Proteomic Insights Into Anaplastic Meningioma Heterogeneity

Meningiomas are the most common primary tumors of the central nervous system and show clinical heterogeneity across WHO grades. While grade 1 meningiomas are typically benign, grade 3 (anaplastic) meningiomas are rare, highly aggressive, and associated with poor prognosis. Current histopathological grading does not fully capture tumor biology, therefore molecular approaches are needed to better reflect disease behavior. Proteomic profiling provides insight into functional biological differences between tumor grades, but sample processing and analysis of formalin-fixed paraffin-embedded (FFPE) tissues remains challenging. This study aimed to develop and implement a robust, high-throughput proteomics workflow for FFPE tissues and to apply the workflow to meningioma samples in order to characterize proteomic differences between grade 1 and grade 3 tumors, explore heterogeneity within high-grade meningiomas, and investigate associations with EZH2 expression. Multiple deparaffinization, protein extraction, and digestion methods were systematically evaluated using pilot tissues to optimize protein yield, reproducibility, and proteome coverage. The final workflow consisted of n-heptane/methanol deparaffinization, BeatBox-based protein extraction, and automated magnetic bead-based digestion on the KingFisher Apex platform. The optimized protocol was applied to a cohort of 43 FFPE meningioma samples analyzed by data-independent acquisition mass spectrometry. Proteomic analysis revealed clear differences between grade 1 and grade 3 meningiomas, with high-grade tumors showing increased abundance of proteins involved in proliferation, DNA replication, RNA processing, and metabolic reprogramming. Pathway enrichment analyses confirmed a predominance of proliferative and biosynthetic pathways in grade 3 tumors, whereas grade 1 meningiomas were associated with homeostatic and cytoskeletal processes. Unsupervised clustering further identified three distinct proteomic subgroups within grade 3 meningiomas, each associated with different biological pathways, EZH2 expression levels, and clinical outcomes. These findings demonstrate pronounced biological heterogeneity within high-grade meningiomas and highlight the potential of FFPE-based proteomics to refine tumor stratification beyond histological grading.