Expression of Phosphorylated STAT5 in Acute Myeloid Leukemia

Transcription

1 Expression of Phosphorylated STAT5 in Acute Myeloid Leukemia Thesis Submitted for Partial Fulfillment of Master Degree in Clinical and Chemical Pathology By Engy Mohamed Sabry M.B.,B.Ch. (Cairo University) Supervisors Prof. Dr. Iman Mansour Professor of Clinical Pathology Faculty of Medicine Cairo University Prof. Dr. Heba Hassan Abou-Elew Professor of Clinical Pathology Faculty of Medicine Cairo University Faculty of Medicine Cairo University 2012

2 Acknowledgements First and foremost, thanks are due to ALLAH, the beneficent and merciful. This study would not have been possible without the continuous support and encouragement of so many people. As a person usually too full of words, I find myself overwhelmed in offering them all my thanks. In the first place I would like to record my gratitude to Prof. Dr. Iman Mansour Professor of Clinical and Chemical Pathology, Faculty of Medicine, Cairo University; whose encouragement and knowledge have supported me over the whole way of this research. I am much indebted to her for her cooperation, advice, guidance and kind supervision. I am greatly thankful to Prof. Dr. Heba Hassan Abou-Elew Professor of Clinical and Chemical Pathology, Faculty of Medicine, Cairo University; for her scientific supervision and guidance. Her willingness to motivate me contributed tremendously to the study. She always kindly granted me her time, guidance, advice and valuable comments. Furthermore, an honorable mention goes to my family and friends for their understanding and support in completing this thesis. At last but certainly not least my special thanks to my patients and their families for their cooperation without which this work would have never been accomplished and asking GOD to help them and giving them good quality of life. Engy Mohamed Sabry

3 ABSTRACT Background: AML is characterized by maturation arrest of a malignant clone of myeloid cells. Signal transducer and activator of transcription 5 (STAT5) is a transcription factor that regulates many aspects of cell growth, survival and differentiation. Constitutive activation of STAT5 has been identified in a number of hematopoietic malignancies including AML. Aim: The aim of this study was to assess the protein expression of phosphorylated-stat5 in leukemic blasts of newly diagnosed AML patients to correlate its expression with the clinical outcome in these patients. Subjects and Methods: Thirty patients suffering from de novo AML were included in the study. Assessment of phosphorylated-stat5 protein in AML blasts of bone marrow aspirate/peripheral blood smear films was performed by immunocytochemistry using rabbit anti-phospho-stat5a/b (Y694/Y699) and fluorescence-labelled anti-rabbit IgG antibodies. Results: Expression of total p-stat5 was detected in 23/30 (76.7%) of cases. Expression of cytoplasmic and nuclear p-stat5 was detected in 24/30 (80%) and in 7/30 (23.3%) of cases, respectively. Median blast count percentage in peripheral blood and bone marrow at presentation was significantly higher in the total p-stat5 positive AML patients than the p- STAT5 negative AML patients (median 30% vs. 10%, P=0.028 and median 60% vs. 45%, P=0.048; respectively). No statistically significant difference was found between the response to induction therapy and p-stat5 expression. Conclusion: The present study confirmed the expression of total phosphorylated-stat5 in about two-thirds of newly diagnosed AML cases. Cytoplasmic p-stat5 was detected in a larger set of AML patients than nuclear p-stat5. No statistically significant impact of p-stat5 expression was encountered on the clinical outcome in these patients. Key words: AML, phosphorylated STAT5, nuclear p-stat5, cytoplasmic p-stat5, FLT3.

4 Contents Pages List Abbreviations List of Tables List of Figures I IV VI Introduction & Aim of Work. 1 Review of Literature. 4 subjects & Methods. 60 Results 68 Appendix 91 Discussion. 94 summary. 102 Conclusion & Recommendations 105 References. 106 Arabic Summary.

