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Clonal Expansion of B and T Lymphocytes Defines a Spectrum of Monoclonal Lymphocytosis

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Clonal Expansion of B and T Lymphocytes Defines a Spectrum of Monoclonal Lymphocytosis Clonal Expansion of B and T lymphocytes Defines a Spectrum of Monoclonal Lymphocytosis By Sadaf Shaukat Memon A thesis submitted in conformity with the requirements for the degree of Master of Science Laboratory Medicine & Pathobiology University of Toronto © Copyright by Sadaf Shaukat Memon 2011 Clonal Expansion of B and T lymphocytes Defines a Spectrum of Monoclonal Lymphocytosis Sadaf Shaukat Memon Master of Science Laboratory Medicine & Pathobiology University of Toronto 2011 ABSTRACT Monoclonal B lymphocytosis (MBL) has been recognized as a novel diagnostic condition. This study aims at the identification of clonal lymphocytosis in the patients with asymptomatic lymphocytosis. A total of 203 patients were evaluated for clonal B and T lymphocytosis by using flow cytometry and multiplex-PCR. Among them clonal B- or T-cells were detected in 54.2% of the cases, of which 38.4% were clonal B-cells and 15.8% were clonal T-cells cases. By immunophenotype, MBL was classified into the chronic lymphocytic leukemia (CLL) type (21.7%) and non-CLL-type (7.4%). Flow cytometry analysis and cell counts were used to determine the size of clonal population, and the data indicate that MBL and CLL are present in a continuous spectrum of clonal expansion. The findings may contribute to the current understanding of MBL and evaluation of incidental lymphocytosis. Further studies are required to evaluate clonal progression as a precursor stage of lymphoid malignancy. ii ACKNOWLEDGEMENTS I would first like to express my sincere gratitude to my mentor and research supervisor Dr. Chen Wang for his continuous support throughout the term of this project. His guidance, enthusiasm and immense knowledge motivated me to pursue further knowledge and improve my skill set in science. I whole heartedly thank my committee members Dr. B. Fernandes, Dr. M. Minden, Dr. K. Pritzker for their kind guidance, suggestions and critiques. You are leading minds and I will always be indebted for your kind advice and directions during the completion of my thesis project. I also wanted to thank G.S.Yang for teaching me PCR techniques and providing me with technical support on my project work and my fellow lab members especially Jianing Zheng for teaching me cell culturing techniques and Edward Parker for his help in editing my thesis writing. I would pay my sincere gratitude to the department of LMP and U of T for giving me this opportunity to explore this wonderful field of science. I really feel proud to be a part of this wonderful program. I would like to thank my Dad and Mom for their boundless efforts and love in bringing me up and making me the person I am today. Special thanks also goes to my mom for taking care of my sweet little one year old daughter, without her kind support this task would have been very difficult. I would also like to thank my brother for his help in editing and formatting my thesis, and my sister for criticizing my presentations. Finally I would thank my loving husband for always standing beside me at each and every step of my life. iii TABLE OF CONTENTS List of Abbreviations………………………………………………………………...………….viii List of Tables……………………………………………………………………………………..vi List of Figures……………………………………………………………………………......…..vii Abstract………………………………………………………………...........................……….....ii CHAPTER: I Clonal lymphocytosis and its relevance to lymphocytic neoplasia…….....…1 1 Clonal lymphocytosis and discovery of MBL………………………………………………..1 2 MBL as a novel diagnostic entity…………………………………………………………….3 2.1 Clinical and population-screening MBL………………………………………….…8 2.2 CD5-ve MBL (MLUS)……………………………………………………………...9 3 CD5+ MBL as a precursor condition of CLL……………………………………………….11 3.1 Prognostic factors of CLL……………………………………………………….…14 4 Clonal T-Lymphocytosis and T-cell lymphoproliferative disorders………………………..17 4.1 T-cell immunophenotype & T-cell receptor (TCR)……………………………...17 4.2 Precursor T-cell neoplasms and mature/peripheral T-cell neoplasms……….......20 4.2.1 Precursor T-cell neoplasm……………………………………….…….20 4.2.2 Mature/peripheral T-cell neoplasms…………………………….……..21 CHAPTER: II Laboratory methods for the detection of clonal lymphocytosis………...…24 1 Overview……………………………………………………………………………………..24 2 Flow cytometry……………………………………………………………………………....26 3 Molecular clonality tests…………………………………………………………………..…28 iv 3.1 Southern blot………………………………………………………………………....29 3.2 Polymerase Chain Reaction (PCR)…………………………………………………..29 3.3 BIOMED-2 multiplex PCR……………………………………………………..……30 CHAPTER: III Identification of clonal B and T lymphocytes among patients with peripheral lymphocytosis………………………………………………………………………34 1. Introduction………………………………………………………………………………..34 1.1 Rationale and hypotheses…………………………………………………………..35 2. Patients & Methods……………………………………………………………..