Superantigen-SEA gene modified tumor vaccine for hepatocellular carcinoma:An in vitro study

AIM:To construct an eukaryotic superantigen gene expressionvector containing the recombinant gene of SEA and CD80molecule transmembrane region (CD80TM),and to expressstaphylococcus enterotoxin A (SEA) on the membrane ofhepatocellular carcinoma (HCC) cell to form a superantigengene modified tumor vaccine for HCC.METHODS:SEA and linker-CD80TM gene were amplifiedthrough PCR from plasmid containing cDNA of SEA and CD80.Gene fragments were then subcloned into the multiplecloning sites of retroviral vector pLXSN.Recombinant plasmidwas transferred into HepG2 cells mediated with lipofectamine,positive clones were selected in culture medium containingG418.RT-PCR and indirect immunofluorescence studiesconfirmed that SEA was expressed specifically on HCC cellmembrane.INFγ-ELISPOT study demonstrated that SEAprotein was expressed on the membrane of HCC cells.Cytotoxicity of HepG2-SEA primed CTLs (SEA-T) wasanalyzed by ~(51)Cr release assay.T cells cultured with rhIL-2(IL-2-T) were used as control.RESULTS:Restriction digestion and sequence analysesconfirmed the correctness of length,position and orientationof inserted fusion genes.SEA was expressed on the surfaceof HepG2 cells,HepG2-SEA had strong stimulating effect onproduction of HepG2 specific CTL (P<0.001).SEA-T hadenhanced cytotoxicity to HepG2 cells (P<0.05).CONCLUSION:Tumor cell membrane expressed superantigencan be used to reinforce the immune effect of tumor cellvaccine for HCC,which provides a new method of theenhanced active immunotherapy for HCC.     

Activation of c-Yes in hepatocellular carcinoma： A preliminary study

TO THE EDITOR Hepatocellular carcinoma (HCC) is thought to develop through a multistep process[1]. A long history of viral hepatitis or prolonged exposure to environmental toxins predisposes liver cells to mutations of the genes critical in the control of hepatocyte growth. In fact, both activation of cellular oncogenes and inactivation of tumorsuppressor genes are involved in the development of HCC. Activation of oncogenes by hepatitis virus integration has been shown in the woodchuck animal model[2],although the significance of this finding in human hepatocarcinogenesis is still under investigation.     

Systemic chemotherapy for hepatocellular carcinoma in non-cirrhotic liver： A retrospective study

AIM： To investigate the efficacy and toxicity of systemic chemotherapy in a retrospective study of patients with hepatocellular carcinoma （HCC） occurring in normal or fibrotic liver without cirrhosis.
METHODS： Twenty-four patients with metastatic or locally advanced HCC in a normal or a fibrotic liver were given systemic chemotherapy （epirubicin, cisplatin and 5-fluorouracil or epirubicin, cisplatin and capecitabine regimens）. Tumor response, time to progression, survival, and toxicity were evaluated.
RESULTS： There were 7 women and 17 men, mean age 54 ± 10 years; 18 patients had a normal liver and 6 had a fibrotic liver （F1/F2 on biopsy）. Mean tumor size was 14 cm, 5 patients had portal vein thrombosis and 7 had metastasis. Patients received a median of 4 chemotherapy sessions. Overall tolerance was good. There were 5 partial responses （objective response rate = 22%）, and tumor control rate was 52%. Second line surgical resection was possible in two patients. Median survival was 11 mo, and 1- and 2-year overall survival rates were 50% ± 10% and 32 ± 11%, respectively.
CONCLUSION： In patients with HCC in a non-cirrhotic liver, chemotherapy was well tolerated and associated with an objective response rate of 22%, including two patients who underwent secondary surgical resection.     

