Heterogeneity of Epigenetic and Epithelial Mesenchymal Transition Marks in Hepatocellular Carcinoma with Keratin 19 Proficiency

<b><i>Objective:</i></b> Keratin 19 (K19) expression is a potential predictor of poor prognosis in patients with hepatocellular carcinoma (HCC). To clarify the feature of K19-proficient HCC, we traced epigenetic footprints in cultured cells and clinical materials. <b><i>Patients and Methods:</i></b> In vitro, <i>KRT19</i> promoter methylation was analyzed and 5-aza-2’-deoxycytidine with trichostatin A (TSA) treatment was performed. Among 564 surgically resected HCCs, the clinicopathological relevance of K19-proficent HCCs was performed in comparison with hepatocytic (HepPar-1 and arginase-1), epithelial-mesenchymal transition (E-cadherin and vimentin), biliary differentiation-associated (K7 and NOTCH-1) markers, and epigenetic markers (<i>KRT19</i> promoter/long interspersed nucleotide element-1 [<i>LINE-1</i>] methylation status). <b><i>Results:</i></b> <i>KRT19</i> promoter methylation was clearly associated with K19 deficiency and 5-aza-2’-deoxycytidine with TSA treatment-stimulated K19 re-expression, implicating DNA methylation as a potential epigenetic process for K19 expression. After excluding HCCs with recurrence, TNM stage as IIIB or greater, preoperative therapy, transplantation, and combined hepatocellular cholangiocarcinoma, we assessed 125 of 564 HCC cases. In this cohort, K19 expression was found in 29 HCCs (23.2%) and corresponded with poor survival following surgery (<i>p</i> = 0.025) and extrahepatic recurrence-free survival (<i>p</i> = 0.017). Compared with K19-deficient HCCs, lower <i>KRT19</i> promoter methylation level was observed in K19-proficient HCCs (<i>p</i> < 0.0001). Conversely, HCC with genome-wide <i>LINE-1</i> hypermethylation was frequently observed in K19-proficient HCCs (<i>p</i> = 0.0079). Additionally, K19 proficiency was associated with K7 proficiency (<i>p</i> = 0.043), and reduced E-cadherin and HepPar-1 expression (<i>p</i> = 0.043 and <i>p</i> < 0.0001, respectively). <b><i>Conclusions:</i></b> K19-proficient HCC exhibited poor prognosis owing to extrahepatic recurrence, with molecular signatures differing from those in conventional cancer stem cells, providing novel insights of the heterogeneity underlying tumor development.