Advanced Insights into Competitive Endogenous RNAs (ceRNAs) Regulated Pathogenic Mechanisms in Metastatic Triple-Negative Breast Cancer (mTNBC).

Triple-negative breast cancer is aggressive and challenging to treat because of a lack of targets and heterogeneity among tumors. A paramount factor in the mortality from breast cancer is metastasis, which is driven by genetic and phenotypic alterations that drive epithelial-mesenchymal transition, stemness, survival, migration and invasion. Many genetic and epigenetic mechanisms have been identified in triple-negative breast cancer that drive these metastatic phenotypes; however, this knowledge has not yet led to the development of effective drugs for metastatic triple-negative breast cancer (mTNBC). One that may not have received enough attention in the literature is post-translational regulation of broad sets of cancer-related genes through inhibitory microRNAs and the complex competitive endogenous RNA (ceRNA) regulatory networks they are influenced by. This field of study and the resulting knowledge regarding alterations in these networks is coming of age, enabling translation into clinical benefit for patients. Herein, we review metastatic triple-negative breast cancer (mTNBC), the role of ceRNA network regulation in metastasis (and therefore clinical outcomes), potential approaches for therapeutic exploitation of these alterations, knowledge gaps and future directions in the field.

Prognostic significance of the neutrophil to lymphocyte ratio in locally advanced breast cancer.

The present study aimed to clarify the prognostic role of the pre-treatment neutrophil-to-lymphocyte ratio (NLR) for the response to neoadjuvant chemotherapy (NAC) in locally advanced breast cancer (LABC). Due to conflicting results in currently available data, the specific focus of the present study was on evaluating the associations between the pre-treatment NLR and the rate of achieving a pathological complete response (pCR) and survival outcomes. For the present study, data from a cohort of 465 consecutive patients with LABC who underwent NAC at King Feisal Specialist Hospital and Research Center (Riyadh, Saudi Arabia) between 2005 and 2014 were obtained from a prospective BC database and analyzed. Patients were stratified into two groups based on an optimal NLR cut-off determined using the receiver operating characteristic curve. Logistic regression analyses were conducted to assess variables associated with pCR, and Cox regression analyses were used to assess variables associated with survival outcomes. The low pre-treatment NLR group (≤2.2) was found to exhibit a higher likelihood of achieving a pCR (odds ratio, 2.59; 95% CI, 1.52-4.38; P<0.001), along with higher 5-year disease-free survival (DFS) [75.8 vs. 64.9%; hazard ratio (HR), 0.69; 95% CI, 0.50-0.94; P=0.02] and 5-year overall survival (OS; 90.3 vs. 81.9; HR, 0.62; 95% CI, 0.39-0.98; P=0.04) rates compared with those in the high NLR group (>2.2). Sub-group analysis revealed that the observed significance in survival outcomes was driven by the triple-negative BC (TNBC) subgroup. Patients with residual TNBC disease and a high pre-treatment NLR were observed to have lower 5-year DFS (44.4 vs. 75.0%; P=0.02) and 5-year OS (55.9 vs. 84.5%; P=0.055) rates compared with those with residual TNBC disease and a low NLR. To conclude, data from the present study suggest that the pre-treatment NLR can serve as a viable independent prognostic factor for pCR following NAC in patients with LABC and for survival outcomes, particularly for patients with TNBC.

Long noncoding RNA DLEU2 and ROR1 pathway induces epithelial-to-mesenchymal transition and cancer stem cells in breast cancer.

Breast cancer (BC) patient who receives chemotherapy for an extended length of time may experience profound repercussions in terms of metastases and clinical outcomes due to the involvement of the epithelial-to-mesenchymal transition (EMT) mechanism and enriched cancer stem cells (CSCs). BC cells that express high levels of lncRNA deleted in lymphocytic leukemia-2 (lncRNA DLEU2) and type I tyrosine kinase-like orphan receptor ROR1 (ROR1) may play roles in the enhanced ability of the activation EMT and CSC induction. Here we find that lncRNA DLEU2 and ROR1 are specifically upregulated in tumor tissues compared to their normal counterparts in TCGA, PubMed GEO datasets, and samples from archived breast cancer tumor tissues. Following chemotherapy, lncRNA DLEU2 and ROR1 were enhanced in BC tumor cells, coupled with the expression of CSCs, EMT-related genes, and BMI1. Mechanistically, ROR1 and lncRNA DLEU2 overexpression led to enhanced tumor cell proliferation, inhibition of apoptosis, cell-cycle dysregulation, chemoresistance, as well as BC cell's abilities to invade, migrate, develop spheroids. These findings imply that the role of lncRNA DLEU2 and ROR1 in BC therapeutic failure is largely attributed to EMT, which is intricately linked to enriched CSCs. In conclusion, our findings indicate that a lncRNA DLEU2 and ROR1-based regulatory loop governs EMT and CSC self-renewal, implying that targeting this regulatory pathway may improve patients' responses to chemotherapy and survival.

High AUF1 level in stromal fibroblasts promotes carcinogenesis and chemoresistance and predicts unfavorable prognosis among locally advanced breast cancer patients.

