| Gastric cancer is one of the main malignant tumors threaten human health. The morbidity and mortality of gastric cancer in China’s Jiangsu Province is much higher than the national average. Despite the increasing means and level of treatment, the therapeutic effect is still not ideal. To find a new prognostic marker of gastric cancer and to explore new treatment strategies is a very urgent scientific proposition.Recurrence and metastasis is one of the main causes of gastric cancer treatment failure, and in-depth study of tumor microenvironment is an important way to improve 5-year survival rate of gastric cancer. In an effective tumor immune response, not only the first signal is required, but also the costimulatory molecules participate and provide an auxiliary second signal in order to make T cells reach the threshold for physiological activation. Appropriate costimulatory signals can reduce the demand on the first signal during T cell activation, and the inhibitory costimulatory signals can reduce immune response or lead to immune tolerance. Costimulatory molecule B7-H4 is a new member of B7 family, mainly expressed in macrophages, mature dendritic cells, some tumor cells and activated T cells. It can negatively regulate T cell immune response by inhibiting T cell proliferation, cytokine production and cell cycle progression, and play an important role in the process of mediating tumor immune response. B7-H4 expressed on the surface of tumor cells participate in tumor formation and progression. Therefore, to control and interfere with the expression of costimulatory molecules B7-H4 will become a new strategy for gastric cancer treatment. Cytokine-induced Killer Cells (CIK) is a kind of new anti-tumor effector cells which can regulate and enhance immune function, with stronger tumoricidal activity and more extensive tumor killing spectrum than the anti-tumor effector cells previously reported.In this article we discussed the relationship between costimulatory molecules B7-H4 expression in gastric tumor tissues and gastric cancer prognosis, analyzed the clinical significance of B7-H4 in the occurrence and development of gastric cancer, further studied different expression of B7-H4 in tumor tissues of gastric cancer and the influence on the therapeutic efficacy of gastric cancer patients treated by CIK cell immunotherapy, and established the inclusion criteria of CIK cell therapy in clinical treatment, which laid the foundation for further exploring the mechanism of costimulatory molecules B7-H4 in gastric cancer.PART I Expression of B7-H4 in the gastric carcinoma tissues and in correlation to its prognosisObjective: To study the expression of costimulatory molecules B7-H4 in the gastric carcinoma tissues and further to evaluate the correlation between the B7-H4 expression and the patients’prognosis.Methods: B7-H4 expressions in the surgical specimens of gastric cancer tissues were determined by using immunohistochemistry assay in 156 patients who had obtained either autologous cytokine-induced killer cell biotherapy or chemotherapy.Results: Positive expression rate of B7-H4 in gastric carcinoma tissues was 44.9%. Single-factor analysis demonstrated that there was no correlation between the B7-H4 expression and sex, age, histological type, pathological grade or tumor size. However, there was a statistical significant correlation between the B7-H4 positive expression and the invasion depth and lymph node metastasis. The positive expression rate of B7-H4 was obviouslty higher in the invasive group than in the non-invasive group (49.1% vs 16.7%) (χ~2=8.467, P=0.004). And the positive expression rate of B7-H4 was higher in the lymph node metastasis group than in the non-metastasis group (χ~2=17.339, P<0.0001). The median survival time of gastric cancer patients with B7-H4 low expression was 13 months longer than those with high expression (χ~2=12.38, P<0.0001). Multi-factor COX model analysis demonstrated that B7-H4 expression may be considered as the prediction of survival time in the gastric cancer patients (RR=1.85, 95%CI=1.15~2.96). Moreover, the risk of death in gastric cancer patients was significantly lower in the CIK treatment group than in the chemotherapy group (RR=0.53, 95%CI=0.33~0.85).Conclusion: There was a negative correlation between the high expression of B7-H4 and the survival time of gastric cancer patients, which may suggest that B7-H4 is one of negative regulatory molecules and may be considered as a predictive index of gastric cancer patients. CIK cells treatment can significantly prolong the survival time. PART II Establishment of real-time PCR method on detecting costimulatory molecule B7-H4 expressionObjective: To establish a real-time PCR method to detect costimulatory molecule B7-H4 gene expression.Methods: The mRNA levels of costimulatory molecule B7-H4 and the reference gene GAPDH were measured with real-time PCR using TaqMan technology. The primers and TaqMan probes of B7-H4 and GAPDH were designed by ourselves. The B7-H4 and internal reference gene GAPDH fragments in pure form from classical PCR amplification was cloned to pMD19-T vector, and recombinant plasmids were used as the standard substances of B7-H4 and GAPDH quantitative detection. Standard curves were established using a serial dilution of quantified plasmids to measure B7-H4 and GAPDH. The reaction systems were optimized, and the sensitivity, specificity and reproducibility were evaluated.Results: The amplification products were confirmed as