Дисертації: "Liquide biopsy"

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Abbou, Samuel. "Liquid Biopsy in Pediatric Sarcoma." Thesis, université Paris-Saclay, 2022. http://www.theses.fr/2022UPASL037.

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Résumé : Les biopsies liquides ouvrent de nouveaux champs d'applications dans la prise en charge des patients au diagnostic, au cours de leur suivi et également en recherche translationnelle. Ces dernières années, de nombreux efforts ont été consacrés au développement de l'ADN tumoral circulant (ADNct) et des cellules tumorales circulantes (CTC). Il y a malgré tout de nombreux terrains encore en friches dans les cancers de l'enfant, et parmi eux dans les sarcomes pédiatriques.Nous avons souhaité explorer dans ce travail différents aspects des applications cliniques éventuelles et d'utilisation de ces technologies pour la compréhension de la biologie des tumeurs. La première partie de ce projet est une revue de la littérature qui se rapporte à l'application de l'ADNct dans les cancers solides pédiatriques. Nous présentons ensuite un travail de recherche qui vise à utiliser les CTC à visée diagnostique dans les sarcomes à translocation. Cette étude présente une approche permettant d'identifier des fusions pathognomoniques de sarcome à partir de très faibles quantités de tissu fixé, de CTC sur des modèles murins ou chez des patients. La deuxième étude présentée s'intéresse à la détection de l'ADNct au diagnostic de rhabdomyosarcome en utilisant les altérations de nombre de copie de chromosome, les réarrangements chromosomiques et les variants de nucléotide. Nous avons démontré que la détection au diagnostic est faisable et a un impact pronostique fort sur le devenir des patients. La dernière partie de ce manuscrit présente le développement d'un processus de traitement d'échantillons de patient pour détecter et isoler des cellules tumorales circulantes dans le but d'analyser les particularités génomiques de cette population à une résolution cellulaire.Ce travail explore certains aspects de l'utilisation de la biopsie liquide dans les sarcomes pédiatriques, parmi de nombreux autres. Il est crucial dans le développement de ce champ de recherche, de maîtriser les particularités intrinsèques de chaque type tumoral et des technologies disponibles. Nous avons démontré l'utilité d'une telle approche au diagnostic dans deux applications. Cette aire de recherche ouvre de nombreuses possibilités qui appellent à poursuivre les efforts afin d'élargir les applications en recherche et en clinique
Abstract: Liquid biopsy is an opportunity for improved diagnosis, treatment monitoring and genomic studies in oncology. Substantial effort in recent years has focused on circulating tumor DNA (ctDNA) and circulating tumor cells (CTC). However, pediatric cancer, including sarcomas, are still largely unexplored disease areas in this field.In this work, we sought to explore several aspects of liquid biopsy applied to pediatric sarcomas including their clinical use at diagnosis and as a tool to understand tumor biology. We first present a review of the literature demonstrating the feasibility of applying liquid biopsy to pediatric solid malignancies. Then, we report a methodological study using CTC for diagnostic purposes in translocation driven sarcomas. This approach identified fusions from as little as two unstained slides of FFPE tumor biopsy tissue, from CTC collected from tumor-bearing mice, and from liquid biopsy samples from patients with known fusion-positive cancers. The second study focuses on ctDNA for prognostication at the time of diagnosis in rhabdomyosarcoma by detecting copy number alterations, rearrangements, and single-nucleotide variants. Our study demonstrates that baseline ctDNA detection is feasible and has prognostic value. The last part of this work presents the development of a workflow to isolate single sarcoma cancer cells for sequencing, with an ultimate goal to analyze CTC genomic features at a single-cell resolution.This work explores several clinically and scientifically relevant aspects of liquid biopsy in pediatric sarcoma. We showed that liquid biopsy has utility at diagnosis in two different applications. Further development in this field will require a strong knowledge of tumor-specific biology, the clinical care of patients with these diseases, and the adaption of new technologies. My findings demonstrate the transformative possibilities this research may bring to the care of patients with pediatric sarcomas

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Cayron, Helene. "Sélection et capture de biomarqueurs moléculaires et cellulaires à partir d'un fluide complexe." Thesis, Toulouse, INSA, 2016. http://www.theses.fr/2016ISAT0001.

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Ce travail de thèse s'est axé autour de deux approches technologiques issues du domaine de la microfabrication pour la sélection et la capture de biomarqueurs circulants dans le sang. A l'échelle moléculaire, un module d'assemblage capillaire dirigé a été implémenté dans un automate de tamponnage moléculaire puis validé en utilisant un modèle simple, permettant l'isolement et l'étirement de biomolécules individuelles de manière entièrement contrôlée et automatisée à large échelle. Nous avons ensuite appliqué cette technologie à des biomarqueurs moléculaires d'intérêt tels que les ADN libres contenus dans du sang complet, démontrant la capacité de la technique à isoler des acides nucléiques dans un fluide complexe contenant de nombreux éléments cellulaires . A l'échelle cellulaire, une approche innovante pour la sélection et la capture de Cellules Tumorales Circulantes a été développée. Le microdispositif mis au point est fabriqué par écriture laser à 3 dimensions et permet le piégeage physique de ces cellules dans du sang complet non traité tout en les préservant pour une récupération et analyse ultérieure. Après adaptation du microdispositif pour maximiser son efficacité de capture in vitro, une première preuve de concept de capture sélective de cellules cancéreuses dans du sang complet non traité a été réalisée. U n premier prototype pour une utilisation in vivo a été mi s au point et validé in vitro sur la capture de cellules cancéreuses dans du milieu de culture
This research project focused on two technological approaches emerging from microfabrication for the selection and capture of circulating biomarkers from blood. At the molecular scale, this work was based on the automation of a directed capillary assembly protocol. A dedicated module was implemented into an automate for molecular stampin g and validated using a simple molecular model, allowing the elongation and large-scale assembly of single biomolecules in a controlled and automatized manner. The developed technology was then used for the assembly of relevant molecular biomarkers such as cell -free DNA (cf DNA) from untreated whole blood , evidencing the capabilities of this technology to single out nucleic acids from complex fluids composed of other cellular elements. At the cellular scale, an innovative concept for Circulating Tumor Cell s (CTCs) selection and capture was developed . The developed microdevice is fabricated using 30 direct laser writing and allows for a physical capture of cell s from untreated whole blood while preserving them for further recovery and analysis. After having optimized the design in vitro to maximize the capture efficiency of the system, a selective capture of cancer cell s from untreated whole blood was achieved . A first prototype for the in vivo use of this system was also developed and validated in vitro with cancer cells spiked into culture medium

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Jimenez, Zenteno Alejandro Kayum. "Micro-dispositifs pour l'isolement des cellules tumorales circulantes en routine clinique." Thesis, Toulouse 3, 2018. http://www.theses.fr/2018TOU30154.

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Les cellules tumorales circulantes (CTCs) sont la principale voie de dissémination du cancer dans le corps humain au travers de la circulation sanguine. Ces cellules ont la capacité de se détacher de la tumeur primaire, de rejoindre la circulation sanguine et de survivre dans cet environnement. Une sous-population spécifique de ces cellules a la capacité de coloniser de nouveaux tissus et de former des métastases. L'importance de ces cellules rares dans la circulation sanguine a été intensément étudiée au cours des dernières décennies, et il a été constaté que les informations phénotypiques et génomiques qu'elles contiennent pourraient être corrélées avec celles obtenues à partir d'une biopsie tissulaire. De plus, le nombre et l'incidence des CTC chez les patients métastatiques pourraient être utilisés comme indicateurs pronostics. Ainsi, leur isolement à partir d'échantillons sanguins et leur analyse a été proposé en remplacement des biopsies conventionnelles, comme une alternative moins invasive et permettant un échantillonnage plus répété. In fine, la détection et l'analyse des CTC en routine clinique pourraient être utilisées pour le suivi en temps réel des thérapies et de leur efficacité pour améliorer la prise en charge des patients, un pas de plus vers une médecine de précision. Dans ce projet de thèse, nous avons développé de nouveaux micro-dispositifs pour la capture, sous flux, de cellules cancéreuses à partir de sang complet humain. Nous avons exploité les propriétés physiques des CTC, plus grandes et moins déformables que les cellules sanguines normales, pour discriminer ces cellules rares (<1 cellule par mL aux premiers stades de la maladie). Des micro-dispositifs ont été conçus tels des tamis à trois dimensions pour filtrer sélectivement les cellules cancéreuses tout en préservant l'intégrité et la viabilité des cellules. De plus, les dispositifs ont été conçus pour permettre l'accès au matériel biologique isolé et effectuer ainsi une identification des cellules in situ, e.g. par immunocytochimie, mais aussi potentiellement pour servir de plateforme pour une analyse fonctionnelle de ces cellules. Nous avons proposé deux approches totalement compatibles avec la routine clinique. La première consiste en un guide équipé de microdispositifs, conçu pour être introduit directement dans la circulation sanguine au travers d'un cathéter médical et effectuer la capture des cellules cancéreuses in vivo. La deuxième approche vise à réaliser l'isolement des CTCs en utilisant des microdispositifs intégrés à des plateformes ex vivo compatibles avec les consommables médicaux de prélèvement sanguin.[...]
Circulating tumor cells (CTCs) are believed to represent the main pathway of cancer dissemination in the human body through the circulatory system. These cells have the ability to detach from the primary tumor, enter into the bloodstream, and survive in this environment. A specific subpopulation of these cells possesses the capacity of colonizing new tissues and forming metastases. The relevance of these rare cells in the bloodstream has been intensively investigated during the last decades, finding that phenotypic and genomic information they carry could be correlated with that of solid biopsies. Moreover, the number and incidence of CTCs in metastatic patients could be used as an indicator for prognosis. Thus, their isolation from blood samples and analysis has been proposed as a surrogate to solid biopsies, having the added value of being a less invasive procedure and allow a more repeated measure. In fine, the routine analysis of CTCs in clinical practice could be used for the real-time monitoring of therapies and the adaptation of treatment in order to improve the outcome of patients, a step forward towards so-called precision medicine. In this PhD project, we have developed novel micro- devices for the capture, in flow conditions, of tumor-derived cells from human whole blood. CTCs being larger and less deformable than normal blood cells, we exploited theses physical traits to discriminate them. Sieve-like micro-devices were engineered to selectively sort out tumor-derived cells having as a priority the preservation of cell integrity and viability. In addition, devices were designed to allow direct access to the isolated biological material and thus perform in situ cell identification, such as immunocytochemistry, but also to potentially serve as a platform for functional analysis. We proposed two approaches compatible with clinical routine. The first approach consists in a customized guiding-strip equipped with integrated microfilters, designed to be introduced directly within the bloodstream through a conventional medical catheter to perform the capture of tumor-derived cells in vivo. The second approach aims to perform CTC isolation ex vivo through the integration of microfilters into a platform compatible with blood collection medical sets. [...]

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Heeke, Simon. "Développement et implémentation de nouveaux biomarqueurs prédictifs dans le cancer du poumon non à petites cellules - du tissu à la biopsie liquide." Electronic Thesis or Diss., Université Côte d'Azur (ComUE), 2019. http://www.theses.fr/2019AZUR6015.

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Le cancer du poumon est la principale cause de décès liés au cancer dans le monde, tant chez les hommes que chez les femmes. Cependant, le traitement du cancer du poumon a radicalement changé au cours de ces dernières années avec la mise en place de chimiothérapies plus efficaces, mais surtout le développement de traitements ciblés qui permettent une approche thérapeutique personnalisée et avec l'introduction de l'immunothérapie qui a considérablement prolongé la survie de certains patients atteints d’un cancer du poumon non à petites cellules (CPNPC). Bien que ces nouvelles approches thérapeutiques aient permis d'obtenir des réponses parfois spectaculaires, un nombre assez important de patients sont réfractaires à ces traitements. Dans ce contexte, le développement de nouveaux biomarqueurs permettant de sélectionner le meilleur traitement pour le bon patient et au bon moment est crucial pour améliorer les résultats cliniques des patients atteints de CPNPC. Tous les biomarqueurs actuellement à l’étude ne sont pas en mesure d'améliorer cette prédiction, en particulier, la mise en place de certains biomarqueurs en routine clinique est souvent difficile, alors que les résultats préliminaires obtenus in vitro ou même dans les essais cliniques initiaux étaient prometteurs. L'objectif de la thèse a été d’évaluer et d’implémenter de nouveaux biomarqueurs prédictifs de la réponse à l’immunothérapie et aux thérapies ciblées pour la sélection thérapeutique des patients atteints de CPNPC. Dans la première partie de la thèse, est abordé l’importance des biobanques et de la maitrise des ressources biologiques comme pierre angulaire du développement de ces nouveaux biomarqueurs. Nous avons mis en place un mode de fonctionnement qui permet d'entreposer en toute sécurité des collections biologiques d'intérêt et de les utiliser pour les études de recherche de biomarqueurs. Nous décrivons comment une biobanque dédiée à une seule pathologie peut être instaurée et utilisée à des fins de recherche.Au cours de cette thèse est abordée l'évaluation génomique de l'ADN libre plasmatique (cell-free DNA : cfDNA) pour la détection des mutations spécifiques du récepteur du facteur de croissance épidermique (Epidermal growth Factor Receptor : EGFR) est étudiée et évaluée. Nous avons analysé rétrospectivement 324 patients sur une période de trois ans à partir de trois tests biologiques utilisés en routine clinique et nous avons pu démontrer que ces tests sont très « robustes » mais doivent être étroitement contrôlés afin d’éviter de faux résultats positifs ou négatifs. Nous avons ensuite évalué le séquençage à haut débit de l’ADN plasmatique chez ces patients à l'aide d'un test interne développé au laboratoire et d'un test externe et nous avons pu démontrer que ces deux tests étaient fiables pour la détection des altérations génomiques du plasma en routine clinique. Dans la dernière partie de la thèse, je décris comment l'évaluation de grands panels de séquençage ciblés capables d'évaluer la charge tumorale mutationnelle peut être utilisée pour sélectionner les patients pouvant bénéficier d’une immunothérapie anti-tumorale et quels pièges doivent être évités afin d’utiliser ce biomarqueur en routine clinique.En résumé, cette thèse montre la place croissante des nouveaux biomarqueurs permettant la stratification des patients atteints CPNPC pour adapter rapidement leur traitement et décrit les différentes étapes de l’implémentation de ces biomarqueurs tissulaires et circulants dans les soins cliniques courants
Lung cancer is the leading cause of cancer-related deaths worldwide for both men and women. However, the treatment of lung cancer has changed radically in recent years with the introduction of more effective chemotherapies, but above all the development of targeted treatments that allow a personalized therapeutic approach and the introduction of immunotherapy that has considerably prolonged the survival of some patients with non-small cell lung cancer (NSCLC). Although these new therapeutic approaches have made it possible to obtain sometimes spectacular responses, a fairly large number of patients are resistant to these treatments. In this context, the development of new biomarkers to select the best treatment for the right patient at the right time is crucial to improving clinical outcomes for NSCLC patients. Nevertheless, not all biomarkers currently under study are able to improve this prediction, in particular, the implementation of some biomarkers in clinical routine is often difficult, whereas preliminary results obtained in vitro or even in initial clinical trials were promising.The objective of the thesis was to evaluate and implement new biomarkers that predict the response to immunotherapy and targeted therapies for the therapeutic selection of NSCLC patients. The first part of the thesis discusses the importance of biobanks and the control of biological resources as a cornerstone for the development of these new biomarkers. We have implemented an operating procedure that allows us to safely store biological collections of interest and use them for biomarker research studies. We describe how a biobank dedicated to a single pathology can be established and used for research purposes.Additionally, the genomic evaluation of cell-free DNA (cfDNA) for the detection of specific mutations of the Epidermal growth Factor Receptor receptor (EGFR) is studied and evaluated. We retrospectively analyzed 324 patients over a three-year period from three biological tests used in routine clinical practice and were able to demonstrate that these tests are very robust but must be closely controlled to avoid false positive or negative results. We then evaluated the next-generation sequencing (NGS) of plasma DNA using an internal test developed in the laboratory and an external test and were able to demonstrate that both tests were reliable for the detection of genomic alterations in plasma in clinical routine. In the last part of the thesis, I describe how the evaluation of large targeted sequencing panels capable of assessing mutation tumor load can be used to select patients for anti-tumor immunotherapy and what pitfalls should be avoided in order to use this biomarker in clinical routine.In summary, this thesis demonstrates the importance of novel biomarkers for the stratification ofpatients undergoing therapy in NSCLC and contributed to the implementation of tissue and liquidbiopsy-based biomarkers in routine clinical care

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Silva, Luciana Sanches. "Pesquisa de células tumorais circulantes em pacientes com câncer de próstata por método de filtração celular." Botucatu, 2018. http://hdl.handle.net/11449/155896.

