A comprehensive evaluation of full-spectrum cell-free RNAs highlights cell-free RNA fragments for early-stage hepatocellular carcinoma detection

BACKGROUND: Various studies have reported cell-free RNAs (cfRNAs) as noninvasive biomarkers for detecting hepatocellular carcinoma (HCC). However, they have not been independently validated, and some results are contradictory. We provided a comprehensive evaluation of various types of cfRNA biomarkers and a full mining of the biomarker potential of new features of cfRNA. METHODS: We first systematically reviewed reported cfRNA biomarkers and calculated dysregulated post-transcriptional events and cfRNA fragments. In 3 independent multicentre cohorts, we further selected 6 cfRNAs using RT-qPCR, built a panel called HCCMDP with AFP using machine learning, and internally and externally validated HCCMDP's performance. FINDINGS: We identified 23 cfRNA biomarker candidates from a systematic review and analysis of 5 cfRNA-seq datasets. Notably, we defined the cfRNA domain to describe cfRNA fragments systematically. In the verification cohort (n = 183), cfRNA fragments were more likely to be verified, while circRNA and chimeric RNA candidates were neither abundant nor stable as qPCR-based biomarkers. In the algorithm development cohort ([Formula: see text] = 287), we build and test the panel HCCMDP with 6 cfRNA markers and AFP. In the independent validation cohort ([Formula: see text] = 171), HCCMDP can distinguish HCC patients from control groups (all: AUC = 0.925; CHB: AUC = 0.909; LC: AUC = 0.916), and performs well in distinguishing early-stage HCC patients (all: AUC = 0.936; CHB: AUC = 0.917; LC: AUC = 0.928). INTERPRETATION: This study comprehensively evaluated full-spectrum cfRNA biomarker types for HCC detection, highlighted the cfRNA fragment as a promising biomarker type in HCC detection, and provided a panel HCCMDP. FUNDING: 10.13039/501100001809National Natural Science Foundation of China, and The National Key Basic Research Program (973 program).