Dissertations / Theses on the topic 'Clonal heterogeneity'

The classical myeloproliferative disorders (MPD), comprising essential thrombocythaemia (ET), polycythaemia vera (PV) and idiopathic myelofibrosis (IMF), are clonal premalignant haematopoietic neoplasms associated with activating mutations in signalling pathway molecules and a variable tendency to develop acute myeloid leukaemia (AML). This thesis examined genotype-phenotype associations of JAK2 and MPL mutations, the presence of clonal diversity in the MPD and the genetic events associated with progressive disease. Mutations in MPL were identified in 4% of ET and 7% of IMF but not in PV. Three different acquired MPL mutations were identified, one of which had been reported as an inherited allele. Although MPL mutations did not delineate a distinct clinical or histopathological subtype of ET, molecular testing provides an important new tool in the diagnostic armamentarium. Clones homozygous for the JAK2 V617F mutation were identified in female but not male patients with ET, suggesting that gender differences may be important in the determination of disease phenotype. In patients with two acquired genetic alterations, a signalling pathway mutation and a cytogenetic abnormality were usually present within the same clone. By contrast, coexistence of two signalling pathway mutations indicated the presence of biclonal disease that in two patients had arisen independently and not from a shared founder clone. RAS mutations were identified as potential cooperating events in patients with JAK2 or MPL mutant IMF. In patients developing AML following a JAK2 V617F-positive MPD, those with V617F-positive leukaemia had progressed via an accelerated phase of disease and harboured acquired alterations of RUNX1 or EVI1. V617F-negative leukaemias tended to follow directly from ET or PV, and loss of the JAK2 mutation by reversion to wild-type due to mitotic recombination, gene deletion or gene conversion was excluded. The thesis concludes with a discussion of how clonal heterogeneity can be integrated into current models of MPD disease pathogenesis.

The normal mammary gland contains “stem cells” with extensive in vivo growth and bi-lineage differentiation potential and a surface phenotype of basal cells (BCs). BCs also contain cells with more limited growth and differentiation activity in vitro. An analogous luminal-restricted progenitor (LPs) subset has surface characteristics of both basal and luminal cells. I hypothesized that the growth and differentiation activity displayed by individual mammary epithelial cells from both subsets would be highly diverse, and that the properties of tumours produced from these cells would be affected by their cell of origin. To address this hypothesis, I first developed a lentiviral-mediated barcoding strategy that involves transducing each cell with a unique 27-base pair non-coding DNA sequence so that the number of its clonal progeny can be inferred from high-throughput sequencing data obtained on the progeny of bulk-transduced populations. The use of “spiked-in” control cells carrying a known barcode provided an internal calibration for clone size calculations and allowed clones of ≥100 cells to be reliably detected. Application of this strategy to normal mouse and human mammary cells identified expected bi-lineage clones but an unanticipated predominance of lineage-restricted clones produced in primary transplants. These experiments also revealed that many clones apparent in secondary hosts were not detected in the primary hosts, indicating their origin from cells with very delayed growth activity. Application of the barcoding strategy to normal human BCs and LPs transduced with lentiviruses encoding KRASG¹²D ± PI3KCAH¹⁰⁴⁷R ± TP53R²⁷³C showed tumour formation in subsequently transplanted immunodeficient mice was rapid (within 8 weeks) and efficient from both cell types (8-12/18 donors, 1/200-1/4,000 transduced cells). However, tumours generated from LPs contained larger clones than tumours generated from BCs. Surprisingly, none of the LP-derived tumours were ERα⁺ (typical of luminal-like breast cancers) whereas 60% of the BC-derived tumours were. Earlier analysis of xenografts of similarly transduced cells revealed changes in both the number and phenotype of the cells present. Taken together, these findings underscore the diverse regenerative activity of normal mammary cells and provide definitive evidence that the cell of origin can affect the properties of human breast tumours generated using identical oncogenes.

Thesis: Ph. D., Massachusetts Institute of Technology, Computational and Systems Biology Program, 2016.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 119-121).
Cancer is a clonal evolutionary process. This results in complex clonal architecture and intratumoral heterogeneity in each patient. This also presents challenges for effective therapeutic intervention - with constant selective pressure to induce or select pre-existing resistant subclones toward drug resistance. Mathematical/computational modeling from population genetics, evolutionary dynamics, and engineering are being utilized to a greater extent in recent times to study tumor progression, intratumoral heterogeneity, drug resistance, and rational drug scheduling/combinations design. In this thesis we present several joint quantitative and experimental approaches for the rational design of drug combinations to tackle the issue of intratumoral heterogeneity and clonal evolution. Using a tractable experimental system with pre-defined tumor compositions, we derived computational approaches to rationally design drug combinations with the goal of minimizing a given heterogeneous tumor. We found that the best drug combinations can oftentimes be non-intuitive as they do not contain component drugs most effective for the individual subpopulations. This was the result of a need for combinatorial considerations on the effects of each drug on all subpopulations, hence at times leading to non-intuitive drug regimens. We validated our computational model predictions in vitro and in vivo in a preclinical model of Burkitt's lymphoma, with predictable evolutionary trajectories upon treatment. Next, we extended this methodology to study the effects of more complex tumor heterogeneity on combinatorial drug design, with similar conclusions. Sampling and statistical analyses over a range of tumor compositions can further inform effective drug combinations under some uncertainty in initial tumor heterogeneity. Moving beyond a model where we have control of initial tumor composition, we sought to examine collateral resistance and sensitivity during clonal evolution. Using a murine model of Ph+ acute lymphoblastic leukemia, we performed drug selection and pharmacological screen experiments. We observed important evolutionary processes of selection and drift in giving rise to resistance to clinically used BCR-ABL1 inhibitors. Remarkably, the resistant population also became hyper-sensitized to nonclassical BCR-ABL1 inhibitors at intermediate stages of the clonal evolution, in this so-called 'temporally collateral sensitivity'. Mathematical modeling and experimentation brought additional insight into the evolutionary dynamics and mechanism of action, with demonstrated in vivo efficacy.
by Boyang Zhao.
Ph. D.

