Liquid biopsy by combining 5-hydroxymethylcytosine signatures of plasma cell-free DNA and protein biomarkers for diagnosis and prognosis of hepatocellular carcinoma

BACKGROUND: Liquid biopsy based on 5-hydroxymethylcytosine (5hmC) signatures of plasma cell-free DNA (cfDNA) originating from tumor cells provides a novel approach for early diagnosis in hepatocellular carcinoma (HCC). Here, we sought to develop a reliable model using cfDNA 5hmC signatures and protein biomarkers for diagnosis and prognosis of HCC. PATIENTS AND METHODS: We carried out genome-wide 5hmC sequencing of cfDNA samples collected from 165 healthy volunteers, 62 liver cirrhosis (LC) patients and 135 HCC patients. A sensitive 5hmC diagnostic model was developed based on 5hmC signatures selected by sparse Partial Least Squares Discriminant Analysis and cross-validation to define the weighted diagnostic score (wd-score). Then, we combined protein biomarkers with the wd-score to build a more robust score (HCC score) by logistic regression. RESULTS: The distribution pattern of differential 5hmC regions could clearly distinguish HCC patients, LC patients and healthy volunteers. The wd-score based on 64 5hmC signatures in cfDNA achieves 93.24% of area under the curve (AUC) to distinguish HCC patients from non-HCC patients, and the HCC score by combing protein biomarkers achieves 92.75% of AUC to distinguish HCC patients from LC patients. Meanwhile, the HCC score showed high capacity for screening high recurrence risk patients after receiving surgical resection, and appeared to be an independent indicator for both relapse-free survival (P = 0.00865) and overall survival (P = 0.000739). Furthermore, the values of the HCC score in patients' longitudinal plasma samples were positively associated with tumor burden dynamics during follow-up. CONCLUSION: We have developed and validated a novel non-invasive liquid biopsy strategy for HCC diagnosis, prognosis and surveillance during HCC progression.