| Objective:Immunotherapy targeting immune checkpoints has made a breakthrough in cancer treatment including human lung adenocarcinoma(LUAD)and colorectal cancer(CRC).VISTA(V-domain Ig inhibitor of T cell activation)and HHLA2(Human endogenous retrovirus-H long terminal repeat-associating protein 2)are two recently identified members of the B7 homologue family of immune-regulatory molecules.This study was designed to investigate the expression patterns and clinical significance of VISTA and HHLA2 in LUAD and CRC.Understanding the immunological functions of VISTA and HHLA2 pathways and the heterogeneity of tumor microenvironment will facilitate the optimization of immune checkpoint therapy.Methods:Immunohistochemistry was performed to examine the expression of VISTA,HHLA2,PD-L1,and CD8+tumor-infiltrating lymphocytes(TILs)in 74 LUAD and 62 CRC cases,and the corresponding noncancerous tissues.We measured expression patterns and location of these targets,evaluating their association with clinicopathological features and overall survival.In addition,Opal/TSA multiplex immunofluorescence technique was used to simultaneously detect the co-expression of the above indicators in lung cancer tissues.Results:1.VISTA and HHLA2 were overexpressed in the tumor tissues of LUAD and CRC,compared with noncancerous tissues,and displayed more frequent expression than PD-L1.2.In LUAD patients,VISTA expression in ICs was correlated with smoking history(P=0.029).However,in CRC patients,VISTA expression in TCs displayed a significant correlation with TNM stage(P=0.005).3.In CRC patients,HHLA2 expression in ICs and TCs were correlated with lymph node metastasis(P=0.022,P=0.006).PD-L1 expression in ICs was linked with age(P=0.001);PD-L1 expression in TCs demonstrated a significant correlation with TNM stage(P=0.004).4.In LUAD patients,VISTA in ICs was positively associated with HHLA2 in ICs(r_s=0.371,P=0.001),PD-L1 in ICs(r_s=0.445,P<0.001),PD-L1 in TCs(r_s=0.302,P=0.009),stromal CD8(r_s=0.380,P<0.001)and intratumoral CD8(r_s=0.469,P<0.001).5.In LUAD cohort,high VISTA expression in ICs predicted poorer survival and high mortality rate(P=0.042).High PD-L1 expression in ICs and TCs showed poor survival(P=0.029,P=0.043).Patients with high stromal CD8~+TILs demonstrated significantly higher OS(P=0.020).6.In CRC cohort,high VISTA expression in ICs was significantly correlated with a favorable survival(P=0.018).High HHLA2 expression in ICs and TCs demonstrated better overall survival(P=0.049,P=0.034).PD-L1 upregulation in ICs predicted better survival(P=0.030),while PD-L1overexpression in TCs indicated worse survival(P=0.025).Patients with high intratumoral CD8~+TILs also demonstrated significantly higher OS(P=0.002).7.The independent prognostic value was detected by multivariate analysis.The results suggested that stromal CD8 was independent prognosis factor in the overall survival of LUAD cohort(P=0.017).Likewise,VISTA in ICs,PD-L1 in ICs and TCs and lymph node metastasis were four independent predictors in the overall survival of CRC cohort(P=0.040,0.020,0.002,and 0.006).8.Two distinctive LUAD patient clusters were obtained by hierarchical clustering analysis,according to VISTA in ICs,PD-L1 in ICs,PD-L1 in TCs and stromal CD8~+TILs.The low-risk subgroup consisted of 58 patients,and high-risk subgroup contained 16 patients.The Kaplan-Meier curves and log-rank test demonstrated that overall survival was significantly distinct between these two patient clusters stratified by these four immune biomarkers(P=0.015).9.Multiplex immunofluorescence combined with multispectral imaging technology can display the expression patterns and correlations of the above various markers in situ.Conclusion:1.High expression of VISTA in ICs was significantly associated with poor prognosis in LUAD.2.High expression of VISTA in ICs and high expression of HHLA2 in ICs and TCs were significantly associated with favorable prognosis in CRC.VISTA expression in ICs acted as an independent prognostic predictor of CRC.3.VISTA and HHLA2 are potential immunotherapeutic targets,which possess different prognostic significance between LUAD and CRC. |