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Orientador: Adriana Polachini Valle
Resumo: Introdução: O câncer de próstata (CP) é o mais incidente entre os homens em todas as regiões do Brasil. A detecção e caracterização de células tumorais circulantes (CTCs) tem sido apontada como uma alternativa para melhor compreensão da biologia dos tumores, incluindo câncer de próstata. Objetivo: Este estudo tem como objetivo avaliar a detecção de CTCs em pacientes com tumor de próstata localizado e metastático por teste rápido de filtração celular. Metodologia: Foram incluídos pacientes com diagnóstico anatomopatológico de câncer de próstata ou neoplasia intraepitelial prostática. Os dados demográficos, laudos anatomopatológicos e de Cintilografia Óssea e valores do antígeno prostático especifico ( PSA) foram obtidos pelo estudo dos prontuários médicos dos pacientes. Os pacientes foram classificados como portadores de tumor metastático quando apresentavam evidência de imagem metastática pela Cintilografia Óssea. As CTS foram isoladas por teste rápido de filtração celular com posterior imunocitoquímica utilizando-se anticorpos monoclonais anti-PSA para caracterização câncer de próstata específica das células. Resultados: As CTCs foram detectadas em 9 dos 21 pacientes (43%) com positividade de 60% no grupo metastático e 36% no grupo de tumor localizado. Não foram observadas associações entre os valores de PSA e tratamento instituído com a detecção de CTCS. Discussão: A positividade das CTCs no presente estudo mostrou-se semelhante aos dados da literatura, embora possam ser ci... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Introduction: Prostate cancer (PC) is the most frequent among men in all regions of Brazil. The detection and characterization of circulating tumor cells (CTCs) has been pointed out as an alternative for a better understanding of the biology of tumors, including prostate cancer. Objective: This study aims to evaluate the detection of CTCs in patients with localized and metastatic prostate tumor by rapid cell filtration test. Methodology: Patients with anatomopathological diagnosis of prostate cancer or prostatic intraepithelial neoplasia were included. Demographic data, anatomopathological and bone scintigraphy reports and prostate specific antigen (PSA) values were obtained by the study of patients' medical records. Patients were classified as having metastatic tumor when they presented evidence of metastatic image by Bone Scintigraphy. The CTS were isolated by rapid cell filtration test with subsequent immunocytochemistry using anti-PSA monoclonal antibodies for cell-specific prostate cancer characterization. Results: CTCs were detected in 9 of the 21 patients (43%) with 60% positivity in the metastatic group and 36% in the localized tumor group. No associations were observed between PSA values and treatment established with CTCS detection. Discussion: The positivity of the CTCs in the present study was similar to the data in the literature, although some limitations of the study may be cited, such as a small number of patients included, difficulties encountered by research... (Complete abstract click electronic access below)
Mestre

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Buscail, Etienne. "Intérêt diagnostique de la biopsie liquide dans la prise en charge de l'adénocarcinome canalaire du pancréas à un stade précoce." Thesis, Bordeaux, 2019. http://www.theses.fr/2019BORD0081.

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Introduction:Un des problèmes du cancer du Pancréas (CP) est le temps de latence entre la suspicion du CP et la mise en place des traitements. Les méthodes de biopsie liquide pourraient accélérer la mise en évidence d’éléments tumoraux et le diagnostic.Objectif :L’objectif principal de l’étude était de comparer la performance diagnostique de plusieurs techniques de biopsie liquide chez des patients atteint d’un CP résécable d’emblé. L’objectif secondaire était la corrélation avec le taux de récidive post-opératoire.Méthodes:Tout d'abord, nous avons testé 2 méthodes d'enrichissement CTC pour estimer la sensibilité de la détection CTC avec des expériences de cell-spiking de deux lignées de cellules tumorales pancréatiques dans des échantillons de sang de 24 volontaires sains en utilisant la méthode en gradient de densité OncoQuick® et la méthode de sélection négative RosetteSep™. De plus, les mutations KRAS ont été quantifiées dans l'ADN génomique de cellules purifiées par digital droplet PCR (dd-PCR) avec des amorces spécifiques des allèles.Nous avons conçu un essai clinique prospectif (NCT03032913) visant à détecter les cellules tumorales circulantes (CTC), l’ADN tumoral circulant (ADNct) et les onco-exosomes chez les patients atteint de CP et chez les patients d’un groupe témoin. Pour les CTCs : enrichissement et détection de CTCs par la méthode CellSearch©, méthode d’enrichissement de CTCs RosetteSep® et OncoQuick® puis quantification de l’ADN tumoral par dd-PCR. Les exosomes ont été isolés puis caractérisés avec le taux d’expression de Glypican-1. Tous les patients de l’étude ont eu un prélèvement de sang périphérique, les patients du groupe CP ont eu un prélèvement de sang portal peropératoire.Résultats:La sensibilité analytique était de 100 % pour OncoQuick®, quelle que soit la lignée cellulaire, et se situait entre 70 et 100 % pour RosetteSep™. Le taux moyen de récupération des cellules était de 56±23% pour OncoQuick® contre 39±27% pour RosetteSep™ (p<0,001). Les cellules tumorales de la population de cellules sanguines enrichies ont été détectées par dd-PCR après enrichissement par RosetteSep™ et OncoQuick® La détection des allèles K-RAS mutants par ddPCR après enrichissement de RosetteSepTM était 3 à 4 fois plus sensible qu'après OncoQuick®. Ainsi, RosetteSep™ est plus fiable en termes d'efficacité de récupération et de détection des mutants KRAS que OncoQuick®.De février à novembre 2017, 22 patients atteints de CP résécable et 28 patients témoins ont été inclus. Tous les patients ont été détectés positifs par au moins une méthode. Les CTCs ont été détectées chez 9 patients avec la méthode cellsearch (70% dans le sang portal exclusif) et 13 avec la méthode Rosettesep (60%). Les onco-exosomes ont été détecté chez 14 patients sur 22. L’ADNct n’a été détecté que chez deux patients métastatiques. La détection combinée des CTCs et des onco-exosomes était significativement corrélée à la survie sans récidive.Conclusion:Cette étude suggère que la biopsie liquide combinée peut être un outil prometteur à fois diagnostique et pronostique dans le CP à un stade précoce
Introduction:One of the problems of pancreatic ductal adenocarcinoma (PC) is the latency time between the suspicion of PC and the initiation of treatments, especially neo-adjuvants that require histological evidence. Liquid biopsy methods could be a companion test for diagnosis.Objective :The main objective of the study was to compare the diagnostic performance of several liquid biopsy techniques in patients with resectable pancreatic without neo-adjuvant therapy cancer. The secondary objective was the correlation between the quantification of liquid biopsy parameters and clinic-pathologic features.Methods:First, we tested 2 CTC enrichment methods to estimate the sensitivity of CTC detection with cell spiking experiments of two pancreatic tumour cell lines in blood samples from 24 healthy volunteers using the onco-specific density gradient OncoQuick® and the negative selection enrichment method RosetteSep™. Additionally, KRAS mutations were quantified in genomic DNA of purified cells by digital droplet Q-PCR (dd-PCR) with allele specific primers.We designed a prospective clinical trial (PANC-CTC# NCT03032913) to detect circulating tumour cells (CTC), circulating tumour DNA (ADNct) and onco-exosomes in patients with pancreatic cancer and in patients in a control group using different methods. For CTCs, it was the enrichment and detection of CTCs by the CellSearch© method (reference method), the RosetteSep® and OncoQuick® CTC enrichment method and the quantification of tumor DNA by dd-PCR. Exosomes were isolated and characterized with the expression rate of Glypican-1. All patients in the study had a peripheral blood sample, patients in the PDAC group had a portal blood sample during surgery.Results:Analytical sensitivity was 100% for OncoQuick®, regardless of the cell line, and ranged between 70 and 100% for RosetteSep™. Mean recovery rate of cells was 56±23% for OncoQuick® versus 39±27% for RosetteSep™ (p<0.001). Molecular detection of mutant K-RAS alleles by ddPCR after RosetteSepTM enrichment was 3- to 4-fold more sensitive than after OncoQuick®. Thus, RosetteSep™ is more reliable in terms of recovery efficiency and KRAS mutant detection than OncoQuick®.From February to November 2017, 22 patients with resectable pancreatic cancer and 28 control patients were included. All patients were positive by at least one method. CTCs were detected in 9 patients with the cellsearch method (70% in the exclusive portal blood) and 13 with the Rosettesep method (59%), Onco-exosomes were detected in 14 out of 22(64%) patients in peripheral and/or portal blood. DNAct was detected in only two metastatic patients. The combined detection of CTCs with cellsearch and onco-exosomes was significantly correlated with progression free survival and overall survival when CTC cluster were found.Conclusion: This study suggests that combined liquid biopsy can be a promising tool for both diagnosis and prognosis in early pancreatic cancer

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Sanz, García Enrique. "Análisis de RAS en plasma en cáncer colorrectal metastásico: impacto de la fracción mutante alélica en pronóstico." Doctoral thesis, Universitat Autònoma de Barcelona, 2018. http://hdl.handle.net/10803/666000.

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Introducción: A pesar de los importantes avances en el tratamiento del cáncer de colon metastásico (mCRC), la supervivencia sigue siendo corta. Existen diversos factores pronósticos y predictivos que se han de tener en cuenta, entre ellos la mutación del oncogén RAS presente en más del 40% de tumores. Esta mutación suele ser determinada en tejido sólido pero dada la imposibilidad en ocasiones de obtener suficiente muestra y la heterogeneidad tumoral, la determinación de dicha mutación en el DNA circulante en sangre (biopsia líquida) puede ser realizada por ejemplo mediante BEAMing (basada en la PCR digital). La hipótesis principal de este estudio es que la determinación cuantitativa de esta mutación (fracción mutante alélica –MAF-) podría ser un factor pronóstico o predictor en mCRC. Material y métodos: Se trata de un estudio retrospectivo que incluye 110 pacientes procedentes de dos centros. Se han recogido las principales variables clínicas, patológicas y de supervivencia de estos pacientes así como la determinación de RAS en tejido sólido por la técnica habitual. La determinación de la MAF en plasma se ha realizado mediante la técnica del BEAMing y se ha establecido la correlación con la mutación en tejido sólido mediante PCR en tiempo real. Se ha analizado el impacto pronóstico en supervivencia global (OS) y libre de progresión (PFS) de la MAF de RAS en una población homogénea tratada de forma similar, así como correlación con distintas variables. Resultados: En el total de la población de estudio, se han detectado 62 pacientes (56.4%) que presentaban en plasma alguna mutación de RAS mediante BEAMing. La concordancia entre la determinación mediante PCR en tejido sólido y el BEAMing en plasma fue del 90% con un índice estimado Kappa de Cohen 0.80 (IC 95% 0.68-0.91). No se han objetivado diferencias estadísticamente significativas en OS entre la población RAS mutada y no mutada en tejido sólido y en plasma. Con el fin de homogeneizar la población para el análisis pronóstico de la MAF de RAS se han seleccionado un total de 42 pacientes que no habían recibido cirugía de lesiones metastásicas. No se ha observado correlación estadísticamente significativa con la mayoría de las variables clínicas analizadas salvo localización de metástasis. El análisis de la MAF de RAS basal previo al tratamiento de primera línea reveló una correlación significativa con OS (HR = 3.514; p = 0.00066), siendo los pacientes con menores niveles de MAF de RAS los que tienen una OS mayor. Asimismo, los pacientes con menores niveles de MAF presentaron una mayor PFS aunque no fue estadísticamente significativa dicha tendencia. En el análisis multivariante en primera línea la MAF fue un factor pronóstico independiente en OS (HR = 2.73; p = 0.006) y PFS (HR = 3.74; p = 0.049). Asimismo, la MAF fue más alta en aquellos pacientes con progresión de enfermedad como mejor respuesta (p= 0.007). Conclusiones: La MAF en plasma de RAS puede ser un factor pronóstico en los pacientes con mCRC RAS mutados pudiendo ayudar al clínico a tomar decisiones sobre el tratamiento de dicha enfermedad. No obstante, dada la naturaleza del estudio, es necesario estudios más amplios y prospectivos que puedan validar el uso de esta técnica en la práctica clínica.
Introduction: Despite recent major advances in metastatic colorrectal cancer (mCRC), survival is still poor. There are different prognostic and predictive factors to be taken into account, among them, RAS mutation which is observed in 40% of all tumors. This mutation is determined in solid biopsy but sometimes, as it is not possible to get enough sample for this determination and due to tumor heterogeneity, this mutational status can be analyzed from circulating DNA in blood (liquid biopsy) using BEAMing for instance (a digital PCR-based technology). The main hypothesis of this study is to analyze whether quantitative determination of this mutation (mutant allele fraction-MAF-) could be a prognostic or predictive factor for RAS mutant mCRC. Material and Methods: This is a retrospective study comprising a total of 110 patients from two different sites. Main clinical, pathological and survival data have been recorded as well as determination of RAS mutational status in solid biopsy using routine techniques. MAF determination in plasma has been determined using BEAMing and correlation with mutational status in solid tissue using real time PCR has been analyzed. Prognosis impact in overall survival (OS) and progression free survival (PFS) of RAS MAF in a homogenous cohort has been analyzed as well its correlation with different variables. Results: In the whole population, RAS mutation in plasma has been detected with BEAMing in a total of 62 patients (56.4%). Concordance between real time PCR in solid biopsy and BEAMing in plasma is 90% with an estimated Cohen Kappa index of 0.80 (95% CI 0.68-0.91). No statistical significant differences in OS have been detected between RAS mutant and wild type in solid and liquid biopsy. In order to make population homogenous regarding prognosis impact of RAS MAF, a total of 42 patients who have not been operated for metastatic disease have been selected. There are not statistical significant correlations with the most part of the clinical variables except for metastases location. RAS MAF prior to first line therapy shows a significant correlation with OS (HR = 3.514; p = 0.00066), as RAS MAF is lower in patients with longer OS. Moreover, patients with lower MAF show a trend to longer PFS that is not statistically significant. In the multi-variant analysis, RAS MAF is an independent prognosis factor for OS (HR = 2.73; p = 0.006) and PFS (HR = 3.74; p = 0.049). Moreover, patients with higher MAF tend to have progressive disease as best response to treatment (p = 0.007). Conclusions: RAS MAF in plasma could be an independent prognosis factor in patients with RAS mutant mCRC and may help clinicians to make decisions about management of this disease. However, due to the characteristics of this study, prospective studies are needed to validate this technique for the use in the daily practice.

