Detection of Mutations in FLT3 and Jak-2 Genes in Patients with Different Subtypes of Acute Myeloid Leukemia

Transcription

1 Detection of Mutations in FLT3 and Jak-2 Genes in Patients with Different Subtypes of Acute Myeloid Leukemia Presented by Aida Kamal Ahmed A Thesis submitted To Faculty of Science In Fulfillment of the Requirements for the Master degree in science (Genetic, Histology and Cell Biology) Zoology Department Faculty of Science Cairo University 2012

2 TO WHOM IT MAY CONCERN This is to certify that Aida Kamal Ahmed. Has attended and passed successfully the following Postgraduate Courses as a Partial Fulfillment of the requirements of the degree of Master of Science (Cytology, Histology & Genetics), Cell Biology and Cancer Biology 2- Tissue Biology 3- Histopathology & Toxicology 4- Molecular Biology and Biotechnology 5- Tissue Culture & Electron Microscopy 6- Experimentation and Data Analysis 7- Cytogenetics and Molecular Genetics. 8- Developmental Biology. 9- German Language. This Certificate is issued at his own request Controller Head of Zoology Department Prof. Dr. Anwar Bakr Mansour Prof. Dr. Abdel-Rahman Bashtar

3 Chapter I Introduction and Aim of the Work Chapter I Introduction and Aim of the Work Acute myeloid leukemia (AML) is a broad range of disorders that are all characterized by an arrest of maturation along with uncontrollable proliferation of hematopoietic progenitor cells, with numerous cytogenetic abnormalities and mutations within key signaling pathways involved in cell differentiation, proliferation and survival. One of these key signaling molecules is FLT3 (FMS-like tyrosine kinase 3), belongs to the group of class 3 receptor tyrosine kinases. It is expressed in murine hematopoietic stem cells and, when activated by its ligand (FL), supports survival, proliferation, and differentiation of primitive hematopoietic progenitor cells (Kelly, 2004 and Fröhling et al., 2005). One type of somatic mutation of FLT3 gene is internal tandem duplication (ITD) in the juxtamembrane (JM) domain, which was first reported by Nakao et al. (1996), and is present in about 20% of patients with AML. These mutations cluster in exons 14 and 15 of the human FLT3 gene on chromosome band 13q12. The lengths of the duplicated segments have been reported to range in size from 6 to 180 bases and are always in frame (Carow et al., 1996, and Stirewalt and Radich, 2003). Several studies showed that FLT3/ITD mutations are associated with an increased risk of relapse, decreased disease-free survival, and overall survival. It is therefore very important to identify FLT3 mutations so as to provide prognostic information and to choose appropriate treatment options. FLT3 expression in leukemia and its clinical significance have been widely studied, but little information is available about Egyptian patients with AML, including the prevalence of disease, the mutant to wild-type ratio, and the mutant patterns and their prognostic effects (Zhong et al., 2012). Moreover, an acquired mutation in the JAK2 gene has recently been described in human myeloproliferative disorders (MPD). JAK2 is a cytoplasmic tyrosine kinase that plays an essential role in the signaling pathways of 1

4 Chapter I Introduction and Aim of the Work cytokines and growth factors. The mutation 1849 G>T, which leads to amino acid substitution of phenylalanine for a highly conserved valine (V617F), renders JAK2 kinase constitutively active and leads to cell proliferation in the absence of the growth factors (Chen et al., 2007). Now there is evidence in the literature that patients with AML with an antecedent MPD often have JAK2 V617F mutations (Fröhling et al., 2006). Only recently, a few patients with AML without previous hematologic disorders were found to have the JAK2- V617F mutation. It can be assumed that mutations of JAK2-V617F lead to a more aggressive subtype of leukemia because of the activation of the JAK2- STATs cascade which substantially alters apoptotic response, self-renewal and proliferative capacity of myeloid cells (Illmer et al., 2007). It is of note that a high prevalence of co-operating mutations of FLT3, KIT, or N-RAS in AML patients with the JAK2 mutation has been reported. Accordingly, the present study was planned to assess the frequency and prognostic impact of FLT3/ITD and JAK2 V617F gene mutations in de novo AML patients and to correlate these mutations with the clinical picture and disease outcome. Two assays (PCR and ARMS-PCR respectively) were used to detect the frequency of FLT3 and JAK2 mutations. 2

