Tumorigenesis and Neoplastic Progression Keratin 19 Epithelial Patterns in Cirrhotic Stroma Parallel Hepatocarcinogenesis

Cirrhotic septa harbor vessels and inflammatory, fibrogenic, and ductular epithelial cells, collectively referred to as the ductular reaction (DR). Lack of the DR in the stromal compartment around hepatocellular carcinoma (HCC) has been documented; however, the relationship of epithelial keratin 19 (K19) structures to progression of intralesional carcinogenesis has not been explored. K19 immunoreactivity in the stromal compartment around 176 nodules in cirrhotic explants was examined. Quantitative differences (P < 0.0001) were manifested in three distinct histologically identifiable patterns: “complex” around cirrhotic nodules (CN), “attenuated” around dysplastic nodules (DN), and “absent” around HCC. Markers of necrosis or apoptosis could not explain the perinodular K19 epithelial loss; however, multicolor immunolabeling for K19, vimentin, E-Cadherin, SNAIL, and fibroblast-specific protein 1 (FSP-1) demonstrated discrepancies in immunophenotype and cytomorphologic features. Variability of cellular features was accompanied by an overall decrease in epithelial markers and significantly increased fractions of SNAILand FSP-1–positive cells in the DR around DN when compared with CN (P < 0.0001). Immunolabeling of transforming growth factorsignaling components (TGF R1, SMAD3, and pSMAD2/3) demonstrated increased percentages of pSMAD2/3 around DN when compared with CN (P < 0.0001). These findings collectively suggest marked alterations in cellular identity as an underlying mechanism for the reproducible extralesional K19 pattern that parallels progressive stages of intranodular hepatocarcinogenesis. Paracrine signaling is proposed as a link that emphasizes the importance of the epithelial-stromal compartment in malignant progression of HCC in cirrhosis. (Am J Pathol 2011, 179:1015–1029; DOI: 10.1016/j.ajpath.2011.04.040) In advanced chronic liver disease (cirrhosis) from virtually any cause, cirrhotic nodules (CN) containing hepatocytes are surrounded by perinodular stroma, that is, cirrhotic septa, that consists of an expanded fibrous matrix harboring lymphovascular structures, mixed inflammatory cells, and epithelial cells with ductular phenotype, the “ductular-reaction” (DR) (Figure 1A). The presence of these biliary-type epithelial cells between mesenchymal elements makes perinodular cirrhotic septa by definition an epithelial-stromal compartment (ESC) (Figures 1B; see also Supplemental Figure S1 at http://ajp. amjpathol.org). Although this ESC is the primary site for adaptations to sustained injuries (eg, fibrogenesis in chronic inflammation), the number of investigations of the ESC in hepatocarcinogenesis is limited. This is surprising because stromal invasion is a key, if not challenging, histopathologic feature used to differentiate premalignant dysplastic nodules (DN) from malignant hepatocellular carcinoma (HCC). Furthermore, one of the proposed human stem cell niches resides in the ESC and includes epithelial cells that express type-1 keratin 19 (K19) (Figure 1A), a more specific biliary marker than is type-2 keratin 7. Based on the relatively limited characterization of perinodular ESC surrounding hepatocellular nodules (HCN) in cirrhosis the present study initially correlated perinodular K19 patterns with the intranodular hepatocellular pathologic alterations and quantified a compelling relationship of intranodular and extranodular findings to demonstrate that the ESC demonstrates significant alterations in the progression from CN to DN to HCC that can func-