8,163 research outputs found

    The interplay between Natural Killer cells and Pancreatic Stellate cells in Pancreatic Ductal Adenocarcinoma

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    Pancreatic ductal adenocarcinoma (PDAC) is a disease with dismal prognosis. With five-year survival rates of less than 11%, PDAC is set to become the second leading cause of cancer related deaths by 2040. The role of pancreatic stellate cells in pancreatic ductal adenocarcinoma has been well established. However, to date, little remains know about the interaction between these crucial stromal cells and the innate lymphocytes, natural killer (NK) cells, in PDAC. Herein we demonstrate that naĂŻve NK cells possess the functional efficacy to target and kill both quiescent (qPSC) and activated (aPSC) pancreatic stellate cells. Furthermore, qPSC, but not aPSC education of NK cells resulted in decreased NK cell-mediated cancer cell cytotoxicity. NK-PSC direct co-culture was found to modulate both PSC and NK phenotype, as well as functional changes within NK cells, an effect not observed with TranswellTM separation. Multiplex Luminex ELISA further revealed upregulation of IFN-Îł and related chemokines in NK cells co-cultured with PSC (activated/quiescent), suggesting that this pathway may be involved in phenotypic modulation. Through global proteomic analysis we demonstrate NK cell-induced differential protein changes in aPSC versus qPSC. Furthermore, we demonstrate changes in intracellular NK pathways as a result of direct contact with PSCs, indicating a dynamic, bidirectional interaction between these two key players. Using multiplex immunohistochemical analysis, we demonstrate that NK cell proximity to CAFs, and not total NK cell infiltrate is correlated with overall survival in PDAC. Consequently, we suggest that the spatial biology of NK/CAFs may play a prognostic role in PDAC and may potentially be used as a tool for patient stratification Taken together, our results demonstrate a significant bidirectional relationship between NK cells and PSC/CAFs in the context of PDAC, providing novel insight into this crucial cell-cell interaction

    Molekulare Charakterisierung lokalisierter und systemischer FollikulÀrer Lymphome: Neue Einblicke in die Pathogenese und Identifizierung potentieller therapeutischer Zielgene

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    Das FollikulĂ€re Lymphom (FL) ist ein B-Zell-Lymphom und wird klinisch in vier Stadien unterteilt. Aufgrund seines indolenten klinischen Verlaufs wird die ĂŒberwiegende Mehrheit der FL erst in systemischen klinischen Stadien (III und IV; sFL) diagnostiziert, weshalb auch bisherig erhobene molekularbiologische Charakteristika ĂŒberwiegend auf den Daten der sFL basieren. Entsprechende Daten FL lokalisierter klinischer Stadien (I und II; lFL) existieren bislang kaum. Da der klinische Verlauf des FL stark unterschiedlich sein kann und erste Hinweise auf biologische Unterschiede zwischen den lFL und sFL bzw. FL mit (BCL2+) und ohne (BCL2-) charakteristischer t(14;18)-Translokation des BCL2-Gens gefunden wurden, wurde im Rahmen dieser Arbeit eine große Kohorte an lFL und sFL molekularbiologisch charakterisiert. HierfĂŒr wurden die KopienzahlverĂ€nderungen (CNA) und somatischen Mutationen der Tumoren detektiert und analysiert. Ein Vergleich der molekularbiologischen Profile von lFL und sFL ergab erstaunlicherweise keine großen Unterschiede. Bei den lFL und sFL zeigten sich lediglich unterschiedliche Frequenzen in Zugewinnen in den Chromosomen 17, 18 und X bzw. der Mutationen des Gens ARID1A, mit jeweils hĂ€ufigerem Auftreten in den sFL, signifikant. Vergleichende Analysen der BCL2+ und BCL2- FL ergaben keine signifikanten Unterschiede in ihrem CNA-Profil, zeigten aber signifikante Unterschiede auf Basis der somatischen Mutationen. Hierbei traten STAT6-Mutationen signifikant hĂ€ufiger in BCL2- FL auf, wĂ€hrend BCL2+ signifikant hĂ€ufiger Mutationen in BCL2, KMT2D, ABL2 und IGLL5 aufwiesen. ErgĂ€nzend zu den detektierten Unterschieden zwischen den Subkohorten wurden bisher fĂŒr das FL noch nicht beschriebene CNA und Mutationen identifiziert. Sowohl fĂŒr lFL als auch sFL wurden signifikante Zugewinne in einzelnen interessanten Zielgenen in 1q23.1 (FCRL5), 6p21.32, 7p12.2 (IKZF1) sowie 11q24.3 (ETS-1) nachgewiesen. Signifikant auftretende Verluste wurden in 8p11.22 identifiziert. Eine erhöhte Proteinexpression in FĂ€llen mit Zugewinn konnte fĂŒr IKZF1 beobachtet werden. ViabilitĂ€tsassays an Zelllinien (ZL) mit und ohne Zugewinnen in ETS-1 bzw. IKZF1, die mit den bereits verfĂŒgbaren spezifischen Inhibitoren behandelt wurden, ergaben ein differentielles Ansprechen der ZL. Zudem zeigte sich ein Zugewinn von BCL2 als signifikant mit einem kĂŒrzeren progressionsfreien Überleben assoziiert und bildet damit den ersten prognostischen molekularbiologischen Marker im lFL. Die neu identifizierten, rekurrenten Aberrationen sollen in weiterfĂŒhrenden Analysen systematisch charakterisiert werden, um potentielle neue Zielstrukturen fĂŒr therapeutische AnsĂ€tze im FL zu definieren und die Risikostratifizierung von Patienten mit FL zu optimieren

