Expression Of PD-L1 On Tumor-associated Macrophages Of High Grade Serous Ovarian Cancer And Its Clinical Significance

Background and objectivesOvarian cancer is one of the three major malignant tumors of the female reproductive system,most patients have been diagnosed in the advanced stage,in which serous cancer accounts for more than 80%.70%of patients will relapse within three years after treatment.The 5 year survival rate was only 30%,and the proportion of patients who remained tumor-free within 10 years after treatment was less than 15%.Immunotherapy brings new dawn to the treatment of ovarian cancer as a new treatment method with great potential.The current application of inhibitors of the programmed cell death receptor-1(PD-1)/programmed cell death ligand-1(PD-L1)pathway in ovarian cancer therapy is still under study.Tumor associated macrophages(TAMs)have become a new target for the treatment of epithelial ovarian cancer because of its important role in tumor development in epithelial ovarian cancer.Therefore,exploring the expression of PD-L1 on TAMs will help to improve the effect of immunotherapy in the treatment of ovarian cancer.Studies have shown the PD-L1 expression on TAMs of both primary and metastatic ovarian cancer,but the clinical significance and expression mechanism in high-grade serous ovarian cancer(HGSOC)are rarely reported at home and abroad.The purpose of this study was to clarify the relationship between the expression of PD-L1 on TAMs with prognostic and clinicopathological factors,and to provide theoretical and experimental basis for its use as a prognostic evaluation index;to analyze the regulatory effect of ovarian cancer cells on the expression of PD-L1 on TAMs,and to provide new ideas for the combined application of targeted TAMs therapy and PD-L1 inhibitors in ovarian cancer therapy.Methods1.Immunohistochemistry and immunofluorescence:Patients who underwent surgical treatment in the first affiliated hospital of zhengzhou university from january 2015 to december 2016 were collected,including 83 patients with HGSOC(average age 52.8±8.3 years),15 patients with ovarian borderline serous cystadenoma(average age 39.7±11.6 years),and 15 patients with benign serous cystadenoma of ovary(average age 48.4 ± 19.5 years).PD-L1 expression on tumor cells and CD 163(TAMs markers)expression in tumor stroma were analyzed by immunohistochemistry,the expression of PD-L1 on TAMs was analyzed by immunofluorescence double staining.The patients were divided into high expression group and low expression group according to staining intensity and positive rate.The correlation between the expression of PD-L1,CD163 with prognosis and clinicopathological factors(menopause,surgical stage and lymph node metastasis)was analyzed.2.Cellular experiments:A co-culture system of SKOV-3 and M2-macrophages was build.The positive rate of PD-L1 expression on M2-macrophages was detected by flow cytometry before the co-culture,1 day,2 days and 3 days after the co-culture.The SKOV-3 medium was used to culture M2-macrophages,changes in the positive rate of PD-L1 expression were detected by flow cytometry at 1 day,2 days and 3 days after culture,respectively.3.Statistical analysis:Survival analysis using Kaplan-Meier method,multivariate Cox proportional risk model was used to analysis the prognostic factors.χ2 test was used to analyze the correlation between the expression of PD-L1 and CD 163 with clinicopathological factors.The Wilcoxon rank sum test was used to compare the differences of the PD-L1 positive rate between groups.The significance level was set at 0.05.Results1.Expression and clinical significance of PD-L1 on tumor cells:The positive rates of PD-L1 expression on tumor cells in HGSOC group,borderline group and benign group were 88%,33%and 13%,respectively.PD-L 1 expression was significantly upregulated in tumor cells in HGSOC compared with borderline and benign serous cystadenomas(all P are<0.05).PD-Ll expression positive rates were 90%and 87%in premenopausal and postmenopausal patients,respectively.The positive rates were 71%,67%,92%,89%in patients of Ⅰ-Ⅳ period,respectively.The positive rates were 89%and 87%with or without lymph node metastasis,respectively.According to menopause,pathological stage and lymph node metastasis,the patients were divided into two groups,and there was no significant difference in the expression of PD-L1 in tumor cells between the two groups(all P are>0.05).Expression of PD-L1 on tumor cells was not related to total survival(OS)and disease-free survival(DFS)(all P are>0.05).2.Expression and clinical significance of CD163 in tumor stroma:The positive rates of CD163 expression in the stroma of HGSOC group,borderline group and benign group were 95%,53%and 13%,respectively.The expression of CD163 in the stroma of HGSOC tumors increased significantly compared with borderline and benign serous cystadenomas(all P are<0.05).CD163 expression positive rates were 100%and 92%in premenopausal and postmenopausal patients,respectively.The positive rates were 86%,83%,79%,100%in patients of Ⅰ-Ⅳ period,respectively.The positive rates were 98%and 92%with or without lymph node metastasis,respectively.CD163 expression was not statistically significant between groups with different menopausal conditions and pathological stages(all P are>0.05),but it was associated with lymph node metastasis(59.1%VS33.3%,χ2=5.51,P=0.019).CD163 high expression group had shorter PFS(P=0.004),and CD 163 expression was an independent risk factor for postoperative DFS(P=0.017,HR=0.493,95%CI:0.276-0.883).3.Expression and clinical significance of PD-L1 on TAMs:The expression of PD-L1 on TAMs was found in 92%of HGSOC patients’ tissues.PD-L1 expression on TAMs positive rates were 93%and 91%in premenopausal and postmenopausal patients,respectively.The positive rates were 100%,83%,92%,89%in patients of Ⅰ-Ⅳperiod,respectively.The positive rates were 91%and 92%with or without lymph node metastasis,respectively.The expression of PD-L1 on TAMs was associated with lymph node metastasis(63.6%VS35.9%,χ2=6.37,P=0.012),but it has no correlation with the condition of menopause,the stage of operation and pathology(all P are>0.05)PD-L1 high expression group on TAMs had shorter DFS than the low expression group(P=0.022),TAMs PD-L1 expression was an independent risk factor for postoperative DFS(P=0.035,HR=0.503,95%CI:0.266-0.952).4.Effects of tumor cells on PD-L1 expression on TAMs:In the co-culture system of SKOV-3 and M2-macrophages,the positive rates of PD-L1 expression on the surface of M2-macrophages were(2.2 ± 0.7)%before co-culture,(16.9 ± 2.0)%,(42.9±1.7)%,(58.2 ± 1.6)%on 1,2 and 3 days after co-culture,respectively.The difference of positive rate between two adjacent days was statistically significant(all P are<0.05).The positive rates of expression on the surface of M2-macrophages were(25.7±1.2)%,(62 ±1.8)%,(84 ±2.0)%,respectively,after 1 day,2 days and 3 days of culture in the SKOV-3 conditions medium.The difference of positive rate between two adjacent days was statistically significant(all P are<0.05).Conclutions1.The TAMs infiltration in HGSOC tumor stroma increased significantly,and the high infiltration of TAMs is an independent risk factor for postoperative DFS,TAMs may become a new marker to predict the prognosis of ovarian cancer.2.Ovarian cancer cells can up-regulate the expression of PD-L1 on M2-macrophages,the high expression of TAMs PD-L1 indicates a poor prognosis,suggesting that the expression of PD-L1 on TAMs is expected to be a new direction for ovarian cancer therapy.