Publications by authors named "Marie-Anne Debily"

29 Publications

  • Page 1 of 1

Radiogenomics of diffuse intrinsic pontine gliomas (DIPGs): correlation of histological and biological characteristics with multimodal MRI features.

Eur Radiol 2021 May 18. Epub 2021 May 18.

Pediatric Radiology Department, AP-HP, Hôpital Universitaire Necker-Enfants Malades, 149 rue de Sèvres, F-75015, Paris, France.

Objectives: The diffuse intrinsic pontine gliomas (DIPGs) are now defined by the type of histone H3 mutated at lysine 27. We aimed to correlate the multimodal MRI features of DIPGs, H3K27M mutant, with their histological and molecular characteristics.

Methods: Twenty-seven treatment-naïve children with histopathologically confirmed DIPG H3K27M mutant were prospectively included. MRI performed prior to biopsy included multi-b-value diffusion-weighted imaging, ASL, and dynamic susceptibility contrast (DSC) perfusion imaging. The ADC and cerebral blood flow (CBF) and blood volume (CBV) were measured at the biopsy site. We assessed quantitative histological data, including microvascular density, nuclear density, and H3K27M-positive nuclear density. Gene expression profiling was also assessed in the samples. We compared imaging and histopathological data according to histone subgroup. We correlated MRI quantitative data with histological data and gene expression.

Results: H3.1K27M mutated tumors showed higher ADC values (median 3151 μm/s vs 1741 μm/s, p = 0.003), and lower perfusion values (DSC-rCBF median 0.71 vs 1.43, p = 0.002, and DSC-rCBV median 1.00 vs 1.71, p = 0.02) than H3.3K27M ones. They had similar microvascular and nuclear density, but lower H3K27M-positive nuclear density (p = 0.007). The DSC-rCBV was positively correlated to the H3K27M-positive nuclear density (rho = 0.74, p = 0.02). ADC values were not correlated with nuclear density nor perfusion values with microvascular density. The expression of gated channel activity-related genes tended to be inversely correlated with ADC values and positively correlated with DSC perfusion.

Conclusions: H3.1K27M mutated tumors have higher ADC and lower perfusion values than H3.3K27M ones, without direct correlation with microvascular or nuclear density. This may be due to tissular edema possibly related to gated channel activity-related gene expression.

Key Points: • H3.1K27M mutant DIPG had higher apparent diffusion coefficient (p = 0.003), lower α (p = 0.048), and lower relative cerebral blood volume (p = 0.02) than H3.3K27M mutant DIPG at their biopsy sites. • Biopsy samples obtained within the tumor's enhancing portion showed higher microvascular density (p = 0.03) than samples obtained outside the tumor's enhancing portion, but similar H3K27M-positive nuclear density (p = 0.84). • Relative cerebral blood volume measured at the biopsy site was significantly correlated with H3K27M-positive nuclear density (rho = 0.74, p = 0.02).
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http://dx.doi.org/10.1007/s00330-021-07991-xDOI Listing
May 2021

A subset of pediatric-type thalamic gliomas share a distinct DNA methylation profile, H3K27me3 loss and frequent alteration of EGFR.

Neuro Oncol 2021 01;23(1):34-43

Department of Neuropathology, Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany.

Background: Malignant astrocytic gliomas in children show a remarkable biological and clinical diversity. Small in-frame insertions or missense mutations in the epidermal growth factor receptor gene (EGFR) have recently been identified in a distinct subset of pediatric-type bithalamic gliomas with a unique DNA methylation pattern.

Methods: Here, we investigated an epigenetically homogeneous cohort of malignant gliomas (n = 58) distinct from other subtypes and enriched for pediatric cases and thalamic location, in comparison with this recently identified subtype of pediatric bithalamic gliomas.

Results: EGFR gene amplification was detected in 16/58 (27%) tumors, and missense mutations or small in-frame insertions in EGFR were found in 20/30 tumors with available sequencing data (67%; 5 of them co-occurring with EGFR amplification). Additionally, 8 of the 30 tumors (27%) harbored an H3.1 or H3.3 K27M mutation (6 of them with a concomitant EGFR alteration). All tumors tested showed loss of H3K27me3 staining, with evidence of overexpression of the EZH inhibitory protein (EZHIP) in the H3 wildtype cases. Although some tumors indeed showed a bithalamic growth pattern, a significant proportion of tumors occurred in the unilateral thalamus or in other (predominantly midline) locations.

Conclusions: Our findings present a distinct molecular class of pediatric-type malignant gliomas largely overlapping with the recently reported bithalamic gliomas characterized by EGFR alteration, but additionally showing a broader spectrum of EGFR alterations and tumor localization. Global H3K27me3 loss in this group appears to be mediated by either H3 K27 mutation or EZHIP overexpression. EGFR inhibition may represent a potential therapeutic strategy in these highly aggressive gliomas.
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http://dx.doi.org/10.1093/neuonc/noaa251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7850075PMC
January 2021

[Identifying essential non-oncogenic addictions to define new therapeutic targets in pediatric cancer].

Med Sci (Paris) 2020 Apr 1;36(4):326-329. Epub 2020 May 1.

Inserm U981, Molecular predictors and new targets in oncology, Gustave Roussy, Université Paris-Saclay, 94805 Villejuif, France - Univ. Évry, Université Paris-Saclay, 91000 Évry, France.

