they could pass the polio virus on to the subject.  Subjects will be instructed to avoid persons 
who have taken oral polio vaccine within the last several months, not to get close to them, or stay in 
the same room with them for very long. If they cannot take these precautions, they will be instructed 
to consider wearing a protective face mask that covers the nose and mouth.  
 
Regarding subsequent monoclonal antibody therapy: Subjects who develop hypersensitivity to 
alemtuzumab (Campath®) will be advised they may have an allergic or hypersensitivity reactions to 
other monoclonal antibodies. 
 


 
 
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Regarding herbal sublement and Campath: Subjects may not initiate or resume herbal supplements 
(Echinacea purpurea or Usnea barbata [Old Man’s Beard]) while they are participating on this trial.  
 
Regarding concomitant medications and cyclosporine:  Certain other medications can change the 
level of cyclosporine in the blood.  Some of these medications are erythromycin, ketoconazole, 
diltiazem, rifampin, phenytoin and phenobarbital.  We will ask subjects to inform us of any 
medication taking concomitantly while on the study.  
 
Regarding prohibited food and cyclosporine: Grapefruit and grapefruit juice may increase the effects 
of cyclosporine by increasing the amount of this medicine in the body. Subjects will be advised not to 
eat grapefruit or drink grapefruit juice while taking this medicine. 
 
 
6.0 
       CLINICAL EVALUATION   
 
Bone marrow aspirate will be read by a pathologist and used for diagnostic purposes.  Samples will 
be ordered and tracked through the CRIS Screens.  Should a CRIS screen not be available, the NIH 
form 2803-1 will be completed and will accompany the specimen and be filed in the medical record. 
 
6.1 
Pre-study Evaluation:  Subjects baseline status will be evaluated as follows:  
 Medical history and physical examination 
 MDS eligibility panel (done at screening or diagnostic workup, not repeated on study) 
Folate level  
B12 level  
Coagulation screen (PT, PTT) 
Iron panel (ferritin, transferrin, % saturation) 
 
Baseline laboratory studies  
 
 
 
 
Complete blood count with differential 
Reticulocyte count 
DAT (direct antiglobulin test) 
Type and Screen 
Acute Care (Na, K, Cl, CO2, Creatinine, Glucose, and Urea Nitrogen), Mineral 
(Phosphorus, Magnesium, Albumin, and Calcium), Hepatic (Alk Phosphatase, ALT, 
AST, Total Bilirubin, and Direct Bilirubin) , and Other (Total Protein, CK, Uric Acid, 
and LD) panel.Pregnancy test (urine HCG in women of child bearing potential) 
 
Thyroid function tests  
 
Viral serologies for hepatitis A, B, C, HIV, HSV, VZV 
 
EBV and CMV PCR in the blood prior to treatment 
 
Bone marrow aspirate and biopsy with cytogenetic analysis (morphology, cellularity, percentage 
of blast cells, and/or chromosomal analysis by PCR) as appropriate to stage and classify 
underlying disease  
 
Flow cytometry of the peripheral blood for GPI-cells  
 
HLA typing (if not already available) 
 
Chest X-ray 
 
Serum troponin-I before first dose of alemtuzumab (Campath®),  
 
ECHO, 24 hour Holter monitor before first dose of alemtuzumab (Campath®)  
 
EKG  on the day of admission 
 
Lymphocyte phenotyping 
 
 


 
 
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6.2 
On Study Drug Monitoring    
 
On treatment monitoring will consist of: 
 
Physical examination 
 
CBC with differential (daily) 
 
Acute care, mineral, hepatic and other panel (every other day)  
 
Reticulocyte count (weekly +/- 3 days)  
 
Vital signs (daily)  
 
ECHO and 24 hour Holter monitor after last dose alemtuzumab (Campath®)  
 
Serum troponin-I at day 5 and after the last dose of alemtuzumab (Campath®) 
 
EBV and CMV PCR in the blood (weekly +/- 3 days) 
 
Chest x-ray, CT scans or appropriate imaging studies (only as medically indicated) 
 
6.3 
Post Treatment Monitoring Discharge Home Through 3 Months 
 
After completing treatment, subjects will be followed by their home physician or at the Clinical 
Center and have blood work done as detailed below. Progress notes and laboratory results from home 
physicians will be faxed to the Research nurse, Barbara Weinstein, 301 402-3088.  Subjects must be 
evaluated at the Clinical Center at the 1 month (+/- 7 days) and 3 month (+7 days) time points 
 
 
Physical examination 
 
Complete blood counts with differential (weekly +/- 3 days) 
 
DAT (direct antiglobulin test) (months 1 and 3 +/- 2 weeks) 
 
Type and screen (at 1 month and 3 months only +/- 2 weeks or if clinically indicated)  
 
Acute care, mineral, hepatic and other panel  (Home MDs: electrolytes, transaminases, urea 
nitrogen (BUN), serum creatinine, total bilirubin) (weekly +/- 3 days) 
 
EBV and CMV PCR in the blood (weekly +/- 3 days) for the first month, then every other 
week +/- 3 days) in the second month, and then monthly (+/- 2 weeks) starting in month 3 
 
Reticulocyte count (at 1 and 3 months only, Home MDs only if available) 
 
ECHO (at 3 months only +/- 2 weeks)  
 
Serum troponin-I (at 3 months only +/- 2 weeks)  
 
Thyroid function tests (at 3 months only +/- 2 weeks) 
 
Flow cytometry of the peripheral blood for GPI-cells  (at 3 months only +/- 2 weeks) 
 
6.4 
Post Treatment Monitoring 4 Months to 6 Months  
 
After the 3 month visit, subjects will be followed by their home physician or at the Clinical Center 
and have blood work done as detailed below.  Progress notes and laboratory results from home 
physicians will be faxed to the Research nurse, Barbara Weinstein, 301 402-3088.  Subjects must be 
evaluated at the Clinical Center at the 6 month (+7 days) time point 
 
 
Physical examination 
 
Complete blood counts with differential (every other week +/- 3 days) 
 
DAT (direct antiglobulin test) (6 months +/- 1 month) 
 
Type and screen (at 6 months only +/- 2 weeks) 
 
Acute care, mineral, hepatic and other  panel  (every other week +/- 3 days)  (Home MDs: 
electrolytes, transaminases, urea nitrogen (BUN), serum creatinine, total bilirubin) 
 
EBV and CMV PCR in the blood monthly (+/- 2 weeks) to 6 months. 
 
Reticulocyte count (at 6 months only, Home MDs only if available) 
 
Thyroid function tests (at 6 months only (+/- 1 month) 


 
 
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21
 
Bone marrow biopsy and aspiration with cytogenetics (6 months (+/- 1 month only) 
 
Flow cytometry of the peripheral blood for GPI-cells  (6 months (+/- 1 month only) 
 
If Cyclosporine (CsA) is added: CsA levels will be monitored every week (+/- 3 days) for the first 
month and then every other week (+/- 3 days) for the remainder of the treatment period. Less frequent 
monitoring may occur if CsA drug level is therapeutic and steady. Dosing will be adjusted to maintain 
blood level of 200 – 400 ng/ml. More frequent drug serum levels may be obtained as needed to 
achieve constant therapeutic levels. 
 
6.5   
Long Term Follow Up After 6 Months 
 
After the 6 month visit, subjects must be evaluated at the Clinical Center (12 months (+/- 2 month) 
and then yearly (+/- 2 month) thereafter for 5 years and have blood work done as detailed below.   
Subjects will be seen by their home physician as clinically indicated and the Clinical Center and 
home physician will continue to communicate.  
 
Physical examination 
 
Complete blood counts with differential  (annually +/- 1 month) 
 
Acute care, mineral, hepatic and other panel(annually +/- 1 month) 
 
Reticulocyte counts (annually +/- 1 month) 
 
DAT (direct antibody titer) (annually +/- 1 month) 
 
Flow cytometry of the peripheral blood for GPI-cells (annually +/- 1 month )  
 
EBV and CMV PCR in the blood (at 12 months only  +/- 1 month )  
 
Thyroid function tests (annually +/- 1 month)  
 
Bone marrow biopsy and aspiration with cytogenetics (at 12 months +/- 1 month and annually 
thereafter, as clinically indicated) 
 
Type and screen (annually +/- 1 month or if clinically indicated) 
 
Lymphocyte phenotyping (12 months (+/- 1 month) and as clinical indicated) 
 
If cyclosporine (CsA) is added: CsA levels will be monitored every week (+/- 3 days) for the first 
month and then every other week (+/- 3 days) for the remainder of the treatment period. Less 
frequent monitoring may occur if CsA drug level is therapeutic and steady. Dosing will be 
adjusted to maintain blood level of 200 – 400 ng/ml. More frequent drug serum levels may be 
obtained as needed to achieve constant therapeutic levels 
  
7.0 
LABORATORY RESEARCH STUDIES 
 
Intended use:  During the course of participating on this study, an additional 105 cc of blood (at 
baseline) , 5 cc (Day 5), 10 cc (Day 10), or 50 cc of blood (at 1 month post treatment), 45 cc of blood 
(3months, 6 months and 12 months post treatment), 20 cc of blood (annually thereafter) and 5 cc of 
bone marrow aspirate (baseline, 6 months, 12 months, and annually thereafter as clinically indicated) 
may be collected for exploratory ancillary laboratory research studies which have been approved by 
the IRB and are listed in Appendix A. These studies will not be used to assess the primary endpoint, 
may or may not be done, and if done, may be correlated with the presence or absence of response. 
These specimens will not be read by a pathologist or used for diagnostic purposes.   Campath research 
studies may include:  
 
 
Anti-alemtuzumab (Campath®) antibody may be measured. 
 
Drug sensitivity testing assessment may be collected. In order to assess the mechanisms of 
infusion related toxicities (fever, hypotension, wheezing, rash, etc.) following alemtuzumab 
(Campath®). Blood sample will be collected at baseline and 2-4 hours after alemtuzumab 
(Campath®) infusion and correlated with clinical adverse events: 
 


 
 
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22
o 
Tryptase (2.5 ml blood) 
o 
Inflammatory cytokines (e.g., TNF-a, IL-1ra, IL-6) and IL-10  
 
Storage:  Research samples will be stored with identifiers (subject initials, data time point, and date 
of sample collection) in the secure laboratory of the principal investigator.  
Tracking:  Samples will be ordered and tracked through the CRIS Research Screens.  Should a CRIS 
screen not be available, the NIH form 2803-1 will be completed and will accompany the specimen 
and be filed in the medical record.  Specimens will be entered in the NHLBI Biospecimen Inventory 
System (BSI). Samples will not be sent outside NIH without IRB notification and an executed MTA.  
 
End of study procedures:  Samples from consenting subjects will be stored until they are no longer 
of scientific value or if a subject withdraws consent for their continued use, at which time they will be 
destroyed.  
Loss or destruction of samples:  Should we become aware that a major breech in our plan for 
tracking and storage of samples has occurred, the IRB will be notified. 
  
8.0 
BIOSTATISTICAL CONSIDERATIONS 
 
8.1 
Primary Endpoint 
 
The primary endpoint is hematologic response measured at 3 months after first dose alemtuzumab 
(Campath®) sustained on at least two serial measurements at least one week apart at landmark time 
points (3, 6 and 12 months)  will  be defined using standard hematologic and cytogenetic response 
criteria for MDS  (See Appendix B for detailed description)77. 
 
Complete response:   Normalization of all affected cell lines (Hemoglobin, Reticulocyte count, 
Platelets, ANC) 
 
Partial response:  At least one minor response 
 
1. Hemoglobin Response  
Major response: For subjects with pretreatment hemoglobin less than 11 g/dL, greater than 2 
g/dL increase in hemoglobin; for RBC transfusion-dependent subjects, transfusion 
independence or  an absolute reticulocyte count to > 60,000/µL of blood in transfusion-
dependent patients. 
 
Minor response: For subjects with pretreatment hemoglobin less than 11 g/dL, 1 to 2 g/dL 
increase in hemoglobin; for RBC transfusion-dependent patients, 50% decrease in transfusion 
requirements. 
 
2. Platelet response  
Major response: For subjects with a pretreatment platelet count less than 100,000/mm3, an 
absolute increase of 30 000/mm3 or more; for platelet transfusion-dependent subjects, 
stabilization of platelet counts and platelet transfusion independence. 
 


 
 
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23
Minor response: For subjects with a pretreatment platelet count less than 100,000/mm3, a 
50% or more increase in platelet count with a net increase greater than 10,000/mm3 but less 
than 30,000/mm3. 
 
