Thèses sur le sujet « High grade gliomas »

Reseksjon av høygradige gliomer Høygradige gliomer er den vanligste formen for primær hjernesvulst. Glioblastomer (Verdens Helseorganisasjon grad IV) og anaplastiske astrocytomer (Verdens Helseorganisasjon grad III) utgjør mellom 70 og 85 % av høygradige gliomer. Høygradige gliomer er assosiert med både høy morbiditet og mortalitet. Nærmest alle pasienter med høygradige gliomer opplever tilbakefall og dør som følge av sykdommen. Til tross for kirurgi, strålebehandling og cellegift, er median overlevelse for pasienter med glioblastom fremdeles under 12 måneder. For pasienter med anaplastisk astrocytom er median overlevelse 2 til 3 år. Høygradige gliomer infiltrerer omkringliggende hjernevev, og hensikten med kirurgi er, foruten å histopatologisk verifisere diagnosen, å fjerne så mye av svulsten som mulig uten å påføre pasienten nye eller økte nevrologiske utfall. I vår avdeling benytter vi et navigasjonssystem under operasjonene som nyttiggjør tredimensjonale preoperative MR-bilder og tredimensjonal ultralydavbildning under operasjonen. Dette navigasjonssystemet gjør at kirurgen til en hver tid kan se posisjonen til sine instrumenter i forhold til hjernen og svulsten. Ved hjelp av funksjonell MR (eller mer presist blood-oxygenation-level-dependent functional magnetic resonance imaging) og diffusjon tensor traktografi (DTT) kan en henholdsvis kartlegge viktige områder i hjernens grå og hvite substans før operasjonen. Disse undersøkelsene utføres som regel når svulster ligger i nær relasjon til ekstra følsomme områder av hjernen (for eksempel språkområder og viktige områder for bevegelse). Informasjon fra disse undersøkelsene kan også importeres i navigasjonssystemet som benyttes under operasjonen. I de to første studiene i denne avhandlingen ønsket vi å undersøke hvordan funksjonell MR og DTT ble brukt i preoperative vurderinger. Vi evaluerte om funksjonell MR og DTT i kombinasjon med tredimensjonal ultralydavbildning under operasjonen la forholdene til rette for skånsom fjerning av høygradige gliomer beliggende i ekstra følsomme områder av hjernen. I den tredje studien undersøkte vi konsekvensene av kirurgiske komplikasjoner og nevrologiske utfall som følge av kirurgi på glioblastompasienters funksjonsnivå og overlevelse. Videre gjorde vi volumetriske analyser for å beregne hvor mye svulstvev vi klarte å fjerne hos pasienter med primære glioblastomer behandlet i vår avdeling. I den fjerde studien undersøkte vi om det var noen sammenheng mellom overlevelse og fall i selvrapportert livskvalitet kort tid etter kirurgi hos pasienter med glioblastomer. Den femte studien var basert på data fra Kreftregisteret og undersøkte overlevelse og behandling blant eldre pasienter (≥66 år) med glioblastomer over en tyve års periode. Hovedfunnene i denne avhandlingen er: - Kombinasjonen av funksjonell MR, DTT og tredimensjonal ultralydavbildning kan være nyttig når en utfører kirurgisk reseksjon av høygradige gliomer beliggende i ekstra følsomme områder av hjernen. - Pasienter som opplevde komplikasjoner og nevrologiske utfall som følge av kirurgi hadde lavere sannsynlighet for å motta strålebehandling og kjemoterapi. - Tidlig fall i helserelatert livskvalitet etter kirurgi synes å være en sterk og uavhengig negativ prognostisk faktor for pasienter med glioblastom. - Økende alder er en sterk og uavhengig negativ prognostisk faktor for pasienter med glioblastom. Selv om det har vært en intensivering av behandling over tid, har gevinsten i den eldste aldersgruppen vært begrenset. Prognosen for de eldste er fremdeles svært dårlig til tross for multimodal behandling
High-grade gliomas are the most common primary brain tumour. Glioblastomas (World Health Organization Grade IV) and anaplastic astrocytomas (World Health Organization Grade III) account for 70-85% of high-grade gliomas. High-grade gliomas are associated with high morbidity and mortality. Virtually all patients with high-grade glioma will experience recurrence and will eventually die from progressing disease. Despite surgery, radiotherapy, and chemotherapy, median survival in patients with glioblastoma still does not exceed 12 months. The median survival for patients with anaplastic astrocytoma (AA) has been reported to be between 2 and 3 years. According to current guidelines, surgery is warranted to establish a histopathologic diagnosis and to achieve safe, maximal, and feasible resection. However, these aggressive tumours cannot be cured and overly aggressive resection is not recommended due to the risk of new neurological deficits. High-grade glioma surgery is a delicate balance between achieving maximal tumour resection and inducing new deficits. In our department a neuronavigation system based on preoperative 3D magnetic resonance imaging (MRI) and intraoperative 3D ultrasound is utilised when resecting high-grade gliomas. Blood-oxygenation-level-dependent functional magnetic resonance imaging (BOLD fMRI) and diffusion tensor tractography (DTT) are specialized MRI techniques for imaging eloquent cortices and neural tracts in grey and white matter, respectively. The neuronavigation system allows the integration of BOLD fMRI and DTT data if the tumours are located in eloquent regions. In the two first studies of this thesis we sought to investigate the use of BOLD fMRI and DTT for preoperative assessments and determine whether using these data together with 3D intraoperative ultrasound enabled safe resection of high-grade gliomas situated in eloquent regions. In the third study we wanted to explore the impact of surgical morbidity on functional outcome and survival in GBM patients. Further, we sought to determine extent of tumour resection achieved in a consecutive sample of primary GBM from our own department. In the fourth study we wanted to determine if changes in health related quality of life early after surgery could be a predictor for survival in patients with glioblastoma. The aims of the fifth study were to explore survival and the treatment provided to elderly patients (≥66 years) diagnosed with glioblastoma during a 20-year time period in a population-based cohort using the Norwegian Cancer Registry. This thesis investigated the role of surgical resection in the treatment of high-grade gliomas and the following conclusions can be drawn: - The combination of BOLD fMRI, DTT, and 3D intraoperative ultrasound may facilitate resection of high-grade gliomas harboured in eloquent areas while preserving motor and language function. - Functional neuronavigation combined with intraoperative 3D ultrasound can, in most patients, enable resection of brain lesions with general anaesthesia without jeopardizing neurological function. - Patients with perioperative complications and surgically acquired deficits were less likely to receive adjuvant therapy. - Early deterioration in HRQL after surgery was independently and markedly associated with impaired survival in patients with glioblastoma. - Advancing age remains a very strong and independent negative prognostic factor in glioblastoma. Although there has been an increase in the aggressiveness of treatment provided to elderly with glioblastoma, the gain for the oldest age group seems at best very modest. The prognosis of the oldest age group remains very poor, despite multimodal treatment.

Several biomarkers had been proposed as useful parameters to better define the prognosis or to delineate new target therapy strategies for glioblastoma (GBM) patients. MicroRNAs could represent interesting molecules, for their role in tumorigenesis and cancer progression and for their specific tissue expression. Although many studies have tried to identify a specific microRNAs signature for glioblastoma, by now an exhaustive GBM microRNAs profile is far to be well defined. In this work we set up a real-time qPCR, based on LNA primers, to investigate the expression of 19 microRNAs in brain tumors, focusing our attention on GBMs. MiRNAs expression in 30 GBM paired FFPE-Fresh/Frozen samples was firstly analyzed. The good correlation obtained comparing miRNAs results confirmed the feasibility of performing miRNAs analysis starting from FFPE tissues. This leads to many advantages, as a good disposal of archival tumor and normal brain specimens and the possibility to verify the percentage of tumor cells in the analyzed sample. In the second part we compared 3 non-neoplastic brain references to use as control in miRNAs analysis. Normal adjacent the tumor, epileptic specimens and a commercial total RNA were analyzed for miRNAs expression and results showed that different non-neoplastic controls could lead to important discrepancies in GBM miRNAs profiles. Analyzing 50 FFPE GBMs using all 3 non-neoplastic references, we defined a putative GBM miRNAs signature: mir-10b, miR-21 and miR-27a resulted upregulated, while miR-7, miR-9, miR-26a, miR-31, miR-101, miR-137, miR-222 and miR-330 were downregulated. Comparing miRNAs expression among GBM group and gliomas of grade I, II and III, we obtained 3 miRNAs (miR-10b, mir-34a and miR-101) showing a different regulation status between high grade and low grade gliomas. Intriguingly, miR-10b was upregulated in high grade and significantly downregulated in low grade gliomas, suggesting that could be a candidate for a GBM target therapy.

Several biomarkers had been proposed as useful parameters to better define the prognosis or to delineate new target therapy strategies for glioblastoma (GBM) patients. MicroRNAs could represent interesting molecules, for their role in tumorigenesis and cancer progression and for their specific tissue expression. Although many studies have tried to identify a specific microRNAs signature for glioblastoma, by now an exhaustive GBM microRNAs profile is far to be well defined. In this work we set up a real-time qPCR, based on LNA primers, to investigate the expression of 19 microRNAs in brain tumors, focusing our attention on GBMs. MiRNAs expression in 30 GBM paired FFPE-Fresh/Frozen samples was firstly analyzed. The good correlation obtained comparing miRNAs results confirmed the feasibility of performing miRNAs analysis starting from FFPE tissues. This leads to many advantages, as a good disposal of archival tumor and normal brain specimens and the possibility to verify the percentage of tumor cells in the analyzed sample. In the second part we compared 3 non-neoplastic brain references to use as control in miRNAs analysis. Normal adjacent the tumor, epileptic specimens and a commercial total RNA were analyzed for miRNAs expression and results showed that different non-neoplastic controls could lead to important discrepancies in GBM miRNAs profiles. Analyzing 50 FFPE GBMs using all 3 non-neoplastic references, we defined a putative GBM miRNAs signature: mir-10b, miR-21 and miR-27a resulted upregulated, while miR-7, miR-9, miR-26a, miR-31, miR-101, miR-137, miR-222 and miR-330 were downregulated. Comparing miRNAs expression among GBM group and gliomas of grade I, II and III, we obtained 3 miRNAs (miR-10b, mir-34a and miR-101) showing a different regulation status between high grade and low grade gliomas. Intriguingly, miR-10b was upregulated in high grade and significantly downregulated in low grade gliomas, suggesting that could be a candidate for a GBM target therapy.