Traditional classifications and treatment of human cancers have operated with limitations surrounding tumor homogeneity and mutational stasis. Clinical metrics of malignant tumors focused on descriptive and behavioral properties such as tissue of origin, cellular morphologic features and extent of spread. Missing has been an understanding of the dynamics of cellular subpopulations that underpin divergent functional properties in space and time. This dissertation is focused on the development and application of methods, including next generation DNA sequencing, computational modeling, and single-cell genotyping protocols to elucidate breast tumor heterogeneity and clonal evolution at single nucleotide and single-cell resolution. First, I present advances in our knowledge of the mutational spectrum that may occur and evolve in an individual epithelial cancer, namely a lobular breast cancer metastases and matched primary tumor separated by a nine year interval. This seminal study demonstrated clonal evolution in a patient’s breast cancer and the successful application of targeted deep sequencing for determining digital allelic prevalences and clonal genotypes in bulk tumors. Second, I describe the diversity of genomic sequence and clonal heterogeneity in tumors of the triple-negative breast cancer subtype. The study uncovered wide clonal diversity in these primary tumors at first diagnosis. Third, I demonstrate via genotyping single tumor cells, that computational inferences of tumor clonal architecture can be made reliably from bulk tissue-derived data sets. This was performed using both somatic point mutations and loss of heterozygosity loci as clonal marks. And fourth, I applied single-cell analysis to study the clonal evolution in breast tumor murine xenografts following engraftment and serial passaging. This research uncovered a range of outcomes in tumor clonal composition upon initial engraftment and serial passaging. The same clonal groups were found to arise independently in separate xenografts derived from the same primary tumor, suggesting selection of functionally significant genotypes. Comprehensive capabilities in the measurement and analysis of clonal structure in cancers offers improved classification and combinatorial treatments of subpopulations in heterogeneous tumors and better use of murine xenograft models. Functionally relevant subpopulations of tumor cells, irrespective of numerical abundance or spatiotemporal persistence, can thereby be targeted using clonally informative genomic profiles.
Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate

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%.

Background: Clonal competition in cancer describes the process in which the progeny of a cell clone supersedes or succumbs to other competing clones due to differences in their functional characteristics, mostly based on subsequently acquired mutations. Even though the patterns of those mutations are well explored in many tumors, the dynamical process of clonal selection is underexposed. Methods: We studied the dynamics of clonal competition in a BcrAbl-induced leukemia using a γ-retroviral vector library encoding the oncogene in conjunction with genetic barcodes. To this end, we studied the growth dynamics of transduced cells on the clonal level both in vitro and in vivo in transplanted mice. Results: While we detected moderate changes in clonal abundancies in vitro, we observed monoclonal leukemias in 6/30 mice after transplantation, which intriguingly were caused by only two different BcrAbl clones. To analyze the success of these clones, we applied a mathematical model of hematopoietic tissue maintenance, which indicated that a differential engraftment capacity of these two dominant clones provides a possible explanation of our observations. These findings were further supported by additional transplantation experiments and increased BcrAbl transcript levels in both clones. Conclusion: Our findings show that clonal competition is not an absolute process based on mutations, but highly dependent on selection mechanisms in a given environmental context.

The ability to culture individual cells provides a unique method to assess the heterogeneity of mammalian cell populations. However, there are many challenges when scaling down culture systems due to the complexity of re-creating a stimulating environment at the clonal level. Small volume culture systems such as integrated microfluidic platforms offer the potential to radically alter the throughput of clonal screening through the use of time-lapse imaging, dynamic stimulus control and economy of scale. In particular, the use of automated fluidic control allows for the characterization of single cells in a dynamic microenvironment similar to large-scale culture. This thesis describes how small volume cell culture practices such as the use of conditioned medium and microfluidic technology can be implemented to isolate large numbers of cells in small volumes and evaluate clonal populations under precise medium conditions. For a Chinese Hamster Ovary (CHO) cell system normal growth kinetics and specific productivity were sustained in small volumes. When exposed to conditioned medium from a parental CHO line, clones cultured at sub-mL scales matched the performance of large-scale cultures. A microfluidic bead assay was developed to detect Immunoglobulin G titers secreted from clones in nL volumes. The combination of microfluidic conditioned medium perfusion with the magnetic bead assay allowed for clonal productivity to be evaluated under simulated fed-batch conditions. Lastly, microfluidic cell culture was demonstrated on a human embryonic stem cell (hESC) system through the robust generation of colonies derived from single cells. hESCs propagated in the microfluidic system were observed to match the growth kinetics, marker expression and colony morphologies of larger cultures, while resolving response heterogeneity during differentiation induction. This thesis demonstrates how high-throughput, small volume culture systems can be used to screen clonal populations for therapeutic applications under complex culture conditions.
Applied Science, Faculty of
Graduate

