Dissertations / Theses on the topic 'Tumour sequencing'
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Burns, Alice Sin Ying Wai. "The role of the p53 tumour suppressor pathway in central primitive neuroectodermal tumours." Thesis, University of Newcastle Upon Tyne, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.300357.
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Duarte, Antonio. "Regulation of gene expression by the Wilms' tumour suppressor, WT1." Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.389178.
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Murtaza, Muhammed. "Identification and monitoring of somatic mutations in solid cancers by sequencing circulating tumour DNA." Thesis, University of Cambridge, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708647.
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Ross, Edith. "Inferring tumour evolution from single-cell and multi-sample data." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/274604.
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Tumour development has long been recognised as an evolutionary process during which cells accumulate mutations and evolve into a mix of genetically distinct cell subpopulations. The resulting genetic intra-tumour heterogeneity poses a major challenge to cancer therapy, as it increases the chance of drug resistance. To study tumour evolution in more detail, reliable approaches to infer the life histories of tumours are needed. This dissertation focuses on computational methods for inferring trees of tumour evolution from single-cell and multi-sample sequencing data. Recent advances in single-cell sequencing technologies have promised to reveal tumour heterogeneity at a much higher resolution, but single-cell sequencing data is inherently noisy, making it unsuitable for analysis with classic phylogenetic methods. The first part of the dissertation describes OncoNEM, a novel probabilistic method to infer clonal lineage trees from noisy single nucleotide variants of single cells. Simulation studies are used to validate the method and to compare its performance to that of other methods. Finally, OncoNEM is applied in two case studies. In the second part of the dissertation, a comprehensive collection of existing multi-sample approaches is used to infer the phylogenies of metastatic breast cancers from ten patients. In particular, shallow whole-genome, whole exome and targeted deep sequencing data are analysed. The inference methods comprise copy number and point mutation based approaches, as well as a method that utilises a combination of the two. To improve the copy number based inference, a novel allele-specific multi-sample segmentation algorithm is presented. The results are compared across methods and data types to assess the reliability of the different methods. In summary, this thesis presents substantial methodological advances to understand tumour evolution from genomic profiles of single cells or related bulk samples.
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Fewings, Eleanor Rose. "The use of whole exome sequencing data to identify candidate genes involved in cancer and benign tumour predisposition." Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/285963.
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The development of whole exome sequencing has transformed the study of disease predisposition. The sequencing of both large disease sets and smaller rare disease families enables the identification of new predisposition variants and potentially provide clinical insight into disease management. There is no standard protocol for analysing exome sequencing data. Outside of extremely large sequencing studies including thousands of individuals, statistical approaches are often underpowered to detect rare disease associated variants. Aggregation of variants into functionally related regions, including genes, gene clusters, and pathways, allows for the detection of biological processes that, when interrupted, may impact disease risk. In silico functional studies can also be utilised to further understand how variants disrupt biological processes and identify genotype-phenotype relationships. This study describes the exploration of sequencing datasets from cancers and benign tumour diseases including: i) hereditary diffuse gastric cancer, ii) sweat duct proliferation tumours, iii) adrenocortical carcinoma, and iv) breast cancer. Each set underwent germline whole exome sequencing followed by additional tumour or targeted sequencing to identify associated predisposition genes. Variants within a cluster of risk genes that are involved in double strand break repair were identified as associated with hereditary diffuse gastric cancer risk via gene ontology enrichment analysis. This cluster included PALB2 within which, using externally collated data, loss of function variants were identified as significantly associated with hereditary diffuse gastric cancer risk. Germline protein-affecting variants in the myosin gene MYH9 were identified in all individuals with a rare sweat duct proliferative syndrome, suggesting a role for MYH9 in skin development, regulation and tumorigenesis. These MYH9 variants were analysed in silico to identify a genotype-phenotype relationship between the clinical presentation and variants in the ATP binding pocket of the protein. Tumour matched normal sequence data from adrenocortical carcinoma cases was used to elucidate the role of Lynch syndrome genes in disease pathogenesis. Within the breast cancer set, candidate genes were selected to undergo targeted sequencing in a larger set of cases to further explore their role in breast cancer risk. Risk associated genes identified within this study may ultimately aid in diagnosis and management of disease. This thesis has also generated multiple novel tools and sequencing analysis techniques that may be of use for further studies by aiding in the prioritisation of candidate variants. The described techniques will provide support to researchers working on rare, statistically underpowered datasets and to provide standard analysis pipelines for a range of dataset sizes and types, including familial data and unrelated individuals.
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Asante, Du-Bois. "Development and evaluation of methodologies for analysis of CTC and ctDNA in patients with ovarian carcinoma." Thesis, Edith Cowan University, Research Online, Perth, Western Australia, 2022. https://ro.ecu.edu.au/theses/2570.
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About 70% of patients with ovarian cancer (OC) are diagnosed at an advanced stage (III-IV), with associated poor prognosis, even after chemotherapeutic interventions (mostly platinum taxane-based), leading to poor survival rates. The current biomarkers available in the clinic are not enough for efficient prognostication and surveillance of OC. Hence, more accurate and reliable biomarkers of patient disease status are urgently required.
Currently, histopathological evaluation of tumour biopsies is the gold standard in clinical practice with the purpose of evaluating specific biomarkers to predict therapeutic response. However, this is invasive, and associated complications may occur. Liquid biopsy is a minimally invasive test and has the advantage of allowing real-time monitoring of treatment response and comprehensive serial/repetitive phenotypic and genotypic profiling of the primary, metastatic and recurrent tumours. Circulating tumour cells (CTC) and circulating tumour DNA (ctDNA) represent a new generation of biomarkers that can be used for predicting therapy responsiveness and longitudinal monitoring of OC patients undergoing therapy.
This thesis describes a series of investigations in OC which include, a methodological study to improve phenotypic characterization of detected CTCs, genomic validation of putative CTCs, and the evaluation of the clinical validity of ctDNA as biomarker of response to neoadjuvant chemotherapy (NACT). This thesis consists of six chapters: a general introduction to OC (Chapter 1); a thorough review of the literature regarding CTCs and ctDNA in OC (Chapter 2); three results chapters (Chapters 3 - 5); and a final chapter that comprises of a general discussion of the main findings, the limitations of the study and future directions (Chapter 6).
The first chapter (Chapter 1) of the thesis includes a review of the literature, which consist of the characterisation and clinical landscape of OC, while briefly introducing the topic of liquid biopsy. This is then followed by a comprehensive review (Chapter 2) that highlights the progress in the field of liquid biopsy for OC from 2011 to 2019, focusing specifically on CTCs and ctDNA as potential biomarkers for the management of the disease.
Based on the limitations identified in this review (in Chapter 2), a multi-marker antibody staining protocol was developed to target epithelial (cytokeratin (CK) and EpCAM), mesenchymal (vimentin), and OC specific (PAX8) markers for detection of CTCs (Chapter 3). CD45/16 and CD31 were used for the exclusion of white blood and vascular endothelial cells, respectively. PD-L1 was used for CTC characterisation. Optimisation of the CTC markers were done using OC cell lines, SKOV-3 and OVCA432. CTCs were enriched using the ParsortixTM (Angle plc.) system, and further detected using the multifluorescent markers from 16 OC patients. Patients were found to have heterogeneous CTCs, with 113/157 (72%) cells positive for CK/EpCAM (epithelial marker), 58/157 (37%) positive for vimentin (mesenchymal marker), and 17/157 (11%) positive for both (hybrid). Positivity for PD-L1 was significantly associated with the hybrid phenotype when compared with the epithelial (p = 0.0009) and mesenchymal (p = 0.007) expressing CTCs.
In Chapter 4, we performed copy number alteration (CNA) profiling on putative CTCs that were previously identified in Chapter 3. CNA were detected in both CK/EpCAM and vimentin positive CTCs. However, a proportion of cells expressing CK/EpCAM, PD-L1 and CD31 were found not to carry CNAs. Further characterisation of CTCs in combination with clinical outcomes is needed to provide insight into the role that epithelial to mesenchymal plasticity and vasculogenic mimicry plays in OC and its relationship with PD-L1 expression.
In addition, ctDNA was analysed in 58 patients with high grade serous ovarian cancer (HGSOC) enrolled in a neoadjuvant chemotherapy (NACT) plus immune checkpoint inhibitor clinical trial (iPRIME, ACTRN12618000109202) (Chapter 5). Cell free DNA (cfDNA) was sequenced using the Oncomine™ Breast cfDNA Research Assay V2, and selected mutations were validated using droplet digital PCR (ddPCR). For ctDNA, the frequencies of genes affected by non-synonymous somatic mutations included TP53 (67%), KRAS (11%), PIK3CA (2%) and SF3B1 (2%). A total of 41 gene variants were identified in either pre- or post-NACT samples; 36 (87.8%) of them had variants in TP53 alone, and 3 (7.4%) in KRAS and 1 (2.4%) each for PIK3CA and SF3B1. Undetectable levels of ctDNA post, but not prior to NACT was associated with chemotherapy response score 3 (CRS3) (p = 0.04) when compared with CRS1/2. Comparison of mutant copies per mL of plasma quantified by NGS and ddPCR demonstrated a strong correlation (Pearson’s r = 0.929; p < 0.0001) between the two platforms. Longitudinal monitoring of ctDNA specific mutant gene variants using ddPCR, showed a predictive lead time, and anticipates disease progression earlier (7-12 weeks) compared to monitoring patients by analysis of serum levels of CA-125. These results suggest that ctDNA could be used as a biomarker to determine patients’ clinical outcomes and serve as a potential endpoint for clinical trials.
Finally, Chapter 6 provides a general discussion of the studies covered in this thesis, highlighting potential contributions to the growing field of liquid biopsy and its application for the clinical management of OC patients. The limitations inherent to both CTCs and ctDNA analysis, and suggestions for improvements before its successful implementation in routine clinical practice, are comprehensively discussed.
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Naven, Marc. "Development of a pipeline and protocols for next generation sequencing of blood and formalin-fixed, paraffin-embedded tumour DNA samples." Thesis, Cardiff University, 2015. http://orca.cf.ac.uk/91435/.
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Using existing software and six novel scripts, we developed a pipeline for variant calling using exome re-sequencing data of germline blood deoxyribonucleic acid (DNA) samples. We observed >99% (6,723/6,739) concordance between calls from our pipeline and previous genotyping. We identified >93% of single nucleotide variants (SNVs) and >94% of insertion/deletions called by a commercial partner using the same sequencing reads. Using a subset of genes, we showed that around half of predicted pathogenic variants could be validated in vitro. Together, these data showed that our pipeline was reliable for variant calling. Next generation sequencing (NGS) of DNA from formalin-fixed, paraffin-embedded (FFPE) tumours is technically challenging. We sought to determine the sensitivity of NGS to detect known somatic hotspot mutations (n=25) in 19 FFPE advanced colorectal cancers and to optimise it for the identification of novel somatic variants. Analysis using Illumina’s MiSeq software identified 100% of somatic mutations with 93% specificity, whereas the Genome Analysis Tool Kit’s (GATK) HaplotypeCaller identified 80-92% of somatic mutations with 100% specificity. We investigated the background mutator profile and found that normal FFPE DNA had 3-fold more SNVs than matched blood DNA, with an excess of FFPE-associated C:G>T:A substitutions (27.1 vs. 5.3%). Uracil DNA glycosylase treatment reduced this excess. Only ~5% of variants were consistently called in replicate runs and were likely to be genuine somatic variants. We detected potential candidates for genetic biomarkers of cetuximab-resistance in colorectal cancers that were previously shown to be wild type for KRAS, NRAS, BRAF and PIK3CA. We found that 7/21 (33%) of the CRCs analysed harboured mutations at either codon 12 or 61, whileother CRCs carried truncating KRAS mutations. NRAS also carried a codon 12 mutation in 1 CRC, and PIK3CA possessed mutations at codons 542 and 545. PTEN was found to harbour 5 truncating mutations at codons 71, 140, 155, 178 and 189, potentially leading to loss of function.
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Bucher, Katharina M. I. "The tumour suppressor gene p53 in the horse : identification, cloning and sequencing : its possible role in the pathogenesis of equine sarcoid /." [S.l.] : [s.n.], 1995. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
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Choudhry, Hani. "Genome-wide analysis of the hypoxic breast cancer transcriptome using next generation sequencing." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:9a66b553-a66c-4164-a854-5881be65ca45.
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Hypoxia pathways are associated with the pathogenesis of both ischaemic and neoplastic diseases. In response to hypoxia the transcription factor hypoxia‐inducible factor (HIF) induces the expression of hundreds of genes with diverse functions. These enable cells to adapt to low oxygen availability. To date, pan-genomic analyses of these transcriptional responses have focussed on protein-coding genes and microRNAs. However, the role of other classes of non-coding RNAs, in particular lncRNAs, in the hypoxia response is largely uncharacterised. My thesis aimed at improving understanding of the transcriptional regulation of the non-coding transcriptome in hypoxia. I performed an integrated genomic analysis of both non-coding and coding transcripts by massively parallel sequencing. This was interfaced with pan-genomic analyses of DNAse hypersensitivity and HIF, H3k4me3 and RNApol2 binding in hypoxic cells. These analyses have revealed that hypoxia profoundly regulated all RNA classes. snRNAs and tRNAs are globally downregulated in hypoxia, whilst miRNAs, mRNAs and lncRNAs are both up- and downregulated with an overall trend towards slight upregulation. In addition, a significant number of previously non-annotated (and largely hypoxia upregulated) transcripts were identified, including novel intergenic transcripts and natural antisense transcripts. HIF bound close to genes for mRNAs, miRNAs and lncRNAs that were upregulated by hypoxia, but was excluded from binding at genes for RNA classes that showed global downregulation. This suggests that HIF acts as a transcriptional activator (but not repressor), of lncRNAs as well as mRNAs and miRNAs. Consistent with direct regulation by HIF, many of these hypoxia-inducible, HIF-binding lncRNAs were downregulated following HIF knockdown. Analysis of RNApol2 binding and DNAse HSS signals at HIF transcriptional target genes indicated that HIF-dependent transcriptional activation occurs through release of RNApol2 that is pre-bound to open promoters of lncRNAs as well as mRNAs. In these datasets, NEAT1 was the most hypoxia-upregulated, HIF-targeted lncRNA in MCF-7 cells and, despite binding of both HIF-1 and HIF-2 isoforms at its promoter, was selectively regulated by HIF-2 alone. Furthermore, NEAT1 was induced by hypoxia in a wide range of breast cancer cell lines and in hypoxic xenograft models. Functionally, NEAT1 is required for the assembly of nuclear paraspeckle structures. Increased nuclear paraspeckle formation was observed in hypoxia and was dependent on both NEAT1 and HIF-2. Knockdown of hypoxia-induced NEAT1 significantly reduced cell proliferation and survival and induced apoptosis. Finally, high expression of NEAT1 correlated with poor clinical outcome in a large cohort of breast cancer patients. These findings extend the role of the hypoxic transcriptional response in cancer into the spectrum of non-coding transcripts and provide new insights into molecular roles of hypoxia-regulated lncRNAs, which may provide the basis for novel therapeutic targets in the future.
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Strakova, Andrea. "Genome diversity and evolution in canine transmissible venereal tumour." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/276037.
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The canine transmissible venereal tumour (CTVT) is a contagious cancer that is naturally transmitted between dogs by the allogeneic transfer of living cancer cells during coitus. CTVT first arose several thousand years ago and has been reported in dog populations worldwide. The goals of this Thesis were (1) to gain further understanding of CTVT distribution patterns and prevalence around the world, (2) to use genetics to trace the historical spread of CTVT and (3) to map the genetic as well as phenotypic diversity of CTVT tumours around the world. To understand the distribution patterns of CTVT, I obtained information from 645 veterinarians and animal health workers in 109 countries, and generated a snapshot of the locations in which this disease is found. Additionally, as preparation for further genetic analysis, I collected samples from over one thousand CTVT cases from more than 50 countries, optimised methods for high-throughput DNA extraction and quantification and optimised a qPCR-based assay for CTVT diagnosis and host contamination detection. With the goal of tracing the historical spread of CTVT and learning about the genetic diversity of this disease, I sequenced complete mitochondrial genomes of 449 CTVT tumours and their matched hosts. The analysis of the CTVT mitochondrial diversity revealed that CTVT has captured mitochondrial DNA (mtDNA) through horizontal transfer events at least five times during the history of the lineage, delineating five tumour clades. CTVT appears to have spread rapidly around the world within the last 2,000 years, perhaps transported by dogs travelling along historic maritime trade routes. This work indicated that negative selection has operated to prevent accumulation of deleterious mutations in captured mtDNA, and that recombination has caused occasional mtDNA re-assortment. A histology-based screen of CTVT clades did not show any significant phenotypic differences between groups. In order to determine how the five mtDNA clades relate to each other, I analysed data from 539 CTVT exomes. This revealed that a single canine mtDNA haplogroup has recurrently and recently undergone multiple horizontal transfer events. Analysis of this haplotype highlighted a number of candidate genetic variants which may be conferring a selective advantage to this haplotype in CTVT, possibly by influencing mtDNA transcription or replication. Overall, genetic and phenotypic analysis of CTVT tumours from across the globe has broadened our understanding of CTVT diversity, and provided important insights into the biology of a unique transmissible cancer.
