Transcript Presentation

Hematopoiesis: Part II
 Abnormal hematopoiesis:
-Malignant – Leukemia & Myelodysplasia
-Failure – Aplastic Anemia
-Lymphoma
 Clinical Uses of Hematopoietic Stem Cells
The Hematopoietic Development Tree & Disease
eosinophil
Normal
MYELOID
(CMP)
Acute Leukemia
LTHSC
STHSC
neutrophil
GMP
monocyte
basophil
MPP
MEP
erythrocytes
platelets
Chronic Leukemia
megakaryocyte
LYMPHOID
(CLP)
Stem Cells
Multipotent
Progenitor Cells
Committed
Precursor Cells
T
T lymphocyte
B
B lymphocyte
Mature Cells
Leukemia Classification
Acute
(>20% blasts in marrow)
Chronic
Lymphoid
Acute Lymphoblastic Leukemia
Precursor B-cell ALL
Precursor
T-cell ALL
Rapid progression
Chronic Lymphoid Leukemias
Chronic Lymphocytic Leukemia
Prolymphocytic Leukemia
Slow progression
Hairy Cell Leukemia
Multiple Myeloma
Waldenstrom’s Macroglobulinemia
Myeloid
Acute myeloid leukemia (AML)
• AML recurrent cytogenetic
abnorm
•AML, minimally differentiated
Arrested
maturation
•AML,
without maturation
•AML, with maturation
•Acute promyelocytic leukemia
•Acute myelomonocytic leukemia
•Acute monoblastic leukemia
•Acute erythroid leukemia
Myeloproliferative Disorders
Chronic myeloid leukemia
Polycythemia vera
Essential Thrombocythemia
Full
maturation….
Chronic
idiopathic
myelofibrosis
just too much
of everything
Myelodysplastic
Syndromes
Refractory anemia
Refractory anemia with RS
Refractory anemia with excess blasts
Chronic myelomonocytic leukemia
•Acute megakaryoblastic leukemia
Acute Leukemias
(mature/maturing cells)
Lymphoid
(immature cells)
Chronic Leukemias
CLL
Myeloid
ALL
AML
CML
Leukemia Classification
Acute
Chronic
Lymphoid
Acute Lymphoblastic Leukemia
•
Precursor B-cell ALL
•
Precursor T-cell ALL
Chronic Lymphoid Leukemia
• Chronic Lymphocytic Leukemia
• Prolymphocytic Leukemia
• Hairy Cell Leukemia
• Multiple Myeloma
• Waldenstrom’s Macroglobulinemia
Myeloid
Acute myeloid leukemia (AML)
• AML recurrent cytogen abnorm
•AML, minimally differentiated
•AML, without maturation
•AML, with maturation
•Acute promyelocytic leukemia
•Acute myelomonocytic leukemia
•Acute monoblastic leukemia
•Acute erythroid leukemia
Myeloproliferative Disorders
• Chronic myeloid leukemia (CML)
• Polycythemia vera (PV)
• Essential Thrombocythemia (ET)
• Chronic idiopathic myelofibrosis (MF)
Myelodysplastic Syndromes
• Refractory anemia
• Refractory anemia with RS
• Refractory anemia with excess blasts
• Chronic myelomonocytic leukemia
•Acute megakaryoblastic leukemia
Myelodysplastic Syndromes - myelodysplasia
WHO Classification 2008
Myelodysplastic Syndromes - myelodysplasia
Prognostication
Examples of bone marrow biopsies
Normal
Lymphoma
Hypocellular
lymphoid nodule
Hoffbrand et al Essential Hematology 2001
Aplastic Anemia (AA)
• Failure of the marrow to produce adequate numbers of blood cells in
the absence of malignancy or dysplasia.
• Patients have pancytopenia [anemia, neutropenia, & thrombocytopenia]
AND a hypocellular marrow.
