EGFR and TP53 mutations refine prognostic stratification of IDH-wildtype astrocytomas, not elsewhere classified

IDH-wildtype astrocytomas of histological grade 2 or 3 without the WHO 2021 molecular criteria for glioblastoma-TERT promoter mutation, EGFR amplification, or combined whole-chromosome 7 gain and 10 loss (+ 7/- 10)-are designated "Not Elsewhere Classified" (NEC), a residual category with clinical behavior ranging from indolent disease to courses clinically indistinguishable from glioblastoma (GBM). However, reliable molecular markers capable of resolving this heterogeneity have not been established. We retrospectively profiled a multi-institutional cohort comprising 38 adult IDH-wildtype astrocytoma NEC cases and 429 GBM controls using a bimodal DNA/RNA next-generation sequencing panel and, in 30 NEC cases, genome-wide DNA methylation profiling. NEC tumors were markedly heterogeneous: 21 of the 30 methylation profiles (70%) failed to match any reference class family, and the remaining nine were distributed across six categories, with Uniform Manifold Approximation and Projection (UMAP) showing scattered embedding adjacent to multiple high- and low-grade reference clusters, rather than a coherent NEC cluster. Against this heterogeneous background, two point mutations emerged as candidate markers for aggressive disease: EGFR mutations (3/38, 7.9%), all clustered in the extracellular domain, and TP53 mutations (8/38, 21.1%), predominantly in the DNA-binding domain. Both retained independent prognostic significance in Firth-penalized multivariate Cox regression (EGFR: HR 1.61, 95% CI 1.02-2.57, P = 0.028; TP53: HR 1.55, 95% CI 1.00-2.42, P = 0.041). NEC patients harboring EGFR and/or TP53 mutations showed an overall survival curve closely paralleling that of GBM (P = 0.9605) and significantly diverged from EGFR/TP53-wildtype NEC (P = 0.0011). Thus, despite the marked genomic and epigenomic heterogeneity within NEC, EGFR and TP53 point mutations may identify a high-risk NEC subgroup. This stratifier is deployable with routine targeted sequencing panels already widely used in diagnostic practice, and provides a practical refinement complementary to the cIMPACT-NOW Update 11 framework, pending prospective validation.