肝动脉化疗栓塞术对肝癌患者细胞免疫功能的影响

目的探讨肝癌患者细胞免疫功能及肝动脉化疗栓塞术(TACE)对其影响。方法对2005年3月-8月在我科行TACE的肝癌患者于治疗前1天、治疗后1周和2周分别测定外周血T细胞亚群,进行比较,同时采用健康志愿者作为对照组进行治疗前比较。结果治疗前CD3+、CD4+、CD4+/CD8+水平较对照组显著降低,治疗后1周CD3+、CD4+、CD4+/CD8+水平均较治疗前略升高,但无统计学差异。治疗后2周CD3+、CD4+、CD4+/CD8+水平均较治疗前显著升高。结论肝癌患者细胞免疫功能处于抑制状态,TACE对细胞免疫功能影响较小,随着肿瘤负荷减小,细胞免疫功能显著提高。

肝动脉化疗栓塞术对原发性肝癌患者T淋巴细胞亚群的影响

目的探讨肝动脉化疗栓塞术(TACE)对原发性肝癌患者T淋巴细胞亚群的影响。方法对2005年3月至2006年4月在我科行TACE的肝癌患者于治疗前1d、治疗后1周和2周分别测定外周血T细胞亚群,进行比较,同时采用健康志愿者作为对照组进行治疗前比较。结果治疗前CD3+、CD4+、CD4+/CD8+水平较对照组显著降低,治疗后1周CD3+、CD4+、CD4+/CD8+水平均较治疗前略升高,但差异无统计学意义。治疗后2周CD3+、CD4+、CD4+/CD8+水平均较治疗前显著升高。结论肝癌患者细胞免疫功能处于抑制状态,TACE对细胞免疫功能影响较小,随着肿瘤负荷减小,细胞免疫功能显著提高。

Relationship between survivin expression and recurrence,and prognosis in hepatocellular carcinoma

AIM： To study the expression of the inhibitor of apoptosis protein survivin in hepatocellular carcinoma （HCC）, and its correlation with clinicopathological factors, cell proliferation, recurrence and prognosis after hepatectomy. METHODS： Immunohistochemical staining of survivin and Ki-67 was performed by the standard streptavidin- peroxidase technique on paraffin sections of 55 cases of HCC. RESULTS： The positive rate of survivin in HCC was 52.7% （29/55）. Significant correlation was found between survivin expression with portal vein thrombi and intrahepatic matastasistic nodes （P 〈 0.05）. The recurrent rate in survivin-positive HCC was significantly higher than that in survivin-negative HCC after hepatectomy, the 1- and 3-year survival rate in patients with survivin-positive tumors was significantly lower than that in patients with survivin-negative tumors （58.62 and 10.34% vs 76.92 and 30.77%, P 〈 0.05, log-rank test）. The proliferation index （Ki-67） in survivin-positive HCC （33.83% ± 18.90%） was significantly higher than that in survivin-negative HCC （19.60% ± 19.35%） （P 〈 0.05）. CONCLUSION： Survivin may play an important role in progression of HCC by promoting cell proliferation, and may be positively correlated with high risk of disease recurrence and poor prognosis in HCC. Its expression may serve as a prognostic factor for patients with HCC after hepatectomy.