5 Chapter II 2.1. Acute Myeloid Leukemia (AML): Definition: The terms acute myeloid leukemia (AML), acute myelogenous leukemia, and acute nonlymphocytic leukemia (ANLL) refer to a group of marrow-based neoplasms that have clinical similarities but distinct morphologic, immuno-phenotypic, and cytogenetic features. AML should be distinguished from acute lymphoblastic leukemia (ALL) and may follow myelodysplasia (MDS) (Cheson et al., 1990). (AMLs) represent a clinically and biologically heterogeneous group of diseases caused by the malignant transformation of a hematopoietic stem cell or myeloid progenitor cell. The proliferative advantage of the leukemic stem cell, coupled with impairments in differentiation and inhibition of apoptosis, is thought to arise from acquired genetic alterations that lead to accumulation of immature or blast cells in the bone marrow. The blasts eventually suppress normal hematopoiesis and infiltrate other organs and tissues (Yagi et al, 2003 and Lacayo et al, 2004) Pathophysiology: The hematopoietic system produces appropriate levels of blood cells over an individual's life time through a careful balance of differentiation, proliferation and self-renewal. The acquisition of genetic and epigenetic alterations leads to deregulation of these processes and the development of acute leukemia. Numerous investigations have provided evidence that AML 3

6 arises from genetic mutations. It is not currently known for certain whether the mutations occur in the normal stem cells or in more differentiated myeloid cell progenitor, which then acquire stem cell-like features (Misaghian et al., 2009). The pathogenesis of AML is uncertain, but cytogenetic abnormalities are present in most patients. Chromosome translocations result in the formation of fusion proteins, which are a common pathway in leukemogenesis. New diagnostic tools, including fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), comparative genomic hybridization, and micro-array analysis, have improved the sensitivity of detection of genetic abnormalities and the ability to sub-classify AML and to detect minimal residual disease (Yunis et al., 1981). Because of the importance of cytogenetics in diagnosis and prognosis in AML, the World Health Organization (WHO) has incorporated cytogenetic findings into AML classification (Brunning et al., 2001). Genetic syndromes and toxic exposures contribute to the pathogenesis in some patients. The underlying pathophysiology consists of a maturation arrest of bone marrow cells in the earliest stages of development. This developmental arrest results in 2 disease processes. First; marked decrease in normal blood cells production, which results in varying degrees of anaemia, thrombocytopenia, and neutropenia. Second; the rapid proliferation of these cells, along with a reduction in their ability to undergo programmed cell death (apoptosis), results in their accumulation in the bone marrow, blood, and frequently the spleen and liver (Henderson et al., 2002). 4

7 Epidemiology: Acute leukemia are rare disease but have a disproportionately large effect on cancer survival statistics among children and younger adults. The reported frequency of leukemia increased in the first half of the twentieth century, began slowing in its rate of acceleration in the 1940 and has stabilized over the last 30 or 50 years. Improved diagnostic technology presumably accounts for this peculiar trend (Scheinberg et al., 2005) General incidence: Although the acute leukemia account for less than 2% of all cancer, these diseases are the first and second leading causes of death due to cancer in the United States in men and women, under 40 years of age. AML accounts for approximately 15% to 30% of acute leukemia in children and adolescents and 90% in adults. The overall annual incidence is 3.4 per 100,000 (Ries et al., 2002). In National Cancer Institute (NCI), Cairo University, there were 1300 cases of leukemia attending the NCI between January 2002 and December These cases account for 7% of all newly diagnosed proven malignant cases. AML accounts for approximately 41.5% of all 840 newly diagnosed acute leukemia and for 4.6% of all incident cancers (NCI Cancer Registry, 2004) Age incidence: The age-incidence of AML is subtly biomodal. Between early childhood and age 45, the annual incidence of AML remains constant at 0.8 cases/105 population. The incidence rises exponentially after the age of 45, exceeding 15 cases/105 populations by age 75. AML has been extensively 5