5 List of Abbreviations ABL Abelson gene AIDS Acquired immunodeficiency syndrome ALL Acute lymphoblastic leukemia AML Acute myeloid leukemia AMML Acute myelomonocytic leukemia APL Acute Promyelocytic Leukemia (M3) ATRA All trans- retinoic acid BAALC Brain and acute leukemia cytoplasmic gene BCR Breakpoint cluster region BMA Bone marrow aspirate BMSC bone marrow stromal cell BMT Bone marrow Transplantation BUN Blood urea nitrogen CBC Complete blood count CBFB Core Binding Factor Beta gene CD Cluster of differentiation CEBPA CCAAT/enhancer-binding protein α CLL Chronic lymphocytic leukemia CXCR4 CXC chemokine receptor 4 DIC Disseminated Intravascular Coagulation DN Dominant-negative DNA Deoxyribonucleic acid EDETA Ethylene diamine tetra acetic acid EPO Erythropoietin ETO Eight-Twentyone gene EVI1 Ecotropic virus integration-1 FAB French American British FISH Fluorescence Insitu Hybridization FLT3 Fetal liver Tyrosine Kinase 3 G-CSF Granulcoytoe colony stimulating factor GH Growth Hormone I

6 GM GvHD GvL HB HLA-DR HNSCCs HSC HTLV-I IFN IHC IL ITD JAKs LCL LDH M3v M4Eo MAP MAPK MDR-1 MDS MGG MLL MPD MPO MYH11 NPM1 ODNs PAS PCR PI3K PML PRL Granulocyte monocyte Graft-versus-Host Disease Graft versus Leukemia Haemoglibin Human leucocyte antigen class II Human head and neck squamous cell carcinomas Hematopoietic stem cell Human T-cell lymphotrophic virus type I Interferon Immunohistochemical staining InterLeukin internal tandem duplication Janus family tyrosine kinases Cherry lymphoblastoid cells Lactate dehydrogenase M3 variant M4 associated with Eosinophilia Mitogen-activated protein Mitogen-activated protein kinase Multi Drug Resistance-1 Myelodysplastic syndrome May Grunwald-Giemsa Mixed lineage Leukemia gene Myeloproliferative Disorders Myeloperoxidase Smooth Muscle Myosine Heavy Chain 11gene Nucleophosmin1 Oligodeoxynucleotides Periodic Acid Schiff Polymerase Chain Reaction Phosphatidylinositol 3 kinase Promyelocytic leukemia Prolactin Hormone II

7 P value Probability value RARα Retinoic Acid Receptor α gene RB1 Retinoblastoma1 RBM15 RNA-binding protein 15 RT Reverse Transcription RUNX1 Runt-related transcription factor 1 SBB Sudan Black B SD Standard Deviation STAT Signal transducer and activator of transcription STAT3 Signal transducer and activation of transcription 3 STAT5 Signal transducer and activation of transcription 5 STK-1 Stem cell tyrosine kinase TdT Terminal deoxynucleotide transferase TLC Total leucocytic count TPO Thrombopoietin WBC White Blood Cells WHO World Health Organization III

8 List of Tables Tables Pages 1 Cytochemistry for AML 18 2 The most common immunological markers used in immunophenotypic analysis of AML cases 3 Cytogenetic/Molecular genetic entities not included in the WHO classification of AML 4 Summary of clinical relevance of genetic abnormalities in AML Prognostic factors of AML 33 6 STAT chromosomal localization 40 7 Phenotypes of STAT knock-out mice 41 8 STATs structure 43 9 STAT3 isoforms Age and sex distribution of AML patients Clinical data of AML patients Peripheral blood data of AML patients FAB subtypes of AML in the studied patients Response to induction therapy in the studied AML patients 15 Results of phosphorylated-stat5 and CD34 expression in the studied AML patients IV

9 List of Tables (Cont.) 16 Blast percentage showing cytoplasmic p-stat5 expression in the studied AML patients 17 Blast percentage showing nuclear p-stat5 expression in the studied AML patients 18 Comparison between p-stat5 negative and total p- STAT5 positive AML patients regarding clinical parameters 19 Comparison between p-stat5 negative and total p- STAT5 positive AML patients regarding hematological parameters 20 Relation between CD34 positivity and p-stat5 expression in the studied AML patients 21 Relation between AML FAB subtype and p-stat5 expression in the studied AML patients 22 Relation between phosph-stat5 expression and response to induction therapy in the studied AML patients 23 Blast percentage showing cytoplasmic and nuclear p- STAT5 expression with respect to CD34 status in the studied AML patients 24 Blast percentage showing cytoplasmic and nuclear p- STAT5 expression with respect to AML FAB subtype in the studied AML patients 25 Blast percentage showing cytoplasmic and nuclear p- STAT5 expression with respect to induction response in the studied AML patients V