Expression and hypermethylation of p27^(kip1) in hepatocarcinogenesis

AIM： To study the expressions of p27^kip1 protein and p27mRNA, the hypermethylation of p27^kip1 and the relation between them in various stages of hepatocarcinogenesis. METHODS： p27 protein and p27mRNA were detected by immunohistochemical staining and in situ hybridization respectively in 68 cases of normal liver, liver cirrhosis, pericancerous cirrhosis and hepatocellular carcinoma （HCC）. The hypermethylation of p27^kip1 was detected by methylation-specific PCR （MSP） in 44 cases of normal liver, liver cirrhosis, and HCC. RESULTS： The positive rate of p27 protein was 66.7% （4/6） in normal liver, 60.0% （6/10） in liver cirrhosis, 50.0% （12/24） in pericancerous cirrhosis and 21.4% （6/28） in HCC. There were no statistical differences in normal liver, liver cirrhosis and pericancerous cirrhosis, but the positive rate of p27 protein significantly decreased in HCC compared to that in the other groups （P = 0.006, %2 = 7.664）. The positive rate of p27^kip1 mRNA was 83.3% （5/6） in normal liver, 70.0% （7/10） in liver cirrhosis, 75.0% （18/24） in pericancerous cirrhosis and 25.0% （7/28） in HCC. There were no statistical differences in normal liver, liver cirrhosis and pericancerous cirrhosis, but the positive rate of p27^kip1 mRNA also significantly decreased in HCC compared to that in the other groups （P = 0.000, %2 = 16.600）. In addition, there was a significant correlation between the expression of p27 protein and p27mRNA in the integrated group of normal liver and liver cirrhosis. However, no significant correlation was found between pericancerous cirrhosis and HCC. Using MSP, we found that 1 HCC in 44 cases （including 6 cases of normal liver, 10 cases of liver cirrhosis and 28 cases of HCC） was methylated, whose p27 protein and p27mRNA were negative. CONCLUSION： The reduction or loss of p27 protein and p27mRNA are potentially involved in hepatocarcinogenesis. The hypermethylation of p27 might lead to the loss of p27mRNA transcription.

Reactive lymphoid hyperplasia of the liver:A case report and review of literature

A case of a 53-year-old female patient with reactive lymphoid hyperplasia （RLH）, clinically designated as pseudolymphoma of the liver is described in this article. The patient was admitted to our hospital for further evaluation of hepatic tumors incidentally discovered at another hospital. Various diagnostic methods, including ultrasonography （US）, computerized tomography （CT）, magnetic resonance imaging （MRI） and hepatic angiography displayed three small lesions in the liver with outstanding findings consistent with hepatocellular carcinoma （HCC）. Surgical resection was performed and the three lesions were microscopically diagnosed as RLH of the liver. The lesions comprised a massive infiltration of lymphoid cells with follicles and hyalinized inter- follicular spaces. Immunohistochemical examination revealed that infiltrating lymphocytes had no prominent nuclear atypia and polyclonality. RLH of the liver is a very rare condition and only twelve cases have been reported in the English literature. Majority of the reported cases were middle-aged women and about half of them had some immunologic abnormalities such as autoimmune thyroiditis, Sjogren＇s syndrome, primary immunodeficiency, primary biliary cirrhosis. Since they are olden clinically misdiagnosed as HCC, surgery is the choice of treatment for these patients. Although their pathology resembles malignant lymphoma, the clinical course is completely benign. The authors propose that RLH of the liver can be discriminated from HCC by its clinical features.

Immunohistochemical analysis of p53,cyclinD1,RB1,c-fos and N-ras gene expression in hepatocellular carcinoma in Iran

AIM： TO study the effect of some genes especially those involved in cell cycle regulation on hepatocellular carcinoma. METHODS： Paraffin-embedded tissue samples of 25 patients （18 males and 7 females） with hepatocellular carcinoma were collected from 22 pathology centers in Tehran during 2000-2001, and stained using immunohistochemistry method （avidin-biotin-peroxidase） for detection of p53, cyclinD1, RB1, c-los and N-ras proteins. RESULTS： Six （24%）, 5 （20%）, 12 （48%） and 2 samples （8%） were positive for p53, cyclinDl, C-los and N-ras expression, respectively. Twenty-two （88%） samples had alterations in the （31 cell-cycle checkpoint protein expression （RBI or cyclinD1）. P53 positive samples showed a higher （9 times） risk of being positive for RBI protein than p53 negative samples. Loss of expression of RBI in association with p53 over-expression was observed in 4 （66.7%） of 6 samples. Loss of expression of RBI was seen in all cyclinD1 positive, 20 （90.9%） N-ras negative, and ii （50%） C-fos positive samples, respectively. CyclinD1 positive samples showed a higher （2.85 and 4.75 times） risk of being positive for c-los and N-ras expression than cyclinD1 negative samples. CONCLUSION： The expression of p53, RB1 and c-los genes appears to have a key role in the pathogenesis of hepatocellular carcinoma in Iran. Simultaneous overexpression of these genes is significantly associated with their loss of expression during development of hepatocellular carcinoma.

