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Skeletal metastasis occurs in around 75% of advanced breast cancers, with the disease incurable once cancer cells disseminate to bone, but there remains an unmet need for biomarkers to identify patients at high risk of bone recurrence. This study aimed to identify such a biomarker and to assess its utility in predicting response to adjuvant zoledronic acid (zoledronate). We used quantitative proteomics (stable isotope labelling by amino acids in cell culture‐mass spectrometry; SILAC‐MS) to compare protein expression in a bone‐homing variant (BM1) of the human breast cancer cell line MDA‐MB‐231 with parental non‐bone‐homing cells to identify novel biomarkers for risk of subsequent bone metastasis in early breast cancer. SILAC‐MS showed that dedicator of cytokinesis protein 4 (DOCK4) was upregulated in bone‐homing BM1 cells, confirmed by western blotting. BM1 cells also had enhanced invasive ability compared with parental cells, which could be reduced by DOCK4‐shRNA. In a training tissue microarray (TMA) comprising 345 patients with early breast cancer, immunohistochemistry followed by Cox regression revealed that high DOCK4 expression correlated with histological grade (p = 0.004) but not oestrogen receptor status (p = 0.19) or lymph node involvement (p = 0.15). A clinical validation TMA used tissue samples and the clinical database from the large AZURE adjuvant study (n = 689). Adjusted Cox regression analyses showed that high DOCK4 expression in the control arm (no zoledronate) was significantly prognostic for first recurrence in bone (HR 2.13, 95%CI 1.06–4.30, p = 0.034). No corresponding association was found in patients who received zoledronate (HR 0.812, 95%CI 0.176–3.76, p = 0.790), suggesting that treatment with zoledronate may counteract the higher risk for bone relapse from high DOCK4‐expressing tumours. High DOCK4 expression was not associated with metastasis to non‐skeletal sites when these were assessed collectively. In conclusion, high DOCK4 in early breast cancer is significantly associated with aggressive disease and with future bone metastasis and is a potentially useful biomarker for subsequent bone metastasis risk. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Breast cancer can occur in either gender; however, it is rare in men, accounting for

Purpose: Breast cancer (BC) affects both genders, but is understudied in men. Although still rare, male BC is being diagnosed more frequently. Treatments are wholly informed by clinical studies conducted in women, based on assumptions that underlying biology is similar. Experimental design: A transcriptomic investigation of male and female BC was performed, confirming transcriptomic data in silico. Biomarkers were immunohistochemically assessed in 697 MBCs (n=477, training; n=220, validation set) and quantified in pre- and post-treatment samples from a male BC patient receiving Everolimus and PI3K/mTOR inhibitor. Results: Gender-specific gene expression patterns were identified. eIF transcripts were up-regulated in MBC. eIF4E and eIF5 were negatively prognostic for overall survival alone (Log rank; p=0.013; HR=1.77, 1.12-2.8 and p=0.035; HR=1.68, 1.03-2.74, respectively), or when co-expressed (p=0.01; HR=2.66, 1.26-5.63), confirmed in the validation set. This remained upon multivariate Cox regression analysis (eIF4E p=0.016; HR 2.38 (1.18-4.8), eIF5 p=0.022; HR 2.55 (1.14-5.7); co-expression p=0.001; HR=7.04 (2.22-22.26)). Marked reduction in eIF4E and eIF5 expression was seen post BEZ235/Everolimus, with extended survival. Conclusions: Translational initiation pathway inhibition could be of clinical utility in male BC patients overexpressing eIF4E and eIF5. With mTOR inhibitors which target this pathway now in the clinic, these biomarkers may represent new targets for therapeutic intervention, although further independent validation is required.

Carcinoma‐associated fibroblasts (CAFs) influence the behaviour of cancer cells but the roles of microRNAs in this interaction are unknown. We report microRNAs that are differentially expressed between breast normal fibroblasts and CAFs of oestrogen receptor‐positive cancers, and explore the influences of one of these, miR‐26b, on breast cancer biology. We identified differentially expressed microRNAs by expression profiling of clinical samples and a tissue culture model: miR‐26b was the most highly deregulated microRNA. Using qPCR, miR‐26b was confirmed as down‐regulated in fibroblasts from 15 of 18 further breast cancers. Next, we examined whether manipulation of miR‐26b expression changed breast fibroblast behaviour. Reduced miR‐26b expression caused fibroblast migration and invasion to increase by up to three‐fold in scratch‐closure and trans‐well assays. Furthermore, in co‐culture with MCF7 breast cancer epithelial cells, fibroblasts with reduced miR‐26b expression enhanced both MCF7 migration in trans‐well assays and MCF7 invasion from three‐dimensional spheroids by up to five‐fold. Mass spectrometry was used to identify expression changes associated with the reduction of miR‐26b expression in fibroblasts. Pathway analyses of differentially expressed proteins revealed that glycolysis/TCA cycle and cytoskeletal regulation by Rho GTPases are downstream of miR‐26b. In addition, three novel miR‐26b targets were identified (TNKS1BP1, CPSF7, COL12A1) and the expression of each in cancer stroma was shown to be significantly associated with breast cancer recurrence. MiR‐26b in breast CAFs is a potent regulator of cancer behaviour in oestrogen receptor‐positive cancers, and we have identified key genes and molecular pathways that act downstream of miR‐26b in CAFs. © 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

IntroductionBreast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice.MethodsMore than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer ‘stem’ cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account.ResultsThe 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working.ConclusionsWith resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years.