• Disease severity:
Non-severe: Does not meet criteria for severe
Severe:
Has 2 or 3 of the following:
RBC:
retic < 60,000/ml
Neut: < 500/ml
Plt:
<20,000/ml
Very Severe:
Neut: < 200/ml
• Bimodal age distribution:
teens/20’s & > 60
Aplastic Anemia
Table 1. A Classification of Aplastic Anemia:
Acquired (80%):
Primary:
Idiopathic – most often auto-immune
Secondary:
Ionizing radiation – accidental exposure
Chemicals: benzene, DTT,
organophosphates, ecstasy
Rx: chemotherapy agents, psychotropic agents,
anticonvulsants, NSAIDs, chloramphenicol
Viruses: hepatitis – usually nonA, nonB, nonC, nonG
EBV
CMV [in immunosuppressed host]
Paroxysmal Nocturnal Hemoglobinuria (PNH)
Congenital (20%):
Fanconi’s anemia
Dyskeratosis Congenita
Amegakaryocytic Thrombocytopenia
Other rare disorders
Aplastic anemia and telomere length…..
1998
1999
The chromosome end & the telomerase complex
TERT enzymatically adds TTAGGG repeats to the telomere 3’ end using TERC as a template.
Dyskerin NOP10, NHP2, and GAR also bind TERC to stabilize the complex.
Calado, R. T. et al. Blood 2008;111:4446-4455
Telomerase complex mutations and human disease
Green: Acquired Aplastic Anemia
Red: Dyskeratosis Congenita
Black: Pulmonary fibrosis
Blue: Polymorphisms
Calado, R. T. et al. Blood 2008;111:4446-4455
Clinical Consequences of Telomere Erosion
Dyskeratosis Congenita
Nail dystrophy
Hypo pigmentation
Marrow Aplasia
Pulmonary Fibrosis
Oral Leukoplakia
Leukemia & dysplasia
Hepatic Fibrosis
TPO/MPL Signaling and Human Hematopoietic
Development:
•
MPL is the thrombopoietin (TPO) receptor
•
Homodimeric; no other known ligand
•
mpl-/- mice implicate a critical role of TPO/MPL in HSC biology:
•
Inactivating Mpl mutations =
-Congenital Amegakaryocytic Thrombocytopenia (CAMT)
-Progressive aplastic anemia and death very early in life
•
mpl-/- mice live a full life without cytopenias except for
up to a 25-fold reduction in HSCs!
platelets.
The phenotypic difference between CAMT patients and mpl-/mice suggests that mpl-/- mice may be a sub-optimal model for
studying the role of Mpl in human
MPL protein & gene exon structure
: nonsense mutation
hematopoietic stem cell biology.
: missense mutation
S : splice site mutation
: frame shift deletion
Lymphoma – A Disease of Lymphoid Tissues
Non Hodgkin vs Hodgkin (7:1)
NHL - ~95% are B cell
Continuous spread (HD) vs Hematogenous spread (NHL)
The pathologist is everything for making Dx, which
in turn, dictates therapy.
Indolent (not curable) to aggressive (curable)
B-cell NHLs correspond in morphology and
B-Cell
Maturation
immunophenotype
to normal developing B-cells
Plasma cells
Lymphoblast
Small round
lymphocytes
Memory B-cells
TdT
Lymphoblastic
lymphoma
CD5
Large noncleaved
lymphocytes
(centroblasts)
Mantle cell
lymphoma
CD5, CD23
Small lymphocytic
lymphoma
*DLBCL can likely arise from other stages of B-cell diff.
Small noncleaved
lymphocytes
Small cleaved
lymphocytes
(centrocytes)
CD10, BCL6
Burkitt
CD10, BCL6
CD10, BCL6 lymphoma
Diffuse large
Follicular
B-cell
lymphoma
lymphoma*
None of these!
Marginal
zone
lymphoma
P. Treseler, UCSF
Low Grade
(Indolent)
Small Cells
High Grade
(Aggressive)
Histiocyte
Intermediate Cells
Same disease
as ALL!Cells
Large
Lymphoblastic Lymphoma
Small round
Same disease
as CLL!
Small cleaved
Lymphoblast
Large non-cleaved
Diffuse Large
B-Cell
Lymphoma
Burkitt Lymphoma
Small non-cleaved
Immunoblast
•Small lymphocytic lymphoma
•Follicular lymphoma
•Mantle cell lymphoma
•Marginal zone lymphoma
P. Treseler, UCSF
B-Cell Non-Hodgkin Lymphomas
with characteristic translocations
•
•
•
•
•
•
•
Small lymphocytic lymphoma (CLL/SLL)
t(11;14)
Mantle cell lymphoma (MCL)
t(14;18)
Follicular lymphoma (FL)
Marginal Zone Lymphoma (MZL)
t(8;14)
Burkitt lymphoma
Lymphoblastic lymphoma
Diffuse large B-cell lymphoma
P. Treseler, UCSF
Clinical Uses of Hematopoietic Stem Cells
I.
