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Objective: To identify the phenotype and immune function of dendritic cells derived from HBV-related HCC patients's peripheral blood monocytes pulsed with soluble tumor antigen, and their relation to immune escape., Methods: Peripheral blood monocytes were isolated from 18 HBV-related hepatocellular carcinoma (HCC) patients, 11 HBV-related liver cirrhosis patients (LC) and 10 health blood donors; DCs were induced in the completed medium containing GM-CSF and IL-4. The morphology of DCs was studied using a confocal microscope and scanning electronic microscope, and the phenotype of DCs were detected by flow cytometric analysis. The mixed leucocyte reaction test was employed to determine the stimulatory capacity of DCs before and after being pulsed with soluble tumor antigen (prepared from HCCLM6 cell line). IL-12 ELISA kit was used to investigate IL-12 secretion of DCs in the supernate of MLR., Results: The amount of PBMC and DCs was significantly lower in LC and HCC compare to those in the healthy subjects; the expression levels of HLA-DR, CD1a, CD80 and CD86 on DC surfaces were lower in LC and HCC patients than those of the healthy group; the stimulating capacity of DC in MLR and levels of IL-12 in supernate of MLR were also lower in LC and HCC, but were enhanced after tumor antigen pulsed in all three groups, particularly in the LC group; the secretion of IL-12 in MLR supernate was still lower than that of the healthy group., Conclusion: The phenotype and function defects of DC derived from PBMC of LC and HCC patients might play a key role in immune escape in HBV infection and HCC. The function of DC of LC patients can be enhanced after the tumor was antigen-pulsed.

The HCCLM3 cell line was established at the authors' institute from the lung metastatic lesions of BALB/c nude mice bearing human hepatocellular carcinoma (HCC) from the metastatic HCC cell line MHCC97-H. It has been shown to have a high potential for lung metastases and extensive metastases when the cells are inoculated subcutaneously or orthotopically in athymic nude mice. In the present study, the molecular cytogenetic characteristics of this cell line were evaluated with conventional G-banding, comparative genomic hybridization, and multiplex fluorescence in situ hybridization. A hyperdiploid karyotype of 53-58 chromosomes with 10 marker chromosomes was identified. The chromosomal aberrations such as i(X)(q10), der(Y)t(Y;18)(q12;p11), der(3)t(3;20) (p25;q13), der(4)t(4;8)(q31;q22)5, der(9)t(9;13)(p21;q22), der(14)t(14;22)(p13;q13), and der(15) t(15;21)(q11;q22) were described for the first time in human HCC cells. The analysis of this cell line through a combination of molecular cytogenetic techniques provides information on the possible molecular mechanisms involved in the metastatic process of HCC.

Objective: To investigate the relationship between the distribution and quantity of dendritic cells (DCs) and memory T lymphocytes in the tumor site and the prognosis of hepatocellular carcinoma (HCC)., Methods: Hematoxilin and eosin staining was done on 123 specimens of pathologically proven HCC to observe the infiltration of lymphocytes. Immunohistochemistry was used to count the S-100 protein positive DCs and CD45RO antigen positive memory T cells in ten high power fields within a specimen. Double immunohistochemical staining with double enzyme renovated by microwave for S-100 protein and CD45RO antigens was used to observe relationship between the positivity of S-100 protein and positivity of CD45RO antigen. The specimens were divided into 4 groups according to the averaged numbers of DCs and memory T cells: group I with the DCs and T cells both >/= average numbers, group II with the DCs >/= T cells and T cells < average number, group III with the DCs < average number and memory T cells >/= average, and group IV with both DCs and T cells < average numbers., Results: HE staining showed that the grade of intratumoral or peritumoral lymphocytes was not correlated with the tumor-free survival rate (P = 0.054, 0.071, both P > 0.05), the number of DCs in the cancerous node (DC >/= 25/10 high power field) was positively correlated with the tumor-free survival rate (P = 0.005) and the number of T cells in the cancerous node was positively correlated with the tumor-free survival rate (P = 0.003). Double staining method of immunohistochemistry showed that in the cancerous nodes the number of CD-100 positive DCs and the number of CD45RO positive memory T cells was significantly correlated (r = 0.531, P = 0.000). A greater number of DCs in the cancerous nodes showed a longer tumor-free survival rate of the patients (P = 0.000 between group I and group IV, P = 0.009 between group II and group IV, and P = 0.545 between group I and group II)., Conclusion: The tumor-free survival rate of the patients who had more intratumoral DCs is higher, which indicates that DCs play an important role in activation of T lymphocytes and immune response and can be used as an independent prognostic factor.

Background: The adrenal gland is a common site of extrahepatic metastases from hepatocellular carcinoma. However, it has been the subject of few studies, and the optimal treatment remains unclear. Methods previously tried for the management of adrenal gland metastasis of hepatocellular carcinoma included surgical resection, transarterial chemoembolization or percutaneous ethanol injection, on the basis of case reports. External beam radiation therapy has seldom been applied for patients with adrenal gland metastases., Methods: We retrospectively studied 22 patients with adrenal metastases from hepatocellular carcinoma who were treated with limited-field external beam radiation therapy. The radiation dose to the adrenal lesion ranged from 36 to 54 (median 50) Gy, while the intrahepatic lesions were treated with either surgical resection or transarterial chemoembolization., Results: Among the 14 patients who had pain related to adrenal metastases, 11 (78.6%) had complete pain relief without medication that lasted until death. Two (14.3%) patients had marked pain relief, but still required analgesics. Partial responses were observed in 73% of the patients. The median survival period for all patients was 10 months. No patient died from complications related to adrenal metastasis. Adverse effects were mild., Conclusion: Adrenal metastases from hepatocellular carcinoma are sensitive to radiation treatment. Radiation therapy with 50 Gy for adrenal gland metastases is a good palliative therapy with reasonable safety.

Purpose: Cell-matrix adhesive interaction has an important role in the invasive process of tumor cells, and integrins are the major receptors mediating cell-matrix adhesion. The current study is to investigate the modulation of basement membrane (BM) proteins, especially collagen IV (C IV), laminin (LN), and fibronectin (FN) in the invasive processes of human hepatocellular carcinoma (HCC) cells in vitro, and to reveal the roles of beta1 integrins and RGD-containing oligopeptide in the cell-matrix interaction., Methods: Static adhesion assay was performed to study the rates of adhesion of MHCC97-H cells, treated or untreated with anti-beta1 (2 microg ml(-1)) and GRGDS, to C IV (50 microg ml(-1)), LN (50 microg ml(-1)) or FN (50 microg ml(-1)). Gelatin zymography was used to detect the secretion of MMPs in the conditioned medium of MHCC97-H cells incubated 24 h by C IV, LN or FN, and interactions with anti-beta1 and GRGDS. Transwell chamber assay was used to investigate the influence of C IV, LN or FN, interacting with anti-beta1 and GRGDS, on the cellular mobility of MHCC97-H cells., Results: Compared with blank control group, MHCC97-H cells showed significantly higher rates of adhesion to C IV, LN, and FN. Pretreatment with anti-beta1 could suppress adhesion to C IV, LN or FN, but GRGDS inhibited adhesion to FN (P<0.05) only. LN and FN could stimulate the secretion of MMPs by MHCC97-H cells cultured in vitro, especially MMP-9 and its activated type. Treatment with anti-beta1 could partly counteract the effects of LN and FN. GRGDS could prominently induce the secretion of MMPs, but the effect could be inhibited by pretreatment of anti-beta1. The results of Transwell chamber assay showed that LN, FN, and GRGDS could increase the number of tumor cells penetrating the microporous membrane, but the data of C IV did not reach significance. The effects were partly counteracted by anti-beta1., Conclusion: BM proteins play an active role in the invasive process of human hepatocellular carcinoma cells. Integrin beta1 is an important molecule which mediates the cell-matrix adhesive interaction of tumor cells. RGD-containing peptides competitively combine with the binding site of integrin beta1, and the effects of FN are RGD sequence-dependent.

Aim: To evaluate the effect of postoperative adjuvant transcatheter arterial chemoembolization (TACE) on the prognosis of hepatocellular carcinoma (HCC) patients with or without risk factors for the residual tumor., Methods: From January 1995 to December 1998, 549 consecutive HCC patients undergoing surgical resection were included in this research. There were 185 patients who underwent surgical resection with adjuvant TACE and 364 patients who underwent surgical resection only. Tumors with a diameter more than 5 cm, multiple nodules, and vascular invasion were defined as risk factors for residual tumor and used for patient stratification. Kaplan-Meier method was used to analyze survival curve and Cox proportional hazard model was used to evaluate the prognostic significance of adjuvant TACE., Results: In the patients without any risk factors for the residual tumor, the 1-, 3-, 5-year survival rates were 93.48%, 75.85%, 62.39% in the control group and 97.39%, 70.37%, 50.85% in the adjuvant TACE group, respectively. There was no significant difference in the survival between two groups (P = 0.3956). However, in the patients with risk factors for residual tumor, postoperative adjuvant TACE significantly prolonged the patients' survival. There was a statistically significant difference in survival between two groups (P = 0.0216). The 1-, 3-, 5-year survival rates were 69.95%, 49.86%, 37.40% in the control group and 89.67%, 61.28%, 44.36% in the adjuvant TACE group, respectively. Cox proportional hazard model showed that tumor diameter and cirrhosis, but not the adjuvant TACE, were the significantly independent prognostic factors in the patients without risk factors for residual tumor. However, in the patients with risk factors for residual tumor adjuvant TACE, and also tumor diameter, AFP level, vascular invasion, were the significantly independent factors associated with the decreasing risk for patients' death from HCC., Conclusion: Postoperative adjuvant TACE can prolong the survival of patients with risk factors for residual tumor, but can not prolong the survival of patients without risk factors for residual tumor.