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http://dx.doi.org/10.1051/medsci/2020049DOI Listing
April 2020

High-grade gliomas in adolescents and young adults highlight histomolecular differences from their adult and pediatric counterparts.

Neuro Oncol 2020 08;22(8):1190-1202

Paris Descartes University, Sorbonne Paris Cité, Paris, France.

Background: Considering that pediatric high-grade gliomas (HGGs) are biologically distinct from their adult counterparts, the objective of this study was to define the landscape of HGGs in adolescents and young adults (AYAs).

Methods: We performed a multicentric retrospective study of 112 AYAs from adult and pediatric Ile-de-France neurosurgical units, treated between 1998 and 2013 to analyze their clinicoradiological and histomolecular profiles. The inclusion criteria were age between 15 and 25 years, histopathological HGG diagnosis, available clinical data, and preoperative and follow-up MRI. MRI and tumoral samples were centrally reviewed. Immunohistochemistry and complementary molecular techniques such as targeted/next-generation sequencing, whole exome sequencing, and DNA-methylation analyses were performed to achieve an integrated diagnosis according to the 2016 World Health Organization (WHO) classification.

Results: Based on 80 documented AYA patients, HGGs constitute heterogeneous clinicopathological and molecular groups, with a predominant representation of pediatric subtypes (histone H3-mutants, 40%) but also adult subtypes (isocitrate dehydrogenase [IDH] mutants, 28%) characterized by the rarity of oligodendrogliomas, IDH mutants, and 1p/19q codeletion and the relative high frequency of "rare adult IDH mutations" (20%). H3G34-mutants (14%) represent the most specific subgroup in AYAs. In the H3K27-mutant subgroup, non-brainstem diffuse midline gliomas are more frequent (66.7%) than diffuse intrinsic pontine gliomas (23.8%), contrary to what is observed in children. We found that WHO grade has no prognostic value, but molecular subgrouping has major prognostic importance.

Conclusions: HGGs in AYAs could benefit from a specific classification, driven by molecular subtyping rather than age group. Collaborative efforts are needed from pediatric and adult neuro-oncology teams to improve the management of HGGs in AYAs.
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http://dx.doi.org/10.1093/neuonc/noaa024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594566PMC
August 2020

TP53 Pathway Alterations Drive Radioresistance in Diffuse Intrinsic Pontine Gliomas (DIPG).

Clin Cancer Res 2019 11 3;25(22):6788-6800. Epub 2019 Sep 3.

UMR8203, "Vectorologie & Thérapeutiques Anticancéreuses," CNRS, Gustave Roussy, Université Paris-Sud, Université Paris-Saclay, Villejuif, France.

Purpose: Diffuse intrinsic pontine gliomas (DIPG) are the most severe pediatric brain tumors. Although accepted as the standard therapeutic, radiotherapy is only efficient transiently and not even in every patient. The goal of the study was to identify the underlying molecular determinants of response to radiotherapy in DIPG.

Experimental Design: We assessed response to ionizing radiations in 13 different DIPG cellular models derived from treatment-naïve stereotactic biopsies reflecting the genotype variability encountered in patients at diagnosis and correlated it to their principal molecular alterations. Clinical and radiologic response to radiotherapy of a large cohort of 73 DIPG was analyzed according to their genotype. Using a kinome-wide synthetic lethality RNAi screen, we further identified target genes that can sensitize DIPG cells to ionizing radiations.

Results: We uncover mutation as the main driver of increased radioresistance and validated this finding in four isogenic pairs of DIPG cells with or without knockdown. In an integrated clinical, radiological, and molecular study, we show that DIPG patients respond less to irradiation, relapse earlier after radiotherapy, and have a worse prognosis than their counterparts. Finally, a kinome-wide synthetic lethality RNAi screen identifies CHK1 as a potential target, whose inhibition increases response to radiation specifically in cells.

Conclusions: Here, we demonstrate that mutations are driving DIPG radioresistance both in patients and corresponding cellular models. We suggest alternative treatment strategies to mitigate radioresistance with CHK1 inhibitors. These findings will allow to consequently refine radiotherapy schedules in DIPG.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-0126DOI Listing
November 2019

A kinome-wide shRNA screen uncovers vaccinia-related kinase 3 (VRK3) as an essential gene for diffuse intrinsic pontine glioma survival.

Oncogene 2019 09 19;38(38):6479-6490. Epub 2019 Jul 19.

UMR8203, Vectorologie & Thérapeutiques Anticancéreuses, CNRS, Gustave Roussy, Univ. Paris-Sud, Université Paris-Saclay, Villejuif, France.