3. ANC response  
Major response: For absolute neutrophil count (ANC) less than 1500/mm3 before therapy, at 
least a 100% increase, or an absolute increase of more than 500/mm3 whichever is greater. 
 
Minor response: For ANC less than 1500/mm3 before therapy, ANC increase of at least 
100%, but absolute increase less than 500/mm3. 
 
 
8.2 Secondary Endpoints 
 
8.2.1 
Transfusion-independence for red blood cells and/or platelets: ability to maintain a 
hemoglobin >8gm/dl and a platelet count > 20 000/µL without the need for transfusions for a period 
of 6 weeks regardless of subsequent events. 
 
8.2.2 
Overall survival which is defined as the time elapsed between time of treatment and death 
and will be compared between responders and non-responders.  Any such comparison may be biased 
and needs to be interpreted cautiously.   
 
8.2.3 
Life threatening toxicity.  Subjects who do not complete the treatment course because of 
toxicity or voluntary withdrawal will be included in analyses. 
 
8.2.4 
Overall survival and transformation-free survival, which is defined as the time elapsed 
(reported at 3 month intervals for the first four years, then yearly thereafter) between time of 
treatment, and bone marrow morphologic evidence for progression (as determined by FAB criteria), 
development of acute leukemia, or death will be compared between responders and non-responders.  
Any such comparison may be biased and needs to be interpreted cautiously.   
 
8.2.5 
Response duration will be determined as the interval between time of onset of response as 
outlined above, and the time of relapse or loss of response characteristic (reported at 3 month 
intervals for the first four years, then yearly thereafter).   
 
8.2.6 
Affects of the addition of cyclosporine. Subjects who relapse after responding to Campath 
will be given CsA to determine if CsA will be beneficial under these circumstances.  For this 
protocol, CsA is investigational, subjects will be followed, data will be collected and analyzed as 
secondary endpoints.  The number of subjects who will receive CsA cannot be predicted since it will 
depend on the initial response to Campath which is unknown.  
 
Response proportion, response duration, overall survival, and transformation free survival will be 
determined and compared within the FAB sub-groups and International Prognostic Scoring System 
categories of MDS and with the results of previous studies at the NIH using h-ATG alone or CsA 
alone to treat the cytopenia of MDS.   
 
8.3 
Sample Size   
 
Because the efficacy of this treatment is almost completely unknown, we would like to reject the 
treatment as quickly as possible with a small number of subjects if the treatment is not effective.  


 
 
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Christopher Hourigan, M.D.  
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24
A response probability of 30% or less would warrant terminating the treatment on this subject 
population, and we hypothesize that the actual response probability using this treatment would reach 
50% or more. 
 
Let p be the probability of complete or partial response at 3 months. Our sample size is determined by 
testing the null hypothesis H0: p≤30% versus the alternative H1: p ≥50% at 0.05 significance level and 
0.80 of the power. We determine the sample size using the Two-Stage Minimax Design outlined in 
Table 1 of Simon (1989) 75 This design is selected because it requires a smaller total number of 
subjects (n=39) than the Simon’s Two-Stage Optimal Design (n=46). At the first stage, 19 subjects 
will be accrued and the null hypothesis will be accepted (i.e., the treatment will be terminated) if 6 or 
less subjects respond to the treatment at 3 months. If 7 or more subjects respond to the treatment  at 3 
months in the first stage, then an additional 20 subjects will be accrued, bringing the total number of 
subjects to n=39. The null hypothesis of p ≤ 30% will be accepted if the total number of complete and 
partial responders at 3 months is 16 or less.   
 
An additional 39 subjects will be enrolled to allow for statistically valid subgroup comparisons of 
secondary endpoints such as response to cyclosporine in subjects who relapse within six months of 
treatment. 
 
8.4  
Statistical Methods 
 
The planned analyses will include descriptive statistics on the proportions of responses (i.e. % subjects 
with partial or complete response) and the time to response. The response probabilities will be 
estimated using the sample proportions and their inferences including confidence intervals and 
hypotheses testing will be evaluated using Binomial distributions. The time to responses will be 
analyzed using appropriate tools in survival analysis such as Kaplan-Meier estimates and Cox 
regression taking consideration of random censoring. In addition methods based on survival analysis, 
cumulative incidence rates for response and other competing risk models will be used to evaluate the 
treatment effects. Graphical tools will be used to display the appropriate estimates (i.e. estimated 
proportions and Kaplan-Meier curves) and their corresponding 95% confidence intervals. Methods 
based on multiple regression, analysis of variance and logistic regression will also be employed if 
deemed appropriate. 
 
8.5 
Stopping Rules   
 
The following two types of treatment related severe adverse events will be monitored and considered 
for early stopping for all or part of the study:  
 
 
Death  considered to be definitely related to  alemtuzumab (Campath-1H®)  
 
Any grade IV toxicity considered to be definitely related to alemtuzumab (Campath-1H®), 
i.e. opportunistic infection such as tissue-invasive CMV or Pneumocystis carinii, with 
exception of temporary cytopenias.  
 
We will monitor the numbers of subjects who have developed one or more of the above treatment 
related severe adverse events (TRSAE).  The study will be seriously considered for early stopping if 
the corresponding number of subjects who have developed one or more of the above TRSAE is over a 
pre-specified threshold value.  
 
From our experience using this agent in other clinical settings, we anticipate the rate of developing at 
least one of the above TRSAE to be 10% or less. Following Geller et al. (2003, “Design of Early 


 
 
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Christopher Hourigan, M.D.  
April 26, 2018 
25
Trials in Stem Cell Transplantation: A Hybrid Frequentist-Bayesian Approach”), our stopping rule is 
determined by a Bayesian approach.76 The stopping boundary is reached if the Bayesian posterior 
probability that the true probability of developing one or more of the above TRSAE exceeds this 
benchmark rate of 10% is at least 90%. We take our prior distribution to be a beta distribution with 
the sum of the two beta parameters to be  6, i.e. the parameters of the beta prior distribution are  1.0 
and  5.0. Since we have seen in the past that the first few subjects to be accrued are possibly sicker 
than the rest of the subjects in the sample, we will start safety monitoring when 3 or more subjects 
have developed TRSAE. The following table summarizes the threshold numbers for stopping an 
experiment:  
   
Number of Subjects in the 
experiment 
Stop if the number of subjects who have developed any of 
the TRSAE reaches: 
≤  9 
3 
≤  16 
4 
≤   23 
5 
≤   31 
6 
≤  38 
7 
≤  46 
8 
≤  54 
9 
≤  62 
10 
≤  70 
11 
≤  78 
12 
 
We investigated the performance of the above stopping rule by a simulation study. In each simulation 
run, we generated a study with  78 independent Bernoulli trials, each had a probability p for having 
the above TRSAE and q=1-p for not having such TRSAE and compared the TRSAE outcomes with 
the above stopping boundary to determine whether the study was stopped. We repeated the simulation 
100,000 times and computed the proportion of stopped studies (i.e. “number of stopped 
studies”/100,000) which were stopped using the above stopping rule. The following table summarizes 
the proportions of stopped studies under a number of scenarios for p: 
 
Prob of TRSAE = p 
2% 
5% 
10% 
15% 
20% 
 
25% 
Proportion of Stopped Studies 
      0.7% 
     1.7% 
    21.8% 
     65.4% 
    92.8% 
 
 99.6% 
Average Number of Subjects 
 77.95 
 76.93 
 67.32 
 46.97 
 28.46 
 18.17 
Average Numbers of Subjects 
Suffering TRSAE 
 1.55 
 3.86 
 6.73 
 7.03 
 5.70 
 4.55 
 
These results suggest that our stopping rule has a low probability of stopping a study when the 
proportion of the above TRSAE is below the benchmark value of 10%, and the probability of stopping 
a study is high when the true proportion of the above TRSAE exceeds this benchmark value. Based 
on these results, we believe that our Bayesian stopping rule has satisfactory statistical properties. 
  
8.6 
Off Study Criteria 
 
• 
Per Subject choice:  Subjects may be removed from study at their request.  The risks of 


 
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26
withdrawing will be discussed, as will alternative treatment options.  Those subjects who choose to 
withdraw while taking alemtuzumab (Campath®) will continue to have labs monitored through the 
6 month off drug time point.    
• 
Per principal investigator decision:  Alemtuzumab (Campath®) administration will be 
discontinued, the subject will be followed until the event has resolved and labs have been 
monitored through the 6 month off drug time point if the subject develops:  
 
Intolerance of alemtuzumab (Campath®) infusion as manifested by hypotension, fever, chills 
and/or rash that is refractory to pre-medication with anti-histamines and acetaminophen. 
 
Life threatening acute hypersensitivity reactions 
 
Lymphoproliferative disease or malignancy. 
 
Worsening of MDS with increased numbers of blasts fulfilling a diagnosis of RAEBt or 
leukemia 
 
Pregnancy or unwillingness to refrain from pregnancy  
 
Initiation of additional immunosuppressive therapy (except steroids or CsA) 
 
Initiation of the herbal supplement Echinacea purpurea or Usnea barbata (Old Man’s Beard)  
 
Failure to respond to Campath-1H therapy 
 
Failure to improve with CsA after relapse. 
 
Once off study, subjects will be referred back to their referring physician or consented to the 
Hematology Branch evaluation and treatment protocol (94-H-0010) for consideration for standard 
therapy or evaluation for eligibility for another branch protocol, depending on what is considered to 
be in the best interest of the subject. 
 
8.7 
Data Management 
 
 
The PI will be responsible for overseeing entry of data into an in-house password protected 
electronic system and ensuring data accuracy, consistency and timeliness. The principal 
investigator, associate investigators/research nurses and/or a contracted data manager will assist 
with the data management efforts.  All human subjects personally identifiable information (PII) as 
defined in accordance to the Health Insurance Portability and Accountability Act,  eligibility and 
consent verification will  be recorded in DIR’s Clinical Data System (CDS) or the Laboratory of 
Cardiac Energetics (LCE) database.  Primary data obtained during the conduct of the protocol will 
be kept in secure network drives or in approved alternative sites that comply with NIH security 
standards.  Primary and final analyzed data will have identifiers so that research data can be 
attributed to an individual human subject participant, e.g., study-specific identifying number 
(SSPIN) generated by CDS or other unique code or minimum PII required for subject identification. 
 
End of study procedures:  Data will be stored in locked cabinets and in a password protected 
database until it is no longer of scientific value.  
 
Loss or destruction of data:  Should we become aware that a major breech in our plan to protect 
patient confidentiality and trial data has occurred, the IRB will be notified. 
 
No data will be sent outside NIH without prior IRB approval and an executed MTA or CTA. 
 
Publication Policy: Given the research mandate of the NIH, patient data including the results of 
testing and responses to treatment will be entered into an NIH-authorized and controlled research 
database. Any future research use will occur only after appropriate human subject protection 


 
05-H-0206 
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27
institutional approval such as prospective NIH IRB review and approval or an exemption from the 
NIH Office of Human Subjects Research (OHSR). 
 
9.0 
DATA AND SAFETY MONITORING  
 
9.1 
Safety Monitoring   
 
Principal investigator:  Accrual, efficacy and safety data will be monitored by the PI.  
 
NHLBI IRB: Accrual and safety data will be reviewed annually by the Institutional Review Board 
(IRB).  Prior to implementation of this study, the protocol and the proposed patient consent and 
assent forms will be reviewed and approved by the properly constituted Institutional Review Board 
(IRB) operating according to the Title 45 CFR 46.  This committee must approve all amendments to 
the protocol or informed consent, review all SAEs, and conduct continuing annual review so long 
as the protocol is open to accrual or follow up of subjects. 
 
NHLBI DSMB: The NHLBI Data safety and Monitoring Board will review the protocol at 6 to 12 
month intervals. A progress report will be forwarded to the DSMB at these times and their 
recommendations will be expeditiously implemented.  The DSMB may recommend early 
termination of the study for considerations of safety and efficacy.  
 
9.2  
Event Characterization and Reporting 
 
Events include adverse events (AE), serious adverse events (SAE), protocol deviations (PD), 
unanticipated problems (UP), and non-compliance.   
 
The principal investigator will review all events (AEs, protocol deviations, UPs, SAEs) to 
determine the seriousness, expectedness, and reportability of the event.  As required and/or 
needed, the principal investigator will review the events with the Sponsor to make the final 
determination of seriousness and reportability. 
 
9.2.1  Definitions 
Adverse Event (AE):  Any untoward or unfavorable medical occurrence in a human subject, 
including any abnormal sign (e.g., abnormal physical exam or laboratory finding), symptom, or 
disease, temporally associated with the subject’s participation in the research, whether or not 
considered related to the research. 
 