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Brown, David Norman. "Application of phylogenetic inference methods to quantify intra-tumour heterogeneity and evolution of breast cancers." Doctoral thesis, Universite Libre de Bruxelles, 2017. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/260251.
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Cancer related mortality is almost always due to metastatic dissemination of the primary disease. While research into the biological mechanisms that drive the metastatic cascade continues to unravel its molecular underpinnings, progress in our understanding of biological phenomena such as tumour heterogeneity and its relevance to the origins of distant recurrence or the emergence of resistance to therapy has been limited.In parallel to major breakthroughs in the development of high throughput molecular techniques, researchers have begun to utilise next generation sequencing to explore the relationship between primary and matched metastatic tumours in diverse types of neoplasia. Despite small cohort sizes and often, a limited number of matched metastases for each patient, pioneering studies have uncovered hitherto unknown biological processes such as the occurrence of organ specific metastatic lineages, polyclonal seeding and homing of metastatic cells to the primary tumour bed. While yet other studies continue to highlight the potential of genomic analyses, at the time this thesis was started, an in-depth knowledge of disease progression and metastatic dissemination was currently lacking in breast cancers.Herein, we employed phylogenetic inference methods to investigate intra-tumour heterogeneity and evolution of breast cancers. A combination of whole exome sequencing, custom ultra-deep resequencing and copy number profiling were applied to primary tumours and their associated metastases from ten autopsied breast cancer patients. Two modes of metastatic progression were observed. In the majority of cases, all distant metastases clustered on a branch separate from their primary lesion. Clonal frequency analysis of somatic mutations showed that the metastases had a monoclonal origin and descended from a common metastatic precursor. Alternatively, the primary tumour was clustered alongside metastases with early branches leading to distant organs. This dichotomy coincided with the clinical history of the patients whereby multiple seeding events from the primary tumour alongside cascading metastasis-to-metastasis disseminations occurred in treatment naïve de novo metastatic patients, whereas descent from a common metastatic precursor was observed in patients who underwent primary surgery followed by systemic treatment. The data also showed that a distant metastasis can be horizontally cross-seeded and finally revealed a correlation between the extent of somatic point mutations private to the distant lesions and patient overall survival. In an unrelated dataset of relapsed breast cancer patients with matched primary and distant lesions profiled using whole genome sequencing, the landscape of somatic alterations confirmed the time dependency of copy number aberrations implying that cancer phylogenies can be dated using a molecular clock.The work presented here harnesses the strength of high throughput genomic techniques and state of the art phylogenetic tools to tell the evolutionary history of breast cancers. Our results show that the linear and parallel models of metastatic dissemination which have been held as near doctrines for many years are overstated point of views of cancer progression. Beyond the biological insights, these results suggest that surgical excision of the primary tumour in de novo metastatic breast cancers might reduce dissemination in selected cases hence providing a potential biological rationale for this practice. Similarly, there is no strong evidence of benefit in overall survival from surgical resection of oligo-metastases in breast cancer. From our analyses, metastatic lesions constitute an additional source of seeding and heterogeneity in advanced breast cancer. The data presented here is too small to derive practice-changing evidence, but supports the concept that resecting isolated metastases may be of clinical benefit in oligo-metastatic breast cancer patients. In both cases, results from larger prospective studies are warranted.Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)
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Marass, Francesco. "Latent feature models and non-invasive clonal reconstruction." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/267784.
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Intratumoural heterogeneity complicates the molecular interpretation of biopsies, as multiple distinct tumour genomes are sampled and analysed at once. Ignoring the presence of these populations can lead to erroneous conclusions, and so a correct analysis must account for the clonal structure of the sample. Several methods to reconstruct tumour clonality from sequencing data have been proposed, spanning methods that either do not consider phylogenetic constraints or posit a perfect phylogeny. Models of the first type are typically latent feature models that can describe the observed data flexibly, but whose results may not be reconcilable with a phylogeny. The second type, instead, generally comprises non-parametric mixture models, with strict assumptions on the tumour’s evolutionary process. The focus of this dissertation is on the development of a phylogenetic latent feature model that can bridge the advantages of these two approaches, allowing deviations from a perfect phylogeny. The work is recounted by three statistical models of increasing complexity. First, I present a non-parametric model based on the Indian Buffet Process prior, and highlight the need for phylogenetic constraints. Second, I develop a finite, phylogenetic extension of the previous model, and show that it can outperform competing methods. Third, I generalise the phylogenetic model to arbitrary copy-number states. Markov chain Monte Carlo algorithms are presented to perform inference. The models are tested on datasets that include synthetic data, controlled biological data, and clinical data. In particular, the copy-number generalisation is applied to longitudinal circulating tumour DNA samples. Liquid biopsies that leverage circulating tumour DNA require sensitive techniques in order to detect mutations at low allele fractions. One method that allows sensitive mutation calling is the amplicon sequencing strategy TAm-Seq. I present bioinformatic tools to improve both the development of TAm-Seq amplicon panels and the analysis of its sequencing data. Finally, an enhancement of this method is presented and shown to detect mutations de novo and in a multiplexed manner at allele fractions less than 0.1%.
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Leventoux, Nicolas. "Etude des foyers d’hétérogénéité tumorale dans les gliomes diffus de bas grade de l’adulte mutés IDH1." Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTT037.
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Les gliomes sont les principales tumeurs primitives du cerveau affectant environ 4000 nouveaux patients par an en France. La moitié des gliomes est détectée au stade avancé de glioblastome (grade IV) tandis que 15% des tumeurs sont diagnostiquées au stade II de gliomes diffus dit de bas grade. Ces tumeurs affectent des patients jeunes et présentent des mutations caractéristiques, notamment une mutation pour l’enzyme IDH1 communément retrouvée dans les glioblastomes secondaires. Ces tumeurs de bas grade sont traitées par une chirurgie, idéalement en condition éveillée mais du fait de leur nature diffuse, la partie résiduelle progressera inexorablement vers un stade III ou IV avec une survie globale entre 5 ans et 15 ans après diagnostique. La progression tumorale est hautement variable et non prédictible d’un patient à l’autre. Des foyers de progression tumorale chez 20% des patients atteints de gliome diffus de bas grade ont été identifiés. Ces foyers montrent une densité cellulaire plus élevée ainsi qu’un Ki67 augmenté. Mon travail de thèse aura consisté à étudier les modifications cellulaires et moléculaires associées à ces foyers de progression tumorale. À partir du profil ARN des foyers et des territoires adjacents, j’ai pu mettre en évidence par des techniques haut-débit la baisse d’expression significative de gènes dans les foyers notamment de AGXT2L1/ETNPPL, carboxypeptidase E, EDNRB, SFRP2. J’ai émis l’hypothèse que SFRP2 et ETNPLL pourraient s’opposer à la prolifération cellulaire et que leur diminution dans les foyers ouvrirait la voie à la transformation tumorale. Une corrélation inverse entre la quantité d’ETNPPL enzyme et la survie de patients atteints d’hépatocarcinomes a été publiée. En limitant la quantité de précurseurs de phospholipides dans la cellule, ETNPPL pourrait agir comme un frein en s’opposant à la prolifération et de fait, sa diminution dans les foyers de transformation des gliomes pourrait lever cette inhibition. Mes travaux auront été innovants tant dans leur approche comparative des différents compartiments tumoraux pour chaque patient étudié et auront révélés ETNPPL comme corrélé à la gliomagenèse et potentielle cible thérapeutiqueGliomas are the main primary brain tumours affecting around 4000 new patients in France each year. Half of gliomas are detected in the advanced stage of glioblastoma (grade IV) while 15% of tumours are diagnosed in stage II (diffuse low-grade gliomas-DLGG). These tumors affect young patients and bear characteristic mutations, including a mutation for the enzyme IDH1 commonly found in secondary glioblastomas. These low-grade tumours are treated by surgery, ideally in awake condition but due to their diffuse nature, the residual part will progress inexorably to stage III or IV with overall survival between 5 and 15 years after diagnosis. Tumor progression is highly variable and unpredictable from one patient to another. Foci of tumor progression have been identified in 20% of patients with DLGG. These foci show a higher cell density and an increased Ki67. My thesis work consisted in studying the cellular and molecular changes associated with tumor progression. From the RNA profile of the foci and adjacent territories, I was able to highlight through high-throughput techniques significant decrease in gene expression in the foci, particularly of AGXT2L1/ETNPPL, carboxypeptidase E, EDNRB, SFRP2. I hypothesized that SFRP2 and ETNPLL could oppose cell proliferation and that their decrease would pave the way for tumor transformation. An inverse correlation between the amount of ETNPPL and the survival of patients with hepatocarcinoma has been published. By limiting the amount of phospholipid precursors in the cell, ETNPPL could act as a brake against proliferation and indeed, its decrease in glioma transformation foci could remove this inhibition. My PhD work will have been innovative in the comparative approach of the different tumors’ compartments for each patient studied and will have revealed ETNPPL as correlated to gliomagenesis and as potential therapeutic target
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Behjati, Sam. "Massively parallel sequencing of benign and malignant human tumours." Thesis, University of Cambridge, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708693.
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Fishler, Kristen B. S. "“It’s the Wild, Wild West Out There” Experiences of a Multidisciplinary Genomic Breast Cancer Tumor Board Implementing Tumor Sequencing in Clinical Care." University of Cincinnati / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1525169475571341.
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Anamaria, Crisan. "Mutation discovery in regions of segmental cancer genome amplifications from next generation sequencing of tumours." Thesis, University of British Columbia, 2010. http://hdl.handle.net/2429/29454.
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Next generation sequencing has now enabled a cost-effective enumeration of the full mutational complement of a tumour genome - in particular single nucleotide variants (SNVs). However, most current computational and statistical models for analyzing next generation sequencing data do not account for cancer-specific biological properties, including somatic segmental copy number alterations (CNAs), which require special treatment of the data.
Here we present CoNAn-SNV (Copy Number Annotated –SNV): a novel algorithm for the inference of single nucleotide variants (SNVs) that overlap copy number alterations. The method is based on modelling the notion that genomic regions of segmental duplication and amplification induce an extended ‘genotype space’ where a subset of genotypes will exhibit heavily skewed allelic distributions in SNVs (and therefore render them undetectable by methods that assume diploidy). CoNAn-SNV introduces the concept of modelling allelic counts from sequencing data using a panel of Binomial mixture models where the number of mixtures for a given locus in the genome is informed by a discrete copy number state given as input. We applied CoNAn-SNV to a previously published whole genome shotgun data set obtained from a lobular breast cancer and show that it is able to detect 24 experimentally revalidated somatic non-synonymous mutations that were not detected using copy number insensitive SNV discovery algorithms. Importantly, ROC analysis shows that the increased sensitivity of CoNAn-SNV does not result in disproportionate loss of specificity. Our results indicate that in genomically unstable tumours, copy number annotation for SNV detection will be critical to fully characterize the mutational landscape of cancer genomes.
The Binomial mixture model framework, however, is unable to fully cope with the complexity of tumour sequence data. We explore substituting the Binomial mixture model framework with the Beta-Binomial framework to overcome this limitation. The effectiveness of this substitution is compared against the lobular breast carcinoma and the 30 exon capture data sets all derived from triple negative breast cancers. The performance of Binomial and Beta-Binomial mixture model is evaluated against a cohort of exon capture test cases and we report ROC and f-measures.
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Finetti, Martina Anna. "Next-generation sequencing identifies mechanisms of tumourigenesis caused by loss of SMARCB1 in Malignant Rhabdoid Tumours." Thesis, University of Newcastle upon Tyne, 2015. http://hdl.handle.net/10443/3891.
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Introduction: Malignant Rhabdoid Tumours (MRT) are unique malignancies caused by biallelic inactivation of a single gene (SMARCB1). SMARCB1 encodes for a protein that is part of the SWI/SNF chromatin remodelling complex, responsible for the regulation of hundreds of downstream genes/pathways. Despite the simple biology of these tumours, no studies have identified the critical pathways involved in tumourigenesis. The understanding of downstream effects is essential to identifying therapeutic targets that can improve the outcome of MRT patients. Methods: RNA-seq and 450K-methylation analyses have been performed in MRT human primary malignancies (n > 39) and in 4 MRT cell lines in which lentivirus was used to re-express SMARCB1 (G401, A204, CHLA-266, and STA-WT1). The MRT cell lines were treated with 5-aza-2 -deoxycytidine followed by global gene transcription analysis (RNA-seq and 450K-methylation) to investigate how changes in methylation lead to tumourigenesis. Results: We show that primary Malignant Rhabdoid Tumours present a unique and distinct expression/methylation profile which confirms that MRT broadly constitute a single and different tumour type from other paediatric malignancies. However, despite their common cause MRT can be can sub-group by location (i.e. CNS or kidney). We observe that re-expression of SMARCB1 in MRT cell lines determines activation/inactivation of specific downstream pathways such as IL-6/TGF beta. We also observe a direct correlation between alterations in methylation and gene expression in CD44, GLI2, GLI3, CDKN1A, CDKN2A and JARID after SMARB1 re-expression. Loss of SMARCB1 also promotes expression of aberrant isoforms and novel transcripts and causes genome-wide changes in SWI/SNF binding. Conclusion: Next generation transcriptome and methylome analysis in primary MRT and in functional models give us detailed downstream effects of SMARCB1 loss in Malignant Rhabdoid Tumours. The integration of data from both primary and functional models has provided, for the first time, a genome-wide catalogue of SMARCB1 tumourigenic changes (validated using systems biology). Here we show how a single V deletion of SMARCB1 is responsible for deregulation of expression, methylation status and binding at the promoter regions of potent tumour-suppressor genes. The genes, pathways and biological mechanisms indicated as key in tumour development may ultimately be targetable therapeutically and will lead to better treatments for what is currently one of the most lethal paediatric cancers.
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Chicard, Mathieu. "Utilisation de l’ADN circulant pour l’étude de l'hétérogénéité tumorale et l'évolution clonale en oncologie pédiatrique Whole Exome Sequencing of Cell-Free DNA Reveals Temporo-Spatial Heterogeneity and Identifies Treatment-Resistant Clones in Neuroblastoma." Thesis, université Paris-Saclay, 2020. https://tel.archives-ouvertes.fr/tel-03154451.
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Les cancers pédiatriques représentent un défi thérapeutique, et une compréhension des mécanismes d’échappement aux traitements est nécessaire pour pouvoir développer de nouvelles approches thérapeutiques. L'ADN circulant peut être libéré par une tumeur dans les fluides corporels et permet de détecter et suivre des altérations génétiques tumorales par des prélèvements successifs peu invasifs tels qu'une prise de sang. Dans ce travail, une technique de séquençage de type « whole exome » (séquençage de l’ensemble des exons) de l’ADN circulant a été mis au point pour permettre l’étude des altérations génétiques tumorales dans le plasma chez des enfants atteints de cancer.Ces analyses soulignent la grande hétérogénéité génétique spatiale et temporelle des cancers pédiatriques. De plus, un rôle important de l’évolution clonale dans la progression de la maladie a ainsi pu être mis en évidence. Des approches utilisant les caractéristiques particuliers de l'ADN circulant ont également permis d’inférer le profil d’expression, basées sur l’empreinte des sites de début de transcription, ou le profil épigénétique de la tumeur. En plus d'une aide à une classification des tumeurs, ces particularités pourront aider à l'observation d'un changement d'identité cellulaire sous traitement. L'ADN circulant est donc un formidable outil pour mieux comprendre l'échappement aux traitements d'une tumeur par son hétérogénéité spatiale et temporelle et sa plasticitéPediatric cancers represent a therapeutic challenge, and an understanding of the mechanisms of escape from treatment is necessary in order to be able to develop new therapeutic approaches. Circulating DNA can be released by a tumor in bodily fluids and enable to detect and follow genetic alterations in tumors by successive minimally invasive samples such as a blood test. In this work, a “whole exome” sequencing technique for circulating DNA was developed to allow the study of genetic tumor alterations in plasma in children with cancer. These analyzes highlight the frequent spatial and temporal genetic heterogeneity of pediatric cancers. In addition, an important role of clonal evolution in the progression of the disease was thus highlighted. Approaches using the particular characteristics of circulating DNA have also made it possible to infer the expression profile, based on the imprint of the transcription start sites, or the epigenetic profile of the tumor. In addition to an aid in the classification of tumors, these features may help to observe a change in cellular identity under treatment. Circulating DNA is therefore an important tool for better understanding mechanisms of escape from treatment of a tumor based on spatial and temporal heterogeneity and cellular plasticity
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Batra, Rajbir Nath. "Decoding the regulatory role and epiclonal dynamics of DNA methylation in 1482 breast tumours." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/274923.
