Abstract 3746: Identification of cancer specific extracellular vesical biomarkers to aid in diagnosis of cancer
Authors: Hembrough Todd A. , Opheim Zachary , Bandoski Cheryl , Isaac Issa , Mehnert Martin , Ezrin Alan 
Published: March 2024

The limitations of serial tumor biopsies are well known, thus liquid biopsies which allow for repeated plasma-based biomarker analysis are urgently needed. Extracellular vesicles (EVs) represent an extremely stable and emerging proteomic substrate which allows for deep enrichment of biologically relevant signals from plasma, and broad analysis of tumor associated and host response proteins. To identify novel biomarkers, we performed proteomics analysis on EVs purified using size exclusion chromatography and a proprietary buffer system which increases EV and corona protein recovery. Initial DIA mass spectrometry (MS) analysis was performed on EVs from 125 patients (25 each from CRC, breast, NSCLC, ovarian, and normal patients). A subset of 25 normal and 25 ovarian was subjected to a second deeper analysis on a much more powerful DIA platform. Initial analysis identified 204 unique proteins differentially expressed in ovarian cancer patients (&gt;2-fold change; p&lt;0.05). Repeat analysis of the ovarian vs normal cohorts on the Biognosys TrueDiscovery™ platform identified 645 differentially expressed, a significant increase in depth of analysis. These cancer exosome associated proteins represent a broad range of host response signals. Taking ~20 of the top proteins, we extended our analysis using ELISAs to determine if the results by MS were reproducible in a simpler format. The ELISA analysis recapitulated the MS results. This confirms that the TrueDiscovery™ platform can easily be translated into a simple, quick and distributable ELISA platform. Extending these results, a blinded cohort of ovarian and normal EVs was analyzed using a predefined cutoff for a host immune response biomarker. This blinded analysis demonstrated sensitivity of 1.0 and specificity of 0.9, exceptionally high values for a single analyte assay, underscoring the potential value of analyzing the host/immune response proteins in the context of the EV microenvironmentKey biomarkers from cancer patient EVs suggests it is possible to track host immunosuppressive environment via antigen presenting cell and check point antagonist biomarkers. In both cases, highly significant differences are seen comparing EVs from normal patient plasma versus cancer patient plasma. Interestingly, when these biomarkers are evaluated in pre-EV enriched plasma, no significant differences are seen. This underscores the unique nature of the EV microenvironment, and suggests that host immune response and tumor microenvironment may be recapitulated in the EV/host response proteome using our unique methods. These data confirm that the EV microenvironment represents a unique oncological ecosystem with highly specific and sensitive disease biomarkers which are not dysregulated in complete plasma. This novel approach to assessing cancer proteomics has great promise for real time, longitudinal analysis of cancer patient tumor/host immune biology.

Citation Format: Todd A. Hembrough, Zachary Opheim, Cheryl Bandoski, Issa Isaac, Martin Mehnert, Alan Ezrin. Identification of cancer specific extracellular vesical biomarkers to aid in diagnosis of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3746.