TERT promoter mutation analysis for blood-based diagnosis and monitoring of gliomas

Liquid biopsy offers a minimally invasive tool to diagnose and monitor the heterogeneous molecular landscape of tumors over time and therapy. Gliomas have been challenging to sample with blood-based liquid biopsy due to low levels of circulating tumor-derived nucleic acid, with existing methods showing plasma-derived cell-free DNA (cfDNA) detectable in less than 5% of patients. Detection of TERT promoter mutations (C228T, C250T) in cfDNA has been successful for some systemic cancers but has yet to be demonstrated in gliomas, despite the high prevalence (>60% of all tumors). Here, we developed a digital droplet PCR (ddPCR) assay for the simultaneous detection and longitudinal monitoring of two TERT promoter mutations (C228T and C250T) in cfDNA from the plasma of glioma patients. In provisioning across n = 157 samples, our assay in tumor tissue showed perfect concordance with pathology (95% CI 94%-100%). Extending to matched plasma samples, we detect TERT mutations in discovery and blinded multi-institution validation cohorts with an overall sensitivity of 62.5% (95% CI 52%-73%) and a specificity of 90% (95% CI 80%-96%). Upon longitudinal monitoring in 5 patients, we report that peripheral TERT mutant allele frequency reflects the clinical course of the disease with levels decreasing after surgical intervention and therapy and increasing with tumor progression. Our results demonstrate the utility of a novel blood-based ddPCR assay which allows for the detection of TERT promoter mutations in tissue and circulating cfDNA with high sensitivity and specificity in the context of glioma diagnosis and monitoring. The functionality of our assay establishes the potential role of a plasma-derived cfDNA analysis to complement tissue-based histopathologic and molecular characterization for diagnosis and spatiotemporal monitoring of brain tumors.