Purpose: This study evaluated the effect of transcatheter arterial chem-oembolization combined with external beam radiotherapy on the response rates and sur vival of patients with unresectable hepato-cellular carcinoma. Transcatheter arterial chemoembolization is frequently used for the treatment of this cancer, but complete or massive necrosis is seldom observed. Historically, radiotherapy for hepatocellular carcinoma has yielded poor long-term survival. Multimodality therapy has been initiated in an effort to improve survival statistics., Patients and Methods: We retrospectively studied 203 patients with unresectable hepa-tocellular carcinoma, who were free of tumor thrombus, lymph node involvement, or extrahepatic metastasis based on computed tomography scans of the chest and abdomen. Among the 203 patients who received transcatheter ar terial chemoembolization as initial therapy, 54 also received combination therapy with external beam radiotherapy. Tumor response rate, survival, and failure patterns were analyzed and compared between the two groups., Results: Objective responses (complete and partial responses) on computed tomography study were obser ved in 31% and 76% of patients in the non-radiotherapy and radiotherapy groups, respectively. Overall survival rates in the patients in the radiotherapy group were 71.5%, 42.3%, and 24.0% at 1, 2, and 3 years, respectively, improved over the non-radiotherapy group rates of 59.6%, 26.5%, and 11.1% at 1, 2, and 3 years, respectively. Intrahepatic failure was lower in the radiotherapy group than in the non-radiotherapy group, but the difference was not significant. Side effects from radiotherapy were common, but rarely severe., Conclusions: This retrospective study suggests that the outcome of unresectable hepatocellular carcinoma can be influenced by radiation therapy, but a prospective randomized trial would be necessary to draw definitive conclusions.

Purpose: To investigate the antitumor effect of interferon-alpha 2a (IFN-alpha2a) combined with capecitabine on hepatocellular carcinoma (HCC) in nude mice in relation to thymidine phosphorylase (TP) expression., Methods: Thirty nude mice bearing orthotopic xenografts of a human HCC tumor (LCI-D20) were divided into control, capecitabine, IFN-alpha2a, and combination (capecitabine plus IFN-alpha2a) groups. Tumor growth was determined by measuring the tumor volume. An enzyme-linked immunosorbent assay (ELISA) was used to study the TP expression in the cancer tissues of the liver., Results: IFN-alpha2a enhanced the sensitivity of the LCI-D20 tumor response to capecitabine treatment. The tumor volume was significantly reduced in the capecitabine (455+/-236 mm(3)), IFN-alpha2a (248 +/- 114 mm(3)) or combination (46 +/- 29 mm(3)) treatment groups as compared to the control (1,033 +/- 146 mm(3)) (P < 0.01). A significant difference was also found between the single treatment (capecitabine or interferon) and combination treatment group (P < 0.01 and P < 0.05, respectively). IFN-alpha2a up-regulated TP expression in LCI-D20 tumor. An approximate 1.5-fold increase in TP expression was observed in the mice which received IFN-alpha2a treatment compared to the control mice., Conclusion: IFN-alpha2a enhanced the antitumor effect of capecitabine on HCC in nude mice, which might be ascribed to the up-regulation of TP expression in liver cancer tissues by IFN-alpha2a.

Background & Objective: As a new technique of local therapy for liver cancer, radiofrequency ablation (RFA) was widely used these years in China. This study was to evaluate the treatment efficacy of RFA on primary liver cancer (PLC), identify the risk factors of recurrence, and determine the indications of RFA for PLC., Methods: Records of 94 primary small liver cancer patients underwent a single session of percutaneous RFA in Liver Cancer Institute/Zhongshan Hospital from Jan. 2001 to Dec. 2003 were reviewed retrospectively. Data analyses were performed using SPSS for windows Ver. 11.5 software., Results: With a median follow-up of 16 months affer RFA treatment, the cumulative survival rate of 94 patients was 85.5% at 1 year, and 75.6% at 2 years. The cumulative recurrence-free survival rate was 31.3% at 1 year, and 10.4% at 2 years. The total recurrence rate was 66.0% (62/94). Log-rank test revealed that tumor with diameter of >3 cm (P< 0.05), proximity to intrahepatic vessels (P < 0.01), and subcapsular location (P< 0.05) were related to tumor recurrence,while gender, Child-pugh class, alpha fetoprotein (AFP) concentration, and combination therapy with percutaneous ethanol injection were not related to tumor recurrence. Cox regression analysis indicated that tumor proximity to intrahepatic vessels (95%CI, 2.102-7.899; P=0.000), and subcapsular location (95%CI, 1.672-6.289; P=0.001)were associated independently with recurrence after RFA treatment. Severe complications occurred in 2 cases (2.1%), including 1 case of bile duct hemorrhage, 1 case of sub-diaphragm effusion. No RFA related death and other severe complications occurred., Conclusions: RFA appears to be a safe treatment for liver cancer. Patients with tumor diameter of

Purpose: To establish a "stepwise metastatic human hepatocellular carcinoma (HCC) cell model system" for in-depth study of the underlying mechanisms of HCC metastasis., Methods: Using MHCC97- a metastatic human hepatocellular carcinoma (HCC) cell line reported in 1999-as the parent cells, we subsequently established three cell lines (MHCC97-L, HMCC97-H, and HCCLM3) with increasing spontaneous metastatic potential. Now, the fourth cell line with unique multiple metastatic characteristics has been established by six rounds of in vivo selection., Results: This cell line, designated as HCCLM6, is a polygonal epithelial cell with hypotriploid karyotype, the modal chromosomes are 55-58, and marker chromosomal abnormalities include i(1) (q10), i(8)(q10), der (4) t(4;8)(q31;q22), i(X)(q10). The cell population doubling time was 32 h. Fluorescent PCR showed HBV DNA integration in the cellular genome. Thirty-five days after HCCLM6 was injected subcutaneously into BALB/c nude mice, prominent lung metastases occurred in 100% of the recipient animals. When tumor tissue was orthotopically implanted into the liver of nude mouse, widespread loco-regional and pulmonary metastases occurred. Inoculation of this cell into the footpad of nude mice also produced 75% regional lymph node metastasis. Compared with MHCC97-L which was not metastastatic via subcutaneous or footpad inoculation and 40% metastatic via orthotopic inoculation, HCCLM6 had increased expression of matrix metalloproteinase (MMP-2 and MMP-9) and cytokeratin 19 (CK19), and decreased expression of Rb2/p130. The establishment of this new cell line has completed our stepwise metastatic HCC cell mode system, which was characterized by a similar genetic background but with significant differences in spontaneous metastasis behavior., Conclusions: The study supports the theory that cancer metastasis is a highly selective dynamic process and the cell model system could be a useful platform for the study of HCC metastasis.

Objective: To investigate the role of dendritic cells (DCs) in host defense against aspergillus, and how corticosteroids hydrocortisone (HC) affects host anti-aspergillus immunity at the DCs level., Methods: Mouse bone marrow-derived DCs were generated ex vivo and the uptake of inactivated Aspergillus fumigatus conidia (iAfc) by DCs was assayed using McFarland standard. Cytokine production by DCs or by splenic T cells were measured using ELISA method., Results: DCs were capable of ingesting iAfc efficiently. HC significantly inhibited the capacity of DCs to phagocytose iAfc. After stimulated with iAfc in vitro, DCs secreted IL-12 and IFN-gamma, but did not secret IL-10. 10(-5) mol/L HC down-regulated IL-12 and IFN-gamma production by DCs, but it didn't affect IL-10 production. After the mice were inoculated with iAfc-pulsed DCs, their splenic T cells secreted IFN-gamma upon the re-stimulation with iAfc in vitro. In contrast, after the mice were inoculated with iAfc-HC-pulsed DCs, the splenic T cells showed significantly decreased IFN-gamma secretion upon the same stimulation, but the secretion of IL-10 was increased., Conclusions: DCs might act as effector cells in host defense against aspergillus, and initiate anti-aspergillus Th1 type immunity. HC inhibited the above functions of DCs and skew the immunity response towards Th2 type. iAfc-pulsed DCs might be useful for promoting the anti-aspergillus immunity of immunocompromised host.

