Showing papers in "Breast Cancer Research in 2015"

Human breast cancer cells educate macrophages toward the M2 activation status

Abstract: The immune system plays a major role in cancer progression. In solid tumors, 5-40 % of the tumor mass consists of tumor-associated macrophages (TAMs) and there is usually a correlation between the number of TAMs and poor prognosis, depending on the tumor type. TAMs usually resemble M2 macrophages. Unlike M1-macrophages which have pro-inflammatory and anti-cancer functions, M2-macrophages are immunosuppressive, contribute to the matrix-remodeling, and hence favor tumor growth. The role of TAMs is not fully understood in breast cancer progression. Macrophage infiltration (CD68) and activation status (HLA-DRIIα, CD163) were evaluated in a large cohort of human primary breast tumors (562 tissue microarray samples), by immunohistochemistry and scored by automated image analysis algorithms. Survival between groups was compared using the Kaplan-Meier life-table method and a Cox multivariate proportional hazards model. Macrophage education by breast cancer cells was assessed by ex vivo differentiation of peripheral blood mononuclear cells (PBMCs) in the presence or absence of breast cancer cell conditioned media (MDA-MB231, MCF-7 or T47D cell lines) and M1 or M2 inducing cytokines (respectively IFN-γ, IL-4 and IL-10). Obtained macrophages were analyzed by flow cytometry (CD14, CD16, CD64, CD86, CD200R and CD163), ELISA (IL-6, IL-8, IL-10, monocyte colony stimulating factor M-CSF) and zymography (matrix metalloproteinase 9, MMP-9). Clinically, we found that high numbers of CD163+ M2-macrophages were strongly associated with fast proliferation, poor differentiation, estrogen receptor negativity and histological ductal type (p<0.001) in the studied cohort of human primary breast tumors. We demonstrated ex vivo that breast cancer cell-secreted factors modulate macrophage differentiation toward the M2 phenotype. Furthermore, the more aggressive mesenchymal-like cell line MDA-MB231, which secretes high levels of M-CSF, skews macrophages toward the more immunosuppressive M2c subtype. This study demonstrates that human breast cancer cells influence macrophage differentiation and that TAM differentiation status correlates with recurrence free survival, thus further emphasizing that TAMs can similarly affect therapy efficacy and patient outcome.

Invasive lobular carcinoma of the breast: morphology, biomarkers and ’omics

Abstract: Invasive lobular carcinoma of the breast is the most common ‘special’ morphological subtype of breast cancer, comprising up to 15% of all cases. Tumours are generally of a good prognostic phenotype, being low histological grade and low mitotic index, hormone receptor positive and HER2, p53 and basal marker negative, and with a generally good response to endocrine therapy. Despite this, clinicians face countless challenges in the diagnosis and long-term management of patients, as they encounter a tumour that can be difficult to detect through screening, elicits a very invasive nature, a propensity for widespread metastatic colonisation and, consequently, in some studies a worse long-term poor outcome compared with invasive carcinoma of no special type. Here we review the morphological and molecular features that underpin the disparate biological and clinical characteristics of this fascinating tumour type.

Benefits and harms of mammography screening

Abstract: Mammography screening for breast cancer is widely available in many countries. Initially praised as a universal achievement to improve women's health and to reduce the burden of breast cancer, the benefits and harms of mammography screening have been debated heatedly in the past years. This review discusses the benefits and harms of mammography screening in light of findings from randomized trials and from more recent observational studies performed in the era of modern diagnostics and treatment. The main benefit of mammography screening is reduction of breast-cancer related death. Relative reductions vary from about 15 to 25% in randomized trials to more recent estimates of 13 to 17% in meta-analyses of observational studies. Using UK population data of 2007, for 1,000 women invited to biennial mammography screening for 20 years from age 50, 2 to 3 women are prevented from dying of breast cancer. All-cause mortality is unchanged. Overdiagnosis of breast cancer is the main harm of mammography screening. Based on recent estimates from the United States, the relative amount of overdiagnosis (including ductal carcinoma in situ and invasive cancer) is 31%. This results in 15 women overdiagnosed for every 1,000 women invited to biennial mammography screening for 20 years from age 50. Women should be unpassionately informed about the benefits and harms of mammography screening using absolute effect sizes in a comprehensible fashion. In an era of limited health care resources, screening services need to be scrutinized and compared with each other with regard to effectiveness, cost-effectiveness and harms.

Patient-derived xenograft models of breast cancer and their predictive power

Abstract: Despite advances in the treatment of patients with early and metastatic breast cancer, mortality remains high due to intrinsic or acquired resistance to therapy. Increased understanding of the genomic landscape through massively parallel sequencing has revealed somatic mutations common to specific subtypes of breast cancer, provided new prognostic and predictive markers, and highlighted potential therapeutic targets. Evaluating new targets using established cell lines is limited by the inexact correlation between responsiveness observed in cell lines versus that elicited in the patient. Patient-derived xenografts (PDXs) generated from fresh tumor specimens recapitulate the diversity of breast cancer and reflect histopathology, tumor behavior, and the metastatic properties of the original tumor. The high degree of genomic preservation evident across primary tumors and their matching PDXs over serial passaging validate them as important preclinical tools. Indeed, there is accumulating evidence that PDXs can recapitulate treatment responses of the parental tumor. The finding that tumor engraftment is an independent and poor prognostic indicator of patient outcome represents the first step towards personalized medicine. Here we review the utility of breast cancer PDX models to study the clonal evolution of tumors and to evaluate novel therapies and drug resistance.

Dysregulation of microRNAs in breast cancer and their potential role as prognostic and predictive biomarkers in patient management.

Abstract: MicroRNAs (miRNAs) are an emerging class of gene expression modulators with relevant roles in several biological processes, including cell differentiation, development, apoptosis, and regulation of the cell cycle. Deregulation of those tiny RNA molecules has been described frequently as a major determinant for the initiation and progression of diseases, including cancer. Not only miRNAs but also the enzymes responsible for miRNA processing could be deregulated in cancer. In this review, we address the role of miRNAs in the pathogenesis of breast cancer, since there are oncogenic, tumor-suppressive, and metastatic-influencing miRNAs. Additionally, the different detection platforms and normalization strategies for miRNAs will be discussed. The major part of this review, however, will focus on the capability of miRNAs to act as diagnostic, predictive, or prognostic biomarkers. We will give an overview of their potential to correlate with response to or benefit from a given treatment and we will consider their ability to give information on prognosis in breast cancer. We will focus on miRNAs validated by more than one study or verified in independent cohorts or where results rely on preclinical as well as clinical evidence. As such, we will discuss their potential use in the personalized management of breast cancer.