10 List of Figures Figures Pages 1 JAK-STAT signal transduction pathway 39 2 Structure and functional domains of STAT molecules Response to induction therapy in the studied AML patients 4 Results of total p-stat5 expression in the studied AML patients. 5 Blast percentage showing cytoplasmic p-stat5 expression in the studied AML patients 6 Blast percentage showing nuclear p-stat5 expression in the studied AML patients 7 Bone marrow aspiration smear showing mild positivity (1+) of nuclear phophorylated-stat5 (40X) 8 Bone marrow aspiration smear showing profound positivity (3+) of nuclear phophorylated-stat5 (20X) 9 Bone marrow aspiration smear showing mild positivity (1+) of cytoplasmic phophorylated-stat5 (40X) 10 Bone marrow aspiration smear showing moderate positivity (2+) of cytoplasmic phophorylated-stat5 (20X) 14 Comparison of peripheral blood blast % between AML cases with total positive p-stat5 and negative p- STAT5 expression 12 Comparison of bone marrow blast % between AML cases with total positive p-stat5 and negative p- STAT5 expression 11 Relation between total p-stat5 expression and response to induction therapy in the studied AML patients (P>0.05) VI

11 Introduction & Aim of the Work Introduction The signal transducers and activators of transcription (STAT) gene family consist of seven proteins. STAT mediated signal transduction pathways influence neoplastic processes on a broad basis by perturbing control over survival, differentiation, proliferation, and apoptosis (Krebs and Hilton, 2001). STAT5 is one of seven members of the STAT family of transcription factors. Aberrant activation of STAT5 has been demonstrated in diverse groups of neoplasms, including AML. Activation of STAT is caused by tyrosine phosphorylation followed by the consecutive translocation of p-stat into the nucleus. In normal hematopoiesis, STAT5 is activated by receptor ligation of Fms-like tyrosine kinase 3 (FLT3) to its ligand as well as by several other cytokines as IL3, GM-CSF and IL5. STAT5 is considered the preferred second messenger of FLT3-mediated signaling (Meier et al., 2009). FLT3 and its downstream STAT-related pathways play a fundamental role in AML. Expression of p-stat5 is significantly correlated to FLT3-internal tandem duplications (FLT3-ITD) in myeloid blasts. Strong adverse prognostic impact of FLT3-ITD in AML was reported in literature, with an association with an adverse clinical outcome with poor overall survival and a higher risk of relapse, particularly in AML with normal karyotypes. Therefore, assessment of FLT3 mutations as well as addressing their functional significance by measurement of p-stat5 might be important (Kottaridis et al., 2001). Analysis of p-stat has been attempted by different methodologies such as electrophoretic mobility shift assays (Xia et al., 1998), western blot analysis (Spiekermann et al., 2002), flowcytometry - 1 -

12 Introduction & Aim of the Work (Pallis et al., 2003), immunofluorescence (Bodo et al., 2009) and immunohistochemistry (Obermann et al., 2010). Interestingly, the percentage of cases with p-stat expression varied between 7% and 95%. Some discrepancies can probably be attributed to the applied methodologies as well as the sensitivity and specificity of the antibodies. Assessment of STAT5 gene expression by quantitative PCR would not be helpful as well since the phosphorylated (active) protein amount and its microtopographic distribution is of relevance.this raises the question of whether immunocytochemistry of peripheral blood or bone marrow smears, which has the potential for delivering reproducible evaluation of cellular protein levels, would be a suitable methodology for the measurement of phosphorylated-stat5 in AML

13 Introduction & Aim of the Work Aim of the Work The aim of this study was to assess the protein expression of phosphorylated-stat5 in leukemic blasts of newly diagnosed AML patients by immunocytochemistry using rabbit anti-phospho-stat5a/b (Y694/Y699) and fluorescence-labelled anti-rabbit IgG antibodies to correlate its expression with the clinical outcome in these patients

14 Review of Literature Acute Myeloid Leukemia Acute leukemias are clonal malignant disorders resulting from genetic alterations in hematopoietic stem cells that limit the ability of stem cells to differentiate into red cells, granulocytes, and platelets, and lead to the proliferation of abnormal leukemic cells or blasts. Acute myeloid leukemias (AML), also referred to as acute nonlymphocytic leukemias, are heterogeneous disorders. Most AML subtypes are distinguished from other related blood disorders by the presence of more than 20% blasts in the bone marrow. It is characterized by the rapid growth of abnormal white blood cells that accumulate in the bone marrow and interfere with the production of normal blood cells (Smith et al., 2004). Epidemiology Approximately 11,000 new cases of AML are diagnosed annually in the United States, with an overall annual incidence of 3.4 new cases per 100,000 people (Jemal et al., 2008). The incidence of AML is higher in males than in females and in whites than in blacks. The incidence of AML increases with age; the median age at diagnosis is 63 years. AML accounts for about 90% of all acute leukemias in adults, but is rare in children (Jemal et al., 2002). Congenital leukemia is usually AML rather than ALL (acute lymphoblastic leukemia) and is often monocytic, with high incidence of extramedullary disease particularly involving the skin and the central nervous system (Greer et al., 2004)