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Breast cancer is a clinically and molecularly heterogeneous disease displaying distinct therapeutic responses. Although recent studies have explored the genomic and transcriptomic landscapes of breast cancer, the epigenetic architecture has received less attention. To address this, an optimised Reduced Representation Bisulfite Sequencing protocol was performed on 1482 primary breast tumours (and 237 matched adjacent normal tissues). This constitutes the largest breast cancer methylome yet, and this thesis describes the bioinformatics and statistical analysis of this study. Noticeable epigenetic drift (both gain and loss of homogeneous DNA methylation patterns) was observed in breast tumours when compared to normal tissues, with markedly higher differences in late replicating genomic regions. The extent of epigenetic drift was also found to be highly heterogeneous between the breast tumours and was sharply correlated with the tumour’s mitotic index, indicating that epigenetic drift is largely a consequence of the accumulation of passive cell division related errors. A novel algorithm called DMARC (Directed Methylation Altered Regions in Cancer) was developed that utilised the tumour-specific drift rates to discriminate between methylation alterations attained as a consequence of stochastic cell division errors (background) and those reflecting a more instructive biological process (directed). Directed methylation alterations were significantly enriched for gene expression changes in breast cancer, compared to background alterations. Characterising these methylation aberrations with gene expression led to the identification of breast cancer subtype-specific epigenetic genes with consequences on transcription and prognosis. Cancer genes may be deregulated by multiple mechanisms. By integrating with existing copy number and gene expression profiles for these tumours, DNA methylation alterations were revealed as the predominant mechanism correlated with differentially expressed genes in breast cancer. The crucial role of DNA methylation as a mechanism to target the silencing of specific genes within copy number amplifications is also explored which led to the identification of a putative tumour suppressor gene, THSZ2. Finally, the first genome-wide assessment of epigenomic evolution in breast cancer is conducted. Both, the level of intratumoural heterogeneity, and the extent of epiallelic burden were found to be prognostic, and revealed an extraordinary distinction in the role of epiclonal dynamics in different breast cancer subtypes. Collectively, the results presented in this thesis have shed light on the somatic DNA methylation basis of inter-patient as well as intra-tumour heterogeneity in breast cancer. This complements our genetic knowledge of the disease, and will help move us towards tailoring treatments to the patient's molecular profile.
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Sivertsson, Åsa. "Detection and analysis of genetic alterations in normal skin and skin tumours." Doctoral thesis, KTH, Biotechnology, 2002. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3432.
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The investigation of genetic alterations in cancer-relatedgenes is useful for research, prognostic and therapeuticpurposes. However, the genetic heterogeneity that often occursduring tumour progression can make correct analysischallenging. The objective of this work has been to develop,evaluate and apply techniques that are sufficiently sensitiveand specific to detect and analyse genetic alterations in skintumours as well as in normal skin.
Initially, a method based on laser-assisted microdissectionin combination with conventional dideoxy sequencing wasdeveloped and evaluated for the analysis of the p53 tumoursuppressor gene in small tissue samples. This method was shownto facilitate the analysis of single somatic cells fromhistologic tissue sections. In two subsequent studies themethod was used to analyse single cells to investigate theeffects of ultraviolet (UV) light on normal skin. Single p53immunoreactive and nonimmunoreactive cells from differentlayers of sunexposed skin, as well as skin protected fromexposure, were analysed for mutations in the p53 gene. Theresults revealed the structure of a clandestine p53 clone andprovided new insight into the possible events involved innormal differentiation by suggesting a role for allele dropout.The mutational effect of physiological doses of ultravioletlight A (UVA) on normal skin was then investigated by analysingthe p53 gene status in single immunoreactive cells at differenttime-points. Strong indications were found that UVA (even atlow doses) is indeed a mutagen and that its role should not bedisregarded in skin carcinogenesis.
After slight modifications, the p53 mutation analysisstrategy was thenused to complement an x-chromosomeinactivation assay for investigation of basal cell cancer (BCC)clonality. The conclusion was that although the majority ofBCCs are of monoclonal origin, an occasional tumour withapparently polyclonal origin exists. Finally, apyrosequencing-based mutation detection method was developedand evaluated for detection of hot-spot mutations in the N-rasgene of malignant melanoma. More than 80 melanoma metastasissamples were analysed by the standard approach of single strandconformation polymorphism analysis (SSCP)/DNA sequencing and bythis pyrosequencing strategy. Pyrosequencing was found to be agood alternative to SSCP/DNA sequencing and showed equivalentreproducibility and sensitivity in addition to being a simpleand rapid technique.
Keywords:single cell, DNA sequencing, p53, mutation,UV, BCC, pyrosequencing, malignant melanoma, N-ras
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Fermey, Pierre. "Identification de nouvelles bases moléculaires des cancers précoces par séquençage à haut débit." Thesis, Normandie, 2017. http://www.theses.fr/2017NORMR110/document.
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Une des plus grandes avancées en cancérologie et en génétique au cours des vingt dernières années fût l'identification des formes héréditaires de cancer et des gènes deprédisposition impliqués. Chez une majorité de patients soupçonnés de présenter une formehéréditaire de cancer, les analyses centrées sur les gènes connus pour être impliqués dansles prédispositions mendéliennes au cancer restent bien souvent négatives. Aujourd'hui,grâce à l'émergence du séquençage à haut-débit (NGS), il est possible de séquencerl'ensemble des exons (exome) d'un individu ou plusieurs centaines de gènes dans un lapsde temps court et à des coûts raisonnables. Dans ce contexte, nous avons appliqué plusieurs stratégies basées sur ces nouveaux outils, avec l'objectif d'identifier de nouvellesbases moléculaires des cancers héréditaires à survenue précoce. Tout d’abord, nous avons employé une stratégie d'analyse exomique intrafamiliale dans une famille atypique présentant des chondrosarcomes de localisation thoracique pour lesquels aucune base moléculaire n'avait pu être mise en évidence. Grâce à cette stratégie, nous avons pu identifier une altération tronquante du gène EXT2 (NM_000401.3; c.237G>A; p.Trp79*). Les altérations perte de fonction documentées pour ce gène sont impliquées dans la maladie des ostéochondromes multiples (OM), des tumeurs bénignes. Or, dans cette famille, aucun signe clinique d'OM n'était présent. Ces travaux nous ont donc permis d'étendre le spectre phénotypique des mutations EXT2 et de modifier la prise en charge clinique de cette famille. Nous avons ensuite employé une stratégie d'analyse exomique soustractive de trio enfant malade / parents sains dans le but d’identifier des mutations de novo potentiellement responsables de la prédisposition génétique au cancer observée chez un jeune patient ayant développé un médulloblastome du cervelet à l’âge de 8 ans, suivi d’un méningiome à 22 ans. L’analyse exomique du trio a révélé l’existence chez ce patient d'une mutation de novo faux-sens affectant un acide aminé très conservé de la protéine HID-1. Cette dernière est particulièrement exprimée dans les cellules neuronales et sécrétrices, et semble fonctionner autour de l’appareil de Golgi pour réguler le tri des vésiculesnouvellement formées. Ainsi, notre hypothèse est qu’un défaut de la protéine HID-1, lié à une mutation du gène HID-1, perturberait la voie de sécrétion et participerait à la genèse du médulloblastome. Ces travaux, toujours en cours, démontrent à la fois la force de la stratégie exomique de trio pour identifier rapidement des mutations de novo et illustre toute la difficultéd'interprétation des variants détectés dans des gènes non impliqués dans le cancer. Par ailleurs, nous avons appliqué une stratégie exomique soustractive et interfamiliale à une cohorte de dix patients ayant développé un corticosurrénalome à un âge très précoce et pour lesquels aucune base moléculaire n'a pu être mise en évidence. Malheureusement, nous n'avons pas pu identifier de nouvelles bases moléculaires du corticosurrénalome de l'enfant par ces techniques. Enfin, sous l'hypothèse que des mutations rares ou privées dans un nombre limité de gènes impliqués dans le cancer contribueraient à des formes héréditaires de cancer, nous avons entrepris un projet visant à séquencer à haut débit 201 gènes fortement impliqués dans le cancer chez des patients ayant développé des tumeurs à un âge pédiatrique. Les premiers résultats de ce projet toujours en cours ont permis de confirmer la robustesse de cette technique et suggèrent une extension phénotypique du spectre des mutations DICER1 ainsi qu'une contribution oligogénique des gènes de réparation de l'ADN dans les tumeurs pédiatriques. L'ensemble de ces résultats seront bientôt compilés au sein d'une base de données et bénéficieront d'une analyse statistique fine avec l'objectif d'identifier des enrichissements en variants rares dans des gènes ou voies biologiquesOne of the greatest advances in oncology and genetics over the past 20 years has been the identification of hereditary forms of cancer and of the cancer genes. Nevertheless, in a majority of patients suspected to present an inherited form of cancer, analyses of the genes known to be involved in the Mendelian predispositions to cancer often remain negative. Today, thanks to the emergence of high-throughput sequencing (NGS), it is now possible to sequence all exons of an individual (exome) or several hundred genes in a short period of time and for a reasonable cost. In this context, we have applied several strategiesbased on these new tools in order to identify new molecular basis of early-onset cancers. First, we applied an intra-familial exome analysis strategy to an atypical family with chondrosarcomas of the chest, for which no molecular basis could be identified. Using this strategy, we were able to identify a truncating alteration of the EXT2 gene NM_000401.3; c.237G> A; p.Trp79 *). The documented loss of function alterations of this gene are implicated in a disease called multiple osteochondromas (OM), associated with benign lesions. Interestingly, these patients showed no clinical signs of OM indicating a potential phenotypic extension of EXT2 mutations. Plus, this work allowed us to change the clinical management of this family. We then used a strategy of subtractive exomic analysis of trio sick child/healthy parents in order to identify de novo mutations in a young patient who developed a medulloblastoma of the cerebellum at 8 years-old followed by a meningioma at 22 years-old. The analysis of the trio revealed the existence of a de novo mutation affecting a highly conserved amino acid of the HID-1 protein. HID-1 is specifically expressed in neuronal and secretory cells, and seems to function around the Golgi apparatus to regulate the sorting of newly formed vesicles. Our hypothesis is that a defect of the HID-1 protein linked to a mutation of the HID-1 gene, could alter the secretory pathway therefore contributing to the development of the tumor. This work, which is still ongoing, demonstrates both the strength of the trio strategy for the rapid identification of de novo mutations and illustrates all the difficulty of interpreting variants detected in genes not yet involved in cancer. Then, thanks to the recruitment of the Laboratory of Molecular Genetics of the CHU of Rouen, we have collected a cohort of 10 patients who developed an adrenocortical carcinoma (ACC) at a very early age and for which no molecular basis could be identified. Despite subtractive and inter-familial exomic analyses, we were unable to highlight new molecular bases for these cases of pediatric ACC. Finally, under the assumption that rare or private mutations in a limited number of genes involved in cancer could contribute to inherited forms of cancer, we undertook a project to sequence 201 genes involved in cancer in patients who developed tumors at a pediatric age. The first results of this project confirmed the robustness of this technique and suggested a phenotypic extension of the DICER1 mutation spectrum as well as an oligogenic contribution of DNA repair genes in pediatric tumors. Soon, these results will be compiled in a database and will benefit from a statistical analysis with the objective to identify enrichment of rare variants in specific genes or biological pathways in these patients compared to control individuals
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Goldstein, Ellen Sara. "Estimating the Incidence of Germline Mutations in Patients with Bone and Soft Tissue Sarcoma using Clinical Tumor Sequencing." The Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1592844958063832.
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Oliveira, Juliana Gonçalves de [UNESP]. "Detecção de mutação no gene supressor de tumor p53 e associação e cepas patogênicas do Helicobacter pylori em câncer gástrico." Universidade Estadual Paulista (UNESP), 2008. http://hdl.handle.net/11449/87824.
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oliveira_jg_me_botfm.pdf: 1162046 bytes, checksum: 81511cad148a738a034b784dbc6eec91 (MD5)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O câncer gástrico é o quarto tipo mais comum e o segundo em mortalidade. Apesar da diminuição do número de casos nos últimos tempos ainda é um grande problema mundial. E mais, sua associação com o Helicobacter pylori (HP) está cada vez mais preocupante, devido à alta prevalência de cepas patogênicas dessa bactéria. Além disso, mutações em genes que agem no ciclo celular podem levar ao câncer. O gene que codifica a proteína p53 é responsável pelo reparo de lesões no DNA durante o ciclo celular funcionando como um regulador transcricional. Quando o reparo não é viável a proteína p53 induz a entrada da célula danificada em apoptose. Mutações mais freqüentes ocorrem nos exons 5, 6, 7 8 e 9. No atual trabalho investigou-se a possível associação da mutação no gene p53, correlação com presença da cepa patogênica CagA+ e estadiamento do tumor. Foram estudadas 55 amostras de câncer gástrico extraídas por biópsia durante gastrectomia. PCR foi utilizada para os exons 5 ao 9, análise de mutação por sequenciamento automático e dados clínicos foram coletados incluindo infecção por Helicobacter pylori/CagA+. Trinta e dois casos apresentaram mutações em p53 sendo que 6 deles apresentaram mutação em mais de um exon. 53% apresentaram-se infectados por HP CagA+. 87% dos pacientes estavam em estadiamento avançado do tumor (II, III e IV) e 80% eram do tipo intestinal. Quanto a correlação de p53 e cagA+ a casuística é relativamente pequena, e assim a correlação de p53 e cagA não foi vista. Os resultados confirmam a relevância das mutações em p53 e da presença da cepa patogênica CagA nos casos de câncer gástrico, contudo estudos devem ser realizados com um maior número amostral. Neste caso, o uso da p53 mutada pode ser utilizada em diagnóstico indicando, assim um melhor tratamento, desde que há casos em que sua alteração impõe resistência...
The gastric cancer is the fourth most common type and the second in mortality. Despite the decrease in the number of cases in recent times is still a major problem worldwide. Moreover, its association with Helicobacter pylori (HP) is increasingly worrying, because of the high prevalence of pathogenic strains of this bacterium. Furthermore, mutations in genes that act in the cell cycle can lead to cancer. The gene encoding the p53 protein is responsible for repair of lesions in DNA during cell cycle working as a transcriptional regulator. When the repair is not viable in the p53 protein induces the entry of the cell into apoptosis damaged. Change frequently occur in exons 5, 6, 7 8 and 9. In the current work investigated is the possible association of the mutation in the p53 gene, correlation with the presence of pathogenic strain CagA + and staging of the tumor. We studied 55 samples of gastric cancer extracted by biopsy taken during gastrectomy. PCR was used to the exons 5 to 9, analysis of mutation by sequencing automatic and clinical data were collected including infection by Helicobacter pylori / CagA +. Thirty-two patients had mutations in p53 is that 6 of them had more than one mutation in exon. 53% showed up infected HP CagA +. 87% of the patients were in advanced staging of the tumor (II, III and IV) and 80% were of the intestinal type. As the correlation of p53 and cagA + the casuistic is relatively small, and thus the relationship of p53 and cagA was not seen. The results confirm the relevance of mutations in p53 in the presence of pathogenic strain CagA in cases of gastric cancer, but studies must be conducted with greater sample. In this case, the use of p53 mutant can be used in diagnosis indicating thus a better treatment, since there are cases where its amendment requires resistance to certain types of treatment, regardless of the presence of H pylori... (Complete abstract click electronic access below)
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Le, Loarer François. "Dérégulation du complexe BAF dans les sarcomes épithélioïdes et leur variants génétiques." Thesis, Lyon 1, 2015. http://www.theses.fr/2015LYO10127.