8 characterized using cytogenetic since the mid The increased incidence of AML in the elderly is probably related to improved diagnosis, the recognition of AML after MDS and longer life expectancy, resulting in increased environmental exposures (Tallman, 2005). In NCI, Cairo University the median age of AML patients was 22 years, with a range from less than 10 years and up to 80 years (NCI Cancer Registry, 2004) Sex incidence: The incidence of AML is higher in males than in females (1.3:1.0) (Scheinberg et al., 2005). In NCI, Cairo University, the male: female ratio in AML is 1.37:10 (NCI Cancer Registry, 2004) Geographical variations: For most types of cancers, striking differences in frequency exist between countries. This is less true for hematological malignancies, but some international variation is seen for each of the main diagnostic groups of leukemia. The leukemias seem to occur at the highest rates in certain areas of Canada (Quebec, Ontario, Saskatchewan and Yukon), among whites in the USA, Australia and Denmark. Leukemias are also relatively more common in other Western countries than registered in most parts of the world (Olsen, 2005). There is a somewhat lower incidence is seen in persons of Asian descent. An increase in the frequency of AML is seen in Jews, especially those of Eastern or European descent (Liesveld and Lichtman, 2005) Etiology: The precise molecular origins of AML are unknown. The patho-physiologic mechanisms are multiple, act in concert, and probably are distinct in different types 6

9 of AML. Environmental, inherited genetic and occupational factors play a role in the pathogenesis of AML (Linet and Devesa, 2002 and Deschler and Lubbert, 2006). Table 1 and 2 represented different environmental and genetic factors that contribute to AML. Table 1: Environmental factors contributing to Acute Myeloid Leukemia (Henderson et al., 2002). Solvents (benzene) Smoking Ionizing radiation Atomic bomb exposure Nuclear power exposure Medical radiation Non-ionizing radiation Chemotherapy Alkylating agents Topoisomerase II inhibitors Other drugs Chloramphenicol Phenylbutazone Table 2: Genetic Disorders Implicated in the Pathogenesis of Acute Myeloid Leukemia (Taylor and Birch, 1996). Congenital Defects Down syndrome Bloom syndrome Monosomy 7 syndrome Klinefelter syndrome Turner syndrome Neurofibromatosis Congenital dysmorphic syndromes Marrow Failure Syndromes Fanconi anemia Dyskeratosis congenital Schwachman-Diamond syndrome Amegakaryocytic thrombocytopenia Blackfan-Diamond syndrome Kostmann agranulocytosis Familial aplastic anaemia Familial platelet disorder 7

10 Diagnostic workup: Morphology: Microscopic examination of a properly prepared and stained bone marrow or peripheral blood smear remains the cornerstone of AML diagnosis. Morphologic findings can direct the appropriate selection of additional diagnostic tests. A- Peripheral blood: Blood counts vary widely among patients with AML. The leukocyte count is elevated in more than one half of patients but is more than 100,000 cells/mm 3 in less than 20%. Blasts usually are identified on peripheral smear; Auer rods and Phi bodies are considered pathognomonic of AML. Phi bodies are fusi-form or spindle-shaped rods similar to Auer rods and require special stains for hydro-peroxidases (Hanker et al., 1978). Cytopenias result from hematopoietic failure and contribute to symptoms and signs. Neutropenia is present in most AML patients. A normal neutrophil count is more common in patients with monocytic variants of AML and suggested that a correlation exists between the normal neutrophil count and maturation of the leukemia cells in AMoL (Medical Research Council Leukaemia Committee, 1975). Anaemia is common in AML and is predominantly normochromic and normocytic. Thrombocytopenia, which may be severe at diagnosis. Thrombocytosis is rarely identified (Goad and Gralnick, 1996). 8

