Myeloproliferative disorders Clonal hematopoiesis (stem cell disorder) Marrow hypercellularity Overproduction of one or more cell lines (effective hematopoiesis) Exception: myelofibrosis Differentiation nearly normal

The “common” bcr-abl negative myeloproliferative disorders Polycythemia vera Essential thrombocythemia Myelofibrosis/myeloid metaplasia

Less common conditions Undifferentiated myeloproliferative disorder Mast cell disease Hypereosinophilic syndrome Chronic neutrophilic leukemia Myelodysplastic/myeloproliferative disorders CMML Atypical CML (BCR-ABL negative) Juvenile CML “Unclassifiable” MDS/MPD

JAK-2 Tyrosine kinase involved in signaling pathways initiated by EPO, TPO, G-CSF and other growth factors V617F mutations in most cases of PV, ET, MF Other JAK-2 mutations (exon 12, etc) in a minority of PV cases (almost all cases of PV have a JAK-2 mutation of some type) Some cases of ET, MF lack JAK-2 mutation Mutation releases cells from dependence on growth factors Homozygosity for mutation seen mainly in P vera, associated with disease progression Specific inhibitors of the kinase now in clinical trials

Structure-function relationships in the JAK2 receptor Science 2014; 344:703

In Panel A, in the absence of ligand, the erythropoietin receptor (EPOR) binds JAK2 as an inactive dimer. In cells with wild-type JAK2 protein, the binding of erythropoietin (Epo) to its receptor induces conformational changes in the receptor, resulting in phosphorylation (P) of JAK2 and the cytoplasmic tail of the receptor. This leads to signaling through pathways made up of Janus kinases and signal transducers and activators of transcription (JAK–STAT), phosphatidylinositol 3 kinase (PI3K), and RAS and mitogen-activated protein kinase (RAS–MAPK). In cells with the V617F mutation, the signaling is constitutively increased, even in the absence of erythropoietin. In Panel B, the JAK2 protein binds to multiple cytokine receptors — EPOR, thrombopoietin receptor (MPL), granulocyte colony-stimulating factor receptor (G-CSFR), and probably others — that are important for hematopoietic stem-cell biology and differentiation. Therefore, the JAK2 protein with the V617F mutation exerts its effects at various stages of differentiation and in various lineages. In Panel C, the development of homozygosity for the V617F mutation is a two-step process, with the initial point mutation followed by mitotic recombination of chromosome 9p between the JAK2 locus and the centromere. This results in the loss of heterozygosity but a diploid DNA copy number. NEJM 2006; 355:2452

NEJM 2006; 355:2452

Polycythemia vera Elevated RBC mass, typically high platelets and WBC Some patients present with thrombocytosis and develop erythrocytosis subsequently Hypercellular marrow with variable degree of reticulin fibrosis Morphology fairly normal, some clustering and dysmorphism of megas Splenomegaly in 70%, constitutional sx, increased risk of arterial & venous thrombosis (esp portal vein), microvascular disease (erythromelalgia, pruritus, headache, etc), hypermetabolic sx & gout

Differential diagnosis of erythrocytosis H&P COPD or other possible causes of hypoxemia? Smoker? Splenomegaly, pruritus, erythromelalgia? Concomitant thrombocytosis and/or leukocytosis? Serum EPO JAK-2 mutation testing

Polycythemia Vera Natural History Life expectancy decreased (3 deaths/100 pts/yr) Cardiovascular mortality increased 1.4x Major thrombosis in 3/100pts/yr Risk of death from leukemia increased 36-fold Progressive myelofibrosis

NEJM 2004; 350:99

NEJM 2004; 350:99

Oxygen delivery vs Hematocrit J Clin Invest 1963;42:1150

Vaso-occlusion in P vera

Thrombosis in myeloproliferative disorders 41% of deaths in PV from cardiovascular causes 15% coronary dz 8% CHF 8% non hemorrhagic stroke 8% PE Complex pathophysiology Abnormal RBC/WBC/plt function Activated PMNs/cytokines affect vascular endothelium Prothrombotic microparticle release Increased whole blood viscosity

Risk factors for thrombosis in myeloproliferative disorders Hx of thrombosis Disease category (PV > ET, MF) Cardiovascular risk factors (lipids, blood pressure, smoking, diabetes) Age OCP therapy (splanchnic vein clots) JAK2 mutation status/allele burden High hematocrit (PV) & WBC Extreme thrombocytosis increases bleeding risk Thrombophilic genetic traits?

