Cell-free DNA and circulating TERT promoter mutation for disease monitoring in newly-diagnosed glioblastoma

Abstract

The clinical implications of plasmatic cell-free and tumor DNA (cfDNA and ctDNA) are challenging in glioblastoma. This prospective study included 52 consecutive newly diagnosed glioblastoma (n = 49) or gliosarcoma (n = 3) patients treated with concomitant temozolomide and radiotherapy (RT-TMZ), followed by a TMZ maintenance phase. Plasma samples were collected at baseline, before RT-TMZ (pre-RT-TMZ) and at the end of adjuvant TMZ, or at the time of progression in cases of progressive disease (PD). The cfDNA concentration was measured with a fluorometric method, and ctDNA was detected using targeted droplet digital PCR. The main objectives were to analyze the associations between cfDNA and ctDNA measurements during the course of treatment with PD and survival. There was a significant decrease in median cfDNA concentration from baseline to pre-RT-TMZ-19.4 versus 9.7 ng/mL (p < 0.0001)-in the entire cohort. In patients with PD, a significant increase in cfDNA concentration from pre-RT-TMZ to time of PD was observed, from 9.7 versus 13.1 ng/mL (p = 0.037), respectively, while no difference was observed for nonprogressive patients. Neither the cfDNA concentration at baseline nor its kinetics correlated with survival. ctDNA was detected in 2 patients (3.8%) and only in gliosarcoma subtypes.Trial registration ClinicalTrial, NCT02617745. Registered 1 December 2015, https://ichgcp.net/clinical-trials-registry/NCT02617745?term=glioplak&draw=2&rank=1 .

Keywords: Cell-free DNA; Circulating tumor DNA; Glioblastoma; Liquid biopsy; TERT promoter mutation.

Conflict of interest statement

All the authors report no disclosure.

Figures

Fig. 1

Correlations between cell-free DNA concentration at baseline and MRI characteristics. The cfDNA concentration is plotted against three tumor volumes: a T2-Flair; b T1-enhanced volume with necrosis; c T1 enhanced volume without necrosis (p = 0.018); and d rCBV (p = 0.03). rCBV had the highest association with cfDNA concentration. Gado: gadolinium; W/: with; W/O: without; rCBV: relative cerebral blood volume

Fig. 2

Variations of circulating cell-free DNA concentration during treatment. a Median cfDNA concentration (ng/mL of plasma) at baseline is higher than in the control group (healthy subjects) and decreases between baseline and pre-RT-TMZ in both the biopsy group (b) and the tumor resection group (c). RT: radiotherapy; TMZ: temozolomide

Fig. 3

Survival curves. a Overall survival and progression-free survival b according to baseline cfDNA concentration below or above the median. OS (c) and PFS (d) according to the increase or decrease of cfDNA between baseline and pre-RT-TMZ

Fig. 4

Patient (#059) with TERTp C228T gliosarcoma and TERTp detection in plasma at diagnosis. Patient #059 was a 64-year-old man suffering from frontal C228T TERTp, IDH wild-type gliosarcoma spreading into the corpus callosum. a The lesion was predominantly necrotic: 43 cm3 of T1-weighted gadolinium volume with necrosis versus 11 cm3 without necrosis; the percentage of necrosis was 74%. In contrast, the rest of the cohort had an average rate of necrosis of 33%. The T2-FLAIR volume was also consequential, with a total volume of 184 cm3. b Circulating C228T TERTp mutation was detected both pre- and postoperatively. Two assays were performed (assay 1 and 2). The presented results are the mean of the two assays, with error bar (± standard deviation). c The kinetics of cfDNA concentration correlated with ctDNA MAF and decreased after tumor resection

Fig. 5

Plasma longitudinal follow-up of the TERTp C228T mutation in a patient (#082) with TERTp-mutated gliosarcoma. a Patient #082 was a 69-year-old woman suffering from left temporal unmethylated MGMTp, IDH wild-type (wt) gliosarcoma with C228T TERTp mutation (MAF 28.6% in initial resected tumor using ddPCR). Partial tumor resection was initially performed. The plot shows the evolution of MAF and plasma cfDNA concentration as a function of time. C228T TERTp mutation was not detected in plasma until cerebral relapse, with sphenoid bone invasion. The longitudinal plasma samples taken during the first-line treatment schedule revealed a correlation between circulating C228T TERTp MAF, cfDNA concentration, and the evolution towards metastatic status. b Two representations of the search for TERTp C228T mutation in plasma at baseline (left) and at progression (right). Channel 1 fluorescence (vertical axis, FAM™) is plotted against channel 2 (horizontal axis, HEX™). Droplets are grouped as clusters: FAM™/HEX™negative (double-negative droplets, blue points), FAM™positive/HEX™negative (green points), FAM™negative/HEX™positive (pink points), and FAM™/HEX™positive (double-positive droplets, orange points)