…………...36 2.1 Patients…………………………………………………………………………..…36 2.2 Flow cytometry analysis of B & T-lymphocytes……………………………….….37 2.3 PCR amplification for IgH, TCR-β and TCR-γ genes………………………….....40 2.3.1 DNA extraction……………………………….............................................40 2.3.2 Primers design for IgH, TCR-β and TCR-γ and multiplex PCR………..…40 2.3.3 PCR product analyses…………………………………………..………….42 3. Results…………………………………...……………………………………………….47 3.1 CD5+ MBL and CLL……………………………………………..……...…………50 3.2 CD5- MBL…………………………………………………………………...…….51 3.3 T-cell clonal……………………………………………………………….……….52 3.4 Comparison of PCR and flow cytometry for B-cell clonality detection…………...53 3.5 The presence of clonal T-cells in Monoclonal B lymphocytosis…………………...53 4 Discussions…………………………………………………………………………….….54 CHAPTER: IV Conclusion & Future Studies…………………………………...…….…....59 REFERENCES………………………………………………………………………………….62 v LIST OF TABLES 1. Table 1: Prevalence of MBL in population studies by using flow cytometry ….…….…5 2. Table 2: Primer numbers in each PCR reaction tube………………………………….…41 3. Table 3: The primer design and multiplex combinations of the standardized BIOMED-2 multiplex PCR protocol ………………………………...……………………………….42 4. Table 4: Summary of 203 patients with asymptomatic peripheral lymphocytosis ……...49 5. Table 5: T-cell clonal cases characteristics………………………………………………53 6. Table 6: The presence of clonal T-cells in MBL ………………………………………..53 7. Table 7: Comparison of PCR and flow cytometry for B-cell clonality detection…….…54 vi LIST OF FIGURES 1. Figure 1: PCR analysis of IgH (VH-JH) rearrangements and BIOMED-2 PCR primers.................................................................................................................................31 2. Figure 2: TCR-β and TCR-γ gene complex and primers…………………………..……...32 3. Figure 3: Detection of non-clonal lymphocytosis & B-lymphocyte clonality by Flow- Cytometry Figure 5: Typical negative patterns for TCR- β, A, B and C…………………39 4. Figure 4: Gel electrophoresis of the PCR products derived from amplification at the IgH locus in clonal and polyclonal controls of well-defined clonality status………………….43 5. Figure 5: Size of PCR products and a typical GeneScan image for VDJ of IgH gene.…..44 6. Figure 6: Positive and negative image patterns for VDJ of IgH gene……………….……45 7. Figure 7: Typical Negative Patterns for TCR- β, A, B and C………………….…….….46 8. Figure 8: Typical Positive Pattern for TCR-β, A, B and C……………………………….46 9. Figure 9: Distribution of lymphocyte counts CLL-type MBL & CLL…………………....51 10. Figure 10: Distribution of lymphocyte counts of CD5-MBL cases……………………….52 vii LIST OF ABBREVIATIONS ALC: Absolute lymphocyte counts ADCC: Antibody dependent cytotoxicity ATLL: Adult T-cell leukemia/lymphoma B-ALC: Total B-cell count C-ALC: Total clonal B- cells CLL: Chronic lymphocytic leukemia CD: Cluster of Differentiation CC: Colorectal Carcinoma D: Diversity EBV: Epstein - Barr virus FISH: Fluorescence in situ hybridization FAP: Familial Adenomatous Polyposis HTLV-1: Human T-cell leukemia virus type 1 Ig: Immunoglobulin IgH: Immunoglobulin heavy chain IGHV: Immunoglobulin heavy variable gene IWCLL: International Workshop on CLL J: Joining viii κ/ λ: Kappa/Lambda LDT: Lymphocyte doubling time LPDs: Lymphoproliferative disorders MBL: Monoclonal B-cell lymphocytosis MHC: Major histocompatibility complex MF: Mycosis fungoides MGUS: Monoclonal gammopathy of undetermined significance MLUS: Monoclonal lymphocytosis of undetermined significance MM: Multiple Myeloma NCI: National Cancer Institute NHL: Non-Hodgkin lymphoma PB: Peripheral blood PBS: Phosphate buffered saline RBC: Red blood cell SS: Sézary Syndrome T-ALC: Total T-cell count TCR: T-cell receptor TCR-α: T-cell receptor alpha TCR-β: T-cell receptor beta TCR-γ: T-cell receptor gamma ix TCR-δ: T-cell receptor delta TdT: Terminal deoxynucleotidyl transferase T-LGL: T-cell large granular lymphocytic leukemia T-PLL: T-cell prolymphocytic leukemia V: Variable ZAP-70: Zeta-chain-associated protein kinase 70 x CHAPTER: I Clonal Lymphocytosis and its relevance to lymphocytic neoplasia 1. Clonal lymphocytosis and discovery of MBL Clonal lymphocytosis refers to the presence of a population of B or T lymphocytes derived from a single common precursor. This phenomenon may involve the lymphocyte populations in any or all of the blood, bone marrow or lymphoid tissues. Lymphoid cells (B- and T-cells) at almost any stage of differentiation or maturation can form a clone of cells which are arrested at a particular stage of development. Thus, clonal cell populations may bear the immunophenotypic markers that one would expect of normal lymphocytes reaching the stage at which maturation has been arrested. However, the significance of detecting clonal lymphocytosis
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