Immune Cell Infiltration in Primary and Recurrent Glioblastoma, <scp>IDH</scp> ‐Wild Type: Validation of an Immunohistochemistry‐Based Scoring System for Research and Clinical Practice

Significant heterogeneity in the immune microenvironment of Glioblastoma, IDH-wildtype (GBM), has been reported, necessitating a standardized approach to evaluate immune infiltration in the context of emerging immunotherapies. To address this, we developed and validated a standardized immunohistochemistry-based scoring system for quantifying immune cell infiltration in formalin-fixed, paraffin-embedded (FFPE) tissue. Paired primary and recurrent GBM specimens from 20 adult patients were labeled for CD3, CD8, CD45, CD68, and PD-1, and scored across six anatomical regions, including intra- and peritumoral, meningeal, and normal brain areas. The scoring system demonstrated excellent interrater (ICC = 0.932) and intrarater (ICC = 0.953) reliability. CD68+ and CD45+ cells were most numerous across all regions. CD3+ and CD8+ cells concentrated more in the perivascular area rather than within the parenchyma. No significant differences in immune infiltration were observed between primary and recurrent GBM. Cox proportional-hazards analysis showed worse survival with higher CD8+ and CD45+ infiltration in primary GBM, and higher CD45+ and CD68+ infiltration in recurrent GBM. In conclusion, we propose a feasible, cost-efficient, and robust method to assess immune infiltration on FFPE material, enabling standardized comparison of inflammation, with applications for ongoing clinical trials.