Heterogeneity within the PF-EPN-B ependymoma subgroup

Abstract

Posterior fossa ependymoma comprise three distinct molecular variants, termed PF-EPN-A (PFA), PF-EPN-B (PFB), and PF-EPN-SE (subependymoma). Clinically, they are very disparate and PFB tumors are currently being considered for a trial of radiation avoidance. However, to move forward, unraveling the heterogeneity within PFB would be highly desirable. To discern the molecular heterogeneity within PFB, we performed an integrated analysis consisting of DNA methylation profiling, copy-number profiling, gene expression profiling, and clinical correlation across a cohort of 212 primary posterior fossa PFB tumors. Unsupervised spectral clustering and t-SNE analysis of genome-wide methylation data revealed five distinct subtypes of PFB tumors, termed PFB1-5, with distinct demographics, copy-number alterations, and gene expression profiles. All PFB subtypes were distinct from PFA and posterior fossa subependymomas. Of the five subtypes, PFB4 and PFB5 are more discrete, consisting of younger and older patients, respectively, with a strong female-gender enrichment in PFB5 (age: p = 0.011, gender: p = 0.04). Broad copy-number aberrations were common; however, many events such as chromosome 2 loss, 5 gain, and 17 loss were enriched in specific subtypes and 1q gain was enriched in PFB1. Late relapses were common across all five subtypes, but deaths were uncommon and present in only two subtypes (PFB1 and PFB3). Unlike the case in PFA ependymoma, 1q gain was not a robust marker of poor progression-free survival; however, chromosome 13q loss may represent a novel marker for risk stratification across the spectrum of PFB subtypes. Similar to PFA ependymoma, there exists a significant intertumoral heterogeneity within PFB, with distinct molecular subtypes identified. Even when accounting for this heterogeneity, extent of resection remains the strongest predictor of poor outcome. However, this biological heterogeneity must be accounted for in future preclinical modeling and personalized therapies.

Keywords: Clustering; Ependymoma; PFA; PFB; Posterior fossa; Subgrouping.

Figure 1:

Clustering of PFB reveals five distinct subtypes. A) t-SNE plot showing the relative distribution of the five subtypes. B) Heatmap obtained from spectral clustering (SpC) where by yellow represents more similar samples and red represents disparate ones. Color bars at the top indicate the t-SNE cluster at k=5 and the spectral clustering clusters (SpC) at k=5 C) t-SNE plot showing PFB clustered concomitantly with PF-SE and PFA. D) Heatmap representing the expression levels of the ten most differentially expressed genes per subtype. Each column represents one sample, and each lane represents one gene. Gene expression levels are represented by a color scale as indicated.

Figure 2:

Clinical characteristics across PFB subtypes. A) Boxplot of age at diagnosis across PFB subtypes. B) Frequency of male and female gender within the five subtypes. P-values for age determined using the Kruskal Wallis test and frequency of gender using the Fisher Exact Test.

Figure 3:

Arm-level cytogenetic events across PFB subtypes. A) Frequency and significance of arm-level gains and losses across the five PFB subtypes. Darker bars show significant arm level events (q-value≤ 0.1, chi-squared test). B) Frequency and significance of whole chromosome gain and losses across the five PFB subtypes. Darker bars show significant arm level events (q-value≤ 0.1, chi-squared test).

Figure 4:

Progression Free Survival stratified by A)1q gain and B)13q status. p-values determined using the log-rank test.