Preliminary Experimental And Clinical Study Of Combination Therapy With Microwave Ablation And Cellular Immunotherapy For Hepatocellular Carcinoma

BACKGROUND Hepatocellular carcinoma(HCC)is a malignant tumor with poor prognosis.Current treatment methods include resection,liver transplantation,local thermal ablation and TACE.The long-term results of thermal ablation for HCC are similar with resection.Thermal ablation therapy had been generally acknowledged as one of the radical methods for HCC.However it still has an urgent demand for new methods to solve the problem of high rate of recurrence and metastasis.In the last twenty years,adoptive cellular immunotherapy developed quickly and is expected to be an effective adjuvant therapy for HCC.OBJECTIVE Research the condition of temperature and energy which could kill tumor cells and release neoantigen effectively.Observe the influence of combination therapy with microwave ablation(MWA)and peritumoral injection of immature dendritic cells on survival and tumor growth in mice.Observe clinical safety of combination therapy with MWA and adoptive immunotherapy of dendritic cells and eJector cells.Research the change of percentage of lymphocyte subsets and concentration of cytokine in peripheral blood after combination therapy.Initially observe the short-term result of combination therapy.MATERIAL AND METHODS 1.Simulate the condition of temperature and energy control in percutanous MWA for HCC to ablate SMMC-7721 cell strain suspension.There were five experiment groups:A.control group,B.45℃10 min group,C.50℃3min group,D.54℃3min group and E.60℃instant group.Detection for apoptosis was performed at 30 minutes and 2 hours after ablation.2.Thirty-two C57 mice were undertaken hypodermic inoculation of Hepal-6 tumor piece at right groin as ablation tumor and at left groin as observation tumor one week later.The mice with double tumors were separated into four groups:A.control group with no treatment,B.MWA group treated with MWA only,C.mDC group treated with MWA and subcutaneous injection of mature dendritic cells sensitized by neoantigen of Hepal-6 cell strain,D.iDC group treated with MWA and peritumoral injection of immature dendritic cells without neoantigen sensitization.The observation tumors were not treated.Tumors were measured every 7-8 days after ablation until mice died.3.With informed consent,10 HCC patients accepting combination treatment of radical MWA and adoptive immunotherapy with multiple immunocytes were included in the combination group.121 patients undertaken MWA alone with similar indication were included in the MWA group.The immunotherapy was designed as three treatment courses.Dendritic cells and effector cells separated from peripheral blood of patient were injected into the junctional zone of ablation region and hyperemia band with the guidance of contrast-enhanced sonography or into gorin lymph nodes or abdominal cavity of right upper quadrant under sonography guidance. 10 patients in the combination group and 24 patients in the MWA group accepted detection of lymphocyte subset and cytokine in peripheral blood before and 1,3,6 months after therapy.RESULT 1.In vitro experiment,the major state of tumor cells was dead and fewer cells were apoptotic after MWA.The percentage of live cells decreased in groups with higher temperature and energy.The percentage of live cells decreased markedly at 2h after ablation comparing with 30min after ablation.94.36%tumor cells were dead or apoptotic in 60℃instant group while many live cells were detected in other groups at 2h after ablation.2.In vivo experiments,the mean maximum diameter of ablation tumors in three treatment groups was markedly smaller than that of control group however had no statistical difference among three treatment groups at the 12^th day after ablation.The mean maximum diameter of observation tumors in three treatment groups was markedly smaller than that of control group and the size of observation tumors in iDC group was markedly smaller than that of mDC group (p＜0.05)at the 12^thday after ablation.After 120 days observation,the rates of complete response of ablation tumors were 37.5%in all treatment groups;the rate of disappearance of observation tumors was 87.5%,75%and 62.5%in iDC,mDC and MWA group separately which the iDC group was the highest.Comparing with control group,the mean survival time was markedly prolonged in all treatment groups which the iDC group was the longest.The ratio of life extension in iDC and mDC group was higher than MWA group which the iDC group was the highest.3.In clinical research,there were no serious adverse effects higher than gradeⅡafter fitly-four times of cellular injection and the results of hepatic,renal and blood coagulation function and blood routine had no statistical significance at 1,3,6 months after treatment comparing with those before treatment in the combination group.At 1,3,6 months after combination therapy,the percentage of CD8+CD28+ subset of peripheral blood lymphocytes in the combination group increased which was markedly higher(p＜0.05)than that of MWA group and CD4+/CD8+ decreased which was markedly lower(p＜0.05)than that of MWA group.The percentage of CD8+CD28-subset of peripheral blood lymphocytes in the combination group at 1 and 3 months after therapy markedly increased comparing with that of before treatment.The percentage of CD4+CD25high Treg subset of peripheral blood lymphocytes in the combination group markedly decreased(p＜0.05)which was remarkably lower than that of MWA group(p＜0.05)at 1 month after treatment, however increased which had no significant difference with that of MWA group at 3 and 6 months after treatment although was still lower than that of before treatment. The concentration of IL-2 and IL-2/IL-4 in peripheral blood of the combination group were increased at 1 month after treatment but returned at 3 and 6 months after treatment.The rate of local tumor progression or the rate of intrahepatic recurrence and extrahepatic metastasis in the combination group were lower than that of MWA group although had no statistical difference.57.14%patients obtained virological response at 1 month after combination therapy.CONCLUSIONS 1)94.36%tumor cells were dead or apoptotic at the second hours after microwave ablation under ablation temperature control of 60℃.The condition of ablation temperature control of 60℃and time control of 2 hours after ablation was presumed to be a good choice for combination therapy with microwave ablation and iDC injection at the marginal area of ablation.2)Combination therapy with microwave ablation and iDC injection at the marginal area of ablation could prolong the survival of HCC mice and the distance tumors were contracted or vanished.3)Combination therapy with microwave ablation and adoptive immunotherapy was safe in clinical application.4)Combination therapy improved the immune state of patients and obtained high rate of virological response,however could not last long duration.The duration between immunotherapy courses could be shortened and the number of treatment course be increased to upgrade current program.Antiviral immunotherapy would be needed after ablation to ameliorate whole immune state.Clinical result should be further evaluated after expanding the number of cases and extending the duration of follow-up.