Pharmacokinetics of radioimmunotherapeutic agent of direct labeling mAb ^(188)Re-HAb18

AIM:To labed Anti-hepatoma monoclonal antibody(mAb) fragment HAb18 F(ab')_2 was labeled with 188 Re for the pharmacokinetic model of ^(188)Re-HAb18 F(ab')_2 and to evaluate its pharmacokinetic parameters in hepatoma- bearing nude mice. METHODS:HAb18 F(ab')_2 was directly labeled with ^(188)Re using 2-mercaptoethanol(2-ME)as reducing agents. Labeling efficiency and immunoreactivity of ^(188)Re-HAb18 F (ab')_2 were evaluated by Whatman 3MM paper chromatography and live cell assay,respectively. Biodistribution analysis was also conducted in nude mice bearing human hepatoma in which animals were sacrificed at different time points(1,4,18,24 and 24h)after ^(188)Re-HAb18 F(ab')_2 was injected through tail-vein into hepatoma-bearing nude mice.The blood and radioactivity of organs and mass were measured.The concentrations of ^(188)Re-HAb18 F(ab')_2 were evaluated with a pharrnacokinetic 3P97 software. RESULTS:The optimum labeling efficiency and immunoreactive fraction were 91.7% and 0.78%, respectively.The parameters of ^(188)Re-HAb18 F(ab')_2 were: T_(1/2),2.29h;Vd,1.49×10^(-9)L·Bq^(-1);AUC,20.49×10~9Bq·h· L^(-1);CL,0.45×10^(-3)L·h^(-1).^(188)Re-HAb18 F(ab')_2 could locate specially in hepatoma with high selective reactivity of HAb18 F(ab')_2.^(188)Re-HAbl8 F(ab')_2 was mainly eliminated by kidney.The maximal tumor to blood ratio was at 48h,and maximal tumor to liver ratio was at 18h. CONCLUTION:The pharmacokinetics of ^(188)Re-HAb18 F(ab')_2 fit a I-compartment model.^(188)Re-HAb18 F(ab')_2 can be uptaken selectively at the hepatoma site.

Immunohistochemical assessment of angiogenesis in hepatocellular carcinoma and surrounding cirrhotic liver tissues

AIM: To investigate whether vascular endothelial growth factor (VEGF) was over-expressed in hepatocellular carcinoma (HCC) or in surrounding cirrhotic liver tissues.METHODS: Immunohistochemistry was performed to investigate the expression of VEGF proteins in HCC tissues from 105 consecutive patients undergoing curative resection for HCC. The immunostaining results and related clinicopathologic materials were analyzed with statistical methods. Kaplan-Meier method was used to calculate survival curves, and Log-rank test was performed to compare differences in survival rates of the patients with positive HCC staining and negative VEGF.RESULTS: VEGF-positive expression was found in 72 of105 HCC patients (68.6%). Capsular infiltration (P= 0.005),vascular invasion (P = 0.035) and intrahepatic metastasis(P=0.008) were observed more frequently in patients with VEGF-positive expression than in those with VEGFnegative expression. Kaplan-Meier curves showed that VEGF-positive expression was associated with a shorter overall survival (P = 0.014). VEGF-positive expression was found in 47 of tissues 68 HCC (69.1%), and VEGF-positive expression was found in 54 of 68 surrounding cirrhotic liver tissues (79.4%). VEGF-positive expression was significantly higher in surrounding cirrhotic liver tissues than in HCC (P= 0.017).CONCLUSION: VEGF may play an important role in the angiogenesis and prognosis of HCC, as well as in the angiogenesis of liver cirrhosis.

