Análise da expressão da proteína de checkpoint imunológico CTLA-4 em biópsias de pacientes com câncer de mama e seu impacto na produção sistêmica de mediadores da resposta imune

Abstract

Purpose: Breast cancer is the leading cause of women’s death among all cancers. It is a heterogeneous disease consisting of subgroups with different molecular features and clinical outcomes. Cancer cells can evade antitumor T- cell responses by expressing some immune inhibitory molecules as the programmed cell death protein 1 (PD-1) and/or the cytotoxic T-lymphocyte associated protein 4 (CTLA-4), known as immune checkpoints, which have the function of reducing or limiting the cellular immune responses in a physiological scenario. Recent evidences have pointed out that the expression of the CTLA-4 in tumors is a poor prognosis factor, but the systemic impact of its expression in tumor or infiltrating leukocytes is not clear for breast cancer. In this context, it is important to investigate CTLA-4 expression in BC microenvironment and its potential role in systemic inflammation modulation. Based on this, we analyzed CTLA-4 expression by immunofluorescence, and according to its expression in tumor or TILs, evaluated the circulating levels of interleukins and oxidative stress mediators known as players of the inflammatory response. This study was approved by the Ethics Committee on Research of State University of Western Paraná under the number CAAE 35524814.4.0000.0107. Methods: Paraffin- embedded breast tumors and whole blood samples were collected from 117 women diagnosed with breast cancer. Clinicopathological data were obtained through medical records. In blood samples, oxidative stress parameters were evaluated by measuring its plasmatic lipoperoxidation by high-sensitivity chemiluminescence and estimating nitric oxide metabolites (NO) by the cadmium-copper system coupled to Griess reaction. Interleukins 12 (IL-12) and 4 (IL-4) were measured in plasma samples by ELISA kits. CTLA-4 expression was determined by immunofluorescence and evaluated by its labeling in tumor- infiltrating leukocytes (TILs) or breast tumors. Statistical analyses were performed using the GraphPad Prism 7.0 software package (GraphPad Software, San Diego, CA, USA). Also, SPSS 22.0 software (IBM, USA) was used to obtain the clinicopathological data frequencies and Spearman’s correlations. Results: CTLA-4 expression in TILs significantly correlated to triple negative breast tumors, Patients carrying CTLA-4 positive tumors exhibited lower plasmatic NO levels, while those with CTLA-4 expression only in TILs exhibited reduced levels of IL-12 in plasma. No variations were observed in IL-4 or lipid peroxidation profiles in any CTLA4 status. Conclusion: CTLA-4 expression in BC is a putative marker of clinical significance as well as a rationale therapeutic target in the emerging field of immunotherapy. Moreover, our findings suggest that CTLA-4 expression in both tumor and TILs can affect the systemic inflammatory status of breast cancer patients, affecting directly the levels of antitumor molecules as IL12 and NO, and correlating to most aggressive disease.