The Research On Immune Microenvironment Of Breast Cancer And Its Association Analysis With Prognosis

Part 1Objective Tumor immune microenvironment plays an important role in the development of breast cancer.The aim of this study is based on the analysis of the immunology indexexpression differences in different types of breast cancer,to explore the immune the relationship between microenvironment and breast cancer to provide data for further prognosis and immunotherapy.Methods According to the inclusion and exclusion criteria,the clinical pathology characteristics,follow-up data and paraffin tissue samples of the 256 operable breast cancer patients was from the department of general surgery in Changzheng Hospital between January,2008 and December,2008.We detect 6 kinds of immunological makers with Tissue microarray technique and immunohistochemical method and record the positive cell percentage of PD-L1(programmed death ligand-1)and TGF-β1(transforming growth factor-beta 1)in both tumor cells and tumor stroma cells,the positive cell percentage of CTLA 4(Cytotoxic T cell antigen-4),IFN-γ(interferon-gamma)and PD1(programmed death-1)in lymphocytes,and the density of FOXP3 + lymphocytes infiltrated in tumor stroma.The expression of PDL1 and PD1 will be divided into positive expression and negative expression with 5% as cut-off.The other markers can be divided into high expression(or dense infiltration)and low(or low density infiltrates)with the cut-off calculated by the minimum p value method;Then we analysis the expression differences of each marker between breast cancer with different age,tumor size,axillary lymph node status,Ki-67,ER,PR,Her-2,molecular subtyping and the pathological staging.Results1,205 cases of PD-L1 were available,200 cases of PD-1,IFN-gamma and TGF-beta was available and 199 cases of CTLA – 4 and 194 cases of FOXP3 + lymphocytes were available.2,The positive cell percentage of PD-1,PD-L1 in tumor cells and tumor stroma cells was 52%,81.46% and 49.76% respectively;The high expression rate of CTLA-4,IFN –gamma in lymphocytes,and TGF-β1in both tumor cells and tumor stroma cells was 56.06%,63.5%,56.06% and 63.5% respectively.The average density of FOXP3+lymphocyte infiltrated in intraepithelial and itrastroma was(2.73 + 3.77)/ 0.0625mm2 and(15.14 + 15.08)/0.0625mm2 respectively.3,In tumor cells,the positive expression rate of PD-L1 in ER negative was 27.14%,significantly higher than that of ER positive breast cancer(P = 0.0261);The positive expression rate of PD-L1 was 26.39% in HER-2 positive breast cancer,significantly higher than HER-2 negative ones(P = 0.0483).In tumor stroma cells,the positive expression rate of PD-L1 in the patients with ER negative rate was 68.57%,significantly higher than that in ER positive ones(P = 0.0002);In the PR negative breast cancer,positive expression rate of PD-L1 was 62.20%,significantly higher than PR positive ones(P = 0.0065);In HER-2positive breast cancer,positive expression rate of PD-L1 was 63.89%,significantly higher than HER-2 negative ones(P = 0.0067).4,In tumor cells,the high expression rate of TGF-β1 in ER negative breast cancer was 97.10%,significantly higher than that in ER positive ones(P = 0.0109);Compared to the PR positive breast cancer,the high expression rate of TGFβ-1 in negative PR ones(96.34%)was higher(P = 0.0059).In Lymphocytes,the expression of TGFβ-1 had no statistical differences between different groups in all clinical pathologic factors(P >0.05).5,The expression of PD-1 in breast cancer had no statistical differences between different groups in all clinical pathologic factors(P > 0.05).6,In tumor epithelial,compared patients with age<50 years old,the proportion of high-density infiltration of FOXP3+ lymphocytes in patients with age ≥ 50 years old was significantly higher(23.75%)(P = 0.0392);In patients with Ki-67 ≥1 4%,the high-density infiltration of FOXP3+ lymphocytes in patients with a proportion of 20% was higher than the Ki-67 < 14% ones(P = 0.0460);In ER negative patients,proportion of high-density infiltration of FOXP3 + lymphocytes was 25.76%,significantly higher than that with ER positive ones(P = 0.0237);In patients with Ⅲ B ~ Ⅲ C,the percentage of high-density infiltrating FOXP3 + lymphocyte was 32.00%,significantly higher than patients with stageⅠ~ ⅢA(P = 0.0379).7,In lymphocyte,high expression rate of CTLA-4 in tumor stroma in ER negative breast cancer was 66.67%,higher than that in ER positive ones(P = 0.0256);In