Diffuse intrinsic pontine glioma (or DIPG) are pediatric high-grade gliomas associated with a dismal prognosis. They harbor specific substitution in histone H3 at position K27 that induces major epigenetic dysregulations. Most clinical trials failed so far to increase survival, and radiotherapy remains the most efficient treatment, despite only transient tumor control. We conducted the first lentiviral shRNA dropout screen in newly diagnosed DIPG to generate a cancer-lethal signature as a basis for the development of specific treatments with increased efficacy and reduced side effects compared to existing anticancer therapies. The analysis uncovered 41 DIPG essential genes among the 672 genes of human kinases tested, for which several distinct interfering RNAs impaired cell expansion of three different DIPG stem-cell cultures without deleterious effect on two control neural stem cells. Among them, PLK1, AURKB, CHEK1, EGFR, and GSK3A were previously identified by similar approach in adult GBM indicating common dependencies of these cancer cells and pediatric gliomas. As expected, we observed an enrichment of genes involved in proliferation and cell death processes with a significant number of candidates belonging to PTEN/PI3K/AKT and EGFR pathways already under scrutiny in clinical trials in this disease. We highlighted VRK3, a gene involved especially in cell cycle regulation, DNA repair, and neuronal differentiation, as a non-oncogenic addiction in DIPG. Its repression totally blocked DIPG cell growth in the four cellular models evaluated, and induced cell death in H3.3-K27M cells specifically but not in H3.1-K27M cells, supporting VRK3 as an interesting and promising target in DIPG.
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http://dx.doi.org/10.1038/s41388-019-0884-5DOI Listing
September 2019

Transcriptomic and epigenetic profiling of 'diffuse midline gliomas, H3 K27M-mutant' discriminate two subgroups based on the type of histone H3 mutated and not supratentorial or infratentorial location.

Acta Neuropathol Commun 2018 11 5;6(1):117. Epub 2018 Nov 5.

UMR8203,Vectorologie et Nouvelles Thérapies Anticancéreuses, CNRS, Gustave Roussy, Univ. Paris-Sud, Université Paris-Saclay, 94805, Villejuif, France.

Diffuse midline glioma (DMG), H3 K27M-mutant, is a new entity in the updated WHO classification grouping together diffuse intrinsic pontine gliomas and infiltrating glial neoplasms of the midline harboring the same canonical mutation at the Lysine 27 of the histones H3 tail.Two hundred and fifteen patients younger than 18 years old with centrally-reviewed pediatric high-grade gliomas (pHGG) were included in this study. Comprehensive transcriptomic (n = 140) and methylation (n = 80) profiling was performed depending on the material available, in order to assess the biological uniqueness of this new entity compared to other midline and hemispheric pHGG.Tumor classification based on gene expression (GE) data highlighted the similarity of K27M DMG independently of their location along the midline. T-distributed Stochastic Neighbor Embedding (tSNE) analysis of methylation profiling confirms the discrimination of DMG from other well defined supratentorial tumor subgroups. Patients with diffuse intrinsic pontine gliomas (DIPG) and thalamic DMG exhibited a similarly poor prognosis (11.1 and 10.8 months median overall survival, respectively). Interestingly, H3.1-K27M and H3.3-K27M primary tumor samples could be distinguished based both on their GE and DNA methylation profiles, suggesting that they might arise from a different precursor or from a different epigenetic reorganization.These differences in DNA methylation profiles were conserved in glioma stem-like cell culture models of DIPG which mimicked their corresponding primary tumor. ChIP-seq profiling of H3K27me3 in these models indicate that H3.3-K27M mutated DIPG stem cells exhibit higher levels of H3K27 trimethylation which are correlated with fewer genes expressed by RNAseq. When considering the global distribution of the H3K27me3 mark, we observed that intergenic regions were more trimethylated in the H3.3-K27M mutated cells compared to the H3.1-K27M mutated ones.H3 K27M-mutant DMG represent a homogenous group of neoplasms compared to other pediatric gliomas that could be further separated based on the type of histone H3 variant mutated and their respective epigenetic landscapes. As these characteristics drive different phenotypes, these findings may have important implication for the design of future trials in these specific types of neoplasms.
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http://dx.doi.org/10.1186/s40478-018-0614-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6219253PMC
November 2018

Diagnostics of pediatric supratentorial RELA ependymomas: integration of information from histopathology, genetics, DNA methylation and imaging.

Brain Pathol 2019 05 28;29(3):325-335. Epub 2018 Nov 28.

Department of Neuropathology, Sainte-Anne Hospital, Paris, France.

Ependymoma with RELA fusion has been defined as a novel entity of the revised World Health Organization 2016 classification of tumors of the central nervous system (CNS), characterized by fusion transcripts of the RELA gene and consequent pathological activation of the NFkB pathway. These tumors represent the majority of supratentorial ependymomas in children. The validation of diagnostic tools to identify this clinically relevant ependymoma entity is essential. Here, we have used interphase fluorescent in situ hybridization (FISH) for C11orf95 and RELA, immunohistochemistry (IHC) for p65-RelA and the recently developed DNA methylation-based classification besides conventional histopathology, and compared the precision of the methods in 40 supratentorial pediatric brain tumors diagnosed as ependymomas in the past years. Reverse transcription PCR (RT-PCR) and RNA sequencing were performed to explore discordant cases. Furthermore, we integrated imaging and clinical features as additional layers of information. The concordance between nuclear RelA expression by IHC and RELA FISH was 100%. Concordance between IHC and DNA methylation profiling, and between FISH and DNA methylation profiling was also high (96.4% and 95.2%, respectively). Thirty-four out of 40 (85%) cases were confirmed by integrated diagnoses as ependymal tumors, including 22 RELA-fused ependymomas (71% of ependymal tumors), two YAP1-fused ependymomas (6%), six non-RELA/non-YAP1 ependymomas (18%) and four ependymal/subependymal mixed tumors (12%). Ependymal/subependymal mixed tumors had an excellent clinical outcome despite the presence of histopathological signs of malignancy, suggesting that these tumors should not be diagnosed as classic ependymomas. DNA methylation profiling helped in the differential diagnosis of RELA-fused ependymomas. IHC and FISH, which are available in the majority of pathology laboratories, are valuable tools to identify RELA-fused ependymomas.
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http://dx.doi.org/10.1111/bpa.12664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7379587PMC
May 2019

Cerebral blood flow changes after radiation therapy identifies pseudoprogression in diffuse intrinsic pontine gliomas.

