High expression of CD34-positive sinusoidal endothelial cells is a risk factor for hepatocellular carcinoma in patients with HCV-associated chronic liver diseases

CD34 has been widely used for the assessment of sinusoid-like neoangiogenesis in hepatocellular carcinoma (HCC). Recently, it was demonstrated that CD34-positive cells isolated from human peripheral blood differentiate into endothelial cells and contribute to neoangiogenesis in adults. We investigated the localization and the substantial role of CD34-positive endothelial cells in the liver with hepatitis C virus (HCV)--associated chronic liver diseases. Liver tissue sections obtained by biopsy from 56 patients with HCV-associated chronic liver diseases by were examined immunohistochemically using anti-CD34, anti-von Willebrand factor (vWF), and anti-vascular endothelial growth factor (VEGF) antibodies. CD34 was stained in the sinusoid, showing dotty, linear, semicircular, or circular patterns. However, sinusoidal expression of vWF was not substantially identified in the same specimens, indicating the existence of sinusoidal CD34-positive but vWF-negative endothelial cells. We classified these cells as CD34 LI and found that CD34 LI was correlated with the expression of VEGF. Among 34 patients with advanced-stage disease, the cumulative incidence of HCC was significantly higher in patients with CD34 LI >or= 12 (n = 16) than in those with CD34 LI < 12 (n = 18; P = .009). Moreover, among several clinicopathologic risk factors, CD34 LI could be recognized as an independently significant factor for development of HCC (relative risk, 7.36; P = .019). We conclude that CD34-positive endothelial cells are regulated by several factors, such as VEGF, and might play a substantial role in hepatocarcinogenesis. Furthermore, high expression of CD34-positive sinusoidal endothelial cells is a risk factor for HCC in patients with HCV-associated chronic liver diseases.     

Expression of tumour necrosis factor-related apoptosis-inducing ligand and caspase-3 in relation to grade of inflammation and stage of fibrosis in chronic hepatitis C

AIM: To assess whether the distribution of the recently described proapoptotic ligand, tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), and the apoptosis effector, caspase-3 alters with the degree of inflammation and fibrosis present in liver biopsy specimens from patients with chronic hepatitis C virus infection. METHODS AND RESULTS: Expression of TRAIL and caspase-3 was assessed immunohistochemically in liver biopsy specimens obtained from 89 adults with chronic hepatitis C. Expression of TRAIL in hepatocytes correlated inversely with stage of fibrosis (P = 0.001), classified according to the Scheuer score; expression of caspase-3 in hepatocytes correlated with grade of inflammation (P = 0.012). Expression of TRAIL in hepatocytes was not correlated with grade of inflammation (P > 0.05); expression of caspase-3 was not correlated with stage of fibrosis (P > 0.05). Maximum expression of proapoptotic TRAIL protein was observed in cases with low grade inflammation (G0) and low stage fibrosis (S1). Maximum expression of caspase-3 in hepatocytes was observed in cases with high grade inflammation (G3-4) and high stage fibrosis (S3), but not with liver cirrhosis (S4). CONCLUSIONS: There is a significant decrease in TRAIL expression with increasing grade of inflammation, whereas caspase-3 expression is significantly increased with advanced fibrosis, short of cirrhosis.     

Expression of CD34-positive sinusoidal endothelial cells in patients with HBV-associated chronic liver diseases

It has been demonstrated that CD34-positive cells isolated from human peripheral blood differentiate into endothelial cells and contribute to neoangiogenesis in adults. We investigated the role of CD34-positive endothelial cells in liver samples from patients with hepatitis B virus (HBV)-associated chronic liver diseases. Tissue sections were obtained by liver biopsy from 25 patients with HBV-associated chronic liver diseases and were examined by immunohistochemistry using anti-CD34, anti-von Willebrand factor (vWF), and anti-vascular endothelial growth factor (VEGF) antibodies. CD34-positive, but vWF-negative endothelial cells were observed, particularly in the sinusoids and vascular endothelial cells. We counted these cells and expressed the results as a CD34-labeling index (LI). The CD34 LI did not correlate with VEGF expression and the CD34 LI of patients who progressed to hepatocellular carcinoma (HCC) tended to increase compared to those that did not progress to HCC. CD34 LI was an independent risk factor for development of HCC (relative risk, 35.689; P = 0.033). We conclude that CD34-positive endothelial cells in patients with HBV-associated chronic liver diseases might play a role in hepatocarcinogenesis.     