    Charting genomic heterogeneity in tumours : from bulk to single cell

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    Tumours do not consist of a single homogeneous population but are complex heterogeneous systems that contain billions of ever-evolving cells with no two tumours being the same. Tumour heterogeneity is present at three levels, 1) inter-patient heterogeneity; 2) intra-patient heterogeneity; and 3) intra-tumour heterogeneity (ITH). Understanding all levels of heterogeneity is crucial for patient prognosis and treatment choice. To this end, we aimed to improve our understanding of all three levels of tumour heterogeneity. In paper I we investigated the prevalence, type, length, and genomic distribution of 853.218 somatic copy number alterations (SCNAs) across 20.249 tumours belonging to 32 cancer types. Based on the 1) number of SCNAs; 2) percentage of the genome altered; and 3) average SCNA size, we found high levels of inter-patient heterogeneity, both between and within cancer types. We found that specific chromosomes were preferentially lost or gained depending on cancer type. Lastly, we detected co-alterations of key oncogenes and TSGs. Taken together, we provided a comprehensive analysis on SCNAs across many cancer types as a valuable resource for the community. In paper II we sought to elucidate intra-patient heterogeneity in non-small cell lung cancer (NSCLC) and their matched brain metastasis (BM). We performed shallow wholegenome sequencing (WGS) on 51 primary NSCLC and matched BM, whole exome sequencing on 40 of the pairs, multi-region sequencing of 15 BMs, and shallow WGS on an additional cohort of 115 BMs. We showed that there is significant intra-patient heterogeneity at the SCNA level, with BM samples showing, on average, more SCNAs compared to their matched NSCLC. In contrast, multi-region sequencing of 15 BMs did not show significant ITH at the level of SCNAs. Finally, we identified putative metastatic driver SCNAs and singlenucleotide variants in key tumour suppressor genes (TSGs) and oncogenes. In paper III we aimed to assess the level of ITH in early localized prostate cancer. We performed organ-wide, multi-region, single-cell DNA sequencing on two prostate midsections. We found transient chromosomal instability (CIN) both in tumour and normal prostate tissue, evidenced by a large number of cells with unique chromosomal (arm) losses and or gains. Furthermore, we found three distinct groups of cells within the prostate: 1) diploid cells; 2) pseudo-diploid cells; and 3) monster cells. We observed an enrichment of diploid cells in normal regions and pseudo-diploid cells in tumour-rich regions, while monster cells were equally distributed over the entire prostate, again suggesting that there were elevated CIN levels across the prostate. Lastly, we detected highly localized subclones that were exclusive to tumour-rich regions and harboured deletions in TSGs that are known to be frequently deleted in prostate cancer. Taken together, with this thesis, I have contributed to advance the understanding of inter-patient, intra-patient, and intra-tumour heterogeneity

    Molekulargenetische Charakterisierung von Sarkomen zur Identifizierung prognostischer Risikogruppen und potentieller therapeutischer Angriffspunkte