Gene-expression molecular subtyping of triple-negative breast cancer tumours: importance of immune response

Abstract: Triple-negative breast cancers need to be refined in order to identify therapeutic subgroups of patients. We conducted an unsupervised analysis of microarray gene-expression profiles of 107 triple-negative breast cancer patients and undertook robust functional annotation of the molecular entities found by means of numerous approaches including immunohistochemistry and gene-expression signatures. A triple-negative external cohort (n = 87) was used for validation. Fuzzy clustering separated triple-negative tumours into three clusters: C1 (22.4%), C2 (44.9%) and C3 (32.7%). C1 patients were older (mean = 64.6 years) than C2 (mean = 56.8 years; P = 0.03) and C3 patients (mean = 51.9 years; P = 0.0004). Histological grade and Nottingham prognostic index were higher in C2 and C3 than in C1 (P < 0.0001 for both comparisons). Significant event-free survival (P = 0.03) was found according to cluster membership: patients belonging to C3 had a better outcome than patients in C1 (P = 0.01) and C2 (P = 0.02). Event-free survival analysis results were confirmed when our cohort was pooled with the external cohort (n = 194; P = 0.01). Functional annotation showed that 22% of triple-negative patients were not basal-like (C1). C1 was enriched in luminal subtypes and positive androgen receptor (luminal androgen receptor). C2 could be considered as an almost pure basal-like cluster. C3, enriched in basal-like subtypes but to a lesser extent, included 26% of claudin-low subtypes. Dissection of immune response showed that high immune response and low M2-like macrophages were a hallmark of C3, and that these patients had a better event-free survival than C2 patients, characterized by low immune response and high M2-like macrophages: P = 0.02 for our cohort, and P = 0.03 for pooled cohorts. We identified three subtypes of triple-negative patients: luminal androgen receptor (22%), basal-like with low immune response and high M2-like macrophages (45%), and basal-enriched with high immune response and low M2-like macrophages (33%). We noted out that macrophages and other immune effectors offer a variety of therapeutic targets in breast cancer, and particularly in triple-negative basal-like tumours. Furthermore, we showed that CK5 antibody was better suited than CK5/6 antibody to subtype triple-negative patients.

Efficient treatment of breast cancer xenografts with multifunctionalized iron oxide nanoparticles combining magnetic hyperthermia and anti-cancer drug delivery

Abstract: Tumor cells can effectively be killed by heat, e.g. by using magnetic hyperthermia. The main challenge in the field, however, is the generation of therapeutic temperatures selectively in the whole tumor region. We aimed to improve magnetic hyperthermia of breast cancer by using innovative nanoparticles which display a high heating potential and are functionalized with a cell internalization and a chemotherapeutic agent to increase cell death. The superparamagnetic iron oxide nanoparticles (MF66) were electrostatically functionalized with either Nucant multivalent pseudopeptide (N6L; MF66-N6L), doxorubicin (DOX; MF66-DOX) or both (MF66-N6LDOX). Their cytotoxic potential was assessed in a breast adenocarcinoma cell line MDA-MB-231. Therapeutic efficacy was analyzed on subcutaneous MDA-MB-231 tumor bearing female athymic nude mice. All nanoparticle variants showed an excellent heating potential around 500 W/g Fe in the alternating magnetic field (AMF, conditions: H = 15.4 kA/m, f = 435 kHz). We could show a gradual inter- and intracellular release of the ligands, and nanoparticle uptake in cells was increased by the N6L functionalization. MF66-DOX and MF66-N6LDOX in combination with hyperthermia were more cytotoxic to breast cancer cells than the respective free ligands. We observed a substantial tumor growth inhibition (to 40% of the initial tumor volume, complete tumor regression in many cases) after intratumoral injection of the nanoparticles in vivo. The proliferative activity of the remaining tumor tissue was distinctly reduced. The therapeutic effects of breast cancer magnetic hyperthermia could be strongly enhanced by the combination of MF66 functionalized with N6L and DOX and magnetic hyperthermia. Our approach combines two ways of tumor cell killing (magnetic hyperthermia and chemotherapy) and represents a straightforward strategy for translation into the clinical practice when injecting nanoparticles intratumorally.

Multigene prognostic tests in breast cancer: past, present, future

Abstract: There is growing consensus that multigene prognostic tests provide useful complementary information to tumor size and grade in estrogen receptor (ER)-positive breast cancers. The tests primarily rely on quantification of ER and proliferation-related genes and combine these into multivariate prediction models. Since ER-negative cancers tend to have higher proliferation rates, the prognostic value of current multigene tests in these cancers is limited. First-generation prognostic signatures (Oncotype DX, MammaPrint, Genomic Grade Index) are substantially more accurate to predict recurrence within the first 5 years than in later years. This has become a limitation with the availability of effective extended adjuvant endocrine therapies. Newer tests (Prosigna, EndoPredict, Breast Cancer Index) appear to possess better prognostic value for late recurrences while also remaining predictive of early relapse. Some clinical prediction problems are more difficult to solve than others: there are no clinically useful prognostic signatures for ER-negative cancers, and drug-specific treatment response predictors also remain elusive. Emerging areas of research involve the development of immune gene signatures that carry modest but significant prognostic value independent of proliferation and ER status and represent candidate predictive markers for immune-targeted therapies. Overall metrics of tumor heterogeneity and genome integrity (for example, homologue recombination deficiency score) are emerging as potential new predictive markers for platinum agents. The recent expansion of high-throughput technology platforms including low-cost sequencing of circulating and tumor-derived DNA and RNA and rapid reliable quantification of microRNA offers new opportunities to build extended prediction models across multiplatform data.