Objective: As our previous comparative proteomics study on high and low metastasis human hepatocellular carcinoma (HCC) cell strains revealed that cytokeratin 19 (CK19) was related to higher metastasis potential, we further investigated the relationship between serum CK19 level in HCC patients and their clinico-pathologic characteristics., Methods: Serum CK19 levels of 101 normal controls and 108 pathology-proven HCC patients were determined using radioimmunoassay, and the their correlation with clinico-pathologic parameters were studied., Results: The upper limit of one-side 98% confidence interval of normal serum CK19 level was 2.3 microg/L. Among 108 HCC patients, 24 (22.2%) had increased serum CK19 level, ranging from 2.4 to 45.5 microg/L. There were 12 patients (11.1%) with increased CK19 level but normal AFP level. The percentage of poor differentiated tumor was higher in CK19 increased cases (37.5%, 9/24) than in CK19 normal cases (20.2%, 17/84). Moreover, the presence of portal vein tumor emboli was significantly higher in CK19 increased cases (25.0%, 6/24) than in CK19 normal cases (6.0%, 5/84). (Chi-square = 7.403, P < 0.01) In addition, the percentage of TNM stage III/IV tumor was significantly higher in CK19 increased patients (54.2%, 13/24) than in CK19 normal cases. (chi-square = 13.300, P < 0.005), Conclusion: Some HCC patients do have increased serum CK19 level, which could be related to portal vein tumor emboli, poor tumor differentiation and advanced tumor stages.

Metastasis remains one of the major challenges before hepatocellular carcinoma (HCC) is finally conquered. This paper summarized a decade's studies on HCC metastasis at the Liver Cancer Institute of Fudan University. We have established a stepwise metastatic human HCC model system, which included a metastatic HCC model in nude mice (LCI-D20), a HCC cell line with high metastatic potential (MHCC97), a relatively low metastatic potential cell clone (MHCC97L) and several stepwise high metastatic potential cell clones (MHCC97H, HCCLM3, and HCCLM6) from their parent MHCC97 cell. Endeavors have been made for searching human HCC metastasis-related chromosomes/proteins/genes. Monogene-based studies revealed that HCC invasion/metastasis was similar to that of other solid tumors, and the biological characteristics of small HCC were only slightly better than that of large HCC. Using comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), genotyping, cDNA microarray, and 2-dimensional gel electrophoresis, we obtained some interesting results. In particular, in collaboration with the National Institute of Health (NIH) in the United States, we generated a molecular signature that can classify metastatic HCC patients, identified osteopontin as a lead gene in the signature, and found that genes favoring metastasis progression were initiated in the primary tumors. We also found that chromosome 8p deletion, particularly in the region of 8p23, was associated with HCC metastasis. Cytokeratin 19 was identified as one of the proteins, which was found in MHCC97H, but not in MHCC97L cells. Experimental interventions using the high metastatic nude mice model have provided clues for the prevention of HCC metastasis. Translation from workbench to bedside demonstrated that serum VEGF, microvessel density, and p53 scoring may be of value for the prediction of postoperative metastatic recurrence. Interferon alpha proved effective for the prevention of recurrence both experimentally and clinically. In conclusion, HCC metastasis that probably initiated in the primary tumor is a multigene-involved, multistep, and changing process. The further elucidation of the mechanism underlying HCC metastasis will provide a more solid basis for the prediction and prevention of the metastatic recurrence of HCC.

Objective: To evaluate the effect of postoperative adjuvant transcatheter arterial chemoembolization (TACE) on hepatocellular carcinoma (HCC) patients with residual tumor., Methods: The patients were classified into intervention group (with adjuvant TACE) and control group (without adjuvant TACE) who were further stratified to those with high risk (patients with single tumor > 5 cm in diameter, or with multiple tumors, invasion to blood vessels), and low risk factors. Univariate analysis and Cox model were used to analyse prognostic factors., Results: In low risk patients with residual tumor, the 1-, 2-, 3-, 4-year survival rate was 97.2%, 78.0%, 66.5% and 66.5% in the intervention group, and 91.2%, 81.4%, 70.3% and 54.4% in the control group, respectively. There was no statistical difference between the two groups in survival (log-rank P = 0.7667). Comparing with the control group, the 1-, 2-, 3-, 4-year survival rate was 89.5%, 73.4%, 59.2% and 53.8% in the intervention group, and 70.5%, 61.9%, 46.8% and 46.8% in the control group, respectively. Postoperative adjuvant TACE significantly prolonged the survival in high risk patients with residual tumor (P = 0.0029). Cox model revealed that the benefit of adjuvant TACE was significantly increased by the high risk factors in HCC patients with residual tumor., Conclusion: The beneficial effect of postoperative TACE was only observed in high risk patients with residual tumor but not in the low risk patients with residual tumor.

Background & Objective: Invasion and metastasis are obstacles to successful treatment of hepatocellular carcinoma. Lung is the most common site of distant metastasis from hepatocellular carcinoma. In in vitro chemoinvasion assay that different tissue extracts from mice were used to induce human hepatocellular carcinoma cells with different metastatic potentials, lung extracts show the strongest inducing activity. This study was designed to investigate the mechanism of migration and invasion in human hepatocellular carcinoma with highly metastatic potential mediated by lung extracts from mice., Methods: The changes of cytoskeleton were tested using F-actin polymerization assay and flow cytometry (FCM). Correlation between matrix metalloproteinas-9 and F-actin was analyzed by fluorescence double staining in human hepatocellular carcinoma MHCC97-H cells induced by lung extracts., Results: When MHCC97-H cells were incubated with serum-free medium or spleen extracts, the cells showed elongated or polygonal morphology. When MHCC97-H cells were incubated with lung extracts, the formation of lamelliopodia or filopodia became more and more obvious and distinct as time increasing. Results of FCM showed that 1.9-fold increase in intracellular F-actin within 30s after MHCC97-H cells were incubated with lung extracts. Confocal laser scan microscopy of MHCC97-H cells stimulated in suspension showed intense F-actin staining in the periphery of the cells and redistribution of F-actin towards a leading edge. MMP-9 and F-actin were mainly localized in the perinuclear pool when the cells were incubated with serum-free medium. After stimulation with lung extracts, MMP-9 and F-actin were localized at the front of extending pseudopodia of MHCC97-H cells., Conclusion: The mechanism that lung extracts promote migration and invasion of MHCC97-H may correlate with the pseudopodia formation and reorganization of MMP-9. Lung extracts may contribute to organ-specific metastasis of hepatocellular carcinoma.

To better understand the mechanism underlying the hepatocellular carcinoma (HCC) metastasis and to search potential markers for HCC prognosis, differential proteomic analysis on two well-established HCC cell strains with high and low metastatic potentials, MHCC97-H and MHCC97-L, was conducted using two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/time-of-flight mass spectrometry. Cytokeratin 19 (CK19) was identified and found to be overexpressed in MHCC97-H as compared with MHCC97-L. This result was further confirmed by two-dimensional Western blot analysis and immunofluorescence assay. Furthermore, one-dimensional Western blot analysis showed consistently increased CK19 expression in progressively more metastatic cells. Immunohistochemical study on 102 human HCC specimens revealed that more patients in the CK19-positive group had overt intrahepatic metastases (satellite nodules, p < 0.05; vascular tumor emboli, p < 0.001; tumor node metastatis staging, p < 0.001). CK19 fragment CYFRA 21-1 levels measured in sera from nude mice model of human HCC metastasis with radioimmunoassay increased in parallel with tumor progression and rose remarkably when pulmonary metastases occurred. The results demonstrated that overexpression of CK19 in HCC cells is related to metastatic behavior. Serum CK19 level might reflect the pathological progression in some HCC and may be a useful marker for predicting tumor metastasis and a therapeutic target for the treatment of HCC patients with metastases.

Purpose: This study was to investigate the effect of capecitabine on recurrent tumor and metastasis after curative resection of liver cancer, xenograft of a highly metastatic human hepatocellular carcinoma (HCC) tumor (LCI-D20), with special reference to the expression of platelet-derived endothelial cell growth factor (PD-ECGF)., Experimental Design: LCI-D20 and LCI-D35 (a low metastatic human HCC model) liver tumors were orthotopically implanted in 96 nude mice and divided into a treatment group (24 LCI-D20 mice and 24 LCI-D35 mice) and a prevention group (48 LCI-D20 mice). In the prevention group, curative resection of liver tumors was done 10 days after the orthotopic implantation of LCI-D20 tumor. Arabic gum (control), 5-fluorouracil (5-FU), and capecitabine were administrated respectively to all of the 96 mice., Results: In the treatment group, tumor volume was 468 +/- 138, 442 +/- 81, and 240 +/- 119 mm3 (P<0.01) in the control, 5-FU, and capecitabine subgroups, respectively, in LCI-D20 mice, whereas it was 168 +/- 35, 164 +/- 23, and 144 +/- 21 mm3 (P>0.05), respectively, in LCI-D35 mice. In the prevention group, incidence of liver recurrence in the control, 5-FU, and capecitabine subgroups was 100, 100, and 50%; lung metastasis being 100, 100, and 17%; and life span being 31 +/- 5, 37 +/- 5, and 77 +/- 19 days, respectively. PD-ECGF was highly expressed in HCC and its metastatic tissues in LCI-D20 mice and hardly expressed in HCC tissues in LCI-D35 mice., Conclusions: Capecitabine inhibits tumor growth and metastatic recurrence after resection of HCC in highly metastatic nude mice model. The effect of capecitabine may be attributed to the high expression of PD-ECGF in tumors.