Serious Adverse Event (SAE):  A serious adverse event that: 
• 
results in death; 
• 
is life-threatening (places the subject at immediate risk of death from the event as it occurred); 
• 
results in in-patient hospitalization or prolongation of existing hospitalization; 
• 
results in a persistent or significant incapacity; 
• 
results in a congenital anomaly/birth defect; or 
• 
based upon appropriate medical judgment, may jeopardize the subject’s health and may require 
medical or surgical intervention to prevent one of the other outcomes listed in this definition. 
 
Suspected adverse reaction: Suspected adverse reaction means any adverse event for which there is 
a reasonable possibility that the drug caused the adverse event. A suspected adverse reaction 
implies a lesser degree of certainty about causality than adverse reaction, which means any adverse 
event caused by a drug. 


 
05-H-0206 
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28
 
Serious event: An event is serious if it meets the definition of a serious adverse event (above) or if it 
requires immediate corrective action by a PI and/or IRB to protect the safety, welfare or rights of 
subjects. 
 
Unexpected adverse reaction: An adverse event or suspected adverse reaction is considered 
“unexpected” if it is not listed in the investigator brochure or is not listed at the specificity or 
severity that has been observed; or, if an investigator brochure is not required or available, is not 
consistent with the risk information described in the general investigational plan or elsewhere in the 
current application. "Unexpected”, also refers to adverse events or suspected adverse reactions that 
are mentioned in the investigator brochure as occurring with a class of drugs or as anticipated from 
the pharmacological properties of the drug, but are not specifically mentioned as occurring with the 
particular drug under investigation. 
 
Unanticipated Problem (UP):  Any incident, experience, or outcome that meets all of the following 
criteria: 
1. unexpected in terms of nature, severity, or frequency in relation to 
a. the research risks that are described in the IRB-approved research protocol and informed 
consent document; Investigator’s Brochure or other study documents; and 
b. the characteristics of the subject population being studied; and 
2. related or possibly related to participation in the research; and 
3. places subjects or others at a greater risk of harm (including physical, psychological, economic, 
or social harm) than was previously known or recognized. 
 
Unanticipated Problem that is not an Adverse Event:  An unanticipated problem that does not fit 
the definition of an adverse event, but which may, in the opinion of the investigator, involves risk to 
the subject, affect others in the research study, or significantly impact the integrity of research data.  
For example, report occurrences of breaches of confidentiality, accidental destruction of study 
records, or unaccounted-for study drug. 
 
Protocol Deviation (PD):  Any change, divergence, or departure from the IRB approved research 
protocol. 
 
Non-compliance: The failure to comply with applicable NIH HRPP policies, IRB requirements, or 
regulatory requirements for the protection of human research. Noncompliance may be further 
characterized as: 
 
1. Serious non-compliance: Non-compliance that: 
a. Increases risks, or causes harm, to participants. 
b. Decreases potential benefits to participants. 
c. Compromises the integrity of the NIH HRPP. 
d. Invalidates the study data. 
 
2. Continuing non-compliance: Non-compliance that is recurring. An example may be a pattern of 
non-compliance that suggests a likelihood that, absent an intervention, non-compliance will 
continue. Continuing noncompliance could also include a failure to respond to IRB requests to 
resolve previous allegations of non-compliance. 
 
3. Minor (non-serious) non-compliance: Non-compliance that, is neither serious nor continuing. 
 
 


 
05-H-0206 
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29
9.2.2  Adverse Events  Management: 
 
.  All adverse events occurring during the study, including those observed by or reported to the 
research team, will be recorded  The AEs will be attributed (unrelated, unlikely, possibly, probably 
or definitely) to study medication and/or disease and graded by severity utilizing CTC version 2.0.  
A copy of the criteria can be down-loaded from the CTEP home page at  
http://ctep.cancer.gov/reporting/ctc.html.   
 
Abnormal laboratory findings used to evaluate the safety of this protocol regimen will be 
collected to include any change from laboratory assessments done prior to first dose of study 
medication that results in a progression to a grade 3 or 4 laboratory toxicity and/or are characterized 
by any of the following: 
• 
Results in discontinuation from the study 
• 
Is associated with clinical signs or symptoms 
• 
Requires treatment or any other therapeutic intervention 
• 
Is associated with death or another serious adverse event, including hospitalization 
• 
Is judged by the Investigator to be of significant clinical impact 
• 
If any abnormal laboratory result is considered clinically significant, the investigator will 
provide details about the action taken with respect to the test drug and about the patient’s 
outcome.  
 
The laboratory toxicities will be attributed (unrelated, unlikely, possibly, probably or definitely) to 
study medication and/or disease and graded by severity utilizing CTC version 2.0. 
 
Length of Adverse Event reporting: Adverse event collection will start at the baseline and 
continue for 6 months after the last dose of Campath and when applicable cyclosporine, after which 
the events will be recorded in the medical record but not abstracted to toxicity tables.   
 
9.2.3     Serious Adverse Events Management 
 
 
Serious adverse events will be attributed as definitely (clearly related to the research), probably 
(likely related to the research), possibly (may be related to the research), unlikely (doubtfully 
related to the research) and unrelated (clear not related to the research).    
 
Treatment related SAEs (TRSAEs) are those attributed as definitely, probably, or possibly. As 
detailed in section 8.5 stopping rules, Death and any grade IV toxicity considered to be definitely 
related to alemtuzumab (Campath-1H®) will be monitored and considered for early stopping the 
study according to statistically determined criteria.  John Tisdale, MD, NHLBI will serve as the 
independent monitor who reviews the attribution of TRSAEs  
 
Hospitalization (overnight admission) for routine supportive care (platelet or RBC transfusions) or 
admission from the NIH inpatient unit to the NIH ICU for routine monitoring will not be reported 
as a serious adverse event.  
 
Duration of Serious Adverse Event collecting and reporting: The collection of SAEs will begin 
on the first day of initiation of the study drug and will continue along as the subject is on study.  
SAE reporting will continue as long as the subject remains on study. 
 
9.2.4 
Reporting of Events 


 
05-H-0206 
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April 26, 2018 
30
 
 
 
PI:  All  events will be reported to the Principal Investigator of this study  
 
Christopher Hourigan, M.D., NHLBI, HB  
10 Center Dr. Building 10, Room CRC 6C103C 
Bethesda, MD 20892-1452 
Tel:  301-451-0257 
 
 
Reporting Timeframes to IRB Chair, Clinical Director, and/or NHLBI IRB 
 
Serious Events 
Reports to the IRB and CD: The PI must report Serious UPs, and Serious PDs to the IRB and CD as 
soon as possible but not more than 7 days after the PI first learns of the event via PTMS using the 
NIH Problem Report Form. 
 
Reports to the IRB Chair and CD: The PI must report all SAEs that do not meet the definition of 
UP to the IRB chair and CD not more than 14 days after the PI first learns of the event via PTMS, 
using the SAE submission form. 
 
Non-serious Events   
Reports to the IRB and CD: The PI must report all UPs that are not Serious to the IRB and CD, and 
PDs that are not Serious to the IRB, not more than 14 days after the PI first learns of the event via 
PTMS using the NIH Problem Report Form. 
 
Deaths 
The PI must report all deaths (that are not UPs) to the CD as soon as possible, but not more than 7 
days after the PI first learns of the event. 
 
At continuing review, the PI will provide to the IRB a summary of:   
• 
All UPs 
• 
All PDs  
• 
All AEs (except for those granted a waiver of reporting) 
• 
If, while preparing the continuing review, the PI identifies a greater frequency or level of 
severity of expected adverse events than was previously identified in the protocol or 
investigational brochure (IB), these should be reported separately as a UP.  If such an 
observation occurs before the time of continuing IRB review, it should be reported to the IRB 
and CD as a UP in the time frames noted above, and summarized at the time of continuing 
review. 
 
Exclusions to data reporting: 
The following Adverse Events will be captured only in the source documents and will not be 
reported to the IRB at the time of continuing review. 
• 
Laboratory values that do not meet the definition of a laboratory AE listed in Section 9.2.2. 
• 
All grade 1 events listed as expected in the investigator’s brochure, package insert, and/or 
anticipated events. 
 
NHLBI DSMB Reporting: 
Reports of serious adverse events that are unexpected and thought to be related to the experimental 
drug will also be forwarded no later than seven (7) days in the case of death or life-threatening 


 
05-H-0206 
Christopher Hourigan, M.D 
April 26, 2018 
31
serious adverse events or within fifteen (15) days after the occurrence of all other forms of serious 
adverse events to the Data and Safety Monitoring Board (DSMB). Asummary of events will be 
included in DSMB reports for review by the DSMB.  
 
 
 
If the serious adverse event is thought to be due to the experimental component of the protocol, 
accession to the protocol will be stopped until a full discussion with the IRB has been held.   
 
 
10.0 
HUMAN SUBJECTS PROTECTION  
 
10.1 
 Rationale for Subject Selection 
 
MDS is not known to show a racial or gender preference in incidence, as such, there will be no 
restrictions based on ethnicity or gender in patient selection. One of the difficulties clinicians face is 
distinguishing between hypoplastic MDS and aplastic anemia when faced with a hypoplastic 
marrow.  For the purposes of this protocol, patients with hypocellular marrows (<30%) will be 
classified as MDS.   A cytogenetic abnormality in the setting of a hypoplastic marrow makes a 
diagnosis of MDS. When there are no cytogenetic abnormalities the distinction between MDS and 
aplastic anemia needs to be made on morphologic grounds.  However, dyserythropoiesis, which is a 
feature of both AA and MDS will not be used as a sole diagnostic criterion. 
 
The Hematology Branch has a robust source of patients with bone marrow failure, and we do not 
anticipate recruitment to be problematic. We have screened over 950 patients with MDS since 
1997.  Because the inclusion/exclusion reflects a prognostic index that incorporates age, we 
anticipate the study population will be slightly younger.  
 
Reimbursement: Reimbursement for protocol participation, travel, food, and lodging will be 
consistent with NIH guidelines.  In determining reimbursement, the following factors are 
considered applicable to this protocol:   the patients are diagnosed with a rare disease; the patient 
population is sick; the protocol offers the potential for direct benefit; the protocol regimen is 
demanding; and in order to complete accrual in a reasonable timeframe a geographically dispersed 
participant population is required.  
 
Recruitment efforts: The study will be listed on clinicaltrials.gov, Clinical Center research studies, the 
NHLBI patient recruitment website, and the Aplastic Anemia and MDS Foundation websites.  If 
recruitment goals are not met, a recruitment plan will be developed by the Office of Patient Recruitment.  
 
Competition among other Branch protocols: 
 


 
05-H-0206 
Christopher Hourigan, M.D 
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32
MDS Subjects are screened for 
appropriateness for protocol 
participation
 05-H-0206
Campath-1H 
MDS 
RA,RARS, 
RCMD-RS, 
RCMD & 
RAEB-1
Anemia 
requiring 
RBCs or 
Hgb<9 or 
Plts<50000
or ANC <500
Not suitable 
for PBSCT
07-H-0159
WT1/PR1 
Vaccine 
Low risk MDS 
RA, RARS, 
HLA-A0201 
or HLA-
A2402
06-H-0248
07-H-0136
99-H-0050
Transplant 
protocols
MDS RA 
RARS, 
RAEB, 
RAEBt or 
transformed 
to leumemia 
HLA 
matched, 
single 
mismatched 
donor 
07-H-0225
Cyclin D 
Inhibitor
MDS RAEB -
1 or 2 and 
anemia 
requiring 
RBCs or 
HgB<9 or Plts 
< 50,000 or 
ANC <500 
not suitable 
for PBSCT
07-H-0091 
WT1 Vaccine
Refractory 
MDS REAB, 
REABt ,  
HLA-A0201 
or HLA-
A2402  with 
>20% bone 
marrow 
cellularity
08-H-0046
Umbilical cord 
transplant 
3/6 or 4/6 HLA 
match family
 
 
 
 
10.2 
 Participation of Children.   
 
Pediatric MDS patients less than 18 yrs old are excluded from protocol participation. Less than 3% of 
patients with the diagnosis of MDS are under the age of 30.  MDS may also be biologically a different 
disease in children.  The treatment of choice in an MDS patient under age twenty with MDS is a bone 
marrow transplant, even from a matched unrelated donor.  Furthermore, at this time, we do not believe 
treatment with Campath® will be curative which should be the goal of therapy in a young patient.   
 