Neuro Oncol 2018 06;20(7):994-1002

Hôpital Necker Enfants Malades, Pediatric Radiology Department, Paris, France.

Background: The interval between progression and death in diffuse intrinsic pontine glioma (DIPG) is usually <6 months. However, reports of longer patient survival following radiotherapy, in the presence of radiological signs of progression, suggest that these cases may be comparable to pseudoprogression observed in adult glioblastoma. Our aim was to identify such cases and compare their multimodal MRI features with those of patients who did not present the same evolution.

Methods: Multimodal MRIs of 43 children treated for DIPG were retrospectively selected at 4 timepoints: baseline, after radiotherapy, during true progression, and at the last visit. The patients were divided into 2 groups depending on whether they presented conventional MRI changes that mimicked progression. The apparent diffusion coefficient, arterial spin labeling cerebral blood flow (ASL-CBF), and dynamic susceptibility contrast perfusion relative cerebral blood volume (DSCrCBV) and flow (DSCrCBF) values were recorded for each tumor voxel, avoiding necrotic areas.

Results: After radiotherapy, 19 patients (44%) showed radiological signs that mimicked progression: 16 survived >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.
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http://dx.doi.org/10.1093/neuonc/nox227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6007230PMC
June 2018

Integrated Molecular Meta-Analysis of 1,000 Pediatric High-Grade and Diffuse Intrinsic Pontine Glioma.

Cancer Cell 2017 10 28;32(4):520-537.e5. Epub 2017 Sep 28.

Department of Cytogenetics and Reproductive Biology, Farhat Hached Hospital, Sousse, Tunisia.

We collated data from 157 unpublished cases of pediatric high-grade glioma and diffuse intrinsic pontine glioma and 20 publicly available datasets in an integrated analysis of >1,000 cases. We identified co-segregating mutations in histone-mutant subgroups including loss of FBXW7 in H3.3G34R/V, TOP3A rearrangements in H3.3K27M, and BCOR mutations in H3.1K27M. Histone wild-type subgroups are refined by the presence of key oncogenic events or methylation profiles more closely resembling lower-grade tumors. Genomic aberrations increase with age, highlighting the infant population as biologically and clinically distinct. Uncommon pathway dysregulation is seen in small subsets of tumors, further defining the molecular diversity of the disease, opening up avenues for biological study and providing a basis for functionally defined future treatment stratification.
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http://dx.doi.org/10.1016/j.ccell.2017.08.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5637314PMC
October 2017

New avatars of diffuse intrinsic pontine gliomas (DIPG) from stereotactic biopsies performed at diagnosis.

Oncotarget 2017 Aug 2;8(32):52543-52559. Epub 2017 Feb 2.

UMR8203 "Vectorologie & Thérapeutiques Anticancéreuses", CNRS, Gustave Roussy, Université Paris-Sud, Université Paris-Saclay, Villejuif, France.

Diffuse Instrinsic Pontine Glioma is the most aggressive form of High Grade Gliomas in children. The lack of biological material and the absence of relevant models have hampered the development of new therapeutics. Their extensive infiltration of the brainstem renders any surgical resection impossible and until recently biopsies were considered not informative enough and therefore not recommended. Thus, most models were derived from autopsy material. We aimed to develop relevant DIPG models that mimic this specific disease and its molecular diversity from tumor material obtained at diagnosis. Eight patient-derived orthotopic xenograft models were obtained after direct stereotactic injection of a mixed cell suspension containing tumor cells and stromal cells in the brainstem or thalamus of nude mice and serially passaged thereafter. In parallel, we developed 6 cell-derived xenograft models after orthotopic injection of tumor-initiating cells cultured from stereotactic biopsies. Cells were modified to express luciferase to enable longitudinal tumor growth monitoring, and fluorescent reporter proteins to trace the tumor cells in the brain. These models do not form a tumor mass, they are invasive, show the H3K27 trimethylation loss and the tumor type diversity observed in patients in terms of histone H3 mutations and lineage markers. Histological and MRI features at 11.7 Tesla show similarities with treatment naïve human DIPG, and in this respect, both direct and indirect orthotopic xenograft looked alike. These DIPG models will therefore constitute valuable tools for evaluating new therapeutic approaches in this devastating disease.
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http://dx.doi.org/10.18632/oncotarget.15002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5581049PMC
August 2017

Histone H3 genotyping refines clinico-radiological diagnostic and prognostic criteria in DIPG.

Acta Neuropathol 2016 May 1;131(5):795-6. Epub 2016 Apr 1.