Cryoablation Combined with TACE for Treating Large Hepatocellular Carcinoma： Tumor Load and Cellular Immunity

OBJECTIVE To study the effectiveness on the tumor load and cellular immune function of percutaneous cryoablation （argon-helium cryoablative system, AHCS） combined with transarterial chemoembolization （TACE） for treating large hepatocellular carcinomas （HCCs） with diameters over 10 ca. METHODS A total of 48 HCC patients were treated with AHCS after TACE. Tumor sizes ranged from 10 to 14 cm. All cases were a hypervascular type. There were 38 Child A cases and 10 Child B cases. Forty were AFP positive and 8 negative. The patients were randomized with therapy group consisting of 26 cases and the control group 22 cases. The therapy group received AHCS 4 weeks following TACE treatment. Reexamination included pathology, tumor markers, T-lymphocyte subgroup levels and computed tomography or MRI. The necrosis rate of the tumor load was calculated by Cavalieri＇s theory. EORTC QLQ-C30 was used in quality of life evaluation. RESULTS The average tumor-load reduction rate （necrosis rate） was 8.07% after TACE, and 28.65% after AHCS. Coagulation necrosis was produced in the target area. The tumor markers deceased significantly after AHCS. Tumor-load reduction after AHCS was more significant than after TACE. Suppression of cellular immunity after TACE was significant. In contrast, CD3^＋, CD4^＋ and NK increased after AHCS and an abnormal T-lymphocyte distribution was corrected. Quality of life after AHCS increased according to the EORTC QLQ-C30 evaluation. No severe complications occurred. CONCLUSION Percutaneous AHCS cryoablation after TACE reduced the tumor load in the short term. At the same time, cellular immune function was increased after AHCS. TACE was critical in increasing the therapeutic efficacy of AHCS because of its embolisation of blood vessels preventing a Flow Effect. Reduction of the tumor load in the short term may conduce to increase cellular immunity. Percutaneous AHCS cryoablation combined with TACE can reduce the tumor load, improve cellular immunity and increase quality of life of HCC patients. This type of therapy deserves to be studied further research.

Overexpression of DNA methyltransferase 1 and its biological significance in primary hepatocellular carcinoma

AIM： To explore the relationship between DNA methyltransferase 1 （DNMT1） and hepatitis B virus （HBV）-related hepatocellular carcinoma （HCC） and its biological significance in primary HCC. METHODS： We carried out an immunohistochemical examination of DNMT1 in both HCC and paired nonneoplastic liver tissues from Chinese subjects. DNMT1 mRNA was further examined in HCC cell lines by real-time PCR. We inhibited DNMT1 using siRNA and detected the effect of depletion of DNMT1 on cell proliferation ability and cell apoptosis in the HCC celt line SMMC-7721. RESULTS： DNMT1 protein expression was increased in HCCs compared to histologically normal nonneoplastic liver tissues and the incidence of DNMT1 immunoreactivity in HCCs correlated significantly with poor tumor differentiation （P = 0.014）. There were more cases with DNMT1 overexpression in HCC with HBV （42.85%） than in HCC without HBV （28.57%）. However, no significant difference in DNMT1 expression was found in HBV-positive and HBV-negative cases in the Chinese HCC group. There was a trend that DNMT1 RNA expression increased more in HCC cell lines than in pericarcinoma cell lines and normal liver cell lines. In addition, we inhibited DNMT1 using siRNA in the SMMC-7721 HCC cell line and found depletion of DNMT1 suppressed cells growth independent of expression of proliferating cell nuclear antigen （PCNA）, even in HCC cell lines where DNMT1 was stably decreased. CONCLUSION： The findings implied that DNMT1 plays a key role in HBV-retated hepatocellular tumorigenesis. Depletion of DNMT1 mediates growth suppression in SMMC-7721 cells.

Systematic Investigation of Berberine for Treating Hepatocellular Carcinoma Based on Network Pharmacology

