Entwicklung und Implementierung von Auswertungswerkzeugen für Hochdurchsatz-DNA-Kopienzahl-Analysen und deren Anwendung auf Lymphomdaten

Aberrationen in der DNA-Kopienzahl sind häufige genetische Veränderungen bei malignen Lymphomerkrankungen. Zugewinne sowie Deletionen stellen dabei Mechanismen zur Onkogen-Aktivierung sowie Tumorsuppressorgen-Inaktivierung dar und tragen somit zur Pathogenese der Erkrankung bei. Array-CGH und SNP-Array sind Messplattformen, die die genomweite Bestimmung von Kopienzahlaberrationen in einem Experiment ermöglichen. Die bei der Analyse entstehenden Datensätze sind komplex und erfordern automatische Methoden zur Unterstützung der Analyse und Interpretation der Messergebnisse. In dieser Promotionsarbeit wurden Methoden entwickelt, welche die Analyse von Array-CGH- und SNP-Array-Messungen ermöglichen. Diese Methoden wurden für die Auswertung umfangreicher Datensätze von malignen Non-Hodgkin-Lymphomen verwendet. Dabei wurden Lymphome der Entitäten Burkitt-Lymphom, diffus großzelliges B-Zell-Lymphom, Mantelzelllymphom, primäres ZNS-Lymphom und peripheres T-Zell-Lymphom – nicht anderweitig spezifiziert – analysiert. Für die untersuchten Lymphom-Entitäten konnten hierbei zahlreiche neue rekurrente Kopienzahlaberrationen sowie uniparentale Disomien gezeigt werden, die neue Einblicke in die Pathogenese der jeweiligen Erkrankungen erlauben. Darüber hinaus erfolgte ein Vergleich beider Messplattformen anhand eines Datensatzes mit gepaarten Array-CGH- und SNP-Array-Daten. Für die eingesetzten Plattformen (2800k-BAC-Array vs. Affymetrix 250k-Sty-SNP-Array) konnte eine circa zwölffach höhere effektive Auflösung der SNP-Array-Plattform gezeigt werden. Die wesentlichen Ergebnisse dieser Arbeit sind in sieben Publikationen eingeflossen.:Inhaltsverzeichnis Abkürzungsverzeichnis Tabellenverzeichnis Abbildungsverzeichnis 1. Einführung 1.1 Biologischer Hintergrund 1.1.1 Aberrationen der DNA-Kopienzahl und Tumorentstehung 1.1.2 Lymphome 1.2 Motivation und Rationale für die Arbeit 1.3 Array-CGH Analyse 1.4 SNP-Array-Analyse 1.5 Vergleich von Array-CGH und SNP-Array-Analyse 1.6 Assoziationen von DNA-Kopienzahlaberrationen mit RNA-Expression, Lymphomentität sowie klinischen und phänotypischen Faktoren 2.Publikationen 2.1 Publikation 1: “Development and implementation of an analysis tool for array-based comparative genomic hybridization” Methods Inf Med. 2007;46(5):608-13 2.2 Publikation 2: “Recurrent loss of the Y chromosome and homozygous deletions within the pseudoautosomal region 1: association with male predominance in mantle cell lymphoma” Haematologica. 2008 Jun;93(6):949-50 2.3 Publikation 3: “GeneChip analyses point to novel pathogenetic mechanisms in mantle cell lymphoma” Br J Haematol. 2009 Feb;144(3):317-31 2.4 Publikation 4: “Chromosomal imbalances and partial uniparental disomies in primary central nervous system lymphoma.” Leukemia. 2009 Oct;23(10):1875-84 2.5 Publikation 5: “High resolution SNP array genomic profiling of peripheral T cell lymphomas, not otherwise specified, identifies a subgroup with chromosomal aberrations affecting the REL locus” Br J Haematol. 2010 Feb;148(3):402-12 2.6 Publikation 6: “Detection of genomic aberrations in molecularly defined Burkitt\''s lymphoma by array-based, high resolution, single nucleotide polymorphism analysis” Haematologica. 2010 Dec;95(12):2047-55 2.7 Publikation 7: “Patient age at diagnosis is associated with the molecular characteristics of diffuse large B-cell lymphoma” Blood. 2012 Feb 23;119(8):1882-7 2.8 Kennzeichnung des Eigenanteils für alle eingeschlossenen Publikationen 3. Diskussion und Ausblick 4. Zusammenfassung 5. Referenzen 6. Eigene Publikationen 7. Erklärung 8. Danksagung 9. Curriculum vita

Hereditary angioedema (HAE) in children and adolescents : a consensus on therapeutic strategies

