Myeloproliferative Neoplasms - MPN

Key Points

Molecular Genetics of BCR-ABL1 Negative Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) are associated with dysregulation of tyrosine kinases which leads to abnormal downstream signaling of pathways and increased cellular proliferation. The presence of the BCR-ABL1 mutation characterizes chronic myelogenous leukemia. For BCR- ABL1-negative MPNs, new molecular markers have implications for further classifying and diagnosing MPNs. 

Content in tables below based on Chaligné R et al, 2007; Guglielmelli P et al, 2009;  Pardanani AD et al, 2006; Schmidt AE et al, 2012; Vainchenker W et al, 2011; Vakil E et al, 2011.

JAK2

JAK2 (janus kinase 2)
Mutations	Chromosome location – 9p24 JAK2 V617F Prototypic tyrosine kinase mutation   Gain-of-function in exon 14 Exon 12 Next most common mutation site Missense, insertion, or deletion mutations Observed in JAK2 V617F negative clones Occurs less frequently  in essential thrombocythemia (ET) or primary myelofibrosis (PMF)
Mutation Prevalence	JAK2 V617F mutation Polycythemia vera (PV), 90-95%; ET, ~50%; PMF, ~50% Mutations rarely found in non-MPNs such as AML, MDS Mutation is common in the rare MDS/MPN subtype RARS-T (refractory anemia with ring sideroblasts and thrombocytosis) JAK2 exon 12 mutation Present in the majority of patients with PV who are JAK2 V617F-negative, but rarely occurs in PMF, ET
Biology	Cytoplasmic tyrosine kinase activates JAK-STAT pathway leading to cytokine-independent growth
Disease Association	PV ET PMF
Indications	JAK2 V617F Unexplained erythrocytosis, thrombocytosis; bone marrow fibrosis, BCR-ABL1 negative granulocytosis; unexplained monocytosis or splenomegaly, aquagenic pruritus Blood and bone marrow testing are equivalent – performing both is unnecessary JAK2 exon 12 mutation Same symptoms as for JAK2 V617F Subnormal EPO and negative JAK2 V617F testing Quantitative testing May be useful in assessment of residual disease after stem cell transplant Enhances diagnostic certainty in cases with low mutant allelic burden
Clinical Implications	JAK2 inhibitors are in development Decrease in mutant JAK2 V617F allelic burden may not be correlated with clinical response   Utility of monitoring mutant allelic burden is under investigation JAK2 V617F Useful in classifying the process as MPN Not useful in classifying specific MPN subtypes. High mutant allelic burden more likely PV Diagnosis based on clinical factors Lower JAK2 V617F allele burden in primary myelofibrosis (PMF) is associated with worse outcome (Guglielmelli P, 2009)
ARUP Available Tests	JAK2 Gene, V617F Mutation, Qualitative  0051245 JAK2 Gene, V617F Mutation, Quantitation  0040168 JAK2 Exon 12 Mutation Analysis by PCR  2002357 Myeloproliferative Neoplasms Reflex Panel 2007418 (If V617F not detected, test reflexes to JAK2 Exon 12; if JAK2 Exon 12 not detected, test reflexes to MPL codon 515)

MPL

MPL (myeloproliferative leukemia virus oncogene)
Mutations	Chromosome location – 1p34 MPL W515L or MPL W515K  Resides on exon 10 Most common mutation Activates JAK-STAT signaling Observed in JAK2 V617F-negative clones
Mutation Prevalence	Essential thrombocythemia (ET) – 3-4% Primary myelofibrosis (PMF) – 8-10% Polycythemia vera (PV) – rare
Biology	Activating mutation of the thrombopoietin receptor causes activation of downstream signaling Contributes to megakaryocytic myeloproliferation
Disease Association	ET PMF
Indications	Screening is appropriate in JAK2 V617F-negative where suspicion for a MPN is high Examples Thrombocytosis and morphologically equivocal for ET Marrow fibrosis and morphologically equivocal for PMF
Clinical Implications	Diagnostic for  JAK2-negative MPN
ARUP Available Tests	MPL codon 515 Mutation Detection by Pyrosequencing, Quantitative  2005545 Myeloproliferative Neoplasms Reflex Panel 2007418 (If V617F not detected, test reflexes to JAK2 Exon 12; if JAK2 Exon 12 not detected, test reflexes to MPL codon 515)

