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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. [ABSTRACT FROM AUTHOR]

Background: Up to 30% of breast cancer (BC) patients treated with neoadjuvant chemotherapy (NCT) will relapse. Our objective was to analyze the predictive capacity of several markers associated with immune response and cell proliferation combined with clinical parameters. Methods: This was a single-center, retrospective cohort study of BC patients treated with NCT (2001-2010), in whom pretreatment biomarkers were analyzed: neutrophil-to-lymphocyte ratio (NLR) in peripheral blood, CD3+ tumor-infiltrating lymphocytes (TILs), and gene expression of AURKA, MYBL2 and MKI67 using qRT-PCR. Results: A total of 121 patients were included. Median followup was 12 years. In a univariate analysis, NLR, TILs, AURKA, and MYBL2 showed prognostic value for overall survival. In multivariate analyses, including hormone receptor, HER2 status, and response to NCT, NLR (HR 1.23, 95% CI 1.01-1.75), TILs (HR 0.84, 95% CI 0.73-0.93), AURKA (HR 1.05, 95% CI 1.00-1.11) and MYBL2 (HR 1.19, 95% CI 1.05-1.35) remained as independent predictor variables. Conclusion: Consecutive addition of these biomarkers to a regression model progressively increased its discriminatory capacity for survival. Should independent cohort studies validate these findings, management of early BC patients may well be changed. [ABSTRACT FROM AUTHOR]

Simple Summary: Stromal tumor infiltrating lymphocytes (sTIL) are a validated predictive and prognostic biomarker in non-luminal breast cancer. Our aim was to evaluate their clinical relevance in luminal (hormone receptor positive, HER2 negative) early breast cancer. Our results show that, although sTIL are associated with a better response to neoadjuvant chemotherapy, they are also associated with worse biological features (proliferation, higher stage) and poorer prognosis in luminal B breast cancer. TIL might improve prognostic stratification and contribute to therapeutic decision-making in the early high-risk setting of luminal B breast cancer. Luminal breast cancer (BC) is associated with less immune activation, and the significance of stromal lymphocytic infiltration (sTIL) is more uncertain than in other BC subtypes. The aim of this study was to investigate the predictive and prognostic value of sTIL in early luminal BC. The study was performed with an observational design in a prospective cohort of 345 patients with predominantly high-risk luminal (hormone receptor positive, HER2 negative) BC and with luminal B features (n = 286), in which the presence of sTIL was analyzed with validated methods. Median sTIL infiltration was 5% (Q1–Q3 range (IQR), 0–10). We found that sTIL were associated with characteristics of higher biological and clinical aggressiveness (tumor and lymph node proliferation and stage, among others) and that the percentage of sTIL was predictive of pathologic complete response in patients treated with neoadjuvant chemotherapy (OR: 1.05, 95%CI 1.02–1.09, p < 0.001). The inclusion of sTIL (any level of lymphocytic infiltration: sTIL > 0%) in Cox regression multivariable prognostic models was associated with a shorter relapse-free interval (HR: 4.85, 95%CI 1.33–17.65, p = 0.016) and significantly improved its performance. The prognostic impact of sTIL was independent of other clinical and pathological variables and was mainly driven by its relevance in luminal B BC. [ABSTRACT FROM AUTHOR]

Simple Summary: The miR200 family is involved in breast cancer progression. Our aim was to evaluate the predictive and prognostic role of miR-200c-3p, both in plasma and in tumor, in women with locally advanced and metastatic breast cancer. Our results show that plasma levels of miR-200c-3p are higher in these patients and might be used as a breast cancer marker. In patients treated with neoadjuvant chemotherapy, miR-200c-3p expression may also improve prognostic stratification beyond pathologic response and clinical stage. While the role of miR-200c in cancer progression has been established, its expression and prognostic role in breast cancer is not completely understood. The predictive role of miR-200c in response to chemotherapy has also been suggested by some studies, but only limited clinical evidence is available. The purpose of this study was to investigate miR-200c-3p in the plasma and primary tumor of BC patients. The study design included two cohorts involving women with locally advanced (LABC) and metastatic breast cancer. Tumor and plasma samples were obtained before and after treatment. We found that miR-200c-3p was significantly higher in the plasma of BC patients compared with the controls. No correlation of age with plasma miR-200c-3p was found for controls or for BC patients. MiR-200c-3p tumor expression was also associated with poor overall survival in LABC patients treated with neoadjuvant chemotherapy, independently of pathological complete response or clinical stage. Our findings suggest that plasmatic miR-200c-3p levels could be useful for BC staging, while the tumor expression of miR-200c-3p might provide further prognostic information beyond residual disease in BC treated with neoadjuvant chemotherapy. [ABSTRACT FROM AUTHOR]

