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Asia has a disproportionate share of the world's burden of hepatocellular carcinoma (HCC). However, the highly regarded clinical practice guidelines and recommendations for the design and conduct of clinical trials for HCC largely reflect Western practice. In order to design mutually beneficial international clinical trials of promising targeted therapies, it is imperative to understand how the aetiology, staging and treatment of HCC differ between Asian and Western countries. Our group, comprising experts in oncology and hepatology from countries that constitute the Eastern Asian region, convened to compare and contrast our current practices, evaluate potential compliance with the clinical trial recommendations, and offer suggestions for modifications that would enhance international collaboration. Here, we describe the results of our discussions, including recommendations for appropriate patient stratification based on potentially important differences in HCC aetiology, identification of practices that may confound interpretation of clinical trial outcomes (traditional Chinese medicine; antivirals that target hepatitis B virus; heterogeneous embolization procedures), suggestions for utilizing a common staging system in study protocols, recognition that sorafenib usage is limited by financial constraints and potentially increased toxicity in Asian patients, and expansion of patient populations that should be eligible for initial clinical trials with new agents., (© 2010 John Wiley & Sons A/S.)

Background/aims: The metastasis of hepatic carcinoma is correlated with the body's immune status. T lymphocytes play a big part role in tumor immune. The aim of the present study is to investigate the inhibition effects of metastatsis in nude mice bearing hepatic carcinoma after T lymphocytes reconstitution., Methodology: An immune reconstitution model was established in nude mice. Then, 42 nude mice were distributed into 4 groups for T lymphocytes reconstitution. The lymph nodes of each group were obtained to investigate the tumor metastasis. And the secretion of cytokines and the apoptosis of tumor cells in each group were also detected., Results: The ratio of CD3, CD4, CD8, CD4/CD8 in reconstituted groups were higher than controlled groups. The average time of tumor formation in Balb/c nu/nu mice was 7.7 +/- 0.6 days and in Balb/c mice was 11.5 +/- 1.3 days. After active T lymphocytes reconstitution, the extent lymph nodes metastasis in reconstitution groups was lower than control groups (p < 0.05), but proximal metastasis has no significant difference. The level of serum IL-10 in nude mice after immune reconstitution was significantly lower and VEGF was significantly higher than that of the control group (p < 0.05). Apoptosis of the hepatic carcinoma cells was increased significantly after immune reconstitution (p < 0.05)., Conclusions: The metastasis of hepatic carcinoma can be inhibited by reconstitution of actived T lymphocytes in nude mice, which indicated that tumor metastasis can be affected by the immune status in host body.

Background and Aim: Osteopontin (OPN) has been linked to clinical outcomes in several solid tumors. However, it has not been fully evaluated whether OPN could be used as a single marker for the prognosis of patients with hepatocellular carcinoma (HCC), particularly in patients of the tumor-node-metastasis (TNM) stage I., Methods: A total of 151 patients with HCC who underwent surgical resection were enrolled, including 112 patients of the TNM stage I. OPN expression was evaluated using immunohistochemistry in the tissue microarrays derived from these patients. Immunoreactivity was classified according to the percentage and intensity of staining: negative (-), weak (+) and strong (++). The impact of OPN expression on survival of patients was analyzed., Results: In total, 65.6% (99 of 151) of HCC tissues expressed OPN. Overall survival in patients of OPN (-) group was significantly higher than those of OPN (+) or OPN (++) group (P = 0.049 and P = 0.001). Interestingly, in patients of the TNM stage I, OPN expression was correlated with the early recurrence after surgical resection (P = 0.001). Multivariate analysis showed that OPN expression was an independent prognostic factor for overall survival and disease-free survival in patients with the TNM stage I HCC (hazard ratio, 2.272, P = 0.014 and 1.982, P = 0.037)., Conclusions: These results suggest that OPN is commonly expressed in HCC and is a useful marker for predicting the prognosis of patients with the TNM stage I HCC, contributing to determining which individual patient needs adjuvant therapy to prevent the early recurrence after surgical resection.

Objective: To identify the effect of postoperative adjuvant transarterial chemoembolization (TACE) on late recurrence of hepatocellular carcinoma (HCC) patients after radical resection., Methods: From year 2001 to 2007, 2436 HCC patients underwent radical resection were retrospectively selected. Patients underwent resection only were classified into control group, while those received adjuvant TACE within 2 months after operation were classified into intervention group. Patients were further stratified into those with tumor5 cm and presenting low or high risk factors for recurrence. Patients with single tumor and without microscopic tumor thrombus were defined as low risk for recurrence; otherwise they were defined as high risk. The effect of adjuvant TACE on late (>2 years) recurrence was evaluated., Results: Recurrence rates of tumor5 cm and presenting low, high risk factors for recurrence at 2-year after resection were 20.38%, 33.06%, 30.54% and 50.82%, respectively in the control group, compared with 25.41%, 39.61%, 40.55% and 51.57%, respectively in the intervention group; there were no significant differences between intervention group and control group in each stratum. For patients recurred or died within the first 2 years after resection, the median survival of tumor>5 cm and presenting high risk factors for recurrence was 24 months in the intervention group and 12 months in the Control group. Only in this subgroup, the survival curve of the intervention group was significantly higher than that compared to the control group. For patients who remained recurrence free and survived within the first 2 years after resection, there were no significant differences in the recurrence curves between the intervention group and control group in each stratum; while cumulative survival rates in the subgroup of patients with tumor size is less than or equal to 5 cm and presenting low risk factors for recurrence were 93.95%, 91.50% and 88.42% respectively in the control group, compared with 91.70%, 81.32% and 78.19% respectively in the intervention group at 3-, 4- and 5-year after resection (P=0.0062); for other subgroups, there were no significant differences in the survival curves between the intervention group and the control group in each stratum. Cox regression model suggested adjuvant TACE was not an independent risk factor for late recurrence; however, it might have negative effect on survival [hazard ratio (HR)=1.50, P=0.062] for those patients (especially patients with tumor is less than or equal to 5 cm and presenting low risk factors for recurrence)., Conclusions: The value of adjuvant TACE was mainly due to its therapeutic actions on residual tumor or early recurrence. It had no effect on postponing or eliminating late recurrence; moreover, it could be a risk rather than a benefit in patients at low risk for recurrence (especially those with tumor is less than or equal to 5 cm and presenting low risk factors for recurrence).

Objective: To evaluate the efficacy and analyze the prognostic factors of sorafenib treatment in patient with unresectable primary hepatocellular carcinoma (HCC)., Methods: During the period from December 2005 to March 2009, 50 patients with unresectable primary HCC of Child-Pugh status A were treated with sorafenib (400 mg, Bid). The tumor response was evaluated with CT or MRI imaging every 6 - 8 weeks according to the RECIST criteria. The overall survival (OS) and time to progression (TTP) were defined as the time from administration of sorafenib to the death or the last follow up and were evaluated by Kaplan-Meier method., Results: There was no PR or CR, but 28 patients (56.0%) achieved stable disease. The median follow up time was 15 months with a median OS of 14 months and median TTP of 4 months. The common adverse events were dermal reaction (68.0%, 34/50), diarrhea (52.0%, 26/50), hypertension (4.0%, 2/50), hair loss (14.0%, 7/50), myelosuppression (16.0%, 8/50), and liver dysfunction (20.0%, 10/50). However, most of the drug-related adverse events were grade I-II and reversible. The patients with lower tumor burden and without distant metastasis had better prognosis., Conclusion: Soafenib is effective for unresectable primary HCC with tolerable toxicity. Tumor stage is a predominant prognostic factor.

