The TeloDIAG: how telomeric parameters can help in glioma rapid diagnosis and liquid biopsy approaches.

BACKGROUND: In glioma, TERT promoter mutation and loss of ATRX (ATRX loss) are associated with reactivation of telomerase or alternative lengthening of telomeres (ALT), respectively, i.e. the two telomere maintenance mechanisms (TMM). Strangely, 25% of gliomas have been reported to display neither or both of these alterations. MATERIALS AND METHODS: The C-circle (CC) assay was adapted to tumor (formalin-fixed paraffin-embedded and frozen) and blood samples to investigate the TMM. RESULTS: We constructed a CC-based algorithm able to identify the TMM and reported a sensitivity of 100% and a specificity of 97.3% (n = 284 gliomas). By combining the TMM, the mutational status of the isocitrate dehydrogenase 1/2 (IDH) gene (IDHmt), and the histological grading, we propose a new classification tool: TeloDIAG. This classification defined five subtypes: tOD, tLGA, tGBM_IDHmt, tGBM, and tAIV, corresponding to oligodendroglioma, IDHmt low-grade astrocytoma, IDHmt glioblastoma, and IDHwt glioblastoma (GBM), respectively; the last class gathers ALT+ IDHwt gliomas that tend to be related to longer survival (21.2 months) than tGBM (16.5 months). The TeloDIAG was 99% concordant with the World Health Organization classification (n = 312), and further modified the classification of 55 of 144 (38%) gliomas with atypical molecular characteristics. As an example, 14 of 69 (20%) of TERTwt, ATRXwt, and IDHwt GBM were actually tAIV. Outstandingly, CC in blood sampled from IDHmt astrocytoma patients was detected with a sensitivity of 56% and a specificity of 97% (n = 206 gliomas and 30 healthy donors). CONCLUSION: The TeloDIAG is a new, simple, and effective tool helping in glioma diagnosis and a promising option for liquid biopsy.

[Elaboration of a national biobank for the study of gestational trophoblastic diseases]. / Création d'une biobanque nationale pour l'étude des maladies trophoblastiques gestationnelles.

AIM: To generate a national biobank made up of samples of the highest quality for the purpose of inciting basic research on gestational trophoblastic diseases (GTD). MATERIAL AND METHODS: Three priority axes of research were defined to optimize the nature, method of collection, and storage of the samples. These are: to enhance our understanding of GTD, develop new diagnostic tests, and identify new therapeutic targets. The protocol for patient inclusion and sample processing was determined after extensive literature review and collaboration with international experts in the field of GTD. RESULTS: For each patient with a GTD and for control patients (legally induced abortions), chorionic villi, decidua and tumor samples (fresh, immersed in RNA-protective solution and fixed in formaldehyde), blood (serum, plasma, RNA, and peripheral blood mononuclear cells), urine (supernatant), and cell cultures of villous cytotrophoblasts are prospectively collected. Associations are then made between the collected samples and numerous clinical and biological data, such as human chorionic gonadotropic plasma levels following curettage in the case of a hydatidiform mole. CONCLUSION: Such a collection of high quality samples and their associated data open up new perspectives for both national and international collaborative research projects.

Semaphorin 3A-vascular endothelial growth factor-165 balance mediates migration and apoptosis of neural progenitor cells by the recruitment of shared receptor.

The dynamic and coordinated interaction between cells and their microenvironment controls cell migration, proliferation, and apoptosis, mediated by different cell surface molecules. We have studied the response of a neuroectodermal progenitor cell line, Dev, to a guidance molecule, semaphorin 3A (Sema3A), described previously as a repellent-collapsing signal for axons, and we have shown that Sema3A acts as a repellent guidance cue for migrating progenitor cells and, on prolonged application, induces apoptosis. Both repulsion and induction of cell death are mediated by neuropilin-1, the ligand-binding component of the Sema3A receptor. The vascular endothelial growth factor, VEGF165, antagonizes Sema3A-induced apoptosis and promotes cell survival, migration, and proliferation. Surprisingly, repulsion by Sema3A also depends on expression of VEGFR1, a VEGF165 receptor, expressed in Dev cells. Moreover, we found that these repulsive effects of Sema3A require tyrosine kinase activity, which can be attributed to VEGFR1. These results indicate that the balance between guidance molecules and angiogenic factors can modulate the migration, apoptosis (or survival), and proliferation of neural progenitor cells through shared receptors.

Soluble factors, including TNF alpha, secreted by human T cells are both cytotoxic and cytostatic for medulloblastoma cells.

We studied the effect of the treatment of a medulloblastoma cell line by human T cells derived soluble factors. Medulloblastoma is one of the more common aggressive solid neoplasms in children for which there is no adequate therapy. Cell lines established from such tumours may be helpful to test the effect of various molecules on cell proliferation. Previous studies have suggested that T cell-derived factors may be toxic for the medulloblastoma cell line Dev. Cytokines were thought to mediate this effect. In this paper, we described changes in morphology, survival and cell cycle induced in Dev cells cocultured with human T cell lines chronically infected with a retrovirus (HTLV-I) and known to secrete high level of cytokines TNF alpha, IL1alpha and IL6. Such cocultures resulted in the death of a part of Dev cells and in decreased proliferation of surviving cells, associated with morphological changes and increase in vimentin expression. Treatment with conditioned medium from infected Dev cells, containing virus induced cytokines, triggered the same effect. Reduction of these effects by TNF alpha deprivation of conditioned medium suggested that this cytokine may be implicated. Direct treatment of Dev cells with recombinant cytokines indicated that TNF alpha, but not IL1 or IL6, is associated with Dev cell alterations. TNF alpha was shown to induce the death of Dev cells by an apoptotic pathway. Furthermore, TNF alpha had a bimodal effect on the cell cycle of surviving Dev cells. These differential effects of such cytokines on medulloblastoma cells could be therefore of interest for immunotherapy of these tumours.