Background: A prognostic model based on the results of molecular analysis of sentinel lymph nodes (SLN) is needed to replace the information that staging the entire axilla provided. The aim of the study is to conduct an external validation of a previously developed model for the prediction of 5-year DFS in a group of breast cancer patients that had undergone SLN biopsy assessed by the One Step Nucleic Acid Amplification (OSNA) method. Methods: We collected retrospective data of 889 patients with breast cancer, who had not received systemic treatment before surgery, and who underwent SLN biopsy and evaluation of all SLN by OSNA. The discrimination ability of the model was assessed by the area under the ROC curve (AUC ROC), and its calibration by comparing 5-years DFS Kaplan–Meier estimates in quartile groups of model predicted probabilities (MPP). Results: The AUC ROC ranged from 0.78 (at 2 years) to 0.73 (at 5 years) in the training set, and from 0.78 to 0.71, respectively, in the validation set. The MPP allowed to distinguish four groups of patients with heterogeneous DFS (log-rank test p < 0.0001). In the highest risk group, the HR were 6.04 [95% CI 2.70, 13.48] in the training set and 4.79 [2.310, 9.93] in the validation set. Conclusions: The model for the prediction of 5-year DFS was successfully validated using the most stringent form of validation, in centers different from those involved in the development of the model. The external validation of the model confirms its utility for the prediction of 5-year DFS and the usefulness of the TTL value as a prognostic variable. [ABSTRACT FROM AUTHOR]

Purpose: The analysis of breast cancer residual tumors after neoadjuvant chemotherapy (nCT) may be useful for identifying new biomarkers. MicroRNAs are known to be involved in oncogenic pathways and treatment resistance of breast cancer. Our aim was to determine the role of miR-18a, a member of the miR-17-92a cluster, in breast cancer behavior and outcome after nCT. Methods: Pre- and post-nCT tumor miR-18a expression was retrospectively assessed by qRT-PCR in 121 patients treated with nCT and was correlated with survival outcomes and with clinical and pathological characteristics. Breast cancer-derived MCF-7 and MDA-MB-231 cell lines were transfected with miR-18a and anti-miR-18a to evaluate the biological effects of this molecule. In addition, whole-transcriptome expression analysis was performed. Results: High miR-18a expression in post-nCT residual tumors was found to be associated with a significantly worse overall survival [hazard ratio (HR): 2.80, 95% confidence interval (CI): 1.01–7.76] and a strong trend towards a poorer disease-free survival (HR: 2.44, 95% CI: 0.99–5.02) compared to low miR-18a expressing post-nCT residual tumors. Clinical and experimental data were found to be in conformity with the proliferative effects of miR-18a, which showed a significant correlation with Ki67 and MYBL2 expression, both in pre- and post-nCT tumors and in public databases. In vitro analysis of the role of miR-18a in breast cancer-derived cell lines showed that a high expression of miR-18a was associated with a low expression of the estrogen receptor (ER), a decreased sensitivity to tamoxifen and an enrichment in luminal B and endocrine resistance gene expression signatures. Conclusions: From our data we conclude that post-nCT miR-18a expression in breast cancer serves as a negative prognostic marker, especially in luminal tumors. Clinical, in vitro and in silico data support the role of miR-18a in breast cancer cell proliferation and endocrine resistance and suggest its potential utility as a biomarker for additional adjuvant treatment in patients without a pathologic complete response to neoadjuvant therapy. [ABSTRACT FROM AUTHOR]

Background: 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. Methods: 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. Results: 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. Conclusions: 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. [ABSTRACT FROM AUTHOR]

The effects of breast cancer conventional chemotherapy on tumor angiogenesis need to be further characterized. Neoadjuvant chemotherapy is an ideal model to evaluate the results of chemotherapy, allowing intra-patient direct comparison of antitumor and antiangiogenic effects. We sought to analyze the effect of neoadjuvant chemotherapy on tumor angiogenesis and its clinical significance in breast cancer. Breast cancer patients ( n = 108) treated with neoadjuvant sequential anthracyclines and taxanes were studied. Pre- and post-chemotherapy microvessel density (MVD) and mean vessel size (MVS) were analyzed after CD34 immunohistochemistry and correlated with tumor expression of pro- and antiangiogenic factors (VEGFA, THBS1, HIF1A, CTGF, and PDGFA) by qRT-PCR. Angiogenic measures at diagnosis varied among breast cancer subtypes. Pre-treatment higher MVS was associated with triple-negative subtype and more advanced disease. Higher MVS was correlated with higher VEGFA ( p = 0.003), while higher MVD was correlated with lower antiangiogenic factors expression (THBS1, p < 0.0001; CTGF, p = 0.001). Increased angiogenesis at diagnosis (high MVS and glomeruloid microvascular proliferation) and higher VEGFA expression were associated with tumor recurrence ( p = 0.048 and 0.009, respectively). Chemotherapy-induced angiogenic response (defined as decreased MVD) was present in 35.2 % of patients. This response correlated with an increase in antiangiogenic factors (THBS1) without changes in VEGFA expression, and it was associated with tumor downstaging, but not with clinical response, pathologic complete response, or prognosis. Global effects of chemotherapy mainly consisted in an increased expression of antiangiogenic factors (THBS1, CTGF), with significant changes neither of tumor VEGFA nor of MVS. Conventionally scheduled neoadjuvant chemotherapy exerts antiangiogenic effects, through an increase in antiangiogenic factors, THBS1 and CTGF, but the expression of VEGFA is maintained after treatment. Better markers of angiogenic response and a better understanding of the cooperation of chemotherapy and antiangiogenic therapy in the neoadjuvant clinical scenario are needed. [ABSTRACT FROM AUTHOR]