1. Myeloproliferative Disoreders, Myelodysplastic, AML and Lymphomas: Recent Advances. Dr A S Akanmu Professor Of Haematology & Blood Transfusion. College Of Medicine University of Lagos

2. The Basics of Recent Advances Advancement In Cell Biology Particularly Cell Membrane Proteins Advancement In Diagnostic Tools ▫Cytogenetic  The banding technology  Gene Localisation by FISH ▫Molecular genetic  Detection of RNA Transcript by RT-PCR  Mutation detection by Sequencing  Real Time PCR for quantitation RNA transcript ▫Availability of monoclonal antibodies to detect cell membrane and cytoplasmic expression of proteins

3. Advances In Diagnosis of the Myeloproliferative Disorders CML PPP Myelofibrosis Essential Thrombocythaemia

4. The Basic Pathology

5. Flourescent Insitu Hybridisation Localising BCR-ABL1 on Chromosome22 THE NORMAL BCR-ABL FUSION GENE

6. Types of BCR/ABL1 Fusion Protein

7. The Pathway To Neoplasia

8. Morphology: Still Important Low Power High Power Trephine Chronic phase high Point: Myelocyte Metamyelocyte Bulge

9. Small Hypolobated Megakarryocytes are Common Findings In CML

10. Diagnosing Accelerating CML Blasts 10-19% of WBC in blood or bone marrow Basophilia >20% Platelet ▫Persistent Thrombocytopaenia <100x10 9/ L. unrelated to therapy ▫Thrombocytosis >1000x10 9 /L unresponsive to therapy Increasing splenic size Increasing WBC unresponsive to therapy Cytogenetic evidence of clonal evolution ▫A second philadelphia chromosome ▫Trisomy 8 ▫Isochromosome 17q

11. Diagnosing Blast Phase CML Blasts ≥20% of WBC in blood or nucleated bone marrow cells ▫60-70% Myeloid ▫30-40% Lymphoid Extramedullary blast cell proliferation Large foci or cluster of blasts in the bone marrow biopsy

12. Blastic Transformation Myeloid Blasts Lymphoid Blasts

13. Typing Blastic Phase CML PhenotypeMyeloid BlastLymphoid Blast Philadelphia ChromosomePos Inv(16)(p13.1q22)Pos/NegNeg Myeloid Antigens: MPOPosNeg CD13PosNeg CD33PosNeg CD117PosNeg Precursor B-Cell Phenotype: PAX5NegPos CD10NegPos CD19NegPos TdTNegPos

14. Polycythemia Vera Study Group Diagnostic Criteria: Significance and Proposed Alternatives/Additions M1. Identifies actual polycythemia vs. spurious polycythaemia. ▫Increased RCM  Male >36 ml/kg  Female >32 ml/kg Or  RCM >125% of predicted M2. Rules out most common aetiology of secondary polycythemia ▫Arterial O2 saturation>92% Or ▫Serum erythropoietin concentration not elevated M3. Evidence of a myeloproliferative state ▫Clinical splenomegaly or two of the following:

15. Polycythemia Vera Study Group: Minor Criteria m1-Thrombocytosis >400,000/ μl m2-Leukocytosis >12,000/ μl m3-Leukocyte alkaline phosphatase activity >100 (no fever or infection) m4-Serum vit. B12 (>900 pg/ml) or unsaturated B 12 binding capacity (>2200 pg/ml) The diagnosis of PV requires the presence of: ▫all three major criteria (M) or ▫the first two major criteria and two minor criteria (m).

16. Polycythaemia: Normal Activation of EPOR CFU-E and Erythroid blasts are rich in Erythropoietin receptors. EPOR EPOR has activation and regulatory domains EPO bind to the activation domain ▫Dimerisation of EPOR results ▫This activates JAK2 Haematopoietic cell phosphatase binds to regulatory domain to cause ▫dephosphorylation of JAK2 Mutations in EPOR regulatory domain causes hypersensitivity to EPO ▫Results in congenital Polycythaemia.