UMR8203 "Vectorologie and Thérapeutiques Anticancéreuses", CNRS, Gustave Roussy, Univ. Paris-Sud, Université Paris-Saclay, 94805, Villejuif, France.

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http://dx.doi.org/10.1007/s00401-016-1568-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4835508PMC
May 2016

Histone H3F3A and HIST1H3B K27M mutations define two subgroups of diffuse intrinsic pontine gliomas with different prognosis and phenotypes.

Acta Neuropathol 2015 Dec 23;130(6):815-27. Epub 2015 Sep 23.

UMR8203 "Vectorologie et Thérapeutiques Anticancéreuses", CNRS, Gustave Roussy, Univ. Paris-Sud, Université Paris-Saclay, 94805, Villejuif, France.

Diffuse intrinsic pontine glioma (DIPG) is the most severe paediatric solid tumour, with no significant therapeutic progress made in the past 50 years. Recent studies suggest that diffuse midline glioma, H3-K27M mutant, may comprise more than one biological entity. The aim of the study was to determine the clinical and biological variables that most impact their prognosis. Ninety-one patients with classically defined DIPG underwent a systematic stereotactic biopsy and were included in this observational retrospective study. Histone H3 genes mutations were assessed by immunochemistry and direct sequencing, whilst global gene expression profiling and chromosomal imbalances were determined by microarrays. A full description of the MRI findings at diagnosis and at relapse was integrated with the molecular profiling data and clinical outcome. All DIPG but one were found to harbour either a somatic H3-K27M mutation and/or loss of H3K27 trimethylation. We also discovered a novel K27M mutation in HIST2H3C, and a lysine-to-isoleucine substitution (K27I) in H3F3A, also creating a loss of trimethylation. Patients with tumours harbouring a K27M mutation in H3.3 (H3F3A) did not respond clinically to radiotherapy as well, relapsed significantly earlier and exhibited more metastatic recurrences than those in H3.1 (HIST1H3B/C). H3.3-K27M-mutated DIPG have a proneural/oligodendroglial phenotype and a pro-metastatic gene expression signature with PDGFRA activation, while H3.1-K27M-mutated tumours exhibit a mesenchymal/astrocytic phenotype and a pro-angiogenic/hypoxic signature supported by expression profiling and radiological findings. H3K27 alterations appear as the founding event in DIPG and the mutations in the two main histone H3 variants drive two distinct oncogenic programmes with potential specific therapeutic targets.
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http://dx.doi.org/10.1007/s00401-015-1478-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4654747PMC
December 2015

Functionally defined therapeutic targets in diffuse intrinsic pontine glioma.

Nat Med 2015 Jun 4;21(6):555-9. Epub 2015 May 4.

1] Department of Pediatric Laboratory Medicine, University of Toronto, Toronto, Ontario, Canada. [2] Labatt Brain Tumor Research Centre, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada.

Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood cancer. We performed a chemical screen in patient-derived DIPG cultures along with RNA-seq analyses and integrated computational modeling to identify potentially effective therapeutic strategies. The multi-histone deacetylase inhibitor panobinostat demonstrated therapeutic efficacy both in vitro and in DIPG orthotopic xenograft models. Combination testing of panobinostat and the histone demethylase inhibitor GSK-J4 revealed that the two had synergistic effects. Together, these data suggest a promising therapeutic strategy for DIPG.
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http://dx.doi.org/10.1038/nm.3855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4862411PMC
June 2015

Learning a Markov Logic network for supervised gene regulatory network inference.

BMC Bioinformatics 2013 Sep 12;14:273. Epub 2013 Sep 12.

IBISC EA 4526, Université d'Évry-Val d'Essonne, 23 Boulevard de France, 91037, Évry, France.

Background: Gene regulatory network inference remains a challenging problem in systems biology despite the numerous approaches that have been proposed. When substantial knowledge on a gene regulatory network is already available, supervised network inference is appropriate. Such a method builds a binary classifier able to assign a class (Regulation/No regulation) to an ordered pair of genes. Once learnt, the pairwise classifier can be used to predict new regulations. In this work, we explore the framework of Markov Logic Networks (MLN) that combine features of probabilistic graphical models with the expressivity of first-order logic rules.

Results: We propose to learn a Markov Logic network, e.g. a set of weighted rules that conclude on the predicate "regulates", starting from a known gene regulatory network involved in the switch proliferation/differentiation of keratinocyte cells, a set of experimental transcriptomic data and various descriptions of genes all encoded into first-order logic. As training data are unbalanced, we use asymmetric bagging to learn a set of MLNs. The prediction of a new regulation can then be obtained by averaging predictions of individual MLNs. As a side contribution, we propose three in silico tests to assess the performance of any pairwise classifier in various network inference tasks on real datasets. A first test consists of measuring the average performance on balanced edge prediction problem; a second one deals with the ability of the classifier, once enhanced by asymmetric bagging, to update a given network. Finally our main result concerns a third test that measures the ability of the method to predict regulations with a new set of genes. As expected, MLN, when provided with only numerical discretized gene expression data, does not perform as well as a pairwise SVM in terms of AUPR. However, when a more complete description of gene properties is provided by heterogeneous sources, MLN achieves the same performance as a black-box model such as a pairwise SVM while providing relevant insights on the predictions.