Objective Liver cancer is the 4th leading cause of cancer death worldwide,and hepatocellular carcinoma(HCC)accounts for the largest proportion of these deaths.Berberine is a quaternary amine compound extracted from plants such as Coptidis Rhizoma(Huang Lian,黄连)and Phellodendri Chinensis Cortex(Huang Bo,黄柏)and is considered as a potential candidate for treating HCC.This study used network pharmacology methods,reveal the core mechanism of action of berberine in the treatment of HCC,clarify its medicinal value,and locate the anti-tumor mechanism of berberine.Methods Structural information of Berberine(PubChem CID:2353)was obtained from the NCBI PubChem;ADME parameter were obtained from the Traditional Chinese Medicine Systems Pharmacology(TCMSP)database;Berberine prediction targets were collected from symmap,stitch and targetnet databases;HCC significant targets were retrieved from OncoDB.HCC and Liverome;A PPI network was established at STRING,Prediction target of berberine therapy for HCC are collected by gene mapping;The core target,pathway,biological process(BP),cellular component(CC),and molecular function(MF)of berberine in the treatment of HCC were predicted by topological analysis and enrichment analysis;the visualized"target pathway"network diagram of berberine in the treatment of HCC was established by the software of Cytoscape.Results Through PubChem and tcmsp databases,the good drugforming properties of berberine were identified;32 prediction targets of berberine were collected in symmap,stitch and targetnet databases;566 related targets of HCC were collected in oncodb.hcc and liverome databases;10 targets of berberine treatment for HCC were predicted by gene mapping,and a PPI with 10 nodes and 34 edges was established Through topological analysis and enrichment analysis,6 topologically important targets,6 related pathways and 16 BP,6 cc and 7 MF involved in Berberine treatment of HCC were obtained.Conclusions The anticancer effect of berberine is mainly involved in the regulation of cells of hepatoma cells through complex interactions between the TB52,MAPK1,CCND1,PTGS2,ESR1 that act on Hub nodes and their associated 6 pathways.The cycle is related to the immune inflammatory response,including biological processes such as proliferation and apoptosis of liver cancer cells.

Overexpression of metastasis-associated in colon cancer 1 predicts a poor outcome of hepatitis B virus-related hepatocellular carcinoma

AIM: To investigate the intratumoral expression of metastasis-associated in colon cancer 1 (MACC1) and c-Met and determine their clinical values associated with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). METHODS: A retrospective study admitted three hundred fifty-four patients with HBV-related HCC. The expression and distribution of MACC1 and c-Met were assessed by quantitative real-time polymerase chain reaction and immunohistochemistry staining. Prognostic factors influencing survival, metastasis and recurrence were assessed. RESULTS: Intratumoral MACC1 level was found to be associated with HCC disease progression. Both median tumor-free survival (TFS) and overall survival (OS) were significantly shorter in the postoperative HCC patients with high intratumoral MACC1 expression, as compared to those with low intratumoral MACC1 levels (TFS: 34 mo vs 48.0 mo, P < 0.001; OS: 40 mo vs 48 mo, P < 0.01). Multivariable analysis indicated that high MACC1 expression or co-expression with c-Met were independent predictors for HCC clinic outcome (P < 0.001). CONCLUSION: High intratumoral MACC1 expression can be associated with enhanced tumor progression and poor outcome of HBV-related HCC. MACC1 may serve as a prognostic biomarker for postoperative HCC.

Vasculogenic mimicry formation in hepatocellular carcinoma

Objective： To explore possibility of vasculogenic mimicry （VM） in hepatocellular carcinoma （HCC） by constructing tumor cell three-dimensional culture system and liver cancer tissues. Methods： Based on three-dimensional cell culture system developed by matrigel, liver cancer cell lines HepG2 were tested for evidence of VM. Fifteen HCC simples were collected. Potential formation of tumor channels and their characterization of network were observed by immunohistochemical and histological double staining of CD31 and PAS, or Ferritin and PAS. Results： Three-dimensional culture model of HCC cell line proved the liver cancer cells stretch out thin and long tubers at the second day, and the cells linked each other to form wreath and network structure at the seventh day. In fifteen HCC simples, endothelial cells were all stained by CD31, and tumor cells were all stained by Ferritin. The immunohistochemical and histological double staining also exhibited evidence of VM in seven simples of HCC, CD31-negative and Ferritin-positive tumor cells were observed to form tubal structure. Tumor cells were separated by PAS-positive matter like basement membrane from the tube. Red blood cells could be seen in the tube. In well-differentiated simples, VM was less than that in poorly differentiated ones, and several CD31-positive tumor cells could be observed in poorly differentiated simples. Conclusion： HepG2 cells have the capacity of self-metamorphose and vascularized trend. The tumor cells can obtain oxygen and nutrition through this structure.