BACKGROUND: Locally advanced breast cancer (LABC), the most aggressive form of the disease, is a serious threat for women's health worldwide. The AU-rich RNA-binding factor 1 (AUF1) promotes the formation of chemo-resistant breast cancer stem cells. Thereby, we investigated the power of AUF1 expression, in both cancer cells and their stromal fibroblasts, as predictive biomarker for LABC patients' clinical outcome following neoadjuvant treatment. METHODS: We have used immunohistochemistry to assess the level of AUF1 on formalin-fixed paraffin-embedded tissues. Immunoblotting was utilized to show the effect of AUF1 ectopic expression in breast stromal fibroblasts on the expression of various genes both in vitro and in orthotopic tumor xenografts. Cytotoxicity was evaluated using the WST1 assay, while a label-free real-time setting using the xCELLigence RTCA technology was utilized to assess the proliferative, migratory and invasive abilities of cells. RESULTS: We have shown that high AUF1 immunostaining (≥ 10%) in both cancer cells and their adjacent cancer-associated fibroblasts (CAFs) was significantly associated with higher tumor grade. Kaplan-Meier univariate analysis revealed a strong correlation between high AUF1 level in CAFs and poor patient's survival. This correlation was highly significant in patients with triple negative breast cancer, who showed poor disease-free survival (DFS) and overall survival (OS). High expression of AUF1 in CAFs was also associated with poor OS of ER+/Her2- patients. Similarly, AUF1-positive malignant cells tended to be associated with shorter DFS and OS of ER+/Her2+ patients. Interestingly, neoadjuvant therapy downregulated AUF1 to a level lower than 10% in malignant cells in a significant number of patients, which improved both DFS and OS. In addition, ectopic expression of AUF1 in breast fibroblasts activated these cells and enhanced their capacity to promote, in an IL-6-dependent manner, the epithelial-to-mesenchymal transition and stemness processes. Furthermore, these AUF1-expressing cells enhanced the chemoresistance of breast cancer cells and their growth in orthotopic tumor xenografts. CONCLUSIONS: The present findings show that the CAF-activating factor AUF1 has prognostic/predictive value for breast cancer patients and could represent a great therapeutic target in order to improve the precision of cancer treatment.

High DNMT1 Expression in Stromal Fibroblasts Promotes Angiogenesis and Unfavorable Outcome in Locally Advanced Breast Cancer Patients.

Background: Active breast cancer-associated fibroblasts (CAFs) play a leading role in breast carcinogenesis through promoting angiogenesis and resistance to therapy. Consequently, these active stromal cells have significant influence on patient outcome. Therefore, we explored here the role of the DNA methyltransferase 1 (DNMT1) protein in CAF-dependent promotion of angiogenesis as well as the prognostic power of DNMT1 level in both cancer cells and their adjacent CAFs in locally advanced breast cancer patients. Methods: We applied immunohistochemistry to evaluate the level of DNMT1 in breast cancer tissues and their adjacent normal counterparts. Quantitative RT-PCR and immunoblotting were performed to investigate the role of DNMT1 in regulating the expression of pro-angiogenic genes in active CAFs and also their response to the DNMT1 inhibitors decitabine (DAC) as well as eugenol. Results: We have shown that DNMT1 controls the pro-angiogenic potential of CAFs both in vitro and in vivo through positive regulation of the expression/secretion of 2 important pro-angiogenic factors VEGF-A and IL-8 as well as their upstream effectors mTOR and HIF-1α. To confirm this, we have shown that these DNMT1-related pro-angiogenic effects were suppressed by 2 DNMT1 inhibitors decitabine and eugenol. Interestingly, in a cohort of 100 tumors from locally advanced breast cancer patients (LABC), we have shown that high expression of DNMT1 in tumor cells and their adjacent stromal fibroblasts is correlated with poor survival of these patients. Conclusion: DNMT1 upregulation in breast stromal fibroblasts promotes angiogenesis via IL-8/VEGF-A upregulation, and correlates well with poor survival of LABC patients.

Association Between Obesity and Clinicopathological Profile of Patients with Newly Diagnosed Non-Metastatic Breast Cancer in Saudi Arabia.

Purpose: Obesity is prevalent in Saudi Arabia and is associated with adverse clinical features and poor breast cancer (BC) outcomes. We determined the distribution of body mass index (BMI) and evaluated its association with disease characteristics and outcomes in women with non-metastatic BC. Patients and Methods: We conducted a retrospective analysis of a prospectively collected database of consecutive patients treated for non-metastatic BC between 2002 and 2014. Patients were categorized into the following groups: underweight/normal weight (BMI <25 kg/m2), overweight (BMI 25-29.9 kg/m2), and obese (BMI ≥30 kg/m2). Regression analysis was used to evaluate clinicopathological factors associated with BMI and clinical stage. Results: A total of 2212 patients were enrolled. The median age was 45 years (interquartile range [IQR], 39-52 years), and the median BMI was 30 kg/m2 (IQR, 26-34 kg/m2). Most patients were premenopausal (63.6%), nearly half of the patients had stage III disease, and 11.2% were screen-detected. The prevalence of obesity was 53.4%, with a significant difference between the peri/premenopausal (49.4%) and postmenopausal (61.7%) groups (p < 0.001). Obese patients were more likely to be aged >40 years, be postmenopausal, have a history of oral contraceptive pills, have advanced-stage disease, and have undergone radiation therapy, and were less likely to have human epithelial growth factor 2 (HER2)+ disease than non-obese patients. Premenopausal obese women had fewer hormone receptor-positive and more triple-negative cancers than postmenopausal obese women did. Obesity, non-screening-detected BC, and HER+ status were independent prognostic factors for advanced-stage presentation. Conclusion: The prevalence of obesity and its significant association with advanced BC justify the upscaling of screening services and instituting weight-reduction strategies.

