High resolution profiling of EGFR mutations in glioblastoma patients using an ultrasensitive digital PCR approach

Glioblastoma Multiforme (GBM) is the most aggressive type of adult brain cancer. The average survival time after GBM diagnosis is 14.6 months even with current tri-modality therapy. The Epidermal Growth Factor Receptor (EGFR) is amplified in 57% of GBM. Mutations in EGFR such as EGFR variant III, A289V, and R108K lead to more aggressive tumors, and diminished survival. We are in dire need of a molecular assay that rapidly profiles these alterations in EGFR since other assays currently available clinically, like Next Generation Sequencing, may take up to 4 weeks due to the batching of samples in current workflows. Our lab has established a very sensitive and novel digital Polymerase Chain Reaction (dPCR) assay that detects EGFRvIII in patient tumors within 24 hours of resection. This dPCR assay utilizes RNA extracted from microgram quantities of resected tumor from GBM patients, which is then converted to complementary DNA (cDNA). cDNA is then pre-amplified and subjected to the dPCR assay using specific primers and probes for EGFRvIII and EGFR WT. The assay is multiplexed with an internal reference control, RNaseP. The same starting material can be used to detect the presence or absence of two other mutations, R108K and A289V, with exquisite sensitivity and specificity. We have utilized this assay and tested the platform on patient derived organoids and patient tumor samples. We have also validated this assay on exosomal RNA extracted from media used for culturing U87 WT and U87 vIII cell lines, as well as patient-derived glioma stem cell lines like NS039 and T4213. This assay allows for rapid and ultrasensitive detection of EGFRvIII, EGFRWT, R108K, and A289V mutations in patient tumors and patient derived organoids. The workflow for this assay allows results within 24 hours of tumor resection, which facilitates early initiation of novel investigational therapeutic agents. It is possible that molecular characterization of tumor tissue, biofluids, microvesicles, platelets, and cfRNA would help to elucidate genomic variations that occur during disease recurrence. In the future, we plan to test this assay on RNA extracted from various microvesicles and platelets derived from blood to facilitate non-invasive tumor characterization and usefully complement conventional follow-up and imaging methods.This poster was presented at the first annual Celebration of Undergraduate Research and Creative Activity while the author was an undergraduate student at Rutgers University-Camden