Study of tumor heterogeneity in IDH1 mutated-diffuse low-grade gliomas in adults

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

NK Cells Acquire CCR5 and CXCR4 by Trogocytosis in People Living with HIV-1

International audienceNK cells play a major role in the antiviral immune response, including against HIV-1. HIV-1 patients have impaired NK cell activity with a decrease in CD56dim NK cells and an increase in the CD56−CD16+ subset, and recently it has been proposed that a population of CD56+NKG2C+KIR+CD57+ cells represents antiviral memory NK cells. Antiretroviral therapy (ART) partly restores the functional activity of this lymphocyte lineage. NK cells when interacting with their targets can gain antigens from them by the process of trogocytosis. Here we show that NK cells can obtain CCR5 and CXCR4, but barely CD4, from T cell lines by trogocytosis in vitro. By UMAP (Uniform Manifold Approximation and Projection), we show that aviremic HIV-1 patients have unique NK cell clusters that include cells expressing CCR5, NKG2C and KIRs, but lack CD57 expression. Viremic patients have a larger proportion of CXCR4+ and CCR5+ NK cells than healthy donors (HD) and this is largely increased in CD107+ cells, suggesting a link between degranulation and trogocytosis. In agreement, UMAP identified a specific NK cell cluster in viremic HIV-1 patients, which contains most of the CD107a+, CCR5+ and CXCR4+ cells. However, this cluster lacks NKG2C expression. Therefore, NK cells can gain CCR5 and CXCR4 by trogocytosis, which depends on degranulation

Leukemic non-nodal mantle cell lymphomas have a distinct phenotype and are associated with deletion of PARP1 and 13q14

International audienceLeukemic non-nodal mantle cell lymphoma (lMCL) is a particular subtype of mantle cell lymphoma (MCL), characterized by leukemic non-nodal disease and slow progression. Recognition of this entity is relevant to avoid overtreatment. Despite indolent clinical behaviour, lMCL might transform to a more aggressive disease. The purpose of this study was to compare lMCL with classical MCL (cMCL) and aggressive MCL (aMCL) using immunohistochemistry, interphase fluorescence in situ hybridization (FISH), and array-based comparative genomic hybridization, in order to identify biomarkers for lMCL diagnosis and prognosis. Seven lMCL patients were included. All had bone marrow involvement without lymphadenopathy. An lMCL phenotype was distinct from that of cMCL and aMCL: SOX11-, ATM+, PARP1+/-, and low KI67 (average 2 %). Beyond the t(11;14) translocation, fewer secondary cytogenetic alterations were found in lMCL compared to cMCL and aMCL, including deletion of PARP1 and 13q14. At last follow-up, one patient with lMCL had died of disease and another had progressive disease. These patients were respectively 13q14 deletion- and PARP1-positive. One other case of lMCL harbored a 13q14 deletion associated with PARP1 deletion. This patient had indolent disease. lMCL has a particular phenotype and fewer secondary cytogenetic alterations than cMCL and aMCL. PARP1 protein expression and 13q14 deletion are associated with a progressive clinical course of lMCL and should be included in initial diagnostic studies as predictors of unfavorable outcome. PARP1 deletion is involved in lMCL pathogenesis and might confer advantage

Urine-derived cells from the aged donor for the 2D/3D modeling of neural cells via iPSCs

Human neural cell models derived from induced pluripotent stem cells (iPSCs) have been widely accepted to model various neurodegenerative diseases such as Alzheimer’s disease (AD) in vitro. Although the most common sources of iPSCs are fibroblasts and peripheral blood mononuclear cells, the collection of these cells is invasive. To reduce the donor’s burden, we propose the use of urine-derived cells (UDCs), which can be obtained non-invasively from a urine sample. However, the collection of UDCs from elderly donors suffering from age-related diseases such as AD has not been reported, and it is unknown whether these UDCs from the donor aged over 80 years old can be converted into iPSCs and differentiated into neural cells. In this study, we reported a case of using the UDCs from the urine sample of an 89-year-old AD patient, and the UDCs were successfully reprogrammed into iPSCs and differentiated into neural cells in four different ways: (i) the dual SMAD inhibition with small-molecules via the neural progenitor precursor stage, (ii) the rapid induction method using transient expression of Ngn2 and microRNAs without going through the neural progenitor stage, (iii) the cortical brain organoids for 3D culture, and (iv) the human astrocytes. The accumulation of phosphorylated Tau proteins, which is a pathological hallmark of AD, was examined in the neuronal models generated from the UDCs of the aged donor. The application of this cell source will broaden the target population for disease modeling using iPS technology

PD1 and PDL1 expression in primary central nervous system diffuse large B-cell lymphoma are frequent and expression of PD1 predicts poor survival

International audiencePrimary central nervous system diffuse large B-cell lymphoma (PCNS-DLBCL) is a rare and aggressive type of diffuse large B-cell lymphoma (DLBCL) whit poorly understood pathogenesis. Finding biomarkers associated with patient survival may be important for understanding its physiopathology and to develop new therapeutic approaches. We investigated 32 PCNS-DLBCL from immunocompetent patients for BCL2, CMYC, LMO2, and P53 expression and for cytogenetic aberrations of BCL2, BCL6, and MYC genes, all known for their prognostic value in systemic DLBCL (s-DLBCL). We analyzed PD1 and PDL1 protein expression in both tumor infiltrating lymphocytes (TILs) and tumor cells. Finally, we searched for correlation between biological data and clinical course. The PCNS-DLBCL expressed BCL2, CMYC, LMO2, and P53 at similar frequency than s-DLBCL but without significant prognostic on survival. None cases harbored aberrations involving BCL2 and MYC gene whereas BCL6 abnormalities were present in 20.7% of cases but without value on survival. Expression of PD1 in TILs and PDL1 in tumor cells was observed at higher rates than in s-DLBCL (58% and 37%, respectively). The PD1 expression in TILs correlated with PDL1 expression in tumor cells (P = .001). Presence of PD1 positive TILs was associated with poorer overall survival (P = .011). Patients with PDL1 overexpression tended to better response to chemotherapy (P = .23). In conclusion PCNS-DLBCL pathogenesis differs from s-DLBCL without prognostic value of the phenotypic and cytogenetic parameters known for their pejorative impact in the latter. The PD1/PDL1 pathway plays a strong role in PCNS-DLBCL and represents an attractive target for this aggressive lymphoma