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Yap, Soo Ann [Verfasser]. "Extracellular vesicles as cancer liquid biopsy biomarker / Soo Ann Yap." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2021. http://d-nb.info/1234982889/34.

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Bracht-Loman, Jillian Wilhelmina Paulina. "Validation of liquid biopsy-based analysis on the NanoString nCounter platform." Doctoral thesis, Universitat Autònoma de Barcelona, 2021. http://hdl.handle.net/10803/672549.

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L'avaluació dels marcadors moleculars en teixit tumoral per predir el pronòstic del càncer i la resposta al tractament, també coneguda com a tractament personalitzat, ha transformat la pràctica clínica per a molts tipus de càncer. Es va descobrir que aquesta teràpia dirigida per genotipar, millora la supervivència del pacient i per tant s'han introduït diverses plataformes tècniques en els laboratoris clínics. No obstant això, no tots els tumors es poden biopsiar i, sovint, les quantitats de teixit són insuficients per a la caracterització del tumor. L'ARN, l'ADN i les proteïnes lliures circulants de biòpsies líquides, es poden extreure dels fluids corporals i poden reemplaçar o complementar les biòpsies de teixit. Les biòpsies líquides tenen diversos avantatges: la possibilitat de realització d’ estudis de serie o mínimament invasiva i permet analitzar l'heterogeneïtat tumoral. Malauradament, encara hi ha una gran bretxa entre la recerca bàsica i la implementació clínica de biòpsies líquides, principalment a causa de la manca de metodologies estandarditzades. A més, les plataformes tècniques que s'utilitzen actualment, no sempre són adequades per analitzar la baixa quantitat i qualitat de material del tumor derivat d'una biòpsia líquida. En conseqüència, la validació i implementació dels assajos de biomarcadors en biòpsies líquides en els laboratoris clínics, requereixen una plataforma tècnica estandarditzada que sigui sensible, ràpida, fàcil d'utilitzar, relativamente econòmica, flexible i amb poca quantitat de mostra. La plataforma nCounter es pot utilitzar per analitzar tota classe de molècules, inclosos ARN, ADN i proteïnes. La hibridació de codis de barres codificats per colors pels objectius d'interès permet una lectura directa dels nivells d'expressió de gens i proteïnes o la detecció de mutacions. El desenvolupament d'assaigs de biomarcadors en teixits, usant el nCounter, va conduir a l'aprovació per la FDA de l'assaig Prosigna™ per a ús clínic en la tipificació del càncer de mama. Els esforços anteriors també han destacat el potencial d'aquesta plataforma per analitzar molècules derivades i amplificades de biòpsies líquides, encara que es necessiten estudis de validació en l'entorn clínic. En aquesta tesi validem l'ús de la plataforma NanoString nCounter per analitzar material de biòpsies líquides i desenvolupar assajos de biomarcadors clínicament rellevants.
La evaluación de los marcadores moleculares en tejido tumoral para el pronóstico del cáncer y la predicción de respuesta al tratamiento (lo que habitualmente se conoce como tratamiento personalizado) ha transformado la práctica clínica a la hora de tratar muchos tipos de cáncer. Son numerosos los trabajos que desde hace tiempo respaldan el efecto que esta terapia dirigida por genotipo tiene sobre los pacientes oncológicos mejorando la supervivencia del paciente; consecuentemente, un amplio rango de plataformas técnicas han sido implementadas en los laboratorios clínicos en los últimos años. Sin embargo, no todos los tumores se pueden biopsiar y, a menudo, las cantidades de tejido son insuficientes para la caracterización del tumor. Las biopsias líquidas, como el ARN, el ADN o las proteínas circulantes tanto libres como encapsuladas en una membrana, pueden extraerse de los fluidos corporales reemplazando o complementando de este modo las tradicionales biopsias de tejido. Las biopsias líquidas tienen varias ventajas: ofrecen la posibilidad de realizar estudios seriados, son mínimamente invasivas y permiten analizar la heterogeneidad tumoral. Desafortunadamente, todavía existe una gran brecha entre la investigación básica y la implementación clínica de las biopsias líquidas, principalmente debido a la falta de metodologías estandarizadas. Además, las plataformas técnicas que se utilizan actualmente no siempre son adecuadas para analizar la baja cantidad y calidad de material del tumor procedente de una biopsia líquida. En consecuencia, la validación e implementación de los ensayos de biomarcadores en biopsias líquidas en los laboratorios clínicos requieren una plataforma técnica estandarizada que sea sensible, rápida, fácil de usar, viable económicamente, flexible y que requiera un aporte inicial de ácidos nucleicos bajo, debido a la baja concentración que normalmente se obtiene en las biopsias líquidas. La plataforma nCounter se puede utilizar para analizar todo tipo de moléculas, incluyendo ARN, ADN y proteínas. La hibridación de diferentes códigos formados por moléculas de colores siguiendo patrones específicos con secuencias de interés permite una lectura directa de los niveles de expresión de genes y proteínas o la detección de mutaciones. El desarrollo de ensayos de biomarcadores en tejidos usando nCounter condujo a la aprobación por la administración de fármacos y alimentos de los Estados Unidos (FDA) del ensayo Prosigna ™ para su uso clínico en la tipificación del cáncer de mama. Numerosos estudios han destacado el potencial de esta plataforma para analizar moléculas derivadas y amplificadas de biopsias líquidas, aunque estudios de validación en el entorno clínico aun son necesarios. El objeto de esta tesis es la validación del uso de la plataforma NanoString nCounter para analizar material de biopsias líquidas y desarrollar ensayos de biomarcadores clínicamente relevantes.
The assessment of predictive- and prognostic molecular markers in tumor tissue, also known as personalised treatment, has transformed clinical practice for many cancer types. This genotype-directed therapy was found to improve patient survival, and several technical platforms have been introduced in clinical laboratories since then. However, not all tumors can be biopsied and tissue quantities are often insufficient for tumor characterisation. Liquid biopsies, such as membrane-encapsulated- or circulating free RNA, DNA and proteins, can be derived from body fluids and can replace or complement tissue biopsies. They have several advantages, such as repeated sampling, a minimally invasive character and heterogeneous profiling. Unfortunately, there is still a big gap between basic research and clinical implementation of liquid biopsies, mainly due to the lack of standardised methodologies. In addition, currently used technical platforms are not always suitable to analyze the low quantity and quality of tumor-derived material that can be found in a liquid biopsy. In consequence, large-scale validation and clinical implementation of liquid biopsy-based biomarker assays requires a sensitive, quick, easy-to-use, relatively cheap, flexible and standardized technical platform with low input requirements. The nCounter platform can be used to analyze all types of molecules, including RNA, DNA and proteins. Binding of color coded barcodes to targets of interest allows for either a direct read-out of gene- or protein expression levels or the detection of mutations. Tissue-based biomarker assay development on nCounter led to the FDA approval of the Prosigna™ assay for clinical use in breast cancer subtyping. Previous efforts have also highlighted the potential of this platform to analyze amplified liquid biopsy-derived molecules, although validation studies in the clinical setting are needed. In this thesis we validated the use of the NanoString nCounter platform to analyze material from liquid biopsies and develop clinically relevant biomarker assays.
Universitat Autònoma de Barcelona. Programa de Doctorat en Bioquímica, Biologia Molecular i Biomedicina

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Rauof, Goran, and Jonas Jägerback. "Utvecklingen av ett produktsystem för bättre och billigare cancerdiagnostik : Framtagning av engångskassett och tillhörande basenhet för isolering av cirkulerande och andra suspenderade tumörceller." Thesis, KTH, Maskinkonstruktion (Inst.), 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-99301.

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Det här examensarbetet består i ett produktutvecklingsprojekt som utfördes i samarbete med Liquid Biopsy AB. Syftet med arbetet var att utveckla ett engångskassettsbaserat produktsystem baserat på företagets patentsökta metod för isolering av cancer celler i suspension, inklusive cirkulerande tumörceller. Liquid Biopsy AB är ett svenskt utvecklingsbolag som baserat på ny och unik teknik, är oberoende av proteinmarkörer, använder cirkulerande tumörceller och andra suspenderade tumörceller för att möjliggöra bättre och billigare cancerdiagnostik. Examensarbetet har fokuserat på utvecklingen av engångskassetten, men parallellt arbete har även utförts med tillhörande basenhet. Ulrich och Eppingers produktutvecklingsprocess har utgjort grunden för den process som följts i arbetet, dock med ökat fokus på testning och utvärdering. För att få en bredare kunskapsbas inleddes arbetet med en marknads- och omvärldsanalys samt informationsinsamling om utmaningar och medicintekniska krav. För att tydligt definiera produktvisionen utfördes även undersökningar med potentiella användarna, om företagets patentsökta metod och befintliga prototyper samt framtida förbättringspotential. Det kassettkoncept som utvecklats bygger på användning av provrör av existerande standard, få tillverkningsprocesser och god användarvänlighet, något som samtliga varit av hög prioritet under arbetet. För att säkerställa att produktens flödessystem fungerar som tänkt utfördes tester under prototypframtagningen. Testningen visade att konceptet fungerar i stort sett som tänkt med avseende på flöden, dock förekom vissa toleransproblem som följd av den valda prototypframtagningsprocessen, och vissa andra viktiga egenskaper återstår att testa. Resultatet av utvecklingsprocessen är en första fysisk prototyp av engångskassetten och en funktionell partiell prototyp av basenheten, motsvarande gränssnittet mot engångkassetten, för att möjliggöra testning av engångskassetten. Slutsatsen av arbetet är att det framtagna produktsystemet har tydliga fördelar gentemot företagets befintliga prototyper: inklusive att en engångskassett framtagits, att denna kan utgöra underlag för en produkt, och att denna bland annat har väsentligt kortare processväg vilken i sin tur borde kunna leda till förkortad processtid. Utförd finansiell analys visar även att framtaget produktsystem kan säljas till konkurrenskraftiga priser och med en betydligt lägre instegskostnad än dagens konkurrerande produkter.
This thesis consists of a product development project conducted in collaboration with Liquid Biopsy AB. The purpose of this work was to develop a disposable cartridge-based product system based on the company’s patent-pending method for isolation of circulating tumor cells and other suspended tumor cells. Liquid Biopsy AB is a Swedish medical technology research company with a unique new rheological technology, that is independent of protein markers, using suspended cancer cells, including circulating tumor cells, allows better and cheaper cancer diagnostics than today. The thesis work has focused on the development of the disposable cassette, but parallel work has also been performed with the associated base unit. Ulrich and Eppingers product development process has made up the basis for the process being followed in the thesis work, with increased focus on testing and evaluation. The work began with a market analysis and information gathering on challenges and medical requirements. Several activities were also carried out in order to clearly define the product vision, including user-surveys, analysis of the company's existing prototypes, as well as potential for future improvements. The developed cartridge concept is based on the use of standard test tubes, few manufacturing processes and user-friendliness which all have been high priorities in this work. The cartridge concept consists essentially of various plastic materials and is adapted for manufacturing by injection molding. To ensure that the product’s flow system was operating as intended, tests were conducted during the prototype phase. Testing showed that the concept design flows largely as intended, yet with some tolerance problems as a result of the selected rapid prototyping process, while other essential properties remain to be tested. The result of the development process is a first physical prototype of the disposable cartridge and a partial functional prototype of the base unit to allow testing with the disposable cartridge. The conclusion of this thesis work is that the developed product system has strong advantages over the company’s existing prototypes, including a first version of a disposable cassette that has potential to form the basis of a mass-producible product, significantly shorter processing route which in turn should allow a reduction of the processing time. Financial analysis also indicates that the designed product systems can be sold at competitive prices and with a significantly lower entry cost than today's rivaling products.

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Taus, García Álvaro. "Estudio de mutaciones de EGFR y KRAS en ADN circulante en pacientes afectos de cáncer de pulmón de célula no pequeña." Doctoral thesis, Universitat Autònoma de Barcelona, 2021. http://hdl.handle.net/10803/671917.

