Background. Approximately half of the isocitrate dehydrogenase (IDH)-wildtype glioblastomas (GBMs) exhibit EGFR amplification. Additionally, genomic changes that occur in the extracellular domain of EGFR can lead to ligand-hypersensitivity (R108K/A289V/G598V) or ligand-independence (EGFRvIII). Unlike in lung adenocarcinoma (LUAD), clinical trials with epidermal growth factor receptor (EGFR) inhibitors showed no survival benefit for GBM and it remains unclear why. We aimed to elucidate differences in molecular mechanisms of EGFR activation and regulation between GBM and LUAD. Methods. We used RNA-sequencing (RNA-seq) data to find EGFR co-regulated genes and pathways in GBM and compare EGFR signaling patterns between GBM and LUAD. Cellular origins of expression signals were determined by analyzing single-cell RNA-seq data. Results. We identified 2 ligands (BTC/EREG) among the significant EGFR predictor genes (TCGA-GBM: n = 169, Intellance-2: n = 166). Their expression was inversely correlated with EGFR amplification and incidence of ligand-sensitive mutations. Ligands were expressed by nonmalignant cells and differed in their primary source of expression (BTC: neurons, EREG: myeloid). High expression of MDM2 and CDK4 was less common in EGFR-amplified GBMs with ligand-sensitive mutations compared with those without these mutations. Our analyses revealed distinct transcriptional profiles between GBM and LUAD when comparing tumors carrying activating mutations. Conclusions.BTC and EREG are negatively associated with EGFR expression in GBM. These findings emphasize the role of ligands in regulating EGFR, where EGFR activation seems to be modulated by the highly varying levels of EGFR amplification, the sensitivity of the receptor toward ligands, and ligand expression levels. Ligand expression levels and EGFR mutations could refine patient stratification for EGFR-targeted therapies in GBM.

A better understanding of transcriptional evolution of IDH-wild-type glioblastoma may be crucial for treatment optimization. Here, we perform RNA sequencing (RNA-seq) (n = 322 test, n = 245 validation) on paired primary-recurrent glioblastoma resections of patients treated with the current standard of care. Transcriptional subtypes form an interconnected continuum in a two-dimensional space. Recurrent tumors show preferential mesenchymal progression. Over time, hallmark glioblastoma genes are not significantly altered. Instead, tumor purity decreases over time and is accompanied by co-increases in neuron and oligodendrocyte marker genes and, independently, tumor-associated macrophages. A decrease is observed in endothelial marker genes. These composition changes are confirmed by single-cell RNA-seq and immunohistochemistry. An extracellular matrix-associated gene set increases at recurrence and bulk, single-cell RNA, and immunohistochemistry indicate it is expressed mainly by pericytes. This signature is associated with significantly worse survival at recurrence. Our data demonstrate that glioblastomas evolve mainly by microenvironment (re-)organization rather than molecular evolution of tumor cells.

Consensus molecular subtyping is an RNA expression-based classification system for colorectal cancer (CRC). Genomic alterations accumulate during CRC pathogenesis, including the premalignant adenoma stage, leading to changes in RNA expression. Only a minority of adenomas progress to malignancies, a transition that is associated with specific DNA copy number aberrations or microsatellite instability (MSI). We aimed to investigate whether colorectal adenomas can already be stratified into consensus molecular subtype (CMS) classes, and whether specific CMS classes are related to the presence of specific DNA copy number aberrations associated with progression to malignancy. RNA sequencing was performed on 62 adenomas and 59 CRCs. MSI status was determined with polymerase chain reaction-based methodology. DNA copy number was assessed by low-coverage DNA sequencing (n = 30) or array-comparative genomic hybridisation (n = 32). Adenomas were classified into CMS classes together with CRCs from the study cohort and from The Cancer Genome Atlas (n = 556), by use of the established CMS classifier. As a result, 54 of 62 (87%) adenomas were classified according to the CMS. The CMS3 ‘metabolic subtype’, which was least common among CRCs, was most prevalent among adenomas (n = 45; 73%). One of the two adenomas showing MSI was classified as CMS1 (2%), the ‘MSI immune’ subtype. Eight adenomas (13%) were classified as the ‘canonical’ CMS2. No adenomas were classified as the ‘mesenchymal’ CMS4, consistent with the fact that adenomas lack invasion-associated stroma. The distribution of the CMS classes among adenomas was confirmed in an independent series. CMS3 was enriched with adenomas at low risk of progressing to CRC, whereas relatively more high-risk adenomas were observed in CMS2. We conclude that adenomas can be stratified into the CMS classes. Considering that CMS1 and CMS2 expression signatures may mark adenomas at increased risk of progression, the distribution of the CMS classes among adenomas is consistent with the proportion of adenomas expected to progress to CRC.