Fumagillin treatment of hepatocellular carcinoma in rats： An in vivo study of antiangiogenesis

AIM: To investigate the effect and possible mechanisms of antiangiogenesis therapy for HCC in rats. METHODS: Adult male LEW/SsN rats were divided into 3 groups, 25 animals each. Group A was the control group. Groups B and C were given diethylnitrosamine, 5 mg/kg/d. In addition, group C rats received an intraperitoneal injection of fumagillin, 30 mg/(kg·d). Five animals in each group were killed at 6th, 12th, 18th, 20th and 24th wk to evaluate the development of HCC and metastasis. Weight of the rats, liver tumors, and number of organs involved by HCC were measured at each stage. We compared methionine aminopeptidase-2 (MetAP-2) mRNA, Bcl-2 mRNA, telomerase mRNA, and telomerase activity at 24th wk in the liver tissue of group A rats and tumor tissue of HCC from group B and C rats. RESULTS: No HCC developed in group A, but tumors were present in group B and C rats by the 18th wk. At wk 20 and 24, the median liver weight in group B was 0.64 g (range: 0.58-0.70 g) and 0.79 g (range: 0.70-0.90 g) (P= 0.04), and that in group C was 0.37 g (range: 0.35-0.42 g) and 0.39 g (range: 0.35-0.47 g) (P= 0.67). The liver weight in group C rats was significantly lower than that in group B rats (P= 0.009). At the same time, the median metastasis score (number of organ systems involved) was 3 (range2-3) in group B, and 1 (range 1-2) in group C, a significant difference between the groups (P= 0.007, 0.004). The levels of MetAP-2 mRNA were significantly higher in groups B and C than in group A (P = 0.025), and significantly higher in group C than in group B (P= 0.047). The level of Bcl-2 mRNA was significantly higher in group B than in group A (P=0.024), but lower in group C than in group B, although not significantly (P = 0.072). Telomerase mRNA was significantly higher in group B than in group A (P = 0.025), but significantly lower in group C than in group B (P= 0.016). The same inter-group relationship was also true for telomerase activity (P= 0.025 and 0.046). CONCLUSION: Fumagillin effectively inhibits both liver tumor growth and metastasis in rats in vivo. A possible mechanism is fumagillin-induced inhibition of MetAP-2, which plays an essential role in endothelial cell proliferation. Inhibition of MetAP-2 also results in inhibition of Bcl-2 and telomerase activity.     

Role of immunosuppression and tumor differentiation in predicting recurrence after liver transplantation for hepatocellular carcinoma： A multicenter study of 412 patients

INTRODUCTION Hepatocellular carcinoma (HCC) is one of the most common neoplasms and its incidence is currently rising worldwide[1-3]. HCC usually occurs in cirrhotic livers and less than 30% of patients presenting with HCC are considered candidates for re…     

Postoperative adjuvant arterial chemoembolization improves survival of hepatocellular carcinoma patients with risk factors for residual tumor： A retrospective control study

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.     

Intratumoral sampling variability in hepatocellular carcinoma： A case report

The differential diagnosis between hepatocellular carcinoma （HCC） and regenerative liver nodules and other primary liver tumors may be very difficult, particularly when performed on liver biopsies. Difficulties in histological typing may be often minimized by immunohistochemistry. Among the numerous markers proposed, CK18, Hep Par1 and glypican 3 （GPC3） are considered the most useful in HCC diagnosis. Here we report a case of HCC in a 72-year-old male with HBV- related chronic liver disease, characterized by a marked morphological and immunohistochemical intratumoral variability. In this case, tumor grading ranged from areas extremely well differentiated, similar to regenerative nodule, to undifferentiated regions, with large atypical multinucleated cells. While almost all sub nodules were immunostained by Hep Par 1, immunoreactivity for glypican 3 and for Ckl8 was patchy, with negative tumor region adjacent to the highly immunoreactive areas. Our case stresses the relevance of sampling variability in the diagnosis of HCC, and indicates that caution should be taken in grading an HCC and in the interpretation of immunohistochemical stains when only small core biopsies from liver nodules are available.     