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L'anàlisi molecular de l'tumor s'ha convertit en un procediment fonamental per a definir el maneig dels pacients amb càncer de pulmó. Encara que l'anàlisi de el teixit tumoral es considera la tècnica d'elecció per a l'estudi molecular, aquesta aproximació té una sèrie de limitacions, com l'absència de material tumoral suficient o la impossibilitat per capturar l'heterogeneïtat tumoral. L'ús de la biòpsia líquida podria ajudar a superar aquestes limitacions. En aquest treball analitzem la capacitat de la biòpsia líquida de detectar mutacions de EGFR o KRAS en plasma. Utilitzant la biòpsia líquida hem detectat mutacions de EGFR en un 83% dels casos (100% en casos amb disseminació extratorácica) i en un 82% dels casos KRAS mutats (96% en casos amb més d'una localització metastàtica). A més, el nostre treball demostra la capacitat de l'anàlisi de la dinàmica de la càrrega mutacional circulant de preveure la resposta o progressió amb antelació. En els casos EGFR mutats, la dinàmica de la càrrega mutacional va permetre predir la resposta en un 83% de els casos i la progressió en un 89% amb una antelació mitjana de 38 i 80 dies respectivament. En els casos amb mutacions de KRAS, la taxa de predicció de la resposta i progressió radiològiques va ser de el 93% i el 63% respectivament amb una antelació mitjana de 33 i 50 dies. D'altra banda, tant en els casos EGFR com en els KRAS mutats, la supervivència lliure de progressió va ser significativament superior en els casos en què, després del inici de tractament, les mutacions van deixar de ser detectables a ADN tumoral circulant. En resum, el nostre treball demostra, en un entorn de pràctica clínica habitual, la utilitat de la biòpsia líquida per al maneig de pacients amb càncer de pulmó.
El análisis molecular del tumor se ha convertido en un procedimiento fundamental para definir el manejo de los pacientes con cáncer de pulmón. Aunque el análisis del tejido tumoral se considera la técnica de elección para el estudio molecular, esta aproximación tiene una serie de limitaciones, como la ausencia de material tumoral suficiente o la imposibilidad para capturar la heterogeneidad tumoral. El uso de la biopsia líquida podría ayudar a superar estas limitaciones. En este trabajo analizamos la capacidad de la biopsia líquida de detectar mutaciones de EGFR o KRAS en plasma. Utilizando la biopsia líquida hemos detectado mutaciones de EGFR en un 83% de los casos (100% en casos con diseminación extratorácica) y en un 82% de los casos KRAS mutados (96% en casos con más de una localización metastática). Además, nuestro trabajo demuestra la capacidad del análisis de la dinámica de la carga mutacional circulante de prever la respuesta o progresión con antelación. En los casos EGFR mutados, la dinámica de la carga mutacional permitió predecir la respuesta en un 83% de los casos y la progresión en un 89% con una antelación mediana de 38 y 80 días respectivamente. En los casos con mutaciones de KRAS, la tasa de predicción de la respuesta y progresión radiológicas fue del 93% y 63% respectivamente con una antelación mediana de 33 y 50 días. Por otro lado, tanto en los casos EGFR como en los KRAS mutados, la supervivencia libre de progresión fue significativamente superior en los casos en los que, tras el inicio de tratamiento, las mutaciones dejaron de ser detectables en ADN tumoral circulante. En resumen, nuestro trabajo demuestra, en un entorno de práctica clínica habitual, la utilidad de la biopsia líquida para el manejo de pacientes con cáncer de pulmón.
Molecular profiling of the tumor has become a crucial procedure in the management of patients with lung cancer. Although the analysis of the tumor tissue is considered the gold standard for the molecular profiling, this approach has some limitations, such as the absence of the required amount of tissue or the inability to capture the tumor heterogeneity. The use of liquid biopsy may help to overcome these limitations. In this work we analyze the ability of the liquid biopsy to detect EGFR or KRAS mutations in plasma. Using liquid biopsy, we detected EGFR mutations in 83% of cases (100% in cases with extrathoracic metastases) and in 82% of mutated KRAS cases (96% in cases with more than one metastatic location). Furthermore, our work demonstrates the ability of the analysis of the circulating mutational load dynamics to predict the response or progression well in advance. In the EGFR mutated cases, the dynamics of the mutational load allowed predicting the response in 83% of the cases and the progression in 89% with a median advance of 38 and 80 days respectively. In the cases with KRAS mutations, the radiological response and progression prediction rate was 93% and 63%, respectively, with a median lead time of 33 and 50 days. On the other hand, in both EGFR and KRAS mutated cases, progression-free survival was significantly higher in cases in which, after starting treatment, the mutations became undetectable in circulating tumor DNA. In summary, our work demonstrates, in a routine clinical practice setting, the utility of liquid biopsy for the management of patients with lung cancer.
Universitat Autònoma de Barcelona. Programa de Doctorat en Medicina

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Afrogheh, Amir. "An evaluation of Shandon Papspin liquid based oral test utilizing a novel cytologic scoring system." Thesis, University of the Western Cape, 2010. http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_4355_1360592750.

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Background and Aims: While a single &ldquo
high quality&rdquo
oral liquid based cytology (LBC) study has shown a high sensitivity and specificity for the technique in detection of oral dysplasia and malignancy, the high unit cost of this technology cannot be borne by the developing African countries. This study aims to evaluate the efficiency of an alternative cost-effective technique, Shandon PapSpin (PS) LBC in
diagnosis of oral and oropharyngeal dysplasia and malignancy. Materials and Methods.We compared the diagnostic accuracy of Shandon PS LBC with that of scalpel biopsy in 69 patients. Transepithelial cytology specimens were obtained using a cervical Cytobrush. The cytology specimens were graded and scored using a novel oral cytologic grading and scoring system respectively. Results: Histological diagnosis of dysplasia or invasive squamous cell carcinoma was made in 51 of the 69 cases. Histology confirmed the cytological diagnosis of dysplasia or malignancy in 49 of the 51 cases. There were two false negative and no false positive cases. The sensitivity was 96% and the specificity 100%. The cytologic grade correlated positively with histologic grade. The best cut off value for distinguishing reactive/mildly dysplastic lesions from high 9 grade/invasive squamous cell carcinoma was determined as a cytologic score of 3, representing a sensitivity of 95% and a specificity of 96%. Conclusion: The Shandon PS LBC in association with transepithelial brush biopsy technique (TBBT) is a highly sensitive, specific and economical screening test in detection of oral and oropharyngeal dysplasia and malignancy. The proposed oral cytologic grading system correlates well with histology. The novel oral cytologic scoring system shows promise as a simple, reliable and reproducible scoring system. In addition, the liquid residual allows for immunocytochemical (Podoplanin) testing.

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De, Mattos Arruda Leticia. "Genomic characterisation of brain malignancies through liquid biopsies: The cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain tumours than plasma." Doctoral thesis, Universitat Autònoma de Barcelona, 2016. http://hdl.handle.net/10803/394019.

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Los recientes avances en la secuenciación masiva en paralelo y en las técnicas genómicas digitales apoyan la validez clínica del ADN libre tumoral circulante (ctDNA) como una "biopsia líquida” en el cáncer humano. La presencia de ctDNA en el plasma puede ser útil para identificar alteraciones genómicas, monitorizar la respuesta al tratamiento, identificar la resistencia terapéutica, y potencialmente caracterizar la heterogeneidad del tumor. El estudio de prueba de concepto en el campo de las biopsias líquidas titulado “Capturing intra-tumor genetic heterogeneity by de novo mutation profiling of circulating cell-free tumor DNA: a proof-of-principle” publicado en la revista Annals of Oncology en julio de 2014, es el artículo complementario analizado en esta tesis. Este artículo es uno de los primeros en demostrar que la secuenciación masiva en paralelo del ctDNA derivado del plasma constituye una herramienta potencial para la identificación y el seguimiento de las alteraciones genómicas somáticas durante el curso de la terapia dirigida, y que esta herramienta no invasiva se puede emplear para superar los retos planteados por la heterogeneidad del tumor. El ctDNA derivado del plasma ha demostrado ser capaz de identificar las alteraciones genómicas de los pacientes con cáncer. Sin embargo, los pacientes con tumores cerebrales tienen cantidades bajas o indetectables de ctDNA en el plasma lo que excluye la caracterización genómica del cáncer de cerebro a través del ctDNA en el plasma. La prueba de concepto en el campo de las biopsias líquidas del sistema nervioso central, que es el artículo fundamental analizado en este tesis: “Cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain tumours than plasma”, fue publicado en Nature Communications en noviembre de 2015. El ctDNA derivado de tumores malignos del sistema nervioso central primario y secundario esta enriquecido en el líquido cefalorraquídeo (LCR) y retrata las alteraciones genómicas de las enfermedades del sistema nervioso central mejor que el plasma. Los niveles de CSF ctDNA fluctúan longitudinalmente en el tiempo y siguen los cambios en la carga tumoral cerebral, proporcionando biomarcadores para monitorizar los canceres cerebrales. Además, el LCR ctDNA ha demostrado facilitar y complementar el diagnóstico del carcinomatosis leptomeníngea. El ctDNA presente en el LCR de neoplasias cerebrales y el ctDNA presente en el plasma de los cánceres de mama con metástasis sistémicas extra-craneales podría ser utilizado para caracterizar las alteraciones genómicas de las metastasis de estos cánceres. El uso de los CSF ctDNA representa una herramienta mínimamente invasiva que puede cambiar el paradigma para el manejo clínico de los pacientes con tumores malignos en el sistema nervioso central. Las biopsias líquidas tienen el potencial de proporcionar la información genómica completa del tumor, secuencial y en tiempo real y que permitirá mejorar el manejo terapéutico de los pacientes con cáncer.
Recent developments in massively parallel sequencing and digital genomic techniques support the clinical validity of cell-free circulating tumour DNA (ctDNA) as a ‘liquid biopsy’ in human cancer. ctDNA in plasma may be useful to identify actionable genomic alterations, monitor treatment responses, unravel therapeutic resistance, and potentially to characterise tumour heterogeneity. The proof-of-principle study in the field of liquid biopsies, which is the ancillary article analysed in this thesis entitled: “Capturing intra-tumor genetic heterogeneity by de novo mutation profiling of circulating cell-free tumor DNA: a proof-of-principle” was published in Annals of Oncology in July 2014. This article is one of the first to demonstrate that high-depth targeted massively parallel sequencing of plasma-derived ctDNA constitutes a potential tool for de novo mutation identification and monitoring of somatic genomic alterations during the course of targeted therapy. This article demonstrated that plasma ctDNA may be employed to overcome the challenges posed by tumour heterogeneity. Plasma-derived ctDNA has been shown to be informative of the genomic alterations of patients with cancers. Nevertheless, patients with brain tumours have low or undetectable amounts of ctDNA in plasma precluding the genomic characterisation of brain cancer through plasma ctDNA. The proof-of-principle in the field of central nervous system liquid biopsies, which the fundamental article analysed in this thesis entitled: “Cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain tumours than plasma” was published in Nature Communications in November 2015. ctDNA derived from primary and secondary central nervous system malignancies was shown to be more abundantly present in the cerebrospinal fluid (CSF) than in plasma and it portrayed the genomic alterations of central nervous system disease better than plasma. CSF ctDNA levels longitudinally fluctuated in time and followed the changes in brain tumour burden providing biomarkers to monitor brain malignancies. Additionally, CSF ctDNA was shown to facilitate and complement the diagnosis of leptomeningeal carcinomatosis. Taken together, ctDNA present in the CSF of brain malignancies and ctDNA present in the plasma of breast cancers with extra-cranial systemic metastases may be used to characterise metastasis-specific genomic alterations. CSF ctDNA represents a minimally invasive tool that may change the paradigm for the clinical management of cancer patients with central nervous system malignancies. Liquid biopsies have the potential to provide comprehensive, sequential and real-time tumour-derived genomic information that will improve the therapeutic management of cancer patients.

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Hilke, Franz Joachim [Verfasser]. "Genetische Charakterisierung und Therapieüberwachung von fortgeschrittenen Tumorerkrankungen mit Hilfe der Hochdurchsatzsequenzierung und Liquid Biopsy / Franz Joachim Hilke." Tübingen : Universitätsbibliothek Tübingen, 2021. http://d-nb.info/1230796525/34.

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Notarangelo, Michela. "Exploiting extracellular vesicles for ultrasensitive detection of cancer biomarkers from liquid biopsies." Doctoral thesis, Università degli studi di Trento, 2019. http://hdl.handle.net/11572/243195.

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Extracellular vesicles (EVs) are small membrane-surrounded structures containing transmembrane proteins and enclosing cytosolic proteins and nucleic acids. They are released in the extracellular space by both normal and neoplastic cells and play an important role in cell-cell communication in numerous physiological processes and pathological conditions, through the transfer of their functional cargo to recipient cells. EVs are highly abundant in biological fluids, and even more represented in cancer patients’ biofluids, therefore many studies suggested that they can be instrumental in liquid biopsies as prognostic markers or for early detection of tumors. Moreover, being secreted by potentially all the cells, they can serve in oncology to represent the tumor heterogeneity, which is underestimated by the current diagnostic tools. Given their small size, EVs are difficult to isolate in a high-throughput way and, therefore, one of the main obstacles to their clinical application, is that the existing isolation methods are impractical. During these years, I worked at the development and optimization of a novel technique that allows purification of heterogeneous EVs from biological fluids in an efficient, fast and reproducible way. This technique, named Nickel-Based Isolation (NBI), is a biochemical assay that allows obtaining polydisperse EVs in a physiological pH solution, therefore, preserving their morphology, heterogeneity, and stability. We tested and optimized this assay in protein-enriched systems and comparing it to the techniques currently used to characterize and measure EVs, such as flow cytometry and Tunable Resistive Pulse Sensing. We challenged the reproducibility of this method by isolating EVs from different biological fluids. Interestingly, the EVs purified with NBI result more intact and stable compared to the ones obtained with other methods, and can be studied in a clinical setting and used as an innovative tool for detection of molecules associated with diseases. We demonstrated the specificity of the procedure by using individual isolated vesicles in biochemical and molecular assay, optimized to characterize the biological content of EVs. We were able to detect picomolar concentration of PSMA on 105 EVs isolated from plasma of prostate cancer patients and BRAF-V600E transcript in just 103 EVs from the plasma of colon cancer patients, reaching unprecedented matching with tissue biopsy results. We also investigated the transcriptome of EVs isolated from glioblastoma cancer stem cells, in order to exploit the potential of EVs as diagnostic markers.

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Chudasama, Dimple. "Discovery and development of liquid biomarkers for ovarian and lung cancer." Thesis, Brunel University, 2018. http://bura.brunel.ac.uk/handle/2438/16174.

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Survival rates in cancers have improved vastly over the years. However, some survival rates remain extremely low, as is the case for ovarian and lung cancer. The lack of robust and reliable biomarkers is strongly reflected in the absence of pre-screening programs, and as such, most patients in these cancer types are diagnosed only in advanced stages, leaving few treatment options. Moreover, relapse and resistance to therapies adds to the complexities of treating these diseases, even in the era of targeted drug development. Research has shown the presence of cancer material, in the form of circulating cancer cells (CTCs) and genomic material in the blood of patients, opening the possibility of 'liquid biopsies'. Liquid biopsies allow sampling of the disease to provide phenotypic and genomic data on the cancer in real-time and on a routine basis. Moreover, they overcome obstacles currently faced by conventional tissue biopsies. In this work we evaluate the use of a novel CTC imaging flow-cytometry platform, and report the ability to characterise and quantify these cells in blood samples. Moreover, we report significantly higher levels of CTCs in cancer patients compared to controls, and found them to be associated with a poorer prognosis. In particular, in lung cancer we observe these findings even in the early stages, suggesting a potential diagnostic use for this assay. We detect a similar trend in when analysing the ctDNA and suggest the possibility of using this technique with a prognostic value in the advanced setting. We also report on the analysis of existing microarray data by use of unique gene regulatory networks to identify biomarkers of interest. RAD51AP1 was identified by this process. Clinical validation revealed an over-expression of this gene in both tissue and blood of ovarian and lung cancers. Moreover, the gene over-expression was associated with a poor overall survival. Functional analysis in vitro revealed silencing RAD51AP1 suppressed tumour growth, in addition, various tumorigenic proteins were down-regulated, whilst apoptotic and immune genes were up-regulated. These results suggest a role for RAD51AP1 as a potential therapeutic target. In this study, we also demonstrate the ability to further exploit tumour genomic material in the blood by means of RNAseq, cancer panels, and CNI scoring to identify novel markers, that play an important role in disease genesis and evolution. RNAseq analysis identified XIST as a gene up-regulated in the blood and tissue of lung cancers. The ovarian cancer panels revealed 2 unique gene signatures in the ovarian cancer patients. With the CNI analyses also highlighting chromosomal aberrations from plasma analysis of cancer patients. Collectively, the use of all these techniques and exploitation of available blood based biomarkers could see significant improvements to survival rates in these, currently devastating diseases.

