The variability in the prognosis of individuals with hepatocellular carcinoma (HCC) suggests that HCC may comprise several distinct biological phenotypes. These phenotypes may result from activation of different oncogenic pathways during tumorigenesis and/or from a different cell of origin. Here we address whether the transcriptional characteristics of HCC can provide insight into the cellular origin of the tumor. We integrated gene expression data from rat fetal hepatoblasts and adult hepatocytes with HCC from human and mouse models. Individuals with HCC who shared a gene expression pattern with fetal hepatoblasts had a poor prognosis. The gene expression program that distinguished this subtype from other types of HCC included markers of hepatic oval cells, suggesting that HCC of this subtype may arise from hepatic progenitor cells. Analyses of gene networks showed that activation of AP-1 transcription factors in this newly identified HCC subtype might have key roles in tumor development.

D espite decades of research, the processes that govern liver development and regeneration are only partially understood. A good understanding of the mechanisms that play a role in these processes is important because it may lead to new treatments of human liver diseases. Several in vivo experimental conditions have been used to study liver regeneration and repair and the interaction of different cell types in these processes. These include partial hepatectomy, administration of toxic compounds, or a combination of both, and transgenic expression of certain proteins. 1,2 In contrast to in vitro experiments, these approaches raise fewer questions concerning the influence of artificial matrices on the function and behavior of hepatocytes and other liver cell types.Although these procedures have generated useful information on rodent liver regeneration, stem cell activation, and other processes, extrapolating these findings to the Abbreviations: uPA, urokinase plasminogen activator; HBV, hepatitis B virus; HCV, hepatitis C virus; HBsAg, hepatitis B surface antigen; HBeAg, hepatitis B e antigen; PAS, periodic-acid-Schiff. From the

In our series, 28% of HCCs contained cells expressing CK7 and/or CK19. They potentially derive from HPCs. The higher recurrence rate of CK19+ HCC after transplantation suggests a worse prognosis for these HCCs compared with CK19- HCC.

Distinguishing small hepatocellular carcinoma (HCC) from other types of small focal lesions that occur in a cirrhotic liver can be difficult on the basis of morphologic features alone. We investigated whether the expression of glypican-3 (GPC3) could be an ancillary tool in the histopathologic diagnostic process. We performed immunohistochemistry for GPC3 on 16 low-grade dysplastic nodules, 33 high-grade dysplastic nodules, 13 focal nodular hyperplasia-like nodules, and 59 HCCs with a diameter less or equal to 3 cm present in the cirrhotic liver of 66 patients. Both resected lesions and lesions biopsied by needle were included and nonlesional cirrhotic parenchyma was also stained. In a subset of cases (23 samples of cirrhosis, 4 low-grade dysplastic nodules, 5 high-grade dysplastic nodules, 2 focal nodular hyperplasia-like nodules, and 18 HCCs), real time reverse transcriptase-polymerase chain reaction for GPC3 was performed. GPC3 expression was, both on immunohistochemistry and by real time reverse transcriptase-polymerase chain reaction, much higher in small HCCs than in cirrhosis and other types of small focal lesions, indicating that the transition from premalignant lesions to small HCC is associated with a sharp increase of GPC3 expression in a majority of cases. The sensitivity and specificity of a positive GPC3-staining for the diagnosis of HCC in small focal lesions was 0.77 and 0.96, respectively, in resected cases, and 0.83 and 1, respectively, for needle biopsies. Because the result of the staining was easily interpretable, immunohistochemistry for GPC3 is valuable ancillary tool in the histopathologic diagnosis of small focal lesions in cirrhosis.

