Frequency and relevance of genetic alterations of the ID3-TCF3-CCND3 pathway in pediatric mature B-cell Non-Hodgkin lymphoma Inauguraldissertation In zur Erlangung des Grades eines Doktors der Medizin troduction des Fachbereichs Medizin der Justus-Liebig-Universität Gießen 1 vorgelegt von Marius Rohde aus Lengerich Gießen 2013 Aus dem Zentrum für Kinderheilkunde und Jugendmedizin Abteilung für Pädiatrische Hämatologie und Onkologie am Fachbereich Medizin der Justus-Liebig-Universität Gießen Leiter: Prof. Dr. med. A. Reiter Introduction 2 Gutachter: PD Dr. B. Burkhardt Gutachter: Prof. Dr. M. Rummel Tag der Disputation: 16.02.2015 Table of content 1 Introduction ....................................................................................................... 1 1.1 The human Immune System ............................................................................. 1 1.2 B lymphocytes ................................................................................................... 2 1.2.1 Commitment to the B cell lineage ........................................................................ 3 1.2.2 Somatic recombination ........................................................................................ 3 1.2.3 Differentiation stages of B cell maturation ........................................................... 4 1.3 Germinal center reaction .................................................................................. 4 1.3.1 Antigen-activation of B lymphocytes .................................................................... 4 1.3.2 Affinity maturation ............................................................................................... 5 1.3.3 Class switching ................................................................................................... 5 1.3.4 Malignant transformation ..................................................................................... 5 1.4 Mature B cell lymphoma ................................................................................... 7 1.4.1 NHL-BFM study group ......................................................................................... 7 1.4.2 Burkitt Lymphoma ............................................................................................... 7 1.4.3 Diffuse large B-cell lymphoma ............................................................................. 9 1.4.4 Unclassifiable B-NHL ........................................................................................ 10 1.5 Recurrently mutated genes in B-NHL ............................................................ 10 1.6 Candidate genes ID3, TCF3 and CCND3 ........................................................ 11 1.7 Study objectives .............................................................................................. 14 2 Material and methods ..................................................................................... 15 2.1 Materials .......................................................................................................... 15 2.1.1 Equipment ......................................................................................................... 15 Introduction 2.1.2 Kits .................................................................................................................... 16 2.1.3 Reagents and Enzymes .................................................................................... 16 2.2 Methods ........................................................................................................... 18 2.2.1 Recurrent mutations in 13 pediatric BL in the ICGC-MMML-Seq project ........... 18 3 2.2.2 Validation of ID3, TCF3 and CCND3 mutations ................................................. 18 2.2.3 Patient samples ................................................................................................. 18 2.2.4 DNA extraction from B-NHL samples ................................................................ 19 2.2.5 Polymerase chain reaction ................................................................................ 21 2.2.6 Purification of PCR products ............................................................................. 23 2.2.7 Bacterial culture, subcloning and plasmid DNA preparation .............................. 24 2.2.8 Agarose gel electrophoresis .............................................................................. 27 2.2.9 UV spectrometry ............................................................................................... 28 2.2.10 Cycle sequencing .............................................................................................. 28 2.2.11 Ethanol precipitation .......................................................................................... 29 2.2.12 Capillary electrophoresis ................................................................................... 30 2.2.13 Analysis of electropherograms .......................................................................... 32 2.2.14 Data analysis and interpretation of sequencing results ...................................... 33 2.2.15 Definition and terminology of mutations ............................................................. 33 2.2.16 Statistical analysis ............................................................................................. 36 3 Results ............................................................................................................. 37 3.1 Recurrent mutations in 13 pediatric BL in the ICGC-MMML-Seq project .... 37 3.2 Validation of ID3, TCF3 and CCND3 mutations in pediatric B-NHL ............. 39 3.2.1 Characterization of analyzed samples ............................................................... 39 3.2.2 ID3, TCF3 and CCND3 sequencing in B-NHL ................................................... 43 3.2.3 Clinical characteristics and outcome according to ID3, TCF3 and CCND3 mutation status 55 3.3 ID3, TCF3 and CCND3 mutations in B-NHL patients who relapsed ............. 65 3.3.1 Patient characteristics ....................................................................................... 65 3.3.2 Results of ID3, TCF3 and CCND3 sequencing .................................................. 65 3.4 ID3 sequencing in pB-ALL .............................................................................. 66 4 Discussion ....................................................................................................... 67 4.1 Patients, samples and methods ..................................................................... 67 4.2 Frequency of ID3, TCF3 and CCND3 mutations in Burkitt lymphoma ......... 69 4.3 Functional background of ID3, TCF3 and CCND3 mutations ....................... 71 4.4 Clinical relevance of ID3, TCF3 and CCND3 mutations in MYC rearrangement positive B-NHL............................................................................................................... 73 4.5 ID3 mutations in pB-ALL and the role of MYC rearrangement ..................... 75 4.6 Consequence of ID3, TCF3 and CCND3 mutations in pediatric BL ............. 76 4.7 Conclusion of the study.................................................................................. 78 5 Summary (English/German) ........................................................................... 79 6 List of abbreviations ....................................................................................... 81 7 List of figures .................................................................................................. 85 Introduction 8 List of tables .................................................................................................... 87 9 References ....................................................................................................... 89 10 Appendix ........................................................................................................ 104 4 10.1 Overall sequencing results on B-NHL patients ........................................... 105 10.2 Correlation between certain mutational patterns and clinical data ........... 110 10.3 ID3 gene segmented into sections ............................................................... 114 10.4 List of SNP identifiers ................................................................................... 115 11 List of publications ....................................................................................... 116 12 Declaration (in German) ................................................................................ 117 13 Acknowledgment (in German) ...................................................................... 118 14 Curriculum vitae ............................................................................................ 119 Introduction 1 1 Introduction The term ‘Non-Hodgkin lymphoma’ (NHL) is a still widely used, but historical collective name for malignant diseases of the lymphoid system, including any kind of lymphoma except Hodgkin lymphomas. There have been different approaches to classify this group of heterogeneous malignancies, like the Kiel classification (Stansfeld et al., 1988), the Working Formulation of Non-Hodgkin’s Lymphoma for Clinical Usage (Robb-Smith, 1982) and the current WHO classification of Malignant Lymphoma (Swerdlow et al., 2008). The WHO classification pursues an approach in which lymphomas are differentiated by the cell type from which they are originating from. Of all malignancies listed in the annual report