Pathology Research International and may soon allow better patient stratiﬁcation for treatment than current clinical HCC staging systems. In 2004, a group led by Dr. Thorgeirsson at the National Cancer Institute published the results of a genome-wide expression study of 91 HCCs showing that the tumors fell into two subclasses with distinct mRNA expression proﬁles and dramatically diﬀerent patient survival [72]. The poor survival group, designated A, displayed gene expression signatures of high cellular proliferation and low apoptotic rate. A subsequent study, in which human HCC expression proﬁles were compared with those of rodent hepatocytes (fetal hepatoblasts, adult hepatocytes, and genetically engi- neered murine HCC), led to further subdivision of group A HCCs into hepatoblast-like (HB) and mature hepatocyte-like (HC) subgroups [73]. The hepatoblast gene signature was associated with earlier recurrence and worse survival than the hepatocyte signature, and this association was independent of other pathologic variables. The authors speculated that the HB phenotype may reﬂect the origin of this subset of tumors from adult hepatic progenitor cells (HPCs), which, like fetal hepatoblasts, are able to diﬀerentiate into both cholan- giocytes and hepatocytes. Consistent with this hypothesis, several well-known HPC markers such as cytokeratins 7 and 19 (CK7 and CK19) were found to be included in the HB gene signature. The existence of two broad categories of HCCs, one with and the other without a “high-proliferation” gene expression signature, has been conﬁrmed in multiple, independent studies [71]. Most have found that tumors of the high- proliferation group are more aggressive and less histolog- ically diﬀerentiated than the group lacking this expression signature. Boyault et al. presented a remarkably detailed reﬁnement of this molecular classiﬁcation system, in which three subgroups of each major category of HCC were identiﬁed (yielding a total of six HCC subtypes) through expression analysis followed by correlation with various genetic, genomic, and clinical factors [74]. Although not yet clinically applicable, this sort of analysis helps to elucidate the biological basis of HCC heterogeneity and provides a foun- dation for future prospective studies aimed at correlating treatment responses with molecular subtype classiﬁcation. The epithelial cell adhesion molecule, EpCAM, is a known marker of fetal hepatoblasts and adult hepatic pro- genitor cells as well as biliary epithelium. In 2004, Kim et al. performed microarray studies of cirrhotic liver and found that EpCAM is dramatically overexpressed in premalignant lesions as well as in a subset of HCC [75]. Molecular characterization of the EpCAM-expressing HCC subgroup demonstrated a signature of coexpressed genes that included other HPC markers, such as c-kit and CK19 [76]. The further stratiﬁcation of tumors into four groups according to EpCAM expression and the patients’ α-fetoprotein (AFP) status (positive or negative for elevated serum AFP) was shown to deﬁne four prognostic categories, each with a characteristic gene expression pattern. Importantly, this result was replicated in three independent cohorts of HCC (all HBV-related), in one of which the EpCAM analysis was performed by immunohistochemistry, rather than mRNA hybridization. Thus, the EpCAM-AFP classiﬁcation system 9 has the potential for easy application in clinical practice. It is not yet clear whether the stratiﬁcation of AFP-positive cases according to EpCAM expression will be clinically useful as a prognostic indicator, since AFP positivity is already well- known to be associated with aggressiveness in HCC [77]. However, there is evidence that EpCAM expression correlates with biological variables that can be targeted therapeutically, and, therefore, it may prove valuable as a predictive marker of responsiveness to antiangiogenic therapies [78], Wnt/β- catenin pathway inhibitors, and/or stem-cell targeted agents such as anti-EpCAM antibodies [79]. Gene expression proﬁling has yielded novel insights into the biological heterogeneity of HCCs and has particularly highlighted the importance of hepatic progenitor cell/stem- cell characteristics in deﬁning its aggressiveness. In addition, these studies have focused attention on the roles of “cancer stem-cells” in HCCs [79]. However, much remains to be learned about the origins and behavior of hepatic progenitor cell lineages in benign and premalignant conditions, as well as their relationships to cancer stem-cells, cell-population dynamics within tumors, and the clinicopathologic rami- ﬁcations of stem-cell marker expression in diﬀerent HCC molecular subtypes (Figure 5). Although it is not yet clear which gene signatures and molecular markers will have the broadest applicability, recent rapid progress in this ﬁeld implies that the incorporation of molecular data into HCC classiﬁcation and staging systems in the near future will lead to continued improvements in the clinical management of HCC. 4.6. MicroRNA Proﬁling of HCCs. MicroRNAs (miRNAs) are short, noncoding RNA molecules that regulate gene expression by binding to speciﬁc messenger RNAs and pre- venting their translation into protein. Because each type of miRNA is able to downregulate hundreds of genes at a time, miRNAs often control entire transcriptional programs that determine fundamental cellular properties and behavior. Accordingly, miRNA proﬁling has emerged as an extremely valuable method for phenotyping and subclassifying tumors [80]. Compared to conventional gene expression proﬁling (in which protein-coding, messenger RNAs are examined), miRNA analysis has several advantages. Due to the stability of miRNAs, formalin-ﬁxed samples (rather than frozen tis- sue) can be used. Furthermore, the interrogation of hundreds of miRNAs (and often signiﬁcantly fewer) yields as much information as might be gleaned from examining thousands of messenger RNAs. Many independent groups have conducted compre- hensive analyses of miRNAs in HCC, and a plethora of informative miRNA markers have been identiﬁed. Many of these miRNA signatures correlate with important biological parameters, such as metastasis [81–83], diﬀerentiation [83– 85], HBV or HCV infection [86, 87], tumor recurrence [88], and patient survival [89–91]. In addition to providing new candidates for investigation as possible diagnostic, prognostic, and/or predictive molecular markers in HCCs, these studies are opening new avenues in basic research on the mechanisms of hepatocarcinogenesis. For example,