PDGFRA, PDGFRB, and FGFR1

PDGFRA (platelet derived growth factor receptor, alpha-polypeptide) PDGFRB (platelet derived growth factor receptor, beta-polypeptide) FGFR1 (fibroblast growth receptor 1)
Mutations	Chromosome location 4q12 (PDGFRA) 5q31-33  (PDGFRB)  8p11 (FGFR1)  Translocations cause fusion genes
Mutation Prevalence	FIP1L1-PDGFRA fusion, PDGFRB translocations, or FGFR1 translocations are most common in MPNs associated with eosinophilia FGFR1 also associated with both myeloid and lymphoid neoplasms   PDGFR mutations also found in CMML, atypical CML, and rarely CEL
Biology	PDGFRA and PDGFRB are receptor kinases that regulate cell growth and proliferation FGFR1 promotes proliferation and survival of eosinophils
Disease Association	Eosinophilic disease
Indications	Unexplained persistent eosinophilia Presence of any of these mutations precludes the diagnosis of eosinophilic leukemia or hypereosinophilic syndrome and cases should be assigned to the corresponding molecular-defined categories Cytogenetics may be negative for FIP1L1-PDGFRA Use fluorescence in situ hybridization (FISH) analysis for detection of mutations if high suspicion FGFR1 usually identified by conventional cytogenetics or FISH
Clinical Implications	Tyrosine kinase inhibitors (TKI) sensitivity  (Imatinib) FIP1L1-PDGFRA fusion – sensitive PDGFRB translocation –  sensitive FGFR1 mutation – not enough data to determine Acquired resistance is common; however, Sorafenib may be alternative inhibitor to use
ARUP Available Tests	Myeloproliferative Disorders Panel by FISH 2002360 Eosinophilia Panel by FISH 2002378 Chromosome Analysis, Bone Marrow 2002292

KIT

KIT (v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog or mast/stem cell growth factor receptor)
Mutations	Chromosome location -  4q12 D816V mutation of the tyrosine kinase domain of the KIT gene Rare(< 5%) juxtamembrane KIT mutations
Mutation Prevalence	80% of patients with systemic mastocytosis (KIT D816V mutation)
Biology	KIT (CD117) is a type III tyrosine kinase KIT expression is down-regulated upon differentiation of hematopoietic cells except for mast cells KIT binding to its ligand (stem cell factor) regulates proliferation and maturation
Disease Association	Systemic mastocytosis
Indications	Clinical suspicion for systemic mastocytosis Bone marrow with IHC positive for CD117 and flow cytometry positive for CD117, CD25, and CD2 confirm diagnosis
Clinical Implications	Tyrosine kinase inhibitors (TKI) sensitivity (Imatinib) Sensitive KIT D816V-negative Rare KIT juxtamembrane domain mutations Resistant KIT D816V positive
ARUP Available Tests	Myeloproliferative Disorders Panel by FISH 2002360 Eosinophilia Panel by FISH 2002378 Chromosome Analysis, Bone Marrow 2002292 CD117 (c-Kit) by Immunohistochemistry 2003806 Leukemia/Lymphoma Phenotyping by Flow Cytometry 2008003

Diagnosis

Indications for Testing

• Deep vein thrombosis
• Abnormal CBC – erythrocytosis and/or thrombocytosis

Criteria for Diagnosis

• Polycythemia vera (PV)

  Must exclude secondary causes of erythrocytosis 2008 WHO diagnostic criteria – must meet both major and 1 minor criteria or the first major and 2 minor criteria Major criteria Evidence of increased RBC volume, including ≥1 of the following Hgb>18.5 g/dL (men), >16.5 g/dL (women) Hgb or Hct >99th percentile of reference Elevated red cell mass >25% above mean predicted Hgb>17 g/dL (men), Hgb>15 g/dL (women) if associated with sustained increase ≥2 g/dL not attributed to correction of iron deficiency anemia Presence of JAK2 (V617F) (90-95%) or JAK2 exon 12 mutations (5-10%) Minor criteria Bone marrow trilineage proliferation Subnormal serum erythropoietin level Endogenous erythroid colony growth in vitro

  Essential thrombocythemia (ET)

  Must exclude other causes of thrombocytosis Reactive Anemias Iron deficiency Hemolytic Acute blood loss Post splenectomy Inflammatory disorders WHO criteria 2008 – must meet all 4 criteria Sustained elevation of platelets ≥450x109/L  Defined as at least 2 measurements 2 months apart Bone marrow – megakaryocyte proliferation with little or no granulocyte/erythrocyte proliferation Does not meet WHO criteria for BCR-ABL1 positive chronic myelogenous leukemia, PV, primary myelofibrosis, myelodysplastic syndromes, or other myeloid neoplasm Demonstration of JAK2 (V617F) (50%), MPL gene mutations (eg, W515L and W515K), or no evidence of reactive thrombocytosis Other criteria Bone marrow biopsy Normal to hypercellular with increased megakaryocytes Stainable iron Normal red cell blood mass No significant collagen or reticulin fibrosis

  Primary myelofibrosis (PMF)