Metastasis is the most lethal attribute of cancer, which severely affects the effectiveness and prognosis of cancer patients. The discovery of metastasis suppressor genes will provide important clues for the predictive diagnosis and interferential therapies of metastasis. However, there have been few metastasis suppressor genes discovered till now. And this kind of research has not been reported domestically yet. In order to promote this research, this paper reviewed the theoretical principles and technical approaches for the functional localization and cloning strategy for metastasis suppressor genes, which mainly include microcell mediated chromosome transfer, PCR analysis of site tagged sites, and spontaneous metastasis analysis. The metastasis suppressor genes, KAI-1, KiSS-1, MKK4, and BRMS1, discovered by this technique and the application of this technique in prostate cancer, melanoma, and liver cancer are also reviewed.

Purpose: The MHCC97 cell line contains two clones with high (MHCC97-H) and low (MHCC97-L) metastatic potentials, for which the pulmonary metastatic rate was 100% vs 40% between the two human hepatocellular carcinoma (HCC) clones. In an effort to elucidate the mechanism of organ-specific metastasis, we studied the effect of lung extracts from C57BL/6 mice on migration and invasion using the MHCC97 cell line., Methods: Determination of migration and invasion induced by lung extracts to MHCC97 cell lines was examined by chemoinvasion assay. The organization of cytoskeleton was tested using filamentous actin (F-actin) polymerization assay and flow cytometry. The activity of matrix metalloproteinase (MMPs) was analyzed by zymogram. Fluorescence double staining was employed for MMPs and F-actin colocalization in MHCC97-H cells induced by lung extracts., Results: The number of cells in response to extracts of lung, liver, kidney, and spleen in MHCC97-H cells was 64+/-10, 6+/-2, 22+/-4, and 3+/-1, respectively. Of the extracts, lung extracts showed significant differences to promote the migratory and invasive ability for MHCC97-H cells( p<0.001). The number of cells in response to lung extracts was threefold higher in MHCC97-H than that of cells in MHCC97-L ( p<0.001). Confocal laser scan microscope of MHCC97-H cells and MHCC97-L cells stimulated with lung extracts revealed pseudopodia formation around the cell front at the indicated time point. With the time increasing, the pseudopodia formation became increasingly more obvious and distinct. Compared with unstimulated cells, analysis of FACS showed a transient 1.9-fold and 1.7-fold increase in F-actin within 30 s in MHCC97-H cells and MHCC97-L cells, respectively. Confocal laser scan microscopy of MHCC97-H cells stimulated in suspension with lung extracts revealed intense F-actin staining in the periphery of the cells and redistribution of F-actin towards a leading edge. After the cells were incubated with lung extracts, not only expressions of active and latent form of MMP-9 were upregulated, but that of latent form of MMP-2 was increased in the MHCC97-H cells and MHCC97-L cells. The levels of latent and active form of MMP-9(18.8+/-1.2, 100.1+/-1.1), and latent form of MMP-2(22.4+/-1.3) were much higher in MHCC97-H cells than those of MHCC97-L cells, which were 7.8+/-0.3, 40.8+/-2.2, and 8.2+/-0.4, respectively. MMP-9 was mainly localized perinuclear pool when the MHCC97-H cells were incubated with serum-free medium. After the cells were stimulated with lung extracts, MMP-9 were expressed and colocalized with F-actin at the front of extending pseudopodia., Conclusions: Our results indicate that the expression of matrix metalloproteinase and the pseudopodia formation in MHCC97-H cells may correlate to the metastatic potential; thus, the host environment may contribute to the preferential metastasis of HCC cells to the lung depending on the high level of MMP-9 and migratory ability.

Aim: To study the immunoprotective effect of liver cancer vaccine with co-transfected IL-2 and B7-1 genes on hepatocarcinogenesis in mice., Methods: The murine liver cancer cell line Hepal-6 was transfected with IL-2 and/or B7-1 gene via recombinant adenoviral vectors and the liver cancer vaccines were prepared. C57BL/6 mice were immunized with these vaccines and challenged with the parental Hepal-6 cells afterwards. The immunoprotection was investigated and the reactive T cell line was assayed., Results: The immunoprotection of the tumor vaccine was demonstrated. The effect of IL-2 and B7-1 genes co-transfected Hepal-6 liver cancer vaccine (Hep6-IL2/B7 vaccine) on the onset of tumor formation was the strongest. When attacked with wild Hepal-6 cells, the median survival period of the mice immunized with Hep6-IL2/B7 vaccine was the longest (68 days, chi2=7.70-11.69, P<0.05) and the implanted tumor was the smallest (z =3.20-44.10, P<0.05). The effect of single IL-2 or B7-1 gene-transfected vaccine was next to the IL2/B7 gene co-transfected group, and the mean survival periods were 59 and 54 days, respectively. The mean survival periods of wild or enhanced green fluorescence protein gene modified vaccine immunized group were 51 and 48 days, respectively. The mice in control group all died within 38 days and the implanted tumor was the largest (z=3.20-40.21, P<0.05). The cellular immunofunction test and cytotoxicity study showed that the natural killer (NK) cell, lymphokine activated killer (LAK) cell and cytotoxic T lymphocyte (CTL) activities were significantly increased in mice immunized with the Hep6-IL2/B7 vaccine, (29.5+/-2.5 %, 65.0+/-2.9 %, 83.1+/-1.5 % respectively, compared with other groups, P<0.05)., Conclusion: The Hep6-IL2/B7 liver cancer vaccines can induce the mice to produce activated and specific CTL against the parental tumor cells, and demonstrate stronger effect on the hepatocarcinogenesis than single gene modified or the regular tumor vaccine. Therefore, the vaccines may become a novel potential therapy for recurrence and metastasis of HCC.

Background & Objective: The authors' previous study suggested there could be metastasis suppressor gene for liver cancer on human chromosome 8p. TEY1 was a newly discovered metastasis suppressor gene for prostate cancer located on human chromosome 8p. In order to explore whether TEY1 gene could be a metastasis suppressor gene for liver cancer, the expression of TEY1 was quantitated in liver cancer cell strains with different metastatic potential., Methods: RNA was extracted from liver cancer cell strains, MHCC97-H, MHCC97-L, and HCCLM3, and normal liver cell line L02. The first strand cDNA was synthesized by reverse transcription, which was then used as the template to perform fluorescent quantitative polymerase chain reaction (FQ-PCR). The quantity of the expression of TEY1 gene was normalized by dividing with that of the housekeeping gene, GAPDH for each sample. One-way analysis of variance (ANOVA) was performed for the normalized values., Results: The normalized initial cDNA concentrations of TEY1 in MHCC97-H, MHCC97-L, HCCLM3, and L02 were (3.57+/-2.62) x 10(-4), (5.02+/-1.95) x 10(-4), (2.82+/-0.78) x 10(-5), and (15.20+/-0.58) x 10(-4), respectively. The expression level of TEY1 in L02 was significantly different from those of liver cancer strains (P< 0.001). The expression level of TEY1 in HCCLM3 was also significantly different from those in MHCC97-H and MHCC97-L (P< 0.05). But there was no significant difference between MHCC97-H and MHCC97-L although the expression level of TEY1 gene in MHCC97-L was 1.4 times higher than that in MHCC97-H., Conclusion: The expression levels of TEY1 gene in liver cancer cells were negatively correlated with their different metastatic potentials; it could be conjectured that TEY1 may possibly be a metastasis suppressor gene for liver cancer.

To understand the molecular mechanisms of metastasis in hepatocellular carcinoma (HCC), it is necessary to identify the accumulating genetic alterations during its progression as well as those responsible for the acquisition of metastatic potential in cancer cells. In our previous study, using comparative genomic hybridization (CGH), we found that loss on chromosome 8p is more frequent in metastatic lesions than in matched primary tumors of HCC. Thus, 8p deletion might contribute to HCC metastasis. To narrow the location of metastasis-related alteration regions, we analyzed 22 primary and matched metastatic lesions of HCC by genome-wide microsatellite analysis. Common regions with high levels of allelic imbalance (AI) were identified on 17p, 8p11-cen, 8p21-23, 4q32-qter, 4q13-23, 16q, and 1p33. Regions with increased AI in metastatic lesions were 8p23.3, 8p11.2, 17p11.2-13.3, 4q21-22, 4q32-qter, 8q24.1, 9p11, 9q31, 11q23.1, 13q14.1-31, 13q32-qter, 16p13.3, 16q13, 16q22, and 19p13.1, and these were considered to be related to the metastasis phenotype. Among them, loss on 8p was again proved to be related to progression and metastasis of HCC, and 8p23.3 and 8p11.2 were two likely regions harboring metastasis-related genes. It was also shown for the first time in HCC that AI of 19p13.1 might also be related to metastatic potential. These results provide some candidate regions for further study to identify putative genes suppressing metastasis of HCC.

