La diffusion dans le sang des cellules tumorales circulantes (CTC) à partir de la tumeur primitive est un signe précoce d'invasivité tumorale et du risque de développer des métastases. Par conséquent, la capacité à les détecter de façon très sensible et spécifique est censée constituer un test cliniquement important pour le pronostic du cancer, le suivi des patients et la personnalisation de la thérapie. Les CTC sont des cellules rares, et plusieurs méthodes ont été proposées pour leur détection. La technique ISET (Isolation by Size of Epithelial/Tumor cells) se base sur la différence de taille des CTC par rapport aux cellules leucocytaires et a montré une très grande sensibilité d'isolement et spécificité d'identification des CTC. Elle permet l'analyse cytopathologique, immunologique et moléculaire des cellules isolées.Le cancer du rein représente 3% des cancers de l'adulte, dans 75% des cas il s'agit d'un carcinome rénal à cellules claires (RCC). Sur le plan génétique, il est un des rarissimes cancers solides caractérisé par des variations de l'ADN, il s'agit de mutations au niveau du gène VHL.Ce projet de recherche vise l'analyse comparative, moléculaire et cytopathologique, des CTC isolées à partir des patients avec RCC dans le but d'évaluer, par une approche moléculaire, les critères cytopathologiques diagnostiques des CTC. Notre étude a porté sur 29 patients ayant bénéficié de l'isolement des CTC par ISET avant toute intervention chirurgicale.L'analyse cytopathologique a été réalisée utilisant les critères décrits par l'équipe de P. Hofman pour définir les CTC (CNHC-MF) et les Cellules Atypiques Circulantes " CAC " (CNHC-UMF). L'analyse génétique par séquençage du gène VHL a été réalisée avec succès sur l'ADN de 205 cellules individuelles, sur l'ADN issu du tissu tumoral et sur l'ADN génomique de chaque patient.Sur les 29 tumeurs étudiées, 25 étaient caractérisées par des mutations du gène VHL. Cent soixante et une cellules, CTC et CAC, isolées à partir du sang de ces 25 patients, ont présenté des variations génétiques du gène VHL identiques à l'ADN issu du tissu tumoral. Il s'agit de 18 mutations différentes affectant les 3 exons de ce gène. Nous avons trouvé des CTC/CAC dans 29/30 des patients avec CCRC analysés. Des mutations VHL ont été trouvées dans 25 des 29 tumeurs CCRC correspondantes. Nous avons obtenu des résultats spécifiques VHL dans 205 des 327 CTC/CAC microdisséquées, comprenant 64 CTC et 141 CAC, selon l'analyse cytopathologique. Les mutations VHL ont été détectées en aveugle dans 57/64 CTC et dans 125/141 CAC. Cependant, nous avons observé que les 8 et 16 CTC et CAC restantes, respectivement, avaient été isolées de patients sans mutations VHL détectables dans le tissu tumoral.Conclusion : Ceci est la première étude comparative de diagnostic génétique et cytopathologique des CTC/CAC chez des patients avec un cancer solide, le CRCC. Nos résultats suggèrent que des critères cytopathologiques élargis pourraient être appliqués au diagnostic des CTC chez les patients avec CCRC. Bien que des études complémentaires et plus élargies soient maintenant nécessaires, cette méthode ouvre la voie à une approche génétique pour le diagnostic des Cellules Tumorales Circulantes

Oral cancer is the sixth most common cancer worldwide and has been marked by high morbidity and poor survival rates that have changed little over the past few decades. Beyond prevention, early detection is the most crucial determinant for successful treatment and survival of cancer. Yet current methodologies for cancer diagnosis based upon pathological examination alone are insufficient for detecting early tumor progression and molecular transformation. Development of new diagnostic tools incorporating tumor biomarkers could enhance early detection by providing molecular-level insight into the biochemical and cellular changes associated with oral carcinogenesis. The work presented in this doctoral dissertation aims to address this clinical need through the development of new automated cellular analysis methods, incorporating lab-on-a-chip sensor techniques, for examination of molecular and morphological biomarkers associated with oral carcinogenesis. Using the epidermal growth factor receptor (EGFR) as a proof-of-principle biomarker, the sensor system demonstrated capacity to support rapid biomarker analysis in less than one-tenth the time of traditional methods and effectively characterized EGFR biomarker over-expression in oral tumor-derived cell lines. Successful extension from in vitro tumor cell lines to clinically relevant exfoliative brush cytology was demonstrated, providing a non-invasive method for sampling abnormal oral epithelium. Incorporation of exfoliative cytology further helped to define the important assay and imaging parameters necessary for dual molecular and morphological analysis in adherent epithelium. Next, this new sensor assay and method was applied in a small pilot study in order to secure an initial understanding of the diagnostic utility of such biosensor systems in clinical settings. Four cellular features were identified as useful indicators of cancerous or pre-cancerous conditions including, the nuclear area and diameter, nuclear-to-cytoplasm ratio, and EGFR biomarker expression. Further examination using linear regression and ROC curve analysis identified the morphological features as the best predictors of disease while a combination of all features may be ideal for classification of OSCC and pre-malignancy with high sensitivity and specificity. Further testing in a larger sample size is necessary to validate this regression model and the LOC sensor technique, but shows strong promise as a new diagnostic tool for early detection of oral cancer. / text