Life history traits such as growth, survival, and clonality can vary within a population. When such variation exists in a population of an invasive species, it can affect population dynamics, and if any part of the variation has a genetic basis the population can evolve in response to control regimes. Evolutionary responses to control efforts may shift the population towards a few more resilient genotypes, or towards different types in different microenvironments, depending on the scale of gene flow with respect to the patchiness of the environment. The purpose of this study is to examine whether the application of stress similar to control efforts (light level manipulation and biomass removal) results in varying emergence, growth, and survival rates between samples taken from spatially separated patches of the invasive clonal grass Imperata cylindrica. Accelerated Failure Time (AFT) and logistic regression models were fit to survival, emergence and growth data collected from two experiments in which samples collected from four spatially separated Imperata cylindrica patches were exposed to light level manipulation and biomass removal. Patch identity plays a large role in explaining variation in time-to-emergence, time-to-death, and probabilities of emergence and survival, especially under stressed conditions. Rhizome and above ground biomass characteristics also play substantial roles in explaining variation in emergence, survival, and growth, though more so under non-stressed conditions. Our results warrant further study of heterogeneous responses to stressful conditions, especially those imposed under control and management regimes. This heterogeneity may have important impacts on population processes such as maintenance, expansion, and gene flow.

Depuis sa découverte en 1948, l’analyse de l’ADN circulant (ADNcir) a démontré son potentiel clinique en oncologie.Mon premier objectif de thèse a été d’étudier l’hétérogénéité clonale de tumeurs de patients atteints de cancer colorectal métastatique (CCRm) par l’analyse de l’ADNcir au cours de leur prise en charge thérapeutique. Seuls les patients ayant des tumeurs RAS non-mutées peuvent bénéficier des thérapies anti-EGFR. Avant traitement par anti-EGFR, l’analyse de l’ADNcir a montré la détection d’un plus grand nombre de mutations RAS/BRAF (76%, n=119) que l'analyse du tissu tumoral (55%, n=89) ainsi qu’une proportion élevée de mutations co-occurrentes (45%) chez les patients atteints de CCRm (étude KPLEX2). Nous avons montré que la médiane du délai entre la réception des échantillons et la communication des résultats était de 12 jours pour l’analyse du tissu tumoral (n=83; 3-273 jours) contre seulement de 2 jours pour l’analyse de l’ADNcir (n=121; 0-10 jours). Ces résultats indiquent que l’ADNcir pourrait remplacer l’analyse du tissu tumoral en pratique clinique.Dans l’étude rétrospective KPLEXR, l’analyse longitudinale des patients atteints de CCRm traités par FOLFOX-dasatinib avec ou sans cetuximab a montré qu’à l’arrêt des traitements 98% des patients réfractaires aux traitements (n=42) sont mutés RAS/BRAF. Ce résultat démontre que les mutations RAS/BRAF induisent des résistances au traitement par anti-EGFR. L’analyse quantitative et longitudinale de l’ADNcir mime étroitement les changements des niveaux sanguins de l’ACE et l’évolution de la masse tumorale observée par CT-scan. De plus, 21% des mutations RAS/BRAF sont retrouvées avec des fréquences alléliques inférieures à 0.1% montrant la nécessité d'utiliser une méthode ultrasensible pour détecter les mutations ponctuelles avant et durant les traitements par anti-EGFR. Ces résultats indiquent le potentiel théranostique de l’ADNcir durant un traitement par anti-EGFR.Les patients réfractaires à l’ensemble des thérapies standards peuvent être traités au regorafenib. Cependant, aujourd’hui aucun biomarqueur ne permet de prédire un bénéfice ou une résistance au regorafenib. C’est dans ce contexte qu’a été réalisé l’étude prospective TEXCAN. Nous avons montré que le statut muté RAS/BRAF des patients atteints de CCRm lourdement prétraités et réfractaires aux thérapies standards n’influe pas la survie de ces patients traités au régorafénib. Par contre, les resultats sugerent qu’il est possible d’identifier avant le traitement les patients qui auront un bénéfice clinique à cette molécule. En effet, nous avons pu établir des seuils de concentrations d’ADNcir total (26 ng/mL de plasma), d’ADNcir tumoral muté (2 ng/mL de plasma) et de fréquence allélique (6%) qui permettraient de prédire les patients qui auront une meilleure survie globale. L’ADNcir pourrait être un biomarqueur pour prédire les résistances ou les tolérances des tumeurs des patients au regorafenib.Il est admis depuis longtemps que l’apoptose est le mécanisme de libération de l’ADNcir le plus pertinent puisque de nombreuses études ont montré que l’ADNcir a un profil de taille aux alentours de 150-180 bp. Le deuxième objectif de ma thèse a été d’évaluer l’association entre la formation des NETs (neutrophil extracellular traps) et la production des ADNcir chez les patients atteints de CCRm. Nous montrons qu’une fraction importante de l’ADNcir des patients CCRm proviendrait du phénomène de NETose. Ces travaux redéfinissent les paradigmes existants sur les mécanismes de libération de l'ADNcir. La dégradation des NETs pourrait expliquer la grande amplitude de la fréquence allélique dans le sang des patients atteints de CRCm. Cependant, il reste encore à révélerer l’ensemble des acteurs conduisant à la production des ADNcir par la dégradation des Nets in vivo
Since its discovery in 1948, circulating DNA (cirDNA) analysis has demonstrated its clinical potential in oncology.My first thesis objective was to study the clonal heterogeneity of tumors from metastatic colorectal cancer (mCRC) patients by cirDNA analysis during their therapeutic management. Only patients with non-mutant RAS tumors can benefit from anti-EGFR therapies. Prior to anti-EGFR therapy, cirDNA analysis showed the detection of higher RAS/BRAF mutations (76%, n=119) than tumor tissue analysis (55%, n=89) as well as a high proportion of co-occurring mutations (45%) in mCRC patients (KPLEX2 study). We showed that the median data turn-around time was 12 days for tumor tissue analysis (n=83; 3-273 days) versus only 2 days for cirDNA analysis (n=121; 0-10 days). These results indicated that cirDNA could replace tumor tissue analysis in clinical practice.In the retrospective KPLEXR study, longitudinal analysis of mCRC patients treated with FOLFOX-dasatinib with or without cetuximab showed that 98% of treatment-refractory patients (n=42) were RAS/BRAF mutant at the end of the treatment. This result suggested that RAS/BRAF mutations induce resistance to anti-EGFR therapy. Quantitative and longitudinal analysis of cirDNA closely mirrored variations in blood CEA levels and tumor mass as observed by CT-scan. Moreover, 21% of RAS/BRAF mutations were found with allelic frequencies lower than 0.1% revealing the need of the use of an ultrasensitive method to detect point mutations before and during anti-EGFR treatments. These results highlighted the theranostic potential of cirDNA during anti-EGFR therapy.Refractory patient tumors to all standard therapies can be treated with regorafenib. However, there is currently no biomarker to predict benefit or resistance to regorafenib. Inthe prospective TEXCAN study we observed that the RAS/BRAF mutant status of heavily pretreated mCRC patients refractory to standard therapies does not influence the survival of these patients treated with regorafenib. On the other hand, we showed that it is possible to identify prior to treatment those patients who will benefit clinically from the drug. Indeed, we were able to establish thresholds for total cirDNA concentration (26 ng/mL plasma), mutant cirDNA concentration (2 ng/mL plasma) and allelic frequency (6%) that would predict which patients will have better overall survival. The cirDNA could be a biomarker for predicting patient tumor resistance or tolerance to regorafenib.It has long been recognized that apoptosis is the most relevant mechanism of cirDNA release since many studies have shown that cirDNA has a size profile around 150-180 bp. The second objective of my thesis was to evaluate the association between NETs (neutrophil extracellular traps) formation and cirDNA production in mCRC patients. We show that a significant fraction of cirDNA in mCRC patients is derived from the NETosis mecanism. This work redefines existing paradigms on the mechanisms of cirDNA release. NET degradation and resultant cirDNA release could explain the large variation of allelic frequency in the blood of mCRC patients. However, the full set of enzymes leading to the production of cirDNA by the in vivo degradation of NETs remains to be demonstrated

