MYELOPROLIFERATIVE NEOPLASMS

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1 9 : 2 MYELOPROLIFERATIVE NEOPLASMS Introduction William Dameshek in 1951 introduced the term Myeloproliferative disorders (MPD). This included polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), chronic myelogenous leukemia (CML) and Di Guglielmo s syndrome (erythroleukemia) 1. Later on in 1960 Philadelphia (Ph1) chromosome could be associated with CML and erythroleukemia was found to be a variant of acute myeloid leukemia (AML). So the rest of the three disorders were redesigned as classic Ph1 negative MPD. The first systematic attempt to classify MPD and MPDlike clinicopathologic entities was undertaken by the World Health Organization (WHO) committee for the classification of hematologic malignancies. According to the 2001 WHO classification system, CML, PV, ET, and PMF were included under the category of chronic myeloproliferative diseases (CMPD) 2. The identification of BCR-ABL as a CML specific genetic event in the context of CMPD has facilitated accurate molecular diagnosis and effective targeted therapy. Specific genetic defects of the other disorders were not known until recently. The last five years has witnessed advances in the understanding of molecular pathogenesis of some of the BCR-ABL negative CMPD and specific molecular abnormalities associated with PV, ET and PMF were identified. As a result WHO diagnostic criteria and classification has been revised and the term CMPD has been changed to myeloproliferative neoplasms (MPN). CLASSIFICATION OF MPN The 2008 WHO classification of myeloid neoplasms of which MPN forms a separate group is as follows 3-6 : 1. Acute myeloid leukemia AML and related precursor neoplasms 2. Myelodysplastic syndromes)(mds) 3. Myeloproliferative neoplasms (MPN) 3.1 Chronic myelogenous leukemia (CML) BCR-ABL positive 3.2 Polycythemia vera (PV ) 3.3 Essential thrombocytopenia (ET ) K Pavithran, Mathew Thomas, Cochin 3.4 Primary myelofibrosis(pmf) 3.5 Chronic neutrophilic leukemia (CNL) 3.6 Chronic eosinophilic leukemia, not otherwise specified (CEL, NOS) 3.7 Mastocytosis. 3.8 Myeloproliferative neoplasm, unclassified (MPN-U) 4. Myelodysplastic/myeloproliferative neoplasms (MDS/MPN). 4.1 Chronic myelomonocytic leukemia (CMML) 4.2 Juvenile myelomonocytic leukemia (JMML). 4.3 Atypical chronic myeloid leukemia BCR ABL negative. 4.4 Myelodysplastic /myeloproliferative neoplasms unclassifiable. 4.5 Refractory anemia with ringed sideroblasts associated with marked thrombocytosis. 5. Myeloid/ lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA,PDGFRB or FGFR Myeloid and lymphoid neoplasms associated with PDGFA rearrangement 5.2 Myeloid neoplasms with PDGFB rearrangement 5.3 Myeloid and lymphoid neoplasms with FGFR1 abnormalities. MOLECULAR BASIS OF MPN The members of the Janus Kinase family of receptors are JAK1, JAK2, JAK3 and tyrosine kinase 2 TyK2. JAK2V617F mutation is a somatically acquired G to T nucleotide shift at position 1849 in exon 14 (of chromosome 9) which results in a valine to phenylalanine substitution at codon 617 located in the JH2 pseudo kinase domain of JAK2 receptor. As a result the auto inhibitory control of JAK2 is lost. The mutated JAK2 is in a constitutively phosphorylated state. This results in continuous signalling by STAT leading to uncontrolled proliferation of the erythroid cells.