Omega-3 fatty acids for breast cancer prevention and survivorship

Abstract: Women with evidence of high intake ratios of the marine omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) relative to the omega-6 arachidonic acid have been found to have a reduced risk of breast cancer compared with those with low ratios in some but not all case–control and cohort studies. If increasing EPA and DHA relative to arachidonic acid is effective in reducing breast cancer risk, likely mechanisms include reduction in proinflammatory lipid derivatives, inhibition of nuclear factor-κB-induced cytokine production, and decreased growth factor receptor signaling as a result of alteration in membrane lipid rafts. Primary prevention trials with either risk biomarkers or cancer incidence as endpoints are underway but final results of these trials are currently unavailable. EPA and DHA supplementation is also being explored in an effort to help prevent or alleviate common problems after a breast cancer diagnosis, including cardiac and cognitive dysfunction and chemotherapy-induced peripheral neuropathy. The insulin-sensitizing and anabolic properties of EPA and DHA also suggest supplementation studies to determine whether these omega-3 fatty acids might reduce chemotherapy-associated loss of muscle mass and weight gain. We will briefly review relevant omega-3 fatty acid metabolism, and early investigations in breast cancer prevention and survivorship.

Prognostic significance of tumor-infiltrating CD8+ and FOXP3+ lymphocytes in residual tumors and alterations in these parameters after neoadjuvant chemotherapy in triple-negative breast cancer: a retrospective multicenter study

Abstract: The status of tumor-infiltrating lymphocytes (TILs) has been recently proposed to predict clinical outcome of patients with breast cancer. We therefore studied the prognostic significance of CD8+ TILs and FOXP3+ TILs in residual tumors after neoadjuvant chemotherapy (NAC) and the alterations in these parameters before and after NAC in patients with triple-negative breast cancer (TNBC). One hundred thirty-one TNBC patients who received NAC at three institutions were examined. CD8+ TIL and FOXP3+ TIL in residual tumors and biopsy specimens were evaluated by double-staining immunohistochemistry. The CD8+ TIL and FOXP3+ TIL status of the residual tumors was assessed, and the rates of their changes before and after NAC were calculated. TNBC patients with high CD8+ TIL levels or a high CD8/FOXP3 ratio in residual tumors had significantly better recurrence-free survival (RFS) and breast cancer-specific survival (BCSS) than patients with low values of these parameters. In multivariate analyses, CD8+ TIL exhibited strong prognostic significance for RFS, with a hazard ratio (HR) of 3.09 (95 % confidence interval (CI) 1.537–6.614, P=0.0013). The CD8/FOXP3 ratio was also significantly correlated with RFS (HR=2.07, 95 % CI 1.029–4.436, P=0.0412). TNBC with larger residual tumor size and positive lymph node status, which are known prognostic factors, was independently associated with worse RFS (P=0.0064 and P=0.0015, respectively). High CD8+ TIL levels were a markedly powerful indicator of improved BCSS, with an HR of 3.59 (95 % CI 1.499–9.581, P=0.0036). Nodal status was also associated with BCSS (P=0.0024). TNBC with a high rate of CD8+ TIL changes was associated with significantly better RFS compared with the low group (P=0.011). Higher rates of changes in the CD8/FOXP3 ratio were significantly correlated with both better RFS and BCSS compared with lower rates (P=0.011 and P=0.023, respectively). This is the first study to demonstrate that high CD8+ TIL and a high CD8/FOXP3 ratio in residual tumors and increment of these parameters following NAC and accurately predict improved prognosis in TNBC patients with non-pathological complete response following NAC. These parameters could serve as a surrogate one for adjuvant treatment in patients with residual disease in the neoadjuvant setting.

Mammographic density adds accuracy to both the Tyrer-Cuzick and Gail breast cancer risk models in a prospective UK screening cohort

Abstract: The Predicting Risk of Cancer at Screening study in Manchester, UK, is a prospective study of breast cancer risk estimation. It was designed to assess whether mammographic density may help in refinement of breast cancer risk estimation using either the Gail model (Breast Cancer Risk Assessment Tool) or the Tyrer-Cuzick model (International Breast Intervention Study model). Mammographic density was measured at entry as a percentage visual assessment, adjusted for age and body mass index. Tyrer-Cuzick and Gail 10-year risks were based on a questionnaire completed contemporaneously. Breast cancers were identified at the entry screen or shortly thereafter. The contribution of density to risk models was assessed using odds ratios (ORs) with profile likelihood confidence intervals (CIs) and area under the receiver operating characteristic curve (AUC). The calibration of predicted ORs was estimated as a percentage [(observed vs expected (O/E)] from logistic regression. The analysis included 50,628 women aged 47–73 years who were recruited between October 2009 and September 2013. Of these, 697 had breast cancer diagnosed after enrolment. Median follow-up was 3.2 years. Breast density [interquartile range odds ratio (IQR-OR) 1.48, 95 % CI 1.34–1.63, AUC 0.59] was a slightly stronger univariate risk factor than the Tyrer-Cuzick model [IQR-OR 1.36 (95 % CI 1.25–1.48), O/E 60 % (95 % CI 44–74), AUC 0.57] or the Gail model [IQR-OR 1.22 (95 % CI 1.12–1.33), O/E 46 % (95 % CI 26–65 %), AUC 0.55]. It continued to add information after allowing for Tyrer-Cuzick [IQR-OR 1.47 (95 % CI 1.33–1.62), combined AUC 0.61] or Gail [IQR-OR 1.45 (95 % CI 1.32–1.60), combined AUC 0.59]. Breast density may be usefully combined with the Tyrer-Cuzick model or the Gail model.

Inhibition of iNOS as a novel effective targeted therapy against triple-negative breast cancer

Abstract: Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with no effective targeted therapy. Inducible nitric oxide synthase (iNOS) is associated with poor survival in patients with breast cancer by increasing tumor aggressiveness. This work aimed to investigate the potential of iNOS inhibitors as a targeted therapy for TNBC. We hypothesized that inhibition of endogenous iNOS would decrease TNBC aggressiveness by reducing tumor initiation and metastasis through modulation of epithelial-mesenchymal transition (EMT)-inducing factors. iNOS protein levels were determined in 83 human TNBC tissues and correlated with clinical outcome. Proliferation, mammosphere-forming efficiency, migration, and EMT transcription factors were assessed in vitro after iNOS inhibition. Endogenous iNOS targeting was evaluated as a potential therapy in TNBC mouse models. High endogenous iNOS expression was associated with worse prognosis in patients with TNBC by gene expression as well as immunohistochemical analysis. Selective iNOS (1400 W) and pan-NOS (L-NMMA and L-NAME) inhibitors diminished cell proliferation, cancer stem cell self-renewal, and cell migration in vitro, together with inhibition of EMT transcription factors (Snail, Slug, Twist1, and Zeb1). Impairment of hypoxia-inducible factor 1α, endoplasmic reticulum stress (IRE1α/XBP1), and the crosstalk between activating transcription factor 3/activating transcription factor 4 and transforming growth factor β was observed. iNOS inhibition significantly reduced tumor growth, the number of lung metastases, tumor initiation, and self-renewal. Considering the effectiveness of L-NMMA in decreasing tumor growth and enhancing survival rate in TNBC, we propose a targeted therapeutic clinical trial by re-purposing the pan-NOS inhibitor L-NMMA, which has been extensively investigated for cardiogenic shock as an anti-cancer therapeutic.

