Corticosteroid-dependent association between prognostic peripheral blood cell-free DNA levels and neutrophil-mediated NETosis in patients with glioblastoma

Non-invasive prognostic biomarkers to inform clinical decision-making are an urgent unmet need for the management of patients with glioblastoma (GBM). We previously showed that higher circulating cell-free DNA concentration [ccfDNA] is associated with worse survival in GBM. However, the biology underlying this is unknown. We prospectively enrolled 129 patients with treatment-naïve GBM with blood drawn prior to initial resection (baseline) and at time of first post-radiotherapy MRI. We performed ccfDNA methylation deconvolution to determine cellular sources of ccfDNA. ELISA was performed to detect citrullinated H3 (citH3), a marker of neutrophil extracellular traps (NETs). Multiplex proteomic analysis was used to measure soluble inflammatory proteins. We found that neutrophils contributed the highest proportion of prognostic ccfDNA. The percentage of ccfDNA derived from neutrophils was correlated with total [ccfDNA], but only in patients receiving pre-operative corticosteroids. At baseline and on-therapy, [citH3] was significantly higher in the plasma of patients with GBM receiving corticosteroids compared to corticosteroid-naïve GBMs or no-cancer controls. Unsupervised hierarchical clustering of ccfDNA methylation patterns yielded two clusters, with one enriched for patients with the NETosis phenotype and who received corticosteroids. Unsupervised clustering of circulating inflammatory proteins yielded similar results. These data suggest neutrophil-mediated NETosis is the dominant source of prognostic ccfDNA in patients with GBM and may be associated with glucocorticoid exposure. If further studies show that pharmacological inhibition of NETosis can mitigate the deleterious effects of corticosteroids, these plasma markers will have important clinical utility as non-invasive correlative biomarkers.