Human primitive neuroectodermal tumour cells behave as multipotent neural precursors in response to FGF2.

Primitive neuroectodermal tumours (PNET) are thought to derive from the malignant transformation of pluripotent CNS precursors although this hypothesis has yet to be tested in vitro. Here we show that cells of a human PNET cell line 'Dev' express functional fibroblast growth factor (FGF) receptors (FGFR) and respond to FGF2 as multipotent CNS precursors. FGF2 induces tyrosine phosphorylation of FGFR-1 and FGFR-2 and many cellular substrates including MAP kinases and stimulates proliferation. Cells detach from plastic substrates and proliferate to form large clusters of undifferentiated cells. After adhesion to polylysine, cells migrate out from the clusters and differentiate. The majority of differentiated cells express neuronal phenotypes but distinct subpopulations express oligodendrocytic and astrocytic markers. Mature neural differentiation markers are not otherwise detected in Dev cells in defined medium. Identical results were obtained with 12 monoclonal subclones as well as the parent cell line, confirming that Dev cells are multipotent. This extends evidence that multipotent CNS precursors are the cellular substrate from which certain PNET develop and shows that FGF2 is a potent proliferation and differentiation inducer for PNET cells in vitro, suggesting that FGF2 may also modulate the evolution of PNET in vivo. Finally our results suggest that PNET cell lines may provide models to elucidate the biology of human multipotent CNS precursors.

Retroviral infection (HTLV-I) induces cytokine-regulated immunomodulation and cytotoxicity of medulloblastoma cells.

Primitive neuroectodermal tumors are thought to result from disturbed differentiation of neuroepithelial stem cells. These tumor cells retain the capacity to differentiate toward the neuron or glia phenotype under extrinsic stimuli. Previously, we have developed a model for the differentiation of a medulloblastoma cell line (Dev cells) induced by infection with the human retrovirus HTLV-I. This virus delivers signals which trigger the Dev cells to differentiate toward an astrocytic lineage. The aim of this study was to characterize the time course of viral infection, to identify the soluble factors released and to analyze their effects on Dev cells. The early phase of viral replication is followed by latent infection. Viral infection induces glial differentiation in a subpopulation of cells and results in the death of others. The inflammatory cytokines TNF alpha, IL1 alpha and IL6 were detected in medium conditioned by infected Dev cells. TNF alpha was cytotoxic and cytostatic for subpopulations of Dev cells. Furthermore, TNF alpha treatment reproduced the modulation of expression of the major histocompatibility complex antigens (MHC class I) observed in infected Dev cells. These observations support the view that HTLV-I infection, which triggers glial differentiation of medulloblastoma Dev cells, also causes the release of soluble factors capable of downregulating proliferation of dividing tumor cells and of modifying their recognition by cellular immune effectors.

Cholera toxin beta subunit induces the differentiation of human medulloblastoma cell line DEV in a neuronal pathway.

Medulloblastomas are primitive neuroectodermal tumours that are thought to arise from multipotent precursor cells in the cerebellum. Medulloblastoma cells may be undifferentiated or exhibit glial, neuronal or ependymal characteristics, suggesting that they may conserve their ability to differentiate in appropriate circumstances. Medulloblastoma cell lines may thus provide models to study the commitment and differentiation of multipotent CNS progenitor cells. A human medulloblastoma cell line, DEV, has previously been shown to differentiate in an astrocytic pathway after infection by the retrovirus HTLV-1. In this study immunofluorescence flow cytometry shows that cholera toxin beta subunit (CT beta), which binds to the ganglioside GM1, induces a twofold increase in the number of DEV cells differentiating towards a neuronal pathway, as shown by the increased proportion and labelling intensity of cells stained by an anti-neurofilament antibody. Immunocytochemistry shows that after 3 days in culture with CT beta, DEV cells develop processes which stain positive for neurofilaments and MAP-1. This suggests that CT beta induces DEV cells to express a more neuronal phenotype.

Differentiation of a medulloblastoma cell line towards an astrocytic lineage using the human T lymphotropic retrovirus-1.

Constituent cells of medulloblastoma, the most common brain tumor occurring in childhood, resemble the primitive neuroepithelial cells normally found in the developing nervous system. However, mutational events prevent their further differentiation. We used the human T cell lymphotrophic virus type 1 to activate these deregulated immature cells by means of its transactivating protein Tax. Concomitant with viral infection was a decrease in cell proliferation characterized by inhibition of [3H]thymidine incorporation and in the number of cells in the G2/M phase of the cell cycle. Morphological changes suggested that medulloblastoma cells differentiated along the astrocytic lineage. The glial phenotype was confirmed by the induction of the glial fibrillary acidic protein and the glial enzyme glutamine synthetase. A direct viral effect and/or secondary effects to viral infection via paracrine/autocrine pathways could counterbalance the maturational defect in these medulloblastoma cells.