10.3 
Risks and Discomforts  
 
10.3.1 Related to alemtuzumab (Campath®)  
 
 
WARNING: CYTOPENIAS, INFUSION REACTIONS, and INFECTIONS 
Cytopenias: Serious, including fatal, pancytopenia/marrow hypoplasia, autoimmune idiopathic 
thrombocytopenia, and autoimmune hemolytic anemia can occur in patients receiving Campath. Single 
doses of Campath greater than 30 mg or cumulative doses greater than 90 mg per week increase the 
incidence of pancytopenia. 
Infusion Reactions: Campath administration can result in serious, including fatal, infusion reactions. 
Carefully monitor patients during infusions and withhold Campath for Grade 3 or 4 infusion reactions. 
Gradually escalate Campath to the recommended dose at the initiation of therapy and after interruption of 
therapy for 7 or more days. 
Infections: Serious, including fatal, bacterial, viral, fungal, and protozoan infections can occur in patients 
receiving Campath. 
Administer prophylaxis against Pneumocystis jiroveci pneumonia (PCP) and herpes virus infections. 
 


 
05-H-0206 
Christopher Hourigan, M.D 
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33
The most commonly reported infusion related adverse events were rigors (86%), drug-related 
fever (85%), nausea (54%), vomiting (41%), and hypotension (32%). Hematologic toxicities 
included pancytopenia/marrow hypoplasia (6%), anemia (80%), thrombocytopenia (72%), 
neutropenia (85%), and profound lymphopenia, and should be monitored. Infections reported 
included sepsis (15%), pneumonia (16%), and opportunistic infections such as CMV (8% - Study 
1), Candidiasis (5% - Study 1), Aspergillosis (2% -Study 1), and Mucormycosis (2% - Study 1). 
 
Infusion-related symptoms and signs:  Alemtuzumab (Campath®) can result in serious infusion 
reactions which include nausea, vomiting, fever, chills, rigors, hypotension, rash, fatigue, headache, 
diarrhea, pruritus, urticaria, bronchospasm and dyspnea.   An attempt to prevent these reactions is made 
by using premedication. Patients will be carefully monitored during infusions.  In case of mild reaction, 
the patient will be treated according to symptoms (antiemetics, IV fluid hydration, acetaminophen, 
antihistamines, inhaled bronchodilators). In case of moderate or severe reaction, the infusion will be 
discontinued and restarted at a lower rate once symptoms have subsided. When alemtuzumab is 
administered subcutaneously, rare and usually mild or moderate injection site reactions can result  in 
pain, erythema, itching, or edema at the injection site. These reactions are usually Grade 1 or 2 and can 
be minimized by applying ice before and after the injection and with medications like Tylenol and 
Benadryl. 
 
Infections:  Serious, sometimes fatal bacterial, viral, fungal and protozoan infections have been 
reported in patients receiving alemtuzumab (Campath®).  Patients will be at risk for opportunistic 
infections. Therefore patients will receive prophylaxis for Herpes virus and Pneumocystis carinii.   
 
Cardiac toxicities: The following were reported in at least one patient treated on studies where 
alemtuzumab (Campath®) was used as a single agent: cardiac failure, cyanosis, atrial fibrillation, 
cardiac arrest, ventricular arrhythmia, ventricular tachycardia, angina pectoris, coronary artery 
disorder, myocardial infarction, pericarditis. Some of these cardiac abnormalities may be 
irreversible.  For this reason, we will monitor subjects with an echocardiogram and a 24 hour holter 
monitor before treatment begins and after the last dose of alemtuzumab (Campath®).  The ECHO 
will also be repeated at the 3 month follow up visit.   We will closely monitor subjects for cardiac 
symptomology and ask them to immediately report any cardiac symptoms (palpitations, irregular 
pulse, difficulty in breathing, dizziness, swelling in the ankles, chest discomfort or pain).  
 
Hematologic toxicities: Myelosuppression involves all 3 cell lineages.  Serious and in rare 
instances fatal pancytopenia/marrow hypoplasia, autoimmune idiopathic thrombocytopenia, and 
autoimmune hemolytic anemia have occurred in patients receiving alemtuzumab (Campath®).   
 
Other side effects (< 5% in CLL studies) include headache, anorexia, asthenia, skeletal pain, 
myalgias, peripheral edema, dysthesias, dizziness, tremor, stomatitis, mucositis, dyspnea, cough, 
pneumonitis, rhinitis, abdominal pain, back pain, dyspepsia, constipation, insomnia, depression, 
somnolence, diarrhea, and EBV related lymphoproliferative disease.   
 
Associated with pregnancy and breast feeding: Animal reproductive studies have not been 
conducted.  It is not known whether alemtuzumab (Campath®) can affect reproductive capacity or 
cause fetal harm.  Human IgG is known to pass the placental barrier and thus may potentially cause 
fetal B-cell depletion; therefore alemtuzumab (Campath®) should only be given to pregnant women 
only if clearly needed.   It is not know whether alemtuzumab (Campath®) is excreted in human 
milk.  Because human IgG is excreted in human milk and the potential for absorption and 
immunosuppression in the infant is unknown, women should be advised to discontinue nursing for 
at least 3 months following last dose of alemtuzumab (Campath®). 


 
05-H-0206 
Christopher Hourigan, M.D 
April 26, 2018 
34
 
 
10.3.2 Related to Cyclosporine:   
 
 
Potential serious side effects include 
 
 
Potential side effects:  
 
Although it is metabolized primarily in the liver, CsA major toxicity is renal.  CsA causes a 
decrease in creatinine clearance, which almost always returns to normal range on cessation of the 
drug or lowering of the dose.  Rare development of a hemolytic-uremic syndrome has been reported 
in patients with CsA after allogeneic bone marrow transplant. In our patients with SAA, frequent 
creatinine measurements have allowed prompt adjustment of dose and serious renal complications 
are infrequent. 
 
Evidence of hepatotoxicity is common, usually as transient increases in bilirubin and transaminases.  
These levels often normalize with continued administration of the drug; reduction of the dose is 
uniformly associated with a return to normal levels. 
 
Additional complications include hypertrichosis, gingival hypertrophy (possibly related to pre-
existing poor dental hygiene), hyperesthesia, hirsutism, tremors, headaches, nausea and nonspecific 
gastrointestinal complaints.  Hypertension may occur, and be high enough to require treatment.   
 
Neurologic complications include insomnia, dizziness, anxiety, confusion, and vertigo. We have 
observed seizures in patients receiving CsA, when drug levels were within the therapeutic range. 
Posterior Reversible Encephalopathy Syndrome (PRES) is an increasingly recognized neurologic 
disorder seen in 1% of patients on cyclosporine following solid organ transplantation which manifest 
with acute to subacute hypertension and/or seizures.84  In the event of hypertension, subjects will be 
prescribed 1 or more medications to control blood pressure in an effort to decrease the risk of this 
complication. 
 
Hypomagnesemia and hyperkalemia may occur but are asymptomatic. Increases in uric acid may 
occur and attacks of gout have been rarely reported. Cyclosporine therapy may be associated with a 
modest increase of serum triglycerides or cholesterol. 
 
Less frequent adverse events include: 
 
Autonomic Nervous System:   dry mouth, increased sweating 
Infection related: Because of low white blood cell counts, patients with MDS are susceptible to 
infections.  By further blocking the immune system, CsA further increases this risk.   
 
Cancer related:  When used at high doses in transplant patients, CsA may be associated with an 
increased risk of cancer, especially lymphoma (4 of every 10, 000 patients who receive the 
medication).  Transplant patients receive higher doses than you will be given and are treated for 
longer periods than the duration of this study.  However, because of the way that CsA acts on 
the body, there is a chance that it may cause effects that may not occur until years after the 
medicine is used. 
 
Blindness:  In very rare instances (less that .01%), CsA has been reported to cause blindness. 


 
05-H-0206 
Christopher Hourigan, M.D 
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35
Systemic:   allergy, asthenia, hot flushes, malaise, weight decrease, weight increase  
Cardiovascular:   abnormal heart sounds, cardiac failure, myocardial infarction, peripheral ischemia 
Central and Peripheral Nervous System:   hypoesthesia, neuropathy, vertigo 
Endocrine:   goiter 
Gastrointestinal:   constipation, dysphagia, enanthema, eructation, esophagitis, gastric ulcer, 
gastritis, gastroenteritis, gingival bleeding, glossitis, peptic ulcer, salivary gland enlargement, 
tongue disorder, tooth disorder 
Infection:   abscess, bacterial infection, cellulitis, folliculitis, fungal infection, herpes simplex, 
herpes zoster, renal abscess, moniliasis, tonsillitis, viral infection 
Hematologic:   anemia, epistaxis, leukopenia, lymphadenopathy  
Liver and Biliary System:   bilirubinemia 
Metabolic and Nutritional:   diabetes mellitus, hyperkalemia, hyperuricemia, hypoglycemia 
Musculoskeletal System:   arthralgia, bone fracture, bursitis, joint dislocation, myalgia, stiffness, 
synovial cyst, tendon disorder 
Neoplasms:   breast fibroadenosis, carcinoma 
Psychiatric:   anxiety, confusion, decreased libido, emotional lability, impaired concentration, 
increased libido, nervousness, paroniria, somnolence 
Reproductive (Female):   breast pain, uterine hemorrhage 
Respiratory System:   bronchospasm 
Skin and Appendages:   abnormal pigmentation, angioedema, dermatitis, dry skin, eczema, nail 
disorder, pruritus, skin disorder, urticaria 
Special Senses:   abnormal vision, cataract, conjunctivitis, deafness, eye pain, taste perversion, 
tinnitus, vestibular disorder, blindness 
Urinary System:   abnormal urine, hematuria, increased BUN, micturition urgency, nocturia, 
polyuria, pyelonephritis, urinary incontinence 
 
10.3.3 Related to bone marrow aspirate and biopsy 
 
No major risks are involved with bone marrow aspirate and biopsy. However, a small risk of 
infections, pain, bleeding, and hematoma formation at the site of the aspiration exists with the 
procedure. 
 
10.3.4  Related to blood draws:  No major risks are involved with blood draws. Minor 
complications including bleeding, pain, and hematoma formation at the site of blood draws or 
infections may rarely occur. 
 
10.3.5 Related to central line placement:  A catheter may be placed in a large vein of the neck, 
chest, or arm using local anesthetic. Patients will sign a separate consent for the placement 
procedure. Intravenous line placement carries a small risk of bleeding, bruising or pain and a very 
low risk of accidental injury to the adjacent artery and nerve.  Some patients may experience a 
vasovagal reaction (lightheadedness, or, rarely, fainting due to temporary lowering of blood 
pressure). Only trained experienced staff will place the line in order to minimize these procedure 
related risks.   
 
 
10.3.6  Related to cardiac monitoring 
 
• 
EKG:  An electrocardiogram (EKG) is a test that measures the electrical activity of the 
heartbeat.  With each beat, an electrical impulse (or “wave”) travels through the heart.  This 
wave causes the muscle to squeeze and pump blood from the heart.  A technician will put 


 
05-H-0206 
Christopher Hourigan, M.D 
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36
patches (electrodes) on the chest, arms and legs.  The electrodes are soft and don’t cause any 
discomfort when they’re put on or taken off by the technician.  The machine only records the 
EKG.  It doesn’t send electricity into the body.  There’s no pain or risk associated with having 
an electrocardiogram. 
 
• 
ECHO:   The ECHO uses sound waves to visualize and evaluate the function of the heart. 
There are no associated risks. 
 
• 
Holter monitor: The Holter involves wearing a monitor for 24 hours during which time the 
electrical activity of the heart is recorded. There are no associated risks other than the 
inconvenience of wearing the apparatus.  
 
10.3.7 Related to infection prophylaxis medications 
 
• 
Cirpofloxacin (Cipro, Cipro XR, Proquin XR) The most frequent side effects include nausea, 
vomiting, diarrhea, abdominal pain, rash, headache, and restlessness. Rare allergic reactions 
have been described, such as hives and anaphylaxis (shock). Rare instances of seizures, other 
neurologic sequella and tendon rupture have been reported. Drug interactions with other 
medications have been reported and these will be reviewed before antibiotic start. 
 
 
 
 
 
 
 
 
 
 
 
 
• 
Pentamidine: cough (31-47%), bronchospasm (10-23%), decreased appetite (53-72%), fatigue, 
metallic taste, shortness of breath, decreased appetite, dizziness, rash, nausea, pharyngitis, chest 
pain/congestion, night sweats, chills, vomiting.  
 
• 
Valacyclovir:  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 at greater than the recommended dose. 
 
