Transcript ppt

An Ongoing Story of Discovery:
Pathophysiology of Chronic
Myeloproliferative Disorders
Katy Moran MD
August 30, 2005
“Imagination is more important than
knowledge, for knowledge is limited
while imagination embraces the entire
world.”
Albert Einstein
First,
some cases. . .

Case #1: 63 yo woman presents to clinic with increasing abdominal girth,
physical – hepatosplenomegaly. CBC reveals a Hct 52% and Platelet count
900,000 cells/mm3. Diagnosis?

Polycythemia vera

Case #2: 46 yo man presents to clinic with painful unilateral swelling of the
right lower extremity for 48 hours. No known risk factors for DVT,
ultrasound reveals femoral vein DVT. CBC reveals platelet count 1,200,000
cells/mm3. Diagnosis?

Essential thrombocytosis

Case #3: 65 yo man of Jewish ancestry presents with fatigue, low grade
fever. Mild pancytopenia and teardrop-shaped rbcs are noted on blood
smear. Bone marrow biopsy shows atypical megakaryocytes and stromal
stranding. Diagnosis?

Agnogenic Myeloid Metaplasia ≈ Idiopathic Myelofibrosis

Case #4: 55 yo man presents with complaints of generalized fatigue,
weight loss and abdominal discomfort with early satiety. Physical exam –
afebrile, thin, massive splenomegaly. No adenopathy is identified, liver is
normal in size. CBC reveals neutrophilic leukocytosis. Diagnosis?

Chronic myelogenous leukemia
Tefferi, A. N Engl J Med 2000;342:1255-1265
Disease
Characteristics
Transformation
CML
Genetic translocation
Philadelphia chromosome
t(9;22) resulting in fusion
of bcr-abl oncogene
>90% will transform to
acute leukemia if
untreated
Polycythemia vera
Elevated red cell mass,
hypercellular marrow,
independent of
erythropoietin
10% myelofibrosis @10
yrs
25% myelofibrosis @ 25
yrs
Essential thrombocytosis
Clonal or autonomous
thrombocytosis
<5% will transform to
acute leukemia
Agnogenic myeloid
metaplasia
(Chronic idiopathic
myelofibrosis)
Bone marrow fibrosis not
associated with CML or
MDS
Mean survival <5 yrs
Atypical
Atypical CML, chronic
neutrophilic leukemia,
systemic mast cell
disease, chronic
eosinophilic leukemia
Variable
Chronic Myeloproliferative Disorders

Common features:






Overproduction of one or more formed elements in
the blood in the absence of an obvious stimulus
Clonal disorders arising in a single, multipotent
progenitor or stem cell  proliferates  dominates
the marrow and blood
Extramedullary hematopoiesis
Hypercellular marrow
Hyperplastic megakaryocytes  myelofibrosis
Clinical tendency toward thrombotic and hemorrhagic
complications

1892 Louis Vasquez of Paris described a pt with cyanotic
polycythemia, autopsy massive enlargement liver and spleen

1903 William Osler at Johns Hopkins reported four patients with
polycythemia, two with splenomegaly


Osler-Vasquez disease ( polycythemia vera)
1951 William Dameshek writes an article in Blood grouping PV,
idiopathic myelofibrosis, ET, CML, and ‘erythroleukemia’ into a
general category termed myeloproliferative disorders

“Perhaps it is possible…not that the various conditions listed are
different, but that they are closely interrelated. It is possible that these
various conditions – “myeloproliferative disorders”-are all somewhat
variable manifestations of proliferative activity of the bone marrow cells,
perhaps due to a hitherto undiscovered stimulus.”
Dameshek W. Some Speculations on the Myeloproliferative Syndromes. Blood 1951. Adaptation from Table 1:
Syndrome
Erythroblasts

Chronic
Granulocytic
Leukemia
(CML)
+/-
PV
+++
Agnogenic
Myeloid
Metaplasia
Megakaryocytic
Leukemia
Granulocyte
Megakaryocytes
Myelostimulatory Factor (s)
+++
+
Fibroblasts
Spleen and
liver

