Search

BACKGROUND & METHODS. The BIOlogical MEdicines for DIPG Eradication (BIOMEDE) trial is a randomized multi-arm multi-stage program (drop-the-loser adaptive design). The first trial was an open-label phase-II trial comparing three drugs (everolimus, dasatinib, erlotinib) combined with irradiation, allocated according to the presence of their specific targets. All patients underwent a biopsy at diagnosis to confirm the DIPG molecularly and establish the expression of pre-specified biomarkers. Patient samples were explored by WES with a sequencing depth average >100X for the tumor and >60X for the blood. RESULTS. Unsupervised clustering of copy-number-variations (CNV) identified 4 groups corresponding to a new stratification of DIPG: cluster 1 (n=42) with high CNV, cluster 2 (n=23) with chromosome 1q + chromosome 2 gains, cluster 3 (n=9) with low CNV and cluster 4 (n=21) with isolated 1q gain. Clusters 1 had higher hazard-ratio for death than the others (mean HR 1.959, range 1.054–3.643, p< 0.0001, adjusted Cox model). Extensive structural rearrangements/chromotrypsis were significantly more frequent in TP53-mutated samples. Indeed, when stratiftying patients in 4 groups based on type of histone H3 mutated at K27 and the presence of a TP53 pathway alteration the subgroup with TP53 altered pathway had a significantly higher hazard-ratio for death than the others (mean HR 3.450, p=0.0017, adjusted Cox model). Additional drug targets were identified, especially in the DNA repair machinery that could be exploited for new targeted therapies. CONCLUSION. WES at diagnosis was feasible in most patients and brings new prognostic and theranostic informations. This allows a better patients stratification and the development of personalized medicine in DIPG.

Diffuse intrinsic pontine gliomas (DIPG) are the most severe pediatric brain tumours. Though accepted as the main therapeutic, radiotherapy is only transiently efficient and not even in every patient. We previously identified an heterogeneous response to radiotherapy at diagnosis depending on the type of histone H3 mutated (Castel et al., 2015). We aimed at defining possible molecular determinants of this response to radiotherapy. We assessed in vitro response to ionizing radiations in a collection of DIPG cellular models derived from treatment-naïve biopsies reflecting the variability encountered in patients and correlated it to their principal molecular alterations. The in vitro 50% lethal dose of these DIPG cellular models ranged from 0.5 to 7 Gy and were correlated to time to progression after radiotherapy in the corresponding patients. We uncovered TP53 mutation as the main driver of increased radioresistance. We validated this finding in 4 isogenic pairs of DIPG cells with TP53(WT) and TP53 knock-down, confirming the pivotal role of TP53 inactivation in inducing DIPG radioresistance irrespective of the type of canonical or variant histone H3 mutated. Clinical and radiological response to radiotherapy and overall survival of an extended cohort of 73 DIPG was analysed according to their genotype and we demonstrated that mutated TP53 patients had a poor response to radiotherapy. Using a kinome-wide synthetic lethality RNAi screen, we further identified target genes that can sensitize TP53(MUT) DIPG to ionizing radiations. CHK1 inhibition increases response to radiation specifically in TP53(MUT) cells and could be considered as a new therapeutic approach in this setting. In all, TP53 alterations drive radioresistance in DIPG cells and TP53 mutational status is a biomarker to predict poor response to radiotherapy in patients. These findings will allow defining more tailored radiotherapy in DIPG and suggesting alternative treatment strategies to mitigate radioresistance with for example CHK1 inhibitors.

Diffuse Intrinsic Pontine Glioma (DIPG) and more largely Diffuse Midline Gliomas H3 K27M-mutant (DMG) harbor a unique property of infiltration. Our objective is to elucidate/describe the cellular and molecular determinants of micro-environmental modifications resulting from the tumour/stroma dialogue as it might provide pro-invasive conditions that favour the development of the disease. To this end, we performed RNA-seq analyses to characterize exhaustively the bidirectional molecular modifications of the stroma/tumour in DIPG xenograft models. Gene expression changes in murine microenvironment compartment were investigated as continuous or semi-continuous traits of tumor load by measuring transcriptome in zone with high vs. low infiltration. We observed substantial modulations in gene expression in the microenvironment associated with increasing tumor cell content, pointing to a modification of the macrophage/microglial infiltrate. The expression or overexpression of several modulated genes was validated by IHC in the stroma of DMG primary tumors. Among them, overexpression of the cytokine CCL3 was confirmed, reflecting the activation status of microglial cells. Moreover, we observed in patients that the density of IBA-1 positive microglial cells increases according to the extent of tumor infiltration and that a significant part of them harbor a mitotic status, supporting their interaction with DMG cells. The involvement of this interaction in DMG development needs further evaluation and might represent opportunity to slow down DIPG extension.