Wound healing and carcinogenesis are defined as complex, adaptive processes which are controlled by intricate communications between the host and the tissue microenvironment. A number of phenotypic similarities are shared by wounds and cancers in cellular signaling and gene expression. Radiotherapy is the second most effective modality of cancer treatment after surgery and can be used, either alone or in combination with chemotherapy. Recent findings suggest that radiotherapy apart from tumor cell death also rapidly and persistently modifies the tissue microenvironment. These modifications affect cell phenotype, tissue metabolism, bidirectional exchanges and signaling events between cells. The complex interactions between stromal cells and cancer cells are of immense interest and in The First Part of My Thesis, I tried to explore the crosstalk between stromal and carcinoma cells in response to radiotherapy by genetic modulation of the stroma and irradiation. We found that fibroblasts, irrespective of their RhoB status, do not modulate intrinsic radiosensitivity of TC-1 but produce diffusible factors able to modify tumor cell fate. Then we found that Wt and RhoB deficient fibroblasts stimulated TC-1 migration through distinct mechanisms respectively, TGF-β1 and MMP-mediated. We also found that co-irradiation of fibroblasts and TC-1 abrogated the pro-migratory phenotype by repression of TGF-β and MMP secretion. This result is highly relevant to the clinical situation and suggests that conversely to, the current view; irradiated stroma would not enhance carcinoma migration and could be manipulated to promote anti-tumor immune response. Secondly, our in vivo experiments, tends to confirm the in vitro data showing that irradiated tumor bed does not stimulate tumor growth and escape. Our results also challenges the view that irradiated stroma would promote migration of carcinoma cells as we show that independently from their genotype co-irradiation of fibroblasts and carcinoma cells repressed carcinoma cell migration and confirmations studies are currently performed in vivo. The Third Part of My Project, was dedicated to investigate the effect on CTC release after radiotherapy. Consistently with the results reported after surgery , the number of CTC increases in the blood stream after radiotherapy probably due to radiation-induced vascular injury induced or/and by EMT induction in tumor cells but these cells seemed to be entrapped into the cardiac cavity. The significance of these CTC to metastatic development is still under investigation but there is evidence for a metastasis-promoting effect of RT from animal studies.Thus the microenvironment can exert antagonist stimulatory or inhibitory effects on malignant cells.

Objective: The aim of this study was to develop an immunomagnetic/real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay and assess its clinical value for the molecular detection of circulating tumor cells (CTCs) in peripheral blood of pancreatic cancer patients. Methods: The presence of CTCs was evaluated in 34 pancreatic cancer patients before systemic therapy and in 40 healthy controls, through immunomagnetic enrichment, using the antibodies BM7 and VU1D9 [targeting mucin 1 and epithelial cell adhesion molecule (EpCAM), respectively], followed by real-time RT-PCR analysis of the genes KRT19, MUC1, EPCAM, CEACAM5 and BIRC5. Results: The developed assay showed high specificity, as none of the healthy controls were found to be positive for the multimarker gene panel. CTCs were detected in 47.1% of the pancreatic cancer patients before the beginning of systemic treatment. Shorter median progression-free survival (PFS) was observed for patients who had at least one detectable tumor-associated transcript, compared with patients who were CTC negative. Median PFS time was 66.0 days [95% confidence interval (CI) 44.8–87.2] for patients with baseline CTC positivity and 138.0 days (95% CI 124.1–151.9) for CTC-negative patients (p = 0.01, log-rank test). Conclusion: Our results suggest that in addition to the current prognostic methods, CTC analysis represents a potential complementary tool for prediction of outcome in pancreatic cancer patients. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Diss. Linköping : Univ., 2004.

