Multiomic and clinical analysis of multiply recurrent meningiomas reveals risk factors, underlying biology, and insights into evolution

Abstract

An important subset of meningiomas behaves aggressively and is characterized by multiple recurrences. We identify clinical, genetic, and epigenetic predictors of multiply recurrent meningiomas (MRMs) and evaluate the evolution of these meningiomas in patient-matched samples. On multivariable binomial logistic regression, MRMs were significantly associated with male sex (P = 0.012), subtotal resection (P = 0.001), higher number of meningiomas on presentation (P = 0.017), and histopathological sheeting (P = 0.002). Multiomic analysis of primary meningiomas revealed that MRMs have greater copy number losses (P = 0.0313) and increased DNA methylation (P = 0.0155). In meningioma cells with knockdown of EDNRB, a locus with greater promoter methylation and decreased gene expression in MRMs had increased proliferation (P < 0.0001). MRM recurrences were found to be similar to primaries but have a greater burden of copy number gains (P < 0.0001) and increased methylation (P = 0.0045). This clinical and multiomic investigation of MRMs harbors implications for the future development of biomarkers and therapeutic agents for these challenging tumors.

Fig. 1.. PSM of patients from two institutions reveals clinical predictors of MRMs.

(A) Schematic representing patient institutional distribution before and after PSM. (B) Histogram depicting WHO grade distribution for nonrecurrent meningioma (NRM) and multiply recurrent meningioma (MRM) groups before and after propensity score matching (PSM). (C) Density plot depicting the follow-up time (months) distribution for NRM and MRM groups before and after PSM. (D) Representative non-contrast head computed tomography and associated T1-weighted post-contrast magnetic resonance imaging highlighting dystrophic calcification and bone invasion. (E) Clinical characteristics of 115 patients with meningioma included in clinical cohort after PSM separated by multiple recurrent (left, dark green) and nonrecurrent (right, light green) meningiomas. (F) Univariate analysis revealed a significant association between MRMs and male sex (*P = 0.042), subtotal resection (***P < 0.001), sheeting (***P < 0.001), macronucleoli (*P = 0.035), necrosis (**P = 0.009), hypercellularity (*P = 0.015), and bone invasion on imaging (*P = 0.015). NRMs were significantly associated with psammoma bodies (**P = 0.005) and dystrophic calcification on imaging (**P = 0.006).

Fig. 2.. Sample representation across omic methods.

(A) Oncoprint representing sample distribution across omic methods. (B) Venn diagram showing omic sample distribution across methods.

Fig. 3.. WES reveals a greater burden of chromosomal losses in MRMs.

(A) Schematic representing the MRM and NRM MenG C meningiomas that underwent WES and methylation array analysis from formalin-fixed paraffin-embedded (FFPE) tissue. t-SNE, t-Distributed Stochastic Neighbor Embedding. (B) Patient information for the MenG C meningioma samples including molecular class, institution, recurrence status, sex, and WHO grade. (C) Status of non-synonymous genomic variants commonly reported in the meningioma literature from WES in MRM (n = 12) and NRM (n = 17). (D) Arm-level copy number differences between MRMs (n = 12) and NRMs (n = 16) (two-sample t test; losses, P = 0.0313; gains, P = 0.7894; and alterations, P = 0.1377). (E) Unsupervised clustering based on chromosomal arm alterations calculated from WES of all MenG C tumors is unable to separately cluster MRMs and NRMs. n.s., not significant.

Fig. 4.. Methylation array profiling elucidates differences between MRMs and NRMs.

(A) Supervised clustering heatmap comparing top 10,000 most variable probes (MVPs) for MRMs (n = 12) versus NRMs (n = 17) in MenG C meningiomas. (B) Comparison of average beta values of top 10,000 MVPs between MRMs (n = 12) and NRMs (n = 17). Two-sample t test performed (P = 0.0155). (C) Quantification of differentially methylated probes (DMPs) between MRMs and NRMs focused on promoter site probes associated with genes. (D) Overlap of genes from methylation analysis in (C) and RNA-seq analysis (MRM, n = 3; and NRM, n = 63) comparing MRMs and NRMs. Up-regulated RNA expression was defined as differentially expressed genes (DEGs) with P < 0.05 and log₂ fold change (log₂FC) > 1, and down-regulated DEGs had P < 0.05 and log₂FC < −1. (E) Reverse transcription–quantitative polymerase chain reaction (qPCR) validation of knockdown efficiency for three distinct EDNRB small interfering RNA (siRNA) constructs in the CH157 meningioma cell line (n = 3 independent experiments). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (F) Cell proliferation of each siRNA construct in CH157 cells (n = 4 independent experiments). All comparisons ****P < 0.0001 on one-way analysis of variance (ANOVA) with multiple comparisons.

Fig. 5.. Recurrent MRMs exhibit no change in genomic variants or subclones but bear an increased burden of copy number gains.

(A) WES and methylation array profiling was performed on longitudinal matched primary and recurrent FFPE samples from patients with MRM. (B) Patient information for the 11 primary, 11 first recurrence, and 2 second recurrence lesions. (C) Non-synonymous genomic variants commonly reported in the meningioma literature from WES of 10 primary and matched recurrent pairs. (D) Quantification of NF2 variants from (C) showing no significant difference between primary and first recurrence for any specific variant type. (E) Arm-level copy number differences between primary (n = 10) and matched first recurrence (n = 10) tumors (paired t test; losses, P = 9302; gains, P < 0.0001; and alterations, P = 0.0836). (F) Chromosomal arm gains and losses for primary and matched recurrent tumors of 10 patients. (G) Histogram representing total chromosomal arm gains and losses between primary and first recurrence of MRMs. (H) Subclonal analysis of whole-exome data using variant allele frequencies (VAFs) from a representative patient comparing primary versus recurrence 1, recurrence 1 versus recurrence 2, and primary versus recurrence 2.

Fig. 6.. Matched primary and recurrent meningiomas have similar methylation profiles.

(A) Unsupervised clustering heatmap comparing 10,000 MVPs for primary and matched recurrence from patients with MRM (n = 10). (B) Heatmap of percent of 10,000 MVPs with conserved methylation status across samples (n = 10 patients). Unsupervised clustering groups primary and recurrent samples together. (C) Pair-wise comparison of average methylation ß values of the 10,000 MVPs between matched primary (n = 10) and recurrence 1 (n = 10) of each MRM tumor. Paired analysis was performed using paired t test (P = 0.0045).