17. Polycythaemia: Gain of Function mutation JAK2 -A tyrosine kinase protein ▫A point mutation on chr9 1849 G T ▫V617F where valine is replaced by phenylalanie It occurs at a negative regulatory domain of the protein resulting in ▫Constitutive activation of the protein Found in 90-95% of PV patients 80% of JAK2 V617F negative patients HAVE Jak2 Mutation on exon 12 of JAK2 gene 99% of pts therefore have JAK2 mutation

19. WHO 2008 Major Criteria M1 ▫Hb >18g/dl in men or 16.5g/dl in women Or Hb and or Ht > 99 th percentile reference range for age/sex/altitude ▫Hb >17g/dl and 15g/dl in M and F respectively if associated with 2g/dl increase baseline not attributable to correction of iron deficiency. ▫ Red cell mass>25% above mean predicted value M2 ▫JAK2 Mutation Or ▫JAK2 Exon 12 Mutation

20. WHO 2008 Minor Criteria m1- Bone marrow biopsy showing Panmyelosis: ▫Erythroid, granulocytic and megakaryocytic proliferation m2-Serum Erythropoietin below the reference range of normal m3-Endogenous erythroid colony formation in vitro

21. JAK2 Positive Monocytes May Mimic Gauchers Disease

22. Diagnosing Myelofibrosis

23. Leucoerythroblastosis

24. Aggregates of platelets

25. The atypical megakarryocytes

26. Cluster of atypical Megakarryocytes and Patchy Fibrosis

27. Reticulosis to fibrosis to osteonecrosis Silver Stain Showing Reticulin Fiber Fibrosis with Osteonecrosis

28. Major Clinical and Lab Features PMF FindingsPrefibrotic StageFibrotic Stage ClinicalNone Or Mild Splenomenomegaly Mod spleno/hepato megaly HaematologyMild/Mod anaemiaMod/Marked anaemia Mild/Mod LeucopaeniaWBC L or N or Increase Mild/Mod thromboPlate L or N or Increase Blood SmearN/mild Leucoerythroblastosis Leucoerythroblastosis Poikilocytes N/mildTear-drop cells

29. Major Clinical and Lab Features PMF FindingsPrefibrotic StageFibrotic Stage Bone MarrowHypercellularDecreased Cellularity Neut Proliferation Megakaryo Proliferation Megakarryocytic atypia Clustering Nuclear lobulation Abn Bare megakaryo nuclei Reticulin Normal/mild Reticulin/collagen fibrosis New bone formation Dilated snuses.

30. The Neoplastic Cell A marrow stem cell with JAK2 V617F mutation in 50% And Thrombopoietin Receptor gene (MPL gene) mutation in a small number of cases MPL mutation is W515K/L where ▫Tryptophan is replaced by Lysine or leucine at position 515 of the TPOR The major proliferating Cells are the abnormal megakarryocytes  The stem cell niche that was primarily affected  Yet unidentified primary insult

31. The Reactive Marrow Fibrosis The Neoplastic megakarryocytes elaborate cytokines that Induce polyclonal fibroblast proliferation and Fibroblast synthesise reticulin and collagen The Identifiable Cytokines are ▫Platelet derived growth factor (PDGF) ▫Transforming growth factor  (TGF-  ▫ Basic fibroblast growth factor (b-FGF  ▫Calmodulin. Also produced is angiogenic protein VEGF

32. The Extramedullary haemopoiesis The normal structure of the bone marrow is distorted by the fibrosis ▫CD34+ marrow cells leak to the peripheral blood in very large numbers unlike in other MPNs ▫These cells may express JAK2 Mutations ▫They home to the spleen largely ▫Establish primordial haemopoiesis The abnormal leucuerythroblastosis is largely from the extramedullary haemopoiesis

33. Secondary (Reactive) Thrombocytosis Infection hyposplenism Inflammatory disease Chronic inflammatory bowel disease Collagen vascular diseases Malignancy (Non-hematopoietic or non-myeloid hematopoietic Blood loss /hemorrhage Chronic iron deficiency anaemia Post-surgical procedures Trauma (especially brain trauma) Rebound following chemotherapy or replacement vitamin B12 or folic acid therapy Differentials of Thrombocytosis

