Circulating Extracellular Vesicle Protein Biomarkers for the Early Detection of High-Grade Serous Ovarian Cancer

Small extracellular vesicles (sEVs), lipid-bilayer delimited particles (50-200 nm) released by cells, are emerging as a promising class of liquid biopsy biomarkers for elusive cancers, such as high-grade serous ovarian cancer (HGSOC). HGSOC originates from the fallopian tube (FT), progressing from p53 signatures to a precursor lesion known as serous tubal intraepithelial carcinoma (STIC). We hypothesize that sEVs contribute to ovarian cancer pathogenesis, carry cargo reflective of their site of origin, and serve as diagnostic biomarkers for early detection. To test this, we established a case-control cohort using archival plasma samples from 30 HGSOC patients (10 early stage [ES] and 20 late stage [LS]) and 40 healthy controls (HC). sEVs were enriched by size-exclusion chromatography and profiled by LC-MS/MS. Across all samples, 1078 EV-associated proteins (exoproteins) were identified, including 52 upregulated in ES HGSOC versus HC and 59 upregulated in LS HGSOC versus HCs (log₂ fold change >1, p < 0.05). Upregulated EV proteins were prioritized based on FT origin and tissue expression in STIC lesions. Seven candidate biomarkers (MYL6, GSTP1, TTYH3, PRDX6, MUC1, MYH14, and PTGS1) were validated by immunohistochemistry in FT tissue harboring STIC lesions and in HGSOC tissues, as well as by Western blotting in FT/HGSOC cell-derived EVs. These findings suggest that circulating exoproteins upregulated in ES cancer disease reflect precursor lesions. A four-protein combinatorial panel (MUC1, MYL6, TTYH3, and GSTP1), selected using Akaike Information Criterion, yielded an area under the curve (AUC) of 0.975 and 90% sensitivity at 95% specificity for distinguishing ES HGSOC versus HC. In addition, increased MUC1 levels in circulating sEVs were confirmed by immunoassay (AUC = 0.840 for ES HGSOC versus HC; AUC = 0.860 for LS HGSOC versus HC, p < 0.05). In summary, our sEV proteomic analysis of ES HGSOC reveals exobiomarkers associated with early FT lesions, offering a promising avenue for detecting disease while it remains confined to the FT.