Abstract Glioblastoma multiforme (GBM) is the most aggressive form of primary brain tumor, with no curative treatment options. Multiple studies have characterized at single cell resolution the GBM as being composed of transcriptional cell states interconnected with components in the tumor immune microenvironment (TME). Our group proposed and validated the first single cell guided functional classification of GBM in four tumor-intrinsic cell states which informed clinical outcome and delivered therapeutic options. However, single cell technologies are unable to unravel the spatial relationships among the cell states of GBM and between GBM cell states and TME. Spatially resolved transcriptomic technologies are emerging as powerful tools to reconstruct the spatial architecture of a tissue. We performed spatial transcriptomics of multicellular regions of interest (ROI) in 6 primary IDH wild-type GBM and 2 recurrent GBM with both CosMx Spatial Molecular Imager, which analyzes 1,000 RNA probes and 64 proteins at single cell resolution, and GeoMx Digital Spatial Profiler which profiles the whole transcriptome (~18,000 genes) at ROI resolution. The development of computational tools aimed to integrate spatial proximity and CosMx derived single-cell transcriptomics revealed spatial segregation of the tumor cell clones and cellular states and highlighted recurrent patterns of cell states, distinct TME cell types associated with coherent histopathological features across multiple samples. The development of a spatial informed intercellular communication algorithm and the reconstruction of ligand-receptor-target networks will allow the discovery of tumor cell states-TME cross-talks and the biological signaling regulated by these interactions that are driving the heterogeneity of GBM and therefore potentially therapeutically targetable. Analysis of matched regions of interest profiled by GeoMx and spatial proteomics with CosMx further cross-validated the spatial ecosystem of glioblastoma as reconstructed at single-cell resolution. Our studies established a scalable approach to resolve the transcriptional heterogeneity of GBM and reconstruct the architecture of GBM cell states and tumor microenvironment. Citation Format: Bruno Adabbo, Simona Migliozzi, Luciano Garofano, Fulvio D'Angelo, Pedro Davila, Sakir H. Gultekin, Daniel Bilbao Cortes, Benjamin Currall, Sion L. Williams, Marc Sanson, Franck Bielle, Anna Luisa Di Stefano, Michele Ceccarelli, Anna Lasorella, Antonio Iavarone. Reconstruction of the spatial ecosystem of glioblastoma reveals relationships between tumor cell states and microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1151.
Abstract Neurofibromatosis type 1 (NF-1) is the most common cancer predisposition syndrome in which 15-20% of affected individuals develop glioma. Large scale DNA and RNA bulk profiling showed the molecular complexity of NF-1 glioma with the tumor cellular ecosystem constituted by multiple malignant phenotypes and heterogenous immune microenvironment. However, the composition and function of infiltrating cells was hidden in the bulk tumor, and the extended granularity of NF-1 glioma tumor microenvironment (TME) remained still unexplored. Here, we collected glioma samples from 46 NF-1 patients including 22 high-grade (HGG) and 24 low-grade (LGG) tumors, and we analyzed their gene expression by single nuclei RNA sequencing. A total of 239,044 single cells were classified into tumor and non-tumor components by integrating multiple computational approaches (including genomic copy number inference, gene signature enrichment, and clustering). We defined the pattern of intra-tumor heterogeneity of NF-1 glioma cells using non-negative matrix factorization and derived 7 malignant meta-programs (MPs) that we respectively defined as Neuronal-like, EMT, Astrocyte-like, Dividing Radial Glia-like, Ependymal-like, Immune, and Glycolytic/Hypoxic-like. These MPs recapitulated normal brain cell subtypes, thereby reflecting broad cell plasticity. The non-tumor cell compartment (121,364 cells, 51%) was dissected for the characterization of the cell types that populate the TME of NF-1 glioma. We identified different subpopulations exhibiting specific immune functions within myeloid and lymphoid components. Different glioma ecomodules were highlighted by comparing the relative composition of the TME across the tumors. Recruitment and activation of cytotoxic CD8+ T cells and natural killers by an active crosstalk with dendritic and pro-inflammatory myeloid cells defined an immune-supportive phenotype that could mediate a potential anti-tumor response in low-grade NF-1 glioma (LGG immune high). Conversely, regulatory T cell infiltration and effector T cell exhaustion induced immune suppression in a low-grade glioma immune dysfunctional ecomodule. The absence of lymphocytes characterized a large set of cold tumors, mostly including high-grade glioma. Together, the complex interplay of tumor cell states with different TME compartments elucidated the existence of separate ecomodules in NF-1 glioma, with the LGG immune high TME associated with Neuronal-like and the LGG immune dysfunctional with Ependymal-like tumor cells. The Ependymal-like state also exhibits maximal association with brain-specific normal cells, including oligodendrocytes, neurons and astrocytes, whereas the HGG are enriched with Dividing Radial Glia- and Glycolytic/Hypoxic-like tumor cell states. The elucidation of different ecomodules provides novel insights for the application of targeted therapies in NF-1 glioma patients. Citation Format: Luciano Garofano, Fulvio D'Angelo, Michael Oh, Michele Ceccarelli, Franck Bielle, Marc Sanson, Anna Lasorella, Antonio Iavarone. Identification of distinct tumor-TME ecomodules in glioma from neurofibromatosis type 1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6938.