History/Background for HSC transplant
II.
Disorders treated
III.
Types of HSC transplants
IV.
Source of cells for HSC transplant
V.
HLA matching: rejection and more – the immunology of BMT
VI.
Gene Therapy
VII. hESCs - A new HSC source?
VIII. Some pressing questions in the field
History of Hematopoietic Stem Cell Transplantation
A report in 1950 that included classified studies performed in early 1940s by the atomic energy commission.
History of Hematopoietic Stem Cell Transplantation
Surgically mobilized
spleens are
blocked from
irradiation
by lead blocks
J Lab Clin Med 34:1538, 1949
Bone Marrow Protects Against Radiation Injury
Are the
protective effects
cellular or humoral?
J Natl Cancer Inst. 12:197, 1951
Evidence Favoring the Cellular Theory of BM
Protection
DBA/2JN mouse [dark] with BALB/cAnN skin
graft [white] after administration of high dose
radiation (800 r) and DBA/2JN x BALB/cAnN F1
bone marrow. Image at 150 days after skin grafting
J Natl Cancer Inst. 15:1023, 1955
Hematopoietic Stem Cell Transplantation The Start of Human Studies
 Mid 1950s: realized that BM transplant could be
used to treat diseases of the marrow and not just
to provide radiation protection
 Mid 1950s - mid 1960s: Lots of failed attempts
for allo transplant -- interest drops
 1968: First successful transplant to treat an
inherited immunodeficiency using HLA matched sibling
 1975: NEJM paper by Thomas* et al reviewed the
state of BMT, and helped define critical issues in
the field, including HLA matching, infectious
complications, GVHD, etc. and showed clearly that
one could use this therapy to cure people with leukemia.
 1977: Thomas et al report on 100 patients with
end stage leukemia treated with HSC transplantation,
with 13 long term survivors.
From E. Donnall Thomas A Hx of BMT
 1979: Better results if treat earlier, i.e., in first remission.
*E. Donnall Thomas awarded the Nobel Prize in 1990 for BMT
Timeline History for Blood and Marrow Transplantation
2008
~50-60,000/yr
worldwide
Appelbaum NEJM 2007
Diseases Commonly Treated with HSC
Transplantation
AUTOLOGOUS*
Cancers
Multiple Myeloma
Non-Hodgkin’s lymphoma
Hodgkin’s disease
Acute myeloid leukemia
Neuroblastoma
Ovarian cancer
Germ-cell tumors
Other Diseases
Autoimmune disorders
Amyloidosis
*>30,000 annually worldwide;
2/3 for lymphoma and multiple myeloma
ALLOGENEIC**
Cancers
Acute myeloid leukemia
Acute lymphoblastic leukemia
Chronic myeloid leukemia
Chronic lymphocytic leukemia
Myeloproliferative disorders
Lymphoma (Non-HD & HD)
Multiple myeloma
Myelodysplastic syndromes
Other Diseases
Aplastic Anemia
Sickle cell anemia
Thalassemia major
Paroxysmal nocturnal hemoglobinuria
SCIDs
Inborn errors in metabolism
**>15,000 annually; >50% for acute leukemias
Copelan NEJM 2006
Transplant activity worldwide: 1980-2009
35,000
Autologous
30,000
Allogeneic
Transplants
25,000
20,000
15,000
10,000
5,000
0
'80 '81 '82 '83 '84 '85 '86 '87 '88 '89 '90 '91 '92 '93 '94 '95 '96 '97 '98 '99 '00 '01 '02 '03 '04 '05 '06 '07 '08 '09
Year
Transplant activity in the U.S.: 1980-2009
18,000
16,000
Transplants
14,000
Autologous
Related Donor
Unrelated Donor
12,000
10,000
8,000
6,000
4,000
2,000
0
'80 '81 '82 '83 '84 '85 '86 '87 '88 '89 '90 '91 '92 '93 '94 '95 '96 '97 '98 '99 '00 '01 '02 '03 '04 '05 '06 '07 '08 '09
Year