Abstract Background and aims. High grade gliomas (HGGs) are aggressive brain tumours characterized by a poor prognosis and the ability to promote an immunosuppressive tumour microenvironment that impairs anti-tumor immune responses. Therefore, there is increasing interest in developing new immunotherapeutic approaches, aimed at boosting anti-tumor immune responses in HGG patients. Because HGG has shown the highest susceptibility to dendritic cell (DC) vaccines amongst other human cancers, DC-based immunotherapeutic strategies may be particularly promising in these patients. DCs are antigen presenting cells that have the unique ability to initiate antitumor immune responses, making these cells crucial in cancer immunosurveillance. They are a rare population composed of different subsets that differ each other in origin, immunophenotype and function. The differential role of different DC subsets in HGG, and in particular the subsets specifically recruited into the tumour site and the impact of HGG on the activatory/tolerogenic properties of DCs have been poorly investigated, so far. For these reasons, in this study we performed a deep characterization of circulating and tumour-infiltrating DC subsets, and investigated possible correlations between DC parameters and histopathological and molecular HGG features, patient outcome and response to treatment. To this aim, we used multiparameter flow-cytometry and single-cell RNA sequencing (scRNAseq), which allow complex analyses on high-dimensional data. Materials and methods. In this cross-sectional study we enrolled HGG patients undergoing surgery at their first diagnosis, and we applied an 18-colour flow-cytometry panel that allows the identification of DC-lineage DCs (pDCs, cDC1s, cDC2s) and inflammatory DCs (slanDCs, moDCs), and the characterisation of their activatory/inhibitory state. This panel was applied to DC characterization in the peripheral blood (n=23) and the tumour lesion (n=10) of HGG patients. Twelve whole blood samples obtained from healthy donors (HDs) and 3 healthy brain tissue samples were included as controls. scRNAseq experiments were performed on 7 tumoral samples and 2 healthy brain tissues obtained from HGG patients, by using 10x Genomics technology. Ingenuity Pathway Analysis (IPA) software was used to investigate the pathways and functions differentially activated or inhibited in infiltrating DCs. We also performed a longitudinal study on a second cohort of patients, diagnosed with recurrent HGG and enrolled in different immunotherapeutic early clinical trials (ieCTs), mainly containing immune checkpoint inhibitors (n=17). In these patients, we assessed the count and phenotype of circulating DC subsets before and at different time points after immunotherapy, by using the same 18-colour flow-cytometry panel described above. Multivariate analyses were used to correlate DC parameters with the patient outcome. Results. In the cross-sectional study, we observed by flow-cytometry that the frequency of circulating pDCs, cDC1s, cDC2s and slanDCs was significantly lower in HGG patients than HDs. DC reduction was evident only in patients affected by the most severe form of HGGs (IDHwt IV grade gliomas). The analysis of tissue DCs revealed that DC subsets were absent in healthy brain parenchyma, whereas they infiltrated HGG tumour tissues. In particular all subsets of myeloid DCs (including cDC1s, cDC2s, slanDCs, and moDCs) were observed in the tumours, whereas pDCs were observed only in a few patients. Tumour-infiltrating DCs were markedly reduced in corticosteroid-receiving patients. By performing scRNAseq, we confirmed that DCs were mostly absent in healthy brain parenchyma whereas they were present in tumour samples and could be sub-divided in 2 sub-clusters. By IPA analysis, we observed a functional dichotomy between these clusters, with the largest one being characterised by an impaired/dormancy state, as assessed by the down-regulation of pathways and functions related to pro-inflammatory responses, cell motility and cell interactions, compared with the smallest cluster characterised, on the contrary, by a more active profile. In the longitudinal study performed on relapsed HGG patients enrolled in ieCTs, we observed that patients with a positive clinical response to immunotherapeutic agents, as assessed by an increased overall survival, showed an increase in the number of circulating cDCs. Conclusions. This study demonstrated that different subsets of DCs infiltrate human HGGs, but are mainly characterized by a transcriptomic profile suggestive of a functional impairment. These results provide novel insights into the comprehension of the molecular mechanisms of DC impairment in HGG microenvironment, and pave the way for the development of novel strategies aimed at restoring the ability of DCs to activate cytotoxic anti-tumour immune cells. Our observation in the longitudinal study that an increase of cDCs correlated with a better clinical response to immunotherapy seems to support the relevant role played by DCs in the control of HGG growth. On the other hand, our study also demonstrated that corticosteroid treatment, commonly used in HGG patients for the management of cerebral oedema, reduces the number of tumour-infiltrating DCs. Based on the above considerations, this finding may suggest a negative impact of corticosteroid treatment on anti-tumour immune responses, thus supporting the use of alternative approaches to control this clinical complication. Altogether, our results support and encourage the study of DCs in HGG, in order to improve our knowledge on the role played by DCs within the immunosuppressive tumour microenvironment that characterizes this human cancer. To this aim, in the near future we plan to apply new bioinformatic tools to the analysis of single-cell data collected in HGG tumour environment that may be particularly useful for investigating the intricate interactions occurring between DCs and other HGG-infiltrating immune cells or malignant glioma cells.

The survival for high-grade glioma patients is poor and the treatment may cause severe side effects. A common obstacle in the treatment is chemoresistance. To improve the quality of life and prolong survival for these patients prognostic biomarkers and new approaches for chemotherapy are needed. To this end, a strategy to evade chemoresistance was evaluated by combining chemotherapeutic drugs with agents inhibiting resistance mechanisms identified by a bioinformatic analysis (paper I). The prognostic value of 13 different proteins was analyzed in this thesis (papers II-IV). Two of them, p38 mitogen-activated protein kinase (MAPK) and protein tyrosine phosphatase non-receptor type 6 (PTPN6, also known as SHP1) were analyzed for their potential as targets in combination chemotherapy (in paper III and IV, respectively).   We found that: PTPN6 expression and methylation status may be important for survival of anaplastic glioma patients, p38 MAPK phosphorylation may be a potential negative prognostic biomarker for high-grade glioma patients and FGF2 expression may be a potential negative prognostic biomarker for proneural glioma patients. PTPN6 may be a useful target for combination chemotherapy with cisplatin, melphalan or bortezomib in high-grade gliomas. The following drug combinations; camptothecin combined with an EGFR or RAC1 inhibitor, imatinib combined with a Notch or RAC1 inhibitor, temozolomide combined with an EGFR or FAK inhibitor and vandetanib combined with a p38 MAPK inhibitor may be useful combination chemotherapy for high-grade gliomas.

Les gliomes sont les tumeurs cérébrales primaires les plus communes chez l’adulte. Parmi eux, le glioblastome (GBM) représente la tumeur cérébrale la plus fréquente avec le pronostic le plus sombre. Son incidence annuelle est d'environ 3 à 5 cas pour 100 000 personnes (environ 3000 nouvelles chaque année en France). La survie médiane varie entre 11 et 13 mois selon la qualité de la résection tumorale.Le standard de soins inclue une résection chirurgicale et est suivie d'une radiothérapie et d'une chimiothérapie. Une résection maximale est souhaitée afin de diminuer les risques de récidive. Bien que l’utilisation de la technique de diagnostic photodynamique peropératoire, appelée résection fluoroguidée (FGR), améliore la qualité de résection, une récidive survient dans ces berges de la cavité opératoire dans 85% des cas.Des thérapies alternatives doivent être développées pour améliorer la survie globale des patients. Dans ce contexte, la thérapie photodynamique (PDT) semble pertinente. La PDT est basée sur la synergie de trois paramètres : une molécule, la photosensibilisateur (PS) qui se concentre préférentiellement dans les cellules tumorales, la lumière laser et l'oxygène. La lumière laser induit une réaction entre le PS et l’oxygène de la cellule. Cette réaction produit des molécules cytotoxiques (dont l'oxygène singulet) et conduit à la mort de cellules tumorales. Deux modalités de traitement sont étudiées : la PDT interstitielle (iPDT) ou la PDT peropératoire.L'objectif principal de cette thèse est de fournir des outils technologiques afin développer la PDT pour le traitement du GBM. Ainsi, les deux modalités de traitement ont été étudiées.Lorsque la résection n'est pas réalisable (environ 20% à 30% des cas), l'iPDT peut être privilégiée. Cette modalité vise à insérer des fibres optiques dans la cible thérapeutique pour éclairer les tissus tumoraux. Ainsi, la simulation de la propagation de la lumière dans les tissus est nécessaire pour planifier la localisation des fibres optiques. Considérée comme méthode de référence, un modèle Monte-Carlo accéléré par processeurs graphiques a été développé. Ce modèle calcule la propagation de la lumière émise par un diffuseur cylindrique dans des milieux hétérogènes. La précision du modèle a été évaluée avec des mesures expérimentales. L'accélération fournie par la parallélisation permet son utilisation dans la routine clinique.L'iPDT doit être planifiée à l'aide d'un système de planification de traitement (TPS). Une preuve de concept d'un TPS dédié au traitement stéréotaxique iPDT du GBM a été développée. Ce logiciel fournit des outils de base pour planifier l'insertion stéréotaxique de diffuseurs cylindriques et calculer la dosimétrie associée. Le recalage stéréotaxique et la précision du calcul dosimétrique ont été évalués avec des méthodologies spécifiques.Lorsque la résection est réalisable, la PDT peropératoire peut être appliquée au début de la FGR. Celle-ci profite de la présence du PS (la protoporphyrine IX) utilisé pour la FGR et qui s’est déjà concentrée dans les cellules tumorales. Ainsi, la stratégie de traitement proposée peut s’inclure facilement au standard de soin. Un dispositif médical a été conçu pour s'adapter à la cavité et éclairer de façon homogène les berges de la cavité opératoire. Le dispositif est constitué de deux parties : un trocart couplé à un ballon gonflable et un guide de fibre optique développé au sein du laboratoire ONCO-THAI permettant d'insérer la source lumineuse. Des méthodologies spécifiques ont été développées pour étalonner et évaluer l'appareil en termes de contrainte mécanique et de dosimétrie. L'étalonnage a permis la création d’une fonction de transfert permettant une prescription de durée de traitement rapide, robuste et facile. De plus, de nombreux tests ont été réalisés en amont de l'essai clinique qui évalue la sécurité de la procédure
Gliomas are the most common primary brain tumors in adults. Among them, glioblastoma (GBM) represents the most frequent primary brain tumor and have the most dismal prognosis. Its annual incidence is about 3 to 5 cases for 100,000 persons (about 3000 news cases each year in France). Median survival varies between 11 to 13 months according the extent of tumor resection.The standard of care includes surgery and is followed by radiation therapy and chemotherapy. Maximal resection is expected to delay recurrence. Despite of using intraoperative photodynamic diagnosis, or fluorescence guided resection (FGR), which improves the extent of resection, relapse still occurs in these resection margins in 85% of cases.Alternatives therapies have to be developed to enhance patients’ overall survival. In this context, Photodynamic Therapy (PDT) seems relevant. PDT is based on the synergy of three parameters: a photosensitizing molecule, the photosensitizer (PS) that concentrates preferentially into the tumor cells, laser light and oxygen. Laser light induces a reaction between the PS and the oxygen of the cell. This reaction produces highly cytotoxic molecules (including singlet oxygen) and leads to death of tumor cells. Two treatment modalities are investigated: interstitial PDT (iPDT) or intraoperative PDT.The main goal of this thesis is to provide technological tools to develop the PDT for GBM treatment. Thus, the two treatment modalities have been investigated.When tumor resection is non-achievable (about 20% to 30% of cases), iPDT may be preferred. This modality aims to insert optical fibers directly into the target to illuminate tumor tissues. Thus, simulation of light propagation in brain tissues is required to plan the location of optical fibers. Considered as reference method, a Monte-Carlo model accelerated by graphics processing unit was developed. This model computes the light propagation emitted by a cylindrical diffusor inside heterogeneous media. Accuracy of the model was evaluated with experimental measurements. The acceleration provided by the parallelization allows its use in clinical routine.The iPDT has to be planned using a Treatment Planning System (TPS). A proof of concept of a TPS dedicated to the stereotactic iPDT treatment of GBM was developed. This software provides basic tools to plan the stereotactic insertion of cylindrical diffusors in patient’s brain and to compute the associated dosimetry. The stereotactic registration and the dosimetry computation’s accuracy were evaluated with specific methodologies.When tumor resection is achievable, the intraoperative PDT may be applied early after the FGR. It takes advantage of the presence of the PS (the protoporphyrin IX) used for FGR purpose and that is already concentrates into the tumor cells. Thus, the proposed treatment strategy fits into the current standard of care. A medical device was designed to fit to the resection cavity and illuminate homogeneously the cavity’s margins. The device is constituted of two parts: a trocar coupled to an inflatable balloon and a fiber guide developed in the ONCO-THAI laboratory allowing to insert the light source. Specific methodologies were developed to calibrate and assess the device in terms of mechanical properties and dosimetry. The calibration process leaded to a transfer function that provides fast, robust and easy treatment duration prescription to induce a PDT response in cavity margins. Furthermore, a comprehensive experimental design has been worked out prior to the clinical trial that evaluate the safety of the procedure