15 Review of Literature Etiology Although several factors have been implicated in the causation of AML, most patients who present with de novo AML have no identifiable risk factor. Risk factors for AML (Lichtman and Liesveld, 2001): 1- Environmental factors - Radiation - Benzene - Alkylating agents and cytotoxic drugs 2-Acquired diseases A- Clonal hematopoietic diseases Chronic myelogenous leukemia Idiopathic myelofibrosis Primary thrombocythemia Polycythemia vera Acquired sideroblastic anemia Paroxysmal nocturnal hemoglobinuria B- Other hematopoietic diseases Aplastic anemia 3-Inherited conditions o Down's syndrome o Bloom's syndrome o Ataxia telangiectasia o Dyskeratosis congenita o Combined immunodeficiency syndrome o Neurofibromatosis and Schwachman-Diamond syndrome - 5 -

16 Review of Literature Pathophysiology The malignant cell in AML is the myeloblast. In normal hematopoiesis, the myeloblast is an immature precursor of myeloid white blood cells; a normal myeloblast will gradually mature into a mature white blood cell. However, in AML, a single myeloblast accumulates genetic changes and prevents differentiation. Such a mutation alone does not cause leukemia; however, it is combined with other mutations which disrupt genes controlling proliferation, the result is the uncontrolled growth of an immature clone of cells, leading to the clinical entity of AML (Fialkow et al., 1991). The underlying pathophysiology in AML consists of a maturational arrest of bone marrow cells in the earliest stages of development, it involves the activation of abnormal genes through chromosomal translocations and other genetic abnormalities. This developmental arrest results in 2 disease processes. First, the production of normal blood cells markedly decreases, which results in varying degrees of anemia, 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 (Smith et al., 2004). A well-accepted concept states that for an AML to develop, at least two broad mutations are necessary, referred to as Class I and Class II mutations. Class I mutations, including RAS and FLT3 mutations, activate signal-transduction pathways and confer a proliferation advantage to hematopoietic cells. Class II mutations complement Class I mutations and affect transcription factors and serve primarily to impair hematopoietic differentiation. Other molecular mechanisms have been - 6 -

17 Review of Literature implicated in leukemogenesis. For example, gene amplification may result in gene overexpression. In addition, numerical gains or losses of chromosomes, such as trisomy or monosomy as a result of nondisjunction, are detected in a large subset of acute and chronic leukemias. Gene deletions, such as those arising from partial chromosomal deletions or unbalanced translocations, may result in tumor-suppressor gene inactivation or loss. Hypermethylation is another mechanism of gene inactivation. Often more than one of these mechanisms is involved in leukemogenesis, leading to the accumulation of genetic lesions that culminates in leukemogenesis or subsequently contributes to disease progression (Gilliland et al., 2002). Clinical Presentation Most signs and symptoms of AML are caused by the replacement of normal blood cells with leukemic cells. A lack of normal white blood cell production makes the patient susceptible to infections; while the leukemic cells themselves are derived from white blood cell precursors, they have no infection-fighting capacity. A drop in red blood cell count (anemia) can cause fatigue, paleness, and shortness of breath. A lack of platelets can lead to easy bruising or bleeding with minor trauma. The early signs of AML are often vague and non-specific, and may be similar to those of influenza or other common illnesses. Some generalized symptoms include fever, fatigue, weight loss or loss of appetite, shortness of breath, anemia, easy bruising or bleeding, petechiae (flat, pin-head sized spots under the skin caused by bleeding), bone and joint pain, and persistent or frequent infections (Hoffman et al., 2005)

