Les cellules souches cancéreuses (CSC) représentent une sous-population de cellules tumorales à l’origine de l’hétérogénéité et de la croissance tumorale. Les CSC sont plus résistantes aux traitements, et à l’origine de la rechute et des métastases. L’identification des CSC constitue actuellement un enjeu majeur dans le développement de nouvelles thérapies ciblées pour inhiber la croissance tumorale et éradiquer le cancer. Dans ce travail, nous avons cherché à identifier, caractériser, et cibler les CSC dans l’adénocarcinome gastrique. Des modèles murins de xénogreffe de tumeurs primaires de patients atteints d'adénocarcinome gastrique hors cardia de types intestinal et diffus ont été développés, ainsi qu’un modèle de tumorsphere in vitro afin d’évaluer les capacités tumorigéniques de sous-populations tumorales. Nous avons identifié CD44 et l'aldéhyde déshydrogénase (ALDH) comme marqueurs d’enrichissement des CSC dans les 2 types d’adénocarcinomes gastriques, l’ALDH représentant un marqueur plus spécifique que CD44. Nous avons ensuite étudié l'effet de l’acide rétinoïque tout trans (ATRA), et nous avons montré que l'ATRA inhibe la formation et la croissance des tumorspheres in vitro ainsi que la croissance tumorale in vivo. Cet effet de l’ATRA passe par l’inhibition de l’expression des marqueurs souches et des capacités d'auto-renouvèlement des CSC. En conclusion, CD44 et ALDH sont des marqueurs de CSC dans les adénocarcinomes gastriques hors cardia de types intestinal et diffus, et le traitement par l’ATRA constituerait une stratégie commune de traitement pour cibler spécifiquement les CSC et inhiber la croissance tumorale dans ces deux types de cancer gastrique. / Cancer stem cells (CSCs) are a subpopulation of tumor cells at the origin of the heterogeneity and growth of tumors. CSCs are more resistant to treatment, and are responsible for relapse and metastasis. The identification of CSCs is a major challenge for the development of new targeted therapies to inhibit tumor growth and eradicate cancer. In this work, we aimed to identify, characterize, and target CSCs in gastric adenocarcinoma. Mouse models of primary tumor xenografts from intestinal and diffuse type non-cardia gastric adenocarcinomas from patients were developed, as well as an in vitro tumorsphere assay, to assess the tumorigenic capacity of subpopulations of tumor cells. We identified CD44 and aldehyde dehydrogenase (ALDH) as CSC enrichment markers in the two types of gastric adenocarcinoma, ALDH representing a more specific marker than CD44. We then studied the effect of All-trans retinoic acid (ATRA), and showed that it inhibited the formation and growth of tumorspheres in vitro and tumor growth in vivo. This effect of ATRA is due to the inhibition of stem marker expression and the self-renewal capacity of CSCs. In conclusion, CD44 and ALDH are effective CSC markers in intestinal and diffuse type non-cardia gastric adenocarcinomas, and treatment with ATRA provides a common treatment strategy to specifically target CSCs and inhibit tumor growth in both subtypes of this gastric cancer.

No description available.

Gastric and esophagogastric junction cancers are heterogeneous and aggressive tumors with an unpredictable response to cytotoxic treatment. New methods allowing for the analysis of drug resistance are needed. Here, we describe a novel technique by which human tumor specimens can be cultured ex vivo, preserving parts of the natural cancer microenvironment. Using a tissue chop- per, fresh surgical tissue samples were cut in 400 μm slices and cultivated in 6-well plates for up to 6 days. The slices were processed for routine histopa- thology and immunohistochemistry. Cytokeratin stains (CK8, AE1/3) were ap- plied for determining tumor cellularity, Ki-67 for proliferation, and cleaved caspase-3 staining for apoptosis. The slices were analyzed under naive conditions and following 2–4 days in vitro exposure to 5-FU and cisplatin. The slice culture technology allowed for a good preservation of tissue morphology and tumor cell integrity during the culture period. After chemotherapy exposure, a loss of tumor cellularity and an increase in apoptosis were observed. Drug sensitivity of the tumors could be assessed. Organotypic slice cultures of gastric and es- ophagogastric junction cancers were successfully established. Cytotoxic drug effects could be monitored. They may be used to examine mechanisms of drug resistance in human tissue and may provide a unique and powerful ex vivo platform for the prediction of treatment response.

Gastric cancer ranks the fifth most common and third leading cause of cancer‐related deaths worldwide. Alterations in the RTK/MAPK, WNT, cell adhesion, TP53, TGFβ, NOTCH, and NFκB signaling pathways could be identified as main oncogenic drivers. A combination of altered pathways can be associated with molecular subtypes of gastric cancer. In order to generate model systems to study the impact of different pathway alterations in a defined genetic background, we generated three murine organoid models: a RAS‐activated (KrasG12D, Tp53R172H), a WNT‐activated (Apcfl/fl, Tp53R172H), and a diffuse (Cdh1fl/fl, Apcfl/fl) model. These organoid models were morphologically and phenotypically diverse, differed in proteome expression signatures and possessed individual drug sensitivities. A differential vulnerability to RTK/MAPK pathway interference based on the different mitogenic drivers and according to the level of dependence on the pathway could be uncovered. Furthermore, an association between RTK/MAPK pathway activity and susceptibility to HDAC inhibition was observed. This finding was further validated in patient‐derived organoids from gastric adenocarcinoma, thus identifying a novel treatment approach for RTK/MAPK pathway altered gastric cancer patients.

Bypass or resection of the stomach and oesophagus, has long been recognised to result in profound changes in the handling of ingested nutrients. This results in significant morbidity after radical surgery for oesophago-gastric cancer, in particular post-prandial hypoglycaemia, altered appetite, early satiety and noxious post-prandial symptoms. By profiling and challenging the gut hormone axis in healthy volunteers and patients who had undergone total or subtotal gastrectomy, or oesophagectomy, this thesis explores the possible causative mechanisms for the challenges faced by this patient population. In the surgical groups, an oral glucose tolerance test (OGTT) resulted in enhanced secretion of satiety and incretin gut hormones (GLP-1, GIP, PYY) and insulin, followed by hypoglycaemia in a cohort of patients. Continuous glucose monitoring of gastrectomy participants over two weeks of normal lifestyle identified an increased incidence of day and night time hypoglycaemia. RNAseq and mass spectrometry based peptidomics of human and murine enteroendocrine cells in the pre- and post-operative populations revealed no significant change in the underlying cellular pathways for nutrient sensing and gut hormone secretion, indicating that the altered hormone secretion is primarily driven by accelerated nutrient transit, rather than adaptive changes in the gut. Finally, specific blockade of the GLP-1 receptor in post-gastrectomy patients using Exendin 9-39 normalised insulin secretion and prevented reactive hypoglycaemia after an OGTT. In conclusion, profound changes in gut hormone secretion as a result of enhanced nutrient transit after foregut surgery likely underlie the early and late post-prandial symptoms seen in this group, and therapies specifically targeting the gut hormone axis, and GLP-1 in particular, could be the first targeted treatments for post-gastrectomy syndromes.

Toll-like Rezeptoren (TLRs) sind essentielle angeborene Rezeptoren, die konservierte Strukturen von Krankheitserregern oder Gefahrsignale, die von beschädigten Zellen freigesetzt werden, erkennen können. Genetische Variationen in TLRs wie Einzel-Nukleotid-Polymorphismus (SNP) können die Funktion von TLRs beeinträchtigen und erste Studien zeigen, dass dies zu einer erhöhten Anfälligkeit gegenüber Virusinfektionen oder einem erhöhten Krebsrisiko führen kann. In dieser Studie haben wir einen Multicolor-Durchflußzytometrie-Test entwickelt, um die TLR-Funktionen in verschiedenen Subpopulationen unseparierter peripherer mononukleärer Blutzellen (PBMCs) simultan analysieren zu können. Wir konnten beobachten, dass das Ausmaß der TLR-Antworten zwischen den Probanden stark variierte, jedoch über einen Zeitraum von einem Monat gut reproduzierbar war. Zunächst untersuchten wir TLR Reaktionen bei Patienten mit rezidivierenden Herpes labilalis (HL). Im Vergleich zu asymptomatischen Personen war eine HL- Anamnese mit einer signifikant verminderten TLR3-IFN-Gamma-Antwort nach Stimulation mit poly(I:C) in NK Zellen assoziiert. Weitere molekulare Untersuchungen zeigten eine mögliche Beteiligung von TLR3 L412F SNP, welcher die oberflächliche TLR3 Expression und die IFN-Gamma-Antworten in NK-Zellen reduzierte. Einige Studien zeigen, dassTLR1 I602S, ein weiterer sehr verbreiteter SNP, in der Lage ist die TNF-Alpah-Antworten von Monozyten gegen den TLR2/1-Agonisten (Pam3Cys) zu verringern. In der hier vorliegenden Arbeit konnten wir zudem nachweisen, dass TLR1 I602S SNP auch die Funktion von NK-Zellen und CD8+ T-Zellen beeinträchtigt. Wir konnten keine Assoziation zwischen TLR2/1-Defizienz und reaktivierendem HL feststellen. Jedoch konnten wir an einer großen Kohorte von über 326 Patienten zeigen, dass der TLR1 SNP sowohl ein Risikofaktor für Magenkarzinomentstehung als auch für die Metastasierung ist. Zusammenfassend weisen unsere Ergebnisse darauf hin, dass genetische Polymorphismen von TLRs die Funktion von NK-Zellen beeinträchtigen und zu einer erhöhten Anfälligkeit für HSV-1 Erkrankung und Magenkarzinom führen können. / Toll-like Receptors (TLRs) are essential innate receptors which recognize conserved structures of pathogens, or danger signals released from damaged cells. Alterations of TLR responses might result in severe viral infections or a higher risk of cancer. Therefore, development of clinical assays to evaluate TLR functions could provide personalized information about susceptibility to these diseases. Since TLRs are differentially expressed on different subsets of human peripheral blood mononuclear cells (PBMCs), a multi-color flow cytometry-based assay was developed to detect TLR responses of individual cell types simultaneously. We observed that the magnitude of TLR responses largely varied between human subjects, but was highly reproducible over one month. To evaluate the potential role of differences in natural killer (NK) cell TLR response we studied the association of NK cell TLR function and TLR single nucleotide polymorphisms (SNPs) with susceptibility to recurrent herpes labialis (HL) and gastric cancer. Using our assay, impaired TLR3 response of NK cells was found in people with recurrent HL. In addition, we have identified enhanced levels of homozygous TLR3 L412F SNP in people with recurrent HL, which results in lower surface expression and reduced NK cell response to poly(I:C). TLR1 I602S, another common SNP, has been reported to decrease TNF-Alpha responses of monocytes toward TLR2/1 agonist, Pam3CSK4 (Pam3Cys), stimulation. In our study, we found that TLR1 I602S homozygosity also contributes to impaired IFN-Gamma responses of NK cells and CD8+T cells. Although we did not observe an association of TLR2/1 deficiency with recurrent HL, association of TLR1 I602S with risk for primary as well as metastatic gastric cancer was found in a cohort of 326 patients. To sum up, our results suggest that genetic polymorphisms of TLRs can impair TLR function of NK cells, which contribute to the increased susceptibility to HSV-1 diseases and gastric cancer.

Helicobacter pylori est associée à des pathologies gastro-duodénales sévères mais est également un marqueur bactérien de migrations humaines. Nous avons montré que des populations génétiques distinctes de H. pylori ont accompagné au moins quatre migrations en Asie du sud-est et en Océanie : i) une expansion des ancêtres des austronésiens il y a 5000 ans à partir de Taiwan en Océanie, ii) une migration d’Inde en Asie du sud-est depuis 2000 ans,iii) une migration des ancêtres des locuteurs des langues austro-asiatiques au Vietnam et auCambodge il y a 4000 ans, i) une migration des ancêtres des Thaïs du sud de la Chine vers l’actuelle Thaïlande au début du second millénaire. Ces données confirment la résolution plus élevée de la diversité génétique de H. pylori pour retracer les anciennes migrations humaines par comparaison aux marqueurs génétiques humains traditionnels. Nous avons ensuite investigué les facteurs de virulence de souches isolées de patients présentant des symptômes gastriques au Sénégal et au Cambodge. Au Sénégal, une association significative a été observée entre le cancer gastrique et le gène cagA, deux motifs EPIYA-C et l’allèle vacA s1. De multiples segments EPIYA-C étaient observés moins fréquemment que dans les autres régions du monde, contribuant probablement à la faible incidence du cancer gastrique. Au Cambodge, une introgression fréquente d’allèles cagA et vacA européens dans des souches d’Asie de l’est a été observée. CagA et VacA ayant des effets antagonistes, cette expansion pourrait entraîner la rupture de l’équilibre entre les effets biologiques de ces deux protéines et être responsable de conséquences graves sur l’évolution de la maladie. / Helicobacter pylori is associated with severe gastroduodenal disorders but is also a bacterial genetic marker of human migrations. First, we provide evidence that distinct H. pylori genetic populations accompanied at least four ancient human migrations into Oceania and Southeast Asia: i) an expansion of Austronesian speaking people about 5000 years ago from Taiwan into Oceania, ii) a migration from India into Southeast Asia within the last 2000 years, iii) a migration of Austro-Asiatic speaking people into Vietnam and Cambodia about 4000 years ago, and iv) a migration of the ancestors of the Thai people from Southern China into Thailand during the early second millennium AD. These findings demonstrate that H. pylori genetic diversity has more discriminatory power than traditional human genetic markers for tracing old human migrations. Second, we investigated virulence factors of H. pylori strains isolated from patients with gastric symptoms in Senegal and Cambodia. In Senegal, a significant association was observed between gastric cancer and the cagA gene, two EPIYA-C segments, and the s1 vacA allele in Senegal. Multiple EPIYA-C segments were less frequent than reported in other countries, possibly contributing to the low incidence of gastric cancer. In Cambodia, the frequent introgression of European vacA and cagA alleles into East Asian H. pylori strains was observed. As VacA and CagA have opposing effects, this expansion may disrupt the balancing activities on epithelial cells which might result in severe consequences for individual disease outcome.

Made available in DSpace on 2016-01-26T12:51:44Z (GMT). No. of bitstreams: 1 lucianadesouzapenhalbelsilva_dissert.pdf: 1343193 bytes, checksum: e6aec2f7449d341b83eb617b4ddfb1ff (MD5) Previous issue date: 2013-06-07 / Introduction: Malnutrition adversely affects the patients who have gone through oncological surgery, leading to greater surgical morbidity and mortality. However, there is still the need to establish a golden rule for nutritional evaluation as a predictive factor in the morbimortality of these patients. The objective of this study was to analyze and compare the capacity of nutritional assessment methods in the prognosis of complications in patients submitted to gastric cancer gastrectomy. Methods: The method used was the prospective observational study of 66 gastric cancer patients who were submitted to gastrectomies. Assessment was made through the following nutritional evaluation methods: Subjective Global Assessment of Nutritional Status (SGA), Objective Nutritional Evaluation (ONE) and the Nutritional Risk Index (NRI). Beyond the surgical risk by means of a prognostic index of Sheffield (SPI). The results were compared to surgical morbimortality, total duration of hospitalization and stay in the Intensive Care Unit (ICU). The stage of the disease was based on the American classification for gastric cancer. For the estatiscal analyses were used Kaplan-Meier method, log-rank test, t test or non-parametric Kruskal-Wallis test. Associations were evaluated by the Pearson chi-square test, or by the Fisher exact test whenever recommended. Results: Malnutrition was present in 80.7%, 66.7% and 85% in the SGA, ONE and NRI, respectively. The SPI demonstrated a high surgical risk in 92% of patients. There was a slight agreement in the SGA and NRI (Kappa=0.30). The SGA results showed that the malnourished patient (p<0.05) remained in the hospital for a longer period time. Most of the patients (62.9%) were at an advanced stage of the disease which was associated to an increase in complications (p<0.01) and isolated cases of pneumonia that were associated with lymphopenia (p<0.05). Conclusion: The SGA and the NRI were the methods that had the closest agreement in the results. The SGA was the only method that demonstrated an association between malnutrition and longer stay in hospital. Pneumonia was observed in the presence of advanced tumors and lymphopenia, thus increasing the duration of hospitalization and stay in the ICU (p<0.05). / Introdução: A desnutrição afeta adversamente o paciente cirúrgico oncológico, levando-o a maiores morbidades e mortalidade operatórias. Porém, ainda há necessidade de se estabelecer um padrão ouro de avaliação nutricional como fator preditivo de morbimortalidade nesses pacientes. O objetivo deste estudo foi analisar e comparar a capacidade dos métodos de avaliação nutricional no prognóstico de complicações de pacientes submetidos a gastrectomias por câncer gástrico. Métodos: Estudo observacional prospectivo envolvendo 66 pacientes com câncer gástrico e submetidos a gastrectomias. Foram analisados os métodos de avaliação nutricional subjetiva global (ANSG), avaliação nutricional objetiva (ANO) e o índice de risco nutricional (IRN), além do risco cirúrgico por meio do índice prognóstico de Sheffield (IPS). Os resultados foram comparados com a morbimortalidade cirúrgica, tempo de internação total e em UTI. O estadio da doença foi obtido pela classificação americana para câncer gástrico. Resultados: A desnutrição esteve presente em 80,7%, 66,7% e 85% nas ANSG, ANO e IRN, respectivamente. O IPS demonstrou alto risco cirúrgico em 92% dos pacientes. Houve concordância leve entre a ANSG e IRN (Kappa=0,30). A ANSG demonstrou maior tempo de internação hospitalar nos desnutridos (p<0,05). A maioria dos pacientes (62,9%) apresentaram-se com estágio avançado e associado à maior incidência de complicações (p<0,01) e isoladamente a pneumonia esteve associada com a linfopenia (p<0,05). Conclusão: ANSG e o IRN foram os métodos que tiveram uma concordância mais próxima. A ANSG foi o único método que demonstrou associação entre desnutrição e maior tempo de internação hospitalar. A pneumonia foi observada no tumor avançado e na presença de linfopenia, aumentando os dias de internação hospitalar e em UTI (p<0,05).

Gastric cancer (GC) is one of leading causes of death in the world, and peritoneal metastases (PM) are a major site of recurrence. PM from GC implies a poor prognosis, with median overall survival (mOS) approximately 3 months and no survival at five years. The aims of this thesis were to explore the incidence and evaluate prognostic factors for mOS of PM from GC in a defined population; to investigate the outcome of a new multimodal treatment; to analyse the treatment costs, and to investigate differences in drug sensitivity between individual patient samples and between various tumours. The incidence of loco-regional advanced GC was 3.8 per 100,000 person-years. Synchronous loco-regional GC in combination with synchronous distant metastasis was a negative prognostic factor while chemotherapy and good performance status, and radiotherapy plus chemotherapy were positive prognostic factors . There were no significant differences in mOS for the group of patients included during the period 2000-2004 versus 2005-2009, and this lack of improvement in mOS during the past decade justifies new treatment approaches. In a Phase II study of patients treated with neoadjuvant systemic chemotherapy followed by cytoreductive surgery + hyperthermic intraperitoneal chemotherapy, mOS was 14.3 months and for patients with macroscopically radical surgery mOS was 19.1 months. The mean overall cost of the loco-regional treatment was $145,700 compared to $59,300 with systemic chemotherapy treatment. In an ex vivo chemo-sensitivity test, it was determined that GC samples were equivalent to colorectal cancer in chemo-sensitivity to standard drugs and targeted drugs, whereas ovarian cancer samples were more sensitive. The individual GC samples varied considerably in sensitivity to increasing concentrations of the drugs, arguing for individualized drug selection. The incidence of loco-regional advanced GC was more common than previously reported and there were no improvements in mOS over the past decade. The mOS for patients with neoadjuvant systemic chemotherapy followed by macroscopically radical cytoreductive surgery + hyperthermic intraperitoneal chemotherapy was better than in recent reports on treatment with systemic chemotherapy. Treatment of advanced GC patients is costly irrespective of treatment modality. The GC samples varied considerably between individuals in terms of sensitivity to increasing concentrations of the drugs and were comparable to colorectal cancer in chemo-sensitivity.

La evidencia sobre el efecto de las nitrosaminas (NA) sobre el cáncer gástrico (CG) es insuficiente. El objetivo fue evaluar el efecto de las NA (exógenas y endógenas ) y sus alimentos fuente sobre el riesgo de CG. El estudio incluyó 521.457 individuos del estudio EPIC (European prospective Investigation into Cancer and Nutrition) y 314 casos incidentes de C. Estimamos la exposición exógena aplicando una tabla de composición que desarrollamos para tal fin. Para estimar las NA endógenas creamos un índice de formación endógena (ENOC) basado en datos previamente publicados. Observamos un efecto de carnes rojas, procesadas y ENOC restringida a la región distal y en individuos positivos el Helicobacter Pylori. No se observó ningún efecto de las NA exógenas sobre el CG. Se observo una interacción entre ENOC y vitamina C. En resumen, el riesgo de CG se ve afectado por la interrelación entre ENOC, Hp y vitamina C. / Evidence relating nitrosamines (NA) and gastric cancer (GC) risk is not conclusive. The risk of GC associated with meat and NA intake and endogenous formation of NA (ENOC) was investigated in the European Prospective Investigation into Cancer and Nutrition with a population of 521.457 subjects and 314 incident cases of GC. To estimate NA intake we developed a food composition database. An index of ENOC was created using information from previous published studies. The risk of CG increases with intake of red and processed meat and ENOC restricted to non-cardia site and mainly in Helicobacter pylori (Hp) infected subjects. There were no association between NA intake and GC. We observed an interaction between ENOC and vitamin C. In summary, Hp infection, vitamin C and ENOC play an important role on GC risk.