Analysis of histological and immunohistochemical pattern of hepatocellular carcinoma expressing cytokeratin 7 by computerized morphometry

Background and Aims: Recent studies have identified a subset of HCC containing cells expressing cytokeratin 7 (CyK7). These CyK7 positive cells potentially originates from hepatic progenitor cells and are activated in chronic liver disease. Furthermore, they could give rise to HCC. Our aim was to apply a computerized morphometric model to evaluate and quantify the morphologic features, including hepatic progenitor cells, in hepatocellular carcinoma (HCC) expressing CyK7. Methods: This study included 12 HCC expressing CyK7 from 12 patients submitted to LTx for HCV-related cirrhosis at the Niguarda Hospital in Milan. For each case, five consecutive sections 4 mm thick were obtained from the selected block. All specimens were stained with hematoxylin-eosin and immunohistochemically for CD34 to assess the degree of capillarization and reticulin to assess the cell plate architecture. Moreover, we have done a dual immunohistochemical labeling with CyK7 and Ki67 to identify respectively neoplastic cells expressing biliary marker and tumor cells that are actively dividing. We determined by a computerized morphometric model the volume fractions occupied by hepatocyte nuclei and cytoplasm, sinusoids, portal triads, capillarised sinusoids, tumor cells respectively expressing biliary marker and actively dividing. Lastly, the surface fraction occupied by reticulin was calculated. Results have been compared with a control group of 10 high-grade dysplastic nodules (DN) from 10 patients submitted to LTx for HCV-related cirrhosis. Results: Volume fractions of capillarised sinusoids, CyK7 positive cells and Ki67 positive cells were more prominent in HCC when compared to DN; surface fraction of reticulin was markedly decreased in HCC. Moreover, in HCC the dual immunohistochemical labeling performed for CyK7 and Ki67 identified a group of cells expressing both immunostains: the number of these cells correlated with both the number of cells expressing CyK7psitive and Ki67 positive (p < 0.0001). Other features most discriminatory between DN and HCC were: volume fractions occupied by hepatocytes nuclei (p < 0.0001) and cytoplasm (p < 0.001), nuclear/cytoplasmic ratio (p < 0.0001). Conclusion: Our morphometric model is an objective method of quantification of the morphologic changes in HCC expressing CyK7; moreover, cells expressing both immunostains (CyK7/Ki67) could represent cells in continuous proliferation potentially derived from hepatic progenitors cells.