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Title: An Ongoing Story of Discovery: Pathophysiology of Chronic Myeloproliferative Disorders


1
An Ongoing Story of Discovery Pathophysiology
of Chronic Myeloproliferative Disorders
  • Katy Moran MD
  • August 30, 2005

2
  • Imagination is more important than knowledge,
    for knowledge is limited while imagination
    embraces the entire world.
  • Albert Einstein

3
  • First, some cases. . .

4
  • 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

5
Tefferi, A. N Engl J Med 20003421255-1265
6
Disease Characteristics Transformation
CML Genetic translocation Philadelphia chromosome t(922) resulting in fusion of bcr-abl oncogene gt90 will transform to acute leukemia if untreated
Polycythemia vera Elevated red cell mass, hypercellular marrow, independent of erythropoietin 10 myelofibrosis _at_10 yrs 25 myelofibrosis _at_ 25 yrs
Essential thrombocytosis Clonal or autonomous thrombocytosis lt5 will transform to acute leukemia
Agnogenic myeloid metaplasia (Chronic idiopathic myelofibrosis) Bone marrow fibrosis not associated with CML or MDS Mean survival lt5 yrs
Atypical Atypical CML, chronic neutrophilic leukemia, systemic mast cell disease, chronic eosinophilic leukemia Variable
7
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

8
  • 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 possiblenot 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.

9
Dameshek W. Some Speculations on the
Myeloproliferative Syndromes. Blood 1951.
Adaptation from Table 1
Syndrome Erythroblasts Granulocyte Mega-karyocytes Fibroblasts Spleen and liver
Chronic Granulocytic Leukemia (CML) /- to
PV to to to
Agnogenic Myeloid Metaplasia /- /- to
Mega-karyocytic Leukemia /- /- to
? Myelostimulatory Factor (s) ?
10
  • 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

11
  • In the middle of difficulty lies opportunity.
  • Albert Einstein

12
  • 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 DAndrea Cloning of EPO receptor
  • No recognizable intracellular signals/pathway
    compared with other known receptors such as
    insulin

13
  • 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, GM-CSF, IGF-1
  • ? Downstream effect

14
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

15
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16
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

17
  • 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)

18
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19
  • 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 gtAmino acid ?
    valine to phenylalanine
  • Termed V617F

20
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 20053521744-1746

21
Schwartz, R. N Engl J Med 2002347462-463
22
  • 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.

23
Mechanism of Loss of Heterozygosity at Chromosome
9p
24
  • What are the implications of this mutation among
    the chronic myeloproliferative disorders?

25
Study Purpose PV ET MF
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. Focused on the key role of JAK2 in signal transduction from multiple hematopoietic growth factor receptors 97 N73 57 N51 50 N16
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). DNA sequence analysis of activation loops and autoinhibitory domains of 85 tyrosine kinases 74 N164 32 N115 35 N46
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). Endogenous erythroid colonies inhibitors 88 N45 small small
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. Observed patients with PV had loss of heterozygosity in chromosome 9p that included the site of the JAK2 gene 65 N128 23 N93 57 N23
Carriers of the mutation had more complications
such as fibrosis, hemorrhage, and thrombosis and
were more likely to receive cytoreductive therapy.
26
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 Hyper-eosinophilic 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)
27
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

28
  • 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.

29
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30
  • 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. . .

31
  • If the facts don't fit the theory, change the
    facts.
  • Albert Einstein

32
  • 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
33
  • 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

34
  • 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

35
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

36
  • If we knew what we were doing, it wouldn't be
    called research, would it?
  • Albert Einstein

37
References
  • Tefferi, A. N Engl J Med 20003421255-1265
  • Dameshek W. Some Speculations on the
    Myeloproliferative Syndromes. Blood 1951.
  • Goldman, J. M. N Engl J Med 20053521744-1746
  • Schwartz, R. N Engl J Med 2002347462-463
  • 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.
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