Molecular classification has transformed pediatric brain tumor diagnosis but provides only a static view of highly dynamic diseases, overlooking tumor evolution, intra-tumoral heterogeneity, and functional therapeutic vulnerabilities. We established a living pediatric brain tumor atlas by prospectively generating patient-derived cell lines (PDCLs) and xenografts (PDXs) from 118 pediatric brain tumors and integrating genomic, epigenomic, histopathological, spatial transcriptomic, and functional drug-response profiling. Patient-derived models faithfully preserved the molecular and epigenomic features of their parental tumors while capturing clonal evolution and the emergence of aggressive subpopulations. Spatial transcriptomics demonstrated conservation of malignant cellular programs despite remodeling of stromal and vascular compartments in PDXs. Functional drug screening revealed clinically relevant therapeutic heterogeneity, distinguished intrinsic from acquired resistance, and identified actionable signaling dependencies beyond molecular classification. This living atlas provides a clinically relevant framework for functional precision neuro-oncology by integrating molecular, spatial, and functional data to improve therapeutic stratification and accelerate personalized treatment strategies for pediatric brain tumors.

Pediatric diffuse midline gliomas (DMGs), defined by H3K27 alterations, remain devastating brain tumors with marked biological and clinical heterogeneity. Although hypoxia has been implicated in tumor adaptation, the role of the hypoxia-inducible factor HIF-2α in DMG biology remains poorly defined. We analyzed HIF-2α expression in a monocentric cohort of pediatric high-grade gliomas enriched for H3K27-altered DMGs, integrating clinical, imaging, and metabolomic data. Functional relevance was investigated using matched patient-derived DMG models combining chromatin profiling, transcriptomics, metabolomics, and pharmacological or genetic HIF-2α inhibition under hypoxia. HIF-2α overexpression defined a subgroup of tumors enriched for TP53-mutant DMGs, associated with reduced overall survival and impaired radiotherapy response. HIF-2α–positive tumors displayed reduced apparent diffusion coefficient values and increased glucose uptake, consistent with metabolic reprogramming toward glycolysis. At the molecular level, HIF-2α in DMG models drove context-dependent transcriptional programs, activating RAS/MAPK/mTOR and Wnt signaling, as well as neuronal and synaptic pathways in a brain location-specific manner. Metabolomic analyses confirmed enhanced glycolysis, glutaminolysis, and lipid remodeling in HIF-2α-positive tumors and models. Functionally, HIF-2α inhibition suppressed hypoxia-driven metabolic programs, reduced neuron-dependent tumor proliferation and invasion, and increased radiation-induced DNA damage in DMGs. Notably, HIF-2α activation was dynamic, emerging during tumor progression and passaging. Together, these findings identify HIF-2α as a central regulator of hypoxia-driven oncogenic plasticity in DMGs. Beyond a biomarker of aggressive disease, HIF-2α represents a context-dependent vulnerability linking metabolic, transcriptional, and microenvironmental adaptation. These results might support its integration as a therapeutic target in combination strategies for pediatric DMGs.

Detecting homozygous deletion (HD) of CDKN2A is critical in BRAF-altered gliomas, as this molecular alteration has both diagnostic and prognostic significance. It is predominantly associated with BRAF-altered high-grade gliomas and has been associated with poorer prognosis in certain BRAF-altered low-grade glioma tumor types. The 2021 WHO classification of central nervous system tumors therefore recommends screening for this alteration in most BRAF-altered gliomas, but it does not recommend one specific technique over another. Here, we compare the performance of several detection methods, including p16 immunohistochemistry, fluorescence in situ hybridization (FISH), droplet digital PCR, next-generation sequencing and DNA methylation profiling-derived copy-number variation (CNV) analysis, in a retrospective cohort of 25 BRAF-altered gliomas. Ten cases showed diffuse p16 immunohistochemical expression (10/25) with no associated CDKN2A HD, whereas 15 cases had complete absence of p16 expression (15/25). In the latter group, a high level of discrepancy in CDKN2A HD detection when considering FISH versus other techniques was observed, suggesting a high false-negative rate with FISH. Using an original bioinformatic pipeline leveraging genome alignment of routinely available CNV raw data, we identified among most false-negative cases (4/5) a large and undeleted region encompassing MTAP, which is targeted by most commercial CDKN2A FISH probes. This is likely due to non-specific probe hybridization. Our finding suggests that FISH probes targeting the entire 9p21 locus may have lower sensitivity than anticipated among BRAF-altered gliomas and emphasizes the critical need for appropriate probe selection.