10.4 
Risks in Relation to Benefits  
 
 
 
For adult subjects: 
 
The potential benefits to the subject include reduction or even abolition of transfusion requirements 
and/or improvement of cytopenia, resulting in improved quality of life and also decreased 
morbidity and mortality from transfusion-associated viral agents, iron overload, and/or a 
susceptibility to infections.  For patients with MDS who have failed initial immunosuppression 
therapy and do not have a histocompatible matched sibling donor, therapeutic options are limited. 
Although unrelated donor stem cell transplantation has been reported with success, the morbidity 
 Fluoroquinolones, including CIPRO®, are associated with an increased risk of tendinitis 
and tendon rupture in all ages. This risk is further increased in older patients usually over 
60 years of age, in patients taking corticosteroid drugs, and in patients with kidney, heart or 
lung transplants. Fluoroquinolones, including CIPRO, may exacerbate muscle weakness in 
persons with myasthenia gravis. Avoid CIPRO in patients with known history of myasthenia 
gravis." 


 
05-H-0206 
Christopher Hourigan, M.D 
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37
and mortality are still considerably higher than a transplant with a matched sibling donor. 
Potentially, treatment with other more toxic therapies could also be avoided or postponed.  
 
Therefore, the research involves greater than minimal risk to subjects with the prospect of direct 
benefit (45 CRF 46.102) 
 
As of January 27, 2017, this study will no longer be in subject follow-up and will continue in 
data analysis only; the level of risk is now minimal. 
 
 
10.5  
Consent Processes and Procedures  
 
The investigational nature and research objectives of this trial, the procedures and treatments 
involved and their attendant risks and discomforts and benefits, and potential alternative therapies 
will be carefully explained to the patient during the initial clinic evaluation. A copy of the consent 
form will be presented to the patient, with instructions to note down questions or underline areas of 
the consent form they do not properly comprehend. The principal investigator or an associate 
investigator on this protocol from the Hematology Branch will lead this discussion and obtain the 
informed consent. Investigators who may obtain consent are listed on page 1 of the protocol.  The 
consent form will be signed in the presence of the investigator and a witness prior to 
commencement of the treatment plan. The treatment plan and risks will be discussed again and in 
detail during their hospital visit for treatment.  
  
If at any time during participation in the protocol, new information becomes available relating to 
risks, adverse events, or toxicities, this information will be provided orally or in writing to each 
enrolled or prospective patient.  Documentation will be provided to the IRB and if necessary the 
informed consent amended to reflect relevant information. 
 
We anticipate enrollment of non-English speaking participants into this study.  The IRB approved 
consent will be translated into the subject’s language in accordance with Clinical MAS Policy M77-
2.  If there is an unexpected enrollment of a research participant for which there is no translated 
IRB approved consent document, the principal investigator and/or those authorized to obtain 
informed consent will use the short form oral consent process as described in MAS Policy M77-2, 
45 CFR 46.117 (b) (2), 21 CFR 50.27 (b)(a).  The summary that will be used is the English version 
of the IRB approved consent document.  We request prospective IRB approval of the use of the 
short form for up to a maximum of 5 participants in a given language and will notify the IRB at the 
time of continuing review of the frequency of the use of the short form.  Should we reach the 
threshold of 5, we will notify the IRB of the need for an additional use of the short form and that we 
will have the consent document translated into the given inherent language. 
 
 
10.6    Conflict of Interest 
 
The Principal Investigator assured that each associate investigator listed on the protocol title page 
received a copy of the NIH’s Guide to preventing conflict of interest.  Investigators added subsequent 
to the initial circulation were provided a copy of the document when they were added. Copies of the 
Conflict of Interest Statement were forwarded to the Clinical Director.  No initial or subsequent 
members of the research team reported a potential conflict of interest.  
 


 
05-H-0206 
Christopher Hourigan, M.D 
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38
This protocol has no associated patents, CRADAs or CTAs.   No transfer of material will be 
accomplished until a Material Transfer Agreement is fully executed through the NHLBI Office of 
Technology Transfer and Development (OTTAD). 
 
FWA Coverage Agreement 
Dr. Catherine Lai has recently undertaken the role of Director of Leukemia at MedStar Georgetown 
University Hospital’s Lombardi Comprehensive Cancer Center, Washington DC. She still maintain 
her work at NIH as an associate investigator and will continue to analyze identifiable data for the 
purpose of publishing a manuscript based on subject outcomes. Dr. Lai’s role in the research will be 
limited to data analysis. An FWA coverage agreement to cover this activity will be executed by Dr. 
Lai and Dr. Hourigan, once this amendment is approved. 
 
11.0     PHARMACEUTICALS 
 
11.1 
ALEMTUZUMAB (CAMPATH®)  
 
 
Generic: Alemtuzumab  
Classification: Monoclonal antibody  
Action: Monoclonal antibody directed against CD52 antigen, a surface glycoprotein expressed by 
lymphocytes  
Availability: Commercial, Genzyme Corporation 
Product Description: Alemtuzumab injection is available in single-use, clear glass vials containing 
30 mg of alemtuzumab in 1 ml of solution 
Storage: Stored at 2 to 8 degrees Celsius (36 to 46 degrees Fahrenheit) and protected from direct 
sunlight. Protect from freezing; discard if frozen. 
Stability: Stability data is not available for undiluted alemtuzumab drawn into a syringe. Once the 
prescribed dose is drawn into a syringe the syringe, the dose should be administered within 4 hours 
of syringe preparation (i.e. 4 hour expiration) whether at room temperature or refrigerated. Protect 
from light. 
Preparation for Administration: Parenteral drug products should be inspected for visible particulate 
matter and discoloration prior to administration. If particulate matter is present or the solution is 
discolored, the vial should not be used. DO NOT SHAKE VIAL PRIOR TO USE. As with all 
parenteral drug products, aseptic technique should be used during the preparation and 
administration of alemtuzumab (Campath®).  
Withdraw the protocol prescribed amount of alemtuzumab from the vial (solution concentrated to 
30mg/ml) into a 1 ml syringe calibrated in increments of 0.1 ml.  
 
11.2 
CYCLOSPORINE (Gengraf, Sandimmune, Neoral) 
 
Supply:  Cyclosporine will be obtained by the NIH Clinical Center Pharmacy Department from 
commercial sources and is available in capsules (25 mg and 100 mg), USP [MODIFIED], oral 
solution (100 mg/ml), USP [MODIFIED], and as a parenteral concentrate for injection (50 mg/ml).  
When oral capsules are prescribed for this protocol, the cyclosporine capsules, USP [NON-
MODIFIED] should NOT be used. 
Preparation:  For parenteral doses, each milliliter of concentrate (50mg/ml) should be diluted in 20 
to 100ml of dextrose 5% in water or sodium chloride 0.9%.  Parenteral doses of cyclosporine will be 
prepared in non-PVC containers and infused with non-PVC administration sets/tubing.  Oral 
cyclosporine solution may be mixed in orange juice or other beverages, but not milk. 
Storage and Stability:  Capsules, oral solution, and ampules of parenteral concentrate bear expiration 
dates and are stored at room temperature and protected from light.  Cyclosporine concentrate for 


 
05-H-0206 
Christopher Hourigan, M.D 
April 26, 2018 
39
injection that has been diluted to a final concentration of approximately 2mg/ml is stable for 24 hours 
in 5% dextrose or 0.9% sodium chloride injection in glass, PVC or non-PVC plastic containers.  To 
minimize the potential for sorption to PVC plastic bags and tubing as well the leaching of phthalate 
plasticizer (DEHP) into the solution, only non-PVC plastic bags and intravenous administration sets 
should be utilized. 
 
Administration: Cyclosporine may be given intravenously or orally. 
Adverse Reactions: see section 10.3.2, Risks and discomforts.  
 
11.3 
CIPROFLOXACIN (CIPRO) 
 
Drug Class and Mechanism: Many common infections in humans are caused by single cell 
organisms, called bacteria. Bacteria can grow and multiply, infecting different parts of the body. 
Medicines that control and eradicate these bacteria are called antibiotics. Ciprofloxacin is an 
antibiotic that stops multiplication of bacteria by inhibiting the reproduction and repair of their 
genetic material (DNA). 
Supply: Commercially available 
Drug interactions: Ciprofloxacin administered together with theophylline can lead to elevated 
blood levels of theophylline. Theophylline is used to open airways in the treatment of asthma. 
Toxic levels of theophylline can lead to seizures, and disturbances in heart rhythm. If concurrent 
use of ciprofloxacin and theophylline cannot be avoided, frequent blood tests to monitor 
theophylline blood levels are performed. Ciprofloxacin should be used with caution in patients with 
central nervous system diseases such as seizures, because rare seizures have been reported in 
patients receiving this medication. Ciprofloxacin should be avoided in children and adolescents 
under 18 years old, as safe use in these patients have not been established. 
Many antibiotics, including ciprofloxacin, can alter the normal bacteria in the colon and encourage 
overgrowth of abacteria responsible for the development of inflammation of the colon 
(pseudomembranous colitis). Pseudomembranous colitis can cause fever, abdominal pain, diarrhea, 
and sometimes even shock. Patients taking ciprofloxacin can develop sensitivity of the skin to 
direct sunlight. Ciprofloxacin can enhance the action of the anticoagulant warfarin (Coumadin), and 
increase the risk of bleeding. 
Storage and Stability: Ciprofloxacin should be stored at below 86 degrees F. 
Administration: Ciprofloxacin may be taken with or without food. Ciprofloxacin is partially 
metabolized by the liver and excreted by the kidney. Dosages require adjustment in patients with 
severely abnormal liver or kidney function. Antacids block the absorption of ciprofloxacin and they 
should not be taken together.  
Adverse reactions: see section 10.3.8, Risks and discomforts 
 
11.3     PENTAMIDINE (NebuPent®) 
 
Supply: Commercially available (NebuPent®, American Pharmaceutical Partners, Inc.) 
Product description:   Pentamidine isethionate is available as a 300 mg single dose vial containing 
300 mg of lyophilized powder in a 15 mL capacity vial. The contents of one vial must be dissolved 
in 6 mL of sterile water for injection, USP. It is important to use only sterile water, saline solution 
will cause the drug to precipitate. 
Storage and stability:  Store dry product at controlled room temperature 15-30oC (59-86oF). 
Route of administration:  Inhalation; Once reconstituted, the entire contents of a vial should be 
placed into the Respigard® II nebulizer (Marquest) reservoir for administration by inhalation Do not 
mix the pentamidine solution with any other drugs.  
Adverse Reactions: see section 10.3.8, Risks and discomforts.  
 
11.4 
VALACYCLOVIR (VALTREX) 


 
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40
 
Generic name: valacyclovir 
Brand Name:  VALTREX 
Supply: Commercially available. 
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. 
Product description: Valacyclovir is available in 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. 
Storage and Stability: Oral tablets should be stored at 15o to 25oC (59o to 77oF).  
Route of administration: Oral  
Adverse Reactions: see section 10.3.8, Risks and discomforts. 
 
 
 


 
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12.0 REFERENCES 
 
1 Nydegger E. Suppressive and substitutive immunotherapy:  an essay with a review of recent literature. Immunol.Lett. 
1985;9:185-190. 
 
2 Sulecki M, Shadduck RK, Zeigler Z. Anti-thymocyte globulin for hypoplastic myelodysplastic syndrome. Blood 
1988;72 (Suppl. 1):229a. 
 
3 Tichelli A, Gratwohl A, Wuersch A, Nissen C, Speck B. Antilymphocyte globulin for myelodysplastic syndrome? 
Br.J.Haematol. 1988;68:139-140. 
 
4 Biesma DH, van den Tweel JG, Verdonck LF. Immunosuppressive therapy for hypoplastic myelodysplastic 
syndrome. Cancer 1997;79:1548-1551. 
 
5 Jonasova A, Neuwirtova R, Cermak J et al. Cyclosporin A therapy in hypoplastic MDS patients and certain 
refractory anaemias without hypoplastic bone marrow. Br.J.Haematol. 1998;200:304-309. 
 
6 Miescher PA, Favre H, Beris P. Autoimmune myelodysplasias. Semin Hematol. 1991;28:322-330. 
 
7 Enright H, Jacob HS, Vercellotti G et al. Paraneoplastic autoimmune phenomena in patients with myelodysplastic 
syndromes: response to immunosuppressive therapy. Br.J.Haematol. 1995;91:403-408. 
 
8 Porta F, Facchetti F, Tettoni K et al. Myelodysplastic syndrome in an infant: induction of remission by cyclosporin. 
Lancet 1998;352:1600-1601. 
 