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Hisey, Colin Lee Hisey. "Microfluidic Devices for Clinical Cancer Sample Characterization." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1525783108483419.

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Julich-Haertel, Henrike [Verfasser], and Veronika [Akademischer Betreuer] Lukacs-Kornek. "Microparticles – A novel liquid biopsy tool to identify and classify primary hepatic cancer / Henrike Julich-Haertel ; Betreuer: Veronika Lukacs-Kornek." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2018. http://d-nb.info/1173703349/34.

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Kalubowilage, Madumali. "Liquid biopsies of solid tumors: non-small-cell lung and pancreatic cancer." Diss., Kansas State University, 2017. http://hdl.handle.net/2097/35385.

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Doctor of Philosophy
Department of Chemistry
Stefan H. Bossmann
Cancer is a group of diseases that are characterized by uncontrolled growth and spread of cells. In order to treat cancer successfully, it is important to diagnose cancers in their early stages, because survival often depends on the stage of cancer detection. For that purpose, highly sensitive and selective methods must be developed, taking advantage of suitable biomarkers. The expression levels of proteases differ from one cancer type to the other, because different cancers arise from different cell types. According to the literature, there are significant differences between the protease expression levels of cancer patients and healthy people, because solid tumors rely on proteases for survival, angiogenesis and metastasis. Development of fluorescence-based nanobiosensors for the early detection of pancreatic cancer and non-small-cell lung cancer is discussed in this thesis. The nanobiosensors are capable of detecting protease/arginase activities in serum samples over a broad range. The functionality of the nanobiosensor is based on Förster resonance energy transfer and surface energy transfer mechanisms. The nanobiosensors for protease detection feature dopamine-coated Fe/Fe₃O₄ nanoparticles, consensus (cleavage) peptide sequences, meso-tetra(4-carboxyphenyl)porphine (TCPP), and cyanine 5.5. The consensus peptide sequences were synthesized by solid-supported peptide synthesis. In this thesis, improved consensus sequences were used, which permit faster synthesis and higher signal intensities. TCPP, which is the fluorophore of the nanoplatform, was connected to the N-terminal end of the oligopeptides while it was still on the resin. After the addition of TCPP, the TCPP-oligopeptide was cleaved off the resin and linked to the primary amine groups of Fe/Fe₃O₄-bound via a stable amide bond. In the presence of a particular protease, the consensus sequences attached to the nanoparticle can be cleaved and release TCPP to the aqueous medium. Upon releasing the dye, the emission intensity increases significantly and can be detected by fluorescence spectroscopy or, similarly, by using a fluorescence plate reader. In sensing of arginase, posttranslational modification of the peptide sequence will occur, transforming arginine to ornithine. This changes the conformational dynamics of the oligopeptide tether, leading to the increase of the TCPP signal. This is a highly selective technology, which has a very low limit of detection (LOD) of 1 x 10⁻¹⁶ molL⁻¹ for proteases and arginase. The potential of this nanobiosensor technology to detect early pancreatic and lung cancer was demonstrated by using serum samples, which were collected from patients who have been diagnosed with pancreatic cancer and non-small cell lung cancer at the South Eastern Nebraska Cancer Center (lung cancer) and the University of Kansas Cancer Center (pancreatic cancer). As controls, serum samples collected from healthy volunteers were analyzed. In pancreatic cancer detection, the protease/arginase signature for the detection of pancreatic adenocarcinomas in serum was identified. It comprises arginase, MMPs -1, - 3, and -9, cathepsins -B and -E, urokinase plasminogen activator, and neutrophil elastase. For lung cancer detection, the specificity and sensitivity of the nanobiosensors permit the accurate measurements of the activities of nine signature proteases in serum samples. Cathepsin -L and MMPs-1, -3, and -7 permit detecting non-small-cell lung-cancer at stage 1.

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Schneegans, Svenja [Verfasser], and Susanne [Akademischer Betreuer] Dobler. "The putative role of HERC5 in NSCLC metastasis and its potential utility as a liquid biopsy marker / Svenja Schneegans ; Betreuer: Susanne Dobler." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2020. http://d-nb.info/121481154X/34.

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Schneegans, Svenja Verfasser], and Susanne [Akademischer Betreuer] [Dobler. "The putative role of HERC5 in NSCLC metastasis and its potential utility as a liquid biopsy marker / Svenja Schneegans ; Betreuer: Susanne Dobler." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2020. http://nbn-resolving.de/urn:nbn:de:gbv:18-105564.

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Fuh, Marceline Manka [Verfasser], and Hartmut [Akademischer Betreuer] Schlüter. "Investigation of extracellular vesicles from glioblastoma multiforme and meningioma patients for cancer liquid biopsy by differential quantitative proteomics / Marceline Manka Fuh ; Betreuer: Hartmut Schlüter." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2019. http://d-nb.info/1197801480/34.

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Schier, Wiebke Henrike [Verfasser], and Arne [Akademischer Betreuer] Trummer. "Etablierung einer Liquid Biopsy zur Detektion einer BTK Resistenzmutation bei Patienten unter Ibrutinib-Therapie / Wiebke Henrike Schier ; Akademischer Betreuer: Arne Trummer ; Städtisches Klinikum Braunschweig." Hannover : Bibliothek der Medizinischen Hochschule Hannover, 2019. http://d-nb.info/1201824826/34.

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Kehagias, Pashalina. "Sensitivity and Resistance to Regorafenib Therapy in Advanced Colorectal Cancer: ctDNA Monitoring and Molecular Mechanisms." Doctoral thesis, Universite Libre de Bruxelles, 2020. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/312523.

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Résumé en Français:Le régorafénib est une des dernières options thérapeutiques pour les patients atteints d’un cancer colorectal chimio-réfractaire de stade avancé (CCRa). Cet inhibiteur de multiples tyrosine kinases n’est pas dépourvu de toxicité ce qui limite son utilisation en dernière ligne chez ces patients dont l’état général est dégradé, alors même que son efficacité n’est pas certaine chez tous les patients. Son mécanisme d'action reste largement inconnu, ce qui rend difficile l'identification de biomarqueurs prédictifs cliniquement utiles. C’est dans ce contexte que se situe ce projet de thèse, dont l’objectif est d’identifier les patients qui pourraient ou pas bénéficier du regorafenib. Pour atteindre cet objectif, nous avons développé à la fois nous avons entamé des projets de recherche translationnelle et mécanistiques.Nos études translationnelles se sont portées sur le monitoring de la réponse thérapeutique dans des biopsies liquides récoltées dans le cadre d’un essai clinique prospectif incluant des patients souffrant d’un CCRa et traités au régorafénib (RegARd-C). Nous avons d’abord confirmé la valeur pronostique de l’ADN libre circulant (ADNlc) avant traitement. De même, nous avons démontré qu'un haut volume tumoral métaboliquement actif avant traitement était associé à un mauvais pronostic. Combinés, ces marqueurs sont restés des prédicteurs de la survie sans progression (SSP) et globale (SG) des patients. Par la suite, nous avons mesuré l’ADN tumoral circulant (ADNtc) en suivant les mutations tumorales pré-identifiées dans le tissu tumoral des patients par séquençage. Nous avons démontré qu’une augmentation de plus de 50% de l’ADNtc après seulement 14 jours de traitement quel que soit le nombre de mutations suivies, indiquait un moins bon pronostic en termes SSP et SG. En outre, nos données suggèrent que ADNtc et ADNlc sont indépendants mais complémentaires dans leur valeur prédictive. Aussi, nous avons mis en évidence le rôle important de l’antigène carbohydrate 19-9 en tant que marqueur pronostique et prédictif précoce de la survie de ces patients.Ayant observé un profil général de résistance chez les patients, nous avons investigué les mécanismes potentiels de résistance intrinsèque et acquise au régorafénib dans des lignées cellulaires représentatives de CCR et avons trouvé différents degrés de résistance intrinsèque dans ces lignées. Nous avons dès lors exploré les mécanismes de résistance au régorafénib dans des cellules que nous avons rendues plus résistantes que les lignées parentales. L’investigation des voies de signalisation des MAPK et de PI3K/AKT a montré que cette dernière est un acteur majeur dans la résistance acquise dans la lignée HCT-116. Une autre observation basée sur des changements morphologiques particuliers des cellules nous a conduits à investiguer en détail un phénotype sénescent induit par le régorafénib, la sénescence étant reconnue comme causant la résistance aux médicaments. A cet égard, le comportement des deux lignées était différent. Les cellules SW480 étaient capables d'acquérir des propriétés de sénescence stables suite à la sécrétion de facteurs favorisant celle-ci ainsi qu’un arrêt du cycle cellulaire. En accord avec le fait que ces cellules tumorales dormantes peuvent avoir un effet positif sur le devenir du patient, tant que celui-ci reste sous traitement, elles pourraient contribuer à la rechute de la maladie dès l’arrêt du traitement. Les cellules HCT-116 quant à elles présentent des propriétés de sénescence à court terme et développent une résistance acquise sous traitement au régorafénib continu via une transition épithélio-mésenchymateuse (TEM), également associée à une résistance aux médicaments. Ce dernier mécanisme pourrait avoir un effet délétère sur le patient comme l’apparition de sous-clones plus agressifs qui induise la progression de la maladie et assombrit le pronostic.Nous avons également investigué la p-glycoprotéine comme un possible mécanisme de résistance additionnel et avons montré que dans les cellules SW480 traitées au régorafénib, ce dernier serait capable de surmonter la résistance induite par cette protéine. Cependant, une augmentation de l'expression de la p-glycoprotéine n’est observée dans les cellules HCT-116 qu'après une courte exposition au régorafénib et plus dans les cellules traitées en continu.En conclusion, nous avons contribué à une meilleure compréhension des différents mécanismes de résistance au régorafénib dans le CCR. Nous avons indiqué la sénescence, la TEM et probablement la p-glycoprotéine comme acteurs majeurs potentiels; et avons souligné l’hétérogénéité de cette maladie. Est-ce que le régorafénib gagnera-t-il une meilleure place comme traitement efficace dans le CCR ?Notre travail propose plusieurs pistes pour répondre à cette question.
Abstract:Regorafenib is one of the last treatment options for patients with chemo-refractory metastatic colorectal cancer (mCRC), associated with some efficacy but with important toxicities impairing its use in patients with poor general condition. As a multi-targeted tyrosine kinase inhibitor, its mechanism of action remains largely unknown, challenging the identification of clinically useful predictive biomarkers. The ultimate aim of our work was to contribute to the identification of mCRC patients unlikely to benefit from regorafenib. To achieve this objective, we moved from translational to mechanistic studies.Our translational studies focused on therapy response monitoring using liquid biopsies obtained from a prospective clinical trial in mCRC patients treated with regorafenib (RegARd-C trial). We first, confirmed the prognostic value of cell-free DNA (cfDNA) level before treatment. Similarly, we found that high level of pre-treatment metabolically active tumor volume was associated with poor prognosis. When combined, these markers remained predictors of patients’ progression-free (PFS) and overall survival (OS).Then, we measured circulating tumor DNA (ctDNA) based on tumor-specific mutations already identified in patients’ tumor tissue by NGS. We demonstrated that an increasing ctDNA level more than 50% after 14 days of treatment, either based on one or on multiple mutations, is correlated with patients’ clinical outcome in terms of PFS and OS. Furthermore, our data strongly suggested that both baseline cfDNA and ctDNA dynamics are strong complementary predictors of both PFS and OS. Also, we highlighted the leading role of Carbohydrate Antigen 19-9 as a prognostic and early predictive biomarker of mCRC patients’ outcome.Having observed an overall resistance to regorafenib in the majority of patients, we investigated the potential related intrinsic and acquired resistance mechanisms in representative CRC cell lines and found rather different degrees of intrinsic resistance to regorafenib in these cell lines. We then explored potential mechanisms of resistance after short and long-term exposure to regorafenib. The investigation of MAPK and PI3K/AKT pathways pointed to the latter as a major player in acquired resistance.Another observation based on particular cell morphological changes led us to investigate in deep a drug-initiated senescence-like phenotype that is also known to cause drug resistance. SW480 cells were able to acquire stable senescent-like properties, also promoted by a specific senescence-associated secretome, and cell cycle arrest. In line with tumor cell dormancy this phenotype may have a positive impact on patient’s outcome as long as he is under treatment. However, dormant cells contribute to disease recurrence after drug withdrawal. In contrast, HCT-116 cells undergo senescent properties after short exposure and develop acquired resistance triggering EMT, which is also associated with drug resistance. This latter mechanism could be deleterious for the patient as the appearance of more aggressive tumor subclones may induce disease progression and worsen clinical outcome.We also investigated Multi-Drug Resistant protein 1 (MDR1) as a possible additional mechanism of resistance and we found that regorafenib seems to overcome MDR in SW480 treated cells while in HCT-116, an increase of MDR1 expression was observed after short and long exposure compared to parental cells.In conclusion, we contributed to a better understanding of different mechanisms of resistance to regorafenib in CRC. We pointed to cell plasticity such as senescence and EMT in addition to possible MDR as major players; and certainly highlighted the high heterogeneity of the disease. Will regorafenib gain a better place as an efficient drug in CRC? Our work provides some insights for answering this question.
Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)
info:eu-repo/semantics/nonPublished

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Kuhlmann, Jan Dominik [Verfasser], Sabine [Akademischer Betreuer] Kasimir-Bauer, Stephan [Akademischer Betreuer] Hahn, and Ralf [Akademischer Betreuer] Küppers. "Identifizierung neuer Biomarker für das Ovarialkarzinom – : Primärtumorbasierte Analysen und blutbasierte „Real-Time-Liquid-Biopsy“ / Jan Dominik Kuhlmann. Gutachter: Stephan Hahn ; Ralf Küppers. Betreuer: Sabine Kasimir-Bauer." Duisburg, 2013. http://d-nb.info/1035066416/34.

