Dissertations / Theses on the topic 'Neuroblastoma'

Eines der charakteristischen Merkmale des Neuroblastoms stellt seine einzigartige biologische Heterogenität dar, die eine genaue Ausage des weiteren klinischen Verlaufes stark erschwert. Bestimmte prognostisch wirksame klinische, molekularbiologische und genetische Faktoren, wie zum Beispiel Alter bei Erstdiagnose, Tumorstadium, MYCN-Amplifikation und 1p Deletion, werden seit längerem zur Risikostratifizierung genutzt. Bereits in anderen Tumorerkrankungen konnte nun der Einfluß einer Telomerlängenveränderung auf das Gesamtüberleben von Patienten nachgewiesen werden. Telomere sichern die genomische Integrität und bestimmen maßgeblich die proliferative Kapazität jeder somatischen Zelle. Aktuelle Forschungsergebnisse legen die Vermutung nahe, dass Veränderungen der Telomerlänge auch in Neuroblastomen einen prognostischen Effekt auf das Gesamtüberleben haben. In diesem Kontext untersucht die vorliegende Arbeit den Zusammenhang zwischen Telomerlänge und Gesamtüberleben in 420 MYCN nicht-amplifizierten primären Neuroblastomen mit Erstdiagnosen von 1983-2001. Hierfür wurden die relativen Telomerlängen mithilfe einer neu etablierten monochromen multiplex q-RT-PCR ermittelt. Anschließend wurden diese sowohl mit ausgesuchten klinischen Variablen (Alter bei Erstdiagnose, Tumorstadium, Primärlokalisation des Tumors, Histologie, Geschlecht und Rezidivauftreten) korreliert als auch auf ihren Einfluß auf das Gesamt- und ereignisfreie Überleben untersucht. In Korrelation mit den klinischen Parametern konnte zwischen Alter bei Erstdiagnose und Telomerlänge ein eindeutiger Zusammenhang nachgewiesen werden. Je älter die Patienten bei Erstdiagnose, desto höher war sowohl der Anteil verlängerter Telomere als auch der extremer Telomerlängenveränderungen. Neuroblastome mit verlängerten Telomeren zeigten in der gleichen Altersgruppe ein verringertes Gesamtüberleben der betroffenen Patienten verglichen mit Neuroblastomen mit verkürzten Telomeren. Somit könnte eine Telomerlängenveränderung, insbesondere verlängerte Telomere, im klinischen Alltag als Hinweis auf einen prognostisch ungünstigen Verlauf genutzt werden.

Le neuroblastome est le cancer solide extra-cranial le plus fréquent chez l’enfant. Il est caractérisé par une très grande hétérogénéité tant au niveau clinique que moléculaire. Alors que certains patients rentrent spontanément en rémission, on peut se demander quels facteurs permettent la réémergence du cancer chez d’autres malgré traitement. Pour répondre à cette question, il convient d’identifier chez les patients ayant rechuté, les différentes populations clonales coexistant au diagnostic et/ou à la rechute. Cela permet, entre autre, d’étudier les voies différemment altérées entre ces deux temps. Dans cette optique, nous présentons ici QuantumClone, un algorithme de reconstruction clonal à partir de données de séquençage, ainsi que son application à une cohorte de patients souffrant d’un neuroblastome. Sur ces données, l’application de notre méthode a permis d’identifier des différences dans le ratio de variants prédits fonctionnels par rapport à ceux prédits passagers entre les populations ancestrales, enrichies à la rechute ou appauvries à la rechute
Neuroblastoma is the most frequent solid extra-cranial cancer of childhood. This cancer displays a high heterogeneity both at clinical and molecular levels. Even though in some patients spontaneous remission can be observed, some others relapse despite treatment and surgical resection. It may be wondered which are the factors that distinguish these two cases. In order to answer this question, identification of populations coexisting at diagnosis and/or relapse in the patients which have relapsed is a prerequisite. This would allow, between other things, to study the pathways differently altered in clones that are specific to each time point. With this in mind, we hereby present QuantumClone, a clonal reconstruction algorithm from sequencing data. In addition, we applied this method to a cohort of patients suffering from neuroblastoma. On these data, our method identified differences in the functional mutation rate, i.e. the number of putative functional variants by total number of variants, between the ancestral clones, clones expanding at relapse, and clones shrinking at relapse

Le neuroblastome (NB) est une tumeur pédiatrique du système nerveux sympathique. Des mutations activatrices du gène ALK (Anaplastic Lymphoma Kinase) ont été identifiées dans 8 % des formes sporadiques et dans des formes familiales de NB. Le gène ALK code pour un récepteur tyrosine kinase appartenant à la famille des récepteurs à l’insuline, principalement exprimé dans le système nerveux central et périphérique. Le récepteur ALK représente une cible thérapeutique pertinente dans ce cancer. Des mutations de novo du gène ALK ont également été rapportées dans une forme syndromique associant NB congénital et encéphalopathie sévère avec dysmorphie du tronc cérébral, suggérant un rôle développemental du gène ALK en plus de son implication dans l’oncogenèse.Dans ce contexte, mon projet de thèse avait pour but de déterminer le rôle du récepteur ALK muté dans l’oncogenèse du NB et le développement, principalement à l’aide de modèles murins originaux obtenus au laboratoire. J’ai ainsi largement caractérisé deux lignées de souris KI (Knock-In) Alk pour les deux mutations les plus fréquemment observées dans le NB: F1174L et R1275Q chez l’homme, correspondant à F1178L et R1279Q chez la souris.Une analyse détaillée de ces deux lignées de souris n’a pas révélé de phénotype majeur chez les souris KI AlkR1279Q hétérozygotes et homozygotes ainsi que chez les hétérozygotes KI AlkF1178L. Par contre, nous avons documenté une forte létalité post-natale des animaux KI AlkF1178L homozygotes et montré que ces nouveaux-nés présentent des troubles majeurs d’alimentation. Les homozygotes KI AlkF1178L phénocopient donc partiellement les patients encéphalopathes. La différence d’effet observé entre les animaux hétérozygotes et homozygotes suggère fortement qu’il existe un seuil d’activation du récepteur Alk compatible avec la survie.Nous avons ensuite exploré le rôle du récepteur ALK muté dans le système nerveux sympathique des souris KI Alkmut. Cette analyse a montré que l’activation du récepteur induit un excès de prolifération des neurones sympathiques de E14.5 à la naissance. Néanmoins, nous n’avons pas observé de NB chez ces animaux. En croisant ces souris avec la lignée TH-MYCN, nous avons documenté une coopération des mutations Alk avec l’oncogène MYCN pour le développement de NB. La comparaison des profils transcriptomiques des tumeurs murines MYCN et MYCN/Alkmut a révélé que l’expression de l’oncogène Ret (codant également un récepteur à activité tyrosine kinase) était fortement induite par l’activation du récepteur Alk. Le traitement des souris par un inhibiteur de l’activité kinase du récepteur Ret a montré une diminution de la taille des tumeurs suggérant que le gène Ret joue un rôle majeur dans l’oncogenèse induite par le récepteur Alk muté. Par ailleurs, l’induction de l’expression du gène RET par le récepteur ALK muté dans les NB a été confirmée dans des lignées et des tumeurs humaines.Afin de déterminer le mécanisme par lequel l’activation du récepteur ALK aboutit à la régulation de l’expression du gène RET des expériences ont été effectuées sur des lignées humaines de NB dans lesquelles le récepteur ALK peut être activé ou inactivé. Ce travail a montré que l’expression du gène RET est dépendante de l’axe ALK-ERK-ETV5. En effet, la modulation de l’activité du récepteur ALK affecte l’expression des gènes ETV5 et RET. Cet effet est dépendant de l’activation de la voie MEK/ERK. Par ailleurs, ETV5 active l’expression du gène RET. Afin de confirmer le rôle de Ret dans l’oncogenèse dépendante du récepteur Alk, nous avons croisé des souris portant une mutation activatrice de Ret avec les souris TH-MYCN. Nous avons ainsi mis en évidence que le récepteur Ret activé coopère avec l’oncogène MYCN dans le développement de tumeurs et que ces tumeurs sont des NB présentant des caractéristiques très semblables à celles des tumeurs MYCN/Alkmut. Le gène Ret apparaît donc comme une cible essentielle du récepteur Alk muté dans l’oncogenèse du NB
Neuroblastoma (NB) is a pediatric tumor arising from the sympathetic nervous system. Activating mutations of the ALK gene have been observed in around 8 % of sporadic neuroblastoma as well as in familial cases. The ALK gene encodes a tyrosine kinase receptor of the insulin receptor super-family. It is mainly expressed in the central and peripheral nervous system. The ALK receptor represents a therapeutic target in this cancer. De novo ALK mutations have also been reported in a syndrome associating congenital NB and severe encephalopathy with abnormal shape of the brainstem, suggesting a developmental role for the ALK gene in addition to its implication in oncogenesis.In this context, my PhD project was to determine the role of the mutated ALK receptor in NB oncogenesis and in development, mainly with original mouse models obtained in the laboratory. I extensively characterized two knock-in (KI) Alk mouse lines with the two mutations that are most frequently observed in NB: F1174L and R1275Q in human and F1178L and R1279Q in mouse.A detailed analysis of these two mouse lines showed that the KI AlkR179Q heterozygous and homozygous mice as well as the KI AlkF1178L heterozygous mice do not show striking clinical signs. On the contrary, we documented a high postnatal lethality for KI AlkF1178L homozygous mice and showed that these pups presented with a dramatic reduced milk intake. Thus, the KI AlkF1178L homozygous mice partially phenocopy the human patients with encephalopathy. The difference of phenotype between the heterozygous and the homozygous KI AlkF1178L mice highly suggest a threshold of activity of the Alk receptor compatible with survival.We then explored the role of the mutated ALK receptor in the sympathetic nervous system of the KI Alkmut mice. This analysis showed that the activation of the receptor induces an excess of proliferation in sympathetic neurons from E14.5 to birth. However, we could not observe NB in these animals. We next bread these mice with the transgenic TH-MYCN line. We documented cooperation between Alk mutations and the MYCN oncogene to induce NB. Comparison of transcriptomic profiles of MYCN vs MYCN/Alkmut tumors revealed that the expression of the Ret oncogene (encoding a tyrosine kinase receptor) was strongly induced by the activation of the Alk receptor. Besides, the induction of the expression of the RET gene by the mutated ALK receptor in NB was confirmed in human cell lines and tumors.In order to determine the mechanism by which the activation of the ALK receptor regulates RET gene expression, experiments were done on human NB cell lines in which the ALK receptor can be activated or inactivated. This work showed that RET gene expression is dependent of the ALK-ERK-ETV5 axis. Indeed, the modulation of the ALK receptor activity affects gene expression of ETV5 and RET. This effect is dependent of the activation of the MEK/ERK pathway. Besides, ETV5 increases RET gene expression. In order to confirm the role of the Ret receptor in oncogenesis driven by the mutated Alk receptor, we bread mice bearing an activating mutation of the Ret gene with the TH-MYCN mice. We showed that the activated Ret receptor cooperates with the MYCN oncogene in tumor formation and that these tumors are NB presenting with characteristics very close to MYCN/Alkmut tumors. Thus, the Ret gene appears to be an essential target of the mutated Alk receptor in NB oncogenesis