the specific fragments of B7-H4 and GAPDH by DNA sequencing instrument. For B7-H4, the sensitivity was 527copies/ml. The linear range was found from 5.27×102 to 5.27×107 copies/ml, the standard curve equation was Y=-3.1395X+41.805, the correlation coeffecient was 0.995, the interassay coefficient of variations was 2.39%~3.59%, and the amplification efficiency was 108.2%; For GAPDH, the sensitivity was 386 copies/ml. The linear range was found from 3.86×102 to 3.86×107 copies/ml, the standard curve equation was Y=-3.2436X+41.083, the correlation coeffecient was 0.999, the interassay coefficient of variations was 2.26%~3.86%, and the amplification efficiency was 103.4%.Conclusions: The real-time PCR system for quantifying costimulatory molecule B7-H4 mRNA levels has been established successfully with high specificity, sensitivity and good repeatability.PART III Influence of B7-H4 expression on survival time of gastric cancer patients treated with cytokine-induced killer cellObjective: To study the influence of different expression levels of costimulatory molecules B7-H4 in gastric cancer tissues on survival time of gastric cancer patients treated with CIK cell biotherapy, and to provide new strategies for gastric cancer diagnosis and therapy.Methods: Cytokine-induced killer cells (CIK cells) were induced in vitro through biotechnology for clinical application. Follow-up investigation of related clinical data of 156 cases of gastric cancer patients were collected by the method of retrospective cohort study. The B7-H4 expression was detected in the surgical specimens of gastric cancer patients by immunohistochemistry assay. And the impact of different expression levels of B7-H4 in gastric cancer tissues on therapeutic efficacy of gastric cancer patients in the chemotherapy group and in the CIK treatment group were compared.Results: In the chemotherapy group, the median survival time of gastric cancer patients in B7-H4 low expression group was significantly higher than those in B7-H4 high expression group (38 vs 16), and the difference was statistically significant (χ~2=13.99, P<0.0001); In the CIK treatment group, the median survival time of gastric cancer patients in B7-H4 low expression group was significantly higher than those in B7-H4 high expression group (55 vs 34), and the difference was statistically significant (χ~2= 4.679, P< 0.05); In the B7-H4 high expression group, the median survival time of gastric cancer patients in the CIK treatment group was 18 months longer than those in the chemotherapy group; In the B7-H4 low expression group, the median survival time of gastric cancer patients in the CIK treatment group was 17 months longer than those in the chemotherapy group. After confounding factors such as age, sex and tumor site were adjusted, risk of death of gastric cancer patients in B7-H4 high expression group was significantly higher than in B7-H4 low expression group (RR=1.73,95%CI=1.09~2.76).Conclusion: Costimulatory molecules B7-H4 high expression can significantly increase the risk of death of gastric cancer patients. CIK cell treatment can prolong the median survival time of gastric cancer patients, and it will control and interfere in the B7-H4 expression, which will become a new method of gastric cancer treatment.PART IV Interdependent multi-factor analysis of gastric cancer treated with CIK cell therapy and survival timeObjective: To study the interdependent multi-factor dependablity of gastric cancer treated with cytokine-induced killer cell adoptive immunotherapy and survival time.Methods: Immunotherapy was provided for gastric cancer patients by the induction in vitro of autologous cytokine-induced killer cells with biotechnology. In the retrospective cohort study, median survival time and survival rate was estimated using the Kaplan-Meier method, the impact of clinical factors on survival rate was compared by Log-rank test, the contact intensity of CIK cell therapy and outcome of death and survival time was estimated with RR and 95% CI through multi-factor COX model.Results: The difference was not statistically significant in the equilibrium analysis between the chemotherapy group and the CIK treatment group (P>0.05). The recurrence rate of gastric cancer patients in the CIK treatment group was significantly lower than that in the chemotherapy group (53.3% vs 71.6%), and the difference was statistically significant (χ~2=5.566,P=0.018). The median survival time (months) of gastric cancer patients in the CIK treatment group was significantly longer than that in the chemotherapy group (49 vs 27), and the difference was statistically significant (χ~2=10.907,P=0.001). With the increase of the number of CIK infusion, the difference among survival rate curves of the four groups was statistically significant (χ~2=14.534,P=0.002). The 2-year survival rate of gastric cancer patients in the CIK treatment group was significantly higher than that in the chemotherapy group (P<0.05). After confounding factors such as age, sex, tumor stage and depth of invasion were adjusted, the risk of death in gastric cancer patients in the CIK treatment group was significantly lower than in the chemotherapy group (RR=0.52,95%CI=0.34-0.82). And the risk of death in gastric cancer patients gradually degraded with the increase of the number of CIK infusion. (trend test, P=0.0001). Compared to those in the chemotherapy group, gastric cancer patients treated with CIK infusion for more than 25 times, RR=0.14, 95%CI=0.03-1.58. The number of CIK