The Expression And Clinical Significance Of B7 Family Co-stimulatory Molecule In The Distinct Stage Of Gastric Carcinoma

With the further researching on tumor microenvironment and immune-escape of cancer,a series of molecular mediated cancer immune-regulation which called immune checkpoints,such as PD-L1(B7-H1)/PD-1,B7-H3,B7-H4,abnormal express in series of tumor tissues and tumor cells.These molecular take parts in the process of immune-escape and are related with tumor clinical pathology and prognosis.They are the important component of tumor microenvironment.The immune checkpoints are series of inhibitor molecular which maintain the self-immune-tolerance and steady states of immunology.They protect the body from tissue damage which original from abnormal immune response.With the inducer of immune microenvironment,tumor cells take advantage of immune checkpoints to counteract the anti-tumor immune response especially the cytotoxicity T cells which specially target the tumor immunogen.This is the crucial mechanism of tumor immune escape.Several immune checkpoints need to bind the ligand to be activity,so the antibody or recombination protein may regulate the signal pathway of cancer;help the tumor microenvironment get the immune potential to prevent the tumor.More importantly,the α-PD-L1,α-PD-1 and α-CTLA-4 antibodies showed the outstanding efficacy in the treatment of a variety of tumors.Therefore,the prospect of blocking the immune checkpoints strategy in tumor immunotherapy has received unprecedented attention,and the related mechanisms and key molecules will provide a new way for the intervention of tumor immunotherapy.Gastric cancer is one of the most common malignant diseases in China.It is urgent to find and establish a new method to improve the survival rate of 5 years.In recent years,with the understanding of the tumor microenvironment and immune escape mechanism of tumor immune escape,and the immune checkpoints of intervention getting the great progress in cancer immunotherapy,tumor immune therapy has become the most promising intervention strategies.In view of this,the use of advanced researchand diagnostic treatment technology,the integration of multidisciplinary resources to tumor microenvironment as the starting point,to explore the key cancer cells and immune evolution of immune homeostasis and immune escape of tumor in different stages to control points of molecular,exploring new therapeutic targets and biomarkers.The construction of joint immunosuppressive therapy is more effective and has important transformation value.Under this background,we used flow cytometry,immunohistochemistry,ELISA analysis,Real Time PCR and other technical methods of gastritis / gastric cancer clinical samples for the detection and analysis of the comprehensive,aims to explore 1.the negative co-stimulatory molecule expression of gastric cancer evolution;2.negative co-stimulatory expression and clinical value of molecular in clinical gastric cancer patient’s blood cells,plasma and tumor tissues;and 3.the expression of negative co-stimulatory molecules and infiltration of T cells and macrophages influence on clinical prognosis of patients.In our study,we analyzed the expression of negative co-stimulatory molecules in gastric cancer patients,which has the frontier and innovation of the theoretical research and the high clinical value.Part 1 The expression of negative co-stimulatory molecules PD-L1,B7-H3 and B7-H4 in the distinct stage of gastric cancerObjective: to investigate the meaning of the expression of PD-L1,B7-H3 and B7-H4 in the process of gastric carcinogenesis by detecting the PD-L1,B7-H3,B7-H4 expression of superficial gastritis,atrophic gastritis,low grade neoplasia,high-grade intraepithelial neoplasia and early gastric cancer patients,and analyzing the intrinsic relationship of the expression of gastric cancer from inflammation to tumor.Methods: collected endoscopy diagnosed pathology specimens,superficial gastritis,atrophic gastritis,low grade neoplasia,high-grade intraepithelial neoplasia in20 cases,and 30 cases of early gastric cancer surgical specimens.by immunohistochemistry staining,detected the expression of PD-L1,B7-H3 analysis and B7-H4 molecules in various tissues,and statistical analyzed the differences of the expressions.Results: immunohistochemistry showed that the expression of PD-L1 ininflammatory tissue and tumor tissue were expressed in the process of gastric carcinogenesis,and did not appear as the disease evolution enhanced expression,and IHC showed strong expression in gastric carcinoma stage in gastric cancer tissue and tumor edge.But in the stage of gastric cancer,expression of PD-L1 in the infiltration of inflammatory cells is much higher than the inflammatory stage.Almost no expression in superficial and atrophic gastritis,the expression of B7-H3 expression increased began with neoplasia grade,while in gastric cancer,the expression of B7-H3 increased significantly and widely expressed in tumor tissue and interstitial tissue.The expression of B7-H4 was strongly expressed in the inflammatory cells in the inflammatory tissues,and the expression of B7-H4 in the tumor tissues was diffusely expressed.In the tumor tissues,B7-H4 was diffusely expressed in the infiltrating cells and tumor cells.Conclusion: PD-L1 expressed in the