Sagar Rayamajhi, Jared Sipes, Bidii Ngala, Amrita Mitra, Meizhang Li, Camille V. Trinidad, Wei Cui, Mohammod Mahmudur Rahman, Foyez Ahmmed, Leonidas E. Bantis, Mihaela E. Sardiu, Dennis Province, Harsh B. Pathak, Andrew K Godwin
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 and 20 late-stage) and 40 healthy controls (HC). sEVs were enriched by size exclusion chromatography and profiled by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Across all samples, 1,078 EV-associated proteins (exo-proteins) were identified, including 52 upregulated in early-stage HGSOC vs HC, and 59 upregulated in late-stage HGSOC vs healthy controls (HC) (log 2 FC>1, p-value<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 exo-proteins upregulated in early-stage cancer disease reflect precursor lesions. A four-protein combinatorial panel (MUC1, MYL6, TTYH3, GSTP1), selected using Akaike Information Criterion (AIC), yielded an AUC of 0.975 and 90% sensitivity at 95% specificity for distinguishing early-stage HGSOC vs HC. Additionally, increased MUC1 levels in circulating sEVs were confirmed by immunoassay (AUC = 0.840 for early-stage HGSOC vs HC; AUC=0.860 for late-stage HGSOC vs HC, p-value<0.05). In summary, our sEV proteomic analysis of early-stage HGSOC reveals exo-biomarkers associated with early FT lesions, offering a promising avenue for detecting disease while it remains confined to the fallopian tube.