Microarray-based analysis for hepatocellular carcinoma： From gene expression profiling to new challenges

Accumulation of mutations and alterations in the expression of various genes result in carcinogenesis,and the development of microarray technology has enabled us to identify the comprehensive gene expression alterations in oncogenesis.Many studies have applied this technology for hepatocellular carcinoma(HCC),and identified a number of candidate genes useful as biomarkers in cancer staging,prediction of recurrence and prognosis,and treatment selection.Some of these target molecules have been used to develop new serum diagnostic markers and therapeutic targets against HCC to benefit patients.Previously,we compared gene expression profiling data with classification based on clinicopathological features,such as hepatitis viral infection or liver cancer progression.The next era of gene expression analysis will require systematic integration of expression profiles with other types of biological information,such as genomic locus,gene function,and sequence information.We have reported integration between expression profiles and locus information,which is effective in detecting structural genomic abnormalities,such as chromosomal gains and losses,in which we showed that gene expression profiles are subject to chromosomal bias.Furthermore,array-based comparative genomic hybridization analysis and allelic dosage analysis using genotyping arrays for HCC were also reviewed,with comparison of conventional methods.     

Coexistence of hepatocellular carcinoma and gastrointestinal stromal tumor： A case report

Malignant gastrointestinal stromal tumors(GIST)are raremesenchymal tumors originating from the wall of thegastrointestinal tract.Their coexistence with other tumorsoriginating from other germ layers is unique.We havereported a case of a 63-year-old GIST patient presentingas an epigastric mass associated with hepatic tumor.Histologically,the mesenteric tumor was composed ofspindle cells showing both neural and smooth muscledifferentiation.Immunohistochemical examinationshowed positive staining for CD117,vimentin,S-100,and SMA,while CD34 antigen was negative.The hepatictumor was diagnosed as hepatocellular carcinoma(HCC).To the best of our knowledge,this is the first case ofGIST and HCC coexistence.The rarity of the case,however,should not lead to ignoring such a possibility indifferential diagnosis.     

肝癌疫苗的研究现状及进展

近年来，肿瘤疫苗备受关注，已成为肿瘤免疫治疗的研究热点之一。其研究主要涉及树突状细胞(dendritic cells，DC)疫苗、基因修饰疫苗、高强度聚焦超声(high—intensityfo—cusedultra—sound，HIFU)固化疫苗、化学修饰肝癌细胞疫苗、人细胞融合肝癌疫苗和细胞因子—微粒肝癌疫苗等方面。有关DC疫苗和基因修饰疫苗的报道较多，其它肝癌疫苗的报道相对较少。     

细胞因子缓释微球免疫激活剂预防肝细胞癌术后复发

目的观察肿瘤免疫激活剂预防肝细胞癌（HCC）切除术后复发的效果。方法1999年3月2003年6月80例HCC根治切除术后患者采用随机、对照的方法分为免疫激活剂组和对照组，进行肿瘤免疫激活剂注射，该免疫激活剂成分包括同定的HCC细胞或组织碎片、粒细胞巨噬细胞集落刺激因子和白细胞介素2的生物可降解缓释微球和免疫辅助物。观察肿瘤免疫激活剂皮内接种激发的迟发型超敏反应（DTH）和预防HCC切除术后复发的效果。结果免疫激活剂组中8例和对照组中5例失访，平均随访34．3个月（15～55个月）。在HCC免疫激活剂治疗中未见不良反应。32例患者中23例出现抗HCC DTH阳性。免疫激活剂组术后l、2、3年复发率分别为l2．6％、35．9％、54．0％；而对照组HCC切除术后1、2、3年复发率分别为31．6％、61．3％、72．1％，免疫激活剂组的复发率低于对照组（P〈0．05）。结论这种细胞因子缓释微球的肿瘤免疫激活剂具有很强的抗肿瘤和预防肝癌切除术后复发的效果。     

Autologous tumor vaccine lowering postsurgical recurrent rate of hepatocellular carcinoma

