Enhanced glypican-3 expression differentiates the majority of hepatocellular carcinomas from benign hepatic disorders

Abstract

Background/aims: Hepatocellular carcinoma (HCC) is a common malignant tumour worldwide, and its differential diagnosis from benign lesions of the liver is often difficult yet of great clinical importance. In the present study, we analysed whether glypican-3 is useful in differentiating between benign and malignant liver diseases and whether it influences the growth behaviour of HCC.

Methods: Northern blot analysis and in situ hybridisation.

Results: Northern blot analysis indicated that expression of glypican-3 mRNA was either low or absent in normal liver, in focal nodular hyperplasia (FNH), and in liver cirrhosis. In contrast, expression of glypican-3 mRNA was markedly increased in 20 of 30 and moderately increased in five of 30 HCC samples. The average increase in glypican-3 mRNA expression in HCC was significant compared with expression in normal liver (21.7-fold increase, p<0.01). In comparison with FNH or liver cirrhosis, glypican-3 mRNA expression in HCC was increased 7.2- (p<0.05) and 10.8-fold (p<0.01), respectively. In addition, pushing HCCs exhibited significantly higher glypican-3 mRNA expression than invading tumours (p<0.05). In situ hybridisation analysis demonstrated weak expression of glypican-3 mRNA in normal hepatocytes and bile ductular cells, and weak to occasionally moderate signals in hepatocytes forming nodules of liver cirrhosis and in regenerated hepatic nodules of FNH. In contrast, glypican-3 in situ hybridisation signals were intense in hepatic cancer cells with even higher levels in pushing HCCs than in invading HCCs.

Conclusions: These findings suggest that glypican-3, in many cases, has the potential to differentiate between benign and malignant liver diseases.

Figure 1

Northern blot analysis of glypican-1, -3, -4, and -6 in normal liver, liver cirrhosis, hepatocellular carcinoma (HCC), and focal nodular hyperplasia (FNH). Total RNA (20 µg) was size fractionated, blotted, and hybridised with the indicated ³²P labelled probes.

Figure 2

Northern blot analysis. (A) Glypican-3 mRNA in normal liver, focal nodular hyperplasia (FNH), liver cirrhosis, and hepatocellular carcinoma (HCC). (B) Northern blot analysis of glypican-3 mRNA in normal liver and HCC. Total RNA (20 µg) was size fractionated, blotted, and hybridised with 1×10⁶ cpm/ml ³²P CTP labelled glypican-3 cRNA probe and 1×10⁵ cpm/ml ³²P dCTP 7S cDNA probe.

Figure 3

Densitometric analysis. The ratio of the optical density of glypican-3 mRNA to the corresponding 7S signals was calculated and expressed as mean (SEM). FNH, focal nodular hyperplasia; HCC, hepatocellular carcinoma. p<0.01 (HCC v normal); p<0.05 (HCC v FNH); p<0.01 (HCC v liver cirrhosis). Insert: comparison of expression of glypican-3 mRNA between pushing and invading tumours.

Figure 4

In situ hybridisation of glypican-3 in normal liver tissue (A, B), focal nodular hyperplasia (FNH) (C, D), liver cirrhosis (E, F), and hepatocellular carcinoma (HCC) (G, H). In normal liver tissues, weak glypican-3 mRNA signals were present in bile ductal cells and in hepatocytes (magnification: A ×50, B ×100). In FNH (magnification: C ×50, D ×100; D insert, bile duct adjacent to FNH tissue ×100) and liver cirrhosis (magnification: E ×50, F ×100), glypican-3 mRNA signals were weakly to occasionally moderately present in hepatocytes and weakly to moderately present in bile ductal cells in liver cirrhosis and in bile ductal cells adjacent to FNH tissue. In HCC, glypican-3 mRNA was more intensely present in tumours with a pushing growth behaviour (magnification: H ×50, H insert ×200) than in those with an invading growth behaviour (magnification: G ×50, G upper left insert ×200). In situ hybridisation in HCC with the glypican-3 sense probe showed no signal (magnification: G insert ×100).