Background: Hepatocellular carcinoma (HCC) is a complicated condition influenced by multiple confounding factors, making optimum patient management extremely challenging. Ethnicity, stage at diagnosis, comorbidities and tumour morphology affect outcomes and vary from region to region, and there is no common language to assess patient prognosis and make treatment recommendations. Despite recent efforts to reduce the incidence of HCC, most patients present with unresectable disease. Non-surgical treatments include ablation, transarterial chemoembolisation and the multikinase inhibitor, sorafenib, but their effects in all patient subgroups are not known and further information is needed to optimise the use of these treatments., Aims: The Global Investigation of Therapeutic DEcisions in Hepatocellular Carcinoma and Of its Treatment with SorafeNib (GIDEON) study (ClinicalTrials.gov identifier NCT00812175; http://clinicaltrials.gov/) is an ongoing global, prospective, non-interventional study of patients with unresectable HCC who are eligible for systemic therapy and for whom the decision has been taken to treat with sorafenib under real-life practice conditions. The aim of this study is to evaluate the safety and efficacy of sorafenib in different subgroups, especially Child-Pugh B where data are limited., Discussion: This study will recruit 3000 patients from > 40 countries and follow them for approximately 5 years to compile a large and robust database of information that will be used to analyse local, regional and global differences in baseline characteristics, disease aetiology, treatment practice patterns and treatment outcomes, with a view to improve the knowledge base used to guide physician treatment decisions and to improve patient outcomes.

Sodium was incorporated into Cu(In,Ga)Se(2) (CIGS) thin films by evaporating NaF before the second and third stages and after the third stage of CIGS deposition. The Na-doping dosage was precisely controlled by a light-controlling method based on reflected light signals. X-ray diffraction and scanning electron microscopy results reveal that CIGS crystalline growth and microstructure can be strongly influenced by periods of Na incorporation, which leads to different device performance characteristics. Based on the analysis of results, incorporating Na before the second stage of the standard three-stage process is suggested.

Objective: To investigate the effects of palliative liver resection on metastatic potential and its gene function net of residual hepatocellular carcinoma (HCC) in nude mice model., Methods: Orthotopic HCC model was established by implantation of human HCC cell line MHCC97H xenografts with a high metastatic potential. Thirty-six HCC-bearing nude mice were randomized into 3 groups at 14 days post-operation, including palliative resection group (HCC samples A, B), control group 1 (sham operation, HCC sample C1) and control group 2 (without intervention, HCC sample C2). Six mice in each group were sacrificed by cervical dislocation at 14 days after palliative resection. Oligo tumor metastasis microarray and GEArray expression analysis suite software were employed for gene analysis. The methods of support vector machine (SVM), gene significance analysis and gene correlation degree analysis were used to search the markers capable of differentiating the metastatic potential of HCC. Gene function net was constructed based on special gene clustering analysis and multi-dimensional scale. Real-time PCR was applied to detect the mRNA expression. The remaining mice were sacrificed at 35 days after palliative resection for the examination of pulmonary metastasis. SAS 8.2 software was used for statistical analysis., Results: Gene analysis showed that for different gene expression among groups, the density of gene net in palliative group (B, 0.0670) were higher than those in control groups (A, 0.0145; C1, 0.0210; C2, 0.0146), the condensation degree of gene net in residual HCC (B, 0.1940) was higher than that in its own control group (A, 0.0098). Human gene metastasis suppressor 1 (MTSS1) that encodes a protein of missing in metastasis B (MIM-B) was situated in the central position of gene function net of residual HCC. The relative MIM-B mRNA expression examined by Real-time PCR was significantly up-regulated in residual HCC (B, 0.283 +/- 0.023) as comparison was made among groups (A, 0.142 +/- 0.018; C1, 0.177 +/- 0.054; C2, 0.156 +/- 0.017, all P < 0.05). The number of lung metastatic nodules in the palliative group (14.3 +/- 4.7) was more than that in the sham group (8.7 +/- 3.6) at 35 days post-resection (P < 0.01)., Conclusion: Palliative liver resection enhances the pulmonary metastatic potential of residual HCC in nude mice model. MIM-B may be one of the key therapeutic targets for HCC patients.

The aim of this study was to observe the effect of interferon alpha (IFNalpha) on tumor endothelial cells (TECs) in highly metastatic hepatocellular carcinoma (HCC) model, and to investigate the underlying mechanism. Nude mice with HCC xenograft were treated with IFNalpha. Gene expression profiles of TECs were analyzed by utilizing cDNA microarray. The differentiation of tumor blood vessels was evaluated by CD31/alphaSMA dual immunohistochemistry. Apoptosis of TECs was determined by CD31/TUNEL double staining. The functions of TECs in adhesion and uptake of acetylated low-density lipoprotein were observed in vitro. Results showed that IFNalpha effectively inhibited HCC tumor growth, with decreased microvessel density, increased apoptosis in TECs and normalized tumor blood vessels. cDNA microarray analysis revealed differential gene expression patterns in TECs under the treatment of IFNalpha. The cell-cell contact distribution of VE-Cadherin and uptake of acetylated low-density lipoprotein were significantly inhibited by IFNalpha in cultivated TECs. These results suggest that IFNalpha may induce apoptosis and interfere with hemophilic adhesion of TECs. The changes of gene expression in TECs contribute essentially to its effect of anti-angiogenesis and the subsequent inhibition of tumor progression., (2009 Elsevier Ireland Ltd. All rights reserved.)

Objective: To explore the effect of postoperative adjuvant transarterial chemoembolization (TACE) upon early recurrence of hepatocellular carcinoma (HCC) patients after radical resection., Methods: Between November 2000 and December 2007, 2591 HCC patients undergoing radical resection were retrospectively recruited. Patients undergoing resection alone were selected as control group while those receiving post-operative adjuvant TACE as intervention group. The patients were further stratified into tumor < or = 5 cm with low or high risk factors for residual tumor and tumor > 5 cm with low or high risk factors for residual tumor. A low risk factor for residual tumor was defined as single tumor and without microscopic tumor thrombus while a high risk factor for residual tumor was defined as 2 - 3 nodules or with the presence of microscopic tumor thrombus. The effect of adjuvant TACE upon early (< or = 2 years) recurrence was evaluated., Results: Recurrent rates of tumor < or = 5 cm with low or high risk factors for residual tumor and tumor > 5 cm with low or high risk factors for residual tumor at Month 3 post-resection were 1.34%, 3.17%, 5.33% and 8.43% in the control group versus 4.14% (P = 0.002), 8.15% (P = 0.011), 12.88% (P = 0.002) and 14.29% (P = 0.045) respectively in the intervention group; recurrence rates at Month 6 post-resection were 4.63%, 8.73%, 11.50% and 19.46% in the control group versus 6.71% (P = 0.133), 13.48% (P = 0.070), 21.02% (P = 0.052) and 23.94% (P = 0.210) respectively in the intervention group. For patients remaining recurrence free within the first 6 months post-resection, there were no significant differences in recurrence rates at Months 9, 12, 18 and 24 post-operation between each intervention group and control group., Conclusion: Postoperative adjuvant TACE has no preventive effect upon early recurrence, but may be of benefit to detect residual tumor and early recurrence.

