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BackgroundUp 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.MethodsThis 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.ResultsA 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.ConclusionConsecutive 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.

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.

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.

Aim To determine predictive factors of axillary lymph node dissection (ALND) results in breast cancer (BC) patients undergoing neoadjuvant chemotherapy (NACT), and subsequent staging using Targeted Axillary Dissection (TAD). Material and method Case-control study between January 2016 and August 2019. Patients with BC, cN1 staging, marked with a metallic clip prior to NACT, and subsequently staged with TAD and ALND were included. They were divided into 2 groups: ALND patients with or without metastatic involvement (group 1 and group 2, respectively). We carried out a univariate analysis comparing clinical, radiological, surgical and pathological variables, and a logistic regression, (dependent variable: positive result of ALND; independent variables: number of suspicious lymph nodes in diagnostic ultrasound, positive hormone receptors, HER2 positive, complete clinical-radiological response to NACT, positive TAD, and biopsy of ≤2 nodes in TAD). A score for prediction of a metastatic ALND was proposed, with an internal validation study. Results 60 patients were included: Group 1: 33 (55.0%); Group 2: 27 (45.0%). Tumor size (Odds Ratio (OR) = 1.67; 95%CI 1.02–2.74), number of suspected nodes in ultrasound (OR = 2.20; 95%CI 1.01–4, 77), HER2 positive (OR 0.04; 95%CI 0.003–0.54), clinical-radiological response to NACT (OR = 0.07; 95%CI 0.01–0.75), and positive TAD (OR 15.48; 95%CI 1.68–142.78) were independent predictors of a positive result in ALND. We developed a “positive ALND predictive score”, with good calibration (Hosmer-Lemeshow test: p = 0.65), and discrimination (AUC = 0.93; 95% CI 0, 87–0.99), with highest Youden index (0.7) at cut-off point of 17% risk of positive ALND (sensitivity = 100%; specificity = 70%). Conclusion Tumor size, number of suspected nodes, positive HER2, response to NACT, and metastatic TAD are independent predictors of ALND. The predictive score for positive ALND would be a good indicator to safely omit ALND.

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., This study was supported by Sysmex España S.L.

The possibility of avoiding axillary lymphadenectomy (AL) in patients with breast cancer (BC) after positive sentinel lymph node biopsy (SLNB) and low metastatic burden (ó = 2 positive lymph nodes) has put into question the role of axillary ultrasound due to the risk of overtreatment after positive axillary lymph node biopsy with low metastatic burden. Our aim was to identify clinical and ultrasound features to detect low and high metastatic burden.A retrospective study of 405 BC patients with primary surgical treatment with axillary ultrasound examination and subsequent AL after positive fine needle aspiration (FNA) or SLNB. The low and high tumor burdens after AL were correlated with clinical and ultrasound variables: lymph node morphology (UN1 to UN5), number of suspicious lymph nodes, and Berg level.Positive FNA, lymph node morphology UN4 (focal thickening with displacement of the fatty hilum) or UN5 (complete replacement of the fatty hilum) and2 suspicious lymph nodes were significantly associated with "high metastatic burden". Lymph node morphology UN2 and UN3, even after FNA+, lymph node morphology UN4 after FNA-, and suspicious lymph nodes at Berg level I were low metastatic burden criteria. Lymph node morphology UN5, lymph node morphology UN4 after FNA+, two nodes or more with UN4/UN5 morphology, and suspicious lymph nodes at Berg levels II and III with FNA+ were associated with high metastatic burden.Axillary lymph node ultrasound data for patients with early BC allows predicting the axillary metastatic burden, guiding the optimal clinical management of the axilla.