Bevacizumab has shown activity in different tumor types, including high grade gliomas (HGG). However, the use of bevacizumab and other antiangiogenic drugs in the clinical setting limited by the lack of markers to predict responses. We report that the combined treatment with bevacizumab and irinotecan is effective in recurrent HGG patients, particularly in those with local disease, with mild toxicity. Median OS and PFS were 33 and 18 weeks, respectively. PFS at 6 and 12 months were 32% and 12%. OS at 6 months was 60%. Patients with distant intracerebral disease or leptomeningeal dissemination at baseline magnetic resonance had shorter PFS and OS. We analyzed circulating endothelial cells (CECs) and their progenitors (CEPs), as previous studies supported their involvement in responses to bevacizumab. Higher levels of CD109+ CECs, CEPs and CD45dimCD34+CD133+ hematopoietic committed progenitors before treatment were associated with longer PFS. Moreover, long-term responders showed higher baseline CD109+ CECs and CD45dimCD34+VEGFR2+ hematopoietic progenitors. These findings pave the way for larger studies further addressing the potential of CECs and CEPs as biomarkers to target patient populations that may benefit from bevacizumab and possibly other antiangiogenic drugs.

Introduction: Brain tumours are the 17th most common cancer worldwide. Gliomas are the most common of the primary brain tumours and are highly malignant. / Objectives: (a) To undertake gene expression profiling of the blood of glioma patients to determine key genetic components of signalling pathways (b) To develop a panel of genes that could be used as a potential blood-based biomarker to differentiate between high and low grade gliomas, non-glioma and control samples. / Methods: Blood samples were obtained from glioma patients, non-glioma and control subjects. Ten samples each were obtained from patients with high and low grade tumours respectively, ten samples from non-glioma patients and twenty samples from control subjects. Total RNA was isolated from each sample after which first and second strand synthesis was performed. The resulting cRNA was then hybridized with the Agilent Whole Human Genome (4x44K) microarray chip according to the manufacturer's instructions. Universal Human Reference RNA and samples were labeled with Cy3 CTP and Cy5 CTP respectively. Microarray data were analyzed by Agilent Gene Spring 12.1V software using stringent criteria which included at least a 2-fold difference in gene expression between samples. Statistical analysis was performed using the unpaired student T-test with a p-value < 0.01. Pathway enrichment was also performed with key genes within these pathways selected for validation with ddPCR. / Results: The gene expression profiling indicated that were a substantial number of genes that were differentially expressed with more than a 2-fold change (FDR corrected value < 0.01) between each of the four different conditions. We selected key genes within significant pathways that were analyzed through pathway enrichment. These key genes included regulators of cell proliferation, transcription factors, cytokines and tumour suppressor genes. / Conclusion: In this study, we have shown that key genes involved in significant and well established pathways, could possibly be used as a potential blood-based biomarker to differentiate between high and low grade gliomas, non-gliomas and control samples.

Introduction: Fluorescein is widely used as a fluorescent tracer for many applications. Its capability to accumulate in cerebral areas with blood-brain barrier damage makes it an ideal dye for intraoperative visualization of high-grade gliomas (HGG). This study presents a new fluorescein-guided technique to remove HGG with a dedicated filter on the surgical microscope (FLUOGLIO trial). Methods: The FLUOGLIO study is a prospective phase II-trial to evaluate safety and obtain initial indications about efficacy of fluorescein-guided surgery for HGG. Since September 2011, 24 patients (age range 45-74 years) were enrolled in the study. Fluorescein was intravenous injected after intubation (5-10 mg/Kg). Tumor was removed with microsurgical technique and fluorescence visualization by BLU400 or YELLOW560 filters on the Pentero microscope (Carl Zeiss, Germany). Degree of tumor resection was calculated on an early (within 72 hours of surgery) postoperative MRI. In 11 patients, biopsies were performed at the tumor margin in order to evaluate sensitivity and specificity of fluorescein in tumor tissue identification. The study was approved by our Ethical Committee and registered on the European Regulatory Authorities website (EudraCT No. 2011-002527-18) Results: Median pre-operative tumor volume was 33.1 cm3 (1.3-87.8 cm3). No adverse reaction related to the administration of fluorescein was registered. Contrast-enhanced tumor was completely removed in 83% of the patients on early postoperative MRI. The remaining patients had a mean tumor resection of 92.6%. Median follow-up was 12 months. The biopsies at the tumor margins gave a preliminary estimation of sensitivity and specificity of fluorescein in identifying tumor tissue of 95% and 86% respectively. Conclusion: The presented data suggest that fluorescein-guided technique with a dedicated filter on the surgical microscope is safe and allows a high-rate of complete resection of HGG at the early post-operative MRI.

In the radiation therapy of high-grade gliomas, T1-weighted magnetic resonance imaging (MRI) with contrast enhancement does not accurately represent the extent of the tumour. Functional imaging techniques, such as positron emission tomography (PET) and diffusion tensor imaging (DTI), can potentially be used to improve tumour localization and for biologically-based treatment planning. This project investigated tumour localization using 3,4-dihydroxy-6-[?????F]fluoro-L-phenylalanine (?????F-FDOPA) PET and interhemispheric difference images obtained from DTI, and determined whether radiation therapy of high-grade gliomas using dose painting was feasible with volumetric modulated arc therapy (VMAT). First, radiation therapy target volumes obtained from five observers using ?????F-FDOPA PET and MRI were compared with the location of recurrences following radiotherapy. It was demonstrated with simultaneous truth and performance level estimation that high-grade glioma radiation therapy target volumes obtained with PET had similar interobserver agreement to MRI-based volumes. Although PET target volumes were significantly larger than volumes obtained using MRI, treatment planning using the PET-based volumes may not have yielded better treatment outcomes since all but one central recurrence extended beyond the PET abnormality. The second study characterized the distribution of fractional anisotropy (FA) and mean diffusivity (MD) values obtained from DTI, as well as FA and MD interhemispheric differences. It was demonstrated that FA, MD, and interhemispheric differences approached those of contralateral normal brain as the distance from the tumour increased, consistent with the expectation of a gradual and decreasing presence of tumour cells. Lastly, a treatment planning study compared VMAT for high-grade gliomas obtained from dose painting using ?????F-FDOPA PET images. Dose constraints for each contour were specified by a radiobiological model. VMAT planning using dose painting for high-grade gliomas was achieved using biologically-guided thresholds of ?????F-FDOPA uptake with no significant change in the dose delivered to critical structures.

Biomarkers for the classification, clinical management and prognosis of pediatric brain tumors (ependymoma and high grade glioma, (HGG)) are lacking. To address this, biomarkers were developed and explored in view of classification, prognostication, target identification and prediction of the efficacy of treatment for patients with such tumors.We show that overexpression of neuronal markers distinguishes supratentorial from infratentorial ependymoma, and among the former higher immunoexpression of neurofilament 70 (NEFL) is correlated with better progression free survival (PFS). Tenascin-C (TNC) is significantly overexpressed in infratentorial ependymoma. A multi-institutional European ependymoma collaboration group was established and analyses were performed in a pediatric cohort of 250 patients, where immunohistochemistry (IHC) for TNC showed to be a robust marker of poor overall survival (OS) and PFS, particularly among children under 3 years, this being further validated in an independent cohort. Techniques and scoring performed in different laboratories were highly reproducible. IHC for NEFL and TNC could be used for prognostication of pediatric ependymoma.The analysis of putative predictive markers for the response to targeted therapies in pediatric HGG in the setting of a clinical trial with the anti-EGFR agent erlotinib was performed by IHC and fluorescent in situ hybridization. The frequent loss of PTEN in diffuse intrinsic pontine glioma (DIPG) and the confirmation of the biological singularity of the certain subgroups (expressing EGFR, displaying oligodendroglial differentiation) which seem to be associated with better response to erlotinib have helped our group to establish the design of the next Phase III protocol for this disease at our institution. We report mutations in PI3KCA constituting the first identification of oncogene mutations in some DIPG, which further highlight their biological heterogeneity. Further studies are needed to define the interaction between PTEN loss, EGFR overexpression, oligodendroglial differentiation, PI3KCA mutations and other recent findings such as PDGFRA/MET gains/amplification and TP53 mutations in these heterogeneous lesions and their relationship to the outcome of patients under new targeted therapies for this largely fatal disease.This thesis has allowed us to explore the molecular pathology in the context of biology and clinical setting of pediatric brain tumors.