11 B- Bone marrow examination: Bone marrow examination is a very important investigation for establishing diagnosis in hematological neoplasms. Bone marrow aspirate should be evaluated for cellularity, number and morphology of megakaryocytes, myeloid to erythroid ratio (M : E), cellular maturation, and presence of dysplasia or asynchronous maturation. The blast percentage should be determined on a 500-cell differential of the bone marrow aspirate. Iron stores should be assessed with Prussian blue stain and the presence or absence of ringed sideroblasts specifically noted. The original FAB classification recognized three types of myeloblasts: I, II, and III (Table 3) (Bennett et al., 1976). Table 3: Type of Blasts in Acute Myeloid Leukemia (Bennett et al., 1976). Myeloblast Features Type I Type II Type III Agranular basophilic cytoplasm, nucleus with fne chromatin, high nuclear/cytoplasmic ratio, two to four distinct nucleoli Granulated basophilic cytoplasm with few ( 20) azurophilic granules, nuclear features similar to those of type I blasts but lower nuclear-to-cytoplasmic ratio Heavily granulated basophilic cytoplasm with numerous (>20) azurophilic granules, nuclear features similar to those of type I blasts Cytochemistry: Myeloid cells exhibit characteristic staining profiles for esterase, myeloperoxidase (MPO), and periodic acid Schiff (PAS) reagent (Table 4) Hayhoe and Quaglino, 1988). 9

12 Table 4: Cytochemical stains used for diagnosis and classification of acute leukemia (Hayhoe and Quaglino, 1988). Cytochemical stain Specificity Stains primary and secondary granules of cells of myeloid/neutrophil lineage, eosinophil granules Myeloperoxidase (MPO) (granules appear solid), granules of monocytes, Auer rods Granules of normal mature basophils do not stain Stains primary and secondary granules of cells of myeloid/neutrophil lineage similar to MPO Eosinophil granules, granules of monocytes, Auer Sudan black B (SBB) rods are positive Basophil granules usually are negative but sometimes show metachromatic staining (red/purple) Stains neutrophil and mast cell granules Auer rods usually are negative except in acute Naphthol AS-D chloracetate myeloid leukemia associated with granules), some T esterase ( specific esterase) and B lymphocytes Abnormal eosinophils are positive α-naphthyl acetate esterase ( nonspecific esterase) α-naphthyl butyrate esterase ( nonspecific esterase) Periodic (PAS) Acid phosphatase acid Schiff Toluidine blue Perls stain, Prussian blue stain Monocytes and macrophages, megakaryocytes, platelets, most T lymphocytes, some T lymphoblasts (focal) Monocytes and macrophages Variable staining of T lymphocytes Neutrophil lineage (granular, increasing with maturation), leukemic promyelocytes (diffuse cytoplasmic), eosinophil cytoplasm but not granules, basophil cytoplasm (blocks), monocytes (diffuse plus granules), megakaryocytes, platelets (diffuse plus granules), erythroblasts positive in block-like pattern Negative in normal erythroid precursors Neutrophils, most T lymphocytes, T lymphoblasts (focal) Variable staining of eosinophils, monocytes, platelets Strong staining of macrophages, plasma cells, megakaryocytes, some leukemic megakaryoblasts Metachromatic granules in basophils, mast cells Hemosiderin, and iron in erythroblasts, macrophages occasionally, plasma cells 10

13 Chemistry profile: Blood chemistries are typically normal but in advanced disease, or in infiltrative cases such as with monocytic leukemias, so liver function tests and BUN/ceatinine level tests are necessary prior to the initiation of therapy. Hyper-uricemia has been noted in up to 50% of patients with AML and can also be associated with tumor lysis, although the latter is more common in all (O Regan et al., 1977). Appropriate hydration and administration of allopurinol can prevent complications of tumor lysis, which may occur during induction chemotherapy, most often in the setting of hyper-leukocytosis. Serum lactate dehydrogenase levels may be elevated, particularly in monocytic (M4/M5) subtypes, but to a lesser degree than is observed in ALL (Wiernik, 2003). Levels of lysozyme are elevated, particularly in variants of AML with a predominantly monocytic component, including AMoL and AMMoL. Excess lysozyme (muramidase) may cause proximal renal tubular damage, which results in hypokalemia (Pickering and Catovsky, 1973). Other factors that can contribute to hypo-kalemia in AML include potassium uptake by rapidly proliferating cells, as well as drugs, particularly antibiotics and diuretics. Hyper-kalemia can occur in association with hyperuricemia and tumor lysis. Patients with AML may have hyper-calcemia (Gewirtz et al., 1983), but hypo-calcemia is more common and may rarely be caused by acceleration of bone formation by leukemia cells (Schenkein et al., 1986). 11