Prothrombotic effects of erythrocytosis Barbui et al. Blood 2013;122:2176-2184

Hematology 2005:201

Recommendations for treatment of patients with polycythemia vera Keep Hct < 45 (plebotomy) Low dose ASA unless contraindicated Manage reversible risk factors (BP, lipids, smoking) Consider cytoreduction if: Intolerant to phlebotomy Thrombocytosis or substantial leukocytosis Symptomatic splenomegaly Choice of cytoreductive therapy: Age < 40: Interferon-alpha Age > 40: hydroxyurea Anagrelide or busulfan for patients intolerant of, or not responsive to, above choices Hematology 2005:201

Aspirin vs placebo in P vera

Essential thrombocytosis Thrombocytosis (typically > 600K) with variable leukocytosis, normal RBC count About 50% have JAK2 mutation Normal to mildly hypercellular marrow with marked increase in megakaryocytes, often with clustering Minimal reticulin fibrosis Splenomegaly in about 50% Many patients asymptomatic at diagnosis Increased risk of arterial and venous thrombosis as well as hemorrhage

Thrombocytosis Differential diagnosis Essential thrombocythemia Other MPD P vera Myelofibrosis CML Myelodysplasia (5q- et al) Reactive Inflammation Surgery/trauma Non-myeloid malignancy Iron deficiency Hemolysis Acute blood loss Absence of spleen Rebound following thrombocytopenia

Hematology 2005:201

Essential Thrombocytosis Natural History 1-2 cases/100,000/yr Most patients >50, but occurs in young adults Woman more often affected than men Life expectancy normal to slightly decreased Patients > 60, or with prior thrombosis, at increased risk for thrombotic events Very high platelet counts (>1.5 million) increase risk of bleeding, not thrombosis Risk of myelofibrosis about 8% at 10 years Risk of AML about 1% overall

Essential Thrombocytosis Treatment Low-risk patients (young, no vascular risk factors) may not require treatment Aspirin decreases thrombotic events Hydroxyurea Anagrelide Interferon Other: busulfan (leukemogenic)

Recommendations for treatment of patients with essential thrombocytosis Manage reversible risk factors (BP, lipids, smoking) If hx of thrombosis, or age >60, or plts > 1.5 million: Hydroxyurea Low dose ASA unless for plts > 1.5 m 2nd line: Anagrelide or interferon-alpha Age < 60, no other risk factors: Low dose ASA Hematology 2005:201

Myelofibrosis 0.5-1.5 cases/100,000/yr Most patients >60 Marrow fibrosis with extramedullary hematopoiesis in spleen, liver, many other tissues “Prefibrotic” stage with hypercellular marrow & abnormal megakaryocytes Peripheral leukoerythroblastosis, teardrop cells, often with anemia. WBC and platelet counts may be high, normal or low Splenomegaly, constitutional symptoms Poor prognosis: marrow failure, AML in 5-30%

Improving survival in MF Median survival 6.5 yrs Median survival 4.5 yrs J Clin Oncol 2012;30:2981

DDx of Marrow Fibrosis Myeloproliferative disorders MF > > P vera, ET, CML Other heme neoplasm Megakaryocytic leukemia Hodgkins Hairy cell leukemia Non-heme cancer Non-malignant conditions Renal osteodystrophy Autoimmune disease Vitamin D deficiency

SPLENOMEGALY IN MYELOFIBROSIS Mayo Clin Proc 2004;79:503 SPLENOMEGALY IN MYELOFIBROSIS

Myelofibrosis Treatment Supportive care Transfusions Splenectomy Thalidomide/lenalidomide (low dose) Reduce transfusion requirements, reduce spleen size Chemotherapy (limited data; myelosuppression often dose-limiting) Cladribine Azacytidine/decitabine Low dose cytarabine Marrow transplant in selected patients JAK inhibitor therapy: Ruxolitinib (inhibits JAK-1 and JAK-2)

Ruxolitinib treatment of myelofibrosis Spleens shrink Symptoms improve Better survival NEJM 2012;366:799

Eosinophilia Non-clonal/secondary (common) Clonal Idiopathic hypereosinophilic syndrome Familial (very rare) Mild: AEC 500-1500 Moderate: AEC 1500-5000 Severe: AEC > 5000

Hypereosinophilia (AEC > 5000) Parasitic infection Drug reaction Allergic gastroenteritis Eosinophilic fasciitis Churg-Strauss syndrome Pulmonary eosinophilia Eosinophilia-myalgia syndrome IL-2 therapy Hyper-IgE syndrome AML Eosinophilic leukemia Hypereosinophilic syndrome

Clinical manifestations of hypereosinophilia Skin: pruritus, angioedema, ulcers, papular or nodular lesions Heart: endocardial fibrosis, valvular disease, mural thrombi, cardiomyopathy, incr troponin Lungs: infiltrates, nodules, pleural effusion Neurologic: poly- or mononeuropathy, transverse myelitis, optic neuritis, CNS vasculitis GI: hepatosplenomegaly, gastroenteritis, sclerosing cholangitis Heme: marrow fibrosis, cytopenias Kidney: thrombotic microangiopathy

