Development of biofluid-based molecular diagnostic tests for cancer is an important step towards tumor characterization and real-time monitoring in a minimally invasive fashion. Extracellular vesicles (EVs) are released from tumor cells into body fluids and can provide a powerful platform for tumor biomarkers because they carry tumor proteins and nucleic acids. Detecting rare point mutations in the background of wild-type sequences in biofluids such as blood and cerebrospinal fluid (CSF) remains a major challenge. Techniques such as BEAMing (beads, emulsion, amplification, magnetics) PCR and droplet digital PCR (ddPCR) are substantially more sensitive than many other assays for mutant sequence detection. Here, we describe a novel approach that combines biofluid EV RNA and BEAMing RT-PCR (EV-BEAMing), as well droplet digital PCR to interrogate mutations from glioma tumors. EVs from CSF of patients with glioma were shown to contain mutant IDH1 transcripts, and we were able to reliably detect and quantify mutant and wild-type IDH1 RNA transcripts in CSF of patients with gliomas. EV-BEAMing and EV-ddPCR represent a valuable new strategy for cancer diagnostics, which can be applied to a variety of biofluids and neoplasms.
Bibliographical noteFunding Information:
This work was supported by NIH/NCI grants CA069246 (F.H., X.O.B., B.C.); CA141226 (X.O.B.); CA156009 (X.O.B.); and CA141150 (X.O.B.); Brain Tumor Funders’ Collaborative (B.C., X.O.B.); American Brain Tumor Association (ABTA; J.S.); Harvard Catalyst (B.C.); A Reason To Ride research fund (E.T.W.); Hyugens Scholarship NL (L.B.), the Richard Floor Biorepository and the Accelerate Brain Cancer Cure (ABC2). All processing of samples by Exosome Diagnostics Inc and RainDance Technologies was carried out on de-identified samples. We thank Suzanne McDavitt for skilled editorial assistance. We thank N Agrawal for his advice and for providing us with BEAMing reagents and A Mandinova for providing access to the TissueLyser. We thank Karen Messer for help with statistical analysis. We also thank Winston Patrick Kuo and Fatemeh Momen-Heravi for help with TEM and Mikkel Noerholm for help with probe design. J.S. is an inventor on the microvesicle technology used in this study which has been licensed to Exosome Diagnostics, Inc. He holds equity in, and is an employee of that company. Breakefield and Carter are on the Scientific Advisory Board of the company for which they receive cash compensation. M.L.S. and S.K.K. are employees of RainDance Technologies Inc. The other authors declared no conflict of interest.
- BEAMing PCR
- Droplet digital PCR
- Extracellular vesicles