The Study Of Carcinoma-associated-fibroblasts And Tumor-infiltration Lymphocytes In Tumor Microenvironment Of Breast Cancer

Part1ObjectiveInvasive micropapillary carcinoma (IMPC) is a rare histological special type of breast cancer. Due to its high tendency for lymphovascular invasion and axillary lymph node metastasis, clinicians and pathologists pay more attention to IMPC. Recently, Caveolin-1(Cav-1) and Monocarboxylate transporter4(MCT4) expression by carcinoma-associated fibroblasts (CAFs) in tumor stroma are implicated in tumor progression and clinical outcomes. Our previous study found that myofibroblasts and newly formed capillaries were rich in the stroma of IMPC.The current study was undertaken to evaluate the expression of Cav-1and MCT4in stromal CAFs of IMPC.MethodsCav-1and MCT4expression was studied by immunohistochemistry in a group of86cases of IMPC along with a control group of105cases of invasive ductal carcinoma, not otherwise specified (IDC-NOS). The relationship between stromal Cav-1, MCT4and various clinicopathologic parameter of IMPC were analyed. And we also analyzed the univariate Kaplan-Meier and Cox-regression survival.Results1. Compared to the group of IDC-NOS, IMPC had extremely higher lymph node stage (P<0.001).2. Cav-1expression in stromal CAFs was significantly higher than that in tumor cells both in IMPC group and IDC-NOS group.43%of IMPC demonstrated positive expression of stromal Cav-1, while63.8%in IDC-NOS (P=0.004), indicating a higher negative rate in IMPC.3. MCT4was also expressed in cancer cells and stromal cells (CAFs). S(++) MCT4expression in stromal CAFs was observed in68.6%cases of IMPC, while48.6%in IDC-NOS (P=0.005), indicating a significant up-regulation in IMPC. The difference of tumor cell MCT4expression between IMPC and IDC-NOS was not proven to be statistically significance. Meanwhile, no significant correlation was observed between stromal MCT4expression and tumor MCT4expression both in the groups of IMPC and IDC-NOS.4. The expression of stromal Cav-1was negatively correlated with stromal MCT4.5. The expression of stromal Cav-1was negatively correlated with tumor size and LN metastasis (P<0.05), and the up-regulation of stromal MCT4was correlated with higher lymph node stage of IMPC.6. Univariate Cox proportional hazards models analyses indicated that absence of stromal Cav-1expression were significantly associated with worsen progression-free survival (PFS) of patients with IMPC (P=0.001); S(++) MCT4expression were significant associated with shortened PFS of patients (P=0.001). In contrast, Cav-1and MCT4expression in cancer cells failed to prove its prognostic significance. Multivariate analysis with respect to PFS indicated that stromal Cav-1and MCT4expression were independent prognostic factors for PFS of IMPC. In patients with IMPC spreading to local lymph nodes, loss of stromal Cav-1was confirmed to be associated with increased hazard ratio (HR) of4.365(95%CI,1.787-10.662) for RFS.Conclusions1. Cav-1and MCT4expression in tumor stroma CAFs are associated with clinical outcome of IMPC, they could be severed as prognostic factor for IMPC patients.2. Absence of Cav-1expression of CAFs and up-regulation suggests CAFs may play a role in tumor initiation and progression and might be a promoter of the mechanism of lymph node metastasis and poor outcome of IMPC.3. The research on protein of CAFs suggest that CAFs a not only for cancer diagnosis but also for the development of novel therapeutic agents. Part2ObjectiveTumor immune microenvironment plays an important role in the evolution of breast cancer. The purpose of this study is to analysis differences of infiltrating immune cells in the groups of different clinical outome in breast cancer, explore the relationship between the immunosuppressive effects of tumor associated B7-H1and induced Treg cells, and to explore the prognostic significance of the two cytokines for breast cancer patients. Screen out cell immune cells and immune factors, and provide new theoretical basis for the personalized immunotherapy of breast cancer.Methods40cases of different prognosis were detected by immunohistochemistry for the expression infiltrating immune cells in different locations, including tumor intraepithelial, tumor stroma, and tumor periphery.501cases of primary invasive breast cancer tissue with not receive any preoperative anti-tumor therapy were selected. We observed the expressions of B7-H1and Foxp3+Treg, analyzed the associations with clinicopathological features, breast cancer patient survival and molecular subtypes, and further explore the relationship between the expressions of B7-H1and Foxp3+Treg.Results1. We detected the expression of infiltrating immune cells in different locations in breast cancer patients with differences prognostic. And found that the density of infiltrate GrB+lymphocytes in tumor intraepithelial, T-bet+lymphocyte in tumor intraepithelial and tumor stroma, and CD68+macrophages in tumor periphery were higher than the patients with poor prognostic (P<0.01), while PD-1+lymphocyte in periphery were lower (P=0.044).2. The expression of B7-H1was found to be associated with the tumor histologic features known to be related with unfavorable prognosis of patients, including higher histological grade (rs=0.114, P=0.012), higher lymph stage node(rs=0.102, P=0.027), negative ER (rs=-0.110, P=0.014), negative PR (rs=-0.141, P=0.002) and negatively related to OS and PFS as well (P<0.001).3. In multivariate analyses, the expression of B7-H1was an independent prognostic factors of OS and RFS (P<0.05).4. The expression of B7-H1was found to be associated with the molecular subtypes of breast cancer (P<0.001). In the Basal-like subtypes breast cancer, the expression of B7-H1was up-regulated, and its expression in this subtype was.still has a significant value of prognostic (PFS, P=0.030; OS, P=0.020).5. It was identified that expression of B7-H1was positively correlated with the intratumoral Foxp3+Treg infiltration(rs=0.287, P<0.001). The prognostic value of combined B7-H1expression/Foxp3+Treg was also clarified. Our results indicated that B7-H1hign/Foxp3+Treg high were the most strongly influenced clinical outcomes, and has a poor clinical outcome (PFS, P<0.001; OS, P<0.001). In addtion, its association with PD-1+T lymphocyte was not proven to be significant (rs=0.092, P=0.465).ConclusionsThe expression of B7-H1is an independent prognostic predictor of breast cancer patients, and it therefore may have the potential to serve as a target for immunotherapeutic strategy for breast cancer patients, and it may be provide a potential immune target for Basal-like BC patients whom were not suit for endocrine and HER-2targeted therapy. B7-H1probilily promote the differentiation of T cells towards Treg cells. Treg cells negatively regulate all of adaptive immune, so they contribute more to tumor escape. In addtion, screen out cell immune cells in different locations may provide new theoretical basis for the personalized immunotherapy of breast cancer.