Immune Checkpoint and EMT-Related Molecules in Circulating Tumor Cells (CTCs) from Triple Negative Breast Cancer Patients and Their Clinical Impact

SIMPLE SUMMARY: A better understanding of the molecular mechanisms that govern metastasis and the identification of early therapeutic approaches to prevent the dissemination of tumor cells in triple negative breast cancer (TNBC) patients is highly important. The present study focuses on investigating the expression of immune checkpoint molecules (PD-L1, CTLA-4) and epithelial to mesenchymal transition (EMT)-related proteins (detyrosinated α-tubulin (GLU) and vimentin (VIM)) in TNBC patients’ CTCs and assess their relations to disease severity and clinical outcome. All the examined biomarkers were found to be expressed in CTCs, whereas PD-L1, GLU, and VIM were related to worse overall survival (OS) in TNBC patients. Our data demonstrate the importance of these four biomarkers for TNBC patients and provide an interesting tool for stratifying patients that could benefit from a potential combination of novel therapies. ABSTRACT: Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype. There are few targeted therapies for these patients, leading to an unmet need for new biomarkers. The present study aimed to investigate the expression of PD-L1, CTLA-4, GLU, and VIM in CTCs of TNBC patients. Ninety-five patients were enrolled in this study: sixty-four TNBC and thirty-one luminal. Of these patients, 60 were in the early stage, while 35 had metastatic disease. Protein expression was identified by immunofluorescence staining experiments and VyCAP analysis. All the examined proteins were upregulated in TNBC patients. The expression of the GLU(+)VIM(+)CK(+) phenotype was higher (50%) in metastatic TNBC compared to early TNBC patients (17%) (p = 0.005). Among all the BC patients, a significant correlation was found between PD-L1(+)CD45(−)CK(+) and CTLA-4(+)CD45(−)CK(+) phenotypes (Spearman test, p = 0.024), implying an important role of dual inhibition in BC. Finally, the phenotypes GLU(+)VIM(+)CK(+) and PD-L1(+)CD45(−)CK(+) were associated with shorter OS in TNBC patients (OS: log-rank p = 0.048, HR = 2.9, OS: log-rank p < 0.001, HR = 8.7, respectively). Thus, PD-L1, CTLA-4, GLU, and VIM constitute significant biomarkers in TNBC associated with patients’ outcome, providing new therapeutic targets for this difficult breast cancer subtype.