Glioma are frequently seen in the second decade of life in children with constitutional mismatch repair deficiency (CMMRD) syndrome. They are often associated with a high mutation load but their characteristics have not been extensively described. Your study describes 15 cases including 2 siblings from the radiological, histological et molecular (Whole Exome Sequencing) points of view. Tumors were frequently associated with lesions of different ages (multicentric) or vascular malformations. Different histologies were observed with a majority of glioblastomas (n=9) and giant tumor cells were almost always present. Immunohistochemistry was positive for p53 in 14/15 (concordant with mutations detected with sequencing) and was negative for ATRX in 11/15 (concordant with mutations detected with sequencing in all but one patient). PDL1 expression was only seen in 6/15. Mutation load was above 100/Mb in all but four; all hypermutated tumors had secondary mutations in POLE or POLD1. PMS2 and MSH2 were the two most frequently MMR genes mutated. The distribution of the variant allele frequency was most frequently bi- or tri-modal suggesting different waves of mutations gains. SomaticSignatures and DeconstructSig identified various combinations of signatures in the different exomes. Qualitatively mutations observed in the CMMRD samples were different from those observed in common pediatric high-grade gliomas (pHGG): absence of K27M and G34R/V mutations in H3F3A, absence of BRAF V600E mutation, absence of IDH1 R132H mutation, TP53 mutations were present in all but one. In conclusion, CMMRD-associated gliomas represent a distinct entity of pHGG with a specific oncogenesis.

BACKGROUND: The interval between progression and death in diffuse intrinsic pontine glioma (DIPG) is usually 6 months following so-called pseudoprogression, with a median of 8.9 months and a maximum of 35.6 months. All 43 patients exhibited increased blood volume and flow after radiotherapy, but the 90th percentile of those with signs of pseudoprogression had a greater increase of ASL-CBF (P < 0.001). Survival between the 2 groups did not differ significantly. During true progression, DSCrCBF and DSCrCBV values increased only in patients who had not experienced pseudoprogression. CONCLUSIONS: Pseudoprogression is a frequent phenomenon in DIPG patients. This condition needs to be recognized before considering treatment discontinuation. In this study, the larger increase of the ASL-CBF ratio after radiotherapy accurately distinguished pseudoprogression from true progression.

Grade I or III ganglioglioma (GG) is characterized by alterations in the MAPK pathway, including BRAF mutation. Diffuse midline glioma H3 K27M mutated was added to the WHO 2016 classification as a new grade IV entity. A co-occurrence of H3F3A/BRAF mutations has been reported in midline tumors. Firstly, we searched for BRAF V600E and H3 K27M mutations in a series of 54 pediatric midline grade I GG to determine the frequency of double mutations in grade I GG. We then studied the clinico-radiological and histo-molecular features of 12 grade I to III double-mutated tumors to explore the therapeutic and prognostic relevance. The frequency of the co-occurrence of H3F3A/BRAF mutations in grade I midline GG at diagnosis was 9.3%. Double-immunostaining revealed that BRAF V600E and H3 K27M mutant proteins colocalized in both the glial and neuronal components, The series of double-mutated GG included 8 grade I, and 4 grade III at diagnosis. The median survival was 8 years. Three grade I tumors showed a malignant transformation at 0.5, 3.5 and 7 years and two of them showed a loss of the BRAF mutation in recurrent tumor samples. Five patients with grade I were alive with stable disease without adjuvant therapy at the last follow-up. One grade III showed a paradoxical response under BRAF inhibitor. In this work we stress that H3F3A mutation can occur, in association with the BRAF mutation, in grade I GG, and that these have a better spontaneous outcome than classic diffuse midline H3 K27M-mutant gliomas. These data highlight the importance of an integrated histomolecular diagnosis especially in the context of targeted therapy.

As more genomes are sequenced, we are facing the challenge of rapidly unraveling the functions of genes. To that end, cell microarrays have recently been described that transfect thousands of nucleic acids in parallel and can be used to analyze the phenotypic consequences of such perturbations. As many parameters can influence the efficacy of transfection in such a format, we describe some important features in manufacturing cell microarrays that may improve reliability and efficiency of both plasmid DNA and siRNA transfection. We have also developed image analysis software that allows automatic detection of cell clusters, quantification of transfection efficiency and levels of expression/extinction of genes. Along with cell microarrays, this bioinformatic tool should expedite functional exploration of the human genome.