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

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.

Connective tissue growth factor in human liver cirrhosis.

BACKGROUND: Connective tissue growth factor (CTGF) belongs to a family of factors that regulate fibrogenesis and wound healing. While the significance of transforming growth factor beta (TGF-beta) in liver fibrosis is well established, the role of CTGF in fibrosing hepatopathy is still unknown. METHODS: CTGF was analyzed in 10 normal and in 16 cirrhotic liver tissue samples. Northern blot analysis was used to examine the concomitant expression of CTGF and TGF-beta1 mRNAs, and the cellular localization of CTGF mRNA was studied by in situ hybridization. For identification of myofibroblasts and activated hepatic stellate cells, alpha-smooth muscle actin (alpha-SMA) immunohistochemistry was used. RESULTS: Northern blot analysis showed 6.5-fold enhanced expression of CTGF mRNA and 7.8-fold enhanced expression of TGF-beta1 mRNA in liver cirrhosis in comparison with normal controls (p<0.01). By in situ hybridization, CTGF mRNA was detectable in only a few spindle cells in the portal tracts in normal liver samples. In contrast, there was strong expression of CTGF mRNA in fibroblasts and myofibroblast-like cells present in fibrous septa surrounding the cirrhotic nodules, in stellate cells, in endothelial cells and in mesenchymal cells around ductular proliferations, and in ductular epithelial cells. There was a strong correlation between CTGF mRNA and TGF-beta1 mRNA as well as the degree of fibrosis (p<0.01). CONCLUSIONS: Overexpression of CTGF in liver cirrhosis, especially in fibroblasts/myofibroblasts and stellate cells, suggests that this novel factor may play an important role in hepatic fibrosis.

Group II and IV phospholipase A(2) are produced in human pancreatic cancer cells and influence prognosis.

BACKGROUND: Phospholipase A(2) (PLA(2)) is involved in regulating biosynthesis of arachidonic acid and its metabolites. There are three major structurally different forms of PLA(2): group I, also called pancreatic PLA(2) (PLA(2)-I); group II, referred to as secretory non-pancreatic or synovial or platelet PLA(2) (PLA(2)-II); group IV, referred to as cytosolic PLA(2) (PLA(2)-IV). AIMS: To examine PLA(2)-I, PLA(2)-II, and PLA(2)-IV in normal and pancreatic cancer tissues. Patients-PLA(2) was studied in 58 pancreatic adenocarcinomas, obtained from 25 women and 33 men undergoing pancreatic resection. Normal organ donor pancreas served as control. METHODS: The enzymes were analysed by northern blot, in situ hybridisation, and immunohistochemistry. The molecular findings were correlated with clinical variables of the patients. RESULTS: Northern blot analysis of total RNA showed enhanced PLA(2) group II and IV mRNA expression in 52% and 55% of the pancreatic cancer samples respectively compared with the normal controls (p = 0.0013 and p = 0.0025). On immunohistochemical analysis, intense PLA(2)-I immunoreactivity was seen in acinar cells, but not in ductal cells, in the normal pancreas. In pancreatic cancer cells, PLA(2)-I immunostaining was absent. PLA(2)-II immunostaining was visible only in some acinar and ductal cells in the normal pancreas, whereas in pancreatic cancer increased PLA(2)-II immunoreactivity was present in 65% of the cancer samples. On in situ hybridisation, weak PLA(2)-IV mRNA signals were detected in acinar and ductal cells of normal samples; these signals were present to a much greater extent in pancreatic cancer cells. The presence of PLA(2)-II in pancreatic cancer was associated with a higher degree of fibrosis (p<0.01). Furthermore, there was a significant correlation between the enhanced expression of PLA(2)-II and longer survival after surgery (p<0.03), but not of PLA(2)-IV and longer postoperative survival. CONCLUSION: These data suggest that PLA(2)-II and PLA(2)-IV are upregulated in human pancreatic cancer, and that upregulation of PLA(2)-II in pancreatic cancer covariates negatively with cancer cell growth.