Mesial Temporal Lobe Epilepsy-associated Hippocampal Sclerosis (MTLE-HS) is a syndrome associated with various aetiologies. We previously identified CD34-positive extravascular stellate cells (CD34+ cells) possibly related to BRAFV600E oncogenic variant in a subset of MTLE-HS. We aimed to identify the BRAFV600E oncogenic variants and characterise the CD34+ cells.We analysed BRAFV600E oncogenic variant by digital droplet Polymerase Chain Reaction in 53 MTLE-HS samples (25 with CD34+ cells) and nine non-expansive neocortical lesions resected during epilepsy surgery (five with CD34+ cells). Ex vivo multi-electrode array recording, immunolabelling, methylation microarray and single nuclei RNAseq were performed on BRAFwildtype MTLE-HS and BRAFV600E mutant non-expansive lesion of hippocampus and/or neocortex.We identified a BRAFV600E oncogenic variant in five MTLE-HS samples with CD34+ cells (19%) and in five neocortical samples with CD34+ cells (100%). Single nuclei RNAseq of resected samples revealed two unique clusters of abnormal cells (including CD34+ cells) associated with senescence and oligodendrocyte development in both hippocampal and neocortical BRAFV600E mutant samples. The co-expression of the oncogene-induced senescence marker p16INK4A and the outer subventricular zone radial glia progenitor marker HOPX in CD34+ cells was confirmed by multiplex immunostaining. Pseudotime analysis showed that abnormal cells share a common lineage from progenitors to myelinating oligodendrocytes. Epilepsy surgery led to seizure freedom in eight of the 10 patients with BRAF mutant lesions.BRAFV600E underlies a subset of MTLE-HS and epileptogenic non-expansive neocortical focal lesions. Detection of the oncogenic variant may help diagnosis and open perspectives for targeted therapies.
Primary central nervous system lymphoma (PCNSL) represents a particular entity within non-Hodgkin lymphomas and is associated with poor outcome. The present study addresses the potential clinical relevance of chimeric transcripts in PCNSL discovered by using RNA sequencing (RNA-seq).Seventy-two immunocompetent and newly diagnosed PCNSL cases were included in the present study. Among them, 6 were analyzed by RNA-seq to detect new potential fusion transcripts. We confirmed the results in the remaining 66 PCNSL. The gene fusion was validated by fluorescence in situ hybridization (FISH) using formalin-fixed paraffin-embedded (FFPE) samples. We assessed the biological and clinical impact of one new gene fusion.We identified a novel recurrent gene fusion, E26 transformation-specific translocation variant 6-immunoglobulin heavy chain (ETV6-IgH). Overall, ETV6-IgH was found in 13 out of 72 PCNSL (18%). No fusion conserved an intact functional domain of ETV6, and ETV6 was significantly underexpressed at gene level, suggesting an ETV6 haploinsufficiency mechanism. The presence of the gene fusion was also validated by FISH in FFPE samples. Finally, PCNSL samples harboring ETV6-IgH showed a better prognosis in multivariate analysis, P = 0.03, hazard ratio = 0.33, 95% CI = 0.12-0.88. The overall survival at 5 years was 69% for PCNSL harboring ETV6-IgH versus 29% for samples without this gene fusion.ETV6-IgH is a new potential surrogate marker of PCNSL with favorable prognosis with ETV6 haploinsufficiency as a possible mechanism. The potential clinical impact of ETV6-IgH should be validated in larger prospective studies.
Abstract BACKGROUND Adult glioma patients rarely benefit from immunotherapy. This lack of efficacy has been related to gliomas immune “cold” microenvironment, which is commonly observed in these tumors and reduces lymphocyte-mediated antitumor responses. Here, we investigated the immune microenvironment of rare adult gliomas with high lymphocytic infiltration in order to evaluate the biology of this unusual immune response, its prognostic value and its potential role as predictive biomarker for immunotherapy strategies. MATERIAL AND METHODS Two retrospective cohorts of adult gliomas were analyzed: a supervised cohort of 206 samples enriched for tumors with high lymphocytic infiltration (including glioblastoma IDH wildtype [GBM], gemistocytic variant of astrocytoma IDH-mutant, ganglioglioma [GGG] and pleomorphic xanthoastrocytoma [PXA], and hypermutated mismatch repair deficient gliomas [MMRd]), and a consecutive cohort of 112 samples as control of the usual epidemiology. Immune microenvironment was characterized through (i) an analysis of spatial patterns and semi-automated quantification of T lymphocytes on CD3 immunolabeling and (ii) deconvolution of immune cells content from bulk RNA sequencing of FFPE tumor tissue. Cellular and spatial patterns of immune infiltration were correlated with demographic, histomolecular and outcome features. We investigated the underlying biology through multiplex immunolabeling and single-cell RNAseq of FACS-sorted intratumoral immune cells. Results: We identified three main spatial patterns (perivascular, peritumoral, diffuse intratumoral) of T cells infiltration in addition to the most usual aspect of scarce tumor-infiltrating lymphocytes. T cell proportion exhibited a wide range of distribution and was significantly associated with tumor type and IDH-status, with GBM, PXA and GGG exhibiting the highest proportions. Using transcriptomic signatures of immune cells and deconvolution of bulk RNAseq data, we observed recurrent patterns of associated immune cell types across diverse tumor types. We analyzed the correlations of these spatial and cellular immune patterns with tumor types and genetic alterations. Multiplex immunolabeling and single cell RNAseq revealed distinct T cells subpopulations, as well as potential functional states and cell-cell communication pathways driving these states. T cell infiltration had a significant prognostic impact among GBM patients. CONCLUSION Adult gliomas with high lymphocytic content stands for a population of interest to better understand immune responses in the brain parenchyma and develop novel immunotherapy clinical trials. Our results suggest that therapeutic evaluation requires an adjustment due to the prognostic impact of immune microenvironment.