Mechanisms of endocrine resistance in breast cancer: an overview of the proposed roles of noncoding RNA

Abstract: Endocrine therapies such as tamoxifen and aromatase inhibitors are the standard treatment options for estrogen receptor-positive breast cancer patients. However, resistance to these agents has become a major clinical obstacle. Potential mechanisms of resistance to endocrine therapies have been identified, often involving enhanced growth factor signaling and changes in the expression or action of the estrogen receptor, but few studies have addressed the role of noncoding RNA (ncRNA). Two important types of ncRNA include microRNA (miRNA) and long noncoding RNA (lncRNA). miRNAs are small RNA molecules that regulate gene expression via translational inhibition or degradation of mRNA transcripts, while lncRNAs are larger RNA molecules that have been shown to play a role in multiple cellular maintenance functions such as protein scaffolding, chromatin looping, and regulation of mRNA stability. Both miRNA and lncRNA have recently impacted the field of breast cancer research as important pieces in the mechanistic puzzle of the genes and pathways involved in breast cancer development and progression. This review serves as an overview of the roles of miRNA and lncRNA in breast cancer progression and the development of endocrine resistance. Ideally, future experiments in the field should include identification of ncRNAs that could be potential therapeutic targets in endocrine-resistant tumors, as well as ncRNA biomarkers that facilitate more tumor-specific treatment options for endocrine-resistant breast cancer patients.

The clinical and functional significance of c-Met in breast cancer: a review

Abstract: c-Met is a receptor tyrosine kinase that upon binding of its ligand, hepatocyte growth factor (HGF), activates downstream pathways with diverse cellular functions that are important in organ development and cancer progression. Anomalous c-Met signalling has been described in a variety of cancer types, and the receptor is regarded as a novel therapeutic target. In breast cancer there is a need to develop new treatments, particularly for the aggressive subtypes such as triple-negative and basal-like cancer, which currently lack targeted therapy. Over the last two decades, much has been learnt about the functional role of c-Met signalling in different models of breast development and cancer. This work has been complemented by clinical studies, establishing the prognostic significance of c-Met in tissue samples of breast cancer. While the clinical trials of anti-c-Met therapy in advanced breast cancer progress, there is a need to review the existing evidence so that the potential of these treatments can be better appreciated. The aim of this article is to examine the role of HGF/c-Met signalling in in vitro and in vivo models of breast cancer, to describe the mechanisms of aberrant c-Met signalling in human tissues, and to give a brief overview of the anti-c-Met therapies currently being evaluated in breast cancer patients. We will show that the HGF/c-Met pathway is associated with breast cancer progression and suggest that there is a firm basis for continued development of anti-c-Met treatment, particularly for patients with basal-like and triple-negative breast cancer.

The benefit of HER2-targeted therapies on overall survival of patients with metastatic HER2-positive breast cancer – a systematic review

Abstract: This study aimed at evaluating the overall survival (OS) gain associated with human epidermal growth factor receptor 2 (HER2)-directed therapies in patients with metastatic breast cancer (mBC). A bibliographic search was conducted in PubMed and Cochrane databases. Only phase III randomized controlled trials (RCTs) including HER2-positive (HER2+) mBC patients were included in this review. OS was defined as time from randomization until the occurrence of death from any cause. Studies have been grouped according to the line of treatment, i.e., first-line or second-line or beyond. Nineteen RCTs were eligible for inclusion, of which 12 assessed therapies targeting HER2+ mBC in the first-line setting. OS improved from 20.3 months in the first RCT (standard chemotherapy; Slamon et al. (N Engl J Med 344:783–92, 2001)) evaluating HER2-targeting therapies to 48 months in the study of Swain et al. (Lancet Oncol 14:461–71, 2013), with triple combination of pertuzumab, trastuzumab and docetaxel. Seven RCTs evaluated the OS of HER2-targeting therapies in the second-line setting and beyond. The OS in second-line setting improved from 15.3 months (capecitabine; Cameron et al. (Breast Cancer Res Treat 112:533–43, 2008)) to 30.7 months (trastuzumab emtansine; Verma et al. (N Engl J Med 367:1783–91, 2012)). In the third-line setting, the association of lapatinib and trastuzumab has demonstrated to improve OS to 4.5 months compared with lapatinib alone (14 months vs. 9.5 months; Blackwell et al. (J Clin Oncol 30:2585–92, 2012)). HER2-directed therapies had an undeniable beneficial impact on the OS of patients with HER2+ mBC. The triple combination of docetaxel, pertuzumab and trastuzumab is associated with a survival extent of more than 4.5 years, compared with a life expectancy of 1.5 years achieved 14 years ago.

Leptin produced by obese adipose stromal/stem cells enhances proliferation and metastasis of estrogen receptor positive breast cancers.