Objective: To explore the effects of manipulation and traction combined with Nimodipine on the blood flow velocity of vertebrobasilar artery (VBA) in cervical vertigo of high flow velocity,and to evaluate clinical therapeutic effects between two methods., Methods: From March 2008 to Feburary 2009,70 patients who were diagnosed as high flow velocity of cervical vertigo were randomly divided into treatment group (35 cases) and control group (35 cases). Among 70 patients, 32 were male and 38 were female. The age ranged from 21 to 45 years with an average of 37.6 years. The disease course ranged from one day to two years with an average of 12.6 days. Patients of the treatment group were treated with manipulation for total three weeks, three times once week. The patients in the control group were treated with traction (weight ranged from 5 to 6 kg, 20 minutes each time, once every other day) and Nimodipine for total three weeks (three times each day, and with a dose of 40 mg each time). After three weeks, the changes of flow velacity of VBI and score before and after treatment were observed using transcranil Doppler (TCD) and Evaluation Scale for Cervical Vertigo. After six weeks, the therapeutic effects were assessed., Results: The mean velocity in left vertebral artery (LVA), right vertebral artery (RVA) and basilar artery (BA) were obviously lower than those before treatment in two groups (P < 0.01). The LVA, RVA and BA of the treatment group was lower than those of control group after 3 weeks (P < 0.01). There was significant difference in vertigo score after treatment between the two groups. The improvement rate of double-sides sign in X-ray image and the therapeutic effects of treatment group was superior to that of control group (P < 0.01)., Conclusion: The effect of manipulation on flow velocity of VBA is superior to that of traction combined with Nimodipine, and there are better therapeutic effects in treating cervical vertigo of high flow velocity in comparison with traction combined with Nimodipine. But there are more higher demands for manipulation's application.

For patients with early-stage hepatocellular carcinoma (HCC), potentially curative treatment options exist, including liver transplantation, surgical resection, and ablation therapy. These treatments are associated with survival benefits, and outcomes are optimized by identification of appropriate patients. However, further studies are needed to definitively confirm optimal treatment approaches for all patients. Treatment patterns vary in different parts of the world as a result of geographic differences in the incidence and presentation of the disease. In particular, because of successful screening programs, a high proportion of tumors that are identified in Japan are amenable to curative treatments, which are appropriate in a smaller proportion of patients in the west, although screening is now widely carried out in industrialized countries. Differences in the applicability of transplantation are also evident between the west and Asia. Although existing treatments for early-stage HCC are supported by considerable evidence, there remain significant data gaps. For example, further data, ideally from randomized controlled trials, are needed regarding: the use of neoadjuvant and adjuvant therapy to decrease the rate of recurrence after resection or ablation, further investigation of the role of chemoprevention following resection, and prospective analysis of outcomes of living donor compared with deceased donor liver transplantation.

Purpose: To clarify clinicopathologic differences between patients with intrahepatic cholangiocarcinoma (ICC) and hepatocellular carcinoma (HCC), and identify potential factors influencing survival after hepatectomy for ICC., Methods: Comparison of clinicopathologic data was made between patients who underwent hepatectomy for ICC (n = 272) and HCC (n = 5,829) during the same period. Twenty-five clinicopathologic variables were selected for univariate and multivariate analyses to evaluate their influence on prognosis of ICC., Results: Compared with patients with HCC, ICC patients were more common in females and more elderly, had a lower proportion of asymptomatic tumors, lower serum alpha-fetoprotein, higher serum carcinoembryonic antigen, carbohydrate antigen 19-9 and alkaline phosphatase levels; lower incidence of hepatitis history, associated cirrhosis and serum hepatitis B surface antigen; lower proportion of small tumors, well-encapsulated tumors and tumor emboli in the portal vein; higher proportion of single tumor, perihila lymph node involvement and poor differentiation; and less frequency of limited resection (all, P < 0.0001). Distant metastasis was less frequent in patients with ICC (P = 0.027). A total of 5-years overall and disease-free survival (in brackets) after resection was 26.4% (13.1%) and 44.5% (33.1%) (P < 0.0001, P < 0.0001) for patients with ICC and HCC, respectively. Factors influencing survival after resection of ICC can be divided mainly into two categories: early detection of asymptomatic ICC (P < 0.0001) and curative resection (P = 0.002)., Conclusion: ICC Patients have distinct clinicopathologic features as compared with HCC patients. Surgery remains the only effective treatment for ICC. Early detection of asymptomatic ICC and curative resection were the key to achieve optimal survival.

Background: A majority of studies predicting the foetal RhD blood group in free foetal DNA from RhD-negative maternal plasma have been conducted in Caucasian populations, whereas limited data have been accumulated for Asian populations. In this study, we assessed the feasibility of prenatal genotyping of RHD in RhD-negative Chinese pregnant women., Materials and Methods: Cell-free plasma DNA was extracted from 78 RhD-negative Chinese women carrying a singleton foetus (gestation between 14 and 40 weeks). Foetal DNA was confirmed by testing SRY or nine different polymorphic STR loci in the maternal plasma and buffy coat. Foetal RHD exons 5, 7 and 10 and intron 4 were successfully amplified with RQ-PCR. The RHD1227A allele was examined in all RhD-positive individuals. The foetal RHD genotyping results were compared with the infant cord blood serological analysis., Results: Among the 78 specimens, RHD genotyping results of 70 cases were in complete concordance with serological results from foetal umbilical cord blood. Sixty of these cases were identified as RhD-positive, and 10 cases were typed as RhD-negative. In addition, five cases were 'false-positives', while three cases were considered inconclusive. The detection rate was 89.7% (70/78). In four of the five 'false-positive' cases, the RhDel phenotype was assessed by detecting the RHD1227A allele. Thus, this method yielded a 94.9% (74/78) accuracy rate., Conclusions: The correct foetal RhD phenotype may be accurately predicted from RhD-negative maternal plasma in Chinese subjects. The RHD1227A allele proved to be an important genetic marker in the RhDel Chinese population.

Background and Objective: Systemic chemotherapy would suppress the immune system and cause reactivation of hepatitis B virus (HBV) in the tumor patients with HBV infection, which seriously affect the prognosis. Our study was to investigate the effect of transcatheter arterial chemoembolization (TACE) on HBV DNA level in primary liver cancer patients, and explore related factors., Methods: Clinical data of 162 patients with primary liver cancer who underwent TACE from December 2004 to July 2008 were analyzed. All patients' HBV DNA level, alpha-fetoprotein (AFP) and liver function before and after TACE were evaluated. Correlation of HBV DNA alteration to AFP was analyzed., Results: The positive rate of HBV DNA was decreased significantly after TACE (55.6% vs. 71.6%, P<0.01). HBV DNA level was inclined to decrease after TACE in the patients with HBV DNA of > or =1 x 10(5)/mL (odds ratio = 2.7, P<0.01). The decrease of HBV DNA was also related with the decrease of AFP level (odds ratio = 2.6,P<0.05)., Conclusion: TACE can decrease HBV DNA level in primary liver cancer patients, especially for those with preoperative HBV DNA level of > or =1 x 10(5)/mL and those with postoperative AFP decline.

Objective: Hepatic stellate cells (HSC) in hepatocellular carcinoma (HCC) transdifferentiate into extracellular matrix-producing myofibroblasts. Activated HSC can promote invasion and metastasis of HCC. To understand the differences of HSC in normal liver and HCC, we compared the gene expression patterns in HCC cell induction-activated and culture-activated rat HSC., Methods: HSC were isolated by density centrifugation and exposed to conditioned medium from rat HCC cell line C5F. Expression of 22 012 genes in quiescent HSC, culture-activated HSC and HCC induction-activated HSC was analyzed by cDNA microarray and confirmed by real-time RT-PCR and Western blot., Results: 1672 genes were differentially expressed in culture-activated HSC, including proinflammatory factors, cell adhesion molecules, cell surface receptors, signaling transduction molecules and immune factors. 711 genes were differentially expressed in HCC induction-activated HSC. Some of them were identical to those in culture-activated HSC. HCC Induction-activated HSC showed specific gene expression patterns, including Raf1, Rac2, Adam17, Wnt6, MMP-9 and TNF, suggesting that HCC cells can specifically induce HSC activation., Conclusion: The gene expression patterns in HCC induction-activated HSC are different from those in culture-activated HSC. HCC induction-activated HSC may play a major role in the invasion and metastasis of HCC. In vivo activation should be considered as the standard for the study of HSC biology. HCC induction-activated HSC should be considered as the standard for HSC biology studies.

