1. MYELOPROLIFERATIVE DISORDERS
Riadi Wirawan
Departemen Patologi Klinik FKUI-RSCM

2. Diagrammatic representation of the BM pluripotent stem cell and the cell line that arise from it.
Hoffbrand AV, Moss PAH, Pettit JE. Essential haematology .5th ed. Oxford : Blackwell Publishing; 2006.p.2.

3. MYELOPROLIFERATIVE DISORDERS (MPD)
JAK2 MUTATION
Polycythemia Vera (PV)
Essential thrombocythemia (ET)
Myelofibrosis (MF
BCR-ABL FUSION GENE
Chronic myeloid leukemia (CML)

4. MYELOPROLIFERATIVE DISORDERS (MPD)
MPD : a group of conditions characterized by clonal proliferation of one or more hematopoietic components in the BM and in many cases the liver and spleen
Disorders are include :
Polycythemia vera (PV)
Essential thrombocythemia (ET)
Myelofibrosis

5. Relationship between the three myeloproliferative diseases
Relationship between the three myeloproliferative diseases. They may all arise by somatic mutation in the pluripotential stem and progenitor cells. Many transitional cases occur showing features of two conditions and, in other cases, the disease transforms during its course from one of these diseases to another or to acute myeloid leukaemia. The three diseases, polycythaemia rubra vera, essential thrombocythaemia and myelofibrosis, are characterised by JAK2 mutation in a varying proportion of cases.
Hoffbrand AV, Moss PAH, Pettit JE. Essential haematology .5th ed. Oxford : Blackwell Publishing; 2006.p.230.

6. This disorders are closely related to each other
Transitional form occur
A single acquired mutation of the cytoplasmic tyrosine kinase Janus-associated kinase 2 (JAK-2)(Val617Phe) occurs in the marrow and blood of almost all patients with PV, 50% with ET and myelofibrosis

7. The role of JAK2 mutation in the generation of myeloproliferative diseases. (a) (i) Most haemopoietic growth factor receptors do not have intrinsic kinase activity but associate with a protein kinase such as JAK2 in the cytoplasm. (ii) When the receptor binds a growth factor the cytoplasmic domains move closer together and the JAK2 molecules can activate each other by phosphorylation. (iii) The V617F JAK2 mutation allows the JAK protein to become activated even when no growth factor is bound.
Hoffbrand AV, Moss PAH, Pettit JE. Essential haematology .5th ed. Oxford : Blackwell Publishing; 2006.p.231.

8. The role of JAK2 mutation in the generation of myeloproliferative diseases. (c) JAK2 activation leads to cell survival and proliferation through activation of three major pathways; the STAT transcription factors, the PI3K pathway acting through Akt and Ras activation which subsequently activate ERK and MAPK. The net result is production of a diverse range of proteins that promote cell survival and proliferation.
Hoffbrand AV, Moss PAH, Pettit JE. Essential haematology .5th ed. Oxford : Blackwell Publishing; 2006.p.231.

9. Total red cell volume and total plasma volume
Normal
Primary or secondary polycythaemia
Relative polycythaemia
Total red cell volume (51Cr)
♂ mL/kg
♀ mL/kg  N
Total plasma volume
(125I-albumin)
40-50 mL/kg 
Hoffbrand AV, Moss PAH, Pettit JE. Essential haematology .5th ed. Oxford : Blackwell Publishing; 2006.p.232.

10. POLYCYTHEMIA
Is define as an increased in the Hb concentration above the upper limit of normal for the patient age and sex

11. Causes of polycythemia
Primary
Polycythemia (rubra) vera
Familial (congenital) polycythemia
Secondary
Caused by compensatory erythropoietin increase in:
high altitudes
pulmonary disease and alveolar hypoventilation (sleep apnoe)
cardiovascular disease, especially congenital with cyanosis
increased affinity haemoglobin (familial polycythemia
heavy cigarette smoking
Caused by inappropriate erythropoietin increased in:
renal diseases (e.g. hydronephrosis, vascular impairment, cysts, carcinoma)
tumours such as uterine leiomyoma, hypernephroma, hepatocellular carcinoma,
cerebellar haemangioblastoma
Relative
Stress or pseudopolycythemia
Cigarette smoking
Dehydration: water deprivation, vomiting
Plasma loss: burns, enteropathy
Hoffbrand AV, Moss PAH, Pettit JE. Essential haematology .5th ed. Oxford : Blackwell Publishing; 2006.p.232.