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Drelich, Lauranne. "De l’hétérogénéité intra-tumorale à la recherche de vésicules extracellulaires au sein de biopsie liquide en vue d’une médecine personnalisée." Thesis, Lille 1, 2020. http://www.theses.fr/2020LIL1S105.

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Les gliomes représentent 80% des tumeurs cérébrales malignes primitives et sont classés selon différents grades de malignité. Les glioblastomes, groupe le plus agressif, représentent plus de la moitié de tous les gliomes. Il s'agit de tumeurs dont la composition est hétérogène, présentant notamment de zones de nécrose, de prolifération vasculaire, de prolifération cellulaire. La survie des patients peut aller de quelques mois à quelques années après la chirurgie et la chimiothérapie. L’imagerie par spectrométrie de masse MALDI est une technique intéressante pour l’étude de ces tumeurs, car elle permet de prendre en considération l'hétérogénéité intra-tumorale. Dans cette étude, l’imagerie MALDI MS est couplée à la microprotéomique localisée sur tissu dans l'objectif d'identifier des sous-groupes de glioblastomes afin d'aider au diagnostic et au pronostic. Les images moléculaires ont été générées à partir de coupes fines de tissus dans le but de déterminer la localisation de peptides digérés. Ensuite, des analyses statistiques non supervisées nous ont permis de générer un regroupement hiérarchique de régions moléculaires homogènes au sein du tissu. Une analyse restreinte à ces différentes régions à nous a donné accès à leur contenu protéique. Le regroupement hiérarchique a révélé trois régions moléculaires dans l’ensemble des échantillons. À partir de ces régions, 3 groupes de patients ont été identifiés : le groupe A (13/50 patients), le groupe B (9/50 patients) et le groupe C (23/50 patients) . Cinq patients sont restés non classables. Chaque groupe présentait des signatures moléculaires spécifiques. Les analyses microprotéomiques ont montré un panel de protéines spécifiquement surexprimées dans chaque groupe : les protéines surexprimées dans le groupe A sont associées à la neurogenèse ; celles du groupe B sont associées à une activation du système immunitaire et celles du groupe C sont impliquées dans des processus viraux. Enfin, nous avons identifié 6 nouveaux marqueurs pronostiques des glioblastomes qui pourraient aider à stratifier les patients et aider à la décision thérapeutique.En parallèle, nous nous sommes intéressés au développement d’une nouvelle technique de microprotéomique localisée basée sur l’expansion de tissu. Agrandir le tissu permet, tout en conservant les méthodes de protéomique conventionnelle, d’analyser des régions plus petites que celles atteignables actuellement. Jusqu’à 655 protéines ont pu être identifiées dans une région de 460 µm de diamètre, ce qui correspond à une moyenne de 940 cellules. De plus, cette stratégie est intéressante pour l'imagerie MS, car elle donne la possibilité de cartographier facilement un grand nombre de protéines sur la base de leur quantification avec une résolution spatiale inférieure à 350 µm. En ce sens, l’expansion de tissu est d’un grand intérêt pour augmenter la résolution spatiale de la protéomique localisée.Finalement, étudier les vésicules extracellulaires circulant dans le sang pourrait à terme permettre d'aider au dépistage, au diagnostic ainsi qu'au suivi des glioblastomes. L’analyse protéomique des vésicules du sang des patients pourrait permettre d’identifier des protéines ou des réseaux déjà identifiés dans les échantillons tumoraux. Les résultats préliminaires à partir de plasma témoin montrent qu’il est possible d’isoler des vésicules et de réaliser des analyses protéomiques. Ainsi, il serait intéressant de pouvoir associer un profil de vésicule extracellulaire pour chaque groupe de tumeurs. De plus, nous avons été capables d’isoler spécifiquement les vésicules contenues dans une coupe fine de cerveau de rat. Ces résultats sont encourageants et à terme il sera possible d’identifier les vésicules provenant de la tumeur
Gliomas account for 80% of all malignant brain tumors and are classified within different maligniy grades. Glioblastomas, the most aggressive group, represent more than half of all gliomas but remain a heterogeneous group. Indeed, patient survival ranges from several months to a few years after surgery and chemotherapy. To study gliomas, MALDI mass spectrometry imaging is a technique of choice, allowing for tumor heterogeneity analysis. In this study, MALDI-MSI is coupled with spatially-resolved microproteomic aiming at identifying subgroups of glioblastomas patients in order to help for diagnosis and prognosis. Molecular images are generated from thin tissue sections to determine digested peptides spatial localizations. Based on unsupervised statistical analysis, we generated hierarchical clustering of homogeneous molecular regions. According to these regions, spatially resolved proteomic provided a broad range of protein identification and their relative quantifications. The hierarchical clustering reveals three molecular regions within all the tumor samples. Based on these regions, three groups of patients can be determined: group A (13/50 patients), group B (9/50 patients) and group C (23/50 patients) and 5 non-classifiable patients, each group with specific molecular signatures. Microproteomic analyzes show a panel of proteins specifically overexpressed in each group: proteins overexpressed in group A are associated with neurogenesis; those of group B are linked to immune system activation; and those of group C are involved in viral processes. Finally, we have identified 6 new prognosis markers for glioblastomas that could help stratifying patients and orient clinician in choosing therapeutics.In parallel, we were interested in developing a novel spatially resolved microproteomic technique based on tissue expansion. Tissue enlargement allows, while maintaining conventional proteomics methods, to analyze regions smaller than those currently achievable. Up to 655 proteins were identified within a region of 460 µm diameter, which corresponds to an average of 940 cells. In addition, this strategy is relevant for MS imaging as it gives the possibility to easily map a large number of proteins based on their quantification within a spatial resolution of less than 350 μm. Thus, tissue expansion is of great interest in increasing the spatial resolution of localized proteomics.Finally, studying circulating extracellular vesicles in blood may ultimately lead to an earlier diagnosis of glioblastomas. Proteomic analysis of patients' blood vesicles can identify proteins or networks already identified in tumor mass. Preliminary results from control plasma show that it is possible to isolate and perform proteomic analyzes on circulating vesicles. Thus, it would be interesting to be able to associate an extracellular vesicle profile for each group of tumors. In addition, we have been able to specifically isolate the vesicles embedded in a thin section of rat brain. These results are encouraging and, in the long term, will make it possible to identify vesicles originating from the tumor

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Dörner, Natalie Regina [Verfasser], Bernd [Gutachter] Schmidt, Dirk [Gutachter] Vordermark, and Christoph [Gutachter] Schäper. "Der DNA-Integritäts-Index K-ras 300 / 60 in der "liquid biopsy" und sein diagnostischer Nutzen bei Lungentumoren / Natalie Regina Dörner ; Gutachter: Bernd Schmidt, Dirk Vordermark, Christoph Schäper." Halle (Saale) : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2019. http://d-nb.info/1210730707/34.

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Dörner, Natalie Regina [Verfasser], Bernd Gutachter] Schmidt, Dirk [Gutachter] [Vordermark, and Christoph [Gutachter] Schäper. "Der DNA-Integritäts-Index K-ras 300 / 60 in der "liquid biopsy" und sein diagnostischer Nutzen bei Lungentumoren / Natalie Regina Dörner ; Gutachter: Bernd Schmidt, Dirk Vordermark, Christoph Schäper." Halle (Saale) : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2019. http://nbn-resolving.de/urn:nbn:de:gbv:3:4-1981185920-323140.

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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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Brychta, Nora [Verfasser], Matthias U. [Gutachter] Kassack, and Nikolas Hendrik [Gutachter] Stoecklein. "Analyse tumorspezifischer Mutationen in der Flüssigbiopsie (liquid biopsy) - Vergleich von zirkulierenden Tumorzellen und zirkulierender Tumor-DNA für die Frühdiagnose im Pankreaskarzinom / Nora Brychta ; Gutachter: Matthias U. Kassack, Nikolas Hendrik Stoecklein." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2017. http://d-nb.info/1149330546/34.

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Keup, Corinna [Verfasser], and Sabine [Akademischer Betreuer] Kasimir-Bauer. "Comprehensive molecular characterization of circulating tumor cells, extracellular vesicles and cell-free DNA as matched multi-parametric liquid biopsy for therapy management in metastatic breast cancer patients / Corinna Keup ; Betreuer: Sabine Kasimir-Bauer." Duisburg, 2020. http://d-nb.info/1221960350/34.

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Mijnes, Jolein Verfasser], Edgar [Akademischer Betreuer] Dahl, Ralph [Akademischer Betreuer] [Panstruga, and Gabriele [Akademischer Betreuer] Pradel. "Identification, validation and characterization of novel DNA methylation biomarkers for liquid biopsy based early breast cancer detection and therapy monitoring in non-small cell lung cancer / Jolein Mijnes ; Edgar Dahl, Ralph Panstruga, Gabriele Pradel." Aachen : Universitätsbibliothek der RWTH Aachen, 2020. http://d-nb.info/1225401690/34.

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Mijnes, Jolein [Verfasser], Edgar Akademischer Betreuer] Dahl, Ralph [Akademischer Betreuer] [Panstruga, and Gabriele [Akademischer Betreuer] Pradel. "Identification, validation and characterization of novel DNA methylation biomarkers for liquid biopsy based early breast cancer detection and therapy monitoring in non-small cell lung cancer / Jolein Mijnes ; Edgar Dahl, Ralph Panstruga, Gabriele Pradel." Aachen : Universitätsbibliothek der RWTH Aachen, 2020. http://d-nb.info/1225401690/34.

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34

Muyas, Remolar Francesc 1992. "Highly accurate variant detection for identification of tumor mutations and mosaic variants." Doctoral thesis, Universitat Pompeu Fabra, 2019. http://hdl.handle.net/10803/668325.

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The rapid development of high-throughput sequencing technologies pushed forward the fields of medical genomics and precision medicine, creating many new applications for diagnostics and clinical studies that require high quality data and highly accurate analysis methods. Distinguishing errors from real variants in Next Generation Sequencing data is a challenge when systematic errors, random sequencing errors, germline variants or somatic variants at very low allele frequency are present in the same data. In the first part of this thesis, we developed a genotype callability filter (ABB) able to identify systematic variant calling errors that were not found by state-of-the art methods. This tool cleans false positive calls from somatic and germline variant callsets, as well as detects false gene-disease associations in case-control studies. Secondly, we developed a set of novel methods able to distinguish and correct sequencing and PCR errors with the use of molecular barcodes, permitting us to build error rate models for the detection of somatic mutations at extremely low allele frequencies in liquid biopsies. As final part of this thesis, we characterized mosaic mutations in a multi-tissue, multi-individual study using a cohort of thousands of samples from hundreds of healthy individuals.
El ràpid desenvolupament de les tecnologies de seqüenciació d’alt rendiment ha impulsat els camps de la genòmica mèdica i la medicina d’alta precisió, creant una gran varietat de noves aplicacions, les quals requereixen dades d’una qualitat excel·lent i mètodes d’anàlisi altament precisos. La distinció entre errors i variants reals en dades de seqüenciació de propera generació (NGS) és un repte quan hi ha errors sistemàtics o aleatoris mesclats amb variants germinals o somàtiques a freqüències al·lèliques molt baixes. En la primera part d'aquesta tesi, hem desenvolupat un filtre per al genotipatge de variants (ABB) capaç d'identificar errors sistemàtics durant el procés de detecció de variants que altres mètodes convencionals no poden trobar. Aquesta eina filtra falsos positius del conjunt de variants finals en estudis de variacions somàtiques i germinals, així com també detecta falses associacions de malalties gèniques en estudis de casos-controls. En segon lloc, hem desenvolupat un conjunt de nous mètodes capaços de distingir i corregir els errors de seqüenciació i PCR amb l’ús d’identificadors moleculars. Aquests ens permeten modelar les taxes d’error i conseqüentment detectar mutacions somàtiques a freqüències al·lèliques extremadament baixes en l’anàlisi de biòpsies líquides. Per finalitzar aquesta tesi, hem caracteritzat les mutacions mosaiques en un estudi multi-teixit multi-individu utilitzant una cohort de centenars d'individus sans amb milers de mostres.

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Meunier-Komosa, Nathalie. "Recherche moléculaire de Tropheryma Whippelii dans le liquide articulaire de patients affectés de rhumatismes inflammatoires inclassés." Bordeaux 2, 2001. http://www.theses.fr/2001BOR23015.

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Ferreira, Guilherme José Bolzani de Campos. "Desenvolvimento e adaptação de tecnologia para obtenção de amostras de material intra uterino durante a gestação em cães SRD (Canis familiares - Linnaeus, 1758)." Universidade de São Paulo, 2005. http://www.teses.usp.br/teses/disponiveis/10/10132/tde-24092007-162634/.

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Foram utilizadas 10 cadelas prenhez (SRD), oriundas de Centros de Controle de Zoonoses de São Paulo e Região, em diferentes estágios de gestação. Os animais foram submetidos a exame clinico geral e a uma avaliação ultrasonográfica para determinação do tempo de gestação. O desenvolvimento de tecnologias que possam auxiliar o diagnóstico prematuro de alterações genéticas em animais é algo ainda novo, contudo fundamental para o futuro. Uma destas técnicas é a Biopsia de corio fetal a qual nos permite coletar de modo pouco invasivo amostra de tecido fetal e submeter este a exames cromossõmicos e de DNA. Através deste conseguimos identificar o sexo do feto com total exatidão. Também realizamos um estudo sobre a composição dos líquidos fetais, o qual obtivemos os seguintes resultados para líquido amniótico e alantoideano respectivamente: pH 8,19 e7,13, proteína 144,44 e 121,11, densidade 1007,5 e 1011,44, Creatinina 2,21 e 22,71, Albumina 0,28 e 0, 13, Fosfatase alcalina 432,94 e 22,73, proteína total 0,22 e 0,24, concluindo assim a grande semelhança dos líquidos fetais com a urina. A macroscopia do cordão umbilical nos forneceu alguns dados interessantes tais como a relação de tamanho entre o feto e o cordão umbilical a qual é de 2:1, outro dado interessante que é a relação de peso entre a placenta e o cordão umbilical que fica na proporção de 11,5:1. Microscopicamente o cordão umbilical do cão possui características próprias como a confluência das veias umbilicais ocorrendo próximo a cavidade abdominal, as paredes das veias (2) são delgadas e das artérias (2) são musculares e espessas, o dueto alantoideano localiza-se entre os quatro vasos. O saco vitelínico apresenta grande quantidade de capilares, parede delgada e vitelo.
We used pregnant female mongrel dogs, froco Control centers of Zoonoses of São Paulo and region, in different periods of gestation. The animais had been submïtted the general clinic examination and to a ultrasonographyc evaluation for determination of the time of pregnancy period. The development of technologies can assist the prematura diagnoses of genetic alterations, in animais is something new, however still basic for the future. One of these techniques and the biopsy of fetal chorion which in allows them to collect in little invasive fetal tissue and to submit this the chromosomic and DNA examïnations. Through this we obtain to identify the embryo sex with total exactness. Also carry through a study on the composition of the fetal fluids, which we found the respectìvely he following results to amniotic and allantois liquida pH 8,19 and 7,13, protein 144,44 and 121.11, density 1007,5 and 1011,44, Creatinina 2.21 and 22.71, Albumina 0,28 and 0,13, alkaline Fosfatase 432,94 and 22,73, total protein 0,22 and 0,24, thus concluding the great similarity of the fetal fluids with urine. The gross shape of the umbilical cord in supplied to some interesting data such to them as the relation of size between the embryo and the umbilical cord which is of 2:1, another interesting data that are the relation of weight between the placenta and the umbilical cord is the ratio of 11,5:1. Microscopicolly the umbilical cord of the dog possess proper aspects as the confluente of the umbilicais veins occurring next the abdominal cavity, the walis of the veins (2) are thin and arteries (2) are muscular and thick, allantois duct are between situated the four vasas. The yoik sac presents great amount of capillaries, thin wall and yoik.

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Shaheed, Sadr-ul, C. Tait, K. Kyriacou, J. Mullarkey, W. Burrill, Laurence H. Patterson, R. Linforth, M. Salhab, and Chris W. Sutton. "Nipple aspirate fluid - a liquid biopsy for diagnosing breast health." 2017. http://hdl.handle.net/10454/11980.

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Yes
Purpose: Nipple secretions are protein-rich and a potential source of breast cancer biomarkers for breast cancer screening. Previous studies of specific proteins have shown limited correlation with clinicopatholigical features. Our aim, in this pilot study, was to investigate the intra- and inter-patient protein composition of nipple secretions and the implications for their use as liquid biopsies. Experimental design: Matched pairs of NAF (n=15) were characterised for physicochemical properties and SDS PAGE. Four pairs were selected for semi-quantitative proteomic profiling and trypsin-digested peptides analysed using 2D LC Orbitrap Fusion mass spectrometry. The resulting data was subject to bioinformatics analysis and statistical evaluation for functional significance. Results: A total of 1990 unique proteins were identified many of which are established cancer associated markers. Matched pairs shared the greatest similarity (average Pearson correlation coefficient of 0.94), but significant variations between individuals was observed. Conclusions: This was the most complete proteomic study of NAF to date providing a valuable source for biomarker discovery. The high level of milk proteins in healthy volunteer samples compared to the cancer patients was associated with galactorrhoea. Using matched pairs increased confidence in patient-specific protein levels but changes relating to cancer stage require investigation of a larger cohort.
Proteomics research was supported by Yorkshire Cancer Research projects, BPP047 and B381PA.

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38

Ascenção, Íris Joana da Costa. "Liquid Biopsy in Rectal Cancer: Applications in Neoadjuvant Therapy Monitoring." Dissertação, 2020. https://hdl.handle.net/10216/128881.

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Introdução: No que concerne ao carcinoma do reto localmente avançado, uma resposta clínica completa à terapia neoadjuvante é, atualmente, prevista com dificuldade pelas modalidades de imagem convencionais. Os métodos de avaliação da resposta começam agora a assumir um papel central no desenvolvimento das estratégias de tratamento. Todavia, o potencial clínico da análise do ctDNA no contexto do cancro do reto não metastático continua por esclarecer. Assim, este trabalho visa proceder à revisão da evidência estabelecida sobre o papel das biópsias líquidas como método de avaliação da resposta à terapêutica neoadjuvante e no progresso do tratamento dos tumores localmente avançados do reto. Métodos: Foi conduzida uma revisão da literatura nas bases de dados da MEDLINE (através do motor de busca da PubMed), Web of Science e Scopus. Um total de 45 artigos foram incluídos neste trabalho. Resultados/Discussão: Os resultados explanados neste trabalho sugerem que, apesar de nenhum biomarcador preditivo se ter revelado suficientemente robusto para ter utilidade clínica por si só, foram encontradas diferenças significativas entre os doentes com boa e escassa resposta ao tratamento neoadjuvante. Parâmetros como a variação dos níveis de DNA, o índice de integridade do DNA ou o estado de metilação e mutação de genes específicos, integrados numa abordagem multidisciplinar ao tratamento, provaram ser uma ferramenta valiosa na avaliação da resposta terapêutica e uma oportunidade para aprimorar as estratégias de tratamento. Conclusão: A inclusão das biópsias líquidas na monitorização da resposta ao tratamento neoadjuvante pode melhorar a acuidade da avaliação clínica e radiológica dos doentes com cancro do reto e providenciar, num futuro próximo, uma oportunidade concreta para um tratamento mais personalizado destes doentes.
Introduction: Currently, regarding locally-advanced rectal cancer, a complete pathological response after neoadjuvant therapy is poorly predicted by conventional imaging modalities. Methods of response assessment are now starting to have a pivotal role in improving treatment strategies. However, the clinical potential of ctDNA analysis in non-metastatic rectal cancer remains unsettled. Thus, this work aims to review the established evidence on the role of liquid biopsies as a method of assessing the response to neoadjuvant therapy and refining the current treatment strategies of locally-advanced rectal tumors. Methods: A literature search was conducted in the MEDLINE database, using PubMed search engine, the Web of Science and Scopus databases. A total of 45 articles were comprised in this literature review. Results/Discussion: The results evidenced in this work suggest that, although no predictive molecular biomarker for response to nCRT has been proved to be sufficiently robust to have clinical utility on its own, significant differences between response and nonresponse could be found during treatment. Biomarkers such as DNA level variations, DNA integrity index, methylation or mutation status of specific genes, in the context of a multidisciplinary approach to treatment, have proven to be a valuable tool in assessing treatment response and refining therapy strategies. Conclusion: The inclusion of liquid biopsies in the monitorization of neoadjuvant treatment response can improve the accuracy of the clinical and radiological assessment of rectal cancer patients and offer, in the near future, a concrete opportunity for a more personalized treatment approach.

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Ascenção, Íris Joana da Costa. "Liquid Biopsy in Rectal Cancer: Applications in Neoadjuvant Therapy Monitoring." Master's thesis, 2020. https://hdl.handle.net/10216/128881.

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Анотація:

Introdução: No que concerne ao carcinoma do reto localmente avançado, uma resposta clínica completa à terapia neoadjuvante é, atualmente, prevista com dificuldade pelas modalidades de imagem convencionais. Os métodos de avaliação da resposta começam agora a assumir um papel central no desenvolvimento das estratégias de tratamento. Todavia, o potencial clínico da análise do ctDNA no contexto do cancro do reto não metastático continua por esclarecer. Assim, este trabalho visa proceder à revisão da evidência estabelecida sobre o papel das biópsias líquidas como método de avaliação da resposta à terapêutica neoadjuvante e no progresso do tratamento dos tumores localmente avançados do reto. Métodos: Foi conduzida uma revisão da literatura nas bases de dados da MEDLINE (através do motor de busca da PubMed), Web of Science e Scopus. Um total de 45 artigos foram incluídos neste trabalho. Resultados/Discussão: Os resultados explanados neste trabalho sugerem que, apesar de nenhum biomarcador preditivo se ter revelado suficientemente robusto para ter utilidade clínica por si só, foram encontradas diferenças significativas entre os doentes com boa e escassa resposta ao tratamento neoadjuvante. Parâmetros como a variação dos níveis de DNA, o índice de integridade do DNA ou o estado de metilação e mutação de genes específicos, integrados numa abordagem multidisciplinar ao tratamento, provaram ser uma ferramenta valiosa na avaliação da resposta terapêutica e uma oportunidade para aprimorar as estratégias de tratamento. Conclusão: A inclusão das biópsias líquidas na monitorização da resposta ao tratamento neoadjuvante pode melhorar a acuidade da avaliação clínica e radiológica dos doentes com cancro do reto e providenciar, num futuro próximo, uma oportunidade concreta para um tratamento mais personalizado destes doentes.
Introduction: Currently, regarding locally-advanced rectal cancer, a complete pathological response after neoadjuvant therapy is poorly predicted by conventional imaging modalities. Methods of response assessment are now starting to have a pivotal role in improving treatment strategies. However, the clinical potential of ctDNA analysis in non-metastatic rectal cancer remains unsettled. Thus, this work aims to review the established evidence on the role of liquid biopsies as a method of assessing the response to neoadjuvant therapy and refining the current treatment strategies of locally-advanced rectal tumors. Methods: A literature search was conducted in the MEDLINE database, using PubMed search engine, the Web of Science and Scopus databases. A total of 45 articles were comprised in this literature review. Results/Discussion: The results evidenced in this work suggest that, although no predictive molecular biomarker for response to nCRT has been proved to be sufficiently robust to have clinical utility on its own, significant differences between response and nonresponse could be found during treatment. Biomarkers such as DNA level variations, DNA integrity index, methylation or mutation status of specific genes, in the context of a multidisciplinary approach to treatment, have proven to be a valuable tool in assessing treatment response and refining therapy strategies. Conclusion: The inclusion of liquid biopsies in the monitorization of neoadjuvant treatment response can improve the accuracy of the clinical and radiological assessment of rectal cancer patients and offer, in the near future, a concrete opportunity for a more personalized treatment approach.

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40

Yacoub, Nicole. "Development and evaluation of a pre-analytical device for liquid biopsy." Thesis, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-354483.

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In this project, a novel method to discover and monitor cancer was studied. Tumoureducated platelets (TEPs) have shown the ability to take up tumour-derived secretedmembrane vesicles, which contain tumour RNA. Therefore they are of great interestfor detecting cancer and could potentially work as biomarkers. One problem withusing blood platelets as a source of biomarkers is that clinical implementation isprevented by pre-analytical procedures that are difficult to perform in clinicallaboratories. In this study, human blood samples have been used to investigatewhether density gradient centrifugation followed by microfiltration could result inpure platelet (PLT) fractions. The idea is to enable parallel extraction of multiplesources of biomarkers from a single liquid biopsy, namely peripheral bloodmononuclear cells (PBMCs), PLTs and plasma. The procedure was done by firstresuspending the buffy coat in plasma after the centrifugation, where some cells werefixed and then filtered through a combination of membranes. From that, PLT fractionsfree from any nucleated cell could be obtained both for fixed and unfixed cells. Thenext step will be to detect tumour-specific transcripts in PLTs by investigatingwhether the purity of the extracted mRNA is sufficient for these kinds of procedures.This method holds great promise for creating a procedure to isolate, extract andanalyse several biomolecule fractions from a single liquid biopsy. Further studies couldinclude other biomarkers of interest such as circulating tumour DNA (ctDNA),extracellular vesicles (EVs) and plasma proteins.

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41

Cheng, Yuhsiang, and 鄭宇翔. "A prospection observation study on liquid biopsy in cancer diagnosis and prognosis." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/u5d4hb.

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碩士
國立臺灣大學
生物科技管理碩士在職學位學程
107
Precision medicine is an indispensable part of future medical service.This study advocated that a treatment plan in target of its tumor molecular type must be developed based on the cancer patient’s liquid biopsy to yield a better response and prognosis. It would serve as a better supplement to follow up therapies, by tracking the efficacy and tumor recurrence. The liquid biopsy does show considerable potential in the future market. In order to verify the argument,this study would explore such expectation in cancer patients who had liquid biopsy exami nation. The entire research was conducted in two phases: First, market survey with in depth clinical interview was performed to understand the product positioning of liquid biopsy for cancer patients and its possible future direction in the market.The study found: 1) The genetic sequencing of liquid biopsy for patients selected by clinical physicians could indeed help in customization of individual therapy and showed significant improvement in therapeutic outcome for some tumor cases. 2) For patients with poor tolerance to chemotherapy who might even experience some side effects, it benefited them in finding the most appropriate target drugs to minimize any adverse reactions and even maximize the chance of getting treatment as early as possible. 3) Clinical experts believed that liquid biopsy is not only suitable for some people, but its ability to analyze the patient cluster and direct marketing focus shows great potential.

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42

Marques, Joana Rita Fernandes. "Breaking the ctDNA sensitivity barrier." Master's thesis, 2017. http://hdl.handle.net/10316/83284.

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Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e Tecnologia
Over the years, advances in the molecular and biological understanding of cancer has changed the paradigm of clinical practice from a systematic to a personalized and targeted therapeutic approach. However, an accurate molecular characterization of the tumor is only achieved through tissue biopsy, an invasive strategy that comprises many limitations. In this context, liquid biopsy, peripheral blood sample, comes as an alternative. Technological advances for detection and characterization of tumor-specific mutations in plasma have revealed its potential clinical relevance as biomarkers for tumor detection, response to therapy and disease follow-up. Despite the exciting breakthroughs, the intrinsic low abundance of circulating tumor DNA (ctDNA) makes the detection of such mutations in plasma a challenging task. This problem has been tackled by the development of high sensitive technologies but their complexity makes them difficult to implement in clinical routine. In alternative, our hypothesis was that pre-treatment of patients with cytotoxic drugs could increase the levels of ctDNA allowing the use of routine methodologies for the detection of tumor derived mutations in plasma. The major goal of this study was to test the effect of cytotoxic chemotherapeutic drug treatment on the levels of ctDNA. More specifically, we aimed to determine the effective therapeutic concentration with effect on ctDNA release and the ideal time point for blood collection after treatment. In order to address our hypothesis, in vitro assays were performed in lung cancer cell lines to establish the dose and time dependent effect of widely used cytotoxic chemotherapeutics. Liquid biopsies strategies were adopted to assess the effect of single drug treatment in DNA release levels both in vitro and in vivo using xenografted mice models. Additionally, identification of ctDNA was achieved through the detection of specific sequences of DNA derived from the plasma of xenografted mice.The results have shown that docetaxel was the most effective drug to reduce cell viability. This effect correlated to significant increase levels of late apoptosis in cells 48h after treatment with docetaxel. Additionally, in vitro drug treatment induced an increase in ctDNA release levels, with a greater effect at the 48h time point, suggesting an impact of docetaxel in cfDNA release. In vivo, a single dose treatment of docetaxel (25 mg/Kg) resulted on increased tumor apoptosis at 48h which correlates with increased levels of cfDNA detected in plasma. The specific detection of increased levels of tumor-derived DNA confirmed the influence of docetaxel treatment on ctDNA release. Furthermore, xenograft mice treated with docetaxel revealed increased mutational load when compared to untreated conditions, where the mutation was not detected. Despite preliminary, these findings provide evidence that a single treatment with docetaxel approximately 48h prior to liquid biopsy, in vivo, can contribute to higher levels of ctDNA and consequently overcome the problem of low sensitivity of detection. Moreover, the present work indicate apoptosis as the major release mechanism of ctDNA. Altogether, this study provided new insights into a novel strategy which might accelerate the implementation of ctDNA detection as a liquid biopsy strategy on the clinical routine and have an impact on clinical management of patients.
O estudo da biologia molecular do cancro tem permitido avanços científicos com impacto na prática clínica, mudando o paradigma de abordagem terapêutica convencional, sistémica, para uma abordagem personalizada. No entanto, a caracterização molecular de tumores sólidos só é possível através da biopsia do tecido, uma estratégia altamente invasiva com diversos riscos e limitações associadas. Neste contexto, a biopsia líquida, colheita de sangue, surge como uma alternativa. Avanços tecnológicos para deteção e caracterização de mutações específicas do tumor presentes no plasma permitiram a descoberta da relevância clínica do ADN circulante como biomarcador no diagnóstico, resposta a terapia e monitorização da doença. Apesar destas descobertas, a baixa abundância do ADN tumoral em circulação torna difícil a deteção de mutações relevantes. Estratégias para a resolução deste problema incluem o desenvolvimento de novas metodologias, mais sensíveis, para a deteção de mutações raras. No entanto estas estratégias são ainda muito complexas e dispendiosas para serem aplicadas na prática clínica. Assim, a nossa hipótese é que o pré-tratamento dos pacientes com quimioterápicos poderá aumentar os níveis de ADN tumoral em circulação permitindo o uso das metodologias disponíveis para deteção, no plasma, de mutações derivadas do tumor. O principal objetivo desta dissertação foi testar o efeito do uso de quimioterápicos nos níveis de ADN tumoral circulante de forma a ultrapassar a baixa sensibilidade de deteção das metodologias disponíveis na clínica. Especificamente, determinamos a dose terapêutica efetiva com impacto nos níveis de ADN circulante e o momento ideal para realização da biopsia líquida após tratamento de forma a maximizar a deteção de ADN tumoral circulante.Foram realizados ensaios em linhas celulares de cancro do pulmão de forma a determinar o efeito da concentração e do tempo do tratamento com quimioterápicos usados na prática clínica. De modo a determinar o efeito do tratamento com docetaxel na libertação de ADN circulante, estratégias de biopsia líquida foram aplicadas tanto in vitro como in vivo. Por fim, a deteção de ADN tumoral circulante foi feita com a utilização de sondas específicas para sequências de interesse do ADN tumoral.Verificámos que o docetaxel foi o quimioterápico mais eficiente a reduzir a viabilidade celular, o que foi concordante com o aumento dos níveis de apoptose verificados 48h após tratamento. Os níveis aumentados de ADN circulante, observados in vitro, após 48h de exposição ao docetaxel, sugerem o impacto do tratamento nos níveis de apoptose e consequentemente na quantidade de ADN circulante. In vivo, o tratamento com uma dose de docetaxel (25 mg/Kg) provocou o aumento dos níveis de apoptose 48h após tratamento, um aumento que também se verificou relativamente aos níveis de ADN circulante. Especificamente, foi possível detetar níveis aumentados de ADN proveniente do tumor no plasma de ratinhos tratados com docetaxel, quando comparado com ratinhos não tratados. Foi detetado um aumento na sensibilidade de deteção da mutação específicas do tumor no plasma de ratinhos após tratamento com docetaxel, o que não se verificou para os animais não tratados.Este estudo apresenta evidências preliminares de que uma dose única de docetaxel 48h antes da realização de biopsia líquida, in vivo, pode contribuir para o aumento dos níveis de ADN em circulação e consequentemente, ultrapassar o problema de sensibilidade de deteção de mutações raras. Neste estudo também foi possível verificar a importância do processo de apoptose como um mecanismo de libertação de ADN tumoral. Esta estratégia inovadora poderá agilizar a aplicação da deteção de ADN circulante (biopsia líquida) como biomarcador, na prática clínica, para a monitorização do cancro.
Outro - FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the projects "Institute for Research and Innovation in Health Sciences" (POCI-01-0145-FEDER-007274), and "Horizontal transmission of drug resistance: a game changer in the clinical management of cancer patients" (PTDC/DTP-PIC/2500/2014); and by Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER), in the framework of the project NORTE-01-0145-FEDER-000029.

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43

Sequeira, José Pedro Leite. "LiKidMiRS: unveiling the diagnostic potential of liquid biopsy-based circulating microRNAs in renal cell tumors." Master's thesis, 2021. https://hdl.handle.net/10216/138114.

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44

Oliveira, Inês Domingues. "Detection of BRAF V600E mutation in ctDNA by ddPCR: from laboratory to market." Master's thesis, 2018. http://hdl.handle.net/10362/53897.

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Cutaneous melanoma is the most aggressive form of skin cancer and has a high mortality rate. The current methods used to stage melanoma and detect metastases are imaging techniques and sentinel lymph node biopsies (SLNB). However, these have limited sensitivity for the detection of early stage melanoma and often can’t provide timely clinical evidence of disease recurrence or for monitoring therapies. Cell-free tumour DNA (ctDNA) could be a specific biomarker in melanoma patients which present V600E mutation in BRAF gene. ctDNA is released by cancer cells into the bloodstream and then excreted in urine, allowing it to be analysed by liquid biopsy. This analysis provides a real-time snapshot of tumour burden and represents a non-invasive, cost-effective alternative that can be performed repeatedly. In this work, a surveillance tool was implemented to help patients with melanoma through the detection of BRAF V600E mutation in ctDNA by Droplet Digital PCR (ddPCR). Firstly, we focused on design and optimization of the assay to ensure a low limit of detection and a distinction between false-positives and true-positives. Secondly, the assay was tested in order to show the possibility to detect V600E mutation in BRAF gene in plasma and midstream urine samples from melanoma patients. In addition to the test implementation, a go-to-market strategy was explored for the commercialization of the test through a collection kit. This test is directed to laboratories, clinics and hospitals aiming to help doctors in the diagnosis, prognosis and treatment of their patients. After a market analysis, we concluded that the new test has advantages over existing competitors in the market and has a potential to improve the melanoma management.

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45

Huang, Pei Ying, and 黃珮穎. "Industrial Analysis and Technology Valuation of the Liquid Biopsy: A Case Study of the Circulating Tumor Cell Enumeration Technology." Thesis, 2019. http://ndltd.ncl.edu.tw/cgi-bin/gs32/gsweb.cgi/login?o=dnclcdr&s=id=%22107CGU05105004%22.&searchmode=basic.

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46

Surani, Arif A., and Krzysztof Poterlowicz. "Circulating tumour DNA: a minimally invasive biomarker for tumour detection and stratification." 2016. http://hdl.handle.net/10454/11183.

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Ye
Genetic and epigenetic alterations significantly contribute to development of human cancer. Genotyping tumour tissue in search for these actionable genetic and epigenetic changes has become routine practice in oncology. However, sampling tumour tissue has significant inherent limitations. It provides only a single snapshot in time, prone to selection bias due to intra-tumour heterogeneity, and cannot always be performed owing to its invasive nature. Circulating tumour DNA (ctDNA) based liquid biopsy provides an effective alternative to invasive tissue sampling and have emerged as a minimally invasive, real-time biomarker. Recent advancements in DNA sequencing technologies have revealed enormous potential of ctDNA to improve tumour detection and stratification. In this review, we critically appraise the role of ctDNA as a liquid biopsy for cancer and evaluate the role of circulating tumour DNA as a diagnostic, prognostic and predictive biomarker. We also highlight some technical challenges and constraints associated with circulating DNA analysis.

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47

Shaheed, Sadr-ul, C. Tait, K. Kyriacou, R. Linforth, M. Salhab, and Chris W. Sutton. "Evaluation of nipple aspirate fluid as a diagnostic tool for early detection of breast cancer." 2017. http://hdl.handle.net/10454/14660.

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Yes
There has been tremendous progress in detection of breast cancer in postmenopausal women, resulting in two-thirds of women surviving more than 20 years after treatment. However, breast cancer remains the leading cause of cancerrelated deaths in premenopausal women. Breast cancer is increasing in younger women due to changes in life-style as well as those at high risk as carriers of mutations in high-penetrance genes. Premenopausal women with breast cancer are more likely to be diagnosed with aggressive tumours and therefore have a lower survival rate. Mammography plays an important role in detecting breast cancer in postmenopausal women, but is considerably less sensitive in younger women. Imaging techniques, such as contrast-enhanced MRI improve sensitivity, but as with all imaging approaches, cannot differentiate between benign and malignant growths. Hence, current well-established detection methods are falling short of providing adequate safety, convenience, sensitivity and specificity for premenopausal women on a global level, necessitating the exploration of new methods. In order to detect and prevent the disease in high risk women as early as possible, methods that require more frequent monitoring need to be developed. The emergence of “omics” strategies over the last 20 years, enabling the characterisation and understanding of breast cancer at the molecular level, are providing the potential for long term, longitudinal monitoring of the disease. Tissue and serum biomarkers for breast cancer stratification, diagnosis and predictive outcome have emerged, but have not successfully translated into clinical screening for early detection of the disease. The use of breast-specific liquid biopsies, such as nipple aspirate fluid (NAF), a natural secretion produced by breast epithelial cells, can be collected non-invasively for biomarker profiling. As we move towards an age of active surveillance, home-based liquid biopsy collection kits are increasingly being applied and these could provide a paradigm shift where NAF biomarker profiling is used for routine breast health monitoring. The current status of established and newly emerging imaging techniques for early detection of breast cancer and the potential for alternative biomarker screening of liquid biopsies, particularly those applied to high-risk, premenopausal women, will be reviewed.
Proteomics research was supported by Yorkshire Cancer Research projects, BPP047 and B381PA, and co-funded by the European Regional Development Fund and the Republic of Cyprus through the Research Promotion Foundation projects ΥΓΕΙΑ/ΒΙΟΣ/0311(ΒΙΕ/07) and NEKYP/0311/17.

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48

Carneiro, Adriana Filipa Ribeiro. "Circulating Tumour Cells: Paving the Way Towards the Identification of Epithelial and Mesenchymal Phenotypes in the Clinic." Master's thesis, 2020. http://hdl.handle.net/10316/92925.

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Dissertação de Mestrado em Biotecnologia Farmacêutica apresentada à Faculdade de Farmácia
O cancro colorretal (CCR) é o terceiro cancro mais incidente e o segundo com maior taxa de mortalidade a nível mundial. A principal causa de morte nestes doentes continua a ser a metastização, cerca de 60% dos mesmos são diagnosticados numa fase mais avançada da doença (fase III e IV). Há, por essa razão, uma necessidade urgente no desenvolvimento de estratégias que permitam o diagnóstico precoce e monitorização da doença metastática em CCR. Nesta perspetiva, as células tumorais circulantes (CTCs), libertadas pelo tumor para a corrente sanguínea, revelaram ter um papel importante como ferramenta para o diagnóstico e monitorização em tempo real do cancro. Estas refletem a heterogeneidade tumoral e a evolução clonal, bem como o seu isolamento e análise podem ser efetuados repetidamente ao longo do tratamento, de forma fácil, rápida e minimamente invasiva. Assim, o desenvolvimento de tecnologias que permitam a aplicação destas células na prática clínica é imperativo quer para a deteção precoce de potenciais recidivas, quer para a monitorização atempada da resposta do paciente ao tratamento. Este estudo teve como objetivo avaliar a eficiência de captura de um dispositivo de microfluídica, o RUBYchip™, desenvolvido no International Iberian Nanotechnology Laboratory (INL) com o propósito de capturar e caracterizar CTCs com base em duas características, o tamanho e a deformabilidade celular. A fim de otimizar o desempenho deste dispositivo, foram adicionadas concentrações definidas de células de diferentes linhas celulares de CCR (SW480, Caco2, RKO e HT-29) a 7,5 ml de sangue de voluntários saudáveis para posterior processamento a diferentes velocidades de fluxo de modo a aferir a eficiência de captura nestes tipos celulares. Posteriormente, a identificação e análise fenotípica de CTCs no RUBYchip™ foi efetuada por ensaios de imunocitoquímica com anticorpos que reconhecem a Citoqueratina, para uma seleção positiva dos CTCs; a Vimentina, expressa por CTCs em transição epitélio mesenquimatosa; e ainda, o CD45, para identificar e excluir as populações de células sanguíneas. Após otimização, as condições ótimas definidas foram aplicadas em amostras de sangue periférico de pacientes recrutados no Instituto Português de Oncologia do Porto, com o propósito de identificar subpopulações de CTCs, que potencialmente podem predizer diferentes respostas clínicas.
Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second with highest mortality worldwide. Metastases are the main cause of cancer-related mortality and 60% of patients are diagnosed at stages III and IV. Hence, there is an urgent need for the development of methodologies for the early diagnosis and monitoring of metastatic disease in colorectal cancer.Circulating tumour cells (CTC) are shed from tumours into the bloodstream and are emerging as a fundamental tool for cancer diagnosis and monitoring. Just like solid tumours, CTCs are very heterogeneous, hence reflecting tumour heterogeneity and clonal evolution of cancer in real time. Adopting CTCs into the clinical practice, particularly in CRC, is imperative for early detection of potential recurrences, and in individualized monitoring of the disease during treatment. However, current tools in the clinic have shown limited sensitivity and clinical utility. To overcome this limitation, a microfluidic technology for unbiased CTC isolation and characterization was developed at INL, the RUBYchip™, designed to capture CTC based on two important characteristics, cellular size and deformability. To optimize the performance of the device, four CRC cell lines (SW480, Caco2, RKO and HT-29) were spiked into 7.5 mL of healthy whole blood samples and processed at different flow rates to assess capture efficiency. Further identification and phenotypical analysis of CTCs in the microfluidic device was achieved by immunostaining with antibodies against Cytokeratin, to positively select CTCs; Vimentin, to identify CTCs that undergone epithelial-mesenchymal transition and CD45, to identify and exclude white blood cells.Subsequently to optimization studies, whole blood samples from metastatic CRC patients, recruited at IPO do Porto, were processed in the microfluidic device and further analysed, aiming to elucidate and identify CTC subpopulations that could be related to different clinical outcomes.

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49

Barreira, Ana Margarida Bento Cardoso Barros. "Extracellular Vesicle-based liquid biopsy in bladder cancer: establishing a protocol for the detection of urothelial carcinoma-derived EVs in the urine." Dissertação, 2020. https://hdl.handle.net/10216/131403.

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50

Barreira, Ana Margarida Bento Cardoso Barros. "Extracellular Vesicle-based liquid biopsy in bladder cancer: establishing a protocol for the detection of urothelial carcinoma-derived EVs in the urine." Master's thesis, 2020. https://hdl.handle.net/10216/131403.

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