Pathologists are often faced with similar problem with specimens derived from hepatic tumors or tumor like lesions. Hepatocellular carcinoma (HCC) is the most common type of primary malignant liver tumor. Less differentiated HCCs can be mistaken for biliary or metastatic carcinomas. It also can be challenging, especially in case of highly differentiated tumors, to distinguish these from dysplastic nodules or hepatocellular adenomas. Hepatocyte paraffin (Hep Par)-1 and CD 10 antibodies stain hepatocytes and hepatocyte derived tumors in paraffin embedded tissue [1,2]. Thus, they help to identify the hepatocytic origin of a tumor but do not reflect whether it is benign or malignant. In addition, they may be negative in poorly differentiated HCC [3]. Glypican-3, alpha-fetoprotein (AFP), heat shock protein 70 (Hsp70) glutamine synthetase, clathrin heavy chain [3-7] antibodies are reported to be distinctive for HCC, but none of these antibodies are flawless. They are occasionally positive in non malignant liver or in non HCC tumors [8]. Delta like protein (DLK) is a new sensitive and specific marker, which can be used together with AFP to diagnose hepatoblastoma [9]. Cholangiocarcinomas, (CC) also pose difficulties in diagnosis. They are usually adenocarcinomas and therefore their distinction from metastatic tumors or sometimes from HCC is a common problem. It can also be difficult to distinguish highly differentiated cholangiocarcinomas from ductular reactions or biliary hamartomas. Recently αvβ6 integrin has been reported to be a highly specific imunohistochemical marker for cholangiocarcinoma [10] but this antigen is also present in reactive biliary proliferations. In summary, the panel of antibodies available for immunohistochemical diagnosis of hepatic tumors has expanded substantially in the last few years. Due to focal staining patterns and cross-reactions with other tissues, diagnostic difficulties are still commonly encountered in this field, and form the basis of the ongoing search for newer and better immunomarkers.

Formalin-fixed paraffin-embedded tissue was used for the immunohistochemical reactions. Staining was performed using an automated Leica Bond immunostainer, with the Leica Bond Polymer refine detection system and 3,3' Diaminobenzidine (DAB) as the chromogen. Antigen retrieval was achieved with Bond Epitope Retrieval Solution 2 (high pH) for 20 minutes. The primary antibody was a mouse monoclonal anti-EZH2 (clone 11/EZH2) from BD Biosciences (San Jose CA, USA) (dilution 1:100). The reaction resulted in nuclear staining. Scores were assigned based on the density of positivity by using negative (score=0, no staining), weak (score=1, <25% of nuclei staining), moderate (score=2, 25-75% of nuclei staining) and strong (score=3, >75% of nuclei staining).

Knockdown of EZH2 reversed the tumorigenicity in experimental liver tumors, suggesting that EZH2 plays an important role in HCC tumorigenesis [23,24]. Increased expression of EZH2 was correlated with unfavourable outcome of HCC [25] and metastatic capacity [26]. Cai et al. [11] demonstrated convincingly that EZH2 is a highly sensitive diagnostic biomarker of HCC, which can be used to distinguish it from benign liver lesions such as hepatocellular adenomas, focal nodular hyperplasias, dysplastic and regenerative nodules. There is a complete agreement between the findings of Cai et al’s [11] and our study that all the investigated regenerative nodules and adenomas were negative. Our results also support that EZH2 is a sensitive marker of HCC. Although not reaching statistical significance, EZH2 appeared less able to recognize well differentiated HCCs in both studies. There seems to be a borderline or “grey zone” in highly differentiated hepatocellular tumors, in which EZH2 similarly to the other markers, is not absolutely reliable. It requires further direct comparative studies, which one of the recently applied markers (Glypican3, AFP, Hsp70, EZH2) is the most sensitive [3-7]. Most likely however, a panel of these antibodies would provide the most trustworthy information and EZH2 could be a useful member of this battery. All of the adenomas in our study were beta-catenin negative. The beta-catenin status of the adenomas in the study of Cai et al. [11] is not indicated. It would be of interest to examine EZH2 expression in beta-catenin positive hepatocellular adenomas, which have a higher tendency for malignant transformation [27].

In addition to hepatocellular lesions, there are other hepatic tumors, which may raise differential diagnostic problems. For this reason we investigated EZH2 expression in the most common other tumor types of liver. EZH2 staining was positive in 96% of cholangiocarcinomas, including 3 highly differentiated Klatskin tumors. As far as we know EZH2 expression has not previously been examined in this type of tumor. We have studied a few biliary cystadenomas and tumor mimicking ductal plate malformations, all of them were negative, as well as the ductular reactions. CC also must be differentiated from metastatic liver tumors. Practically all the metastatic adenocarcinomas, regardless of their origin, were positive for EZH2. Although our case number is low, the primary tumors of colon, pancreas, breast, lung [14-21] have already been described to express this tumor marker, so the few metastases we report probably do reflect reality. That is, EZH2 similarly to other CC markers e.g. CK7, CK19, claudin 4 [28] does not provide major help in distinguishing cholangiocarcinomas from metastatic tumors, but it does seem to be able to differentiate reactive/hamartomatous biliary structures and benign biliary tumors from malignant ones. This is important because so far no such marker has been reported. Even the recently described highly specific cholangiocarcinoma marker, αvβ6 integrin [10] is positive in proliferating bile ducts. The combination of these two antibodies may facilitate the diagnosis of cholangiocarcinomas.