Clonality analysis in B-cell chronic lymphocytic leukemia (B-CLL) associated with Richter's syndrome [Elektronische Ressource] / vorgelegt von Zhengrong Mao

Bp 70072

Sujets

• Gesundheit

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2006
• Nombre de lectures: 23
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Clonality Analysis in B-Cell Chronic Lymphocytic
Leukemia (B-CLL) Associated with Richter’s Syndrome





Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Bayerischen Julius-Maximilians-Universität Würzburg







vorgelegt von

Zhengrong Mao

aus Hangzhou, China





Würzburg 2006























Eingereicht am: 11. Sep. 2006

Mitglieder der Prüfungskommission:
Vorsitzender: Prof. Dr. M. Müller
Gutachter: Prof. Dr. Hans Konrad Müller-Hermelink
Gutachter: PD. Dr. Andreas Rosenwald
Gutachter: PD. Dr. Robert Hock

Tag des Promotionskolloquiums: 28. Nov. 2006
Doktorurkunde ausgehändigt am:





Contents

Abstrac 1

Chapter 1
Introduction 6

1.1 B-cell Chronic Lymphocytic Leukemia (B-CLL) and Richter’s Syndrome 6

1.2 Definition of Richter’s Syndrome 8
1.2.1 Incidence of Richter’s Syndrome 9
1.2.2 Morphology of Richter’s Syndrome 10
1.2.3 Treatment and Clinical Outcome of Richter’s Syndrome 11

1.3 Pathogenetic Mechanisms in B-CLL 13
1.3.1 Chromosomal Abnormalities
1.3.2 Immunoglobulin Heavy Chain Variable Region (IgVH) Genes and their
Mutational Status 14
1.3.3 DNA Mismatch-repair and Microsatellite Instability 16
1.3.4 Epstein-Barr Virus 16

1.4 Recent Studies on Richter’s Syndrome: A Short Review 17
1.4.1 Study of B-CLL and DLBCL in Richter’s Syndrome 17
1.4.2 IgVH Gene Analysis in the Hodgkin’s Lymphoma (HL) Variant
of Richter’s Syndrome 20

1.5 Aims ofthe Present Study 22
1.5.1 Richter’s Syndrome: Lymphoma Progression or Composite Lymphoma? 22
1.5.2 IgVH-Mutational Status of B-CLL Cells in Richter’s Syndrome: Mutated
or Unmutated? 23
1.5.3 ZAP70, a Surrogate Marker for the IgVH Mutation Status of B-CLL
in Richter’s Syndrome? 24

IChapter 2
Materials nd Methods 26

2.1 Materils 26

2. Reagnts 6
2.2.1 Reagents for Immunohistochemical Staining (IHC) 26
2.2.1.1 Immunohistochemical Staining Kit 26
2.2.1.2 Primary Antibodies for Immunohistochemical Staining 26
2.2.1.3 HistoGripTM and Peroxidase Blocking Solution 27
2.2.2 Reagents for DNA Extraction 27
2.2.2.1 Digestion Buffer and Enzyme 27
2.2.2.2 Phenol-Chloroform Reagent 28
2.2.3 Reagents for PCR Reactions 28
2.2.3.1 Primers for PCR-GeneScan Analysis and Sequencing 28
2.2.3.2 PCR Enzymes 28
2.2.3.3 dNTP 28
2.2.4 Reagents for Electropherosis 29
2.2.4.1 Preparation of 5x TBE Buffer 29
2.2.4.2 Ethidium Bromide (EB) Solution 29
2.2.4.3 Preparation of 1.5 % Low-Melting Agarose 29
2.2.4.4 DNA Markers 29
2.2.5. Reagents for GeneScan Analysis 30
2.2.5.1 Chemical Reagents 30
2.2.5.2 Preparation of 10 % Ammonium Persulfate (APS) 30
2.2.5.3ion of 4.5 % Polyacrylamide Gel Solution 30
2.2.5.4 GeneScan Marker 30
2.2.5.5 Preparation of Loading Buffer 30
2.2.6 Reagents for Subcloning 31
2.2.6.1 PCR Product Purification Kit 31
2.2.6.2 Ligation and Transformation Kit 31
2.2.6.3 Plasmid DNA Purification Kit 31
2.2.6.4 Restriction Enzyme 31
2.2.6.5 Preparation of Antibiotics 31
2.2.6.6ion of LB Medium and Agar Plates 31
2.2.6.7 Preparation of X-gal Plates 32
II 2.2.7 Reagents for Sequencing 32
2.2.7.1 Polymer 32
2.2.7.2 Buffer
2.2.7.3 Loading Buffer
2.2.7.4 Sephadex™G50