9 Bynoe AG, Scott CS, Ford P, Roberts BE. Decreased T helper cells in the myelodysplastic syndromes. 
Br.J.Haematol. 1983;54:97-102. 
 
10 Knox SJ, Greenberg BR, Anderson RW, Rosenblatt LS. Studies of T-lymphocytes in preleukemic disorders and 
acute nonlymphocytic leukemia: in vitro radiosensitivity, mitogenic responsiveness, colony formation, and 
enumeration of lymphocytic subpopulations. Blood 1983;61:449-455. 
 
11 Colombat PH, Renoux M, Lamagnere JP, Renoux G. Immunologic indices in myelodysplastic syndromes. Cancer 
1988;61:1075-1081. 
 
12 Zoumbos NC, Djeu JY, Young NS. Interferon is the suppressor of hematopoiesis generated by stimulated 
lymphocytes in vitro. J.Immunol. 1984;133:769-774. 
 
13 Molldrem J, Jiang YZ, Stetler-Stevenson M et al. Haematological response of patients with myelodysplastic 
syndrome to antithymocyte globulin is associated with a loss of lymphocyte-mediated inhibition of CFU-GM and 
alterations in T-cell receptor Vbeta profiles. Br J Haematol 2000;28:148-155. 
 
14 Shetty V, Mundle S, Alvi S et al. Measurement of apoptosis, proliferation and three cytokines in 46 patients with 
myelodysplastic syndromes. Leuk.Res. 1996;20:891-900. 
 
15 Raza A, Mundle S, Shetty V et al. A paradigm shift in myelodysplastic syndromes. Leukemia 1996;10:1648-1652. 
 
16 Baumann MA, Milson TJ, Patrick CW, Libnoch JA, Keller RH. Immunoregulatory abnormalities in myelodysplastic 
disorders. Am J Hematol 1986;22:17-26. 
 
17 Rosenfeld SJ, Kimball J., Vining D, Young NS. Intensive immunosuppression with antithymocyte globulin and 
cyclosporine as treatment for severe acquired aplastic anemia. Blood 1995;85:3058-3065. 
 
                                                 


 
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42
                                                                                                                                                                
18 Frickhofen N, Kaltwasser JP, Schrezenmeier H et al. Treatment of aplastic anemia with antilymphocyte globulin and 
methylprednisolone with or without cyclosporine.  The German Aplastic Anemia Study Group. N.Engl.J.Med. 
1991;324:1297-1304. 
 
19 Silverman LR, Holland JF, Weinberg RS et al. Effects of treatment with 5-azacytidine on the in vivo and in vitro 
hematopoiesis in patients with myelodysplastic syndromes. Leukemia 1993;7 (Suppl.1):21-29. 
 
20 Young NS, Alter BP. Aplastic Anemia, Acquired and Inherited. Philadelphia: Saunders; 1994. 
 
21 Raefsky E, Gascon P, Gratwohl A, Speck B, Young NS. Biological and biochemical characterization of anti-
thymocyte globulins (ATG) and anti-lymphocyte globulins (ALG). Blood 1986;68:712-719. 
 
22 Bourdage JS, Hamlin DM. Comparative polyclonal antithymocyte globulin and antilymphocyte/ antilymphoblast 
globulin anti-CD antigen analysis by flow cytometry. Transplantation 1995;59:1194-1200. 
 
23 Zeng W, Nakao S, Takamatsu H et al. Characterization of T-cell repertoire of the bone marrow in immune-mediated 
aplastic anemia: Evidence for the involvement of antigen-driven T-cell response in cyclosporine-dependent aplastic 
anemia. Blood 1999;93:3008-3016. 
 
24 Sloand E, Kim S, Maciejewski JP et al. Presence of intracellular interferon-gamma (IFN-gamma) in circulating 
lymphocytes and response to immunosuppressive therapy in patients with aplastic anemia [abstract]. Blood 1998;10, 
Suppl. 1:158a. 
 
25 Genestier L, Fournel S, Flacher M et al. Induction of Fas (Apo-1, CD95)-mediated apoptosis of activated 
lymphocytes by polyclonal antithymocyte globulin. Blood 1998;91:2360-2368. 
 
26 Gascon P, Zoumbos N, Djeu J, Scala G, Young N. Lymphokine abnormalities in aplastic anemia.  Implications for 
the mechanism of action of ATG. Blood 1985;65:407-413. 
 
27 Barrett AJ, Longhurst P, Rosengurt N, Hobbs JR, Humble JG. Crossreaction of antilymphocyte globulin with human 
granulocyte colony forming cells. J.Clin.Pathol. 1978;31:129-135. 
 
28 Greco B, Bielory L, Stephany D et al. Antithymocyte globulin reacts with many normal human cell types. Blood 
1983;62:1047-1054. 
 
29 Shevach EM. The effects of cyclosporin A on the immune system. Annu.Rev.Immunol. 1985;3:397-423. 
 
30 Nussenblatt RB, Palestine AG. Cyclosporine:  immunology, pharmacology, and therapeutic uses. Surv.Ophthalmol. 
1986;31:159-169. 
 
31 Orosz GG, Roopenian DC, Widmer MB, Bach FH. Analysis of cloned T cell function.  II. Differential blockade of 
various cloned T cell functions by cyclosporine. Transplantation 1983;36:706-711. 
 
32 Bickel M, Tsuda H, Amstad P et al. Differential regulation of colony-stimulating factors and interleukin 2 
production by cyclosporin A. Proc.Natl.Acad.Sci.U.S.A. 1987;84:3274-3277. 
 
33 Kronke M, Leonard WJ, Depper JM et al. Cyclosporine A inhibits T-cell growth factor gene expression at the level 
of mRNA transcription. Proc.Natl.Acad.Sci.U.S.A. 1984;81:5214-5218. 
 
34 Cohen DJ, Loertscher R, Rubin MF et al. Cyclosporine:  a new immunosuppressive agent for organ transplantation. 
Ann.Intern.Med. 1984;101:667-682. 
 
35 Horigome A, Hirano T, Oka K et al. Glucocorticoids and cyclosporine induce apoptosis in mitogen-activated human 
peripheral mononuclear cells. Immunopharmacology 1997;27:87-94. 
 


 
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36 Bacigalupo A, Frassoni F, Podesta' M et al. Cyclosporin A (CyA) does not enhance CFU-C growth in patients with 
severe aplastic anaemia. Scand.J.Haematol. 1985;34:133-136. 
 
37 Saunthararajah Y, Nakamura R, Wesley R, Wang QJ, Barrett AJ. A simple method to predict response to 
immunosuppressive therapy in patients with myelodysplastic syndrome. Blood 102: 3025-7, 2003. 
 
38 Hale G, Xia M-Q, Tighe HP, et al. The Campath-1 antigen (CDw52). Tissue Antigens 35:188-27, 1990 
 
39 Takeda J, Miyata T, Kawagoe K, et al. Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A 
gene in paroxysmal nocturnal hemoglobinuria. Cell 73; 703-11, 1993. 
 
40 Rosse WF, Ware RE. The molecular basis of paroxysmal nocturnal hemoglobinuria. Blood 86: 3277-3286, 1995. 
 
41 Hertenstein B, Wagner B, Bunjes D, et al. Emergence of CD52-, phosphatidylinositolglycan-anchor-deficient T 
lymphocytes after in vivo application of Campath-1H for refractory B-cell non-Hodgkin’s lymphoma. Blood 86: 1487-
92, 1995. 
 
42 Taylor VC, Sims M, Brett S, et al. Antibody selection against CD52 produces a paroxysmal nocturnal 
haemoglobinuria phenotype in human lymphocytes by a  novel mechanism. Biochem J 322: 919-25, 1997. 
 
43 Rawstron AC, Rollinson SJ, Richards S, et al. The PNH phenotype cells that emerge in most patients after 
CAMPATH-1H therapy are present prior to treatment. Br J Hematol 104: 148-153, 1999. 
 
44  Brett SJ, Baxter G, Cooper H, et al. Emergence of CD52-, glycosylphosphatidylinositol-anchor-deficient 
lymphocytes in rheumatoid arthritis patients following Campath-1H treatment. International Immunology  8: 325-334, 
1996.  
 
45 Finnegan A, Schnitzer TJ. Persistence of CD4+ T cells in arthritic joint after CAMPATH-1H treatment [letter]. J 
Rheumatol 24: 1448-9, 1997. 
 
46 Issacs JD, Watts RA, Hazleman BL, at al. Humanized monoclonal antibody therapy for rheumatoid arthritis. Lancet 
340: 748-752, 1992. 
 
47 Coles AJ, Wing M, Smith S, et al. Pulsed monoclonal antibody treatment and autoimmune thyroid disease in 
multiple sclerosis. Lancet 354: 1691-1695, 1999. 
 
48 Moreau T, Coles A, Wing M, et al. CAMPATH-1H in multiple sclerosis. Multiple Sclerosis 1: 357-365, 1996. 
 
49 Coles AJ, Wing MG, Molyneaux P, et al. Monoclonal antibody treatment exposes three mechanisms underlying the 
clinical course of multiple sclerosis. Ann Neurol 46: 296-304, 1999. 
 
50 Dick AD, Meyer P, James T, et al. Campath-1H therapy in refractory ocular inflammatory disease. Br J Ophthalmol 
84: 107-109, 2000. 
 
51 Isaacs JD, Hale G, Waldmann H, et al. monoclonal antibody therapy of chronic intraocular inflammation using 
Campath-1H [letter]. Br J Ophthalmol 79: 1054-1055, 1995. 
 
52 Hale G, Cobboid S, Waldmann H. T cell depletion with Campath-1H in allogeneic bone marrow transplantation. 
Transplantation 45: 753-759, 1988. 
 
53 Hale G, Waldmann H. CAMPATH-1H monoclonal antibodies in bone marrow transplantation. J Hematother 3: 15-
31, 1994. 
 
54 Hale G, Waldmann H. Recent results using CAMPATH-1H antibodies to control GVHD and graft rejection. Bone 
Marrow Transplant 17: 305-308, 1996. 
 


 
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55 Willemze R, Richel DJ, Falkenburg JHF, et al. In vivo use of Campath-1G to prevent graft-versus-host disease and 
graft rejection after bone marrow transplantation. Bone Marrow Transplant 9: 255-261, 1992. 
 
56 Jacobs P, Wood L, Fullard L, et al. T cell depletion by exposure to Campath-1G in vitro prevents graft-versus-host 
disease. Bone Marrow Transplant 13: 763-769, 1994.  
 
57 Watts RA, Isaacs JD, Hale G, et al. Peripheral blood lymphocytes subsets after CAMPATH-1H therapy for RA – a 3 
year followup [abstract]. Arthritis Rheum 37 (suppl 9): S338, 1994. 
 
58 Weinblatt ME, Maddison PJ, Bulpitt KJ, et al. CAMPATH-1H, a humanized monoclonal antibody, in refractory 
rheumatoid arthritis. An intravenous dose-escalation study. Arthritis Rheum 38: 1589-94, 1995. 
 
59 Matteson EL, Yocum DE, St Clair EW, et al. Treatment of active refractory rheumatoid arthritis with humanized 
monoclonal antibody CAMPATH-1H administered by daily subcutaneous injection. Arthritis Rheum 38: 1187-93, 
1995.   
 
60 Brett S, Baxter G, Cooper H, et al. Repopulation of blood lymphocyte sub-populations in rheumatoid arthritis 
patients treated with the depleting humanized monoclonal antibody, CAMPATH-1H. Immunology 88: 13-9, 1996. 
 
61 Schnitzer TJ, Yocum DE, Michalska M, et al. Subcutaneous administration of CAMPATH-1H; clinical and 
biological outcomes. J Rheumatol 24: 1031-6, 1997. 
 
62 Isaacs JD, Manna VK, Rapson N, et al. Campath-1H in rheumatoid arthritis-an intravenous dose-ranging study. Br J 
Rheumatology 35: 231-40, 1996. 
 
63 Wing MG, moreau T, Greenwood J, et al. Mechanism of first dose cytokine-release syndrome by CAMPATH-1H: 
involvement of CD16(FcgammaRIII) abd CD11a/CD18 (LFA-1) on NK cells. J Clin Invest 98; 2819-2826, 1996. 
 
64 Keating MJ, Flinn I, Jain V, et al. Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed 
fludarabine: results of a large international study. Blood 99: 3554 – 3561, 2002.  
 
65 Rai KR, Freter CE, Mercier RJ et al. Alemtuzumab in previously treated chronic lymphocytic leukemia patients who 
also had received fludarabine. J Clin Oncol 20: 3891-3897, 2002.  
 