2 Medicine Update 2010 Vol. 20 When this mutation is introduced into erythroid cell lines, growth of erythroid cells occur independent of Epo. This may be the explanation of endogenous erythroid colony formation (EEC) 7. JAK2V617F mutational frequency is found in more than 95% of PV, 60% of ET or PMF, 40-50% in refractory anemia with ringed sideroblasts and thrombocytosis. On the other hand it is vey rare in AML or MDS. Mutations in exon 12 of JAK2 have been discovered in PV who is negative for the classical mutation. Whether JAK2V617F is actually the cause of the disease or only a disease modifier is not yet known. How a mutation of a single gene can be responsible for three different clinical phenotypes is also not entirely understood. Another recurrent molecular abnormality of MPN is a somatic mutation at codon 515 of MPL (named after myeloproliferative leukemia virus oncogene homology). This is found in 5-11% of PMF patients and 9% of ET-JAK2 negative patients. The gene encoding for the receptor of platelet derived growth factor A (PDGFA) is involved in at least 4 different genetic abnormalities associated with eosinophilia. c-kit is a receptor for stem cell factor and is an important cytokine in the generation and proliferation of mast cells. A D816V mutation of this tyrosine kinase receptor occurs in systemic mastocytosis. DIAGNOSIS OF MPN The diagnosis of MPN has been always difficult in the past. However, the availability of the new molecular markers is expected to facilitate the diagnosis of MPN. Molecular phenotyping has become an integral part of the diagnostic criteria put forward by WHO in Tests for JAK2 and MPL have already become a standard tool in the diagnostic work up of MPN. Detection of one of the molecular abnormalities establishes a clonal abnormality and it is going to be unlikely to be reactive erythrocytosis thrombocytosis or myelofibrosis. Chronic myeloid leukemia Although there are characteristic findings in the blood CBC, peripheral blood smear and the bone marrow supported by low leukocyte alkaline phophotase, the diagnosis of CML must be confirmed by Ph1 chromosome or BCR/ABL transcript using PCR technique 3. Polycythemia vera In patients with increased hemoglobin or red cell mass according to the WHO criteria, demonstration of JAK2V617F allows a definite diagnosis in more than 95% of cases. Less than 2% of PV patients will have the JAK2 exon 12 abnormalities. Revised WHO criteria for diagnosis of PV is as follows: Accordingly there are 2 major and 3 minor criteria 3. Major criteria 1. Hemoglobin level above 18.5g/dl for men and 16.5g/dl for females OR Hemoglobin or hematocrit > 99 th percentile of reference range for age, sex, or altitude of residence OR elevated red cell mass >25% above mean normal predicted value. 2. Presence of JAK2 gene mutation (V617F) or other functionally similar mutation such as JAK2 exon 12 mutation. Minor criteria 1. Bone marrow showing hypercellularity for age and trilineage growth (panmyelosis) 2. Subnormal Erythropoietin level 3. EEC (endogenous erythroid colony formation) in vitro Diagnosis requires the presence of both major criteria + one minor criterion or the presence of first major criterion + 2 minor criteria. Essential thrombocythemia (ET) A sustained platelet count of greater than 450,000/cumm is a compelling criterion by WHO. This is lower than the 2001 recommendation of 600,000/cumm because cases of ET with lower counts and positive JAK2 mutation have been discovered. CML should be excluded by FISH or PCR analysis for BCR-ABL re arrangement. Since JAK2V617F mutation and MPL mutation are found in only 60-70% of ET cases bone marrow morphology is important for the diagnosis. A normal CRP almost rules out secondary causes of thrombocytosis WHO criteria for Essential thrombocythemia diagnosis-requires all four criteria 3 1. Sustained platelet count 450x 10 9 /L 2. Bone marrow biopsy specimen showing proliferation of the megakaryocytic lineage with increased numbers of enlarged, mature megakaryoytes. No significant increase or left-shift of neutrophil granulopoiesis or erythropoiesis 3. Not meeting WHO criteria for polycythemia vera, primary myelofiboris or BCR-ABL