34. Primary Thrombocytosis (Myeloid Neoplasm Related) Chronic myeloproliferative neoplasia Chronic myeloid leukemia Polycythemia vera Primary myelofibrosis, mainly prefibrotic stage Essential thrombocythemia Acute myeloid leukemia associated with t(3;3) (q21;q26.2) or inv (3) (q21q26.2) Myelodysplastic syndromes associated isolated del (5q) abnormality Myelodysplastic syndromes/myeloproliferative neoplasms unclassifiable (refractory anemia with ring sideroblasts with marked thrombocytosis (RARS-T)

35. Features Helpful in Distinguishing Essential Thrombocythemia and secondary Thrombocytosis FindingEssential thrombocythemia Secondary thrombocytosis Persistent platelet increase+- Splenomegaly/hepatomegaly+- History of thrombosis/ hemorrhage +- Virtually all giant megakaryocytes+- JAK2 v617f mutation, MPL W515K/L mutation +- Elevated acute phase reactants (e.g. CRP) -+ Known inflammatory or infectious disease or non-myeloid malignancy -+

36. Revised WHO (2008) Diagnostic criteria for the Diagnosis of Essential Thrombocythemia Diagnosis requires meeting all four criteria 1.Sustained platelet count >450 x 10 3 /mm 3 (>450 x 10 9 /L) 2.Bone marrow biopsy specimen showing proliferation mainly of the megakaryocytic lineage with increase numbers of enlarged, mature megakaryocytes. No significant increase or left shift or neutrophil granulocytes or erythropoiesis. 3.Not meeting WHO criteria for polycythemia vera, primary myelofibrosis, BCR-ABL1 + chronic myelogenous leukaemia, myelodysplastic syndrome or other myeloid neoplasm 4.Demonstration of JAK2 V617F or other clonal marker, or in the absence of JAK2 V617F, no evidence of reactive thrombocytosis

37. Morphologic Features of ET Peripheral Blood ▫Marked thrombocytosis with prominent platelet anisocytosis ▫Neutrophils usually normal ▫No basophilia ▫No significant red blood cell anisopoikilocytosis Bone Marrow ▫Normocellular, hypercellular, occassional hypocellular ▫Marked megakaryocytic proliferation; found in loose clusters and/or dispersed ▫Megakaryocytes large to giant, deeply lobulated nuclei but not bizarr ▫Large aggregates of platelets on aspirate smears ▫No reticulin fibrosis ▫Erythroid lineage usually normal, may be hyperplastic if bleeding present ▫Granulocytic lineage usually normal

38. Comparison of features of Essential Thrombocythemia with Other Chronic Myeloproliferative Disorders featuresETPMF (Prefibrotic PMF (fibroitc) CMLPolycythemia Vera CellularityNormocellular hypercellular Hypercellular Normocellular hypercellular Megakaryocy te morphology Large, giant forms, large amount of cytoplasm, multilobated nuclei Bizarre, abnormal chromatin but often normal in size Dwarf forms often hypolobated Variable morphology Megakaryocy te architecutre Loose clusters or individual Tight clusters common Clusters or individual FibrosisAbsent-mild Moderate- severe Absent- severe

39. Comparison of features of Essential Thrombocythemia with Other Chronic Myeloproliferative Disorders granulocyte s NormalIncreased ErythroidNormal Often decreased Increased Cytogenetic abnormalitie s RareFrequent T(9;22) almost always present Occassional Molecular abnormalitie s JAK2 V617F 50% MPLW515K/ L 1% JAK2 V617F 50% MPLW515K/L 5% BCR-ABL1, always present JAK2 V617F 95% exon 12 mutations 4%

40. Diagnosing Mastocytosis Major molecular lesion is c-kit (CD117) mutation ▫Found in 50-80% of patient ▫C-Kit is normally activated by kit ligand (stem cell factor) PDGFR  and F1P1L1 fusion gene that results in constitutive activation of PDGFR  tyrosine kinase activity ▫Found in 50% of patients Diagnostic Immunochemistry ▫Tryptase-highly specific for mast cell ▫Chymase ▫Heparin –absent in basophils