early stage of inflammation,as the disease progresses without increased expression.B7-H3 is not expressed in inflammation,and it is gradually expressed in neoplasia tissues and extensively expressed in gastric cancer.B7-H4 in inflammatory tissues showed strong expression of a single cell,and showed diffuse expression in neoplasia and gastric cancer.PD-L1,B7-H3 and B7-H4 were all expressed in the tumor infiltrating immune cells.Part 2 Expression and clinical significance of PD-L1,B7-H3 and B7-H4 in blood cells,plasma and tumor tissues of patients with gastric cancerObjective: Study of negative B7 family molecules in patients with gastric cancer overall expression patterns,finding the correlation between the expression of different tissues / cells and the expression of different types and analysis of the clinical value of different cell types expression.Methods: Flow cytometry detected the peripheral blood CD4,CD8 and CD14 cells of negative co-stimulatory molecules PD-L1,B7-H3,B7-H4 expression;ELISA detected soluble molecular of PD-L1,B7-H3 and B7-H4 in plasma;immunohistochemistry detected negative co-stimulatory molecules PD-L1,B7-H3B7-H4 expression in paraffin sections;Real-Time PCR detected the expression of Th1 and Th2 cells secreted cytokines CCR7,CXCR4,IFN-γ,IL-4,IL-10,TNF-αin freshtissue.The correlation between the expression and the pathological data was analyzed.Results: Negative co-stimulatory molecule PD-L1 expression by immunohistochemistry was in related to IFN-γ expression level.Expression of plasma soluble B7-H3 was associated with the expression of B7-H3 in tumor tissue.PD-L1 and B7-H4 in tumor tissue had no correlation with soluble PD-L1 and B7-H4.Soluble B7-H3 in plasma expression was in positive related to proliferation of Ki67 cells of patients.There was no correlation between the expression of CD14+PD-L1+/CD14+B7-H3+/ CD14+B7-H4+ in peripheral blood cells and the expression of PD-L1/B7-H3/B7-H4 in tumor tissue.Conclusion: Immunohistochemistry expression of PD-L1 was in positive related with IFN-γ expression level;show that the possible relationship between the mutual regulation IFN-γ and PD-L1 protein,PD-L1 is likely to be associated with activation of Th1 cells.The plasma soluble B7-H3 expression was positively correlated with tissue B7-H3 expression indicates that B7-H3 may promote the proliferation of tumor cells.Also,tissue B7-H3 expression secretes into the serum to form the secretory form.Part 3 The expression of negative co-stimulatory molecules PD-L1,B7-H3,B7-H4 and CD8,CD68 in gastric cancer tissue and clinical relevanceObjective: to detect the expression of PD-L1,B7-H3,B7-H4,CD8 and CD68 in160 cases of specimens with gastric cancer,statistical analysis of the relationship between the combination expression of each kind of negative co-stimulatory molecules and cytokines molecules and the prognosis in gastric cancer patients or pathological information,to investigate the impact of the expression of PD-L1,B7-H3,B7-H4 and infiltration of T cells and macrophages on survival of patients with gastric cancer.Methods: collected 160 cases gastric cancer tissue slides pathologically diagnosed by immunohistochemistry staining,detected the expression of PD-L1,B7-H3,B7-H4 analysis,CD8 and CD68 molecules in gastric cancer tissues,statistically analyzed negative co-stimulatory molecules / associated inflammatory molecules in combination with patient information and the influence on prognosis of the patients.Results: the combination of PD-L1/CD8 was in correlation with the patients withlymph node metastasis(P=0.046),and the low expression of CD8 with high PD-L1 expression are more likely to have multiple lymph node metastasis(more than 4).Combined with the patient B7-H3/CD8 or B7-H3/CD68,had significant correlation with death(P=0.003,P=0.01),the prognosis of the patients with low B7-H3 expression and high expression of CD8 or CD68 was significantly better than other patients,but on the contrary,the prognosis of the patients with the high expression of B7-H3 and the low expression of CD8 or CD68 was significantly lower than other patients.B7-H4/CD8 and B7-H4/CD68 are no correlation with the any pathological information of the patients(age,gender,tumor size,tumor stage,lymph node metastasis and distant metastasis death,etc.),but the low expression of B7-H4 with high expression of CD8 or CD68 patients had good survival than other patients.Conclusion: the expression of combined CD8 and PD-L1 was in correlation with the patient’s lymph node metastasis.High expression of PD-L1 with low expression of CD8 patients was prone to multiple lymph node metastases.B7-H3/CD8 and B7-H3/CD68 combination were significant predictors of patient survival.High expression of B7-H3 with low expression of CD8 or CD68 had worst prognosis of the patients.The prognosis of the patients was best in low B7-H3 expression with high CD8 or CD68 expression.In summary,this paper expounds the expression and clinical value of comprehensive expression of negative B7 family molecules in the process of gastric carcinogenesis and discusses the expression of negative B7 family molecules in gastric cancer,provides a new theoretical basis and potential application value for the early diagnosis of gastric cancer and gastric cancer biological indicators to design effective interventions.