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Les sarcomes épithélioides sont caractérisés dans 85% des cas par une perte d'expression nucléaire de la protéine SMARCB1, codée par un gène suppresseur de tumeurs situés en 22q11 impliqué dans la génèse des tumeurs rhabdoides malignes. L'exploration par BAC-FISH (Bacterial Artificial Chromosome- Fluorescence In Situ Hybridization) d'une série de 40 sarcomes épithélioides a permis d'établir que cette perte d'expression était secondaire dans 85% des cas à des délétions homozygotes et a mis en évidence le premier cas de sarcome épithélioide associé à une délétion germinale de SMARCB1, altération jusqu'alors uniquement identifiée dans les tumeurs rhabdoides malignes. Nous avons par la suite testé le gène suppresseur de tumeurs SMARCA4 comme gène candidat impliqué dans les sarcomes épithélioides SMARCB1-conservés à partir d'une série rétrospective de 16 cas. SMARCA4 code la sous-unité ATPase du complexe BAF dont SMARCB1 représente une sous unité. Ce screening initial a permis d'identifier 6 cas de sarcomes SMARCA4-inactivés dont la localisation était exclusivement thoracique et dont les caractéristiques clinique et anatomopathologique stéréotypées ont permis le recrutement prospectif et rétrospectif de nouveaux cas. L'étude par RNA-sequencing d'une fraction de notre cohorte (n=13/19) a confirmé leur homogénéité transcriptomique et souligné leur parenté avec les tumeurs rhabdoides SMARCB1 et SMARCA4 déficientes. L'absence de mutation germinale fréquente (n=1/11) a fait proposer le terme de sarcome thoracique SMARCA4-déficient (SMARCA4-DTS) en proscrivant l'utilisation du qualificatif « rhabdoide ». La parenté transcriptomique de ces tumeurs laisse entrevoir des vulnérabilités thérapeutiques communes qui restent à identifierEpithelioid sarcomas (ES) display loss of SMARCB1 nuclear expression in 85% of cases. SMARCB1 is encoded by a tumor suppressor gene located in 22q11 which was first linked to cancer in malignant rhabdoid tumors. While investigating a series of 40 epithelioid sarcomas with BAC-FISH (Bacterial Artificial Chromosome-Fluorescence In Situ Hybridization), we demonstrated that SMARCB1 loss in ES occurred through genomic deletions in 85% of cases. We were also able to highlight the first case of ES associated with a heterozygous SMARCB1 deletion in the germ line, which feature was previously thought to be restricted to malignant rhadboid tumors (MRT). We subsequently investigated a series of 16 SMARCB1-retained ES to identify its underlying culprit gene with a focus on the candidate tumor suppressor gene SMARCA4. SMARCA4 encodes one of the ATPase subunit of BAF complexes. Interestingly, SMARCB1 is also a core submit of these complexes which regulate chromatin remodeling. We were able to identify a set of 6 cases displaying SMARCA4 inactivation with this discovery cohort. The review of medical records highlighted these cases had similar presentation : all tumors presented with large compressive and aggressive mediastinopulmonary masses. We further recruited 13 cases based on these characteristics including 5 prospective cases. The characterization of their transcriptomes by RNA-sequencing (n=13/19) confirmed their remarkable homogeneity, all our samples clustering together with MRT. However our variant diverge from malignant rhabdoid tumors as it lacks SMARCA4 alteration in the germline (n=0/11) and displays complex polyploidy genetic profiles. We therefore called this new tumor variant “SMARCA4-deficient thoracic sarcoma” (SMARCA4-DTS). The transcriptomic vicinity of SMARCA4-DTS and MRT let foresee they share common therapeutic vulnerabilities
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Rodríguez, Sodupe Pau 1993. "Liquid biopsy for early tumor relapse detection : Development of hypersensitive genomic sequencing methodologies for the detection of ultra-rare genetic variants in the blood plasma of pediatric cancer patients." Doctoral thesis, TDX (Tesis Doctorals en Xarxa), 2022. http://hdl.handle.net/10803/673741.
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The profiling of circulating-tumor DNA (ctDNA) in blood plasma, often known as liquid biopsy, has recently
been of great interest in cancer research. This minimally-invasive strategy relies on the fact that ctDNA
represents, at least partially, the real-time state of the tumor genome and holds great potential for early
cancer detection and patient care. However, the fraction of ctDNA in total cell-free DNA (cfDNA) is often very
low and requires ultra-sensitive and specific methods for its detection. This thesis focuses on the
development and implementation of ultra-sensitive Next Generation Sequencing (NGS) methods that enable
the identification of rare variants in plasma in a specific way. In summary, this work presents a
comprehensive and cost-effective strategy for monitoring tumor mutations in the plasma of pediatric patients
in a personalized manner. Our strategy aims to provide a tool to anticipate the detection and treatment of
tumor relapses.L’anàlisi d'ADN tumoral circulant (ADNtc) al plasma sanguini, sovint conegut com a biòpsia líquida, ha despertat recentment gran interès en la recerca del càncer. Aquesta estratègia mínimament invasiva es basa en que l’ADNtc representa, almenys parcialment, l'estat en temps real del genoma del tumor i té un gran potencial per a la detecció precoç del càncer. Tanmateix, la fracció d'ADNct en l'ADN lliure circulant (ADNlc) total és sovint molt baixa i es requereixen mètodes ultra-sensibles i específics per a la seva detecció. Aquesta tesi se centra en el desenvolupament i la implementació de mètodes ultra-sensibles basats en tecnologies de seqüenciació massiva (NGS) que permetin la identificació de variants rares en plasma de forma específica. En resum, aquest treball presenta una estratègia integral i rendible pel seguiment de mutacions tumorals en el plasma de pacients pediàtrics de manera personalitzada. La nostra estrategia pretén oferir una eina que permeti anticiparse en la detecció i tractament de recidives tumorals.
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Akhavanfard, Sara. "NEXT-GENERATION SEQUENCING APPROACHES TO CHARACTERIZE GENOMIC PREDISPOSITION OF SOLID TUMORS IN CHILDREN, ADOLESCENTS, AND YOUNG ADULTS (C-AYA)." Case Western Reserve University School of Graduate Studies / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=case1575571085728229.
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Oliveira, Juliana Gonçalves de. "Detecção de mutação no gene supressor de tumor p53 e associação e cepas patogênicas do Helicobacter pylori em câncer gástrico /." Botucatu : [s.n.], 2008. http://hdl.handle.net/11449/87824.
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Orientador: Nídia Alice PinheiroBanca: Elaine Sbroggio de Oliveira Rodini
Banca: Daniel Araki Ribeiro
Resumo: O câncer gástrico é o quarto tipo mais comum e o segundo em mortalidade. Apesar da diminuição do número de casos nos últimos tempos ainda é um grande problema mundial. E mais, sua associação com o Helicobacter pylori (HP) está cada vez mais preocupante, devido à alta prevalência de cepas patogênicas dessa bactéria. Além disso, mutações em genes que agem no ciclo celular podem levar ao câncer. O gene que codifica a proteína p53 é responsável pelo reparo de lesões no DNA durante o ciclo celular funcionando como um regulador transcricional. Quando o reparo não é viável a proteína p53 induz a entrada da célula danificada em apoptose. Mutações mais freqüentes ocorrem nos exons 5, 6, 7 8 e 9. No atual trabalho investigou-se a possível associação da mutação no gene p53, correlação com presença da cepa patogênica CagA+ e estadiamento do tumor. Foram estudadas 55 amostras de câncer gástrico extraídas por biópsia durante gastrectomia. PCR foi utilizada para os exons 5 ao 9, análise de mutação por sequenciamento automático e dados clínicos foram coletados incluindo infecção por Helicobacter pylori/CagA+. Trinta e dois casos apresentaram mutações em p53 sendo que 6 deles apresentaram mutação em mais de um exon. 53% apresentaram-se infectados por HP CagA+. 87% dos pacientes estavam em estadiamento avançado do tumor (II, III e IV) e 80% eram do tipo intestinal. Quanto a correlação de p53 e cagA+ a casuística é relativamente pequena, e assim a correlação de p53 e cagA não foi vista. Os resultados confirmam a relevância das mutações em p53 e da presença da cepa patogênica CagA nos casos de câncer gástrico, contudo estudos devem ser realizados com um maior número amostral. Neste caso, o uso da p53 mutada pode ser utilizada em diagnóstico indicando, assim um melhor tratamento, desde que há casos em que sua alteração impõe resistência... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The gastric cancer is the fourth most common type and the second in mortality. Despite the decrease in the number of cases in recent times is still a major problem worldwide. Moreover, its association with Helicobacter pylori (HP) is increasingly worrying, because of the high prevalence of pathogenic strains of this bacterium. Furthermore, mutations in genes that act in the cell cycle can lead to cancer. The gene encoding the p53 protein is responsible for repair of lesions in DNA during cell cycle working as a transcriptional regulator. When the repair is not viable in the p53 protein induces the entry of the cell into apoptosis damaged. Change frequently occur in exons 5, 6, 7 8 and 9. In the current work investigated is the possible association of the mutation in the p53 gene, correlation with the presence of pathogenic strain CagA + and staging of the tumor. We studied 55 samples of gastric cancer extracted by biopsy taken during gastrectomy. PCR was used to the exons 5 to 9, analysis of mutation by sequencing automatic and clinical data were collected including infection by Helicobacter pylori / CagA +. Thirty-two patients had mutations in p53 is that 6 of them had more than one mutation in exon. 53% showed up infected HP CagA +. 87% of the patients were in advanced staging of the tumor (II, III and IV) and 80% were of the intestinal type. As the correlation of p53 and cagA + the casuistic is relatively small, and thus the relationship of p53 and cagA was not seen. The results confirm the relevance of mutations in p53 in the presence of pathogenic strain CagA in cases of gastric cancer, but studies must be conducted with greater sample. In this case, the use of p53 mutant can be used in diagnosis indicating thus a better treatment, since there are cases where its amendment requires resistance to certain types of treatment, regardless of the presence of H pylori... (Complete abstract click electronic access below)
Mestre
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Tirier, Stephan Marius [Verfasser], and Roland [Akademischer Betreuer] Eils. "Dissecting tumor cell heterogeneity in 3D cell culture systems by combining imaging and next generation sequencing technologies / Stephan Marius Tirier ; Betreuer: Roland Eils." Heidelberg : Universitätsbibliothek Heidelberg, 2018. http://d-nb.info/1198484292/34.
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Rubio, Pérez Carlota 1990. "Understanding the genomic makeup of tumors to guide personalized medicine." Doctoral thesis, Universitat Pompeu Fabra, 2017. http://hdl.handle.net/10803/664287.
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Cancer is a disease of the genome. The study of tumor genomic alterations is used to guide several precision medicine strategies, some approved and a large number under clinical development. On the other hand, the study of tumor immunity is recently becoming the key for the success of other personalized strategies, named immunotherapies. Along this thesis I have made several contributions towards the advance of cancer precision medicine, based on the study of tumor “omics” data. First, I evinced the landscape of genomic-guided anti-cancer therapies. Second, I developed OncoPaD, a tool for the rational design of cost-effective cancer gene panels. Third, I contributed to the development of Cancer Genome Interpreter, a tool for the biological and therapeutic interpretation of variants found in newly sequenced tumors. Forth, I identified tumor intrinsic molecular mechanisms involved in tumor immune evasion.El càncer és una malaltia del genoma. L'estudi de les alteracions genòmiques dels tumors s’utilitza com a guia en varies estratègies de medicina de precisió, algunes d'elles aprovades i d'altres en assajos clínics. D'altra banda, l’estudi de la immunitat tumoral està esdevenint una peça clau per l’èxit d’altres estratègies terapèutiques, anomenades immunoteràpies. Al llarg d'aquesta tesi, mitjançant l'estudi de les dades “òmiques” tumorals, he contribuït de varies maneres cap a l'avenç de la medicina de precisió pel càncer. Primer, he identificat el panorama de les teràpies anticanceroses guiades per alteracions genòmiques. Segon, he desenvolupat OncoPaD, una eina pel disseny cost-efectiu i racional de panells de seqüenciació per càncer. A més, he contribuït al desenvolupament del Cancer Genome Interpreter, una eina que ajuda a la interpretació biològica i terapèutica de les variants presents a tumors novament seqüenciats. Per últim, he identificat diversos mecanismes mitjançant els quals els tumors són capaços d'evadir l’atac del sistema immunològic.
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Tomasini, Pascale. "Établissement d'un profil génomique spécifique des métastases cérébrales des adénocarcinomes bronchiques." Thesis, Aix-Marseille, 2019. http://www.theses.fr/2019AIXM0692.
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Le cancer bronchique est la première cause de mortalité par cancer, notamment parce qu’il est le plus grand pourvoyeur de métastases cérébrales (MC). Une meilleure connaissance de la biologie des cancers bronchiques non à petites cellules (CBNPC) a amélioré le pronostic des patients. Cependant, l’efficacité cérébrale des traitements est variable. Ce travail avait pour objectif une meilleure connaissance de la biologie des MC de CBNPC et de l’hétérogénéité génomique entre la MC et les autres sites tumoraux pour guider la prise en charge thérapeutique des malades.Nous avons montré que l’efficacité intracérébrale de l’immunothérapie était variable et que l’incidence et l’évolution des MC étaient associées au profil mutationnel. Nous avons ensuite comparé le séquençage pan-exomique de paires d’échantillons de tumeurs pulmonaires (TP) et MC de patients atteints de CBNPC et identifié 13 gènes spécifiques des MC.Nous avons ensuite constitué une cohorte prospective de patients atteints de CBNPC avec MC opérées. Dans ces MC, nous avons trouvé un nombre de mutations très élevé, dont 2 mutations des 13 gènes. De plus, l’ADNtc dans le LCR était représentatif des mutations des MC. Ces travaux soulignent l’importance de l’hétérogénéité tumorale entre les MC, les TP et l’ADNtc. Il est difficile d’établir une signature spécifique des MC, notamment du fait du faible nombre d’échantillons disponibles et de la difficulté d’obtenir des paires TP/MC mais l’étude de l’ADNtc dans le LCR peut être une piste. Nous allons ensuite étudier le microenvironnement cérébral et utiliser d’autres approches comme la modélisation mathématique pour une meilleure compréhension de la biologie des MCLung cancer is the leading cause of cancer-related deaths, partly because it is the first cause of brain metastases (BM). A better knowledge of non-small cell lung cancer (NSCLC) molecular biology and the development of targeted therapies have improved patients’ outcomes. However, the intracranial efficacy of these new treatments is inconstant. The objective of this work was a better knowledge of BM biology in lung adenocarcinoma and a better knowledge of genomic heterogeneity between BM and PT to guide patients’ treatment strategy.We showed that intracranial efficacy of immunotherapy was inconstant and that BM incidence and recurrence after local treatment was associated with mutation profile. We then compared whole exome sequencing of paired frozen samples from PT and BM of patients with NSCLC and identified 13 genes with recurrent mutations in BM never mutated in PT samples. We then analyzed a prospective cohort of patients with CBNPC and resected BM. In these BM, the number of mutations was high, including 2 genes among the 13 genes identified. Moreover, CSF ctDNA was representative of BM mutation status.This work highlights the importance of tumor heterogeneity between BM, PT and ctDNA. Whereas it is difficult to establish a specific signature of BM because of the poor number of samples available and the difficulty to have paired PT/BM samples for each patient, CSF ctDNA study may be a way to assess BM biology. We plan to study brain microenvironment and use new approaches such as mathematical modeling for a better understanding of BM biology
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Abecassis, Judith. "Statistical methods for deciphering intra-tumor hereterogeneity : challenges and opportunities for cancer clinical management." Thesis, Université Paris sciences et lettres, 2020. http://www.theses.fr/2020UPSLM065.
