Update of Molecular Genetics of Myeloproliferative Neoplasms Kathleen Finnegan MS MT(ASCP)SHCM Stony Brook University Kathleen.finnegan@stonybrook.edu

Objectives Describe the classification and characteristics of the MPNs Discuss the molecular mutations associated with the MPNs and their role in the disease process. Discuss the molecular mutations for clinical management and better understanding of treatment

Case Study HISTORY A 49 year old male police officer was rushed to the Emergency Room at University Hospital after a bad motorcycle accident. Upon examination an enlarge spleen was noted along with many bruises and a possible broken arm and leg.

Laboratory Results WBC 386.5 RBC 3.98 HGB 11.8 HCT N/A MCV N/A MCH N/A MCHC N/A RDW N/A PLT 303,000 MPV 7.2

Additional Laboratory Tests Bone Marrow Hypercellular marrow M:E Ratio: 25: 1 BCR/ABL 1: Positive

Acute vs Chronic Leukemia

Myeloproliferative Neoplasms MPNs Increase in RBC’s, WBC’s and platelets Characterized by excess proliferation of one or more normal cells Overproduction or deregulation of one or more blood cell lines Difficult classifying due to overlap of clinical presentations Bone marrow can be hypercellular and often becoming fibrotic Tend to transform into an Acute Leukemia Includes CML, PM, PV, and ET

WHO Classification 2008 Chronic Myelogenous Leukemia BCR-ABL 1 Positive Chronic Neutrophilic Leukemia (CNL) Polycythemia Vera (PV) Primary Myelofibrosis (PM) Essential Thrombocythemia (ET) Chronic Eosinophilic Leukemia (CEL) Mastocytosis Cutaneous Mastocytosis Systemic Mastocytosis Mast Cell Leukemia Mast Cell Sarcoma Extracutaneous Mastocytoma Myeloproliferative Neoplasm, Unclassifiable

WHO Classification Revision Last update in 2008 Looking at unique Biomarkers Gene expression analysis Next gene sequencing Significance: Improve diagnostic criteria Prognostic relevance of entities Suggest new entities Incorporate new clinical, prognostic, morphologic, immunophenotypic, and genetic data

Myeloproliferative Revisions Mastocytosis no longer considered a subgroup Mastocytosis is now a separate disease category CML suggest a bone marrow aspirate to be performed to confirm the phase of the disease Accelerated phase of CML now includes hematologic, morphologic and cytogenetic parameters Blast phase CML requires at least 20% blasts Standardize morphologic criteria of the MPN’s The 2016 revision to the WHO classification of myeloid neoplasms and acute leukemia: Blood, May 19, 2016, Volume 127, Number 20

WHO Classification 2016 Chronic Myelogenous Leukemia BCR-ABL 1 Positive Chronic Neutrophilic Leukemia (CNL) Polycythemia Vera (PV) Primary Myelofibrosis (PM) Prefibrotic Overt Fibrotic Essential Thrombocythemia (ET) Chronic Eosinophilic Leukemia not otherwise specified (NOS) Mastocytosis Myeloproliferative Neoplasm, Unclassifiable

MPNs General Characteristics: Patients usually over the age of 40 Hypercellularity of the bone marrow Disease advances slower Exrtramedullary hematopoiesis Bizarre RBC morphology Platelets usually elevated

Chronic Myelocytic Leukemia CML Clonal stem cell disorder Marked leukocytosis with all stages of granulocytic maturation BCR/ABL 1 Fusion Gene Ph’ positive in 90% - 95% of the cases Hepatosplenomegaly Chronic phase Accelerated / Blast phase

Groups of CML CML Common: Juvenile: Chronic Neutrophilic: BCR/ABL 1, Ph +, < 2% blasts Juvenile: < 5 yrs old, > 10% blasts, resistant to therapy, very large spleen, BCR/ABL 1 negative, PH - Chronic Neutrophilic: Ph absent, rare, usually adults, more bands than other Atypical CML: Features of CML and MDS Ph absent, no basophilia, less favorable prognosis