Abstract: The steady increase in the incidence of obesity among adults has been paralleled with higher levels of obesity-associated breast cancer. While recent studies have suggested that adipose stromal/stem cells (ASCs) isolated from obese women enhance tumorigenicity, the mechanism(s) by which this occurs remains undefined. Evidence suggests that increased adiposity results in increased leptin secretion from adipose tissue, which has been shown to increased cancer cell proliferation. Previously, our group demonstrated that ASCs isolated from obese women (obASCs) also express higher levels of leptin relative to ASCs isolated from lean women (lnASCs) and that this obASC-derived leptin may account for enhanced breast cancer cell growth. The current study investigates the impact of inhibiting leptin expression in lnASCs and obASCs on breast cancer cell (BCC) growth and progression. Estrogen receptor positive (ER+) BCCs were co-cultured with leptin shRNA lnASCs or leptin shRNA obASCs and changes in the proliferation, migration, invasion, and gene expression of BCCs were investigated. To assess the direct impact of leptin inhibition in obASCs on BCC proliferation, MCF7 cells were injected alone or mixed with control shRNA obASCs or leptin shRNA obASCs into SCID/beige mice. ER+ BCCs were responsive to obASCs during direct co-culture, whereas lnASCs were unable to increase ER+ BCC growth. shRNA silencing of leptin in obASCs negated the enhanced proliferative effects of obASC on BCCs following direct co-culture. BCCs co-cultured with obASCs demonstrated enhanced expression of epithelial-to-mesenchymal transition (EMT) and metastasis genes (SERPINE1, MMP-2, and IL-6), while BCCs co-cultured with leptin shRNA obASCs did not display similar levels of gene induction. Knockdown of leptin significantly reduced tumor volume and decreased the number of metastatic lesions to the lung and liver. These results correlated with reduced expression of both SERPINE1 and MMP-2 in tumors formed with MCF7 cells mixed with leptin shRNA obASCs, when compared to tumors formed with MCF7 cells mixed with control shRNA obASCs. This study provides mechanistic insight as to how obesity enhances the proliferation and metastasis of breast cancer cells; specifically, obASC-derived leptin contributes to the aggressiveness of breast cancer in obese women.

Molecular characterization and targeted therapeutic approaches in breast cancer

Abstract: Despite the wide improvements in breast cancer (BC) detection and adjuvant treatment, BC is still responsible for approximately 40,000 deaths annually in the United States. Novel biomarkers are fundamental to assist clinicians in BC detection, risk stratification, disease subtyping, prediction of treatment response, and surveillance, allowing a more tailored approach to therapy in both primary and metastatic settings. In primary BC, the development of molecular profiling techniques has added prognostic and predictive information to conventional biomarkers - estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Moreover, the application of next-generation sequencing and reverse-phase protein microarray methods in the metastatic setting holds the promise to further advance toward a personalized management of cancer. The improvement in our understanding on BC biology associated with the study of the genomic aberrations characterizing the most common molecular subtypes allows us to explore new targets for drug development. Finally, the integration of cancer stem cell-targeted therapies and immune therapies in future combination regimens increases our chances to successfully treat a larger proportion of women with more aggressive and resistant metastatic disease. This article reviews the current state of novel biological markers for BC, the evidence to demonstrate their clinical validity and utility, and the implication for therapeutic targeting.

RANK-ligand (RANKL) expression in young breast cancer patients and during pregnancy

Abstract: RANKL is important in mammary gland development during pregnancy and mediates the initiation and progression of progesterone-induced breast cancer. No clinical data are available on the effect of pregnancy on RANK/RANKL expression in young breast cancer patients. We used our previously published dataset of 65 pregnant and 130 matched young breast cancer patients with full clinical, pathological, and survival information. 85% of patients had available transcriptomic data as well. RANK/RANKL expression by immunohistochemistry using H-score on the primary tumor and adjacent normal tissue was performed. We examined the difference in expression of RANK/RANKL between pregnant and non-pregnant patients and their association with clinicopathological features and prognosis. We also evaluated genes and pathways associated with RANK/RANKL expression on primary tumors. RANKL but not RANK expression was more prevalent in the pregnant group, both on the tumor and adjacent normal tissue, independent of other clinicopathological factors (both P <0.001). 18.7% of pregnant and 5.3% of non-pregnant patients had tumors showing ≥10% of cells with 3+ RANKL expression. RANKL expression was significantly higher in progesterone receptor-positive, and luminal A-like tumors, with negative correlation with Ki-67 (all P <0.001). On the contrary, RANK expression was higher in triple negative tumors (P <0.001). Using false discovery rate <0.05, 151 and 1,207 genes were significantly correlated with tumor-expressed RANKL and RANK expression by immunohistochemistry, respectively. High RANKL expression within primary tumor was associated with pathways related to mammary gland development, bone resorption, T-cell proliferation and regulation of chemotaxis, while RANK expression was associated with immune response and proliferation pathways. At a median follow-up of 65 months, neither RANK nor RANKL expression within tumor was associated with disease free survival in pregnant or non-pregnant group. Pregnancy increases RANKL expression both in normal breast and primary tumors. These results could guide further development of RANKL-targeted therapy.

Histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), enhances anti-tumor effects of the poly (ADP-ribose) polymerase (PARP) inhibitor olaparib in triple-negative breast cancer cells

Abstract: Olaparib, a poly (ADP-ribose) polymerase (PARP) inhibitor, has been found to have therapeutic potential for treating cancers associated with impaired DNA repair capabilities, particularly those with deficiencies in the homologous recombination repair (HRR) pathway. Histone deacetylases (HDACs) are important for enabling functional HRR of DNA by regulating the expression of HRR-related genes and promoting the accurate assembly of HRR-directed sub-nuclear foci. Thus, HDAC inhibitors have recently emerged as a therapeutic agent for treating cancer by inhibiting DNA repair. Based on this, HDAC inhibition could be predicted to enhance the anti-tumor effect of PARP inhibitors in cancer cells by blocking the HRR pathway. We determined whether suberoylanilide hydroxamic acid (SAHA), a HDAC inhibitor, could enhance the anti-tumor effects of olaparib on breast cancer cell lines using a cytotoxic assay, cell cycle analysis, and Western blotting. We evaluated how exposure to SAHA affects the expression of HRR-associated genes. The accumulation of DNA double strand breaks (DSBs) induced by combination treatment was assessed. Induction of autophagy was monitored by imaging green fluorescent protein-tagged microtubule-associated protein 1A/1B-light chain 3 (LC3) expression following co-treatment with olaparib and SAHA. These in vitro data were validated in vivo using a human breast cancer xenograft model. Triple-negative breast cancer cell (TNBC) lines showed heterogeneous responses to the PARP and HDAC inhibitors. Co-administration of olaparib and SAHA synergistically inhibited the growth of TNBC cells that expressed functional Phosphatase and tensin homolog (PTEN). This effect was associated with down-regulation of the proliferative signaling pathway, increased apoptotic and autophagic cell death, and accumulation of DNA damage. The combined anti-tumor effect of olaparib and SAHA was also observed in a xenograft model. These data suggest that PTEN expression in TNBC cells can sensitize the cell response to simultaneous inhibition of PARP and HDAC both in vitro and in vivo. Our findings suggest that expression of functional PTEN may serve as a biomarker for selecting TNBC patients that would favorably respond to a combination of olaparib with SAHA. This provides a strong rationale for treating TNBC patients with PTEN expression with a combination therapy consisting of olaparib and SAHA.