Objective: To evaluate the relationship between epithelial mesenchymal transition (EMT) and lung metastasis in hepatocellular carcinoma., Methods: There were 100 patients who underwent surgical resection of hepatocellular carcinoma between January 2000 and March 2004. They were classified with non-distance metastasis and lung metastasis depend on the close following up till March 2007. Their hepatocellular carcinoma specimens were retrospectively examined for EMT markers (E-cadherin, Vimentin, Fibronectin) with immunochemistry staining in tissue microarray. Univariate and multivariate analysis were used for study the relationship between EMT and lung metastasis., Results: Univariate analysis showed that down regulation of E-cadherin, overexpression of fibronectin, cytosolic expression of vimentin, AFP >or= 400 ng/ml, tumor size more than 10 cm, portal vein involvement, poorly differentiated of tumor had close correlation with lung metastasis. Multivariate analysis indicated that overexpression of fibronectin was independent factor for lung metastasis apart from tumor size more than 10 cm, portal vein involvement and poorly differentiated of tumor., Conclusion: The results proposed that EMT has close relation with lung metastasis in hepatocellular carcinoma.

Objective: To evaluate the efficacy of radiofrequency ablation for the treatment of postoperative recurrence of hepatocellular carcinoma and whether radiofrequency ablation can be used as first line treatment for recurrent hepatocellular carcinoma., Methods: There were 213 patients with small recurrent hepatocellular carcinoma (tumor size of 3 cm or less and no more than 3 nodules) who treated in Liver Cancer Institute, Fudan University from January 2000 to December 2005. Among these patients 68 were treated with radiofrequency ablation and 145 were treated with repeated surgical resection. Kaplan-Meier method was used to evaluate the overall survival or disease free survival. Log-rank used to determine the survival difference between groups and COX proportional hazard was used for multivariate analysis to evaluate the risk factors for prognosis. The overall survival or disease free survival was calculated from the time treated with radiofrequency or repeated surgical resection., Results: The 1-, 3-, 5-years overall survival rates were 94.7%, 65.1%, 37.3% and 88.1%, 62.6%, 41.0% in radiofrequency ablation group and surgical repeated resection group, respectively. There was no significant difference between two groups (P = 0.693). However, the disease free survival was better in repeated surgical resection than in radiofrequency ablation, which were 79.4%, 48.1%, 34.4% and 58.0%, 27.8%, 12.4% in repeated surgical resection and radiofrequency ablation, respectively (P = 0.001). The interval between recurrence and initial hepatectomy with more than 2 years was independent factor favor to good prognosis., Conclusions: Radiofrequency ablation seems to be as effective as repeated surgical resection owing to comparable overall survival and can be considered as alternative therapy for surgical resection treatment of small recurrent hepatocellular carcinoma.

Objective: To study survivin expression in human hepatoma cells and the effects of survivin siRNA on the malignant phenotypes of human hepatocellular cell line HCCLM6., Methods: Four hepatocellular carcinoma (HCC) cell lines were used. Semi-quantitative RT-PCR and Western blot were used to measure and compare their survivin expressions. The siRNA expression vector pshRNA-survivin targeting the mRNA of survivin and vector pGPU6/GFP/Neo-NC (as a control) were constructed, and then transfected into HCCLM6 cells. FQ-PCR was used to quantify the mRNA levels of survivin. The malignant phenotypes of transfected HCCLM6 cells, including invasive activities and adhesive capabilities, were analyzed., Results: Survivin expression gradually increased with the increase of the invasion and metastasis behaviors of the four HCC cell lines (P<0.05). The expression of survivin was highest in cell line HCCLM6. Survivin mRNA level was decreased by 93.500%+/-3.117% after the pshRNA-survivin transfection. The cell adhesion rates significantly decreased in the cells transfected with pshRNA-survivin (cell adhesion rates were 11.403%+/-1.256% vs 32.545%+/-1.367%, t=20.732, P<0.01). The migrating number of HCCLM6 cells (13.5+/-0.9) transfected with pshRNA-survivin was also significantly decreased (t=14.5, P<0.01) as compared with the control group (32.6+/-1.4)., Conclusion: The expression of survivin in HCC might have a close relationship to their invasion and metastasis properties. Sequence-specific shRNA can significantly reduce the survivin expression in the HCCLM6 cell line. Suppression of survivin expression in HCCLM6 cells transfected with pshRNA-survivin can reduce their invasive and adhesive capabilities.

Objective: We previously showed that introduction of a normal, neomycin-tagged human chromosome 8 reduced the metastatic capacity of C5F rat liver cancer cell line, which had high metastatic potential without affecting tumorigenicity, suggesting the presence of one or more metastasis suppressor genes encoded on human chromosome 8. We proceeded to define further the region harboring the metastasis suppressor gene(s) and to determine the random loss of human chromosome 8 by PCR amplification of sequence tag site (STS) markers., Methods: The national Center for Biotechnology Information (NCBI) databases were used as references of the relative genetic distances of the STS markers. C5F genomic DNA and A9/neo8 genomic DNA were used as negative and positive controls for chromosome 8 amplification, respectively. Genomic DNA was isolated and quantified from cultured hybrid clones (A9/C5F-1 and A9/C5F-2 microcell hybrid clones served as metastasis-unsuppressed groups; A9/C5F-4, A9/C5F-8 and A9/C5F-10 microcell hybrid clones served as metastasis suppressed groups). STS-PCR products were separated by electrophoresis through 2% agarose gel., Results: Metastasis-suppressed microcell hybrid clones (A9/C5F-4, A9/C5F-8 and A9/C5F-10) conserved STS markers between D8S542 --> D8S1973 (8p21.1-23.1). In contrast, metastasis-unsuppressed clones (A9/C5F-1 and A9/C5F-2) lacked several markers in this region. In attempts to refine the region retained in the microcell suppressed clones, more densely spaced STS markers in the human chromosome 8p21.1-23.1 were used. We found that the metastasis-suppressed clones retained 18cM region between D8S542 and D8S1973 (8P21.1-23.1), where as the metastasis-unsuppressed clones lacked the region., Conclusion: Our results suggest that a metastasis suppressor gene is located within the interval between D8S542 and D8S1973 on human chromosome 8p21.1-23.1.

Objective: To study the tumor cell killing function of T lymphocytes stimulated by dendritic cells (DC) and to analyze the differences of protein contents of exosomes in each type of cell., Methods: The exosomes of hepatic cell lines with high (P group) or low (F group) metastatic potentials were isolated by a process of four-step centrifugation and the collected exosomes were observed under an electron microscope (EM). The tumor cell killing experiment was performed by adding T lymphocytes activated by DC loaded with exosomes from corresponding P and F group cells and was studied using 3H-TdR experiments. The proteomic analysis was performed by surface-enhanced laser desorption/ ionization time of flight mass spectrometry (SELDI-TOF-MS ) on the exosomes of P and F group cells., Results: The density distribution and content of exosomes in the P group were not equal to those in the F group observed by EM. The CD80, CD86, MHC-I and MHC-II in the P group were 64.27+5.00, 44.89+10.11, 84.35+19.89 and 59.03+19.37, and those in the F group were 71.53+4.85, 50.01+9.50, 80.68+29.87 and 58.86+21.11, respectively (P>0.05, compared with the control group). The counts per minute value in the P group was 528.40+179.06 and 78.80+24.44 in the F group after being loaded with exosomes (P<0.01, compared with the control group). There were significant differences between the proteins in the exosomes of hepatic cancer cell lines with high or low metastatic potentials., Conclusion: Exosomes have potential values of application in immunotherapy and in biotherapy for recurrences and metastases of hepatic carcinomas.