Background: 3D cell cultures are emerging as more physiologically meaningful alternatives to monolayer cultures for many biological applications. They are attractive because they more closely mimic in vivo morphology, especially when co-cultured with stromal fibroblasts. Methodology/Principal Findings: We compared the efficacy of 3 different 3D cell culture systems; collagen I, low attachment culture vessels and a modification of Fibrolife®, a specialised humanised cell culture medium devoid of animal-derived components, using breast cancer cell lines representative of the different molecular subtypes of breast cancer, cultured alone or with human mammary fibroblasts with a view to developing matrix-free humanised systems. 3D collagen I culture supported the growth of a range of breast cancer cell lines. By modifying the composition of Fibrolife® to epiFL, matrix-free cell culture was possible. During sequential transfer to epiFL breast cancer cells gradually detached from the flask, growing progressively as spheroids. Phenotype was stable and reversible with cells remaining actively proliferating and easily accessible throughout culture. They could also be revived from frozen stocks. To achieve co-culture with fibroblasts in epiFL required use of low attachment culture vessels instead of standard plastic as fibroblasts remained adherent in epiFL. Here, cancer cell spheroids were allowed to form before adding fibroblasts. Immunohistochemical examination showed fibroblasts scattered throughout the epithelial spheroid, not dissimilar to the relationship of tumour stroma in human breast cancer. Conclusions: Because of its ease of handling, matrix-free 3D cell culture may be a useful model to study the influence of fibroblasts on breast cancer epithelial cells with use of epiFL culture medium taking this a step further towards a fully humanised 3D model. This methodology could be applied to other types of cancer cell lines, making this a versatile technique for cancer researchers wishing to use in vitro systems that better reflect cancer in vivo.

Background: Bone is the predominant site of metastasis from breast cancer, and recent trials have demonstrated that adjuvant bisphosphonate therapy can reduce bone metastasis development and improve survival. There is an unmet need for prognostic and predictive biomarkers so that therapy can be appropriately targeted. Methods: Potential biomarkers for bone metastasis were identified using proteomic comparison of bone-metastatic, lung-metastatic, and nonmetastatic variants of human breast cancer MDA-MB-231 cells. Clinical validation was performed using immunohistochemical staining of tumor tissue microarrays from patients in a large randomized trial of adjuvant zoledronic acid (zoledronate) (AZURE-ISRCTN79831382). We used Cox proportional hazards regression, the Kaplan-Meier estimate of the survival function, and the log-rank test to investigate associations between protein expression, clinical variables, and time to distant recurrence events. All statistical tests were two-sided. Results: Two novel biomarker candidates, macrophage-capping protein (CAPG) and PDZ domain–containing protein GIPC1 (GIPC1), were identified for clinical validation. Cox regression analysis of AZURE training and validation sets showed that control patients (no zoledronate) were more likely to develop first distant recurrence in bone (hazard ratio [HR] = 4.5, 95% confidence interval [CI] = 2.1 to 9.8, P < .001) and die (HR for overall survival = 1.8, 95% CI = 1.01 to 3.24, P = .045) if both proteins were highly expressed in the primary tumor. In patients with high expression of both proteins, zoledronate had a substantial effect, leading to 10-fold hazard ratio reduction (compared with control) for first distant recurrence in bone (P = .008). Conclusions: The composite biomarker, CAPG and GIPC1 in primary breast tumors, predicted disease outcomes and benefit from zoledronate and may facilitate patient selection for adjuvant bisphosphonate treatment.

background: The proposed involvement of CD151 in breast cancer (BCa) progression is based on findings from studies in invasive ductal carcinoma (IDC). The IDC and invasive lobular carcinoma (ILC) represent distinct disease entities. Here we evaluated clinical significance of CD151 alone and in association with integrin α3β1 in patients with ILC in context of the data of our recent IDC study. methods: Expression of CD151 and/or integrin α3β1 was evaluated in ILC samples (N=117) using immunohistochemistry. The findings were analysed in relation to our results from an IDC cohort (N=182) demonstrating a prognostic value of an expression of CD151/integrin α3β1 complex in patients with HER2-negative tumours. results: Unlike in the IDCs, neither CD151 nor CD151/α3β1 complex showed any correlation with any of the ILC characteristics. Lack of both CD151 and α3β1 was significantly correlated with poor survival (P=0.034) in lymph node-negative ILC N(−) cases. The CD151−/α3β1− patients had 3.12-fold higher risk of death from BCa in comparison with the rest of the ILC N(−) patients. conclusions: Biological role of CD151/α3β1 varies between ILC and IDC. Assessment of CD151/α3β1 might help to identify ILC N(−) patients with increased risk of distant metastases.

Background MicroRNA (miR) expression is commonly dysregulated in many cancers, including breast. MiR–92 is one of six miRs encoded by the miR-17-92 cluster, one of the best-characterised oncogenic miR clusters. We examined expression of miR–92 in the breast epithelium and stroma during breast cancer progression. We also investigated the role of miR–92 in fibroblasts in vitro and showed that down-regulation in normal fibroblasts enhances the invasion of breast cancer epithelial cells. Methodology/Principal Findings We used laser microdissection (LMD) to isolate epithelial cells from matched normal, DCIS and invasive tissue from 9 breast cancer patients and analysed miR–92 expression by qRT-PCR. Expression of ERβ1, a direct miR–92 target, was concurrently analysed for each case by immunohistochemistry. LMD was also used to isolate matched normal (NFs) and cancer-associated fibroblasts (CAFs) from 14 further cases. Effects of miR–92 inhibition in fibroblasts on epithelial cell invasion in vitro was examined using a Matrigel™ assay. miR– 92 levels decreased in microdissected epithelial cells during breast cancer progression with highest levels in normal breast epithelium, decreasing in DCIS (p