12. POLYCYTHEMIA VERA

13. Polycythemia vera (PV)
Increased in red cell volume caused by a clonal malignancy of the marrow stem cell
The disease result from somatic mutation of a single hematopoietic stem cell (JAK-2 mutation is present in almost 100 % of patients)
Increased in red cells volume is the diagnostic finding and an overproduction of granulocyte and platelet
Chromosome abnormality : del 9p, del 20q

14. Clinical features
Common in older subjects, with an equal sex incidence
Hyperviscosity :
Headache, dispnoe, blurr vision
Phletoric appearance : ruddy cyanosis
Bleeding : GI, uterine, cerebral
Thrombosis : arterial (cardiac, cerebral, peripheral) venous (leg veins, cerebral, portal, hepatic)
Hypervolemia : retinal venous engorgement, hypertension
Hypermetabolism
Night sweat
Pruritus
Gout
Peptic ulcers
Splenomegali : 75% patients

15. Gouty tophi on the index and middle fingers.
Hoffbrand AV, Pettit JE. Color atlas of clinical hematology. 2nd ed. London : Mosby-Wolfe; 1998.p.198.

16. Laboratory findings
Increased hemoglobin concentration, hematocrit, RBC, total red cell volume (TRCV), blood viscosity and low serum erythropoietin
Leucocyte :
Neutrophil leucocytosis
Basophilia
Increased Neutrophil Alkaline Phosphatase (NAP) score
Increased vit B12 and vit B12 binding capacity
Increased uric acid serum and normal LDH
Thrombocyte count raise in about 50 % patients

17. Laboratory findings
Bone marrow
Hypercellular
Prominent megakaryocyte

18. Criteria for diagnosis of polycythemia (rubra) vera
Total red cell mass
male > 35 mL/kg
female > 32 mL/kg A2
Arterial oxygen saturation normal (> 92%) A3
Splenomegaly A4
JAK2 mutation B1
Platelets > 400 x 109/L B2
White cells > 12 x 109/L B3
Increased NAP score (n : ) B4
Raised serum vitamin B12 level
A1 + A2 + A3 or
A1 + A2 + A4 or
A1 + A2 + another two B
NAP = neutrophil alkaline phosphatase.
Hoffbrand AV, Moss PAH, Pettit JE. Essential haematology .5th ed. Oxford : Blackwell Publishing; 2006.p.232.

19. Treatment
Maintain a normal blood count, Ht should be maintain at 45 % and platelet count < /uL
Venesection :
To reduce hematocrit < 45%
Rapid reduction of red cell volume
Resulting IDA, does not control the platelet count
Cytotoxic myelosuppression
Poor tolerance of venesection, symptomatic, progressive splenomegaly, weight loss, night sweat
Hydroxyurea for controlling the blood count, side effects myelosuppression, skin toxicity and nausea

20. Treatment Phosphorus32 therapy
Use for older patients with severe disease
Late development of leukemic
Interferon alpha
Suppress excess proliferation in the marrow
Low dose aspirin : reduce thrombotic complication

21. Course and prognosis Prognosis is good, median survival 10-16 years
Major clinical problems : thrombosis and hemorrhage
Transformation PV to :
Myelofibrosis ( 30 %)
Acute leukemia (5 %)

22. ESSENTIAL THROMBOCYTHEMIA

23. Essential thrombocythemia
Megakaryocyte proliferation
Increased platelet count
Overproduction of platelet
Normal hematocrite
Philadelphia chromosome or BCR-ABL fusion gene are absent
No collagen fibrosis in the marrow

24. Essential thrombocythemia
Persisting platelet count > /uL, exclude thrombocytosis by other causes before diagnosis can be made
JAK-2 mutation in 50 % cases

25. Causes of a raised platelet count
Reactive
Haemorrhage, trauma, postoperative
Chronic iron deficiency
Malignancy
Chronic infections
Connective tissue disease (e.g. rheumatoid arthritis)
Post-splenectomy
Endogenous
Essential thrombocythaemia (JAK2 mutation + or -)
In some cases of polycythaemia vera, myelofibrosis and chronic myeloid leukaemia
Hoffbrand AV, Moss PAH, Pettit JE. Essential haematology .5th ed. Oxford : Blackwell Publishing; 2006.p.237.