+
++
+++
++
+
+
to
to
to
+++
+++
+++
+
+++
+++
to
+/-
++
+/-
to
+/-
+/-
+++
+++
+
+
to
+++
 “Myelostimulatory


Factor”
Highly potent since it causes not only normal
bone marrow to become highly proliferative
but also causes activation of sites embryonic
or potential hematopoeisis such as spleen
and liver
Theorized of a hormonal or steroid type of
factor
“In the middle of difficulty lies
opportunity.”
Albert Einstein

1974 NEJM Prchal and Axelrad demonstrate that in
patients with PV erythroid progenitor cells from marrow
or peripheral blood proliferate in serum-containing
culture in the absence of exogenous erythropoietin
termed “Endogenous Erythroid Colony” formation

1977 J Clin Invest Zanjani shows this phenomenon really
is hypersensitivity to erythropoietin in the culture serum
rather than a erythropoietin independent respose

1989 Cell D’Andrea – Cloning of EPO receptor
 No recognizable intracellular signals/pathway
compared with other known receptors such as insulin

1989 Research continues on a new class of receptors, called type I
cytokine receptors

GM-CSF, multiple interleukin receptors, and others are identified

Mechanism via novel kinase/signal transduction pathway

1992 Cell Valezquez describe this novel pathway as JAK
receptor/signal transducer and activator of transcription (STAT)

JAK – “Just another kinase”

Janus kinase – named for Roman god of gates and passages

Studies in 1992-1994 demonstrate hypersensitivity of PV erythroid
progenitor cells with a variety of growth factors such as IL-3, GMCSF, IGF-1

? Downstream effect
Tyrosine Kinases
 Enzymes
that catalyze transfer of
phosphate from ATP to tyrosine residues
in polypeptides
 2 Classes


Receptor TK – Transmembrane Protein with
extracellular domain
Nonreceptor TK – Intracellular - found in
cytosol, nucleus
Janus Kinase Protein

Kinase domain (JH1)+ catalytically inactive
pseudokinase domain (JH2) which acts as a
regulator
 Intermediate between membrane receptors and
signaling molecules
 Cytoplasmic region of a membrane receptor –
when receptor is activated (for example a
cytokine binds) JAK is phosphorylated and
activated initiating signalling cascade via the
STAT molecules

STAT molecules enter the nucleus  transcription
 Four


members of JAK family
JAK 1
JAK 2
• Activated particularly when receptor binds to
hematopoietic growth factors, including
erythropoietin, GM-CSF, G-CSF, and
thrombopoietin


JAK 3
TYK 2 (tyrosine kinase 2)
 Region
of JH2 interacts with the activation
loop of the kinase domain. A specific site
mutation in the JH2 domain results in
constitutive kinase activity of JH1
 Mutation
has been mapped to position 617
on the pseudokinase domain


Guanine to thiamine substitution –>Amino
acid Δ valine to phenylalanine
Termed V617F
Addition of
pseudokinase
JH2 domain
greatly reduces
the level of
autoactivation

Expression of an
isolated JAK-2 JH1
kinase domain
leads to its
constitutive activity
Goldman, J. M. N Engl J Med 2005;352:1744-1746

Schwartz, R. N Engl J Med 2002;347:462-463
 Mutation

found only in hematopoietic cells
Acquired somatic mutation
• Present in DNA from granulocytes but absent in T
cells

