Abstract Background: Non-invasive biomarkers for diagnosing and prognosing hepatocellular carcinoma (HCC) are urgently needed. Cirrhosis, a consequence of deposition and cross-linking of collagens, underlies HCC in 80-90% of cases, and associates with HCC initiation and progression. We evaluated circulating cross-linked pro-peptides of type III collagen (PC3X) as a diagnostic and prognostic biomarker for HCC. Methods: PC3X was measured by ELISA in plasma from patients with HCC (n=79), cirrhosis (n=86), non-cirrhotic hepatitis-B infection (n=74) and from healthy controls (n=44). PC3X was compared to the liver fibrosis marker PRO-C3 and the HCC tumor-cell derived marker alpha-fetoprotein (AFP). Diagnostic and prognostic potential was evaluated by AUROC and by calculating hazard ratios (HR) for progression free survival (PFS) and overall survival (OS). Results: PC3X, PRO-C3 and AFP were significantly elevated in patients with HCC compared to other liver diseases and healthy controls (p=0.0002, p<0.0001). In patients with normal AFP (<20 IU/ml), PC3X and PRO-C3 separated HCC from cirrhosis with an AUROC of 0.69 and 0.67, respectively. High PC3X and AFP predicted for poor PFS (HRPC3X=1.80, p=0.032; HRAFP=1.70, p=0.031) and OS (HRPC3X=2.12, p=0.024; HRAFP=2.55; p=0.003), whereas PRO-C3 did not (PFS: HR=1.19, p=0.059 and OS: HR=1.12, p=0.324). PC3X was independent of AFP (PFS: HR=1.74, p=0.045 and OS: HR=2.21, p=0.018) and combining the two improved prognostic value (PFS: HR=2.66, p=0.004 and OS: HR=5.86, p<0.0001). Conclusions: PC3X is associated with HCC independent of AFP and provides diagnostic and prognostic value for HCC patients. If validated, this suggests that PC3X has biomarker potential for HCC. Citation Format: Christina Jensen, Signe H. Nielsen, Mohammed Eslam, Federica Genovese, Mette J. Nielsen, Roslyn Vongsuvanh, Raj Uchila, David van der Poorten, Jacob George, Morten A. Karsdal, Diana J. Leeming, Nicholas Willumsen. Cross-linked multimeric pro-peptides of type III collagen (PC3X) in hepatocellular carcinoma - A biomarker that provides additional prognostic value in AFP positive patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6484.