Une élévation majeure de la charge mutationnelle (hypermutation) est observée dans certains gliomes. Néanmoins, les mécanismes de ce phénomène et ses implications thérapeutiques notamment concernant la réponse à la chimiothérapie ou à l'immunothérapie sont encore mal connus. Sur le plan du mécanisme, une association entre hypermutation et mutations des gènes de la voie de réparation des mésappariements de l'ADN (MMR) a été rapportée dans les gliomes, cependant la plupart des mutations MMR observées dans ce contexte n'étaient pas fonctionnellement caractérisées, et leur rôle dans le développement d’hypermutation restait de ce fait incertain. De plus, l'impact de l'hypermutation sur l'immunogénicité des cellules gliales et sur leur sensibilité au blocage des points de contrôles immunitaires (par exemple par traitement anti-PD-1) n’est pas connu. Dans cette étude, nous analysons de manière exhaustive les déterminants cliniques et moléculaires de la charge et des signatures mutationnelle dans 10 294 gliomes, dont 558 (5,4%) tumeurs hypermutées. Nous identifions deux principales voies responsables d'hypermutation dans les gliomes : une voie "de novo" associée à des déficits constitutionnels du système MMR et de la polymérase epsilon (POLE), ainsi qu'une voie "post-traitement", plus fréquente, associée à l'acquisition de déficits MMR et de résistance secondaire dans les gliomes récidivant après chimiothérapie par temozolomide. Expérimentalement, la signature mutationnelle des gliomes hypermutés post-traitement (signature COSMIC 11) était reproduite par les dommages induits par le témozolomide dans les cellules MMR déficientes. Alors que le déficit MMR s'associe à l'acquisition de résistance au témozolomide, des données cliniques et expérimentales suggèrent que les cellules MMR déficientes conservent une sensibilité à la nitrosourée lomustine. De façon inattendue, les gliomes MMR déficients présentaient des caractéristiques uniques, notamment l'absence d'infiltrats lymphocytaires T marqués, une hétérogénéité intratumorale importante, une survie diminuée ainsi qu’un faible taux de réponse aux traitements anti-PD-1. De plus, alors que l'instabilité des microsatellites n'etait pas détectée par des analyses en bulk dans les gliomes MMR déficients, le séquençage du génome entier à l'échelle de la cellule unique de gliome hypermuté post-traitement permettait de démontrer la presence de mutations des microsatellites. Collectivement, ces résultats supportent un modèle dans lequel des spécificités dans le profil mutationnel des gliomes hypermutés pourraient expliquer l’absence de reconnaissance par le système immunitaire ainsi que l’absence de réponse aux traitements par anti-PD-1 dans les gliomes MMR déficients. Nos données suggèrent un changement de pratique selon lequel la recherche d’hypermutation par séquençage tumoral lors de la récidive après traitement pourrait informer le pronostic et guider la prise en charge thérapeutique des patients
High tumor mutational burden (hypermutation) is observed in some gliomas; however, the mechanisms by which hypermutation develops and whether it predicts chemotherapy or immunotherapy response are poorly understood. Mechanistically, an association between hypermutation and mutations in the DNA mismatch-repair (MMR) genes has been reported in gliomas, but most MMR mutations observed in this context were not functionally characterized, and their role in causing hypermutation remains unclear. Furthermore, whether hypermutation enhances tumor immunogenicity and renders gliomas responsive to immune checkpoint blockade (e.g. PD-1 blockade) is not known. Here, we comprehensively analyze the clinical and molecular determinants of mutational burden and signatures in 10,294 gliomas, including 558 (5.4%) hypermutated tumors. We delineate two main pathways to hypermutation: a de novo pathway associated with constitutional defects in DNA polymerase and MMR genes, and a more common, post-treatment pathway, associated with acquired resistance driven by MMR defects in chemotherapy-sensitive gliomas recurring after temozolomide. Experimentally, the mutational signature of post-treatment hypermutated gliomas (COSMIC signature 11) was recapitulated by temozolomide-induced damage in MMR-deficient cells. While MMR deficiency was associated with acquired temozolomide resistance in glioma models, clinical and experimental evidence suggest that MMR-deficient cells retain sensitivity to the chloroethylating nitrosourea lomustine. MMR-deficient gliomas exhibited unique features including the lack of prominent T-cell infiltrates, extensive intratumoral heterogeneity, poor survival and low response rate to PD-1 blockade. Moreover, while microsatellite instability in MMR-deficient gliomas was not detected by bulk analyses, single-cell whole-genome sequencing of post-treatment hypermutated glioma cells demonstrated microsatellite mutations. Collectively, these results support a model where differences in the mutation landscape and antigen clonality of MMR-deficient gliomas relative to other MMR-deficient cancers may explain the lack of both immune recognition and response to PD-1 blockade in gliomas. Our data suggest a change in practice whereby tumor re-sequencing at relapse to identify progression and hypermutation could inform prognosis and guide therapeutic management

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumour. Despite advances in GBM treatment, there is a high frequency of local relapse due to the acquisition of drug resistance. Investigation of glioma cell lines will help us to understand the molecular basis of this hard to treat tumour. In this study, the rat C6 glioma cell line was used as a model alongside two drug selected derivatives (C6-etoposide and C6-irinotecan) to investigate the mechanisms of chemo-resistance in glioma by identifying candidate proteins, genes, and key signalling pathways. Proteomic (2D gel electrophoresis) and genomic (gene array) analyses were performed to determine protein and gene expression changes. Integration of this data with cellular pathway analysis resulted in the prediction that cellular migration and the response to oxidative stress would be distinct in the drug selected C6 cell lines. Cell migration was subsequently assessed using wound scratch repair and transwell migration assays, whilst the response to oxidative stress produced by reactive oxygen species was determined fluorimetrically. The C6 cell line exposed to irinotecan (DNA topoisomerase I inhibitor) showed reduced migration, even under the influence of chemoattractant, compared to other cell lines, consistent with alterations in the expression of collagen genes. The C6 cell line exposed to etoposide (DNA topoisomerase II inhibitor) showed greater resistance to oxidative stress which was proposed to be due to alterations in the signalling pathways downstream of the PTEN/PI3Kinase. Future studies, investigating the effect of PI3Kinase pathway inhibitors are considered and it is proposed that further research into this signalling pathway will be able to uncover the molecular basis of distinct chemo-resistance in this important model cell system for aggressive glioma.

Paediatric high grade gliomas remain poor prognosis tumours, with a median survival of only 15 months following diagnosis. Current investigation of antiangiogenic strategies, an approach which holds considerable therapeutic promise, has focused on adult glioblastoma multi forme with phase III trials targeting vascular endothelial growth factor continuing. In this study the level of neo-vascularisation within pHGG was assessed by immunohistochemistry against vascular markers such as CD31 and CD lO5. Levels of known mediators associated with increased angiogenesis were also measured and tumours segregated into low and high vascularity samples. Gene expression changes between tumours of differing vascularity were then assessed using genome wide expression profiling and validated, thus identifying novel angiogenesis related genes in pHGG. Tumours were also profiled for their levels of micro-ribonucleic acids (microRNAs) using the Nanostring array based system. Again, microRNAs were found that were significantly differentially expressed between high and low vascularity tumours. Differences were also observed in microRNA expression levels between high and low grade astrocytomas and between long and short term survivors. Modelling a complex three dimensional process such as angiogenesis is difficult in conventional monolayer cell culture. A three dimensional rotary cell culture system was optimised for the growth of macroscopic brain tumour aggregates. Characterisation of the cultures from this system revealed enhanced similarity between the cultures and actual brain tumours (compared to monolayer cultures of the same cell lines). Three dimensional aggregates showed increased drug resistance and significant up-regulation of angiogenic pathways, including fibroblast growth factor and transforming growth factor beta pathways, on real-time polymerase chain reaction and immunohistochemistry. Up-regulation of endothelial markers was also demonstrated in pure tumour cell cultures, in vitro evidence of the process of vasculogenic mimicry.

Malignant gliomas (MG), most notably glioblastoma multiforme (GBM), are among the most aggressive of all malignancies. High-grade variants of this type of brain cancer are generally considered incurable with singular or multimodal therapies. Many patients with GBM die within one year of diagnosis, and the 5-year survival rate in people is approximately 10%. Despite extensive research in diagnostic and therapeutic technologies, very few developments have emerged that significantly improve survival over the last seven decades. Irreversible electroporation (IRE) is a new non-thermal focal tissue ablation technique that uses low-energy electric pulses to destabilize cell membranes, thus achieving tissue death. The procedure is minimally invasive and is performed through small electrodes inserted into the tissue with treatment duration of about one minute. The pulses create an electric field that induces an increase in the resting transmembrane potential (TMP) of the cells in the tissue. The induced increase in the TMP is dependent on the electric pulse parameters. Depending on the magnitude of the induced TMP the electric pulses can have no effect, transiently increase membrane permeability or cause spontaneous death. In this dissertation we hypothesize that irreversible electroporation is capable of ablating normal (gray and white matter) and pathological (MG and/or GBM) brain tissue in a highly focused non-thermal manner that is modulated through pulse parameters and electrode configuration. Through a comprehensive experimental and numerical investigation, we tested and attained results strongly supporting our hypothesis. Specifically, we developed numerical models that were capable of simulating an entire IRE treatment protocol and would take into account pulse parameters (e.g. duration, frequency, repetition rate and strength) in addition to the dynamic changes in tissue electrical conductivity due to electroporation and joule heating, as well as biologically relevant processes such as blood perfusion and metabolic heat. We also provided a method to isolate the IRE effects from undesired thermal damage in models that were validated with real-time temperature measurements during the delivery of the pulses. Finally we outlined a procedure to use 3D volumetric reconstructions of IRE lesions using patient specific MRI scans in conjunction with the models described for establishing field thresholds or performing treatment planning prior to the surgical procedure; thus supplying the readers with the tools and understanding necessary to design appropriate treatment protocols for their specific application. Experimentally we presented the first systematic in vivo study of IRE in normal canine brain and the multimodal treatment of a canine MG patient. We confirmed that the procedure can be applied safely in the brain and was well tolerated clinically. The lesions created with IRE were sub-millimeter in resolution and we achieved 75% tumor volume reduction within 3 days post-IRE in the patient. In addition to the sharp delineation between necrotic and normal brain, the treatments spared the major blood vessels, making it appropriate for treatment of tumors adjacent to, or enveloping critical vascular structures. We believe that irreversible electroporation will play a key role in the treatment of intracranial disorders including malignant brain cancer in which the intent is to focally kill undesired tissue while minimizing damage to surrounding healthy tissue.
Ph. D.

