Angiogenesis is a key process for tumor progression and a target for treatment. However, the regulation of breast cancer angiogenesis and its relevance for clinical resistance to antiangiogenic drugs is still incompletely understood. Recent developments on the contribution of microRNA to tumor angiogenesis and on the oncogenic effects of miR-17-92, a miRNA cluster, point to their potential role on breast cancer angiogenesis. The aim of this work was to establish the contribution of miR-20a, a member of miR-17-92 cluster, to tumor angiogenesis in patients with invasive breast carcinoma. Tube-formation in vitro assays with conditioned medium from MCF7 and MDA-MB-231 breast cancer cell lines were performed after transfection with miR-20a and anti-miR20a. For clinical validation of the experimental findings, we performed a retrospective analysis of a series of consecutive breast cancer patients (n = 108) treated with neoadjuvant chemotherapy and with a full characterization of their vessel pattern and expression of angiogenic markers in pre-treatment biopsies. Expression of members of the cluster miR-17-92 and of angiogenic markers was determined by RT-qPCR after RNA purification from FFPE samples. In vitro angiogenesis assays with endothelial cells and conditioned media from breast cancer cell lines showed that transfection with anti-miR20a in MDA-MB-231 significantly decreased mean mesh size and total mesh area, while transfection with miR-20a in MCF7 cells increased mean mesh size. MiR-20a angiogenic effects were abrogated by treatment with aflibercept, a VEGF trap. These results were supported by clinical data showing that mir-20a expression was higher in tumors with no estrogen receptor or with more extensive nodal involvement (cN2-3). A higher miR-20a expression was associated with higher mean vessel size (p = 0.015) and with an angiogenic pattern consisting in larger vessels, higher VEGFA expression and presence of glomeruloid microvascular proliferations (p<0.001). This association was independent of tumor subtype and VEGFA expression. Transfection of breast cancer cells with miR-20a induces vascular changes in endothelial tube-formation assays. Expression of miR-20a in breast invasive carcinomas is associated with a distinctive angiogenic pattern consisting in large vessels, anomalous glomeruloid microvascular proliferations and high VEGFA expression. Our results suggest a role for miR-20a in the regulation of breast cancer angiogenesis, and raise the possibility of its use as an angiogenic biomarker.

The prognostic impact of neutrophil-lymphocyte ratio (NLR) in metastatic breast cancer (MBC) has been previously evaluated in early and metastatic mixed breast cancer cohorts or without considering other relevant prognostic factors. Our aim was to determine whether NLR prognostic and predictive value in MBC was dependent on other clinical variables. We studied a consecutive retrospective cohort of patients with MBC from a single centre, with any type of first line systemic treatment. The association of NLR at diagnosis of metastasis with progression free survival (PFS) and overall survival (OS) was evaluated using Cox univariate and multivariate proportional hazard models. In the full cohort, that included 263 MBC patients, a higher than the median (>2.32) NLR was significantly associated with OS in the univariate analysis (HR 1.36, 95% CI 1.00–1.83), but the association was non-significant (HR 1.12, 95% CI 0.80–1.56) when other clinical covariates (performance status, stage at diagnosis, CNS involvement, visceral disease and visceral crisis) were included in the multivariate analysis. No significant association was observed for PFS. In conclusion, MBC patients with higher baseline NLR had worse overall survival, but the prognostic impact of NLR is likely derived from its association with other relevant clinical prognostic factors.

Purpose: The prognostic utility and biological correlates of neutrophil to lymphocyte ratio (NLR), a potential biomarker of the balance between immune response and the inflammatory status, are still uncertain in breast cancer (BC). Methods: We analysed a cohort of 959 women with early breast cancer, mostly treated with neoadjuvant or adjuvant chemotherapy. Clinical and pathological data, survival, NLR (continuous and categorical) and stromal tumor infiltrating lymphocytes (sTIL) were evaluated. Results: NLR was only weakly associated with Ki67, while no association was found for grade, histology, immunohistochemical subtype or stage. Lymphocyte infiltration of the tumor did not correlate with NLR (Rho: 0.05, p = 0.30). These results were similar in the whole group and across the different BC subtypes, with no differences in triple negative BC. Relapse free interval (RFI), breast cancer specific survival (BCSS) and overall survival (OS) changed according to pre-treatment NLR neither in the univariate nor in the multivariate Cox models (RFI: HR 0.948, p = 0.61; BCSS: HR 0.920, p = 0.57; OS: HR 0.96, p = 0.59). Conclusion: These results question the utility of NLR as a prognostic biomarker in early breast cancer and suggest the lack of correlation of NLR with tumor microenvironment immune response.