Highly sensitive EGFRvIII detection in circulating extracellular vesicle RNA of glioma patients

Liquid biopsy offers an attractive platform for non-invasive tumor diagnosis, prognostication and prediction of glioblastoma clinical outcomes. Prior studies report that 30-50% of GBM lesions characterized by EGFR amplification also harbor the EGFRvIII mutation.

Researchers at Massachusetts General Hospital have developed a novel digital droplet PCR (ddPCR) assay for high GC content amplicons and optimized it for sensitive detection of EGFRvIII in tumor tissue and circulating extracellular vesicle RNA (EV RNA) isolated from the plasma of glioma patients.

This optimized qPCR assay detected EGFRvIII mRNA in 81% (95% CI, 68% – 94%) of EGFR amplified glioma tumor tissue, indicating a higher than previously reported prevalence of EGFRvIII in glioma. Using the optimized ddPCR assay in discovery and blinded validation cohorts, the researchers detected EGFRvIII mutation in 73% (95% CI, 64% – 82%) of patients with a specificity of 98% (95% CI, 87% – 100%), compared with qPCR tumor tissue analysis. Additionally, upon longitudinal monitoring in 4 patients, they report detection of EGFRvIII in the plasma of patients with different clinical outcomes, rising with tumor progression, and decreasing in response to treatment.

Detection of EGFRvIII mutation in plasma samples of patient cohort (n=3)

(a) Schematic depicting experimental workflow, including isolation of plasma, extraction platform, reverse transcription, purification and subsequent ddPCR amplification. (b) Copies / ml of EGFRvIII mutation in tested plasma samples are graphed according to sample number. Oncoprint depicting the demographic characteristics and genomic landscape of each sample are plotted underneath. (c) Contingency tables were constructed from data obtained, and sensitivity and specificity were calculated and graphed above. Overall sensitivity and specificity across all three cohorts are also reported.  

This study demonstrates the feasibility of detecting EGFRvIII mutation in plasma using a highly sensitive and specific ddPCR assay. We also show a higher than previously reported EGFRvIII prevalence in glioma tumor tissue. Several features of the assay are favorable for clinical implementation for detection and monitoring of EGFRvIII positive tumors.