8,163 research outputs found
The interplay between Natural Killer cells and Pancreatic Stellate cells in Pancreatic Ductal Adenocarcinoma
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
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 groĂe 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 groĂen 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 Ăberleben 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
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Inference of Population Splits and Mixtures from Genome-Wide Allele Frequency Data
Many aspects of the historical relationships between populations in a species are reflected in genetic data. Inferring these relationships from genetic data, however, remains a challenging task. In this paper, we present a statistical model for inferring the patterns of population splits and mixtures in multiple populations. In our model, the sampled populations in a species are related to their common ancestor through a graph of ancestral populations. Using genome-wide allele frequency data and a Gaussian approximation to genetic drift, we infer the structure of this graph. We applied this method to a set of 55 human populations and a set of 82 dog breeds and wild canids. In both species, we show that a simple bifurcating tree does not fully describe the data; in contrast, we infer many migration events. While some of the migration events that we find have been detected previously, many have not. For example, in the human data, we infer that Cambodians trace approximately 16% of their ancestry to a population ancestral to other extant East Asian populations. In the dog data, we infer that both the boxer and basenji trace a considerable fraction of their ancestry (9% and 25%, respectively) to wolves subsequent to domestication and that East Asian toy breeds (the Shih Tzu and the Pekingese) result from admixture between modern toy breeds and âancientâ Asian breeds. Software implementing the model described here, called TreeMix, is available at http://treemix.googlecode.com.</p
Charting genomic heterogeneity in tumours : from bulk to single cell
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
Sarkome sind seltene Tumore, die sich durch eine erhebliche HeterogenitÀt 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 VerÀnderungen
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 HeterogenitÀt von Sarkomen wurden mehrere histopathologische
Subtypen analysiert, wobei das Leiomyosarkom (17 %) am hÀufigsten 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 FunktionalitÀt der homologen Rekombination (HRD)
auf. Bei 30,8% wurde ein Fusionsgen nachgewiesen, wobei EWSR1-FLI1 und
EWSR1- WT1 am hÀufigsten waren. Insgesamt wurden 38 KopienzahlverÀnderungen
(CNAs) gefunden, was auf eine erhebliche genomische InstabilitÀt hinweist.
Bei 15 Patienten wurden Keimbahnmutationen gefunden, die alle behandlungsrelevant
sind, wobei die Mutation im MUTYH-Gen die hÀufigste ist. Therapierelevante
somatische Mutationen wurden bei 47 Patienten gefunden (3,2 Mutationen/
Patient). Die am hÀufigsten 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 gemÀà 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
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
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: âAdvancing European Aquaculture by Genome Functional Annotationâ. 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
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
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Origination of an X-Linked Testes Chimeric Gene by Illegitimate Recombination in <i>Drosophila</i>
The formation of chimeric gene structures provides important routes by which novel proteins and functions are introduced into genomes. Signatures of these events have been identified in organisms from wide phylogenic distributions. However, the ability to characterize the early phases of these evolutionary processes has been difficult due to the ancient age of the genes or to the limitations of strictly computational approaches. While examples involving retrotransposition exist, our understanding of chimeric genes originating via illegitimate recombination is limited to speculations based on ancient genes or transfection experiments. Here we report a case of a young chimeric gene that has originated by illegitimate recombination in Drosophila. This gene was created within the last 2â3 million years, prior to the speciation of Drosophila simulans, Drosophila sechellia, and Drosophila mauritiana. The duplication, which involved the BĂ€llchen gene on Chromosome 3R, was partial, removing substantial 3âČ coding sequence. Subsequent to the duplication onto the X chromosome, intergenic sequence was recruited into the protein-coding region creating a chimeric peptide with ~ 33 new amino acid residues. In addition, a novel intron-containing 5âČ UTR and novel 3âČ UTR evolved. We further found that this new X-linked gene has evolved testes-specific expression. Following speciation of the D. simulans complex, this novel gene evolved lineage-specifically with evidence for positive selection acting along the D. simulans branch.</p
Intratumoral heterogeneity and clonal evolution induced by HPV integration
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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