Superantigens trigger the polyclonal activation of human T cells. We exploited this property to gain insight into the mechanisms governing CD4 T cell expansion and to study the functional heterogeneity of naive and memory CD4 T cell subsets. We show that the amount of TCR ligand affects the evolution of a T cell response in two ways: by shaping the diversity of the T cell population recruited in the proliferative pool and by affecting the progression of these precursors into cell cycle. These two processes characterize a hierarchy of recruitment of cells that strongly correlates with the efficiency of TCR engagement but not with the relative precursor frequency in the non-immune repertoire. Remarkably, once established, the distribution of T cell clones within a selected repertoire is maintained by the characteristic of T cells to expand at a rate that is independent of quantitative differences in ligand exposure. Moreover, at optimal ligand concentrations that lead to the simultaneous expansion of all responsive T cell clones, we observed a marked clone-specific heterogeneity in the capacity to secrete cytokines. The functional heterogeneity of different CD4+ T cell subpopulations was also studied using a superantigen model. Based on the expression of CD45RA and CCR7, four distinct subsets of CD4+ T lymphocytes can be identified: naive (CD45RA+ CCR7+), central memory (TCM, CD45RA- CCR7+), effector memory (TEM, CD45RA- CCR7-) and a previously uncharacterized subset (CD45RA+ CCR7-). In CD8 T cells, this subset has been shown to comprise "terminally differentiated" effector memory cells. The four subsets show different functional sensitivities for the superantigens, the TEM population being the most sensitive and the naive cells being the least sensitive, as measured by the upregulation of activation markers. We show that the CD4+ CD45RA+CCR7- subpopulation, which is rarely detectable in healthy individuals, is enriched in T cells having str