Paediatric high grade glioma (HGG), including diffuse intrinsic pontine glioma (DIPG) are highly aggressive tumours with no effective cures. Lack of understanding of the molecular biology of these tumours, in part due to lack of well-characterised pre-clinical models, is a great challenge in the development of novel therapies. Analysis of paired cell culture/biopsy samples in this study revealed that paediatric HGG short-term cell cultures retain many of the tumour characteristics in vivo. Using a genome-wide approach, copy number, gene and miRNA expression, and methylation changes were characterised in 17 paediatric HGG-derived short-term cell cultures including 3 from DIPG. The majority of the genomic changes were unique from those arising in adult HGG. Approximately 65% (11/17) of paediatric HGG short-term cell cultures had balanced genetic profiles resembling normal karyotypes. The most frequent copy number gain and loss were detected at 14q11.2 (94%) and 8p11.23-p11.22 (59%), respectively. H3F3A (K27M) mutation was present in 2/17 (12%) cases and concurrent loss of CDKN2A and BRAFV600E in 1/17 (6%) case. Genes involved in reelin/PI3K signaling (DAB1), RTK signaling (PTPRE), and arginine biosynthesis (ASS1 and ASL) were frequently deregulated by methylation in these tumours. The anti-growth and anti-migratory properties of DAB1 and PTPRE were demonstrated in vitro. Preliminary investigations validated the therapeutic potential of ADI-PEG20 (arginine depletion), and PI-103 (PI3K/mTOR inhibition) in a subset of paediatric HGG short-term cell cultures. This study has identified novel genetic and epigenetic changes in paediatric HGG that may, following further validation, be translated into potential biomarkers and/or therapeutic targets.

Les gliomes sont les principales tumeurs primitives du cerveau affectant environ 4000 nouveaux patients par an en France. La moitié des gliomes est détectée au stade avancé de glioblastome (grade IV) tandis que 15% des tumeurs sont diagnostiquées au stade II de gliomes diffus dit de bas grade. Ces tumeurs affectent des patients jeunes et présentent des mutations caractéristiques, notamment une mutation pour l’enzyme IDH1 communément retrouvée dans les glioblastomes secondaires. Ces tumeurs de bas grade sont traitées par une chirurgie, idéalement en condition éveillée mais du fait de leur nature diffuse, la partie résiduelle progressera inexorablement vers un stade III ou IV avec une survie globale entre 5 ans et 15 ans après diagnostique. La progression tumorale est hautement variable et non prédictible d’un patient à l’autre. Des foyers de progression tumorale chez 20% des patients atteints de gliome diffus de bas grade ont été identifiés. Ces foyers montrent une densité cellulaire plus élevée ainsi qu’un Ki67 augmenté. Mon travail de thèse aura consisté à étudier les modifications cellulaires et moléculaires associées à ces foyers de progression tumorale. À partir du profil ARN des foyers et des territoires adjacents, j’ai pu mettre en évidence par des techniques haut-débit la baisse d’expression significative de gènes dans les foyers notamment de AGXT2L1/ETNPPL, carboxypeptidase E, EDNRB, SFRP2. J’ai émis l’hypothèse que SFRP2 et ETNPLL pourraient s’opposer à la prolifération cellulaire et que leur diminution dans les foyers ouvrirait la voie à la transformation tumorale. Une corrélation inverse entre la quantité d’ETNPPL enzyme et la survie de patients atteints d’hépatocarcinomes a été publiée. En limitant la quantité de précurseurs de phospholipides dans la cellule, ETNPPL pourrait agir comme un frein en s’opposant à la prolifération et de fait, sa diminution dans les foyers de transformation des gliomes pourrait lever cette inhibition. Mes travaux auront été innovants tant dans leur approche comparative des différents compartiments tumoraux pour chaque patient étudié et auront révélés ETNPPL comme corrélé à la gliomagenèse et potentielle cible thérapeutique
Gliomas are the main primary brain tumours affecting around 4000 new patients in France each year. Half of gliomas are detected in the advanced stage of glioblastoma (grade IV) while 15% of tumours are diagnosed in stage II (diffuse low-grade gliomas-DLGG). These tumors affect young patients and bear characteristic mutations, including a mutation for the enzyme IDH1 commonly found in secondary glioblastomas. These low-grade tumours are treated by surgery, ideally in awake condition but due to their diffuse nature, the residual part will progress inexorably to stage III or IV with overall survival between 5 and 15 years after diagnosis. Tumor progression is highly variable and unpredictable from one patient to another. Foci of tumor progression have been identified in 20% of patients with DLGG. These foci show a higher cell density and an increased Ki67. My thesis work consisted in studying the cellular and molecular changes associated with tumor progression. From the RNA profile of the foci and adjacent territories, I was able to highlight through high-throughput techniques significant decrease in gene expression in the foci, particularly of AGXT2L1/ETNPPL, carboxypeptidase E, EDNRB, SFRP2. I hypothesized that SFRP2 and ETNPLL could oppose cell proliferation and that their decrease would pave the way for tumor transformation. An inverse correlation between the amount of ETNPPL and the survival of patients with hepatocarcinoma has been published. By limiting the amount of phospholipid precursors in the cell, ETNPPL could act as a brake against proliferation and indeed, its decrease in glioma transformation foci could remove this inhibition. My PhD work will have been innovative in the comparative approach of the different tumors’ compartments for each patient studied and will have revealed ETNPPL as correlated to gliomagenesis and as potential therapeutic target

Glioblastoma is a highly malignant and aggressive high grade glioma with a poor prognosis. The low survival rates stem from tumour progression, late intervention, ineffective therapies and drug resistance, requiring new therapeutic and diagnostic approaches. Lipid droplets are dynamic organelles suggested to be influential facets of cancer metabolism and biology in many tumours. In glioblastoma lipid droplets have been associated with hypoxia higher clinical grades and poor survival however the cellular pathways underlying lipid droplet metabolism remain unclear. Using a publically available database of grade 2 ta 4 glioma gene expression, we observed that genes associated with lipid droplet metabolism were important prognostic survival and tumour progression indicators. Moreover, through confocal microscopy, flow cytometry and NMR-based methods, we observed that uptake of exogenous lipids and adipose triglyceride lipase-mediated lipid shuttling produced lipid droplets whilst autophagy was vital to lipid droplet breakdown. ATGL-mediated lipid shuttling was further observed to prevent unsaturated fatty acid oxidative damage. Finally, we investigated the effect of pharmacological lipid droplet manipulation and observed that autophagy inhibition can improve temozolomide and irradiation cytotoxicity. Taken together our data suggests that understanding lipid droplet metabolic pathways may generate prognostic bio-markers of survival and progression and improve current therapies.

Paediatric high grade glioma (pHGG) and diffuse intrinsic pontine glioma (DIPG) are highly aggressive brain tumours. Their invasive phenotype contributes to their limited therapeutic response and novel treatments that block brain tumour invasion are needed. Here, two novel anti-invasive therapeutic strategies are evaluated: glycogen synthase kinase-3 (GSK-3) inhibitors and oncolytic viruses (OVs). Small molecule GSK-3 inhibitors, lithium chloride (LiCl) and the indirubin derivative 6-bromoindirubin-oxime (BIO), reduced migration and invasion of pHGG cell lines (SF188 and KNS42) and one patient-derived DIPG cell line (HSJD-DIPG-007) in 2D and 3D in vitro assays. Following drug treatment, pHGG cells demonstrated loss of polarity and altered morphology as seen by live cell imaging and cytoskeletal rearrangement of actin fibres and focal adhesions as seen by immunofluorescence. OVs (herpes simplex virus (HSV), reovirus and vaccinia virus (VV)) were able to inhibit the migration and invasion of pHGG and DIPG cell lines. Oncolytic HSV was the most interesting candidate, as anti-migratory and anti-invasive effects did not appear to be a consequence of cytotoxicity or altered proliferation. Oncolytic HSV altered pHGG cytoskeletal dynamics, stabilising microtubules through the accumulation of post-translational tubulin modifications, as evaluated by Western blotting and immunofluorescent labelling. Furthermore, oncolytic HSV treatment of pHGG cell lines inhibited GSK-3β activity and prevented the localised clustering of adenomatous polyposis coli to the leading edge of the cell. These observations are highly novel and begin to document the molecular mechanisms by which oncolytic HSV may inhibit pHGG migration and invasion. In conclusion, this study is the first to demonstrate that it is possible to target migration and invasion of pHGG and DIPG in vitro using either small molecule GSK-3 inhibitors or OVs, such as HSV. These agents warrant further in vivo pre-clinical investigation as potential anti-invasive therapeutics and have the potential to improve outcomes of these devastating childhood diseases.

Overall paediatric high grade glioma (pHGG) has a poor prognosis, in part due to the lack of understanding of the underlying biology. We therefore used high resolution 244K oligo array comparative genomic hybridisation (oligo aCGH) (Agilent Technologies) to analyse DNA from 38 formalin-fixed paraffin embedded pHGG samples, including 13 DIPG. The pattern of gains and losses were distinct from those seen in HGG arising in adults. In particular we found 1q gain in 21% of our cohort compared to 9% in adults. Homozygous loss at 8p12 was seen in 6/38 (15%) of pHGG. This deletion has not been previously reported in adult or paediatric high grade gliomas. The minimal deleted region is of the gene ADAM3A and homozygous deletion of ADAM3A was confirmed by quantitative real time PCR (qPCR). This novel homozygous deletion of ADAM3A in pHGG merits further study. Amplification of the 4q11-13 region was detected in 8% of cases and included PDGFRA and KIT and subsequent qPCR analysis was consistent with amplification of PDGFRA. MYCN amplification was seen in 2/38 samples (5%) and was shown to be significantly associated with anaplastic astrocytomas (p=0.03). Loss of CDKN2A/B was seen in 4/38 (10%) samples by oligo a CGH, confirmed by FISH on TMAs, and was restricted to supratentorial tumours. ~50% of supratentorial tumours were positive for CDKN2B expression by IHC, whilst ~75% of brainstem gliomas were positive for CDKN2B expression (p = 0.03). Overall DIPG shared a similar spectrum of changes to supratentorial HGG with some notable differences including high frequency of 17p loss and 14q loss, low occurrence of 10q loss and lack of CDKN2A/B deletion.