BACKGROUND/AIMS: A tumor vaccine consisting of formalin-fixed hepatocellular carcinoma (HCC) tissue fragments, biodegradable sustained-releasers of granulocyte-macrophage-colony stimulating factor (GM-CSF) and interleukin-2 (IL-2), and an adjuvant was developed. The aim of this study was to evaluate the effects of autologous tumor vaccine for protective immunity against HCC. METHODOLOGY: C57BL/6J mice were immunized intradermally with the Hepa1-6 tumor vaccine on day 0 and 7, followed by intrahepatic challenge with live Hepa1-6 cells. On day 21, the tumor volumes were measured and the effect of tumor vaccine was evaluated. Lymphocytes from the immunized mice were cultured and the specific cytotoxicity against Hepa1-6 was accessed. Then from March 1999 to June 2003, 67 patients with HCC undergoing curative resection were randomly divided into a tumor vaccine group (n = 32) and a control group (n = 35). Patients in the tumor vaccine group received 3 vaccinations at a 2-week interval and the control group only adjuvant treatment for symptoms. A delayed-type-hypersensitivity test was performed before and after vaccination. Primary endpoint was time to first recurrence and recurrent rates were analyzed. RESULTS: The tumor vaccine protected 87% of syngeneic mice from Hepa1-6 cells inoculation. In an in vitro experiment, splenocytes from the vaccinated mice exhibited a 56% lytic activity against the Hepa1-6 cells at an effector/target (E/T) ratio of 5, whereas they did not exhibit such activity against other tumor cells. The cytotoxic activity was inhibited by the treatment with anti-CD3, anti-CD8, and anti-MHC-class II monoclonal antibodies but not with anti-CD4 and anti-MHC-class I antibodies. In clinical trial, thirty-two patients had completed the tumor vaccine procedure and no essential adverse effect occurred. The follow-up averaged 33.6 months (range from 15 to 54 months). The recurrent rate was significantly better in the tumor vaccine group (1 year, 12.6%; 2 years, 35.9%; 3 years, 54%) than in the control group (1 year, 31.6%; 2 years, 61.3%; 3 years, 72.1%; P = 0.037). 23/32 patients developed a DTH response against the fragments of HCC and DTH-response-positive patients had a lower recurrent rate than DTH-response-negative patients (7/23 vs. 5/9). CONCLUSIONS: The autologous tumor vaccine is a promising adjunctive modality to prevent recurrence of human HCC.     

Neutrophils enhance invasion activity of human cholangiocellular carcinoma and hepatocellular carcinoma cells: an in vitro study

BACKGROUND AND AIM: Tumor-mesenchymal interactions are involved in the mechanism of tumor invasion in several types of carcinoma. Mutual interactions between carcinoma cells and neutrophils, however, have been poorly understood. In the present study we examined the effect of neutrophils on invasion activities of carcinoma cells in vitro. Role of hepatocyte growth factor (HGF) as a mediator was also evaluated. METHODS: Using a Matrigel invasion chamber, invasion activities of HuCC-T1 human cholangiocellular carcinoma cells and HepG2 hepatocellular carcinoma cells in response to recombinant HGF or neutrophils were evaluated. RESULTS: Recombinant HGF dose-dependently increased invasion activities of HuCC-T1 and HepG2 cells. Neutrophils significantly enhanced invasion activities of these cells, which were suppressed to the respective basal levels with anti-HGF antibody. The carcinoma cells did not secrete HGF. Neutrophils cultivated in tumor condition medium (TCM) of HuCC-T1 or HepG2 cells secreted a significant level of HGF protein without increasing HGF mRNA expression. Treatment with heat or ultrafiltration of TCM of HuCC-T1 or HepG2 cells suggested carcinoma cell-derived HGF inducer(s) to be certain protein(s) with a molecular weight of more than 30 000. CONCLUSIONS: The present study suggests the presence of mutual interactions between HuCC-T1/HepG2 carcinoma cells and neutrophils in tumor invasion via paracrine regulation mediated by neutrophil-derived HGF.     

