Abstract 3301: Ultrasensitive compartment-free digital phenotyping of circulating extracellular vesicles for diagnosis of Ewing sarcoma
Authors: Wen Yunjie , Vishwakarma Vikalp , Godwin Andrew K. , Zeng Yong 
Published: April 2023

Abstract
Ewing sarcoma (EWS) is the second most common pediatric osseous malignancy in the US. Vague symptoms and high prevalence of metastasis and recurrence present serious obstacles to its clinical management. Current diagnostic and monitoring modalities for EWS, including tissue biopsy, imaging, and cytogenetics have limitations in the early diagnosis and disease monitoring. Thus, EWS patients, as do other solid pediatric cancers, face unique challenges in the lack of non-invasive tests to aid with patient management. Extracellular vesicles (EVs), including exosomes, are emerging as a new paradigm of liquid biopsy for non-invasive cancer diagnosis and monitoring owing to their informative cargoes and imperative biological functions. However, limited progresses have been reported in the field of EWS-related EV diagnostics. To address this gap, we developed a new microfluidics-based digital immunoassay platform, termed μTUNER (microfluidic Topographic modUlation and iNtensification of Enzymatic Reaction), for ultrasensitive single-molecular analysis of EV protein biomarkers and liquid biopsy-based diagnosis of EWS. Through constructing nanoconfinements with tunable sizes, this technology affords programmable patterning and modulation of surface enzymatic reactions in a non-contact manner. Such unique capacity enables us to develop a compartment-free digital immunoassay platform which is conceptually distinct from the existing digital assays. As a proof-of-concept for clinical applications, we adapted the μTUNER digital immunoassay platform to quantitatively evaluate four exo-protein biomarkers (CD99, ENO-2, NGFR, and Ezrin) that we identified by extensive proteomics analysis of EWS cell line-derived EVs (PMID: 32821345). Using protein standards and EWS cell line-derived EVs, we demonstrated that our technology affords sensitive and quantitative detection of exo-protein biomarkers with LODs at the fg/mL level, which warrants subsequent assessment of its adaptability to clinical EWS diagnosis. EV profiling of plasma samples from adult and pediatric EWS patients (n = 16) and matched healthy controls (n = 17) reveals unique expression patterns of the four markers. These exo-protein biomarkers individually afforded excellent diagnostic performance (AUC, CD99: 0.992; ENO-2: 0.985; NGFR: 0.996; Ezrin: 0.978) to differentiate the EWS and control groups. The unweighted SUM signature of the four proteins further improves the diagnostic power to yield an AUC of 1.00. Meanwhile, the distinctive EV signature obtained from the machine learning-based analysis permits further classification of control, adult, and pediatric patients with an overall accuracy of 97%. Based on these results, we envision that our technology combined with the novel exo-protein biomarker signature can be useful clinically to improve the diagnosis of patients with Ewing sarcoma.
Citation Format: Yunjie Wen, Vikalp Vishwakarma, Andrew K. Godwin, Yong Zeng. Ultrasensitive compartment-free digital phenotyping of circulating extracellular vesicles for diagnosis of Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3301.