Expression of Bax, Bcl-xL and Bcl-2 proteins in relation to grade of inflammation and stage of fibrosis in chronic hepatitis C

AIMS: To determine the expression of regulators of apoptosis in chronic hepatitis C. METHODS AND RESULTS: Expression of Bax, Bcl-xL and Bcl-2 proteins was assessed immunohistochemically in liver biopsy specimens obtained from 89 adults with chronic hepatitis C. Expression of Bax in hepatocytes correlated inversely with grade of inflammation (P < 0.001) and stage of fibrosis (P = 0.011), classified according to the Scheuer score; expression of Bcl-xL in hepatocytes did not correlate with grade of inflammation (P = 0.106) or stage of fibrosis (P = 0.078); maximum Bcl-xL expression was observed in grade 3 inflammation and stage 4 fibrosis. Expression of Bcl-2 protein in hepatocytes was present in only two cases (both with advanced disease); the expression of Bcl-2 protein in interlobular bile duct epithelial cells correlated with the grade of inflammation (P = 0.018), but not with stage of fibrosis (P = 0.154). The expression of Bcl-2 protein in lymphoid cells infiltrating portal zones and lobules did not correlate with grade of inflammation (P = 0.113) or stage of fibrosis (P = 0.815). CONCLUSION: Major differences in expression of studied proteins were observed in relation to grade of inflammation and stage of fibrosis in chronic hepatitis C.     

原发性肝癌及慢性肝脏病变端粒酶活性的研究

目的比较原发性肝细胞癌（ＨＣＣ）及其癌旁不同慢性病变组织端粒酶活性的异同,探讨端粒酶活性在恶性肿瘤诊断中的意义。方法用ＴＲＡＰ方法检测３８例ＨＣＣ及其癌旁不同慢性病变肝组织的端粒酶活性。同时以４例正常肝组织为对照。结果３８例ＨＣＣ中３２例显示端粒酶活性（８６８％）。端粒酶活性与ＨＣＣ的细胞分化、类型、大小及有无乙型肝炎病毒（ＨＢＶ）感染无关,但与病人血清中甲胎蛋白（ＡＦＰ）水平相关。端粒酶活性阴性组ＡＦＰ水平明显低于各阳性组（Ｐ＜００１）。正常肝（４例）、先天性胆管闭锁（４例）、肝小叶间纤维增生（２例）、慢性病毒性肝炎（２例）及未见明显病变肝组织（７例）均未见有端粒酶活性。２５例肝硬化中４例端粒酶呈弱活性。结论端粒酶活性见于大多数ＨＣＣ及个别肝硬化组织,其活性可能在ＨＣＣ的发生、发展中起重要作用,有希望成为恶性肿瘤诊断的标记物。     

Hepatic Stellate Cells in Inflammation‐Fibrosis‐Carcinoma Axis

Almost 80% of hepatocellular carcinoma (HCC) cases are associated with chronic hepatitis and cirrhosis resulting from inflammation and fibrosis. A three‐step process of “inflammation‐fibrosis‐carcinoma” is believed to be involved in hepatocarcinogenesis. The activation of hepatic stellate cells (HSCs) may serve as an important mediator in the process of inflammation‐fibrosis‐carcinoma axis, even in tumor metastasis. A remarkable knowledge of activated HSCs in the pathology of HCC development is mostly focused on the liver fibrosis. The molecular links that connects inflammation and cancer in the activation of HSC are not completely known. This highlights urgent need to increase our understanding of the cellular and molecular mechanisms, by which activation of HSCs is involved in the hepatic inflammation, carcinogenesis, and metastasis. Anat Rec 293:1492–1496, 2010. © 2010 Wiley‐Liss, Inc.     

Bile ductular cell reaction with senescent hepatocytes in chronic viral hepatitis is lost during hepatocarcinogenesis

Cellular senescence is defined as irreversible cell arrest and could work as a safeguard against tumorigenesis. This mechanism was examined in chronic viral hepatitis-related hepatocarcinogenesis. By using surgical resected or wedge biopsied liver specimens from 87 chronic viral hepatitis patients in whom 35 neoplastic nodules (dysplastic nodules and hepatocellular carcinoma) were complicated, P21 expression and senescence-associated β galactosidase activity, a marker of senescence, were examined. All of these neoplastic nodules harbored portal tracts within the tumors. Hepatocytes expressing senescence markers and cytokeratin (CK)7-positive bile ductules including hepatic progenitor-like cells were increased in periseptal areas in cirrhosis. Interestingly, these cells appeared to form an anatomical complex that was completely lost in the periportal areas within the neoplastic nodules. In one-third of the neoplastic nodules, CK7-positive small neoplastic hepatocytes resembling hepatic progenitor cells proliferated zonally around the portal tracts. In conclusion, loss of a complex of senescent hepatocytes and ductular cell including hepatic progenitor-like cells in the periportal or periseptal areas may be associated with emergence of neoplastic hepatocytes and their proliferation followed by neoplastic nodules arising in liver cirrhosis. Zonal proliferation of CK7-positive small neoplastic hepatocytes resembling hepatic progenitor cells may develop during early hepatocarcinogenesis.     