  WHO 2008 criteria – must meet 3 major and 2 minor criteria Major criteria Megakaryocyte proliferation and atypia accompanied by reticulin or collagen fibrosis or, in the absence of reticulin fibrosis, megakaryocyte changes must be accompanied by increased marrow cellularity Does not meet WHO criteria for BCR-ABL1 positive chronic myelogenous leukemia, PV, myelodysplastic syndromes, or other myeloid neoplasm JAK2 (V617F) or other clonal markers (eg, MPL W515K/L) or no evidence of reactive fibrosis Minor criteria Leukoerythroblastosis Increased serum lactate dehydrogenase Anemia Splenomegaly

Laboratory Testing

• Initial testing – CBC with differential, erythropoietin level, uric acid, lactate dehydrogenase
• Rule out most common causes of anemia
  • Nutritional – vitamin deficiency (B₁₂, folate, iron)
  • Gastrointestinal blood loss – fecal blood testing
  • Thyroid disease – TSH
  • Chronic disease – BUN, creatinine, liver function
• Follow with JAK2 (V617F) qualitative, JAK2 (V617F) quantitative, or MPL molecular testing
  • Presence of JAK2 mutation virtually rules out reactive PV
    • If JAK2 (V617F) testing negative, JAK2 exon 12 mutation testing indicated
    • If no JAK2 mutations are found and PV is still suspected, perform p50 and VHL mutation gene testing
  • MPL mutation
    • MPL mutation testing can be used in diagnosis of MPN and suggests either ETE or PMF
    • Majority of patients with MPL mutations test negative for JAK2 (V617F) mutation but possess a phenotype consistent with MPN

Histology

• Noneosinophilic MPN
  • Bone marrow examination is generally performed after JAK testing
    • Cytogenetic analysis
• Eosinophilic MPN
  • Molecular testing
    • Myeloid neoplasms associated with eosinophilia and abnormalities in PDGFA, PDGFB, or FGFR1
    • Cytogenetic and fluorescence in situ hybridization analysis for detection of FIP1L1-PDGFA fusion, PDGFB (5q33) translocations, or FGFR1 (8p11) translocations
    • In the absence of these molecular markers, chronic eosinophilic leukemia (CEL), not otherwise specified (NOS) (CEL-NOS), or hypereosinophilic syndrome (HES) is considered
    • Diagnosis in both CEL-NOS and HES requires the following
      • Presence of ≥1.5x10⁹/L eosinophil count – peripheral blood
      • Exclusion of secondary eosinophilia
      • Exclusion of other acute or chronic myeloid neoplasm
      • No evidence for phenotypically abnormal or clonal T lymphocytes
    • Diagnosis of HES requires the absence of both cytogenetic abnormality and ≥2% peripheral blasts or ≥5% bone marrow blasts
  • Mast cell MPNs – CD117 (c-Kit), CD25 testing
• Further testing based on presumed diagnosis

Prognosis

• JAK2 (V617F) – quantitative testing may predict degree of fibrosis, thrombotic tendencies, or survival
• Karyotyping in PMF
  • Unfavorable – complex karyotype or single or two abnormalities, including +8, -7/7q-, i(17q), -5/5q-, 12p-, inv(3), or 11q23

Differential Diagnosis

• Thrombocytosis
  • Primary pulmonary fibrosis and hypertension
  • Malignancy
  • Infection
  • Leukemia
    • Hairy cell leukemia
    • CML
    • Chronic myelomonocytic leukemia
  • Connective tissue diseases
  • Drugs – corticosteroids, adrenaline
  • Hemolysis
  • Hyposplenism
• Anemia
  • Vitamin deficiency – B₁₂, iron, folate
  • Chronic disease – renal, liver
  • Thyroid disease
  • Gastrointestinal bleeding – peptic ulcer disease, malignancy
• Thrombocytopenia
  •  Myelodysplastic syndrome
  • Immune mediated
  • Connective tissue disease
  • Vasculitis
  • Leukemia – AML, ALL
• Thromboses
  • Hereditary thrombophilias
  • Antiphospholipid syndrome
  • Malignancy

Clinical Background

Myeloproliferative neoplasms (MPN) are a group of slow-growing blood cancers, including chronic myelogenous leukemia (CML). MPNs present with clonal proliferation of abnormal hematopoietic cells that involve bone marrow and peripheral blood.