Hereditary angioedema due to C1 inhibitor (C1 esterase inhibitor) deficiency (types I and II HAE-C1-INH) is a rare disease that usually presents during childhood or adolescence with intermittent episodes of potentially life-threatening angioedema. Diagnosis as early as possible is important to avoid ineffective therapies and to properly treat swelling attacks. At a consensus meeting in June 2011, pediatricians and dermatologists from Germany, Austria, and Switzerland reviewed the currently available literature, including published international consensus recommendations for HAE therapy across all age groups. Published recommendations cannot be unconditionally adopted for pediatric patients in German-speaking countries given the current approval status of HAE drugs. This article provides an overview and discusses drugs available for HAE therapy, their approval status, and study results obtained in adult and pediatric patients. Recommendations for developing appropriate treatment strategies in the management of HAE in pediatric patients in German-speaking countries are provided.Conclusion Currently, plasma-derived C1 inhibitor concentrate is considered the best available option for the treatment of acute HAE-C1-INH attacks in pediatric patients in German-speaking countries, as well as for short-term and long-term prophylaxis

High-Resolution Cartography of the Transcriptome and Methylome Landscapes of Diffuse Gliomas

Molecular mechanisms of lower-grade (II–III) diffuse gliomas (LGG) are still poorly understood, mainly because of their heterogeneity. They split into astrocytoma- (IDH-A) and oligodendroglioma-like (IDH-O) tumors both carrying mutations(s) at the isocitrate dehydrogenase (IDH) gene and into IDH wild type (IDH-wt) gliomas of glioblastoma resemblance. We generated detailed maps of the transcriptomes and DNA methylomes, revealing that cell functions divided into three major archetypic hallmarks: (i) increased proliferation in IDH-wt and, to a lesser degree, IDH-O; (ii) increased inflammation in IDH-A and IDH-wt; and (iii) the loss of synaptic transmission in all subtypes. Immunogenic properties of IDH-A are diverse, partly resembling signatures observed in grade IV mesenchymal glioblastomas or in grade I pilocytic astrocytomas. We analyzed details of coregulation between gene expression and DNA methylation and of the immunogenic micro-environment presumably driving tumor development and treatment resistance. Our transcriptome and methylome maps support personalized, case-by-case views to decipher the heterogeneity of glioma states in terms of data portraits. Thereby, molecular cartography provides a graphical coordinate system that links gene-level information with glioma subtypes, their phenotypes, and clinical context

Genomic basis of a social polymorphism in a halictid bee

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GeneChip analyses point to novel pathogenetic mechanisms in mantle cell lymphoma

The translocation t(11;14)(q13;q32) is the genetic hallmark of mantle cell lymphoma (MCL) but is not sufficient for inducing lymphomagenesis. Here we performed genome-wide 100K GeneChip Mapping in 26 t(11;14)-positive MCL and six MCL cell lines. Partial uniparental disomy (pUPD) was shown to be a recurrent chromosomal event not only in MCL cell lines but also in primary MCL. Remarkably, pUPD affected recurrent targets of deletion like 11q, 13q and 17p. Moreover, we identified 12 novel regions of recurrent gain and loss as well as 12 high-level amplifications and eight homozygously deleted regions hitherto undescribed in MCL. Interestingly, GeneChip analyses identified different genes, encoding proteins involved in microtubule dynamics, such as MAP2, MAP6 and TP53, as targets for chromosomal aberration in MCL. Further investigation, including mutation analyses, fluorescence in situ hybridisation as well as epigenetic and expression studies, revealed additional aberrations frequently affecting these genes. In total, 19 of 20 MCL cases, which were subjected to genetic and epigenetic analyses, and five of six MCL cell lines harboured at least one aberration in MAP2, MAP6 or TP53. These findings provide evidence that alterations of microtubule dynamics might be one of the critical events in MCL lymphomagenesis contributing to chromosomal instability

Mantle cell lymphomas with concomitant MYC and CCND1 breakpoints are recurrently TdT positive and frequently show high-grade pathological and genetic features

Chromosomal breakpoints involving the MYC gene locus, frequently referred to as MYC rearrangements (MYC - R+), are a diagnostic hallmark of Burkitt lymphoma and recurrent in many other subtypes of B-cell lymphomas including follicular lymphoma, diffuse large B-cell lymphoma and other high-grade B-cell lymphomas and are associated with an aggressive clinical course. In remarkable contrast, in MCL, only few MYC - R+ cases have yet been described. In the current study, we have retrospectively analysed 16 samples (MYC - R+, n = 15, MYC - R-, n = 1) from 13 patients and describe their morphological, immunophenotypic and (molecular) genetic features and clonal evolution patterns. Thirteen out of fifteen MYC - R+ samples showed a non-classical cytology including pleomorphic (centroblastic, immunoblastic), anaplastic or blastoid. MYC translocation partners were IG-loci in 4/11 and non-IG loci in 7/11 analysed cases. The involved IG-loci included IGH in 3 cases and IGL in one case. PAX5 was the non-IG partner in 2/7 patients. The MYC - R+ MCL reported herein frequently displayed characteristics associated with an aggressive clinical course including high genomic-complexity (6/7 samples), frequent deletions involving the CDKN2A locus (7/10 samples), high Ki-67 proliferation index (12/13 samples) and frequent P53 expression (13/13 samples). Of note, in 4/14 samples, SOX11 was not or only focally expressed and 3/13 samples showed focal or diffuse TdT-positivity presenting a diagnostic challenge as these features could point to a differential diagnosis of diffuse large B-cell lymphoma and/or lymphoblastic lymphoma/leukaemia

