1. HEMATOPOIETIC STEM CELL TRANSPLANTATION
FOR THE TREATMENT OF PEDIATRIC PATIENTS
WITH
CONGENITAL OR ACQUIRED
HEMATOPOIETIC DISORDERS
OVERVIEW

2. NUMBERS TO PUT THINGS IN CONTEXT
Number of patients diagnosed with cancer in the US – 2007
~30,000/yr potentially treatable with SCT
• Adult Cancer
-Overall 1,445,000
-Prostate 218,000
-Breast 178,000
-Lung 214,000
-Colon 154,000
-Leukemia 44,000
• Pediatric Cancer
-Overall 13,400
-Leukemia 3,400

3. HISTORICAL BACKGROUND
Animal Studies
Early 1950s
High doses of total body irradiation caused
fatal damage to the GI and CNS systems
lower doses resulted in late death from hemorrhage and infection
Marrow from animals genetically identical to treated animals averted death
Marrow from non-identical animals led to an immunologic reaction
methotrexate prevented that reaction
induced tolerance to skin grafts
Cyclophosphamide also permitted engraftment of allogeneic marrow.

4. HISTORICAL BACKGROUND
1939 Osgood Infusion of a few ml of marrow into patients
with aplastic anemia no benefit
1959 Thomas Infusion of large volumes of marrow into
patients with refractory leukemia
one patient with transient engraftment
First paper on BMT in humans
1960’s van Rood Human Leukocyte Antigens (HLA) defined
Dupont Attempts at sibling donor transplants
1968 Good First successful BMT in 2 patients
Bortin (SCID and WAS)

5. THE FIRST 100 ALLO TRANSPLANTS
Fred Hutchinson Cancer Research Center - Seattle

6. WHAT HAPPENED – 1980s to 2007
Progress in the field of transplantation
- Early 1980’s: Autologous and matched sibling SCT
- Mid 1980s GvHD prophylaxis w/CSA and MTX
First T cell depletion techniques
- Late 1980’s: Unrelated Donor Allogeneic SCT
- Mid 1990’s: Mobilized stem cells from peripheral blood
Stem cells from placental cord blood
- Late 1990’s: Mismatched related family donors
Non-myeloablative (mini) transplants

7. ANNUAL NUMBERS OF TRANSPLANTS
WORLDWIDE
1970-2006
40000
35000
30000
autologous
25000
20000
15000
allogeneic
10000
5000
0
1970 1975 1980 1985 1990 1995 2000 2005

8. NUMBER OF TRANSPLANTS AT MSKCC
ADULT ALLO PEDIATRIC ALLO
2002 58 2002 41
2003 60 2003 31
2004 58 2004 37
2005 68 2005 51
2006 97 2006 41
ADULT AUTO PEDIATRIC AUTO
2002 128 2002 31
2003 154 2003 14
2004 127 2004 13
2005 195 2005 7
2006 142 2006 3

9. DEFINITIONS
&
BASIC PRINCIPLES
OF
TRANSPLANTATION

10. AUTOLOGOUS AND ALLOGENEIC STEM CELL TRANSPLANT
PURPOSE
• ALLOGENEIC
– Replace marrow that has a cancer (ex: leukemia) or 1 or more
abnormal hematopoietic lineages (ex: SCID or AA)
– Give a patient allogeneic (immune) T-cells to create a graft-
versus-tumor effect (ex: renal cell carcinoma)
– Donor can be
• Related – syngeneic, HLA matched or mismatched
• Unrelated – HLA matched or mismatched
• AUTOLOGOUS
– Treat a cancer that responds to high dose therapy (dose
intensity) and rescue marrow suppression by giving
autologous stem cells afterwards (ex: neuroblastoma or
lymphoma)
– harvested prior to therapy and frozen

11. BASIC PRINCIPLES OF HEMATOPOIETIC
STEM CELL TRANSPLANTATION

12. HEMATOPOIETIC SCT
Leukemias
Lymphomas Allogeneic Stem cells
Aplastic Anemia
SCID
= Treatment
Other marrow disorders
some solid tumors
Brain Tumors
Retinoblastoma
Breast CA (no longer) Autologous Stem cells
Ovarian CA
Wilms Tumor = Rescue
Lymphomas HD-NHL

13. ALLOGENEIC STEM CELL TRANSPLANTATION
INDICATIONS
Eliminate a defective hematopoietic system
and replace it with a normal/healthy one
Defective hematopoietic system:
Any disease in which the pathology is caused by a
1. A hematologic malignancy
Ex: Leukemias, MDS
2. A defective marrow with multilineage involvement
Ex: AA, Fanconi anemia
3. A defective single hematopoietic cell lineage
Ex: SCID, Hemoglobinopathies