66 Osterborg A, Dyer MJS, Bunjes D, et al. Phase II multicenter study of human CD52 antibody in previously treated 
chronic lymphocytic leukemia. J Clin Oncol 15: 1567-1574, 1997.  
 
67 Kottaridis PD, Milligan DW, Chopra R, et al. In vivo CAMPATH-1H prevents graft-versus-host disease following 
nonmyeloablative stem cell transplnatation. Blood 96: 2419-2425, 2000. 
 
68 Hale G, Zhang MJ, Bunjed D, et al. Improving the outcome of bone marrow transplantation by using monoclonal 
antibodies to prevent graft-versus-host disease and graft rejection. Blood 92: 4581-90, 1998. 
 
69 Calne R, Friend P, Moffatt S, et al. Prope tolerance, peri-operative Campath-1H, and low dose cyclosporine 
monotherapy in renal allograft recipients [letter]. Lancet 352: 408, 1998. 
 
70 Vassiliou GS, Webb DKH, Pamphilon D, et al. Improved outcome of alternative donor bone marrow transplantation 
in children with severe aplastic anemia using a conditioning regimen containing low-dose total body irradiation, 
cyclophosphamide and Campath. Br J Hematol 114: 701-705, 2001. 
 
71 Hamblin M, Marsh JCW, Lawler M, et al. Campath-1G in vivo confers a low incidence of graft-versus-host disease 
associated with high incidence of mixed chimaerism after bone marrow transplnatation for severe aplastic anemia 
using HLA-identical sibling donors. Bone Marrow Transplantation 17: 819-824, 1996. 
 
72 Willis F, Marsh JCW, Bevan DH, et al. The effect of treatment with Campath-1H in patients with autoimmune 
cytopenias. Br J Haematol 114: 891-898, 2001. 


 
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73 Killick SB, Marsh JCW, Hale G, et al. Sustained remission of severe resistant autoimmune neutropenia with 
Campath-1H. Br J Haematol 97; 306-308, 1997. 
 
74 Rosenfeld S, Follmann D, Nunez O, et al. Antithymocyte globulin and cyclosporine for severe aplastic anemia: 
association between hematologic response and long term outcome. JAMA 289: 1130 – 5, 2003.  
 
75 Simon R. Optimal two-stge designs for phase II clinical trials. Controlled Clinical Trials 10:1-10, 1989. 
 
76 Geller NL, Leifer ES, Follmann D. and Carter SL. Design of Early Trials in Stem Cell Transplantation: A Hybrid 
Frequentist-Bayesian Approach. Advances in Clinical Trials Biostatistics. 2003. 
77Cheson BD, Bennett JM, Kantarjian H et al. Report of an international working group to standardize response criteria 
for myelodysplastic syndromes. Blood 2000;96:3671-3674. 
 
78. Hellstrom-Lindberg E. Approach to anemia associated with myelodysplastic syndromes. Curr.Hematol.Rep. 
2003;2:122-129. 
79. Hellstrom-Lindberg E, Negrin R, Stein R et al. Erythroid response to treatment with G-CSF plus erythropoietin for 
the anaemia of patients with myelodysplastic syndromes: proposal for a predictive model. Br.J.Haematol. 
1997;99:344-351. 
80  Solomon SR, Savani BN, Childs R et al. Improved outcome for peripheral blood stem cell transplantation for 
advanced primary myelodysplastic syndrome. Biol Blood Marrow Transplant. 2005;11:619-626. 
81Ma X, Does M, Raza A, Mayne ST. Myelodysplastic syndromes: incidence and survival in the United States. Cancer 
2007;109:1536-1542 
82 Atallah E, Kantarjian H, Garcia-Manero G. The role of decitabine in the treatment of myelodysplastic syndromes. 
Expert Opinion on Pharmacotherapy 2007;8:65-73. 
83.Kantarjian H, Issa JP, Rosenfeld CS et al. Decitabine improves patient outcomes in myelodysplastic syndromes: 
results of a phase III randomized study. Cancer 2006;106:1794-1803.84 Bartynski WS, Tan HP, Boardman JF, Shapiro 
R, Marsh JW.  Posterior reversible encephalopathy syndrome after solid organ transplantation. AJNR Am J 
Neuroradiol. 2008 May;29(5):924-30. Epub 2008 Feb 13.) 
85 Hwang WYK et al., Outpatient-Based Therapy of Oral Fludarabine and Subcutaneous Alemtuzumab for Asian Patients with 
Relapsed/Refractory Chronic Lymphocytic Leukemia. Advances in Hematology, vol. 2009, Article ID 547582, 4 pages, 2009. 
86 Wierda WG et al., Self-Administered, Subcutaneous Alemtuzumab to Treat Residual Disease in Patients With Chronic 
Lymphocytic Leukemia. Cancer Volume 117, Issue 1, pages 116–124, 1 January 2011 
87 Risitano AM et al., Alemtuzumab is safe and effective as immunosuppressive treatment for aplastic anaemia and single-lineage 
marrow failure: a pilot study and a survey from the EBMT WPSAA. Br J Haematol. 2010 Mar;148(5):791-6.  
88 Gómez-Almaguer D et al. Subcutaneous alemtuzumab plus cyclosporine for the treatment of aplastic anemia. Ann Hematol. 2010 
Mar; 89(3):299-303. 
 


 
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APPENDIX A NHLBI HEMATOLOGY BRANCH LABORATORY RESEARCH STUDIES 
-2/5/2013 
DESCRIPTION OF LABORATORY STUDY BY BRANCH SECTION 
Does this test 
pose a greater 
than minimal 
risk to 
pediatric 
subjects per 45 
CFR 46.404? 
Does this test 
pose a 
greater than 
minimal risk 
to healthy 
pediatric 
donors per 45 
CFR 46.404? 
 
 
A 
Stem Cell Allotransplantation Section (Dr. A. John Barrett) 
 
 
A.1 
Measurement of lymphocyte function and immune responses directed toward 
allogeneic tissues, malignant cells, and infectious agents.  Assay of a variety of 
antigens, including standard proliferation, cytotoxicity, and intracellular cytokine  
detection including GVHD predictive markers. Measurement of antigen-specific 
responses including employment of tetramers, ELISPOT technique, gene 
amplification-based assays, and flow cytometry.  Selection of cells using 
immunomagnetic beads or flow cytometry.  Culture, expansion, and selection of 
cells. Surface marker analysis of PB MC using flow cytometry. Cytokine/chemokine 
analysis of plasma/serum samples using ELISA and/or Luminex techniques. 
 
No 
No 
A.2 
Generation of cell lines for the study of immune cell interactions with other cells.  
Transformation of B-lymphocytes using Epstein-Barr virus.  Derivation of malignant 
cell lines from patient leukemic or solid tumor samples. 
No 
No 
A.3 
Infection of cells and cell lines with recombinant genes to ascertain the effects of 
expressed molecules on immune responses and on growth and development. 
Transfection of cell lines with specific molecules to study antigen-specific responses. 
No 
No 
A.4 
Assays of peripheral blood and bone marrow progenitor cells including primitive and 
late erythroid progenitor-derived colonies, myelomonocytic colonies, and primitive 
multi- potential progenitor-derived colonies. 
No 
No 
A.5 
Injection of human cells into experimental animals to study the immune system and 
the growth of normal and malignant cells under varying conditions. 
No 
No 
A.6 
Testing of selection methods, cell isolation, and cell expansion leading to the 
development of new cell-based therapies requiring scale-up for clinical application. 
No 
No 
A.7 
Identification of individual T cell clones by their T cell receptor sequence. 
No 
No 
A.8 
Measurement of tumor and tissue specific antigens in cells of subjects and donors by 
mRNA,protein, or peptide expression in cells or fluids. 
No 
No 
A.9 
Laser capture micro dissection of cells from biopsies for GVHD to determine 
clonotypes. 
No 
No 
A.10 
DNA and RNA typing of genes that control immune responses in lymphocytes. 
No 
No 
A.11 
Microassay studies utilizing cellular DNA, cDNA, and RNA for neoplasia and host-
tumor interactions. 
No 
No 
 
 
B 
Molecular Hematopoiesis  Section  (Dr. Cynthia Dunbar)  
 
 
B.1 
Flow cytometric analysis of cell surface and cytoplasmic proteins, including cell 
adhesion molecules, putative retroviral receptors, and markers of differentiation, 
using bone marrow and mobilized peripheral blood cells. 
No 
No 
B.2 
Hematopoietic progenitor-derived colony ascertainment in vitro (as described above), 
and engraftment of immunodeficient mice for detection of human stem cell number 
and function. 
No 
No 


 
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B.3 
Testing ability of hematopoietic progenitor cells to be transduced with retroviral, 
lentiviral, and novel gene transfer vectors in vitro. 
No 
No 
B.4 
Reprogramming of adult mature cells, including skin fibroblasts and blood cells, into 
induced pluripotent stem cells in vitro. 
No 
No 
 
 
C 
Cell Biology Section (Dr. Neal Young)  
 
 
C.1 
Studies of blood and bone marrow hematopoietic progenitor numbers, including 
early and late erythroid progenitors, myelomonocytic progenitors, and multi-potential 
progenitor cells.  In addition, bone marrow may be placed in long-term bone marrow 
culture to assess the function of stroma and stem cells and to assay more primitive 
progenitors, as well as organelle culture.  Whole or selected bone marrow 
populations are cultured short-term for CD34 cell expansion. 
No 
No 
C.2 
Assays of apoptosis in hematopoietic cells and their progeny, using flow cytometric 
methods such as annexin and caspase-3 staining, propidium iodide uptake, and 
mitochondrial permeability tests. 
No 
No 
C.3 
Separation and functional study of cell populations characteristic of paroxysmal 
nocturnal hemoglobinuria, identified by absence of glycosylphosphatidylinositol 
anchored proteins. 
No 
No 
C.4 
Studies of mutation rates in hematopoietic cells and in buccal mucosa cells, using 
conventional hypoxanthine phosphoribosyltransferase activity functional assays, 
sequencing of mitochondrial DNA after specific gene amplification, and 
measurement of GPI-anchored deficient cells in blood and bone marrow. 
No 
No 
C.5 
Assays of immune function of T-cells, including intracellular cytokine staining, 
ELISPOT, semiquantitative gene amplification for gamma-interferon, tumor necrosis 
factor, interleukin-2, and other cytokines, and functional assessment in co-culture 
using specific neutralizing monoclonal antibodies.  In addition, peripheral blood 
lymphocytes are subjected to spectratyping for CDR3 size distribution as well as 
nucleotide sequence of CDR3 peaks obtained. 
No 
No 
C.6 
Studies of engraftment of human normal and diseased bone marrow and peripheral 
blood in immunodeficient mice in order to determine the presence of hematopoietic 
repopulating stem cells as well as functional differences among selected populations. 
No 
No 
C.7 
Flow cytometric analysis of blood and bone marrow for lymphocyte phenotype, 
especially for evidence of activation of lymphocytes, for markers of apoptosis, and 
for antigens associated with primitive and mature hematopoietic cell populations. 
No 
No 
C.8 
Flow cytometric analysis of blood and bone marrow for hematopoietic stem cell 
progenitors and CD34 positive cells. 
No 
No 
C.9 
Studies of chromosomal instability in myelopdysplastic syndromes including BM 
cell and CD34 cell response to PAS crosslinking and examination of the cytotoxic 
effect of lymphocytes to the abnormal clone of cells. 
No 
No 
C.10 
Surface Enhanced Laser/Desorption Ionization (SELDI)  time-of-flight mass 
spectrometry (Ciphergen)  (proteomics methodology). 
No 
No 
C.11 
Mitochondrial DNA (mtDNA) sequence heterogeneity. 
No 
No 
C.12 
Measurement of EBV viral load. 
No 
No 
C.13 
Measurement of EBV LMP-1 via RT-PCR for LMP-1 RNA or flow cytometry for 
LMP-1. 
No 
No 
C.14 
Outgrowth assay of EBV transformed B cells. 
No 
No 
C.15 
Quantification of serumchemokines and cytokines (e.g. SDF-1, IL-10, IL-6, CXCR4, 
CXCL12). 
No 
No 
C.16 
Quantification of EBV cytotoxic T cells (tetramerstaining). 
No 
No 
C.17 
Telomere length measurement by Southern blot, Q-PCR, flow-fish, in situ 
hybridization and STELA 
No 
No 