positive Chronic myelogenous leukaemia, myelodysplastic syndrome or other myeloid neoplasm 4. Demonstration of JAK2V617F or other clonal marker, or in the absence of JAK2V617F, no evidence for reactive thrombocytosis ( causes of reactive thrombocytosis include iron deficiency, splenectomy, surgery, infection, inflammation, connective tissue disease, metastatic cancer and lymhoproliferatie disorders. Primary myelofibrosis (PMF) PMF is the least common of the MPDs and the most difficult to diagnose in part because of the inability to obtain an adequate marrow aspirate for evaluation and in part because marrow fibrosis is a nonspecific reactive process that complicates many benign as well as malig nant disorders. Criteria for primary myelofibrosis (PMF) Diagnosis requires meeting all 3 major criteria and 2 584

3 Myeloproliferative Neoplasms minor criteria Major criteria 3 1. Presence of megakaryocyte proliferation and atypia, usually accompanied by either reticulin or collagen fibrosis, or, in the absence of significant reticulin fibrosis, the megakaryocyte changes must be accompanied by an increased bone marrow cellularity characterized by granulocytic proliferation and often decreased erythropoiesis (ie, prefibrotic cellular-phase disease) 2. Not meeting WHO criteria for polycythemia vera, BCR- ABL1 positive chronic myelogenous leukemia, myelodysplastic syndrome, or other myeloid disorders 3. Demonstration of JAK2 V617F or other clonal marker (eg, MPLW515K/L), or, in the absence of the above clonal markers, no evidence that bone marrow fibrosis is secondary to infection, autoimmune disorder or other chronic inflammatory condition, hairy cell leukemia or other lymphoid neoplasm, metastatic malignancy, or toxic (chronic) myelopathies Minor criteria 1. Leukoerythroblastosis 2. Increase in serum lactate dehydrogenase level 3. Anemia 4. Palpable splenomegaly Age above 60 years, hemoglobin level of <10g/dl, platelet count <100,000/cumm and >3% blast cells in the peripheral blood indicate poor prognosis. Chronic neutrophilic leukemia (CNL) This is a rare MPN disorder found in elderly people, characterised by sustained peripheral blood neutrophilia, bone marrow hypercellularity and hepatosplenomegaly. Only 150 cases have been published and the disease not fully understood. Diagnostic criteria 3 : 1. Peripheral blood leucocytosis, WBC 25 x 10 9 /L (Segmented neutrophils and band forms are >80% of white blood cells) 2. Bone marrow biopsy showing hyperplasia of granulocytic lineage without involvement of other series, absence of fibrosis and myelodysplsia, myeloblasts <5% of nucleated marrow cells 3. Hepatosplenomegaly 4. No identifiable cause for physiologic neutrophilia 5. No Philadelphia chromosome or BCR-ABL fusion gene 6. No rearrangement of PDGFRA, PDGFRB or FGFR1 7. No evidence of polycythemia vera, primary myelofibrosis or essential thrombocythemia. 8. No evidence of a myelodysplastic syndrome or MDS/MPN Chronic eosinophilic leukemia and hyper eosinophilic syndrome Patients with a persistent absolute eosinophil count of 1500/cumm should be investigated for any form of reactive eosinophilia or any other hematologic disorder. If these conditions are excluded they fall under one of the following entities 3 1. Idiopathic hyper eosinophilia which is a T cell mediated disorder and has an increased level of interleukin 5. A definite diagnosis requires T cell immuno phenotyping and T cell receptor antigen gene rearrangement studies. 2. A clonal/ myeloproliferative disorder which requires demonstration of a cytogenetic or molecular abnormality. Chronic eosinophilic leukaemia, not otherwise specified. CEL, NOS is an MPN in which an autonomous, clonal proliferation of eosinophilic precursors results in persistently increased numbers of eosinophils in the peripheral blood, bone marrow and peripheral tissues, with eosinophilia being the dominant hematological abnormality. CEL, NOS excludes patients with a Ph chromosome, BCR-ABL1 fusion gene or rearrangement of PDGFRA, PDGFRB and FGFR1. To make a diagnosis of CEL, there should be evidence for clonality of the eosinophils or an increase in myeloblasts in the PB or BM. In many cases however, it