MYELOPROLIFERATIVE DISORDERS Riadi Wirawan Departemen Patologi Klinik FKUI-RSCM

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.

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

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

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.

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

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.

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.

Total red cell volume and total plasma volume Normal Primary or secondary polycythaemia Relative polycythaemia Total red cell volume (51Cr) ♂ 25-35 mL/kg ♀ 22-32 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.

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

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.

POLYCYTHEMIA VERA

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

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

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.

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

Laboratory findings Bone marrow Hypercellular Prominent megakaryocyte

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 : 10-100) 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.

Treatment Maintain a normal blood count, Ht should be maintain at 45 % and platelet count < 400.000/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

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

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 %)

ESSENTIAL THROMBOCYTHEMIA

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

Essential thrombocythemia Persisting platelet count > 400.000/uL, exclude thrombocytosis by other causes before diagnosis can be made JAK-2 mutation in 50 % cases

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.

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)

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.

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

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

Treatment To control platelet count so as to reduce the risk of thrombosis (risk > 60 years, thrombocyte > 1.000.000/uL, with previous episode of thrombosis or hemorrhage, smoking history and hypertension) Keep the platelet count below 600.000/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

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

MYELOFIBROSIS

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

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

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

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

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.

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

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 < 30.000/ul Presence of abnormal chromosome

CHRONIC MYELOID LEUKEMIA (CML)

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

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.

(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.

Clinical features Occur in either sex (male : female ratio = 1,4 : 1) Most frequency between age 40-60 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

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.

Laboratory findings Normochromic normocytic anemia Leukocytosis 50.000 - > 500.000/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 (+)

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.

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.

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

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 (< 100.000/uL) unrelated to therapy or persistent thrombocytosis (> 1.000.000/uL) unresponsive to therapy Increasing spleen size and increasing WBC count unresponsive to therapy Cytogenetic evidence of clonal evolution

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

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

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