Cellular and molecular characterization of IDH1-mutated diffuse low grade gliomas reveals tumor heterogeneity and absence of EGFR/PDGFRα activation

Diffuse low grade gliomas (DLGG, grade II gliomas) are slowly-growing brain tumors that often progress into high grade gliomas. Most tumors have a missense mutation for IDH1 combined with 1p19q codeletion in oligodendrogliomas or ATRX/TP53 mutations in astrocytomas. The phenotype of tumoral cells, their environment and the pathways activated in these tumors are still ill-defined and are mainly based on genomics and transcriptomics analysis. Here we used freshly-resected tumors to accurately characterize the tumoral cell population and their environment. In oligodendrogliomas, cells express the transcription factors MYT1, Nkx2.2, Olig1, Olig2, Sox8, four receptors (EGFR, PDGFRα, LIFR, PTPRZ1) but not the co-receptor NG2 known to be expressed by oligodendrocyte progenitor cells. A variable fraction of cells also express the more mature oligodendrocytic markers NOGO-A and MAG. DLGG cells are also stained for the young-neuron marker doublecortin (Dcx) which is also observed in oligodendrocytic cells in nontumoral human brain. In astrocytomas, MYT1, PDGFRα, PTPRZ1 were less expressed whereas Sox9 was prominent over Sox8. The phenotype of DLGG cells is overall maintained in culture. Phospho-array screening showed the absence of EGFR and PDGFRα phosphorylation in DLGG but revealed the strong activation of p44/42 MAPK/ERK which was present in a fraction of tumoral cells but also in nontumoral cells. These results provide evidence for the existence of close relationships between the cellular phenotype and the mutations found in DLGG. The slow proliferation of these tumors may be associated with the absence of activation of PDGFRα/EGFR receptors

Transformation Foci in IDH1-mutated Gliomas Show STAT3 Phosphorylation and Downregulate the Metabolic Enzyme ETNPPL, a Negative Regulator of Glioma Growth

International audienceIDH1-mutated gliomas are slow-growing brain tumours which progress into high-grade gliomas. The early molecular events causing this progression are ill-defined. Previous studies revealed that 20% of these tumours already have transformation foci. These foci offer opportunities to better understand malignant progression. We used immunohistochemistry and high throughput RNA profiling to characterize foci cells. These have higher pSTAT3 staining revealing activation of JAK/STAT signaling. They downregulate RNAs involved in Wnt signaling (DAAM2, SFRP2), EGFR signaling (MLC1), cytoskeleton and cell-cell communication (EZR, GJA1). In addition, foci cells show reduced levels of RNA coding for Ethanolamine-Phosphate Phospho-Lyase (ETNPPL/AGXT2L1), a lipid metabolism enzyme. ETNPPL is involved in the catabolism of phosphoethanolamine implicated in membrane synthesis. We detected ETNPPL protein in glioma cells as well as in astrocytes in the human brain. Its nuclear localization suggests additional roles for this enzyme. ETNPPL expression is inversely correlated to glioma grade and we found no ETNPPL protein in glioblastomas. Overexpression of ETNPPL reduces the growth of glioma stem cells indicating that this enzyme opposes gliomagenesis. Collectively, these results suggest that a combined alteration in membrane lipid metabolism and STAT3 pathway promotes IDH1-mutated glioma malignant progression

RNA Profiling of the Human and Mouse Spinal Cord Stem Cell Niches Reveals an Embryonic-like Regionalization with MSX1+ Roof-Plate-Derived Cells

Summary: Anamniotes, rodents, and young humans maintain neural stem cells in the ependymal zone (EZ) around the central canal of the spinal cord, representing a possible endogenous source for repair in mammalian lesions. Cell diversity and genes specific for this region are ill defined. A cellular and molecular resource is provided here for the mouse and human EZ based on RNA profiling, immunostaining, and fluorescent transgenic mice. This uncovered the conserved expression of 1,200 genes including 120 transcription factors. Unexpectedly the EZ maintains an embryonic-like dorsal-ventral pattern of expression of spinal cord developmental transcription factors (ARX, FOXA2, MSX1, and PAX6). In mice, dorsal and ventral EZ cells express Vegfr3 and are derived from the embryonic roof and floor plates. The dorsal EZ expresses a high level of Bmp6 and Gdf10 genes and harbors a subpopulation of radial quiescent cells expressing MSX1 and ID4 transcription factors. : A niche of stem cells is present around the central canal of the adult spinal cord. A better description of cell diversity and genes expressed in this niche may help to use it to promote spinal cord regeneration after lesions. In this article, based on several techniques, Ghazale and colleagues provide a cellular and molecular resource for the adult human and mouse stem cell niches. Keywords: spinal cord, niche, neural stem cells, regionalization, ependyma, ependymal cells, radial glial cells, transcription factors, Msx1, roof plate, floor plat