Indications for hematopoietic stem cell
transplant in North America - 2008
5,500
Allogeneic (Total N=6,672)
5,000
Autologous (Total N=10,302)
Number of Transplants
4,500
4,000
3,500
3,000
2,500
2,000
1,500
1,000
500
0
Multiple
Myeloma
NHL
AML
HD
ALL
MDS/MPD Aplastic
Anemia
CML
Other
Leuk
Other
Cancer
NonMalig
Disease
Number of allogeneic transplants, by disease,
registered with CIBMTR - 1998-2008
3,000
AML
ALL
CML
AA
LYM / MM / CLL
Transplants
2,500
2,000
1,500
1,000
500
0
1999
2000
2001
2002
2003
2004
Year
2005
2006
2007
*
2008 *
* Data incomplete
Hematopoietic Stem Cell Transplantation:
Type & Stem Cell Source
1. Autologous - BM (>1,000 ml) or PBSC (~300 ml)
2. Allogeneic - BM or PBSC:
Related
Unrelated - NMDP
3. Umbilical Cord (<100 ml)
Hematopoietic Stem Cell Source for Transplants:
Unrelated Donors
~45%
~15%
The Hematopoietic Stem Cell Circulation
Peripheral
Circulation
Mobilize/Release
Circulate
Retain
Lodge
Home
*NOTE: This just shows the endosteal niche for simplicity, but there is also a perivascular niche.
Wilson & Trumpp 2006
HSC sources for clinical application - Bone Marrow
1.
General anesthesia in operating room
2.
200- 300 entries into posterior
iliac crest yielding >1,000 ml
3.
Overnight stay (or more)
4.
Sore for a while
5.
Net loss of a couple units of blood
6.
Dose: > 2 x 108 nucleated cells/Kg
HSC sources for clinical application:
Peripheral Blood Stem Cells
1.
Mobilization - G-CSF
[CXCR-4 antagonist]
2.
Two IVs & a cell separator
3.
3-4 hours
4.
Cytokine can give flu-like symptoms
5.
Product is ~250-300 ml
6.
“Dose”: 2-5 x 106 CD34+ cells/Kg
HSC sources for clinical application: Cord Blood
1.
No pain
2.
No cytokines
3.
<100 ml
4.
Dose: >2 x 107 nucleated cells
5.
Permits greater HLA variation
6.
A readily available unrelated source
Histocompatibility - Are You a Match?
MHC [Major Histocompatibility Complex] region:
encodes antigens that provoke the strongest transplant rejection
HLA [human leukocyte antigens] genes:
The key genes of the MHC cluster,
Located at p21.3 on chromosome 6.
3 classes - I, II, and III
HLA genes are extremely polymorphic and play a key role in T cell activation
associated with the immune response to foreign tissue.
Class I: >17 loci
Ia = A, B, C, the most important for tissue transplantation
Class II: 9 distinct genes; 5 families DR, DQ, DO, DN, & DP
Key for HSC transplant: DRB1 and DQB1
FOR HSC Transplant: HLA typing of A, B, C, DR, DQ; 10/10 is the best
But clearly more to it than this….
Histocompatibility - Are You a Match?
Unrelated allo donor with 10/10 match
Preventing and treating Graft vs. Host Disease
Currently used drugs to modulate the process
Tacrolimus (aka FK-506): binds immunophilin FKBP-12 (FK506 binding protein)
creating a new complex, FKBP12-FK506, which interacts with and inhibits calcineurin,
thereby inhibiting both T-lymphocyte signal transduction and IL-2 transcription
Mycophenolic acid mafatil (MMF): a noncompetitive, selective and reversible inhibitor
of inosine monophosphate dehydrogenase (IMPDH), a key enzyme for nucleotide
biosynthesis. Unlike other cell types that can use the salvage biosynthesis pathway,
B and T lymphocytes depend on the IMPDH-requiring de novo biosynthesis pathway.
Methotrexate: DNA synthesis inhibitor, an anti-folate, and one of the original
anti-GVHD drugs.