Vascular smooth muscle cell (VSMC) accumulation is a hallmark of atherosclerosis and vascular injury. However, fundamental aspects of proliferation and the phenotypic changes within individual VSMCs, which underlie vascular disease remain unresolved. In particular, it is not known if all VSMCs proliferate and display plasticity, or whether individual cells can switch to multiple phenotypes. To assess whether proliferation and plasticity in disease is a general characteristic of VSMCs or a feature of a subset of cells, multi-colour lineage labelling is used to demonstrate that VSMCs in injury-induced neointimal lesions and in atherosclerotic plaques are oligo-clonal, derived from few expanding cells, within mice. Lineage tracing also revealed that the progeny of individual VSMCs contribute to both alpha Smooth muscle actin (aSma)-positive fibrous cap and Mac-3-expressing macrophage-like plaque core cells. Co-staining for phenotypic markers further identified a double-positive aSma+ Mac3+ cell population, which is specific to VSMC-derived plaque cells. In contrast, VSMC-derived cells generating the neointima after vascular injury generally retained expression of VSMC markers and upregulation of Mac3 was less pronounced. Monochromatic regions in atherosclerotic plaques and injury-induced neointima did not contain VSMC-derived cells expressing a different fluorescent reporter protein, suggesting that proliferation-independent VSMC migration does not make a major contribution to VSMC accumulation in vascular disease. Similarly, VSMC proliferation was examined in an Angiotensin II perfusion model of aortic aneurysm in mice, oligo-clonal proliferation was observed in remodelling regions of the vasculature, however phenotypic changes were observed in a large proportion of VSMCs, suggesting that the majority of VSMCs have some potential to modulate their phenotype. To understand the mechanisms behind the inherent VSMC heterogeneity and observed functionality, the single cell transcriptomic techniques Smart-seq2 and the Chromium 10X system were optimized for use on VSMCs. The work within this thesis suggests that extensive proliferation of a low proportion of highly plastic VSMCs results in the observed VSMC accumulation after injury, and the atherosclerotic and aortic aneurysm models of cardiovascular disease.

Os pacientes com melanomas, em geral, apresentam extrema quimiorresistência e prognóstico ruim, com taxa de sobrevida de aproximadamente seis meses; portanto, novas estratégias terapêuticas são necessárias. As células deste tipo de tumor acumulam alterações na expressão gênica que contribuem para a proliferação descontrolada, evasão de senescência e inibição de morte celular em múltiplas rotas intracelulares. No Capítulo 1, foi explorado o controle epigenético do supressor tumoral RECK. Esse gene é largamente expresso em tecidos normais, com correlação de sua expressão com melhor prognóstico. Em tumores, RECK é inibido, incluindo em melanoma. Neste estudo, sua inibição por hipermetilação do promotor e remodelamento da cromatina por HDACs não foi o único fator inibitório nas linhagens de melanoma analisadas. Mesmo após a utilização a remoção de marcas epigenéticas associadas ao silenciamento gênico, a expressão proteica não pôde ser recuperada nas linhagens utilizadas neste trabalho. No Capítulo 2, isolou-se subpopulações clonais ao acaso a fim de modelar a heterogeneidade tumoral intrínseca. Neste modelo experimental, caracterizamos a presença de dois perfis tumorais subclonais: uma mais proliferativa, invasiva e sensível a vemurafenibe; e outra menos proliferativa, mais resistente e com maior expressão de RECK e fatores ligados a EMT. Nossos resultados, em conjunto, mostraram que as linhagens celulares parentais utilizadas apresentam diferenças entre si quanto à viabilidade celular, indução de apoptose e fosforilação de ERK após o tratamento com vemurafenibe. Segundo o nosso modelo, a resistência intrínseca ao vemurafenibe está presente e reflete a heterogeneidade do tumor inicial. Com estes resultados, pretendemos contribuir para o conhecimento sobre a composição clonal presente na heterogeneidade tumoral, além de contribuir para a função de RECK na biologia do melanoma.
Patients with melanoma often present extreme chemoresistance and poor prognosis, with survival rates of approximately six months; therefore, new therapeutic strategies are necessary. Melanoma cells accumulate genotypic changes that contribute to uncontrolled proliferation, evasion of senescence and cell death inhibition in multiple cellular pathways. In Chapter 1, we have explored the epigenetic control of the tumor suppressor RECK. This gene is widely expressed in normal tissues, with correlation of its expression with better prognosis. In tumors, RECK is inhibited, including melanoma. Here, RECK inhibition by promoter hypermethylation and chromatin remodeling by HDACs was not the only inhibiting factor in the melanoma cell lines analyzed. Even after removing the epigenetic marks associated to gene expression silencing, the protein expression could not be recovered in the cell lines used in this study. In Chapter 2, it has been isolated several tumor clonal subpopulations in order to modulate the intrinsic tumor heterogeneity. In this experimental model, we have characterized the presence of two tumor subclonal profiles: a more proliferative, invasive and sensitive to Vemurafenib; and other less proliferative, less sensitive to Vemurafenib and presenting more expression of RECK and factors associated to EMT. Our results together have showed the parental cell lines used here differed from each other in terms of cell viability, induction of apoptosis, and ERK phosphorylation after treatment with Vemurafenib. According to our model, the intrinsic resistance to Vemurafenib is present and reflects the heterogeneity of the initial tumor. With these results, we intend to contribute to the knowledge of the tumor clonal subpopulations composition in tumor heterogeneity, and contributes to the RECK function in melanoma biology.

