Plasma cell-free DNA (cfDNA) concentration. (A) Concentration of plasma cfDNA in CCA was significantly higher than normal control and patients with benign biliary disease (BBD) (p < 0.0001, Kruskal−Wallis test). (B) Concentration of cfDNA in CCA stage IV was significantly higher than stage I, II and III (p < 0.0001, Kruskal−Wallis test). (C) CCA patients with lymph node metastasis exhibited a significantly higher cfDNA concentration than CCA patients without lymph node metastasis (p = 0.005, Mann−Whitney U Test). (D) Concentration of cfDNA in CCA patients with tumor size greater than 5 cm was higher than those with tumor size lower than 5 cm (p = 0.033, Mann−Whitney U Test). (E) Concentration of cfDNA was positively correlated with tumor size of CCA patients (Spearman r = 0.28; p = 0.031). A p-value < 0.05 was considered statistically significant.