14 Immunophenotyping: The availability of well-characterized antibodies classified under the uniform terminology of clusters of differentiation (CD) has expanded the application of flow cytometry and made phenotyping of AML more precise, reproducible, and comprehensive. Immuno-phenotyping now is routinely used to distinguish AML from ALL, in defining hybrid or bi-phenotypic leukemias and for lineage assignment (Table 5). Individual cells can be stained with multiple antibodies directly labeled with various fluorophores. Several "gating" strategies specifically identify blast cells by combining side (orthogonal) scatter and CD45 staining (Figure 1). This strategy identifies blasts by cytoplasm complexity (granularity) on the side scatter channel Y- axis and "brightness" of staining with CD45 on the X-axis, creating specific zones or "gates" of the most common morphologic variants of AML blasts. This strategy permits the gating of even a small group of blasts in a background of numerous more mature cells (Borowitz et al., 1993). 12

15 Table 5: Monoclonal antibodies commonly used for immunophenotyping of AML (Kita et al., 1992). Precursor antigen B lineage Immuno-histochemistry (Core Biopsy) CD34, CD34, TdT CD20, CD79a T lineage CD2, CD3, CD5, CD7 CD3, CD5, CD7 Flow Cytometry (Blood/Bone Marrow) CD45, TdT, CD34, HLA- DR CD19, CD20, CD22, CD79a, CD10 Myeloid lineage Monocyticmonocytic Erythroid Megakaryocytic CD13, CD33, CD117, CD15 CD14, CD4, CDllb, CDllc, CD64, CD36 Glycophorin A CD41, CD42, CD61 (cytoplasmic) Myeloperoxidase, CD117, CD15 Lysozyme, CD68 Glycophorin A, hemoglobin A von Willebrand factor, factor VIII 13

16 Fig. 1: Side scatter versus CD45 flow cytometry ploy with approximate areas of "gates" fig Fig. 1: Side scatter versus CD45 flow cytometry ploy with approximate areas of gates where myeloblasts of the FAB acute myeloid leukemia subtypes usually are positioned. Gates are named according to the predominant normal peripheral blood cell population that normally inhabits them. Side scatter is a measure of "cytoplasm granularity," and CD45 represents the log of the intensity of staining with a common leukocyte antigen antibody (Borowitz et al., 1993) Cytogenetics and molecular genetics: AML is a very heterogeneous disease regard to clinical features and acquired genetic alterations, both those detectable microscopically as structural and numerical chromosome aberrations, and those detected as submicroscopic gene mutations and changes in gene expression. At present, cytogenetic aberration detected at the time of AML diagnosis constitute the most common basis predicting clinical outcome (Mròzek and bloomfield, 2006). 14

17 Several chromosomal rearrangements, and the molecular abnormalities that they produce, identify distinct clinical subgroups with predictable clinical features and therapeutic responses. Recurrent patterns of numerical changes (gain and loss) or structural changes (translocation, inversion, deletion and duplication) have been identified and found to be characteristic of specific subtypes of AML (Mecucci and Hagemeijer, 2005). The pathogenesis of AML involves an array of molecular alterations that disrupt almost every facet of cell transformation. These processes include the inappropriate proliferation in the absence of normal growth signals, indefinite self-renewal in a manner analogous to a stem cell, escape from programmed cell death, and inhibition of differentiation, aberrant cell cycle checkpoint control, genomic instability and multi-organ dissemination of leukemic cells. These properties may be directly linked to focal genetic lesions in AML and represent discrete processes amenable to therapy. However, may of these properties are intertwined and result from integration of multiple anomalies in the genetic program of the leukemic cell (Licht and Sternberg, 2005). The purpose of a cytogenetic investigation is to determine whether the karyotype of the affected cells is abnormal and if so, in what way. Different techniques are used: (1) classical karyotype analysis using banding techniques; (2) molecular cytogenetic, e.g fluorescence in situ hybridization (FISH), multicolor FISH and spectral karyotyping (SKY); (3) molecular techniques to analyze DNA, RNA or proteins directly, e.g. polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), quantitative real-time PCR (RQ-PCR), southern blotting and micro-array analysis (Gorczyca, 2006). 15