Cette thèse a pour but de développer des techniques en imagerie par résonance magnétique(IRM) afin de détecter des altérations neurologiques à l’échelle microscopique dans des modèlesanimaux. Deux modèles chez la souris ont été étudiés en particulier: le modèle Glio6 de glioblastomehumain et le modèle MAP6, apparenté à la schizophrénie. Les méthodologies développées ont étécentrées autour de l’IRM du tenseur de diffusion (DTI) 3D rapide et à haute résolution spatiale pourdes applications ex vivo et in vivo chez le rongeur. Dans le modèle Glio6, la migration de cellulestumorales dans le corps calleux a été précocement détectée et quantifiée alors qu’aucun signe n’étaitvisible sur les IRM anatomiques classiques. La tractographie, imagerie des fibres de la matièreblanche, a permis d’identifier des déficits de certains tracts et de leurs connectivités dans le modèleMAP6. Des altérations inhomogènes ont été détectées, avec en particulier une réduction drastique dela voie cortico-spinale, résultats mettant en exergue le rôle primordial de la protéine MAP6 lors de laneuromorphogénèse. La méthode « Super Résolution » développée puis appliquée in vivo aux sourisMAP6, a permis d’obtenir en moins d’une heure une imagerie de tractographie comparable à celleobtenue ex vivo (en 59h), ce qui ouvre la voie à des suivis longitudinaux in vivo pour des études dudéveloppement du cerveau ou de l’évaluation de nouvelles thérapies. D’autre part, une méthode IRMcellulaire in vivo quantitative a été mise en place. Le principe repose sur la mesure combinée desrelaxivités cellulaires in vitro (pouvoir à réduire les temps de relaxation T2*, T2 et T1) pour convertir lestrois paramètres de la relaxation in vivo en concentrations cellulaires. En utilisant le modèle de gliomeU87 et des cellules U937 marquées magnétiquement, les résultats ont montré qu’une très large gammede concentrations cellulaires peut être quantifiée et que la biodistribution des cellules U937 autour dela tumeur est hétérogène, information essentielle pour étudier l’efficacité d’une thérapie cellulaire. / This thesis aims to develop magnetic resonance imaging (MRI) techniques to detectneurological damage at the microscopic level in animal models. Two mouse models were examined inparticular human glioblastoma model (Glio6) and a schizophrenia mouse model (MAP6 model). Themethodologies developed were centered around 3D fast diffusion tensor imaging (DTI) with highspatial resolution for ex vivo and in vivo applications in rodents. In Glio6 model, the migration oftumor cells in the corpus callosum was early detected and quantified while no signs were visible onconventional anatomical MRI. Tractography identified deficits of some tracts and their connectivity inthe MAP6 model. Inhomogeneous alterations were detected, especially with a drastic reduction of thecorticospinal pathway. Theses results highlight the crucial role of the MAP6 protein in the braindevelopment. The "Super Resolution" post-proccesing was developed and applied in vivo to MAP6mouse model. Tractography imaging comparable to that obtained ex vivo (in 59h) was obtained in lessthan one hour, paving the way for in vivo longitudinal studies as brain development studies orevaluation of new therapies. On the other hand, a in vivo cellular MRI method has been established.The principle is based on the combined measurement of cell relaxivities in vitro, to obtain in vivo cellconcentrations based on relaxation parameters. Using the U87 glioma model and U937 magneticallylabeled cells, the results showed that a wide range of cell concentrations can be quantified and thebiodistribution of U937 cells around the tumor is heterogeneous, information essential to study theeffectiveness of cell therapy.

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Introduction: Together with the introduction of new therapeutic options in castration- resistant prostate cancer (CRPC), an advance in individual disease characterization is required. Since common biopsy methods are not suitable for the majority of CRPC patients, one possible solution is the liquid biopsy that is, the analysis of circulating tumor cells (CTCs) isolated from the cancer patients' blood. Methods: A method based on the immunomagnetic enrichment of CTCs and subsequent PCR detection of tumor-associated genes (AdnaTest, Qiagen) was characterized and used for the detection of CTCs in 41 CRPC patients. Each patient was screened at the time of CRPC diagnosis and after the 3rd cycle of docetaxel therapy. A panel of genes associated with therapeutic decision-making was established and validated. Quantitative PCR (qPCR) method on a BioMark platform (Fluidigm, USA) was used to determine the expression of the gene panel in the CTC-enriched and primary tumor samples and the results were analyzed. Results: CTCs were found in 85% and 45% of CRPC patients before and during the therapy, respectively. The presence of CTCs, as well as EGFR and AR PCR fragments, was associated with a decreased sPSA response and lower survival. The gene expression of the CTC- enriched and primary tumor samples differed...

O desenvolvimento da imunoterapia do câncer baseada em células dendríticas (DCs) é alvo de vários estudos. Para tumores sólidos, a abordagem baseada no uso de DCs alogenêicas fundidas com células tumorais tem se mostrado relativamente eficaz. Por outro lado, esta estratégia necessita uma massa tumoral considerável de cada paciente para a geração das células híbridas. Para contornar este problema o uso de DCs transfectadas com mRNA tumoral, o qual pode ser amplificado in vitro a partir de uma pequena amostra inicial do tumor, tem sido investigado. Para isto, uma transfecção eficiente e tradução correta do mRNA tumoral nas DCs são etapas críticas. Sabendo-se que as DCs de pacientes com câncer possuem atividade aloestimuladora defeituosa, DCs derivadas de doadores saudáveis poderiam ser uma alternativa para induzir uma resposta imune mais eficiente. Assim, este trabalho pretendeu aprimorar a metodologia de transfecção de DCs alogenêicas, derivadas de monócitos de doadores saudáveis, com mRNA de antígenos tumorais (survivina e RPSA) super-expressos na leucemia linfóide crônica (LLC) e avaliar sua capacidade em estimular a resposta linfocitária. Ao mesmo tempo, foram estabelecidas as metodologias para amplificação e síntese do RNA destes antígenos tumorais específicos, assim como do RNA mensageiro total, contidos nas células tumorais de pacientes com LLC. Os resultados mostraram ser possível a amplificação do mRNA total extraído das células leucêmicas com manutenção da expressão dos antígenos tumorais. Ainda, várias condições de transfecção com mRNA da survivina, transcrito in vitro, foram testadas, encontrando-se na lipofecção, a melhor maneira de transfectar as DCs. A lipofecção mostrou-se com baixa toxicidade quando comparada à técnica de eletroporação. Observou-se uma eficiência em torno de 40% de células transfectadas num intervalo de tempo entre 12 e 48 horas. Estas células foram usadas como estimuladoras em ensaios de proliferação usando-se linfócitos T alogenêicos como células respondedoras. As células transfectadas com mRNA da survivina foram capazes de estimular resposta linfoproliferativa com maior produção de IFN-gama, avaliado por ELISA. Além disso, a transfecção não alterou o padrão de expressão dos marcadores de superfície característicos das DCs. Estes dados mostram que a transfecção das DCs com mRNA pode afetar a resposta imune induzida por estas APCs. Nossos resultados suportam o uso de DCs transfectadas com mRNA para produção de vacinas anti-tumorais e mostram a survivina como um potente antígeno indutor da resposta linfocitária. / The development of dendritic cell (DC)-based cancer immunotherapy has been the target of many studies. For solid tumors, a promising approach based in allogeneic DCs fused to tumor cells has been relatively effective. On the other hand, this approach needs large tumor samples to generate enough DC-tumor cell hybrids. To overcome this problem, tumor mRNA-transfected DCs can be used, since mRNA can be amplified in vitro and allow unlimited vaccine production. For this, efficient transfection and optimal translation of tumor mRNA in DCs are critical. Moreover, DC derived from cancer patients has defective alostimulatory activity. In this case, DC derived from healthy donors may be an alternative to induce immune response more efficiently. Here, we established the methodology of allogeneic DC transfection with mRNA for tumor antigens (survivin and RPSA) overexpressed in chronic lymphocytic leukemia (CLL), and evaluated their ability for T cell stimulation. At the same time, we established mRNA amplification and mRNA in vitro transcription methodology for specific tumor antigens and total messenger RNA, present in CLL tumor cells. Our results showed it to be possible to amplify total mRNA derived from leukemic cells maintaining tumor antigen expression. Moreover, several transfection conditions using survivin mRNA obtained from in vitro transcript reactions were evaluated, defining lipofection as the better way to transfect DC. We obtained nearly 40% of transfected DCs between 12 and 48 hours. Transfected DCs were used as stimulator cells in proliferation assays using allogeneic T cells as responder cells. Survivin mRNA transfected DCs were able to stimulate T cell proliferation with increased IFN-gama production, measured by ELISA. Furthermore, the transfection did not change the pattern of surface molecules expression characteristic of DC. These data show that mRNA DC transfection can affect immune responses induced by these APCs. These findings support the use of tumor mRNA transfected DCs for anti-cancer vaccine production and show survivin as a potent antigen to induce T cell responses.