Rapid and ultrasensitive digital PCR (dPCR) profiling of EGFRvIII in tumor cells and tissues.

backgroundAmplification of the epidermal growth factor receptor (

methodsThe unique sequence created by the fusion of exon 1 and exon 8 in EGFRvIII was used to guide the design of primers and a Minor Groove Binder (MGB) probe. Extracted total RNA was reverse transcribed and pre-amplified by PCR, followed by detection of the EGFRvIII mutation by dPCR.

resultsThe lowest limit of quantification of our EGFRvIII assay was 0.003%. The EGFRvIII variant was identified in patient-derived glioma neurosphere cell lines, xenograft mouse model, and patient-derived tumor specimens. The overall workflow can be accomplished within 24 hours. In certain samples, EGFRvIII was detected when next-generation sequencing was unable to identify the variant. This finding highlights the ability of the dPCR assay to identify EGFRvIII mutations in heterogeneous solid tumors such as GBM in a rapid fashion by profiling samples from spatially distinct areas of tumors from the same patient.

conclusionsIn this study, we developed a highly sensitive digital PCR (dPCR) platform and leveraged our assay to detect the variant III alteration of EGFR (EGFRvIII) and amplified EGFR in patient-derived glioma neurosphere cell lines, orthotopic xenograft GBM mouse models, and patient-derived tumor specimens in less than 24 hours from minute quantities of starting material.