Immunophenotypic Profile of Adult Glioblastoma IDH-Wildtype Microenvironment: A Cohort Study

<b>Background</b>: Glioblastoma <i>IDH</i>-wildtype (GBM <i>IDH</i>-wt) is the most aggressive brain tumor in adults and is characterized by an immunosuppressive microenvironment. Different factors shaping its tumor microenvironment (TME) regulate tumor progression and treatment response. The aim of this study was to characterize the main immunosuppressive elements of the GBM <i>IDH</i>-wt TME. <b>Methods:</b> Immunohistochemistry for CD3, CD4, CD8, CD163, programmed death ligand 1 (PD-L1) and programmed death 1 (PD1) was performed on surgical tumor specimens from patients diagnosed with GBM <i>IDH</i>-wt, according to the CNS WHO 2021 criteria. The impact of categorical variables on time-dependent outcomes such as overall survival (OS) and progression-free survival (PFS) has been estimated through the Kaplan-Meier method. <b>Results:</b> We included 30 patients (19 males and 11 females), median age of 59.8 years (range 40.2-69.1 years). All patients underwent surgery followed by temozolomide concurrent with and adjuvant to radiotherapy. MGMT was methylated in 14 patients (47%) and unmethylated in 16 patients (53%). The overall absolute percentages of CD4+ lymphocytes, both intratumoral and perivascular, were significantly more represented than CD8+ lymphocytes in the TME (<i>p</i> = 0.02). A low density of CD4+ lymphocytes (≤10%) was found to be a favorable prognostic factor for GBM outcome (<i>p</i> = 0.02). Patients with MGMT methylated and unmethylated tumors exhibited a distinct TME composition, with a significant higher number of perivascular CD8+ lymphocytes (<i>p</i> = 0.002), intratumoral CD8+ lymphocytes (<i>p</i> = 0.0024) and perivascular CD4+ lymphocytes (<i>p</i> = 0.014) in MGMT unmethylated tumors. PD-L1 expression in tumor cell surface was observed in four tumors (13.3%), and PD1 expression in infiltrating T lymphocytes was observed in nine (30%) tumors, with predominantly perivascular distribution. <b>Conclusions:</b> MGMT methylated and unmethylated tumors exhibit different immune profiles, likely reflecting the different biology of these tumors. The expression of PD-L1 in GBM <i>IDH</i>-wt patients is confined to a small subpopulation. While we found a significant association between low CD4+ lymphocyte density (≤10%) and survival, given the small numbers of our cohort, the prognostic value of CD4+ lymphocyte density will need to be validated in large-scale studies.