Brain cancers are the most frequently diagnosed tumor types in pediatric and young adult populations. Unfortunately, they often carry a poor prognosis and face common challenges such as treatment resistances due to intra- and intertumoral heterogeneity. To fully understand the intrinsic and extrinsic mechanisms which might hinder proper care, there is an emerging need to develop precise models of brain tumors to understand resistances and to predict therapeutic responses. While tumor-derived primary cell lines are relatively easy to produce, fully replicate the complexity of tumors as they grow without interactions with and from the microenvironment. Therefore, the advent of organoid cultures represents a significant advancement in modelling development. Derived from this technology, tumoroids offer a promising solution for modeling 3D tumors and their complex microenvironment. We develop tumoroids from fresh biopsies and patient-derived tumor xenografts. These models were rigorously validated against to initial tumor using confocal microscopy, methylome analyses, metabolomics and single-cell RNAseq. To assess the intrinsic hypoxic environment and its spatial heterogeneity, characteristic of aggressive cancers, we employed fluorescent oxygen-sensitive dye loaded polymeric nanorods. We successfully generated tumoroids using PDX of ependymomas (BT39) and it paired relapse (BT42), two H3.3 mutated high-grade gliomas (BT35 and BT69), one ganglioglioma (BT63) and directly from fresh biopsies of DIPG sample (BT140). Several passages were obtained after primary derivation, and cryopreservation demonstrating the stability of the models over time. Tumoroids displayed similar protein and omics markers as their parental tumors and harbored specific stem cell and neural progenitor markers. The methylation patterns were entirely preserved comparing 3D models and their parental tumors, and other multiomics and metabolic approaches confirmed the accurate reconstitution of tumor complexity. The study of oxygen-sensitive nanorods highlighted the heterogeneous oxygen gradient within tumoroids. All these initial characterized tumoroids helped us to develop radioresistant paired models.

Faced to the growing development of collecting systematic molecular analyses in relapsed pediatric cancers to transform their targeted matched therapies, this study aimed to assess the clinical and therapeutic indications of systematic diagnostic genomic explorations performed in pediatric solid cancers to determine which type of screening and if it afford at relapse time an accurate targeted strategy. A total of 280 patients less than 22 years, referred at the University Hospitals of Strasbourg for a newly diagnosed solid tumor from January 2015 to December 2021, were prospectively genomically investigated since diagnosis. Using 7 different molecular tests going from single-gene methods (IHC, FISH, RT-PCR, Sanger sequencing, droplet digital PCR) to largescale analyses (Next-Generation sequencing, RNAsequencing and FoundationOne®CDx), we explored retrospectively the molecular findings in those pediatric solid tumors (except hematolymphoid cancers) to improve diagnosis, prognosis assessment and relapse therapeutics. One hundred and ninety-eight patients (71

PDF file - 131K, The effect of Alpha5 integrin on p53 activity and resistance to temozolomide is restricted to p53wt expressing cells

PDF file - 23K, Proportion of grade III and grade IV glioma in the subgroups of patients with high and low Alpha5 integrin mRNA expression in the 3 cohorts of patients included in this study