CML Laboratory Findings WBC count ranges from 100,000 - 200,000 N/N Anemia Increased platelets, giant and fragments Differential: complete spectrum < 2% myeloblasts Absolute basophilia Increase Eos and Baso Bone Marrow: hypercellular 25:1 Philadelphia Chromosome t(9:22)(arm of 22 is translocated to 9) positive in 90 - 95% of the cases BCR/ABL 1 Fusion Mutation

Three Phases of CML Initial Phase or Chronic: 2 - 3 years, highly treatable Accelerated or transitional: rising peripheral WBC count with increase of the blast, 6 - 18 months, resistant to treatment Blast Crisis: about 3/4 of the patients enter this phase, 1- 20% blasts, 3 - 4 months, unresponsive to treatment Looks like M2 2 - 6 years from diagnosis

BCR – ABL 1 Fusion Gene BCR: Breakpoint Cluster Region DNA fragment localized on chromosome 22 Defines the chromosomal break DNA fragment localized on chromosome 9 ABL: protein possessing increased tyrosine kinase activity Abnormal chromosome forms a fusion gene

BCR – ABL Gene Chromosome 9 q34 ABL Gene Ia II XI 1b 20 1 Chromosome 22 q11 1 2 3 4 BCR Gene Ia II 1 XI BCR/ABL Fusion Gene BCR/ABL Fusion Gene p210 Ph + Chromosome BCR ABL

BCR-ABL 1 Fusion gene produces a protein that dysregulates tyrosine kinase activity Mutation activates signal transduction pathways that increase cell proliferation and decrease cell apoptosis Increase in tyrosine kinase activity drives deregulated growth More seen in the chronic phase

Philadelphia Chromosome Long arm of chromosome 22 is translocated to the distal end of the long arm of chromosome 9 A small part of chromosome 9 is reciprocally translocated to the broken end of 22

CML Morphology Complete spectrum of myeloid cells Segs and the myelocyte are more numerous Basophilia Blast 1- 3%

Treatment Gleevec Tyrosine kinase inhibitor (TKI) Imatinub mesylate Tyrosine kinase inhibitor (TKI) Targets the fusion gene and inhibits the proliferation of abnormal cells Molecular targeted drug

Polycythemia General term used to describe erythrocytosis Increase in both hemoglobin and hematocrit Increase in red cell mass and total blood volume Accelerated erythropoiesis

Classification of Polycythemia Polycythemia Vera or Primary Polycythemia Secondary Polycythemia or Absolute Erythrocytosis Relative Polycythemia or Stress Erythrocytosis

Polycythemia Vera Hematological stem cell disorder Panmyelosis Accelerated erythropoiesis Increase in all three cell lines Cell maturation, function and life span normal Absolute increase in red cell mass Normal O2 saturation Enlarged spleen Decreased serum erythropoietin

Clinical Findings Onset is gradual Patients complain of headaches, vertigo, ringing in the ears, and blurred vision Skin has a ruddy appearance Feeling of being full due to the enlarged spleen Thrombotic events Cardiovascular disease

Laboratory Findings Elevated RBC, WBC, and platelet Stainable iron lacking in most cases Decreased serum erythropoietin BRC-ABL 1 Negative >95% exhibit the JAK 2 Mutation

JAK2V617F Mutation Valine to a phenylalanine at condon 617 Janus kinases are non receptors of tyrosine kinase which regulate the phosphorlation of several signaling pathways Mutation results in a “gain of function” Resulting in elevated granulocyte-macrophage stimulation factor receptor Proliferation of myeloid cells Eight distinct mutations in exon 12 of JAK 2 have been associated with hematopoietic cell independent growth Current information on disease – specific prognostic relevance of JAK2V617F is inconclusive Drug Target Therapy

JH7 JH6 JH5 JH4 JH3 JH2 JH1 Exon 12 Mutation V617F JAK2 V617F Mutation Cytokine Receptor Binding Pseudokinase Domain JH7 JH6 JH5 JH4 JH3 JH2 JH1 Kinase Domain Exon 12 Mutation V617F JAK2 V617F Mutation

Polycythemia Vera Bone marrow: hypercellular Hypervolemic or Hyperviscosity 80 – 95% exhibit the mutation 15% progress to Acute Leukemia