Molecular aspects of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly malignant tumor with a poor prognosis. The success of its established treatment modalities is frequently limited by the advanced stage of the tumor at the time of diagnosis. Therefore, it is important to understand the mechanisms that control its growth behavior. In the present study, we review some aspects of molecular and cellular processes involved in growth control and metastatic potential of HCC. These include some growth factors and their receptors, oncogenes and tumor suppressor genes, and factors that control angiogenesis and extracellular matrix formation. These factors may be important targets for novel therapeutic approaches in the future.

Transforming growth factor betas and their signaling receptors in human hepatocellular carcinoma.

BACKGROUND: Transforming growth factor betas (TGF-betas) are multifunctional polypeptides that have been suggested to influence tumor growth. They mediate their functions via specific cell surface receptors (type I ALK5 and type II TGF-beta receptors). The aim of this study was to analyze the roles of the three TGF-betas and their signaling receptors in human hepatocellular carcinoma (HCC). METHODS: HCC tissue samples were obtained from 18 patients undergoing partial liver resection. Normal liver tissues from 7 females and 3 males served as controls. The tissues for histological analysis were fixed in Bouin's solution and paraffin embedded. For RNA analysis, freshly obtained tissue samples were snap frozen in liquid nitrogen and stored at -80 degrees C until used. Northern blot analysis was used in normal liver and HCC to examine the expression of TGF-beta1, -beta2, -beta3 and their receptors: type I ALK5 (TbetaR-I ALK5), type II (TbetaR-II), and type III (TbetaR-III). Immunohistochemistry was performed to localize the corresponding proteins. RESULTS: All three TGF-betas demonstrated a marked mRNA overexpression in HCC in comparison with normal controls, whereas the levels of all three TGF-beta receptors showed no significant changes. Intense TGF-beta1, TGF-beta2, and TGF-beta3 immunostaining was found in hepatocellular carcinoma cells and in the perineoplastic stroma with immunohistochemistry, whereas no or mild immunostaining was present in the normal liver. For TbetaR-I ALK5 and TbetaR-II, the immunostaining in both HCC and normal liver was mild to moderate, with a slightly higher intensity in the normal tissues. CONCLUSION: The upregulation of TGF-betas in HCC suggests an important role for these isoforms in hepatic carcinogenesis and tumor progression. Moreover, the localization of the immunoreactivity in both malignant hepatocytes and stromal cells suggests that TGF-betas act via autocrine and paracrine pathways in this neoplasm.

Laparoscopic partial hepatectomy in the rat: a new resectional technique.

BACKGROUND: Rats are widely used for basic research in laparoscopic surgery. We have developed a new technique of laparoscopic partial hepatectomy in the rat. METHODS: 40 American Cancer Institute rats were randomized into 3 groups. Group A (n = 14) underwent laparoscopic liver resection using a CO2 pneumoperitoneum. Group B (n = 14) was operated on with a gasless laparoscopic technique using a lifting device. A control group C (n = 12) underwent conventional open liver resection. In each group half of the animals underwent single lobectomy and the other half bilobectomy. RESULTS: The liver resection was performed successfully in all 40 rats. No conversion to open surgery was necessary. No mortality or morbidity was observed. CONCLUSIONS: This new technique of laparoscopic partial hepatectomy proved to be feasible and safe. It is the first description of a laparoscopic hepatic resection in the rat that could prove valuable in further investigations of liver physiology and pathology.

KAI1, a new metastasis suppressor gene, is reduced in metastatic hepatocellular carcinoma.