京都大学
新制・課程博士
博士(人間健康科学)
甲第23385号
人健博第92号
新制||人健||6(附属図書館)
京都大学大学院医学研究科人間健康科学系専攻
(主査)教授 田村 恵子, 教授 稲富 宏之, 教授 溝脇 尚志
学位規則第4条第1項該当
Doctor of Human Health Sciences
Kyoto University
DFAM

High-grade gliomas (HGG) are a collection of lethal brain tumours characterised by aggressive, diffuse spread. Clinically-approved therapies lack efficacy and mean patient survival rates have not improved for ~50 years. The importance of the tumour microenvironment (TME) in influencing tumour biology and response to therapies has been long appreciated, and the lack of effective HGG treatments is in part due to a dearth of pre-clinical models which account for tumour-TME interactions. Work described in this thesis therefore aimed to develop and characterise in vitro models to investigate HGG-TME interactions and test new therapies. Hydrogel-scaffold and tumour spheroids were first used to characterise a novel HGG-targeting chimeric antigen receptor (CAR)-T cell therapy. Data show that CAR-T cells effectively trafficked to- and killled tumour cells in a hydrogel scaffold model. Data further showed CAR-T cells were able to infiltrate and retain cytotoxicity inside spheroids. However, hydrogel-embedded spheroid models highlighted striking differential TME-dependent responses, prompting the development of an organotypic human embryonic stem cell-derived brain organoid model. Characterisation of a dorsal cortical organoid model by showed that organoids recapitulate ‘neural tissue’ and were capable of hosting HGG cells. For the first time, a proteomic approach was used to interrogate the effect of HGG contact with the brain tumour TME-mimetic provided by the brain organoids on HGG protein expression. Analysis of proteomic data revealed that contact with a neural TME alone promotes mitotic pathways, increased protein production and maintenance of a stem-phenotype in HGG cells. Taken together, results showcase the development of an HGG-organoid co-culture model, and highlights the need for mimetic models for basic biology and therapeutics testing.

This research was a longitudinal mixed methods study that involved the delivery of an exercise intervention in brain cancer patients undergoing treatment. Three studies were conducted to examine post-surgical functioning, feasibility and safety, and a qualitative review of patients’ acceptability and experiences. This research highlighted functional deficits after surgery, limiting independence. The delivery of an exercise intervention during treatment is both feasible and safe. Clinically important improvements in aerobic functioning, lower-body strength and mobility were observed after cancer treatment. Participating in the exercise intervention provided patients with respite from their cancer journey and promoted positive coping ability towards future outlook.

Introduction: At the core of UK policy for improving outcomes in cancer are goals for a healthcare where patients are empowered through information enabling engagement in shared care decisions with clinicians. Interventions to support patients’ engagement in shared decision making are lacking within colorectal cancer and high grade glioma care despite intensive treatment regimens with uncertain outcomes. Navigation, a communication and decision support intervention, has been successfully piloted with prostate and breast cancer patients who demonstrated significantly more confidence and less uncertainty in their treatment decisions. With healthcare policy advocating patients be educated and engaged in their care, the applicability of this intervention to other cancer settings is required. The Navigation intervention includes: consultation planning with a Navigator, formulation of a consultation plan and recording (summary and CD) of the medical consultation. Objectives: To determine the effectiveness of the Navigation intervention in enhancing decision-making quality over time when compared with usual care, in patients with colorectal cancer. To explore repeated experiences of the Navigation intervention from the perspective of colorectal cancer (CRC) patients, patients with high grade glioma (HGG), and consulting clinicians. Design and Studies: A mixed methods study using a pragmatic randomised controlled trial and qualitative evaluation was undertaken during November 2010 – December 2013. The intervention was trialled separately with two cohorts of cancer patients (CRC and HGG). A longitudinal parallel-group pragmatic randomised controlled trial was conducted. Study 1 consisted of a longitudinal parallel-group pragmatic randomised control trial. Participants with colorectal cancer were openly randomised after completion of baseline measures to receive the intervention or usual care (no intervention). The intervention was administered to patients at three particular time points during first line cancer treatment. Participants completed tools collecting primary outcome (decision self-efficacy) and secondary outcomes (decision conflict, decision regret, anxiety and depression) measured prior to baseline, post consultation and at follow-up. Mean change in scores overtime and between groups were compared using Mixed ANOVAS. Study two was a prospective qualitative study undertaking serial in-depth semi-structured evaluation interviews with patients with High Grade Glioma. Study three undertook interviews with the consulting HGG and CRC clinicians. Framework analysis was undertaken. Setting: Two oncology settings within a tertiary cancer centre in Scotland. Participants: 132 patients with colorectal cancer (65 intervention, 67 control) participated in the randomised controlled trial. For the qualitative study, 17 colorectal trial participants (8 intervention, 9 control), 11 high grade glioma patients and 7 clinicians were interviewed. Evaluation Results: No significant difference was found between the control and Navigation intervention participants over time in the primary outcome of decision self-efficacy, or in the following secondary outcomes; decision conflict or anxiety and depression scores. At follow-up, the intervention group reported significantly less decision regret than the controls (p=0.039). In the qualitative data, Navigated participants reported being well prepared for medical consultations, able to actively engage in information exchange during consultation and enabled to recall and understand information provided. This was in contrast to participants receiving usual care who described being less prepared for medical consultations and experienced barriers to gathering information, such as time pressures, forgetting questions, and gaps in understanding. Clinicians identified that patients benefitted from preparing for, and having a written summary of, the consultation. Whereas neuro-oncology clinicians were supportive of Navigation as a tool to tailor information to patients; colorectal clinicians felt Navigation was a disruption to their normal consultation routine. Concern was expressed regarding the extra resource required by Navigated patients and therefore about the feasibility and sustainability of the intervention. Conclusions: Whilst models of shared decision making remain highly profiled in cancer strategies, information exchange and use of interventions in context is problematic. This evaluation of Navigation has demonstrated more impact on the process of decision making, rather than outcome per se, and has raised questions about its sustainability in clinical practice. A more nuanced understanding of different cancer pathways and the specific decisions to be made, may inform a more targeted use of decision support in cancer care.

Purpose: The prognosis for malignant gliomas is poor, and overall survival remains less than 15 months despite improved diagnostic and treatment techniques, underscoring the need for novel therapeutic options. This multicenter, single-arm Phase Ib/II trial was conducted to assess the safety and efficacy of AdV-TK, an adenoviral vector containing the herpes simplex virus thymidine kinase gene, in patients newly diagnosed with high grade glioma. Patients & Methods: Patients were recruited between 2005 and 2010 at four clinical sites. In addition to the standard of care (SOC), which included surgery, adjuvant XRT + temozolomide (TMZ), AdV-TK was administered to the tumor bed at the time of resection followed by valacyclovir prodrug. A total of 48 patients completed therapy per protocol, and were stratified based on extent of resection (total vs. subtotal) and pathological diagnosis (GBM vs. AA/AO). Data from 134 matched patients, who underwent similar SOC at a fifth clinical site during the same time period, were used for descriptive comparison. Results: There were no dose-limiting toxicities or significant adverse events considered related to AdV-TK. Median overall survival (OS) was 17.05 months, with 67% and 35% at 1- and 2- years respectively. Median OS for patients with total resection was 25 months, compared with 13.5 months for patients with subtotal resection. This difference was more pronounced in patients with a pathological diagnosis of GBM (25.05 vs. 10.6 months). In the comparison group median OS was 13.5 months, with 57% and 22% at 1- and 2- years respectively. Median OS for those with total resection was 16.9 months and 12.47 months for subtotal resection. Progression free survival (PFS) was also improved in the AdV-TK group (8.3 vs. 6.43 months). Conclusions: Results from this multicenter Phase Ib/II trial demonstrate that AdV-TK plus valacyclovir can be safely delivered at the time of surgical resection without added toxicity to patients with newly diagnosed high grade glioma. The median OS and the 1- and 2-year survival rates of AdV-TK study patients compare favorably to historical reports and a matched comparison set. Survival outcomes are significantly better in patients having undergone total resections versus subtotal resections. These data strongly support launching a statistically powered randomized clinical study of AdV-TK for the treatment of high grade glioma.

Gliomas represent a very heterogeneous group of tumors that affect the cellular components of the glial system. Pediatric High Grade Gliomas (pHGGs) are a rare, malignant and diffusively infiltrating neoplasms. They show an extremely broad range of clinical behaviour with only few patients achieving long-term survival. Notch signaling is an evolutionarily conserved pathway that regulates many cellular and developmental processes. Its dysregulation has been reported in many pathological contexts, including brain tumours, but little is known about the relevance of Notch signaling in pHGGs. The study of epigenetic mechanism engaged in its regulation could allow a better understanding of mechanisms at the basis of its deregulation. MicroRNAs are small molecules of 21-24 nucleotides in length and are emerging as major regulators of cancer-related gene expression. The identification of specific microRNAs signature is one of the latest hallmarks of scientific efforts. High-throughput microRNA profiling of pHGGs showing that several dysregulated microRNAs characterized these tumors. The present project intends to investigate the functional role of the Notch signalling unveiling epigenetic networks between dysregulated microRNAs and the Notch pathways. For the purpose, immunohistochemistry (IHC) on pHGG patients’ derived tissues and immunofluorescence (IF) on a patient derived pHGG cell line (KNS42) were performed to assess Notch receptors expression level. RT-qPCR was used to validate microRNAs levels in pHGG tissues and cells in respect to healthy brain tissues. Cell growth and viability were evaluated on KNS42 cells after microRNAs re-expression or pharmacological (GSI IX) and genetic (siRNA N2) treatments with by trypan blue exclusion assays. Luciferase reporter assay was performed to validate the binding of miR-29a-3p to the 3’-UTR of Notch2. IHC and IF analysis have demonstrated that Notch2 is highly expressed in pHGG tissues and cells. Pharmacologic and genetic treatments have highlighted its pivotal role in the control of pHGGs cell growth. Studies on patients’ derived pHGG tissues and pHGG cell line, KNS42, revealed down-regulation of three miRNAs, specifically miR-107, miR-181c and miR-29a-3p. Their re-expression in KNS42 cell line has effect on Notch2 protein levels and cell proliferation. The same effect was obtained after pharmacological inhibition of Notch signaling pathway with GSI IX for 96 h. Functional luciferase assay provides the evidence that miR-29a-3p is a direct negative regulator of Notch2 expression. The study reported suggests that Notch2 pathway activation plays a critical role in pHGGs growth and reveals a direct epigenetic mechanism that controls Notch2 expression. These findings identify new molecular targets for more effective treatment of these devastating pediatric brain tumors.