Purpose: Two hepatocellular carcinoma (HCC) cell clones named MHCC97-H and MHCC97-L with different metastatic potential have recently been established from the same parent cell line MHCC97 in our institute. The cytogenetic alterations of these two clones were investigated in this study to explore the possible clues to the mechanism involved in HCC metastasis., Methods: Their chromosomal aberrations were analyzed with comparative genomic hybridization (CGH), chromosome-specific painting, and two-color fluorescence in situ hybridization (FISH)., Results: The aberrations were found in a total of 17 chromosomes, and six kinds of the aberrations including gains of 1q, 7q, 8q, 20, and the losses of 8p23, 21q were found both in the two cell clones and their parent cell line MHCC97. Using modified CGH, with the DNA of MHCC97-L as control to test the MHCC97-H clone, the loss of 8p23 and the gain of 1q31-32, 8q21.3-22, 13q22, 17q22 were highlighted, and the most significant finding was on chromosome 8. Dual color FISH combining a pericentromeric probe and a BAC probe mapping at 8q23.1 was then performed to verify this result, and the signal ratios of the BAC to centromere were 1.43 in MHCC97-H and 1.45 in MHCC97-L, confirming the over-representations at 8q in both cells. Another interesting finding in the dual-color metaphase FISH was the intrachromosomal translocation of 8q to 8p (looked like an isochromosome 8) and non-reciprocal translocation of part of 8q to 4q, which was further clarified and proved by the FISH with whole chromosome 8 painting probe., Conclusions: The high copies amplification on 8q, the formation of isochromosome 8, non-reciprocal translocation of partial 8q to 4q, and loss of 8p occurred at the same time and are the characteristic chromosomal aberrations of the two cell clones. The chromosome 8p, especially 8p23, might harbor some novel genes related to the HCC metastasis.

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Its high mortality rate is mainly a result of intra-hepatic metastases. We analyzed the expression profiles of HCC samples without or with intra-hepatic metastases. Using a supervised machine-learning algorithm, we generated for the first time a molecular signature that can classify metastatic HCC patients and identified genes that were relevant to metastasis and patient survival. We found that the gene expression signature of primary HCCs with accompanying metastasis was very similar to that of their corresponding metastases, implying that genes favoring metastasis progression were initiated in the primary tumors. Osteopontin, which was identified as a lead gene in the signature, was over-expressed in metastatic HCC; an osteopontin-specific antibody effectively blocked HCC cell invasion in vitro and inhibited pulmonary metastasis of HCC cells in nude mice. Thus, osteopontin acts as both a diagnostic marker and a potential therapeutic target for metastatic HCC.

Aim: Our previous research on the surgical samples of primary liver cancer with CGH showed that the loss of human chromosome 8p had correlation with the metastatic phenotype of liver cancer. In order to seek the functional evidence that there could be a metastatsis suppressor gene(s) for liver cancer on human chromosome 8, we tried to transfer normal human chromosome 8 into rat liver cancer cell line C5F, which had high metastatic potential to lung., Methods: Human chromosome 8 randomly marked with neo gene was introduced into C5F cell line by MMCT and positive microcell hybrids were screened by double selections of G418 and HAT. Single cell isolation cloning was applied to clone microcell hybrids. Finally, STS-PCR and WCP-FISH were used to confirm the introduction., Results: Microcell hybrids resistant to HAT and G418 were obtained and 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. STS-PCR detected a random loss in the chromosome introduced and WCP-FISH found a consistent recombination of the introduced human chromosome with the rat chromosome., Conclusion: The successful introduction of human chromosome 8 into highly metastatic rat liver cancer cell line builds the basis for seeking functional evidence of a metastasis suppressor gene for liver cancer harboring on human chromosome 8 and its subsequent cloning.

We have developed a new class of adenovirus vectors that selectively replicate in tumor cells. The vector design is based on our recent observation that a variety of human tumor cell lines support DNA replication of adenovirus vectors with deletions of the E1A and E1B genes, whereas primary human cells or mouse liver cells in vivo do not. On the basis of this tumor-selective replication, we developed an adenovirus system that utilizes homologous recombination between inverted repeats to mediate precise rearrangements within the viral genome resulting in replication-dependent activation of transgene expression in tumors (Ad.IR vectors). Here, we used this system to achieve tumor-specific expression of adenoviral wild-type E1A in order to enhance viral DNA replication and spread within tumor metastases. In vitro DNA replication and cytotoxicity studies demonstrated that the mechanism of E1A-enhanced replication of Ad.IR-E1A vectors is efficiently and specifically activated in tumor cells, but not in nontransformed human cells. Systemic application of the Ad.IR-E1A vector into animals with liver metastases achieved transgene expression exclusively in tumors. The number of transgene-expressing tumor cells within metastases increased over time, indicating viral spread. Furthermore, the Ad.IR-E1A vector demonstrated antitumor efficacy in subcutaneous and metastatic models. These new Ad.IR-E1A vectors combine elements that allow for tumor-specific transgene expression, efficient viral replication, and spread in liver metastases after systemic vector application.

Aim: To improve the preparation of adherent lymphokine-activated killer (A-LAK) cells and to study the effects of cryopreservation and phenylacetate (PA) on biological characters of A-LAK cells., Methods: A-LAK cells were obtained from peripheral blood mononuclear cells (PBMCs) of the patients with hepatocellular carcinoma (HCC) by using L-phenylalanine methyl ester (PME) to deplete immunosuppressive monocytes. Proliferative activity of SMMC7721 cell line after treatment with phenylacetate (PA) was observed. A-LAK cells were treated with the supernatant of SMMC7721 cells that had been pretreated with PA. The changes of proliferation, cytotoxicity and phenotype of A-LAK cells were investigated after cryopreservation., Results: The expansion of A-LAK cells (96.79 +/- 69.10 folds on Day 14) was significantly higher than that of non-adherent LAK (NA-LAK) cells (22.77 +/- 13.20) as well as conventional LAK cells (4.64 +/- 0.91). PA significantly suppressed the growth of SMMC7721 cells, and the inhibitor ratio was 46%. The supernatant of cultured tumor cells intensively suppressed the proliferation and cytotoxicity of A-LAK cells, but the suppressive effect of the supernatant was previously decreased after treatment with PA. Impairments in proliferation and cytotoxicity of A-LAK cells immediately after thawing of cryopreservation and recovery after reincubation with IL-2 were observed. The cytotoxicity of thawed A-LAK cells on Day 5 was significantly higher than that of fresh A-LAK before freezing (54.8 +/- 10.2% vs 40.5 +/- 6.4%). No significant change in the percentage of lymphocyte subsets was identified in frozen A-LAK cells as compared with that in the fresh control cells., Conclusion: A-LAK cells can be simply prepared by using PME, and showed a synergistic anti-tumor effect with the combination of PA. Cryopreservation can increase the immunoactivities of A-LAK cells from the patients with hepatocellular carcinoma.

Among various immunotherapeutic approaches, interleukin-12 (IL-12) is particularly appealing because of its superior antitumor effects, which have been demonstrated in preclinical as well as clinical studies. However, IL-12 therapy was often accompanied by severe side effects due mainly to the supranormal induction of interferon-gamma. To optimize the therapeutic efficacy and lower the side effects of IL-12, we have investigated the antitumor activity of combined IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene therapy in a highly malignant and poorly immunogenic murine hepatocellular carcinoma model. Using a versatile hydrodynamics-based DNA delivery method, we showed that the combined gene delivery of IL-12 and GM-CSF induced very strong antitumor cellular immunity and achieved significant therapeutic efficacy, whereas each cytokine gene alone yielded appreciable but less effects. We also observed that the combined therapy induced lower levels of interferon-gamma than did IL-12 alone. These results suggest that combined IL-12 and GM-CSF therapy can render a stronger antitumor effect as well as lowering potential side effects.

Aim: To establish clone cells with different metastatic potential for the study of metastasis-related mechanisms., Methods: Cloning procedure was performed on parental hepatocellular carcinoma (HCC) cell line MHCC97, and biological characteristics of the target clones selected by in vivo screening were studied., Results: Two clones with high (MHCC97-H) and low (MHCC97-L) metastatic potential were isolated from the parent cell line. Compared with MHCC97-L, MHCC97-H had smaller cell size (average cell diameter 43 microm vs 50 microm) and faster in vitro and in vivo growth rate (tumor cell doubling time was 34.2h vs 60.0h). The main ranges of chromosomes were 55-58 in MHCC97-H and 57-62 in MHCC97-L. Boyden chamber in vitro invasion assay demonstrated that the number of penetrating cells through the artificial basement membrane was (37.5 +/- 11.0) cells/field for MHCC97-H vs (17.7 +/- 6.3)/field for MHCC97-L. The proportions of cells in G0-G1 phase, S phase, and G2-M phase for MHCC97-H/MHCC97-L were 0.56/0.65, 0.28/0.25 and 0.16/0.10, respectively, as measured by flow cytometry. The serum AFP levels in nude mice 5wk after orthotopic implantation of tumor tissue were (246 +/- 66) microg.L(-1) for MHCC97-H and (91 +/- 66) microg.L(-1) for MHCC97-L. The pulmonary metastatic rate was 100% (10/10) vs 40% (4/10)., Conclusion: Two clones of the same genetic background but with different biological behaviors were established, which could be valuable models for investigation on HCC metastasis.