26. Clinical features Thrombosis : Hemorrhage :
Venous
Arterial
Hemorrhage :
Result of abnormal platelet function
May cause chronic or acute bleeding
Erythromelalgia : burning sensation felt in the hands or feet and reliaved by aspirin
Splenomegaly or splenic atrophy (infarction)

27. Gangrene of the left fourth toe in thrombocythaemia essential.
Hoffbrand AV, Pettit JE, Moss PAH. Essential haematology .4th ed. Oxford : Blackwell Science; 2001.p.230.

28. Laboratory findings Peripheral blood
Thrombocytosis with abnormal large platelet
Megakaryocyte fragment may be seen in PB
Abnormal platelet aggregation with adrenaline
Bone marrow : excess of abnormal megakaryocyte
Cytogenetics : negative BCR-ABL fusion gene

29. Essential thrombocythaemia : peripheral blood film showing a gross increase in platelet numbers.

30. Treatment
To control platelet count so as to reduce the risk of thrombosis (risk > 60 years, thrombocyte > /uL, with previous episode of thrombosis or hemorrhage, smoking history and hypertension)
Keep the platelet count below /uL
Hydroxyurea most widely use
Alpha interferon valuable in younger patient
Anagrelide for reducing the platelet count
Plateletpheresis helpful in short term management
Aspirin to reduce thrombotic risk

31. Course Stationery  10 – 20 years
Transform to myelofibrosis or acute leukemia

32. MYELOFIBROSIS

33. Myelofibrosis
Myelofibrosis is a clonal stem cell disease, in some patient there is osteosclerosis
Progressive generalized reactive fibrosis of the bone marrow in association with the development of hemopoiesis in the spleen and liver (myeloid metaplasia)
JAK mutation occurs in ± 50 % cases
No specific cytogenetic abnormalities
1/3 patients have a previous history of PV

34. Clinical feature Insidious onset in older people as usual with anemia
Massive splenomegaly
Hypermetabolic symptom : loss of weight, anorexia, fever and night sweat
Bleeding problem, bone pain or gout

35. Laboratory findings
Anemia but normal or increase hemoglobin level may be found
Leucocytosis and thrombocytosis are frequent at the time of presentation, later leucopenia and thrombocytopenia are common
Leucoerythroblastic blood film : immature granulocyte with NRBC
Tear drop cells

36. Laboratory findings
Bone marrow : unobtainable by aspiration, trephine biopsy showed fibrotic hypercellular marrow, increase megakaryocyte, increase bone formation
Increased NAP score
High serum urate and LDH level  increase turn over cell
JAK-2 mutation in 50 % cases

37. Peripheral blood film in essential thrombocythaemia showing increased numbers of platelets and a nucleated megakaryocytic fragment.
Hoffbrand AV, Moss PAH, Pettit JE. Essential haematology .5th ed. Oxford : Blackwell Publishing; 2006.p.237.

38. Treatment
Palliative and reducing the effects of anemia and splenomegali
Blood transfusion
Regular folic acid therapy
Hydroxyurea : reduce splenomegaly and hypermetabolic symptom
Splenic radiation
Allopurinol : prevent gout and urate nephropathy

39. Course Transformation to acute myeloid leukemia (AML) 10 – 20 % cases
Median survival 3,5 years
Cause of death : heart failure, infection and leukemic transformation
Poor prognosis if :
Hb < 10 g/dL
WBC < 4000/uL
Thrombocyte < /ul
Presence of abnormal chromosome

40. CHRONIC MYELOID LEUKEMIA (CML)

41. CML Clonal disorders of a pluripotent stem cell
15% of leukemia & and may occur at any age
CML characteristic present of Philadelphia (Ph) chromosome, t(9;22) (q34;q11)  resulting
BCR-ABL fusion gene  codes for a fusion protein of size p210

42. The Philadelphia chromosome
The Philadelphia chromosome. (a) There is translocation of part of the long arm of chromosome 22 to the long arm of chromosome 9 and reciprocal translocation of part of the long arms of chromosome 9 to chromosome 22 (the Philadelphia chromosome). This reciprocal translocation brings most of the ABL gene into the BCR region on chromosome 22 (and part of the BCR gene into juxtaposition with the remaining portion of ABL on chromosome 9). (b) The breakpoint in ABL is between exons 1 and 2. The breakpoint in BCR is at one of the two points in the major breakpoint cluster region (M-BCR) in CML or in some cases of Ph+ ALL. (c) This results in a 210-kDa fusion protein product derived from the BCR-ABL fusion gene. In other cases of Ph+ ALL, the breakpoint in BCR is at a minor breakpoint cluster region (m-BCR) resulting in a smaller BCR-ABL fusion gene and a 190-kDa protein.
Hoffbrand AV, Moss PAH, Pettit JE. Essential haematology .5th ed. Oxford : Blackwell Publishing; 2006.p.175.