Lobular breast cancer: incidence and genetic and non-genetic risk factors

Abstract: While most invasive breast cancers consist of carcinomas of the ductal type, about 10% are invasive lobular carcinomas. Invasive lobular and ductal carcinomas differ with respect to risk factors. Invasive lobular carcinoma is more strongly associated with exposure to female hormones, and therefore its incidence is more subject to variation. This is illustrated by US figures during the 1987 to 2004 period: after 12 years of increases, breast cancer incidence declined steadily from 1999 to 2004, reflecting among other causes the decreasing use of menopausal hormone therapy, and these variations were stronger for invasive lobular than for invasive ductal carcinoma. Similarly, invasive lobular carcinoma is more strongly associated with early menarche, late menopause and late age at first birth. As for genetic risk factors, four high-penetrance genes are tested in clinical practice when genetic susceptibility to breast cancer is suspected, BRCA1, BRCA2, TP53 and CDH1. Germline mutations in BRCA1 and TP53 are predominantly associated with invasive ductal carcinoma, while BRCA2 mutations are associated with both ductal and lobular cancers. CDH1, the gene coding for the E-cadherin adhesion protein, is of special interest as mutations are associated with invasive lobular carcinoma, but never with ductal carcinoma. It was initially known as the main susceptibility gene for gastric cancer of the diffuse type, but the excess of breast cancers of the lobular type in CDH1 families led researchers to identify it also as a susceptibility gene for invasive lobular carcinoma. The risk of invasive lobular carcinoma is high in female mutation carriers, as about 50% are expected to develop the disease. Carriers must therefore undergo intensive breast cancer screening, with, for example, yearly magnetic resonance imaging and mammogram starting at age 30 years.

High mammographic density is associated with an increase in stromal collagen and immune cells within the mammary epithelium

Abstract: Mammographic density (MD), after adjustment for a women’s age and body mass index, is a strong and independent risk factor for breast cancer (BC). Although the BC risk attributable to increased MD is significant in healthy women, the biological basis of high mammographic density (HMD) causation and how it raises BC risk remain elusive. We assessed the histological and immunohistochemical differences between matched HMD and low mammographic density (LMD) breast tissues from healthy women to define which cell features may mediate the increased MD and MD-associated BC risk. Tissues were obtained between 2008 and 2013 from 41 women undergoing prophylactic mastectomy because of their high BC risk profile. Tissue slices resected from the mastectomy specimens were X-rayed, then HMD and LMD regions were dissected based on radiological appearance. The histological composition, aromatase immunoreactivity, hormone receptor status and proliferation status were assessed, as were collagen amount and orientation, epithelial subsets and immune cell status. HMD tissue had a significantly greater proportion of stroma, collagen and epithelium, as well as less fat, than LMD tissue did. Second harmonic generation imaging demonstrated more organised stromal collagen in HMD tissues than in LMD tissues. There was significantly more aromatase immunoreactivity in both the stromal and glandular regions of HMD tissues than in those regions of LMD tissues, although no significant differences in levels of oestrogen receptor, progesterone receptor or Ki-67 expression were detected. The number of macrophages within the epithelium or stroma did not change; however, HMD stroma exhibited less CD206+ alternatively activated macrophages. Epithelial cell maturation was not altered in HMD samples, and no evidence of epithelial–mesenchymal transition was seen; however, there was a significant increase in vimentin+/CD45+ immune cells within the epithelial layer in HMD tissues. We confirmed increased proportions of stroma and epithelium, increased aromatase activity and no changes in hormone receptor or Ki-67 marker status in HMD tissue. The HMD region showed increased collagen deposition and organisation as well as decreased alternatively activated macrophages in the stroma. The HMD epithelium may be a site for local inflammation, as we observed a significant increase in CD45+/vimentin+ immune cells in this area.

Lobular breast cancer series: imaging

Abstract: The limitations of mammography in the detection and evaluation of invasive lobular carcinoma (ILC) have long been recognized, presenting real clinical challenges in treatment planning for these tumors. However, advances in mammography, ultrasound, and magnetic resonance imaging present opportunities to improve the diagnosis and preoperative assessment of ILC. The evidence supporting the performance of each imaging modality will be reviewed, specifically as it relates to the pathology of ILC and its subtypes. Further, we will discuss emerging technologies that may be employed to enhance the detection rate and ultimately result in more effective screening and staging of ILC.

Effect of physical activity on sex hormones in women: a systematic review and meta-analysis of randomized controlled trials

Abstract: Introduction Exposure to high levels of endogenous estrogens is a main risk factor for breast cancer in women, and in observational studies was found to be inversely associated with physical activity. The objective of the present study is to determine the effect of physical activity interventions on sex hormone levels in healthy women.

Changes in insulin receptor signaling underlie neoadjuvant metformin administration in breast cancer: a prospective window of opportunity neoadjuvant study

Abstract: The antidiabetic drug metformin exhibits potential anticancer properties that are believed to involve both direct (insulin-independent) and indirect (insulin-dependent) actions. Direct effects are linked to activation of AMP-activated protein kinase (AMPK) and an inhibition of mammalian target of rapamycin mTOR signaling, and indirect effects are mediated by reductions in circulating insulin, leading to reduced insulin receptor (IR)-mediated signaling. However, the in vivo impact of metformin on cancer cell signaling and the factors governing sensitivity in patients remain unknown. We conducted a neoadjuvant, single-arm, “window of opportunity” trial to examine the clinical and biological effects of metformin on patients with breast cancer. Women with untreated breast cancer who did not have diabetes were given 500 mg of metformin three times daily for ≥2 weeks after diagnostic biopsy until surgery. Fasting blood and tumor samples were collected at diagnosis and surgery. Blood glucose and insulin were assayed to assess the physiologic effects of metformin, and immunohistochemical analysis of tumors was used to characterize cellular markers before and after treatment. Levels of IR expression decreased significantly in tumors (P = 0.04), as did the phosphorylation status of protein kinase B (PKB)/Akt (S473), extracellular signal-regulated kinase 1/2 (ERK1/2, T202/Y204), AMPK (T172) and acetyl coenzyme A carboxylase (S79) (P = 0.0001, P < 0.0001, P < 0.005 and P = 0.02, respectively). All tumors expressed organic cation transporter 1, with 90% (35 of 39) exhibiting an Allred score of 5 or higher. Reduced PKB/Akt and ERK1/2 phosphorylation, coupled with decreased insulin and IR levels, suggest insulin-dependent effects are important in the clinical setting. These results are consistent with beneficial anticancer effects of metformin and highlight key factors involved in sensitivity, which could be used to identify patients with breast cancer who may be responsive to metformin-based therapies. ClinicalTrials.gov identifier: NCT00897884 . Registered 8 May 2009.