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L'obtention du répertoire des gènes de cancer mutés a été déterminant pour notre compréhension de la tumorigénèse. Cependant, les efforts menés pour caractériser les cancers au niveau génétique ne sont pas suffisants pour prédire la survie des patients, ou leur réponse aux traitements, ce qui est essentiel pour améliorer leur prise en charge. Cet échec est en partie attribué au caractère évolutif des cancers. En effet, comme toute population biologique capable d'acquérir des changements héréditaires, les cellules tumorales sont soumises à la sélection naturelle et la dérive génétique, résultant en une structure mosaique, dans laquelle coexistent plusieurs sous-clones ayant des génomes et des propriétés différentes. Cela a d'importantes conséquences sur les traitements anti-cancéreux, puisque ces sous-populations peuvent être sensibles ou résistantes à différentes thérapies, et de nouveaux phénotypes résistants peuvent continuer d'apparaître alors que la maladie continue à progresser. Un nombre importants de méthodes mathématiques ou statistiques a été développé pour détecter et mesurer l'hétérogénéité intra-tumorale (ITH), mais aucune évaluation systématique de leurs performances et de leur application clinique potentielle n'a été effectué. Notre première contribution a donc été de réaliser une étude des approches existantes pour détecter l'hétérogénéité intra-tumorale, pour permettre de naviguer plus facilement entre les idées sous-tendant ces approches. Nous avons aussi proposé un cadre pour analyser la robustesse de ces approches, et leur usage potentiel pour la stratification des patients. Cette enquête approfondie nous a aussi permis d'identifier un type de données encore non exploité pour la reconstruction de l'hétérogénéité intra-tumorale, et notre seconde contribution vise à combler ce manque. En effet, au-delà de la fréquence observée d'une mutation somatique dans un échantillon tumoral, qui permet de distinguer plusieurs clones, le contexte nucléotidique d'une mutation révèle les processus mutationnels causaux et non observables. Nous montrons, à la fois avec des données simulées et réelles la possibilité de modéliser ces deux aspects de l'évolution tumorale conjointement. En conclusion, nous mettons en évidence le besoin de renforcer l'intégration de données de nature ou d'origine multiples pour exploiter pleinement le potentiel de l'évolution tumorale dans la prise en charge clinique du cancerAccessing the repertoire of cancer somatic alterations has been instrumental in our current understanding of carcinogenesis. However, efforts in genomic characterization of cancers are not sufficient to predict a patient's outcome or response to therapy, which is key to inform their clinical management. This failure is partly attributed to the evolutionary aspect of cancers. Indeed, as any biological population able to acquire heritable transformations, tumor cells are shaped by natural selection and genetic drift, resulting in a mosaic structure, where several subclones with distinct genomes and properties coexist. This has important implications for cancer treatment as those subpopulations can be sensitive or resistant to different therapies, and new resistant phenotypes can keep emerging as the diseases progresses further. An important number of mathematical or statistical methods have been developed to detect and quantify the intra-tumor heterogeneity (ITH), but no systematic evaluation of their performances and potential for clinical application has been performed. Our first contribution consists in a survey of existing approaches to decipher ITH, that allows to navigate the different underlying ideas easily. We have also proposed a framework to assess the robustness of those approaches, and their potential for use in patient stratification. This survey has allowed us to identify an unexploited type of data in the process of ITH reconstruction, and our second contribution fills remedies to this shortfall. Indeed, besides observed prevalences of somatic mutations within a tumor sample that allow us to distinguish several clones, the nucleotidic context of those mutations reveals the unknown causative mutational processes. We illustrate on both simulated and real data the opportunity to jointly model those two aspects of tumor evolution. In conclusion, we highlight the need to reinforce data integration from several sources or samples to harness the potential of tumor evolution for cancer clinical management
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Giner-Calabuig, Mar. "Novel Germline and somatic processes in mismatch repair deficient tumors." Doctoral thesis, Universidad de Alicante, 2020. http://hdl.handle.net/10045/113914.
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La inestabilidad de microatélites (MSI) está presente en diferentes tipos tumorales. Generalmente se explica por metilación del promotor de MLH1 o mutaciones germinales en los genes de reparación mismatch repair (MMR) del ADN, causando el conocido síndrome de Lynch. Recientemente, con el cribado universal se ha dado a conocer que un gran porcentaje de tumores con sospecha de síndrome de Lynch, no presentan estas mutaciones. Hasta la fecha, la hipótesis más aceptada para explicar este fenómeno conocido como síndrome Lynch-like, es la doble inactivación de estos genes a nivel somático. Aun así, estos pacientes presentan cáncer colorectal y otros tumores a edades tempranas y en muchos casos, se acompaña de una historia familiar de cáncer colorectal, que sugiere un origen hereditario. Objetivos: Elucitar las bases moleculares responsables de la pérdida de función del sistema MMR en casos de sospecha de Síndrome de Lycnh sin mutaciones germinales en estos genes. Estudio germinal: Identificar nuevas mutaciones germinales en genes reparadores del ADN que puedan estar implicadas en el desarrollo de inestabilidad de microsatélites. Estudio somático: Realizar una caracterización molecular de los tumores con MSI para identificar los eventos moleculares que pueden impactar el comportamiento tumoral y el manejo clínico. Materiales y métodos: Se analizó el exoma germinal de 100 muestras de pacientes Lynch-like y 30 Lynch. Adicionalmente se secuenciaron 37 tumores Lynch-like, 25 Lynch y se utilizó la información de 31 MSI/BRAF del TCGA. Las muestras fueron secuenciadas en Illumina HiSeq y analizadas en un High-performance computer siguiendo los estándares del Yale Center for Genome Analysis (YCGA). Resultados: Estudio germinal: Entre los Lynch-like encontramos 7 Lynch no identificados previamente. El 50% del MSI detectado en Lynch-like podía explicarse por dobles inactivación de los mismos a nivel somático. Además entre los Lynch-like encontramos 15 variantes germinales patogénicas que podían explicar la inestabilidad genética. Dos de ellas con inactivación del otro alelo a nivel somático. Además demostramos que la inactivación de un solo alelo es suficiente para tener un fenotipo deficiente. Estudio somático: no todos los tumores clasificados como MSI por MSI-PCR son presentan esta inestabilidad a nivel global. Al analizar diferentes tumores MSI descubrimos dos clústeres bien diferenciados en base a los perfiles mutacionales detectados. Buscamos mutaciones en genes reparadores y de predisposición al cáncer que pudieran estar marcando esas diferencias en las signatures. Discusión y conclusiones: Estudio germinal: Este estudio confirma el enriquecimiento existente en variantes germinales en los genes reparadores del ADN en pacientes con Síndrome Lynch-like. Variantes en estos genes, conferirían una deficiencia media heredada que promovería y podría inducir una inestabilidad genómica con el paso del tiempo. LA identificación de la variante en RECQL5 como potencial mutación asociada a un fenotipo de cáncer colorrectal familiar. Lynch-like es un grupo heterogéneo a nivel genético, con la característica común de ser MSI. Los tumores Lynch-like pueden ser causados por doble inactivación somática u otros defectos en otros genes reparadores. Además, el grupo incluye Lynch no identificados por los métodos de detección actuales. Estudio somático: Este estudio proporciona un valorable nueva perspectiva en los tumores con deficiencia en el sistema MMR. Muestra la correlación entre los diferentes niveles de MSI global y los perfiles mutacionales y clínicos, lo cual puede tener grandes implicaciones a nivel de diagnóstico y tratamiento de los pacientes.
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Tatsuoka, Hisato. "Single-cell Transcriptome Analysis Dissects the Replicating Process of Pancreatic Beta Cells in Partial Pancreatectomy Model." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/263543.
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Zhu, Yan. "Microfluidic Technology for Low-Input Epigenomic Analysis." Diss., Virginia Tech, 2018. http://hdl.handle.net/10919/83402.
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Epigenetic modifications, such as DNA methylation and histone modifications, play important roles in gene expression and regulation, and are highly involved in cellular processes such as stem cell pluripotency/differentiation and tumorigenesis. Chromatin immunoprecipitation (ChIP) is the technique of choice for examining in vivo DNA-protein interactions and has been a great tool for studying epigenetic mechanisms. However, conventional ChIP assays require millions of cells for tests and are not practical for examination of samples from lab animals and patients. Automated microfluidic chips offer the advantage to handle small sample sizes and facilitate rapid reaction. They also eliminate cumbersome manual handling.
In this report, I will talk about three different projects that utilized microfluidic immunoprecipitation followed by next genereation sequencing technologies to enable low input and high through epigenomics profiling. First, I examined RNA polymerase II transcriptional regulation with microfluidic chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) assays. Second, I probed the temporal dynamics in the DNA methylome during cancer development using a transgenic mouse model with microfluidic methylated DNA immunoprecipitation followed by next generation sequencing (MeDIP-seq) assays. Third, I explored negative enrichment of circulating tumor cells (CTCs) followed by microfluidic ChIP-seq technology for studying temporal dynamic histone modification (H3K4me3) of patient-derived tumor xenograft on an immunodeficient mouse model during the course of cancer metastasis.
In the first study, I adapted microfluidic ChIP-seq devices to achieve ultrahigh sensitivity to study Pol2 transcriptional regulation from scarce cell samples. I dramatically increased the assay sensitivity to an unprecedented level (~50 K cells for pol2 ChIP-seq). Importantly, this is three orders of magnitude more sensitive than the prevailing pol2 ChIP-seq assays. I showed that MNase digestion provided better ChIP-seq signal than sonication, and two-steps fixation with MNase digestion provided the best ChIP-seq quality followed by one-step fixation with MNase digestion, and lastly, no fixation with MNase digestion.
In the second study, I probed dynamic epigenomic changes during tumorigenesis using mice often require profiling epigenomes using a tiny quantity of tissue samples. Conventional epigenomic tests do not support such analysis due to the large amount of materials required by these assays. In this study, I developed an ultrasensitive microfluidics-based methylated DNA immunoprecipitation followed by next-generation sequencing (MeDIP-seq) technology for profiling methylomes using as little as 0.5 ng DNA (or ~100 cells) with 1.5 h on-chip process for immunoprecipitation. This technology enabled me to examine genome-wide DNA methylation in a C3(1)/SV40 T-antigen transgenic mouse model during different stages of mammary cancer development. Using this data, I identified differentially methylated regions and their associated genes in different periods of cancer development. Interestingly, the results showed that methylomic features are dynamic and change with tumor developmental stage.
In the last study, I developed a negative enrichment of CTCs followed by ultrasensitive microfluidic ChIP-seq technology for profiling histone modification (H3K4Me3) of CTCs to resolve the technical challenges associated with CTC isolation and difficulties related with tools for profiling whole genome histone modification on tiny cell samples.Ph. D.
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Hubert, Jean-Noël. "Génomique des populations appliquée : détection de signatures de sélection au sein de populations expérimentales." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS141/document.
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La génomique des populations rend possible la mise en évidence de traces de sélection dans le génome. Les travaux effectués considèrent en général une échelle de temps longue (~ 10³ générations). En comparaison, peu d’intérêt a été porté aux études expérimentales de court terme (~ 10 générations). De telles expériences sont pourtant susceptibles de nous renseigner sur la base génétique de caractères complexes. Nous proposons une méthode de vraisemblance basée sur un modèle de Wright-Fisher pour détecter la sélection à partir d’échantillons génétiques temporels acquis sur une période de dix générations. Nous montrons par simulation que notre méthode permet de différencier les signaux dus à la combinaison de la sélection et de la dérive génétique de ceux dus à la dérive seule. Nous montrons également par simulation qu’il est possible d’estimer le coefficient de sélection appliqué à un locus testé. De plus, nous illustrons l’intérêt de notre méthode pour la détection de marqueurs candidats à la sélection au travers de deux études génomiques sur données réelles, chez le diable de Tasmanie (Sarcophilus harrisii) et chez la truite arc-en-ciel (Oncorhynchus mykiss). Ces applications mettent en évidence des régions génomiques candidates pour des phénotypes complexes dans des contextes différents. Dans l’ensemble, nos résultats montrent qu’il est possible de détecter des gènes sujets à une sélection directionnelle intense à partir d’échantillons génétiques temporels, même si la sélection est de courte durée et si les populations examinées ont un faible effectifPopulation genomics makes it possible to detect traces of selection in the genome. Studies in this field have mainly focused on long time scale (~ 10³ generations). In comparison, short-term experimental studies (~ 10 generations) have attracted much less interest. Such experiments are, however, likely to inform us about the genetic basis of complex characters. We propose a likelihood method based on a Wright-Fisher model to detect selection from genetic temporal samples collected over ten generations. We show through simulation that our method can disentangle signals due to the combination of genetic drift and selection to those due to drift alone. We also show through simulation that it is possible to estimate the selection coefficient applied to a tested locus. In addition, we illustrate the interest of our method for the detection of candidate markers for selection through two genome scans performed on real data, in the Tasmanian devil (Sarcophilus harrisii) and in the rainbow trout (Oncorhynchus mykiss). These practical applications highlight candidate genomic regions for complex phenotypes in different contexts. Collectively, our results show the possibility of detecting genes submitted to strong directional selection from genetic time-series, even if selection is applied on a short time period and if the examined populations are small
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Zapata, Ortiz Luis 1985. "On the evolution of cancer genomes : Signatures of selection reveal cancer genes across multiple tumor types." Doctoral thesis, Universitat Pompeu Fabra, 2016. http://hdl.handle.net/10803/456685.
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Tumors are composed of fast-growing cells that become malignant under selection of biological functions needed for cancer development. In this thesis, I intend to uncover the basic evolutionary principles underlying cancer etiology. The first part constitutes a longitudinal analysis of a single CLL case, which tumor heterogeneity and clonal evolution were revealed by sequencing. The second explores the signatures of positive selection of somatic mutations allowing the identification of driver genes. The last part is an attempt to uncover the essential functions of the cancer cell using signals of purifying selection. Altogether, we have identified a landscape of cancer-related genes that can be used for improving current cancer treatments.El tumor esta compuesto de células que crecen indiscriminadamente, bajo la lupa de selección natural. En esta tesis hemos intentado reconstruir los principios básicos de la evolución del cáncer, como estos describen la adquisición de mutaciones que inician la malignidad tumoral. El primer trabajo es un anaálisis genómico de un paciente con leucemia. El Segundo explora la heterogeneidad intratumoral para identificar genes drivers del cáncer. Y el último trabajo se enfoca en desenmascarar las señales de selección negativa. Nuestros resultados de estos tres trabajos constituyen una fuente de nuevos genes que pueden ser explorados como dianas terapéuticas del cáncer.
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Daher, Tamás [Verfasser]. "Comparative TP53 targeted next generation sequencing analysis as a diagnostic tool for determining lung tumor origin in patients with head and neck squamous cell carcinoma and synchronous / metachronous squamous cell lung carcinoma / Tamás Daher." Gießen : Universitätsbibliothek, 2018. http://d-nb.info/1170582079/34.
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Shinde, Jayendra. "Mutational signatures reveal the dynamic interplay of risk factors and cellular process during liver tumorigenesis." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCC324/document.