Liver transplantation for hepatocellular carcinoma on cirrhosis:Strategies to avoid tumor recurrence

Hepatocellular carcinoma(HCC) is one of the most frequent neoplasms worldwide and in most cases it is associated with chronic liver disease.Liver transplantation(LT) is potentially the optimal treatment for those patients with HCC who have a poor functional hepatic reserve due to their underlying chronic liver disease.However,due to the limited availability of donors,only those patients whose oncologic profile is favorable can be considered for LT.Despite the careful selection of candidates based on strict rules,10 to 20%of liver transplant recipients who have HCC in the native cirrhotic liver develop tumor recurrence after transplantation.The selection criteria presently employed to minimize the risk of recurrence are based on gross tumor characteristics defined by imaging techniques;unfortunately,the accuracy of imaging is far from being optimal.Furthermore,microscopic tumor features that are strictly linked with prognosis can not be assessed prior to transplantation.Pre-transplantation tumor downstaging may allow transplantation in patients initially outside the selection criteria and seems to improve the prognosis;it also provides information on tumor biology.Themain peculiarity of the transplantation setting,when this is compared with other modalities of treatment,is the need for pharmacological immunosuppression:this is based on drugs that have been demonstrated to increase the risk of tumor development.As HCC is an aggressive malignancy,immunosuppression has to be handled carefully in patients who have HCC at the time of transplantation and new categories of immunosuppressive agents should be considered.Adjuvant chemotherapy following transplantation has failed to show any significant advantage.The aim of the present study is to review the possible strategies to avoid recurrence of HCC after liver transplantation based on the current clinical evidence and the more recent developments and to discuss possible future directions.

Expression of Smac and the relationship with cell apoptosis and proliferation in hepatocarcinoma tissues

Objective:The aim of the study was to investigate the expression of Smac protein in human hepatocarcinoma and their relationship with cell apoptosis and proliferation.Methods:The expressions of Smac and the proliferating cell nuclear antigen (PCNA) in 41 cancer tissues,41 adjacent cirrhosis tissues and 9 normal control tissues in hemangioma were assessed by two-step immunohistochemical method and apoptosis was detected by TUNEL method.Results:Smac protein was expressed in 14 (34.14%) of the 41 cases of hepatocarcinoma,in 23 (56.10%) of the 41 cases of the adjacent cirrhosis tissues,and in 7 (77.8%) of the normal tissues in hemangioma.Smac protein positive expression rate in hepatocarcinoma was significantly lower than that in the adjacent cirrhosis tissues and the normal control tissues,χ2 were 3.989 and 4.115,respectively,and P were 0.046 and 0.042,respectively.Smac protein expression in cancer was significantly correlated with the ratio of apoptotic index to proliferative index,t'=2.260,P<0.05,but was not with the clinicopathological indicators such as the age and the histological grade,P>0.05.Conclusion:The relatively lower level of the expression of Smac may in a certain extent break the dynamic balance between apoptosis and proliferation of hepatocarcinoma cells,and then plays an important role in the pathogenesis of hepatocarcinoma.

A case of hepatic angiomyolipoma difficult to distinguish from hepatocellular carcinoma

We report a case of hepatic angiomyolipoma with uncommon clinical features. A 56-year-old man presented with a hepatic tumor in the caudate lobe. The tumor was hypoechoic on ultrasonography, showed early-phase hyperattenuation on enhanced computed tomography and did not absorb iron on superparamagnetic iron oxide-enhanced magnetic resonance imaging. Hepatocellular carcinoma was highly suspected, and the patient underwent hepatic resection. Histologically, the tumor was mainly composed of smooth muscle cells and contained small amounts of adipose cells and blood vessels. On immunohistochemical staining, the smooth muscle cells were positive for a melanocytic cell-specific monoclonal antibody. In cases with uncommon features of angiomyolipoma, it is quite difficult to distinguish angiomyolipoma from hepatocellular carcinoma.

Tumor immunology and immunotherapy: a journey I started from Hangzhou

This short article is dedicated to the 90th Anniversary of the School of Life Sciences at Zhejiang University,China.Immunotherapy of cancer is currently a hot topic in the biomedical field,and a research focus of my laboratory is on developing new and effective combinatorial immunotherapeutic strategies for liver cancer.Of note,my interest in immunotherapy of cancer stems from the training as an undergraduate student at Hangzhou University,China,almost 40 years ago.