Detection of urine circulating tumor DNA using droplet digital PCR for predicting hepatocellular carcinoma recurrence

Aim: This study aims to evaluate the feasibility of examining circulating tumor DNA (ctDNA) in urine samples from hepatocellular carcinoma (HCC) patients by droplet digital PCR (ddPCR) and to assess its value in predicting HCC recurrence after surgery. Methods: HCC cases who accepted surgical resection were included. Perioperative urine, tissue and peripheral blood specimens were collected. Four hotspot mutants [TP53 -rs28934571 (c.747G>T), TRET -rs1242535815 (c.1-124C>T), CTNNB1 -rs121913412 (c.121A>G), and CTNNB1 -rs121913407 (c.133T>C)] were chosen for ctDNA analysis, and mutant allele frequency (MAF) was worked out. Sanger sequencing was performed on matched tumor tissues and peripheral blood mononuclear cells (PBMCs). The patients’ clinicopathologic characteristics were retrospectively analyzed. The predictive abilities of urine ctDNA for postoperative recurrence were evaluated using the Kaplan-Meier method. Results: Forty-six patients were enrolled, and 18 patients (39.1%, 18/46) exhibited detectable circulating mutants, with the MAF in the range of 0.07% to 0.91%. The consistency test indicated moderate to substantial concordance between urine and paired tumor tissue mutations. The mutation level dropped dramatically or disappeared after surgery. Positive urine ctDNA before surgery was closely related to greater tumor size and recurrence. Kaplan-Meier curves revealed significantly shorter disease-free survival (DFS) for ctDNA-positive patients. Multivariate analysis identified detectable urine ctDNA as an independent risk factor for tumor recurrence. More than that, receiver operating characteristic (ROC) curves demonstrated that urine ctDNA had the largest area under the curve (AUC) for predicting HCC recurrence. Conclusion: Detecting ctDNA in urine using ddPCR is feasible and holds significant potential for predicting and monitoring HCC recurrence.