Estimulação Magnética vem sendo utilizada no tratamento de várias patologias do sistema nervoso central, mas a compreensão de sua ação em nível celular precisa ser melhor investigada. Sendo assim, o objetivo principal desta dissertação foi estabelecer, em cultura celular, um método de Estimulação Magnética Estática (E- ME) e avaliar seu efeito em diferentes tipos celulares. Para tanto, foi desenvolvido um suporte de placa de cultura com ímãs NeFeB (neodímio-ferro-boro) com a forma cilíndrica de 12mm de diâmetro por 6mm de altura. Cada suporte apresenta seis ímãs espaçados, de modo que os campos magnéticos não interajam entre si. As células de neuroblastoma humano SH-SY5Y foram plaqueadas 1x106 células por poço e cultivadas em placas de 24 poços. A análise microscópica das placas demonstrou que as células adaptaram-se ao novo ambiente, demonstrando aderência e crescimento adequados à superfície da placa. Após este primeiro passo, as células SH-SY5Y foram estimuladas utilizando 0,1 T, 0,2 T e 0,3 T por 60 minutos, buscando determinar a melhor intensidade de EME. Após, este período de estimulação, para avaliar a viabilidade celular, foi realizado o ensaio de MTT (brometo de [3- (4,5-dimetiltiazol-2yl)-2,5-difenil tetrazolium) nos grupos de células estimuladas e não estimuladas. Não tendo sido observada diferença estatisticamente significativa entre os grupos avaliados. A partir da obtenção destes dados, foram definidos os parâmetros de intensidade e período de estimulação da EME. Os experimentos foram, então, realizados aplicando 24 horas de estimulação com intensidade de 0,3T em culturas de diferentes tipos celulares. Optamos por utilizar, além de células de neuroblastoma humano SH-SY5Y, outro tipo de células tumoral, células de melanoma vaginal; e células de neuroblastoma diferenciado em células neuronais e células mesenquimais derivadas de adipócitos. Com estas escolhas objetivamos determinar a resposta de diferentes tipos celulares a EME. Para tanto, cada tipo celular foi dividido em 4 grupos, 2 grupos não estimulados (controles) avaliadas imediatamente (CI) e 24h após o final do experimento (C24), e 2 grupos estimulados por 24h, que foram avaliados imediatamente (EI) e 24h após o final da exposição a EME (E24). Para verificar a resposta celular a EME foram avaliados parâmetros de toxicidade (MTT, PI (Iodeto de propídeo) e HO (Hoechst), de neuroplasticidade (expressão do gene do receptor de BDNF (PCR em tempo real)) e ciclo celular (citometria de fluxo). Os resultados obtidos demonstram que imediatamente após a EME, houve uma diminuição significativa na viabilidade celular das células SH-SY5Y indiferenciadas (Kruskal Wallis, P<0,05). Já no grupo 24h, não houve diferença estatisticamente significativa em nenhum dos gru 9 pos avaliados (Kruskal Wallis, P>0,05). Visto que, houve uma diminuição da viabili- dade celular nas células SH-SY5Y, imediatamente após a estimulação, na busca de encontrar o mecanismo de ação pelo qual estas células apresentavam uma diminui- ção celular, utilizamos as técnicas de PI e de HO para avaliar apoptose e necrose celular, respectivamente. Adicionalmente avaliamos o ciclo celular. Desta forma e, considerando que apenas as células de neuroblastoma humano SH-SY5Y apresen- taram diminuição significativa na viabilidade celular, somente neste tipo celular foi feito esta avaliação. Não foi encontrada diferença estatisticamente significativa entre os grupos. No entanto, a análise descritiva demonstrou que, 24h após o estímulo, as células SH-SY5Y não diferenciadas apresentaram uma redução de duplicação celular (Kruskal Wallis, P >0,05). Outro fator avaliado nas células SH-SY5Y diferenciadas e não diferenciadas, como parâmetro de neuroplasticidade, foi expressão do gene do receptor Trk-β. Não foi encontrada diferença significativa, no entanto na análise descritiva, as células SH-SY5Y não diferenciadas avaliadas 24h após a aplicação de EME, apresentaram um aumento na expressão deste gene, sugerindo um aumento da neuroplasticidade. Estes resultados demonstram um efeito de longa duração da EME, por pelo menos até 24h após o final da EME, corroborando com dados prévios de nosso grupo de pesquisa, utilizando modelos animais e ETCC (estimulação transcraniana por corrente contínua), outra técnica neuromodulatoria, que mostraram efeito por até 7 dias após o termino da tratamento. Interessantemente, não foram observadas diferenças na viabilidade celular nas de- mais culturas celulares analisadas. Estes resultados são muito relevantes, pois demonstram que, em relação aos parâmetros de viabilidade celular analisados, a EME é uma técnica segura no protocolo utilizado (24h de 0,3T de EME). Os dados desta dissertação demonstram que a EME apresenta diferentes efeitos em relação à toxicidade em células de tumores neuronais, tumores não neuronais e células com morfologia normal. A diminuição da viabilidade celular nas células SH-SY5Y indiferenciadas é um resultado surpreendente e favorável considerando ser linhagem celular tumoral. Desta forma estes resultados avaliados em conjunto sugerem que a EME é uma técnica segura que, em células normais não provocou alterações importantes nos parâmetros avaliados e em tumores de células não neuronais não alterou o crescimento celular. No entanto, ainda se faz necessário aumentar o numero amostral da avaliação das fases do ciclo celular a expressão do gene Trk-β, assim como mais estudos para avaliar outros parâmetros de toxicidade e também diferentes protocolos de estimulação celular utilizando a EME.
Magnetic stimulation has been used in the treatment of various pathologies of the central nervous system, but the understanding of its action at the cellular level needs to be investigated. Thus, the main objective of this dissertation was to establish, in cell culture, a method of Static Magnetic Stimulation (SMS). For this purpose, a cul- ture plate holder with NeFeB (neodymium-iron-boron) magnets with a cylindrical shape of 12mm in diameter by 6mm in height was developed. Cells were plated 1x106 cells per well and cultured in 24-well plates. Microscopic analysis of plaques demonstrated that the cells adapted to the new environment, demonstrating ade- quate adhesion and growth to the plaque surface. This was extremely important to the development of this article. After this first step, human neuroblastoma cells (SH- SY5Y) were stimulated using 0.1 T, 0.2 T and 0.3 T for 60 minutes, in order to de- termine the best intensity of static magnetic stimulation. After this stimulation period, to evaluate cell viability, the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium] assay was performed on stimulated (stimulated group) and non- stimulated (control group) cells. No significant difference was observed between the groups evaluated. From the data obtained, it was defined the use of the highest in- tensity tested, an increase in the period of stimulation and application in other cell types. The experiments were then performed applying 24 hours of stimulation with 0.3T intensity in cultures of different cell types. Considering that the cells initially used were of neuronal tumor, we chose to use, in addition to human neuroblastoma cells, another type of tumor cells (vaginal melanoma cells), and cells with normal character- istics in their morphology, such as SH-SY5Y differentiated into neuronal cells and mesenchymal adipocyte-derived cells. With these choices we aimed to determine if different cell types would respond in the same way to SMS. In order to do so, each cell type was divided into 4 groups, 2 non-stimulated groups (controls) evaluated im- mediately (CI) and 24h at the end of the experiment (C24), 2 groups stimulated for 24h, which were evaluated immediately (SI) and 24h after the end of exposure to SMS (S24). To assess the cellular response to EME, toxicity parameters [MTT, PI (Propidium iodide) and HO (Hoechst)] and cell cycle (flow cytometry) were done. The results obtained demonstrate that immediately after SMS, a significant decrease in cell viability of undifferentiated SH-SY5Y cells was found (Kruskal Wallis, P <0.05). In the 24h group, there was no statistically significant difference in any of the groups evaluated (Kruskal Wallis, P> 0.05). Since there was a decrease in cell viability in SH-SY5Y cells, immediately after stimulation, in the search to find the mechanism of action by which these cells had a cellular decrease, we used the PI and HO tech- niques to evaluate apoptosis and death cell and respectively. Additionally, we evalu- ated the cell cycle. In this way, and considering that only SH-SY5Y human neuroblastoma cells showed a significant decrease in cell viability, only this cell type was evaluated. There were no statistically significant differences between groups. 11 However, the descriptive analysis demonstrated that, 24 h after the stimulus, undif- ferentiated SH-SY5Y cells present a decrease in cytoplasm division in the G1 phase and, in G2 phase, a decrease in nuclear division, leading to a reduction of cell dupli- cation (Kruskal Wallis, P> 0.05). Another factor evaluated in differentiated and undif- ferentiated SH-SY5Y cells as a parameter of neuroplasticity was expression of the Trk-β gene. No significant difference was found, however in the descriptive analysis, the undifferentiated SH-SY5Y cells evaluated 24h after the application of SMS showed an increase in the expression of this gene, suggesting an increase in neuro- plasticity. These results demonstrate a long-term effect of SMS for at least 24 hours after the end of the SMS, supporting previous data from our research group, using animal models and TDCs (transcranial direct current stimulation), and another neuromodulatory technique, which showed effect for up to 7 days after the end of treatment. Interestingly, no differences in cell viability were observed in the other cell cultures analyzed. These results are very relevant because they demonstrate that, in relation to the cell viability parameters analyzed; SMS is a safe technique in the pro- tocol used (24h of 0.3T of SMS). The data from this dissertation demonstrate that SMS has different effects in relation to toxicity in cells of neuronal tumors, non- neuronal tumors and cells with normal morphology. Decreased cell viability in undif- ferentiated SH-SY5Y cells is a surprising and favorable finding considering that it is a tumor cell line. Thus, these results evaluated together suggest that SMS is a safe technique that in normal cells did not induce important changes in the evaluated pa- rameters and in non-neuronal cell tumors did not alter the cell growth. However, it is still necessary to increase the sample number of the evaluation of the phases of the cell cycle and the expression of the Trk-β gene, as well as more studies to evaluate other parameters of toxicity and also different protocols of cellular stimulation using SMS.