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Le cancer est une maladie du génome. La transformation tumorale résulte de l’acquisition de mutations somatiques via divers processus mutagènes opérant tout au long de la vie du patient. Les mécanismes à l’origine des mutations incluent les erreurs de réplication, les défauts de réparation de l’ADN, les modifications de base spontanées ou catalysées par des enzymes cellulaires, et l’exposition à des agents mutagènes endogènes (ROS) ou exogènes (tabac, UV…). Au cours de ma thèse, j’ai analysé des données de séquençage exome et génome complet de tumeurs hépatiques pour décortiquer les mécanismes à l’origine des mutations dans ces tumeurs, leur interaction avec les facteurs de risque, les processus cellulaires, les gènes drivers, et leur évolution au cours de la maladie. J’ai utilisé des méthodes statistiques existantes et dévoloppé des outils bioinformatiques innovants pour:- extraire les signatures de mutations et de réarrangements structuraux à l’aide de données de séquençage à haut débit- identifier les facteurs de risque et/ou les altérations génétiques à l’origine de chacune- prédire les mécanismes mutagènes à l’origine de chaque mutation somatique- explorer les corrélations entre la densité des mutations et les processus cellulaires comme la réplication et la transcription- reconstruire l’histoire clonale des tumeurs et dater l’apparition des signatures mutationnelles et des aberrations chromosomiques.Ces approches innovantes m’ont permis d’identifier 10 signatures mutationnelles: 5 signatures ubiquitaires à l’œuvre dans toutes les tumeurs hépatiques mais modulées par les facteurs de risque (sexe, alcool, tabac), et 5 signatures sporadiques opérant dans moins de 5% des tumeurs et associées à des étiologies connues (aflatoxine B1, acide aristolochique) ou restant à identifier. J’ai aussi mis en évidence 6 signatures de réarrangements structuraux, notamment des phénotypes duplicateurs et déléteurs, spécifiques de petits groupes de tumeurs. Chaque processus mutagène est modulé différemment par la réplication et la transcription. Les signatures liées à des molécules formant des adducts sur l’ADN (hydrocarbures polycycliques aromatiques, aflatoxine B1, acide aristolochique) sont nettement moins actives dans les gènes fortement exprimés suite à l’action du transcription-coupled repair, alors que la signature 16, liée à l’alcool, présente un motif unique de transcription-coupled damage. Une corrélation étonnante entre la densité des petites insertions et délétions (indels) et l’expression des gènes a été identifiée, conduisant à une accumulation considérable d’indels dans les gènes très forterment exprimés dans les cellules hépatiques. Enfin, l’histoire clonale des tumeurs hépatiques montre l’évolution des signatures mutationnelles au cours du temps et identifie l’accumulation de gains chromosomiques multiples comme un évènement tardif entraînant probablement une croissance de la tumeur jusqu’à une taille détactable en clinique. Ces résultats nous éclairent sur les mécanismes à l’origine des altérations génomiques dans l’histoire naturelle des cancers du foieCancer is a disease of the genome. A normal cell goes rogue and is transformed into a cancerous cell due to acquired somatic mutations in its genome. The catalogue of these somatic mutations observed in the cancer genome is the outcome of multiple mutational processes that have been operative over the lifetime of a patient. These mutational processes that have occurred throughout the development of cancer may be infidelity of the DNA replication machinery, impaired DNA repair system, enzymatic modifications of DNA, or exposures to exogenous or endogenous mutagens. Each mutational process leaves a characteristic pattern – a “mutational signature” on the cancer genome. Various genomic features related to genome architecture, including DNA replication and transcription, modulate these mutational processes. During my PhD, I analyzed whole exome and whole genome sequencing data from liver tumors to understand the mutational processes remodeling these tumor genomes, their interaction with risk factors, cellular processes, and driver genes, and their evolution along the tumor histories. For that aim, I used existing statistical methods and I developed innovative computational tools to:- extract mutational and structural variant signatures from next-generation sequencing data- identify risk factors or genetic alterations underlying each process- predict the mutational process at the origin of each somatic mutation- explore correlations between mutation rates and cellular processes like replication and transcription- reconstruct the clonal history of a tumor and the timing of mutational processes and copy-number changes These innovative analytical strategies allowed me to identify 10 mutational signatures: 5 ubiquitous signatures operative in every liver cancer but modulated by risk factors (gender, alcohol, tobacco), and 5 sporadic signatures operative in <5% of HCC and associated with specific known (aflatoxin B1, aristolochic acid) or unknown mutational processes. I also identified 6 structural variant signatures, including striking duplicator or deletor phenotypes in rare tumors. Each mutational process showed a different relationship with replication and transcription. Signatures of bulky DNA adducts (polycyclic aromatic hydrocarbons, aflatoxin B1, aristolochic acid) strongly decreased in highly expressed genes due to transcription-coupled repair, whereas the alcohol-related signature 16 displayed a unique feature of transcription-coupled damage. A striking positive correlation between indel rate and gene expression was observed, leading to recurrent mutations in very highly expressed tissue-specific genes. Finally, reconstructing the clonal history of HCC revealed the evolution of mutational processes along tumor development and identified synchronous chromosome duplications as late events probably leading to fast tumor growth and clinical detection of the tumor. Together, these findings shed new light on the mechanisms generating DNA alterations along the natural history of liver cancers
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Courtier, Frédéric. "Caractérisation moléculaire des néoplasies myéloprolifératives et de leur transformation en leucémie aigüe myéloïde." Thesis, Aix-Marseille, 2019. http://www.theses.fr/2019AIXM0651.
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Les néoplasies myéloprolifératives classiques (NMP) Philadelphie négative, thrombocytémie essentielle, polyglobulie de Vaquez et myélofibrose primaire, sont des hémopathies chroniques de bon pronostic dues à l'altération de l'un de ces trois gènes JAK2, CALR ou MPL. Cependant, d'autres anomalies génétiques peuvent survenir au cours de la maladie et entraîner une aggravation, dont le stade ultime est la transformation en leucémie myéloïde aigüe (LAM).Pour mieux comprendre les mécanismes responsables de cette transformation, j'ai utilisé le séquençage de l'ADN pour identifier les mutations responsables de chaque stade de la maladie.Un grand nombre de mutations est associé à un risque plus élevé d'évolution de la maladie. Des mutations dans les fonctions de l’épigénétique des histones et de l'épissage de l'ARN sont associées à la myélofibrose (MF). Ces mutations de l’épigénétique des histones (mutations d'ASXL1, EZH2) et l'épissage d'ARN (mutations de SRSF2, U2AF1, SF3B1) semblent prédisposer à la transformation en LAM, laquelle nécessite la survenue d'autres anomalies affectant d'autres fonctions, telles que la méthylation de l'ADN (mutations d’IDH1/2, TET2, DNMT3A), des facteurs de transcription (mutations de RUNX1, CUX1, ...) ou des mutations de TP53. Ces mutations pourraient être retrouvées durant la phase chronique avant le diagnostic d'évolution, ce qui pourrait les identifier comme marqueurs prédictifs.La connaissance des mécanismes de transformation et l'identification de marqueurs associés peuvent améliorer la prise en charge des patients et aider à leur proposer un traitement spécifique dans le cadre de la médecine personnaliséeClassical Philadelphia-negative Myeloproliferative Neoplasms (MPN) include essential thrombocythemia, polycythemia vera and primary myelofibrosis and are slow evolving and good prognostic blood cancers due to the alteration of one of three driver genes JAK2, CALR or MPL. However, other genetic abnormalities may occur and lead to an aggravation, of which the worst stage is transformation to Acute Myeloid Leukemia (AML).To better understand the mechanisms responsible for this transformation, I used DNA sequencing to identify mutations and abnormalities responsible for each stage of the disease.Chronic phase of an MPN results from a small number of additional mutations outside the drivers, and a large number of mutations is associated with a higher risk of evolution of the disease. Mutations in the functions of chromatin modifiers and RNA splicing are associated with myelofibrosis. These mutations in chromatin modifiers (mutations in ASXL1, EZH2) and RNA splicing (mutations in SRSF2, U2AF1, SF3B1) seem to predispose to transformation to AML, which requires the occurrence of other abnormalities affecting other functions, such as DNA methylation (mutations in IDH1/2, TET2, DNMT3A), transcription factors (mutations in RUNX1, CUX1,...), or TP53 mutations. These mutations associated with MF or acute phases could be found during chronic phase before the diagnosis of evolution, which could identify them as predictive markers.Knowledge of the mechanisms of transformation and the identification of associated markers may improve the care of patients with poor prognosis and help offer them specific treatment in the context of personalized medicine
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Delhomme, Tiffany. "Using the systematic nature of errors in NGS data to efficiently detect mutations : computational methods and application to early cancer detection." Thesis, Lyon, 2019. http://www.theses.fr/2019LYSE1098/document.
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La caractérisation exaustive des variations de l'ADN peut aider à progresser dans de nombreux champs liés à la génomique du cancer. Le séquençage nouvelle génération (NGS en anglais pour Next Generation Sequencing) est actuellement la technique la plus efficace pour déterminer une séquence ADN, du aux faibles coûts et durées des expériences comparé à la méthode de séquençage traditionnelle de Sanger. Cependant, la détection de mutations à partir de données NGS reste encore un problème difficile, en particulier pour les mutations somatiques présentes en très faible abondance comme lorsque l'on essaye d'identifier des mutations sous-clonales d'une tumeur, des mutations dérivées de la tumeur dans l'ADN circulant libre, ou des mutations somatiques dans des tissus normaux. La difficulté principale est de précisement distinguer les vraies mutations des artefacts de séquençage du au fait qu'ils atteignent des niveaux similaires. Dans cette thèse nous avons étudié la nature systématique des erreurs dans les données NGS afin de proposer des méthodologies efficaces capables d'identifier des mutations potentiellement en faible abondance. Dans un premier chapitre, nous decrivons needlestack, un nouvel outil d'appel de variants basé sur la modélisation des erreurs systématiques sur plusieurs échantillons pour extraire des mutations candidates. Dans un deuxième chapitre, nous proposons deux méthodes de filtrage des variants basées sur des résumés statistiques et sur de l'apprentissage automatique, dans le but de d'améliorer la précision de la détection des mutations par l'identification des erreurs non-systématiques. Finalement, dans un dernier chapitre nous appliquons ces approches pour développer des biomarqueurs de détection précoce du cancer en utilisant l'ADN circulant tumoralComprehensive characterization of DNA variations can help to progress in multiple cancer genomics fields. Next Generation Sequencing (NGS) is currently the most efficient technique to determine a DNA sequence, due to low experiment cost and time compared to the traditional Sanger sequencing. Nevertheless, detection of mutations from NGS data is still a difficult problem, in particular for somatic mutations present in very low abundance like when trying to identify tumor subclonal mutations, tumor-derived mutations in cell free DNA, or somatic mutations from histological normal tissue. The main difficulty is to precisely distinguish between true mutations from sequencing artifacts as they reach similar levels. In this thesis we have studied the systematic nature of errors in NGS data to propose efficient methodologies in order to accurately identify mutations potentially in low proportion. In a first chapter, we describe needlestack, a new variant caller based on the modelling of systematic errors across multiple samples to extract candidate mutations. In a second chapter, we propose two post-calling variant filtering methods based on new summary statistics and on machine learning, with the aim of boosting the precision of mutation detection through the identification of non-systematic errors. Finally, in a last chapter we apply these approaches to develop cancer early detection biomarkers using circulating tumor DNA
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Dupain, jourda Célia. "Découverte de nouveaux transcrits de fusion dans des tumeurs pédiatriques en rechute et caractérisation fonctionnelle d’un nouvel oncogène LMO3-BORCS5." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS310/document.
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Les cancers pédiatriques représentent la première cause de décès par maladie chez les enfants puisque 20% des patients présentent des résistances et rechutent. Les traitements actuels sont à leurs limites d'efficacité mais aussi de toxicité. L'amélioration des connaissances de la biologie des tumeurs résistantes et l'identification de nouvelles cibles moléculaires sont nécessaires pour ces pathologies rares, extrêmement diverses et mal comprises. Les oncogènes de fusion sont responsables de l’oncogenèse de ~ 20% des cancers. En raison de la biologie particulière des cancers pédiatriques, nous avons émis l'hypothèse qu’ils seraient dus à des oncogènes de fusion. Leur exploration, particulièrement à la rechute où les données actuelles sont limitées, permettrait de mieux comprendre ces pathologies et d’introduire de nouvelles cibles thérapeutiques. Notre premier objectif était de détecter de nouveaux transcrits de fusion parmi 48 patients pédiatriques en rechute inclus dans l'essai de médecine de précision MOSCATO-01 mené à Gustave Roussy. A partir de données de RNA-seq, nous avons détecté l’ensemble des fusions retrouvées dans ces tumeurs et classé les plus pertinentes selon leur fonction et leur potentiel thérapeutique. Un nouvel oncogène de fusion nommé LMO3-BORCS5 retrouvé chez un patient atteint de sarcome d'Ewing dans les biopsies tumorales à la rechute et au diagnostic, et dans diverses lignées cellulaires a particulièrement retenu notre attention. Nos études fonctionnelles montrent que LMO3-BORCS5 a un rôle important dans la tumorigenèse et la sensibilité au traitement et que BORCS5 fonctionnerait, in vivo, comme un gène suppresseur de tumeur. Ces résultats montrent le rôle critique des transcrits de fusion dans l'initiation et la progression tumorale ainsi que dans l’acquisition de résistance au traitement et que les fusions isolées ne doivent pas être ignorées. A l'avenir, la prise en charge des cancers réside en une médecine moléculaire de précision, les oncogènes de fusions représentant l'un des outils les plus remarquables en cliniquePediatric cancers represent the first cause of death by disease in children as 20% of patients harbor resistances and die from recurrence of the malignancy. Nowadays treatment are at their limits of efficacy but also toxicity. Improving the knowledge on the biology of the relapsed/resistant tumors and identifying new molecular targets are needed for these rare, extremely diverse and misunderstood pathologies. Fusion oncogenes are known to be major drivers of oncogenesis, responsible for ~20% of cancers. Due to their particular biology, we hypothesized that pediatric tumors would be more likely to harbor fusions and that their exploration, especially at relapse where very limited data are available, could help to understand the particularity of these malignancies and consequently to provide new therapeutic targets. Our first aim was to detect new fusion transcripts in a cohort of 48 pediatric patients at relapse included in the precision medicine trial MOSCATO-01, previously conducted in Gustave Roussy. From the RNA-seq data, we described the landscape of fusions in these tumors and classified the most confident candidates according to their biological function and druggability predictions. A new fusion named LMO3-BORCS5 found in biopsies of a patient with Ewing's sarcoma, at diagnosis and at relapse as well as in various cell lines particularly caught our attention. Functional studies showed that LMO3-BORCS5 has a high impact on tumorigenesis and treatment sensitivity and that BORCS5 would act, in vivo, as a tumor suppressor gene. Our results strengthened the critical role of fusion transcripts in tumor initiation, progression and resistance and reinforces the idea that a private fusion oncogene should not be ignored. Indeed, the future of cancer care lays in precision medicine, with fusion transcripts being one of the most remarkable tool for treatment decision
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Mallaret, Martial. "Identification de nouveaux gènes d'ataxies cérébelleuses récessives et intérêt du séquençage haut débit dans le diagnostic des ataxies d'origine génétique." Thesis, Strasbourg, 2015. http://www.theses.fr/2015STRAJ090/document.
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Les ataxies cérébelleuses héréditaires sont un ensemble de pathologies neurodégénératives ou neuro-développementales rares responsables d’un handicap fonctionnel important. Nous décrivons la découverte dans deux familles consanguines avec une ataxie cérébelleuse, une épilepsie et un retard mental deux mutations homozygotes dans le gène WWOX à l’aide du séquençage de l’exome d’un des patients de chaque famille. Ce gène était connu comme un gène suppresseur de tumeur. Par une stratégie de capture ciblée de 57 gènes d’ataxies cérébelleuses sur une série de 155 patients, nous avons posé un diagnostic dans 20,6% des cas dont des mutations d’ANO10 et SYNE1. Des études multicentriques ont permis d’étendre les connaissances sur ces maladies et montrer l’existence de phénotypes sévères dans ARCA1.A partir de cette série, nous avons validé en aveugle la pertinence d’un algorithme diagnostique clinico-biologique proposé par l’article de Anheim dans le New England Journal of Medicine en 2012Hereditary cerebellar ataxias are a group of neurodegenerative or neurodevelopemental diseases responsible of major disability. We found thanks to exome sequencing mutations in the WWOX gene in two consaguineous families presenting with cerebellar ataxia, epilepsy and mental retardation. This gene was until recently only recognized to be a tumor suppressor.With a 57 ataxia genes targeted capture strategy, next generation sequencing in 155 patients found 20,6% of positive diagnosis, including several new mutations in ANO10 and SYNE1. Multi center studies allow to extend clinical knowledge with severes phenotypes especially in ARCA1.We validate a clinico-biological algorithm for recesssive ataxias diagnosis published by Anheim in the in New England Journal of Medicine, 2012 in a blinded manner
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Thery, Jean-Christophe. "Détection et contribution de variants rares constitutionnels dans les formes précoces de cancer du sein : Apports du Séquençage de Nouvelle Génération. Contribution of de novo and mosaic TP53 mutations to Li-Fraumeni syndrome Germline mutations of inhibins in early-onset ovarian epithelial tumors." Thesis, Normandie, 2019. http://www.theses.fr/2019NORMR111.