Conclusions: The numerical studies show that MLN achieves very good predictive performance while opening the door to some interpretability of the decisions. Besides the ability to suggest new regulations, such an approach allows to cross-validate experimental data with existing knowledge.
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http://dx.doi.org/10.1186/1471-2105-14-273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3849013PMC
September 2013

A synthetic interaction screen identifies factors selectively required for proliferation and TERT transcription in p53-deficient human cancer cells.

PLoS Genet 2012 20;8(12):e1003151. Epub 2012 Dec 20.

Howard Hughes Medical Institute, Programs in Gene Function and Expression and Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

Numerous genetic and epigenetic alterations render cancer cells selectively dependent on specific genes and regulatory pathways, and represent potential vulnerabilities that can be therapeutically exploited. Here we describe an RNA interference (RNAi)-based synthetic interaction screen to identify genes preferentially required for proliferation of p53-deficient (p53-) human cancer cells. We find that compared to p53-competent (p53+) human cancer cell lines, diverse p53- human cancer cell lines are preferentially sensitive to loss of the transcription factor ETV1 and the DNA damage kinase ATR. In p53- cells, RNAi-mediated knockdown of ETV1 or ATR results in decreased expression of the telomerase catalytic subunit TERT leading to growth arrest, which can be reversed by ectopic TERT expression. Chromatin immunoprecipitation analysis reveals that ETV1 binds to a region downstream of the TERT transcriptional start-site in p53- but not p53+ cells. We find that the role of ATR is to phosphorylate and thereby stabilize ETV1. Our collective results identify a regulatory pathway involving ETV1, ATR, and TERT that is preferentially important for proliferation of diverse p53- cancer cells.
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http://dx.doi.org/10.1371/journal.pgen.1003151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3527276PMC
May 2013

Large scale RNAi screen reveals that the inhibitor of DNA binding 2 (ID2) protein is repressed by p53 family member p63 and functions in human keratinocyte differentiation.

J Biol Chem 2011 Jun 10;286(23):20870-9. Epub 2011 Apr 10.

CEA, IRTSV, Laboratoire Biopuces, 17 rue des Martyrs, 38054 Grenoble cedex 9, France.

The inhibitor of DNA binding 2, dominant negative helix-loop-helix protein, ID2, acts as an oncogene and elevated levels of ID2 have been reported in several malignancies. Whereas some inducers of the ID2 gene have been characterized, little is known regarding the proteins capable to repress its expression. We developed siRNA microarrays to perform a large scale loss-of-function screen in human adult keratinocytes engineered to express GFP under the control of the upstream region of ID2 gene. We screened the effect of siRNA-dependent inhibition of 220 cancer-associated genes on the expression of the ID2::GFP reporter construct. Three genes NBN, RAD21, and p63 lead to a repression of ID2 promoter activity. Strikingly NBN and RAD21 are playing on major role in cell cycle progression and mitosis arrest. These results underline the pregnant need to silence ID2 expression at transcript level to promote cell cycle exit. Central to this inhibitory mechanism we find p63, a key transcription factor in epithelial development and differentiation, which binds specific cis-acting sequence within the ID2 gene promoter both in vitro and in vivo. P63 would not suppress ID2 expression, but would rather prevent excessive expression of that protein to enable the onset of keratinocyte differentiation.
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http://dx.doi.org/10.1074/jbc.M110.169433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3121489PMC
June 2011

A functional and regulatory network associated with PIP expression in human breast cancer.

PLoS One 2009 5;4(3):e4696. Epub 2009 Mar 5.

Array s/IMAGE, Genexpress, Functional Genomics and Systems Biology for Health, LGN-UMR 7091-CNRS and Pierre & Marie Curie University, Paris VI, Villejuif, France.

Background: The PIP (prolactin-inducible protein) gene has been shown to be expressed in breast cancers, with contradictory results concerning its implication. As both the physiological role and the molecular pathways in which PIP is involved are poorly understood, we conducted combined gene expression profiling and network analysis studies on selected breast cancer cell lines presenting distinct PIP expression levels and hormonal receptor status, to explore the functional and regulatory network of PIP co-modulated genes.

Principal Findings: Microarray analysis allowed identification of genes co-modulated with PIP independently of modulations resulting from hormonal treatment or cell line heterogeneity. Relevant clusters of genes that can discriminate between [PIP+] and [PIP-] cells were identified. Functional and regulatory network analyses based on a knowledge database revealed a master network of PIP co-modulated genes, including many interconnecting oncogenes and tumor suppressor genes, half of which were detected as differentially expressed through high-precision measurements. The network identified appears associated with an inhibition of proliferation coupled with an increase of apoptosis and an enhancement of cell adhesion in breast cancer cell lines, and contains many genes with a STAT5 regulatory motif in their promoters.

Conclusions: Our global exploratory approach identified biological pathways modulated along with PIP expression, providing further support for its good prognostic value of disease-free survival in breast cancer. Moreover, our data pointed to the importance of a regulatory subnetwork associated with PIP expression in which STAT5 appears as a potential transcriptional regulator.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0004696PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2650800PMC
June 2009

[Inference of transcriptional regulatory networks].

Med Sci (Paris) 2008 Jun-Jul;24(6-7):629-34

CEA, DSV, IRCM, Laboratoire d'Exploration Fonctionnelle des Génomes, Evry, France.