14. INDICATIONS FOR HEMATOPOIETIC SCT
UNITED STATES
2005
5,500
5,000
4,500 Allogeneic (Total N=7,880)
4,000
Transplants
Autologous (Total N=10,840)
3,500
3,000
2,500
2,000
1,500
1,000
500
0
Multiple NHL AML Hodgkin ALL MDS/MPD CML Aplastic Other Other Non-
Myeloma Disease Anemia Leuk Cancer Malig
Disease

15. MARROW, PERIPHERAL BLOOD, AND TISSUE POOLS
MARROW
BLOOD

16. INDICATIONS FOR ALLOGENEIC HEMATOPOIETIC SCT
CML ALL, NHL
AML MDS BMF Syndromes
1. Hematopoietic Malignancies
Leukemias (CML*, AML**, ALL***)
Lymphomas*
Myelodysplastic Syndromes*
2. Bone Marrow Failure Syndromes
- Acquired:
- Aplastic anemia*
- Paroxysmal Nocturnal Hemoglobinuria
- Constitutional:
- Fanconi anemia***
- Dyskeratosis Congenita

17. INDICATIONS OF ALLOGENEIC HEMATOPOIETIC SCT
3. Lineage Specific Defects
Neutrophil –
Kostmann Syndrome (Congenital Agranulocytosis)
CGD, Schwachman Diamond, Leukocyte Adhesion Defect
Chediak Higashi Syndrome
Macrophage
Osteopetrosis, Metabolic Disorders (Gaucher, Hunter, Hurler,
Leukodystrophy)
Lymphocyte
SCID, Wiskott Aldrich Syndrome, Reticular Dysgenesis
Red Cell
Diamond Blackfan Anemia, Thalassemias, Sickle Cell Disease
Platelet
Congenital Amegakaryocytic Thrombocytopenia, TAR
SCA
THAL
CAMT SCID
Osteopetrosis WAS

18. BASIC PRINCIPLES OF HEMATOPOIETIC
STEM CELL TRANSPLANTATION
- Solid Organ Transplant
Donor Host
(Graft) rejection (Patient)
Rejection
T-cells
- Bone Marrow Transplant
Graft-Versus-Host Disease
Donor Host
(Graft) rejection (Patient)
Rejection T-cells
T-cells

19. MAJOR HISTOCOMPATIBILITY COMPLEX
HUMAN LEUCOCYTE ANTIGENS
Graft-Versus-Host Disease
HOST
DONOR HLA
Rejection
• HLA antigens are glycoproteins expressed on the surface of cells
• They are transmitted genetically (chromosome 6)
• They determine the presence or absence of reactions between
T-cells of donor and patient, and therefore, determine the outcome
of allogeneic transplants
• Parallel: HLA antigens are for allogeneic transplants, what ABO
blood types are to transfusion

20. MAJOR HISTOCOMPATIBILITY COMPLEX
HUMAN LEUCOCYTE ANTIGENS
Low A2 B4 DR1 C05 DQB1 01
Resolution A11 B44 DR13 C17 DQB1 03
High A0203 B4101 DRB1 0102 C 0502 DQB1 0101
Resolution A1102 B4402 DRB1 1301 C 1701 DQB1 0301
6 Antigens
10 Antigens

21. MAJOR HISTOCOMPATIBILITY COMPLEX
HUMAN LEUCOCYTE ANTIGENS
Mother Father
a A1 B8 DR7 c A2 B44 DR4
b A3 B7 DR2 d A29 B44 DR7
a a b b
A1 B8 DR7 A1 B8 DR7 A3 B7 DR2 A3 B7 DR2
A2 B44 DR4 A29 B44 DR7 A2 B44 DR4 A29 B44 DR7
c d c d
a
HLA-matched
A1 B8 DR7
Siblings
A2 B44 DR4
c

22. MAJOR HISTOCOMPATIBILITY COMPLEX
HUMAN LEUCOCYTE ANTIGENS
Mother Father
aA01 B08 DR7 A01 B08 DR7 c A02 B44 DR4
b A03 B7 DR2 A02 B44 DR4 d A29 B44 DR7
a A0101 B0803 DR704
c A0202 B4403 DR401
3/6 (haplo) 0/6 3/6
A0104 B0807 DR701 A0104 B0802 DR701 A0101 B0807 DR701
A0202 B4403 DR401 A0201 B4401 DR404 A0201 B4403 DR401
HLA-matched unrelated A0101 B0803 DR704
donor A0202 B4403 DR401