Development of an effective antitumor immune response depends on the appropriate interaction of effector and target cells. Thus, the expression of chemokines within the tumor may induce a more potent antitumor immune response. Secondary lymphoid tissue chemokine (SLC) is known to play a critical role in establishing a functional microenvironment in secondary lymphoid tissues. Its capacity to attract dendritic cells (DCs) and colocalize them with T cells makes it a good therapeutic candidate against cancer. In this study, we used SLC as a treatment for tumors established from a murine hepatocellular carcinoma model. SLC was encoded by recombinant adeno-associated virus (rAAV), a system chosen for the low host immunity and high efficiency of transduction, enabling long-term expression of the gene of interest. As a result, rAAV-SLC induced a significant delay of tumor progression, which was paralleled by a profound infiltration of DCs and activated CD4(+) T cells and CD8(+) T cells (CD3(+) CD69(+) cells) into the tumor site. In addition, rAAV-SLC treatment was also found to reduce tumor growth in nude mice, most likely due to inhibition of neoangiogenesis. In conclusion, local expression of SLC by rAAV represents a promising approach to induce immune-mediated regression of malignant tumors.

Objective: To observe changes of sensitivity to 5'-deoxy-5-fluorouridine (5'-dFUrd), and 5-fluorouracil (5-FU) in SMMC-7721 hepatocellular carcinoma cells by interferon alpha (IFN-alpha), and its relationship with the expression of thymidine phosphorylase (TP)., Methods: TP mRNA expression was determined by RT-PCR. Cytotoxicity of 5'-dFUrd, and 5-FU against SMMC-7721 cells was evaluated by MTT assay., Results: Expression levels of TP mRNA was elevated in SMMC-7721 cells after cultured with IFN-alpha, which was a concentration- and time-dependent increase. After SMMC-7721 cells was treated in the concentration of 5000 U/ml, and 10 000 U/ml IFN-alpha, the level of TP mRNA was significantly higher than that in untreated control (P < 0.05). When SMMC-7721 cells was cultured with the concentration 10 000 U/ml IFN-alpha, the level of TP mRNA rose at 8hr, reached the peak value at 12hr, and remained high level up to 72hr. IFN-alpha enhanced the sensitivity of SMMC-7721 cells to 5'-dFUrd with dose-dependent increase. IFN-alpha (10 000 U/ml) reduced IC(50) of 5'-dFUrd from (102.1 +/- 18.4) micromol/L to (34.2 +/- 4.1) micromol/L (P < 0.05). There was no obvious relation between use of IFN-alpha and 5-FU sensitivity. IC(50) of 5-FU was (5.8 +/- 2.0) micromol/L and (6.3 +/- 1.4) micromol/L in with IFN-alpha (10 000 U/ml) and control group, respectively. Under the use of interferon, sensitivity to 5'-dFUrd increased positively related to up-regulation of TP mRNA expression., Conclusion: IFN-alpha enhanced cytotoxicity of 5'-dFUrd against SMMC-7721 cells positively related to its induction of TP mRNA.

Objective: To investigate the relation between the changes of dendritic cell (DC) function and down-regulation of beta-centractin in hepatocellular carcinoma., Methods: DC derived from peripheral blood were cultured and then pulsed by lysates from hepatocarcinoma cells (HCC) with high, low, or none metastatic potential of the lines HCCLM6, MHCC97L, and Hep3B, and from normal human liver cell of the line Chang liver. DC not pulsed were used as control group. Three days later scanning electron microscopy and inverted microscopy were used to observe the morphology of the DC. Flow cytometry was used to observe the phenotype. The protein expression of beta-centractin was detected by Western blotting and immunocytochemistry., Results: Scanning electron microscopy and inverted microscopy showed no change in the morphology of the DC pulsed by different antigens. The expression levels of HLA-DR, CD80, CD83, and CD86 of the 4 pulsed groups were all significantly higher than that of the un-pulsed group (all P < 0.05). The expression of CD86 of the DC + LM6 group was significantly lower than those of the DC + Chang, DC + Hep3B, and DC + 97L groups (all P < 0.05). The mixed lymphocyte reaction (MLR) levels of the 4 pulsed groups were all significantly higher than that of the control group (all P < 0.05). The MLR level of the DC + LM6 group was significantly lower than those of the DC + Chang, DC + Hep3B, and DC + 97L groups (all P < 0.05). Western blotting showed that beta-centractin was not expressed in the control DC and was expressed in the 3 pulsed DC groups, and the beta-centractin expression levels of the DC + Chang, DC + Hep3B, and DC + 97L groups were all higher than that of the DC + LM6 group. The results of immunocytochemistry were similar to that of Western blotting., Conclusion: The down-regulation of beta-centractin in the DC pulsed with high metastatic potential HVV cell lysates is associated DC dysfunction and may be one of the mechanisms of HCC immune escape.

Secondary lymphoid tissue chemokine (SLC) is strongly expressed in secondary lymphoid organs. Its ability to facilitate chemotaxis of both dendritic cells (DC) and T cells makes it a promising candidate for cancer therapy. In this study, we modified a BMDC vaccine by incorporating the SLC mature peptide gene. The efficacy of this vaccine was evaluated using a mouse hepatocellular carcinoma (HCC) model, with rAAV2 as the gene delivery vector. The rAAV2 encoding SLC (rAAV2-SLC) transfected immature BMDCs at high efficiency and the anti-tumor effects of SLC gene modified BMDCs (rAAV2-SLC/BMDC) were evaluated. In addition, rAAV2-SLC/BMDC vaccine injected directly into tumors attracted more CD4(+) and CD8(+) T lymphocytes into tumors and showed stronger anti-tumor effects than footpad delivery. Moreover, we found that the phenotypic expression of MHC II, the secretion of IL-12 and IFN-gamma, and T cell stimulation were increased in vitro following treatment with rAAV2-SLC/BMDC vaccine and these responses were inhibited by PTX. In vivo, PTX also inhibited the anti-tumor effects of the vaccine. The results suggest that the expression of SLC by rAAV2-SLC/BMDC plays more than a chemotactic role in anti-tumor responses, thus these studies further demonstrate that SLC has potential to be valuable in cancer therapy.

Objective: To compare different expression profiles of all known chemokine receptors in human hepatocellular carcinoma (HCC) cell lines with different metastasis potentials., Methods: Eighteen pairs of chemokine receptor primers were designed using Premier software. Expression profiles of the 18 chemokine receptors on four HCC cell lines of lower to higher potentials of metastasis (SMMC-7721, MHCC97-L, MHCC97-H and HCCLM6) were analyzed by RT-PCR. Expression of CXCR4 was detected by RT-PCR., Results: Expression profiles of chemokine receptors on four HCC cell lines with different metastatic potentials had significant differences (P < 0.01), in which CCR10, CXCR4 and CXCR6 expressions decreased gradually as the metastatic potential of the cell lines increased. The expressions of CCR3, CCR4, CCR10, CCR12 and XCR1 on HCCLM6 were significantly reduced compared with SMMC-7721 (P < 0.01), whereas the expressions of CXCR1 (P = 0.006) and CXCR5 (P = 0.003) exceeded that of SMMC-7721. Except for CXCR2, CXCR6 and XCR1, most of chemokine receptors on MHCC97-H were expressed differently compared with MHCC97-L (P < 0.05), in which expressions of CCR1 (P = 0.002), CCR2 (P = 0.004) and CCR5 (P = 0.046) exceeded MHCC97-L. CXCR4 was detected only on the positive controls and SMMC-7721 when the template of total RNA was reduced one-half in RT-PCR., Conclusion: Chemokine receptors are expressed very differently at mRNA level on HCC cell lines with different metastatic potentials. The different profiles of chemokine receptors in tumor microenvironment and the function of CXCR4 in HCC should be further studied. Our findings have important implications in understanding the relationship between chemokine receptors and the metastatic potential of HCC.

