EGFR gene amplification in IDH-wildtype glioblastomas: an integrative bioinformatic and histopathological analysis using immunohistochemistry and fluorescence in situ hybridization.

Background: Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor in adults, characterized by marked histopathological features such as high proliferative activity, mitoses, necrosis, and microvascular proliferation (MVP), as well as extensive molecular heterogeneity. Among key molecular alterations, EGFR amplification and MGMT promoter methylation hold significant diagnostic and prognostic relevance. In this study, we performed a comprehensive bioinformatic analysis of EGFR and conducted a correlation analysis of EGFR amplification in a cohort of 19 GBM samples using standard fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). These results were further correlated with established molecular hallmarks of glioblastoma.

Methods: We analyzed mRNA expression profiles of EGFR family genes using publicly available datasets from the Gene Expression Omnibus (GEO) and Chinese Glioma Genome Atlas (CGGA) databases. Additionally, we utilized multiple online bioinformatics platforms, including GEPIA, TIMER, TISIDB repository, cBioPortal, Metascape, and GeneMANIA, to explore the expression patterns, immune cell infiltration, correlation of EGFR amplification with immunomodulatory and prognostic significance of the EGFR family in glioblastoma. EGFR amplification and protein expression were assessed using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), respectively. MGMT promoter methylation was determined by methylation-specific qPCR (MS-qPCR).

Results: Bioinformatic analyses revealed that EGFR and ERBB2 were overexpressed and associated with poor prognosis, while ERBB3 and ERBB4 exhibited decreased expression in GBM compared to normal brain tissue. Immune infiltrating level correlation of EGFR family, revealed that EGFR and ERBB4 expression were positively correlated with various immune cell infiltrates in glioblastoma, particularly CD8 + T cells, macrophages, and B cells. Additionally, EGFR expression was negatively correlated with key co-stimulatory molecules including CD28, CD40, CD70, and IL2RA, no significant associations were found with classical immune checkpoints such as PD-L1 and CTLA-4, though EGFR was inversely correlated with TGFBR1. Subtype analysis showed elevated EGFR expression in Classical-like and Mesenchymal-like GBM subtypes, and a trend toward higher levels in the lymphocyte-depleted immune subtype. Genomic analysis revealed that EGFR family alterations in 49% of GBM cases, with EGFR amplification being the most frequent and linked to poorer overall survival. Enrichment and the PPI network analyses demonstrated that the EGFR family is deeply involved in tumorigenic pathways, particularly PI3K/AKT signaling, integrin interactions, and growth factor receptor cascades. We found that EGFR amplification was detected in 36.84% of cases and was significantly associated with EGFR protein overexpression (IHC score 3+). MGMT promoter methylation was present in 47.4% of cases. No significant association was found between EGFR amplification and MGMT methylation, ATRX, or p53 status.

Conclusion: Our integrative bioinformatics and histopathological analyses highlight EGFR as a central oncogenic driver and potential immunomodulatory factor in GBM. EGFR amplification correlates with poor prognosis, immune dysregulation, underscoring its utility as both a diagnostic biomarker and a potential therapeutic target in GBM management.