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Simple Summary  Molecular classification of cancers has the potential to automate and decrease errors in cancer classification. We previously showed that transcriptomic classification is comparable to methylomic and mutation methods for glioma classification and may provide benefit in predicting survival prognosis. Here we validate the transcriptomic classification method on a single molecule counting gene expression platform using formalin-fixed paraffin embedded samples.    Abstract  Gene expression profiling has been shown to be comparable to other molecular methods for glioma classification. We sought to validate a gene-expression based glioma classification method. Formalin-fixed paraffin embedded tissue and flash frozen tissue collected at the Augusta University (AU) Pathology Department between 2000–2019 were identified and 2 mm cores were taken. The RNA was extracted from these cores after deparaffinization and bead homogenization. One hundred sixty-eight genes were evaluated in the RNA samples on the nCounter instrument. Forty-eight gliomas were classified using a supervised learning algorithm trained by using data from The Cancer Genome Atlas. An ensemble of 1000 linear support vector models classified 30 glioma samples into TP1 with classification confidence of 0.99. Glioma patients in TP1 group have a poorer survival (HR (95% CI) = 4.5 (1.3–15.4),  p  = 0.005) with median survival time of 12.1 months, compared to non-TP1 groups. Network analysis revealed that cell cycle genes play an important role in distinguishing TP1 from non-TP1 cases and that these genes may play an important role in glioma survival. This could be a good clinical pipeline for molecular classification of gliomas.

Retrospective Validation of a 168-Gene Expression Signature for Glioma Classification on a Single Molecule Counting Platform