What is clonal rearrangement

Detection of monoclonal rearrangements of the beta chain of the T cell receptor gene using a newly developed polymerase chain reaction in conjunction with the GENESCAN technology


Title page, table of contents 1 Abbreviations 6 2 Introduction 7 2.1 General 7 2.2 Classification of non-Hodgkin lymphomas 8 2.3 The T-cell receptor: structure and function 10 2.4 Configuration of the T-cell receptor genes in lymphatic cells 11 2.5 Analysis of the T Cell receptor genes using the polymerase chain reaction 15 2.6 Issues and objectives of the work 17 3 Material and methods 18 3.1 Sources of supply for the devices used 18 3.2 Sources of supply for the chemicals and consumables used 19 3.3 Control tissue 19 3.4 Malignant lymphomas of the T cell series 19 3.5 Histology and immunohistology 20 3.6 DNA extraction 21 3.6.1 Automatic DANN isolation from patient material 21 3.6.2 DNA extraction from paraffin-embedded tissue 22 3.6.3 Isolation of mononuclear cells (Ficoll gradient) 22 3.7 Determination of the DANN concentration 22 3.8 Development of a PCR method to detect rearrangements of the TCR-ß-chain 23 3.8.1 Criteria for the Pr Selection of primers 23 3.8.2 Primer synthesis 23 3.8.3 Purification of the oligonucleotides using HPLC 24 3.8.4 Optimization of the PCR conditions 24 3.8.5 Control experiments 24 3.9 Polyacrylamide gel electrophoresis (PAGE) 25 3.9.1 Production of a polyacrylamide gel 26 3.9.2 Photo documentation 26 3.10 Sequence analysis of the amplified products 26 3.10.1 Sequence reaction 27 3.10.2 Evaluation of the sequences 29 3.11 Analysis of PCR products (GENESCAN analysis) 29 3.11.1 Preparation of gel electrophoresis 30 3.11.2 Sample preparation for electrophoresis 31 3.11.3 Determining the size of DNA fragments 32 4 Results 33 4.1 Methodological development 33 4.1.1 Establishing a TCR-ß PCR using highly degenerate Jß consensus primers 33 4.1.2 Own development of a family-specific TCR-ß PCR 35 4.1.3 Primer selection for the Jß segments 35 4.1.4 Primer selection for the Vb segments 36 4.1.5 Establishing the reaction conditions 38 4.1.6 Establishing the GENESCAN analysis 40 4.1.7 Specificity of the family-specific TCR-b PCR 41 4.1.8 Sensitivity of the family-specific TCR-b PCR 43 4.1.9 Sequence analysis of TCR-ß PCR production 44 4.1.10 Optimization of a TCR-y PCR 46 4.1.11 Specificity of the TCR -g PCR 46 4.1.12 Sensitivity of TCR-g PCR 48 4.2 Examination of patient material 49 4.2.1 Molecular biological investigation of CTCL patients using TCR-ß PCR compared to TCR-ß PCR 49 4.2.2 TCR-ß analysis of transformed cutaneous T-cell lymphomas 54 4.2.3 Clonality analyzes of non-cutaneous T-NHL using TCR-ß PCR compared to TCR-y PCR 58 4.2.4 Investigation of reactive skin changes using TCR-ß and TCR-y PCR 62 4.2.5 Summary comparison of the family-specific TCR-ß PCR with the TCR-y PCR 64 5 Discussion 65 5.1 Methodological aspects 66 5.1.1 Comparison of previously published TCR-b PCRs 66 5.1.2 Comparison with the family-specific TCR-b PCR 68 5.1.3 Comparison of the family-specific TCR-b PCR using TCR-y PCRs 69 5.1.4 Clonality analysis using the GENESCAN technique 70 5.2 Clinical aspects 71 5.2.1 Examination of normal and reactive tissue 71 5.2.2 Malignant T-cell proliferation 71 5.2.3 Prehistoric T-lymphoblastic lymphomas / leukemia 72 5.2. 4 Peripheral T-cell lymphoma, unspecified 72 5.2.5 Cutaneous T-cell lymphomas 73 5.2.6 Anaplastic large-cell lymphomas 76 5.2.7 Angioimmunoblastic T-cell lymphomas 77 5.2.8 Enteropathy-associated T-cell lymphomas 78 5.2.9 Nasal T / NK cell lymphomas 79 5.2.10 Hodgkin's disease 79 6 Summary 81 7 Bibliography 82 8 Appendix 84
dc. description. abstract