Overexpression of cannabinoid receptors CB1 and CB2 correlates with improved prognosis of patients with hepatocellular carcinoma

CB1 and CB2 are multifunctional cannabinoid-specific receptors considered to be involved in inhibition of tumor development. To elucidate their roles in hepatocarcinogenesis, we analyzed the expression of these receptors in tumor and matched nontumorous tissues of human hepatocellular carcinoma (HCC) samples. In situ hybridization analysis showed overexpression of CB1 mRNAs in 8 of 13 (62%) HCC samples, and of CB2 mRNAs in 7 of 13 (54%). Immunohistochemical analysis of 64 HCC samples showed the expression of CB1 and CB2 receptors to increase from normal liver to chronic hepatitis to cirrhosis. Marked expression of CB1 and CB2 receptors was noted in the majority of cirrhotic liver samples (86 and 78%, respectively). In HCC, high expression of CB1 and CB2 receptors was observed in 29 (45%) and 33 (52%) cases, respectively. Clinicopathological evaluation indicated a significant correlation between CB1 and CB2 expression and two clinicopathological parameters such as the histopathological differentiation (P = 0.021 and 0.001, respectively), portal vein invasion (P = 0.015 and 0.004, respectively). Univariate analysis indicated that disease-free survival was significantly better in HCC patients with high versus those with low CB1 and CB2 expression levels (P = 0.010 and 0.037, respectively). Our results indicate that CB1 and CB2 have potential as prognostic indicators and suggest possible beneficial effects of cannabinoids on prognosis of patients with HCC.     

High expression of vanilloid receptor-1 is associated with better prognosis of patients with hepatocellular carcinoma

The vanilloid receptor-1 (VR1) is a ligand-gated, nonselective cation channel expressed predominantly by sensory neurons, but is also involved in carcinogenesis. To elucidate its role in hepatocarcinogenesis, we analyzed the expression of VR1 receptor in tumor and nontumor tissues from human hepatocellular carcinoma (HCC) samples. In situ hybridization analysis showed overexpression of VR1 mRNAs in 9/15 (60.0%) noncancer and 6/15 (40.0%) HCC samples. Immunohistochemistry of 62 HCC samples showed the expression of VR1 increased from normal liver or chronic hepatitis to cirrhosis. Marked expression of VR1 was noted in the majority [31/38 (81.6%)] of cirrhotic liver samples. In HCC, high expression of VR1 was observed in 30/62 (48.4%) cases. Clinicopathologic evaluation indicated a significant correlation between VR1 expression and histopathologic differentiation (P=0.001). Univariate analysis indicated that disease-free survival was significantly better in HCC patients with high versus those with low VR1 expression levels (P= 0.021). Our results indicate that VR1 has anti-HCC progression effects and can be potentially used as a prognostic indicator of HCC. The results suggest the potential beneficiary effects of VR1 expression on the prognosis of patients with HCC.     

Enhanced CD34 expression of sinusaid-like vascular endothelial cells in hepatocellular carcinoma

The immunohistochemlcal expression of CD34 (human hematopoletic stem cell and endothelial cell marker) and laminin were studied In chronic liver diseases and hepate cellular carcinoma (HCC) to elucidate whether their expression reflected phenotypic differences between non-cancerous slnusoids and sinusoid-like tumor vessels. In normal liver, hepatic sinusoids were always negative for CD34 and lami-nin. In chronic hepatitis and cirrhosis, the two antigens were sparsely expressed in capillarized sinusoids at periportal and petinodular area. In advanced HCC, CD34 was strongly and diffusely expressed by the endothelial lining of sinusoid-like tumor vessels. However, early-stage HCC showed a wide spectrum of CD34 expression from negative to focal and diffuse, strongly positive staining in sinusoid-like vessels. Laminin was strongly expressed in advanced HCC but not in early-stage HCC. The results Indicate that the enhanced expression of CD34 by sinusoidal endothelial cells may reflect the phenotypic change of endothellal cells in chronic liver diseases and HCC, and that the expression may correlate with the processes of angiogenesis induced by hepatocarcinogenesis.     