Objective: To study the effects of osteopontin (OPN) on the phenotypes of human hepatocellular carcinoma cell line SMMC-7721., Methods: Human hepatocellular carcinoma SMMC-7721 cells were transfected with plasmid pcDNA 3.1(-)/OPN and cells transfected with a mock plasmid served as controls. OPN expression was verified by RT-PCR and Western blot, and concentrations of OPN, MMP-2, MMP-9 and uPA were measured by ELISA. A series of functional assays in vitro were used to monitor the changes of SMMC-7721 malignant phenotypes., Results: OPN expression of SMMC-7721 cells was elevated after transfection. Concentrations of OPN, MMP-2 and uPA in the medium of SMMC-7721 cells after transfection were higher than those of the controls [(3.02+/-0.12) ng/ml vs (1.43+/-0.07) ng/ml, (43.04+/-3.06) ng/ml vs (22.15+/-4.34) ng/ml, and (4.78+/-0.70) ng/ml vs (1.61+/-0.34) ng/ml respectively, P less than 0.01], but MMP-9 concentration did not increase [(7.82+/-2.25) ng/ml vs (7.70+/-1.92) ng/ml]. Functional assays in vitro indicated that SMMC-7721 cells after transfection showed higher adhesive, migrant and invasive capabilities than those of the controls (cell adhesion rates were 75.33%+/-10.59% vs 57.34%+/-2.52%; number of outer surface cells in migrant assay was 14.33+/-2.51 vs 6.34+/-1.53; cell number in the invasive assay was 8.23+/-1.53 vs 4.12+/-1.29 respectively, P less than 0.05)., Conclusion: OPN might enhance the expression of MMP-2 and uPA to promote malignant phenotypes of SMMC-7721 cells.

Purpose: Hepatocellular carcinoma (HCC) is an aggressive malignancy and is a devastating clinical complication of chronic liver disease. Therapeutic options are limited mainly because the genetic and biochemical understanding of this disease remains fragmented. We intended to study the role of signal transducer and activator of transcription 3 (STAT3) aberrant signaling in HCC malignancy, and the therapeutic potential of inhibition of STAT3 expression for HCC., Experimental Design: A 2'-O-methoxyethylribose-modified phosphorothioate antisense oligonucleotide (ASO) was used to knock down STAT3 expression in different human HCC cell lines, including the highly metastatic HCCLM3 derived from orthotopic implantation and subsequent lung metastasis in athymic mice. The effects of STAT3 ASO treatment on HCC cells, metastasis, and animal survival following HCCLM3 orthotopic implantation were evaluated., Results: Specific suppression of phosphorylated STAT3 reduced its DNA-binding activity, inhibited the expression of vascular endothelial growth factor, survivin, matrix metalloproteinases 2 and 9, reduced cell proliferation and migratory potential, induced apoptosis in vitro, and inhibited intradermal angiogenesis and s.c. tumorigenesis upon injection in mice. In mice bearing orthotopically implanted HCCLM3, STAT3 inhibition following therapeutic treatment with STAT3 ASO reduced circulating vascular endothelial growth factor and basic fibroblast growth factor, decreased intratumor CD34-positive microvessel density, intrahepatic and intraperitoneal transmission, and lung metastasis. HCC tumor volume and weight were reduced and the survival time of mice bearing orthotopically xenografted HCC was approximately doubled in STAT3 ASO-treated mice (P < 0.05)., Conclusions: Constitutively activated STAT3 is essential for the growth, survival, and metastasis of HCC, suggesting that STAT3-targeted therapy may have utility for HCC.

Objective: In order to seek the functional evidence that there could be metastatsis suppressor gene for liver cancer on human chromosomes, the objective of this study is to establish a method of microcell mediated chromosome transfer (MMCT)., Methods: Human chromosome 8 randomly marked with neo gene was introduced into highly metastatic rat liver cancer C5F cell line by treating the single human chromosome donor cells with sequential steps of micronucleation, enucleation and microcell fusion. Double selections of G418 and HAT were applied to screen positive microcell hybrids, which were cloned by single cell isolation. Microcell hybrid clones were confirmed by STS-PCR and WCP-FISH., Results: Microcell hybrids resistant to HAT and G418 were obtained, from which 15 clones were obtained by single-cell isolation cloning. STS-PCR and WCP-FISH proved that human chromosome 8 had been successfully introduced into rat liver cancer cell line C5F. The human chromosome 8 introduced into C5F was found to have random loss of chromosome fragments by STS-PCR and consistent recombination with rat chromosome by WCP-FISH., Conclusion: The successfulls introduction of human chromosome into highly metastatic rat liver cancer cell line has established the technical basis for functional localization of metastasis suppressor gene(s) for liver cancer on human chromosomes.

Our previous proteomics study on human hepatocellular carcinoma (HCC) cell strains revealed that cytokeratin 19 (CK19) was expressed in cells with high metastasis potential; we further studied serum CK19 fragment CYFRA 21-1 level in HCC patients and nude mice model of HCC metastasis. HCC cell line HCCLM3 was injected subcutaneously into 30 nude mice which were then randomized into 6 groups of 5 mice each. The murine serum CYFRA 21-1 and pulmonary metastases were determined 2, 3, 4, 5, 6, and 7 weeks after injection. Serum CYFRA 21-1 levels of 101 normal controls and 108 HCC patients were also determined. In nude mice model, CYFRA 21-1 level increased significantly when pulmonary metastases occurred. Among 108 HCC patients, 24 (22.2%) had increased serum CYFRA 21-1 level. The presence of portal vein tumor emboli was significantly higher in CYFRA 21-1 increased cases (33.3%, 6/24) than in CYFRA 21-1 normal cases (6.0%, 5/84) (x2=7.403, P < 0.01). In addition, the percentage of TNM stage III/IV tumor was significantly higher in CYFRA 21-1 increased patients (54.2%, 13/24) than in CYFRA 21-1 normal cases (21.4%, 18/84) (x2=9.776, P < 0.005). These results suggest that CK19 may play an important role in HCC metastasis.

Aim: To elucidate the relationship between local immunocompetent cells and prognosis of human hepatocellular carcinoma (HCC) after resection., Methods: HE staining and immunohistochemical study were carried out on specimens from patients underwent surgical resection. Local immunocompetent cells, such as dendritic cells (DCs), memory T cells, CD3+ T lymphocytes and CD8+ T lymphocytes, were counted and their relationships with tumor-free survival rate were analyzed by grouping DCs with the T lymphocytes retrospectively., Results: The number grade of infiltrating immunocompetent cells in HCC nodules and pericancerous tissues under HE staining had no significant correlation with tumor-free survival time (P=0.054, 0.071, respectively). DCs were mainly among tumor cells, encircling tumor cells with their pseudopodia and were in contact with T lymphocytes. A certain number of DCs in HCC nodules (> or =25/10HPF) statistically correlated to tumor-free survival time (P=0.005), while a certain number of DCs in pericancerous tissues (> or =28/10HPF) had no correlation with tumor-free survival time (P=0.329). The number of memory T cells, CD3+ T lymphocytes and CD8+ T lymphocytes in HCC nodules strongly correlated to tumor-free survival time (P=0.003, 0.005, 0.037, respectively). The tumor-free survival rate curves revealed that the more DCs or together with memory T cells/CD3+ T lymphocytes or that the more CD8+ T lymphocytes were detected in HCC nodules, the better the prognosis would be., Conclusions: Marked infiltration of DCs in HCC nodules was closely related to the prognosis of HCC after surgical resection and can be served as a predictive index for recurrence and metastasis of HCC.

