Genome-Scale Profiling of Circulating Cell-Free DNA Signatures for Early Detection of Hepatocellular Carcinoma in Patients with Cirrhosis

Background: Patients with liver cirrhosis are at higher risk of developing hepatocellular carcinoma (HCC). Due to the low accuracy of current methods, new non-invasive strategies for early HCC diagnosis in cirrhotic patients are needed. Methods: A case-control study recruited a total of 2,250 patients with liver cirrhosis (LC), 508 with HCC, and 476 healthy controls (CTRL), from 13 hospitals in 11 provinces of China. Four genomic features of cell-free DNA (cfDNA), including nucleosome footprint, fragmentation, base mismatch and 5-hydroxymethylcytosines were profiled. Training and validation cohorts helped develop the integrated diagnostic model, and a test cohort was used to evaluate its performance. Findings: We established a classifier of each feature for HCC against non-HCC (cirrhosis & healthy control). By integrating these four genomic features, we developed a novel diagnostic model (HIFI, 5-Hydroxymethylcytosines/base mIsmatch/Fragmentation/nucleosome footprInt), and achieved a higher accuracy in differentiating HCCs from liver cirrhosis (AUC=0.997 [0.994-0.999], sensitivity 95.42%, specificity 97.67% in test set) than AFP or PIVKA-II, irrespective of the following demographics and clinical features, age, HBV status, Child-Pugh score, BCLC stage, tumor size, and AFP status. In AFP positive (AFP>20ug/L) liver cirrhosis patients and AFP/PIVKA-II negative (AFP 400μg/L: 100% in liver cirrhosis patients; AFP<20μg/L: 93.9%, PIVKA-II<40mAU/ml: 87.9% in HCC patients). For early-stage HCC (BCLC-0/A, AJCC-I and tumor size<3cm), the accuracies of HIFI method (88.9%/94.4%, 93.3% and 96.7%) significantly outperformed AFP/PIVKA-II (AFP:22.2%/16.7%, 41.3% and 36.7%; PIVKA-II:55.6%/61.1%, 78.7% and 70.0%). Interpretation: HIFI method exhibited an excellent performance for early-stage HCC diagnosis compared with other non-invasive methods, holding great potential of HCC surveillance for cirrhotic patients. Funding Statement: This work was supported by grants from the state Key project for infectious diseases (2018ZX10732202-001), National Research Program of China (2017YFA0505803, 2017YFC0908100), National Natural Science Foundation of China (81790633, 81672860, 81702298, 61922047, 81830054, 91859205 and 81422032), National Natural Science Foundation of Shanghai (17ZR143800). Declaration of Interests: None. Ethics Approval Statement: The study protocol was reviewed and approved by the institutional review board at all participating hospitals. All participants provided written informed consent.