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    Sarkome sind seltene Tumore, die sich durch eine erhebliche HeterogenitĂ€t auf histologischer, molekularer und genetischer Ebene auszeichnen. Trotz aller Fortschritte in der modernen Krebsbehandlung haben Sarkom-Patienten im fortgeschrittenen Stadium weiterhin begrenzte therapeutische Möglichkeiten und eine ungĂŒnstige Prognose. Da die Untersuchung des genetischen Profils nicht nur die Identifizierung prognostischer, sondern auch therapierelevanter VerĂ€nderungen bei heterogenen Erkrankungen ermöglicht, sind genetische Analysen ein unverzichtbarer Bestandteil der modernen Krebsbehandlung geworden. In dieser Studie analysierten wir retrospektiv das genetische Profil einer real-life Kohorte von 53 Sarkom-Patienten anhand eines 720-Gen-Panels. In Anbetracht der HeterogenitĂ€t von Sarkomen wurden mehrere histopathologische Subtypen analysiert, wobei das Leiomyosarkom (17 %) am hĂ€ufigsten vorkam. Das Durchschnittsalter der Patienten zum Zeitpunkt der Analyse betrug 49 Jahre. Die durchschnittliche Zeitspanne von der Erstdiagnose bis zur genetischen Analyse betrug 46,8 Monate. Das GesamtĂŒberleben betrug im Durchschnitt 55,9 Monate. Jeder Patient erhielt eine Tumorgenomsequenzierung mit einem 720-Gene-Panel. Bei 76,9% der Patienten wurde ein niedriger TMB-Wert festgestellt. Keiner der Patienten wurde als mikrosatelliteninstabil identifiziert. 25% der Patienten wiesen einen Mangel an der FunktionalitĂ€t der homologen Rekombination (HRD) auf. Bei 30,8% wurde ein Fusionsgen nachgewiesen, wobei EWSR1-FLI1 und EWSR1- WT1 am hĂ€ufigsten waren. Insgesamt wurden 38 KopienzahlverĂ€nderungen (CNAs) gefunden, was auf eine erhebliche genomische InstabilitĂ€t hinweist. Bei 15 Patienten wurden Keimbahnmutationen gefunden, die alle behandlungsrelevant sind, wobei die Mutation im MUTYH-Gen die hĂ€ufigste ist. Therapierelevante somatische Mutationen wurden bei 47 Patienten gefunden (3,2 Mutationen/ Patient). Die am hĂ€ufigsten betroffenen Gene waren TP53, CDKN2A-C, CDK4, RB1 und ATRX. 93 Auf der Grundlage der NGS-Ergebnisse erhielten 39,6 % der Patienten eine personalisierte Antitumortherapie. Das mediane GesamtĂŒberleben (OS) der Patienten mit einer gemĂ€ĂŸ den Daten der NGS-Analyse ausgerichteten Behandlung betrug 43 gegenĂŒber 33 Monaten bei Patienten ohne zielgerichtete Therapien. Unsere NGS-Daten aus einer heterogenen Kohorte von 53 Sarkom-Patienten deuten darauf hin, dass personalisierte Therapien, die auf den Ergebnissen einer 720 Gen-Panel-Sequenzierung basieren, zu verbesserten klinischen Ergebnissen bei Sarkom-Patienten fĂŒhren könnten

    Investigating the molecular drivers of CNS disease in a murine model of infant leukaemia

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    Infant leukaemia is a rare, aggressive entity with poor outcomes. Infant leukaemia is defined by the age of the patient, less than one year, and is commonly associated with rearrangements of the MLL gene. There are many unique features that distinguishMLLrearranged infant leukaemia from other paediatric leukaemias, including a high rate of central nervous system (CNS) involvement. CNS involvement is typically a leukaemic infiltrate of themeninges, a niche that is very different fromthe primary site of disease, the bone marrow (BM), in terms of nutrient abundance and cellular composition. While our understanding of how leukaemia cells survive and propagate in the CNS has progressed, there are many unanswered questions about infant leukaemia-specific features and the contribution of the niche. This thesis explores the cellular dynamics of leukaemia in the CNS niche, the differential regulation of immune cell interactions and growth factor pathways through transplantation assays, bulk RNA sequencing and functional experiments. In previous studies, two microRNAs (miR-128a and miR-130b) were identified as being upregulated in MLL-AF4+ infant patient samples. Overexpression of these microRNAs individually in mouse fetal liver haematopoietic stem and progenitor cells resulted in microRNA-dependent lineage-specific acute leukaemias in the context of Mll- AF4. Terminal leukaemia development in these immunocompetent models of infant leukaemia was associated with CNS involvement in a leptomeningeal distribution representative of human disease. These mouse models form the foundation of the investigation of CNS niche-specific features in infant leukaemia in this thesis. The functional properties of leukaemia propagating cells (LPC) in both niches, in either model, were explored. Data are presented that show different LPCs are very similar, are able to give rise to one another and are all represented in the CNS niche. Further transplantation assays show a lasting functional impact on LPCs and their ability to repopulate leukaemia following exposure to the CNS niche in both model systems. The thesis goes on to strengthen these findings by describing the transcriptomic differiv ences between these LPCs. The RNA sequencing data generated are also used to validate the miR-128a overexpression Mll-AF4, or pro-B infant acute lymphoblastic leukaemia, model. The next section of the thesis outlines transcriptomic differences between LPCs from the CNS and BM niches in both models. The discussion focuses on comparisons to existing datasets and on two novel differentially regulated processes; interaction with immune cells and growth factor signalling. Subsequent sections present functional followup experiments exploring these two novel themes. The data shown describe nichespecific differences in macrophage, T cell and NK cell dynamics and, of particular interest, imply suppression of anti-leukaemia macrophage and T cell responses in the CNS niche. In the final section, increased activation of the PI3K pathway in CNS-derived compared with BM-derived leukaemia cells is explored through downregulation of the inhibitory regulator PTEN.MiR-93, which targets PTENand CDKN1A, a downstream target of the PI3K pathway, is proposed as a global regulator of this cell-intrinsic niche-specific pathway activation. miR-93 is shown to be upregulated in CNS-derived leukaemia cells in the miR-128a overexpressionMll-AF4 model and three independent infant leukaemia patient-derived xenograft models. To conclude this section, suppression of miR-93 activity is shown to impair leukaemia cell engraftment to the CNS niche. Overall, this thesis will put forward new insights into cell-intrinsic and cell-extrinsic mechanisms of leukaemia cell survival and propagation within the CNS niche in infant leukaemia murine models