Objectives: To study the relationship between lymphangiogenesis and lymphatic metastasis in mice bearing hepatic carcinoma and analyze the mechanism of the lymphatic metastasis., Methods: Hepatic carcinoma cell lines of high and low potentialities of lymphatic metastasis were injected into the footpads of Balb/c mice. Their metastases to lymph nodes were examined. The tumor tissues of each group were stained with 5'-nucleotidase-ALP to observe the lymphoangiogenesis. The total RNA of high and low metastatic potential cell lines were extracted for metastasis gene DNA array. The vascular endothelial cell growth factor C (VEGF-C) and VEGF-D of each cell line were detected using semi-quantitative RT-PCR and were further quantatively analyzed using real time PCR., Results: The para-common iliac a. and renal hilar lymph nodes metastases of the high metastatic potential cells were significantly higher than in the controls (P>0.05). The quantity of lymphatic vessels in the high metastasis group was significantly larger than that of the control group (P<0.05). The expressions of CD44, E-cadherin, HER2/neu, H-Ras and VEGF-C in the high metastasis group were higher than those in the low metastasis group shown by the cDNA micro array experiment but the expressions of nm23A, nm23-E4, p16ink4a, CD61 were lower. The VEGF-C expression was higher and the VEGF-D was lower in the high metastasis group compared to those of the low metastasis group shown by semi-quantitative RT-PCR. The secretion of VEGF-D was significantly lower and the ratio of VEGF-C/VEGF-D was significantly higher in the high metastasis group than the low metastasis group (P<0.05)., Conclusions: The lymphatic metastasis of hepatic carcinoma is related to lymphoangiogenesis. The changes of VEGF-C and VEGF-D expressions might be a cause influencing the lymphoangiogenesis. VEGF-C/VEGF-D might be an effective parameter in affecting lymphatic metastases.

We constructed a novel hepatocellular carcinoma-specific conditionally replicative adenovirus (CRAd). This adenovirus, designated Ad.HS4.AFP.E1A/TRAIL, expresses E1A to mediate viral replication and TRAIL to enhance HCC-killing efficacy under the control of a modified AFP promoter. An insulator HS-4 was placed in front of the AFP promoter to enhance the fidelity of the heterologous promoter. This virus was shown to have specific cytolytic activity in AFP-expressing HCC cells in vitro. Furthermore, the replication efficiency of Ad.HS4.AFP.E1A/TRAIL correlated well with AFP expression of the host cells, showing a 100-fold and 1 000 000-fold decrease in the low-and non-AFP-expressing HCC cells, respectively, compared to the high AFP-expressing HCC cells. An increase in mRNA of TRAIL and the elevated Caspase-3 activity were also observed in Ad.HS4.AFP.E1A/TRAIL-infected HCC cells. These results indicated that TRAIL expression from the viral vector activated the Caspase-3 enzymatic capacity and the HCC cells were sensitive to TRAIL. In vivo, Ad.HS4.AFP.E1A/TRAIL effectively prevented the growth of low AFP-expressing BEL-7404 xenografts. These results indicate that Ad.HS4.AFP.E1A/TRAIL could provide a new strategy of gene therapy for HCC.

Objectives: To examine the activities of transcription factors (TFs) in human hepatocellular carcinoma (HCC) cell lines with different metastatic potentials, so as to identify the TFs associated with HCC metastasis., Methods: Transcription factor activity profile of Hep3B, MHCC97L and MHCC97H, three HCC cell lines with different metastatic potentials, were examined using protein/DNA array. Electrophoretic mobility shift assays (EMSA) and Western blot were used to confirm the results obtained by protein/DNA array., Results: From a total of 345 screened TFs, 7 activity differential TFs were found, of which 5 showed increased activity, including p53, hypoxia inducible factor-1 alpha (HIF-1alpha), signal transducer and activator of transcription 3 (Stat3) and Sp1, and 2 showed decreased activity including Rb and Smad3., Conclusion: The abnormal functioning of transcription factors is closely associated with HCC metastasis. Our present findings could be of help in expanding our understanding of the mechanism of HCC metastasis and identify new predictive biomarkers and therapeutic targets.

Purpose: Little information is available on the heterogeneity of the vascular endothelium in hepatocellular carcinoma. The aim of this study was to identify the differentially expressed genes in tumor endothelial cells from highly metastatic hepatocellular carcinoma., Experimental Design: Magnetic beads conjugated with anti-CD31 antibody were used to isolate vascular endothelial cells from hepatocellular carcinoma xenografts with different metastatic potentials in nude mice. Gene expression profiles for different endothelial cells were compared by use of cDNA microarray. The up-regulated gene was confirmed by reverse transcription-PCR, real-time PCR, and immunohistochemistry., Results: cDNA microarray analysis revealed differential expression patterns in seven genes consistently presented in endothelial cells isolated from hepatocellular carcinoma with different metastatic potentials. Overexpression of platelet-derived growth factor receptor alpha was found only in the endothelium of highly metastatic hepatocellular carcinoma, which was confirmed by reverse transcription-PCR, real-time PCR, and immunohistochemistry. Oral administration of STI571 (imatinib mesylate or Glivec), a protein tyrosine kinase inhibitor of platelet-derived growth factor receptor, combined with s.c. injection of IFN-alpha not only effectively reduced tumor weight (by 81.8%) and microvessel density (by 70.2%) but also inhibited lung metastasis (by 100%). Furthermore, immunohistochemical analysis of platelet-derived growth factor receptor alpha in human hepatocellular carcinoma tissues revealed its correlation with postoperative recurrence, especially in patients without microvessel invasion., Conclusions: The gene expression of hepatocellular carcinoma vascular endothelium is different between tumors with different metastatic potential. Platelet-derived growth factor receptor alpha, which is overexpressed in endothelium of highly metastatic hepatocellular carcinoma, may serve as a biomarker for predicting metastasis and a therapeutic target for highly metastatic hepatocellular carcinoma.

Objective: To further study the impact of interferon-alpha (IFN-alpha) on thymidine phosphorylase (TP) expression and angiogenesis., Methods: Human hepatocellular carcinoma cells of the line SMMC-7721 were cultured and added with IFN-alpha of different doses: 0 (as control group), 1000, 5000 and 10,000 U/ml. Twenty-four hours later RT-PCR was used to detect the TP mRNA expression. Boyden chamber method was used to examine the endothelial cells migration. Suspension of SMMC-7721 cells was inoculated subcutaneously into 30 male BALB/c-nu/nu mice, the mice were randomly divided into 5 equal groups to be subcutaneously injected with IFN-alpha of different doses: 0 (as control group), 1.0 x 10(6), 3.0 x 10(6), 9.0 x 10(6), and 1.5 x 10(7) U.kg(-1).d(-1) for 3 weeks. The eating behavior, activity, body weight, and tumor size were observed. The rats were killed 2 days after the drug injection was stopped. The subcutaneous tumors were taken out to undergo histological examination and TP protein expression by ELISA. The microvessel density (MVD) was detected using anti-CD34 monoclonal antibody., Results: The TP mRNA expression of the SMMC-7721 cells induced by IFN-alpha of the doses of 5000 U/ml and 10,000 U/ml significantly increased in comparison with the un-treated SMMC-7721 cells (0 U/ml, P < 0.05). The endothelial cell migration significantly increased in the IFN-alpha 1000 U/ml group compared with the control group (P < 0.001), and then decreased along with the increase of IFN-alpha dose; there were no significant differences in the epithelial migration among the groups of 0, 5000, and 10,000 U/ml IFN-alpha doses (all P > 0.05). The TP protein expression levels of the tumor in the rats treated with IFN-alpha of the doses of 9.0 x 10(6), and 1.5 x 10(7) U.kg(-1).d(-1) were 48 ng/mg +/- 24 ng/mg and 60 ng/mg +/- 6 ng/mg respectively, 1.9 and 2.4 times that of the control group (both P < 0.01). The MVD of the tumors was 6.0 +/- 1.8 in the 9.0 x 10(6) U.kg(-1).d(-1) IFN-alpha group, significantly higher than that of the control group (P < 0.01); and was 4.0 +/- 1.5 in the 1.5 x 10(7) U.kg(-1).d(-1) group, significantly lower than that of the 9.0 x 10(6) U.kg(-1).d(-1) group (P < 0.05) and not significantly different from that of the control group. The tumor inhibiting rate of the 1.5 x 10(7) U.kg(-1).d(-1) IFN-alpha group was 68%, significantly higher than that of the untreated group (P < 0.05)., Conclusion: IFN-alpha of certain doses up-regulate the TP expression, and inhibit the angiogenesis induced by TP as well.