Abstract HIF-1a and HIF-2a are master regulators operating during tumor hypoxia. Few data are available on their role in pediatric high-grade gliomas (pHGGs), in which recent insights on metabolism and immunotolerance induction through hypoxia-driven mechanisms might opening the path to study further this process and their molecular actors. Our study investigated and confirmed the balanced dependency of both transcription factors in pHGGs at protein levels, but not in their transcriptional reprogramming. We also examined how HIF-1a and HIF-2a are operating during acute and chronic hypoxia in a spatiotemporal cell setting. HIF-1a was clearly correlated within pHGGs samples and models to acute and moderate hypoxia, whereas HIF-2a was associated to H3.3K27M mutant DIPG and chronic hypoxia. The impact of targeting both transcription factors in pHGG-derived models was on cell proliferative and invasive mechanisms, especially when studying this drug testing in engineered neural tissues. We might position those new targets as an innovative way to counterpart their hyperactivation in tumor core hypoxia and to propose them as an innovative approach interfering with cell-to-cell adhesion and synaptic interactions with normal brain cells.

Over the last few decades, deciphering the alteration of molecular pathways in brain tumors has led to impressive changes in diagnostic refinement. Among the molecular abnormalities triggering and/or driving gliomas, alterations in the MAPK pathway reign supreme in the pediatric population, as it is encountered in almost all low-grade pediatric gliomas. Activating abnormalities in the MAPK pathway are also present in both pediatric and adult high-grade gliomas. Across those alterations, BRAF p.V600E mutations seem to define homogeneous groups of tumors in terms of prognosis. The recent development of small molecules inhibiting this pathway retains the attention of neurooncologists on BRAF-altered tumors, as conventional therapies showed no significant effect, nor prolonged efficiency on the high-grade or low-grade unresectable forms. Nevertheless, tumoral heterogeneity and especially molecular alteration(s) associated with MAPK-pathway abnormalities are not fully understood with respect to how they might lead to the specific dismal prognosis of those gliomas and/or affect their response to targeted therapies. This review is an attempt to provide comprehensive information regarding molecular alterations related to the aggressiveness modulation in BRAF-mutated gliomas and the current knowledge on how to use those targeted therapies in such situations.

PDF file - 48K, Nutlin-3a decreases the total expression of Beta1 integrin subunit in U87MG cells but not its expression at the cell membrane

PDF file - 32K, Nutlin-3a does not modulate the Alpha5 integrin expression in U373 cells

PDF file - 25K, Mean fluorescence intensities of Alpha5 and Beta1 integrin subunits at the cell membrane

PDF file - 27K, Relationship between Alpha5 mRNA level and p53 status in human biopsies and in human xenografts in nude mice

CDKN2A is a tumor suppressor gene encoding the p16 protein, a key regulator of the cell cycle. CDKN2A homozygous deletion is a central prognostic factor for numerous tumors and can be detected by several techniques. This study aims to evaluate the extent to which immunohistochemical levels of p16 expression may provide information about CDKN2A deletion. A retrospective study was conducted in 173 gliomas of all types, using p16 IHC and CDKN2A fluorescent in situ hybridization. Survival analyses were performed to assess the prognostic impact of p16 expression and CDKN2A deletion on patient outcomes. Three patterns of p16 expression were observed: absence of expression, focal expression, and overexpression. Absence of p16 expression was correlated with worse outcomes. p16 overexpression was associated with better prognoses in MAPK-induced tumors, but with worse survival in IDH-wt glioblastomas. CDKN2A homozygous deletion predicted worse outcomes in the overall patient population, particularly in IDH-mutant 1p/19q oligodendrogliomas (grade 3). Finally, we observed a significant correlation between p16 immunohistochemical loss of expression and CDKN2A homozygosity. IHC has strong sensitivity and high negative predictive value, suggesting that p16 IHC might be a pertinent test to detect cases most likely harboring CDKN2A homozygous deletion.