Depuis la formulation de la théorie de l’immuno-surveillance en 1957 par Burnet et Thomas, le monde scientifique s’est efforcé d’identifier les cellules immunitaires impliquées dans ce processus. Les lymphocytes Natural Killer (NK) constituent une composant majeure de l’immuno-surveillance innée dans plusieurs cancers hématologiques et solides. L’activité des lymphocytes NK passe principalement par une grande variété de récepteurs avec un rôle activateur ou inhibiteur. Parmi les récepteurs activateurs présents à la surface des lymphocytes NK, le récepteur NCR3/NKp30 a un rôle majeur dans la toxicité directe contre la cellule cible et dans l’activation des cellules dendritiques.Les tumeurs stromales gastrointestinales (GIST) et le Neuroblastome (NB) sont deux tumeurs sensibles à l’immuno-surveillance par les lymphocytes NK. Dans une étude récente notre équipe a démontré que l’épissage alternatif du gène NCR3/NKp30 peut être déterminant dans la fonction NK et dans la survie des patients atteints de GIST.Afin de caractériser les lymphocytes infiltrant le GIST, nous avons effectué une recherche visant à analyser l’infiltrat des lymphocytes CD3+, des lymphocytes T régulateurs (Treg) et des lymphocytes NK dans des tumeurs GIST localisés, et corréler ces résultats à la survie des patients. Nous avons mis en évidence que, avant traitement, les lymphocytes NK sont surtout localisés au niveau des fibres trabéculaires qui entourent la tumeur, alors que les lymphocytes T sont localisé à l’intérieur de la tumeur en contact avec les cellules tumorales qui expriment HLA-I.Nous avons aussi observé que les cellules NK ont un phénotype plutôt CD56bright et migrent à l’intérieur de la tumeur après traitement par Imatinib. L’analyse de survie a mis en évidence que les lymphocytes NK et T peuvent prédire la survie sans progression (PFS). Ces résultats mettent en évidence l’importance de l’infiltrat immunitaire dans la prédiction du risque de rechute dans le GIST et surlignent l’importance de viser une réponse immunitaire dans les protocoles thérapeutiques.Nous avons ensuite déterminé la proportion de lymphocytes NK dans le sang périphérique et dans la moelle dans une cohorte de Neuroblastome (NB) localisé et métastatique : une infiltration plus important par les NK CD56bright a été observé chez les patients présentant une maladie métastatique et chez les patients avec une réponse mineure au traitement d’induction. De plus, les NK présents dans les échantillons de moelle osseuse infiltrés par les neuroblastes, présentaient une expression plus basse du récepteur NKp30. L’expression du ligand de NKp30, B7-H6, a été mise en évidence sur les neuroblastes infiltrant la moelle osseuse, et sa forme soluble, sB7-H6, a été retrouvée être positivement corrélée à l’extension de maladie et inversement à la réponse au traitement d’induction. L’analyse de l’épissage alternatif du gène NCR3/NKp30 a permis de mettre en évidence l’impact des isoformes NKp30 sur la survie sans progression chez les patients atteints de NB de haut risque en maladie minimale résiduelle après chimiothérapie d’induction. En particulier, les patients présentant un taux élevé de l’isoforme pro-inflammatoire (NKp30b) par rapport à l’isoforme immunosuppressive (NKp30c), présentent une meilleure survie sans évènement. Nous avons aussi démontré le rôle des monocytes dans l’amplification de la réponse NKp30 dépendant. Les résultats de notre recherche dans le GIST et dans le NB, deux maladies différentes mais toutes les deux sensibles aux lymphocytes NK, surlignent l’importance d’intégrer de nouvelles options thérapeutiques aptes à cibler le système immunitaire
Since Burnet and Thomas formulated in 1957 the cancer immunosurveillance theory, the scientific world has made tremendous progress to identify the immune cells involved in this process. Natural Killer (NK) cells have emerged as a major component of the innate immunosurveillance of several hematological and solid malignancies. The activity of NK-cells is mainly mediated through their wide variety of receptors with activating and inhibitory functions. Among the versatile receptors present on NK cells, the activating receptor NCR3/NKp30 is a major receptor involved in both direct killing of target cells and mutual NK and dendritic cell activation.Gastrointestinal stromal tumors (GIST) and Neuroblastoma (NB) are known to be tumors sensitive to NK immunosurveillance. In a recent study we showed that alternative splicing of NCR3/NKp30 gene can affect NK cell function and GIST patient’s outcome.In order to better characterize the GIST tumor-infiltrating lymphocytes, we analyzed the CD3+, T regulatory (Treg) and NK lymphocytes infiltration within primary localized GIST tumors and we determined their prognostic value. We described that, before treatment, NK cells are mainly localized in fibrous trabeculae while T lymphocytes are in the tumor nests in HLA-I positive tumor cells contact. Moreover infiltrating NK cells displayed a secreting CD56bright phenotype, and accumulate in tumor nests after Imatinib (IM) treatment. Importantly CD3+ and NK lymphocytes independently predicted progression free survival (PFS). These results highlight the importance of the immune infiltrate in re-define the GIST risk stratification and allow enhancing the immune response in the therapeutic decisions.We next investigated the proportions of NK cells in blood and bone marrow (BM) in a cohort of localized and metastatic NB; a high proportion of CD56bright NK cells was associated with metastatic NB and with poor response to induction treatment within the metastatic NB. Moreover, infiltrated BM presented NKp30 down regulation. The expression of the NKp30 ligand, B7-H6, was found on BM neuroblasts, while the soluble protein, sB7-H6 correlated with resistance to treatment. Furthermore the transcriptional status of NKp30/NCR3 dictated the event-free survival rates of HR-NBs with minimal residual disease post-induction chemotherapy: in particular patients presenting a high proportion of the immunosuppressive isoform (NKp30c) compared to the pro-inflammatory isoform (NKp30b), presented a worse outcome. We further demonstrated the significant role of monocytes to amplify the NKp30 activation response.These researches in GIST and NB, two different but at the meantime NK-sensitive diseases support the effort to define new immunological therapeutic approaches and to determine their optimal use

The solid childhood cancer neuroblastoma arises from cells of the sympathetic nervous system. Neuroblastoma is the most common childhood cancer in children younger than one year old worldwide and accounts for 28% of all cancers diagnosed in Europe and the U.S. in infants. 25% of neuroblastomas bear an amplification of the proto-oncogene MYCN; in addition to this genetic alteration, epigenetic modifications such as transcriptional silencing of tumour suppressor genes by promoter DNA hypermethylation and histone modifications can occur. Previous studies have shown an interaction between MYC and DNA hypermethylation, through recruitment of de novo DNA methyltransferases to the promoter of MYC target genes, in order to repress their transcription. We hypothesised that MYCN might be similarly capable of repressing its target genes. Our results revealed that MYCN interacts with the maintenance and de novo methyltransferase DNMT1 and DNMT3A, respectively in MYCN amplified neuroblastoma cell lines and all three bind the promoter region of the hypermethylated gene RASSF1A. However, only minor changes in DNA methylation of RASSF1A were shown by pyrosequencing analysis upon MYCN knock-down. This probably indicates only a small involvement of MYCN in epigenetically-induced gene silencing by DNA hypermethylation. The important role of MYCN in gene activation was highlighted from DNA methylation microarray studies on neuroblastoma cell lines compared to human neural crest cells. 73% of all hit genes showed hypomethylation, which led to the conclusion that a more open chromatin configuration and thus increased gene expression is favoured in high stage neuroblastoma cell lines, where MYCN and MYC compensate each others protein levels. The same study also led to the discovery of a novel methylated gene MEGF10 in neuroblastoma. The importance of epigenetic silencing in neuroblastoma was also highlighted by the ability of DNA demethylating agent 5-Aza-2'-deoxycytidine to re-sensitise chemotherapeutic resistant neuroblastoma cells to commonly used cytotoxic drugs.