23. A B C DR DQ
Patient a/b 3201 4002 0202 0701 0202
c 0201 5101 1604 1406 0301
Father a 3201 4002 0202 1301 0603
b 2401 4005 0301 0701 0202
1 1 1 1 1
Mother c 0201 5101 1604 1406 0301
d 0101 1302 0608 1101 0301
1 1 1 1 1
Sister b 2401 4005 0301 0701 0202
d 0101 1302 0608 1101 0301
Sister a 3201 4002 0202 1301 0603
c 0201 5101 1604 1406 0301
2 2 2 1 1

24. PRIORITY OF ALLOGENEIC TRANSPLANTS
Patient candidate for allogeneic transplant
Family HLA typing
HLA-matched Sibling No HLA-matched Sibling
(25-30%)
Search for an unrelated donor
(NMDP – Cord Blood Banks)
Transplant
HLA-matched Unrelated Donor No matched
(50-80%) Unrelated Donor
Transplant High Risk Disease Non-High risk Disease
Transplant Transplant No Transplant
Risk (MM Related Donor)

25. HEMATOPOIETIC STEM CELL SOURCES
BONE MARROW
CORD BLOOD
PERIPHERAL BLOOD

26. HEMATOPOIETIC STEM CELL SOURCES
SOURCE OF HEMATOPOIETIC STEM CELLS
Marrow cord blood PBSCs
Average cell dose (TNC/kg) 5 x 108 2 x 106 10 x 108
how harvesting is performed Under GA at birth G-CSF
mobilization
and pheresis
risks/excluded Risk of GA None Hypercoag
quality of states
product SS carriers
cell dose sufficient for Limiting in Higher
conventional or adults
sized matched
can the product be manipulated Yes No Yes
(dose limiting)
risk of GvHD with conventional graft Standard Lower Higher
availability of more cells, cells for CTLs Yes No Yes

27. UNRELATED STEM CELL SOURCES
BY RECIPIENT AGE
1999-2006
100
Bone Marrow (BM)
Bone Marrow (BM)
Peripheral Blood (PB)
Transplants, %
80 Cord Blood (CB) (PB)
Peripheral Blood
Cord Blood (CB)
60
40
20
0
1997-2000 2001-2004 1997-2000 2001-2004
Age ≤20 yrs Age >20 yrs

28. ALLOGENEIC TRANSPLANTS IN PATIENTS <20yo,
REGISTERED WITH THE CMBTR – 1989 -2008
BY DONOR TYPE AND GRAFT SOURCE
5,000
Related
4,500 Unrelated – BM or PB
Unrelated – CB
4,000
Transplants
3,500
3,000
2,500
2,000
1,500
1,000
500
0
1989-90 1991-92 1993-94 1995-96 1997-98 1999-00 2001-02 2003-04 2005-06

29. BASIC PRINCIPLES OF HEMATOPOIETIC
STEM CELL TRANSPLANTATION
CYTOREDUCTION
• In order to perform an allogeneic transplant successfully, we need
to give a cytoreduction prior to the stem cell infusion for:
Immunosuppression
Myeloablation
Anti-leukemic effect
• This is done by using
- Radiation therapy (Total body irradiation – TBI)
and/or
- Chemotherapy (Busulfan, Cyclophosphamide, melphalan,
Thiotepa, fludarabine, …)
• How much of each effect you need varies by the underlying
disease, type of transplant and degree of match

30. BASIC PRINCIPLES OF HEMATOPOIETIC
STEM CELL TRANSPLANTATION
CYTOREDUCTION
1. Myeloablation
2. Immunosuppression Leukemia
3. Anti-leukemia
1. Myeloablation
2. Immunosuppression Aplastic Anemia
3. Anti-leukemia
1. Myeloablation
2. Immunosuppression Hemoglobinopathies
3. Anti-leukemia
1. Myeloablation
2. Immunosuppression SCID
3. Anti-leukemia

31. Stem Cell Transplant Course
Thousands
25
GvHD
20
15
ANC
Platelets
10 BMT
5
0
-14 -10 -7 0 7 14 21 28 90
TBI Cy
Days pre/post SCT
HOST DONOR

33. ACUTE COMPLICATIONS OF ALLOGENEIC SCT
Immunologic
– Graft versus-Host Disease (GvHD) 25-35%
– Graft failure / Rejection < 5%
Infectious 10-20%
– Bacterial / Fungal
– Viral / Parasitic
Organ Toxicity 10%
pneumonitis
veno-occlusive disease
Relapse Variable