18 Classification: The French-American-British (FAB) classification of AML has been widely used since it was described in 1976, pathologists chose to use 30% blasts as the threshold between high-grade myelodysplasia (MDS) and AML, this classification is a lineage-based system that defines the categories primarily on morphologic and cytochemical framework as shown in Table 6 (Bennett et al., 1985). Table 6: FAB classification (Bennett et al., 1985). FAB Classification M0: Undifferentiated leukemia M1: Myeloblastic leukemia without differentiated M2: Myeloblastic leukemia with differentiated M3: Promyelocytic leukemia M4: Myelomonocytic leukemia With marrow eosinophilia (M4Eo) M5: Monoblastic leukemia Monoblastic without differentiated (M5a) Monoblastic differentiated (M5b) M6: Erythroleukemia M7: Megakaryoblastic leukemia However immunophenotyping and other studies have been added in updated versions. A World Health Organization classification was proposed recently and incorporates the FAB classification while adding several distinctive cytogenetic categories of AML as shown in Table 7 (Vardiman et al., 2008). The 2 most significant differences between WHO and FAB classification are: 1. Reduction in the percentage of blasts required for a diagnosis of AML in the WHO classification from 30% to 20% blasts in PB or BM. 2. The categorization of cases of AML into unique clinical and biologic subgroups is present in the WHO classification (these patients should be considered to have AML regardless of the blast percentage). 16

19 Table 7: Acute myeloid leukemia and related myeloid neoplasms (the WHO classification) (Vardiman et al., 2008). I-Acute myeloid leukemia (AML) with recurrent genetic abnormalities AML with t(8;21)(q22;q22), RUNX1-RUNX1T1 AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22), CBFB-MYH11 Acute promyelocytic leukemia (APL) with t(15;17)(q22;q12), PML-RARA AML with t(9;11)(p22;q23); MLLT3-MLL AML with t(6;9)(p23;q34); DEK-NUP214 AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2): RPN1-EVI1 AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1 Provisional entity: AML with mutated NPM1 Provisional entity: AML with mutated CEBPA II-Acute myeloid leukemia with myelodysplasia-related changes III-Therapy-related myeloid neoplasms IV-Acute myeloid leukemia, not otherwise specified AML with minimal differentiation AML without maturation AML with maturation Acute myelomonocytic leukemia Acute monoblastic/monocytic leukemia Acute erythroid leukemias Pure erythroid leukemia Erythroleukemia, erythroid/myeloid Acute megakaryoblastic leukemia Acute basophilic leukemia Acute panmyelosis with myelofibrosis V-Myeloid sarcoma VI-Myeloid proliferations related to Down syndrome Transient abnormal myelopoiesis Myeloid leukemia associated with Down syndrome VII-Blastic plasmacytoid dendritic cell neoplasms 17

20 Prognosis: Clinical features, morphology, surface markers, and cytogenetics are combined to describe clinicopathologic syndromes in AML, and prognosis is usually determined by a combination of specific factors. A single factor can not reliably predict prognosis but must be correlated with all available information, Table 8 summarized the prognostic factors affecting AML (Buchner and Heinecke, 1996). Table 8: Prognostic Factors in Acute Myeloid Leukemia (Buchner and Heinecke, 1996). Factor Favorable Unfavorable Clinical Age <45 y <2 y, >60 y ECOG performance status Leukemia 0 1 >1 De novo 18 Antecedent hematologic disorder; myelodysplasia, myelo-proliferative disorder Infection Absent Present Prior chemotherapy No Yes Leukocytosis <25,000/mm 3 >100,000/mm 2 Serum LDH Normal Elevated Extramedullary disease Absent Present CNS disease Absent Present Cytoreduction Rapid Delayed Morphology Auer rods Present Absent Eosinophils Present Absent Megaloblastic erythroids Absent Present Dysplastic megakaryocytes Absent Present FAB type M2, M3, M4 M0, M6, M7