Neuroblastoma (NB) is the most common extracranial paediatric tumour, and patients with disseminated disease have a poor long-term prognosis. Due to the significant mortality rate, alternative treatments to conventional therapies are continually sought. Using the A/J mouse model it has previously been demonstrated that a cellular vaccine of the syngeneic Neuro-2a NB cell line modified to express IL-2 and IL-12 abrogated the tumourigenicity of Neuro-2a cells, and mediated regression of established tumours. However, establishing cultures of primary NB cells can be problematic, making this approach difficult to implement clinically. This thesis describes the development of an alternative cellular vaccine to treat murine NB using a synthetic vector. Firstly, transfection of primary dendritic cells was optimised. Dendritic cells are potent antigen presenting cells and studies have shown them to generate anti-cancer responses. Optimal transfection levels of 5% were obtained but antigen presentation by these cells was limited. Therefore, an alternative approach was developed using fibroblasts engineered to express IL-2 and IL-12. Cytokine-expressing fibroblasts could be used in place of transfected tumour cells to provide sustained, high-level cytokine expression in the tumour locale. Transfection of syngeneic and allogeneic murine fibroblasts was optimised in vitro to produce therapeutic levels of IL-2 and IL-12. Cytokine-transfected fibroblasts were compared with cytokine-transfected Neuro-2a cells to prevent engraftment of wild-type Neuro-2a cells in vivo. The allogeneic cells prevented tumour engraftment in a nonspecific, cytokine-independent manner. Syngeneic fibroblasts expressing IL-2 and IL-12 inhibited tumour engraftment as effectively as cytokine expressing Neuro-2a cells, and this rejection was cytokine-dependent. The cytokine-transfected syngeneic fibroblasts induced protective immunity against rechallenge with wild-type Neuro-2a cells as effectively as cytokine-transfected-Neuro-2a cells. Intratumoural vaccination of cytokine-transfected syngeneic fibroblasts also demonstrated therapeutic efficacy against Neuro-2a-derived established tumours. Splenocytes from vaccinated mice demonstrated increased IL-2 and IFN-y expression and cytotoxicity compared with controls when co-cultured with wild-type Neuro-2a cells in vitro. Vaccinated tumours showed decreased vascularity and increased infiltration of CD45+ cells compared with controls. Therefore, cytokine-transfected syngeneic fibroblasts are a viable potential alternative vaccine for the treatment of minimal residual NB.

Le neuroblastome est un cancer pédiatrique diagnostiqué chez les très jeunes enfants. afin de pouvir étudier l'impact de signalisation embryonnaire sur le développement de la maladie in vivo, nous avons mis au point un nouveau modèle d'étude du neuroblastome chez l'embryon aviaire. Ce nouveau modèle nous permet d'étudier in vivo la contribution de différentes signalisation à la tumorigénèse neuroblastique. nous avons donc mis en évidece le rôle de Sema3C et HDGF dans la tumorigénèse neuroblastique
Neuroblastoma is a pediatric cancer diagnosed in very young children. In order to study the impact of embryonic signaling on the development of the disease in vivo, we have developed a new model for the study of neuroblastoma in the avian embryo. This new model allows us to study in vivo the contribution of different signaling to neuroblastic tumorigenesis. We have thus highlighted the role of Sema3C and HDGF in neuroblastic tumorigenesis

INTRODUÇÃO: A relevância da expressão MYCN em neuroblastomas sem amplificação, diferentemente do seu aumento quando amplificado, permanece controverso. Neste trabalho, avaliou-se a relação do nível de expressão do transcrito MYCN em neuroblastoma não amplificado com os fatores clínicos e biológicos de prognóstico. MÉTODOS: Neste estudo observacional realizado entre janeiro de 2000 e dezembro de 2004, foram aferidos os valores do nível de expressão MYCN em 29 amostras tumorais, pela técnica RQ-PCR, no Laboratório de Biologia Tumoral da Fundação Pró-Sangue de São Paulo. Seus resultados foram analisados em relação à idade ao diagnóstico, ao estadiamento tumoral, ao grupo de risco, à ocorrência de recaída tumoral e de óbito. RESULTADOS: Foram nove crianças com idade 1 ano, os valores da expressão do transcrito MYCN variaram de 0,041 e 27,569, mediana 3,193. O estadiamento foi: quatro estádio 1; três estádio 2; oito estádio 3 e 14 crianças estádio 4. Entre 20 crianças com classificação patológica, 11 foram favoráveis e nove desfavoráveis. Considerando a mediana dos valores expressos a estratificação em grupos de risco com aumento da expressão foram: cinco crianças baixo risco; quatro risco intermediário e cinco alto risco. Grupos de risco sem aumento da expressão foram: duas crianças baixo risco; quatro risco intermediário e nove alto risco. Vinte e oito crianças obtiveram remissão completa e entre elas 14 apresentaram doença progressiva, sendo que sete morreram. As variáveis clínicas e biológicas não apresentaram freqüências diferentes entre os grupos de risco sem e com aumento de expressão. Entre os grupo alto risco e não alto-risco as variáveis idade, recaída tumoral e óbito apresentaram resultado com significado estatístico quando não se considerou o valor da expressão e quando não houve o seu aumento. Entre os grupos alto risco e nãoalto risco com aumento da expressão apenas a idade apresentou resultado com significado estatístico. CONCLUSÃO: Em crianças com estádio clínico não avançado o nível de expressão parece exercer uma relevância clínica, sugerindo um efeito protetor quanto menor for o aumento da expressão MYCN.
INTRODUTION: MYCN expression value in non-amplified neuroblastosmas remains a controversial issue. In order to add contributions to this field, children with nonamplified neuroblastomas were studied regarding their expression and correlation with clinical and other biological factors. METHODS: Twenty nine tumor samples obtained from non-consecutive patients admitted from January, 2000 through December, 2004, had their MYCN transcript expression levels evaluated according to the RQ-PCR assay, at the Tumoral Biology of Laboratory of the \"Fundação Pró -Sangue Hemocentro de São Paulo\", and compared to the following other factor: age at onset; tumor stage; risk - group; tumoral relapse rate and death. RESULTS: nine under one-year-old children and 20 over one-year-old children, with MYCN transcription expression level between 0.041 and 27.569, mean 3.193. Four children were stage 1, three were stage 2, stage 3 in eight and stage 4 in 14 children. In 20 patients with pathological classifications, 11 were favorable and nine unfavorable histology. Children whose expression level was above the mean were stratified as follows according to risk groups: five low-risk; four intermediate-risk and five high-risk patients. The ones whose expression level was under the mean were two low-risk, four intermediate-risk end nine high-risk patients. Twenty eight children achieved complete remission, with 14 recurrences, with seven deaths. The only factor associated to highly expressed MYCN patients was tumoral state. CONCLUSION: In children with non-advanced-stage disease low levels of expression might be a relevant favorable prognostic factor.

Das Neuroblastom ist der häufigste extrakraniell gelegene solide Tumor der pädiatrischen Onkologie. Der Verlauf der Erkrankung geht von spontaner Regression oder Differenzierung bis hin zu tödlich verlaufenden Erkrankungen. Die Mortalität von Patienten mit Tumoren in fortgeschrittenen Stadien ist immer noch sehr hoch. Die aggressivsten Tumoren sind die, die eine Amplifikation des Protoonkogens MYCN aufweisen. Eine Untergruppe dieser MYCN amplifizierten Tumoren weist eine Coamplifikation von DDX1 auf. Die Prognose dieser Patienten ist besser als die mit allein MYCN amplifizierten Tumoren, wenn auch immer noch schlechter als die von Patienten ohne MYCN Amplifikation. Das DDX1-Protein ist eine putative RNA-Helikase. Über seine genaue Funktion ist noch nicht viel bekannt. Ziel dieser Arbeit war es, potentielle Ziel-mRNAs von DDX1 zu identifizieren, um einen besseren Einblick in die Funktionen von DDX1 und mögliche Wege der Beeinflussung von Tumorverhalten und Prognose zu erhalten. Hierzu wurden eine DDX1 amplifizierte und eine nicht amplifizierte Zelllinie in Kultur genommen und eine Immunopräzipitation mit Zelllysaten der beiden Zelllinien durchgeführt – jeweils mit einem spezifischen Antikörper gegen DDX1 und einem unspezifischen Kontrollantikörper. Die Identifizierung der an DDX1 gebundenen mRNAs erfolgte mittels Microarray. Validiert wurden einige der im Microarray identifizierten RNAs mittels RT-PCR. CDK1, ATM und p18 ließen sich als spezifische Ziel-mRNAs von DDX1 identifizieren.