 
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48
                                                                                                                                                                
C.18 
Telomere repair complex gene mutations by nucleotide sequencing of some or all of 
the following:  DKC1 ,TERC, TERT, SBDS, NOp10 , NHP2. 
No 
No 
C.19 
Analysis of  inflammatory markers and/or bacterial, viral, fungal or protozoal 
elements in plasma or serum using molecular, colorimetric, enzymatic, flow 
cytometric or other assays in subjects receiving immunosuppressive therapy, 
chemotherapy and/or bone marrow transplantation. 
No 
No 
C.20 
Confocal microscopic imaging of bone marrow. 
No 
No 
C.21 
Characterization of intracellular signaling proteins by cell permeabilization and flow 
cytometry, and quantitative immunoblots. 
No 
No 
C.22 
Assays for chromosomal aneuploidy by florescence in situ hybridization (FISH) and 
other molecular techniques. 
No 
No 
C.23 
Conversion of human dermal fibroblasts into hematopoietic progenitors using Oct4 
transfection. 
No 
No 
 
 
D 
Virus Discovery Section (Dr. Neal Young) THESE ASSAYS WILL NOT BE 
PERFORMED ON SAMPLES FROM HEALTHY PEDIATRIC DONORS 
 
 
D.1 
Assays of serum, blood cells, and bone marrow cells for B19 parvovirus and possible 
B19  variants using gene amplification, cell culture, and hematopoietic colony 
inhibition assays. 
No 
N/A 
D.2 
Assays of blood, bone marrow, liver, and other tissues for potentially novel viruses, 
using a variety of techniques including RNA and DNA assays, differential display, 
gene amplification with conserved and random primers, cell culture assays, 
immunohistochemical methods, and inocculation of mice, rabbits, and monkeys, as 
well as antibody measurements. 
No 
N/A 
D.3 
Assays of blood, bone marrow, and liver for known viruses, including herpesviruses 
such as cytomegalovirus, human herpesviruses 6, 7, and 8, enteric viruses such as A-
6, circiviruses, and parvoviruses, using assays as in (2). 
No 
N/A 
D.4 
Spectra-typing of blood cells to determine response to known or putative viral 
infections. 
No 
N/A 
D.5 
HLA typing or subtyping to determine risk factors/determinants for hepatitis-AA 
studies. 
No 
N/A 
D.6 
Cytotoxic lymphocyte assays with intracellular cytokine measurement for 
determining anti-viral response and lymphocyte cloning to obtain clones with 
specific antiviral activity. 
No 
N/A 
 
 
E 
Solid Tumor Section (Dr. Richard Childs) 
 
 
E.1 
Cr51 cytotoxicity assay to evaluating killing of patient tumor cells by patient NK cell 
clones and T-cells. 
No 
No 
E.2 
ELISA for IL-12  maturity of DC's made from subjects monocytes. 
No 
No 
E.3 
ELISA for IFN ã to evaluate specificity of CTL clones. 
No 
No 
E.4 
H thymidine uptake to evaluate proliferation potential of antigen specific T-cells. 
No 
No 
E.5 
PCR of STR to assess chimerism status of cellular subsets grown in-vitro or retrieved 
from subjects post-transplant. 
No 
No 
E.6 
Flow sorting of PBL and/or tissue samples to evaluate chimerism of different subsets. 
No 
No 
E.7 
Surface marker analysis of peripheral blood mononuclear cells using flow cytometry. 
No 
No 
E.8 
cDNA expression arrays to evaluate T-cells expression/gene patterns in subjects with 
GVHD and a GVT effect. 
No 
No 
E.9 
Geno typing of tumor or tissue samples by high density cDNA arrays. 
No 
No 
E.10 
VHL mutation analysis on kidney cancer tissue. 
No 
No 
E.11 
Transduction of dendritic and tissue cells with tumor antigens using plasmids, viral 
vectors and hybrid fusions. 
No 
No 


 
05-H-0206 
Christopher Hourigan, M.D 
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49
                                                                                                                                                                
E.12 
Lasar capture microdisection of cells from tumor biopsies and tissue samples  to 
determine origin (donor vs patient). 
No 
No 
E.13 
Quantification of polyoma virus BK exposure by serology and PCR in stem cell 
transplant donors and recipients from blood and urine samples. 
No 
No 
E.14 
Quantification of polyoma virus BK specific T cells in stem cell transplant donors 
and recipients from peripheral blood samples. 
No 
No 
E.15 
Determination of origin of neovasculature endothelial cells in tumor and tissue 
samples obtained from subjects post transplant. 
No 
No 
E.16 
Quantification of lymphocyte subsets CD34 progenitors and endovasculator 
progenitors in G-CSF mobilized peripheral cell allografts. 
No 
No 
E.17 
Testing for polyoma virus BK latency in CD34 progenitors, B cells and T cells in the 
G-CSF mobilized peripheral cell allografts. 
No 
No 
E.18 
Determination of etiology of membraneous nephropathy using serum from subjects. 
No 
No 
E.19 
Serum Proteomic patterns analysis to diagnose complications related to allogeneic 
transplantation. 
No 
No 
E.20 
Determine cell origin (donor vs patient) of tissue samples using IHC, IF, sorting, and 
FISH. 
No 
No 
 
 
F 
Lymphoid Malignancies Section (Dr. Adrian Wiestner) 
 
 
F.1 
Culture of cells from research subjects to investigate molecular disease mechanisms, 
model host tumor interactions, and to test effect of drugs on cell survival and cellular 
functions. 
No 
No 
F.2 
Generation of stable cell lines for the study of hematologic malignancies. 
No 
No 
F.3 
Modifications of cells using standard expression systems or biologic molecules, e.g. 
interfering RNA, to investigate the effects of candidate genes on cellular functions. 
 
 
F.4 
Identification and monitoring of B or T cell populations as identified by flow 
cytometry and by their B cell or T cell receptor expression. 
No 
No 
F.5 
Measurement of gene expression in cells or tissues. Techniques frequently used 
include gene expression profiling on microarrays, quantitative RT-PCR, Western 
blotting, flow cytometry and ELISA assays. 
No 
No 
F.6 
Analysis of chromosomal abnormalities or mutations in malignant cells and non-
malignant cells including FISH technology and DNA sequencing. 
No 
No 
F.7 
Assays of immune function of B-cells and T-cells, including intracellular cytokine 
staining, ELISPOT, quantitative RT-PCR for cytokines or other immune regulatory 
genes. 
No 
No 
F.8 
Analysis of antibody specificities in serum and antigen specificity of the B-cell 
receptor on cells. Techniques may include expression of antibodies in phage display 
systems, generation of antibodies in cell culture systems and use of such antibodies to 
screen for cognate antigens. 
No 
No 
F.9 
Transplantation of human cells into mice (xenograft model) to study disease biology 
and to investigate the effect of experimental therapy. 
No 
No 
F.10 
Measurements of drug concentrations, biologic molecules and disease markers in 
blood, serum, and plasma. 
No 
No 
 
 
APPENDIX B :INTERNATIONAL WORKING GROUP PROPOSED STANDARDIZED 
CRITERIA FOR RESPONSE IN MDS77 
 
I.    
Altering Disease Natural History 
 
Ia. Complete remission (CR) 
 


 
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Peripheral blood evaluation (absolute values must last at least 2 months)†: 
• 
Hemoglobin greater than 11 g/dL (untransfused, patient not on erythropoietin) 
• 
Neutrophils 1500/mm3 or more (not on a myeloid growth factor) 
• 
Platelets 100 000/mm3 or more (not on a thrombopoetic agent) 
• 
Blasts, 0% 
• 
No dysplasia* 
 
Bone marrow evaluation: Repeat bone marrow showing less than 5% myeloblasts with normal maturation of all 
cell lines, with no evidence for dysplasia.* When erythroid precursors constitute less than 50% of bone marrow 
nucleated cells, the percentage of blasts is based on all nucleated cells; when there are 50% or more erythroid 
cells, the percentage blasts should be based on the nonerythroid cells. 
 
  Ib. Partial remission (PR)   
 
All of the CR criteria (if abnormal before treatment) (absolute values must last at least 2 months): except:  
Bone marrow evaluation: Blasts decreased by 50% or more over pretreatment, or a less advanced MDS FAB 
classification than pretreatment. Cellularity and morphology are not relevant. 
 
Ic. Stable disease:   Failure to achieve at least a PR, but with no evidence of progression for at least 2 months. 
 
Id. Failure:  Death during treatment or disease progression characterized by worsening of cytopenias, increase in 
the percentage bone marrow blasts, or progression to an MDS FAB subtype more advanced than 
pretreatment. 
 
Ie. Relapse after CR or PR:   one or more of the following: 
a) Return to pretreatment bone marrow blast percentage. 
b) Decrement of 50% or greater from maximum remission/response levels in granulocytes or platelets. 
c) Reduction in hemoglobin concentration by at least 2 g/dL or transfusion dependence.§ 
6. Disease progression 
a) For patients with less than 5% blasts: a 50% or more increase in blasts to more than 5% blasts. 
b) For patients with 5% to 10% blasts: a 50% or more increase to more than 10% blasts. 
c) For patients with 10% to 20% blasts: a 50% or more increase to more than 20% blasts. 
d) For patients with 20% to 30% blasts: a 50% or more increase to more than 30% blasts. 
e) One or more of the following: 50% or greater decrement from maximum remission/response levels in 
granulocytes or platelets, reduction in hemoglobin concentration by at least 2 g/dL, or transfusion 
dependence.§ 
 
If.  Disease transformation:  Transformation to AML (30% or more blasts). 
 
Ig.   Survival and progression-free survival 
 
II. 
Cytogenetic response 
 
IIa  Major:  No detectable cytogenetic abnormality, if preexisting abnormality was present. 
 
IIb  Minor: 50% or more reduction in abnormal metaphases. 
 
Requires 20 analyzable metaphases using conventional cytogenetic techniques.Fluorescent in situ hybridization 
may be used as a supplement to follow a specifically defined cytogenetic abnormality. 
 
 
III. Hematologic Improvement (HI) 
 


 
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Improvements must last at least 2 months in the absence of ongoing cytotoxic therapy.†  Hematologic 
improvement should be described by the number of individual, positively affected cell lines (eg, HI-E; HI-E 1 
HI-N; HI-E 1 HI-P 1 HI-N). 
 
IIIa.  Erythroid response (HI-E) 
• 
Major response: For patients with pretreatment hemoglobin less than 11 g/dL, greater than 2 g/dL 
increase in hemoglobin; for RBC transfusion-dependent patients transfusion independence. 
 
• 
Minor response: For patients with pretreatment hemoglobin less than 11 g/dL, 1 to 2 g/dL increase 
in hemoglobin; for RBC transfusion-dependent patients, 50% decrease in transfusion 
requirements. 
 
IIIb.  Platelet response (HI-P) 
• 
Major response: For patients with a pretreatment platelet count less than 100 000/mm3, an 
absolute increase of 30 000/mm3 or more; for platelet transfusion-dependent patients, 
stabilization of platelet counts and platelet transfusion independence. 
 
• 
Minor response: For patients with a pretreatment platelet count less than 100 000/mm3, a 50% or 
more increase in platelet count with a net increase greater than 10,000/mm3 but less than 30, 
000/mm3. 
 
 
IIIc. Neutrophil response (HI-N) 
• 
Major response: For absolute neutrophil count (ANC) less than 1500/mm3 before therapy, at 
least a 100% increase, or an absolute increase of more than 500/mm3, whichever is greater. 
 
• 
Minor response: For ANC less than 1500/mm3 before therapy, ANC increase of at least 100%, 
but absolute increase less than 500/mm3. 
 
IIId.  Progression/relapse after HI:  
One or more of the following: a 50% or greater decrement from maximum response levels in 
granulocytes or platelets, a reduction in hemoglobin concentration by at least 2 g/dL, or transfusion 
dependence.§ 
 
For a designated response (CR, PR, HI), all relevant response criteria must be noted on at least 2 successive 
determinations at least 1 week apart after an appropriate 
period following therapy (eg, 1 month or longer). 
 
* The presence of mild megaloblastoid changes may be permitted if they are thought to be consistent with 
treatment effect. However, persistence of pretreatment abnormalities (eg, pseudo-Pelger-Hu¨et cells, ringed 
sideroblasts, dysplastic megakaryocytes) are not consistent with CR. 
 
† In some circumstances, protocol therapy may require the initiation of further treatment (eg, consolidation, 
maintenance) before the 2-month period. Such patients can be included in the response category into which they 
fit at the time the therapy is started. 
§ In the absence of another explanation such as acute infection, gastrointestinal bleeding, hemolysis, and so on.