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Si le syndrome de prédisposition héréditaire au cancer du sein et de l’ovaire constitue uneentité reconnue et supportée par l’identification de variations délétères sur les gènes BRCA1,BRCA2, PALB2, RAD51C et RAD51D, et si le cancer du sein de la femme jeune (avant 31 ans) estintégré dans le spectre du syndrome de Li-Fraumeni lié aux altérations de TP53, une large fractiondes patientes adressées en consultation d’oncogénétique pour ce motif demeure orpheline dediagnostic moléculaire. La connaissance du génome humain et l’avènement du séquençage denouvelle génération ont permis des avancées considérables, notamment dans l’observation de latrès grande variabilité du génome et de la survenue de variations de novo.Nous avons ainsi appliqué ces outils et ces concepts au cancer du sein de la femmejeune, afin de tenter d’identifier de nouveaux déterminants moléculaires constitutionnels. Dansune première approche basée sur la réalisation d’exomes soustractifs pour des trios parents- enfant, nous avons recherché des variants de novo délétères et effectivement identifié un variantrare de novo et délétère sur le gène INHBA dans le contexte d’un cancer de l’ovaire chez unejeune femme. Cette approche n’a cependant pu être reproduite dans le contexte d’un cancer dusein précoce. Nous avons également tenté une approche par exomes comparatifs dans unefamille remarquable avec survenue de cancers du sein précoces sur trois générations, sansvariation délétère identifiée commune à ces individus. Dans une seconde approche basée surun panel de 201 gènes impliqués dans la cancérogenèse, nous avons tenté d’identifier desvariants délétères ou des enrichissements en variants rares dans une cohorte de cancers du seinprécoces. Nous avons identifié une variation en mosaïque de TP53, sans autre détection devariations formellement délétères parmi 30 patientes atteintes de cancers du sein avant 31 ans.Un enrichissement non significatif en variants rares affectant les voies de la réparation de l’ADN aété néanmoins mis en évidence, suggérant des études plus larges ciblant ces voies. Enfin, nousavons recherché spécifiquement des variants de novo en mosaïque de TP53 dans lecontexte du cancer du sein de la femme jeune ou de cancers pédiatriques, et démontré ainsi laprévalence relativement importante de ces évènements. Ces observations supportent la nécessitéd’user d’un séquençage de forte profondeur et de ne pas restreindre les indications d’analyses deTP53 aux seules situations familiales évocatricesDespite previous identifications of deleterious variants on BRCA1, BRCA2, PALB2,RAD51C and RAD51D supporting the hereditary breast and ovarian cancer syndrom, and thecontribution of TP53 mutations in very early-onset breast carcinomas, a large fraction of patientssuggestive of Medelian disease remains without molecular diagnosis. In the past years,sequencing of the Human genome and next-generation sequencing offered major advances, inparticular in the field of genome variability and de novo variants.We applied these new tools and concepts in the context of very early-onset breastcarcinomas, in order to identify new molecular germline determinants. First, we dealt withsoustractive exomes, in parents - child trios, and succeed in the identification of a deleterious denovo variant in the INHBA gene, in the context of very early-onset of ovarian cancer. However, wehave failed with this approach in a second trio with an index affected by early-onset breastcarcinoma. We also tried a comparative exome sequencing approach in a remarkable pedigreewith multiple probands affected by early-onset breast carcinomas, without identification of ashared deleterious variant. Secondly, we used a home-made 201 genes panel assuming thatgenes somatically affected in cancers might be altered in inherited conditions. We analyzed acohort of very early-onset breast carcinomas, and identified a mosaic TP53 variation. Moreover,we identified some interesting candidate variants and observed a non-significant trend of rarevariants enrichment in the DNA repair pathway. Finally, we designed a specific TP53 gene capturein order to detect mosaic variants in pediatric cancers and very early-onset breast carcinomas.We confirmed the clinically significant prevalence of these alterations, which support TP53analysis in these conditions even in sporadic presentations
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Lode, Laurence. "Etude moléculaire de l'évolution clonalede TP53 des Syndromes Myélodysplasiques avec del(5q) : conséquences sur la résistance au traitement et la progression du cancer." Thesis, Montpellier, 2017. http://www.theses.fr/2017MONTT071/document.
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La protéine p53 (« Gardien du génome ») doit être altérée pour que le cancer puisse se développer. Les nombreuses thérapies anti-cancéreuses disponibles sont très efficaces mais la réponse clinique est souvent transitoire et les cancers disséminés rechutent ou progressent du fait de l'évolution de sous-populations cancéreuses résistantes au traitement, impliquant souvent TP53 qui est le gène le plus muté dans les formes agressives de nombreux cancers. Nous l’avons étudié dans la leucémie lymphoïde chronique (LLC) et les syndromes myélodysplasiques avec délétion 5q (SMD del(5q)). Grâce à l’étude rétrospective longitudinale de 40 patients atteints de SMD del(5q), nous avons généré des données de NGS ciblé et montré que le statut mutationnel de TP53 au diagnostic ne permettait pas de prédire la progression tumorale, contrairement à ce qui avait été publié précédemment (Jädersten et al., JCO 2011). Nous avons montré que c’était l’évolution clonale du gène TP53 qui était l’élément clé de la progression des SMD del(5q). Nous avons observé de nombreuses émergences de clones mutés entre le stade diagnostique et un stade ultérieur de la maladie, toujours après initiation du traitement par lénalidomide.Le lénalidomide a été approuvé comme nouveau traitement spécifique et très efficace contre l’anémie liée aux SMD del(5q), permettant à la plupart des patients d’être indépendants des transfusions sanguines. Le lénalidomide permet souvent d’éradiquer le clone tumoral porteur de l’anomalie génétique del(5q) isolée, induisant une rémission clinique. Malheureusement, cette rémission est courte avec une durée médiane de 2 ans, puis, dans environ 1 cas sur 2, survient une transformation en leucémie aiguë secondaire de pronostic péjoratif.Nous avons étudié un possible lien entre le traitement par lénalidomide et l’évolution clonale de TP53 par annotation clinico-bio-thérapeutiques des résultats de séquençage de TP53 chez les 24 patients dont les échantillons séquentiels avaient été analysés. Dans notre étude, les patients avec progression tumorale (dont 10 évolutions clonales de TP53 et 1 évolution clonale de RUNX1) avaient reçu une dose cumulée de lénalidomide supérieure à celle reçue par les patients dont la tumeur était restée stable (p = 0.036). Nous avons observé chez plusieurs patients que l’éradication de la tumeur n’était pas utile à l’amélioration de la qualité de vie des patients. La non-éradication semblait même permettre un maintien de l’équilibre clonal et une compétition entre les différents sous-clones de la tumeur, résistants ou non au lénalidomide.Nous discutons de l’évolution de l'écologie de la tumeur au cours du traitement, i.e., l’évolution de ses interactions avec son micro-environnement qui se modifie après chaque nouvelle dose de traitement. Un modèle évolutif dit théorie de la thérapie adaptative, développée récemment remet en question les protocoles conventionnels de thérapie anti-cancéreuse qui préconisent souvent d'administrer la dose maximale tolérée par le patient (Gatenby, 2009). Elle suggère que la dose minimale efficace présenterait l’avantage de ne pas éradiquer les cellules cancéreuses sensibles au traitement pour qu'elles restent en compétition avec les cellules cancéreuses résistantes et limiter la progression ou la rechute. Nous suggérons de prendre en compte également la diminution des effets indésirables pour le patient, améliorant ainsi sa qualité de vie, et enfin la diminution des dépenses de santé pour la collectivité. A ce jour, peu d’études cliniques évaluent l’intérêt de l’adoption de tels protocoles de thérapie adaptative.Néanmoins, des modèles in vivo (xénogreffes) et in silico (modèles statistiques) ont permis d’analyser la dynamique évolutive des populations tumorales en fonction du traitement reçu. Ces modèles prédisent que la survie de l’hôte peut être maximisée par la mise en place d’une thérapie adaptativeP53 protein is named «guardian of the genome » because it must be altered to let cancer grow.TP53 is the most mutated gene in agressive cancers.Numerous systemic therapies are successful for treatment of disseminated cancers. However, clinical response is often transient, and cancer undergo relapse or progression due to emergence of resistant populations. These latter often harbour TP53 mutations. We studied TP53 in chronic lymphoid leukemia (CLL) and lower-risk myelodysplastic syndroms with del(5q), MDS del(5q). We conducted a retrospective longitudinal study in 40 patients suffering from MDS del(5q). We obtained targeted NGS data showing that TP53 mutational status at diagnosis could not predict tumor progression, by contrast with previously published data (Jädersten et al., JCO 2011). We show that TP53 clonal evolution is the key feature of tumor progression in MDS del(5q). We observed numerous mutated sub-clones emerging between diagnosis and follow-up. In our study, this emergence always followed onset of lenalidomide treatment. Lenalidomide was recently approved as a new therapy specifically improving anemia in patients with MDS del(5q). It allows most patients to become red-blood-cells-transfusion independent. Lenalidomide often eradicates the major tumor clone harbouring the isolated genetic abnormality deletion (5q) and allows clinical remission. Unfortunately, this remission is short (median, 2 years) and is followed, in 1 case out of 2, by a secondary acute leukemic transformation with a very poor prognosis.We studied the issue of a possible link between lenalidomide therapy and TP53 clonal evolution by annotating TP53 sequencing results with acute biological, clinical and therapeutic features in the 24 patients with sequential samples analyzed. In our study, patients with tumor progression (10 TP53 clonal evolution and 1 RUNX1 clonal evolution) were given a higher cumulative dose of lenalidomide compared to patients with stable disease (p = 0.036). Similarly to « adaptive therapy theory »(Gatenby 2009), we observed that eradication of the tumor wasn’t useful for improvement of quality of life. Absence of eradication might even allow to maintain a clonal equilibrium and a clonal competition between the distinct tumor sub-clones, resistant to lenalidomide or not, and therefore maintain stable disease.This theory of adaptive therapy questions the classical protocols of treatments against cancer, in which the maximal tolerated dose is preferred to the minimal effective dose. The latter might however slow down cancer progression or cancer relapse, with decreased side effects in patients, and decreased health costs.To date, few clinical trials (if any) questions such protocols of adaptive therapy. However, in vivo experiments (xenografts) and in silico statistical models allowed to study evolutionary dynamics of tumor sub-populations with and without therapy.The models predict that host survival can be maximized if “treatment-for-cure strategy” is replaced by “treatment-for-stability.” Specifically, the models predict that an optimal treatment strategy will modulate therapy to maintain a stable population of chemosensitive cells that can, in turn, suppress the growth of resistant populations under normal tumor conditions, Dr Gatenby said
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Faugeroux, Vincent. "Caractérisation moléculaire et fonctionnelle de cellules tumorales circulantes dans le cancer de la prostate et le cancer bronchique non à petites cellules." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS481/document.
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Les cellules tumorales circulantes (CTC) représentent une source de matériel tumoral accessible de manière non invasive, susceptible de fournir des informations cliniques et fondamentales. Ces cellules issues de tumeurs primitives ou métastatiques représentent une population hétérogène d’éléments très rares circulant dans le sang. La personnalisation des traitements en oncologie repose sur la caractérisation moléculaire de biopsies tumorales mais celles-ci peuvent être difficiles à réaliser ou peu informatives. De ce fait, la caractérisation moléculaire et fonctionnelle des CTC présente un double intérêt, clinique pour identifier des biomarqueurs de sensibilité à des traitements, et fondamental pour étudier les mécanismes qui sous-tendent leur potentiel à initier des tumeurs.Les objectifs de ma thèse ont été d’une part de caractériser par séquençage de l’exome (WES) les CTC à l’échelle de cellule unique de patients atteints de cancers de la prostate (PCa) métastatiques et d’autre part d’établir puis caractériser des modèles de xénogreffes dérivés de CTC (CDX) chez des patients atteints de cancers bronchiques non à petites cellules (CBNPC) ou de PCa.Pour répondre au premier objectif, nous avons développé une méthode expérimentale globale incluant trois approches technologiques permettant d’enrichir et d’isoler des CTC individuelles de différents phénotypes (épithélial, épithélio-mésenchymateux et mésenchymateux), d’amplifier la totalité du génome (WGA) et de le séquencer. Le WES a été réalisé pour 34 échantillons de CTC sélectionnés sur des critères de qualité du WGA, ainsi que pour les biopsies de métastases correspondantes chez sept patients. Deux patients présentant une hétérogénéité phénotypique de leurs CTC, ont été analysés en profondeur. Nous avons mis en évidence des mutations partagées entre les CTC et les biopsies tumorales correspondantes ainsi que des mutations uniquement retrouvées dans les CTC. Ces mutations spécifiques aux CTC sont présentes dans tous les phénotypes et affectent particulièrement les gènes impliqués dans le remodelage du cytosquelette, la réparation de l’ADN ou l’invasion. L’existence de mutations communes entre les CTC de différents phénotypes suggère une relation phylogénique entre ces cellules mais une évolution divergente pendant le processus métastatique. Ce travail est soumis pour publication.Dans la seconde partie de ma thèse, nous avons implantés les CTC de 67 patients atteints de CBNPC et 24 patients atteints de PCa chez des souris immunodéprimées. Nous avons établis quatre CDX de CBNPC et un CDX de PCa. La caractérisation de ces modèles, des biopsies tumorales, des CTC collectées au moment de la xénogreffe, des CDX et des lignées cellulaires établies à partir du CDX, ont révélé que les CTC, le CDX et les lignées cellulaires « miment » le phénotype et le profil mutationnel des biopsies tumorales. La caractérisation plus approfondie de l’une des lignées cellulaires montre la présence d’un stress réplicatif et d’une instabilité génomique élevée. Ce résultat nous oriente sur l’hypothèse d’un rôle éventuel de l’instabilité génomique dans la tumorigénicité des CTC.Dans ce travail, nous avons montré que le profil mutationnel des CTC présente de fortes similitudes avec les biopsies tumorales des patients dans les patients atteints de PCa étudiés. De plus, nous avons observé l’existence de mutations spécifiques aux CTC, non détectées dans les biopsies tumorales. Également, nous montrons que des CTC issues de CBNPC et de PCa sont tumorigéniques in vivo et qu’elles reflètent le profil mutationnel des biopsies tumorales des patients. Ces modèles constituent des outils originaux et intéressants pour identifier de nouvelles cibles thérapeutiques et stratégies anti-cancéreuses, et comprendre les mécanismes qui supportent le potentiel des CTC à initier des tumeursCirculating tumor cells (CTCs) represents an non invasive source of tumor material which may provide clinical and basic information. These cells derived from primary or metastatic tumors represents an heterogeneous population of very rare events which circulates in the blood. Oncology personnalized medicine is based on biopsies molecular characterization but these are sometimes which difficult to realize and poorly informative. Thereby molecular and functional characterization of CTCs presents a double interest, clinical to identify treatments biomarkers sensitivity and basic to study mechanisms underlying their tumor inititiating cell (TIC) potential. The two goals of my thesis were on the one hand to characterize by whole-exome sequencing (WES) at the single level the CTCs from patients with metastatic prostate cancers (mPCa) and on the other hand to establish and characterize CTC-derived xenografts (CDX) from patients with non-small-cell lung cancer (NSCLC) or mPCa. For the first goal we developped a global workflow which include three technological approaches to enrich and isolate individual CTCs from different phenotype (epithelial, epithelial and mesenchymal, mesenchymal), to perform whole genome amplification (WGA) and to sequence them. WES was performed on 34 CTC samples selected according to WGA quality and on corresponding metastasis biopsies from seven patients. Two patients with phenotypic heterogeneity of CTCs were deeply analyzed. We highlighted shared mutations between CTCs and matched biopsies as well as mutations only detected in CTCs. These private CTC mutations are detected in all phenotype and particularly affect genes invlved in cytoskeleton remodeling, DNA repair or invasion. The existence of common mutations between CTCs from various phenotype suggests a phylogenic link between these cells but a divergent evolution during metastatic process. This work is submitted for publication. For the second goal, we implanted CTCs from 67 NSCLC patients and 28 mPCa patients in immunocompromised mice. We established four NSCLC CDX and one mPCa CDX. The characterization of tumor biopsies, CTCs collected at the time of xenograft, CDX and CDX-derived cell lines revealed that CTCs, CDX and cell lines miror the phenotype and mutational landscape of tumor biopsies. The more deeply characterization of one cell line show the presence of a high replicative stress and genomic instability. This result directs us to the hypothesis of a possible role of the genomic instability in CTC tumorigenicity.We demonstrated in this work that CTCs mutational landscape harbors high similairities with patients tumor biopsies in mPCa. Furthermore we observed CTC private mutations not detected in tumor biopsies. Also we showed that some CTCs from NSCLC and mPCa are tumorigenic in vivo and that these CTCs mirror mutational profile of patients tumor biopsies. These models are original and interesting tools to identify new therapeutic targets and anti-tumoral strategies and understand mechanisms underlying the TIC potential of CTCs
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Malek, Joël. "Genetic alterations of the metastatic lesions in ovarian carcinoma." Thesis, Paris 11, 2011. http://www.theses.fr/2011PA11T109.
