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We have previously demonstrated an independent association between high levels of plasma circulating cell-free DNA (ccfDNA) concentration and poor survival outcomes in patients with newly diagnosed glioblastoma. Given that somatic mutations are rarely detected in glioblastoma patient plasma, we reasoned that DNA shed by tumor (circulating tumor DNA, ctDNA) was not likely to be a driver of prognostic increases in ccfDNA. To investigate the tissue of origin for this prognostic ccfDNA, we analyzed the plasma ccfDNA methylation signatures of 23 patients with newly diagnosed glioblastoma using the Illumina Infinium EPIC array, a genome-wide DNA methylation technique covering over 850,000 CpG sites. Deconvolution of the ccfDNA methylation signatures based on published reference methylomes revealed an increased proportion of ccfDNA derived from granulocytes in glioblastoma specimens compared to healthy controls (p < 0.0001). Further, this granulocyte proportion was increased in patients with high ccfDNA levels (above median value) compared to patients with low ccfDNA (p = 0.0001). Granulocyte proportion correlated with ccfDNA concentration (Spearman r = 0.64, p = 0.001). The top two gene sets identified by differential methylation analysis followed by gene set enrichment analysis (methylGSA) between high- and low-ccfDNA specimens were “neutrophil activation involved in immune response” (GO:0002283, p = 3.4E-23) and “neutrophil degranulation” (GO:0043312, p = 3.4E-23). Additional analysis of ccfDNA fragment size identified larger fragments ( > 700 bp) as the major source of the prognostic signal (log-rank p = 0.002 for progression-free survival) compared to traditionally sized ccfDNA fragments (50-700 bp, log-rank p = 0.12). Taken together, these data suggest that granulocytes are the primary contributing source of prognostic ccfDNA in glioblastoma. Future studies are needed to determine the mechanism through which granulocyte-derived ccfDNA is associated with clinical outcomes in glioblastoma and to explore related therapeutic opportunities.

Background We aimed to determine whether plasma cell-free DNA (cfDNA) concentration is associated with survival in patients with isocitrate dehydrogenase (IDH) wild-type glioblastoma (GBM). Methods Pre-operative and post-chemoradiotherapy blood samples were prospectively collected from patients with newly diagnosed IDH wild-type GBM. Patients underwent surgical resection or biopsy and received adjuvant radiotherapy with concomitant temozolomide. Cell-free DNA (cfDNA) was isolated from plasma and quantified using SYBR Green-based q polymerase chain reaction (qPCR). Results Sixty-two patients were enrolled and categorized into high vs. low cfDNA groups relative to the pre-operative median value (25.2 ng/mL, range 5.7–153.0 ng/mL). High pre-operative cfDNA concentration was associated with inferior PFS (median progression-free survival (PFS), 3.4 vs. 7.7 months; log-rank P = .004; hazard ratio [HR], 2.19; 95% CI, 1.26–3.81) and overall survival (OS) (median OS, 8.0 vs. 13.9 months; log-rank P = .01; HR, 2.43; 95% CI, 1.19–4.95). After adjusting for risk factors, including O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status, pre-operative cfDNA remained independently associated with PFS (HR, 2.70; 95% CI, 1.50–4.83; P = .001) and OS (HR, 2.65; 95% CI, 1.25–5.59; P = .01). Post-hoc analysis of change in cfDNA post-chemoradiotherapy compared to pre-surgery (n = 24) showed increasing cfDNA concentration was associated with worse PFS (median, 2.7 vs. 6.0 months; log-rank P = .003; HR, 4.92; 95% CI, 1.53–15.84) and OS (median, 3.9 vs. 19.4 months; log-rank P < .001; HR, 7.77; 95% CI, 2.17–27.76). Conclusions cfDNA concentration is a promising prognostic biomarker for patients with IDH wild-type GBM. Plasma cfDNA can be obtained noninvasively and may enable more accurate estimates of survival and effective clinical trial stratification.

Background Plasma cell-free DNA (cfDNA) concentration is lower in glioblastoma (GBM) compared to other solid tumors, which can lead to low circulating tumor DNA (ctDNA) detection. In this study, we investigated the relationship between multimodality magnetic resonance imaging (MRI) and histopathologic features with plasma cfDNA concentration and ctDNA detection in patients with treatment-naive GBM. Methods We analyzed plasma cfDNA concentration, MRI scans, and tumor histopathology from 42 adult patients with newly diagnosed GBM. Linear regression analysis was used to examine the relationship of plasma cfDNA concentration before surgery to imaging and histopathologic characteristics. In a subset of patients, imaging and histopathologic metrics were also compared between patients with and without a detected tumor somatic mutation. Results Tumor volume with elevated (>1.5 times contralateral white matter) rate transfer constant (Kep, a surrogate of blood–brain barrier [BBB] permeability) was independently associated with plasma cfDNA concentration (P = .001). Histopathologic characteristics independently associated with plasma cfDNA concentration included CD68+ macrophage density (P = .01) and size of tumor vessels (P = .01). Patients with higher (grade ≥3) perivascular CD68+ macrophage density had lower volume transfer constant (Ktrans, P = .01) compared to those with lower perivascular CD68+ macrophage density. Detection of at least 1 somatic mutation in plasma cfDNA was associated with significantly lower perivascular CD68+ macrophages (P = .01). Conclusions Metrics of BBB disruption and quantity and distribution of tumor-associated macrophages are associated with plasma cfDNA concentration and ctDNA detection in GBM patients. These findings represent an important step in understanding the factors that determine plasma cfDNA concentration and ctDNA detection.