PV Diagnosis Major Category Minor Category 1. Hgb >16.5 g/dl (M) Hgb>16.o g/dl (F) or Hematocrit >49% in men Hematocrit > 48% in women or Elevated RBC mass > 25% or above normal predicted value 2. BM showing hypercelularity to include erythroid, granulocytic, and megakaryocytic proliferation 3. Presence of JAK2V617F or JAK2 exon 12 mutation Minor Category Subnormal serum erythropoietin level Diagnosis of PV requires meeting all 3 major criteria or the first 2 and the minor

Treatment Therapeutic phlebotomy Repeated phlebotomy tend to diminish iron stores Iron Deficiency Anemia Maintain a HCT below 45% in males, 40% in females Aspirin to reduce thrombosis Cytoreduction pharmaceuticals Hydroxyurea Interferon

Secondary Polycythemia This reflects an increase in erythropoietin production Results from tissue hypoxia: chronic lung disease, heavy smokers or tumors of the kidney Physiologic response to a perceived need for RBC’s

Laboratory Findings WBC and platelet normal RBC mass increased O2 Saturation decreased JAK 2 Negative Bone Marrow: erythroid hyperplasia only

Relative Polycythemia Mild polycythemia Caused by dehydration or excess H2O loss Red cell mass is normal but plasma volume is decreased JAK 2 Negative Bone Marrow: normal cellularity

Primary Myelofibrosis Leukoerythroblastosis Granulocytic hyperplasia in the bone marrow Elevated platelets Fibrosis of the marrow Extramedullary hematopoiesis Peripheral blood shows tear drops, megakaryocyte fragments Enlarged spleen and liver 1st finding

Laboratory Findings Increase WBC’s 30,000 - 100,000 Immature granulocytes: whole spectrum, increase of NRBC’s Platelet overproduction and defective: large in size and megakaryocyte fragments N/N anemia BCR-ABL 1 Negative 50% exhibit JAK 2 mutation

Primary Myelofibrosis Most serious of the BCR-ABL negative MPNs As disease progresses the marrow becomes hypocellular and replaced by fibrotic marrow Limited production of normal cells Median Survival 4 - 5 years 10 - 15% convert to AML Patients with the JAK 2 mutation have a greater risk of transforming into acute leukemia

Laboratory Findings Bone Marrow: Usually a dry tap with collagen deposition found Reticulin stain (Silver stain) for fibrosis

Pre PMF Major Criteria 1. Megakaryocytic proliferation without reticulum 2. Patient does not meeting WHO criteria for CML, PV or MDS or other myeloid 3. Presence of the JAK 2 mutation, CALR or MPL mutation

Pre PMF Minor Criteria Anemia Leukocytosis > 11,000 Palpable Splenomegaly LD increased Diagnosis of pre PMF requires all three criteria and I minor

Overt PMF Major Criteria 1. Megakaryocytic proliferation accompanied by reticulum and /or collagen or 2. Patient does not meeting WHO criteria for CML, PV or MDS or other myeloid 3. Presence of the JAK 2 mutation, CALR or MPL mutation

Overt PMF Minor Criteria Anemia Leukocytosis > 11,000 Palpable Splenomegaly LD increased Leukoerythroblastos Diagnosis of pre PMF requires all three criteria and I minor

Essential Thrombocythemia ET 1. Platelet count >450,000 Often over 1,000,000 2. BM biopsy showing marked thrombocytosis Variable platelet size and megakaryocyte fragments 3. Patient does not meeting WHO criteria for CML, PV or MDS or other myeloid 4. Presence of the JAK 2 mutation, CALR or MPL mutation Minor Presence or absence of reactive thrombocytosis Diagnosis of ET requires meeting all 4 major or 3 and I minor

ET Clinical Findings 50% patients asymptomatic Thrombotic or Bleeding Problems Neurological manifestations due to obstruction of cerebral microvasculature Enlarged spleen in about half of the patients Transition to AML of myelofibrosis Survival 10 years Death usually from thrombosis