T-cell lymphomas occur in various clinical and histological forms, which often make it difficult to differentiate them from non-malignant T-cell lymphoproliferations. The detection of clonal T cell receptor rearrangements offers an important aid for this distinction, since malignant T-NHL are always characterized by a clonal T cell population with identical T cell receptor rearrangements. The aim of the present dissertation was the development of a new method for the detection of clonal T-cell receptor rearrangements. For this purpose, 13 new Jb primers were generated, which were used together with a modified Vb primer. In addition, a new automated separation process (GENESCAN) was established and applied for the analysis of the resulting PCR products, which is able to differentiate PCR products down to a single base pair. Finally, the newly developed TCR-b PCR method was compared with an existing TCR-g method. In a large comparative study with 132 samples from 62 patients with various T-cell lymphoma or leukemia, we were able to detect a monoconal TCR-b rearrangement in 61/62 of the cases (98.4%) using the family-specific TCR-b PCR. In addition, the identical T cell clone could be detected in 15/18 corresponding regional lymph nodes and in 7/11 blood samples. In contrast, the TCR-g PCR showed T cell clonality only in 50/62 (80%) patients and in 63% of the lymph nodes and 44% of the blood samples. The results show that the new TCR-b PCR is able to detect practically all clonal T cell populations. Since this detection is not limited to high quality DNA, but also provides the same results with tissue material embedded in paraffin, the method described here is superior to all other methods used so far. The TCR-b PCR developed and established within the scope of this work is a real alternative to the detection methods used so far.
dc. description. abstract
The distinction between benign polyclonal and malignant monoclonal lymphoid disorders by morphology or immunophenotyping is frequently difficult. Therefore, the demonstration of clonal B-cell or T-cell populations by detecting identically rearranged immunoglobulin (Ig) or T-cell receptor (TCR) genes is often used to solve this diagnostic problem. Whereas the detection of rearranged Ig genes is well established, TCR g (gamma) and b (beta) gene rearrangements often escape detection with the currently available polymerase chain reaction (PCR) assays. To establish a sensitive, specific, and rapid method for the detection of rearranged TCR-b genes, we developed a new PCR approach with family-specific Jb primers and analyzed the resulting PCR products by high-resolution GeneScan technique. The superior efficiency of this new method was demonstrated by investigating 132 DNA samples extracted from lymph node and skin biopsy specimens (mostly formalin fixed) and blood samples from 62 patients who had a variety of T-cell lymphomas and leukemias. In all but 1 of the tumor samples (98.4%) a clonal amplificate was detectable after TCR-b PCR and the same clonal T-cell population was also found in 15 of 18 (83%) of the regional lymph nodes and in 7 of 11 (64%) of the peripheral blood samples. Direct comparison of these results with those obtained currently by the most widely applied TCR-g PCR revealed an approximate 20% lower detection rate in the same set of samples than with the TCR-b PCR method. These results indicate that the new TCR-b PCR is most suitable for a rapid and reliable detection of clonal T-cell populations.
600 Technology, Medicine, Applied Sciences :: 610 Medicine and Health :: 610 Medicine and Health
Detection of monoclonal rearrangements of the beta chain of the T cell receptor gene using a newly developed polymerase chain reaction in conjunction with the GENESCAN technology
dc. contributor. firstReferee
Prof. Dr. med. Harald Stein
dc. contributor. furtherReferee
Prof. Dr. med. Thomas Blankenstein
urn: nbn: de: kobv: 188-2001002586
High detection rate of T-cell receptor beta chain rearrangements in T-cell lymphoproliferations by family specific polymerase chain reaction in combination with the GeneScan technique and DNA sequencing
Charité - University Medicine Berlin
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