Correlation Between The Expression Pattern Of VISTA And Immune Cells In Triple Negative Breast Cancer And Its Effect On Prognosis

Background and objective:Triple-negative breast cancer(TNBC),defining as not expressing estrogen receptor(ER),progesterone receptor(PR),and human epidermal growth factor receptor 2(HER2),is a subtype of breast cancer,which has strong invasive biological characteristics,and due to the lack of targeted therapy and endocrine therapy targets,their prognosis is often poor.In recent years,immunocheckpoint inhibitors have been widely used in the treatment of malignant tumors.V-domain immunoglobulin suppressor of T-cell activation(VISTA)is a new immune checkpoint.It has been confirmed that its expression is increased in prostate cancer and malignant melanoma after immunotherapy.It is speculated that it is related to immunotherapy tolerance and may become a new target of immunotherapy.However,the expression pattern of VISTA in the tumor microenvironment of TNBC and its prognostic significance are still unclear.Purpose:The purpose of this study includes the following three aspects:(1)Analyze the expression of VISTA on tumor cells and immune cells of TNBC,and its relationship with clinicopathological parameters and prognosis.(2)Analyze the correlation between VISTA and CD3,CD4,CD8,CD19 positive tumor infiltrating lymphocytes(TILs)in the tumor microenvironment,and to establish the immunophenotype based on VISTA.(3)TCGA and other databases are used to analyze the relationship between VISTA expression and clinicopathological parameters?prognosis and the expression of CD3,CD4,CD8 and CD 19.The enrichment scores of 28 kinds of immune cells in the tumor microenvironment are analyzed,and the difference of immune cells enrichment between the VISTA high and low expression groups are compared.Analyze the correlation between VISTA and immune cell enrichment and explore the relationship between VISTA and immune cells and its anti-tumor immune response.Methods:(1)A cohort of 254 untreated TNBC patients with complete information is established.Immunohistochemical staining is used to detect the expression of VISTA on tumor cells and immune cells.According to the median percentage of positive cells as the cut-off value,the cohort was divided into tumor/immune cell high/low expression group,and its relationship with clinicopathological parameters and prognosis is analyzed.(2)The density of TILs is analyzed,and the expression of CD3,CD4,CD8 and CD19 is detected,and the correlation between the expression of CD3,CD4,CD8,CD 19 positive TILs and the expression of VISTA on tumor cells and immune cells is analyzed,and the immunophenotype based on VISTA is established,and its predictive significance for prognosis is verified.(3)The interactive data of TCGA,cBioportal,GEPIA,UALCAN are used to obtain the difference between expression of VISTA mRNA in breast cancer,normal breast tissue and different subtypes of breast cancer.To establish a cohort of 139 untreated TNBC patients with complete information from TCGA database.The cohort is divided into VISTA high and low groups based on the median of mRNA expression level as the cut-off value.The relationship between VISTA and CD3,CD4,CD8 and CD 19 mRNA is analyzed.The enrichment scores of 28 kinds of immune cells in the tumor microenvironment are obtained by GSEA enrichment analysis.The enrichment differences of immune cells between the two groups are compared,and the correlations between the enrichment scores of different types of immune cells and VISTA mRNA are analyzed.Result:(1)According to the screening criteria,254 TNBC in stage ?-? were included.The results showed that in TNBC,the expression of VISTA is mainly on immune cells,but less on tumor cells.High expression of VISTA on immune cells is related to lymph node negative(P<0.001)and relatively early TNM stage(stage ? and ?)(P=0.001).Compared with the low expression group,the high expression of VISTA on immune cells group has significantly higher 5-year disease-free survival(DFS)(79.0%VS 64.6%,P=0.011)and 5-year overall survival(OS)(90.0%VS 72.0%,P<0.001).The high expression of VISTA on immune cells is an independent protective factor for OS(Hazard ratio(HR)=0.436,95%Confidenceinterval(CI)=0.228-0.831,P=0.012).In T1-2N0 subgroup,the survival advantage of high expression of VISTA on immune cells is prominent.Compared with low expression group,the high expression of VISTA on immune cells group has significantly higher 5-year DFS(86.8%VS 73.1%,P=0.038)and 5-year OS(95.6%VS 82.7%,P=0.022).The high expression of VISTA on immune cells is an independent protective factor for both DFS(HR=0.281,95%CI=0.093-0.848,P=0.024)and OS(HR=0.462,95%CI=0.218-0.979,P=0.044).(2)In TNBC,the high expression of VISTA on immune cells significantly correlats with the increased density of CD3+TILs,CD4+TILs,CD8+TILs and CD19+TILs(P=0.009,P<0.001,P=0.021,P<0.001),especially with CD4+TILs(R=0.423,P<0.001).According to the expression of VISTA on immune cells and CD4+TILs,the TNBC microenvironment is classified into four types:VISTAH/CD4H,VISTAH/CD4L,VISTAL/CD4H,VISTAL/CD4L.The 5-year OS of VISTAH/CD4H group is significantly higher than that of the other three groups(VISTAH/CD4H VS VISTAH/CD4L VS VISTAL/CD4H VS VISTAL/CD4L:91.5%VS 89.2%VS 77.3%VS 68.7%,P=0.002),the survival benefit of the double high group was significantly higher than that of the other three groups,and the survival advantage in T1-2N0 subgroup is also significant.(3)According to the screening criteria,139 cases of TNBC in TCGA database are included.The results show that there is no significant relationship between VISTA mRNA level and age,menstrual status,tumor stage,lymph node stage,TNM stage,breast and axillary surgery,radiotherapy and chemotherapy,also has no effect on OS.VISTA mRNA is positively correlated with CD3,CD4,CD8 and CD19 mRNA(all P<0.001),and is strongly positively correlated with CD4 mRNA(R=0.764).The enrichment of type 1 helper T cells(Th1),activated dendritic cells(DCs)and auxiliary memory CD8+T cells with positive anti-tumor immunity are significantly higher in the high expression of VISTA mRNA group than in the low group.There is a strongly positive correlation between VISTA and Th1(R=0.751)and activated DCs(R= 0.642).It is speculated that VISTA could stimulate the production of CD4+Th1 cells in TNBC and play a positive role in anti-tumor immunity.Conclusion:(1)In TNBC,high expression of VISTA on immune cells is an independent protective factor for prognosis.(2)The establishment of immune classification of TNBC microenvironment based on VISTA and CD4 has significance in predicting prognosis,and it is better than single indicator of VISTA.(3)In TNBC,there is a significant correlation between VISTA and CD4 expression in both protein and mRNA levels.At the level of cell enrichment,there is a close relationship between VISTA and CD4+Th1 cells.It is speculated that VISTA plays a positive role in anti-tumor immunity by stimulating the production of CD4+Th1 cells.