Aim to study the feasibility and value of “Targeted Axillary Dissection” (TAD) in cN1 breast cancer (BC) patients undergoing neoadjuvant chemotherapy (NACT), in order to avoid unnecessary axillary lymph node dissection (ALND). Materials and methods Design: Prospective observational study. Inclusion criteria: Patients with histologically confirmed cN1 staging BC and treated with NACT between January 2016 and August 2019 who accomplished clinical response. Method: Fine-Needle Aspiration (FNA) positive axillary nodes were marked with a metallic clip prior to neoadjuvant treatment. All patients were summited to TAD and ALND. Analysis of data: We performed [1]: a feasibility analysis of clinical, radiological and pathological variables, as well as difficulties and complications of the TAD [2]; a diagnostic test study of the sentinel lymph node biopsy (SLNB), clipped lymph node biopsy (BCLIP) and their combination (TAD), using ALND as the Gold Standard. Results 60 patients were included. 43 patients (71.7%) had a complete clinical lymph node response to NACT. Neither limitations nor complications in clip placement were found. Intraoperative location of the clipped node was problematic in 7 cases (11.7%). The pathological complete response rate (pCR) was 30.5% (18 patients) and ypN0 staging rate was 38.3% (23 patients). Sensitivity values of each technique were: SLNB: 80.9% (95%CI: 61.8–100); BCLIP: 80.8% (95%CI: 63.7–97.8); TAD: 92.6% (95%CI: 80.9–100) with negative predictive values of: SLNB: 84.6% (95%CI: 68.8–100); BCLIP: 81.0% (95%CI: 63.7–97.8); TAD: 91.3% (95%CI: 77.6–100). Conclusion TAD is feasible and valid to rule out axillary metastatic involvement in cN1 breast cancer patients who respond to NACT.

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. 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. 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. 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.

Aim. To study the feasibility and validity of ultrasound-guided pre-chemotherapy marking of metastatic axillary lymph nodes followed by targeted axillary dissection (TAD), in breast cancer patients undergoing neoadjuvant chemotherapy (NACT). Material and method Prospective diagnostic test study conducted between January 2016 and March 2018. Patients with breast cancer and indication for NACT, cN1 or cN2 axillary staging, were included. A clip was placed in the affected lymph node prior to NACT. A sentinel lymph-node biopsy (SLNB) and a clipped lymph-node biopsy (BCLIP) were conducted, followed by axillary lymph node dissection (ALND). Location rate (LR) and negative predictive value (NPV) were evaluated, taking SLNB, BCLIP and their combination (TAD) as evaluated tests and metastatic involvement in the ALND specimen as the gold standard. Results Twenty-three patients were included in the study. Sentinel lymph node could only be detected in 19 cases (LR = 80.61%), whereas BCLIP was successful in 22 (LR = 95.65%). The sentinel lymph node coincided with the marked lymph node in 14 patients (60.9%). We found a NPV for the SLNB of 0.85 (95%CI: 0.61–1.0), whereas for TAD it was 1.00 (95%CI: 0.74–1.0). Conclusion TAD is a feasible test for axillary restaging after NACT, with a higher success rate than SLNB.

Background: microRNAs (miRNAs) are small ribonucleic acids that regulate protein expression through gene silencing mechanisms and show differential expression between cancer subtypes. Several studies have demonstrated that over- or under-expression of miRNA levels in tumors can modulate the expression levels of their gene/protein targets and thus their behavior. In breast cancer, the members of cluster miR-17-92 increase their expression levels as the histological grade increases, behaving like an oncogene, and are preferentially expressed in basal-like carcinomas. Moreover, mature components of this cluster are involved in estrogen receptor pathways and epithelial-mesenchymal transition, which suggest their implication in resistance to chemotherapy. The aim of this study was to determine the prognostic impact of miR-18a expression in the residual (resistant-to-treatment) tumor after neoadjuvant chemotherapy (nQT) in patients with locally advanced breast cancer (BC). Methods: Small RNA was isolated from FFPE samples of post-chemotherapy residual tumor in patients without pathological complete response (pCR). Quantification of miR-18a was performed by RT-qPCR. Expression levels were determined by 2-DDCt method using snRNAU6 as endogenous control. We used the quartiles of expression as cutoff point. Association of miR-18a levels with clinical and pathological characteristics was evaluated with non-parametric tests. Kaplan-Meier curves, log-rank test and Cox proportional hazard regression multivariate models were used for disease free (DFS) and overall (OS) survival analysis. Statistical analysis was performed with SPSS 18.0 software. Results: 121 consecutive women with invasive BC were included, mostly with stages IIB (28%) or IIIA-C (56.4%). Patients were immunohistochemically (IHC) classified as Her2- hormone-sensitive (HS) in 50.4% of cases; other phenotypes: Her2+ HS, 13.2%; Her2+ non-HS, 10.7%; triple negative, 21.5%. Treatment included sequential anthracyclines and taxanes (80.4% docetaxel) and a pCR was obtained in 17.4% of patients. Median overall survival (OS) and disease free survival (DFS) were not reached after a median follow-up of 60 months. High (above the median) expression levels of miR-18a in post-chemotherapy residual tumor were associated with pre-treatment grade 3 (p = 0.008), cN2-3 (p = 0.004), non-HS (p = 0.006) and triple negative phenotype (p = 0.01). No other associations with clinical or pathologic tumor characteristics (stage, HER2NEU overexpression) were found. High expression levels of miR-18a in residual tumors were also associated with reduced DFS and OS (log-rank test; p = 0.004 and p = 0.041 respectively). This negative prognostic impact was also confirmed for DFS in a multivariate analysis that included IHC phenotype and ypN+ (Table 1). Table 1 OR95%CIPPhenotype1.571.11-2.230.010ypN+7.291.67-31.800.008miR-18a high3.091.01-9.410.047 Conclusion: High expression levels of miR-18a in chemotherapy-resistant residual breast cancer associates with pre-treatment aggressive biological characteristics and independently predicts disease recurrence. Our results suggest that post-treatment miR-18a might serve as a marker of treatment resistance and as a new target for BC treatment. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-07-15.