is impossible to prove clonality of the eosinophils, in which case, if there is no increase in blast cells, the diagnosis of idiopathic hypereosinophilic syndrome is made. Idiopathic HES is defined as eosinophilia persisting more than 6 months, for which no underlying cause can be found, and which is associated with signs of organ involvement and dysfunction; there is no evidence of eosinophil clonality. It is a diagnosis of exclusion. The target organs involved in CEL and hypereosinophilic syndrome (HES) are the heart, muscle, skin, lungs and the nervous system. Diagnostic criteria for chronic eosinophilic leukemia (MPN with prominent eosinophilia) 3 1. There is eosinophilia (eosinophil count of 1500/cumm) 2. There is no Ph chromosome or BCR-ABL fusion gene or other myeloproliferative neoplasm 3. There is no t(5;12) or other rearrangement of PDGFRB 4. There is no FIP1L1- PDGFRA fusion gene or other rearrangement of PDGFRA 5. There is no rearrangement of FGFR1 6. The blast cell count in the peripheral blood and bone marrow is less than 20% and there is no inversion 16 or t(16;16) or other features diagnostic of AML 7. There is a clonal cytogenetic or molecular genetic abnormality, or blast cells are more than 2% in the peripheral blood or more than 5% in the bone marrow. If a patient ahs eosinophilia but these criteria are not met the diagnosis may be reactive eosinophilia, idiopathic hypereosinophilia or idiopathic hypereosinophilic syndrome 585

4 Medicine Update 2010 Vol. 20 Mastocytosis Is due to clonal, neoplastic proliferation of mast cells that accumulate in one or more organ systems. The disease is classified into: 1.Cutaneous mastocytosis (CM) and 2.Systemic mastocytosis (SM) which can be a) indolent or b) aggressive. Life expectancy is normal except in aggressive SM where it is shortened. Diagnostic criteria for systemic Mastocytosis 3 : Diagnosis can be made when the major criterion and one minor criterion or at least three minor criterion are present Major criterion: Multifocal dense infiltrates of mast cells (more than 15 mast cells in aggregates) detected in sections of bone marrow and/or other extracutaneous organ(s) Minor criteria: 1. In biopsy sections of bone marrow or other extracutanoues organs, >25% of the mast cells are spindle shaped or have atypical morphology or of the mast cells in the bone marrow aspirate smears, >25% are immature or atypical 2. High serum level of tryptase (>20ng/ml) 3. Detection of kit D816V molecular mutation by using adequate sensitive techniques (Patients with this mutation are resistant to imatinib). 4. Mast cells in bone marrow, peripheral blood or other extracutanoues organs express CD2 and or CD 25 antigens in addition to normal mast cell markers Significant changes in the diagnosis and classification of MPN 1. The nomenclature has been changed from myeloproliferative disorders (MPDs) to myeloproliferative neoplasms (MPNs). 2. Diagnostic algorithms for PV, ET, and PMF have substantially changed to include information regarding JAK2 V617F and similar activating mutations. Additional clinical, laboratory, and histologic parameters have been included to allow diagnosis and subclassification regardless of whether JAK2 V617F or a similar mutation is or is not present. 3. The platelet count threshold for the diagnosis of ET has been lowered from > 600x 10 9 /L to > 450 x 10 9 /L. 4. Some cases previously meeting the criteria for chronic eosinophilic leukemia (CEL) may be categorized in a new subgroup, Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1. 5. Systemic mastocytosis has been included in the MPN category Treatment for MPDs Overall survival in the 3 MPNs ranges from a normal life expectancy in ET patients to a median of less than 6 years in many patients with PMF. Arterial and venous throm boses contribute to morbidity in these patients. Disease transformation is another major complication of MPNs (eg, PV or ET to PMF or transformation of any of them to acute myeloid leukemia/ myelodysplastic syndromes). Management of PV The mainstay of therapy for PV is phlebotomy, in order to keep the hematocrit below 45% in men and 42% in women. Pruritus is a unique feature of PV and