43. (e)
(d) Karyotype showing the t(9;22) (q34;q11) translocation. The Ph chromosome is arrowed. (e) Visualization of the Philadelphia chromosome on: (i) dividing (metaphase); and (ii) quiscent (interphase) cells by fluorescence in situ hybridization (FISH) analysis (ABL probe in red and BCR probe in green) with fusion signals (red/green) on the Ph and der(9) chromosomes.
Hoffbrand AV, Moss PAH, Pettit JE. Essential haematology .5th ed. Oxford : Blackwell Publishing; 2006.p.176.

44. Clinical features Occur in either sex (male : female ratio = 1,4 : 1)
Most frequency between age yo
Hypermetabolism : weight loss, anorexia or night sweats, gout
Splenomegaly
Anemia : pallor, dyspnoe & tachycardia
Leukostasis : visual disturbances and priapism
Abnormal platelet function : bruising, epistaxis, menorrhagia or haemorhage

45. CML: ocular fundus in the hyperviscosity syndrome shows distended retinal veins and deep retinal haemorrhages at the macular. Hb: 14 g/dL; WBC: 590 x 109/L; platelets: 1050 x 109/L
CML: acute inflammation and swelling of the fourth finger due to uric acid deposition. Hb: 8.6 g/dL; WBC: 540 x 109/L; platelets: 850 x 109/L; serum uric acid: 0.85 mmol/L.
Hoffbrand AV, Pettit JE. Color atlas of clinical hematology. 2nd ed. London : Mosby-Wolfe; 1998.p.198.

46. Laboratory findings Normochromic normocytic anemia
Leukocytosis > /uL with complete spectrum of myeloid cell, dominate neutrophils and myelocytes, basophilia
Thrombocyte : ↑/N/↓
Neutrophil alkaline phosphatase score low. Raised in MPD and infection
Hyperuricemia
BM hypercellular with granulopoietic predominant
Ph chromosome (+)

47. Chronic myeloid leukaemia: peripheral blood showing a vast increase in buffy coat. The white cell count was 532 x 109/L.
Chronic myeloid leukaemia: peripheral blood film showing various stages of granulopoiesis including promyelocytes, myelocytes, metamyelocytes and band and segmented neutrophils.
Hoffbrand AV, Moss PAH, Pettit JE. Essential haematology .5th ed. Oxford : Blackwell Publishing; 2006.p.177.

48. Treatment Tyrosine kinase inhibitor : imatinib, dasatinib, nilotinib
Specific inhibitor of the BCR-ABL fusion protein and block tyrosine kinase activity by competing with ATP binding
Mode of action of the tyrosine kinase inhibitor STI-571. It blocks the adenosine triphosphate (ATP) binding site.
Hoffbrand AV, Moss PAH, Pettit JE. Essential haematology .5th ed. Oxford : Blackwell Publishing; 2006.p.178.

49. Course CML chronic phase CML accelerated phase CML blastic phase :
Acute myeloid leukemia (AML)
Acute lymphoid leukemia (ALL)
Bilineage acute leukemia
Biphenotype acute leukemia

50. Chronic myelogenous leukaemia, accelerated phase
The diagnosis of CML-AP may be made when one or more of the following are present :
Blast 10-19% of WBC in PB and or nucleated BM cells
Basophilia ≥ 20%
Persistent thrombocytopenia (< /uL) unrelated to therapy or persistent thrombocytosis (> /uL) unresponsive to therapy
Increasing spleen size and increasing WBC count unresponsive to therapy
Cytogenetic evidence of clonal evolution

51. Chronic myelogenous leukaemia, blastic phase
Blast phase may be diagnose if ≥ 1 more is present :
Blast 20% of WBC in PB and or nucleated BM cells
Extramedullary blasts proliferation
Large foci or cluster of blast in BM biopsy

52. Prognosis Death occur from : Prognostic according : blast phase or
intercurrent hemorrhage or
infection
Prognostic according :
age
spleen size
platelet count
PB and BM blast cell on presentation

53. thank you