Introdução: Os neuroblastomas apresentam grande diversidade de comportamento clínico, evoluindo desde remissão espontânea à rápida progressão e morte. Heterogeneidade clínica e biológica tem implicado em grande variedade de respostas terapêuticas, inclusive em crianças maiores de 1 ano e em estádios avançados. Objetivo: estudar quadro clínico, aspectos epidemiológicos, características laboratoriais, genéticas e histológicas em crianças maiores de 1 ano portadoras de neuroblastoma disseminado, correlacionando-os com a evolução clínica e tentando definir fatores de risco que possam influir na sobrevida e na possibilidade da indicação do transplante de medula óssea (TMO). Casuística e Métodos: as informações foram obtidas de 53 pacientes admitidos na Unidade de Oncologia (ITACI) do Instituto da Criança do HC-FMUSP no período de 1997 a 2007. Os pacientes foram estudados quanto aos seguintes fatores: sexo, idade, raça, estado nutricional, DHL, Hb, ferritina, VMA, características tumorais (tamanho, localização, metástases, histologia, MYCN), terapêutica utilizada e evolução clínica. Estudamos separadamente também os fatores preditivos para TMO. Resultados: devem ser destacados: 1) crianças submetidas à cirurgia retardada completa apresentam 5 vezes menor chance de óbito do que as demais; 2) pacientes submetidos a TMO apresentam maior sobrevida total e livre de eventos em relação ao grupo não submetido a essa modalidade terapêutica 3) a utilização de retinóides gera 14 vezes menor chance de óbito em relação ao grupo que não os utiliza. 4) surpreendentemente, pacientes com ferritina abaixo de 334 n/ug/l apresentam 8 vezes maior chance de óbito. Conclusões: Não identificamos parâmetros clínicos e laboratoriais, práticos e facilmente disponíveis, de prognóstico para subpopulações de neuroblastomas de prognóstico avançado, sendo o estudo de fatores de ordem molecular e biológicos essenciais, apesar das dificuldades em realizá-lo
Introduction: Neuroblastomas (NB) have widely diverse clinical behavior, moving from spontaneous remission to progression and death. Clinical and biological heterogeneity factors determine different survival, even with children older than 1 year in advanced stages. Objective: to study the clinical presentation, epidemiology, laboratory findings, genetics and histopathologic characteristics in children older than 1 year with advanced neuroblastoma and their correlation with the survival. Also defining prognostic variables for bone marrow transplantation (BMT) indication. Casuistic and Methods: 53 selected medical records from patients older than 1 year with advanced NB admitted to the Instituto da Criança do HC-FMUSP from 1997 to 2007 were reviewed. The following risk factors were analyzed: age, sex, race, nutritional status, LDH, hemoglobin level, ferritin level, urinary VMA, tumor characteristics such as: site, histology, size, metastases, MYCN, treatment and clinical course. Results: it should be mentioned that: 1) possibility of death in children who underwent complete second look is 5 times lower than remaining ones; 2) patients who underwent BMT have better overall survival and event-free survival in comparison to the group not receiving this treatment modality. 3) the use of retinoids generates 14 times less chance of death in comparison to the group not receiving them. 4) patients whose ferritin level was below 334 n/ug/l had surprisingly 8 times more chances to die from diseases Conclusions: no simple and easily obtainable prognostic variables in a subpopulation of patients with advanced stage neuroblastoma, suitable to be widely employed in a country as ours, were identified. The study of molecular and biologics factors remains essential for precise characterization of these patients

La télomérase est une ribonucléoprotéine, constituée d’un composant ARN (hTR) qui sert dematrice à l’addition des séquences télomériques aux extrémités des chromosomes et d’un composantprotéique catalytique à activité de transcriptase inverse (hTERT). La réactivation de la télomérasedans 90% des cancers compense le raccourcissement des télomères, permettant ainsil’immortalisation et la survie des cellules tumorales. Ce rôle canonique de la télomérase estaujourd’hui bien documenté. Cependant des travaux récents suggèrent que la télomérase pourraitavoir d'autres fonctions indépendantes de son rôle sur le maintien de la longueur des télomères dansla tumorigénèse et/ou la progression tumorale.Dans les neuroblastomes (NB), l’augmentation du niveau d’activité télomérase (AT) estassociée à un stade avancé de la maladie et à un mauvais pronostic. En effet, plusieurs études ontmontré que les neuroblastomes agressifs ont un niveau élevé d’AT alors que les tumeurs de bonpronostic, ont peu ou pas d’AT. En effet, les NB de stade 4S ayant la capacité de régresserspontanément ont un très faible niveau d'AT. Ces observations suggèrent que la télomérase peutjouer un rôle crucial dans le développement des NB.Afin de mieux comprendre l’implication de la télomérase dans le phénotype agressif desneuroblastes malins et dans la chimiorésistance, nous avons caractérisé les modificationsphénotypiques et génotypiques induites par l’inhibition de la télomérase via l’expression ectopiqued’un mutant dominant négatif catalytiquement inactif (DN-hTERT) dans la lignée IGR-N-91 induit unedifférenciation cellulaire de type stromale et une sensibilisation à l’apoptose en réponse à trois agentscytotoxiques (cisplatine, staurosporine, TRAIL). Cette chimiosensibilisation n’est pas la conséquenced’un raccourcissement des télomères mais probablement celui d’une modulation de l’expression decertains gènes impliqués dans la réponse apoptotique (ré-expression de la caspase 8 et de p53sauvage), suggérant une fonction non canonique de la télomérase. De plus, nous avons montréqu’hTERT régule activement l’expression de N-Myc. En effet, l’expression ectopique du mutanthTERT-DN entraîne une perte des copies surnuméraires de N-Myc conduisant à l’extinction del'expression de la protéine alors que la surexpression d’hTERT sauvage augmente au contraire lenombre de copies du gène. Cette élimination de la protéine N-Myc pourrait être le signe d’une pertedu caractère agressif des cellules tumorales comme en témoigne la diminution de l’expression de laNSE (marqueur de mauvais pronostique des NB) et l’induction du CD44 dans les cellules hTERT-DN.L’ensemble de nos résultats démontre donc un nouveau rôle majeur de la télomérase,indépendant de sa fonction canonique d’élongation des télomères, dans l’acquisition du phénotypemalin et dans la chimiorésistance des NB. Ces résultats sont importants en termes de connaissancede la biologie du NB et des possibilités thérapeutiques. En effet, ces résultats suggèrent quel’inhibition de la télomérase comme stratégie anti-cancéreuse est une approche qui présente un intérêttout particulier dans les cas de NB de stade 4 dans lesquels le taux de survie des patients reste trèsinsuffisant malgré les thérapeutiques les plus intensives
Telomerase is a ribonucleoprotein consisting of an RNA component (hTR) that serves as atemplate for the addition of telomeric sequences at the ends of chromosomes and a proteincomponent catalytic activity of reverse transcriptase (hTERT). The reactivation of telomerase in 90%of cancers compensates the shortening of telomeres, allowing the immortalization and survival oftumor cells. This canonical role of telomerase is now well documented. However recent studiessuggest that telomerase may have other functions beyond its role in maintaining telomere length intumorigenesis and / or tumor progression.In neuroblastoma (NB), increased levels of telomerase activity (TA) is associated withadvanced disease and poor prognosis. Indeed, several studies have shown that aggressiveneuroblastomas have a high level of TA while favourable tumors, have little or no TA. Therefore, lowtelomerase activity appears to be linked with regression or maturation of NB as it can be seen in theparticular group of 4S stage neuroblastoma. These observations suggest that telomerase may play acrucial role in the development of NB.To better understand the involvement of telomerase in the aggressive phenotype of malignantneuroblasts and drug resistance, we characterized the phenotypic and genotypic changes induced byinhibition of telomerase via ectopic expression of a mutant dominant negative catalytically inactive(DN-hTERT) in a metastatic chemoresistant NB cell line IGR-N-9. Our results show that theexpression of this mutant induces a stromal-type cell differentiation a sensitization to apoptosis inresponse to three cytotoxic agents (cisplatin, staurosporine, TRAIL). The chemosensitization is not theresult of telomere shortening but probably f a modulation of the expression of certain genes involved inthe apoptotic response (re-expression of caspase 8 and wild-type p53), suggesting a noncanonicalfunction of telomerase Furthermore, we showed that hTERT actively regulates the expression ofMYCN. Indeed, ectopic expression of the inactive mutant causes a loss of supernumerary copies ofMYCN leads to the extinction of the expression of the protein, whereas overexpression of wild hTERTincreases the number of copies of the MYCN gene. The elimination of MYCN protein could be a signof a loss of the aggressiveness of the tumor cells as evidenced by the decreased expression of NSE(a marker of poor prognosis of NB) and induction of CD44 in DN-hTERT cells.Overall, our findings thus demonstrate a new role of telomerase independent of its canonicalfunction of telomere elongation in the acquisition of the malignant phenotype and drug resistance inNB. These results are important in terms of knowledge of the biology of NB and therapeuticpossibilities. Indeed, our data suggest that inhibition of telomerase as an anticancer strategy is anapproach that has a particular interest in cases of stage 4 NB in which the survival rate of patientsremains very inadequate despite the therapeutic more intensive