Mechanism for loss of heterozygosity at
chromosome 9p
• Deletion of telomeric part of wild-type chromosome
9p
• Events during mitotic recombination
Kralovics R, Passamonti F, Buser AS, et al. A gain-of-function mutation of JAK2 in
myeloproliferative disorders. N Engl J Med. 2005; 352: 1779-1790.
Mechanism of Loss of
Heterozygosity at Chromosome 9p
What
are the implications of
this mutation among the
chronic myeloproliferative
disorders?
Study
Cambridge, UK
Baxter EJ, Scott LM, Campbell PJ,
et al. Acquired mutation of the
tyrosine kinase JAK2 in human
myeloproliferative diseases. Lancet.
2005; 365: 1054-1061.
Boston
Levine RL, Wadleigh M, Cools J, et
al. Activating mutation in the
tyrosine kinase JAK2 in
polycythemia vera, essential
thrombocytosis, and myeloid
metaplasia with myelofibrosis.
Cancer Cell (in press).
Paris
James C, Ugo V, Le Couedic J-P, et
al. A unique clonal JAK2 mutation
leading to constitutive signalling
causes polycythemia vera. Nature
(in press).
Switzerland-Italy
Kralovics R, Passamonti F, Buser
AS, et al. A gain-of-function
mutation of JAK2 in
myeloproliferative disorders. N Engl
J Med. 2005; 352: 1779-1790.
Purpose
PV
ET
MF
Focused on the key role of
JAK2 in signal transduction
from multiple hematopoietic
growth factor receptors
97%
57%
50%
N=73
N=51
N=16
DNA sequence analysis of
activation loops and
autoinhibitory domains of 85
tyrosine kinases
74%
32%
35%
Endogenous erythroid
colonies inhibitors
88%
N=164 N=115 N=46
small
small
N=45
Observed patients with PV
65%
23%
57%
had loss of heterozygosity in
chromosome 9p that
included the site of the JAK2 N=128 N=93
N=23
gene * Carriers of the mutation had more complications such as fibrosis, hemorrhage, and
thrombosis and were more likely to receive cytoreductive therapy.
Adaptation from Table 1: Jones A, et al. Widespread occurrence of the JAK2
V617F mutation in chronic myeloproliferative disorders. Blood 2005 (in press).
Disease
Subtype
N
V617F
Positive
number (%)
V617F
Negative
number (%)
V617F
homozygotes
number (% of
mutants)
PV
72
58 (81%)
14 (19%)
24 (41%)
ET
59
24 (41%)
35 (59%)
4 (17%)
IMF
35
15 (43%)
20 (67%)
10 (67%)
Idiopathic
Hypereosinophilic
syndrome
134
2 (1.5%)
132 (99%)
2 (100%)
Mastocytosis
28
0
-
-
CML-like
MPDs
99
17 (17%)
82 (93%)
8 (47%)
Unclassified
MPD
53
12 (25%)
40 (75%)
7 (54%)
Total
480
129 (27%)
351 (73%)
55 (43%)
Further evidence of V617 mutation
contribution to CMPDs



Introduction of mutant clone into irradiated mice led to
substantial erythrocytosis
Erythroid progenitor cells carrying the mutation were
able grow in the absence of exogenous erythropoietin
Homozygosity


Arise from recombination of chromatids during mitosis rather
than a second mutation the mutant heterozygous line
Loss of heterozygosity results in a proliferative advantage
• Individuals with one mutant and one wild type gene have reduced
cellular autonomous JAK2 activity and growth factor independent
behavior compared with homozygous individuals
James C, Ugo V, Le Couedic J-P, et al. A unique clonal JAK2 mutation leading to constitutive signalling causes
polycythemia vera. Nature (in press).
Baxter EJ, Scott LM, Campbell PJ, et al. Acquired mutation of the tyrosine kinase JAK2 in human
myeloproliferative diseases. Lancet. 2005; 365: 1054-1061

Duration of disease was significantly longer
among homozygotes compared to
heterozygotes
 Patients testing negative for the mutation had
the shortest duration of disease



Homozygous – mean 48 months
Heterozygous – mean 23 months
Wild type – mean 15 months

Phenotype may be expressed without the
mutation
 Suggests acquiring the mutation and then
homozygosity are likely stepwise processes
Kralovics R, Passamonti F, Buser AS, et al. A gain-of-function mutation of JAK2 in myeloproliferative
disorders. N Engl J Med. 2005; 352: 1779-1790.
 In
patients that are found to be positive for
this mutation by genetic testing, diagnostic
and possibly prognostic information may
be obtained
 Specific
therapeutic target at the level of
the mutant kinase
 More
questions. . .
“If the facts don't fit the theory, change
the facts.”
Albert Einstein

How does one mutation give rise to these various
disorders?



