BackgroundBreast cancer patients under neoadjuvant chemotherapy includes a heterogeneous group of patients who eventually develop distal disease, not detectable by current methods. We propose the use of exosomal miRNAs and circulating tumor cells as diagnostic and predictive biomarkers in these patients.MethodsFifty-three breast cancer women initially diagnosed with localized breast cancer under neoadjuvant chemotherapy were prospectively enrolled in this study. However, six of them were later re-evaluated and diagnosed as metastatic breast cancer patients by PET-CT scan. Additionally, eight healthy donors were included. Circulating tumor cells and serum exosomal miRNAs were isolated from blood samples before and at the middle of neoadjuvant therapy and exosomal miRNA levels analyzed by qPCR.ResultsBefore neoadjuvant therapy, exosomal miRNA-21 and 105 expression levels were higher in metastatic versus non-metastatic patients and healthy donors. Likewise, higher levels of miRNA-222 were observed in basal-like (p = 0.037) and in luminal B versus luminal A (p = 0.0145) tumor subtypes. Exosomal miRNA-222 levels correlated with clinical and pathological variables such as progesterone receptor status (p = 0.017) and Ki67 (p = 0.05). During neoadjuvant treatment, exosomal miRNA-21 expression levels directly correlated with tumor size (p = 0.039) and inversely with Ki67 expression (p = 0.031). Finally, higher levels of exosomal miRNA-21, miRNA-222, and miRNA-155 were significantly associated with the presence of circulating tumor cells.ConclusionLiquid biopsies based on exosomal miRNAs and circulating tumor cells can be a complementary clinical tool for improving breast cancer diagnosis and prognosis.Electronic supplementary materialThe online version of this article (10.1186/s13058-019-1109-0) contains supplementary material, which is available to authorized users.

IntroductionIncreasing evidence supports the view that the detection of circulating tumor cells (CTCs) predicts outcomes of nonmetastatic breast cancer patients. CTCs differ genetically from the primary tumor and may contribute to variations in prognosis and response to therapy. As we start to understand more about the biology of CTCs, we can begin to address how best to treat this form of disease.MethodsNinety-eight nonmetastatic breast cancer patients were included in this study. CTCs were isolated by immunomagnetic techniques using magnetic beads labelled with a multi-CK-specific antibody (CK3-11D5) and CTC detection through immunocytochemical methods. Estrogen receptor, progesterone receptor and epidermal growth factor receptor (EGFR) were evaluated by immunofluorescence experiments and HER2 and TOP2A by fluorescence in situ hybridization. We aimed to characterize this set of biomarkers in CTCs and correlate it with clinical-pathological characteristics.ResultsBaseline detection rate was 46.9% ≥ 1 CTC/30 ml threshold. CTC-positive cells were more frequent in HER2-negative tumors (p = 0.046). In patients younger than 50 years old, HER2-amplified and G1-G2 tumors had a higher possibility of being nondetectable CTCs. Heterogeneous expression of hormonal receptors (HRs) in samples from the same patients was found. Discordances between HR expression, HER2 and TOP2A status in CTCs and their primary tumor were found in the sequential blood samples. Less that 35% of patients switched their CTC status after receiving chemotherapy. EGFR-positive CTCs were associated with Luminal tumors (p = 0.03).ConclusionsThis is the largest exploratory CTC biomarker analysis in nonmetastatic BC patients. Our study suggests that CTC biomarkers profiles might be useful as a surrogate marker for therapeutic selection and monitoring since heterogeneity of the biomarker distribution in CTCs and the lack of correlation with the primary tumor biomarker status were found. Further exploration of the association between EGFR-positive CTCs and Luminal tumors is warranted.

CD133 has been associated with cell properties such as self renewal, migration and vasculogenic mimicry, potentially involved in generation of circulating tumor cells (CTCs). We characterized CD133 expression in CTCs of 98 nometastatic breast cancer (BC) patients. CTCs were isolated by immunomagnetic techniques using magnetic beads labeled with a multicytokeratin(CK)-specific antibody (CK3-11D5) and CTCs and CD133 detection through immunocytochemical methods. CK 1 /CD133 1 CTCs were identified in 65% of patients at baseline and 47.8% after systemic therapy (p 5 0.53). Correlation of CD133 status in CTCs with classical clinicopathological characteristics and response to therapy was performed. Her2 not amplified and low Ki-67 index were positively correlated with presence of CK 1 /CD133 1 CTCs. Before any treatment, CK 1 /CD133 1 CTCs were more frequently isolated in patients with luminal BC subtype. No statistically significant differences were found between proportion of CK 1 /CD133 1 CTCs and BC subtypes after systemic therapy, implying a relative enrichment of CK 1 /CD133 1 CTCs in triple negative and HER2-amplified tumors. While CK 1 /CTCs decreases after chemotherapy when analyzing the whole population, CK 1 / CD133 1 CTCs were enriched in post-treatment samples in nonluminal BC subtypes. These findings suggest the potential role of CD133 as a promising marker of chemoresistance in nonluminal BC patients. Further prospective studies and extensive preclinical modeling will be needed to confirm whether CD133 is a marker of resistance to chemotherapy, and its role as a target for novel anticancer therapies targeting cancer stem cells and tumor vasculature.Breast cancer (BC) is a complex and heterogeneous disease comprising multiple tumor entities associated with distinctive different biological feature, clinical behaviors and response to therapy. The current classification of BC is based on the pattern of expression of the hormone receptors (HR) estrogen receptor and/or progesterone receptor, and human epidermal receptor 2 (Her2) that identify three major BC subtypes: luminal tumors, which are HR positive and Her2 negative; Her2 amplified tumors; and those tumors with lack of expression of the three receptors, referred to as triple negative (TN) BC. 1 Despite advances in BC treatments, primary and acquired resistance to cancer therapies remains a challenge especially in nonmetastatic patients. Cancer stem cells (CSC) have been

Circulating tumor cells (CTCs) are frequently associated with epithelialmesenchymal transition (EMT). The objective of this study was to detect EMT phenotype through Vimentin (VIM) and Slug expression in cytokeratin (CK)-negative CTCs in non-metastatic breast cancer patients and to determine the importance of EGFR in the EMT phenomenon. In CK-negative CTCs samples, both VIM and Slug markers were co-expressed in the most of patients. Among patients EGFR+, half of them were positive for these EMT markers. Furthermore, after a systemic treatment 68% of patients switched from CK- to CK+ CTCs. In our experimental model we found that activation of EGFR signaling by its ligand on MCF-7 cells is sufficient to increase EMT phenotypes, to inhibit apoptotic events and to induce the loss of CK expression. The simultaneous detection of both EGFR and EMT markers in CTCs may improve prognostic or predictive information in patients with operable breast cancer.

Highlights d Characterization of 33 compounds has identified specific inhibitors of LINE-1s d GBS-149, FTC, and 3TC are non-toxic and mammalianspecific LINE-1 inhibitors d GBS-149 is a new and potent RTi of active human LINE-1s