can be extremely disturbing to some of the patients. A variety of remedies have been used to treat these patients- phlebotomy, H1 and H2 blockers and antidepressants such as paroxetine. Cytoreductive therapy is necessary for patients with splenomegaly, very high leu kocyte or platelet counts or high-risk patients (age >60 years or previous history of thrombosis or cardiovascular risk factors). Aspirin is effective in relieving the microvascular complications of erythromelalgia and in preventing thrombotic complications. The Management of ET Low-risk ET patients who are asymptomatic do not need any treatment. For high-risk ET patients (age >60 years, previous history of thrombosis, cardiovascular risk factors or major hemorrhage, platelet count >1, /μL), hydroxyurea has been recommended as the treatment of choice. Other treatments include anagrelide or interferon. There is no established target platelet count, and since there is no correlation between the height of the platelet count and thrombosis, the target platelet count should be one at which the patient is free from microvascular symptoms or from the risk of bleeding. The Management of PMF At present, allogeneic stem cell transplantation is the only therapy with possible curative potential in PMF. Hyperproliferation such as leukocytosis, marked splenomegaly, and thrombocytosis are indications for cytore ductive therapy (eg, hydroxyurea). Splenectomy often alleviates the mechanical symptoms and may also benefit approximately 25% of patients with transfusiondependent anemia, although it is associated with almost a 10% mortality. Low-dose thalidomide in combination with prednisone has also proved to be effective in alleviat ing anemia, thrombocytopenia, and splenomegaly in approximately % of PMF patients 8. Targeted therapy in PV, ET and PMF Currently, with the exception of allogenic stem cell transplantation, no therapy has been shown to be curative, to alter natural history, or to prolong survival in patients MPNs. Therefore, the activating JAK2 mutations have been considered a rational target for drug development. A number of JAK2 inhibitors are currently undergoing clinical trials and are under various phases of development and preliminary results have been encourag ing 9, 10. In summary great changes have occurred in the past 5 years 586

5 Myeloproliferative Neoplasms regarding our knowledge about the familiar myeloproliferative disorders. A new term myeloproliferative neoplasms (MPN) has been coined to designate these disorders. Many molecular abnormalities including JAK2V617F, JAK2 exon 12 mutations, MPL mutations, FIP1L1/PDGFRA fusion gene, kit D816V mutation have given us a better platform on which we can classify and draw diagnostic criteria for MPN. They are also considered as rational targets for drug development. References 1. Dameshek W. Some speculations on the myeloproliferative syndromes. Blood 1951; 6: Jaffe ES, Harris NL, Stein H, Vardiman JW. World Health Organization Classification of Tumors of Hematopoietic and Lymphoid. Tissues. Lyon: IARC Press; Swerdlow SH, Campo E, Harris NL, et al, eds. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC; Tefferi A, Thiele J, Vardiman JW. The 2008 World Health Organization classification system for myeloproliferative neoplasms: order out of chaos. Cancer 2009 ;115: Anastasi J. The myeloproliferative neoplasms: insights into molecular pathogenesis and changes in WHO classification and criteria for diagnosis. Hematol Oncol Clin North Am 2009;23: Tefferi A, Vardiman JW. Classification and diagnosis of myeloproliferative neoplasms: the 2008 World Health Organization criteria and pointof-care diag nostic algorithms. Leukemia. 2008;22: Vannucchi AM, Guglielmelli P, Tefferi A. Advances in understanding and management of myeloproliferative neoplasms. CA Cancer J Clin ;59: Abdel-Wahab OI, Levine RL. Primary myelofibrosis: update on definition, pathogenesis, and treatment. Annu Rev Med 2009;60: Thiele J. Philadelphia chromosome-negative chronic myeloproliferative disease. Am J Clin Pathol 2009 ;132: Atallah E, Verstovsek S. Prospect of JAK2 inhibitor therapy in myeloproliferative neoplasms. Expert Rev Anticancer Ther 2009 ;9: