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Tesis sobre el tema "Liquid biopsy"

Autor: Grafiati
Publicado: 4 de junio de 2021
Última modificación: 7 de julio de 2024

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1

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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2

BARBARESCO, FEDERICA. "Microfluidic devices: application for liquid biopsy". Doctoral thesis, Politecnico di Torino, 2021. http://hdl.handle.net/11583/2903504.

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3

GIORDANI, Elena. "Liquid Biopsy in Real Life Oncology". Doctoral thesis, Università degli studi di Ferrara, 2022. http://hdl.handle.net/11392/2481324.

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Secondo il report “I numeri del cancro in Italia 2020” dell’Associazione italiana registri tumori (AIRTUM) e l’Associazione italiana di oncologia medica (AIOM), il tumore al seno resta la neoplasia più frequente in Italia e la prima causa di morte per tumore nelle donne. Il sottotipo HER2+ rappresenta il 30% dei tumori al seno ed è associato ad una prognosi infausta, sebbene l’utilizzo di terapie mirate abbia reso questo sottotipo curabile come altri biologicamente meno aggressivi. Tuttavia, i pazienti in stadio avanzato, trattati con terapie antiHER2, spesso sviluppano resistenza al trattamento. La biopsia liquida (BL) è una metodica minimamente invasiva, di facile utilizzo, sensibile e specifica, in grado di individuare l’insorgenza della resistenza anche prima che questa si manifesti clinicamente, permettendo così di ridurre tossicità e costi di un trattamento inefficace. I pazienti che presentano alterazioni di HER2 beneficiano di una terapia mirata con molti inibitori. Il monitoraggio longitudinale tramite BL dovrebbe diventare un fattore chiave nella gestione della malattia e nella scelta della strategia terapeutica più appropriata, poiché tiene conto dell'idea di evoluzione del cancro nel contesto dei principi generali dell'oncologia di precisione. Ad oggi, lo stato di HER2 viene assegnato mediante una combinazione di Immunoistochimica e Ibridazione in Situ (IHC/ISH) su tessuto, in una scala binaria che separatamente valuta la sovra espressione e l'amplificazione genica. Ipotizzo che questo schema diagnostico (CDx), statico, una tantum, solo su tessuto, debba essere rivisto o abbandonato poiché assegna dei cut-off definiti per la terapia convenzionale antiHER2 (ad esempio, un tumore è HER2+ oppure no). Questa visione è probabilmente semplicistica, perché i nuovi agenti antiHER2 possono colpire sia i tumori HER2-alti che HER2-bassi, considerando che i livelli di HER2 diminuiscono durante la terapia. In questa tesi, propongo che i livelli di HER2 vengano valutati longitudinalmente, (su una scala continua, e in maniera bimodale), correlando la sovra espressione e la variazione del numero di copie (CNV) di HER2 e co-fattorizzandoli in un nuovo CDx. Questo permetterebbe la riallocazione dinamica dei pazienti ai diversi sottotipi e l’assegnazione di un trattamento non standard in un contesto dinamico che modifichi la pratica clinica comune. Presento dei dati che dimostrano come la BL possa aiutare nel ridisegnare il CDx nel cancro al seno avanzato. Riassumo LiqBreasTrack, un recente studio di NGS, condotto durante la mia tesi (Allegretti, Fabi; Molecular Cancer 2021) e descrivo HER2-2D, un saggio bidimensionale inedito di BL che stima le CNV e il livello di proteina di HER2 nella prima e nella seconda dimensione, rispettivamente. Il test sfrutta saggi custom di dPCR e un ELISA commerciale, è ugualmente applicabile a tessuti e sangue e dà come output finale un punteggio di HER2 cumulativo. L'applicazione principale è nel sangue, e descriverò un sottogruppo di tumori/pazienti con cancro al seno in stadio avanzato elettivamente suscettibili al blocco di HER2 da parte di Trastuzumab Emtansine (T-DM1), un potente anticorpo-coniugato a farmaco (ADC). Accennerò ad una collaborazione con il gruppo del Prof. F. Michelotti (Università Sapienza di Roma), orientata allo sviluppo di un biosensore Surface Plasmon Resonance Imaging (SPRI) per la rilevazione rapida, semplice e senza marcatura estrinseca degli anticorpi antiHER2, per future applicazioni in ambito dell’Health Technology Assessment. In sintesi, attraverso la BL dimostro che HER2 è una caratteristica adattiva dei tumori, e i suoi cambiamenti (tra quelli genomici più generali) nel sangue possono essere studiati per assegnare in modo adattivo terapie bersaglio a coorti definite di pazienti con cancro al seno caratterizzate da diversi gradi di dipendenza oncogenica da HER2.
According to the data contained in the report "I numeri del cancro in Italia 2020" edited by the Italian Association of Tumour Registers (AIRTUM) and the Italian Association of Medical Oncology (AIOM), breast cancer remains the most frequent neoplasm in Italy and the leading cause of death from cancer in women. HER2+ subtype breast cancers represent 30% of all breast cancers and used to be associated with a poor prognosis, although the application of targeted HER2 blockade has rendered this subtype at least as curable as other biologically less aggressive breast cancer subtypes. Unfortunately, patients with advanced breast cancer treated with anti-HER2 therapies almost invariably develop pharmacological resistance. Liquid biopsy (LB) is minimally invasive, easy to perform, highly sensitive and specific. It may detect molecular traits of resistance even before clinical manifestations of progression, which may reduce unnecessary anti-HER2 treatment, abating unwanted side effects, toxicity, and treatment-associated costs. Patients with tumors bearing HER2 alterations benefit from target therapy with many specific inhibitors. Longitudinal monitoring by LB is expected to become a key factor in disease management and the use of these targeted therapies, because it takes into account the idea of cancer evolution in the context of the general principles of precision oncology. The HER2 status is presently assessed by a combination of Immunohistochemistry and In Situ Hybridization (IHC/ISH) to detect gene overexpression and amplification in tissues on a binary scale that separately factors overexpression and gene amplification. I hypothesize that a static, one-time-only, tissue-only HER2 Companion Diagnostics (CDx), like the one we presently use, should be revised, or dismissed. This scale assigns defined cut-offs for conventional HER2 blockade therapy, e.g., tumors are either HER2 or non-HER2. This view is probably simplistic, because novel anti HER2 agents may effectively target both HER2-high and HER2-low tumors, and HER2 levels wane during therapy. In this thesis, I defend the hypothesis that HER2 functional expression should be assessed longitudinally (on a continuous scale, and bimodally), e.g. (over)expression and Copy Number Variation (CNV) should be fully co-factored into a novel CDx scheme, enabling dynamic reallocation of patients to different subtypes and assign non-standard treatment in a potentially practice-changing setting. I present data showing that LB may help redesigning CDx in advanced breast cancer. As an example, I briefly summarize LiqBreasTrack, a recent LB NGS study, carried out during my thesis, published at the time of writing (Allegretti M., Fabi A. et al. Molecular Cancer 2021). I also describe HER2-2D, a bidimensional LB assay that estimates HER2 CNV and HER2 protein level in the first and second dimensions, respectively. This is presently unpublished and personally developed. The assay takes advantage of customized digital PCR and a commercial ELISA, it is equally applicable to tissues and blood, and yields a cumulative HER2 score. HER2-2D main application is in blood, and I will describe a subset of breast cancer tumors/patients electively susceptible to HER2 blockade by Trastuzumab Emtansine (T-DM1), a potent Antibody-Drug Conjugate (ADC) targeting HER2. I will briefly mention a collaboration with the laboratory of Prof. Francesco Michelotti at the University of Sapienza, Rome, aimed at the construction of a novel Surface Plasmon Resonance Imaging (SPRI) biosensor for the rapid, simple and label-free detection of HER2, for future applications in the Health Technology Assessment area. In summary, thorough LB we show that HER2 is an adaptive tumor feature, and that its changes (amongst more general genomic changes) in blood may find application to adaptively assign target therapy to defined, distinct cohorts of breast cancer patients characterized by different degrees of HER2 oncogenic addiction.
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Palmieri, Maria. "CfDNA-NGS Liquid Biopsy for solid cancers and vascular malformations". Doctoral thesis, Università di Siena, 2020. http://hdl.handle.net/11365/1120548.

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The liquid biopsy is a new emerging and repeatable low risky approach able to detect drive mutations that characterize the tumor, to monitor cancer evolution over time, and to overcome the standard tissue biopsy limits. The biomarker par excellence is the circulating cell-free DNA (cfDNA) that was the principal leading actor of this study. The scope of this study was to perform different liquid biopsy analysis both in metastatic cancer and in vascular malformations patients to detect, from a precision medicine perspective, the sniper clone responsible for the tumor evolution or the vascular malformations. The cfDNA was extracted from plasma coming from peripheral and/or efferent vein of vascular malformation. The obtained cfDNA was used to perform the libraries using two different genes panel of 52 and 77 cancer-driver genes, respectively the Oncomine™ Pan-Cancer Cell-Free Assay and AVENIO ctDNA Expanded Kit. The most frequent mutations that we found in metastatic patients were the SNV in TP53, follow by PIK3CA, KRAS, and CNV in FGFR3. In the majority of cases, the mutations found at first liquid biopsy were confirmed by an increased allele frequency at the second one. In vascular anomalies affected patients, the PIK3CA, MET, and KRAS mutated genes were found in Klippel-Trenaunay syndrome, in lymphovenous malformations, and in artero-venous malformations respectively, with a very low allele frequency percentage. In conclusion, repeated analysis of liquid biopsy lead to the identification of key cancer genes and the following of clonal evolution over time. Moreover, the liquid biopsy is suitable not only for cancer patients but also for the diagnosis of vascular malformation. Our data prove that in the new era of precision medicine, this novel approach, based on the combination of NGS and liquid biopsy from the efferent vein at the vascular malformation site, allows to detect even low-grade somatic mosaicism responsible for the vascular phenotype. This approach let to bypassing the need for a highly risky tissue biopsy and lead to a tailored personalized treatment.
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5

Soda, Narshone. "Advanced Liquid Biopsy Technologies for Circulating Cancer Biomarker Detection". Thesis, Griffith University, 2021. http://hdl.handle.net/10072/406071.

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Epithelial ovarian cancer is one of the most prevalent gynaecological cancers in women and is often diagnosed in the late stage due to the mild symptoms. Currently, ovarian cancer screening is reliant on the prevailing usage of blood based CA125 protein biomarker and transvaginal ultrasound which can detect ovarian cancer in the preclinical phase in a substantial portion of cases. However, there are also other elements that can result in elevated CA125 levels such as menstruation, endometriosis or ovarian cysts. As such, the lack of accurate disease risk classification during ovarian cancer screening has led to several health burdens associated with unnecessary biopsies and overtreatment of patients. Thus, new diagnostic methods with improved sensitivity and specificity for ovarian cancer are a clinical priority. To address the enigma associated with ovarian cancer screening, liquid biopsy technologies have been developed. Molecular profiling of liquid biopsies has the potential to detect changes associated with the tumuor in collected, non-invasively body fluid samples. Detection of tumour origin biomolecules such as; circulating tumour cells (CTCs), circulating tumour specific nucleic acids (ctDNA, ctRNA, miRNAs, lnRNAs), exosomes, autoantibodies in blood, saliva, stool, urine etc. has brought about a paradigm shift in the management and diagnosis of cancer. From reliance on painful and hazardous tissue biopsies or sophisticated equipment dependent imaging, cancer management schemes are witnessing rapid evolution towards minimally invasive yet highly sensitive liquid biopsy-based tools. Clinical application of liquid biopsy is already paving the way for precision theranostics and personalised medicine, especially by enabling repeated sampling, which in turn provides a more comprehensive molecular profile of tumours. On the other hand, integration with novel miniaturised platforms, engineered nanomaterials, as well as electrochemical detection has helped in the development of low cost and simple platforms suited for point-of-care application. Despite excellent analytical performances of the existing detection methodologies, electrochemical approaches offer a promising alternative for simple, sensitive, specific, rapid, and cost effective analysis of genetic and epigenetic biomarkers in cancer samples. Therefore, innovative technology using electrochemical approach would be an effective method for the detection of biomarkers in patients with cancer. This thesis focuses on the use of nucleic acids (i.e., genetic and epigenetic) biomarkers, specifically HOX antisense intergenic RNA (HOTAIR) lncRNA and DNA methylation to identify tumour specific changes and their performance as diagnostic biomarkers in non-invasively collected biofluid samples. Novel electrochemical and colourimetric approaches have been demonstrated for the construction of a sensitive, and specific biosensor platform for the complex task of detecting and quantifying circulating ovarian cancer biomarkers. To achieve this goal, first a comprehensive literature review on the biogenesis, significance, and potential role of four widely known biomarkers (CTCs, ctDNA, miRNA and exosomes) in cancer diagnostics and therapeutics has been provided. A detailed discussion of the inherent biological and technical challenges associated with currently available methods and the possible pathways to overcome these challenges is also provided. The recent advances in the application of a wide range of nanomaterials in detecting these biomarkers are also highlighted. Next, an amplification-free electrochemical method for the detection of HOTAIR lncRNA was developed. In this method, HOTAIR sequences were magnetically isolated, purified and detected by a sandwich hybridisation method at a screen-printed gold electrode (SPE-Au). This event was monitored by amperometry using the hydrogen peroxide/horseradish peroxidase/hydroquinone (H2O2/HRP/HQ) system which enabled a catalytic enhancement of the signal. In the following chapter, a more sensitive assay was discussed which utilised colourimetric and electrochemical readout for HOTAIR detection. In this approach, subsequent detection of magnetically purified and isolated sequences was performed using the sandwich hybridisation event coupled with HRP-catalysed reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H2O2 which facilitated the naked eye observation and enabled an alternative amperometric quantification of HOTAIR. We then explored the bioengineering and characterisation of self-assembled superparamagnetic polyhydroxybutyrate (PHB) nanobeads for the development of a platform method for the analysis of circulating biomarkers, where these nanobeads were modified with specific bio-recognition antibodies, dispersed in analyte fluids where they worked as “dispersible capture agents” to bind specific targets. The enormous active sites of PHB nanobeads allow the direct attachment of a larger number of antibodies which can significantly enhance the capture efficiency. Their magnetic property allows magnetic nanoparticle-based mixing, separation and purification which can improve assay performance by reducing the matrix effects of the biological samples, as non-target species can be removed via magnetic isolation and purification steps. Two common circulating biomarkers namely global DNA methylation and exosomes were chosen for this method. After purification and magnetic collection, the isolated targets were directly adsorbed onto a screen-printed gold electrode (SPE-Au) and electrochemically quantified using a catalytic redox cycling system of hydrogen peroxide/horseradish peroxidase/hydroquinone (H2O2/HRP/HQ). In another approach, to simplify the assay protocol, the PHB nanobeads were directly adsorbed onto the SPE-Au electrode via PHB-gold affinity interaction followed by the immune attachment of the methylated DNA targets onto the surface-bound PHB nanobeads/anti 5mC-HRP conjugates. The targets were then quantified using the similar catalytic redox cycling of H2O2/HRP/HQ. Lastly, the clinical utility of these novel technologies was demonstrated using ovarian cancer cell lines and a cohort of well-annotated patient samples. This illustrates an attempt to translate the developed technologies from an academic research phase to patient usage by assessing the clinical performance metrics. To date, there are various ovarian cancer treatment options such as surgery, chemotherapy, targeted therapy, radiation therapy and palliative treatment. Each treatment method is dependent on various factors such as the cancer stage, gene type, overall health and fitness, as well as the desire to bear children. Thus, it is envisioned that the research that integrates new cutting-edge biomarkers and innovative detection strategies (as showcased in this thesis) could advance ovarian cancer diagnosis and risk stratification in clinical settings. This will enable a more personalised treatment approach accustomed to the needs of individual patients.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
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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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Diop, Fary. "Diffuse large B-cell lymphoma genotyping on the liquid biopsy". Doctoral thesis, Università del Piemonte Orientale, 2018. http://hdl.handle.net/11579/105207.

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Accessible and real-time genotyping for diagnostic, prognostic, or treatment purposes is increasingly impelling in diffuse large B-cell lymphoma (DLBCL). Cell-free DNA (cfDNA) is shed into the blood by tumor cells undergoing apoptosis and can be used as source of tumor DNA for the identification of DLBCL mutations, clonal evolution, and genetic mechanisms of resistance. In this study, we aimed at tracking the basal DLBCL genetic profile and its modification upon treatment using plasma cfDNA. Ultra-deep targeted next generation sequencing of pretreatment plasma cfDNA from DLBCL patients discovered DLBCL-associated mutations that were represented in >20% of the alleles of the tumor biopsy with >90% sensitivity and~100% specificity. Plasma cfDNA genotyping also allowed for the recovery of mutations that were undetectable in the tissue biopsy, conceivably because, they were restricted to clones that were anatomically distant from the biopsy site. Longitudinal analysis of plasma samples collected under (R-CHOP) chemotherapy showed a rapid clearance of DLBCL mutations from cfDNA among responding patients. Conversely, among patients who were resistant to R-CHOP, basal DLBCL mutations did not disappear from cfDNA and, moreover, among treatment-resistant patients, new mutations were acquired in cDNA that marked resistant clones selected during the clonal evolution. These results demonstrate that cfDNA genotyping of DLBCL is as accurate as genotyping of the diagnostic biopsy to detect clonally represented somatic tumor mutations and is a real-time and noninvasive approach to tracking clonal evolution and the emergence of treatment-resistant clones
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8

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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MARALANI, Mahafarin. "Liquid Biopsy: A Next Generation Diagnostic And Prognostic Tool In Solid Malignancies". Doctoral thesis, Università degli Studi di Palermo, 2020. http://hdl.handle.net/10447/401539.

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DO, REGO BARROS FERNANDES LIMA MARIA AUGUSTA. "Investigating Label-Free markers at Nanoscale for Liquid Biopsy Using Multimodal Microscopy". Doctoral thesis, Università degli Studi di Trieste, 2021. http://hdl.handle.net/11368/2995896.

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Liquid biopsy emerges as a noninvasive, easily repeatable, and potentially low-cost approach alternative to standard tissue biopsy. In most cases, it can be used to investigate the cause of symptoms or to help diagnose a number of different health conditions. Although originally used to designate analysis of non-solid tissues to screen for cancer cells, liquid biopsy also refers to the investigation of other general body fluids including its constituents characterization and not necessarily related to cancer. In this thesis, three new applications for the usage of label-free markers in the analysis of body fluid cellular constituents will be presented. Digital holographic microscopy and optical tweezers are applied to the characterization of ex-vivo generated and native red blood cells. In a second application, neutrophils precursors are characterized and classified according to its cellular and nuclear morphology during granulocytic differ- entiation. In a third proposed application, morphological markers retrieved by digital holographic microscopy are used to perform fast screening urinalysis, including leukocyturia and bacteriuria. Lastly, although not label-free, fluorescence superresolution microscopy is used to bring insights into why nuclear morphology can be used as a trustful label-free marker and shows the structural arrangement of lamin in the nucleus of neutrophil precursors with unprecedented resolution. Fast screening label-free liquid biopsies integrates the group of emerging approaches that will revolutionize the future of early disease diagnosis and therapeutic choice with disruptive impact on the society. All the investigations described in this Thesis were aimed to contribute to this promising and intriguing new scenario.
Liquid biopsy emerges as a noninvasive, easily repeatable, and potentially low-cost approach alternative to standard tissue biopsy. In most cases, it can be used to investigate the cause of symptoms or to help diagnose a number of different health conditions. Although originally used to designate analysis of non-solid tissues to screen for cancer cells, liquid biopsy also refers to the investigation of other general body fluids including its constituents characterization and not necessarily related to cancer. In this thesis, three new applications for the usage of label-free markers in the analysis of body fluid cellular constituents will be presented. Digital holographic microscopy and optical tweezers are applied to the characterization of ex-vivo generated and native red blood cells. In a second application, neutrophils precursors are characterized and classified according to its cellular and nuclear morphology during granulocytic differ- entiation. In a third proposed application, morphological markers retrieved by digital holographic microscopy are used to perform fast screening urinalysis, including leukocyturia and bacteriuria. Lastly, although not label-free, fluorescence superresolution microscopy is used to bring insights into why nuclear morphology can be used as a trustful label-free marker and shows the structural arrangement of lamin in the nucleus of neutrophil precursors with unprecedented resolution. Fast screening label-free liquid biopsies integrates the group of emerging approaches that will revolutionize the future of early disease diagnosis and therapeutic choice with disruptive impact on the society. All the investigations described in this Thesis were aimed to contribute to this promising and intriguing new scenario.
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11

Rauof, Goran y 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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IANZA, ANNA. "VALIDATION OF PREDICTIVE AND PROGNOSTIC BIOMARKERS AS A GUIDE FOR A PERSONALIZED APPROACH IN SOLID TUMOURS". Doctoral thesis, Università degli Studi di Trieste, 2020. http://hdl.handle.net/11368/2973745.

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Breast cancer (BC), Colorectal Cancer (CRC) and Non-Small Cell Lung Cancer (NSCLC) are among the most commonly diagnosed solid tumors, and occupy the first places in the mortality rankings. Compared to an old fashioned one-size-fits-all approach, precision medicine offers the possibility to accurately choose the most appropriate therapeutic strategy, that fits the patients not only from the clinical (age, comorbidities) but also from a molecular point of view. A genetic and biological understanding of the tumor, integrated with a weighted analysis of results can help the clinician in designing a therapeutic pathway that, ideally from the start, gives the patients the best response rates. The aim of my research is to evaluate the markers that have the greatest impact on the prediction of therapy response. Mutational analysis revolutionized the NSCLC treatment paradigm and, consequently, improved the prognosis. EGFR mutated patients benefit from target therapy with tyrosine kinase inhibitors. A fluid and longitudinal monitoring of mutational status is becoming a key factor in disease management. Firstly we extracted circulating free DNA (cfDNA) from the plasma of 30 patients with EGFR-mutated NSCLC and assessed mutational status with real-time PCR. We then monitored such mutation during target therapy in 19 patients. The liquid biopsy had a sensitivity of 60% in confirming the tissue mutation. Patients whose EGFR mutation was not detectable on plasma had a longer Progression free survival (PFS) and Overall survival (OS). Next step will be assessing if cfDNA analysis allows early detection of resistance mutation such as T790M. Next part of my research focused on luminal BC, working partially retrospectively on data from a phase III study of 90 ER-positive, HER2 negative locally advanced breast cancer patients that were randomly assigned 1:1 to receive Let 2,5 mg daily and metronomic oral Cyc 50 mg daily with (arm B; n=45) or without (arm A, n=45) sorafenib 400 mg/bid daily for six months as neoadjuvant treatment. The predictive role of Ki67, SUV variations and metabolic response and its changes with regards to clinical response and survival was analyzed. The serum of 32 patients was analyzed via Luminex Multiplex Panel technology. 38 analytes (cytokines and growth factors) were simultaneously measured according to arm of treatment and time of sample collection (before and after treatment). Patients were divided into groups according to response to therapy (RECIST). Then we investigated a possible link between chemotherapy-induced RNA disruption and survival/progression. Analysis were performed on 40 biopsies taken at baseline and 15 days after the beginning of the neoadjuvant therapy. The RNA for each sample or subdivided sample was then assessed using the RNA Disruption Assay. The maximum RNA disruption Index (RDI) value for each patient at day 15 was used for all analyses. Finally, We investigated the discordance of mutational status between primary and metastatic site in colorectal cancer.Patients with metastatic CRC who underwent surgery of both primary and metastasis were retrospectively evaluated, and mutational status assessment of K-RAS, N-RAS, BRAF and PIK3CA was performed on 21 patients. Median DFS was 20.5 months (95% CI 9.9-29.6) in patients with concordance in mutational status versus 10.4 months (95% CI 6.1-not reached) in patients with discordance (p=0.01) and median OS was 35.9 months (95% CI 26.3-not reached) in patients with concordance in mutational status 25.6 months (95% CI 6.6-not reached) in patients with discordance (p=0.038). In conclusion discordance seems related to clinical outcome. Overall my results show that new strategies and technologies allow the researchers and the clinicians to strive for a better and more complete understanding of solid tumors complex evolution, an integrated and focused approach to the early disease could become the future of disease management.
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SANTORELLI, LUCIA. "Proteomic analysis of urine-based liquid biopsy to provide new insights into renal diseases". Doctoral thesis, Università degli Studi di Milano-Bicocca, 2020. http://hdl.handle.net/10281/263397.

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L'area di indagine delle paotlogie renali è un campo ampio e complesso e molte delle cause che sono alla loro base non sono completamente comprensibili o curabili. A tal motivo, oltre ad una migliore comprensione della loro eziopatogenesi, è necessario individuare in maniera precisa i fattori di rischio, al fine di migliorare la prognosi e personalizzare il trattamento terapico. Lo sviluppo e l’implementazione di nuovi approcci, come l'analisi proteomica basata sulle urine, come biopsia liquida è attualmente una delle scelte più giuste da compiere. Infatti, l'uso dell'analisi proteomica per la scoperta di proteine clinicamente rilevanti, note come proteomica clinica, individuando ad esempio marcatori diagnostici e prognostici, può effettivamente favorire la traslazione delle scoperte di base in applicazioni cliniche a beneficio del paziente. Un campione biologico facilmente accessibile, come le urine, in tal senso, si rivela essere una preziosa fonte di biomarcatori per patologie renali. Le urine, infatti, possono essere raccolte in grandi quantità e in modo non invasivo; inoltre, sono meno complesse di altri fluidi corporei e per questo più semplici da studiare. Purtroppo, in diversi casi, le preziose informazioni presenti nelle urine sono spesso tecnicamente difficili da estrarre perché le proteine legate alla malattia sono spesso presenti in concentrazioni molto basse e per di più nascoste da proteine urinarie presenti in grande abbondanza, come l'albumina o l'uromodulina. In questo contesto, lo studio proteomico degli esosomi urinari (EU) rappresenta una valida alternativa, per rivelare e scoprire questo interessante paesaggio molecolare nascosto. EU sono vescicole di dimensioni nanometriche (>150 nm), che possono provenire da cellule endoteliali, podociti o cellule epiteliali tubolari. La loro composizione molecolare dipende dal tipo e dalla condizione fisiologica della cellula da cui si originano. Inoltre, l'impiego degli EU permette di ridurre la complessità del proteoma urinario: essi, infatti, contengono solo il 3% delle proteine urinarie totali (>3000 specie). Per tutte queste ragioni, alla stregua delle urine, possiamo considerare anche gli EU come una biopsia liquida, modalità non invasiva, in grado di fornire informazioni diagnostiche e prognostiche relative alle più disparate patologie renali. In questo studio, abbiamo applicato gli approcci di spettrometria di massa (MS) e proteomica applicata allo studio del proteoma e del glicoproteoma di campioni urinari provenienti da pazienti affetti da carcinoma renale a cellule chiare (ccRCC), il più frequente e aggressivo istotipo di carcinoma renale. In aggiunta, abbiamo anche studiato il contenuto proteico di EU di pazienti affetti da sindrome nefrosica idiopatica (INS), la più diffusa malattia glomerulare infantile. Grazie a questo approccio complementare, abbiamo raggiunto il duplice obiettivo di individuare una specifica signature proteica e glicoproteica di progressione tumorale (nel caso di cc-RCC) e di chiarire l'eziopatogenesi della malattia e il meccanismo molecolare che sta alla base della diversa risposta al trattamento farmacologico e dell'insorgenza della farmacoresistenza ai corticosteroidi (nel caso di INS). Combinando i risultati ottenuti, speriamo di sostenere e rafforzare ulteriormente l'impiego della biopsia liquida nella ricerca clinica, sia da un punto di vista tecnico che da un punto di vista applicativo. Inoltre, sfruttando le possibilità legate all'impiego della MS, speriamo di arrivare alla conversione dei nostri risultati in strumenti diagnostici o prognostici che possano migliorare la gestione dei pazienti affetti da ccRCC e INS.
The area of kidney diseases is a wide and complex field, and many conditions leading to them are not fully understood or curable. Along with a better understanding of these pathologies, there is a need for improved risk detection, for determination of prognosis and for improved and personalized treatment. Therefore, it is important to develop and implement new approaches, such as proteomic analysis of urine- based liquid biopsy. Indeed, the use of proteome analysis for the discovery of clinically relevant proteins, known as clinical proteomics, for example for the identification of earlier and prognostic markers may actually foster the translation of basic discoveries into clinical applications for the benefit of the patient. Easily accessible biological sample, such as urine is valuable sources of biomarkers for pathologies related to kidney. In fact, urine can be collected in large quantities and in non-invasive way; additionally, it is less complex than other bodily fluids. Unfortunately, in diverse cases, this information is often technically difficult to be mined because the disease-related proteins are often present in very low concentrations, are frequently labile, and are hidden by high-abundance proteins such as albumin or uromodulin. In this context, the proteomic study of the urinary extracellular vesicles (UEv) represents a valid alternative to reveal and discovery these hidden molecular landscape. UEv are nanometer-sized vesicles (>150 nm), that can originate from endothelial cells, podocytes or tubular epithelial cells. Their molecular composition depends upon the type, and even tatus, of the producer cell. Therefore, the use of UEv allows to reduce the complexity of the urine proteome, because they contain only 3% of total urine proteins (>3000 species). For all these reasons, we can consider also them (as the urine), as a liquid biopsy, non-invasive modality that can provide diagnostic and prognostic information about kidney disease. In this study, we applied the proteomics MS-based approaches to investigate the proteome and glycoproteome in urine samples of patients affected by clear cell Renal Cell Carcinoma (ccRCC), the most frequent and aggressive type of renal carcinoma. Additionally, we also studied the protein content of UEv of Idiopathic Nephrotic Syndrome (INS), the major childhood glomerular disease. By our complementary approaches, we gained the double aim: firstly, to pinpoint a characteristic specific disease protein and glycoprotein signature of tumour progression (in case of cc-RCC); secondly, to clarify the disease etiopathogenesis and the molecular mechanism underling the different response to drug treatment and the onset of pharmacoresistance to corticosteroids (in case of INS). By combining the obtained results, we hope to enforce further the use of liquid biopsy in clinical research, from both a technical and application standpoint. Furthermore, by taking advantage of the possibilities related to the employment of the MS technologies, we also expect that this can lead to the direct translation of our findings into diagnostic or prognostic tools that can improve the clinical management of ccRCC and INS patients.
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14

Hamid, Faysal-Bin. "Genetic profiling of circulating tumour cells and DNAs in patients with colorectal carcinoma". Thesis, Griffith University, 2021. http://hdl.handle.net/10072/404861.

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Over the decades, it has been considered that blood-based biopsy or liquid biopsy could be an alternative approach of tumour biopsy. To date, blood-based biomarkers such as circulating tumour cell (CTC) and circulating tumour DNA (ctDNA) have been widely studied and found their significance in patients with several carcinomas including colorectal carcinoma (CRC). Biologically, both CTC and cell-free DNA (cfDNA) have been released from the tumour and circulate freely in the blood. Although they are present in trivial quantity due to minute scale of discharge and rapid clearance from the blood, the status of the circulating biomarkers at a given time-point can be “snapshot” of the entire tumour landscape. With the advancement of detection techniques, trace quantity of biomarkers can even be detected in the blood while the existing diagnostic biomarkers, e.g., CEA often fails to detect the disease at the early stages of CRC. In addition, an elevation of CTC and ctDNA levels have been observed in patients with advanced stages of CRC suggested the possible role in CRC diagnosis. Molecular analyses of the biomarkers revealed that of CTC and ctDNA have distinct biological, genetic and genomic signatures compared to the primary tumours which can highlight on the critical insights of their lifecycle and pinpoint the novel therapeutic targets. In the thesis, we have presented the isolation techniques, novel biological and molecular characteristics and clinical relevance of CTC and ctDNA in patients with CRC. For CTC detection, we used two techniques- immunomagnetic negative selection and filtration method. Ten millilitres (ml) of the whole blood were collected from the healthy individuals and patients with CRC. To validate the techniques, three colon cancer cell lines SW 48, SW 480 and HCT 116 were spiked in the blood of healthy persons and recovered using the two techniques. After validation, we isolated CTCs from patients with CRC and detected using epithelial cell adhesion molecule (EPCAM) and cytokeratin 18 (CK 18) based immunofluorescence experiments. We have also studied novel morphological characteristics of CTCs from their sizes and phenotypical characteristics of CTCs from different expressions of two proteins in CTCs. In addition, genetic heterogeneities of the CTCs were studied using mRNA expression profiling of a novel multigene panel and compared to the primary tumours. Next, the precise role of the individual CTCs likely could be obscured due to the other contaminated blood cells. Therefore, we have isolated 28 single CTCs from eight patients with CRC to study the genetic heterogeneity of CTCs in single-cell resolution. We have initially validated our single-cell-analysis platform using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression in six single cells from colon cancer cell lines. Then we used a panel of 19 genes to investigate the single CTCs (n=28), primary carcinomas (n=8), and colon cancer cell lines (n=6) using real-time qPCR (RT-qPCR). In addition, we compared the number of CTCs and gene expression patterns of CTCs with pathological stages to assess the correlation with pathological stages of CRC. Moreover, we aimed to study the novel biological characteristics of cell-free DNAs (cfDNA) such as the concentration and DNA integrity in the plasma of patients with CRC. The cfDNA samples were extracted from the plasma samples of patients with CRC and healthy donors. The measurement of cfDNA concentration was performed using two approaches: Alu-based qPCR and Qubit techniques. In the Alu-based qPCR approach, the ratio of the long (Alu 247) and short (Alu 115 and Alu 81) amplicons represented cfDNA integrity (cfDI). To compare the efficiencies of these methods, ROC (Receiver-Operative Characteristic) curve was analysed. Furthermore, only a few of the DNA fragments called ctDNA carry mutations found in the cfDNA pool. Hence, we sought to investigate the genomic status of ctDNA by detecting a KRAS mutation (G12C) using duplex digital PCR (dPCR) in the plasma of patients with CRC. Given that screening KRAS exon 2 and 3 mutations are frequently used to diagnose CRC in clinical practise and G12C mutation is barely (approximately 3%) found in patients with CRC. The rational of KRAS G12C selection was to evaluate the sensitivity and specificity of dPCR technique in KRAS mutation testing in clinical settings. Followed by a validating step with commercially DNA fragment harbouring KRAS G12C mutation, we used the duplex dPCR to detect KRAS wild-type (WT) and G12C mutation from the plasma of patients with CRC. Finally, we compared the concentration, DNA integrity and frequency of KRAS G12C allele of ctDNAs in the plasma with pathological stages to investigate the association with pathological stages of CRC. We have detected CTCs from 61.3% and 69.3% of patients with CRC using negative selection and filtration methods, respectively. The total number and diameter of CTCs were significantly higher (p<0.0001) in the advanced stages of CRC. In addition, the number of the novel EPCAM+CK18 and E-Cadherin+MMP9 phenotypes of CTCs detected by both techniques were significantly higher in patients with advanced pathological stages of CRC. Gene expression profiling of CTCs unravelled three unique CTC subtypes expressing epithelial, epithelium-mesenchymal transition (EMT) and stemness features, which were divergent from the primary tumour specimens. Also, significant alterations in expressions of EPCAM, HRAS, BRAF, TP53, SLUG, TWIST1, CD44 and MMP9 genes of CTCs were observed compared to the primary tumours in patients with CRC. Moreover, gene expression profiling of the single CTCs displayed an extensive heterogeneity of the selected genes among the CTCs. Hierarchical clustering analyses highlighted diverse variations between CTCs and the primary tumours. In addition, the genetic heterogeneities were observed in an individual patient as well as different patients. Our results showed that AKT1 expression in CTCs was significantly (p= 0.0129) higher in advanced pathological stages compared to early stages of CRC. We have also measured the concentration of cfDNA using ALU-sequences and found relatively higher Alu 247 amplicon in patients with CRC compared to the healthy donors. Similarly, fluorometry-based Qubit 2.0 was also showed higher cfDNA concentration in patients with CRC. For the first time, we showed cfDI 2 was significantly higher and had more diagnostic accuracy than cfDI 1. We observed significantly higher concentration of Alu 247 and Alu 115 as well as cfDI 1 and cfDI 2 in patients with advanced pathological stages than early stages of CRC. Furthermore, we have detected G12C allele harbouring ctDNA from 48.53% (33 out of 68) patients with CRC. Our dPCR platform could detect efficiently as low as 0.1% ctDNAs in the validation step. MAF of G12C alleles was observed significantly higher (p= 0.0308) in patients with advanced stages than the early stages of CRC. In conclusion, this study has found that both CTC and ctDNA have the potential to predict and diagnosis of CRC. For CTC isolation, the filtration method is better than immunomagnetic-based negative selection. Also, CTC analyses revealed that CTCs could be heterogeneous in different ways-morphologically, phenotypically, genetically in an individual patient as well as different patients of CRC. Nevertheless, the biological features such as concentration and fragmentation patterns of cfDNA fragments are also distinct in the plasma of patients with CRC. Assessment of dPCR-based KRAS mutation harbouring ctDNA from plasma can be an alternative approach for the early detection of CRC. Therefore, sensitive and accurate detection of CTC and ctDNA will assist in the early screening and progression of CRC. Molecular analyses of CTC and ctDNA can offer critical insights of the drug resistance and offers improved therapeutic targets.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medicine
Griffith Health
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15

Franceschini, Gian Marco. "The DNA methylation landscape of metastatic prostate cancer: from characterization to liquid biopsy applications". Doctoral thesis, Università degli studi di Trento, 2023. https://hdl.handle.net/11572/364210.

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Epigenetic alterations are observed in virtually all cancer types, yet there is limited understanding of their role in tumorigenesis and evolution. The role of DNA methylation has been particularly elusive in this context. While this epigenetic mark has been extensively profiled in healthy and cancerous samples, our ability to understand its relationship with underlying biological processes is still limited. Moreover, recent advancements in the profiling of cell-free DNA in circulation have sparked renowned attention toward tissue-specific and cancer-specific DNA methylation patterns. In this thesis, I present results to improve and refine the computational characterization of DNA methylation in cancer, focusing on metastatic castration-resistant prostate cancer. The first contribution is the development and performance assessment of Rockermeth, a computational methodology to leverage large-scale DNA methylation profiling data to nominate robust differentially methylated regions (DMRs). Rocker-meth can retrieve biologically relevant DNA methylation changes, as demonstrated by extensive integrative analyses with gene expression, chromatin states, and genomic annotations. The second contribution is the generation of a map of DNA methylation changes across prostate cancer progression. The application of Rockermeth and other tailored methodologies can be used to trace the critical evolutionary steps of this disease, from the healthy tissue to the most lethal metastatic AR-independent counterpart. The main result is the evidence of the ability of DNA methylation to capture a snapshot of the active transcription factors in each state of the disease, offering orthogonal information compared to standard genomic sequencing. The third contribution is the design and development of NEMO, a tailored liquid biopsy sequencing panel approach to allow non-invasive neuroendocrine castration-resistant prostate cancer detection in patients with metastatic disease. Based on previous results and the comprehensive analysis of multiple datasets, I designed a set of informative genomic regions to estimate disease burden and evidence of neuroendocrine transdifferentiation. The actual implementation of the NEMO panel produced a scalable and cost-effective strategy, which has been extensively benchmarked using both in silico and in vitro approaches. The application of NEMO to patient-derived cfDNA samples demonstrated accurate tumor content estimation and robust detection of neuroendocrine disease, making it a promising instance of liquid biopsy for CRPC.
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VANNI, VALERIA STELLA. "UTERINE FLUID EXTRACELLULAR VESICLES AS A LIQUID BIOPSY FOR THE DIAGNOSIS OF ENDOMETRIAL RECEPTIVITY". Doctoral thesis, Università Vita-Salute San Raffaele, 2022. http://hdl.handle.net/20.500.11768/133079.

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Introduction. Implantation rates in Assisted Reproduction Technologies (ART) are still suboptimal due to a contribution of both embryonic and endometrial factors. While diagnosis of embryo euploidy can be achieved through preimplantation genetic testing, the diagnosis of a receptive endometrium remains a challenge. The main limitation of current receptivity tests based on the transcriptome of endometrial biopsies are that they: i) overlook the extracellular compartment, which appears to play a major role in the implantation-related cross-talk; ii) cannot be performed in the same cycle of the embryo transfer attempt. Aim of the work. The aim of this project is to use non-invasively collected uterine-fluid derived extracellular vesicles (UF-EVs) as a novel source of transcriptomic markers of endometrial receptivity. Methods. First, fertile volunteer women were included and their UF-EVs were retrieved for physical characterization and transcriptomic analysis, both in a pre-receptive (LH+2) and receptive (LH+7) phase of the cycle. Then, a cohort of ART patients and a further analogous validation cohort were included. UF-EVs were collected during the receptive (LH+7) phase of the cycle immediately preceding that of an euploid blastocyst transfer attempt, for comparison between patients with subsequent successful versus failed implantation. Results. The transcriptome of UF-EVs markedly changes between the nonreceptive (LH+2) and receptive (LH+7) phase, with n=2247 differentially ‘expressed’ genes. The LH+7 transcriptomic content of ART patients who subsequently achieve implantation partly differs from that of patients who subsequently fail implantation, with n=161 differentially ‘expressed’ genes. UF-EVs of ART patients with failed versus successful implantation also show a higher mean size of EVs. After including the validation cohort, a significant ROC curve for prediction of implantation was calculated based on selected genes (AUC=0.86; 95% CI 0.78-0.94, p=2.8x10-8). In the subgroup of patients with a diagnosis of recurrent implantation failure, a two-step cluster analysis model could correctly classify patients as successfully or unsuccessfully treated with a test sensitivity of 94.3% and a specificity of 80.0%. Discussion. These results support the hypothesis that UF-EVs can be used to identify a transcriptomic signature of endometrial receptivity in ART patients. Current/future perspectives will be to validate these findings and to assess the safety of same-cycle, non-invasive sampling of UF-EVs prior to the embryo transfer attempt.
VESCICOLE EXTRACELLULARI DA FLUIDO UTERINO COME BIOPSIA LIQUIDA PER LA DIAGNOSI DI RECETTIVITA' ENDOMETRIALE Introduzione: I tassi di impianto dopo Procreazione Medicalmente Assistita (PMA) sono subottimali a causa di fattori sia embrionari che endometriali. Se la diagnosi di euploidia embrionaria può oggi essere ottenuta grazie alle tecniche di test genetici preimpianto, la diagnosi di recettività endometriale rimane elusiva. I principali limiti degli attuali test di recettività basati sulla trascrittomica di biopsie endometriali sono: i) che non comprendono il compartimento extracellulare, il quale rappresenta un mediatore fondamentale nel cross-talk dell'impianto; ii) che in quanto invasivi non possono venire eseguiti nello stesso ciclo mestruale del tentativo di trasferimento embrionario. Scopo del progetto. Lo scopo di questo progetto è di usare le vescicole extracellulari da fluido uterino (UF-EVs) raccolto non invasivamente come una nuova fonte di marcatori trascrizionali di recettività endometriale. Metodi. Sono state incluse dapprima donne fertili volontarie, le cui UF-EVs sono state confrontate in termini di caratterizzazione fisica e analisi trascrittomica tra la fase pre-recettiva (LH+2) e la fase recettiva (LH+7) del ciclo mestruale. Quindi, è stata inclusa una prima coorte di pazienti sottoposte a PMA e una seconda coorte di validazione, composta da pazienti sottoposte a PMA e selezionata secondo criteri analoghi rispetto alla precedente. Le UF-EVs di queste pazienti sono state raccolte nella fase recettiva (LH+7) del ciclo immediatamente precedente a quello di un trasferimento di blastocisti euploide e i risultati paragonati tra pazienti con successo versus fallimento di impianto. Risultati. Il trascrittoma delle UF-EVs subisce profonde modificazioni nella fase recettiva (LH+7) rispetto a quella pre-recettiva (LH+2), con n=2247 geni differenzialmente 'espressi'. Il trascrittoma delle UF-EVs di pazienti che ottengono l'impianto di blastocisti evidenzia alcune differenze rispetto a quello di pazienti con fallimento di impianto, con n=161 geni differenzialmente 'espressi'. Le UF-EVs di pazienti con fallimento di impianto hanno anche una misura media maggiore di quelle di pazienti con impianto. Dopo aver incluso la coorte di validazione, è stata calcolata una ROC curve significativa per la predizione di impianto basata su un gruppo selezionato di geni (AUC=0.86; 95% CI 0.78-0.94, p=2.8x10-8). Nel sottogruppo di pazienti con diagnosi di fallimento ricorrente di impianto, è stato calcolato un modello two-step cluster analysis con una sensibilità pari a 94.3% e una specificità pari a 80.0% nella corretta classificazione di pazienti con successo versus fallimento di impianto. Discussione. Questi risultati supportano l'ipotesi che le UF-EVs possano essere usate per identificare un profilo trascrittomico associato a recettività endometriale nelle pazienti sottoposte a PMA. Le prospettive attuali e future saranno di validare questi risultati e di studiare la sicurezza del campionamento non invasivo di UF-EVs nello stesso ciclo mestruale del tentativo di trasferimento embrionario.
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17

SAGIRAJU, Sruthi. "Liquid biopsy provides complementary information to tissue biopsies for molecular classification of DLBCL patients". Doctoral thesis, Università del Piemonte Orientale, 2022. http://hdl.handle.net/11579/144258.

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Diffuse large B-cell lymphoma is a molecular heterogeneous disease with a variable clinical course and prognosis. Recent studies have identified different molecular clusters on tissue biopsy. Liquid biopsy is a non-invasive tool that allows the collection of circulating tumor DNA (ctDNA) shed by apoptotic tumor cells potentially deriving from all the different sites of the lymphoma. This may provide an integrative source of tumor DNA for DLBCL genotyping. The aims of the study are: i) to identify new prognostic molecular markers on ctDNA and on lymph node biopsy (LN); and ii) to compare the DLBCL molecular clusters between the LN and the ctDNA. The mutational profiling performed in 77 DLBCL patients, through a NGS approach, allows to identify at least one somatic non-synonymous mutation in 92.2% of patients in the LN biopsy, and in 87.0% in the ctDNA. Mutation analysis of different compartments allowed to identify mutations with potential clinical impact: GRHPR (p=0.035) and SGK1 (p=0.039) mutations in ctDNA, and MYC mutations in LN (p=0.021) were associated with a shorter PFS. Moreover, ctDNA levels harbor prognostic impact since higher levels of ctDNA (22.5 log hGE/mL) showed a significantly worse PFS (p=0.025) and OS (p=0.004). Based on the mutations identified, the LymphGen tool allowed to assign to a specific molecular cluster 46.5% of patients on the LN biopsy, and 40.3% on the liquid biopsy. The combination of mutational data from LN and ctDNA improved DLBCL assignment to a specific cluster, thus classifying 48.7% of cases. From a clinical perspective, patients belonging to the BN2 and ST2 clusters showed a favorable outcome with a 36-month PFS of 100% compared to 62.3% for patients belonging to the MCD or EZB clusters (p=0.040). The combination of mutational data from LN and ctDNA provides complementary information for the molecular classification and prognostic stratification of newly diagnosed DLBCL patients.
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18

Bollu, Bapesh Krishna. "Circulating Tumour Cells in Osteosarcoma". Thesis, The University of Sydney, 2022. https://hdl.handle.net/2123/29874.

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Introduction There is now a growing body of work being conducted in the field of liquid biopsies. A liquid biopsy is a technique to look for biomarkers, arising from cancer, within the blood stream which are accessed in a non-invasive manner. These may include circulating tumour cells (CTCs), circulating tumour DNA, circulating RNA or exosomes. Osteosarcoma is the most common bony malignancy of the paediatric population. Metastatic disease has a poor prognosis with a 20-30% survival. The number of tumour cells present at distant sites when identified by traditional radiological and clinical techniques may be too late to offer reliable cure. Hence techniques to identify the potential for metastatic disease earlier may offer the option to intensify or alter therapy. The aim of this project is to develop a technique to identify osteosarcoma CTCs. Methods Osteosarcoma cell lines were assessed for potential surface markers using flow cytometry. Cell lines used include 143B, HOS and MG63. Following this, normal control blood samples where spiked with osteosarcoma cells and cell recovery was tested using different preanalytical techniques and processing time points. Techniques compared include red blood cell (RBC) lysis and Ficoll density gradient processing. Timepoints included fresh processing, after 24hours and frozen samples. Then patient blood samples were collected at different timepoints of their clinical course. All samples were processed using RBC lysis and stained with dioganglioside (GD2), Ephrin type-a receptor 2 (EphA2) and CD45. All samples were run through BD FACS Symphony and then data was analysed using FlowJo software. Results The use of GD2, EphA2 as positive markers and CD45 as a negative marker was effective in identifying osteosarcoma CTCs. The use of RBC lysis processing with fresh sample processing yielded the best cell recovery rate. Within our patient samples we noted the presence of CTCs in 16/18 samples which were collected from seven patients. There was no correlation between the timepoint of collection and the number of CTCs identified in relation to comparing risk of recurrence and active disease and samples taken when in remission. Conclusion A reproducible technique to identify osteosarcoma CTC was developed. This was seen with both spiked normal control samples and patient samples. Further work on downstream analysis and large sample numbers would assist in further clinical translation of these findings.
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19

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)
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20

Orlando, Francesco. "Unleashing the potential of liquid biopsy: allele-informed evaluation of plasma samples for cancer patients management". Doctoral thesis, Università degli studi di Trento, 2023. https://hdl.handle.net/11572/364264.

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Liquid biopsy and next-generation sequencing of cell-free DNA (cfDNA) in cancer patients’ plasma offer a minimally-invasive solution to detect tumor cell genomic information to aid real-time clinical decision-making. Reliability and sensitivity in the detection of genomic alterations is crucial for patient management and it is particularly relevant in the context of targeted therapies. However, biological and technical factors, such as low cfDNA tumor fraction and sequencing errors, affect the correct interpretation of genomic data limiting the utility of non-invasive cfDNA-based tumor profiling. To address these issues, we designed a prostate cancer bespoke assay, PCF_SELECT, that includes an innovative sequencing panel covering ∼25 000 high minor allele frequency SNPs and tailored analytical solutions to enable allele-informed evaluation of patients’ tumor. The framework also implements ABEMUS, an ad-hoc computational procedure we specifically designed for cfDNA samples to accurately detect somatic point mutations that could emerge under treatment pressure and as drug resistance mechanism. When applied on serial plasma samples from three patients receiving PARP inhibition harboring DNA repair gene aberrations, PCF_SELECT demonstrated high sensitivity in detecting BRCA2 allelic imbalance with decreasing tumor fractions resultant from treatment and identified complex ATM genomic states that may be incongruent with protein losses. As a step towards a more sensitive detection of tumor features in circulation of cancer patients, we next hypothesized that recent WGS-based approaches exploiting cfDNA fragments characteristics could be extrapolated for targeted sequencing data and that gene-region specific measures could improve detection metrics. Preliminary results suggest an increased sensitivity compared to copy-number-based methods supporting the integration at no extra cost in our targeted assay. Overall, this work is relevant to the context of precision oncology. It provides an innovative platform for the management of cancer patients, delivering detailed patient-specific molecular information which could be applied to guide treatment and improve clinical outcomes.
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21

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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22

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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23

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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24

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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25

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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26

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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27

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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28

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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29

Sagot, Matthieu. "Détection électrique In-Situ des événements de microfiltration dans des milieux complexes". Electronic Thesis or Diss., Université de Toulouse (2023-....), 2024. http://www.theses.fr/2024TLSEP028.

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La microfiltration constitue un domaine scientifique et technologique bien documenté, mais qui nécessite encore des recherches approfondies lorsqu'il s'agit de cibler la filtration spécifique et précise d'éléments rares au sein d'un milieu complexe. En effet, des solutions innovantes pour le filtrage d'échantillons dans des milieux complexes pourraient être la clé de multiples problématiques liées à la santé et à l'environnement. Le sang est un exemple concret de milieu complexe : il contient une quantité importante et variée de cellules et de protéines, présentant une viscosité trois à huit fois supérieure à celle de l'eau, ainsi qu'un comportement non newtonien en écoulement. Les applications cliniques de la filtration sanguine nécessitent le traitement de grands volumes de sang, soit en raison de la rareté des éléments ciblés (dans le cas de la capture des cellules tumorales circulantes, la pertinence clinique commence à 5 CTC/mL de sang), soit parce que l'ensemble du sang circulant doit être purgé d'entités indésirables (telles que des agrégats cellulaires ou des microparticules circulantes dans les maladies cardiovasculaires et les accidents vasculaires cérébraux). Enfin, la nature biologique des éléments ciblés peut introduire une variabilité dans leur taille et leur forme, posant ainsi des défis fluidiques pour leur récupération au sein de tels milieux.La filtration sanguine est un processus central dans l'hémodialyse, la surveillance des maladies cardiovasculaires et les applications de biopsie liquide basées sur la capture sélective des cellules tumorales circulantes (CTC), entre autres contextes cliniques. Pour de telles applications, la micro et nano fabrication utilisant des méthodes et des techniques de précision de l'industrie des semi-conducteurs offre la possibilité de contrôler avec une grande précision la taille des pores de filtration par rapport à la taille des éléments ciblés nécessitant une filtration. Ce niveau de précision dans le processus de fabrication ouvre la voie à la rétention exclusive de l'élément ciblé, conduisant à l'information biologique dans le cas d'une application diagnostique, ou à la pathogénicité dans le cas d'applications thérapeutiques, sans altérer la composition du sang élué. Cependant, en raison du traitement d'un grand volume de sang et de la présence de millions de globules blancs et de milliards de globules rouges par millilitre de sang, de tels filtres microfabriqués sont sujets à l'obstruction due à l'accumulation de matériau retenue au fil du temps. Ce désavantage incite au développement d'une méthode in-situ capable de détecter la densité cellulaire à la surface de ces filtres pendant leur utilisation, afin de surveiller leur saturation en vue de nettoyer leur surface ou de procéder à leur remplacement par d’autres filtres. Dans ce contexte, nous proposons des dispositifs microfabriqués en salle blanche capables de répondre à ces exigences. Les dispositifs de détection produits combinent une membrane de filtration avec une méthode de détection cellulaire électrique in-situ à travers des microélectrodes interdigités et des mesures de spectroscopie d'impédance. Malgré l'utilisation de pores de filtration à l'échelle du micron et de dispositifs microfabriqués, nous proposons un design spécifique permettant la filtration sanguine à un débit élevé (11,5 mL/min), bien supérieur à celui des dispositifs microfluidique habituels. Enfin, nous démontrons que des mesures électriques stables peuvent être réalisées dans du sang entier à des débits élevés pour surveiller la saturation du filtre par les cellules retenues. De plus, l'analyse fine des cellules capturées, habituellement confiée à des laboratoires externes, pourrait être transférée au chevet du patient tout au long du traitement des échantillons, si une analyse in-situ et une phénotypisation en temps réel des cellules collectées par leur signature électrique pouvaient être démontrées
Microfiltration is a well-documented scientific and technological domain that still requires research when targeting the specific and accurate filtration of rare elements inside a complex medium. Indeed, innovative solutions for sample filtering of complex media may hold the key to multiple health-related and environmental issues and applications. Blood is a good example of a complex medium: it contains a large quantity and variety of cells and proteins and exhibits a viscosity three to eight times greater than water and non-Newtonian behavior when flowing. Clinical applications of blood filtration require processing large volume of blood either because of the scarcity of the targeted elements (in the case of circulating tumor cell capture, clinical relevance starts at 5 CTCs/mL of blood) or because the whole circulating blood needs to be expurgated from some adverse entities (such as cell aggregates or circulating microparticles in stroke and cardiovascular diseases). Finally, the biological nature of the targeted elements may introduce variability in the targeted element size and shape, therefore bringing fluidic challenges for their retrieval within such media. Blood filtration is a process which is central in hemodialysis, cardiovascular disease monitoring, and liquid biopsy applications based on the selective capture of Circulating Tumor Cells (CTCs), among other clinical contexts. For such applications, micro and nanofabrication using methods and techniques used today in advanced semi-conductor industry, brings the ability to control with great accuracy the size of the filtering pores with respect to the size of the targeted elements that require filtration. This level of accuracy in the fabrication process opens the opportunity to retain only the targeted element driving the biological information in the case of a diagnosis application or driving the pathogenicity in the case of therapeutic applications without impairing the composition of the eluted blood. However, because a large volume of blood is processed and due to the presence of millions of white blood cells (WBC) and billions of red blood cells (RBC) per milliliter of blood, such advanced microfabricated filters are subjected to clogging due to the unwanted accumulation of material unavoidably retained among time. This drawback appeals to the development of an in-situ method capable of sensing the cell density at the surface of these filters during use, to monitor their saturation in order to clean their surface or to proceed to their replacement by fresh ones. In this context, we propose clean room microfabricated devices capable of fulfilling these requirements. The produced sensing devices combine a filtering membrane with an in situ cellular electrical detection method through interdigitated microelectrodes and impedance spectroscopy measurements. Despite using micron-scale filtering pores and microfabricated devices, we propose a specific design that enables blood filtration at a high flow rate (11.5 mL/min), which is much larger than usual microfluidic devices. Finally, we demonstrate that stable electrical measurements can be performed in whole blood at high flow rates to monitor the saturation of the filter by retained cells. Moreover, the fine analysis of the captured cells, usually entrusted to remote laboratories, could be transferred at the patient’s bedside along the sample processing if an in-situ analysis and real-time phenotyping of the collected cells through their electrical signature could be demonstrated. This aspect will be addressed through the conception and fabrication of dedicated filtering devices, thus broadening the application field of electrical sensing on a filtering membrane within a microfluidic chip
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30

AWAN, ZUBAIR ANWAR. "De novo liquid biopsy and radio genomic diagnostic approach with combined deep learning artificial neural networks for NSCLC". Doctoral thesis, Università degli Studi di Palermo, 2022. https://hdl.handle.net/10447/564144.

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Each year, the mortality rate and incidence of non-small cell lung cancer (NSCLC) are dramatically increasing. The introduction of liquid biopsy in the clinical practice of NSCLC has completely revolutionized the approach to such neoplasm since is generally detected through complex and invasive procedures and unfortunately at advanced stages. The importance and innovation of liquid biopsy are linked with the possibility of cancer detection at every stage, adjuvant treatment, resistance genotyping, systematic initiation of treatment, minimal residual disease, early detection of relapse, and screening of NSCLC. Circulating tumor DNA (ctDNA) is now emerging as a non-invasive biomarker that will help to track tumor burden and allow the monitoring of cancer genome in blood across several malignancies. Recently, the combination of liquid biopsy and radiomics seems to deliver an efficient way to study cancer evolution over time providing an important support tool to daily clinical practice. CT (Computed Tomography) images are of particular importance in this context because they convey functional and anatomical information, respectively. Machine learning provides a variety of approaches for dealing with this potentially high-dimensional challenge. In particular, we used Enet neural network for image assessment. This study represents an interesting attempt to explore the usefulness of liquid biopsy, radiomics, and deep learning in the NSCLC clinical routine. We studied a NSCLC patient cohort from the first access to our department to follow-up. Our results showed a promising correlation between the ctDNA quantity and radiomic features evaluated by automated computed tomography according to RECIST criteria with the Enet deep learning method, which allowed us to define more accurately progression-free survival (PFS) and overall survival (OS) of patients during the course of cancer history. Therefore, the above mentioned diagnostic tools including the combination of liquid biopsy, radiomics, and deep learning tools collectively can represent a very robust and new approach in the monitoring and management of NSCLC.
Each year, the mortality rate and incidence of non-small cell lung cancer (NSCLC) are dramatically increasing. The introduction of liquid biopsy in the clinical practice of NSCLC has completely revolutionized the approach to such neoplasm since is generally detected through complex and invasive procedures and unfortunately at advanced stages. The importance and innovation of liquid biopsy are linked with the possibility of cancer detection at every stage, adjuvant treatment, resistance genotyping, systematic initiation of treatment, minimal residual disease, early detection of relapse, and screening of NSCLC. Circulating tumor DNA (ctDNA) is now emerging as a non-invasive biomarker that will help to track tumor burden and allow the monitoring of cancer genome in blood across several malignancies. Recently, the combination of liquid biopsy and radiomics seems to deliver an efficient way to study cancer evolution over time providing an important support tool to daily clinical practice. CT (Computed Tomography) images are of particular importance in this context because they convey functional and anatomical information, respectively. Machine learning provides a variety of approaches for dealing with this potentially high-dimensional challenge. In particular, we used Enet neural network for image assessment. This study represents an interesting attempt to explore the usefulness of liquid biopsy, radiomics, and deep learning in the NSCLC clinical routine. We studied a NSCLC patient cohort from the first access to our department to follow-up. Our results showed a promising correlation between the ctDNA quantity and radiomic features evaluated by automated computed tomography according to RECIST criteria with the Enet deep learning method, which allowed us to define more accurately progression-free survival (PFS) and overall survival (OS) of patients during the course of cancer history. Therefore, the above mentioned diagnostic tools including the combination of liquid biopsy, radiomics, and deep learning tools collectively can represent a very robust and new approach in the monitoring and management of NSCLC.
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31

Martel, Arnaud. "Impact de la biopsie liquide dans le mélanome uvéal primitif et métastatique". Electronic Thesis or Diss., Université Côte d'Azur, 2023. http://www.theses.fr/2023COAZ6050.

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Le mélanome uvéal (MU) est le cancer intraoculaire primitif le plus fréquent chez l'adulte. Malgré une prise en charge médico-chirurgicale adaptée permettant un contrôle local de la tumeur dans plus de 90% des cas, on estime qu'environ 50% des patients présenteront des métastases, principalement hépatiques, dans les 10 années suivant le diagnostic. La dissémination métastatique du MU s'effectue uniquement par voie hématogène. Détecter le plus précocement possible la présence de cellules tumorales dans le sang circulant permettrait une prise en charge très précoce des patients, avant la survenue des métastases hépatiques. Depuis quelques années, la biopsie liquide (BL) s'est imposée comme un moyen non invasif, reproductible et prometteur d'obtenir un diagnostic, un pronostic et un suivi théranostique de patients atteints de tumeurs solides. La BL concerne le sang ainsi que tout liquide humain, et permet entre autres d'identifier plusieurs paramètres tumoraux comme les cellules tumorales circulantes (CTCs) ou l'ADN tumoral circulant.L'objectif de ce travail de thèse était d'évaluer la place de la BL, et notamment des CTCs, dans le MU primitif et métastatique.Dans un premier article, nous avons réalisé une revue de la littérature sur les biopsies liquides afin : (i) de faire un état des lieux des connaissances sur la BL dans le MU, (ii) d'identifier les différents marqueurs évaluables dans le MU, (iii) de recenser les différentes techniques d'identification des marqueurs tumoraux , et (iv) de relever les limites techniques actuelles. Nous avons conclu que monitorer les CTC était actuellement le paramètre le plus pertinent dans la mesure où elles sont le reflet vivant de la dissémination spatio-temporelle des cellules tumorales. Nous avons également observé des discordances entre les résultats des différentes études, dues à une grande diversité des techniques pré analytiques et des cohortes analysées.Afin de déterminer la technique la plus adaptée à l'identification des CTC dans le MU, nous avons dans un deuxième article comparé in vitro 4 dispositifs de capture des CTC en utilisant une lignée de MU métastatique (OMM 2.3). Les cellules étaient filtrées à la fois en utilisant du milieu cellulaire (RPMI) et du sang veineux provenant de volontaires sains. Nous avons successivement comparé le Vortex VTX-1 (Biosciences, Pleasanton, CA, USA, système micro fluidique), le ClearCell FX (ClearBridge, Biolidics, Singapore, système micro fluidique), l'ISET (Rarecells, Paris, France, système de filtration par taille) et le Cellseach (Menarini Silicon Biosystems, Florence, Italie, système immun magnétique). Les taux de capture, étaient de 39,2%, 22,2%, 8,9% et 1,1% pour l'ISET, le Vortex VTX-1, le ClearCell FX et le Cellseach, respectivement. Nous avons également comparé ces dispositifs en termes de reproductibilité, de facilité d'utilisation, de rapidité, d'ergonomie, et de capacité à réaliser des analyses cytogénétiques post analytiques. Ces expérimentations nous ont permis d'identifier le dispositif de capture des CTC ainsi que le protocole analytique les plus adaptés. Nous avons mis au point une technique rapide, fiable et reproductible de capture des CTC en utilisant le système de filtration micro fluidique Vortex VTX-1 suivi de l'immunocytochimie par Melan A.Suite aux conclusions de ce travail in vitro, nous avons maintenant débuté l'application clinique chez nos patients présentant un MU primitif ou métastatique en utilisant le système de filtration micro fluidique Vortex VTX-1.Enfin, nous avons publié un 3e article détaillant la mise en place d'une biobanque dédiée à l'onco-ophtalmologie au CHU de Nice. Une biobanque dédiée, sécurisée et accréditée est essentielle pour collecter des échantillons de haute qualité afin de promouvoir la recherche translationnelle et multicentrique en oncologie oculaire
Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Despite appropriate medical and surgical management allowing local control of the tumor in more than 90% of cases, it is estimated that about 50% of patients will present metastases, mainly hepatic, within 10 years of diagnosis. Metastatic spread occurs only hematogenously. Detecting the presence of tumor cells in circulating blood as early as possible would allow very early management of patients, before the occurrence of liver metastases. In recent years, liquid biopsy (LB) has emerged as a non-invasive, reproducible and promising way to obtain a diagnosis, prognosis and theranostic follow-up of patients with solid malignancies. LB concerns blood as well as any human fluid and allows identifying several tumor parameters such as circulating tumor cells (CTCs) or circulating tumor DNA.The aim of this thesis work was to evaluate the place of LB, and in particular CTCs, in primary and metastatic UM.In a first article, we conducted a review of the literature on liquid biopsies in order to: (i) assess the current status of LB in the UM field, (ii) to identify the different markers evaluable in the MU, (iii) to assess the different techniques for identifying these tumor markers, and (iv) to report the current technical limitations. We concluded that monitoring CTCs is currently the most relevant parameter as they are a living expression of the spatio-temporal dissemination of tumor cells. We also observed discrepancies between the different studies results, due to a wide variety of pre-analytical techniques and cohorts analyzed.In order to determine the most suitable technique for the identification of CTCs in MU, we have in a second article compared in vitro 4 CTC capture devices using a metastatic UM line (OMM 2.3). The cells were filtered using both cellular medium (RPMI) and venous blood from healthy volunteers. We successively compared the Vortex VTX-1 (Biosciences, Pleasanton, CA, USA, microfluidic system), the ClearCell FX (ClearBridge, Biolidics, Singapore, microfluidic system), the ISET (Rarecells, Paris, France, size filtration system) and the Cellseach (Menarini Silicon Biosystems, Florence, Italy, immunomagentism system). Recovery rates were 39.2%, 22.2%, 8.9% and 1.1% for the ISET, Vortex VTX-1, ClearCell FX and Cellseach, respectively. We also compared these devices in terms of reproducibility, ease of use, speed, ergonomics, and ability to perform post-analytical cytogenetic analyses. These experiments allowed us to identify the most suitable CTC capture device and analytical protocol. We have developed a fast, reliable and reproducible CTC capture technique using the Vortex VTX-1 microfluidic filtration system followed by Melan A immunocytochemistry.Following the conclusions of this in vitro work, we have now started clinical application in our patients with primary or metastatic MU using the Vortex VTX-1 microfluidic filtration system.Finally, we published a third article on the establishment of a dedicated biobank to ophthalmic malignancies at the University Hospital of Nice. A secure and accredited dedicated biobank is essential to collect high-quality samples to promote translational and multicenter research in ocular oncology
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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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33

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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34

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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35

Romagnoli, Dario. "Identification and development of epigenetic tumor biomarkers through computational biology approaches". Doctoral thesis, Università di Siena, 2023. https://hdl.handle.net/11365/1227695.

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DNA-methylation alterations are common in cancer and display unique characteristics that make them ideal markers for tumor quantification and classification. In this study, we discuss the development and testing of a computational framework exploiting minimal DNA-methylation signatures composed by a few dozen informative DNA-methylation sites to quantify and classify tumor signals in tissue and cell-free DNA samples. Extensive analyses of multiple independent and heterogenous datasets including >7,200 samples demonstrate the capability of the framework to provide precise estimations of tumor content and to enable accurate classification of tumor type and molecular subtype. To evaluate its applicability in the clinical context, we designed an informed assay incorporating the minimal signatures for breast cancer. Using both artificial samples and clinical serial cell-free DNA samples from patients with metastatic breast cancer, we show that our approach provides accurate estimations of tumor content, sensitive detection of tumor signal and the ability to capture clinically relevant molecular subtype in patients’ circulation. This study provides evidence that an extremely parsimonious approach can be used to develop cost-effective and highly-scalable DNA-methylation assays that could support and facilitate the implementation of precision oncology in clinical practice.
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36

GRISTINA, Valerio. "Circulating cell-free DNA (cfDNA) and extracellular vesicles (EVs) as prognostic and predictive biomarkers in patients with advanced Non-Small Cell Lung Cancer (NSCLC): the LEXOVE prospective study". Doctoral thesis, Università degli Studi di Palermo, 2022. https://hdl.handle.net/10447/571952.

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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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Julich-Haertel, Henrike [Verfasser] y 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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Lázaro, Fortunato Diogo Miguel. "Characterization and selective isolation of human extracellular vesicle subpopulations from simple and complex biosamples for novel clinical liquid biopsy strategies". Doctoral thesis, Università di Siena, 2022. http://hdl.handle.net/11365/1213357.

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All living cells selectively package a wide variety of biomolecules in nanometric entities termed extracellular vesicles (EVs), which actively mediate vital biological events, especially biomolecule recycling and horizontal intercellular communication. To do so, EVs can diffuse through interstitial space, reaching even circulating biofluids to promote long-range paracrine signalling in recipient cells, while sometimes directly functioning as effectors. Despite fundamental knowledge gaps within the field, EVs are frequently dysregulated during disease, often directly sustaining disease hallmarks. Therefore, they are extremely valuable circulating biomarker sources that can provide timed snapshots of individual (patho)physiological status, while carrying cell or tissue-specific cargo, indicative of their origin. Consequently, attention towards EVs has exponentially increased during the last decade, fostered by translational research efforts that have focused on two major EV applications, either as drug delivery vehicles or as analytes in liquid biopsies, which consist in bodily fluid collection for biomarker detection. In contrast to standard tissue biopsies for cancer management, liquid biopsies are heralded as the holy grail of personalized medicine because they are minimally invasive, low-risk, high-throughput and repeated sampling is easy and cheap. These features enable full understanding of tumour heterogeneity, while tumour progression can be tracked longitudinally, as well as resulting alterations in healthy tissues. Liquid biopsies aid clinicians in early disease detection and screening, patient stratification, treatment response monitoring and resistance mechanism identification. Still, the small size and low amount of cargo in biological nanoparticles limit current state-of-the-art technologies employed for EV profiling. These fail to grasp the full complexity of EV heterogeneity, ultimately straggling the establishment of model EV samples and identification of clinically relevant EV subpopulations. Hence, the first central aim of this thesis, further described in the first chapter of the results section, concerns the identification of EVs among particle noise and characterization of relevant subpopulations, using state-of-the-art, dedicated instruments, optimized for high-resolution single nanoparticle detection. Label-free and fluorescence measurements allowed either indiscriminate or selective detection of EV subpopulations and common contaminants. Analysing the expression of classical surface markers facilitated EV sample characterization. Using thoroughly optimized staining procedures, fluorescently-labelled EVs could be reproducibly generated, and later employed as tracer EV models for accurate measurements in downstream spike-recovery experiments. Liquid biopsy studies have often selected blood plasma for EV biomarker discovery and detection, as it potentially transports EVs from any cell type, together with other biomarkers. Since blood collection is a well-established routine clinical practice, it is often considered the ideal biofluid for liquid biopsy tests. The majority of these studies purify bulk EVs with co-isolated contaminants from plasma, failing to investigate the specific EV subsets that actually contain relevant biomarkers, which might end up undetected, diluted among many other macromolecules. Thus, in the second chapter of results, the present thesis aimed to further dissect EV heterogeneity in human plasma samples, and to determine whether distinct EV subpopulations can indeed confer differential clinical utility. To this end, several affinity-based EV isolation approaches were optimized and explored for their efficiency and specificity in simple and complex matrices. However, the complexity of biological matrices, especially plasma, drastically hamper affinity interactions. Hence, additional critical gaps in the field regarding sample pre-analytical processing were addressed. Results demonstrated that target EV subpopulations could be efficiently enriched from plasma, but also, that both complex matrix composition and target EV surface phenotypes can dramatically influence the performance of affinity isolation methods. Furthermore, distinct plasma EV subpopulations were indeed captured when targeting different surface moieties. Platelet-derived EVs, but not other EV subsets, evidenced 8 mRNA expression signatures that could be linked to early-stage lung cancer, proving that distinct EV subpopulations are indeed more relevant than others for liquid biopsy-based biomarker detection. In conclusion, the original experimental work reported in thesis evidenced that EV labelling protocols should be optimized for each single nanoparticle profiling platform. Moreover, fluorescent dyes, affinity reagents and methodologies employed must be carefully selected and tested, as all contribute to accurate EV measurements in simple matrices, but can also promote confounding particle detection and biased analysis. Also, elimination of dyes in excess is extremely important for precise small EV detection, and protocols used to conduct this step must not affect the original subpopulation composition in EV samples. On the other hand, the immunoaffinity-based EV isolation protocol devised in this PhD project purified distinct EV subpopulations from plasma. This protocol is compatible with a wide range of downstream procedures and analytical platforms. It is a simple, quick, scalable and automatable pre- analytical workflow, fitting the requirements of routine clinical assays, as to encourage the inclusion of EVs in novel liquid biopsy tests. Importantly, clinically relevant mRNA expression profiles associated to early-stage lung cancer, were obtained upon isolation of platelet-derived EV subpopulations from plasma, using this immunoaffinity protocol. Furthermore, it was evident that pre- analytical variables, sample matrices and EV surface phenotypes are critical parameters to account for when affinity-based methodologies are employed to target specific EV subsets. As result of the research output throughout this PhD project, ongoing multi-centre collaborations have been established, with the goal of integrating EV multianalyte and multi-omics data. Large-scale validation, prospective and explorative studies will be fundamental for robust biomarker detection and inclusion of EVs in novel liquid biopsy-based assays.
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40

GASPARELLO, Jessica. "Novel trands in microRNA based theranostics". Doctoral thesis, Università degli studi di Ferrara, 2018. http://hdl.handle.net/11392/2478767.

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I miRNA sono una classe di RNA non codificanti in grado di regolare l’espressione genica attraverso l’interazione con la regione 3’UTR del trascritto bersaglio, portando all’inibizione della traduzione o alla degradazione del mRNA bersaglio. A causa della loro breve sequenza (19-25 nucleotidi), i miR sono in grado di riconoscere regioni bersaglio in più trascritti, risultando, coinvolti in più di un processo molecolare. Diverse patologie sono state associate alla disregolazione del profilo di espressione di miR, negli anni sono state quindi elaborate diverse strategie terapeutiche allo scopo di ripristinare i livelli fisiologici di miR. Molecole in grado di mimare la funzione del miR-210 sono state impiegate nella presente tesi allo scopo di ridurre l’espressione del fattore di trascrizione BCL11A, uno dei principali repressori del gene γ-globinico. Lo studio ha permesso di ottenere due risultati chiave: a) miR-210 riconosce una sequenza presente nella regione codificante del trascritto BCL11A, b) l’incremento dei livelli intracellulari di miR-210 sono associati al decremento di trascritto e proteina BCL11A. I dati suggeriscono che l’impiego di molecole in grado di mimare la funzione di miR-210 possono essere impiegate nel trattamento della β-talassemia. Le molecole in grado di modulare i livelli intracellulari di miR richiedono un veicolo per essere internalizzate dalle cellule. Nella presente tesi è stata valutata una molecola a struttura calixarenica (ML122) e precedentemente impiegata per la trasfezione di DNA. ML122 è stato testato, sia per la veicolazione di molecole elettricamente cariche (antimiRNA e premiRNA), sia per molecole elettricamente neutre (PNA). I dati hanno dimostrato a) un’elevata efficienza di internalizzazione cellulare delle molecole veicolate da ML122, associata a b) evidenti effetti biologici delle molecole veicolate, che una volta internalizzate, sono rilasciate dal complesso formato con ML122 e svolgono il loro ruolo biologico. Come dimostrato da Chim nel 2008 i miR sono presenti in diversi fluidi biologici, tra cui il plasma, dove risultano particolarmente stabili, rendendoli ottimi marcatori nell’ambito della diagnostica non invasiva. Partendo da questo assunto i cmiRNA sono stati impiegati come marcatori diagnostici in due differenti ambiti. Nella prima parte i miR sono stati impiegati come marcatori per la diagnosi di carcinoma del colon-retto giungendo alle seguenti conclusioni: a) i miR sono fisiologicamente rilasciati dalle cellule e ogni linea cellulare presenta un proprio profilo di ‘secrezione’ che risulta indipendente dalle concentrazioni intracellulari di miR, b) la comparazione di due diverse metodiche PCR per la quantificazione di miRNA (RTqPCR e ddPCR) ha dimostrato risultati comparabili, nonostante la ddPCR risulti maggiormente performante, specialmente in campioni molto diluiti c) l’analisi di 3 miR selezionati sulla base di analisi riportate in letteratura, in pazienti affetti da CRC e donatori sani ha dimostrato diversi limiti derivanti dall’impiego di un numero limitato di miR suggerendo la necessità di considerare panelli più ampi di miR. L’analisi di miRNA circolanti è stata inoltre applicata al campo dell’identificazioni di frodi sportive, in particolar modo i miR sono stati proposti come marcatori di autoemotrasfusione. L’analisi di un numero seppur limitato di campioni, con metodica microarray ha permesso di identificare due diverse liste di miR candidati marcatori di autoemotrasfusione. Partendo dall’assunto che durante i 2 processi chiave dell’ABT: prelievo del sangue e reinfusione, generano un’alterazione dei livelli di ossigeno, è stata stilata una prima lista di 8 miR, la cui espressione risulta modulata in risposta alla variazione dei livelli di ossigeno. Traendo vantaggio dai dati di microarray è stata inoltre, proposta una seconda lista di miR, la cui espressione è modulata in seguito alla reinfusione.
MiRNAs are a class of small non-coding RNA of about 19-23 nucleotides in length able to act as regulators of gene expression thanks to their ability to bind the 3’UTR which results in inhibition of translation or in mRNA degradation. Due to their short sequence, they can bind more than one transcript, so they may be involved in more than one biological pathway. Since their first identification in 1993, they have been associated to a long list of physiological or pathological conditions. The dysregulation of miRNA profile may be associated to several diseases, so therapies based on the restore of physiological miRNA levels may have huge impact on several pathologies, for this reason molecules able to both increase or decrease miRNA levels have been recently developed. Through miRNAs levels regulation is possible to indirectly regulate their targets levels. This evidence was investigated in this thesis to reduce levels of BCL11A, one of the principal repressor of γ-globin gene. The following key results: a) miR-210 interaction with BCL11A coding region was demonstrated with SPR-based analysis, b) the increase of miR-210 intracellular levels leads to a decrease of BCL11A transcript and protein, encouraged us to consider the employment of miR-210 mimicking molecules as possible therapeutic approach to reduce BCL11A expression in the field of β-thalassemia treatment. Modulation of miRNAs levels into cells can be achieve with different kinds of molecules and most of them generally require an appropriate vehicle. At this propose we investigated with encouraging results a calixarene-based structure compound called ML122, previously used for DNA delivery, to vehicle miRNA-based molecules and PNAs. a) High transfection efficiency, associated with b) evident biological effects obtained when both premiRNA molecules and PNAs are vehicled with ML122, allow us to consider ML122 as a possible alternative to commercial available transfection agents. MiRNAs are present not only into cells but as demonstrated by Chim and colleagues they were found also in several body fluids, including plasma, in which have been demonstrated to be very stable. Several groups employed circulating miRNAs at diagnostic or prognostic propose opening a new important issue in the field of the non-invasive diagnostic techniques. In the present thesis we employed the non-invasive diagnostic technique in two different fields. First, we considered circulating miRNA in colorectal cancer diagnosis and management. In this section, three important massages were obtained a) miRNA are normally released by cells, the release pattern is different in each cell line and often differs from the miRNA pattern into cells, b) a comparison between two different techniques, RTqPCR and ddPCR, demonstrates how the two techniques gave comparable results, even if ddPCR for technical issue is more suitable for miRNA quantification in plasma samples, c) analysis of the three selected miRNAs in CRC patients and healthy controls demonstrates how additional miRNAs (at 6 or 7 miRNAs) are needed to identify patterns associated to CRC patients. The analysis of cmiRNAs was also, not applied to a health problem but to identify an illicit practice in sport such as autologous blood transfusion. The analysis of a limited number of samples using a high-throughput miRNA analysis method, i.e. microarray, allow us to identify two different list of miRNAs possible biomarkers for the detection of ABT: (a) a list of 8 miRNAs which modulation may be related to different oxygen availability immediately after the two key steps of ABT practice i.e. blood withdrawal and blood reinfusion. Moreover, a second list (b) was obtained considering all expressed miRNAs in our plasma samples. Despite the number of analysed samples is limited (6 ABT trained subjects and 3 control pools) preliminary encouraging data were obtained, which of course need to be confirmed increasing the samples size.
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41

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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42

Schneegans, Svenja Verfasser] y 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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Schneegans, Svenja [Verfasser] y 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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PASSIGLIA, Francesco. "Immune-Biomarkers in Advanced Non-Small Cell Lung Cancer". Doctoral thesis, Università degli Studi di Palermo, 2021. http://hdl.handle.net/10447/514194.

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Background: Developing personalized immunotherapy-based approaches, through the identification of predictive immune-related biomarkers, is still a main topic for translational lung cancer research. In the present study we investigated whether baseline tissue “immune-related gene signatures”, as well as plasma chemo-cytokines profiles, could predict sensitivity/resistance to immune-checkpoint inhibitors in pre-treated patients with advanced non-small cell lung cancer (NSCLC). Methods: From September 2015 to September 2018, 150 patients with previously treated advanced NSCLC who received either anti-PD1 or anti-PD-L1 inhibitors in the second-third line setting were included within this translational study. All the patients underwent CT-scan every 6 cycles and responses were evaluated by Response Evaluation Criteria in Solid Tumors (RECIST). Gene expression analysis was performed on 86 FFPE tissue samples by using the nCounter® PanCancer IO360™ Panel applied on NanoString platform, in order to analyze 770 genes involved in key immuno-oncology pathways. Peripheral blood samples were obtained from 57 patients at baseline and 37 patients at the 6th week under IO-therapy. A panel of cytokines and chemokines (IL-6, IL-8, CXCL10, CX3CL1, CCL2, VEGF, and IFN- gamma) were quantified in plasma by Cytokine Bead Array and their association with OS and TTP was assessed by Adaptive Index Modeling multivariable analysis. NLR and PLR were also assessed for potential association with clinical outcomes. Results: The gene expression levels of IL12RB2, ESR1 and CCL8 (p-values = 0.000308, 0.00162 and 0.0398, respectively) resulted significantly higher in responders versus non-responders patients. In patients with 0S > 18 months, a significant upregulation of the ESR1 (p-value = 0.00813) and IL12RB2 (p-value = 2.48e-08) genes, as well as a higher score for lymphoid compartment (p-value = 0.0117), cytokine and chemokine signaling (p-value = 0.0268), costimulatory signaling (p-value = 0.0323), and cytotoxicity (p-value = 0.0412), was observed. As regards peripheral blood samples analysis, an Immune-suppressive blood index score (ISBIS) was identified clustering patients into 3 groups with progressively worsening TTP and OS. The score was composed by higher IL-8 and CCL-2 levels, higher NLR, and lower IFN-gamma level. The differences among both PFS and OS Kaplan Meyers curves across the different subgroups were statistically significant (p < 0.0001). Conclusion: These data suggest that the systemic balance between neutrophil-related inflammation and lymphocyte anti-tumor immunity may condition response to immunotherapy in lung cancer, with clinical benefit primarily occurring in patients with such a preexisting, intra-tumoral T-cell adaptive immune response. Correlative tissue immune gene signatures as well as circulating cyto-chemokine emerged as potential indicators of a T cell– inflamed phenotype necessary for the clinical activity of PD-1/PD-L1 inhibitors.
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45

DEL, BEN FABIO. "A SINGLE-CELL METABOLIC ASSAY FOR THE DETECTION OF CIRCULATING TUMOR CELLS". Doctoral thesis, Università degli Studi di Trieste, 2016. http://hdl.handle.net/11368/2908085.

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The number of circulating tumor cells (CTCs) in blood is strongly correlated with the progress of metastatic cancer and can be used as a diagnostic tool to improve staging, therapy-monitor and recurrence detection. Current FDA-approved device to detect CTCs is based on immunostaining with limitations in sensitivity range, high-cost per analysis and lack of viable cell isolation. Other techniques are emerging, though none of them reached clinical validation. Here, we present an unexplored label-free method exploiting the abnormal metabolic behaviour of cancer cells (extracellular acidification, lactate production, Warburg effect). We demonstrate a single-cell analysis technique to measure the secretion of lactic acid and acidification of extracellular medium of individual, living tumor cells compartmentalized in microfluidically prepared monodisperse, pL droplets and implement the method in a high-throughput semi-automated prototype, able to detect spiked tumor cells in blood. We finally show promising data from blood sample of metastatic cancer patients.
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46

Fuh, Marceline Manka [Verfasser] y 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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47

Schier, Wiebke Henrike [Verfasser] y 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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48

Clark, Michael Edward. "Unravelling the potential applications of extracellular vesicles for the clinical management of melanoma patients". Thesis, Edith Cowan University, Research Online, Perth, Western Australia, 2021. https://ro.ecu.edu.au/theses/2485.

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Metastatic melanoma is the third most common cancer in Australia with global incidence increasing. After decades without effective systemic treatments for advanced melanoma, the advent of targeted and immune therapies has substantially improved patient survival. While this is encouraging, further research is needed as the majority of patients treated with targeted therapy ultimately develop drug resistance. Immunotherapy can achieve durable responses in many patients however, not all patients respond to current single or a combination of immune checkpoint inhibitors. Considering the cost and potential toxicities to patients being treated with these therapies, there is an urgent need to develop biomarkers that can predict patient response to treatment, likelihood of toxicity, and ultimately survival. Extracellular vesicles (EVs) are small particles that contain a diverse array of molecular cargos that represent the cell of origin. EVs have established roles in various hallmarks of cancer, including the mediation of drug resistance and immunosuppression. In addition, EVs have tremendous potential as biomarkers to predict or monitor patient outcomes. This thesis aims to provide a foundation of methodologies to explore the potential of melanoma derived EVs. In turn, this will allow an expansion of our understanding on the role of melanoma derived EVs on therapeutic outcomes. Chapter 1 of the thesis provided a review into the development of melanoma, current treatment strategies, drug resistance and a broad introduction on EVs. Chapter 2 demonstrated the ability to detect the mRNA of BRAF splicing variants in the plasma of melanoma patients who developed resistance to targeted therapy. Further, it showed that these mRNA variants were detected in plasma derived EVs. In Chapter 3, the potential of EVs to transfer resistance to BRAF inhibition was explored utilising a panel of BRAF treatment resistant cell lines. However, no evidence was found that EVs could transfer BRAF resistance. In Chapter 4, the plasma of melanoma patients being treated with pembrolizumab was used to isolate EV-RNA to identify a transcriptional signature predictive of response. Lastly, Chapter 5 provides a general discussion of the studies presented in this thesis. Altogether, the results of these studies underscore the potential of EVs as unique biomarkers to predictive response to treatment in melanoma.
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49

BONO, Marco. "Multigene panel testing in Hereditary Breast and Ovarian Cancer: an effective liquid biopsy approach to identify mutations in genes involved in the Homologous Recombination pathway". Doctoral thesis, Università degli Studi di Palermo, 2021. http://hdl.handle.net/10447/479959.

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Background: Hereditary breast and ovarian cancer (HBOC) syndrome is an autosomal dominant inherited disorder that include 5–7% of all breast cancer (BC) cases and 10-15% of all ovarian cancer (OC) cases. BRCA1 and BRCA2 are the most common genes associated to HBOC syndrome. However, hereditary syndrome could be associated with germline PVs in several high- and moderate-risk genes. In recent years, Next-Generation Sequencing (NGS) has allowed to study multiple genes simultaneously, to reduce analysis costs, to led to an explosion of genetic data, and to offer more information to patients. Methods: We retrospectively collected and analyzed to BRCA1/2 test 876 patients affected by BC and OC (531 of BC, 345 of OC) among January 2016 to August 2020. Successively, we analyzed 192 patients resulted BRCA1/2 negative with a strong personal and/or family history to BC and/or OC by using Multi-gene panel testing. We evaluated 22 genes involved in risk of hereditary breast, ovarian and colorectal cancer, and other inherited tumor syndromes. Results: Analysis conducted with multi-gene panel testing revealed that 28 (14.6%) BC and OC patients showed PVs/LPVs in genes no-BRCA. In particular, we analyzed 165 BC patients and 27 OC patients, and we obtained 27 and 4 patients with PVs/LPVs in genes no-BRCA respectively. BC patients with alteration in gene over BRCA hardly showed TNBC than patients with BRCA1/2 or all wt. Moreover, among BC patients with genes altered beyond BRCA the 45.8% showed a Bilateral Breast Cancer. In OC group we observed that 75% of patients with PVs/LPVs in genes over BRCA showed a previously personal history of BC or other cancer. Conclusion: Our analysis showed that the 14.6% of patients BRCA-negative with a strong personal and/or family history to BC and/or OC presented alteration in genes beyond BRCA1/2. This result highlighted the importance of multi-gene panel testing which should be extended at all these patients and be included in clinical practice
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50

Kuhlmann, Jan Dominik [Verfasser], Sabine [Akademischer Betreuer] Kasimir-Bauer, Stephan [Akademischer Betreuer] Hahn y 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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