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Le cancer de l’ovaire est le cancer gynécologique avec la plus grande mortalité due à un diagnostique tardif au stade de maladie extensive péritonéale. Malgré les progrès de la chirurgie radicale et de la chimiothérapie les récurrences abdominales demeurent la cause la plus fréquente de mortalité. Il existe peu d’études de la maladie métastatique péritonéale. Notre hypothèse de travail est que les différences entre la maladie métastatique et la tumeur primaire sont primordiales dans la survenue d’une maladie résiduelle ou récurrente. Nous avons utilisé une approche exhaustive comprenant des études du transcriptome, des variations du nombre de copie (VNC) et des sequençages des exomes pour caractériser les différences entre lésions primaires, métastases péritonéales et métastases lymphatiques.Résultats: Notre étude démontre que les VNC varient de façon significative entre la tumeur primaire et la métastase peritonéale. Les différences d’expressions géniques bien que mineures permettent de retrouver les voies de signalisation primordiales pour le développement des métastases. Le séquençage des exomes montre très peu de différences en terme de polymorphisme. Par ailleurs la majorité des polymorphismes présents dans les métastases se retrouvent à une faible fréquence dans la tumeur primaire de façon concordante avec la théorie clonale. Conclusion: L’ensemble des résultats montre la possibilité d’une origine clonale de la maladie métastatique des cancers de l’ovaire comportant la majorité des anomalies au niveau des variations du nombre de copie. L’intégration de ces données permettrait d’optimiser les thérapeutiques cibléesOvarian cancer is the most deadly gynecological cancer. The high rate of mortality is due to the large tumor burden with extensive metastatic lesion of the abdominal cavity. There are few studies on genetic alterations and their consequences in peritoneal metastatic tumors when compared to their matched ovarian primary tumors. Our hypothesis is that differences between the metastatic and primary lesions might be the cause of residual disease and, most importantly may have a role in post-chemotherapeutic recurrences. Methods: We conducted integrated genomics analysis on matched primary and metastatic tumors from 9 patients. In the papers presented here we analyze genome-wide Copy Number Variations (CNVs) using SNP Arrays targeting peritoneal metastasis differences, Gene expression differences using Microarrays also targeting peritoneal metastasis differences, and for some patients, Single Nucleotide Polymorphisms (SNPs) in genes through Exome sequencing.Results: Here we show that CNVs vary significantly between primary and metastatic tumors and include genes that have been considered potential chemotherapeutic targets based on primary tumor only data. Gene expression differences, while minor, showed highly statistically significant enrichment of genes in ovarian cancer critical pathways. In agreement with findings in other cancers, exome sequencing data revealed very few SNP differences of which most metastasis enriched SNPs were present at very low levels in the primary tumor. The results presented here should allow better design of therapies to target residual ovarian cancer disease
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Karaderi, Tugce. "Genetics of ankylosing spondylitis." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:8c0e848a-e712-4603-b923-a96a2f1644ac.
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Ankylosing spondylitis (AS) is a common inflammatory arthritis of the spine and other affected joints, which is highly heritable, being strongly influenced by the HLA-B27 status, as well as hundreds of mostly unknown genetic variants of smaller effect. The aim of my research was to confirm some of the previously observed genetic associations and to identify new associations, many of which are in biological pathways relevant to AS pathogenesis, most notably the IL-23/TH17 axis (IL23R) and antigen presentation (ERAP1 and ERAP2). Studies presented in this thesis include replication and refinement of several potential associations initially identified by earlier GWAS (WTCCC-TASC, 2007 and TASC, 2010). I conducted an extended study of IL23R association with AS and undertook a meta-analysis, confirming the association between AS and IL23R (non-synonymous SNP rs11209026, p=1.5 x 10-9, OR=0.61). An extensive re-sequencing and fine mapping project, including a meta-analysis, to replicate and refine the association of TNFRSF1A with AS was also undertaken; a novel variant in intron 6 was identified and a weak association with a low frequency variant, rs4149584 (p=0.01, OR=1.58), was detected. Somewhat stronger associations were seen with rs4149577 (p=0.002, OR=0.91) and rs4149578 (p=0.015, OR=1.14) in the meta-analysis. Associations at several additional loci had been identified by a more recent GWAS (WTCCC2-TASC, 2011). I used in silico techniques, including imputation using a denser panel of variants from the 1000 Genomes Project, conditional analysis and rare/low frequency variant analysis, to refine these associations. Imputation analysis (1782 cases/5167 controls) revealed novel associations with ERAP2 (rs4869313, p=7.3 x 10-8, OR=0.79) and several additional candidate loci including IL6R, UBE2L3 and 2p16.3. Ten SNPs were then directly typed in an independent sample (1804 cases/1848 controls) to replicate selected associations and to determine the imputation accuracy. I established that imputation using the 1000 Genomes Project pilot data was largely reliable, specifically for common variants (genotype concordence~97%). However, more accurate imputation of low frequency variants may require larger reference populations, like the most recent 1000 Genomes reference panels. The results of my research provide a better understanding of the complex genetics of AS, and help identify future targets for genetic and functional studies.
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Zhivagui, Maria. "Genome-wide modeling of mutation spectra of human cancer-risk agents using experimental systems." Thesis, Lyon, 2017. http://www.theses.fr/2017LYSE1278/document.
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Les génomes du cancer présentent une mosaïque de types de mutations. Trente signatures mutationnelles ont été identifiées à partir d'un grand nombre de tumeurs humaines primaires. Déchiffrer l'origine de ces signatures mutationnelles pourrait aider à identifier les causes du cancer humain. Environ 40% des signatures décrites sont d'origine inconnue, soulignant la nécessité de modèles expérimentaux contrôlés pour étudier l'origine de ces signatures. Au cours de mon travail de doctorat, j'ai caractérisé et utilisé des modèles in vitro et in vivo d'exposition aux cancérogènes, caractériser les signatures mutationnelles au niveau de génome entier de plusieurs composés cancérogènes pour lesquels le spectre de mutations n'était pas connu ou controversé. Tout d'abord, les conditions de cytotoxicités et genotoxicités pour chaque composé ont été établies et la formation d'adduits d'ADN a été évaluée. Suite au séquençage du gène TP53, on a effectué un séquençage au niveau du génome des clones MEF immortalisés dérivés de l'exposition à l'acrylamide, au glycidamide et à l'ochratoxine A. Le travail suggère une nouvelle signature mutationnelle unique pour l'acrylamide et médiée par son métabolite actif, le glycidamide. En fait, le motif des mutations de glycidamide, correspondant au profil de sa signature mutationnelle, a récapitulé les types de mutations attendus en fonction de l'analyse des adduits d'ADN. En outre, une analyse intégrée utilisant des modèles in vitro et in vivo suggère un manque de mutagénicité directe pour l'OTA avec une contribution potentielle d'un mode d'action lié à la production des radicaux libres à la signature mutationnelle OTA dans les MEF. Cette stratégie expérimentale simple et puissante peut faciliter l'interprétation des empreintes de mutations identifiées dans les tumeurs humaines, élucider l'étiologie du cancer et finalement soutenir la classification des cancers du CIRC en fournissant des preuves mécanistesCancer genomes harbour a mosaic of mutation patterns from which thirty mutational signatures have been identified, each attributable to a particular known or yet undetermined causal process. Deciphering the origins of these global mutational signatures in full could help identify the causes of human cancer, especially for about 40% of those signatures identified thus far that remain without a known etiological factor. Thus, well-controlled experimental exposure models can be used to assign particular mutational signatures to various mutagenic factors.During the time frame of my PhD work, I characterized and employed innovative in vitro and in vivo models of carcinogen exposure, namely, primary Hupki MEF cells, HepaRG and lymphoblastoid cell lines as well as rodent tumors. The cytotoxic and genotoxic conditions for each tested exposure compound were established and DNA adduct formation was assessed in select cases. Following a pre-screen by TP53 gene sequencing, genome-wide sequencing of immortalized Hupki MEF clones derived from exposure to acrylamide, glycidamide and ochratoxin A was performed, alongside whole genome sequencing of ochratoxin A induced rat renal tumors. The results reveal a novel mutational signature of acrylamide mediated by its active metabolite, glycidamide, a pattern that can be explained by the parallel analysis of individual glycidamide-DNA adducts. In addition, an integrative mutation analysis using in vitro and in vivo models suggests a lack of direct mutagenicity for OTA and possible indirect effects due to the ROS-mediated mode-of-action in MEF cells. The presented robust experimental strategy can facilitate the interpretation of mutation fingerprints identified in human tumors, thereby elucidating cancer etiology, elucidating the relationship between mutagenesis and carcinogenesis and ultimately providing mechanistic evidence for IARC’s carcinogen classification
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Labreche, Karim. "Genetic Susceptibility and Molecular Characterization of Glioma." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS161/document.
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Les gliomes constituent les plus fréquentes des tumeurs malignes primaires du système nerveux central. Les liens qui existent entre ces tumeurs et un certain nombre de cancers rares héréditaires, comme les Neurofibromatoses I et II ou les syndromes de Turcot et de Li-Fraumeni, attestent d’une prédisposition génétique aux gliomes. L’observation d’un risque deux fois plus élevé de développer un gliome chez les parents de premier degré de patients atteints suggère aussi une possible prédisposition génétique dans les gliomes sporadiques. Par ailleurs, l’analyse à haut débit permet de préciser le profil somatique des gliomes et d’identifier des biomarqueurs pronostiques voire prédictifs et s’inscrire dans une démarche de traitement personnalisé du patient. Durant ma thèse, je me suis focalisé sur deux axes de recherches complémentaires; l’identification de gènes de susceptibilité et la découverte de nouveaux gènes fréquemment mutés dans les gliomes, afin de déterminer les voies de signalisation contribuant à la gliomagenèse. Dans leur ensemble, les résultats obtenus dans cette thèse apportent non seulement des informations importantes sur la nature de la prédisposition génétique aux gliomes mais également de son association spécifique pour les différents sous-types de tumeurs. La découverte d’un nouveau gène muté, offre la perspective à plus long terme d’un traitement personnalisé pour chaque patient sur la base du profil génétique de sa tumeurGliomas are the most common adult malignant primary tumour of the central nervous system. Thus far, no environmental exposures has been linked to risk except for ionizing radiation, which only accounts for a very small number of cases. Direct evidence for inherited predisposition to glioma is provided by a number of rare inherited cancer syndromes, such as Turcot's and Li–Fraumeni syndromes, and neurofibromatosis. Even collectively, these diseases however account for little of the twofold increased risk of glioma seen in first-degree relatives of glioma patients. My research was centred on two complementary research activities: Identifying susceptibility genes for glioma to delineate key biological pathways contributing to disease pathogenesis and to identify new recurrent mutated genes for glioma to provide for further insights into glial oncogenesis and suggesting targets for novel therapeutic strategies. Collectively the findings in this thesis provide increased insight into the nature of genetic predisposition to glioma and substantiate the often distinct associations between susceptibility variants and glioma molecular groups. In addition the discovery of a new mutated gene in glioma offers the potential to support drug development and advance precision medicine for this tumours
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Jacob, Arthur. "Apport du séquençage d'exome pour le diagnostic et le traitement des maladies complexes." Thesis, Lille 2, 2020. http://www.theses.fr/2020LIL2S011.
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Depuis l'achèvement du projet du génome humain en 2001, le domaine de lagénomique a progressé de façon exponentielle, en grande partie grâce àl'introduction du séquençage de nouvelle génération (NGS), une technique deséquençage qui a révolutionné les méthodes d'investigation des maladiesgénétiques. L’accessibilité croissante de ces technologies permet l’essor de lamédecine de précision, basée sur une prise en charge spécifique de chaque patienten fonction de son profil génétique. Le séquençage peut être utilisé pour lediagnostic de maladies, la recherche de prédispositions génétiques, ou encore pourle choix thérapeutique en particulier en cancérologie. Le séquençage de l'exome(WES), en particulier, offre une méthode efficace pour étudier les maladies, car lesrégions exoniques représentent 2% du génome entier, mais peuvent contenir jusqu'à85% de variants fonctionnels responsables des maladies. Or, l’analyse génétiquedes patients en milieu clinique est principalement effectuée par le séquençage cibléde panels de quelques gènes choisis en fonction du contexte clinique. Le travaileffectué dans le cadre de cette thèse CIFRE en partenariat avec la sociétéPrenostics a été de développer des analyses WES pertinentes permettant decaractériser le profil génétique de patients atteints de maladies génétiques et decancers. L’objectif a été d’évaluer leur apport dans le diagnostic et l’établissement destratégies thérapeutiques personnalisées dans trois contextes distincts de pratiqueclinique, en le comparant à l’approche conventionnelle de séquençage de panels.- Dans le domaine des maladies génétiques pédiatriques, le diagnostic moléculairepar les méthodes conventionnelles n’atteint que 25%, laissant la majorité desfamilles sans conseil génétique pertinent. Notre analyse WES d’une cohorte de 26enfants atteints de maladies génétiques non diagnostiqués par les analysesgénétiques conventionnelles, notre approche WES a permis d’établir un diagnosticpositif dans 35% des cas.- Environ 5 à 10% des cancers du sein sont héréditaires, mais plus de la moitiéd’entre eux ne sont pas élucidés par le séquençage de panel classique incluant lesgènes à risque (BRCA1, BRCA2, PALB2, etc) et ne sont pas causés par des gènesde prédisposition connus. En analysant l’exome de quatre familles, nous avons tentéd’identifier les gènes en cause de cas familiaux ne présentant pas d’altération desgènes connus BRCA1/BRCA2 (familles BRCAx). Après filtration des variants à risque transmis parmi les membres atteints, nous avons identifié les gènes candidatsHIST1H1C, TYRO3, TPH1, SLC12A3 et CCNF comme de possibles gènes deprédisposition au cancer du sein. Cependant, sans études fonctionnelles pousséespermettant de valider leur implication, le WES ne semble pas apporter de bénéficepour la prise en charge de ces familles.- Enfin, dans le domaine de la cancérologie, la personnalisation du traitement est aucentre des enjeux actuels. Notre étude d’une cohorte de 35 tumeurs solidesréfractaires a démontré la faisabilité et l’efficacité du WES pour la caractérisation duprofil génétique de tumeurs solides et la prise de décision en oncologie. Ladétermination des pattern mutationnels tumoraux, de la charge tumorale et dessignatures mutationnelles à partir des données de WES a permis d’émettre dessuggestions thérapeutiques pour la moitié d’entre eux et a contribué à la modificationdu traitement d’au moins huit patients sur 35.Cette étude décrit les différentes applications, les limites et les avantages du WEScomme outil d’investigation moléculaire des maladies humaines. En démontrant lebénéfice d’utilisation du WES en clinique, nos résultats contribuent aux efforts encours pour son intégration dans le parcours de soin et le développement de lamédecine de précisionSince the completion of the Human Genome Project in 2001, the field of genomicshas grown exponentially, in large part due to the introduction of Next GenerationSequencing (NGS). This technique has revolutionized the investigation methods ofgenetic diseases, allowing high-throughput genome-wide sequencing to establish thegenetic basis of diseases. The increasing accessibility of these technologies allowsthe development of precision medicine, based on the specific care of each patientaccording to his genetic profile. Sequencing can be used for the diagnosis ofdiseases, the search for genetic predispositions to a disease, or for the therapeuticchoice, in particular in oncology. Exome sequencing (WES), in particular, offers aneffective method for studying diseases, since exonic regions represent 2% of theentire genome, but can contain up to 85% of functional variants responsible fordiseases. However, the genetic analysis of patients in a clinical setting is mainly carried out by the targeted sequencing of panels of a few genes chosen according tothe clinical context. The work carried out during this CIFRE thesis in partnership withthe company Prenostics was to develop relevant WES analyzes to characterize thegenetic profile of patients with rare genetic diseases and cancers. The objective wasto assess their contribution to the diagnosis and establishment of personalizedtreatment strategies in three distinct contexts of clinical practice, by comparing it tothe conventional approach of panel sequencing.- In the field of pediatric genetic diseases, molecular diagnosis by conventionalmethods only reaches 25%, leaving the majority of families without precise geneticcounseling. Our WES analysis of a cohort of 26 children with genetic diseases notdiagnosed by conventional genetic analyzes, our WES approach resulted in apositive diagnosis in 35% of cases.- About 5 to 10% of breast cancers are hereditary, but more than half of them are notelucidated by the genes at risk of breast cancer (BRCA1, BRCA2, PALB2, etc.) thatare included in conventional panel sequencing. By analyzing the exome of fourfamilies, we attempted to identify the genes involved in familial cases non-mutatedfor the known BRCA1 / BRCA2 genes (BRCAx families). After filtering the riskvariants transmitted among affected limbs, we identified the candidate genesHIST1H1C, TYRO3, TPH1, SLC12A3 and CCNF as possible genes of predispositionto breast cancer. However, without in-depth functional studies to validate theirinvolvement, WES does not seem to provide any benefit for patient management.- Finally, in the field of oncology, the personalization of treatment is at the center ofcurrent issues. Our study of a cohort of 35 refractory solid tumors aimed atdemonstrating the feasibility and efficacy of WES for characterizing the geneticprofile of solid tumors and for decision-making in oncology. We were able to maketreatment suggestions for half of them and helped modify the treatment of at leasteight out of 35 patients.This study describes the different applications, limits and advantages of WES as amolecular investigation tool for human diseases. By demonstrating the benefit ofusing WES in the clinic, our results contribute to the ongoing efforts to integrate it intothe care pathway and the development of precision medicine