碩士
國立陽明大學
腦科學研究所
98
Background: Intra-tumoral hypoxia is associated with malignant progression, tumor invasion and resistance to radiotherapy and chemotherapy of cancers. Anti-angiogenic therapies targeting the vascular endothelial growth factor (VEGF) / VEGF receptor (V EGFR) have shown therapeutic effect for malignant tumors. However, they also increase intra-tumoral hypoxia. Anti-angiogenic drug 01 (AAD01), on the other hand, targets endothelial cells directly. Tetramethylpyrazine (TMP) decreased the expression of VEGF and reduced angiogenesis. This study would test these two drugs to decrease tumor hypoxia in malignant gliomas in rat. Hypothesis: It is hypothesized that by limiting the rapid proliferation of endothelial cells, AAD01 increase tissue perfusion in gliomas, which in turn result in the decrease of intra-tumoral hypoxia. This study has also evaluated if TMP has similar effect. Materials and Methods: Suspended 1x107 C6 glioma cells were implanted intracerebrally of the adult female Sprague-Dawley rats. The treatment group received AAD01 (10 µg / every 3 days) or TMP (2.4 mg/day, for 7 days) beginning for the 5th day. At day 13, animals were sacrificed. Tumor hypoxia and angiogenic factors were analyzed by immunohistochemistry and quantified by Western blot. Multiple groups were analyzed by one-way analysis of variance (ANOVA). P-value of less than 0.05 was considered significant. Results: In the AAD01 treated group, tumor size was decreased (60.42%). These rats also showed less body weight loss. The expression of hypoxic inducible factor-1 α (HIF-1α) and VEGF both decreased significantly in the treatment group than in control group. The sprouting vessel, which is evaluated by staining of VEGFR2 was inhibited in the AAD01 group. The density of total vessels was decreased. The contrast, the density of perfused vessels was increased. The peri-tumor infiltration was less prominent in the AAD01 group. On the hand, the expression of HIF-1α and VEGF in the TMP group was decreased but less effect than AAD01 group. The density of perfused vessels was not significant statistically different from control. Conclusion: AAD01 reduced tumor hypoxia by increasing tissue perfusion. Otherwise, it can reduce peri-tumor infiltration.

Trabalho Final do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina
Os tumores cerebrais primários são uma entidade heterogénea, correspondendo 75% destes a tumores malignos da série glial. O diagnóstico estabelece-se mais comummente entre a quinta e a sexta décadas de vida, sendo fatores de risco para esta doença a exposição prévia a radiação ionizante e a presença de história familiar de tumores cerebrais.O prognóstico dos doentes com gliomas de alto grau é reservado apesar da terapêutica multimodal, com a associação de cirurgia a radioterapia e quimioterapia. Neste contexto, a identificação de biomarcadores de diagnóstico e prognóstico tem particular importância, uma vez que poderá conduzir à prática de uma medicina mais personalizada, com adaptação da terapêutica ao doente individual e não apenas às características anatomopatológicas do tumor.A Organização Mundial de Saúde, em 2016, procedeu à revisão da classificação de gliomas, com inclusão de características moleculares para definição de diagnóstico de alguns destes tumores. Nesta revisão inclui-se, entre outros, a pesquisa de mutações do gene da isocitrato desidrogenase, a co-deleção do braço curto do cromossoma 1 e do braço longo do cromossoma 19, assim como a metilação do promotor da O6-metilguanina-metiltransferase, com o objetivo de melhorar a acuidade diagnóstica e identificar subgrupos com melhor resposta à terapêutica. No entanto, carecemos atualmente de informação com impacto clínico na individualização terapêutica.Deste modo, com este trabalho pretendeu-se realizar uma revisão narrativa do papel dos biomarcadores nos tumores gliais de alto grau, com inclusão e análise das publicações dos últimos 5 anos neste contexto e dos ensaios clínicos em curso.Nas publicações identificadas foram analisados diversos biomarcadores, categorizados em histológicos, circulantes e imagiológicos. Dos biomarcadores histológicos realça-se o papel da mutação da isocitrato desidrogenase e da metilação do promotor da O6-metilguanina-metiltransferase no diagnóstico e definição de prognóstico. No contexto de imunoterapia foram identificados diferentes biomarcadores histológicos e circulantes; no contexto de terapêutica antiangiogénica foram identificados potenciais biomarcadores de resposta. É ainda realçada a importância de biomarcadores imagiológicos com ressonância magnética, utilizando ponderações de difusão, administração de contraste paramagnético ou algoritmos mais complexos para estratificação dos doentes. É importante realçar a diversidade de contextos em que foram pesquisados os biomarcadores, que limitam a validação clínica do seu papel nos gliomas de alto grau, sendo essencial o desenvolvimento de estudos dirigidos.
Primary brain tumors are a heterogeneous entity, with 75% corresponding to malignant glial neoplasms. They arise mainly during the fifth and sixth decades of life, and the identified risk factors are previous exposure to ionizing radiation and family history of brain tumors.High grade gliomas present with a poor prognosis, although its treatment is multimodal, with the association of surgery with adjuvant radiotherapy and chemotherapy. Identifying diagnostic and prognostic biomarkers in this context is highly relevant, as it can help in the selection of patients with a particular better prognosis and lead to a more individualized approach regarding their treatment, not only based on pathologic features.The World Health Organization revised their classification of glial tumors in 2016 by including molecular markers in their diagnostic definition, such as mutations in isocitrate dehydrogenase gene, codeletion of the short arm of chromosome 1 and the long arm of chromosome 19, and methylation of the O6-methylguanine-DNA-methyltransferase gene promoter. The main goal for this revision was to increase diagnostic acuity and identify patient subgroups with a better therapeutic outcome. However, we still lack definition of prognostic factors that can lead to an individualized treatment regimen for these patients.We aimed to elaborate a narrative review of the role of biomarkers in high grade gliomas, by including relevant publications to this topic over the last five year, as well as ongoing clinical trials.Several biomarkers were identified and stratified according to their type: pathologic, circulating or imaging biomarkers. Regarding pathology, the role of isocitrate dehydrogenase gene mutations and methylation of the O6-methylguanine-DNA-methyltransferase gene promoter in diagnostic accuracy and prognosis definition were emphasized. The role of immunotherapy and antiangiogenic therapy was assessed in several publications, with several potential biomarkers related to treatment response and survival, either pathologic or circulating. We also identified several reports on imaging biomarkers using magnetic resonance, either with dynamic contrast enhanced, diffusion weighted imaging or more complex algorithms for patient stratification on prognosis. It is rather important to emphasize the heterogeneity of treatment scenarios on which these biomarkers were assessed, limiting clinical relevant conclusions. Therefore, studies directed to assess biomarkers in high grade glial tumors are necessary.

博士
國立陽明大學
臨床醫學研究所
106
High-grade gliomas and ependymomas are two main challenging malignancies in children, and which composed of a majority of central nervous system (CNS) tumors of young patients. In addition to maximal safe surgical resection and limited efficacy of chemotherapy, the applications of adjuvant irradiation play an important role for the tumor treatment. However, in the young ages, the resistance of residual and recurrent tumor, and long-term intellectual sequelae remain the major obstacles of radiotherapy. The advancement of high-throughput next-generation sequencing and molecular clustering technologies largely promoted the study of neuro-oncology from diagnosis based molecular to therapeutic targets in the last decade. In order to identify potential targets for the development of new therapeutic strategies, we compared the miRNome and transcriptome of pediatric low- (pLGGs) and high-grade gliomas (pHGGs) using small RNA sequencing (smRNA-Seq) and gene expression microarray, respectively in the first study. Through integrated bioinformatics analyses and experimental validation, we identified miR-137 and miR-6500-3p are significantly downregulated in pHGGs. Overexpression of miR-137 or miR-6500-3p reduced cell proliferation in two pHGG cell lines, SF188 and UW479. We also identified mRNA/miRNA interactions and confirmed that CENPE, KIF14 and NCAPG levels were direct targets of miR-137 or miR-6500-3p and were significantly higher in pHGGs than pLGGs. Furthermore, knockdown of CENPE, KIF14 or NCAPG combined with temozolomide treatment resulted in a combined suppressive effect on pHGG cell proliferation. These targets combined with chemotherapy are also potential therapeutic strategy. In the secondary study, we focused on the mechanism of therapeutic failure caused by radio-resistance and identified the significance of cyclin D1 overexpression in progression and radio-resistance of pediatric ependymomas. Here we analyze clinic-pathological factors in 82 cases of ependymoma less than 20 years old and 31 out of 82 (37.8%) patients are under 3-year-old. The 10 years PFS and OS are 38% and 60%. Gross-total resection is the single significant prognostic factor for longer 10 years progression free survival (PFS) and overall survival (OS) in the multi-variant analysis (p<0.05). We demonstrated that 24 primary (92%) and 16 recurrent (95%) ependymomas were up-regulated, and 5 out of 7 paired samples exhibited higher CCND1 expression in recurrent tumors. Knocking down CCND1 reduced cell proliferation and repressed genes associated with S-phase and DNA repair. Homologous recombination activities of DNA repair were significantly decreased in CCND1-deficient cells while the level of H2AX was increased after irradiation. We treated ependymoma cells with palbociclib plus irradiation significantly suppressed expression of CDC6, MCM2, MAD2L1, CDK2, BRCA2 and RAD51 genes, and induced higher H2AX level after 24 hours and also reduced cell proliferation. In summary, our findings identify novel mRNA/miRNA interactions in pediatric high-grade gliomas and also suggest a robust role of CCND1 in regulating cell proliferation and radio-resistance in ependymomas, which providing insight of mechanism related to the radio-resistance and the potential therapeutic targets for both pediatric high-grade gliomas and ependymomas.

