| Background:Hepatocellular carcinoma (HCC) is a common malignant tumor in China, and the mortality of it is second to lung cancer. For the focal nodular, both surgery and microwave or frequency ablation could eradicate it. However, the high recurrent rate of HCC decreases the patients' long term survival rate even after the tumor had been completely eradicated. So far, most related researches have showed the radiotherapy and chemotherapy had no reliable effect to prevent recurrence, while the immunotherapy has become focus of clinical and experimental study to prevent recurrence which hold multiple advantages like safety, effective, specially targets of tumor cell. In this study, we improved the methods of immunotherapy including procedure, frequency and evolution the reaction after immunotherapy on the basic of our prior study, as well as summarizing the change of immune state and clinical results after immunotherapy.Objective:1 Research the expression of tumor related antigen of H22 after microwave ablation in cell suspension; 2 Research the expression of tumor related antigen of H22 in different point of ablation zone after microwave ablation of HCC in situ of KunMing mice.3 Study the influence of combination microwave ablation(MWA) with adoptive immunotherapy to the immune state and liver function after MWA of HCC.4 Study the effectiveness of combination MWA with adoptive immunotherapy to decrease the incidence of intrahepatic recurrence and extrahepatic metastasis after MWA of HCC.Method:In vitro experiment Three groups divided according to varies temperature conditions during MWA in this experiment. The control group, the 54℃group with a temperature condition of 54℃and the 60℃group with a temperature condition of 60℃. Two hours after ablation, the cell states was detected by flow cytometry include death, apoptosis and survival, and then the expression of tumor relative antigen (CD 147, HSP70) were detected by fluorescence in 54℃and 60℃groups. In vivo experiment The orthotopic transplantation tumor model of HCC was established by implanting the small tumor block. The HCC in situ was ablated with laparotomy 11 after implantation. The animal special antenna was used and the output power was 5 W, and the temperature in tumor margin was limited at 60℃. After the mice was cut off the head 24 hours after ablation, the tumor relative antigen were detected by fluorescence in different area of ablation zone (the centre of tumor and the margin of tumor).Clinical study 1 Clinical research for immunity reaction Fourteen cases were included in experimental group who underwent combination MWA of HCC with immunotherapy and another 15 cases were included in control group who underwent only MWA of HCC. The basic conditions in both groups had no significant difference. All patients had regularly undergone immunotherapy testing and liver function testing. Both test results were compared between two groups.2 Clinical research:fourteen cases were included in experimental group who underwent combination MWA with immunotherapy, while 42 cases were included in control group who underwent only MWA therapy. The basic conditions in both groups had no significant difference. At lease 3 courses immunotherapy were applied in each case of 14 cases in experimental group in 9 days,30 days and 90 days after MWA. And then, it was decided according to the results of lymphocyte count and various T lymphocyte subgroup ratio whether the immunotherapy was continues applied in 6,9,12,18 and 24 months after ablation. The incidences of intrahepatic recurrence including local tumor progression(LTP) and distant recurrence, extrahepatic metastasis and death were compared between two groups.Results:In vitro experiment According to the results of flow cytometry detection 2 hours after ablation, the survival rates in 54℃group and 60℃group were lower than these in control group(P<0.05) and both apoptosis rate and death rate were higher than these in control group(P<0.05). For same temperature condition, the apoptosis rate in 60℃group is higher than the one in 54℃group(86.0% vs 61.0%)(P <0.05) and survival and death rates were lower than these in 54℃group(P<0.05). The tumor relative antigens(CD147 and HSP70) expressed in 54℃and 60℃groups 2 hours after ablation. The expression rate of both antigens in 60℃group is higher than the one in 54℃group(HSP70:98.33% vs 46.22%, CD147:44.0% vs 19.44%)(P< 0.05), and for the same temperature condition, the expression rate of HSP70 is higher than the one of CD147(P<0.05).In vivo experiment Twenty-four hours after ablation of HCC in situ of KunMing mice, the immunofluorescence displayed that a number of HSP70 and CD147 antigen was expressed in the margin of tumor where the temperature was approximately 60℃, however, only a few of HSP70 was expressed, and no CD 147 expressed in centre area of tumor where the temperature was approximately 85℃; Both antigens mainly expressed in cell membrance, a little expressed in cytoplasm and intracellular substance.Clinical study 1 Clinical research for immunity reaction The lymphocyte count and various T lymphocyte subgroups results had no significant difference preoperationly between two groups(P>0.05), the lymphocyte count and cytotoxicity T lymphocyte subgroup ratio(CD3+/CD8+,CD8+/CD28+,NKT) in experimental group were higher than these in control group 6 months after ablation, and the suppressive T lymphocyte subgroup ratio(CD8+/CD28-,T-reg) was lower than the one in control group(P<0.05). The differences help to deduce the immunotherapy has an up-regulated effect on immune system. The difference of albumin and cholinesterase between both groups have no statistical significance preoperatively(P >0.05), but both in immunotherapy group are higher than that in control group 6 months after ablation (P<0.05). In immunotherapy group, the count of peripheral blood mononuclear cell(PBMC) isolated from 45 ml peripheral blood between preoperation and 3 months after ablation has no significant different (P>0.05), but the count of DC and CIK cultured from PBMC in 3 months after ablation is higher than that in preoperation(P<0.05).2 Clinical research The intrahepatic recurrence encountered in 4 cases(28.6%) in follow-up period(4-26 Months, Medium 19M) which included LTP in 3 cases(21.4%) and distant recurrence in 1 cases(7.1%) in experimental group, while intrahepatic recurrence encountered in 11 cases(26.2%)which included LTP in 2 cases(4.8%) and distant recurrence in 9 cases(21.4%), one extrahepatic metastasis and 1 death encountered in control group. The intrahepatic recurrent rate, the LTP rate, the distant recurrent rate, extrahepatic metastasis rate and survival rate had no significant difference between two groups(P >0.05).Conclusion:The experimental study has shown the two temperature conditions applied in clinic(54℃,60℃) could induce most of tumor cell apoptosis or death in cell suspension, and the cell still expressed tumor antigen after apoptosis or death. There were tumor antigens expression at margin of tumor where the temperature is approximately 60℃, and a little of tumor antigen expression in center area of tumor after MWA of HCC in situ of KM mice. The clinical study has shown the immunotherapy could improve lymphocyte count of periphery blood and the ratio of cytotoxicity T lymphocyte subgroup, the liver function and the proliferation capacity of cytotoxicity immunocyte, decrease the ratio of suppression T lymphocyte subgroup. Because of the limitation of case number in experimental group, the combination MWA with immunotherapy haven't deceased the incidence of intrahepatic recurrence of HCC. It needs a further study which should include more objects and longer follow-up period to making a definitive conclusion.
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