Immunohistochemical evaluation of oxidative stress markers in chronic hepatitis C

Oxidative stress (OS) plays a major role in chronic hepatitis C. Various OS markers have been found to be elevated in hepatitis C virus (HCV)-related liver disease. This study detected the presence of OS in serum and liver biopsy specimens of HCV patients. Reactive oxygen molecules (ROM) in sera of 54 HCV patients were compared with 23 controls. OS markers 8-hydroxydeoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal, malondialdehyde, and thioredoxin were measured in liver biopsy specimens of 18 HCV patients with fibrosis staging F1 (six); F2 (two), F3 (four), and F4 (six). The interferon (IFN) response and hepatocellular carcinoma (HCC) occurrence in the presence of OS markers were also evaluated. The level of ROM in HCV patients was 318 +/- 56.7 Carr compared with 248 +/- 40.8 Carr in controls (p=0.032). Multivariate analysis found age (p=0.0236) to be the only independent variable associated with increase in ROM in sera. In liver biopsy specimens, OS markers were found mainly around the area of piecemeal necrosis or the periportal area. The presence of OS markers seemed to increase with fibrosis staging, although not significantly. The OS DNA damage marker 8-OHdG was detected in the nucleus of hepatocytes. Thirteen patients received IFN therapy. During the 4-year follow-up period, HCC developed in four nonresponders to IFN and in one untreated patient. OS markers were stained in both HCC cells and non-HCC cells in HCC patients. OS markers were found in serum and liver specimens of HCV-associated liver disease and in HCC tissue. Detection of OS markers may be important for monitoring disease progression in HCV patients. Antioxidant therapy in combination with antiviral therapy may minimize liver damage and aid in the prevention and subsequent development of HCC.     

High-throughput tissue microarray analysis of c-myc activation in chronic liver diseases and hepatocellular carcinoma

Amplification of 8q23-qter is common in human hepatocellular carcinoma (HCC). c-myc, an oncogene located on 8q24, may be important in hepatocarcinogenesis. The present study aimed to evaluate c-myc activation in hepatocarcinogenesis and its clinicopathological significance. High-throughput analysis of c-myc gene amplification and expression using dual-color fluorescence in situ hybridization and immunohistochemistry was performed on tissue microarrays consisting of 458 liver samples comprising HCCs, nontumorous livers and normal livers. HCCs demonstrated frequent c-myc amplification (30% when corrected for chromosome 8 aneusomy). In contrast, the noncancerous livers, which were mostly chronic hepatitis and cirrhosis, exhibited no c-myc amplification. Despite c-myc amplification, the HCCs exhibited less nuclear c-myc expression than the livers with chronic liver diseases and normal livers (P <0.001 and 0.004, respectively). The HCCs also had less cytoplasmic c-myc staining than the livers with chronic liver diseases (P = 0.002). Despite their absence of c-myc amplification, however, the livers with chronic disease had significantly increased expression of both nuclear and cytoplasmic c-myc protein compared with normal livers (P = 0.015 and 0.009, respectively). Clinicopathologically, the reduction in nuclear c-myc was more marked in HCCs with venous permeation and absence of tumor encapsulation (P = 0.013 and 0.021, respectively), whereas HCCs with cytoplasmic c-myc were positively associated with larger tumor size (P = 0.027). There was no significant association between c-myc amplification and protein expression levels in HCC. Our results suggest that overexpression of c-myc in chronic liver diseases may play an important role in the predisposition to hepatocarcinogenesis. Although c-myc was amplified in HCC, there appears to be a tight regulation by independent pathways of c-myc activation in hepatocarcinogenesis.     

The prevalence of hepatitis B virus preS deletions occurring naturally in Korean patients infected chronically with genotype C

Although Korea is one of the endemic areas for hepatitis B virus infection (HBV), the prevalence of deletions in HBV preS region occurring naturally have not been determined. In the present study, the prevalence of preS deletions was determined in terms of clinical state and HBeAg serostatus in 120 patients with different clinical features [59 HBeAg positive, 61 HBeAg negative; 38 asymptomatic carriers, 21 patients with chronic hepatitis, 21 patients with liver cirrhosis, 40 patients with hepatocellular carcinoma (HCC)]. A total of 37 strains (30.8%) harbored deletions in the preS region. Overall, the frequencies of preS deletions tended to increase gradually according to the degree of the clinical severity of liver disease. The prevalence of preS1 deletions in HCC patients tended to be higher than in patients with liver cirrhosis (32.5% vs. 19%). The prevalence of preS2 deletions in HBeAg negative patients was significantly higher than in HBeAg positive patients (23% vs. 6.8%). The type of deletion encountered most frequently was one disrupting the preS1 start codon [14/37 strains (37.8%)], which showed a very high prevalence in HCC patients (9/40, 22.5%; HCC vs. asymptomatic carriers, P=0.048). These results suggest that there might be the discrepancy between preS1 and preS2 mutations in the mechanism of enhancing the progression of chronic liver disease, especially the development of HCC and to maintain tolerance during the stage of immune tolerance. Specific deletion of the type disrupting preS1 start codon may play important roles in hepatocarcinogenesis, at least in Korean patients with chronic HBV infection.     