Metastatic human HCC model is needed for the studies on mechanism and intervention of metastatic recurrence. By using orthotopic implantation of histologically intact tissues of 30 surgical specimens, a patient-like metastatic model of human HCC in nude mice (LCI-D20) and a low metastatic model of human HCC in nude mice (LCI-D35) have been established. All mice with transplanted LCI-D20 tumors exhibited extremely high metastatic ability including spontaneous metastasis to liver, lungs, lymph nodes and peritoneal seeding. Remarkable difference was also found in expression of some of the invasiveness related genes and growth factors between the LCI-D20 and LCI-D35 tumors. PAI-1 increased gradually following tumor progression in LCI-D20 model, and correlated with tumor size and AFP level. Phasic expression of tissue intercellular adhesion molecule-1 in this model was also observed. Using corneal micropocket model, it was demonstrated that the vascular response induced by LCI-D20 tumor was stronger than that induced by LCI-D35 tumor. Similar report on metastatic human HCC model in nude mice and human HCC cell line with metastatic potential was rarely found in the literature. This LCI-D20 model has been widely used for the studies on intervention of metastasis, including anti-angiogenesis,antisense approach, metalloproteinase inhibitor, differentiation inducer, etc. It is concluded that the establishment of metastatic human HCC model in nude mice and human HCC cell line with metastatic potential will provide important models for the in vitro and in vitro study of HCC invasiveness, angiogenesis as well as intervention of HCC recurrence.

Purpose: To study the relationship between thrombomodulin (TM) plasma levels and the formation of portal vein tumor thrombus (PVTT) in patients with hepatocellular carcinoma (HCC)., Methods: Pre- and-postoperative plasma TM levels of 45 patients with HCC and six patients with benign liver-occupying lesion were measured by enzyme-linked immunosorbent assay (ELISA), and the expression of TM in human HCC tissues was determined by immunohistochemistry assay., Results: The preoperative plasma TM level of patients with HCC (10.2+/-5.7 ng/ml) was significantly higher than that of those patients with benign liver-occupying lesion (6.1+/-2.2 ng/ml) and that of normal controls (5.7+/-1.0 ng/ml), respectively (P<0.05). The postoperative TM level of 40 patients with HCC whose tumors had been removed decreased significantly than the preoperative TM level (10.8+/-5.3 ng/ml versus 7.6+/-4.2 ng/ ml, P < 0.05), whereas there was no significant difference between the preoperative and postoperative TM level of six patients with benign liver-occupying lesion (6.1+/-2.2 ng/ml versus 5.9+/-1.8 ng/ml, P>0.05). The preoperative plasma TM level of patients with single HCC (11.5+/-5.9 ng/ml) or no PVTT (11.4+/-5.6 ng/ml) was significantly higher than that of those patients with multiple HCC (8.1+/-4.6 ng/ml) or PVTT (6.9+/-4.5 ng/ ml), respectively (P<0.05). The preoperative plasma TM level of the patients with HCC tissue that stained positive for TM was significantly higher than those with tissue that stained negative for TM (12.2+/-6.5 ng/ ml versus 8.7+/-4.6 ng/ml, P<0.05). The postoperative plasma TM level showed no difference between the patients with HCC tissue stained positive and negative for TM (8.3+/-4.1 ng/ml versus 7.6+/-4.4 ng/ml, P>0.05). There was also no significant difference between the plasma TM level and other clinicopathological futures., Conclusions: Plasma TM increases in patients with HCC and can be a biomarker of the formation of PVTT.

Recently, we found that chromosome 8p deletion might be associated with hepatocellular carcinoma (HCC) metastasis by analyzing the differences in chromosomal alterations between primary tumors and their matched metastatic lesions of HCC with comparative genomic hybridization (CGH) (Qin et al. 1999). To further confirm this interesting finding, the genomic changes of two models bearing human HCC with different metastatic potentials (LCI-D20 and LCI-D35), and the new human HCC cell line with high metastatic potential (MHCC97) were analyzed by CGH. Gains on 1q, 6q, 7p, and 8q, and losses on 13p, 14p, 19p, 21, and 22 were detected in both LCI-D20 and LCI-D35 models. However, high copy number amplification of a minimum region at 1q12-q22 and 12q, and deletions on 1p32-pter, 3p21-pter, 8p, 9p, 10q, 14q, and 15p were detected only in the LCI-D20 model. Gains on 1p21-p32, 2p13-p21, 6p12-pter, 9p, 15q, and 16q11-q21, and losses on 2p23-pter, 4q24-qter, 7q31-qter, 12q, 17p, and 18 were detected only in the LCI-D35 model. The chromosomal aberration patterns in the MHCC97 cell line were similar to its parent LCI-D20 model, except that gains on 19q and losses on 4, 5, 10q, and 13q were found only in the cell line. These results provide some indirect clues to the metastasis-related chromosomal aberrations of HCC and further support the finding that 8p deletion is associated with HCC metastasis. 1q12-22 and 12q might harbor a novel oncogene(s) that contributes to the development and progression of HCC. Amplification on 8q and deletions on 4q and 17p may be not necessary for HCC metastasis.

Background: Recently, the implementation of screening programs using alpha-fetoprotein (AFP) and ultrasonography in high risk populations has identified increasing numbers of patients with small hepatocellular carcinoma (small HCC). The aim of this study was to summarize the authors' experience in patients who underwent hepatectomy for small HCC and the factors that influence or improve long term survival., Methods: The study included 1000 patients who underwent hepatectomy for small HCC (< or = 5 cm) and compared them with 1366 patients who underwent hepatectomy for large HCC (> 5 cm) during the same period. A Cox proportional-hazards model was used for multivariate analysis of prognostic factors., Results: Comparison between patients with small HCC (n = 1000 patients) and patients with large HCC (n = 1366 patients) revealed that those with small HCC had a higher resection rate (93.6% [1000 of 1068 patients] vs. 55.7% [1366 of 2451 patients]; P < 0.01), a higher curative resection rate (80.5% [805 of 1000 patients] vs. 60.7% [829 of 1366 patients]; P < 0.01), a lower operative mortality rate (1.5% [15 of 1000 patients] vs. 3.7% [50 of 1366 patients]; P < 0.01), better differentiation of tumor cells (Edmondson Grade 3-4; 14.9% vs. 20.1%; P < 0.01), a higher incidence of single nodule tumors (82.6% vs. 64.4%; P < 0.01), a higher proportion of well encapsulated tumors (73.3% vs. 46.3%; P < 0.01), a lower incidence of tumor emboli in the portal vein (4.9% vs. 20.8%; P < 0.01), and higher survival rates after undergoing resection (5 years: 62.7% vs. 37.1%; P < 0.01; 10 years: 46.3% vs. 29.2%; P < 0.01). No significant difference was found between survival after undergoing minor resection (n = 949 patients) or lobectomy (n = 51 patients) in patients with small HCC (P > 0.05). Reresection for subclinical recurrence or solitary pulmonary metastasis after small HCC resection was undertaken in 84 patients., Conclusions: Resection is still the modality of first choice for the treatment of patients with small HCC. Minor resection instead of lobectomy was the key to increasing resectability and decreasing operative mortality, and reresection for subclinical recurrence or solitary pulmonary metastasis was important approach to prolonging survival further., (Copyright 2001 American Cancer Society.)

Aim: To compare the therapeutic effect and significances of multimodality treatment for hepatocellular carcinoma (HCC) with tumor thrombi in portal vein (PVTT)., Methods: HCC patients (n=147) with tumor thrombi in the main portal vein or the first branch of portal vein were divided into four groups by the several therapeutic methods. There were conservative treatment group in 18 out of patients (group A); and hepatic artery ligation(HAL) and/or hepatic artery infusion (HAI) group in 18 patients (group B), in whom postoperative chemoembolization was done periodically; group of removal of HCC with PVTT in 79 (group C) and group of transcatheter hepatic arterial chemoembolization (TACE) or HAI and/or portal vein infusion (PVI) after operation in 32 (group D)., Results: The median survival period was 12 months in our series and the 1-,3-, and 5-year survival rates were 44.3%, 24.5% and 15.2%, respectively. The median survival times were 2, 5, 12 and 16 months in group A, B, C and D, respectively. The 1-, 3- and 5-year survival rates were 5.6%, 0% and 0% in group A; 22.2%, 5.6% and 0% in group B; 53.9%, 26.9% and 16.6% in group C; 79.3%, 38.9% and 26.8% in group D, respectively. Significant difference appeared in the survival rates among the groups (P < 0.05)., Conclusion: Hepatic resection with removal of tumor thrombi and HCC should increase the curative effects and be encouraged for the prolongation of life span and quality of life for HCC patients with PVTT, whereas the best therapeutic method for HCC with PVTT is with regional hepatic chemotherapy or chemoembolization after hepatic resection with removal of tumor thrombi.

Postoperative recurrence of human hepatocellular carcinoma (HCC) is the major issue that must be addressed to further improve prognosis. This study was undertaken to investigate the effects of interferon-alfa-1b (IFN-alpha-1b) on recurrent tumor and metastasis after curative resection in nude mice bearing an HCC xenograft with high metastatic potential. Tumor tissues from LCI-D20, a metastatic model of HCC in nude mice, were orthotopically implanted in 105 nude mice. Eleven days later, 64 mice underwent curative resection of liver tumors. IFN-alpha at different doses was administered subcutaneously to mice with or without resection. In mice without resection, when comparison was made among control, IFN 7.5 x 10(6) U/kg/day, 1.5 x 10(7) U/kg/day for treated groups, and 3 x 10(7) U/kg/day; tumor volume was 8,475 mm(3) +/- 2,636 mm(3), 7,963 mm(3) +/- 3,214 mm(3), 769 mm(3) +/- 287 mm(3), and 13 mm(3) +/- 9 mm(3); incidence of lung metastasis being 100%, 80%, 40%, and 0%; life span was 45 +/- 4 days, 53 +/- 8 days, 81 +/- 6 days, and 105 +/- 24 days, respectively. In mice with curative resection, when comparison was made among control, IFN 5 x 10(5) U/kg/day, 1 x 10(6) U/kg/day, 4 x 10(6) U/kg/day, 7.5 x 10(6) U/kg/day, 1.5 x 10(7) U/kg/day, and 3 x 10(7) U/kg/day for treated groups; incidence of recurrent tumor was 100%, 100%, 87.5%, 100%, 87.5%, 62.5%, and 12.5%; lung metastasis being 100%, 75%, 87.5%, 50%, 62.5%, 0%, and 0%, respectively. IFN-alpha inhibited neovascularization induced by LCI-D20 tumor specimens implanted into the micropocket of nude mice corneas. In conclusion, high-dose and long-term therapy with IFN-alpha dose-dependently inhibits tumor growth and recurrence after resection of HCC. The effect of IFN-alpha may be attributed to antiangiogenesis in this experiment. These results provide potential clinical implication, particularly for the prevention of recurrence after curative resection of HCC.

