Immunohistochemically Characterized Intratumoral Heterogeneity Is a Prognostic Marker in Human Glioblastoma

Simple Summary Intratumoral heterogeneity is believed to contribute to the immense therapy resistance and recurrence rate of glioblastoma. The aim of this retrospective study was to analyze the heterogeneity of 36 human glioblastoma samples on a morphological level by immunohistochemistry. We confirmed that this method is valid for heterogeneity detection. 115 Areas of Interest were labelled. By cluster analysis, we defined two subtypes ("classical" and "mesenchymal"). The results of epigenomic analyses corroborated the findings. Interestingly, patients with tumors that consisted of both subtypes ("subtype-heterogeneous") showed a shorter overall survival compared to patients with tumor that were dominated by one subtype ("subtype-dominant"). Furthermore, the analysis of 21 corresponding pairs of primary and recurrent glioblastoma demonstrated that, additionally to an intratumoral heterogeneity, there is also a chronological heterogeneity with dominance of the mesenchymal subtype in recurrent tumors. Our study confirms the prognostic impact of intratumoral heterogeneity in glioblastoma and makes this hallmark assessable by routine diagnostics. Tumor heterogeneity is considered to be a hallmark of glioblastoma (GBM). Only more recently, it has become apparent that GBM is not only heterogeneous between patients (intertumoral heterogeneity) but more importantly, also within individual patients (intratumoral heterogeneity). In this study, we focused on assessing intratumoral heterogeneity. For this purpose, the heterogeneity of 38 treatment-naive GBM was characterized by immunohistochemistry. Perceptible areas were rated for ALDH1A3, EGFR, GFAP, Iba1, Olig2, p53, and Mib1. By clustering methods, two distinct groups similar to subtypes described in literature were detected. The classical subtype featured a strong EGFR and Olig2 positivity, whereas the mesenchymal subtype displayed a strong ALDH1A3 expression and a high fraction of Iba1-positive microglia. 18 tumors exhibited both subtypes and were classified as "subtype-heterogeneous", whereas the areas of the other tumors were all assigned to the same cluster and named "subtype-dominant". Results of epigenomic analyses corroborated these findings. Strikingly, the subtype-heterogeneous tumors showed a clearly shorter overall survival compared to subtype-dominant tumors. Furthermore, 21 corresponding pairs of primary and recurrent GBM were compared, showing a dominance of the mesenchymal subtype in the recurrent tumors. Our study confirms the prognostic impact of intratumoral heterogeneity in GBM, and more importantly, makes this hallmark assessable by routine diagnostics.