Evolution of hepatocellular carcinoma associated with chronic hepatitis B virus infection in Alaskan Eskimos

Hepatocellular carcinoma (HCC) associated with chronic hepatitis B virus (HBV) infection in Yupik Eskimos in southwestern Alaska, detected in early stages as a result of screening, appears to be more frequently associated with variants of chronic portal inflammation in the noninvolved liver than with fully developed cirrhosis, otherwise common in HBV-associated HCC from other geographic areas. Of 38 patients diagnosed with HCC since 1969, adequate tissue was available from both the tumor and nontumorous liver in 17. Of the 17 specimens, 14 had chronic portal inflammation and three had advanced cirrhosis; 12 of the 14 were from hepatitis B surface antigen carriers. These 12 cases were studied in detail to examine the features accompanying the development of HCC unobscured by cirrhotic transformation. In the noninvolved parenchyma they included hepatocytic nodules as apparent precursors to HCC and, as markers of phenotypic alterations, dysplastic hepatocytes and hepatitis B surface antigen-laden ground-glass hepatocytes. The latter were observed in eight instances and often accumulated in nodules. Parenchyma within 1 mm of the HCC exhibited increased confluent hyperplasia and frequently conspicuous necroinflammation associated with pericellular and periductular fibrosis, which contributed, in addition to fibrous connections between displaced and heavily inflamed portal tracts, to the capsule that was forming in all cases to varying degrees in the pericarcinomatous region. The HCC was uniformly trabecular and in a few specimens, a continuous transition from hyperplasia and dysplasia near the periphery of the tumor to increasing anaplasia in the center could be made out in addition to pressure effects of the HCC. The pericarcinomatous changes, including hyperplasia progressing to neoplasia and necroinflammation, are also observed in experimental models, particularly the woodchuck HCC induced by a hepadna virus related to HBV. Coordinated morphologic and molecular biologic studies on such animal models and on human HCC detected by screening, as for instance in Eskimos, neither complicated by cirrhosis, should elucidate the direction of the evolution of the HCC and the postulated promoting role of the inflammation.     

Expression of TLR-2 in hepatocellular carcinoma is associated with tumour proliferation,angiogenesis and Caspase-3 expression

AimsUnlike other Toll-like receptors (TLRs), the role of toll like receptor 2 (TLR-2) in the pathogenesis of chronic liver disease and hepatocellular carcinoma (HCC) is not well studied. We, therefore, set out to investigate the expression of TLR-2 in different chronic liver disease states along with other markers of cell death, cellular proliferation and tissue vascularisationMethods and resultsImmunohistochemistry was performed on liver tissue microarrays comprising hepatitis, cirrhosis and HCC patient samples using antibodies against TLR-2, Ki-67, Caspase-3 and VEGF. This was done in order to characterise receptor expression and translocation, apoptosis, cell proliferation and vascularisation. Cytoplasmic TLR-2 expression was found to be weak in 5/8 normal liver cases, 10/19 hepatitis cases and 8/21 cirrhosis patients. Moderate to strong TLR-2 expression was observed in some cases of hepatitis and cirrhosis. Both, nuclear and cytoplasmic TLR-2 expression was present in HCC with weak intensity in 11/41 cases, and moderate to strong staining in 19/41 cases. Eleven HCC cases were TLR-2 negative. Surprisingly, both cytoplasmic and nuclear TLR-2 expression in HCC were found to significantly correlate with proliferative index (r = 0.24 and 0.37), Caspase-3 expression (r = 0.27 and 0.38) and vascularisation (r = 0.56 and 0.23). Further, nuclear TLR-2 localisation was predominant in HCC, whereas cytoplasmic expression was more prevalent in hepatitis and cirrhosis. Functionally, treatment of HUH7 HCC cells with a TLR-2 agonist induced the expression of cellular proliferation and vascularisation markers CD34 and VEGF.ConclusionsOur results demonstrate a positive correlation between the expression of TLR-2 and other markers of proliferation and vascularisation in HCC which suggests a possible role for TLR-2 in HCC pathogenesis