    Advancing fish breeding in aquaculture through genome functional annotation

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    Genomics is increasingly applied in breeding programmes for farmed fish and shellfish species around the world. However, current applications do not include information on genome functional activity, which can enhance opportunities to predict relationships between genotypes and phenotypes and hence increase the accuracy of selection. Here, we review prospects for improving aquaculture breeding practises through the uptake of functional genomics data in light of the EU Horizon 2020 project AQUA-FAANG: ‘Advancing European Aquaculture by Genome Functional Annotation’. This consortium targeted the six major farmed fish species in European aquaculture, producing thousands of functional genomic datasets from samples representing embryos to mature adults of both sexes, and following immunological stimulation. This data was used to catalogue functional activity across the genome of each species, revealing transcribed regions, distinct chromatin states and regulatory elements impacting gene expression. These functional annotations were shared as open data through the Ensembl genome browser using the latest reference genomes for each species. AQUA-FAANG data offers novel opportunities to identify and prioritize causative genetic variants responsible for diverse traits including disease resistance, which can be exploited to enhance selective breeding. Such knowledge and associated resources have the potential to improve sustainability and boost production in aquaculture by accelerating genetic gain for health and robustness to infection, whilst reducing the requirement for animal testing. We further outline directions to advance and leverage genome functional annotation beyond the AQUA-FAANG project. Given the diversity of aquaculture sectors and businesses, the incorporation of functional genomic information into breeding decisions will depend on technological readiness level and scale of operation, with cost-benefit analysis necessary to determine the most profitable approach for each species and production system

    Long-Molecule Assessment of Ribosomal DNA and RNA

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    The genes encoding ribosomal RNA and their transcriptional products are essential for life, however, remain poorly understood. Even with the advent of long-range sequencing methodologies, rDNA loci are difficult to study and remain obscure, prompting the consideration of alternative methods to probing this critical region of the genome. The research outlined in this thesis utilises molecular combing, a fibre stretching technique, to isolate DNA molecules measuring more than 5 Mbp in length. The capture of DNA molecules of this size should assist in exploring the architecture of entire rDNA clusters at the single-molecule level. Combining molecular combing with SNP targeting probes, this study aims to distinguish and assess the arrangement of rDNA promoter variants which have been shown to exhibit dramatically different environmental sensitivity. Additionally, through the application of Oxford Nanopore Technologies direct RNA sequencing, the work here has demonstrated the capture of near full-length rRNA primary transcripts, which will allow for assessing post-transcriptional modification across the length of multiple coding subunits within a single molecule, for the first time. Furthermore, an exploration of RNA modification profiles across sample types representative of different developmental stages has been conducted. This study predicts many sites to be differentially modified across these different developmental conditions, several of which are known to be important for, if not crucial in ribosome biogenesis and function. The work outlined in this thesis provides a framework for future studies to conduct long-molecule, genetic, and epitranscriptome profiling of this vital region of the genome, and its dynamic response to a changing environment

    Intratumoral heterogeneity and clonal evolution induced by HPV integration

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    The human papillomavirus (HPV) genome is integrated into host DNA in most HPV-positive cancers, but the consequences for chromosomal integrity are unknown. Continuous long-read sequencing of oropharyngeal cancers and cancer cell lines identified a previously undescribed form of structural variation, "heterocateny," characterized by diverse, interrelated, and repetitive patterns of concatemerized virus and host DNA segments within a cancer. Unique breakpoints shared across structural variants facilitated stepwise reconstruction of their evolution from a common molecular ancestor. This analysis revealed that virus and virus-host concatemers are unstable and, upon insertion into and excision from chromosomes, facilitate capture, amplification, and recombination of host DNA and chromosomal rearrangements. Evidence of heterocateny was detected in extrachromosomal and intrachromosomal DNA. These findings indicate that heterocateny is driven by the dynamic, aberrant replication and recombination of an oncogenic DNA virus, thereby extending known consequences of HPV integration to include promotion of intratumoral heterogeneity and clonal evolution
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