Abstract 3630: Measuring EGFr on extracellular vesicles in human plasma samples

Abstract Introduction: Extracellular vesicles (EVs), secreted extracellularly by almost all types of cells, play a pivotal role in intercellular communication and have emerged as valuable indicators in disease progression. Their potential for cancer diagnostics and monitoring is particularly noteworthy. Epidermal growth factor receptor (EGFr) is a major driver of several cancers and serve as an important target or co-target in cancer treatment. The purpose of this study was to develop an assay for measuring EGFr on EVs directly in plasma samples. Additionally three assays were developed to measure the total concentration of plasma EVs. Methods: Recognizing the expected low concentrations of EGFr on EVs, the immunoassays were developed using Single molecule array (Simoa) technology, which has shown up to a 1000-fold improvement in sensitivity as compared to conventional immunoassay approaches. Commercial available calibrator material and antibodies against surface markers (EGFr, CD9, CD63 and CD81) were used to develop four assays: CD81-EGFr, CD81-CD9, CD81-CD63, and CD9-CD63. Plasma samples from healthy individuals (n=13) and small cohorts of various cancer patients (n=30, lung, colorectal, ovarian) were used for development, validation and testing. Results: Each assay was tested for dilution linearity, limit of detection, analytical variation and pre-analytical stability of EVs. EGFr on EVs was lower in cancer patients (median: 489 × 106 EVegfr/mL) compared with healthy individuals (median: 663 × 106 EVegfr/mL). Conversely, total EV concentrations were markedly higher in plasma from cancer patients compared with healthy individuals (CD81-CD9: 9157 vs 4088, CD81-CD63: 3634 vs 1582, CD9-CD63: 9538 vs 1210, median values x106 EV/mL). Positive correlations were observed among the total EV assays (r=0.6, p<0.0001). Conclusion: The developed assays allow measurement of EGFr on EVs and total EVs in human plasma samples. Notable, total EV concentrations were highly increased in cancer as compared with healthy individuals. The assays are now ready for a comprehensive validation using large cohorts of cancer samples. Citation Format: Dorte Aa. Olsen, Torben F. Hansen, Ivan Brandslund, Jonna S. Madsen. Measuring EGFr on extracellular vesicles in human plasma samples [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 3630.