Les syndromes myélodysplasiques (SMD) forment un groupe de pathologies clonales de la cellule souche hématopoïétique (CSH) caractérisées par une hématopoïèse inefficace. La présence d’au moins une anomalie génétique (anomalie cytogénétique ou mutation somatique) est observée dans plus de 90% des cas. Ainsi, plusieurs clones moléculaires pouvaient coexister au moment du diagnostic de la maladie. Dans les SMD avec délétion du chromosome 5 (del(5q)), il a récemment été montré que les anomalies étaient présentes dès le stade de la CSH. Dans les SMD, la pénétrance des anomalies génétiques décrites est incomplète. De plus, peu de choses sont actuellement connues sur l’ordre d’apparition des mutations et leur impact fonctionnel sur les différents clones moléculaires dans le cas des SMD non-del(5q). Grâce au séquençage d’exome entier (WES) de patients ne présentant aucune mutation dans les gènes décrits dans les SMD, nous avons décrit l’existence de mutations dans les gènes BCOR et BCORL1, chez respectivement 4,2% et 0,8% des patients. Les mutations du gène BCOR arrivent tardivement au cours de l’évolution de la maladie et affectent le pronostic des patients. Des approches à l’échelle unicellulaire nous ont également permis d’observer que la majeure partie des mutations identifiées chez les patients sont retrouvées dès le stade CD34+CD38-. Chez les patients, plusieurs clones moléculaires coexistent à ce stade. De plus, les mutations des gènes de l’épissage et de la régulation épigénétique sont fréquemment acquises en premier dans les cellules hématopoïétiques les plus immatures des patients porteurs de SMD. Nous avons observé que certaines mutations, acquises secondairement, sont réparties inégalement dans les différents compartiments hématopoïétiques et peuvent avoir un impact sur la différenciation hématopoïétique. Enfin, nous montrons que la répartition des clones moléculaires évolue au cours du temps. En réponse au traitement par Lenalidomide, on observe également une évolution rapide de l’architecture clonale qui peut être liée au statut de réponse des patients. Ces résultats tendent à confirmer l’hétérogénéité génétique mais aussi fonctionnelle des SMD. Nous avons pu identifier de nouvelles mutations impliquées secondairement dans la physiopathologie des SMD. Il existe une dominance clonale précoce dans les SMD du fait de l’acquisition de toutes les mutations dans les cellules hématopoïétiques immatures. Cependant, les différentes populations hématopoïétiques peuvent présenter des génotypes différents. Enfin cette architecture est variable au cours de l’évolution de la maladie
Myelodysplastic syndromes (MDS) are a group of clonal disorders of the hematopoietic stem cell (HSC) characterized by ineffective hematopoiesis. At least one genetic abnormality (cytogenetic abnormality or somatic mutation) is observed in more than 90% of cases. Thus, it has been observed several molecular clones which could coexist at diagnosis of the disease. In MDS with deletion of chromosome 5 (del (5q)), it has recently been shown that defects were present in the HSC. In MDS, the penetrance of genetic abnormalities described is incomplete. In addition, little is currently known about the order of appearance of mutations and their functional impact on different molecular clones in the case of non-del (5q) MDS. Through the whole exome sequencing (WES) of patients without mutation in the genes described in MDS, we described the existence of mutations in genes BCOR and BCORL1, in respectively 4.2% and 0.8% of patients. Mutations in the gene BCOR were acquired lately during the course of the disease and affect the prognosis of patients. Approaches at the single cell level have also allowed us to observe that most of the mutations identified in patients are found at the immature differentiation stage CD34+CD38-. In patients, several molecular clones could coexist at this stage. In addition, mutations in gene splicing and epigenetic regulation are frequently first acquired in the most immature hematopoietic cells of MDS patients. We found that certain mutations, acquired in a second time, are distributed unevenly in different hematopoietic compartment and may have an impact on hematopoietic differentiation. Finally, we showed that the distribution of molecular clones evolves over time. In response to treatment with Lenalidomide, it has also been observed a rapid evolution of clonal architecture that can be linked to patient response status. These results tend to confirm the genetic but also functional heterogeneity in MDS. We have identified new mutations involved in the pathogenesis of MDS. We observed an early clonal dominance in MDS because of the acquisition of all mutations in immature hematopoietic cells. However, different hematopoietic populations can have different genotype. Finally, the architecture of mutations could be modifying during the course of the disease

