Identification of novel biomarkers for high grade serous ovarian cancer screening via extracellular vesicle proteomic profiling

Abstract Introduction: Ovarian cancer is a silent killer that rarely shows symptoms and therefore is detected at later stages when treatments are less effective. Developing non-invasive blood tests for pre-symptomatic screening and early detection is crucial. Recent studies support the paradigm that high grade serous carcinomas (HGSOCs), the most common and deadliest form of ovarian cancers, arise from the epithelial cells within the fimbriated end of the fallopian tubes (FT). The goal of this study was to uncover novel protein biomarkers associated with tumor-derived extracellular vesicles (EVs) originating from the FT epithelial cells to advance liquid-based assays for the early detection of HGSOC. We hypothesize that EV-associated proteins (exo-proteins), i.e., lineage biomarkers, shared between the progenitor cells residing in the FT and tumor cells residing in cancerous tissue will allow for detection of the earliest forms of HGSOC. Methods: Differential ultracentrifugation was used to enrich EVs from FT and HGSOC cell lines (n=3 & n=5) and healthy FT and HGSOC tissue explants (n=6 & n=15), respectively. Unbiased proteomic profiling of EVs was performed by LC-MS/MS. Results: We report for the first time 985 proteins that compose the FT/HGSOC EV core proteome. We used bioinformatic pipelines to identify high-quality candidate exo-biomarkers for further validation. We selected exo-proteins that were predicted to have at least one transmembrane domain which could serve as an antigen for immunoaffinity capture. We prioritized proteins that showed a log2 fold-change ≥ -0.58 to identify potential biomarkers present in FT-associated EVs which were elevated as the disease progresses to HGSOC. A ranked list of 45 predicted transmembrane exo-proteins was screened using Simple Western assays for expression in FT and HGSOC cell line-derived EVs. Expression of the top 6 candidate biomarkers (i.e., ACSL4, IGSF8, ITGA2, ITGA5, ITGB3 and MYOF) were confirmed in FT and HGSOC tissue sample by immunohistochemistry using tissue microarray (n=100). Next, we analyzed plasma samples from a small case-control study using our ExoProfile chip (e.g., PMID 31293733) and found these 6 exo-biomarkers, plus folate receptor alpha exhibited higher levels in the HGSOC plasma relative to matched healthy controls. We performed receiver operating characteristic (ROC) analyses and found that all 6 FT/HGSOC shared exo-biomarkers exhibit an overall classification performance ranging from 0.85-0.98 as reflected by the area under the curve (AUC). Furthermore, when we used a linear combination of IGSF8 and ITGA5 based on logistic regression analysis, we achieved a sensitivity of 80% at 99.8% specificity. Conclusion: Validated lineage-associated exo-protein biomarkers can be used in liquid-based assays to detect ovarian cancer while localized to the FT/ovary and when patient outcomes are more favorable. Citation Format: Camille V. Trinidad, Harsh B. Pathak, Shibo Cheng, Rashna Madan, Mihaela E. Sardiu, Leonidas E. Bantis, Yong Zeng, Andrew K. Godwin. Identification of novel biomarkers for high grade serous ovarian cancer screening via extracellular vesicle proteomic profiling [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 3351.