The cell is a very complex system and although molecular biology allowed spectacular progresses, biologists are currently trying to tackle complexity at the system level, to unravel cell mysteries. The inference of transcriptional regulatory networks, the characterization of their topology, their dynamic, their role in major cell functions is at the heart of this strategy. We are proposing a systemic approach, based on integration of large scale data such as expression profiling, ChIP on chip analysis and high throughput RNA interference using siRNA microarrays to infer these networks and characterize associated phenotypes. Ultimately, the characterization of the properties of these networks should impact our understanding of biology and offer potential applications in medicine and pharmacology.
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http://dx.doi.org/10.1051/medsci/20082467629DOI Listing
September 2008

The H-Invitational Database (H-InvDB), a comprehensive annotation resource for human genes and transcripts.

Nucleic Acids Res 2008 Jan 18;36(Database issue):D793-9. Epub 2007 Dec 18.

Japan Biological Information Research Center, Japan Biological Informatics Consortium, Japan.

Here we report the new features and improvements in our latest release of the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/), a comprehensive annotation resource for human genes and transcripts. H-InvDB, originally developed as an integrated database of the human transcriptome based on extensive annotation of large sets of full-length cDNA (FLcDNA) clones, now provides annotation for 120 558 human mRNAs extracted from the International Nucleotide Sequence Databases (INSD), in addition to 54 978 human FLcDNAs, in the latest release H-InvDB_4.6. We mapped those human transcripts onto the human genome sequences (NCBI build 36.1) and determined 34 699 human gene clusters, which could define 34 057 (98.1%) protein-coding and 642 (1.9%) non-protein-coding loci; 858 (2.5%) transcribed loci overlapped with predicted pseudogenes. For all these transcripts and genes, we provide comprehensive annotation including gene structures, gene functions, alternative splicing variants, functional non-protein-coding RNAs, functional domains, predicted sub cellular localizations, metabolic pathways, predictions of protein 3D structure, mapping of SNPs and microsatellite repeat motifs, co-localization with orphan diseases, gene expression profiles, orthologous genes, protein-protein interactions (PPI) and annotation for gene families. The current H-InvDB annotation resources consist of two main views: Transcript view and Locus view and eight sub-databases: the DiseaseInfo Viewer, H-ANGEL, the Clustering Viewer, G-integra, the TOPO Viewer, Evola, the PPI view and the Gene family/group.
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http://dx.doi.org/10.1093/nar/gkm999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2238988PMC
January 2008

Cell microarray for functional exploration of genomes.

Methods Mol Biol 2007 ;381:375-84

CEA, DSV, DRR, Functional Genomics Department, Evry, France.

As more genomes are sequenced, challenge of rapidly unraveling the functions of genes was faced. To that end, cell microarrays have been recently described that permit transfection of thousands of nucleic acids in parallel and enable the analysis of phenotypic consequences of such perturbations. As many parameters can influence the efficiency of transfection and consequently protein expression or extinction, some important features in manufacturing cell microarrays for functional exploration of genomes were described.
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http://dx.doi.org/10.1007/978-1-59745-303-5_19DOI Listing
December 2007

Cell microarrays in drug discovery.

Drug Discov Today 2006 Jul;11(13-14):616-22

CEA, DSV, DRR, Service de Génomique Fonctionnelle, 2 rue Gaston Crémieux-CP 22, 91057 Evry Cedex, France.

There is an increasing need for systematic cell-based assays in a high-throughput screening (HTS) format to analyze the phenotypic consequences of perturbing mammalian cells with drugs, genes, interfering RNA. Taking advantage of the recent progress in microtechnology, new cell microarrays are being developed and applied to a large range of issues in metazoan cells. This article compares different approaches and evaluates their potential use in the drug discovery process. Although still an emerging technology, cell microarrays hold great promise to optimize the efficiency:cost ratio in cell-based HTS.
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http://dx.doi.org/10.1016/j.drudis.2006.05.015DOI Listing
July 2006

The Human Anatomic Gene Expression Library (H-ANGEL), the H-Inv integrative display of human gene expression across disparate technologies and platforms.

Nucleic Acids Res 2005 Jan;33(Database issue):D567-72

Integrated Database Group, Japan Biological Information Research Center, Japan Biological Informatics Consortium, Time24 Building 10F, 2-45 Aomi, Koto-ku, Tokyo 135-0064, Japan.

The Human Anatomic Gene Expression Library (H-ANGEL) is a resource for information concerning the anatomical distribution and expression of human gene transcripts. The tool contains protein expression data from multiple platforms that has been associated with both manually annotated full-length cDNAs from H-InvDB and RefSeq sequences. Of the H-Inv predicted genes, 18 897 have associated expression data generated by at least one platform. H-ANGEL utilizes categorized mRNA expression data from both publicly available and proprietary sources. It incorporates data generated by three types of methods from seven different platforms. The data are provided to the user in the form of a web-based viewer with numerous query options. H-ANGEL is updated with each new release of cDNA and genome sequence build. In future editions, we will incorporate the capability for expression data updates from existing and new platforms. H-ANGEL is accessible at http://www.jbirc.aist.go.jp/hinv/h-angel/.
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http://dx.doi.org/10.1093/nar/gki104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC540058PMC
January 2005

Integrative annotation of 21,037 human genes validated by full-length cDNA clones.

