Early-stage multi-cancer detection using an extracellular vesicle protein-based blood test→

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In this case-controlled pilot study, the authors explored a cohort of 139 pathologically staged patients with stage 1 and stage 2 pancreatic, ovarian, and bladder cancers. Biological Dynamics’ Verita™ proprietary platform detected 96 percent of stage 1 pancreatic cancers and three-quarters of stage 1 ovarian cancers using isolated exosomes and AI-enabled protein marker analysis. 

Juan Pablo Hinestrosa, Razelle Kurzrock, Jean M. Lewis, Nicholas J. Schork, Gregor Schroeder, Ashish M. Kamat, Andrew M. Lowy, Ramez N. Eskander, Orlando Perrera, David Searson, Kiarash Rastegar, Jake R. Hughes, Victor Ortiz, Iryna Clark, Heath I. Balcer, Larry Arakelyan, Robert Turner, Paul R. Billings, Mark J. Adler, Scott M. Lippman, Rajaram Krishnan. doi.org/10.1038/s43856-022-00088-6

ABSTRACT

Background: Detecting cancer at early stages significantly increases patient survival rates. Because lethal solid tumors often produce few symptoms before progressing to advanced, metastatic disease, diagnosis frequently occurs when surgical resection is no longer curative. One promising approach to detect early-stage, curable cancers uses biomarkers present in circulating extracellular vesicles (EVs). To explore the feasibility of this approach, we developed an EV-based blood biomarker classifier from EV protein profiles to detect stages I and II pancreatic, ovarian, and bladder cancer.

Methods: Utilizing an alternating current electrokinetics (ACE) platform to purify EVs from plasma, we use multi-marker EV-protein measurements to develop a machine learning algorithm that can discriminate cancer cases from controls. The ACE isolation method requires small sample volumes, and the streamlined process permits integration into high-throughput workflows.

Results: In this case-control pilot study, comparison of 139 pathologically confirmed stage I and II cancer cases representing pancreatic, ovarian, or bladder patients against 184 control subjects yields an area under the curve (AUC) of 0.95 (95% CI: 0.92 to 0.97), with sensitivity of 71.2% (95% CI: 63.2 to 78.1) at 99.5% (97.0 to 99.9) specificity. Sensitivity is similar at both early stages [stage I: 70.5% (60.2 to 79.0) and stage II: 72.5% (59.1 to 82.9)]. Detection of stage I cancer reaches 95.5% in pancreatic, 74.4% in ovarian (73.1% in Stage IA), and 43.8% in bladder cancer.

Conclusion: This work demonstrates that an EV-based, multi-cancer test has potential clinical value for early cancer detection and warrants future expanded studies involving prospective cohorts with multi-year follow-up.

Simultaneous Isolation of Circulating Nucleic Acids and EV-associated Protein Biomarkers From Unprocessed Plasma Using an AC Electrokinetics-Based Platform→

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In this article, the authors used Biological Dynamics technology based on AC Electrokinetics (ACE) for the simultaneous, rapid detection and isolation of cell-free DNA (cfDNA), extracellular vesicle RNA (EV-RNA), and EV-associated protein biomarkers directly from human biofluids.

Juan P. Hinestrosa, David J. Searson, Jean M. Lewis, Alfred Kinana, Orlando Perrera, Irina Dobrovolskaia, Kevin Tran, Robert Turner, Heath I. Balcer, Iryna Clark, David Bodkin, Dave S. Hoon and Rajaram Krishnan. doi.org/10.3389/fbioe.2020.581157.

ABSTRACT

The power of personalized medicine is based on a deep understanding of cellular and molecular processes underlying disease pathogenesis. Accurately characterizing and analyzing connections between these processes is dependent on our ability to access multiple classes of biomarkers (DNA, RNA, and proteins)—ideally, in a minimally processed state. Here, we characterize a biomarker isolation platform that enables simultaneous isolation and on-chip detection of cell-free DNA (cfDNA), extracellular vesicle RNA (EV-RNA), and EV-associated proteins in unprocessed biological fluids using AC Electrokinetics (ACE). Human biofluid samples were flowed over the ACE microelectrode array (ACE chip) on the Verita platform while an electrical signal was applied, inducing a field that reversibly captured biomarkers onto the microelectrode array. Isolated cfDNA, EV-RNA, and EV-associated proteins were visualized directly on the chip using DNA and RNA specific dyes or antigen-specific, directly conjugated antibodies (CD63, TSG101, PD-L1, GPC-1), respectively. Isolated material was also eluted off the chip and analyzed downstream by multiple methods, including PCR, RT-PCR, next-generation sequencing (NGS), capillary electrophoresis, and nanoparticle size characterization. The detection workflow confirmed the capture of cfDNA, EV-RNA, and EV-associated proteins from human biofluids on the ACE chip. Tumor specific variants and the mRNAs of housekeeping gene PGK1 were detected in cfDNA and RNA isolated directly from chips in PCR, NGS, and RT-PCR assays, demonstrating that high-quality material can be isolated from donor samples using the isolation workflow. Detection of the luminal membrane protein TSG101 with antibodies depended on membrane permeabilization, consistent with the presence of vesicles on the chip. Protein, morphological, and size characterization revealed that these vesicles had the characteristics of EVs. The results demonstrated that unprocessed cfDNA, EV-RNA, and EV-associated proteins can be isolated and simultaneously fluorescently analyzed on the ACE chip. The compatibility with established downstream technologies may also allow the use of the platform as a sample preparation method for workflows that could benefit from access to unprocessed exosomal, genomic, and proteomic biomarkers.

ARTICLE INFORMATION