Extracellular vesicle-associated noncanonical ORFs as biomarkers of surgical outcome and chemoresistance in ovarian cancer

Background: High-grade serous ovarian cancer (HGSOC) is the deadliest gynecologic malignancy, with recurrence and chemoresistance driving poor survival outcomes. Beyond canonical resistance mechanisms, large portions of the genome encode non-canonical open reading frames (ncORFs) that produce short, intrinsically disordered proteins/peptides (ncPRs), largely undetectable by standard proteomics (“dark proteome”). Emerging evidence implicates ncPRs in tumor plasticity, stress adaptation, intercellular communication, and drug resistance. Their role in HGSOC chemoresistance and packaging into extracellular vesicles (EVs) remain unexplored. Methods: We analyzed RNA-seq datasets from primary tumors of 30 patients and matched EV RNA-seq from 28 patients. Cohorts included patients with complete gross resection (CGR, n = 10) and neoadjuvant chemotherapy (NACT) with excellent (NACT-ER, n = 9) or poor response (NACT-PR, n = 9). ncORF abundances were quantified using a non-canonical ORF database and associations with clinical outcomes were assessed. Results: Primary tumors and EVs showed distinct ncORF biotype distributions. exhibited a 17% decrease in coding and 10% decrease in intronic ncORFs (p < 0.0001), with 64% and 53% increases in pseudogene- and ncRNA-derived ncORFs and 3′UTR-derived ncORFs, respectively (p < 0.0001). Although fewer in number, pseudogene-derived ncORFs showed higher mean abundance than coding-derived ncORFs in EVs. Their abundance patterns trended toward discriminating CGR versus NACT patients (Fishers' exact test p = 0.004) and NACT-ER vs NACT-PR (P = 0.08). Conclusions: ncORFs, particularly pseudogene and ncRNA-derived, are selectively enriched in EVs from HGSOC patients, suggesting roles in intercellular communication and therapy response. These findings highlight EV-associated ncORFs as potential biomarkers and therapeutic targets in ovarian cancer.