Molecular Classification of Ependymal Tumors across All CNS Compartments, Histopathological Grades, and Age Groups

Abstract

Ependymal tumors across age groups are currently classified and graded solely by histopathology. It is, however, commonly accepted that this classification scheme has limited clinical utility based on its lack of reproducibility in predicting patients' outcome. We aimed at establishing a uniform molecular classification using DNA methylation profiling. Nine molecular subgroups were identified in a large cohort of 500 tumors, 3 in each anatomical compartment of the CNS, spine, posterior fossa, supratentorial. Two supratentorial subgroups are characterized by prototypic fusion genes involving RELA and YAP1, respectively. Regarding clinical associations, the molecular classification proposed herein outperforms the current histopathological classification and thus might serve as a basis for the next World Health Organization classification of CNS tumors.

Figure 1. Methylation Profiling Identifies the Existence of Nine Distinct Epigenetic Subgroups of Ependymal Tumors

(A) Heat-map representation of an unsupervised clustering of DNA methylation profiles of 500 ependymal tumors. Each row represents a probe; each column represents a sample. The level of DNA methylation (beta value) is represented with a color scale as depicted. For each sample, subgroup association, anatomical location, histopathological diagnosis, and patient age are indicated. (B) Heat map of methylation levels in primary ependymal tumors and corresponding recurrent diseases. Molecular subgroup, anatomical location, histopathological diagnosis, and tumor type are indicated. Equal numbers at top of dendrogram and arrows at the bottom indicate that samples derive from the same individual. See also Figure S2 and Table S1.

Figure 2. Recurrent YAP1 Fusion Transcripts in a Distinct ST Subgroup

(A) Copy-number variation plots of four ST tumors classified as ST-EPN-YAP1 based on DNA methylation. Red rectangular marks chromosomal arm 11q highlighting existence of global and/or focal aberrations. Sample identifications are indicated. (B) Electropherograms of the fusion transcripts detected in the tumor samples depicted in (A). Numbers flanking the indicated breakpoint (dashed line) represent upstream and downstream fusion sites. (C) Predicted YAP1 fusion products at protein level. Red dashed lines indicate fusion sites. Proteins are drawn to scale, and protein domains are indicated at the bottom.

Figure 3. Molecular Subgroups of Ependymal Tumors Show Distinct Copy-Number Profiles

Overview of chromosomal aberrations in the nine molecular subgroups of ependymal tumors. DNA methylation array-based copy-number variation plots were scored for loss (red), gain (green), no change (= balanced; gray), or chromothripsis (purple) for all chromosomal arms. Additional focal aberrations were scored for chromosome 11q only (blue). Results were plotted as frequencies at which these aberrations occurred within each molecular subgroup; p values on the right indicate whether there was a significant difference in the distribution of these frequencies across the nine subgroups (chi-square test). See also Figure S3 and Table S2.

Figure 4. Transcription Profiles of Ependymal Tumors Reveal Subgroup-Specific Gene Signatures and Pathways

(A) Heat-map representation of signature genes across the molecular subgroups of ependymal tumors generated from supervised gene expression analyses. Subgroup affiliation of samples (column) and signatures (row) is indicated by color codes. SP-SE subgroup cases were not included due to unavailability of RNA. (B) Relative expression levels of two representative signature genes per subgroup as compared with the other subgroups are shown as box plots. Box plots represent the interquartile range (IQR), with the median represented by a solid line. (C) Pathway enrichment analysis comparing each of the molecular subgroups SP-MPE (dark green), SP-EPN (yellow), PF-SE (deep pink), PF-EPN-A (orange), PF-EPN-B (blue), ST-SE (dark violet), ST-EPN-YAP1 (cyan), and ST-EPN-RELA (red) against all other subgroups and a collection of normal brain control samples. Distinct pathways and biological processes between the molecular subgroups of ependymal tumors are illustrated (FDR corrected p < 0.01). Nodes represent enriched gene sets, which are grouped and annotated by their similarity according to related gene sets. Node size is proportional to the total number of genes within each gene set. The illustrated network map was simplified by manual curation to remove general and uninformative sub-networks. SP-SE samples were not included due to lack of RNA. See also Figure S4 and Table S3.

Figure 5. Molecular Subgroups of Ependymal Tumors Correlate with Distinct Clinical Outcome

(A–D) Kaplan-Meier curves for overall (A and C) and progression-free (B and D) survival for infratentorial (A and B) and ST (C and D) molecular ependymal tumor subgroups defined by methylation profiling. The p values were computed by log rank tests between subgroups. Numbers of patients at risk are indicated. See also Figure S5.

Figure 6. Graphical Summary of Key Molecular and Clinical Characteristics of Ependymal Tumor Subgroups

Schematic representation of key genetic and epigenetic findings in the nine molecular subgroups of ependymal tumors as identified by methylation profiling. CIN, Chromosomal instability