RNA sequencing and Immunohistochemistry Reveal ZFN7 as a Stronger Marker of Survival than Molecular Subtypes in G-CIMP-negative Glioblastoma

Glioblastoma is the most aggressive brain tumor in adults and has few therapeutic options. The study of molecular subtype classifications may lead to improved prognostic classification and identification of new therapeutic targets. The Cancer Genome Atlas (TCGA) subtype classification has mainly been applied in U.S. clinical trials, while the intrinsic glioma subtype (IGS) has mainly been applied in European trials. From paraffin-embedded tumor samples of 432 patients with uniformly treated, newly diagnosed glioblastoma, we built tissue microarrays for IHC analysis and applied RNA sequencing to the best samples to classify them according to TCGA and IGS subtypes. We obtained transcriptomic results from 124 patients. There was a lack of agreement among the three TCGA classificatory algorithms employed, which was not solely attributable to intratumoral heterogeneity. There was overlapping of TCGA mesenchymal subtype with IGS cluster 23 and of TCGA classical subtype with IGS cluster 18. Molecular subtypes were not associated with prognosis, but levels of expression of 13 novel genes were identified as independent prognostic markers in glioma-CpG island methylator phenotype-negative patients, independently of clinical factors and methylation. These findings were validated in at least one external database. Three of the 13 genes were selected for IHC validation. In particular, high RNA expression and low ZNF7 protein expression were strongly associated with longer survival, independently of molecular subtypes. TCGA and IGS molecular classifications of glioblastoma have no higher prognostic value than individual genes and should be refined before being applied to clinical trials.