Hepatocarcinogenesis is a multistage process in which precursor lesions progress into early hepatocellular carcinomas (eHCC) by sequential accumulation of multiple genetic and epigenetic alterations. To decode the molecular events during early stages of liver carcinogenesis, we performed gene expression profiling on cirrhotic (regenerative) and dysplastic nodules (DN), as well as eHCC. Although considerable heterogeneity was observed at the regenerative and dysplastic stages, overall, 460 differentially expressed genes were detected between DN and eHCC. Functional analysis of the significant gene set identified the MYC oncogene as a plausible driver gene for malignant conversion of the DNs. In addition, gene set enrichment analysis revealed global activation of the MYC up-regulated gene set in eHCC versus dysplasia. Presence of the MYC signature significantly correlated with increased expression of CSN5, as well as with higher overall transcription rate of genes located in the 8q chromosome region. Furthermore, a classifier constructed from MYC target genes could robustly discriminate eHCC from high-grade and lowgrade DNs. In conclusion, our study identified unique expression patterns associated with the transition of highgrade DNs into eHCC and showed that activation of the MYC transcription signature is strongly associated with the malignant conversion of preneoplastic liver lesions. [Cancer Res 2009;69(7):2775-82]

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An increase in bile ductular structures is observed in diverse human liver diseases. These structures harbour the progenitor cell compartment of the liver. Since ATP-binding cassette (ABC) transporters may have a cytoprotective role in liver disease, an immunohistochemical study was performed on human liver specimens from patients with primary biliary cirrhosis (PBC), chronic hepatitis C virus (HCV) infection, submassive cell necrosis, and normal liver. The expression of MDR1, MDR3, BSEP, MRP1, MRP2, and MRP3 was determined using specific antibodies. Dilution series were constructed to determine the critical staining level in order to estimate the factor of up-regulation. In normal liver, hepatocytes showed canalicular staining for MDR3, BSEP, and MRP2. MDR1 stained the canalicular membrane of hepatocytes as well as that of cholangiocytes. MRP3 showed low immunoreactivity of bile duct epithelial cells and centrilobular hepatocytes only. Normal liver showed no immunoreactivity for MRP1. In diseased liver, the expression of MDR3, BSEP, and MRP2 was relatively stable. In PBC, HCV, and submassive necrosis, the expression levels of MDR1, MRP1, and MRP3 were increased. The strongest immunoreactivity was seen after submassive necrosis, where remaining islands of hepatocytes showed strong canalicular staining for MDR1 and MRP3. Regenerating bile ductules at the interface of portal tracts and necrotic areas stained intensely for MDR1, MRP1, and MRP3. In conclusion, MDR1, MRP1, and MRP3 are up-regulated in hepatocytes in severe human liver disease. Strong MDR1, MRP1, and MRP3 reactivity is seen in regenerating human bile ductules.

Ductular reaction and putative progenitor cells (or 'progenitor cells'), which are presumed to be the human counterpart of the oval cells in rat liver, have been discerned in various human liver diseases, including chronic viral hepatitis. Since in experimental models of chronic hepatitis the activation of oval cells is correlated with the inflammatory infiltrate, this study investigated whether there is a correlation in chronic viral hepatitis between the number of 'progenitor cells' extending into the lobule and the severity of parenchymal inflammation, on the one hand, and the extent of ductular reaction and the severity of interface hepatitis, on the other hand. Liver biopsies of 55 patients with chronic hepatitis B and/or C were used. The severity of parenchymal inflammation and of interface hepatitis was semiquantitatively graded on a haematoxylin and eosin-stained paraffin section, while the number of 'progenitor cells' and the extent of the ductular reaction were assessed on a serial section stained for cytokeratin (CK) 7. In addition, more extensive phenotyping of 'progenitor cells' was performed on sections from frozen material from five patients, using antibodies against CK7, CK8, CK18, CK19, chromogranin-A, and the rat oval cell marker OV-6. The number of more centrally located 'progenitor cells' correlated significantly with the severity of the parenchymal inflammation, while the extent of the ductular reaction correlated significantly with the severity of interface hepatitis. These findings suggest that in chronic viral hepatitis, inflammation plays a role in 'progenitor cell' activation and its topography. In cases with moderate and severe lobular inflammation, 'progenitor cells' were strikingly scattered throughout the parenchyma and surrounded by intermediate hepatocyte-like cells, suggesting their migration into the parenchyma and their differentiation towards the hepatocytic lineage.