6 Pathology Research International (a) (b) Figure 4: Glutamine synthase expression is (a) restricted to perivenular (zone 3) hepatocytes in normal liver but (b) diﬀuse and strong in HCC. which can be scarce in needle biopsies. Furthermore, the method requires the sacriﬁce of a small amount of lesional tissue for RNA extraction and thus necessitates the destruc- tion of some potentially valuable histologic information. Nevertheless, in cases with an adequate amount of tissue, this qRT-PCR-based approach has the potential for considerable practical utility. If its predictive accuracy can be shown to extend to lesions that arise in cirrhosis of all etiologies, and if it can be successfully employed for the analysis of formalin- ﬁxed needle biopsies, it may become a valuable adjunct to histology for diﬀerentiating between DN and early HCCs. The gold standard for diagnosis of DN and early HCCs is still histology; in fact, internationally agreed upon histologic deﬁnitions of these lesions and a standardized nomenclature have only recently been formulated [32]. This morphologic classiﬁcation system was developed and reﬁned on the basis of meticulous histologic analyses of entire lesions within surgical resection specimens. It is recognized that needle biopsies of such lesions will often lack areas that are critical for making a histologic diagnosis of malignancy. In particular, the diagnosis of early HCCs may require identifying the invasion of lesional hepatocytes into portal tracts within the surrounding, benign parenchyma. Because biopsies frequently fail to sample such areas, molec- ular markers capable of distinguishing between dysplastic and malignant hepatocytes are urgently needed. However, because histology is still the gold standard for diagnosis, the immunohistochemical assessment of molecular markers, as an adjunct to standard H&E histology, is generally preferred over nucleic acid analysis in clinical practice. Many immunohistochemical markers have been assessed individually, but what has emerged as most useful currently is a panel of three markers: GPC3, GS, and HSP-70. Glypican- 3 was ﬁrst identiﬁed as a gene whose mRNA is frequently expressed in HCC but not in benign, adult liver, HCA or CC [38]. Numerous laboratories have since conﬁrmed the overexpression of GPC3 in HCC at the mRNA and protein levels. Its utility as an immunohistochemical marker for HCC was ﬁrst demonstrated by Yamauchi et al., who found diﬀusely positive GPC3 staining of malignant hepatocytes in 84% of HCCs and only focal, weak staining in a small set of DN [39]. Similar results were obtained by Llovet et al. in the study cited above. In an eﬀort to augment the sensitivity of GPC3 im- munostaining as a molecular test for HCC, and to retain its speciﬁcity in distinguishing DN from malignant lesions, Di Tommaso et al. examined the value of adding other tumor markers to the analysis [40]. Heat-shock protein 70 (HSP70) is an antiapoptotic, “stress response” gene that had been found through mRNA expression proﬁling to be markedly upregulated in early HCC, as compared with adjacent benign liver tissue [36]. Glutamine synthetase (GS) is a metabolic enzyme (now known to be upregulated by β- catenin signaling [41], as discussed above) that had also been shown to be overexpressed in primary liver cancer (Figure 4) [42]. When these three tumor markers, GPC3, HSP70, and GS, were applied as an immunohistochemical panel to a set of benign and malignant nodules that had been resected from cirrhotic livers, positivity for any two of the three markers was found to indicate malignancy with 72% sensitivity and 100% speciﬁcity. In a recent study, these authors used the same panel of immunostains to retrospectively analyze needle biopsies of a similar set of lesions and found that for distinguishing between high grade dysplastic nodules and well- or very well-diﬀerentiated HCC, the sensitivity of two- marker positivity as an indicator of malignancy decreased from 72% to 49%, while its speciﬁcity remained 100% [43]. This reduction in sensitivity may be attributable to sampling error and the somewhat heterogeneous expression patterns of HSP70 and GPC3 proteins within HCC. It would be