Les neuroblastomes, même de haut risque répondent bien à la chimiothérapie initiale mais deviendront fréquemment résistants au traitement. Les inhibiteurs de topoisomérase I représentent un outil thérapeutique important dans la prise en charge des neuroblastomes réfractaires. Pour étudier la résistance aux inhibiteurs de topoisomérase I acquise dans un contexte thérapeutique, un modèle murin de neuroblastome résistant au CPT-11 a été développé. La chimiorésistance est connue comme un phénomène multifacoriel. Nous avons donc utilisé plusieurs approches pour mieux caractériser les mécanismes à l'origine de la résistance dans notre modèle. Une approche génomique a permis d'identifier la dérégulation de la voie de signalisation formée du récepteur ALK et de deux ligands PTN et MDK. Alors que ALK est décrit comme gène majeur de prédisposition au neuroblastome, principalement par le biais de mutations activatrices, nous avons démontré que l'activation du récepteur survenait par des mécanismes alternatifs aux mutations dans une large majorité de cas et participerait à l'initiation de la maladie. En revanche, nous n'avons pas pu prouver l'implication de ce récepteur dans la progression de la maladie ou dans sa réponse au traitement. Il semble que la régulation de ALK soit complexe et le rôle exact de ce récepteur dans la progression du neuroblastome reste à établir. En revanche, nous avons démontré l'importance du ligand MDK dans la régulation de l'expression et de l'activation de ALK ainsi que dans le contrôle de la survie des cellules neuroblastiques. Inhiber cette cytokine représente une stratégie thérapeutique intéressante, complémentaire des thérapies anti-ALK, actuellement en développement clinique dans le neuroblastome. D’autre part, la caractérisation phénotypique du modèle a permis de mettre en évidence une signalisation altérée des dommages à l'ADN associée à une instabilité génétique accrue dans les tumeurs résistantes. Celles-ci présentent également une modification de progression dans le cycle cellulaire et une proportion plus importante de cellules quiescentes. Au final, ce travail a permis d'identifier différents mécanismes de résistance qui représentent des marqueurs de réponse au traitement et des cibles thérapeutiques intéressantes dans le neuroblastome
Neuroblastoma, including high-risk cases, show a good initial response to chemotherapy but will frequently become resistant to treatment. Topoisomerase I inhibitors represent an important therapeutic option for refractory neuroblastoma. To study the reisitance to topoisomerase I inhibitors acquired in a therapeutic setting, we developed in vivo a resistant model to irinotecan (CPT-11). Chemoresistance is known as a multifactorial phenomenon. We have therefore used several approaches to better characterize mechanisms leading to resistance in our model. A genomic approach enabled us to identify the deregulation of a signaling pathway, constituted with a receptor (ALK) and two lignads (PTN and MDK). While ALK is decsribed as a major neuroblastoma predisposition gene, mainly through activating mutations, we demonstrated that the activation of ALK occurs via mechanisms others than mutation in a large majority of cases. Moreover ALK activation is an important event in the initiation of the disease. However, we couldn’t prouve the implication of the receptor in the progression of the disease or in its response to treatment. It seems that the regulation of ALK is complex and its precise role in the progression of neuroblastoma remains to be precisely defined. Nevertheless, we have demonstrated the importance of MDK, one of ALK ligands in the regulation of the expression and activation of ALK as well as in the control of the neuroblastoma cells survival. The inhibition of the cytokine, MDK represents an interesting therapeutic strategy, complementary to anti-ALK therapies, currently in clinical development in neuroblastoma. On another hand, the phenotypic characterization of the model, showed an alteration of the signaling of DNA damage and an increased genomic instability in the resistant tumors. Those tumors also harbor a modification in the cell cycle progression, particularly an increased proportion of quiescent cells. Finally, this work enables us to identify several resistance mechanism that represent markers of response to chemotherapy and relevant therapeutic targets in neuroblastoma

Les neuroblastomes olfactifs (NBOs) sont des tumeurs rares de la base du crâne. Les outils de classification de ces tumeurs sont insuffisants, il n’existe notamment aucune classification moléculaire des NBO. La biologie de ses tumeurs est mal connue. Nos travaux se sont appuyés sur l’analyse des exomes, du transcriptome, du méthylome et des caractéristiques histopathologiques et immunitaires d’une série de 59 NBOs bien annotés cliniquement. Nous avons mis en évidence l’existence de 2 sous-types de NBO présentant un profil d’expression et un profil anatomo-clinique différent. Le type neural, correspond à une tumeur bien différenciée, peu agressive, présentant des caractéristiques plutôt neuronales et partageant des caractéristiques phénotypiques avec les progéniteurs directs de neurones olfactifs. Le type basal correspond à une tumeur moins différenciée, plus agressive, ayant des caractéristiques plutôt embryonnaires, partageant des caractéristiques phénotypiques avec les cellules basales de renouvellement de l’épithélium olfactif. Nous avons mis en évidence que la charge mutationnelle était plus élevée dans le type basal, avec notamment des mutations IDH2 R172 récurrentes, associées à un phénotype CpG Island Methylator (CIMP). Nous avons également montré que les NBOs de type basal étaient infiltrés par un nombre plus important de lymphocytes T avec, dans certaines tumeurs, une expression plus marquée de checkpoints immunitaires et de facteurs immunosuppresseurs. Ce travail ouvre la perspective d’une classification moléculaire permettant de mieux stratifier les patients et ouvre également le champ de nouvelles stratégies thérapeutiques dans ces tumeurs rares
Olfactory neuroblastomas (ONBs) are rare tumors arising in the skull base. Classification tools are poor, notably; no molecular classification of ONB has been reported. Literature data about their cell of origin, the existence of molecular therapeutic targets or their immune environment being scarce, the biology of these tumors is still poorly understood. Our work was based on exome, transcriptome and methylome analysis, but also on histopathological and immune characteristics of a series of 59 clinically well annotated ONBs. We highlighted 2 sub-types of ONB showing different expression and clinicopathologic patterns. The neural type is a well differentiated, poorly aggressive tumor which shows neurons characteristics and shares phenotypic similarities with olfactory neuron progenitors. The basal type is a less differentiated tumor, displaying an aggressive phenotype, with embryonic phenotypical characteristics, which shares similarities with basal renewing cells of the olfactory epithelium. We showed that the mutational load was higher in basal tumors, with notably recurrent IDH2 R172 mutations associated with a CpG Island Methylator Phenotype (CIMP). We also showed that basal type ONBs were infiltrated by a greater number of T cells with, in some cases, a higher expression of immune checkpoints and immunosuppressive factors. This work paves the way towards a new molecular classification which will allow a better stratification of patients and will open the field of new therapeutic strategies for this rare tumor