Genomic and transcriptomic changes complement each other in the pathogenesis of sporadic Burkitt lymphoma

Burkitt lymphoma (BL) is the most common B-cell lymphoma in children. Within the International Cancer Genome Consortium (ICGC), we performed whole genome and transcriptome sequencing of 39 sporadic BL. Here, we unravel interaction of structural, mutational, and transcriptional changes, which contribute to MYC oncogene dysregulation together with the pathognomonic IG-MYC translocation. Moreover, by mapping IGH translocation breakpoints, we provide evidence that the precursor of at least a subset of BL is a B-cell poised to express IGHA. We describe the landscape of mutations, structural variants, and mutational processes, and identified a series of driver genes in the pathogenesis of BL, which can be targeted by various mechanisms, including IG-non MYC translocations, germline and somatic mutations, fusion transcripts, and alternative splicing

Focal structural variants revealed by whole genome sequencing disrupt the histone demethylase KDM4C in B cell lymphomas

Histone methylation-modifiers, like EZH2 and KMT2D, are recurrently altered in B-cell lymphomas. To comprehensively describe the landscape of alterations affecting genes encoding histone methylation-modifiers in lymphomagenesis we investigated whole genome and transcriptome data of 186 mature B-cell lymphomas sequenced in the ICGC MMML-Seq project. Besides confirming common alterations of KMT2D (47% of cases), EZH2 (17%), SETD1B (5%), PRDM9 (4%), KMT2C (4%), and SETD2 (4%) also identified by prior exome or RNAseq studies, we here unravel KDM4C in chromosome 9p24, encoding a histone demethylase, to be recurrently altered. Focal structural variation was the main mechanism of KDM4C alterations, which was independent from 9p24 amplification. We identified KDM4C alterations also in lymphoma cell lines including a focal homozygous deletion in a classical Hodgkin lymphoma cell line. By integrating RNAseq and genome sequencing data we predict KDM4C structural variants to result in loss-of-function. By functional reconstitution studies in cell lines, we provide evidence that KDM4C can act as tumor suppressor. Thus, we show that identification of structural variants in whole genome sequencing data adds to the comprehensive description of the mutational landscape of lymphomas and, moreover, establish KDM4C as putative tumor suppressive gene recurrently altered in subsets of B-cell derived lymphomas

DNA methylome analysis in Burkitt and follicular lymphomas identifies differentially methylated regions linked to somatic mutation and transcriptional control

Although Burkitt lymphomas and follicular lymphomas both have features of germinal center B cells, they are biologically and clinically quite distinct. Here we performed whole-genome bisulfite, genome and transcriptome sequencing in 13 IG-MYC translocation-positive Burkitt lymphoma, nine BCL2 translocation-positive follicular lymphoma and four normal germinal center B cell samples. Comparison of Burkitt and follicular lymphoma samples showed differential methylation of intragenic regions that strongly correlated with expression of associated genes, for example, genes active in germinal center dark-zone and light-zone B cells. Integrative pathway analyses of regions differentially methylated in Burkitt and follicular lymphomas implicated DNA methylation as cooperating with somatic mutation of sphingosine phosphate signaling, as well as the TCF3-ID3 and SWI/SNF complexes, in a large fraction of Burkitt lymphomas. Taken together, our results demonstrate a tight connection between somatic mutation, DNA methylation and transcriptional control in key B cell pathways deregulated differentially in Burkitt lymphoma and other germinal center B cell lymphomas

Genomic and transcriptomic changes complement each other in the pathogenesis of sporadic Burkitt lymphoma

Burkitt lymphoma (BL) is the most common B-cell lymphoma in children. Within the International Cancer Genome Consortium (ICGC), we performed whole genome and transcriptome sequencing of 39 sporadic BL. Here, we unravel interaction of structural, mutational, and transcriptional changes, which contribute to MYC oncogene dysregulation together with the pathognomonic IG-MYC translocation. Moreover, by mapping IGH translocation breakpoints, we provide evidence that the precursor of at least a subset of BL is a B-cell poised to express IGHA. We describe the landscape of mutations, structural variants, and mutational processes, and identified a series of driver genes in the pathogenesis of BL, which can be targeted by various mechanisms, including IG-non MYC translocations, germline and somatic mutations, fusion transcripts, and alternative splicing