Classification

Selected MPNs

• Polycythemia vera (PV)

  Epidemiology Erythroid dominant trilineage proliferation of hematopoietic precursor cells Incidence – 2.8/100,000 Age – peaks in 50s-60s Sex – M>F (minimal) Clinical Presentation Insidious onset Thrombotic complications Deep vein thrombosis Stroke Myocardial infarction Budd-Chiari syndrome Mesenteric ischemia Transient ischemic attacks Bleeding complications – often associated with acquired von Willebrand disease Epistaxis Oral mucosal hemorrhage Gastrointestinal hemorrhage Ecchymoses Hyperviscosity syndrome – less common Hypertension Headache Dizziness Visual disturbances Claudication Erythromelalgia Redness and burning of palms and plantar areas of feet – sometimes progressing to necrosis of digits Gouty arthritis Pruritus Hepatosplenomegaly Transformation to myelofibrosis (or spent phase) – short survival Transformation to acute leukemia – always fatal without allogeneic bone marrow transplant Treatment Many patients asymptomatic and do not require therapy Hydroxyurea Interferon alpha Phlebotomies – controversial Anagrelide Radioactive phosphorus JAK2 tyrosine kinase inhibitors – experimental Allogeneic bone marrow transplant (preferred nonmyeloablative) in selected instances

  Essential thrombocythemia (ET)

  Epidemiology Thrombocytosis and abnormal megakaryocyte proliferation with clonal proliferation of pluripotent stem cells Incidence – 1.5/100,000 Age – peaks in 50s  Secondary peak in 20s – mainly females; M<F, 1:2 Sex – M:F, equal  Clinical Presentation Arterial thrombosis Brain Cardiac Extremities Venous thrombosis Lower extremity deep vein thrombosis Portal and hepatic vein thrombosis Pulmonary emboli Bleeding complications Epistaxis Oral mucosal hemorrhage Gastrointestinal hemorrhage Ecchymoses Acquired von Willebrand disease Many patients are asymptomatic and do not require therapy Transformation to myelofibrosis (or spent phase) in <10% of cases – short survival Transformation to acute leukemia in <5% of cases – always fatal without allogeneic bone marrow transplantation Treatment  Observation Hydroxyurea Interferon alpha – pegylated form much better tolerated Anagrelide Radioactive phosphorus JAK2 tyrosine kinase inhibitors – experimental Allogeneic marrow transplantation (preferred nonmyeloablative) in selected instances

  Primary myelofibrosis (PMF)

  Epidemiology Clonal stem cell deficit characterized by panmyelosis with intact maturation, progressive bone marrow fibrosis, splenomegaly, multiorgan extramedullary hematopoiesis Incidence – 0.3-1.5/100,000 per year Age – mean is 67 years Sex – M:F, equal Clinical Presentation May be a secondary process in PV and ET Shortest survival of all MPNs Some with insidious onset and stable; others rapidly progressing Acute leukemic transformation common Symptoms, physical and laboratory findings Hypercatabolic state – fever, weight loss, night sweats High output cardiac failure from increased plasma volume Blood abnormality – dacryocytes (tear drops), leukoerythroblastic morphology Other blood abnormalities – anemia, leukocytosis, leukopenia, thrombocytosis, thrombocytopenia, increased circulating blasts Fatigue unrelated to anemia Dyspnea secondary to anemia Petechia secondary to thrombocytopenia Gout Splenomegaly – 100% of patients Hepatomegaly – 50% of patients Treatment Observation Blood transfusions Androgens Erythropoietin Histone deacetylase inhibitors Splenectomy Hydroxyurea JAK2 tyrosine kinase inhibitors – promising in phase II studies Allogeneic bone marrow transplant (preferred nonmyeloablative) in selected instances

Pediatrics

Myeloproliferative neoplasms are extremely rare in children.

Polycythemia vera (PV)

Epidemiology

• Incidence – rare
• Age
  • <0.1% with PV are <20 years
  • Two peaks – 5-6 years, 10-14 years

Clinical Presentation

• Thromboses
  • Arterial – stroke
  • Venous – Budd-Chiari syndrome
• Bleeding disorders
• Splenomegaly common
• Rubor
• Pruritus – especially after hot bath
• Erythromelalgia

Diagnosis

Indications for Testing

• Abnormal CBC

Criteria for Diagnosis

• See Diagnosis tab for PV criteria

Laboratory Testing

• Initial diagnosis – CBC with differential, uric acid, lactate dehydrogenase
  • JAK2 (V617F) testing if suspicion for MPN high
  • Bone marrow examination

Indications for Laboratory Testing

• Tests generally appear in the order most useful for common clinical situations
• Click on number for test-specific information in the ARUP Laboratory Test Directory

Click the plus sign to expand the table of additional tests.

Test Name and Number	Comments
CBC with Platelet Count and Automated Differential 0040003 Method: Automated Cell Count/Differential	Initial screen for MPN
EPOR Mutation Detection by Sequencing 2007914 Method: Polymerase Chain Reaction/Sequencing	Confirm a diagnosis of familial or congenital polycythemia (PFPC)