肝细胞癌组织中肿瘤相关基因的初步筛选

为寻找肝癌早期诊断标志物,本研究用基因芯片技术检测了肝细胞癌(HCC)和正常肝组织中基因群的表达情况,初步筛选出相关的基因群,并对其中的肿瘤相关基因进行验证,以进一步了解这些基因对肝癌诊断的价值.     

肝细胞癌抑癌基因p53突变

目的分析重庆地区肝细胞癌ｐ５３基因突变谱．方法住院肝细胞癌患者２０例，皆经病理证实，长期在重庆地区居住，其中早期小肝癌４例，中期６例，晚期１０例．采用ＰＣＲ－ＳＳＣＰ，ＰＣＲ直接测序技术分析ｐ５３基因５，６，７和８外显子突变．结果ｐ５３基因总的突变率为４０％．其中外显子５和６各占１０％，外显子７占２０％，未发现外显子８的突变；测序证实外显子７为第２４９位密码子Ｇ→Ｔ的颠换突变．突变病例多为晚期肿瘤．结论重庆地区肝细胞癌存在明显的ｐ５３基因突变，反映了该地区肝癌与黄曲霉毒素和ＨＢＶ或ＨＣＶ病毒有关     

肝细胞癌组织中PTEN蛋白表达

探讨抑癌基因PTEN在肝细胞癌 (HCC)组织及癌旁组织的表达、临床意义。采用免疫组织化学SP法检测PTEN。 4例正常肝组织均呈PTEN蛋白阳性 ;HCC及其癌旁肝组织中的阳性率分别为 5 8 8%(2 0 / 34)和 10 0 %(34/ 34) ,两者比较差异有显著性 (P <0 0 5 )。中分化癌阳性率为 77 8%(14 / 18) ,低分化阳性率为 2 5 %(3/ 12 ) ,两者比较差异有显著性 (P <0 0 0 1)。PTEN蛋白表达与年龄、性别、肿瘤大小、有无包膜及门脉癌栓均无明显关系 (P >0 0 5 ) ,但与HCC分化程度明显相关 ,HCC分化愈差 ,PTEN蛋白表达愈弱。PTEN蛋白表达与HCC分化程度明显相关。     

Troglitazone inhibits tumor growth in hepatocellular carcinoma in vitro and in vivo

Peroxisome proliferator-activated receptor gamma (PPARgamma) has been implicated in the differentiation and growth inhibition of cancer cells. We examined the effects of PPARgamma activation by troglitazone on hepatocellular carcinoma (HCC) cell growth, proliferation, and apoptosis in vitro and in vivo. We also studied relationships between PPARgamma activation and cyclooxygenase-2 (COX-2) expression. Human HCC cell lines Huh7 and Hep3B were cultured in the presence or absence of troglitazone. Cell growth was determined via WST-1 assay, proliferation by cell cycle analysis and proliferating cell nuclear antigen (PCNA) Western blotting, and apoptosis by flow cytometry and TUNEL. Tumor growth after subcutaneous implantation of Huh7 cells in nude mice was monitored, and the effects of treatment with troglitazone were determined. In resected HCCs, PPARgamma expression was less compared with the histologically normal surrounding liver. In cultures of Hep3B and Huh7 cells, basal expression of PPARgamma was relatively low, but troglitazone caused dose-dependent induction of PPARgamma expression. Cell cycle analysis revealed a decreased proportion of cells in S phase, with arrest at G0/G1. Concomitant downregulation of PCNA and an increase in TUNEL staining, cells were consistent with decreased proliferation and induction of apoptosis by troglitazaone. Troglitazone-mediated PPARgamma activation also suppressed COX-2 expression and induced p27 in HCC cells. Administration of troglitazone to Huh7 tumor-bearing mice significantly reduced tumor growth and caused tumor regression. In conclusion, collectively, these results indicate that PPARgamma could be a regulator of cell survival and growth in HCC. PPARgamma therefore represents a putative molecular target for chemopreventive therapy or inhibition of liver cancer growth.