To improve the safety and efficiency of human hepatocellular carcinoma (HCC) gene therapy, we explored the use of a liver-specific promoter and a tumor-specific enhancer to achieve regular IL-2 gene expression for treatment of HCC. The human alpha-fetoprotein (AFP) enhancer [E(AFP)] and the albumin promoter [P(ALB)] were amplified from human genomic DNA. We used eukaryotic expression vector pcDNA-3 for the delivery of the IL-2 gene because this plasmid is a non-transient, fast-selection expression vector. A recombinant plasmid was constructed including the selectable marker neoR gene and the human IL-2 gene derived by the E(AFP) - P(ALB). The liver-predominant expression pattern of the IL-2 gene was observed in the medium of the transfected cells. When human HCC cell lines displaying different levels of AFP and non-hepatocyte tumor cell lines were transfected with the recombinant plasmid, IL-2 was expressed highly in AFP and albumin-positive HCC cells, but low in nonhepatocyte tumor cells. Moreover, the expression level of IL-2 gene was positively proportional to the level of AFP expression in the transfected cells.

Background/aims: To investigate the effect of antisense H-ras DNA on tumorigenesity, apoptosis and metastasis of a high metastatic tumor model of human hepatocellular carcinoma in nude mice LCI-D20., Methodology: LCI-D20 cells in primary culture were treated with 10 microns/L antisense oligodeoxynucleotide (ODN) drugs in vitro. 1.5 x 10(6) LCI-D20 cells with or without pretreatment were inoculated into each elevated subcutaneous (s.c.) flap in 14 nude mice, 6 animals for antisense H-ras oligodeoxynucleotide treated cells, 4 for H-ras non-specific antisense oligodeoxynucleotide treated cells, and the rest 4 for cells without pretreatment., Results: In in vitro cell culture study, 5-day continuous suppression of H-ras expression by antisense H-ras oligodeoxynucleotide resulted in significant inhibition of the proliferation of LCI-D20 cells (t = 31.529, P < 0.01). In situ end-labeling detection showed that apoptotic cell death was significantly increased in cells with 5-day treatment of antisense H-ras oligodeoxynucleotide (34.0 +/- 4.5%) in comparing with cells without treatment (2.5 +/- 1.2%, t = 13.434, P < 0.01) or treated with non-specific antisense oligodeoxynucleotide (4.8 +/- 1.4%, t = 12.453, P < 0.01) at the corresponding time. In the in vivo experiment, at week 6, no palpable tumor could be found in 50% (3/6) of animals receiving cells with pretreatment of antisense H-ras oligodeoxynucleotide, while 100% (4/4, 4/4) of animals in the 2 control groups developed palpable tumors. Tumor growth in antisense H-ras treated animals was significantly retarded in comparison with that of the untreated (t = 3.509, P < 0.01) or non-specific antisense oligodeoxynucleotide treated animals (t = 3.452, P < 0.01). 75% to 100% of animals in the 2 control groups developed lung metastases, while in antisense H-ras treated animals lung metastasis foci could not be found by random serial section and microscopy (u = 2.536, P < 0.01; u = 3.162, P < 0.01, respectively)., Conclusions: Specific inhibition of H-ras expression by antisense H-ras oligodeoxynucleotides could not only induce apoptotic cell death, inhibit the growth rate of LCI-D20 cells in vitro and in vivo, but also alter in vivo tumorigenesity and metastatic potential of LCI-D20 cells.

Purpose: Both platelet-derived endothelial cell growth factor (PD-ECGF) and vascular endothelial growth factor (VEGF) are known to promote the development of new blood vessels, which are fundamental to tumor growth and metastasis. We aimed at evaluating the gene expression of PD-ECGF and VEGF in hepatocellular carcinoma (HCC) and portal vein tumor thrombus (PVTT)., Patients and Methods: Surgical specimens (28 HCC, 28 nontumorous liver tissues and 18 PVTT) were studied by Northern blot analysis. The levels of PD-ECGF mRNA and VEGF mRNA expression were measured by densitometric scanning of the autoradiographs, and they were normalized to the level of expression of an internal control (glyceraldehydephosphate dehydrogenase) mRNA., Results: The expression rates of PD-ECGF mRNA in PVTT, HCC and nontumorous liver tissues were 77.8% (14/18), 67.9% (19/28) and 35.7% (10/28), being 88.9% (16/18), 75.0% (21/28) and 17.9% (5/28) respectively for VEGF mRNA. The expressions of PD-ECGF mRNA and VEGF mRNA were higher in HCC with PVTT than when PVTT was absent (P < 0.05). The PVTT was more often seen in patients with positive expression of both PD-ECGF mRNA and VEGF mRNA in HCC than in patients who were positive for only one of these factors or negative for both (P < 0.05)., Conclusion: Both PD-ECGF and VEGF correlated well with the formation of PVTT of HCC.

Purpose: The possibility of tumor rejection antigen, encoded by the MAGE-1 gene, being a target for immunotherapy in hepatocellular carcinoma (HCC) patients and the cloning of the full-length cDNA of this gene for subsequent studies were explored with the aim of discovering new immunotherapeutic strategies for HCC., Methods: Expression of the MAGE-1 gene in HCC specimens and HCC cell lines was examined by the reverse transcription/polymerase chain reaction (RT-PCR) with a pair of specific primers, which gave a 421-bp fragment. Another pair of primers were then used to amplify the full-length MAGE-1 cDNA by the same method. The PCR products were then digested by restriction endonucleases and inserted into the plasmid PUC19. After primary selection of the recombinants by endonuclease digestion, the sequences of the inserted gene fragments were confirmed by DNA sequence analysis., Results: In 45 HCC patients, MAGE-1 mRNA was expressed in 27 tumor tissues (60%) and 5 paratumor tissues (11.1%). All the four HCC cell lines positively expressed the MAGE-1 gene. Owing to the high homology of the MAGE gene family, we obtained three clones of different MAGE genes using the same pair of cloning primers. The three clones were confirmed to be a full-length MAGE-1 gene, a 750-bp fragment of the MAGE-3 gene and a fragment highly homologous to MAGE-6 and MAGE-12 but not identical to any known MAGE genes., Conclusion: The high expression frequency of MAGE-1 gene in HCC suggests the possibility of using it as a target for immunotherapy in HCC patients. Some MAGE genes other than MAGE-1 may also be expressed in HCC together with an unknown MAGE gene that might introduce new targets for immune attacks. The three gene clones obtained in this study can be used in further investigations and especially in the development of new liver cancer vaccines.

To understand the genetic mechanisms underlying the progression of hepatocellular carcinoma (HCC) metastasis, differences of genomic alterations between 10 pairs of primary HCC tumors and their matched metastatic lesions were analyzed by comparative genomic hybridization. Several chromosomal alterations including loss of 8p, 4q, 17p, and 19p, gain of 5p and high-level amplification of 1q12-q22 were detected in two or more cases. The most significant finding is the loss of 8p which was detected in 8 metastatic tumors but only in 3 corresponding primary tumors (P = 0.03). This result suggests that the deletion of chromosome 8p might contribute to the development of HCC metastasis. Another interesting result is the detection of a minimum high-level amplification region at 1q12-q22 in HCC. This result provides a candidate amplification region in HCC for further study to identify amplified oncogenes related to the development or progression of HCC. Finally, this study provides a practicable model to detect specific genetic alterations related to the tumor metastasis through comparing the primary tumor and its corresponding metastatic lesion using comparative genomic hybridization technique.

