Immunohistochemical Expression and Clinical Significance of Suggested Stem Cell Markers in Hepatocellular Carcinoma

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Abstract

MATERIALS AND METHODS

Immunohistochemistry

For immunohistochemical (IHC) staining, 10% formalin-fixed, paraffin-embedded tissue sections of representative areas of tumor were prepared into 4-μm-thick tissue samples. IHC staining was performed using a fully automated IHC system with the Bond Polymer Refine Detection Kit (Leica Bond, Newcastle upon Tyne, UK) according to the manufacturer’s instructions. The sources of antibodies used in this study and conditions of the procedure are listed in Table 1. Peroxidase activity was detected with the enzyme substrate 3-amino-9-ethyl carbazole. The immunoreactivity of the specimens was interpreted according to the intensity of staining and proportion of positive cells. The intensity of cytoplasmic and membranous staining was graded into four levels: no immunostaining (0), weak (1), moderate (2), and intense (3). The proportion of positive cells was scored as follows: 0 (none), 1 (1%), 2 (2%–10%), 3 (11%–33%), 4 (34%–66%), and 5 (67%–100%). The sum index was obtained by totaling the scores of intensity and proportion of staining. If the final score was equal to or greater than 4, the immunoreactivity was considered positive; otherwise, the immunoreactivity was considered negative. For negative controls, sections were treated in the same way, except that they were incubated with Tris-buffered saline instead of primary antibodies.

Table 1.

Summary of antibodies and conditions used for immunohistochemistry

RESULTS

Expression of suggested CSC markers

In cirrhotic livers, all four examined markers showed intense immunoreactivity in proliferating reactive bile ductules. These ductules are thought to originate from hepatic progenitor cells and thus were considered as an internal positive control. EpCAM expression in HCC tissues showed intense cytoplasmic and/or membranous staining (Fig. 1). In 82 HCC samples, 46 were EpCAM-positive (56%) and five were K19-positive (6%). Of the 82 HCCs, only two cases showed CD133 and CD56 expression, respectively.

Fig. 1.

(A) Membranous expression of epithelial cell adhesion molecule (EpCAM) in hepatocellular carcinoma cells. Bile duct epithelial cells also showed strong expression of EpCAM (arrow). (B) Tumor cells with cytoplasmic reactivity for keratin 19. Absence of CD133 (C) and CD56 (D) immunoreactivity in tumor cells (T). Note the positive immunoreactivity for CD133 and CD56 in bile ductular cells (arrows).

Correlations between expression of suggested CSC markers and clinicopathological features

Analysis of the relationships between expression of K19, CD133, and CD56 and clinicopathologic features was not meaningful, since the number of cases with K19, CD133, and CD56 expression was very small. However, EpCAM expression significantly correlated with higher histologic grade (p=.006), elevated AFP level (p=.001), and microvessel invasion of the tumor cells (p=.030) (Table 2). Additionally, EpCAM expression was strongly associated with K19 expression (p=.041).

Table 2.

The relationship between the expression of suggested stem cell marker EpCAM and clinicopathologic characteristics

DISCUSSION

Carcinogenesis is currently explained using two models, one involving a traditional stochastic onset and a second based on the role of CSCs. According to the CSC model, only a small population of tumor cells has the ability to divide and repopulate within the tumor [7,13]. CSCs are responsible for tumor initiation, maintenance, growth, metastasis, and relapse after therapy [3-6]. The expression of several proposed CSC markers in HCC has been reported and might prove to be useful for predicting the prognosis of HCC patients [11,14-16]. However, little is known about the relationships between the IHC expression of CSC markers in paraffin-embedded tissue sections and clinicopathologic factors or clinical outcomes of HCC.

