A Novel Liquid Biopsy Technique for Brain Tumor Diagnosis

A minimally invasive liquid biopsy technique enables diagnosis of glioma, the most common type of brain tumor, by PCR analysis of an easily obtained blood plasma sample.

Detection of TERT (Telomerase reverse transcriptase) promoter mutations (C228T, C250T) in circulating cell free DNA (cfDNA) has been successful for some systemic cancers but has not been demonstrated in gliomas, despite the high prevalence of these mutations in glioma tissue (they are in more than 60% of all gliomas and in 80% of all high-grade gliomas, the most aggressive and life-threatening type). The liquid biopsy approach for brain tumors is complicated, as brain tumor DNA is shed into the bloodstream at much lower levels than that of any other type of tumors.


To improve this situation, investigators at Massachusetts General Hospital (Boston, USA) developed a novel digital droplet PCR assay that incorporated features to improve sensitivity and allowed for the simultaneous detection and longitudinal monitoring of two TERT promoter mutations (C228T and C250T) in cfDNA from the plasma of glioma patients.

Droplet digital PCR (ddPCR) is a method of dPCR in which a 20 microliter sample reaction including assay primers and either Taqman probes or an intercalating dye, is divided into about 20,000 nanoliter-sized oil droplets through a water-oil emulsion technique, thermocycled to endpoint in a 96-well PCR plate, and fluorescence amplitude read for all droplets in each sample well in a droplet flow cytometer.

Results revealed that in baseline performance in tumor tissue, the assay had perfect concordance with an independently performed clinical pathology laboratory assessment of TERT promoter mutations in the same tumor samples. Extending to matched plasma samples, the investigators detected TERT mutations in both discovery and blinded multi-institution validation cohorts with an overall sensitivity of 62.5% and a specificity of 90% compared to the gold standard tumor tissue-based detection of TERT mutations. Upon longitudinal monitoring in five patients, they reported that peripheral TERT mutant allele frequency reflected the clinical course of the disease with levels decreasing after surgical intervention and therapy and increasing with tumor progression.

"Liquid biopsy is particularly challenging in brain tumors because mutant DNA is shed into the bloodstream at much lower level than any other types of tumors," said contributing author Dr. Leonora Balaj, investigator in neurosurgery at Massachusetts General Hospital. "By “supercharging” our ddPCR assay with novel technical improvements, we showed for the first time that the most prevalent mutation in malignant gliomas can be detected in blood, opening a new landscape for detection and monitoring of the tumors. The test is easy to use, quick, and low cost, and could be performed in most laboratories. Importantly, the test can also be used to follow the course of disease."

The glioma liquid biopsy paper was published in the October 13, 2020, online edition of the journal Clinical Cancer Research.

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