1. Update of Molecular Genetics of Myeloproliferative Neoplasms
Kathleen Finnegan MS MT(ASCP)SHCM
Stony Brook University

2. 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

3. 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.

4. 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

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

8. Acute vs Chronic Leukemia

9. 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

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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

17. CML Laboratory Findings
WBC count ranges from 100, ,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 % of the cases
BCR/ABL 1 Fusion Mutation

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

19. 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

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

21. 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

22. 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

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

24. 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

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

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

27. 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

28. 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

29. 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

30. 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

31. 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

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

33. 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

34. 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

35. 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

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

37. 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

38. 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

39. 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

40. 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 years
% convert to AML
Patients with the JAK 2 mutation have a greater risk of transforming into acute leukemia

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

42. 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

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

44. 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

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

46. 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

47. 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

48. 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%

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

50. Myeloproliferative Neoplasms

51. 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

52. 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

53. 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

54. 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

55. 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.

56. 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

57. 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%

58. 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

59. 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

60. Thank You