The Initial Establishment Of DC-CTL For Primary Hepatic Carcinoma And The Evaluation Of Efficacy And Safety For This Therapy

ObjectiveTo evaluate the quality control of DC-CTL therapy system in patients with primary liver cancer, to analyze the changes of immune response, side effect and clinical indexes pre-and post-DC-CTL immunotherapy, to investigate the quality of life in patients with tumor after DC-CTL immunotherapy, so as to preliminary establish a DC-CTL therapy system in patients with primary liver cancer and assess the efficacy and safety.MethodsBased on the DC-CTL therapy system established by our laboratory for the following research:1、Selecting whole blood samples of patients with primary liver cancer patients (including hepatocellular carcinoma and intrahepatic cholangiocarcinoma) for our research, and the patients have enrolled in DC-CTL therapy from February 2012 to April 2013 in our hospital Biotherapy.2、Using a blood cell separator to collect peripheral blood mononuclear cells of patients, using MACS method to isolate CD14+cells, adding cytokine to cultivate and amplify DC cells in vitro, using tumor-associated antigen carrying recombinant non-proliferation adenovirus to infect DC cells, loading multi-antigen’including p53, hTERT and Survivin to induce DC maturation, and detecting the surface markers of DCs by flow cytometry.3、Using MACS method to isolate CD3+cells, freezing the cells to Day 8 and co-culturing with mature DC cells to induce antigen-specific CTL, adding cytokines to amplify CTL until Day 14, and detecting the levels of CD69, IFN-y, TNF-a and IL-4 by flow cytometry. Bacterial, fungal mycoplasma and endotoxin were detected before cell transfusion.4、Detecting the changes of immune indicators in whole blood samples of patients before and after DC-CTL therapy by flow cytometry including CD3+, CD3+CD4+, CD3+ CD8+, CD3+CD56+and Treg.5. Detecting the changes of blood routine, liver and kidney function and serum tumor indicators of patients before and after DC-CTL therapy including white blood cell counts, lymphocyte counts, monocyte counts, TBIL, DBIL, Alb, ALT, AST, GGT, AKP, AFP, CEA, CA199 and CA125.6、Observing the adverse reactions in patients receiving DC-CTL treatment, assessing the quality of life of patients using EORTC QLQ-C30, and performing Kaplan-Meier survival analysis to compare the changes of overall survival in different courses of treatment group and different tumor stage group.Results1、On Day 8, the percentages of CD80+, CD86+, CDllc+and CD83+were 88.55±4.07,91.16±7.32,95.24±4.97 and 87.4±5.08, respectively. The positive rate of DC surface markers detected by flow cytometry reached 85%, indicating a good ability of DC maturation.2、On Day 14, the percentage of CD69+T cell reached 90% via flow cytometry. The detection of cytokines production showed that the levels of IFN-y and TNF-a significantly increased to 17% and 19.3%, compared with the Ad-Blank group, while in both group the proportion of IL-4 is less than 1%, indicating a good ability of DC antigen presenting cells and activated T cell immune response.3、The test of bacteria, fungi mycoplasma and endotoxin results showed that the level of endotoxins and fungal glucans in reinfusion cells were controlled within the standard range.4、DC-CTL treatment system of primary liver cancer can significantly reduce the level of Treg cells in vivo. Prior to DC-CTL treatment, we observed the differences of immune indexes between patient and healthy donors, especially in the proportion of Treg cells. Notably, the proportion of CD3+CD8+cells increased, and the proportion of Treg cells decreased significantly (p<0.05) after treatment.5、DC-CTL treatment system of primary liver cancer can affect the clinical response. The changes of five indicators including WBC, LY, Alb, GGT and AKP were statistically significant. The lymphocyte count in HCC patients was lower than reference value before treatment, while it significantly increased after treatment. Levels of GGT and AKP in HCC group were significantly reduced after treatment (p=0.011, p=0.037) and levels of TBIL in ICC patients after treatment has also significantly decreased (p=0.046).6、DC-CTL treatment system for patients with primary liver has lower side effects. None of the patients had any significant or special adverse reactions, except for fever after cell transfusion. There were no allergies or other situation records, indicating the safety of DC-CTL treatment system.7、DC-CTL treatment system of primary liver cancer can improve the quality of life of patients. In HCC group, QoL score of 5 cases increased after treatment, four patients did not change significantly. Totally, the physical function score increased, and 7 patients showed significant improvement in Dyspnea. In ICC group of patients,2 patients showed pain relief, and three patients showed significant improvement in insomnia.8、Patients receiving a continuous courses of treatment or in an earlier TNM staging have better response of DC-CTL therapy system. The median survival time was 15 months in HCC group and 11 months in ICC group, in which 6 cases survived more than 12 months and OS of 3 HCC cases was longer than 24 months. Kaplan-Meier survival analysis showed that the survival time of patients received more than three courses of DC-CTL treatment was significantly longer than those only enrolled a short-term treatment (within three cycles, p<0.001), and the survival time of patients without regional lymph nodes or distal metastasis was longer than those metastasis cases(p= 0.034, p=0.011).ConclusionDC-CTL treatment system of primary liver cancer has a strict quality control. After DC-CTL therapy it can significantly reduce the level of Treg cells, change the immune function of patients, and improve the quality of life. Severe adverse events were not observed, confirmed the safety of our system.