Additional genetic alterations? Pre-existing or acquired after the
JAK2?
Dependent on the subtype of progenitor cell in which the
mutation first arises?
What is the mechanism for disease in patients who do
not carry the V617 mutation?



Some answers may lie in further exploration of genes that are
activated by STAT (signal transducer and activator of
transcription) cascade
Recently, members of the JAK and STAT families have been
implicated in cellular decisions on whether to proliferate or enter
apoptosis
One family of genes called suppressor of cytokine signaling
(SOCS) encode proteins that bind to JAKs and receptor sites
and then BLOCK further signaling
Receptor  JAK  STAT SOCS  Programmed blockade of further JAK signals
 Why
do some patients progress from
indolent CMPDs such as PV to acute
leukemia?
 Rational


approach to therapy?
Tyrosine kinases as potential targets
Broad spectrum of malignancy mediated via
this family of proteins
• Examples: Fms-like tyrosine kinase 3 (FLT3) in
acute myeloid leukemia, epidermal growth factor
receptor in subset NSCLC, c-KIT mutation in GIST

JAKs mediate intracellular signaling in other
pathways and diseases






Leptin receptor
Growth hormone receptor
Interleukin receptors
Cardiovascular signaling systems
Inherited JAK3 deficiency has been implicated in
cases of severe combined immunodeficiency
Developing inhibitors that act specifically on
V617F without causing side effects in other
signaling systems may be challenging
Summary

Advances in the field of molecular/cell biology
and specifically describing JAK2 have provided
a valuable window into the mechanism of
chronic myeloproliferative diseases including PV,
ET, and IMF among others
 This information has diagnostic and prognostic
clinical relevance
 Tyrosine kinases are vital proteins which have
broad implications
 Ongoing research in this field will impact how
medicine is practiced for years to come
“If we knew what we were doing, it
wouldn't be called research, would
it?”
Albert Einstein
References
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Tefferi, A. N Engl J Med 2000;342:1255-1265
Dameshek W. Some Speculations on the Myeloproliferative Syndromes. Blood 1951.
Goldman, J. M. N Engl J Med 2005;352:1744-1746
Schwartz, R. N Engl J Med 2002;347:462-463
Baxter EJ, Scott LM, Campbell PJ, et al. Acquired mutation of the tyrosine kinase JAK2 in human
myeloproliferative diseases. Lancet. 2005; 365: 1054-1061.
Levine RL, Wadleigh M, Cools J, et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera,
essential thrombocytosis, and myeloid metaplasia with myelofibrosis. Cancer Cell (in press).
James C, Ugo V, Le Couedic J-P, et al. A unique clonal JAK2 mutation leading to constitutive signalling causes
polycythemia vera. Nature (in press).
Kralovics R, Passamonti F, Buser AS, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N
Engl J Med. 2005; 352: 1779-1790.
Jones A, et al. Widespread occurrence of the JAK2 V617F mutation in chronic myeloproliferative disorders. Blood
2005
MKSAP Review Hematology and Oncology
Up to Date
Krause, DS, Etten RA. Tyrosine Kinases as Targets for Cancer Therapy. N. Engl J Med. 2005; 353: 172-187.
Tefferi A, Gilliland DG. The JAK2 Tyrosine Kinase Mutation in MPD: Status report. Mayo Clin. Proc. July 2005:
80 (7): 947-958.
Kaushansky K. On the molecular origins of the chronic myeloproliferative disorders: it all makes sense. Blood.
June 2005. 105: 4187-4190.