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 plasticité
Pediatric 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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Universidade Federal de Sao Carlos
The aims of this thesis were: to investigate the heterogeneity of soils and to evaluate the contribution of clonal growth to the population dynamics of woody species under contrasting soil conditions. In the Cerrado-Pantanal ecotone (15º43 S; 56º04 W), the soil possesses typical features from both the soil of Cerrado in the Central Plateau and the soil of the Pantanal Plain. Given the variability of soil and pluviosity, our hypothesis is that clonal growth is the demographic parameter that most contributes to the population growth rate () of five woody species in this ecotone area. This study was carried out in five Cerrado areas. We identified four soil categories; three of them typic Litoplinthic Petric Plintosol, typic Dystrophic Yellow Latosol and Dystrophic Yellow Latosol with plinthite had not yet been described for the study site. The chemical attributes aluminum saturation, magnesium, calcium, pH and manganese explained 38.7% of the variability of the soils in the study sites. The spatial distribution of the edaphic attributes was heterogeneous. These attributes differed between the soils in the Cerrado Pantanal ecotone and the soils of Cerrado in the Central Plateau and of the Pantanal Plain. The population dynamics of Curatella americana, a dominant species with wide geographical distribution, was evaluated in Petric Plintosol (P) and in Yellow Latosol (L), in the dry and rainy seasons. Sexual reproduction and seedling growth were higher in P, whereas clonal growth was higher in L. The population growth rate (λ) was higher in P. The adult survival rate exerted the strongest effect on λ for the two soil categories and the dry and rainy seasons. The interaction between soil category and pluviosity exerted the strongest effect on λ. We also analyzed the dynamics of species with small populations (Bowdichia virgilioides and Roupala montana) and large populations (Curatella americana and Caryocar brasiliense), to determine which demographic parameters characterize woody clonal species with different sizes. For the species with small populations, the rate of sexual reproduction and the density of all life stages were smaller, whereas the clonal growth was higher. Small populations were more susceptible to variations in soil and pluviosity. The rates that most contributed to the λ of Bowdichia virgilioides and Roupala montana in L and P were: respectively, the survival of young ramets and immatures, in the dry season; and for both species, the survival of adults, in the rainy season. For C. americana and C.brasiliense, the survival of adults was the parameter that most contributed to λ, independently of soil category and season. The study indicated an heterogeneity of the superficial layer of the soil and of the soil categories that occur in this area. Soils with contrasting attributes exerted an important effect on the dynamics of woody clonal species. The survival of young and immature individuals, originated from clonal growth, was the rate that most contributed to the λ of the small-sized populations of woody clonal species B. virgilioides and R. montana , whereas the survival of adults was the rate that most contributed to the λ of the large-sized populations C. americana and C. brasiliense.
Os objetivos desta tese foram: investigar a heterogeneidade de solos e verificar qual a contribuição do crescimento clonal para a dinâmica de populações de espécies lenhosas sob condições edáficas contrastantes. Em Cerrado, zona de ecótono com o Pantanal (15º43 S; 56º04 W), o solo é constituído por características inerentes tanto ao solo de Cerrado do Planalto Central como da Planície do Pantanal. Diante da variabilidade de características edáficas e da pluviosidade, a hipótese é que o crescimento clonal apresente-se como o parâmetro demográfico que mais contribua para a taxa de crescimento populacional () de cinco espécies lenhosas nesta zona de ecótono. O estudo foi realizado em cinco remanescentes de Cerrado. Identificou-se quatro classes de solo, sendo que três destas Plintossolo Pétrico Litoplíntico típico, Latossolo Amarelo distrófico típico e Latossolo Amarelo distrófico com plintita ainda não haviam sido descritas para a região em estudo. Saturação por alumínio, magnésio, cálcio, pH e manganês explicaram 38,7% da variabilidade de solos. Houve heterogeneidade na distribuição espacial dos atributos edáficos. Tais atributos diferiram entre os remanescentes e entre solos de Cerrado do Planalto e da Planície do Pantanal. A dinâmica populacional de Curatella americana, espécie dominante e de ampla distribuição geográfica, foi avaliada em Plintossolo Pétrico (FF) e em Latossolo Amarelo (LAd), nas estações seca e chuvosa. A reprodução sexuada e o crescimento de plântulas foram maiores em FF, enquanto o crescimento clonal foi maior em LAd. A taxa de sobrevivência de adultos exerceu maior efeito sobre para as duas classes de solo e estações seca e chuvosa. A interação entre classe de solo e pluviosidade exerceu maior efeito sobre Também foi avaliada a dinâmica das populações de Bowdichia virgilioides e Roupala montana, Curatella americana e Caryocar brasiliense, para determinar quais parâmetros demográficos caracterizam espécies lenhosas clonais com diferentes tamanhos populacionais. A taxa de reprodução sexuada e a densidade de todos os estádios foram menores, enquanto o crescimento clonal foi maior para pequenas populações de espécies lenhosas. Pequenas populações foram mais suscetíveis às variações de solo e de pluviosidade. As taxas que mais contribuíram para de B. virgilioides e R. montana em LAd e em FF foram a sobrevivência de rametas jovens; na estação seca foi a sobrevivência de rametas jovens e de imaturos para as respectivas espécies e na estação chuvosa foi a sobrevivência de adultos para ambas as espécies. Para C. americana e C.brasiliense, a sobrevivência de adultos foi o parâmetro que mais contribuiu para independente da classe de solo e da estação do ano. O estudo comprovou a heterogeneidade da camada superficial do solo e das classes de solo que constituem esta área. Solos de características contrastantes exercem importante efeito sobre a dinâmica de espécies lenhosas clonais. A sobrevivência de indivíduos jovens e de imaturos, provenientes do crescimento clonal, foi a taxa que mais contribuiu para de populações pequenas de lenhosas clonais B. virgilioides e R. montana - ao passo que a sobrevivência de adultos contribuiu para de populações grandes C. americana e C.brasiliense.