Objectives: To evaluate the role of radiotherapy (RT) for hepatocellular carcinoma (HCC) patients with abdominal lymph node (LN) metastasis at our institution in the past 7 years., Methods and Materials: We identified 125 patients with HCC metastasis to regional LNs treated with or without external beam RT (EBRT) between 1998 and 2004. Clinical characteristics collected included alpha-fetoprotein status, gamma-glutamyltransferase, status of intrahepatic tumors (size and number), previous therapy for intrahepatic tumors, metastatic LN status (location, number, and size), tumor thrombi, and Child-Pugh classification. Of the 125 patients, 62 received local limited EBRT and were classified as the EBRT group. They received locoregional LN irradiation. The tumor dose ranged from 40 to 60 Gy in daily 2.0-Gy fractions, 5 times weekly. Another 63 patients, who did not receive EBRT, were selected from hospitalized patients in the same period and were classified as the non-EBRT group. The parameters studied included survival rates and tumor response to EBRT both as demonstrated by clinical symptoms and as seen on CT. The Kaplan-Meier method was used to evaluate the survival rates, and the Cox regression model was used to identify predictors of outcome., Results: After EBRT, partial responses and complete responses were observed in 37.1% and 59.7% of patients, respectively. The median survival was 9.4 months (95% confidence interval 5.8-13.0) for the EBRT group and 3.3 months (95% confidence interval, 2.7-3.9) for the non-EBRT group (p < 0.001). Multivariate analysis showed that multiple intrahepatic primary tumors, occurrence of tumor thrombi, no therapy for intrahepatic tumors, and greater Child-Pugh classification were related to a poorer prognosis in all patients. In the EBRT group, the survival periods decreased as the distance of LN involvement from the liver increased (following the natural flow of lymph) and was also associated with the intrahepatic primary tumor size. The incidence of death resulting from LN-related complications was lower in the EBRT group., Conclusion: Lymph node metastasis from HCC is sensitive to EBRT. EBRT with 25 fractions of 2 Gy is an effective palliative treatment for patients with LN metastases from HCC presenting with good performance status and may prolong overall survival.

Purpose: To investigate the effects on sensitivity to fluoropyrimidine and endothelial cell (EC) migration by transfection with thymidine phosphorylase (TP) cDNA to a hepatocellular carcinoma (HCC) cell line SMMC-7721., Methods: SMMC-7721 was transfected with pcDNA3.1/zeo (+) with human TP cDNA. TP mRNA expression was determined by RT-PCR. Sensitivity to fluoropyrimidine was determined by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT) assay. Induction of EC migration was detected by Boyden chamber assay., Results: The construction of pcDNA3.1/zeo(+)-TP was verified by digestion with restriction endonuclease Apa1. When comparison was made between SMMC-7721 cell clone transfected with pcDNA3.1/zeo(+)-TP (S-TP) and control clone transfected with pcDNA3.1/zeo(+) (S-vector), we found that TP mRNA expression level was much higher in S-TP, being 2.09+/-0.16 vs 0.48+/-0.06 in S-vector (P < 0.01), sensitivity to 5'-deoxy-5-fluorouridine (5'-dFUrd, a prodrug of 5-fluorouracil) in S-TP was significantly enhanced compared with that in S-vector (IC(50); 56.81+/-9.85 micromol/l vs 162.25+/-11.03 micromol/l, P < 0.01), and the culture medium of S-TP possessed more potential to induce EC migration than that of S-vector (the number of ECs appearing on the outer surfaces of the membrane was 275+/-29 vs 122+/-35 per field, P < 0.01)., Conclusion: Sensitivity to 5'-dFUrd could be enhanced by transfection with TP cDNA for SMMC-7721 cells. However, EC migration was also promoted at the same time. Therefore, transfection with TP alone might have no potential to enhance anti-tumoral effects of fluoropyrimidine in HCC.

Dendritic cells (DC) are specialized antigen-presenting cells with powerful immunostimulatory properties. Their use for induction of anti-tumor immunity has been limited by several factors, including identification of appropriate tumor-associated antigens, delivery of antigens to DC, and maintaining DC in a highly activated state. Here, DC propagated in vitro were transduced with an adenoviral (Ad) vector to express hepatitis B surface antigen (HBsAg), an antigen present in hepatocellular carcinoma (HCC). Many patients with HCC demonstrate evidence of prior HBV exposure, suggesting that the presence of the virus in a quiescent state may promote tumorigenesis. Ad-HBsAg-transduced DC stimulated strong cytotoxic T lymphocyte (CTL) responses to HBsAg-expressing tumor cells, and protected mice from lethal tumor challenge. Immunity was antigen-specific, as wild-type tumor (HBsAg -) grew normally. Furthermore, DC transduced with an irrelevant vector had no effect. Vaccination with HBsAg protein, a clinically utilized preparation that confers immunity to HBV infection, did not protect against tumor challenge even though it induced a strong antibody response. These studies describe for the first time the contributions of humoral and cellular immune responses to tumor immunity induced by Ad-transduced DC compared to protein vaccination.

Objective: To investigate the effect of transcatheter hepatic arterial chemoembolization (TACE) therapy on the survival and prognosis of recurrent hepatocellular carcinoma (HCC) after surgical resection., Methods: The data of 130 surgically resected but recurrent HCC patients treated by TACE were reviewed retrospectively. The survival and influencing factors on the prognosis were analyzed., Results: The overall 1-, 3-, 5-year survival rates of these 130 patients were 83.0%, 45.5% and 17.6% respectively (median survival time 2.4 years). Ninty-four of the series were treated with TACE alone, which gave the 1-, 3- year survival rates of 76.4% and 37.1%, respectively (median survival time 2.1 years). Thirty-six out of 130 patients treated with TACE plus percutaneous ethanol injection (PEI), the 1-, 3-year survival rates were 100.0% and 66.5% respectively with a median survival time (MST) of 3.5 years. The survival of TACE plus PEI group was significantly better, and the mortality risk was significantly lower than that of TACE alone group (P < 0.05). The mortality risk of those with > 5 cm diameter recurrent tumor or with distant metastasis was significantly higher than those with < or = 5 cm diameter tumor or without metastasis (P < 0.05)., Conclusion: TACE combined with PEI may improve the survival of recurrent HCC patients.

Objectives: To study the relationship between the expression level of DLC-1 mRNA (located in 8p) and the invasion/metastasis of human hepatocellular carcinoma (HCC)., Methods: Fifty-one surgical specimens of human HCC were divided into high-invasive and low invasive groups according to their clinicopathological features. DLC-1 mRNA expression was studied in the 51 HCC specimens as well as 5 different metastasis potential cell lines using real-time quantitative PCR (RQ-PCR)., Results: The expression level of DLC-1 mRNA in HCC specimens with high invasiveness was significantly lower than that with low invasiveness (P < 0.05). The expression levels of DLC-1 mRNA were significantly different between non-metastatic (Hep3B and HepG2) and metastatic (MHCC97-H, MHCC97-L and HCCLM3) cell lines (P < 0.05). From MHCC97-L to HCCLM3, with an increase of invasiveness and metastatic potentials, the expression level of DLC-1 decreased correspondingly, and its expression level in HCCLM3 was significantly lower than that in MHCC97-L (P < 0.01)., Conclusion: The expression of DLC-1 mRNA may play an important role in inhibiting the invasiveness and metastasis of HCC.