PLoS Biol 2004 Jun 20;2(6):e162. Epub 2004 Apr 20.

Integrated Database Group, Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan.

The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology.
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http://dx.doi.org/10.1371/journal.pbio.0020162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC393292PMC
June 2004

Expression and molecular characterization of alternative transcripts of the ARHGEF5/TIM oncogene specific for human breast cancer.

Hum Mol Genet 2004 Feb 8;13(3):323-34. Epub 2003 Dec 8.

Génomique Fonctionnelle et Biologie Systémique en Santé, FRE 2571, Centre National de la Recherche Scientifique, 7 rue Guy Moquet, BP 8, 94801 Villejuif Cedex, France.

The ARHGEF5/TIM oncogene belongs to the Dbl family of guanine nucleotide exchange factors (GEFs) for Rho GTPases. It is well established that Rho-GEFs play an important role in tumorigenesis and metastasis through the activation of their substrates, the Rho GTPases. Little is known about ARHGEF5/TIM oncogene expression and cellular functions. Because of its localization close to the common fragile site FRA7I, which has been shown to be responsible for an inverted duplication of the 7q34-q35 region in breast carcinoma cells, we examined the expression of the ARHGEF5/TIM oncogene in normal and tumoral breast tissue. We report here the identification of five novel ARHGEF5/TIM alternative transcripts specifically expressed in breast tumors. These variant transcripts were characterized by the absence of one or several exons, all coding for the catalytic Dbl-homology domain and generating modified or truncated predicted variant proteins. The variant transcripts were predominantly expressed in breast carcinoma cell lines and in the most aggressive primary breast carcinomas, suggesting they may play a role in breast tumor progression. Moreover, we demonstrate that the expression of recombinant ARHGEF5/TIM protein in transfected COS-7 and NIH-3T3 cells generated a loss of actin stress fibers and the formation of membrane ruffles and filopodia. This pattern suggests that ARHGEF5/TIM activates Rac1, Cdc42 or RhoG rather than RhoA, as previously demonstrated in in vitro guanine nucleotide exchange assays. We anticipate that the activation of the ARHGEF5/TIM oncogene, possibly by the variant isoforms detected here, may play an important role in proliferative breast disease.
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http://dx.doi.org/10.1093/hmg/ddh024DOI Listing
February 2004

Initiation of the breakage-fusion-bridge mechanism through common fragile site activation in human breast cancer cells: the model of PIP gene duplication from a break at FRA7I.

Hum Mol Genet 2002 Nov;11(23):2887-94

Génomique Fonctionnelle et Biologie Systémique en Santé, FRE 2571, Centre National de la Recherche Scientifique, 19 rue Guy Moquet, 94801 Villejuif, France.

Gene amplification plays a critical role in tumor progression. Hence, understanding the factors triggering this process in human cancers is an important concern. Unfortunately, the structures formed at early stages are usually unavailable for study, hampering the identification of the initiating events in tumors. Here, we show that the region containing the PIP gene, which is overexpressed in 80% of primary and metastatic breast cancers, is duplicated in the breast carcinoma cell line T47D. The two copies are organized as a large palindrome, lying 'in loco' on one chromosome 7. Such features constitute the landmark of the breakage-fusion-bridge (BFB) cycle mechanism. In hamster cells selected in vitro to resist cytotoxic drugs, common fragile site (CFS) activation has been shown to trigger this mechanism. Here, we characterize FRA7I at the molecular level and demonstrate that it lies 2 Mb telomeric to the PIP gene and sets the distal end of the repeated sequence. Moreover, our results suggest that the BFB process was frozen within the first cycle by healing of the broken chromosome. T47D cells thus offer a unique opportunity to observe the earliest products of the BFB cycle mechanism. Our findings constitute the first evidence that this amplification mechanism can be initiated in vivo by fragile site activation.
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http://dx.doi.org/10.1093/hmg/11.23.2887DOI Listing
November 2002

Intragenic amplification and formation of extrachromosomal small circular DNA molecules from the PIP gene on chromosome 7 in primary breast carcinomas.

Int J Cancer 2002 May;99(3):370-7

Génétique Moléculaire et Biologie du Développement, Centre National de la Recherche Scientifique, Villejuif, France.

The PIP gene is expressed in exocrine glands and, in pathologic conditions, in breast cysts and breast cancers exhibiting apocrine features. It is localized on the long arm of chromosome 7, a region frequently alterated in mammary tumors. We previously described an abnormal restriction pattern of the PIP gene in 33% of prostate carcinomas analyzed. Here, we analyze the structure of the PIP gene in primary breast carcinomas. We report that part of the 3' end, including exon 3, intron C, two-thirds of exon 4 and a small portion of intron B, is amplified and involved in the formation of extrachromosomal spcDNA molecules in 3/14 (21.4%) breast cancers analyzed. The involvement of a well-defined intragenic region of a gene in the formation of spcDNA appears to be unprecedented. Since spcDNA has been suggested to serve as an enhancer of genetic instability, the PIP gene may be the target of genomic variability processes in breast cancer.
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http://dx.doi.org/10.1002/ijc.10368DOI Listing
May 2002
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