Diffuse midline (DMG) and brainstem (DIPG) high-grade gliomas (HGG) bearing H3.3K27M driver mutation are aggressive and uncurable brain tumors. We evidenced by the past HIF-2a and its transcriptional gene, EPAS1, as a marker of resistance when targeting mTor/HIF-1a pathway. To understand HIF-2α potential role in chronically active hypoxic environment in DIPG/DMG, we investigate its status in a cohort of pediatric HGGs comprising notably H3.3K27M mutant tumors and in their paired in vitro derived models. Using concomitantly the low-input chromatin immunoprecipitation sequencing method CUT&RUN, RNAsequencing and metabolomics, we profiled the landscape of promoters and pathways regulated by HIF-2a in HGGs, where H3.3K27M and TP53 mutations are associated. Elucidation of HIF-2a downstream genes reveal statistically significant pathways linked to DIPGs (e.g., mTor and canonical Wnt pathways) and to DMGs (glutamatergic synapse and non-canoncial Wnt signaling). Finally, HIF-2a was evidenced as a new therapeutic target opening the path for its use concomitantly to irradiation.

Abstract CDKN2A is a tumor suppressor gene encoding for p16 protein. Its homozygous deletion is a central prognostic factor for numerous brain cancers. This deletion can be detected by several techniques. Up to now, immunohistochemistry (IHC) using p16 antibody is not recommended as a surrogate test. This study aimed to evaluate to what extent IHC levels of p16 expression may provide information about CDKN2A homozygous deletion. A retrospective study was conducted in 173 gliomas comprising 50 circumscribed low-grade MAPK-altered gliomas, 18 pediatric diffuse high-grade gliomas (HGG), and 105 adult-type diffuse gliomas of all grade, using p16 IHC and CDKN2A fluorescent in situ hybridization. Survival analyses were performed to assess their prognostic impact. Three patterns of p16 expression were observed: absence of expression, focal expression and overexpression. The prognostic impact of p16 overexpression was dependent on the tumor drivers. Indeed, it was associated to a better prognosis in MAPK-induced tumors, but to a worse survival in IDH-wild-type glioblastomas. CDKN2A homozygous deletion present in all type of high grade pediatric or adult gliomas was significantly linked to a worse outcome in the entire population. Regarding specifically to H3K27-altered gliomas, no significant prognostic impact was observed, probably related to the small size of our population. Nevertheless, this p16 lost expression was rarely reported in this pediatric population. The lost expression was observed in 7/9 H3K27M-mutants and in 6/8 wild-type cases. Its implication should be considered more largely and is probably opening the path for therapies targeting the p16 pathway in this population. Regarding the IHC technique itself, beyond the association between absence of p16 protein expression and the CDKN2A loss (p<0.001), this technique seems to have a strong sensitivity and a high negative predictive value. These data suggest that p16 IHC might be a pertinent test to detect cases harboring CDKN2A homozygous deletion.

Abstract Diffuse midline (DMG) and brainstem (DIPG) high-grade gliomas (HGG) bearing H3.3K27M driver mutation are aggressive and uncurable brain tumors. We evidenced by the past HIF-2a and its transcriptional gene, EPAS1, as a marker of resistance when targeting mTor/HIF-1a pathway. To understand HIF-2α potential role in chronically active hypoxic environment in DIPG/DMG, we investigate its status in a cohort of pediatric HGGs comprising notably H3.3K27M mutant tumors and in their paired in vitro derived models. Using concomitantly the low-input chromatin immunoprecipitation sequencing method CUT&RUN, RNAsequencing and metabolomics, we profiled the landscape of promoters and pathways regulated by HIF-2a in HGGs, where H3.3K27M and TP53 mutations are associated. Elucidation of HIF-2a downstream genes reveal statistically significant pathways linked to DIPGs (e.g., mTor and canonical Wnt pathways) and to DMGs (glutamatergic synapse and non-canoncial Wnt signaling). Finally, HIF-2a was evidenced as a new therapeutic target opening the path for its use concomitantly to irradiation.