Adipose cells promote resistance of breast cancer cells to trastuzumab-mediated antibody-dependent cellular cytotoxicity.

Abstract: Trastuzumab has been used in the treatment of human epidermal growth factor receptor 2 (HER2)-expressing breast cancer, but its efficacy is limited by de novo or acquired resistance. Although many mechanisms have been proposed to explain resistance to trastuzumab, little is known concerning the role of the tumor microenvironment. Given the importance of antibody-dependent cellular cytotoxicity (ADCC) in the antitumor effect of trastuzumab and the abundance of adipose tissue in the breast, we investigated the impact of adipocytes on ADCC. We set up a coculture system to study the effect of adipocytes on ADCC in vitro. The results were validated in vivo in a mouse xenograft model. We found that adipocytes, as well as preadipocytes, inhibited trastuzumab-mediated ADCC in HER2-expressing breast cancer cells via the secretion of soluble factors. The inhibition of ADCC was not due to titration or degradation of the antibody. We found that adipose cells decreased the secretion of interferon-γ by natural killer cells, but did not alter natural killer cells’ cytotoxicity. Preincubation of breast cancer cells with the conditioned medium derived from adipocytes reduced the sensitivity of cancer cells to ADCC. Using a transcriptomic approach, we found that cancer cells undergo major modifications when exposed to adipocyte-conditioned medium. Importantly, breast tumors grafted next to lipomas displayed resistance to trastuzumab in mouse xenograft models. Collectively, our findings underline the importance of adipose tissue in the resistance to trastuzumab and suggest that approaches targeting the adipocyte–cancer cell crosstalk may help sensitize cancer cells to trastuzumab-based therapy.

Circulating DNA as biomarker in breast cancer

Abstract: As the release of tumor-associated DNA into blood circulation is a common event in patients with cancer, screening of plasma or serum DNA may provide information on genetic and epigenetic profiles associated with breast cancer development, progression, and response to therapy. Quantitative testing of circulating DNA can reflect tumor burden, and molecular characterization of circulating DNA can reveal important tumor characteristics relevant to the choice of targeted therapies in individual patients. Contrary to circulating DNA from blood that presents molecular changes in tumor DNA in real time, tissue biopsies can deliver only a spatially and temporally limited snapshot of the heterogeneous tumor. Analyses of circulating DNA might provide prognostic and predictive information and therefore advance personalized medicine. However, standardization of different technical platforms as well as the control of pre-analytical and analytical factors is mandatory before its introduction into clinical practice. In the present review, we discussed technical aspects and clinical relevance of the analyses of circulating plasma/serum DNA in patients with breast cancer.

Acquisition of epithelial-mesenchymal transition phenotype in the tamoxifen-resistant breast cancer cell: a new role for G protein-coupled estrogen receptor in mediating tamoxifen resistance through cancer-associated fibroblast-derived fibronectin and β1-integrin signaling pathway in tumor cells

Abstract: Acquired tamoxifen resistance remains the major obstacle to breast cancer endocrine therapy. β1-integrin was identified as one of the target genes of G protein-coupled estrogen receptor (GPER), a novel estrogen receptor recognized as an initiator of tamoxifen resistance. Here, we investigated the role of β1-integrin in GPER-mediated tamoxifen resistance in breast cancer. The expression of β1-integrin and biomarkers of epithelial-mesenchymal transition were evaluated immunohistochemically in 53 specimens of metastases and paired primary tumors. The function of β1-integrin was investigated in tamoxifen-resistant (MCF-7R) subclones, derived from parental MCF-7 cells, and MCF-7R β1-integrin-silenced subclones in MTT and Transwell assays. Involved signaling pathways were identified using specific inhibitors and Western blotting analysis. GPER, β1-integrin and mesenchymal biomarkers (vimentin and fibronectin) expression in metastases increased compared to the corresponding primary tumors; a close expression pattern of β1-integrin and GPER were in metastases. Increased β1-integrin expression was also confirmed in MCF-7R cells compared with MCF-7 cells. This upregulation of β1-integrin was induced by agonists of GPER and blocked by both antagonist and knockdown of it in MCF-7R cells. Moreover, the epidermal growth factor receptor/extracellular regulated protein kinase (EGFR/ERK) signaling pathway was involved in this transcriptional regulation since specific inhibitors of these kinases also reduced the GPER-induced upregulation of β1-integrin. Interestingly, silencing of β1-integrin partially rescued the sensitivity of MCF-7R cells to tamoxifen and the α5β1-integrin subunit is probably responsible for this phenomenon. Importantly, the cell migration and epithelial-mesenchymal transition induced by cancer-associated fibroblasts, or the product of cancer-associated fibroblasts, fibronectin, were reduced by knockdown of β1-integrin in MCF-7R cells. In addition, the downstream kinases of β1-integrin including focal adhesion kinase, Src and AKT were activated in MCF-7R cells and may be involved in the interaction between cancer cells and cancer-associated fibroblasts. GPER/EGFR/ERK signaling upregulates β1-integrin expression and activates downstream kinases, which contributes to cancer-associated fibroblast-induced cell migration and epithelial-mesenchymal transition, in MCF-7R cells. GPER probably contributes to tamoxifen resistance via interaction with the tumor microenvironment in a β1-integrin-dependent pattern. Thus, β1-integrin may be a potential target to improve anti-hormone therapy responses in breast cancer patients.