2.3 Equipment 32

2.4 Methods 3
2.4.1 Immunohistochemical Methods 35
2.4.1.1 Pretreatment of Slides and Heat Induced Epitope Retrieval in Tissue Specimens 35
2.4.1.2 Immunohistochemical Staining (IHC) 35
2.4 Microdisection 35
2.4.2.1 Manual Microdissection 36
2.4.2.2 Immuno-Laser Capture Microdissection (LCM) 36
2.4.2.2.1 Description of Two Types of LCM Systems 36
2.4.2.2.2 Preparation of Slides 37
2.4.2.2.3 Removal of Surrounding Tissue 37
2.4.2.2.4 Capturing the Reed-Sternberg Cells/ CD30-Positive Large Cells 37
2.4.3 DNA Methods 38
2.4.3.1 Proteinase K Digestion 38
2.4.3.2 Phenol-Chloroform Extraction 38
2.4.4 Polymerase Chain Reaction (PCR) 38
2.4.4.1 Standard PCR 38
2.4.4.2 Semi-nested PCR 39
2.4.4.3 PCR for Subcloning 43
2.4.4.4 Sequencing PCR 43
2.4.5 Electrophoresis Methods 43
2.4.5.1 Agarose Gel Electrophoresis 43
2.4.5.2 PCR-based GeneScan Analysis 44
2.4.5.2.1 Preparation of the Gel Cassette 44
2.4.5.2.2 Running of GeneScan 44
2.4.5.2.3 Analysis of Results 45
2.4.6 Subcloning and Sequencing 45
2.4.6.1 DNA Purification 47
2.4.6.2 Ligation and Transformation 47
III 2.4.6.3 Purification of Plasmid DNA 47
2.4.6.4 Restriction Enzyme Digestion 47
2.4.6.5 Purification of Sequencing PCR Products 49
2.4.6.6 Automated DNA Sequencing and Analysis 49
2.4.6.6.1 Preparation of the Instrument 49
2.4.6.6.2 Denaturing and Loading of Samples 50
2.4.6.6.3 Analysi ofResult 50

Chapter 3
Results 51

3.1 Classical Richter’s Syndrome: B-CLL and DLBCL 51
3.1.1 Clinical Characteristics and Histology 51
3.1.2 Immunophenotypic Findings 52
3.1.3 Results of PCR-GeneScan Analysis and Clonal Relationship of B-CLL and DLBCL 58
3.1.4 IgVH Gene Sequencing Results 60
3.1.4.1 Clonality Analyses based on IgVH Gene Sequences 60
3.1.4.2 IgVH Gene Usage in B-CLL and DLBCL 60
3.1.4.3 Mutational Status of IgVH Genes 62
3.1.4.4 Relationship between IgVH Gene Status and Expression of ZAP70 62
3.1.5 Clinical Outcome 63

3.2 Richter’s Syndrome: B-CLL and Hodgkin’s Lymphoma 65
3.2.1 Histology and Immunohistochemical Findings 65
3.2.2 IgVH Gene analysis of B-CLL in the HL and CD30- Positive HRS-like Variants
of Richter’s syndrome 68
3.2.3 Immuno-Laser Capture Microdissection and Clonality Analysis in the HL Variant
of Richter’s Syndrome 69
3.2.4 Clinical Outcome 70

3.3 Summary of Clinical Data in Richter’s Syndrome Patients 71

Chapter 4
Discuion 74

4.1 Classical Richter’s Syndrome (B-CLL and DLBCL) 74
IV 4.1.1 Morphological, Inmmunohistochemical and Clinical Findings in Classical
Richter’s Syndrome 74
4.1.2 78% of B-CLL and DLBCL in Classical Richter’s Syndrome are Clonally Identical 80
4.1.3 Clonal Transformation in Richter’s Syndrome Occurs Predominantly
in B-CLL with Unmutated IgVH Genes 83
4.1.4 Biased Usage of IgVH Genes in Richter’s Syndrome: A Role for the Involvement
of Antigen? 085

4.2 B-CLL and Hodgkin’s Lymphoma (HL) as a Variant of Richter’s Syndrome 087
4.2.1 Clonal Relationship between B-CLL and HL in Richter’s Syndrome and
Association with EBV Infection 087
4.2.2 The HL Variant of Richter’s Syndrome is Mainly Associated with
IgVH-Mutated B-CLL 90

Chapter 5
Conclusion 92

List ofAbreviatons 093

List ofTables 095

List ofFigures 096

Refrnces 097

Acknowledgments 107

VAbstract

B-cell chronic lymphocytic leukemia (B-CLL) comprises 90% of chronic lymphoid
leukemias in Western countries and patients with B-CLL have a heterogeneous clinical
course. Approximately 3-5% of B-CLL patients encounter transformation to an
aggressive lymphoma, mainly diffuse large B-cell lymphoma (DLBCL) or Hodgkin’s
lymphoma (HL) which has been defined as Richter’s syndrome and is associated with a
poor clinical outcome. The mutational status of the immunoglobulin heavy chain variable
region (IgVH) gene not only implies the developmental stage at which the neoplastic
transformation occurs in a given B-cell lymphoma, but also constitutes an important
prognostic factor in B-CLL, since B-CLL patients with unmutated IgVH genes usually
have a poor clinical outcome. Spars