027). There was no significant difference in histologic differentiation between K19-positive and -negative HCCs. mRNA expression levels of K19, three EMT-associated genes (Snail, Slug, and Twist), and four invasion-associated genes (uPAR, VIL2, MMP1, and MMP2) were investigated in 43 HCCs of cohort 2. The log-transformed K19 mRNA levels were significantly Adriamycin clinical trial higher in K19 protein-positive HCCs than in K19 protein-negative HCCs (P = 0.048) (Fig. 3). Patients were classified into two groups (K19 mRNA high and K19 mRNA low), using the median of log-transformed K19 mRNA level as the cut-off value. The log-transformed mRNA expression levels of EMT-associated genes were positively correlated with

each other (P < 0.05), and positive correlations were observed between the log-transformed mRNA levels of invasion-associated genes (P < 0.05) (Supporting Table 4). Positive correlations were also observed between the mRNA levels of EMT-associated genes and invasion-associated genes (P < 0.05). The K19 mRNA high HCCs showed significantly higher mRNA levels of Snail (P = 0.012), Twist (P = 0.069), uPAR (P = 0.040), Ivacaftor in vitro and MMP2 (P = 0.040), whereas Slug, VIL2, and MMP1 mRNA

levels were not significantly different between the two groups. Significant positive correlations were observed between the log-transformed mRNA levels of K19 and Twist (P = 0.012), uPAR (P = 0.005), and MMP2 (P = 0.009). Univariable analysis revealed that AST >50 IU/mL, K19 expression, tumor size >5 cm, multiple tumors, major vascular invasion, microvascular invasion, and AFP >1,000 IU/mL were adverse prognostic factors for disease-free survival after resection (Table 3; Fig. 4). Multivariable analysis indicated that K19 expression, tumor size >5 cm, multiple tumors, major vascular invasion, and 上海皓元 AST>50 IU/mL were independent prognostic factors for disease-free survival after resection (Table 4). When survival analysis was performed

separately for HBV-related (n = 190) and HBV-unrelated HCCs (n = 47), K19 expression was associated with decreased disease-free survival in both etiologic groups, but statistical significance was demonstrated only in the HBV-related HCCs (P = 0.011) (Supporting Fig. 3). No significant differences in overall survival were observed for both groups. HCC with stemness-related marker expression is a recently proposed subtype of HCC in which a fraction of tumor cells (>5%) expresses stem/progenitor cell markers, but is not otherwise recognizable by routine hematoxylin-eosin (H&E) stain.16 Because this subgroup of HCCs has been reported to show more aggressive behavior, compared to conventional HCCs without stemness-related marker expression, it is important that a suitable marker is developed to facilitate its diagnosis. This subtype is different from combined hepatocellular-cholangiocarcinomas—which include the recently described combined hepatocellular-cholangiocarcinoma with stem cell features (i.e.