EpCAM is a transmembrane intercellular protein that was initially posed as a homophilic cell adhesion molecule [17] and is highly up-regulated in most human epithelial cancers, including HCC [14-16,18]. EpCAM has been validated as a marker of stem cells in the liver, and EpCAM-positive HCC is likely to have originated from hepatic stem cells [8,15,16,19]. In the present study, we observed that EpCAM expression significantly correlated with tumor progression factors of HCC, such as elevated AFP level, vascular invasion, and high tumor grade. These findings are in agreement with previous studies showing that expression level of EpCAM correlates with de-differentiation [11] and vascular invasion and is associated with the high AFP level in HCC [20,21]. The prognosis of patients with EpCAM-negative HCC is considered to be better than that of those with EpCAM-positive HCC [11,14-16,20,21]. Although EpCAM expression was not found to be an independent predictor of survival in patients with HCC in this study, EpCAM expression was found to be associated with well-known unfavorable prognostic factors in HCC. Thus, the use of EpCAM expression as an unfavorable prognostic factor of HCC is reasonable. Furthermore, gene expression profile study has demonstrated that EpCAM-positive and AFP-positive HCCs have more aggressive behavior and poor clinical outcome, whereas EpCAM-negative and AFP-negative HCCs have good prognosis [20]. Based on these observations, EpCAM has attracted considerable attention as a possible therapeutic target for patients with HCC [7,8,13]. Taken together, our findings suggest that EpCAM is a critical player in facilitating vascular invasion of tumor cells, leading to de-differentiation in HCC with high serum AFP level, and might be useful for predicting the prognosis of HCC patients. In this study, a proportion of HCCs (56%) expressed EpCAM, and EpCAM expression was associated with K19 expression. These findings are in agreement with previous reports showing that the expression of EpCAM and K19 was positively correlated [15,21]. The proportion of tissues expressing EpCAM in this study was a little higher than in earlier series, which reported EpCAM in 15.9%–48.7% of HCCs [15-17,20-23]. Kimura et al. [23] have demonstrated that EpCAM expression was observed more often in HCC patients with HBV than in those with other etiologies, and the immunoreactivity of EpCAM has been found in up to 78% of HCC patients with HBV. The high proportion of HCC patients with HBV (74%) examined in this study might be a possible explanation for the higher EpCAM-positive rate. It has been hypothesized that the transformation of hepatic stem/progenitor cells (maturation arrest theory) underlies the occurrence of HCC expressing CSC markers. However, the concept of the CSC model is still debated. The expression of CSC markers in HCC can develop as an acquisition of progenitor cell features during the de-differentiation of cancer cells (de-differentiation theory) [5,15]. Kim and Park [24] have proposed that HCC expressing CSC markers should be designated as “HCC with stemness-related marker expression” in order to avoid the implication that these HCCs originated from hepatic stem/progenitor cells.

Either CD133 or CD56 expression in this study was rare. Only two of 82 HCC specimens showed positive staining for CD133 and CD56, suggesting that CD133 and CD56 expression is rare in paraffin-embedded HCC tissues. It has been reported that 0%–88% [11,15,16,25] of human HCC tissue samples express CD133, and the expression of which is negatively correlated with presence of HBV [26]. Several IHC studies have demonstrated CD56 expression in 0%–9.7% of human HCCs [11,25,27,28]. The reason for this high discrepancy of positive rate of CD133 and CD56 in HCC is unclear. In our study, we employed the bond polymer IHC staining method to avoid endogenous biotin contamination, and all four examined markers always showed strong immunoreactivity in reactive bile ductules, which were considered as internal positive controls. The discrepancy in the expression rates of CD133 and CD56 is not easily explained but might be related to the different criteria for positivity, the quality of tissue samples analyzed, and the unique antibodies and immunostaining methods used in different studies. Our negative results in analysis of CD133 and CD56 through the use of specific antibodies in an established automated IHC system does not exclude the presence of these markers and can be linked to the different etiologies according to geographic background. Additional investigations with a larger population of HCC and strict criteria for positive immunoreactivity are necessary to determine the expression and clinical implication of these markers.

In conclusion, we showed that the EpCAM expression in paraffin-embedded tissue sections of HCC is associated with tumor progression factors and might be useful for predicting the prognosis of HCC patients. The high proportion of EpCAM expression in HCCs and its associations with unfavorable prognostic factors of HCC provide a basis for further investigation of anti-EpCAM–targeted therapy.

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