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Ependymoma represents a heterogeneous disease affecting the entire neuraxis. Extensive molecular profiling efforts have identified molecular ependymoma subgroups based on DNA methylation. However, the intratumoral heterogeneity and developmental origins of these groups are only partially understood, and effective treatments are still lacking for about 50% of patients with high-risk tumors. We interrogated the cellular architecture of ependymoma using single cell/single nucleus RNA-sequencing to analyze 24 tumor specimens across major molecular subgroups and anatomic locations. We additionally analyzed ten patient-derived ependymoma cell models and two patient-derived xenografts (PDXs). Interestingly, we identified an analogous cellular hierarchy across all ependymoma groups, originating from undifferentiated neural stem cell-like populations towards different degrees of impaired differentiation states comprising neuronal precursor-like, astro-glial-like, and ependymal-like tumor cells. While prognostically favorable ependymoma groups predominantly harbored differentiated cell populations, aggressive groups were enriched for undifferentiated subpopulations. Projection of transcriptomic signatures onto an independent bulk RNA-seq cohort stratified patient survival even within known molecular groups, thus refining the prognostic power of DNA methylation-based profiling. Furthermore, we identified novel potentially druggable targets such as IGF- and FGF-signaling within poorly prognostic transcriptional programs. Ependymoma-derived cell models/PDXs widely recapitulated the transcriptional programs identified within fresh tumors and are leveraged to validate identified target genes in functional follow-up analyses. Taken together, our analyses reveal a developmental hierarchy and transcriptomic context underlying the biologically and clinically distinct behavior of ependymoma groups. The newly characterized cellular states and underlying regulatory networks could serve as basis for future therapeutic target identification and reveal biomarkers for clinical trials.

Medulloblastoma is a malignant childhood cerebellar tumor comprised of distinct molecular subgroups. Whereas genomic characteristics of these subgroups are well defined, the extent to which cellular diversity underlies their divergent biology and clinical behaviour remains largely unexplored. We used single-cell transcriptomics to investigate intra- and inter-tumoral heterogeneity in twenty-five medulloblastomas spanning all molecular subgroups. WNT, SHH, and Group 3 tumors comprised subgroup-specific undifferentiated and differentiated neuronal-like malignant populations, whereas Group 4 tumors were exclusively comprised of differentiated neuronal-like neoplastic cells. SHH tumors closely resembled granule neurons of varying differentiation states that correlated with patient age. Group 3 and Group 4 tumors exhibited a developmental trajectory from primitive progenitor-like to more mature neuronal-like cells, whose relative proportions distinguished these subgroups. Cross-species transcriptomics defined distinct glutamatergic populations as putative cells-of-origin for SHH and Group 4 subtypes. Collectively, these data provide novel insights into the cellular and developmental states underlying subtype-specific medulloblastoma biology.

Pilocytic astrocytoma (PA) is the most common pediatric low-grade glioma. While PAs exhibit a favorable prognosis compared to higher-grade gliomas, treatment morbidity and tumor recurrence still represent major challenges for some PA patients. PAs frequently harbor rearrangements resulting in expression of oncogenic KIAA1549-BRAF fusion transcripts. We performed scRNAseq of both tumor and non-tumor cells in newly-diagnosed PAs that contained KIAA1549-BRAF rearrangements. To confidently distinguish tumor cells from nontumor cells, we sorted cells by glial progenitor marker A2B5 status and profiled KIAA1549-BRAF fusion status of cells using a quantitative PCR-based assay. Results were validated using RNA in situ hybridization and compared to bulk expression data from 151 pediatric low grade gliomas. When compared to higher-grade gliomas, a higher proportion of the PA tumor cells exhibited a differentiated, astrocyte-like phenotype. A smaller proportion of cells exhibited a progenitor-like phenotype with evidence of proliferation. These progenitor-like tumor cells expressed a mitogen-activated protein kinase (MAPK) gene program that was absent from higher-grade gliomas. Similar patterns of expression of genes associated with the astrocyte-like and MAPK gene programs were also seen in formalin fixed, paraffin embedded PA tissues using RNA in situ hybridization. Immune cells, especially microglia, comprised almost half of all cells in the PAs and accounted for many differences seen in bulk pediatric low grade glioma expression profiles. These single cell transcriptional data reveal a transcriptional developmental hierarchy in a pediatric low-grade glioma that is skewed towards more mature brain cells compared to higher-grade gliomas. The results indicate that future analyses of bulk PA tissues should attempt to account for considerable infiltration by nontumor cells. Finally, the finding that a MAPK gene program is not uniformly expressed in PA tumor cells has implications for ongoing clinical investigations of therapies directed at the MAPK pathway for the treatment of PA.

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) remains a devastating and incurable disease. The DIPG-BATs clinical trial incorporates diagnostic biopsy with molecularly determined treatment stratification. Here we present the initial genomic analysis of the DIPG-BATs cohort. METHODS: Children enrolled on the DIPG-BATs clinical trial underwent upfront diagnostic biopsies prior to commencement of therapy. RNA and DNA were extracted from single core biopsies and subjected to whole-genome sequencing (WGS) and RNA-sequencing. Tumor samples were also collected at autopsy if there was parental consent. RESULTS: Fifty-three patients were enrolled on study, of whom 50 underwent biopsy. There were no biopsy-related deaths. A mean of 5ug of RNA and 10ug of DNA were extracted from single frozen cores. WGS on 41 DIPG samples (including eight autopsy samples) revealed a mean mutation rate of 0.753 mutations per Mb. We confirmed TP53, PIK3CA, H3F3A, ACVR1, PPM1D, and HIST1H3B to be recurrently mutated in DIPG. Additional mutations were found in epigenetic modifiers including ASXL1. Copy-number analysis revealed PDGFRA to meet statistical significance as a recurrent amplification peak in DIPG (q

Human tumors are complex systems containing cells with diverse phenotypes, genotypes and epigenetic states; but current treatment strategies and models do not adequately reflect tumor composition in patients. High-grade gliomas are particularly heterogeneous tumors and currently represent the most common cause of cancer-related death in children. Increasing evidence indicates that treatment failure and resistance are at least in part due to this heterogeneity. We used single-cell RNA sequencing (scRNA-seq) to profile the transcriptome of thousands of single cells in pediatric high-grade glioma and reconstructed their cellular architecture. We found restricted expression of neural stem/progenitor expression programs in distinct subsets of cells, and observed that cellular programs evolve during genetic evolution. This suggests that both genetic and superimposed developmental programs contribute to tumor heterogeneity. These results provide unparalleled insight into the cellular composition of pediatric high-grade gliomas at the level of single-cell resolution, and open up new avenues for discovering novel therapeutic targets.