Tese de doutoramento em Ciências e Tecnologias da Saúde (Pré-Bolonha), Especialidade de Biologia Celular e Molecular, apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra
Os tumores cerebrais pediátricos são a segundo tumor maligno mais comum em crianças. Estes tumores são a principal causa de morte por cancro nos grupos dos 0-14 e dos 15-24 anos. Nesta tese, centramos os nossos estudos em tumores pediátricos malignos de origem glial (grau III e IV): astrocitomas, oligodendrogliomas, e glioblastomas (GBM). Apesar dos gliomas pediátricos de alto grau serem histologicamente semelhantes aos gliomas adultos malignos, estes são doenças biologicamente diferentes, possuindo perfis de número de cópias de ADN e alterações genéticas diferentes. As recentes iniciativas de sequenciação de última geração demonstraram a existência de sub-grupos de gliomas de alto grau que são caracterizados por mutações distintas: nas crianças (H3F3A K27M), nos adolescentes e jovens adultos (H3F3A G34R/V) e nos adultos de meia-idade (IDH1/2). As aberrações estruturais dos cromossomas dão origem à formação de genes de fusão que estão normalmente associados com cancro, existindo vários casos descritos em cancros de crianças e adultos. No entanto, nos gliomas pediátricos de alto grau apenas alguns genes de fusão foram descritos. Estes rearranjos estruturais normalmente dão origem a proteínas quiméricas que potencialmente podem ser importantes na resposta tumoral à terapia dirigida, algo extremamente necessário para o tratamento desta doença. O trabalho resumido nesta tese pretende explorar os mecanismos moleculares que estão por detrás dos gliomas pediátricos de alto grau, contribuindo desta forma para a distinção entre os gliomas malignos pediátricos e adultos. O PDGFRA é um receptor de tirosina cinase (RTK) que activa várias respostas celulares, tais como a proliferação, a migração e a sobrevivência das células. Este gene está normalmente amplificado e é sobre-expresso em gliomas malignos pediátricos, desempenhando um papel muito importante nesta doença. De forma a determinar se o PDGFRA é alvo de mutações que não são detectadas através da análise do número de cópias de ADN, decidimos estudar a presença de alterações moleculares do PDGFRA (mutações pontuais e pequenas inserções/deleções), assim como a deleção Δ8,9 no PDGFRA e o gene de fusão KDR:PDGFRA (KP), anteriormente descritos em gliomas malignos em adultos. Os nossos resultados demonstraram a presença de mutações somáticas de ganho de função, incluindo mutações missense e deleções/inserções in-frame que não tinham sido descritas anteriormente na literatura. Estas alterações estão presentes em ambos gliomas pediátricos supratentoriais de alto grau e em gliomas pontinos difusos (DIPG). Os nossos estudos demonstraram que as alterações no PDGFRA são mais comuns em gliomas supratentoriais malignos do que em DIPG, e 8/18 (44%) dos casos possuem simultaneamente amplificação e mutação no PDGFRA. Os rearranjos estruturais normalmente presentes em adultos (KP e PDGFRAΔ8,9) foram apenas encontrados num caso de glioma pediátrico de alto grau. Estes resultados indicam que as alterações presentes no PDGFRA em crianças são diferentes daquelas presentes em adultos. Enquanto que as alterações do número de cópias intragénicas (iCNA) e de genes de fusão funcionalmente importantes começam a ser identificados em gliomas de alto grau em adultos, o mesmo ainda não ocorreu nos gliomas pediátricos. Ao aplicar o algoritmo iCNA a um conjunto de dados de perfis do número de cópias de ADN em casos de gliomas pediátricos malignos, previamente publicado pelo nosso grupo, identificamos novas alterações intragénicas. Neste estudo, identificamos 288 eventos iCNA em gliomas pediátricos de alto grau, sendo que a presença destes breakpoints intragénicos é por si só um factor de prognóstico negativo. Quando comparadas com crianças mais jovens, os adolescentes possuem maior número de iCNA. Os tumores que têm a mutação H3F3A K27M também apresentam maior número de iCNA quando comparados com os tumores que tem a mutação H3F3A G34R/V ou com os casos normais. No nosso estudo também observamos inúmeras disrupções de genes por iCNA devido a deleções e amplificações. Os genes alvo destas disrupções estão associados a gliomas malignos, tais como RB1 e NF1, a supressores tumorais putativos, como por exemplo o FAF1 e o KIDINS220, e também a novos candidatos tumorais, como o PTPRE e KCND2. Identificamos também dois novos genes de fusão, CSGALNACT2:RET e DHX57:TMEM178:MAP4K3. De forma a explorar a ocorrência de alterações cromossómicas estruturais que dão origem a proteínas de fusão oncogénicas, utilizamos a sequenciação de ultima geração do ADN e ARN de linhas celulares de gliomas pediátricos de alto grau – KNS42, SF188 e UW479. O nosso estudo mostrou pela primeira vez que estas linhas celulares são caracterizadas por vários genes de fusão. Descobrimos ainda a presença de três estruturas extra-cromossomais presentes na linha celular SF188. Estas estruturas envolvem os cromossomas 4q12 (SCFD2, FIP1L1), 8q24 (MYC), 11p11, 11q13 (CCND1), 11p14, 11q23 (MLL) e o 12q14 (CDK4). Estas regiões estão amplificadas e estão presentes em três estruturas muito complexas sob a forma de double minutes (DM). Caracterizamos ainda um gene de fusão por cada linha celular: o GORASP2:CDADC1 na KNS42, o NUBPL:AKAP6 na UW479 e o RPTOR:TULP4 na SF188. O gene de fusão RPTOR:TULP4 descrito na SF188 envolve o gene RPTOR, um importante componente da via de sinalização do mTOR, e leva a expressão de uma forma truncada do RPTOR. Esta quimera foi encontrada em 2.2% dos gliomas pediátricos de alto grau . Concluindo, o trabalho resumido nesta tese amplia o nosso conhecimento sobre os eventos genómicos característicos destes tumores, e representa novos e potenciais targets para a terapia dirigida, numa doença que ainda não tem cura.
Paediatric brain tumours are the second most frequent malignancy in children, and the most common cause of cancer-related deaths in both the 0-14-year and the 15-24-year age group. In this thesis we focused our studies in paediatric malignant brain tumours of glial origin (grade III and IV): astrocytomas, oligodendrogliomas and glioblastomas. Although, paediatric high grade glioma (pHGG) is a histologically similar tumour to that arising in adults, these are distinct biological diseases, differing in copy number profiles and driver genetic alterations. Recent sequencing initiatives have conclusively shown the existence of subgroups of HGG marked by distinct driver mutations, which are significantly enriched in young children (H3F3A K27M), teenagers and young adults (H3F3A G34R/V), and middle-aged adults (IDH1/2). Structural rearrangements resulting in novel fusion genes are strongly associated with cancer, and numerous examples exist in both adult and childhood malignancies. Although, only few have been described in pHGG. Structural variants (SV) frequently result in chimeric proteins targetable by novel therapeutic approaches, an outcome desperately needed in pHGG. The work summarized in this thesis aims to explore novel structural rearrangements in childhood malignant gliomas, contributing to uncover the molecular mechanisms underpinning pHGG, to further contribute to distinguish the adult and childhood disease. PDGFRA is a receptor tyrosine kinase (RTK) that triggers essential cellular responses such as proliferation, migration, and survival. This gene is commonly amplified and overexpressed in paediatric malignant gliomas, playing an important role in this disease. To determine whether PDGFRA was also targeted by more subtle mutations missed by copy number analysis, we screened a large series of cases for single base changes and small indels, as well as the PDGFRAΔ8,9 deletion and KDR:PDGFRA (KP) fusion gene, previously reported in adult high grade glioma (aHGG). Somatic-activating mutations were identified in nonbrainstem pHGG and Diffuse Intrinsic Pontine Gliomas (DIPG), including missense mutations and in-frame deletions and insertions not previously described. In our studies PDGFRA alterations were more common in pHGG arising outside the brainstem (14% vs 6%), and 8/18 (44%) cases had concomitant amplification and mutation of PDGFRA. The adult rearrangements (KP and PDGFRAΔ8,9) were only found in adult cases and one case of pHGG. Thus, a distinct spectrum of PDGFRA alterations is present in pHGG. As functionally important intragenic copy number aberrations (iCNA) and fusion genes begin to be identified in aHGG, the same has not yet been done in the childhood setting. We applied an iCNA algorithm to our previously published dataset of DNA copy number profiling in pHGG with a view to identify novel intragenic breakpoints. We reported a series of 288 iCNA events in pHGG, with the presence of intragenic breakpoints itself a negative prognostic factor. We identified an increased number of iCNA in older children compared to infants, and increased iCNA in H3F3A K27M mutant tumours compared to G34R/V and wild-type. We observed numerous gene disruptions by iCNA due to both deletions and amplifications, targeting known HGG-associated genes such as RB1 and NF1, putative tumour suppressors such as FAF1 and KIDINS220, and novel candidates such as PTPRE and KCND2. We further identified two novel fusion genes in pHGG – CSGALNACT2:RET and the complex fusion DHX57:TMEM178: MAP4K3. To further examine whether SVs that lead to the production of oncogenic fusion proteins were present in pHGG, we used whole genome and transcriptome paired end sequencing to detect novel gene fusions in pHGG model cell lines – KNS42, SF188 and UW479. Our study showed for the first time that glioma cell lines are highly rearranged and that they are characterized by several fusion genes. We discovered three extra-chromosomal structural rearrangement structures in SF188, which involve chromosomes 4q12 (SCFD2, FIP1L1), 8q24 (MYC), 11p11, 11q13 (CCND1), 11p14, 11q23 (MLL) and 12q14 (CDK4). The amplified loci represented three very complex structures, which were present in the form of three double minutes (DM). We characterized a fusion gene in each cell line: GORASP2:CDADC1 in KNS42, NUBPL:AKAP6 in UW479 and RPTOR:TULP4 in SF188. The RPTOR:TULP4 fusion gene described in SF188 involved RPTOR, an important component of mTOR signalling. This fusion leads to the expression of a truncated form of RPTOR in SF188. We discovered that RPTOR was disrupted 2.2% of pHGG cases and that patient samples harboured similar truncations to the SF188 cell line. As the roles of these fusion genes are still unclear, further studies need to be performed in order to better understand their role in pHGG. In summary these data expand upon our understanding of the genomic events driving these tumours and represent novel targets for therapeutic intervention in these poor prognosis cancers of childhood.