Secondary Eosinophilia Infection Usually parasitic Allergy Asthma, allergic rhinitis, dermatitis, urticaria/angioedema, drug reaction Autoimmune/inflammatory disorders (many) Paraneoplastic Solid tumors, lymphomas (Hodgkin>NHL) Endocrinopathy Adrenal insufficiency, growth hormone deficiency Clonal T-cell disorder (→ IL-5) Most cases with IL-5 overproduction

Clonal Eosinophilia Acute leukemia (AML>ALL) Chronic myeloid disorders CML (bcr-abl) Systemic mastocytosis (c-Kit) 8p11 myeloproliferative disorder (FGFR-1) PDGFRA and PDGFRB mutations “Classic” MPD (JAK-2) MDS Chronic eosinophilic leukemia

Clonal eosinophilic disorders caused by mutations in tyrosine kinase genes PDGFRA, PDGFRB May be cytogenetically silent→ FISH testing for dx Marrow fibrosis, incr mast cells, elevated tryptase → variant of mast cell dz? Male predominance Imatinib-responsive (100-400 mg/d) FGFR1 Associated with 8p11 translocations Stem cell disorder → aggressive MPD associated with T-lymphoblastic lymphoma High dose chemotherapy + allo-HSCT

Chronic eosinophilic leukemia WHO diagnostic criteria Eosinophil count > 1500 No Ph chromosome or BCR-ABL fusion No PDGFRB, PDGFRA or FGFR1 rearrangement Blast count in blood and marrow < 20% No inv (16) or other variant characteristic of AML Presence of clonal cytogenetic or molecular abnormality OR blasts >2% in blood or >5% in marrow

Idiopathic Hypereosinophilic Syndrome Persistent moderate or severe eosinophilia (AEC > 1500) with end-organ damage Other causes of eosinophilia ruled out Male predominance Can evolve into overt myeloid or lymphoid neoplasm Potentially fatal – 10 yr survival < 50% Treatment Asymptomatic: corticosteroids vs watchful waiting Symptomatic patients: corticosteroids, IFNα, hydroxyurea, imatinib, various myelosuppressive agents

Laboratory evaluation of hypereosinophilia Marrow biopsy with cytogenetics FISH for PDGFR mutations Serum tryptase T-cell immunophenotyping and gene rearrangement study Serum IL-5 Serum IgE Screen for organ damage Echo, troponin level CXR, PFTs

Classification of Mast Cell Disease WHO classification and frequency in Mayo Clinic series Indolent – often limited to skin (urticaria pigmentosa (46%) Associated with myeloproliferative disorder (40%) “Aggressive” mastocytosis – with lymphadenopathy & eosinophilia (12%) Mast cell leukemia (1%) Blood 2009;113:5727

WHO Diagnostic Criteria for Mast Cell Disease Major: multifocal dense infiltrates of mast cells (15+) in marrow or other extracutaneous organs Minor >25% spindle shaped or otherwise atypical MC c-KIT codon 816 mutation MC in marrow or other extracutaneous organ express CD2 and/or CD25 Serum tryptase persistently >20 ng/ml in the absence of another clonal myeloid disorder Diagnosis requires major + 1 minor criterion or at least 3 minor criteria

Stuff in mast cell granules that could make you sick Metcalfe, Blood 2008;112:946

Clinical & laboratory features of mast cell disease Skin: Urticaria pigmentosa, Darier sign (dermatographism), telangiectasias Lymphadenopathy Hepatosplenomegaly Constitutional symptoms Recurrent anaphylaxis GI symptoms (N/V, diarrhea, abd pain, peptic ulcer) Elevated serum/urinary histamine levels Elevated serum tryptase (96%) Cytopenias Eosinophilia Coagulopathy (heparin in mast cells) – rare Lytic or blastic bone lesions

Blood 2009; 113: 5727 ISM: Indolent MCD SM-ANMD: associated with clonal non-mast cell lineage disease ASM: Aggressive MCD MCL: Mast cell leukemia

Mastocytosis in bone marrow H&E Tryptase

Mastocytosis in bone marrow

Skin findings in mast cell disease Dermatographism Urticaria pigmentosa Telangiectasia macularis eruptiva perstans

Molecular biology of mast cell disease A majority of patients have D816V mutation in c-KIT tyrosine kinase gene (imatinib-insensitive) A minority have other mutations, may be imatinib-sensitive Occasionally found together with JAK2 V617F Some patients have eosinophilia & PDGFRA mutation (imatinib-responsive)

Treatment of mast cell disease Antihistamines, PPIs Vit D/Calcium, bisphosphonates Glucocorticoids Epinephrine (for anaphyactic reactions) For aggressive disease: Interferon Imatinib (if there is an imatinib-sensitive mutation) Other TKIs (dasatinib, nilotinib)? Cladribine Combination chemotheapy Allo-HSCT

Survival of patients with MCD Blood 2009; 113: 5727