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2.  A novel role for ezrin in breast cancer angio/lymphangiogenesis 
Introduction
Recent evidence suggests that tumour lymphangiogenesis promotes lymph node metastasis, a major prognostic factor for survival of breast cancer patients. However, signaling mechanisms involved in tumour-induced lymphangiogenesis remain poorly understood. The expression of ezrin, a membrane cytoskeletal crosslinker and Src substrate, correlates with poor outcome in a diversity of cancers including breast. Furthermore, ezrin is essential in experimental invasion and metastasis models of breast cancer. Ezrin acts cooperatively with Src in the regulation of the Src-induced malignant phenotype and metastasis. However, it remains unclear if ezrin plays a role in Src-induced tumour angio/lymphangiogenesis.
Methods
The effects of ezrin knockdown and mutation on angio/lymphangiogenic potential of human MDA-MB-231 and mouse AC2M2 mammary carcinoma cell lines were examined in the presence of constitutively active or wild-type (WT) Src. In vitro assays using primary human lymphatic endothelial cells (hLEC), an ex vivo aortic ring assay, and in vivo tumour engraftment were utilized to assess angio/lymphangiogenic activity of cancer cells.
Results
Ezrin-deficient cells expressing activated Src displayed significant reduction in endothelial cell branching in the aortic ring assay in addition to reduced hLEC migration, tube formation, and permeability compared to the controls. Intravital imaging and microvessel density (MVD) analysis of tumour xenografts revealed significant reductions in tumour-induced angio/lymphangiogenesis in ezrin-deficient cells when compared to the WT or activated Src-expressing cells. Moreover, syngeneic tumours derived from ezrin-deficient or Y477F ezrin-expressing (non-phosphorylatable by Src) AC2M2 cells further confirmed the xenograft results. Immunoblotting analysis provided a link between ezrin expression and a key angio/lymphangiogenesis signaling pathway by revealing that ezrin regulates Stat3 activation, VEGF-A/-C and IL-6 expression in breast cancer cell lines. Furthermore, high expression of ezrin in human breast tumours significantly correlated with elevated Src expression and the presence of lymphovascular invasion.
Conclusions
The results describe a novel function for ezrin in the regulation of tumour-induced angio/lymphangiogenesis promoted by Src in breast cancer. The combination of Src/ezrin might prove to be a beneficial prognostic/predictive biomarker for early-stage metastatic breast cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0438-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-014-0438-2
PMCID: PMC4303119  PMID: 25231728
3.  Automated Quantitative Analysis of p53, Cyclin D1, Ki67 and pERK Expression in Breast Carcinoma Does Not Differ from Expert Pathologist Scoring and Correlates with Clinico-Pathological Characteristics 
Cancers  2012;4(3):725-742.
There is critical need for improved biomarker assessment platforms which integrate traditional pathological parameters (TNM stage, grade and ER/PR/HER2 status) with molecular profiling, to better define prognostic subgroups or systemic treatment response. One roadblock is the lack of semi-quantitative methods which reliably measure biomarker expression. Our study assesses reliability of automated immunohistochemistry (IHC) scoring compared to manual scoring of five selected biomarkers in a tissue microarray (TMA) of 63 human breast cancer cases, and correlates these markers with clinico-pathological data. TMA slides were scanned into an Ariol Imaging System, and histologic (H) scores (% positive tumor area x staining intensity 0–3) were calculated using trained algorithms. H scores for all five biomarkers concurred with pathologists’ scores, based on Pearson correlation coefficients (0.80–0.90) for continuous data and Kappa statistics (0.55–0.92) for positive vs. negative stain. Using continuous data, significant association of pERK expression with absence of LVI (p = 0.005) and lymph node negativity (p = 0.002) was observed. p53 over-expression, characteristic of dysfunctional p53 in cancer, and Ki67 were associated with high grade (p = 0.032 and 0.0007, respectively). Cyclin D1 correlated inversely with ER/PR/HER2-ve (triple negative) tumors (p = 0.0002). Thus automated quantitation of immunostaining concurs with pathologists’ scoring, and provides meaningful associations with clinico-pathological data.
doi:10.3390/cancers4030725
PMCID: PMC3712710  PMID: 24213463
breast cancer; p53/cyclin D1/Ki67/pERK; tissue microarray; automated image analysis; clinico-pathological parameters
4.  Neutropenia Prediction Based on First-Cycle Blood Counts Using a FOS-3NN Classifier 
Advances in Bioinformatics  2012;2011:172615.
Background. Delivery of full doses of adjuvant chemotherapy on schedule is key to optimal breast cancer outcomes. Neutropenia is a serious complication of chemotherapy and a common barrier to this goal, leading to dose reductions or delays in treatment. While past research has observed correlations between complete blood count data and neutropenic events, a reliable method of classifying breast cancer patients into low- and high-risk groups remains elusive. Patients and Methods. Thirty-five patients receiving adjuvant chemotherapy for early-stage breast cancer under the care of a single oncologist are examined in this study. FOS-3NN stratifies patient risk based on complete blood count data after the first cycle of treatment. All classifications are independent of breast cancer subtype and clinical markers, with risk level determined by the kinetics of patient blood count response to the first cycle of treatment. Results. In an independent test set of patients unseen by FOS-3NN, 19 out of 21 patients were correctly classified (Fisher's exact test probability P < 0.00023 [2 tailed], Matthews' correlation coefficient +0.83). Conclusions. We have developed a model that accurately predicts neutropenic events in a population treated with adjuvant chemotherapy in the first cycle of a 6-cycle treatment.
doi:10.1155/2011/172615
PMCID: PMC3290820  PMID: 22454638
5.  Immunohistochemical Assessment of Expression of Centromere Protein—A (CENPA) in Human Invasive Breast Cancer 
Cancers  2011;3(4):4212-4227.
Abnormal cell division leading to the gain or loss of entire chromosomes and consequent genetic instability is a hallmark of cancer. Centromere protein –A (CENPA) is a centromere-specific histone-H3-like variant gene involved in regulating chromosome segregation during cell division. CENPA is one of the genes included in some of the commercially available RNA based prognostic assays for breast cancer (BCa)—the 70 gene signature MammaPrint® and the five gene Molecular Grade Index (MGISM). Our aim was to assess the immunohistochemical (IHC) expression of CENPA in normal and malignant breast tissue. Clinically annotated triplicate core tissue microarrays of 63 invasive BCa and 20 normal breast samples were stained with a monoclonal antibody against CENPA and scored for percentage of visibly stained nuclei. Survival analyses with Kaplan–Meier (KM) estimate and Cox proportional hazards regression models were applied to assess the associations between CENPA expression and disease free survival (DFS). Average percentage of nuclei visibly stained with CENPA antibody was significantly higher (p = 0.02) in BCa than normal tissue. The 3-year DFS in tumors over-expressing CENPA (>50% stained nuclei) was 79% compared to 85% in low expression tumors (<50% stained nuclei). On multivariate analysis, IHC expression of CENPA showed weak association with DFS (HR > 60.07; p = 0.06) within our small cohort. To the best of our knowledge, this is the first published report evaluating the implications of increased IHC expression of CENPA in paraffin embedded breast tissue samples. Our finding that increased CENPA expression may be associated with shorter DFS in BCa supports its exploration as a potential prognostic biomarker.
doi:10.3390/cancers3044212
PMCID: PMC3763419  PMID: 24213134
CENPA; breast cancer; immunohistochemistry

Results 1-5 (5)