Contexte : Le neuroblastome est une tumeur embryonnaire qui se développe à partir du système nerveux sympathique. C’est la tumeur maligne solide extra-cérébrale la plus fréquente chez les enfants de moins d’un an. La cause du neuroblastome est encore inconnue dans la majorité des cas. Cependant, les caractéristiques embryonnaires de la tumeur ainsi que sa courte latence de survenue après la naissance suggèrent l’origine périnatale de ce cancer et l’importance d’étudier les expositions survenant pendant la grossesse et les premières années de vie de l’enfant. Dans ce travail de recherche, nous avons analysé le lien entre certains facteurs périnataux et des expositions pendant la grossesse et le risque neuroblastome chez l’enfant. Matériel et méthodes : les données sont issues des enquêtes ESCALE (2003-2004) et ESTELLE (2010-2011) menées par notre équipe de recherche. Les mères de 357 cas de neuroblastome issus du Registre National des Cancers de l’Enfant (RNCE) ainsi que 1753 témoins recrutés en population générale ont répondu à un entretien qui portait sur les caractéristiques périnatales de l’enfant, les expositions maternelles pendant la grossesse, antécédents médicaux familiaux et personnels de l’enfant ainsi que sur des variables contextuelles et socioéconomiques. La taille de notre échantillon a permis de réaliser des analyses stratifiées sur l’âge au diagnostic et le statut du proto-oncogène MYCN. Résultats : une première analyse sur l’association entre les caractéristiques périnatales et le risque de neuroblastome chez l’enfant a mis en évidence des associations positives avec la présence de malformations congénitales (OR 3.6 [95% CI 1.3–8.9] parmi les enfants de moins de 18 mois) et des altérations de la croissance fœtale tels que le retard de croissance intra-utérine ou la surcroissance fœtale (OR 1.4 [95% CI 1.0-2.0]) et (OR 1.5 [95% CI 1.1–2.2], respectivement). Le fait d’être allaité était inversement associé au risque de neuroblastome (OR 0.7 [95% CI 0.5–1.0]). Des associations inverses ont été également observées avec la supplémentation maternelle préconceptionnelle en acide folique (OR 0.5 [95% CI 0.3–0.9]). La difficulté pour concevoir ou l’utilisation d’assistance médicale à la procréation n’ont pas été associé au risque de neuroblastome dans notre étude. Dans une deuxième partie de ce travail, nous avons analysé les expositions maternelles domestiques et professionnelles aux pesticides pendant la grossesse. Nos résultats suggèrent que l’utilisation domestique de pesticides pendant la grossesse pourrait augmenter le risque de neuroblastome chez l’enfant (OR 1.5 [95% CI 1.2–1.9]). Des associations positives ont été observées avec l’utilisation d’insecticides seulement (OR 1.4 [95% CI 1.1–1.9]), ou en combinaison avec d’autres pesticides (OR 2.0 [95% CI 1.1–3.4]). L’exposition professionnelle de la mère pendant la grossesse était également associée au risque de neuroblastome. La troisième partie de ce travail a porté sur l’analyse du tabagisme parental et la consommation maternelle d’alcool pendant la grossesse. La consommation maternelle de tabac était plus fréquente chez les mères des cas (24.1%) par rapport aux mères des témoins (19.7%) ; (OR 1.3 [95%CI 0.9–1.7]; OR à partir d’une méta-analyse 1.1 [95%CI 1.0–1.3]. Conclusion : nos résultats portant sur les associations entre neuroblastome, surcroissance fœtale et malformations congénitales supportent l’hypothèse d’un rôle des altérations de l’embryogénèse dans la survenue des neuroblastomes de l’enfant. Ce travail contribue à l’évidence en faveur des associations entre neuroblastome et certaines expositions pendant la grossesse, notamment l’utilisation domestique de pesticides et le tabagisme maternelle. Nos résultats soulignent l’importance des recommandations visant à réduire l’exposition aux pesticides et le tabagisme maternel pendant la grossesse. [...]
Background: Neuroblastoma is the most common extra-cranial tumor in children. Little is known about the etiology of neuroblastoma. The early age at onset and the embryonic nature suggest a role for perinatal exposures. In this work, we analyzed whether childhood neuroblastoma was associated with specific perinatal characteristics and environmental exposures around pregnancy. We assessed the following birth-related characteristics: gestational age, birth-weight and fetal growth, and the presence of congenital malformations. The maternal reproductive history before the index pregnancy and maternal intake of folic acid or vitamins/minerals before or during pregnancy was also assessed. With regards to environmental exposures related to parental habits, we focused on maternal use of household pesticides during pregnancy, parental smoking and maternal alcohol consumption. Methods: We conducted a pooled analysis of two French national-based case-control studies. The mothers of 357 neuroblastoma case and 1,783 control children younger than 6 years, frequency-matched by age and gender, completed a telephone interview that focused on sociodemographic and perinatal characteristics, childhood environment and parental lifestyle. Unconditional logistic regression was used to estimate pooled odds ratios (OR) and 95% confidence intervals (CIs), including matching variables, study of origin and potential confounders. A meta-analysis of our findings with those of previous studies was also conducted with regards to maternal smoking and alcohol consumption during pregnancy. We used random effects, precision-based weighting to calculate the summary OR including our results. Results: The first part of the thesis focused on perinatal characteristics. We observed that being born either small (OR 1.4 [95% CI 1.0-2.0]) or large (OR 1.5 [95% CI 1.1–2.2]) for gestational age and, among children younger than 18 months, having congenital malformations (OR 3.6 [95% CI 1.3–8.9]), were significantly associated with neuroblastoma. Inverse associations were observed with breastfeeding (OR 0.7 [95% CI 0.5–1.0]) and maternal use of any supplements containing folic acid, vitamins or minerals (OR 0.5 [95% CI 0.3–0.9]) during the preconception period. The second part of the thesis showed that maternal use of any type of household pesticide during pregnancy was associated with neuroblastoma (OR 1.5 [95% CI 1.2–1.9]). The most commonly used type of pesticides were insecticides and there was a positive association with their use alone (OR 1.4 [95% CI 1.1–1.9]) or with other pesticides (OR 2.0 [95% CI 1.1–3.4]). In the third part, our analyses showed that maternal smoking during pregnancy was slightly more often reported for the cases (24.1%) than for the controls (19.7%) (OR 1.3 [95% CI 0.9–1.7]; Paternal smoking in the year before child’s birth was not associated with neuroblastoma as independent exposure (OR 1.1 [95%CI 0.9–1.4] but the association was stronger when both parents reported having smoked during pregnancy (OR 1.5 [95% CI 1.1–2.1]. Finally, in a meta-analysis of maternal smoking and neuroblastoma the summary OR from meta-analysis was 1.1 [95% CI 1.0–1.3]. Conclusions: Our findings support the hypothesis of a defective embryogenesis in neuroblastoma since fetal growth anomalies and congenital malformations were associated with an increased risk of neuroblastoma. This work also adds to the evidence of an association between neuroblastoma and some exposures during pregnancy, such as maternal use of household pesticides and maternal smoking, which are additional reasons why to advise pregnant women to limit these exposures in this period. Further investigations are needed to clarify the role of folic acid supplementation and breastfeeding, given their potential importance in neuroblastoma prevention

Neuroblastoma is a childhood cancer that originates from neuroblasts in the peripheral nervous system. Neuroblastoma show considerable heterogeneity with respect to location, responsiveness to treatment and prognosis. Since current therapy involves drugs with risk of serious side effects in the growing child, there is a clinical need for more effective and less toxic treatment strategies.

Angiogenesis, the formation of new blood vessels, is critical for tumor progression. Specific inhibition of tumor-induced angiogenesis should restrict growth of most solid tumors and thereby provide a new treatment strategy. The aim of this study was to investigate the effects of angiogenic inhibition in experimental neuroblastoma in mice.

We found that experimental neuroblastomas expressed the perhaps most potent angiogenic growth factor, VEGF-A, and that plasma VEGF-A levels correlated with tumor size. SU5416, a novel antagonist of VEGFR-1 and 2, reduced angiogenesis and tumor growth in our model. We also investigated the properties of SU11657, a new, orally available, synthetic small molecule multi-targeted tyrosine kinase inhibitor. SU11657, at a well-tolerated dose, was more potent than SU5416 in reducing tumor growth rate and angiogenesis, even in MYCN-amplified tumors. Chemotherapeutics can also inhibit angiogenesis, when administrated daily in a non-toxic dose. CHS 828, a new chemotherapeutic, given orally, alone induced complete neuroblastoma regression in 44 % of the animals. Furthermore, the bisphosphonate zoledronic acid, developed to reduce bone resorption, showed anti-tumor activity in our model. Zoledronic acid was more potent than the angiogenic inhibitor TNP-470. Thus bisphosphonates may have other beneficial properties in patients with cancer apart from preventing bone resorption.

In conclusion, SU5416, SU11657, CHS 828, and zoledronic acid represent new drugs with potent anti-tumor effects. Angiogenic inhibition as single therapy or in combination with chemotherapeutics may be beneficial in the treatment of rapidly growing and highly vascularized solid tumors of childhood such as neuroblastoma.

Neuroblastoma is a cancer of the sympathetic nervous system derived from neural crest cells that fail to differentiate during development. Neuroblastoma tumours and cell lines are heterogeneous, comprised of ‘neuroblastic’ N-type cells, precursors to a neuronal neural crest cell lineage and ‘substrate-adherent’ S-type cells, precursors to a non-neuronal neural crest cell lineage. Retinoids, such as retinoic acid (RA), cause both N- and S-type cells to switch from proliferation to differentiation. This underlies the use of RA in the treatment of neuroblastoma disease. The aim of this study was to investigate the role of Ca2+ signalling in the process of differentiation. N- and S-type cell populations were enriched from the SH-SY5Y neuroblastoma cell line to allow characterisation of Ca2+ signalling and differentiation within the two cell phenotypes. The RA-induced switch from proliferation to differentiation was accompanied by a down-regulation in store-operated Ca2+ entry (SOCE) in N-type cells but not in S-type cells. In N-type cells expression of the ER Ca2+ sensor protein STIM1 and the channel protein Orai1 also became down-regulated, whilst expression of the channel protein TRPC1 became up-regulated. Knockdown of STIM1 and Orai1 in proliferating N-type cells down-regulated SOCE. Knockdown of Orai1, but not STIM1, induced differentiation and also enhanced differentiation induced by RA. Overexpression of STIM1 and Orai1 in RA-differentiated cells restored SOCE and reduced the extent of differentiation. Knockdown of TRPC1 had no effect on SOCE or differentiation in proliferating N-type cells but reduced the extent of SOCE down-regulation and differentiation induced by RA. These observations suggest that Orai1 may be a negative regulator of differentiation in N-type cells whereas STIM1 down-regulation may be required to maintain the differentiated state. TRPC1 expression may be required for a fully functional differentiated phenotype. These proteins could represent putative drug targets in the multi-modal treatment of neuroblastoma disease.

