Fig. 1.

Detection of TERT promoter mutations in FFPE DNA extracted from gliomas using ddPCR. a Fluorescent intensity of the droplets after amplification of a 113 bp-fragment of the TERT promoter (TERTp) region using the ddPCR expert design assays (Bio-Rad Laboratories). The individual lanes correspond to: 1, TERTp “C228T” mutation control DNA; 2, TERTp “C250T” mutation control DNA; 3, TERTp wildtype control DNA; 4, no template control; 5, glioblastoma, IDH-wildtyp, CNS WHO grade 4, and 6, oligodendroglioma, IDH-mutant, and 1p/19q-codeleted, both with TERTp “C228T” and “C250T” mutations. X axis, number of droplets with fluorescence; Y axis, fluorescence intensity detected in the FAM-channel (blue dots) and HEX-channel (green dots); pink line, threshold; grey dots, droplets with background fluorescence of non-incorporated probes. b Validation of the two TERT promoter mutations in the IDH-wildtype glioblastoma using Sanger sequencing. Red arrow heads pointing to the “C250T “ (left) and “C228T” (right) mutation. The numbering of the samples corresponds to a. c Mutant allele frequency (MAF) was measured by ddPCR using different amounts of input FFPE DNA generated by serial dilution of samples with either a TERTp “C228T” (mean MAF 37.3%) or a “C250T” (mean MAF 38.1%) mutation with distilled water. d 25 ng of total input FFPE DNA was used for ddPCR. A TERTp “C228T” (mean MAF 34.7%) and a TERTp “C250T” DNA sample (mean MAF 31.7%) were mixed with wildtype DNA resulting in predefined templates with 50%, 25%, 12.5%, 6.3%, 3.1%, 1.6%, 0.8%, and 0.4% TERT promoter-mutant DNA in a TERT promoter-wildtype background