Translational Implications of Dysregulated Pathways and microRNA Regulation in Quadruple-Negative Breast Cancer.

Triple-negative breast cancers (HER2-, ER-, PR-) continue to present a unique treatment challenge and carry unfavorable prognoses. The elucidation of novel therapeutic targets has necessitated the re-evaluation of stratification approaches to best predict prognosis, treatment response and theranostic and prognostic markers. Androgen receptor expression and function have important implications on proliferation, tumor progression, immunity and molecular signaling in breast cancer. Accordingly, there has been increasing support for classification of androgen receptor-negative triple-negative breast cancer or quadruple-negative breast cancer (QNBC). QNBC has unique molecular, signaling and expression regulation profiles, particularly those affected by microRNA regulatory networks. microRNAs are now known to regulate AR-related targets and pathways that are dysregulated in QNBC, including immune checkpoint inhibitors (ICIs), SKP2, EN1, ACSL4 and EGFR. In this review, we explore and define the QNBC tumor subtype, its molecular and clinical distinctions from other subtypes, miRNA dysregulation and function in QNBC, and knowledge gaps in the field. Potential insights into clinical and translational implications of these dysregulated networks in QNBC are discussed.

Peripheral blood eosinophil count is associated with response to chemoimmunotherapy in metastatic triple-negative breast cancer.

Introduction: There is evidence for an association between peripheral blood eosinophil count (PBEC) and response to cancer immunotherapy; however, such data is limited in metastatic triple-negative breast cancer (mTNBC). Patients & methods: This report presents patients (n = 14) who received a combination of durvalumab and paclitaxel for mTNBC (NCT02628132). Results: There was a statistically significant correlation (p = 0.028) between an increase in PBEC (>300/mm3) during treatment and response to the combination therapy. Survival analysis showed a statistically significant association between progression-free survival and increased PBEC, after therapy (p = 0.005). A similar trend existed for overall survival, although it did not reach statistical significance (p = 0.167). Conclusion: This is the first study to report on eosinophilia in mTNBC treated with chemoimmunotherapy and supports a role for eosinophils in immunotherapy for mTNBC.

Plain language summary Previous reports have shown that an increase in peripheral blood eosinophil count is associated with a good response to cancer immunotherapy; however, data on this association is limited in a subtype of breast cancer called metastatic triple-negative breast cancer (mTNBC). The eosinophil count in patients who received the combination of an immunotherapeutic agent, durvalumab and a chemotherapeutic agent, paclitaxel, in mTNBC was assessed. There was a statistically significant association between an increase in eosinophils during treatment and the response to therapy. The progression of the disease was slower or less likely to occur in patients with increased eosinophils. This study supports the role of eosinophils in immunotherapy for mTNBC.

Using the Appropriate Formula for QT Measurement Can Save Lives.

CDK 4/6 inhibitors, in combination with endocrine therapy, are the standard of care for patients with endocrinesensitive advanced breast cancer. This class of drug, however, is associated with QT prolongation, which serves as a surrogate marker for Torsades de Pointes (TdP), a cause of life-threatening ventricular arrhythmias and sudden cardiac death. The ICH E14 guidance document uses the Bazett formula for reporting of cardio-dynamic and safety ECG data in clinical trials. While there is substantial familiarity with the Bazett (QTcB) formula (QT/(RR) 1/2), the Fridericia (QTcF) formula (QT/(RR) 1/3 ) is preferred in the cancer population as it is often more accurate at heart rate extreme. Accordingly, the Fridericia formula is currently the standard adopted by the FDA when submitting QT data for review. At the King Faisal Specialist Hospital and Research Center, a total of 82 patients with advanced breast cancer, had a baseline ECG on day 1 before the initiation of ribociclib based therapy. Of the enrolled 82 patients, 19 (23%) were initially excluded from receiving ribociclib based due to a prolonged QTc >450ms, however, when the QTc-interval was manually measured and recalculated using Fridericia and Framingham formulae using MDCalC (https://www.mdcalc.com),17 of 19 patients successfully received their treatment without any arrhythmogenic effects. Repeat ECG on day14, and day 1 of cycle 2 demonstrated that none of these patients had QTc exceeding 480 ms. Our data highlights the complexities of evaluating the QT interval in oncology patients and the utility of the Fridericia/Framingham formulae in this population. Given these findings, we recommend the adoption of the Fridericia or Framingham formulae for measurement of QTc in all cancer patients exposed to potentially QT-prolonging cancer therapy.