18 Review of Literature Enlargement of the spleen may occur in AML, but it is typically mild and asymptomatic. Lymph node swelling is rare in AML, in contrast to acute lymphoblastic leukemia. The skin is involved about 10% of the time in the form of leukemia cutis. Rarely, Sweet's syndrome, inflammation of the skin, can occur with AML. Some patients with AML may experience swelling of the gums because of infiltration of leukemic cells into the gum tissue. Rarely, the first sign of leukemia may be the development of a solid leukemic mass or tumor outside of the bone marrow, called a chloroma. Occasionally, a person may show no symptoms, and the leukemia may be discovered incidentally during a routine blood test (Abeloff, et al., 2004). Hyperleukocytosis (more than 100,000 white cells per cubic millimeter) can lead to symptoms of leukostasis, such as ocular and cerebrovascular dysfunction or bleeding. There may also be metabolic abnormalities (e.g., hyperuricemia and hypocalcemia), although these are rarely found at presentation (Ryan, 1992). The incidence of CNS disease at diagnosis is difficult to determine because lumbar puncture is not always performed. Meningeal disease has been reported to develop in 5 to 20% of children and up to 16% of adults with AML (Golub and Arceci, 2002). Cardiac abnormalities are usually related to electrolyte imbalances, particularly hypokalemia, but may result from direct involvement of the conduction system or infiltration of vessel walls (Wiernik, 2001). Pulmonary symptoms occur in patients with leukostasis, infections related to neutropenia, or hemorrhage due to thrombocytopenia. Gastrointestinal symptoms also include infections, particularly perirectal abscesses and typhlitis, a necrotizing colitis related to leukemia - 8 -

19 Review of Literature infiltration of the bowel Wall (Keidan et al., 1989). Obstructive jaundice has occurred secondary to granulocytic sarcoma, and patients with AML rarely present with hepatic failure (Wiernik, 2001). Morphological classification Light microscopy supplemented by cytochemistry is the first method for the diagnosis of AML and its further sub-classification. Examination of bone marrow and peripheral blood specimens stained with Wright Giemsa or May Grunwald-Giemsa (MGG) stain provides a rapid initial and frequently conclusive diagnosis. In AML, blasts are classified as myeloblasts, monoblasts, erythroblasts, or megakaryoblasts. Three major types of myeloid blasts have been reported in the literature. Type I myeloblasts have fine nuclear chromatin, 2 4 distinct nucleoli, and a moderate rim of pale to basophilic cytoplasm without azurophilic granules. Type II myeloblasts have nuclear and cytoplasmic features similar to type I myeloblasts, with the addition of up to 20 delicate azurophilic granules in the cytoplasm. Type III myeloblasts have numerous azurophilic granules in the cytoplasm. However, distinguishing between type III myeloblasts and promyelocytes is somewhat arbitrary and may even be impossible (Konoplev and Bueso-Ramos, 2010). One characteristic feature of myeloblasts in AML is the presence of Auer rods, which are seen in 60 70% of all cases and represent abnormal azurophilic granules. Blasts are present in a background of other lineages appearing in varying proportion and degree of maturation. In some cases this can include mature eosinophils and basophils. Evidence of trilineage dysplasia is noted in 10-15% of de novo cases (Vardiman et al., 2002)

20 Review of Literature In some cases of AML, blasts exhibit any of several unusual morphological features. For example, marked nuclear lobulation is seen in cases with the t(15;17)(q22;q21) abnormality. Multinucleation is seen in many types of blasts, especially erythroblasts and megakaryoblasts. Pseudo-Chédiak-Higashitype giant granules result from fusion of primary granules and are occasionally seen in leukemic blasts and granulocytic precursors, especially in cases with the t(8;21) abnormality. Erythrophagocytosis is seen most commonly in cases of AML with monocytic differentiation, especially in cases with the translocation t(8;16). Some myeloblasts have prominent vacuolization, with an appearance similar to the L3 blasts of ALL. To fulfill the diagnostic criterion of 20% blasts established for AML, in some cases it is necessary to combine real blasts with so-called blast equivalents. The term blast equivalent is specific to a particular type of AML. For example, the neoplastic promyelocyte is considered a blast equivalent in cases with the t(15;17)(q22;q21) abnormality, or FAB M3 AML, and has an eccentric, often folded and lobulated nucleus with slightly condensed nuclear chromatin, intense cytoplasmic granularity, and an apparent Golgi zone. The promonocyte is considered a blast equivalent in cases of AML with monocytic differentiation (Konoplev and Bueso-Ramos, 2010). For more than 25 years, the classification system for AML was based on the French American British (FAB) group criteria established in This classification was updated in 1985 and recommended a 500- cell count with accurate morphological and cytochemical quantitation of lineage commitment and differentiation (Bennett et al., 1985)