PR negative breast cancer,high expression rate was 64.20%,higher than in PR positive ones(P= 0.0256).8,In lymphocyte,high expression of IFN-γ was 65.59% in IDC,while was 35.71% in other histological types,the difference was statistically significant(P = 0.0332).In ER negative breast cancer,high expression rate of IFN-γ was 73.53%,while was 58.78% in ER positive ones,the difference was statistically significant(P = 58.78).Conclusions1,The expression of PD-L1 in tumor cells was associated with ER negative,HER-2positive;while the expression of PD-L1 in tumor stroma cells was associated with ER negative,PR negative,HER-2 positive;2,The expression of TGFβ-1 in tumor cells was associated with ER negative,PR negative;3,The expression of PD-1 in breast cancer had no correlation with all all clinical pathologic factors;4,High-density infiltration of FOXP3 + lymphocyte in tumor epithelial was associated with older age,Ki-67≥14%,ER negative,and stage ⅢB ~ ⅢC.And high-density infiltration of FOXP3 + lymphocyte in tumor stromal was associated with IDC,ER negative,PR negative,HER-2 positive;5,The high-expression of CTLA-4 in lymphocytes in breast cance was associated with ER negative,PR negative;6,High expression of IFN-γ in lymphocytes in breast cancer microenvironment was associated with ER negative and IDC.Part 2Objective The prognosis of breast cancer is closely related to tumor immunemicro-enviro nment.This part of the study was to screen immune markers related with prognosis in the immune microenvironment of breast cancer and and further analysis in which type,they have prognostic value in breast cancer.MethodsExperimental methods are the same as the first part,COX univariable model analysis was used to estimate the relationship between immunological markers and DFS;Cox multivariable proportional hazard regression models were used to screen the independent prognostic indicators;Stratified analysis is based on the clinical pathologic factors,the log-rank test was used to assess differences between groups within each layer.This part also analyzes the the correlation between the expression of PD-1and the expression of IFN-γ.Results1,In COX univariable model analysis,the positive expression of PD-1 in lympho--cytes was related with longer DFS survival time(RR = 0.53,95% CI: 0.29 ~ 0.99,P =0.0453).High density of FOXP3+lymphocytes in breast tumor stroma was related with longer DFS survival time longer survival time(RR = 2.35,95% CI: 1.00 ~ 5.53,P =0.0500).2,In Cox multivariable proportional hazard regression models,positive expression of PD-1in lymphocyte was significantly positively related to DFS(OR = 0.47,95% CI: 0.25~ 0.89);The density of FOXP3+lymphocytes infiltrated in the tumor stroma was an inde--pendent risk factor of DFS(OR = 2.76,95% CI: 2.76,7.09).3,In stratified analysis,compared with patients with positive expression of PD-1 in lymphocytes,patients with positive expression of PD-1in lymphocytes was negative expression had longer DFS in patients with age ≥50(P = 0.014),in patients with IDC(P =0.045),in PR positive patients(P = 0.009),in HER-2 positive patients,in non-triple-negative breast cancer(0.0327),and inⅠ~ⅢA patients(P = 0.0238).Compared with patients with low-density infiltration of FOXP3 + lymphocytes in tumor stroma,the high-density infiltration ones had longer DFS in patients with age < 50(P = 0.009),in patients with HER-2(P = 0.009),and in non-triple-negative breast cancer(P = 0.0412).4,Further study found that high expression of IFN-γ was positively associated with PD-1 positive rate(OR = 2.67,95% CI: 1.45 ~ 4.92)(P = 0.0017).Conclusions1,The expression of PD-1 in breast cancer was an independent prognostic factor for DFS;2,The density of FOXP3 + lymphocytes infiltrated in tumor stroma was an independent risk factors for DFS;3,In patients with age ≥ 50 years old,with IDC,with PR positive,with HER-2positive,with non-triple-negtive,and with stageⅠ ~ Ⅲ A,PD-1 positive expression was positively correlated with DFS survival time;4,In patients with age ≥ 50 years old,with HER-2 negative,with non-triple-negtive,the density of FOXP3 + lymphocytes infiltrated in tumor stroma was negatively correlated with DFS survival time,which indicates that FOXP3 + lymphocytes infiltrated in tumor stroma might be an important reason for the poor prognosis of breast cancer;5,Further study found that positive expression of PD-1 infiltrated in lymphocytes had positively correlation with high expression of IFN-γ.