treatment was significantly negatively correlated with the risk of death in gastric cancer patients whose survival time was less than 36 months (RR=0.54, 95%CI=0.36-0.80). In addition, the risk of death in gastric cancer patients will be significantly increased with the increase of tumor stage, recurrence, and age (RR=28.87, 95%CI: 7.05-118.25; RR=2.10, 95%CI: 1.40-3.11; RR=1.66, 95%CI: 1.12-2.46).Conclusions: CIK cell transfusion treatment can reduce the risk of death in gastric cancer patients, and significantly increase the survival time of gastric cancer patients.PART V Multi-factor COX model analysis of impact of costimulatory molecules B7-H4 on gastric cancer prognosisObjecive To investigate the influence of costimulatory molecules B7-H4 expression on prognosis of gastric cancer patients treated by cytokine-induced killer cells(CIK cells) adoptive immunotherapy and to analyze the impact of different expression levels of costimulatory molecules B7-H4 in gastric cancer tissues on survival time of gastric cancer patients treated with cytokine-induced killer cell biotherapy.Methods: Related clinical data of 156 cases of gastric cancer patients were collected by the method of retrospective cohort study, and were divided into chemotherapy group (81 cases) and chemotherapy combine with CIK cell therapy group (75 cases) according to their treatment and conditions. B7-H4 expression was detected in the surgical specimens of gastric cancer patients by immunohistochemistry assay, and the disease free survival of different expression levels of B7-H4 in gastric cancer tissues of gastric cancer patients in the chemotherapy group and in the CIK treatment group were compared.Results: The difference was not statistically significant in all clinical pathological data between the patients in the chemotherapy group and in the CIK treatment group (P>0.05). The postoperative tumor-free median survival time of gastric cancer patients of the two groups were 18.0 months (95%CI: 10.5~25.5) and 45.0 months (95%CI: 19.5~70.5) respectively, and the difference was statistically significant (χ~2= 11.631, P=0.001). The postoperative median survival time of gastric cancer patients of the two groups were 27.0 months (95%CI: 19.6~34.4) and 49.0 months (95%CI: 35.9~62.1) respectively, and the difference was statistically significant (χ~2=10.907, P=0.001). In 86 gastric cancer patients with B7-H4 low expression treated by chemotherapy alone and by chemotherapy combine with CIK cell therapy, the tumor-free median survival time was 32.0 months (95%CI: 26.5~37.5) and 62.0 months (95%CI: 23.0~101.0) respectively, and the difference was statistically significant (χ~2=4.663, P=0.03). In 70 gastric cancer patients with B7-H4 high expression treated by chemotherapy alone and by chemotherapy combine with CIK cell therapy, the tumor-free median survival time was 11.0 months (95%CI: 3.2~18.8) and 18.0 months (95%CI: 7.3~28.7) respectively, and the difference was statistically significant (χ~2=11.971, P=0.001). By comparing between B7-H4 high expression group and B7-H4 low expression group, the median tumor-free survival time of gastric cancer patients(months) and 95%CI of gastric cancer patients was respectively 16 (11.9-20.1) and 47 (22.4-71.6), the median survival time(months) and 95%CI of gastric cancer patients was respectively 25 (19.0-31.1) and 43 (35.2-50.8). In the chemotherapy group, the median survival time of gastric cancer patients in B7-H4 low expression group was significantly longer than those in B7-H4 high expression group (36 vs 16), and the difference was statistically significant (χ~2=14.14, P<0.0001). In the chemotherapy plus CIK treatment group, there was a tendency that the median survival time of gastric cancer patients in B7-H4 low expression group was longer than those in B7-H4 high expression group (55 vs 34), but the difference was not statistically significant (χ~2=1.78, P=0.182). In the chemotherapy group, the median tumor-free survival time of gastric cancer patients in B7-H4 low expression group was significantly longer than those in B7-H4 high expression group (33 vs 11), and the difference was statistically significant (χ~2=18.956, P<0.0001). In the chemotherapy plus CIK treatment group, the median tumor-free survival time of gastric cancer patients in B7-H4 low expression group was significantly longer than those in B7-H4 high expression group(90 vs 18), and the difference was statistically significant (χ~2=3.842, P=0.050). After confounding factors such as sex, age and so on were adjusted, risk of recurrence and death of gastric cancer patients in B7-H4 high expression group was significantly higher than in B7-H4 low expression group (RR=2.30, 95%CI=1.41~3.75; RR=1.90, 95%CI=1.20~3.01,respectively).Conclusion: B7-H4 high expression may be an independent risk factor of increasing the risk of gastric cancer recurrence and death. CIK cell transfusion treatment can control and interfere in the B7-H4 expression, and prolong the median survival time of gastric cancer patients. B7-H4 low expression of gastric cancer can be considered as inclusion criteria of CIK cell biotherapy.In summary, this study investigated the B7-H4 expression and clinical significance in gastric cancer patients, and further explored the new backgrounds for search of the biotheapary and the diagnosis biomarker and means to intervene the gastric cancer progression. Our study is originality and clinical application value. |
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