A novel electrochemical genosensor with modified graphite with poly(4-aminophenol) has been constructed for detection of neuroblastoma, a malignant tumor originating from embryonic precursor cells of the sympathetic nervous system and associated with the amplification MYCN oncogene. The produced genosensor exhibited distinct electric and morphological properties using rhodamine b, specie able to bind with DNA duplex, as indicator of the hybridization process. The detection limit was evaluated to be 0.47 umol.L-1 (N=3) and showed very high selectivity for the complementary DNA using serum sample. This DNA sensing platform was successfully applied to detect the MYCN, an important biomarker for neuroblastoma
Um novo genossensor eletroquímico de grafite modificado com poli (4-aminofenol) foi construído para a detecção de neuroblastoma, um tumor maligno originário a partir de células precursoras embrionárias do sistema nervoso simpático, e associado com a amplificação do oncogene MYCN. O genossensor produzido exibiu propriedades elétricas e morfológicas distintas, utilizando rodamina B, espécie capaz de se ligar com a fita dupla de DNA, como indicador do processo de hibridação. O limite de detecção obtido foi de 0,47 μmol.L-1 (N = 3) e mostrou maior seletividade para o DNA complementar, utilizando amostras de soro. Esta plataforma de detecção de DNA foi aplicada com sucesso para detectar a MYCN, um biomarcador importante para o neuroblastoma
Mestre em Genética e Bioquímica

In the studies described in this thesis, the ability of muscarinic agonists to initiate phosphoinositide metabolism, subsequently leading to the mobilisation of Ca2+ from intracellular stores was examined in permeabilised human neuroblastoma SH-SY5Y cells. Muscarinic receptors, determined as being of the M3 subtype, were found to possess different levels of receptor reserve for the production of the second messenger, inositol 1,4,5-trisphosphate (Ins(l,4,5)P3) and the mobilisation of Ca2+ in this permeabilised cell preparation. The effects of agonist pretreatment of intact cells with the muscarinic agonist carbachol has also been examined. Such pretreatment attenuated the ability of muscarinic agonists to elicit Ca2+ mobilisation in permeabilised cells. The rate of desensitisation was dependent on the dose of agonist used, the temperature at which pretreatment was performed and was affected by the extracellular Ca2+ concentration. The mechanism of such receptor-mediated desensitisation was studied. The structure-activity relationships of a number of inositol phosphates and inositol phosphate analogues have also been studied with regards their interactions with the Ca2+-mobilising Ins(l,4,5)P3 receptor, Ins(l,4,5)P3 5-phosphatase and Ins(l,4,5)P3 3-kinase. This work has identified partial agonists at the Ins(l,4,5)P3 receptor, an inhibitor of Ins(l,4,5)P3 3-kinase which interacts poorly with the Ins(l,4,5)P3 receptor, and a highly potent and selective inhibitor of Ins(l,4,5)P3 5-phosphatase. In conclusion, the results obtained characterise a permeabilised cell preparation in which receptor coupling to phospholipase C is maintained, leading to the formation of Ins(l,4,5)P3 and mobilisation of Ca2+ from intracellular stores, and identifies this as a useful model in which this coupling can be modulated by cell membrane-impermeant agents.

Neuroblastoma is a tumour derived from cells of the nervous system and is the most common solid tumour in childhood. MYCN amplified and 11q-deleted neuroblastoma, two high-risk neuroblastoma were investigated in this study. RAD51 gene family includes six central genes for the dsDNA breaks repair by homologous recombination, which has been reported as important in varying types of cancer. The study aims to investigate if the dysregulation of this gene family could be involved in the unstable genome of 11q-deleted neuroblastoma, and to better understand the link between both high-risk tumours. The RAD51 family genes’ expression level was measured by RT-qPCR in samples of 11q-deleted and MYCN-amplified neuroblastoma that were treated with a UVC treatment and were recovered during varying hours. R2 database and DAVID were used to study the RAD51 family’s expression levels, associated event-free survivability, and altered pathways. RAD51 family is highly dysregulated in these tumours, four genes of six were found to be altered in high-risk neuroblastoma. Four of six genes presented altered expression levels in 11q-loss, and three of six in the MYCN-amplified case after the UVC treatment. The event-free survival probability analysis shown that the levels of expressions associated with high-risk neuroblastoma coincide with those that represent a poor life expectancy. Altered pathways were different in each type of tumour. 11q-deletion neuroblastoma’s pathways were associated with the nervous system development, and MYCN-amplified was related to the immune system. This study suggests that 11q-loss neuroblastoma presents a greater RAD51 family dysregulation compared with MYCN-amplified one, which could explain why its genome is unstable.

Childhood neuroblastoma is the most common solid tumour of infancy and highly refractory to therapy. One of the most powerful prognostic indicators for this disease is the N-Myc gene amplification, which occurs in approximately 25% of all neuroblastomas. N-Myc is a member of transcription factors belonging to a subclass of the larger group of proteins sharing Basic-Region/Helix–Loop–Helix/Leucin-Zipper (BR/HLH/LZ) motif. N-Myc oncoproteins may determine activation or repression of several genes thanks to different protein-protein interactions that may modulate its transcriptional regulatory ability and therefore its potential for oncogenicity. Chromatin modifications, including histone methylation, have a crucial role in transcription de-regulation of many cancer-related genes. Here, it was investigated whether N-Myc can functionally and/or physically interact with two different factors involved in methyl histone modification: WDR5 (core member of the MLL/Set1 methyltransferase complex) and the de- methylase LSD1. Co-IP assays have demonstrated the presence of both N-Myc-WDR5 and N-Myc-LSD1 complexes in two neuroblastoma cell lines. Human N-Myc amplified cell lines were used as a model system to investigate on transcription activation and/or repression mechanisms carried out by N-Myc-LSD1 and N-Myc-WDR5 protein complexes. qRT-PCR and immunoblot assays underlined the ability of both complexes to positively (N-Myc-WDR5) and negatively (N-Myc-LSD1) influence transcriptional regulation of crititical neuroblastoma N-Myc-related genes, MDM2, p21 and Clusterin. Ch-IP experiments have revealed the binding of the N-Myc complexes above mentioned to the gene promoters analysed. Finally, pharmacological treatment pointed to abolish N-Myc and LSD1 activity were performed to test cellular alterations, such as cell viability and cell cycle progression. Overall, the results presented in this work suggest that N-Myc can interact with two distinct histone methyl modifiers to positively and negatively affect gene transcription in neuroblastoma.

Childhood neuroblastoma is the most common solid tumour of infancy and highly refractory to therapy. One of the most powerful prognostic indicators for this disease is the N-Myc gene amplification, which occurs in approximately 25% of all neuroblastomas. N-Myc is a member of transcription factors belonging to a subclass of the larger group of proteins sharing Basic-Region/Helix–Loop–Helix/Leucin-Zipper (BR/HLH/LZ) motif. N-Myc oncoproteins may determine activation or repression of several genes thanks to different protein-protein interactions that may modulate its transcriptional regulatory ability and therefore its potential for oncogenicity. Chromatin modifications, including histone methylation, have a crucial role in transcription de-regulation of many cancer-related genes. Here, it was investigated whether N-Myc can functionally and/or physically interact with two different factors involved in methyl histone modification: WDR5 (core member of the MLL/Set1 methyltransferase complex) and the de- methylase LSD1. Co-IP assays have demonstrated the presence of both N-Myc-WDR5 and N-Myc-LSD1 complexes in two neuroblastoma cell lines. Human N-Myc amplified cell lines were used as a model system to investigate on transcription activation and/or repression mechanisms carried out by N-Myc-LSD1 and N-Myc-WDR5 protein complexes. qRT-PCR and immunoblot assays underlined the ability of both complexes to positively (N-Myc-WDR5) and negatively (N-Myc-LSD1) influence transcriptional regulation of crititical neuroblastoma N-Myc-related genes, MDM2, p21 and Clusterin. Ch-IP experiments have revealed the binding of the N-Myc complexes above mentioned to the gene promoters analysed. Finally, pharmacological treatment pointed to abolish N-Myc and LSD1 activity were performed to test cellular alterations, such as cell viability and cell cycle progression. Overall, the results presented in this work suggest that N-Myc can interact with two distinct histone methyl modifiers to positively and negatively affect gene transcription in neuroblastoma.

Die Abschätzung der Prognose für Patienten, insbesondere Kinder mit onkologischen Erkrankungen stellt eine große Herausforderung an die behandelnden Ärzte dar. Vor Beginn einer Therapie werden daher viele Informationen gesammelt, um einen Patienten möglichst gut in eine vordefinierte Risikogruppe stratifizieren und dementsprechend eine mehr oder weniger intensive Therapie anbieten zu können. Diese Einteilungen sind allerdings für keinen Malignomtyp mit 100%-iger Sicherheit möglich. Das ist die Ursache dafür, dass auch in niedrige Risikogruppen eingeteilte Patienten nicht auf die Therapie ansprechen und einen unvorhergesehen schlechten Verlauf zeigen können. Auf der anderen Seite scheint es Patienten zu geben, die trotz initial schlecht eingeschätzter Prognose einen überaschend guten Verlauf nehmen, auf die Therapie gut ansprechen und letztlich geheilt werden können. Einen Beitrag zu leisten, um die Stratifizierung für Kinder, die an einem Neuroblastom erkrankt sind, zu verbessern und damit zu vermeiden, dass einige Patienten unter- oder andere Patienten übertherapiert werden müssen, ist das Ziel dieser Habilitationsarbeit. Zu diesem Zweck wurden differentielle, molekulare Marker in primären humanen Neuroblastomen identifiziert und deren prognostische Bedeutung dargestellt. Einzelne dieser Marker (differentiell expremierte mRNAs) wurden in Zellkultursystemen funktionell untersucht, um deren zellbiologische Funktion, die der jeweiligen prognostischen Bedeutung zugrunde liegen kann zu erklären. Desweiteren konnten genomische Merkmale des amplifizierten genomischen Abschnittes auf Chromosom 2p25 um MYCN beschrieben werden. Darauf basierend konnte eine patientenindividuelle und tumorzellspezifische PCR entwickelt werden (AFS-PCR), die sich als Marker für den Nachweis einer minimalen Resterkrankung eignet.