Down-regulation of KAI1 expression has been shown to be associated with formation of metastases or disease progression in prostate and pancreatic cancer. In the present study we analyzed the expression pattern of KAI1 in metastatic and nonmetastatic hepatocellular carcinomas (HCCs) in comparison with normal livers to evaluate whether alteration of KAI1 also facilitates the metastatic ability in this malignancy. Thirty-nine primary HCCs and 10 normal liver tissue samples were studied for KAI1 messenger RNA (mRNA) expression with use of Northern blot analysis and in situ hybridization. By Northern blot analysis, moderate to strong KAI1 mRNA expression was present in normal liver samples. In contrast, KAI1 mRNA expression in tissue samples of primary HCCs was markedly decreased compared with normal controls. The normal/tumor ratio of KAI1 mRNA expression was 2.6:1 (P <.01). Primary HCCs that gave rise to metastasis showed significantly lower KAI1 mRNA levels than nonmetastasized HCCs (P <. 05). As seen by in situ hybridization, moderate to strong cytoplasmic KAI1 mRNA staining was present in almost all normal hepatocytes. Bile ducts, blood vessels, and connective tissue showed no or only faint KAI1 mRNA expression in the normal liver samples. In nonmetastatic HCCs, the cancer cells exhibited in situ hybridization signals that were similar to the normal controls. In contrast, most of the primary HCC cells in samples with metastases showed only faint or moderate KAI1 mRNA expression predominantly in the perinuclear regions. When KAI1 mRNA expression of primary hepatocellular cancer cells was compared with metastasized cancer cells in lymph nodes, with intrahepatic satellite metastasis, or with peritoneal metastasis in the same patients, significantly lower (P <.01) KAI1 mRNA levels were present in the metastasized HCC cells. Reduced KAI1 mRNA in HCC cells seems to influence their metastatic ability and thereby enhances the malignant potential of HCC.

Transforming growth factor betas and their receptors in human liver cirrhosis.

BACKGROUND: Transforming growth factor betas (TGF-betas) are a group of homologous polypeptides that exert pleiotropic effects on various cell types and stimulate the formation of extracellular matrix and fibrosis. To evaluate whether TGF-beta isoforms (TGF-beta1, TGF-beta2 and TGF-beta3) and their receptors (types I-III) are also of importance in the pathophysiology of liver cirrhosis, we analysed their concomitant expression and localization in human liver cirrhosis. PATIENTS: Cirrhotic liver tissue samples were obtained from 17 patients (four women, 13 men) with a median age of 41 years (range 22-67). Normal liver tissues from ten patients (seven women, three men) with a median age of 55 years (range 45-75) served as controls. METHODS: The tissues were fixed in Bouin's solution and paraffin-embedded for histological analysis. For RNA analysis, freshly obtained tissue samples were snap-frozen in liquid nitrogen and stored at -80 degrees C until analysed. Northern blot analysis was used to examine the expression of TGF-beta1, beta2 and beta3 and their receptors, type I (TbetaR-I), type II (TbetaR-II) and type III (TbetaR-III). Immunohistochemistry was performed to determine the localization of the corresponding proteins in the normal and the cirrhotic liver. RESULTS: Northern blot analysis revealed enhanced expression (P < 0.05) of TGF-beta1 (twofold increase), TGF-beta2 (threefold increase) and TGF-beta3 (8.5-fold increase) and of TbetaR-II (threefold increase) mRNA in liver cirrhosis in comparison with normal controls. In contrast, TbetaR-I (ALK-5) and TbetaR-III mRNA expression showed no significant changes. No TGF-beta isoform immunoreactivity was present in hepatocytes in either normal livers or in liver cirrhosis. However, in liver cirrhosis, intense TGF-beta1 immunoreactivity was present in bile duct and ductular epithelial cells (including ductular proliferations) and in inflammatory cells. In a few sinusoidal lining cells, faint TGF-beta1 and moderate TGF-beta2 immunoreactivity was present. TGF-beta3 immunostaining was present in bile duct and ductular epithelial cells, in inflammatory cells and in fibroblast-like spindle cells in liver cirrhosis. For TbetaR-I and TbetaR-II, the immunoreactivity was localized in hepatocytes and biliary cells in normal and cirrhotic liver tissues, with higher intensity for TbetaR-II in the cirrhotic liver. CONCLUSION: Enhanced expression of all three TGF-bea isoforms and of TbetaR-II in liver cirrhosis suggests their involvement in this fibrotic disorder. The higher immunoreactivity of the three TGF-beta isoforms in the bile duct epithelial cells in cirrhotic tissues suggests a possible role of these cells in the pathogenesis of liver cirrhosis.