The use of the Gail model, body mass index and SNPs to predict breast cancer among women with abnormal (BI-RADS 4) mammograms

Abstract: Mammography screening results in a significant number of false-positives The use of pretest breast cancer risk factors to guide follow-up of abnormal mammograms could improve the positive predictive value of screening We evaluated the use of the Gail model, body mass index (BMI), and genetic markers to predict cancer diagnosis among women with abnormal mammograms We also examined the extent to which pretest risk factors could reclassify women without cancer below the biopsy threshold We recruited a prospective cohort of women referred for biopsy with abnormal (BI-RADS 4) mammograms according to the American College of Radiology’s Breast Imaging-Reporting and Data System (BI-RADS) Breast cancer risk factors were assessed prior to biopsy A validated panel of 12 single-nucleotide polymorphisms (SNPs) associated with breast cancer were measured Logistic regression was used to assess the association of Gail risk factors, BMI and SNPs with cancer diagnosis (invasive or ductal carcinoma in situ) Model discrimination was assessed using the area under the receiver operating characteristic curve, and calibration was assessed using the Hosmer-Lemeshow goodness-of-fit test The distribution of predicted probabilities of a cancer diagnosis were compared for women with or without breast cancer In the multivariate model, age (odds ratio (OR) = 105; 95% confidence interval (CI), 103 to 108; P < 0001), SNP panel relative risk (OR = 230; 95% CI, 106 to 499, P = 0035) and BMI (≥30 kg/m2 versus <25 kg/m2; OR = 220; 95% CI, 105 to 458; P = 0036) were significantly associated with breast cancer diagnosis Older women were more likely than younger women to be diagnosed with breast cancer The SNP panel relative risk remained strongly associated with breast cancer diagnosis after multivariable adjustment Higher BMI was also strongly associated with increased odds of a breast cancer diagnosis Obese women (OR = 220; 95% CI, 105 to 458; P = 0036) had more than twice the odds of cancer diagnosis compared to women with a BMI <25 kg/m2 The SNP panel appeared to have predictive ability among both white and black women Breast cancer risk factors, including BMI and genetic markers, are predictive of cancer diagnosis among women with BI-RADS 4 mammograms Using pretest risk factors to guide follow-up of abnormal mammograms could reduce the burden of false-positive mammograms

Granulocytic immune infiltrates are essential for the efficient formation of breast cancer liver metastases

Abstract: Breast cancer cells display preferences for specific metastatic sites including the bone, lung and liver. Metastasis is a complex process that relies, in part, on interactions between disseminated cancer cells and resident/infiltrating stromal cells that constitute the metastatic microenvironment. Distinct immune infiltrates can either impair the metastatic process or conversely, assist in the seeding, colonization and growth of disseminated cancer cells. Using in vivo selection approaches, we previously isolated 4T1-derived breast cancer cells that preferentially metastasize to these organs and tissues. In this study, we examined whether the propensity of breast cancer cells to metastasize to the lung, liver or bone is associated with and dependent on distinct patterns of immune cell infiltration. Immunohistocytochemistry and immunohistofluorescence approaches were used to quantify innate immune cell infiltrates within distinct metastases and depletion of Gr1+ (Ly-6C and Ly-6G) or specifically Ly-6G+ cells was performed to functionally interrogate the role of Ly-6G+ infiltrates in promoting metastasis to these organs. We show that T lymphocytes (CD3+), myeloid-derived (Gr-1+) cells and neutrophils (Ly-6G+ or NE+) exhibit the most pronounced recruitment in lung and liver metastases, with markedly less recruitment within bone metastatic lesions. Interestingly, these infiltrating cell populations display different patterns of localization within soft tissue metastases. T lymphocytes and granulocytic immune infiltrates are localized around the periphery of liver metastases whereas they were dispersed throughout the lung metastases. Furthermore, Gr-1+ cell-depletion studies demonstrate that infiltrating myeloid-derived cells are essential for the formation of breast cancer liver metastases but dispensable for metastasis to the lung and bone. A specific role for the granulocytic component of the innate immune infiltrate was revealed through Ly-6G+ cell-depletion experiments, which resulted in significantly impaired formation of liver metastases. Finally, we demonstrate that the CD11b+/Ly-6G+ neutrophils that infiltrate and surround the liver metastases are polarized toward an N2 phenotype, which have previously been shown to enhance tumor growth and metastasis. Our results demonstrate that the liver-metastatic potential of breast cancer cells is heavily reliant on interactions with infiltrating Ly-6G+ cells within the liver microenvironment.

The influence of obesity on survival in early, high-risk breast cancer: results from the randomized SUCCESS A trial.

Abstract: Obese breast cancer patients have worse prognosis than normal weight patients, but the level at which obesity is prognostically unfavorable is unclear. This retrospective analysis was performed using data from the SUCCESS A trial, in which 3754 patients with high-risk early breast cancer were randomized to anthracycline- and taxane-based chemotherapy with or without gemcitabine. Patients were classified as underweight/normal weight (body mass index (BMI) < 25.0), overweight (BMI 25.0–29.9), slightly obese (BMI 30.0–34.9), moderately obese (BMI 35.0–39.9) and severely obese (BMI ≥ 40.0), and the effect of BMI on disease-free survival (DFS) and overall survival (OS) was evaluated (median follow-up 65 months). In addition, subgroup analyses were conducted to assess the effect of BMI in luminal A-like, luminal B-like, HER2 (human epidermal growth factor 2)-positive and triple-negative tumors. Multivariate analyses revealed an independent prognostic effect of BMI on DFS (p = 0.001) and OS (p = 0.005). Compared with underweight/normal weight patients, severely obese patients had worse DFS (hazard ratio (HR) 2.70, 95 % confidence interval (CI) 1.71–4.28, p < 0.001) and OS (HR 2.79, 95 % CI 1.63–4.77, p < 0.001), while moderately obese, slightly obese and overweight patients did not differ from underweight/normal weight patients with regard to DFS or OS. Subgroup analyses showed a similar significant effect of BMI on DFS and OS in patients with triple-negative breast cancer (TNBC), but not in patients with other tumor subtypes. Severe obesity (BMI ≥ 40) significantly worsens prognosis in early breast cancer patients, particularly for triple-negative tumors. Clinicaltrials.gov NCT02181101 . Registered September 2005.