Abstract

Cell Free DNA and Exosome Biomarkers for Liquid Biopsy Cancer Diagnostics and Early Cancer Detection

Michael Heller, Distinguished Scientist - Knight Cancer Institute, Center for Cancer Early Detection and Research (CEDAR), Oregon Health & Science University (OHSU)

Cell free (cf) DNA and exosomal RNA and proteins are now accepted as important biomarkers for liquid biopsy cancer diagnostics, and may also be used for early cancer detection. Nevertheless, the isolation of these biomarkers from patient samples requires relatively complex, time consuming and expensive procedures which greatly limits their practical use for most cancer diagnostic applications. New AC electrokinetic (ACE) microarray/chip devices (Biological Dynamics, La Jolla, CA) now allow 15-20-minute isolation of cancer related cf-DNA, exosomal RNA and protein biomarkers from 20-50ul of blood, plasma or serum. After isolation of the biomarkers, specific fluorescent dyes can be used first to simultaneously detect the different biomarker levels directly on the chip (in-situ). In a subsequent step, immunofluorescent analysis can be carried out to identify specific protein biomarkers on the exosomes. Finally, the cf-DNA and RNA (mRNAs and miRNAs release from the exosomes) can be eluted from the DEP chip, and PCR and sequencing analysis carried out to identify the cancer-related point mutations and other polymorphisms, as well as to further verify the tissue origin of the biomarkers. For glioblastoma exosomes isolated from plasma, exosome-specific surface and interior proteins CD63 and TSG101 could be detected by immuno-fluorescence, and mutated EGFRvlll mRNA was detected by RT-PCR. Exosomal related protein biomarker Glypican-1 could be isolated from pancreatic cancer patient plasma samples by ACE and then detected on-chip by immunofluorescence, and Kras mutations detected in the eluted cf-DNA. Similar results are being obtained for prostate, breast, lung and brain cancer, as well as for TBI patient samples. Thus, ACE technology represents a powerful new minimally invasive technology for cancer diagnostics that is particularly well suited for the rapid isolation of cell free nucleic acid and exosome biomarkers. The technology is setting the stage for seamless sample to answer liquid biopsy, cancer patient therapy monitoring and ultimately for early disease detection.