A new human hepatocellular carcinoma (HCC) cell line with a highly metastatic potential was established from subcutaneous xenograft of a metastatic model of human HCC in nude mice (LCI-D20) by means of alternating cell culture in vitro and growth in nude mice. The line, designated MHCC97, has been cultivated for 18 months and subcultured for more than 90 passages. The line was showed to be of human origin by karyotype analysis. The cells were either grown as compact colonies (in clusters) or as a monolayered sheet with about 31 h of population-doubling time, exhibited typical malignant epithelial in morphology and were positive for alpha-fetoprotein (AFP). Flow cytometric analysis of the cell DNA content showed an aneuploid pattern, and its index was 1.5 as compared to that of normal human peripheral blood lymphocytes. Karyotypic analyses of G- and C-banding techniques revealed that all cells presented chromosome abnormalities in number and structure. The number of cell line MHCC97 chromosome ranged from 59 to 65 with a modal number of 60 and 61. At least two common chromosome markers, i(1q) and der(4)t(4;?)(4pter-->q35::?), were present in all cells, and deletion of Y chromosome also occurred in all cells. The subcutaneous and intrahepatic xenografts were formed and metastatic lesions in lungs were found after the cells were inoculated into nude mice. The rate of metastasis to lungs was 100% using orthotopic inoculation. Reverse transcription polymerase chain reaction products revealed positive expressions of integrin alpha5 and beta1, urokinase type plasminogen activator receptor (uPAR), vascular endothelial growth factor and nm23-H1 mRNAs of cell line MHCC97. Immunostaining of c-Met, uPAR showed strongly positive in both subcutaneous xenografts and lung metastatic lesions; while positive in xenografts and negative in metastatic lesions for integrin alpha5, beta1. E-cadherin and P53 was not expressed either in xenograft or in the metastatic lesions. PCR products of HBsAg and HBxAg were both positive. The cell line MHCC97 still retained some characteristic features of original tumour. Establishment of cell line MHCC97 should be beneficial to the studies of HCC metastatic mechanisms.

AIM:To certify the relationship between AFP mRNA and some pathological parameters of hepatocellular carcinoma (HCC).METHODS:We detected the expression of AFP in mRNA level in tissue samples from 52 patients suffered from HCC by RT-PCR method.RESULTS:The positive rate of AFP mRNA was 76.9% in the HCC tumor tissues,and 69.4% in the paratumor tissues from the HCC patients with severe cirrhosis.However, in HCC patients without cirrhosis, the positive rate reached 50% in tumor tissues, but no AFP mRNA expression was found in the related paratumor tissues.CONCLUSION:The AFP protein was specially expressed by HCC cells and mutated hepatocytes.The AFP mRNA was positively related with cirrhosis, but no significant relationship was found between AFP mRNA and tumor size, capsule status and tumor metastasis.

This study explores the use of a liver-specific albumin promoter and a tumor-specific alpha-fetoprotein (AFP) enhancer to achieve the regulated expression of the cytokine interleukin-2/interferon alpha2b (IL-2/IFNalpha2b) fused gene for treatment of hepatocellular carcinoma (HCC). The human AFP enhancer (E(AFP)) and albumin promoter (P(ALB)) were amplified from human chromosome DNA by the polymerase chain reaction. A recombinant retrovirus was constructed including, as a selectable marker, the neoR gene and the IL-2/IFNalpha2b fused gene controlled by E(AFP)-P(ALB). The liver-targeted expression pattern of the IL-2/IFNalpha2b fused gene was observed when this product was tested in the culture medium of the infected cells (IL-2 activity was 850 IU/10(6) cells, IFNalpha activity was 320 IU/10(6) cells). Moreover, The growth of the IL-2/IFNalpha2b-fused-gene-infected HCC cells, SMMC7721, was clearly suppressed by the second week after innoculation of nude mice compared to the control SMMC7721 cells infected with LXSN and untreated SMMC7721 cells (0.5 +/- 0.1 cm versus 1.4 +/- 0.2 cm and 1.6 +/- 0.2 cm, P < 0.05). The results showed that the combined transcriptional regulatory sequences of E(AFP)-P(ALB) could control the targeted expression of cytokine genes in AFP-positive human HCC cells, and the expression level of the IL-2/IFNalpha2b fused gene was positively correlated to the level of AFP expression in the infected cells. The IL-2/IFNalpha2b fused protein that was expressed has the functions of both IL-2 and IFNalpha. Therefore, this study illustrates the superiority of using transcriptionally targeted recombinant retrovirus vectors in cytokine-based gene therapy.

By 1996, 2898 patients with pathologically proven hepatocellular carcinoma (HCC) had been treated at the Liver Cancer Institute of Shanghai Medical University. The 5 year survival in the entire series was 36.2%, being increased from 4.8% in 1958-70, 12.2% in 1971-83, to 50.5% in 1984-96 and 274 patients had survived more than 5 years. The increase in the survival rate could be attributed to the decreasing mean tumour diameter (11.7, 10.5 and 9.5 cm, respectively) and multimodality treatment. In addition to small HCC resection (5 year survival 64.9%, n = 735) and large HCC resection (5 year survival 37.4%, n = 1050), the following deserves to be mentioned. First, the 5 year survival of unresectable HCC treated by palliative surgery increased from 0% to 7.2% to 20.0%, which was related to the increase in use of multimodality treatment, particularly in those followed by second-stage resection. Second, cytoreduction and sequential resection is a new field with a significant potential in the treatment of localized unresectable HCC in a cirrhotic liver. Cytoreduction can be achieved by surgery, such as hepatic artery ligation, cannulation, cryosurgery and their combination, and followed by intrahepatic arterial chemoembolization, targeting therapy or regional radiotherapy. Ninety of 647 patients with unresectable HCC so treated had marked shrinkage of tumour and received second-stage resection; the 5 year survival was 71.4%. Third, non-surgical cytoreduction was mainly achieved by transcatheter arterial chemoembolization (TACE); for 70 patients with second-stage resection following TACE, the 5 year survival was 56.0%. Finally, re-resection of subclinical recurrence of tumour after curative HCC resection was performed in 155 patients; the 5 year survival calculated from the first resection was 50.9%, which played an important role in increasing the 5 year survival in the resection group (from 13.0% to 29.5% to 56.2%). It is concluded that multimodality treatment with combined and sequential use of different modalities and repeated use of some modalities is of substantial benefit for localized unresectable HCC., (© 1998 The Official Publication of the Asian Pacific Association for the Study of the Liver and the Asian Pacific Association of Gastroenterology.)

Background/aims: The aim of this study was to try to understand the effects of the synthetic matrix metalloproteinase inhibitor Batimastat (BB-94) on hepatocellular carcinoma (HCC)., Methodology: An orthotopic metastatic human hepatocellular carcinoma in nude mice model (LCI-D20) was used to study primary tumor growth, local invasion and metastasis of HCC. MTT assay was used to study the effects of BB-94 on cytotoxin and proliferation of HCC cell line SMMC-7721 in vitro. A gelatine zymograph was used to study the expression of MMPs in the LCI-D20 tumor tissue., Results: BB-94 can inhibit primary tumor growth, local invasion, intrahepatic and lung metastasis, as well as prolong survival. BB-94 did not affect the proliferation of HCC cells in vitro. LCI-D20 tumor tissue expresses MMP-2 and MMP-9., Conclusions: BB-94 has a cytostatic therapeutic effect on HCC.

To evaluate a protocol of combined intramuscular-intradermal (i.m.-i.d.) universal hepatitis B (HB) vaccination of neonates regardless of the mothers' HB surface antigen HBsAg status, 160 full-term newborn babies were sequentially divided into groups A and B (test and control groups). The group A babies were immunized by giving 30 micrograms HB vaccine i.m. within 24 h after birth and 2 micrograms i.d. twice at 1 and 6 months of age. Group B babies were immunized by giving one initial dose of 30 micrograms i.m. and two booster doses of 10 micrograms i.m. after same 0, 1, 6-month schedule. Blood samples were collected at birth before the first dose of the HB vaccine, at 6 months of age before the third dose and at 12 months. The blood samples were tested for HBsAg and anti-HBs by using Abbott RIA reagents. All of the 160 newborns received three doses of vaccine, but in only 96 of them was the blood examination completed. The positive rate of anti-HBs > 10 IU l-1 was 76.27 and 83.08% in groups A and B, respectively at 6 months, and 83.72 and 92.45%, respectively at 12 months. The geometric mean titres (GMT) of anti-HBs was 42.25 and 60.25 IU l-1 in groups A and B, respectively at 6 months, and 74.45 and 87.1 IU l-1, respectively at 12 months. HBsAg was negative in the two groups 6 and 12 months after birth. The chi 2 and t-tests showed there were no significant differences in these data between the two groups. Thus was it demonstrated that the combined i.m.-i.d. protocol using 34 micrograms of HB vaccine can produce a protection similar to that of the conventional 50 micrograms i.m. regimen, while ca on-third of the vaccine can be saved. The new protocol is theoretically rational and may be ideal according to the cost-effectiveness principle.

Vascular endothelial growth factor (VEGF) is an important mediator of vascular development in tumors. We aimed at clarifying the relationship between VEGF mRNA expression and invasion or metastasis of hepatocellular carcinoma (HCC). Reverse transcript-polymerase chain reaction (RT-PCR) was carried out on surgical specimens of 48 patients with HCC. The relative levels of VEGF mRNA expression were measured by determining a ratio between PCR products of VEGF and the endogenous internal standard gene beta-actin. Expression of VEGF mRNA in tumor was found in 76.5% (39/51) of patients, whereas it was 21.69% (11/51) in non tumorous liver. The levels of VEGF mRNA expression in tumors with tumorous emboli and in poor-encapsulated tumors were higher than that without tumorous emboli and in well-encapsulated tumors respectively (p < 0.05 t test). There was no significance in the expression of VEGF mRNA between large HCC (diameter > 5cm) and small HCC (diameter < or = 5cm) (p > 0.05). VEGF may play an important role in the invasion and metastasis of HCC. Angiogenesis in tumor correlates with progression of HCC.