Tumour heterogeneity is a key hurdle for the effective treatment of cancer using oncolytic viruses (OVs). A better understanding of the pathways involved in delineating tumour cell resistance and hypersensitivity to OVs is critical in order to guide the development of new therapeutic strategies to enhance OVs. In this thesis, I performed a comparative genetic and epigenetic study of the murine OV-resistant colon cancer cell line CT26.WT and its hypersensitive subclone CT26.lacZ. This study led to the identification of retroviral insertion sites in AKAP7 and TP53RK genes, that are potentially involved in conveying sensitivity to infection by OVs and the dysregulation of the interferon antiviral response in the CT26.lacZ cell line. Gene overexpression and gene silencing experiments suggest a functional role of these proteins in controlling viral growth. Further investigation of these genes and their relationship to antiviral response pathways is warranted and may lead to novel strategies for improving the therapeutic activity of OVs.

This dissertation aims to identify the functional characteristics and genetic factors present within breast cancers that contribute to intratumoural heterogeneity and therapy resistance. The study utilises breast cancer cell line model systems to address epithelial-mesenchymal plasticity (EMP) at the cellular and functional level and underpins its role in cancer biology and chemoresistance. This research also interrogates the EMP programme in single cell-generated clones and through shRNA mediated functional drug screening assay identifies inhibitors that provides significant synergistic drug combinations. A comprehensive review of the drugs that can clinically target EMP was also consolidated.

Multiple myeloma (MM) is a largely incurable haematological malignancy characterised by the aberrant proliferation of malignant plasma cells (PCs) in the bone marrow (BM). Next generation sequencing (NGS) studies have shown that MM patients display complex mutational landscapes involving intraclonal genetic heterogeneity. While intraclonal heterogeneity is now an established feature of MM, the genomic changes and tumour evolution associated with the transformation from the asymptomatic disease stages of Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smouldering Multiple Myeloma (SMM) to MM remains unknown. This thesis presents a unique assessment of the genomic architecture and subclonal evolution associated with the natural history of disease transformation, with the analyses of a rare cohort of paired BM samples from patients when first diagnosed with MGUS or SMM, who later went on to develop MM (n = 10). Whole exome sequencing (WES) and bioinformatic analyses identified that clonal heterogeneity was present at the asymptomatic MGUS/SMM stages of disease, with a changing spectrum of acquired mutations associated with transition to MM. Subclonality was observed at MGUS/SMM, with the presence of between 5 to 11 subclones. The progression to MM was characterised by a prevailing model of subclonal evolution defined by clonal stability, where the transformed PC subclones of MM were already present at the MGUS/SMM stage. RNA sequencing (RNAseq) revealed that the patterns of expressed genes at MGUS/SMM to MM were found to be relatively homogeneous. Moreover, RNAseq revealed that mutant genes identified by WES were generally not expressed, expressed at low levels, with most genes showing wild-type expression. Analysis of the methylome was carried out using whole genome bisulphite sequencing (WGBS). Significant hypomethylation was observed in PCs recovered at all disease stages (MGUS, SMM and MM) compared to normal PCs. Interestingly, the degree of hypomethylation observed at MGUS was maintained with progression to SMM and MM stages. In addition, the phenomenon of RNA editing in SP140, a recurrently mutated gene in human MM patients, was investigated in the 5TGM1 MM PC line. A high impact C>T (ie. U) RNA editing change was identified in exon 2 of Sp140, resulting in an early STOP codon, which was hypothesised to result in the formation of truncated Sp140 protein that may contribute to MM pathogenesis. In mouse cell lines, Sp140 RNA editing was not restricted to the 5TGM1 cell line, but editing was not observed in any human MM PC lines. CRISPR-Cas9 mediated mutation of the mouse Apobec1 and Apobec3 genes, showed that neither of these cytidine deaminases were responsible for this RNA editing phenomenon. These studies show that MGUS/SMM patients, that progress in a short time frame, appear to be sufficiently genetically complex to be on the threshold of transformation to MM. Furthermore, the intrinsic genomic complexity of MM is present at the asymptomatic stages of disease (MGUS and SMM), suggesting that extrinsic factors from the tumour microenvironment play an important role in mediating progression. Indeed, these studies suggest that early intervention at MGUS/SMM may be possible to prevent progression and result in durable cure for patients.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2018

Clonal plants may be able to cope with spatial heterogeneity due to the physiological integration of ramets. Previous studies demonstrated that benefits of clonal integration increase with patch contrast between individual ramets. However, the same magnitude of contrast may be perceived differently in rich and poor environments. According to the theoretical work of Caraco and Kelly (1991), I expected these benefits to be the greatest in overall poor conditions and high between-patch contrast. To test this hypothesis, I conducted experiments with pairs of ramets of a stoloniferous grass, Agrostis stolonifera, grown in variously nutrient rich conditions. The experiment with pairs of ramet of similar developmental age showed only very weak effect of integration on growth of ramets, although integration significantly improved survival of ramets and also affected root-shoot ratio of ramets. Nevertheless, there were considerable benefits of integration in the experiment with developmentally older mother ramets and their daughter ramets. Contrary to the predictions, the benefits of integration were bigger in rich conditions and they decreased with increasing between-patch contrast. In addition, effect of integration on root-shoot ratio of ramets was opposite to the expected specialization for acquisition...