34. 100 DAY POST-TRANSPLANT MORTALITY
2001-2006
HLA-identical Sibling Autologous
)
GVHD (13%)
y (7%
ic it
Other
Relapse (41%) tox Infection
(16%) (8%)
Relapse (70%)
Infection Other (13%)
(17%)
Toxicity
(13%)

35. ACUTE GRAFT VERSUS HOST DISEASE
• Immune reaction caused by Donor T-cells in the graft, which are
activated by minor or major HLA-disparities between donor and
host, and attack target organs (skin, GI, liver + BM and immune
system)
• Occurs after engraftment - day 14-28 (prior to day 100)
• Reaction dependent upon HLA-disparities
– HLA mismatch: 3 Ag > 2 Ag > 1 Ag
• Related donor: Mismatch > Match
• Donors: Unrelated > Related

36. Acute
Graft-versus-Host Disease

37. GVHD PROPHYLAXIS
GvHD GvL
• Unmodified stem cell transplant
Transplantation of unmodified stem cells +++ ++
(20-40%)
followed by post transplant immunosuppression
+ -
• T-cell depleted stem cell transplant (5-10%)
Transplantation of stem cells after depletion of T-cells

38. Unrelated Donor SCT – NMDP

39. Lancet 2005 Wagner et al Effect of graft-versus-
host disease prophylaxis on 3-year disease-free
survival in recipients of unrelated donor bone
marrow (T-cell Depletion Trial): a multi-centre,
randomised phase II–III trial

40. Acute GvHD (Grades III – IV) Chronic GvHD

41. Incidence of grade II GvHD 8% (no grade III or IV)
Incidence of chronic GvHD 9%
Jakubowski, A. A. et al. Blood 2007;110:4552-4559

42. GRAFT REJECTION
• Immune reaction caused by Host T-cells which survived the
cytoreductive regimen, and are activated by specific minor or
major HLA-disparities between donor and host. They attack the
donor’s stem cells with subsequent aplasia and pancytopenia
• Occurs early (day 10-20) or late (day 40-60)
• Reaction dependent upon HLA-disparities
• HLA mismatch: 3 Ag > 2 Ag > 1 Ag
• Related donor: Mismatch > Match
• Donors: Unrelated > Related
• Multiply transfused patients (AA, Hemoglobinopathies) at higher
risk because of prior T-cell sensitization

43. INFECTIONS POST TRANSPLANT
• 0-30 days Bacterial* All
Fungal Candida
Viral RSV, HSV
• 1-3 months Fungal* Aspergillus
Viral CMV, Adeno, HHV6
VZV, EBV
Parasitic Toxo, PCP
• 3-6 months Viral CMV, Adeno, HHV6
VZV, EBV
Parasitic Toxo, PCP
*After 30 days, the risk of bacterial and fungal infections persists if GvHD

44. OUTCOMES
OVERALL AND DISEASE SPECIFIC

45. THE FIRST 100 ALLO TRANSPLANTS
Fred Hutchinson Cancer Research Center - Seattle

46. VERY HIGH RISK ALL IN CR1
N=21 – OS 84%

47. ALL IN CR2
Overall
Disease-Free Survival
Overall
Risk of Relapse

48. Papadopoulos E, et al., Blood. 91(3):1083-90, 1998

49. BONE MARROW FAILURE SYNDROMES
APLASTIC ANEMIA
• Disorder of hematopoiesis characterized by severe generalized
reduction of all hematopoietic lineages in the bone marrow and
peripheral pancytopenia.
• Two groups:
– ACQUIRED
Idiopathic or secondary to drugs, infections, chemicals or
irradiation
– CONSTITUTIONAL comprises several genetic disorders
Fanconi Anemia Dyskeratosis Congenita
Schwachman Diamond Osteopetrosis
• Indications for SCT:
All patients

50. Kosaka, Y. et al. Blood 2008;111:1054-1059

51. Kosaka, Y. et al. Blood 2008;111:1054-1059
Copyright ©2008 American Society of Hematology. Copyright restrictions may apply.

52. SEVERE APLASTIC ANEMIA
ALLOGENEIC STEM CELL TRANSPLANTATION
vs
IMMUNOSUPPRESSION TREATMENT
Overall Since 1988
BMT
BMT N=25
OS = 75.6%
IST
IST N=23
OS = 73.8%

53. Impact of fludarabine on survival in patients with FA treated with
unrelated donor BMT. Wagner JE, Eapen M, MacMillan ML, Harris RE,
Pasquini R, Boulad F et al. Blood 2007; 109: 2256–2262.