Peripheral blood lymphocyte (PBL) count may reflect the immune status of cancer patients. We retrospectively analyzed the predictive and prognostic impact of baseline and post-chemotherapy PBL counts in a homogeneous group of 103 breast cancer patients treated with neoadjuvant chemotherapy (anthracyclines and taxanes). In univariate analysis, baseline PBL under 1500 × 10(6)/L (p = 0.013; hazard ratio [HR]: 2.80, 95%CI 1.24-6.61), and PBL decrease >200 × 10(6)/L after the first cycle of chemotherapy (p = 0.047; HR: 2.82, 95%CI 1.01-7.86) were significantly related to disease free survival. In multivariate analysis, both baseline PBL count less than 1500 × 10(6)/L (p = 0.034; HR: 3.32, 95%CI 1.09-10.02) and PBL decrease >200 × 10(6)/L after first cycle (p = 0.032; HR: 3.25, 95%CI 1.10-9.56) showed independent prognostic value for worse disease free survival. No effect was observed for overall survival. Our data support the relevance of pre- and post-chemotherapy PBL for breast cancer recurrence after neoadjuvant chemotherapy.

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

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.

Introduction Tumor microenvironment immunity is associated with breast cancer outcome. A high lymphocytic infiltration has been associated with response to neoadjuvant chemotherapy, but the contribution to response and prognosis of immune cell subpopulations profiles in both pre-treated and post-treatment residual tumor is still unclear. Methods We analyzed pre- and post-treatment tumor-infiltrating immune cells (CD3, CD4, CD8, CD20, CD68, Foxp3) by immunohistochemistry in a series of 121 breast cancer patients homogeneously treated with neoadjuvant chemotherapy. Immune cell profiles were analyzed and correlated with response and survival. Results We identified three tumor-infiltrating immune cell profiles, which were able to predict pathological complete response (pCR) to neoadjuvant chemotherapy (cluster B: 58%, versus clusters A and C: 7%). A higher infiltration by CD4 lymphocytes was the main factor explaining the occurrence of pCR, and this association was validated in six public genomic datasets. A higher chemotherapy effect on lymphocytic infiltration, including an inversion of CD4/CD8 ratio, was associated with pCR and with better prognosis. Analysis of the immune infiltrate in post-chemotherapy residual tumor identified a profile (cluster Y), mainly characterized by high CD3 and CD68 infiltration, with a worse disease free survival. Conclusions Breast cancer immune cell subpopulation profiles, determined by immunohistochemistry-based computerized analysis, identify groups of patients characterized by high response (in the pre-treatment setting) and poor prognosis (in the post-treatment setting). Further understanding of the mechanisms underlying the distribution of immune cells and their changes after chemotherapy may contribute to the development of new immune-targeted therapies for breast cancer. Electronic supplementary material The online version of this article (doi:10.1186/s13058-014-0488-5) contains supplementary material, which is available to authorized users.