Expression Of Programmed Death Ligand 1(PD-L1) Is Associated With Poor Survival In Human Breast Cancer

BackgroundBreast cancer is the most common carcinoma that originated from mammary gland epithelial cells in women, and the second most common cause of cancer death among women worldwide.Now the incidence of breast cancer increased year by year. It is reported that the incidence and mortality of breast cancer has became the second one with a rising trend in the cities of China. In addition, breast cancer mortality increase obviously, and the trend of incidence appears to be younger and younger and with age increase. So it is very important to prevent breast cancer in early stage. Breast cancer can be separated into different types based on infiltration degree including carcinoma in situ and infiltrating ductal carcinoma. Currently, the pathogenesis mechanism of breast cancer has not yet been fully understood. Numerous risk factors have been reported to be associated with the development of breast cancer, such as genetic, hormonal, and environmental. With the development of modern molecular science, breast cancer has been defined as a multi-gene disease with the activation of proto-oncogene and inactivation of tumor suppressor genes. It is widely believed that the occurrence, progression of malignant tumor is associated with a series of complicated immune regulation processes. Multiple regulatory genes were involved in this process.The immune system is a complex collection of organs tissues and cells. It has been revealed that human tumors utilized many different mechanisms of immunosuppression to prevent immune cells from exercising their antitumor activities. The tumor-related suppression mechanisms include T cell dysfunction, inhibition ligands such as PD-L1, B7x and low expression of tumor antigens on the tumor cell surfaceand so on.These mechanisms, which enable the tumor to escape from the host immune system and to progress, are being intensively investigated in hope of finding therapeutically safe and effective inhibitors able to counteract tumor-induced immunosuppression.T cells play a critical role in the human immune system, recognizing foreign antigens and orchestrating an effective immune reaction. The interaction between T cells and antigen-presenting cells (APC) is complex and involves the T cell receptor and multiple co-stimulatory receptors, which exert both activating and inhibitory stimuli to the T cell. The interplay between activating and inhibitory signals is especially important in modulating the immune system’s ability to distinguish self from non-self and to prevent autoimmune reactions. The activation of T cell is associated with double signal in immune response.Programmed cell deathl (PD-1), a member of the CD28/CTLA-4 family of co-stimulatory receptors, conveys an inhibitory signal to the T cell and thus impedes immune responses. PD-1 contributes to the immune tolerance of self antigens by peripheral T cells. PD-1 is expressed on natural killer cells, dendriticcells (DCs), activated monocytes as well as on B cells, and T cells.PD-1 has two ligands, programmed death ligandl (PD-L1) and programmed death ligand2 (PD-L2). PD-L1 is expressed not only on APC, DCs, as well as on activated monocytes and B cells, but also on non-lymphoid tissues of different organs. Its normal physiologic role is to bind programmed deathl receptors (PD-1) expressed on the surface of activated cytotoxic T cells. This binding causes inhibition of IL-2, IFN-y production and T cell activation. The PD-1/PD-L1 interaction serves as an important regulatory check against an excessive adoptive immune response to antigens and autoimmunity.Recent evidence suggests that activation of the PD-1/PD-L1 pathway represents one mechanism allowing tumors to elude the host’s immune system. The exertion of this pathway by cancer cells might also explain why there is no tumor growth control despite the induction of cancer specific T cells. These observations provide strong rationale for targeting PD-1 or its ligand PD-L1 with antibodies capable of inhibiting this pathway. Preclinical murine studies with anti-PD-1 antibodies have shown demonstrating that PD-1 blockade in combination with antitumor immunotherapy can prolong survival of mice inoculated with live melanoma cells.The treatment mode of breast cancer has been changed from surgery alone in past to comprehensive treatments including surgery, chemotherapy, radiation therapy, hormonal therapy, biological therapy and so on. Although breast cancer is commonly thought to be less immunogenic than melanomas, there is an increasing evidence of a dynamic crosstalk between the immune system and breast cancer. Recent studies have revealed that the presence of regulatory T cells in peripheral blood as well as in the tumors of breast cancer patients. There is a potential association between activation of the PD-1/PD-L1 pathway and breast cancer.AimTo investigate the expression of PD-L1 in breast cancer, and to evaluate the association between PD-L1 expression and clinicopathologic features and prognosis in patients with breast cancer.Methods1. mRNA extraction, reverse transcription and qRT-PCR were carried out to detect the expression of PD-L1 in breast cancer cell lines.2. The expression of PD-L1 in breast cancer tissues was carried out by Western Blot analysis.3. To analysis the relationship between PD-L1 and clinical outcomes in patients with breast cancer, we detected the expression of PD-L1 in breast cancer tissue microarray by immunohistochemistry. Furthermore, we analyzed the correlation of clinical outcomes and the expression of PD-L1.4. Statistical analysis:Statistical analysis was performed using a SPSS 13.0 software package. Each experiment is repeated at least three times. The 2-△△Ct of qRT-PCR results for each breast cancer cell lines were compared by ANOVA (One-way ANOVA); Comparison between groups for Western-blot were performed by paired sample t test; The chi-square test was used to analyze the relationship between PD-L1 expression and clinicopathological characteristics. Survival times were evaluated with the Kaplan and Meier survival curves, and compared by the log-rank test. The significance of variables for survival was analyzed by multivariate survival analysis using COX’s regression mode. P<0.05 was considered as statistically significant.Results1. Expression of PD-L1 in breast cancer cell linesqRT- PCR data showed that PD-L1 was significantly high expressed in ZR-75-1 (2.7±0.46)、BT-20(3.8±0.35)、MDA-MB-231(5.23±0.35)and　BT-549(4.0±0.56) cell lines,but low expressed in MCF-7、SKBr-3、T47D and BT-474 cells.2. Expression of PD-L1 in breast cancer tissuesTo determine the expression of PD-L1 protein in breast cancer tissues, Western blotting was performed in 10 breast cancer tissues with paired non-cancerous tissues. Among 10 tissues with paired normal tissues, clearly increased levels of PD-L1 expression was detected in all tumors tissues in comparison to paird non-cancerous tissues with significance difference (P<0.05).3. Correlation of PD-L1 expression with Clinicopathological features and outcomesThe expression of PD-L1 in breast cancer tissue microarray was detected by immunohistochemistry. We found that the expression rate of PD-L1 in breast cancer is 22.7%. PD-L1 expression was significantly correlated with tumor size, lymph node status, ER expression and PR expression (P<0.05). In univariate survival analyses, breast cancer cases expressing PD-L1 had a significantly worse OS(HR=0.291,P=0.00). COX regression analysis showed that, PD-L1 was an independent risk factor which affected the prognosis of breast cancer patients.Conclusion1. PD-L1 expression level was increased in the tumor tissues in comparison to the non-cancerous tissues.2. The positive expression rate of PD-L1 is 22.7%, and PD-L1 expression was significantly associated with tumor size, lymph node status, and absence of ER、PR expression.3. In univariate analysis, PD-L1 expression was associated with a significantly worse OS.4. In multivariate analysis, the expression of PD-L1 in breast cancer is an independent negative prognostic factor.