54. FANCONI ANEMIA
OVERALL
SURVIVAL OS 74% (N=23)
Sept 2005

55. OS 94.4%
Overall Survival
β−thalassemia
MSKCC Experience
N = 20
DFS 91.7%
Disease Free Survival
DFS 44.8%

56. OS 91.7%
Overall Survival
Sickle Cell Anemia
MSKCC Experience
N = 12
DFS 91.7%
Disease Free Survival

57. SCID – OVERALL EXPERIENCE
Since 1973: 94 transplants for SCID
14 (2-4/year) over the past 5 years.
Transplant Type
T cell depleted HLA Haplo-disparate related donors
SBA-E- bone marrow 69
CD34+E- PBSC 4
Unmodified HLA matched and partially mismatched
bone marrow grafts 21
Cytoreduction
Matched sibling none
Haplo-disparate TCD 37/69
Basis for cytoreduction
NK function

58. SURVIVAL BY TIME TO DIAGNOSIS OF SCID
1
MREL <3 months = 100% n=8
0.9
0.8
0.7
MREL >3 months = 72.73% n=11
0.6
Proportion
0.5
0.4
0.3
0.2
0.1
0
0 10 20 30 40 50 60
Time Post Transplant (months)

59. LATE COMPLICATIONS OF ALLOGENEIC SCT
Immunologic
– Chronic Graft versus-Host Disease (GvHD)
– Infections
Long term organ toxicity
- Endocrine: Growth, Gonadal, Thyroid
- CNS - Cardiac - Pulmonary - Hepatic
- Renal - Other
Secondary Malignancies
- Solid Tumors
Psychological Late effects

60. CHRONIC GVHD

61. WHAT’S NEW AND EXCITING
NON-MYELOABLATIVE TRANSPLANTS
(MINI- TRANSPLANT)
ADOPTIVE CELL THERAPY
Use of broad unselected or selected donor lymphocytes
prophylaxis or treatment of infections or disease
Use of NK cells
VACCINES
of donor (in the pre-transplant period) or host (in the post-transplant period)
GENE THERAPY
for inborn errors
especially those where a selective advantage may exist for corrected cells

62. NON-MYELOABLATIVE TRANSPLANTS
Principle:
•avoid use of high dose therapy
•decreases toxicity in older or heavily pre-treated
•high doses of donor T-cells
•Cytoreduction limited to immuno-ablation
•Advantages:
•Transplants less toxic
•Graft versus leukemia (or tumor)
•Dis-advantages
•higher incidence of GvHD – especially chronic
•takes time to establish full donor chimerism

63. CONVENTIONAL CYTOREDUCTION
Graft-Versus-Host Disease
Donor Host
(Graft) Rejection
(Patient)
T-cells T-cells
Bone Marrow Bone Marrow
NON-MYELOABLATIVE CYTOREDUCTION
Graft-Versus-Host Disease
Donor Host
(Graft) Rejection (Patient)
T-cells T-cells
Bone Marrow Bone Marrow

64. ADOPTIVE CELL THERAPY – T CELLS
Donor Patient
Donor Lymphocytes (T-cells)
Ex-vivo expanded
Antigen-specific T-cells
T-cells containing
HSV-TK suicide gene
Infection (EBV, CMV)
Leukemia (CML)

65. ADOPTIVE CELL THERAPY – NK CELLS
• Adoptive transfer of NK cells to enhance
engraftment and leukemia resistance.
• Selection of KIR*-mismatched HLA-compatible
donors conferring NK-mediated leukemia
resistance
*KIR = Killer-cell Immunoglobulin-like Receptor

66. GENE THERAPY
Insertion of a normal gene into deficient stem cell
Already trials for X-linked SCID, ADA-SCID, CGD
Other target genes already in or close to trials:
- ADA-deficient SCID
- Thalassemia / Sickle cell disease
- Wiskott Aldrich Syndrome
- Fanconi Anemia
- Hemophilia

67. THALASSEMIA
Gene Therapy
E A
N D
C G-CSF
R M Infusion
a
O I Autologous
t
L S Hematopoietic
h
L S
M
e
I
Busulfan Stem Cells
t Transduced
E O
Pre-SCT e Pre-SCT
Screening N N with THALAGEN
Work-up1 r Work-up 2
Work-up T
PBSCT
Cytotherapy Lab
CD34 selection
Gene Therapy Lab
Lentiviral mediated human β Globin Gene transfer

68. STEM CELL TRANSPLANTATION
PROGRESS IN THE LAST 30 YEARS
2000’s
1977