Differential Diagnosis Platelet count > 450,000 Rule out relative thrombocytosis Increase bone marrow megakaryocytes Absent BCR-ABL gene Normal RBC mass Absence of significant marrow fibrosis Demonstrate Jak-2 40 – 50%

Treatment Therapy to reduce the platelet count Cytoreductive agents Hydroxyurea Current research in developing JAK 2 inhibitors

Myeloproliferative Neoplasms

Other MPN’s Chronic Neutrophilic Leukemia (CNL) Clonal disorder with a hyperproliferation of neutrophilic cells Must be differentiated from CML based on the absence of the BRC/ABL 1 fusion gene and Philadelphia chromosome Peripheral blood neutrophilia Segmented neutrophils and bands Neutrophils often contain toxic granulation Myeloblasts not observed in PB Bone marrow hypercellular RBC’s and Platelets are usually normal CSF3R mutation

CNL Diagnosis WBC count must be greater than 25,000 with greater than 90% mature neutrophils Neutrophilia precusors Must rule out a reactive neutrophilia Hypercellular marrow No evidence of the BCR/ABL mutations or rearrangements of PDGFRA, PDGFRB, or FGRF1 genes. No evidence of PV, PMF, ET, MDS or MDS/MPN disorders A slow smoldering condition Presence of CSF3R mutation

Other MPN’s Chronic Eosinophilic Leukemia Not Otherwise Specified (CEL/NOS) Clonial proliferations of eosinophils and precursors Eosinophil count greater than 1.5 x 109/L Eosinophils are found in heart, lungs and CNS < 20% blasts in the bone marrow Eosinophils appear normal Survival is variable Increase in dysplasia and the blast count are an unfavorable prognosis

Other MPN’s Mastocytosis Clonal neoplastic proliferation of mast cells with the accumulation in one or more organ systems Can occur at any age Cutaneous most of the cases are children Systemic occurs in the second decade of life Presence of multifocal clusters of abnormal mast cells Usually present with skin lesions 80% cases

Diagnosis Skin lesion is the first diagnostic clue Minor Criteria More than 25% of the mast cells must be immature or atypical morphology Mast cells must express a KIT mutation Mast cells must express normal markers and CD2 0r/and CD25 Total serum tryptase must be above 20 mg/dl.

KIT Codon 816 This mutation replaces aspartic acid with valine Ligand-independent activation of KIT tyrosine kinase Alters tyrosine kinase receptor activity Resistant to TK inhibitor Usually stomatic Occurs 95% in adults with systemic mastocytosis 33% in children with systemic mastocytosis

Myeloproliferative Neoplasm Unclassified Express myeloproliferative features but fail to meet the criteria of a specific condition Have overlapping features Patients with early stage PV, ET, of PMF where criteria not fully developed Patients who have clear evidence of an MPN but have a second neoplasm Account for 10 – 15%

Genetic Abnormality Disease Association Molecular Pathogenesis BCR-ABL1 t(9;22) CML Deregulation of ABL tyrosine kinase, effects cell proliferation, apoptosis, adhesion JACK 2 Mutation 9p 24 MPN’s excluding CML Point mutation V617F results in loss of auto-inhibition and cons MPL 1p34 11% PMF 4% ET Encodes the thrombopoietin receptor and interacts with JAK2. LNK 12q13,3-qter 20% seen in patients in the blast phase of the MPN’s Usually not seen in chronic phase Negatively regulates JAK 2 and JAK 2 cytokine signaling TET2 4q24 16% PV 5% ET 17% PMF Epigenetic dysregulation of transcription

Genetic Abnormality Disease Association Molecular Pathogenesis IDH1/IDH2 2q32-qter/15q21-qter 2% PV 1% ET 4% PMF Enzymes that play a role in the citric acid cycle KIT 95% Adult SM 33% Children SM Ligand-independent activation of KIT tyrosine kinase PML-RARA t(15;17) APL Interference with myeloid maturation and differentiation TEL-AML1 t(12;21) Childhood B- Precursor Fusion protein, disruption of the regulatory pathway FLT3 Mutations AML Point mutation activity of cell proliferation MLL 11q23 AML/ALL Gene rearrangement involved in the epigenetic maintenance of transcriptional memory

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