But….some GVHD is good
Probability of Relapse
Probability of Leukemia-free Survival
Better HLA match does not necessarily
mean better outcome
Modern day Allo matching: [10 loci] HLA A, B, C, DR, DQ
Graft vs. leukemia provides part of the cure in allogeneic transplants
Annals of Int. Med 1994
HSC Transplant for Malignant Disorders:
more is not necessarily better
Must overcome two opposing immunologic barriers: GVH and HVG
1.
Intensive conditioning to: (I) immunoablate & (II) eradicate disease
2.
HSC infused to rescue from lethal myeloablation
3.
Immuno-modulate recipient (Rx) post transplant
BUT:
1.
Myeloablation has lots of toxicity and limits patient age
2.
But GVT can be a useful part of GVHD [twins; better outcome w/ some GVHD]
3.
Donor lymphocyte infusion (DLI) -best for CML, CLL, and some lymphomas
SO: Major interest in non-myeloablative conditioning regimens
Gene Marking Studies - The tag sale days
“Tag” autologous cells and allowed one to determine if
Relapses were from infused cells vs. a failure to eradicate
Gene-marking to trace origin of relapse after autologous bone-marrow
transplantation. Brenner MK, et al. Lancet 31:85, 1993.
Pediatric patients with AML; Auto BMT in 1st remission; 2/2 relapses innvoled
the ‘tagged’ cells.
Genetic marking shows that Ph+ cells present in autologous transplants
of CML contribute to relapses after auto BMT in CML.
Deisseroth, et al. Blood 83:3068, 1994.
But…most relapse is due to failure to eradicate disease in host,
and purging autologous samples has not yet proven very useful
Gene Therapy - Correct a Genetic Disease
Gene Therapy of Human Severe Combined Immunodeficiency
(SCID)-X1 Disease Cavazzana-Calvo M. et al. [Science 288:669, 2000]
Sustained Correction of X-linked Severe Combined Immunodefiency
by Ex Vivo Gene Therapy Hacein-Bey-Abina S, et al. [NEJM 346:1185, 2002]
 X-linked SCID = lack of functional
common gamma chain (c) shared with
IL-2, -4, -7, -9, -15, & -21 resulting in
failure to develop B, T, and NK cells
 MLV-based vector, replicationdefective, intact LTRs, uses upstream
LTR to express c; used retrovirus as
supernatant to infect CD34+ cells
CD3=T cells
Gene Therapy - the bubble pops
LMO2-associated clonal T cell proliferation in two patients after
gene therapy for SCID-X1 Hacein-Bey-Abina S, et al. [Science 302:415-9, 2003]
 9/10 children treated are in remission - great
 BUT:
2 developed T-cell leukemia with LMO2-linked retroviral insertions - not great
1 additional patient has an LMO2-associated insertion - not great
What was known about LMO2:
Over-expression in transgenic mice yields T cell leukemias
Blocks T cell differentiation in mice
Acts as a bridging molecule in transcription factor complexes
Expressed in all progenitors and down regulates with differentiation
LMO knockout mice fail to develop any hematopoietic lineage
Retroviruses integrate into promoters and into actively expressed genes
Gene Therapy Insertional Mutagenesis Insights
Dave UP, et al. Science 303:333, 2004 & McCormack et al NEJM 350:913-22, 2004
hESCs as a Therapeutic Source of HSCs?
 Large banks of hESC lines - covers ‘all’ HLA options
 Manipulation of histocompatibility genes in hESC.
 Replacement of immune system of patient with hESC-derived HSC prior
to transplant.
 Manipulation of T cell response with antibodies or drugs.
 Nuclear Transfer involving embryonic stem cells
 Autologous replacement HSCs using induced pleuripotent stem [iPS] cells
Hematopoietic Stem Cell Transplantation:
Areas of Research
 Expanding HS(P)Cs in vitro for therapeutic use
 Understanding where the good cells are
 Purging leukemic/diseased cells from healthy HSCs
 Lots of immunology:
Separating GVHD from GVL effect
Better HLA matching
Treating GVHD
 Improved conditioning Making space - CXCR4 antagonist?
 Novel sources of HSCs
hESCs?
….and that’s all folks!